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Circulating Agonist Autoantibody to 5-Hydroxytryptamine 2A Receptor in Lean and Diabetic Fatty Zucker Rat Strains

DOI: 10.31038/EDMJ.2020435

Abstract

Aims: Circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor were increased in older adult type 2 diabetes in association with certain neurodegenerative complications. The male Zucker diabetic fatty (ZDF) rat is a model system for studies of obese, type 2 diabetes mellitus. The aim of the current study was to test for (and compare) circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor in the Zucker diabetic fatty rat and age-matched lean Zucker rat strains.

Methods: Plasma from lean and Zucker diabetic fatty rat (obtained at different developmental stages) was subjected to protein G affinity chromatography. The resulting immunoglobulin G fraction was tested for neurotoxicity (acute neurite retraction, accelerated neuron loss) in N2A mouse neuroblastoma cells and for binding to a linear synthetic peptide corresponding to the second extracellular loop of the 5-hydroxytryptamine 2A receptor.

Results: The male Zucker diabetic fatty rat (fa/fa) and two Zucker lean strains (+/?) and (fa/+) harbored autoantibodies to the 5-hydroxytryptamine 2A receptor which appeared spontaneously around 7-8.5 weeks of age. The circulating autoantibodies persisted until at least 25 weeks of age in the Zucker diabetic fatty rat and in the Zucker heterozygote (fa/+), but were no longer detectable in 25-week-old lean (+/?) Zucker rats. Autoantibody-induced acute neurite retraction and accelerated loss in mouse neuroblastoma N2A cells was dose-dependently prevented by selective antagonists of the 5-hydroxytryptamine 2A receptor. It was also substantially prevented by co-incubation with antagonists of RhoA/Rho kinase-mediated signaling (Y27632) or Gq11/phospholipase C/inositol triphosphate receptor-coupled signaling.

Conclusions: These data suggest that neurotoxic 5-hydroxytryptamine 2A receptor-targeting autoantibodies increase in the aging male Zucker diabetic fatty rat and in male Zucker lean rats harboring a heterozygous mutation, but not in age-matched, older Zucker lean rats lacking a known leptin receptor mutation. The Zucker genetic strain may be useful in studies of the role of humoral and/or innate immunity in late neurodegeneration.

Introduction

Type 2 diabetes mellitus is associated with an increased risk of late neurodegeneration [1,2] via mechanisms which may involve (in part) increased peripheral and central inflammation. Increased pro-inflammatory cytokines and innate immunity have been associated with early Parkinson’s disease [3] and dementia [4] through complex mechanisms. Leptin is a hormone released by fat cells that is important in energy metabolism. Dysfunction of the leptin system results in classic signs of type 2 diabetes mellitus, such as obesity, insulin resistance and high circulating insulin. Leptin is also a member of the type 1 cytokine family that includes interleukin-6 [5,6]. The leptin receptor is normally expressed on hematopoietic cells where it stimulates helper- T cell, and effector T cell functions and inhibits regulatory T cell function [7].

The male Zucker Diabetic Fatty rat (fa/fa) (ZDF) is a well-recognized animal model of obese type 2 diabetes mellitus and hypertension [8,9] in which hyperphagia causes morbid obesity leading to high level of pro-inflammatory adipocytokines [10]. In the ZDF rat, pro-inflammatory cytokines cause enhanced innate immunity [11], however, owing to its complete leptin receptor deficiency, helper T-cell function is reduced [12]. The lean heterozygous Zucker rat (fa/+) has haplo-sufficiency at the leptin receptor and a two-fold increased plasma leptin concentration [13] compared to the wild-type (+/+) lean Zucker rat. Since hyperleptinemia promoted helper-T cell-mediated autoantibody formation and worsened clinical outcome(s) in a mouse model of systemic lupus erythematosus [14], the lean heterozygote Zucker rat might be expected to exhibit higher autoantibody expression than in the ZDF (fa/fa) rat strain.

In prior studies in older adult (human) obese type 2 diabetes, patients harboring increased circulating autoantibodies to the 5-hydroxytryptamine 2A receptor (5-HT2AR) had an increased prevalence of co-morbid neurodegenerative complications including Parkinson’s disease and dementia [15]. Human plasma autoantibodies targeting the 5-HT2A receptor promoted long-lasting (agonistic) activation of Gq11/phospholipase C/inositol triphosphate receptor/Ca2+ signaling causing accelerated endothelial cell and neuronal cell death [16]. Since the 5-hydroxytryptamine 2A receptor is normally expressed on arterial smooth muscle cells [17] and on neurons in specific brain regions involved in the regulation of mood, thinking, perception, and sleep [18], altered signaling at the 5-HT2A receptor could have diverse peripheral and central effects.

The aim of the present study was to test for spontaneously-occurring autoantibodies to the 5-HT2A receptor in the circulation in male ZDF rat (fa/fa) and age-matched male lean Zucker rats either lacking a known leptin receptor mutation (+/?) or harboring a heterozygous leptin receptor mutation (fa/+). The developmental expression of autoantibody was compared in each genetic strain, and the mechanism of neurotoxicity downstream of 5-HT2AR binding was evaluated in mouse neuroblastoma N2A cells.

Materials and Methods

Animals

All procedures were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Medical Center (East Orange, New Jersey). Male ZDF (N=12) and lean (+/?) Zucker rats (N=12) were obtained from Charles River Laboratories (Kingston, NY) at approximately 6 weeks of age. All rats were single housed upon arrival, with modest enrichment (a PVC tube). Rats were provided ad libitum access to food and water and maintained in a 12 h light/dark cycle with lights on at 0630. All procedures occurred during the light phase of the cycle.

Blood Drawing

EDTA plasma from 10-week ZDF rat and 10-week lean, heterozygous (fa/+) Zucker rats was obtained from Charles River Laboratories. For all other time points, blood was collected from the retro-orbital plexus of ZDF, and lean (+/?) Zucker rats using a retro-orbital blood procedure. The blood was centrifuged at 1400 g x 10 minutes at 4 degrees C. The resulting plasma was stored at -4^oC for up to 2-3 weeks prior to protein G affinity chromatography.

Tail-Nick

Capillary blood glucose was determined from a drop of blood obtained after tail nick at sequential time intervals. A glucose dehydrogenase method (Accucheck Aviva, Roche Diagnostics, Inc) was used for the determination of blood glucose.

Protein G Affinity Chromatography

Protein-G affinity chromatography was carried out as previously reported for protein-A affinity chromatography in human plasma [16]. The pH of the 10 mm Tris binding buffer (6.5) and the 0.1 M citrate elution buffer (2.7) was adjusted to optimize binding and elution of rat immunoglobulin G. Protein-G eluate fractions were stored at 0-4 degrees C.

Mouse N2A Neuroblastoma Cells

Cells were cultured in DMEM with 10% fetal calf serum as previously reported [16].

Neurite Retraction and N2A Cell Survival Assays

Assays were performed as previously described [16].

Enzyme-Linked Immunoassay (ELISA)

The ELISA procedure was carried out as previously reported using an 18-meric linear synthetic peptide Q..N18 corresponding to the second extracellular loop of the human 5-HT2AR as the solid phase antigen [15].

Chemicals

All chemicals were obtained from Sigma Co., Inc. (St. Louis, MO).

Protein determination-Protein levels were determined using a bichinchonic assay method (BioRad, Inc.).

Statistics

Statistical analysis was performed using an unpaired Student’s t-test with an α cutoff level for significance of < 0.05.

Results

Occurrence of Circulating 5-HT2A Receptor Autoantibody in the ZDF Rat: Relation to Obesity and Diabetes

Mean body mass in ZDF rats significantly exceeded that in lean (+/?) Zucker rat at week 7, week 8.5, and week 12 (Figure 1A). There was no longer a significant weight difference between the strains at week 25 (Figure 1A). Diabetes (hyperglycemia) emerged between week 8-10 in the ZDF rat, but at week 7 the ZDF rat was still normoglycemic (Figure 1B). In contrast, lean (+/?) Zucker rats were normoglycemic at all ages tested (Figure 1B), not tested at week 7.

Figure 1: Age-dependent change in body mass(A) and capillary blood glucose concentration (B) in male Zucker fatty (ZDF) vs lean (+/?) (ZDL) rats. A) Body weight increased significantly in the male ZDF rat (starting at week 7) compared to control lean Zucker rat B) capillary blood glucose was determined by tail nick using glucose dehydrogenase method as described in Materials and Methods. Glucose was normal at week 7 in the ZDF rat, but increased significantly compared to ZDL rat starting at week 8.

Detection of plasma immunoreactive 5-HT2AR autoantibody had a similar trajectory to diabetes in ZDF rats. At 7 weeks of age, ZDF rat had no detectable plasma immunoreactive 5-HT2AR autoantibody (Figure 1C). At 10-weeks of age, plasma from ZDF rat contained a two-fold, higher binding to 5-HT2AR (0.14 AU, N=2) compared to background levels (0.07 AU; Figure 2a). Plasma from lean ((+/?) Zucker rats harbored two-fold or slightly higher level of autoantibody binding to 5-HT2AR between 8.5-15 weeks of age which declined to undetectable levels at 25 weeks of age (Figure 2B).

Figure 2: Developmental expression of autoantibody binding to 5-HT2AR synthetic peptide in three Zucker rat strains. A 2 ug/mL concentration of the protein G eluate was tested for binding to the linear synthetic 18-meric 5-HT2A receptor peptide as described in Materials and Methods. Results are “mean +/- SE” in A) ZDF rat: 7-week (N=2); 8.5-week (N=3); 10-week (N=2); 25-week (N=3) B) lean (+/?) Zucker rat: 7-week (N=3); 8.5 week (N=3); 15-week (N=10); 25-week (N=6). C) lean heterozygote (fa/+) Zucker rat: 7-week (N=2); 10-week (N=5); 25-week (N=2).

Spontaneous IgG autoantibody to the 5-HT2A receptor was already present at 7 weeks of age in the heterozygous lean (fa/+) Zucker rat (Figure 2C) and levels at 10-weeks old and 25-weeks old were substantially higher than in age-matched ZDF rat (Figure2C). Autoantibody to the 5-HT2A receptor persisted into older age, i.e. 25 weeks old, in male ZDF and lean heterozygous Zucker rats, but disappeared at 25 weeks of age in the lean (+/?) Zucker rat lacking a known leptin receptor mutation. In a recent report [19], diabetes, obesity and hypertension were associated with substantially increased hazard rates for the occurrence of dementia, Parkinson’s disease and severe depression following traumatic brain injury in older adult veterans.

The present data suggest that male ZDF and heterozygous lean rat strains may be useful in modeling the effects of heightened innate and/or adaptive immunity, respectively, on the occurrence of neurodegeneration.

Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Inhibits N2A Cell Survival

We next tested for neurotoxicity in the autoantibody (from 10- week-old ZDF rat plasma) that displayed increased binding to the 5-HT2A receptor peptide. Autoantibody in plasma from the 10-week old ZDF rat caused dose-dependent inhibition of survival in N2A mouse neuroblastoma cells (Figure 3). A two and one-half microgram per milliliter concentration of the IgG caused approximately 40% neuroblastoma cell loss (after 24 hours incubation) compared to control, untreated cells (P< 0.01, Figure 3).

Figure 3: Dose-dependent neurotoxicity in the protein G eluate fraction from Zucker diabetes fatty rat plasma. The protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells (at the indicated concentrations) for 24 hours. % N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of quadruplicate determinations; * P < 0.01 compared to cell survival in untreated cells.

Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Causes Acute N2A Neurite Retraction

A 120 nanomolar concentration (1.8 g/mL IgG) of the protein G- eluate fraction of plasma from 10-week-old male ZDF fatty rat caused significant 37% acute neurite length-shortening (after 15 minutes of incubation) of N2A mouse neuroblastoma cells (Figure 4A). Co-incubation with a 200 nanomolar concentration of M100907, a highly selective, potent antagonist of the 5-HT2AR, afforded 86% significant (P < 0.01) protection against autoantibody-induced neurite retraction (Figure 4A).

Figure 4: Neurite outgrowth inhibition (A) and accelerated N2A neuroblastoma cell loss (B) in autoantibody from ZDF rat plasma: neutralization by M100907, a highly selective 5-HT2AR antagonist. A) A 120 nM concentration of the protein-G eluate fraction of 10-week-old male ZDF rat plasma was incubated with N2A mouse neuroblastoma cells in the presence or absence of a 200 nM concentration of the selective 5-HT2AR antagonist M100907. % Basal neurite length was determined after 15 minutes as described in Materials and Methods B) A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of M100907 for 24 hours at 37 deg C. Percent N2A cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

Pharmacologic Profile of Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Toxicity

A ~170 nanomolar concentration (2.5 µg/mL) of the protein G-eluate fraction of plasma from 10-week-old male ZDF rat was incubated with N2A mouse neuroblastoma cells in the presence or absence of a (500 or 1000) nanomolar concentration of M100907 for 24 hours at 37 deg C. M100907 caused significant (dose-dependent) protection against accelerated N2A cell loss, after 24 hours incubation with the ZDF rat IgG (Figure 4B).

Spiperone is a potent, but less selective 5-HT2AR antagonist. Spiperone at 500-1000 nanomolar concentrations afforded significant dose-dependent protection against ZDF rat IgG-induced accelerated N2A cell loss (Figure 5). Ketanserin is a less potent 5-HT2AR antagonist compared to M100907 or spiperone. Substantially higher concentration (2.5 µM) of ketanserin was required to significantly protect against ZDF rat IgG-induced N2A cell loss (Figure 6).

Figure 5: Potent, less selective 5-HT2AR antagonist (spiperone) dose-dependently prevented accelerated neuron loss induced by ZDF rat plasma autoantibody. A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of spiperone for 24 hours at 37 deg C. Percent N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

Figure 6: Higher concentration of ketanserin, a less potent, but selective 5-HT2AR antagonist dose-dependently prevented accelerated neuron loss induced by ZDF rat plasma autoantibody A-B) 120 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of ketanserin for 24 hours at 37 deg C. Percent N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

The receptor antagonist profile and concentration associated with significant protection against ZDF IgG autoantibody-induced neuroblastoma cell loss is consistent with relative affinity constants on the 5-HT2A receptor (M100907 < spiperone<< ketanserin).

A one micromolar concentrations of SB-20471, a selective antagonist to the 5-HT2B receptor did not significantly prevent ZDF autoantibody-induced neurite retraction (Table 1). A ten micromolar concentration of either bosentan (an endothelin-1 receptor) or losartan (an angiotensin II type 1 receptor) antagonist did not significantly protect against ZDF IgG-induced acute N2A neurite retraction (Table 1). An 850 nanomolar concentration of prazosin (alpha 1 adrenergic R antagonist) did not afford significant protection against ZDF IgG autoantibody-induced acute neurite retraction (Table 1). Taken together, these data suggest that neurotoxicity in the 10-week old male ZDF rat IgG appears selective for the 5-HT2A receptor.

Table 1: Receptor specificity of prevention of ZDF rat IgG autoantibody-induced acute N2A neurite retraction.

Antagonist	Conc	GPCR	% of ZDF IgG-induced Neurite Retraction
M100907	200 nM	5HT2A	13% + 15%
SB-20471	1 µM	5HT2B	89% + 20%
Bosentan	10 µM	ET-1	95% + 20%
Losartan	10 µM	AT-1	81% + 5%
Prazosin	850 nM	A1-A	89% + 11%

A 160 nanomolar concentration of 10-week-old male Zucker fatty rat IgG autoantibodies was incubated in the presence or absence of the indicated GPCRs. Acute N2A neurite retraction was determined after 15 minutes as described in Materials and Methods. Results are mean +/- SD.

Involvement of Gq11/Phospholipase C/Inositol Triphosphate and Rho Kinase Signaling Pathways

Co-incubation of N2A cells with ZDF plasma IgG autoantibodies together with individual selective antagonists of Gq11 (YM-254890), phospholipase C (U73122), inositol triphosphate receptor (2-APB) signaling or Rho A/Rho kinase (Y27632) mediated signaling completely or nearly completely protected against acute neurite retraction (Table 2) consistent with involvement of Gq11/phospholipase C/inositol triphosphate and Rho kinase signaling pathways in mediating IgG neurotoxicity downstream of 5-HT2A receptor binding.

Table 2: Effect of signaling pathway inhibitors on ZDF rat IgG autoantibody-induced acute N2A neurite retraction.

Antagonist	Conc	Signaling Intermediate	% of ZDF IgG-induced Neurite Retraction
U73122	10 µM	PLC	14% + 13%
YM-254890	1 µM	Gq11	14% + 5%
2-APB	50 µM	IP3R	0% + 0%
Y27632	10 µM	Rho kinase	0% + 0%

Level and Titer of Autoantibody to 5-HT2A Receptor in Male Zucker Heterozygote (fa/+)

An identical (1.5 µg/mL concentration) of the IgG from Zucker heterozygote rat plasma (N=5) displayed two-fold significantly higher binding (to the 5-HT2A receptor peptide) compared to IgG in plasma from five ZDF rat plasmas (N=2, 10-week-old and N=3, 25-week-old rats) (Figure 7A). The titer of autoantibody to the 5-HT2A receptor peptide was more than two-fold higher in the 10-week old lean Zucker heterozygous rat compared to age-matched, 10-week old ZDF rat (Figure 7B).

Figure 7: A) 2 ug/mL concentration of the protein G eluate of ZDF rat and lean Zucker heterozygote plasma was tested for binding to linear, synthetic 18-meric 5-HT2A receptor peptide.
B) Titer of autoantibody binding to 5-HT2A receptor peptide was tested at the indicated concentrations in 10-week lean Zucker heterozygote (N=5) or 10-week ZDF (N=2) protein G eluate. Results are “mean +/- SE”. * P< 0.01

Bioactivity in the Antibody Fraction of Male Zucker Heterozygous (fa/+) Plasma

Dose-dilution curves of the protein-G eluate fraction from three, 10-week-old lean heterozygous Zucker heterozygous plasmas demonstrated potent inhibition of N2A cell survival at all IgG concentrations tested in two of three rats (Figure 8A). In one of the three rats, lean heterozygous Zucker rat #3, a component of significant N2A survival-promoting activity were observed at high IgG dilution (Figure 8A). ‘Functional selectivity’ is a unique property of the 5-hydroxytryptamine 2A receptor [20]-it refers to the ability of different 5-HT2AR ligands to preferentially activate alternative signaling pathways, e.g. PLC/IP3/Ca2+ signaling or a beta-arrestin 2/PI3-kinase survival pathway. Since the selective 5-HT2AR antagonist M100907 significantly protected against lean heterozygous Zucker rat IgG autoantibody-induced accelerated N2A cell loss (Figure 8B) the neurotoxicity is likely due to Gq11/PLC/IP3/Ca2+-mediated signaling occurring downstream of 5-HT2AR binding. More study is needed, however, to determine whether N2A survival-promoting activity in a subset of lean heterozygous Zucker rat IgG autoantibodies may result from selective activation of beta arrestin-2/PI3-kinase-mediated signaling. In a prior report [21], highest level of 5-HT2A receptor-binding occurring in the autoantibodies from a patient with lupus. The lupus IgG autoantibodies were not only neurotoxic, but (at certain concentrations) promoted N2A neuroblastoma cell survival dependent on PI3 kinase-mediated signaling [21].

Figure 8: A) Dose-dilution curves of three Zucker lean heterozygous protein G eluates on survival in N2A mouse neuroblastoma cells; B) Co-incubation of a representative lean heterozygous protein G eluate fraction with 1 µM concentration of the selective 5-HT2AR antagonist M100907. * P< 0.05.

Apparent MW of the Active Component in ZDF Protein G Eluates

The active component in the ZDF protein G eluate (which caused accelerated N2A cell loss) had an apparent MW > 10 kD (Figure 9A) consistent with an antibody or antibody fragment. There was no significant N2A cell survival-inhibitory activity present in the flow-through fraction following membrane dialysis of the ZDF protein G eluate on a 10 kilodalton Molecular Weight (MW) cutoff membrane (Figure 9A). The freshly-isolated, lean heterozygous Zucker rat (fa/+) protein G eluate fraction (SM, starting material) caused significantly increased N2A cell survival. Following dialysis on a 10 kD MW cutoff membrane, more than 90% of the N2A survival stimulatory activity was recovered in the fraction having an apparent MW > 10 kD, i.e. retained on the membrane (Figure 9B). The low MW (< 10 kD) flow-through fractions following membrane dialysis of the ZDF or lean heterozygous Zucker rat protein G eluate did not cause significant inhibition or stimulation of N2A survival (Figure 9A and 9B).

Figure 9: Apparent MW of peak neuroblastoma survival-inhibitory (A) and – stimulatory (B) activity in the plasma protein G eluate from ZDF or heterozygous lean ZDL rat. Apparent MW of peak neuroblastoma survival-inhibitory (A) or -stimulatory (B) activity in the protein G eluate from a representative ZDF (A) and lean heterozygote # 3 Zucker rat (B) plasma. A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker rat plasma was dialyzed on a 10kD MW cutoff membrane. The resulting retained and flow-through fractions or starting material (SM) were incubated with N2a mouse neuroblastoma cells for 24 hours as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations. * P < 0.05 compared to control N2A cell without added test fractions.

Discussion

The male ZDF rat is a well-studied model of human obese type 2 diabetes mellitus. The animals undergo progressive gain in fat mass, with obesity arising around 4-weeks of age [9]. Hyperglycemia commences around week 8- 9, and chronic marked elevation in blood glucose concentrations (400-500 mg/dL) is associated with early hyperinsulinemia followed by later relative insulin deficiency [10]. Hypertension and severe hypertriglyceridemia manifest around week 9 in the ZDF rat [9,10]. In adult, long-standing human obese type 2 diabetes mellitus from the Veterans Affairs Diabetes Trial, the baseline presence of anti-endothelial cell autoantibodies was significantly associated with lack of baseline insulin therapy (i.e. relative insulin deficiency) and lack of baseline triglyceride-lowering medication [22]. Endothelial cell heparanase expression is upregulated in insulin deficiency [23] and by high level of pro-inflammatory cytokines [24]. Heparanase cleaves heparan sulfate side chains abundant on neurons and vascular cells, and heparan sulfate proteoglycan is a known target of humoral autoimmunity [25]. In older adult diabetes patients evidence suggested that autoantibodies targeting the 5-HT2AR were in many cases cross-reactive with heparan sulfate proteoglycan [26]. Taken together it is possible that certain metabolic derangements in the ZDF rat strain, (insulin deficiency, and high level of pro-inflammatory cytokines) may contribute in part to the long persistence of circulating 5-HT2A receptor autoantibodies at older ages.

Although the heterozygous (fa/+) Zucker rat is lean and non-diabetic, it demonstrated substantially higher titer of 5-HT2A receptor autoantibodies compared to age-matched ZDF rat. One possibility is that enhanced helper T-cell immunity in the lean heterozygote Zucker rat drives autoantibody formation which includes a subset (of autoantibodies) targeting the 5-HT2A receptor. Neurotoxicity in the lean heterozygote Zucker IgG autoantibodies (i.e. acute neurite retraction and accelerated N2A cell loss) was as high or higher than in ZDF IgG autoantibodies consistent with a prior report of a significant correlation between antibody binding level and level of acute neurite retraction in N2A cells [15]. Higher level of 5-HT2AR targeting autoantibodies in the lean heterozygote Zucker rat supports a role for upregulated adaptive humoral immunity (in part) due to a heterozygous leptin receptor mutation causing hyperleptinemia. More study is needed to determine whether high titer of autoantibodies in the lean heterozygous Zucker rat may predispose to immune complex formation that could bias signaling pathway activation downstream of 5-HT2A receptor binding.

In summary, 5-HT2A receptor autoantibodies increased in the circulation in male hypertensive ZDF rat between 8.5-10-weeks of age and persisted up until 25-weeks old or longer. The ZDF rat 5-HT2A receptor-targeting autoantibodies caused acute neurite retraction and accelerated mouse neuroblastoma cell loss by a mechanism involving activation of Gq11/PLC/IP3R pathway and RhoA/Rho kinase coupled signaling. The ZDF and lean heterozygous Zucker rat strains may be suitable animal models for investigations of the roles of obesity-associated inflammation and dysregulated humoral immunity, respectively, in the etiology of certain forms of late-onset neurodegeneration.

Funding

This project was supported by grants from the New Jersey Commission on Brain Injury Research (CBIR19PIL007) to MBZ and the Biomedical Laboratory Research and Development Service of the VA Office of Research and Development (I1BX004561) to KCHP. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government.

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DOI: 10.31038/JNNC.2020323

The evidence from randomized controlled trials (RCTs) that surgical facemasks and cloth facemasks are ineffective for preventing transmission of respiratory viruses in public is conclusive [1-8]. In all the meta-analyses of all the existing RCTs [4-7], not a single trial was found in which facemasks provided any protection against virus transmission in public [1,2]. Consistent with the findings of all RCTs comparing transmission rates in public with and without facemasks, Leung et al. compared the rates of detection of three types of viruses in exhalations by infected individuals with and without facemasks: they concluded that, with facemasks, there was: “no significant reduction in detection of influenza virus in aerosols;” “For rhinovirus there were no significant differences between detection of virus with or without facemasks, both in respiratory droplets and in aerosols;” and, for coronavirus there was “no significant reduction in detection in aerosols [3].” They did observe a reduction in detection of viruses in respiratory droplets for influenza virus and coronavirus. There is no doubt that facemasks can reduce the transmission of droplets, but droplets are not the concern for transmission of viruses in public. Significant numbers of droplets are not exhaled by asymptomatic carriers because they are not coughing or sneezing in public. Symptomatic carriers who are coughing and sneezing should be quarantined. The rationale for both recommended and mandated facemasks in public is to reduce transmission by asymptomatic carriers. Since all the RCTs in the literature show no reduction in transmission of viruses in public due to facemasks, one wonders why the CDC, NIH and virtually all medical authorities are stating that there is conclusive scientific evidence that facemasks reduce viral transmission in public.

For example, Brooks, Butler and Redfield [9] stated in the July 14, 2020 issue of JAMA that, “At this critical juncture when COVID-19 is resurging, broad adoption of cloth face covering is a civic duty.” Redfield is the Director of the CDC. Similarly, Gostin, Cohen and Koplan [10] stated in the August 11, 2020 issue of JAMA that, “The ethical justification for face coverings is their utility in preventing transmission of serious disease to community members.” Gostin et al. recommend delivering a public health message of, “When we all mask up, we are all safer [10].” The scientific facts are that facemasks have no effect on the transmission of viruses in public. This is proven by multiple RCTs. And yet, the CDC, writers in JAMA, and many medical authorities, state that it is scientifically proven that facemasks will protect the public from COVID-19. What is the evidence these authorities cite as scientific proof, while ignoring and never citing the RCTs?

Brooks et al. have 14 references that provide their evidence [9]. Their first reference is a study in health care settings that provides no data on transmission in public. The second and third references are to the Massachusetts Department of Public Health website and provide no data. The fourth paper describes a web app for screening healthcare workers for COVID-19. The fifth reference, published in 2019, provides guidance for how to respond to pandemics. The sixth reference says that asymptomatic carriers can transmit the virus to others but does not discuss or provide any data on facemasks. The seventh reference says that undocumented coronavirus infections can result in transmission to others but does not discuss or provide evidence on facemasks. The eighth reference is to a 1994 physics paper that describes reduction of “respiratory jets” by facemasks but provides no evidence on viral transmission. The ninth reference discusses the filtration efficiency of facemasks but provides no data on transmission in public. The tenth reference discusses the “potential utilities” of hand washing and facemasks but provides no evidence that they work. The eleventh reference is a paper by Greenlagh et al. that provides the authors’ responses to their critics but does not provide data on transmission in public [8]. The twelfth reference describes factors affecting whether people use facemasks but no data or evidence on whether facemasks work to reduce transmission in public. The thirteenth paper describes a single situation in which two COVID-positive hair stylists wore masks at work and none of their 104 clients followed up two weeks later reported symptoms of COVID-19; 102 of the 104 clients wore facemasks themselves while at the salon. None of these 104 individuals were tested for COVID-19 and many of them could have been asymptomatic carriers. The fourteenth paper is a discussion of facemasks and Gross National Product in a Goldman Sachs Research publication.

Thus, of Brooks et al.’s fourteen references, none provide any controlled data on the effects of facemasks on transmission of viruses in public [9]. Gostin et al. provide ten references to support their assertion that it is a civic duty to wear facemasks during the COVID-19 pandemic [10]. The first reference is to a CDC website statement that facemasks work in public and should be worn in public [11]. The CDC website provides 19 references to support their recommendation, but those references consist of small or single case uncontrolled studies, discussions of asymptomatic transmission that provide no data on facemasks, papers about infections in residents of long-term care facilities, and papers about the filtration properties of facemasks. None of the CDC references provide any evidence or data that facemasks work in public to reduce the rate of viral transmission.

Returning to the Gostin et al. [10] paper, their second reference is Brooks et al. [9]. Their third reference is to a study that provides no data on reduction of viral transmission rates due to wearing facemasks in public. Their fourth reference is to a study of health care workers not of transmission in public. Their fifth reference is to a study by Lyu et al. [11] that has been previously discussed by Ross and will be discussed below [2]. Their sixth, seventh and eighth references are to single legal cases. Their ninth reference is to a 2007 book by the Institute of Medicine. Their tenth reference discusses the administrate duties of the Social Security Commissioner.

Thus, the CDC website, Brooks et al. and Gostin et al. provide a total of 43 references, none of which provide any controlled evidence that facemasks worn in public reduce the rate of coronavirus transmission [9,10]. None of these three authorities reference a single one of the RCTs, all of which found zero evidence that facemasks reduce viral transmission when worn in public. The authorities in these three examples (the CDC, Brooks et al, and Gostin, et al.), and authorities throughout the United States are virtually unanimous in saying that facemasks work, that this has been proven scientifically, and that the public should wear them. This is put forward as a recommendation at the least, and often as a mandate.

The only paper out of the 43 that provides any data on facemasks in public is a study by Lyu et al. [12]. These authors tracked the rates of COVID-19 infections in 15 states and D.C. from March 31 to May 22, 2020. They found that infection rates declined more in the states that instituted mask wearing in public compared to states that did not. The difference in rates varied from 0.9% to 2.0%. If we assume that during this time period 5% of the population became infected, this would mean that facemasks could have reduced the number of infections in the population by a maximum of (0.05 x 0.02 = 0.001) 0.1%. However, during that time period the COVID-19 curve was flattening in most of the country. More importantly, multiple variables were contributing to the rate of infections including social distancing, hand washing, quarantines, and business and school closures, so facemasks likely contributed only a fraction of 0.1%, which is not clinically meaningful and is well within the margin of error for viral disease epidemiology in the United States. The CDC’s estimate of the number of flu deaths in the United States in a given year is usually stated as a being within a range of tens of thousands of cases. A fluctuation of 0.1% in the infection rate when the best CDC estimate for annual flu deaths is +/-10,000-20,000 is completely meaningless.

If single cases and small uncontrolled observational studies with weak methodology were cited to prove the effectiveness of an alternative treatment for pneumonia, a paper reporting those results would never get published in any mainstream journal; the claim that the alternative treatment was effective would be rejected by medical authorities. Yet this is the quality of evidence cited by the CDC, medical authorities and leading medical journals that facemasks are effective for reducing coronavirus transmission in public. That claim has been disproven by four meta-analyses of RCTs. In any case, there is no reason to expect surgical facemasks to work because their pore size is in the range of 50-100 microns, while the coronavirus is about 0.1 microns and aerosols are about 2-3 microns in size [1,2]. The available science shows conclusively that public facemasks don’t work. This is not a conspiracy theory, an anti-masker ideological statement, or an anti-medical or anti-authority statement. It is a statement of scientific fact. Facemasks may make people feel safer, and may confer a sense of solidarity, but this is just a feeling. Medically and scientifically, facemasks do not protect the public during the COVID-19 pandemic.

Ignoring these facts will not change them. It is not possible to create confidence in organized medicine by mandating public policies that are proven to be ineffective by medical science. The medical profession is putting itself at risk for blowback by endorsing and mandating public wearing of facemasks. Once statements that facemasks work are made enough times by medical authorities, they become common knowledge, and are transmitted throughout the culture. Hopefully, at some point science, data and sound medicine will prevail. The statement that public wearing of facemasks is ineffective for reducing coronavirus infection rates has been made twice by the same group from Harvard in recent issues of the New England Journal of Medicine: “We know that wearing a mask outside health care facilities offers little, if any, protection from infection [13,14].” In conclusion, it should be noted that the author’s mother is in her nineties and living in a long-term care facility. The author is fully supportive of all the COVID-19 precautions instituted at this facility including facemasks, social distancing, quarantining and restriction of visitors during the height of the pandemic and rigorous daily screening of employees. The author is also fully in support of wearing facemasks inside operating rooms. The evidence just does not support wearing facemasks in public.

References

Ross CA (2020) Thoughts on COVID-19. Journal of Neurology and Neurocritical Care 3: 1-3.
Ross CA (2020) Facemasks are not effective for preventing transmission of the coronavirus. Journal of Neurology and Neurocritical Care 3: 1-2.
Leung NHL, Chu DKW, Cowling BY (2020) Respiratory virus shedding in exhaled breath and efficacy of facemasks. Nature Medicine 26: 676-680.
Brainard JS, Jones N, Lake I, Hooper L, Hunter P (2020) Face masks and similar barriers to prevent respiratory illness such as COVID-19: A rapid systematic review. Medrxiv. doi:10.1101/2020.04.01.20049528.
Cowling BJ, Zhou Y, Ip DK, Leung GM, Aiello AE (2010) Face masks to prevent transmission of influenza virus: a systematic review. Epidemiology of Infections138: 449-456.
Xiao J, Shiv EYC, Gao H, Wong JY, Fong MW, Ryu S, et al. (2020) Nonpharmaceutical measures for pandemic influenza in nonhealthcare settings – personal protective and environmental measures. Emerging Infectious Diseases 26: 967-975.
Aggarwhal N, Dwarakananthan V, Gautham N, Ray A (2020) Facemasks for prevention of viral respiratory infections in community settings: A systematic review and meta-analysis. Indian Journal of Public Health64: 192-200.
Greenhalgh T, Schmid MB, Czypionka T, Bassler D, Gruer L (2020) Facemasks for the public during the COVID-19 crisis. British Medical Journal. doi:10.10.1136/bmj.m1435.
Brooks JT, Butler JC, Redfield RR (2020) Universal masking to prevent SARS-CoV-2 transmission – the time is now. JAMA. doi.10.1001/jama.2020.13107.
Gostin LO, Cohen IG, Koplan JP (2020) Universal masking in the United States. The role of mandates, health education and the CDC. JAMA10.1001/jma.2020.15271.
Centers for Disease Control and Prevention (2020) https://www.cdc.gov/coronavirus/ 2019-ncov/prevent-getting-sick/cloth-face-cover- guidance.html.
Lyu W, Wehby GL (2020) Community use of face masks and COVID-19: Evidence from a natural experiment of state mandates in the US. Health Affairs. doi.org/10.1337/hlthaff.2020.00828.
Klompas M, Morris CA, Sinclair J, Pearson M, Shenoy ES (2020) universal masking in hospitals in the COVID-19 era. New England Journal of Medicine 382: e62-63.
Klompas M, Morris CA, Shenoy ES (2020) Universal masking in the COVID-19 era. New England Journal of Medicine 383: e9.

Liver Stiffness Using Transient Elastography Predicts Worse Survival in Patients with Chronic Heart Failure

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DOI: 10.31038/JCRM.2020341

Abstract

Background: Transient elastography using FibroScan is a noninvasive and reliable method to assess liver stiffness. Liver stiffness is influenced not only by fibrosis but also by liver congestion, inflammation and cholestasis. This study aimed to investigate the correlation between liver stiffness, liver congestion and liver fibrosis, and to elucidate the utility of liver stiffness measurement (LSM) in patients with chronic heart failure (CHF).

Methods: We investigated 42 patients with chronic heart failure undergoing right heart catheterization (RHC) from November 2015 to November 2016.LSM was performed with FibroScan. Patients underwent right arterial pressure (RAP) measurement by RHC.

Results: LSM was 10.9 ± 12.6 kPa. RAP was 8.0 ± 5.7 mmHg, and 18 patients had RAP>8 mmHg. LSM was correlated with FIB-4 (r=0.67, p=0.002), HA (r<0.57, p<0.001) and RAP (r=0.67, p9.65kPa) was significantly associated with shorter survival (mean OS; 19.9 vs. 29.9 months, p<0.001).

Conclusion: LSM was directly influenced by liver congestion and liver fibrosis in patients with CHF. Moreover, high LSM was demonstrated to be related with worse survival in patients with CHF.

Keywords

Fibroscan, Hyaluronic acid, Right arterial pressure, Survival

Introduction

Heart failure is a pathologic condition in which impaired pumping function reduces blood flow and leads to congestion of blood and fluids in many organs. Cardiac dysfunction causes liver damage. In particular, right-sided heart failure causes liver congestion, which is known as congestive hepatopathy [1]. Chronic liver congestion progresses to liver fibrosis [2,3]. Histological examination of liver congestion shows sinusoidal engorgement, degeneration, and variable degrees of hemorrhagic necrosis. In patients with chronic or recurrent heart failure, reticulin and collagen accumulation cause liver fibrosis [4].

Despite the seriousness of this condition, only a few studies have reported the progression of liver fibrosis in congestive hepatopathy [5]. Liver biopsy is the gold standard for fibrosis identification. However, it cannot be used as a routine screening tool to detect or monitor liver fibrosis progression due to its inherent shortcomings, which include its invasive nature and the concomitant rare potential risks of bleeding and sampling variability [6].

Because of these disadvantages, several serologic markers that can evaluate the degree of hepatic fibrosis have been used. Hyaluronic acid (HA), which is a highly evolutionarily conserved glycosaminoglycan component of the extracelluar matrix, is generally used as a serum biomarker of liver fibrosis [7]. Moreover, combined assays of multiple markers to improve the predictive ability of liver fibrosis have been also been developed. Among them, fibrosis index based on four factors (FIB-4) is a non-invasive test to stage liver fibrosis in patients with co-infected human immunodeficiency virus (HIV) and hepatitis C virus (HCV) [8]. FIB-4 relies on patient age, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and platelet counts, which are routinely measured and available. Recently, FIB-4 has come to be applied to various liver diseases for its convenience and cost effectiveness [9,10].

In addition to laboratory tests, the method of non-invasive transient elastography (TE) for assessing liver fibrosis has been developed and widely used in the routine clinical setting. FibroScan is a rapid, non-invasive, and reproducible approach for assessing liver fibrosis by measuring liver stiffness [11,12], and has been approved for clinical use in Japan. In various liver diseases such as viral hepatitis, alcoholic liver disease and non-alcoholic fatty liver disease, liver stiffness measurement (LSM) has been found to be strongly associated with the degree of liver fibrosis [13-15]. However, LSM is considerably influenced by liver congestion, inflammation and cholestasis, independent of the degree of fibrosis [16,17]. Colli et al. showed increased LSM in most patients with acute decompensated heart failure in the absence of parenchymal liver disease [18]. Therefore, LSM can not properly reflect liver fibrosis in patients with congestive heart failure [19]. Little is known about LSM in patients with CHF.

The purposes of this study were to assess the relationship between liver stiffness, volume status and liver fibrosis in patients with chronic heart failure (CHF), and to elucidate the utility of TE in those patients.

Methods

Patient Enrollment

We prospectively investigated LSM using transient elastography in 47 patients who were admitted to our hospital with CHF and scheduled for right heart catheterization (RHC) in the Department of Cardiology of Mie University Hospital from November 2015 to November 2016. RHC was performed in patients who required accurate hemodynamic monitoring because of clinically indeterminate volume and in patients who were refractory to initial therapy. The diagnosis of CHF was made clinically based on signs and symptoms derived from patient history and examination. Exclusion criteria included history of alcohol abuse, known chronic liver disease with an etiology other than heart failure, positivity for hepatitis B surface antigen or hepatitis C antibody, severe obesity, and ascites. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee (Authorization Number 2271).

Laboratory Tests and FIB-4

Laboratory data were obtained beginning at the closest date to RHC. FIB-4 was calculated using the formula: FIB-4 = age (years) × AST (IU/l)/[platelet count (Plt) (109/l) × ALT1/2 (IU/l)].

Echocardiography

Transthoracic echocardiography was performed using AplioTM 500 (Toshiba medical systems, Tokyo, Japan). Non-invasive RAP was estimated from diameter of inferior vena and its respiratory motion.

Cardiac Catheterization

RHC was performed in the cardiac catheterization laboratory using a flow-directed pulmonary artery catheter. Pressure calibration was performed before and after pressure measurements. All readings were referenced to the midaxillary line with the patient in the supine position. Pressure measurements were determined at the end-expiratory period, with an average of 3 to 5 cycles obtained. The physician performing the cardiac catheterization was unaware of LSM results.

Liver Stiffness Measurement

LSM was measured with FibroScan (Echosens, Paris, France) in the hepatology unit of our hospital. LSM was obtained within 24 hours before or after RHC. The tip of the probe transducer was placed on the skin between intercostal spaces and the level of the right lobe of the liver. The measurement depth was between 25 and 65 mm below the skin surface. Ten validated measurements were performed on each patient with success rates of at least 60%. The results were expressed in kilopascals (kPa). The only procedures considered reliable were those with at least 10 validated measurements and interquartile range <30% of the median value.

Statistics Analysis

Results are presented as the mean ± standard error of the mean or n, as appropriate. The means or percentages were compared by using independent Student’s t-test or the Mann-Whitney U test for continuous variables. Relationships between variables were determined using the two-sided Pearson’s correlation coefficient. Receiver operator characteristic (ROC) curves and the corresponding area under the curve (AUC) were used to obtain cut-offs for the outcomes. The Youden index was applied to calculate the optimal cutoff point. Overall survival (OS) was measured using the Kaplan-Meier method and compared using the log-rank test. Differences were considered significant at p.

Results

Patient Characteristics

In total, 47 patients were screened for this study. Five patients were excluded before scanning: 5 patients with history of alcoholic abuse, and one patient with positivity for hepatitis C antibody. A total of 42 patients (27 men and 15 women) were enrolled in this study. The distribution of the individual forms of cardiac disease is shown in Table 1. The enrolled patients represented a wide spectrum of cardiac disease. The most dominant form was valvular heart disease. The subjects’ baseline clinical and laboratory characteristics are shown in Table 2. The average duration of heart disease was 79.8 ± 85.7 months. The mean FIB-4 was 2.77 ± 1.72. In the individual components of FIB-4, the mean values were as follows: age (70.0 ± 13.9 yrs), AST (25.6 ± 9.3 IU/l), ALT (17.8 ± 8.9 IU/l), and Plt (205 ± 86 109/l). Aminotransferase levels were only slightly elevated in 2 patients. Mean HA was 102 ± 104 ng/ml, and 25 patients had abnormal HA (>50 ng/ml). In echocardiograph, the mean IVC diameter was 20.5 ± 16.4 mm. IVC did not collapse in only 4 patients. In RHC indications, the mean RAP was 8.0 ± 5.7 mmHg. Successful LSM was obtained in 42 patients. The median IQR was 0.9 and the median IQR/median of liver measurement was 20%, showing reliable results. The mean LSM was 10.9 ± 12.6 kPa, and 28 patients with chronic heart failure were higher than the normal range reported previously (normal, <5.5 kPa).

Table 1: Clinical diagnoses.

Clinical Diagnosis	n=42
Valvular heart disease	18
Ischemic cardiomyopathy	8
Dilated cardiomyopathy	5
Cardiac sarcoidosis	4
Atrial septal defect	3
Pulmonary hypertension	2
HF with preserved ejection fraction	1
Hypertrophic cardiomyopathy	1

HF, heart failure

Table 2: Clinical and laboratory characteristics.

Variable	n=42
Age (years)	70.0 ± 13.9
Sex (F/M)	15/27
Body mass index (kg/m²)	22.3 ± 6.0
Duration of heart disease (months)	79.8 ± 85.7
Laboratory tests
Alb (mg/dl)	3.9 ± 0.5
T-Bil (mg/dl)	0.8 ± 0.3
AST (IU/l)	25.6 ± 9.3
ALT (IU/l)	17.8 ± 8.9
ALP (IU/l)	239.0 ± 76.6
BUN (mg/dl)	22.5 ± 11.3
Cre (mg/dl)	1.07 ± 0.38
WBC	6055 ± 1718
Hb	12.8 ± 2.2
Plt (10⁹/l)	205 ± 86
BNP (pg/ml)	253 ± 326
HA (ng/ml)	102 ± 104
FIB-4	2.77 ± 1.72
Echocardiography
Ejection fraction (%)	51.8 ± 19.5
IVC diameter (mm)	20.5 ± 16.4
Hemodynamics
Systolic aortic pressure (mmHg)	123.5 ± 20.7
Diastolic aortic pressure (mmHg)	68.0 ± 11.8
Heart rate (beats/min)	76 ± 13
Pulmonary capillary wedge pressure (mmHg)	14.7 ± 8.4
Mean pulmonary artery pressure (mmHg)	22.2 ± 11.5
Mean RAP (mmHg)	8.0 ± 5.7
Cardiac index (l/min/m²)	2.83 ± 0.84
Transient elastography
LSM (kPa)	10.9 ± 12.6
Interquartile range/median	15 ± 6

Alb, albumen; T-Bil, total bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; WBC, white blood cells count; Hb, hemoglobin; Plt, platelet count; BUN, blood Urea nitrogen; Cre, creatinine ; Plt, platelet count; BNP, brain natriuretic peptide; HA, hyaluronic acid; FIB-4, fibrosis index based on four factors; IVC, inferior vena cava; RAP, right arterial pressure; LSM, liver stiffness measurement.

Correlation between LSM, FIB-4, HA and RAP

LSM was correlated with FIB-4 (r=0.57, p<0.001, Figure 1A), HA (r=0.67, p<0.001, Figure 1B) and RAP (r=0.67, p<0.001, Figure 1C). FIB-4 was correlated with HA (r=0.68, p<0.001, Figure 1D).

Figure 1: LSM was correlated with fibrotic markers and RAP. Correlation of LSM with FIB-4 (A). Correlation of LSM with HA (B). Correlation of LSM with RAP (C). Correlation of FIB-4 with HA (D). LSM, liver stiffness measurement; FIB-4, fibrosis index based on four factors; HA, hyaluronic acid; RAP, right arterial pressure.

Patient Outcome

Our study period had 8 out of 42 patients (19%) who died in the average follow-up period of 30.0 months. The cause of death for 7 patients included the following conditions related to CHF: heart failure, respiratory failure, and sudden death. One patient died of lymphoma. ROC analyses concerning predictors of survival yielded AUC values of 0.745 for LSM (p=0.04), 0.575 or RAP (p=0.511) (Figure 2A). We calculated the cut-off value of LSM, 9.65 (sensitivity: 0.625, and specificity: 0.647), from our ROC analysis of survival curves. Mean OS was significantly longer in patients with LSM≥9.65 versus those patients with LSM<9.65 (mean OS; 19.9 vs. 29.9 months, p<0.001; Figure 2B). Factors associated with LSM higher than 9.65 kPa are outlined in Table 3. Patients with low LSM were significantly younger (p=0.034) and had lower duration of heart disease, BNP, HA, FIB-4 and RAP but higher hemoglobin and Plt. However, Albumen, T-Bil, AST, ALT and IVC diameter had no significant differences.

Table 3: Characteristics of subjects stratified by LSM.

Variable	LSM>9.65kPa (n=17)	LSM≤9.65kPa (n=25)	p value
Age (years)	75.1 ± 10.2	66.6 ± 15.3	0.037
Duration of heart disease (months)	123 ± 100	50 ± 61	0.013
Alb (mg/dl)	3.8 ± 0.6	4.0 ± 0.4	0.394
T-Bil (mg/dl)	0.9 ± 0.3	0.8 ± 0.3	0.142
AST (IU/l)	26.6 ± 7.9	24.9 ± 10.3	0.543
ALT (IU/l)	17.2 ± 10.0	18.2 ± 8.3	0.746
ALP (IU/l)	259 ± 65	225 ± 82	0.146
BUN (mg/dl)	25.2 ± 12.0	20.7 ± 10.7	0.216
Cre (mg/dl)	1.18 ± 0.40	0.99 ± 0.36	0.131
WBC	5728 ± 2086	6278 ± 1420	0.352
Hb	11.7 ± 2.1	13.5 ± 2.1	0.011
Plt (10⁹/l)	156 ± 68	239 ± 82	0.001
BNP (pg/ml)	418 ± 423	141 ± 172	0.019
HA (ng/ml)	150 ± 130	70 ± 68	0.029
FIB-4	3.89 ± 1.67	2.01 ± 1.30	<0.001
IVC diameter (mm)	20.5 ± 5.3	20.5 ± 21.0	0.998
RAP (mmHg)	11.3 ± 6.4	5.7 ± 3.9	0.004

LSM, liver stiffness measurement; Alb, albumen; T-Bil, total bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood Urea nitrogen; Cre, creatinine ; WBC, white blood cells count; Hb, hemoglobin; Plt, platelet count; BNP, brain natriuretic peptide; HA, hyaluronic acid; FIB-4, fibrosis index based on four factors; IVC, inferior vena cava; RAP, right arterial pressure;

Figure 2: Survival was worsened with LSM>9.65 in patients with chronic heart failure. ROC curves for identification of survival of LSM and RAP (A). Kaplan-Meier curve in patients with chronic heart failure according to LSM (B). LSM, liver stiffness measurement; ROC, receiver operating characteristic; RAP, right arterial pressure.

Discussion

In this study, there was a higher level of liver stiffness in patients with CHF without known pre-existing liver disease. The mean LSM reached 9.2 kPa in those patients, which is a significantly higher value than the normal range reported previously [20]. Several studies have investigated the influence of increased RAP in LSM during heart failure [18,21,22]. Taniguchi et al. showed a close correlation between RAP and LSM with a curvilinear regression equation in patients with heart failure [21]. We also found a good correlation between LSM and RAP in the present study, which is consistent with previous reports. The use of a non-invasive tool for evaluation of RAP should be a great interest for clinical management of patients with CHF. The evaluation of RAP has the potential to improve the hemodynamic profiling of patients, which can lead to better patient management and outcomes.

Millonig et al. showed that the central venous pressure (CVP) value reversibly controls LSM in an animal model [23]. However, Colli et al. used diuresis to demonstrate a small reduction in LSM in patients with chronic heart failure, from 8.8 to 7.2 kPa in 27 patients, with a median reduction of 1.2 kPa [18]. The explanation for the small change in LSM in patients with CHF before and after diuresis was not clear, but the elevated LSM following attainment of euvolemia may be due to the presence of the underlying fibrosis. In the present study, we found that FIB-4 index was increased in patients with CHF. FIB-4 was important biomarkers of liver fibrosis in patients with CHF.

Although little is known about the mechanism of liver fibrosis in patients with CHF, the pathogenesis of congestive hepatic fibrosis is thought to be a reaction of stellate cells following prolonged congestive heart failure or hepatic outflow obstruction. The stellate cells are transformed into alpha-smooth muscle actin-positive myofibroblasts, and these myofibroblasts produce extracellular matrix proteins in centrilobular sinusoidal areas under congestive condition [24]. Fujimoto et al. showed that HA was increased in right heart failure rat model as well as in patients with liver cirrhosis [25]. In the present study, we found that HA was increased in patients with CHF.

As mentioned, liver stiffness is influenced by liver congestion independent of the degree of liver fibrosis. In addition, inflammatory infiltration, tissue edema, and cholestasis can also affect LSM [16,17]. However, total bilirubin, AST, and ALT were within normal range in almost all the patients and there was no significant correlation between LSM and these laboratory parameters in the present study.

Previous studies demonstrated LSM is associated with risk of decompensation, liver cancer, and death in patients with chronic liver disease [26]. In the present study, we found that LSM was also associated with OS in patients with CHF. Moreover, our results showed that LSM was a better prognostic indicator than RAP for OS of CHF patients. The reason is that LSM could be affected not only by RAP but also by liver fibrosis.

There were several limitations in the present study. Firstly, the liver histological data, which would lead to a better proof of the utility of LSM, was insufficient. However, liver biopsy is invasive and considered inappropriate for patients with CHF because of the greater possibility of bleeding complications. Secondly, it is not clear whether the FIB-4 is valuable in patients with CHF. Following congestive heart failure, AST levels also may be routinely increased; however, the liver test abnormalities in our patients were very small, indicating that liver injuries were not severe in our patients. Thirdly, the sample size was not large enough for definitive conclusions. Therefore, further multicenter studies are needed to confirm the present results. Despite these limitations, our findings could be relevant in the future and will stimulate further research in this field.

In conclusion, we suggest the possibility that LSM obtained via Fibroscan may be associated with RAP, but also with the underlying liver fibrosis and survival in patients with CHF. LSM might be used in risk stratification in patients with CHF.

Author Contributions

Concept of the study (K.S, Y.T.), extraction of data (Y.T., K.S.), drafting of the manuscript (Y.T.), writing of the manuscript (Y.T., K.S., K.D), revision for important intellectual content (all authors). All authors approved the final version of the manuscript.

Funding: No external financial support was received.

Conflict of interest: Nothing to declare for all authors.

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The Influence of Glass Content on the Hydraulic Conductance and Tubule Occlusion of Novel Bioactive GlassToothpastes

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DOI: 10.31038/JDMR.2020332

Abstract

Bioactive glasses are widely used as additives in remineralising toothpastes for treating dentine hypersensitivity, which is associated with dentinal fluid flow within exposed dentinal tubules. This study investigates the weight percentage of a fluoride containing bioactive glass in a toothpaste formulation on tubule occlusion and fluid flow of mid coronal dentine discs. Tests were performed after brushing, after immersion in artificial saliva and after a citric acid challenge.

There was a statisticallly significant reduction in fluid flow after application of all the toothpastes but the fluid flow reduction was not statistically significantly above a 5% loading of bioactive glass in the toothpaste. Immersion In artificial saliva after brushing reduced the fluid flow for all loadings but was not statisticaly significant, whilst an acid challenge increased the fluid flow but was again not statistically significant.

SEM observations of mid coronal sections showed improved occlusion of the dentinal tubules up to a 5% loading but minimal further improvement for higher loadings.

In conclusion a 5% loading of bioactive glass is close to optimum for reducing fluid flow and tubule occlusion.

Keywords

Bioactive glass, Glass loading, Hydraulic conductance, Tubular occlusion, Dentine hypersensitivity

Introduction

Dentine Hypersensitivity (DH) is characterized as a sharp dental pain of short duration caused by the reaction of exposed dentine surfaces to stimuli, typically thermal, evaporative, tactile, osmotic or chemical that cannot be associated to any other dental defect of pathology [1]. It is based on the hydrodynamic theory where stimuli applied onto the exposed dentinal tubules cause fluid movement across the dentine, passing them to the intradental pulpal nerves and thereby initiating a painful sensation [2].

DH is known to usually impact on the quality of life of affected individuals by instigating painful sensation during eating and drinking hot and cold food and beverages [3]. The prevalence of DH in UK is estimated at 52% [4]. This figure however drops significantly when the conduct of the study shifted from patients’ self-reported questionnaires to clinical examinations by dentists. In clinical studies, the reported prevalence is 2.8% [5] and 18% [6] respectively.

The hydrodynamic theory is consistent with the observation that when DH is treated with a tubule-occluding agent, this will result in a reduction in DH [7,8]. Occlusion of exposed dentinal tubules is a widely used strategy for treating DH, and many Over-The-Counter (OTC) toothpastes propose tubule occlusion as their mode of action.

Previously, a bioactive glass (NovaMin®, developed originally by NovaMin Technology Inc., Alachua, FL, USA) based on the original 45S5 Bioglass® composition (US Biomaterials Corp. Jacksonville, FL, USA) has been used as a remineralising and occluding ingredient in toothpaste formulations for treating DH [9-17]. This works by precipitating Hydroxycarbonate Apatite (HCA) onto the tooth surface and subsequently occluding the dentinal tubules [12-17]. However, concerns have been expressed over the long-term durability of HCA in the mouth, and formation of Fluorapatite (FAp), rather than HCA is preferable, as it is more resistant to subsequent acid attack and dissolves less readily when exposed to acids (e.g. during consumption of fruit juice and carbonated beverages).

In recent years solid state Nuclear Magnetic Resonance Spectroscopy has been used to understand bioactive glass structure, enabling new bioactive glass compositions to be developed [18,19] with vastly improved bioactivity. Of particular note here are the fluorine containing bioactive glasses developed by Brauer et al. [20,21] and the high phosphate fluorine containing glasses developed by Mneimne et al. [22]. These new glass compositions release fluoride in addition to calcium and phosphate and form fluorapatite and have recently been developed specifically for toothpastes [23-31].

The objective of this paper is to investigate the ability of one of these new fluorine containing glasses to occlude dentinal tubules and to reduce fluid flow as a function of the amount of glass in the paste immediately after treatment, after immersion in saliva and after an acid challenge.

Materials and Methods

The materials and methods consisted of two stages. The first stage involved the preparation of the raw materials to be used for the study whereas the second stage comprised the experimental design, which covered the conduct of the study.

Collection of Teeth

A total of 108 extracted, caries-free human molars were collected from patients attending the walk-in clinic at Tanah Puteh Dental Clinic, Malaysia from May 2016 to August 2016. In accordance with local ethics in the Malaysian clinic verbal consent was obtained from patients who required extraction of their teeth. Following extraction, the teeth were washed and stored in Listerine (Listerine Original) mouthwash solution at room temperature. The collected teeth were then brought to London in September 2016 by SFT under strict health and safety guidelines as required by QMUL. On arrival in the UK laboratory the teeth transferred to a 70% Ethanol solution until the commencement of the study.

Preparation of Mid Coronal Dentine Sections

All the collected human molar teeth were prepared into dentine discs of 1.3 mm thickness using an automatic precision cutting machine (Struers Accutom 5, Denmark). The dentine discs were then ground using a Kemet 4 machine (Kemet Maidstone Kent ME15 9NJ UK) followed by polishing with three different silicon carbide papers in a descending order of abrasive coarseness, starting from carbide paper grade P600, P1000 to P2500. The polishing was considered complete when the discs were polished to the thickness of 1.0 mm. The thickness of the discs was monitored constantly using a digital micrometer to avoid over polishing.

Etching of Dentine Sections

The etching of dentine discs was only performed just before the discs were to be used for the experimental steps. It was undertaken by dipping the discs into 6% w/w citric acid solution for 30 seconds. The purpose of etching was to remove any smear layer on the discs, thereby opening up the tubules [32]. The discs were then ultrasonicated with deionized water in an ultrasonic bath for 30 seconds to remove any residual acid.

Preparation of Artificial Saliva

The artificial saliva is based on a formulation first proposed by Featherstone et al. [32] and consists of 2.24 grams of KCl, 1.36 grams of KH2PO4, 0.76 grams NaCl, 0.44 grams of CaCl2.2H2O, 2.2 grams of porcine Mucin and 0.2 grams of NaN3 (all Sigma-Aldrich, UK) were mixed with 800 grams of deionized water in a 1 litre volumetric flask. The mixture was stirred using a magnetic hotplate stirrer for 30 minutes until all reagents were fully dissolved. The mixture’s pH was then adjusted to 6.5 at room temperature using a pH meter (Oakton, Netherlands) by adding 0.5 M of KOH sequentially until the desired pH was obtained. Separately, 0.5 M of KOH was prepared beforehand by mixing 1.40 grams of KOH (Sigma-Aldrich, UK) in 50 ml deionized water. The final mixture was made up with deionized water to 1 litre. The produced artificial saliva solution was kept in a fridge set at 5°C until required and used within 2 weeks of preparation.

Preparation of Bioactive Glass Toothpaste

The Bioactive Glass BioMinF® was supplied by CDL Ltd Stoke UK and is a fluoride containing bioactive glass. The particle size of the supplied BioMinF® was characterised using a Malvern 3000 Particle Size analyser (Malvern Pananalytical Malvern WR14 1XZ, UK). For comparison a sample of NovaMin® was obtained from 3M (Ceradyne) Seattle USA.

Preparation of the Bioactive Glass Base Paste

This stage involved the preparation of the base paste first, which was then mixed with different loading of bioactive glass. The base paste was made in the laboratory according to the formulation in US Patent US 2009/0324516 [33] with slight modifications. Components such as bioactive glass, fluoride and flavour were omitted. The composition of the active and inactive ingredients required to produce 100 grams of base paste are listed in Table 1. Each ingredient was weighed separately and then mixed together with a metallic mixing spatula in a 100 ml plastic container. The end product was kept in room temperature until use.

Table 1: Composition of active and inactive ingredients required to produce 100 gram of base paste.

Component	Function	Weight Percentage
Glycerol	Humectant	68.75%
Polyethylene Glycol	Dispersant and to reduce stickiness	20.83%
K Acesulfame	Sweetener	0.48%
Polyacrylic Acid	Binder	0.59%
Titanium Oxide	Whitener	1.91%
Syloid 63	Silica	7.15%
Sodium Dedecyl Sulfate	Surfactant	1.01%

Five 10 grams toothpastes with a different loading of bioactive glass of 0.0%, 2.5%, 5.0%, 7.5%, 10.0% and 15.0% by weight were fabricated manually by using a mortar and pestle technique. The composition for each toothpaste is represented in Table 2.

Table 2: Composition of Bioactive Glass in Various Loading.

Type of Toothpaste	Materials incorporated into each toothpaste
Type of Toothpaste	Base Paste (g)	Bioactive glass (g)
0.0% loading	10.00	0.00
2.5% loading	9.75	0.25
5.0% loading	9.50	0.50
7.5% loading	9.25	0.75
10.0% loading	9.00	1.00
15.0% loading	8.50	1.50

To produce a 2.5% bioactive toothpaste, 9.75 grams of base paste were added to a clean mortar. This was followed by adding 0.25 grams of bioactive glass. The two elements were mixed thoroughly for 150 seconds. The same methods were applied to produce 5.0%, 7.5%, 10.0% and 15.0% loading of bioactive toothpaste. All the prepared toothpastes were stored in six separate sealed 50 ml plastic bottles until required.

The experimental design involves two parts:

a) Comparing the dentine permeability by measuring hydraulic conductance (Lp).

b) Comparing the occlusive effect of dentinal tubules using SEM.

Hydraulic Conductance Measurement Procedures

A split chamber device based on the design by Pashley and Galloway [34] was used. The total distance travelled by the air bubble in 240 seconds was designated as the baseline flow rate, which was allocated a value of 100% permeability.

Measuring the Dentinal Permeability after Treatment with Toothpaste

0.4 gram of toothpaste was squeezed onto a brush head (Oral-B Floss Action Replacement) and mounted on an electric rechargeable toothbrush (Oral-B Vitality Plus). Without removing the disc from the Pashley cell, it was treated with the toothpaste for 2 minutes. The brush head bristles were applied on to the discs at an inclination of 90 degrees. Immediately after 2 minutes of brushing, the disc was rinsed with deionized water for 10 seconds.

Measuring the Dentinal Permeability Post-treatment with Artificial Saliva

The dentine disc together with the Pashley cell were immersed in 40 ml of artificial saliva at room temperature for 1 hour and then rinsed with deionized water for 10 seconds. The dentine permeability was then measured again.

Measuring the Dentinal Permeability Post-treatment with Artificial Saliva and an Acid Challenge

The disc was next immersed in 30 ml of a 6% citric acid solution for 2 minutes and rinsed with deionized water for 10 seconds. The dentine permeability was then measured for a final time.

A total of 30 dentine discs were used. They were distributed equally into six groups, with each group treated with a different loading of toothpaste. Each toothpaste was dedicated with a specific brush head to avoid contamination with others. Analysis of the dentinal permeability measurement was conducted as follows:

a) Percentage flow reduction after treatment with toothpaste

b) Percentage flow reduction after treatment with toothpaste and immersion in artificial saliva

c) Percentage flow reduction after treatment with toothpaste, immersion in artificial saliva and acid challenge

where V0 = Dentine permeability at baseline (after acid etch)

V1 = Dentine permeability immediately after toothpaste application

V2 = Dentine permeability following immersion in artificial saliva

V3 = Dentine permeability following acid challenge

Scanning Electron Microscopy was conducted on separate samples according to the same protocols used for the dentine permeability measurements. The discs were sputter coated with gold prior to examination and examined in a FEI Inspect-F SEM.

Results

Particle Size Analysis

Table 3 gives the D90, D50 and D10 values for the particle size for two commercially available Bioactive Glasses. The particle size of the BioMinF® glass is somewhat smaller than the NovaMin® and is thought to have been optimised for tubule occlusion with a larger proportion of particles being smaller than the size of dentinal tubules.

Table 3: Particle Size of Bioactive Glasses.

BioActive Glass

D90

(μm)

D50

(μm)

D10

(μm)

BioMinF®

23.00

5.92

0.62

NovaMin®

45.55

14.47

1.77

Hydraulic Conductance

Figure 1 shows the percentage reduction in hydraulic conductance or fluid flow reduction (FFR) after brushing the toothpaste on to mid coronal dentine discs. There is a small approximately 20% reduction in hydraulic conductance after applying the 0% BG toothpaste that may be a result of silica particles in the paste occluding the tubules. This increases to approximately 30% for the 2.5% glass loading and to just over 60% for a 5% loading of the glass. Above 5% there is a much more limited reduction in the hydraulic conductance with the 15% loading giving a 70% reduction. Above 5% loading of glass there is no statistical increase in the FFR (Tables 4-7). However the FFR is statistically significant up to a 5% loading of glass.

Figure 1: Percentage FFR against BioMinF Loading after Brushing.

Table 4: Statistical Result for Comparison of Percentage Fluid Flow Reduction after Toothpaste Application.

Comparison of Percentage Fluid Flow Reduction after Toothpaste Application
Statistical Data	Bioactive Glass Loading
Statistical Data	0%	2.50%	5.00%	7.50%	10.00%	15.00%
Sample Mean (in %), x	19.72	29.37	61.53	63.63	67.39	71.19
Sample sd, s	13.71	5.31	15.02	3.33	8.61	8.77
Sample Size, n	5	5	5	5	5	5
Confidence Interval, CI	1.96	1.96	1.96	1.96	1.96	1.96
Margin of Error	12.02	4.65	13.16	2.92	7.55	7.69
Upper Bound	31.73	34.02	74.69	66.55	74.94	78.88
Lower Bound	7.70	24.72	48.36	60.70	59.84	63.51
	Paired t-test Comparing Each Group (P<0.05)
T-test comparing to 0%		0.128529	0.000693	0.001276	0.002087	0.0022316
T-test comparing to 2.5%			0.004738	0.000264	0.000104	3.269E-05
T-test comparing to 5.0%				0.386578	0.238683	0.1454652
T-test comparing to 7.5%					0.246252	0.0712444
T-test comparing to 10.0%						0.2436429

Table 5: Percentage FFR in Comparison to the Baseline Control after treatment with the toothpaste after treatment with the toothpaste and immersion in artificial saliva (AS) and then with an acid challenge.

Bioactive Glass Loading	Percentage FFR in Comparison with Baseline/Control (%)
Bioactive Glass Loading	After treatment with toothpaste	After treatment with toothpaste and immersion in artificial saliva	After treatment with toothpaste, immersion in artificial saliva and acid challenge
0.0%	19.72	22.29	16.11
2.5%	29.37	33.97	26.57
5.0%	61.53	63.83	49.02
7.5%	63.63	64.96	56.63
10.0%	67.39	66.29	55.53
15.0%	71.19	71.53	65.26

Table 6: Statistical Result for Comparison of Percentage Fluid Flow Reduction after Toothpaste Application and Saliva Immersion.

Comparison of Percentage Fluid Flow Reduction after Toothpaste Application and Saliva Immersion
Statistical Data	Bioactive Glass Loading
Statistical Data	0%	2.50%	5.00%	7.50%	10.00%	15.00%
Sample Mean (in %), x	22.29	33.97	63.83	64.96	66.29	71.53
Sample sd, s	17.14	5.58	18.70	3.85	6.04	13.59
Sample Size, n	5	5	5	5	5	5
Confidence Interval, CI	1.96	1.96	1.96	1.96	1.96	1.96
Margin of Error	15.03	4.89	16.39	3.38	5.29	11.91
Upper Bound	37.32	38.86	80.22	68.34	71.58	83.44
Lower Bound	7.27	29.07	47.44	61.58	61.00	59.62
	Paired t-test Comparing Each Group (p<0.05)
T-test comparing to 0%		0.1560	0.0011	0.001758	0.003325	0.0073734
T-test comparing to 2.5%			0.019915	0.000576	0.000312	0.0009269
T-test comparing to 5.0%				0.446936	0.389115	0.2486045
T-test comparing to 7.5%					0.357676	0.182191
T-test comparing to 10.0%						0.1308723

Table 7: Statistical Result for Comparison of Percentage Fluid Flow Reduction after Toothpaste Application, Saliva Immersion and Acid Challenge.

Comparison of Percentage Fluid Flow Reduction after Toothpaste Application, Saliva Immersion and Acid Challenge
Statistical Data	Bioactive Glass Loading
Statistical Data	0%	2.50%	5.00%	7.50%	10.00%	15.00%
Sample Mean (in %), x	16.11	26.57	49.02	56.63	55.53	65.26
Sample sd, s	14.39	3.63	20.55	5.17	15.04	10.75
Sample Size, n	5	5	5	5	5	5
Confidence Interval, CI	1.96	1.96	1.96	1.96	1.96	1.96
Margin of Error	12.61	3.18	18.01	4.53	13.18	9.42
Upper Bound	28.72	29.75	67.03	61.16	68.71	74.69
Lower Bound	3.50	23.39	31.01	52.09	42.35	55.84
	Paired t-test Comparing Each Group (P<0.05)
T-test comparing to 0%		0.0734	0.0087	0.0006	0.0093	0.0035
T-test comparing to 2.5%			0.0353	5.46E-05	0.0047	0.0005
T-test comparing to 5.0%				0.2447	0.2922	0.0873
T-test comparing to 7.5%					0.4460	0.1220
T-test comparing to 10.0%						0.0952

Table 5 shows the FFR results after immersion in artificial saliva (AS) and then following an acid challenge to mimic the consumption of an acidic drink. In all cases there is an increase in the FFR from 0% to 2.5% to 5% and these differences are statistically significant (Tables 3 and 4). The results mirror the data before immersion in AS.

Above a 5% loading of BAG in the paste there are no statistically significant increases in the FFR. There was also no significant increase or decrease in the FFR values for any given loading from application to immersion in AS to applying an acid challenge. However, for all glass loadings there is a small decrease in the FFR following an acid challenge, but this was not statistically significant in paired t-tests in regard to either the brushed or the brushed with AS treatments.

Tubule Occlusion

The tubule occlusion was followed using scanning electron microscopy mid coronal dentine discs. Figure 2 shows the SEMs after brushing with the 0% BioMinF toothpaste. There is no visual evidence of any tubule occlusion, although there are a small number of fine particles on the treated surfaces. Figure 3 shows the SEMs of the 2.5% BioMinF toothpaste. There is significant tubule occlusion and a marked reduction in their size. The tubules are more obvious following the acid challenge. For dentine discs treated with 5% or more BioMinF loaded toothpastes (Figures 4-8) there is excellent tubule occlusion. There is slight evidence that this might improve slightly after immersion in AS and may deteriorate slightly on exposure to an acid challenge. There may be very slightly better tubule occlusion on increasing the glass loading above 5%.

Figure 2: SEM Images seen at x10000 Magnification. (A) Control. (B) After 0.0% Bioactive Glass Toothpaste Application. (C) Following Saliva Immersion. (D) Following Acid Challenge.

Figure 3: SEM Images seen at x10000 Magnification. (A) Control. (B) After 2.5% Bioactive Glass Toothpaste Application. (C) Following Saliva Immersion. (D) Following Acid Challenge.

Figure 4: SEM Images seen at x10000 Magnification. (A) Control. (B) After 5.0% Bioactive Glass Toothpaste Application. (C) Following Saliva Immersion. (D) Following Acid Challenge.

Figure 5: SEM Images seen at x10000 Magnification. (A) Control. (B) After 7.5% Bioactive Glass Toothpaste Application. (C) Following Saliva Immersion. (D) Following Acid Challenge.

Figure 6: SEM Images seen at x10000 Magnification. (A) Control. (B) After 10.0% Bioactive Glass Toothpaste Application. (C) Following Saliva Immersion. (D) Following Acid Challenge.

Figure 7: SEM Images seen at x10000 Magnification. (A) Control. (B) After 15.0% Bioactive Glass Toothpaste Application. (C) Following Saliva Immersion. (D) Following Acid Challenge.

Figure 8: SEM Observation of all bioglass loading(s) following brushing (Cross-Sectional View at x5000 magnification).

At a 0.0% glass loading, there was no crystal formation at the surface or within the tubules. However, at 2.5%, a thin layer of crystal deposit (arrow) was observed covering the surface. As the bioactive glass loading increased, the crystal deposits were observed to extend deeper into the tubules (arrows). At the 15.0% bioactive glass loading, large amounts of crystal deposits were observed at the surface of the dentine disc and within the dentinal tubules.

In general the SEM observations fit well with the hydraulic conductance data and FFR values observed.

Discussion

In the absence of the glass in the toothpaste formulation there is a small reduction in hydraulic conductance and the FFR is about 20%, There may be some tubule occlusion due to the presence of fine particles in the paste, notably silica and titanium dioxide particles. There is no evidence of any significant tubule occlusion when examined by SEM. On incorporating 2.5% glass particles into the toothpaste there is a further approximately 10% increase in FFR. The glass has a D50 of 5.92 μm (Table 3) close to the diameter of the larger dentinal tubules and up to 50% of the glass particles are therefore potentially small enough to enter the dentinal tubules and occlude them. On increasing the loading of glass to 5% there will be twice as many particles present that are small enough to occlude the dentinal tubules and the FFR increases to more than 60%. Figure 3 shows much greater tubule occlusion for the 5% glass loading than Figure 2 for the 2.5% loading. More than 90% of the tubules are occluded with the 5% loading. On increasing the bioactive glass content further there were no statistically significant increases in FFR or any observable increase in tubule occlusion in the SEM micrographs. Above 5% there are probably sufficient numbers of particles smaller than the dentinal tubule diameter to fully occlude the tubules and more glass particles present in the higher loadings do not improve the tubule occlusion.

On immersion in AS all the samples show a small increase in the FFR values, however it is not statistically significant. Bioactive glasses react with physiological solutions to form apatite and in the case of fluoride containing bioactive glasses such as BioMinF® fluorapatite is formed. Fluorapatite formation is desirable because fluorapatite is much more resistant to acids than hydroxyapatite or calcium carbonate used to occlude tubules in other proprietary dentine hypersensitivity toothpastes. The micrographs are all very similar to the ones before immersion in AS.

On immersing the samples in 6% citric acid, to mimic the consumption of an acidic drink in the mouth the FFR values all decrease slightly. However statistically there is no significant reduction. The micrographs do show some evidence of more open tubules following the acid treatment, but the effect is small on tubule occlusion. There seems to be evidence that the material of the surface layer and in the entrances of the tubules is being dissolved but that material deeper within the tubules remains, this phenomenon is most marked in with the 2.5% glass loading. The surfaces of the dentine discs also become noticeably smoother and there is generally a loss of the angular glass particles on the surface that is probably a result of the citric acid dissolving the remaining glass particles. Bingel et al. [35] showed that bioactive glasses dissolve much more rapidly under acidic conditions.

Conclusions

A 5% loading of glass is close to being optimal in terms of its effect on FFR reduction immediately after brushing, after immersion in AS and after an acid challenge.
There is no statistically significant increase in FFR after immersion in AS and no statistically significant reduction after an acid treatment.
There is virtually no tubule occlusion for the 0% glass toothpaste and an obvious increasing tubule occlusion up to 5% after this there may be a very slight increase in tubule occlusion with glass loading, but the effect is small.
There is a slight effect of immersion in AS improving tubule occlusion and there may be a slight reduction in tubule occlusion on applying an acid challenge.
It is possible that a higher loading of glass would be desirable, but this would involve a significant cost increase.

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COVID-19 Infection Presenting as Acute Onset Focal Status Epilepticus in a Nine Year Old Boy

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DOI: 10.31038/JNNC.2020322

Abstract

Atypical presentations of COVID-19 in children, such as new onset seizures must be recognized, and warrant liberal testing to prevent spread of the disease. We describe the unique presentation of a child with acute onset focal status epilepticus and vomiting, positive for COVID-19. Patient demographics, history, neurological findings, MRI, treatment, and prognosis were reviewed. The literature was reviewed for prior case reports. This nine year old boy had an episode of vomiting followed by acute onset focal status epilepticus. He was able to walk with assistance but could not speak or follow most commands. He had persistent left eye gaze deviation which could not be overcome and loss of motor function in his right arm. He also developed left arm automatisms. These symptoms lasted for approximately 90 minutes before resolution following administration of lorazepam. Ten hours after the onset of symptoms our patient developed a temperature of 38.6 and began vomiting again. COVID-19 testing was performed and resulted positive. He had no medical history, no known sick contacts, and there was no family history of seizures or epilepsy. Pediatric patients with COVID-19 present with a broader spectrum of symptoms than adults. While fever and cough are the most common presenting symptom in both the pediatric and adult populations, GI symptoms are more common in the pediatric population. It is important to be aware that atypical presentations including neurologic symptoms are being noted in the pediatric population and may indicate the presence of infection rather than another etiology. This is only the second documented pediatric patient in the world to present with seizure and test positive for COVID-19.

Keywords

Children, Novel Corona virus, Status epilepticus

Introduction

Estimates suggest children currently account for 1%-5% of diagnosed COVID-19 cases [1,2]. Available literature focusing on children is limited in comparison to adults but suggests the clinical spectrum of illness in the pediatric population associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes coronavirus disease 2019 (COVID-19) is distinct from and broader than that in the adults. The most frequently reported symptoms in adults with COVID-19 include fever, dry cough, fatigue, and in severe cases dyspnea is common [3]. Children, when symptomatic, also present with fever and cough, but a significant proportion present with atypical symptoms such as vomiting and diarrhea [4,5]. A large proportion of COVID-19 positive children are asymptomatic or have mild cases of disease [6,7]. This difference in presentation and severity may lead to underdiagnosis in children facilitating spread of the disease [8]. This report documents the first reported case of a COVID-19 positive child presenting with focal status epilepticus.

Case History

On the evening of admission our patient, a healthy 9 year old boy of Brazilian Portuguese descent, was at his aunt’s home for dinner. At 2200 hours he became nauseous and vomited one time. He was given an antacid and driven back to his parents’ house where he arrived at 2300 hrs. He required assistance but was able to walk into his house where his mother noticed fixed eye deviation to the left, inability to move the right arm, and was unable to speak. He was able to follow commands and was even able to take a shower. One hour after symptom onset, he was reaching out with his left hand for objects that were not there. He was also producing clicking sounds with his tongue. His mother became concerned that he was not improving and transported him to the Emergency Department for evaluation where he was able to walk under his own power.

In the Emergency Department, he was afebrile and vital signs were normal for age. There were no signs of meningismus. He had fixed left eye deviation with absent oculocephalic reflexes, 4 mm pupils responsive bilaterally, a fine tremor in the distal right upper extremity, which was not suppressible, and left arm automatisms. He was aphasic. Our patient was given 1 mg lorazepam IV which terminated his gaze deviation, abnormal movements, and aphasia with return to baseline mental status shortly afterwards. He was then loaded with levetiracetam 20 mg/kg. This child had no significant medical history, immunizations were up to date, took no medications, had no drug allergies, had no history of hospitalizations or surgeries, and had no family history of seizures or epilepsy. He had no recent head trauma, no fevers, URI symptoms, GI symptoms, sick contacts, or recent foreign travel.

Clinical Findings

Laboratory evaluations included a glucose level of 147 mg/dl. CBC and Chem10 were normal. LFTs showed a mild transaminitis. Urine toxicology screen and chest x-ray were normal. CT of the head without contrast was normal. LTM vEEG was abnormal due to the presence of nearly continuous delta slowing seen broadly throughout the right hemisphere, indicative of cortical and subcortical dysfunction. No epileptiform features were seen. Following admission overnight, our patient had returned to baseline mental status without any further clinical seizure activity. On the morning following admission, ten hours after his initial onset of symptoms, our patient developed a temperature of 38.6 C and experienced multiple episodes of vomiting. He was given ondansetron for nausea and acetaminophen for fever, after which symptoms resolved. In the setting of the COVID-19 pandemic, the acute onset of fever with GI symptoms suggested possible infection with the coronavirus. A test was obtained and resulted positive later on the day of admission. An MRI of the Brain was planned due to the focal neurological findings but was deferred due to his positive coronavirus test. Due to the highly infectious nature of COVID-19, his MRI would require scheduling during a low volume period with additional time required to disinfect the scanning equipment, in turn prolonging his admission and possibly exposing staff and other patients. Our patient was asymptomatic and had a normal examination. Thus the decision was made to provide pharmacologic coverage for seizure control and discharge to home, with outpatient MRI follow up after quarantine.

Our patient was in optimal health prior to the onset of symptoms. His presentation with acute focal status epilepticus and vomiting was typically concerning for an infectious etiology, intracranial process, or structural abnormality. He was afebrile, without leukocytosis, had no signs of meningismus or encephalopathy, and had no known sick contacts, so a lumbar puncture was deferred and antibiotics were not started. Computed tomography of the head was normal, indicating mass or hemorrhages were unlikely. Resolution of his presenting symptoms with lorazepam suggested seizure as an etiology.

Discussion

Prior to the COVID-19 result, thought was also given to norovirus and rotavirus infections as those two viruses are associated with seizures in children. Benign convulsions associated with mild gastroenteritis (CWG) are associated with norovirus and rotavirus, however, the highest incidence occurs in children 12-24 months, and is characterized by generalized tonic-clonic seizure activity lasting < 5 minutes [9]. This was inconsistent with our patient’s presentation. Our patient was outside of the expected age group for febrile seizures or CWG, and his presenting seizure was prolonged and focal in semiology. Furthermore, his electroencephalogram the following day revealed diffuse and continuous right hemispheric slowing. All of these features were atypical for febrile seizures or CWG. Yet, given his rapid return to baseline neurological functioning the following morning, acute infectious encephalitis and stroke were similarly unlikely.

Our patient was discharged to home quarantine. At his follow up visit two weeks later, our patient reported no further seizure activity and had returned to his baseline excellent health. Magnetic resonance imaging of the brain was normal without any signs for ischemic injury or structural lesions to account for his seizure. Anticonvulsant therapy was continued pending a following EEG. The current COVID-19 literature documents the clinical symptoms of over 3000 children world-wide and the spectrum of disease in children is still emerging. Only one two year-old girl in China has previously been reported to present with fever, convulsions, and GI symptoms and test positive for COVID-19 [10]. By contrast, the adult literature contains a growing number of COVID-19 positive patients presenting with central nervous system symptoms, including encephalopathy and seizures. A recent report describes a 78 year old woman presenting with focal status epilepticus as a unique clinical feature of COVID-19 [11]. While the link between COVID-19 infection and epileptogenesis has not been established yet, there is a physiologically logical hypothesis to propose linking the two. First, infection is by far the leading cause of focal status epilepticus in children, even outside the setting of direct infection of the meninges. Coronavirus is also thought to cause a dysregulated immune response which ultimately causes much of the morbidity and mortality associated with the disease. It is reasonable that an infectious etiology such as COVID-19 may cause seizures through alterations of cytokine responses, which are also implicated as an etiologic factor in febrile seizures. In this time of pandemic, it is important to recognize atypical presenting symptoms of COVID-19 such as new onset seizures, test liberally, and isolate those infected to prevent spread of the disease [12]. If focal status epilepticus is a unique presenting feature of COVID-19, our patients’ full outcome suggests that this rare presentation may portend a favorable prognosis.

Competing Interests

The authors declare that they have no competing interests.

Funding Information

No internal or external funding for this manuscript. The authors have indicated they have no financial relationships relevant to this article to disclose.

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An In Vitro Study for the Detection of Breast Cancer by Computed Tomography Using Targeted Gold Nanoparticles

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DOI: 10.31038/NAMS.2020321

Abstract

Breast cancer is the second most commonly diagnosed type of cancer worldwide and one of the leading causes of death in women in developed countries, second only to lung cancer. Standard clinical imaging techniques such as mammography, ultrasound and MRI can readily identify anatomical patterns, tumor location and size, but they cannot distinguish between benign and malignant lesions and are not able to detect metastases smaller than 0.5 cm. We have developed a targeted gold nanoparticle (AuNP) for the CT detection of breast cancer. To enable homing to breast cancer, AuNPs are coated with 2-Deoxy-D-Glucose that facilitates their binding to the Gluocose transporter (Glut-1), a cellular transmembrane receptor linked to the progression of breast cancer. Breast cancer cells MDA-MB-231, macrophages RAW 264.7 and fibroblasts MC 3T3.E1 were scanned using a pre-clinical CT scanner after incubation with AuNPs to analyze for AuNPs uptake. Breast cancer cells showed the most intense signal for 2-DG-AuNPs (916 HU), with almost no signal registered for the bare AuNPs (22 HU) or for the control sample (19 HU). In conclusion, 2-DG-AuNPs are a feasable targeting contrast agents for breast cancer detection, ensuring high contrast enhancement and low toxicity.

Keywords

Gold nanoparticles, Computed tomography, Breast cancer detection, Contrast agents, Glut-1

Introduction

Breast cancer is the second most commonly diagnosed type of cancer worldwide and one of the leading causes of death in women in developed countries, second only to lung cancer [1,2]. Although the overall 5-year survival rate in patients with breast cancer has improved over the past decades, the disease is still a serious public health threat [3,4]. More than 3.5 million women in the United States are living with a history of breast cancer and in 2014 approximately 41,213 deaths caused by this type of cancer were registered [1,2,5].

Despite the undeniable concern posed by this disease, recent advancements in screening and treatment techniques, such as surgery, radiotherapy and adjuvant systemic therapy (endocrine therapy, chemotherapy with anthracyclines and taxanes) have brought little improvement to the life expectancy and quality of life of breast cancer patients [5]. The golden standard for breast cancer imaging is mammography [6]. Early detection and diagnosis strongly correlate with a better outcome and higher survival rates and there is evidence that mammography screening could reduce breast cancer mortality rates in several countries [6,7]. However, in women with dense breasts, the sensitivity of mammography is only 62.9% and the specificity is 89.1%, as opposed to 87% sensitivity and 96.9% specificity in women with fatty breasts. Younger women are more likely to have dense breast tissue and cancer lesions are therefore harder to diagnose with conventional mammography in this patient group [6-8]. Moreover, mammography has a sensitivity of only 55-68% for the diagnosis of locally recurrent breast cancer. This is particularly due to the post-operative benign changes that appear after Breast Conserving Surgery (BCS), such as edema, calcifications, asymmetry and skin thickening [8,9]. Another disadvantage would be the discomfort caused by the compression of the breasts and the exposure to high doses of ionized radiation [8].

Standard clinical imaging techniques such as mammography, ultrasound and MRI are classified as structural imaging modalities because although they can readily identify anatomical patterns and tumor location and size, they cannot distinguish between benign and malignant lesions and are not able to detect metastases smaller than 0.5 cm [10]. Due to these limitations, a new field of imaging has been raising interest in recent years: nanoimaging.

Nanoparticles have unique physiological, optical and magnetic characteristics which bring new possibilities to the field of imaging [11,12]. Their size usually ranges between 1-100 nm and they can have different properties depending on shape, size and surface chemistry [10,13,14]. They can be used for imaging specific receptors, vascular abnormalities and even innate immune responses [13].

Gold nanoparticles (AuNPs) are attractive nanoparticles to study as Computed Tomography (CT) contrast agents [14-16]. Computed Tomography is one of the most common imaging modalities in hospitals as a diagnostic tool, having the advantages of providing superior tissue penetration and spatial resolution [6,17,18]. CT works by visualizing differences between tissue densities, which results in clear anatomical images. However, in order to see the differences between diseased and normal tissues, specific contrast agents need to be used. Radiopaque X-ray contrast agents are usually injected intravenously and exhibit non-specific penetration and binding, as well as rapid renal excretion [19]. These limitations have determined a surge in the research of targeting agents and cellular imaging, using metal-based agents instead of the traditional iodinated ones, such as Omnipaque or Visipaque [17,18,20].

Gold has proven to be an attractive alternative to conventional contrast agents, having a higher atomic number and a higher absorption coefficient compared to iodine. Due to these properties, gold nanoparticles offer a 3-fold increase in contrast per unit weight compared to iodine-based contrast agents [18,19,21]. Moreover, imaging gold nanoparticles at 80-100 kV provides lower soft tissue absorption, as well as allowing the reduction of bone tissue interference [19]. Gold nanoparticles can be prepared in various sizes and can be selected to facilitate specific extravasation through angiogenic endothelium such as those present in the leaking vasculature of cancer cells. Safety is an obvious consideration with any contrast agent. There is extensive experience with the use of gold in the treatment of various inflammatory and infective conditions in humans such as rheumatoid arthritis.

Bare gold nanoparticles exhbit rapid renal clearance. The rapid clearance and aggregation of AuNPs can be prevented by using a coating agent such as Polyethylene Glycol (PEG) [18,19,22,23]. The coating can be done using a short-strand PEG derivative, such as PEG-SH and/or a longer one such as OPSS-PEG-SVA (orthopyridyldisulfide-polyethyleneglycol-N-hydroxysuccinimide). OPSS-PEG-SVA is being used for the covalent couple of a highly specific ligand, resulting in a targeting agent specific for one cellular group.

We have developed a biocompatible targeted radiocontrast agent based on goldnanoparticles (AuNPs) which has the advantages of offering 3-fold greater contrast per unit weight than iodine-based x-ray contrast agents. Glucose transporter (Glut) is a cellular transmembrane receptor linked to the progression of various types of cancer. It had been previously demonstrated that MDA-MB-231 and MCF-7 breast cancer cells are characterized by over-expression of Glut-1. It is also known that 2-deoxy-D-glucose (2-DG) are specific ligands for Glut-1 [24,25]. Our imaging platform consists of 2-deoxy-D-glucose covalently coupled to spherical gold nanoparticles to target Glut-1 over-expressed by breast cancer cells.

Material and Methods

Materials

Sodium citrate, gold chloride and Picrosirius Red stain (Direct Red 80, Picric acid solution and Hematoxylin Solution A according to Weigert) were purchased from Sigma-Aldrich (St. Louis, MO, USA). OPSS-PEG-SVA was purchased from Laysan Bio (Arab, AL, USA). PES membranes (3000 MWCO) were purchased from Fisher Scientific. Silver enhancement staining kit was purchased from Structure Probe, Inc. (West Chester, PA, USA). DMEM, Fibroblast Basal Media and Fibroblast Serum-Free Growth kit were purchased from ATCC (Manassas, VA, USA), Primary anti-Glut1 antibody was purchased from Fisher Scientific, Alexa Fluor 288 goat anti-rabbit IgG (H+L) was purchased from AbCam (Cambridge, MA, USA), Vectashield mounting medium with DAPI was purchased from Vector Laboratories Inc. (Burlingame, CA, USA), Phalloidin was purchased from Invitrogen (Carlsbad, CA, USA), MTT assay kit was purchased from Roche Applied Science (Indianapolis, IN, USA), 2-Deoxy-D-Glucose was purchased from VWR, D (+)-Glucosamine hydrochloride was purchased from Fisher Scientific. Highest grade V1 mica discs 12 mm were purchased from Ted Pella, Inc., (Redding, CA, USA).

Gold Nanoparticle Synthesis

AuNPs were prepared by a method involving the reduction of chloroauric acid with a sodium citrate solution. Nanopure water (500 ml) was filtered through 0.22 μm filter and boiled in a 1 L conical flask. 5 ml of Gold Chloride (10%) was added to the boiling water followed by 4 ml of 1% sodium citrate solution. The solution was boiled for about an hour or until 200 ml of solution was left. The solution has a burgundy color. Next, the AuNPs were pegylated with polyethylene glycol derivatives in order to avoid aggregation. The AuNPs were incubated for 1 hour with a 100:1 molar ratio of PEG-SH to prevent aggregation and with 50:1 OPSS-PEG-SVA for the covalent coupling of the 2-DG. After pegylation, the AuNPs were further concentrated by centrifugation at 3270 rpm for 60 min. The AuNPs collected were further concentrated using PES membrane concentrators (MWCO 10,000) to a final concentration of ~40 mg Au/ml. The AuNPs were characterized in terms of size and polydispersity by UV Spectrophotometry and Dynamic Light Scattering (Malvern Nano-ZS Zetasizer, Malvern Instruments Ltd., a Spectris Company; Worcestershire, UK). The particles were also analyzed by Atomic Force Microscopy.

2-Deoxy-D-Glucose-AuNPs Synthesis

A solution of 2-deoxy-D-glucose (2-DG) (4 mg of 2-DG in 2 mL of nanopure water) was added to 2.2 mL of AuNPs solution (6 nM) and left to stir overnight at room temperature. The unbound 2-DG was removed by centrifugation through PES membrane tubes at 3270 rpm, for 1 hour. The 2-DG-AuNPs were reconstituted with 1 mL of nanopure water.

Assessment of Glut-1 Expression in Cells

The cells were grown overnight on 8-chamber slides. They were fixed with 300 µl of 4% formalin for 10 minutes and then incubated with 500 µl of 1% BSA (10% normal goat serum, 0.3M glycine) in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with 300 µl of antibody (ab652, 1:1000 dilution) overnight at 4ºC. Next, the cells were washed thouroughly with PBS followed by incubation with the secondary antibody, Alexa Fluor® 288 goat anti-rabbit IgG (H+L), used at a 1:1000 dilution for 1 hour at room temperature. The cells were washed with PBS and the slides mounted with DAPI-containing mounting media. Phalloidin was used for staining actin filaments. Fluorescence microscopy was used to determine the Glut-1 expression in cells.

Internalization of AuNPs in Cells

The cells were grown overnight on 8-chamber slides. 50 µl of AuNPs solution was added directly into the media and the cells were incubated for 2, 4 and 24 hours at 37°C and 5% CO2. The cells were then washed three times with warmed PBS and fixated with 300 µl of 4% formaldehyde for 20 minutes at room temperature. The cells were washed three times with PBS and then incubated with 100 µl of Hematoxylin for 15 minutes to stain the nuclei. After the cells were washed again, they were stained for Au with 100 µl of silver staining for 12 minutes. The slides were then washed and dried followed by mounting. Light microscopy was performed to determine the distribution of AuNPs retention in the cells.

Atomic Force Microscopy (AFM) Analysis

Gold nanoparticles were characterized using AFM. Freshly cleaved mica surface was treated with 10 µl of APTES (1 µM in miliQ-water) for 5 min and rinsed with 2 ml of miliQ-water (AP-mica). A drop of 10 µl of AuNPs suspension (to a concentration of 200 µg/ml) was incubated for 15 min at room temperature on functionalized mica (AP-mica) and rinsed with 60 µl of miliQ-water. Excess of liquid was absorbed and let it dry to be immediately scanned after preparation.

Atomic Force Microscopy was conducted at the UT Health – AFM Core Facility using a BioScope II™ Controller (Bruker Corporation; Santa Barbara, CA). This system is integrated to a Nikon TE2000-E inverted optical microscope (Nikon Instruments Inc.; Lewisville, TX). The image acquisition was performed with the Research NanoScope software version 7.30 and analyzed with the NanoScope Analysis software version 1.40 (copyright 2013 Bruker Corporation). High resolution images of AuNPs were obtained using RTESP cantilevers (fo=237-289 kHz, k=20-80 N/m, Bruker Corporation, Santa Barbara, CA). AuNPs size was determined using tapping mode operated in air to a scan rate of 0.5-0.6 Hz. Particle analysis was performed in 2 and 3 µm2 scans.

Computed Tomography Scans

One milliliter of cell suspension (105 cells/mL) was mixed with 1 mL of AuNPs (targeted or non-targeted) and allowed to interact for 4 hours at 37°C. PBS was used as control. Then, the solutions were centrifuged 3 times at 1000 rpm for 5 minutes, to wash out unbound AuNPs. After each centrifugation step the mixture was resuspended in PBS solution (1 mL total volume).

The cell suspensions were then analyzed with computed tomography. CT imaging was performed with a GE Ultra flat panel CT scanner (General Electric, Milwaukee, WI) with the following acquisition settings: 80kVp, 22 mA with 16 s rotation/exposure. Simple back projections were obtained for the 0.154 µm image reconstruction and exported as DICOM images. Image analysis was performed using the OsiriX software.

Results

The gold nanoparticles were synthesized by citrate reduction, using the Turkevich method. All the AuNPs preparations used in this study were analyzed using UV spectrometry, DLS, and atomic force microscopy (AFM). For all the samples, the UV absorbance peaked at 540 nm that corresponds to 60 nm particles. The AFM analysis showed a uniform preparation of AuNPs of 40-50 nm, at 2.0 µm scan zise (Figure 1). The DLS data showed a diameter of 46 nm with a polydispersity index (PDI) value of 0.451 for the bare AuNP samples. 2-DG-AuNPs had a diameter of 43 nm with a PDI value of 0.374.

Colloidal gold has been found to be unstable in a saline environment. The AuNPs were stabilized using a functionalized long chain PEG with a molecular weight of 5 kDa (OPSS-PEG-SVA) to prevent the formation of aggregates and rapid clearance in vivo. Moreover, OPSS-PEG-SVA had functional groups available for covalent bonding which allowed the conjugation via strong gold-thiolate bonds of a specific ligand (a D-glucose analogue).

Glut-1 expression was assessed using immunocytochemistry by staining Glut-1 receptors with green Alexa Fluor® 288 goat anti-rabbit IgG (H+L). Breast cancer cells MDA-MB-231 (image B, Figure 2) and macrophage cells RAW 264.7 (image A, Figure 2) showed Glut-1 receptor expression when compared to fibroblast control cells MC 3T3.E1 (image C, Figure 2). Fibroblasts did not display any Glut-1 expression.

Gold nanoparticle uptake was analyzed after incubating the cells with targeted or non-targeted AuNPs solution for four hours. The gold nanoparticles were stained with silver and appear as small black particles on the light microscopy images (Figure 3).

Figure 1:AFM amplitude image of gold nanoparticles scaned at 2 µm (X-Y) in tapping mode in air.

Figure 2:Fluorescence microscopy images of Glut-1 expression in RAW 264.7 macropages (image A), MDA-MB-231 breast cancer cells (image B) and MC 3T3.E1 fibroblasts (image C); scale bar represents 100 µm. Glut-1 expression is observed in RAW 264.7 and MDA-MB-231 breast cancer cells, but not in MC 3T3.E1 fibroblasts (green color).

Figure 3:Light microscopy images of gold nanoparticle internalization in RAW 264.7 macrophage (A-C), MC 3T3.E1 fibroblast (D-F), and MDA-MB-231 breast cancer cells (G-I) cell lines; scale bar is equal to 100 µm. 2-DG-AuNPs are taken-up by MDA-MB-231 (C) and RAW 264.7 cells (F). Very little non-specific uptake of bare AuNPs is observed in MDA-MB-231 (B) and RAW 264.7 cells (E). No uptake of bare AuNPs (H) or 2-DG-AuNPs (I) is observed in MC 3T3.E1.

Both breast cancer cells MDA-MB-231 and macrophage cells RAW 264.7 displayed AuNPs internalization as compared to fibroblast cells MC 3T3.E1 (Figure 3). Macrophages showed the most uptake of the targeted AuNPs which can be noted because of the presence of darker aggregates inside the cells (image F, Figure 3). Fibroblasts showed no difference in gold nanoparticle uptake between the AuNPs, 2-DG-AuNPs, and control (images G-I, Figure 3).

Breast cancer cells MDA-MB-231, macrophages RAW 264.7 and fibroblasts MC 3T3.E1 were scanned using a pre-clinical CT. The cells were incubated with targeted or non-targeted AuNPs for 4 hours followed by extensive washing. Breast cancer cells showed the most intense signal for 2-DG-AuNPs at 916 HU (Image C, Figure 4) with almost no signal registered for the bare AuNPs at 22 HU (Image B, Figure 4) or the control sample at 19 HU (Image A, Figure 4). No signal was visible on the scan in the control and bare AuNPs sample (Figure 4).

Figure 4:In vitro CT imaging of AuNPs internalization in MDA-MB-231 (A-C), RAW 264.7 (D-F), and MC 3T3.E1 (G-I) cells. Samples that display a higher uptake signal appear red on the CT images. The intensity of the red spots are quantified in Hounsfield Units (HU) included in each image. Approximately 40-fold higher attenuation and 5-fold higher attenuation is observed for the 2-DG-AuNPs as compared to bare AuNPs for the MDA-MB-231 cells and RAW 264.7, respectively.

For the RAW 264.7 macrophages, the 2-DG-AuNPs sample also displayed the highest radiointensity at 767 HU (Image F, Figure 4), followed by bare AuNPs at 140 HU (Image E, Figure 4). No signal was visible on the scan for the control (image D, Figure 4). Control samples registered 20 HU for fibroblast cells. The MC 3T3.E1 registered baseline values for all the samples analyzed (Images G-I, Figure 4).

Discussion

Medical imaging techniques can be divided into structural and functional imaging. Recently, functional imaging has been gaining interest over more conventional anatomical imaging. This came as a response to the need for earlier detection of malignant tissues and metastases, which were not visible through structural scans. This study proposed a novel approach to functional imaging using gold nanoparticles to target a ligand specific to mesenchymal breast cancer cells and macrophages. The imaging platform consisted of 2-deoxy-D-glucose covalently coupled to spherical gold nanoparticles to target Glut-1 over-expression in breast cancer cells. The feasibility of this platform was examined using CT imaging and histological staining.

One of the advantages of the proposed imaging technique is the use of a novel targeting agent (AuNPs) which provides superior X-ray attenuation over conventional iodine-based contrast agents. Moreover, it provides the specific targeting of cancer cells with a ligand against the Glut-1 overexpressed by breast cancer cells and macrophages. CT imaging offers excellent tissue penetration and spatial resolution as well as rapid image acquisition.

Carcinoma cells have been proven to have a higher metabolic rate as well as faster proliferation, which leads to greater demand of glucose. Such metabolic characteristics can be linked to the over-expression of certain glucose transporters, such as Glut-1 or Glut-4, on malignant cell membranes. Glut-1 has been proved to be expressed by almost all cancerous cell types [24,25]. The receptor binds and transports D-glucose within the cell, which is further metabolized into D-glucose-6-phosphate and 1,2-diphosphate [24]. D-glucose analogues can be transported by Glut-1 or Glut-4 receptors, but cannot be fully metabolized, therefore remaining inside the cells for longer periods before excretion. This property allows analogues such as 2-DG to be used as specific ligands for malignant cells. Positron emission tomography (PET) scans with radioactive Fluorodeoxyglucose (FDG) use the glucose pathway described to successfully target cancerous lesions and metastases, which are not always visible through structural imaging techniques. Targeting a metabolic mechanism instead of a cell membrane receptor is one of the most promising developments in cancer research and could eventually lead to better drug delivery systems and overcoming drug-resistance [24,25].

MDA-MB-231 is a breast cancer cell line that is known to overexpress Glucose transporter 1 (Glut-1). These types of cancer cells have a high risk of metastasis and an intermediate response to chemotherapy [25-28]. Previous studies show that RAW 264.7 cells also overexpress Glut-1 transporters and therefore exhibit increased glucose uptake and metabolism [29,30].

We were able to demonstrated Glut-1 expression by Immunocytochemistry (Figure 2) and ELISA (results not shown). Following staining with Glut-1 antibody (green), MDA-MB-231 cells showed the highest Glut-1 expression (most intense green coloration), followed by RAW 264.7. Fibroblasts MC 3T3.E1 were used as a control group and displayed no Glut-1 expression (Figure 2). A pilot study was first conducted to determine the adequate incubation time with gold nanoparticles. Cells were incubated with AuNPs for 2, 4 and 24 hours and the results were analyzed histologically. It was concluded that 2 hours did not allow sufficient gold internalization, while 24 hours allowed too much time for the clearance of the nanoparticles. Therefore the 4-hour time point was used in the following experiments.

The AuNPs internalization was first observed histologically, using a silver staining to stain the nanoparticles. Silver staining gave AuNPs a dark, almost black coloration. Therefore, when comparing MDA-MB-231 and RAW 264.7 slides to MC 3T3.E1, a darker coloration of the Glut-1 expressing cells can be noted due to small dark aggregates present inside said cells. Moreover, MDA-MB-231 and RAW 264.7 cells which were incubated with PBS confirm these results, displaying a light coloration, with no dark particles visible (Figure 3). When comparing bare AuNPs and functionalized 2-DG-AuNPs, a slight difference could be noted in MDA-MB-231 cells, which displayed higher internalization for functionalized nanoparticles (Figure 3). However, for RAW 264.7 cells, the amount of internalization for bare AuNPs and 2-DG-AuNPs was similar (Figure 3). A reason for this could be the presence of other mechanisms (independent of glucose transporters) for internalization in macrophage cells.

The 2-DG-AuNPs were then tested using a pre-clinical CT. As the cells were washed and centrifuged before scanning, the cells gathered at the bottom of the sample tubes, forming cell pellets. The radiointensity measurements were done in triplicates using values from the bottom of the sample tubes, representative for each cell group. It could be noted that 2-DG-AuNPs displayed the highest radiointensity both when measured with the OsiriX software and visually on the scans for the MDA-MB-231 cells (Images A-C, Figure 4) and RAW 264.7 cells (Images D-F, Figure 4). 2-DG-AuNPs registered values of 916 HU for MDA-MB-231 cells and 767 HU for RAW 264.7 cells. The bare AuNPs registered values of 22 HU for MDA-MB-231 cells and 140 HU for RAW 264.7 cells.The PBS controls showed baseline values of 19 HU and 25 HU, respectively. MC 3T3.E1 cells displayed baseline values 20-32 HU.The attenuation coefficient for the 2-DG targeted AuNPs was approximately 40-fold higher than that of the bare AuNPs for MDA-MB-231 breast cancer cells and 5-fold higher for RAW 264.7 cells. The bare AuNPs registered values of 22 HU for MDA-MB-231 cells and 140 HU for RAW 264.7 cells.The PBS controls showed baseline values of 19 HU and 25 HU, respectively. MC 3T3.E1 cells displayed baseline values 20-32 HU.

The attenuation coefficient for the 2-DG targeted AuNPs was approximately 40-fold higher than that of the bare AuNPs for MDAMB-231 breast cancer cells and 5-fold higher for RAW 264.7 cells.

Breast cancer cells express a high level of GLUT-1 receptors. By harnassing the increased metabolic demand and uptake, tracers such as the functionalized 2-DG-AuNPs is an attractive option to detect the cancer cells. This study was the first attempt at using gold nanoparticles functionalized with 2-deoxy-D-glucose to trace and image breast cancer and tumor-associated macrophages. Functionalized 2-DG-AuNPs proved to be a valid targeting contrast agent for cell lines MDA-MB-231 and RAW 264.7, exhibiting high radiointensity upon CT imaging. Gold internalization was verified with histological and radiological techniques and the positive results indicate a need for further research into this topic, such as testing the concept in vivo in a mouse model of breabt cancer. The targeting agent developed, 2-DG-AuNPs, promises to be a cheap and safe alternative for other types of functional imaging techniques such as nuclear imaging with radioactive fluorodeoxyglucose (FDG).

Acknowledgement

We would like to thank Dr. Xiaohong Bi for her help with the cell work. The CT imaging was conducted at the UT Health-Pre-Clinical CT Core Facility/Department of Internal Medicine using a GE Ultra flat panel CT scanner (General Electric, Milwaukee, WI). Atomic Force Microscopy was conducted at the UT Health – AFM Core Facility using a BioScope IITM Controller (Bruker Corporation; Santa Barbara, CA).

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The Profile of Clinically Diagnosed New Type 2 Diabetes among Asian Indians

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DOI: 10.31038/EDMJ.2020434

Abstract

Aim: To study the clinical and metabolic characteristics of newly diagnosed type 2 diabetes (T2DM) in urban clinics (CDD) and also to compare with the screen detected new diabetes cases (SDD) during an urban population survey.

Methods: Newly diagnosed T2DM (aged 20-60 years, n=741), based on blood glucose and Glycosylated haemoglobin (HbA1c) of ≥6.5% (48 mmol/mol) were selected. Demography, anthropometry, blood pressure, glycaemic and lipids profiles were analysed. Relevant statistical tests were used for group comparisons.

Results: Both groups had young age (45.0 ± 8.6 years) at diagnosis. Fasting blood glucose (p<0.05) and HbA1c (p<0.0001) were higher in CDD. Mean values of HbA1c were 9.1 ± 2.3% (76 ± 20 mmol/mol) in CDD and 8.3 ± 2.4% (67 ± 19 mmol/mol) in SDD (p<0.0001). Values of HbA1c were higher than ≥9.0% (75 mmol/mol) in 44.6% of CDD versus 26.4% of SDD (z=4.60, p<0.0001). SDD had higher body mass index (p<0.0001), abdominal obesity (p<0.005), hypertension (p<0.0001), cholesterol (p<0.005) and low density lipoprotein cholesterol (p<0.05) than CDD.

Conclusion: Both groups had young age at diagnosis. CDD had more severe glycaemia than SDD, probably suggesting that the clinic visits were delayed and therefore had longer period of undiagnosed diabetes. In comparison to CDD, SDD had higher metabolic abnormalities although the HbA1c values were lower.

Keywords

Newly diagnosed type 2 diabetes, South Asians, Clinical characteristics, HbA1c, Metabolic abnormalities

Introduction

Many developing countries show a rising trend in the prevalence and also in the incidence of type 2 diabetes T2DM [1-3]. Recently, a systematic review of studies on trends in the incidence of T2DM among adults in developed countries has shown that between 2006 and 2014, 27% of the reported populations had a stable incidence over time, while 36% reported a declining trend and 36% reported an increasing trend in the incidence of T2DM [4]. The declining trend in the incidence has been shown mostly in the developed countries. A huge clinical burden of newly diagnosed T2DM is present in developing countries. There is only limited real-world data describing the clinical characteristics of such patients [5].

The prevalence of diabetes is increasing rapidly in India, which is estimated to be 77 million adults in 2019 [1]. A recent epidemiological study done in urban India showed that, the prevalence and incidence of diabetes have increased significantly in all areas, including the Peri Urban Villages (PUV) of Tamil Nadu [2]. Using the data from the two studies conducted in 2006 [6] and 2016 [2] to estimate the incidence of diabetes, it was noted that, a sharper increase in the incidence occurred in the urban areas when compared with the villages [2]. A similar observation was made in the INdia DIABetes (INDIAB) study conducted in 15 states of India [7] as well as in a cohort study of 10 years follow-up in the urban area in southern India [8].

Several peculiar features are seen among the newly diagnosed South Asian populations with T2DM such as, younger age at onset, delayed clinical diagnosis due to lack of public awareness regarding the disease, reluctance to undergo periodic medical check-up and a long asymptomatic phase of T2DM [3,9]. The diagnosis of diabetes is often delayed, more so in the developing societies. Previous studies have described the characteristics of patients attending the clinics, who are under treatment [3,6,8]. There is sparse data on the profile of clinically diagnosed new T2DM patients in India.

The aim of this project was to study the clinical and metabolic characteristics of newly diagnosed adult T2DM patients from different clinics (CDD). We compared the profile with that of new T2DM identified during an epidemiological screening of urban population (SDD) [2].

Methods and Materials

Study Subjects

This is a study of newly diagnosed adult T2DM from 12 different centres located in four southern states of India. Patients with a previous history of diabetes or had taken anti-diabetes treatment were excluded from the study by the clinicians. Persons with prediabetes were also excluded. Details of men and women in the selected age group, who were treatment naïve were collected and analysed. For comparison, a group of newly detected T2DM subjects from an epidemiological survey of the urban population was taken.

All participants reported on fasting on the day of testing. In the clinics, the diagnosis of diabetes was based on blood glucose (fasting blood glucose ≥7.0 mmol/l and/2 hour Post Prandial Glucose (PPG)≥11.1 mmol/l) [10]. In the epidemiological study, diagnosis was made based on fasting and 2 h Post Blood Glucose (PBG) values. The 2 hour PBG was measured after giving 75 g of glucose load. We selected only patients who had an HbA1c value of ≥6.5% (48 mmol/mol) [11]. The total number of subjects analysed was 741, comprising of 514 from CDD and 227 from SDD; Identifier: NCT03490136 [2].

All centres followed uniform and standardised procedures for metabolic and clinical assessments. The study details and methodology were discussed in a meeting of the investigators, participating physicians and the researchers prior to the commencement of the study.

The Ethics Committee of the India Diabetes Research Foundation and Dr. A. Ramachandran’s Diabetes Hospitals approved the study. A written informed consent was obtained from all participants prior to the enrollment, at all centres. It was also agreed that the identity of the participants would not be disclosed. All study procedures were carried out in accordance to the ethical guidelines.

Clinical Assessments

Age, sex and presence of family history of diabetes were collected. Height was measured to the nearest centimeter using a stadiometer with the patient standing erect. Weight was measured to the nearest 0.1 kg using a digital weighing scale. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Generalized obesity was defined as BMI ≥23.0 kg/m²., BMI between ≥23.0 and 24.9 kg/m² was considered overweight, and BMI≥25.0 kg/m² was defined as obese. Waist circumference (WC), the smallest girth between the coastal margin and the iliac crest, was measured. Abdominal obesity was indicated by WC of ≥90 cm for men and ≥80 cm for women. Blood pressure was measured in the sitting position using the electronic measuring device (Omron HEM 7132; Omron, Tokyo, Japan). An average of two readings taken at 5-minutes’ interval was recorded. Persons with a history of hypertension and those with newly diagnosed diabetes with blood pressure readings ≥140/90 mmHg were categorized as hypertensive. All the clinical and metabolic details were recorded in a standardised questionnaire.

Biochemical Assessments

Glucose was measured in the respective study centres by the glucose oxidase method. Other biochemical parameters such as HbA1c and lipid profile were estimated at the central laboratory (Dr. A. Ramachandran’s Diabetes Hospitals, Chennai). Glycosylated haemoglobin (HbA1c) was measured by immunoturbidimetry (TINA-QUANT II; Roche Diagnostics Corporation, Germany); a procedure certified by the National Glycohemoglobin Standardization Program. Fasting serum lipid profile was estimated by standard enzymatic procedures using the reagents of Roche Diagnostics, Germany. HDLc was estimated by the direct assay. Samples were shipped to the central laboratory between 2° and 8°C on the same day of collection.

Statistical Analyses

Data are presented as mean ± standard deviation (SD) for continuous variables with a normal distribution, as median with interquartile range for skewed continuous variables and as number and frequency (%) for categorical variables. Independent samples t test, chi-square or Z – test were used to assess group differences for continuous variables and categorical variables respectively. Mann Whitney U test was used for skewed variables. A p value of <0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 21.0.

Results

Table 1 shows the demographic and anthropometric characteristics of the two groups with newly diagnosed T2DM. CDD had higher percentage of men than SDD (p<0.05). Both groups had similar mean age at diagnosis (45.2 ± 8 years). The mean BMI was higher in SDD (p<0.0001). Prevalence of overweight and obesity were similar in both the groups. Women in SDD had higher WC (p<0.005) and the percentage of abdominal obesity (≥80 cm) was also higher in SDD (p<0.05). SDD had higher mean diastolic blood pressure (p<0.0001) and higher percentage of hypertension (p<0.005).

Table 1: Demographic and anthropometric characteristics of the study groups with newly diagnosed type 2 diabetes.
	Clinically diagnosed (n=514) (CDD)	Diagnosed during screening (n=227) (SDD)
Men (n, %)	313 (60.9)^*	117 (51.5)
Positive family history of DM (n, %)	175 (34.0)	92 (40.5)
Age (yrs)	45.2 ± 8.6	45.0 ± 8.6
BMI (kg/m2)	25.3 ± 3.7	27.3 ± 4.6^$
Nonobese ≤ 22.9 (n, %)	101 (19.6)	39 (17.2)
Overweight & Obese ≥ 23.0 (n, %)	413 (80.4)	188 (82.8)
Waist Circumference (cm)
Men (cm)	91.9 ± 8.9	93.0 ± 9.8
Women (cm)	88.7 ± 10.6	91.8 ± 10.8^#
Men ≥ 90 (n, %)	195 (37.9)	73 (32.2)
Women ≥ 80 (n, %)	164 (31.9)	94 (41.4)^*
Blood Pressure
Systolic Blood Pressure (mm Hg)	128 ± 13	127 ± 18
Diastolic Blood Pressure (mm Hg)	81 ± 9	86 ± 11^$
BP > 140/90 mm Hg (n, %)	54 (10.5)	44 (19.4)^#

Data are presented as mean ± SD for continuous variables with normal distribution and as frequency (%) for categorical variables. Independent samples t test and chi-square test or Z test were used to test inter group differences for continuous variables and categorical variables.
CDD vs.SDD=^*p< 0.05; ^#p< 0.005; ^$p< 0.0001.
CDD, Clinically Diagnosed Diabetes; SDD, Screen Detected Diabetes; BMI, Body Mass Index; BP, Blood Pressure.

Table 2 shows the metabolic profile of the study groups. SDD had lower FBG (p<0.05) and HbA1c (p<0.0001) when compared with CDD. Among the total cohort of 741, 445 (60.1%) had HbA1c values
≥8.0% (64 mmol/mol), of which 289 (39.0%) had values ≥9.0% (75 mmol/mol).

Table 2 Metabolic profile of the study groups with newly diagnosed type 2 diabetes.
	Clinically diagnosed (n=514) (CDD)	Diagnosed during screening (n=227) (SDD)
Glycemic Parameters
Fasting blood glucose (mmol/l)	9.8 ± 3.3^*	9.3 ± 3.2
2 hour post blood glucose (mmol/l)	15.4 ± 4.4	15.3 ± 4.2
HbA1c % (mmol/mol)	9.1 ± 2.3 (76.1± 19.8)^$	8.3 ± 2.4 (66.8 ± 19.0)
HbA1c ≥ 8.0% (64mmol/mol) (n,%)	349 (67.9)^$	96 (42.3)
HbA1c ≥ 9.0% (75mmol/mol) (n,%)	229 (44.6)^$	60 (26.4)
Lipid Profile (n)	243	222
Triglyceride (mmol/l)	1.8 (1.3 – 2.7)	1.8 (1.3 – 2.8)
Total Cholesterol (mmol/l)	4.8 ± 1.0	5.1 ± 1.1^#
LDLc (mmol/l)	3.4 ± 0.9	3.6 ± 1.0^*
HDLc (mmol/l)	1.0 ± 0.3	1.0 ± 0.2

Data are presented as mean ± SD for continuous variables with a normal distribution, as median with interquartile range for skewedcontinuous variables and as frequency (%) for categorical variables. Independent samples t test and chi-square test or Z testwere usedto test intergroup differences for continuous variables and categorical variables. Mann Whitney U test was used for skewed variables.
CDD vs.SDD=^*p<0.05; ^#p<0.005; ^$p<0.0001
CDD: Clinically Diagnosed Diabetes; SDD: Screen Detected Diabetes; HbA1c: Glycosylated hemoglobin; LDLc: Low Density Lipoprotein Cholesterol; HDLc:HighDensity Lipoprotein Cholesterol.

It was observed that a larger proportion of CDD (67.9%) had values of ≥8.0% (64 mmol/mol) versus 42.3% of SDD (z=6.47, p<0.0001). HbA1c values ≥9.0% (75 mmol/mol) were present in 44.6% in CDD versus 26.4% in SDD (z=4.60, p<0.0001). Among the lipid variables, total cholesterol (p<0.005) and Low Density Lipoprotein cholesterol (LDLc) (p<0.05) were higher in SDD.

Discussion

There have been many reports highlighting the differences in the clinical profile of diabetes between South Asians and the western populations [12,13]. But, there is sparse data on the clinical and metabolic characteristics of newly diagnosed T2DM from India. In this communication we report the clinical and metabolic characteristics of newly diagnosed diabetes patients recruited from different centres in southern India (CDD). The group represents the urban population with diabetes. We compared its clinical profile with a group of undiagnosed diabetes detected during an urban epidemiological survey (SDD).

In our study, the mean age of the newly diagnosed T2DM in both the groups was similar (45.2 ± 8.6 and 45.0 ± 8.6 years in CDD and SDD respectively). Another large, multicentre, cross-sectional study among diabetes patients in India also showed that the mean age at onset of T2DM was 45.4 ± 10.9 years which was similar to the observation made in our study [14]. This was significantly lower than that reported among the newly diagnosed T2DM cases in the United States (55.6 ± 13 years) [5]. Similarly, in two large multi-ethnic studies from UK [15,16], the age at diagnosis of T2DM was found to be lower (51.5 ± 10.42 and 52.6 ± 13.5 years respectively) in the South Asian population when compared to the White population (58.9 ± 10.09 and 63.3 ± 13.8 years respectively). South Asians develop T2DM at least a decade earlier.

Lower BMI with higher HbA1c in the CDD compared to SDD could possibly be due to a reduction in weight usually associated with moderate to high levels of undetected hyperglycaemia. In an earlier study, we had observed that more than 20% of the cases had weight loss prior to the clinical diagnosis of T2DM [17]. Among the Asian populations, Indians have a reluctance to undergo regular health check-up and medical consultation is often sought only when the symptoms or related problems arise due to diabetes [9]. Similar observation had been reported earlier in migrant Asian populations in western countries [15,18]. In the UK, a multiethnic South London study mentioned above showed that HbA1c levels were higher in South Asians than in the Europeans at an early period after the diagnosis. This was partially attributed to a delayed diagnosis in the Asians [15]. In our study, the significantly higher FBG and HbA1c observed in CDD could be related to a delay in the diagnosis of diabetes when compared with those diagnosed during an epidemiological screening. There is a higher likelihood of a few CDD patients having taken treatment for hypertension and hyperlipidaemia prior to visiting a diabetes clinic.

In our study, 44.6% of the patients had significantly higher HbA1c ≥9.0% (75 mmol/mol) when compared with 21.7% of the newly diagnosed patients in the clinics in USA [5]. There may be a number of reasons for high HbA1c in newly detected diabetes. One important reason could be longer periods of undetected T2DM in our population, partially due to the delay on the part of the patients to seek medical help. In India, a larger percentage of men report for medical consultation than women. Therefore, the proportion of men was more among the clinically diagnosed cases.

In an earlier study, we had noted that T2DM remains asymptomatic for a longer period of time and many develop complications such as retinopathy even before diagnosis [19]. We did not record the presence of symptoms or complications in this study.

Hypercholestremia seen among the newly diagnosed patients may also be related to the delayed diagnosis of diabetes and higher blood glucose levels. In SDD, screening for diabetes was done among adults with neither a previous history nor the presence of symptoms of diabetes. There is a higher chance of early detection of diabetes among this group than in the clinic diagnosed cases. The likelihood of early detection of hypertension is also higher when screening is done.

Our study describes the clinical and metabolic features of newly diagnosed T2DM in the clinics. The differences with the screen detected diabetes in the population probably indicate late diagnosis in the clinics.

Declarations

Funding

The study was funded by India Diabetes Research Foundation, Chennai.

Conflict of interest

None

Ethics approval

The study was approved by the Ethics Committee of the India Diabetes Research Foundation and Dr. A. Ramachandran’s Diabetes Hospitals.

Consent to participate

A written informed consent was obtained from all participants prior to the enrollment in the respective studies.

Authors’ contributions

Arun R, AR, CS, AN and RV contributed to the study design, developed the protocol, supervised the study, drafted the manuscript and revised it with critical input. AR, CS, Arun R, PS and KS contributed to data preparation and analyses. KS and PS participated and coordinated the field work and data collection. All authors have read and approved the final draft of the manuscript.

Acknowledgements

The authors acknowledge the support rendered by the study physicians of various hospitals for providing the data source of the clinic patients. The help rendered by Mr.M.Karthikeyan and the epidemiology team of the India Diabetes Research Foundation in coordinating the epidemiological screening is greatly acknowledged. We are grateful to all the participants of the study for their co- operation and support.

Abbreviation

BMI: Body Mass Index

CDD: Clinically Diagnosed Diabetes

FBG: Fasting Blood Glucose

HbA1c: Glycosylated Haemoglobin

PBG: Post Blood Glucose

PPG: Post Prandial Blood Glucose

SDD: Screen Detected Diabetes

T2DM: Type 2 Diabetes

WC: Waist Circumference

References

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Metabolomic Analysis Unfolds Connection of Obesity and Endometrial Cancer

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DOI: 10.31038/EDMJ.2020433

Abstract

In recent years, obesity has been identified as a risk factor for several cancers including endometrial cancer. This paper identifies molecular mechanisms that are potential causes or effects of the correlation between obesity and endometrial cancer. From public sources, metabolites involved in endometrial cancer were extracted and analyzed with pathway analyses. Several elucidated pathways and mechanisms were involved with both obesity and cancer and were studied to better understand the relationship between obesity and endometrial cancer. These pathways include the Adipocytokine signaling pathway, Insulin resistance pathway, and Glucagon signaling pathway. The Adipocytokine signaling pathway showed a relationship between leptin, a body-weight regulating hormone, and the JAK–STAT3 pathway known to promote cancer cell proliferation. The Insulin resistance pathway shows insulin, an obesity-related hormone, activating the STAT3 protein as well as the PI3k–Akt and the mTOR signaling pathways, all of which can lead to increased cancer risk. The Glucagon signaling pathway gave evidence that glucagon may have anticancer properties as it activates the FOXO signaling pathway, which suppresses cancer cell growth. Metabolic syndrome as well as foods with high glycemic loads may hinder the anticancer potential of glucagon and lead to an increase in endometrial cancer risk. Such results can provide targets for reducing the risk or progression of cancer and provides information on the effects of certain lifestyles on endometrial cancer risk.

Introduction

Endometrial cancer is the cancer of the innermost lining layer of the uterus. Endometrial cancer is the most common cancer of the female reproductive organs. In 2020, the American Cancer Society projections for cancer of the uterus in the United States are 65 620 new cases and 12 590 deathsincluding 59 060 and 11 330 for endometrial cancer accordingly (https://www.cancer.org/cancer/endometrial-cancer/about/key-statistics.html, accessed February 17, 2020). Endometrial cancer has a variety of risk factors, such as advanced age, hormonal imbalance, and diabetes. Additionally, one of the most prevalent risk factors of endometrial cancer is obesity. In the US, an estimated 57% of endometrial cancer cases are directly attributable to being overweight or obese [1]. This correlation has been proven by several meta-analyses, such as the American Institute for Cancer Research’s report that for every increase of five Body Mass Index (BMI) units (kg/m²), there was a 50% increase in the risk of developing endometrial cancer [1]. Among the reasons for this correlation is the fact that fat tissue can alter hormonal balance and increase estrogen levels and is related to insulin resistance, all of which have been directly linked to increased risk of endometrial cancer. Furthermore, several mechanisms involved in obesity have been identified as risk factors for endometrial cancer. The identification of obesity-related mechanisms in endometrial cancer networks has shown the oncogenic effects of various pathways and proteins. Such biological functions within the body can activate cancer-causing pathways and genes or inhibit anticancer processes. The Akt signaling pathway, activated by leptin and other obesity-related hormones, was shown to have a correlation with endometrial cancer when cancer cell proliferation decreased as Akt was inhibited in Ishikawa cells [2]. Obesity-caused activation of the STAT3 protein through leptin and insulin resistance, increases endometrial cancer risk as it promotes induction of oncogenic genes such as c-Myc [3]. Insulin resistance, commonly associated with obesity, is related to activation of TNF and mTOR signaling pathways that promote endometrial cancer [4,5]. Obesity-related proteins and genes, such as c-Myc, TNF-ɑ, S6K, and eIF4E, contribute to cell proliferation, protein synthesis, and oncogenesis [6,7]. Other proteins and genes, such as FOXO1, have tumor-suppression properties [8]. Similar studies demonstrated that cancers of the breast, colon, rectum, esophagus, kidney, and pancreas have also been linked to being overweight or obesity (Figure 1) [9,10].

Figure 1. Relationship of Body Mass Index and risk of endometrial cancer. Graph shows a positive correlation between BMI and endometrial cancer risk from several studies. Adapted from reference [10].

Materials and Methods

Approach Overview

Collected from public sources metabolite data were analyzed by Ingenuity^® Pathway Analysis (IPA^®, Santa Clara, CA; Krämer et al. 2014) [11] program. Created by the IPA program metabolite–gene networks were elucidated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) software and analyzed withGeneFriends package (Figure 2).

Figure 2. Approach overview. Data from public sources are inputted to Ingenuity Pathway Analysis (IPA^®) program and results are analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Analysis Tool yielding significant KEGG pathways. Then results analyzed by GeneFriends program to find co-expressed genes and cluster them.

Metabolite Selection and Analysis

Metabolites specific to endometrial cancer were selected from public sources: articles from PubMed and Google Scholar libraries, Human Metabolite Database (https://hmdb.ca/) and BiGG Models (https://bigg.ucsd.edu). To use significant metabolites only they were cut off by p-value and fold change.

Ingenuity^® Pathway Analysis

Ingenuity^® Pathway Analysis (IPA^®; QIAGEN Inc., Redwood City, Calif., USA; Krämer et al. 2014; https://www.qiagenbioinformatics.com/products/ingenuity-pathwayanalysis) [11] is a web-based analysis software that allow to understand complex ‘omics data. Using a list of genes or metabolites, it allows identifying signaling and metabolic pathways, interaction molecular networks, and their functions.

David

The Database for Annotation, Visualization and Integrated Discovery (DAVID) is an online server that consists of the DAVID Knowledgebase and five integrated functional annotation tools for bioinformatics data. It allows, among other functions, functionally classify and annotate genes, convert gene IDs, search for interesting and related genes, highlight them on signaling and metabolic pathways, and view these genes dynamically.

GeneFriends

GeneFriends is a tool that creates a large co-expression map from a wide variety number of datasets. There are two variants to this tool: a dataset consisting of microarray datasets [12] and a dataset consisting of RNA-seq samples [13]. The RNA-seq map currently consists of 46 475 human samples and 34 332 mouse samples while the microarray map currently consists of 4164 human microarray datasets and 3571 mouse datasets. This tool is used to elucidate clustered co-expressed genes since genes that are activated simultaneously tend to share similar transcriptional regulation. Novel genes probably related to the disease of study can be elucidated based on a guilt-by-association approach when observed to be co-expressed with known candidate genes. The microarray version of the program GeneFriends was used to elucidate the genes coexpressed with our genes of interest using over 1000 microarray datasets [12].

Results

Metabolites Selection

Selected metabolites underwent two cutoffs:

• byp-value (p). Metabolites with p<= 0.05 considered as significant.

• by fold change (FC). To reduce noise, the metabolites with 1.33 < FC < 0.67 were used.

The list of metabolites selected is presented in Table 1.

Table 1: List of metabolites from endometrial cancer patients [14].
Metabolite	p-value	Fold change
1-Methylhistidine	0.952	-1.01
2-Hydroxybutyrate	<0.001	1.51
Acetic acid	0.296	-1.19
Betaine	0.988	1.00
L-Carnitine	0.386	-1.31
Creatine	0.214	-1.19
Citric acid	0.612	-1.16
Choline	0.443	1.11
D-Glucose	0.386	1.09
Glycine	0.375	-1.13
Glycerol	0.620	1.09
Formic acid	0.911	-1.01
L-Glutamic acid	0.843	-1.03
L-Tyrosine	0.540	-1.06
L-Phenylalanine	0.779	1.03
L-Alanine	0.816	-1.02
L-Proline	0.320	-1.10
L-Threonine	0.772	-1.04
L-Isoleucine	0.695	-1.04
L-Histidine	0.832	-1.03
Lysine	0.850	1.02
L-Lactic acid	0.601	1.06
Pyruvic acid	0.419	1.08
3-Hydroxybutyric acid	<0.001	2.76
L-Arginine	0.314	-1.10
Creatinine	0.661	1.06
L-Glutamine	0.849	1.01
L-Leucine	0.731	-1.04
L-Methionine	0.002	-1.35
L-Valine	0.702	-1.04
Acetone	<0.001	1.79

Gene-metabolite Network Analysis

The metabolites were analyzed by the IPA^® program, elucidating gene-metabolitenetworks including the selected metabolites, genes, and other entities (Figure 3).

Figure 3. Ingenuity Pathway Analysis networks 1 (a) and 2 (b) including endometrial cancer metabolites. Metabolites from the public sources [14] inputted into IPA, colored in blue. Proteins, colored yellow, were extracted and inputted into DAVID Functional Annotation Tool.

Many genes in these networks have a dual function involved in obesity/insulin resistance/diabetes mechanisms along with clear involvement in cancers including endometrial cancer (Table 2).

Table 2: Genes extracted from IPA networks created on the basis of metabolites in endometrial cancer.
BLMH	PPT1	BDNF
BTK	PRPF38A	FOXO1
C8orf44-SGK3/SGK3	PTK2B	HIF1A
CSK	RPN1	HSPB2
IGF1R	SGK1	MB
JAK2	SLC36A2	PHB
PDHA1	SND1	PPARA
PDPK1	SOCS3	PPARGC1A
PKN2	TTK	STAT5B
PNN	ALB	TNF
		TP53

Network 1

In this network, a number of genes have dual function and are involved in cancer and obesity mechanisms. We will describe some examples of such dual genes (Figure 3a).

PDPK1 (PDK1)

PDPK1 (PDK1) (3-Phosphoinositide Dependent Protein Kinase 1) as a gene phosphorylating AKT1 and involved in MYC1 pathway is very important in mechanisms of various cancer including endometrial cancer [14,15].PDPK1 gene is also a key player in the obesity. It phosphorylates AKT1, which is important in insulin signaling. Inhibiting of PDPK1 affects AKT1 function and consequently insulin signaling and weight gain [16].Intervention of a lifestyle decreasing insulin resistance led to significant degrease of PDPK1 (PDK1) gene expression confirming its dependence on obesity-related parameters [17].

IGFR1

IGFR1 (Insulin-like growth factor 1 receptor) is an important gene activating PI3K/ACT signaling pathway. Interesting to note that its expression growth proportionally to BMI and is leads tocancer prognosis [18]. Levels of activation of IGFR1 were found to be significantly higher in patients with endometrial cancer and insulin-resistant Diabetes II [19].

PPT1

Palmitoyl-protein thioesterase 1 (PPT1) is a lipoprotein;it has been shown that PPT1 gene is overexpressed in insulin-resistant patients [20]. PPT1 also significantly promotes tumor growth; PPT1 plays an important role in autophagy in cancer cells along with activation of mTORC1 [21].

SOCS3

As pointed by Sutherland and colleagues, silencing of the suppressor of cytokine signaling‑3 (SOCS3) by hypermethylationmay promote the oncogenic transformation of epithelial tissues and support growth of tumors [22]. It brings this gene in the set of tumor-suppressing genes. In obesity, SOCS3 plays a role of negative regulator of leptin-activation signaling actually increasing the leptin-resistance [23]. So, we can speculate that in both cases—cancer development and obesity methylation—silencing of SOCS3 would lead to disease enhancement.

BTK

BTK (Bruton’s tyrosine kinase) functions as a dual regulator of apoptosis and plays numerous other roles in cancer development [24]. BTK also plays a significant role in obesityleading to insulin resistance [25].

TTK

TTK protein kinase is a dual specificity protein kinase associatedwith cell proliferation. TTK has been found to be expressed in cancers such as pancreatic ductal adenocarcinoma, where cell proliferation was weakened when TTK was knocked down [26]. High expression levels of TTK was also found in clear cell renal cell carcinoma cells, and cell proliferation and invasion were suppressed with the knockdown of TTK [27].

SGK1

SGK1 (Serum/glucocorticoid regulated kinase 1) is involved in cellular stress response. SGK1 is known to be stimulated by insulin-related diseases as well as hyperglycemia [28]. SGK1 has also been found to have positive correlations with cancer progression and metastasis [29].

Network 2

Network 2 also contains dual-function genes. Several examples are presents below (Figure 3b).

TP53

Function of TP53 as a tumor suppressor is well known and does not need more explanation. In the same time not very obvious its role in obesity. Interesting findings demonstrated Morikawa and colleagues [30]. They found that TP53 expression changes to increase (positive) in patient with close to normal BMI. In the patients in obese category TP53 mostly has decreased expression (negative).Among the patients of invasive bladder cancer normal weight, overweight, and patients with obesity had a TP53 mutation frequency of 68.4%, 44.8%, and 25%, respectively (P < 0.05)” [31]. It means that mutational frequency of this gene is lower in obese patients. These fact that cancer rate is higher and TP53 expression is lowerin patients with lower BMI leads to a suggestion that obesity plays a significant role in cancer independently of the TP53 mutations. It can be related to higher probability of cancer proliferation in the adipose cells, more intensive development of blood vesicles in the tumors of obese patients, and other factors.Interesting to note that we found the same relations in some viral proteins impact to cancer development. We elucidated that presence of HPV viral proteins in patients with ovarian cancer leads to cancer development requiring less cancerogenic mutations than ovarian cancer without HPV (Shang, 2020 in press).

FOXO

FOXO (Forkhead box O3) phosphorylation leads to insulin resistance initiated by ER stress [32]. FOXO genes family has various roles in cancer development. Inactivation of FOXO function leads to uncontrolled cell proliferation and accumulation of DNA damage associated with cancer development [33].

PPARA

PPARA (Peroxisome proliferator-activated receptor-alpha) polymorphisms lead to cancer development [34]. It is also known that liver oncogenesis is significantly dependent on miRNAimpact on PPARA gene [35].

HIF1

HIF-1 (Hypoxia-inducible factor-1) regulates cellular response to hypoxia.As a transcription factors it increases concentration of genes involved in angiogenesis, cell survivaland other cancer-related activities [36]. In the same time HIF1 activation leads to white adipose tissue expansion in obese patients [37].

PHB

PHB (Prohibitin) is a “pleiotropic protein that has roles in both adipocytes and immune cells” [38]. PHB has been found to be upregulated in tumor cells from leukemia and lymphoma and it is associated with cancer progression [39]. PHB expression has been linked to both diabetes and cancer [38].

BDNF

BDNF (Brain Derived Neurotrophic Factor) has been found to play a role in cancer cell growth and progression through the stimulation of oncogenic pathways [40]. BDNF expression and mutation have also been associated with obesity [41].

Signaling Pathways Analysis

Genes elucidated the IPA networks as interacting with the endometrial cancer-related metabolites were analyzed by the DAVID program (Table 2). As expected, several KEGG pathways involving cancer were identified, including proteoglycans in cancer, prostate cancer, and pathways in cancer (Table 3). Moreover, several pathways that were identified were associated with obesity, which could reveal underlying mechanisms involving obesity and endometrial cancer.In order to understand the relationship between obesity and endometrial cancer, the adipocytokine signaling pathway, insulin resistance pathway, and glucagon signaling pathway from the list of these pathways were closely analyzed.

Table 3: KEGG pathways identified by the DAVID program.Count – number of inputted genes in the selected pathway.
Pathway Name	Count	p-Value
Insulin resistance	6	1.60E-05
Adipocytokine signaling pathway	5	6.30E-05
Proteoglycans in cancer	6	3.00E-04
Hepatitis C	5	5.90E-04
Prostate cancer	4	2.10E-03
Glucagon signaling pathway	4	2.90E-03
PI3K-Akt signaling pathway	6	3.20E-03
Thyroid hormone signaling pathway	4	4.50E-03
AMPK signaling pathway	4	5.50E-03
FoxO signaling pathway	4	7.00E-03
Insulin signaling pathway	4	7.60E-03
Hepatitis B	4	8.70E-03
Central carbon metabolism in cancer	3	1.50E-02
Fc epsilon RI signaling pathway	3	1.60E-02
Prolactin signaling pathway	3	2.50E-02
Pathways in cancer	5	2.70E-02
Herpes simplex infection	4	2.80E-02
HIF-1 signaling pathway	3	3.50E-02
Osteoclast differentiation	3	5E-02
Sphingolipid signaling pathway	3	5E-02

Adipocytokine Signaling Pathway

There is a high correlation between obesity and leptin concentration levels. Leptin is a hormone that regulates appetite, body mass composition, and energy expenditure. This hormone, while promoting lower food intake and fat loss, is often found in high concentrations alongside obesity when the hormone fails to induce weight loss. This effect is known as leptin resistance. Hyperleptinemia, where high levels of leptin are present in the bloodstream, is almost exclusively observed in obese animals. Leptin is primarily secreted in adipocytes and its expression is increased by obesity-related factors, such as overfeeding and insulin resistance [42,43]. The expression of leptin can be a risk factor for cancer. Leptin activates JAK, which then phosphorylates STAT3 (Figure 4). The activation of STAT3 is shown to significantly increase cell proliferation in endometrial cancer cells. When the phosphorylation of STAT3 was blocked with an inhibitor, the cell growth of the cancer cells decreased [2]. The activation of STAT3 leads to the induction of several oncogenic proteins, including c-Myc. In endometrial cancer cells, expression of the c-Myc gene increases cancer cell proliferation, and when the gene is knocked down, proliferation of the tumor cells dramatically decreases [6].

Figure 4. KEGG pathway of adipocytokine signaling pathway selected by DAVID program. Leptin is a hormone that regulates obesity-related functions, such as body mass, appetite, and food intake. Leptin is shown to activate JAK–STAT3 pathway known to drive the proliferation of cancer tumor cells.

Insulin Resistance Pathway

Insulin resistance characterized by resistance to the effects of insulin on glucose uptake is a commonly known result of obesity and is a fundamental aspect of type 2 diabetes. In obesity, weight gain causes an increase in islet β cells, which in its turn increases insulin production. It has been shown that with increased insulin production, hyperinsulinemia occurs and induces insulin resistance [44]. Insulin resistance is related toactivation both the STAT3 and PI3k–Akt pathways (Figure 5). Through IL-6, obesity activates the STAT3 protein, which is known to increase cell proliferation in endometrial cancer through induction of the c-Mycprotein [6]. Additionally, insulin resistance is related to activation the PI3k–Akt and the mTOR signaling pathways (Figure 5). mTOR plays an important role in glucose metabolism and is activated with increased levels of insulin and glucose. Hyperactive mTOR occurs with obesity and nutrient overload, likely due to hyperglycemia or hyperinsulinemia—bothinvolved in the development of insulin resistance and obesity [45]. The activation of mTOR can lead to an increased risk of cancer. mTOR activates S6K and eIF4E (Figure 5), which are responsible for protein synthesis and have been shown to promote cancer cell survival [45]. Obesity leads to the expression of TNF-ɑ (Figure 5). TNF-ɑ can stimulate estrogen synthesis and has a direct impact on cell proliferation in the endometrium; overexpression of TNF-ɑ has been linked to endometrial cancer risk [7].

Figure 5. KEGG pathway of insulin resistance from DAVID. Insulin resistance, which is commonly associated with obesity, activates both STAT3 and PI3k–Akt pathways. mTOR, activated by hyperinsulinemia and hyperactive in the obese population, can also lead to increased cancer risk.

Glucagon Signaling Pathway

Several meta-analyses have shown that a high glycemic load diet can increase the risk for endometrial cancer [3,46,47]. Glycemic load (GL) measures the effect a carbohydrate has on blood sugar levels as well as the amount consumed. Diets containing high GL with foods having high glycemic index are commonly associated with weight gain and obesity [48]. High GLs increases blood glucose and insulin levels, which leads to a decline in blood sugar levels within hours, ultimately creating a state of hunger that causes higher food intake and weight gain [49]. During the postprandial stage, a meal with the same nutrition but a higher GL can increase blood sugar levels two-fold, causing hyperglycemia. Hyperglycemia stimulates insulin production to lower blood sugar levels, while inhibiting glucagon release, which in turn would increase blood sugar levels [50]. Increased insulin production could potentially lead to hyperinsulinemia and insulin resistance, increasing the risk of cancer. Furthermore, glucagon has been shown to have anticancer properties on tumor growth as well as breast cancer [51,52]. Glucagon activates the FOXO signaling pathway and phosphorylates FOXO1 (Figure 6). FOXO1 is considered a tumor suppressor gene that is deactivated by insulin and the PI3k–Akt pathway. FOXO1 expression suppressed endometrial cancer cell proliferation in xenograft models and inhibited cell migration in Ishikawa endometrial cancer cells [8]. Diets with a high GL increase insulin levels while inhibiting glucagon release, reducing the activation of FOXO1 and its tumor suppressing properties, causing an increased risk of cancer.

Figure 6. KEGG’s glucagon signaling pathway selected by DAVID program. Foods with high glycemic loads cause hyperinsulinemia and inhibit glucagon release, potentially leading to insulin resistance and obesity. Glucagon, having anticancer properties, can lead to an increased risk of cancer when inhibited.

Similarly Expressed Genes Analysis

To understand more comprehensively the common molecular mechanisms standing behind similarities in endometrial (among the others) cancer with obesity and related events, we performed analysis with the program GeneFriends [12,13]. We submitted to the program our list of genes elucidated by IPA program on the basis of endometrial cancer metabolites. The resulting co-expression networkconsists of four clusters having increased numbers of interconnections (Figure 7). Gene Ontology (GO)—analysis of existing clusters of similarly expressed genes brought the following results.

Figure 7. Similarly expressed genes for the genes elucidated based on endometrial cancer metabolites. Green colored nodes represent genes from the original list.

Cluster 1

This contained mostly the genes related to immune response and related pathways activation (Table 4). These results support already pointed fact that immune response related processes are common for cancer and obesity. As noted by Pérez de Heredia and colleagues [53],“Obesity … is known to impair the immune function, altering leucocyte counts as well as cell-mediated immune responses. In addition, evidence has arisen that an altered immune function contributes to the pathogenesis of obesity”. Here, we see the obvious positive feedback when immune response alterations and obesity enhance each other leading to increase of both manifestations. In all cancers, inflammation is a part of the cancer development mechanism and immune response is common to tumor cells. Obesity is also related to inflammation. It is considered a condition causing chronic low-grade inflammation in patients [54]. Adipose tissue produced pro-inflammatory molecules including interleukins 1 and 6, TNFα, IFNγ, etc. and anti-inflammatory interleukins 3, 4, 10, and Ra [54,55].

Table 4: GO biological processes of the Cluster 1 of similarly expressed genes.
GO biological processes complete	P-value
Response to organic substance (GO:0010033)	2.66E-56
Immune system process (GO:0002376)	2.29E-53
Immune response (GO:0006955)	9.78E-52
Response to cytokine (GO:0034097)	4.18E-51
Cellular response to organic substance (GO:0071310)	1.92E-50
Cellular response to chemical stimulus (GO:0070887)	4.69E-50
Cellular response to cytokine stimulus (GO:0071345)	1.10E-49
Response to chemical (GO:0042221)	1.59E-44
Response to stimulus (GO:0050896)	1.55E-43
Cytokine-mediated signaling pathway (GO:0019221)	7.93E-43
Cell surface receptor signaling pathway (GO:0007166)	1.60E-40
Defense response (GO:0006952)	4.06E-39
Response to external stimulus (GO:0009605)	9.91E-39
Regulation of cell death (GO:0010941)	2.84E-38
Signal transduction (GO:0007165)	1.44E-37
Regulation of immune system process (GO:0002682)	1.08E-36
Positive regulation of immune system process (GO:0002684)	3.40E-36
Signaling (GO:0023052)	7.86E-36
Cell communication (GO:0007154)	1.17E-35
Regulation of cell population proliferation (GO:0042127)	1.21E-35
Immune effector process (GO:0002252)	1.76E-35
Cell activation (GO:0001775)	2.98E-35
Cellular response to stimulus (GO:0051716)	3.31E-35
Regulation of programmed cell death (GO:0043067)	1.07E-34
Leukocyte activation (GO:0045321)	4.09E-34
Regulation of apoptotic process (GO:0042981)	7.09E-34
Response to stress (GO:0006950)	8.63E-34

Cluster 2

This contains a number of biochemical reactions-related processes including interactions of various molecules within the cell environment, metabolic processes (Table 5). We will show in some examples that many ofthese processes are common for obesity and cancer.

Table 5: GO biological processes of the Cluster 2 of similarly expressed genes.
GO biological process complete	P-value
Lipid metabolic process (GO:0006629)	9.98E-12
Steroid metabolic process (GO:0008202)	7.41E-10
Small molecule metabolic process (GO:0044281)	1.66E-09
Regulation of hormone levels (GO:0010817)	1.48E-09
Organic hydroxy compound metabolic process (GO:1901615)	5.50E-09
Cellular response to xenobiotic stimulus (GO:0071466)	1.24E-08
Regulation of blood circulation (GO:1903522)	2.42E-08
Xenobiotic metabolic process (GO:0006805)	3.68E-08
Organic acid metabolic process (GO:0006082)	2.88E-08
Regulation of system process (GO:0044057)	3.87E-08
Monocarboxylic acid metabolic process (GO:0032787)	3.26E-08
Hormone metabolic process (GO:0042445)	3.62E-08
Negative regulation of endopeptidase activity (GO:0010951)	3.57E-08
Negative regulation of proteolysis (GO:0045861)	4.50E-08
Cellular lipid metabolic process (GO:0044255)	4.85E-08
Carboxylic acid metabolic process (GO:0019752)	5.82E-08
Negative regulation of peptidase activity (GO:0010466)	5.53E-08
Oxoacid(ketoacid) metabolic process (GO:0043436)	8.09E-08
Regulation of wound healing (GO:0061041)	1.05E-07
Response to oxygen-containing compound (GO:1901700)	1.61E-07
Negative regulation of hydrolase activity (GO:0051346)	1.73E-07
Response to stress (GO:0006950)	1.78E-07
Platelet degranulation (GO:0002576)	4.45E-07
Response to hormone (GO:0009725)	4.35E-07
Regulation of response to wounding (GO:1903034)	5.55E-07
Response to toxic substance (GO:0009636)	8.87E-07
Regulation of localization (GO:0032879)	8.76E-07
Response to inorganic substance (GO:0010035)	1.43E-06
Regulation of endopeptidase activity (GO:0052548)	1.96E-06
Fatty acid metabolic process (GO:0006631)	2.67E-06
Terpenoidmetabolic process (GO:0006721)	2.88E-06
Secretion (GO:0046903)	3.20E-06
Alcohol metabolic process (GO:0006066)	3.33E-06
Regulation of peptidase activity (GO:0052547)	4.13E-06
Regulation of transport (GO:0051049)	5.69E-06
Regulation of response to external stimulus (GO:0032101)	7.24E-06
Isoprenoidmetabolic process (GO:0006720)	8.05E-06
Tissue homeostasis (GO:0001894)	8.98E-06
Drug catabolic process (GO:0042737)	9.34E-06
Regulation of heart contraction (GO:0008016)	1.01E-05
Drug metabolic process (GO:0017144)	1.17E-05
Negative regulation of response to external stimulus (GO:0032102)	1.30E-05
Oxidation-reduction process (GO:0055114)	1.32E-05

Lipid Metabolic Process(GO:0006629)

Long and colleagues [56] pointed that “…energy metabolism, especially lipid metabolism, is significantly elevated during carcinogenesis.Furthermore, abnormality of lipid metabolism promotes cancer development, invasion and metastasis…”. In breast cancer the concentration of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) are significantly higher than in normal tissue. Moreover, concentration of TC and TG significantly higher in patients with metastases [56-58].

Oxoacid (Ketoacid) Metabolic Process

It was shown that key ketolytic enzymes BDH1 and OXCT1 can be biomarkers for possible ketogenic diet for cancer treatment [59]. Low expression level of these genes leads to sensitivity of cancer cells for low carbohydrates treatment.

Cluster 3

Thiscontains mostly processes-related to chromatin assembly, DNA processing, and epigenetic regulation (Supplemental Table S1). Experimental clinical studies of Ebot and colleagues [60] confirmed a link between obesity and prostate cancer through chromatin regulation. Their study demonstrated that in tumor tissues of very overweight/obese vs. healthy weight men, fifteen gene sets were enriched, while they did not change in normal tissues. They noted that patients with high tumor expression of chromatin-related genes had worse clinical characteristics and increased risk of lethal disease independent of grade and stage [60]. Wang with colleagues found that disruption of the bromodomain containing protein 2 (Bdr2), which plays a role in chromatin remodeling, leads to metabolically healthy obesity, with hyperinsulinemia, but enhanced glucose tolerance and low blood glucose, without T2D [61,62].Obesity causes changes in the acetylation distribution in specific cis-regulatory regions. This was supported by the fact of increased gene expression in the carcinogenic genes [63]. Authors also noted that premalignant lesions in obese patients’ colons have more probability to evolve to malignancy [63].

Cluster 4

.This contains mostly processes related to cell cycle (Supplemental Table S2). For obese patients, a number of cell-cycle-related genes were overexpressed including genes mostly involved in adipogenesis that most probably led to expansion of the adipose tissue during obesity [64]. Overexpressed genes promoting cell cycle were also found by Baranova and colleagues [65]. In cancers,defects of cell cycle is one of the most important molecular mechanisms of the disease: “Deregulation of the cell cycle engine underlies the uncontrolled cell proliferation that characterizes the malignant phenotype” [66]. Otto &Sicinski [67] pointed that cell cycle proteins are a promising target for anticancer drugs.

Discussion

Endometrial cancer is the cancer of the endometrium, contributing to an estimated to more than 10000 deaths in 2020. Obesity is recognized as one of the largest factors of increased risk and severity among different cancers, including endometrial cancer. There have been numerous population studies and meta-analyses supporting that an increase in BMI can significantly increase endometrial cancer risk. However, analysis of the molecular mechanisms involving both obesity and endometrial cancer can help elucidate their relationship and provide possibilities for new endometrial cancer therapies. Metabolites related to endometrial cancer obtained from the public sources were analyzed by the IPA program that created the gene–metabolites networks including the genes that interact with inputted metabolites. From the given list of endometrial cancer related genes and metabolites, DAVID Functional Annotation Tool identified several KEGG pathways involved in obesity, including insulin resistance, glucagon and adipocytokine signaling pathways.These pathways revealed potential targets for cancer treatment. For example, the STAT3 pathway, which was involved in both insulin resistance and adipocytokine signaling, could be targeted or inhibited which may reduce the effects of obesity on cancer risk. Lifestyle alterations, such as avoiding excessive food intake and high GL diets, can reduce metabolic syndrome, hyperinsulinemia, and weight gain, which can reduce endometrial cancer risk.In addition, further study on shared obesity and endometrial cancer biological processes via co-expression GO analysis may elucidate how their disease risks are positively correlated.

Data Availability

All data generated are presented in the article and supplemental materials.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Authors’ Contributions

VLK and IFT designed the study concept; KQW extracted the data; KQW created protein networks and extracted signaling pathways; BKP elucidated the genes with the similar expressions and did GO-analysis of the results; KQW, IFT, and VLK analyzed results; KQW, VLK, and IFT were involved in drafting the manuscript; IFT critically reviewed the manuscript for the intellectual quality of the study. The authors had full access to all data in the study. All authors have given their final approval to the final version of the manuscript.

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Are the Consequences of Gastrointestinal Infections of SARS-CoV-2 Underestimated?

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DOI: 10.31038/IDT.2020121

With more than 7.8 million confirmed cases and over 430,000 deaths COVID-19 has had an impact on the whole world (16.06.2020). In many countries the peak of the first wave seems to be passing and strict lockdown measures are being eased gradually. It is important to implement risk mitigation procedures to ensure the safety of those people returning to the workplace and participating in “public life”. Despite mounting evidence that faeces are infectious there seems to be little discussion of this, no public awareness of risk, or implementation of potential mitigation strategies. In addition, lasting alterations in the gut microbiome of patients infected with SARS-CoV-2 have been observed in at least two pilot studies. Dysbiosis in the gut microbiome influences different biochemical functions and is associated with diverse diseases, effecting the immune system, metabolic states and mental health. In consequence, there could be unidentified long-term health risks of SARS-CoV-2 infections in the gut.

In a meta-analysis of 60 studies, comprising 4243 COVID-19 patients Cheung et al. showed that the pooled prevalence of any gastrointestinal symptoms was 17.6% (95% CI: 12.3-24.5%), with 11.8% occurring with non-severe COVID 19 symptoms and 17.1% with severe symptoms [1]. Not all studies reported all individual gastrointestinal symptoms, consequently the pooled prevalence of anorexia was 26.8% (95% CI: 16.2-40.8), diarrhoea 12.5% (95% CI: 9.6-16.0), nausea/vomiting 10.2% (95% CI: 6.6-15.3), and abdominal pain/discomfort 9.2% (95% CI: 5.7-14.5).

Using immunofluorescent staining, Xiao et al. showed expression of the ACE2 protein in the glandular cells of gastric, duodenal, and rectal epithelia [2]. They also demonstrated staining of viral nucleocapsid protein in the same epithelia cell populations. In addition, the authors showed that of 73 hospitalized patients in the study, 53% tested positive for SARS-CoV-2 RNA in stool. Wu et al. confirmed these findings and went a step further and followed the patients over time showing that respiratory samples remained positive for SARS-CoV-2 RNA for a mean of 16.7 days (SD 6.7) and faecal samples remained positive for a mean of 27.9 days (SD 10.7) after first symptom onset [3]. These findings have been repeated and verified in a number of additional studies, in adults and children, asymptomatic, mild and severe COVID-19 patients. In addition, Wu et al. showed that from 60 patients that were released from hospital after clinical recovery 10% were still positive for SARS-CoV-2 RNA in anal swab [4]. At least four independent studies have been published reporting the isolation of infectious SARS-CoV-2 from stool samples. For example, Wang et al. reported the isolation of live SARS-CoV-2 from stool samples (non-diarrhoea) from two patients [5]. This is of particular interest since various studies have shown the production of bioaerosols during toilet flushing [6]. That is significant since new data shows that airborne transmission plays a major role for the spread of SARS-CoV-2 [7]. Indeed, in a report Liu et al. studied the potential aerosol transmission of SARS-CoV-2 in two Wuhan hospitals [8]. The authors reported elevated levels of viral RNA in the patient toilet areas and recommended proper disinfection and ventilation. In addition, Van Doremalen et al. showed that viable SARS-CoV-2 could be detected in aerosols up to 3 hours after aerosolization, and up to 2-3 days on plastic and stainless steel [9].

First studies of the gut microbiome of COVID-19 patients showed a significant decrease in diversity and abundance compared to healthy control (HC) [10]. This was combined with the increased relative abundance of opportunistic pathogens like Streptococcus and Rothia. The study showed significant disease-specific shifts in the overall microbiota composition between COVID-19 and H1N1 patients and HC. Changes in the gut microbiome have been shown to associate with various diseases, for example IBS, IBD, type 2 diabetes, cardiovascular condition and depression. Further studies are needed to evaluate the long term effects on SARS-CoV-2 infections in the gut and possibly a restorative treatment regime needs to be developed.

Taken together these results strongly indicate that SARS-CoV-2 infections in the gut have short- and long-term effects. The short- term effects are gastrointestinal symptoms and in about 50% of the patients viral shedding in stool occurs. Toilet flushing can create infectious bioaerosols that are a potential risk factor for oral/nasal- faecal transmission of SARS-CoV-2. Cleaning procedures of toilets in hospitals, care facilities, schools, nurseries, work places, restaurants, trains, busses, airplanes and public spaces will need to be looked at and perhaps improved.

Conflict of Interest

I am a founder, director and shareholder of TiKa Diagnostics Ldt., however the company is specialized in diagnostics of Mycobacteria (for example TB) in humans and animals, and has not provided funding or any other support/influence for this review.

Acknowledgement

I thank Martin Cranage, Mark Bodman-Smith, Deborah Baines, Kathleen Kirmer and Ralf Mikut for their feedback.

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Zuo T, Zhang F, Lui GCY, Yeoh YK, Li AYL, et al. (2020) Alterations in Gut Microbiota of Patients With COVID-19 During Time of Hospitalization. Gastroenterologydoi:10.1053/j.gastro.2020.05.048. [crossref]

Food Supplements in the Treatment of Ophthalmic Diseases: Preclinical and Clinical Studies

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DOI: 10.31038/JPPR.2020323

Abstract

At least 2.2 billion people in the world suffer by vision impairment or blindness (World Health Organization, 2019). Apart from infective diseases amenable to antibiotic treatment, cataract and corneal diseases resolvable by surgery, or refractive defects treatable with spectacles, most of the remaining pathologies do not have a cure. They can be treated with drugs or by surgery to alleviate symptoms and to slow down progression, but the underlying cause of the disease is usually not fully resolvable. Therefore, the best strategy to limit the burden of these ophthalmic diseases is prevention. In fact, almost all ophthalmic pathologies have a known etiology and pathway of development (although often not all the elements in the different steps have been elucidated), so that they are amenable to possible preventive treatments. Aim of this review is to illustrate how food supplements might help in the prevention and treatment of these ophthalmic diseases.

Introduction

Among the 2.2 billion people in the world affected by any type of eye condition, the World Health Organization estimates 196 million with Age-related Macular Degeneration (AMD), 146 million with diabetic retinopathy (DR) and 76 million with glaucoma [1]. No clear estimate exists on the global presence of dry eye, though the English Agency known as Global-Data UK Ltd. in a document published on June 2018, considering the 8 more industrialised Countries (US, France, Germany, Italy, Spain, UK, Japan, and China), reports a prevalence of 267,680,785 cases in 2016, destined to raise to 286,308,974 cases in 2026, therefore with an annual growth rate of 0.70% [2]. Approximately 200 million eyes globally are affected by cataracts causing a visual acuity less than 6/60, which become 3-4 times more including cataracts causing an acuity below 6/18 [3], accounting for one third of the worldwide blindness [4]. Globally, over 300 millions adolescents (less than 19 years old) are affected by progressive myopia [1], which in older ages may bring serious complications, like glaucoma or retinal detachment. All these eye pathologies pose a serious threat to vision, and have a strong social and economic impact. It has been estimated in 2014 by the National Eye Institute that the annual economic burden of vision loss, eye diseases and vision disorders in the U.S. amounted to 139 billion $ [5]. Now, what all these pathologies have in common is that there is no efficient pharmacological treatment for any of them. Resolutive surgery is possible in the case of cataract, but it is not devoid of – although minimal – side effects [6]. Surgical approaches are routinary applied also to the other pathologies, but most often they are not curative, and can at best attenuate their progression [7,8]. However, for all of them there is a link with lifestyle and alimentary habits, which becomes more evident in older ages [9,10]. This opens a space for prevention, early treatment and association of dietary habits or use of food supplements with pharmacological or surgical treatments. Aim of this review is to illustrate the scientific evidence supporting the use of food supplements in the management of eye pathologies.

GLAUCOMA

Glaucoma is an optic neuropathy, including a group of similar pathologies affecting the retinal ganglion cells (RGC), the optic nerve (ON) and the connected visual structures, causing vision loss and eventually blindness [11]. Primary open angle glaucoma (POAG) is the most common form of glaucoma (around 90% of the cases). It may occur in association with elevated intraocular pressure (IOP), in which case it is described as hypertensive glaucoma, or with normal IOP, in which case it is designed as normal tension glaucoma (NTG) [12]. In either case, the common pathological marker is the death of RGC by apoptosis, which eventually leads to the degeneration of the whole visual pathway, including the lateral geniculate nuclei and the visual brain cortex through the mechanism known as trans-synaptic degeneration [13,14]. This mechanism is present in other neurological diseases such as Alzheimer, Parkinson and Amyotrophic Lateral Sclerosis [13]. Therefore, also POAG is considered a slow progressing neurological disease [13]. Both extrinsic and intrinsic mechanisms contribute to RGC apoptotic death. Among the first, there are mechanical and metabolic factors. The compression of the ON at the lamina cribrosa (the structure present at the optic disk through which the ON leaves the eye) due to IOP elevation, leads to a decreased axoplasm flow and neurotrophins circulation between the periphery and the nucleus of the cell, necessary for RGC survival [15]. Endothelial dysfunction and decreased blood perfusion of the ON head (ONH) is a common finding in POAG [16], and may become a dominant trigger in NTG [17]. These events may activate or aggravate the intrinsic mechanisms, like glial cell activation and neuroinflammation, glutamate excitotoxicity and calcium release from dying cells, oxidative stress, starting a domino effect of RGC death, and further aggravating the pathology [18-20]. More recently, mitochondrial dysfunction has been added as a main mechanism among the different causes that can possibly lead to RGC death and glaucoma [21]. However, from the clinical perspective, the only pharmacologic therapeutic approach is based on the reduction of the IOP, both for hypertensive POAG and for NTG [22]. However, several clinical studies have shown that IOP reduction is a necessary, but not sufficient condition to arrest POAG progression [23]. Moreover, due to the structure of the retina, and the redundance of the cell network system carrying light signals from the eye to the brain, usually more than 50% of RGCs are dead when the first visual field abnormalities are detectable [24]. Hence, a neuroprotective strategy is becoming more often associated with the classical IOP-lowering treatment to try and improve the therapeutic outcome of POAG patients [25]. We have already reported that neurodegeneration is caused by several different types of insult to RGC, therefore an effective neuroprotective treatment should be endowed with multiple effects on different targets [25]. To this purpose, a single molecule with pleiotropic effects, or an association of molecules joining together their therapeutic mechanisms, should be considered (Figure 1).

Figure 1. Neurodegeneration in PoAG. The main recognized mechanisms of neurodegeneration in POAG are the excessive pressure at the level of the lamina cribrosa, where the optic nerve leaves the eye globe, and/or the decreased blood perfusion at the optic nerve head (ONH) (upper panel). These events may lead to injury and apoptosis of retinal ganglion cells (RGC), and the progressive loss of sight. Indirect neuroprotective agents are those mainly acting on the IOP decrease and the improvement of blood perfusion (bottom left). Direct neuroprotection works by blunting those events directly leading to cell injury and apoptosis (bottom right).

Brimonidine is a drug and it is an α-2A adrenergic receptor agonist, present in eye drops medications either alone, or in association with other hypotonizing drugs (timolol or dorzolamide). It is a paradigm of a multipurpose molecule, being active both on the hypotonizing and the neuroprotective side, and has clearly demonstrated that neuroprotection and hypotonizing effects can be dissociated. In fact, brimonidine, beside the hypotonizing effect on IOP [26], has also shown several neuroprotective effects, such as improved perfusion pressure at the ONH [27]; rescue of RGC from apoptosis by increasing the ratio bcl2/BAX [28]; prevention of the release of pro-apoptotic intracellular calcium [29]; blunting of the excitotoxicity due to excessive glutammate release [30] and upregulation of the neurotrophin BDNF expression by RGC [31]. Brimonidine neuroprotective effects in POAG have been demonstrated both in preclinical animal model systems [32] and in a seminal clinical trial of several years, showing that the neuroprotective effects achieved by brimonidine were indipendent from the hypotonizing effects that could be equally achieved by timolol [33].

Food Supplements

Epigallo-catechin-gallate (EGCG) is the main catechin of green tea, and also displays several activities with neuroprotective value. It has no effect on IOP, but it improves vascular perfusion by stimulating the expression of the endothelial form of nitric oxide synthase [34]; it is an iron chelator and a potent antioxidant, reducing oxidative stress and lipoperoxidation of cell membranes, thus shielding RGC from oxidative damage [35], and it is easily absorbed and distributed throughout the body [36]; it has anti-apoptotic activities, contrasting the activation of proapoptotic genes and favoring the expression of antiapoptotic ones, thus defending RGC from traumatic apoptotic death [37,38]. Its antioxidant and neuroprotective effects on RGC have been demonstrated in a mouse model in which oxidative damage was induced by intravitreal injection of sodium nitroprusside. When EGCG was given together with SNP, apoptotic cell death of RGC was significantly blunted [39]. Accordingly, in a mouse model of retinal ischemia/reperfusion, resembling acute closed angle glaucoma, oral

administration of EGCG could rescue RGC from apoptotic death, as seen by the conserved electroretinogram (ERG) response and thickness of the ganglion cell layer, and by the decreased expression of apoptotic caspases [40]. The antioxidant activity of EGCG has been proposed as a possible remedy to blunt the effects of mytochondrial dysfunction on glaucoma progression [41]. A clinical trial on 18 patients with POAG established that EGCG oral supplementation could improve – although slightly, because of the short treatment time – their pattern electroretinogram (PERG), which is an early alteration detectable in glaucoma subjects [42].

Citicoline is the current name of cytidine-5’-diphosphocholine. Citicoline is naturally present in all cells, serving as an intermediate in the biosynthesis of phosphatidylcholine, a component of biological membranes [43]. Citicoline has been one of the first neuroprotectors proposed [44], and has shown neuroprotective effects in Alzheimer disease, stroke, and Parkinson’s disease, as well as in glaucoma and amblyopia [45]. Some citicoline neuroprotective effects are mediated by the restoration of phosphatidylcholine levels and maintainance of sphingomyelin and cardiolipin levels (constituent of the inner mitochondrial membrane, involved in the cell energetic metabolism); by anti-oxidant effects, due to the increase of glutathione reductase (GR) activity and glutathione (GSH) synthesis and the decrease of PLA2 activity and lipid peroxidation; by the restoration of Na+/ K+ ATPase activity, essential for the proper functioning of neurons and glial cells. The choline provided by citicoline is used for the biosynthesis of the neurotransmitter acetylcholine, the stimulation of tyrosine hydroxylase activity and dopamine release [46]. Moreover, citicoline showed antiapoptotic effects by enhancing antiapoptotic bcl2 expression in a rat model of partial ON crush [47], and by decreasing apoptotic caspases expression in explanted rat retinas cultivated in toxic high glucose cell culture medium [48]. Over the years, citicoline has been studied in several glaucoma clinical trials. Initially, it was given by intramuscular injection, with a notable improvement on both PERG (RGC function) and VEP (Visual Evoked Potentials: gives the function of the whole visual axis) response [49,50]. In a further study, it was shown that oral administration of citicoline to glaucoma patients gave the same protection as the intramuscular injection, thus improving patients’ compliance [51]. Finally, also topical citicoline formulations as eye drops were shown to be effective in improving PERG and VEP of glaucoma patients over a period of 4 months [52].

Coenzyme Q (ubiquinone) neuroprotective activity also appears to be linked with its effects on mitochondria. In fact, coenzyme Q10 (CoQ10: the prevalent form found in humans) is a potent antioxidant, a membrane stabilizer and an important cofactor of the mitochondrial electron transport chain for the production of adenosine triphosphate (ATP) [53]. In the DBA/2J mouse model of spontaneous glaucoma, diet supplementation with CoQ10 significantly decreased the ratio between pro-apoptotic and anti-apoptocic genes in the retina, thus preventing apoptotic cell death. Moreover, mitochondrial DNA and the Tfam transcription factor of the oxydative phosphorylation system in the retina were functionally preserved. This resulted in increased RGC survival and preservation of ON axons, and blunting of astroglial activation in the retina and ONH [54]. Massive RGC death typically results in the release of an excess of glutamate, and a paroxystic activation of glutamate receptors. This leads to the opening of the mitochondrial permeability transition pore (MPTP) through which cytochrome C is released, signaling for the activation of caspase 9 and the apoptotic cascade. CoQ10 showed a further specific antiapoptotic effect by maintaining MPTP in the closed conformation thus preventing the triggering of apoptotic cell death [55]. In a rat model of mechanic ON injury a topical formulation of CoQ10 + vitamin E was administered as eye drops for 4 weeks. Decreased gliosis, increased anti-apoptotic gene expression, and preserved Tfam were observed in the retina of CoQ10 treated mice, which resulted in increased RGC survival [56]. Most recently, a multicenter, controlled clinical trial on 612 POAG patients has started, having visual field progression as the primary outcome, with the aim to evaluate the neuroprotective effects of an ophthalmic solution of CoQ10 and Vitamin E [57].

Saffron (crocus sativus) is a traditional medicinal plant containing in its stigmas many metabolites, among which the carotenoids crocin, crocetin and lycopene and the xantophyl zeaxanthin [58]. Crocin and crocetin are the main bioactive components [59], which can promptly be absorbed through the gastrointestinal tract, diffuse into organs and easily traverse the blood brain barrier (BBB) [60]. They have shown a protective effect on the integrity of the BBB during cerebral ischemia [61], which effect could likely be transposed also on the protection of the blood retinal barrier (BRB) during degenerative retinopathies, such as in AMD or DR [62]. The neuroprotective properties of saffron have been demonstrated in several ophthalmic pathologies and attributed to the important anti-inflammatory, antioxidant, and anti- apoptotic properties of its bioactive components [63]. In glaucoma, retinal ischemia triggers neuronal cell death. Crocin and crocetin blunt oxidative stress and inhibit RGC death in mouse and rat models of ischemia/reperfusion [64-66]. Excitotoxicity by N-methyl-D- aspartate (NMDA) released by dying RGC is a common event during glaucoma progression [19]. Oral administration of crocetin prevented the inner retinal damage induced by intravitreal injection of NMDA in mice, by inhibiting the caspase pathway [67]. Most recently, a mouse model of laser-induced ocular hypertension (OHT) has been used to show the anti-inflammatory and neuroprotective effects of a saffron extract titrated to 3% in crocin. Both gliosis and RGC death were efficiently prevented by crocin oral supplementation in OHT eyes [68]. Clinical studies with saffron used so far IOP as the sole endpoint. Daily oral administration of 30 mg of a saffron infusion for three weeks produced an IOP decrease in POAG patients [69], whilst a discontinuous (twice per week) oral treatment with an even higher amount of 1 gr for a month gave no variation on the IOP [70], suggesting that continuity of treatment rather than dose could be the relevant strategy. The toxic effects of saffron become evident for daily doses higher than 5 g [71]. Therefore, since its human therapeutic use is in the range of milligrams, it has virtually no toxicity [72].

Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a natural phenol, a phytoalexin naturally produced by several plants (it can be found in foods such as grapes, peanuts, blueberries, and dark chocolate) in response to microbiological attacks [73]. It is endowed with potent anti-oxidant and anti-inflammatory properties, which are likely responsible for its cardioprotective [74], neuroprotective [75], and anti- aging effects [76]. In fact, resveratrol has shown neuroprotective effects in neuroinflammatory and neurodegenerative diseases like stroke and Alzheimer’s disease [77,78]. Most interestingly, resveratrol has been shown to blunt the damage of ischemic brain injuries [79], likely by preserving brain mitochondria function after ischemic/reperfusion damage [80], and extend the lifespan of organisms from yeast to mammals [81]. All these beneficial activities of resveratrol appear to be mainly mediated by its stimulating effect on AMPK [82], which in turn stimulates the activity of SIRT1 (the mammal homologue of Sir2), the longevity-associated gene [83]. Several preclinical evidence support the efficacy of resveratrol in controlling the glaucomatous disease. In primary trabecular meshwork (TM) cells subjected in vitro to chronic oxidative stress (40% O2) resveratrol treatment blunted the production of free radicals and inflammatory markers (IL1α, IL6, IL8, and ELAM-1), also exerting antiapoptotic effects [84]. In a mouse model of glaucoma induced by clogging of the TM,

intraperitoneal injections of resveratrol partially protected RGC from apoptotic cell death [85]. RGC dendrites protection by resveratrol was demonstrated in mice fed for one year with a resveratrol supplement, and then subjected to ON crush, which simulates the compression at the ONH further to acute IOP elevation. In this case the protective effect appeared to be associated with increased expression of the nuclear C/EBP homologous protein and the nuclear X-box-binding protein-1 [86]. Intravitreal injection of resveratrol could mitigate the retinal ischemic injury in a rat model system of ischemia/reperfusion obtained by a sudden elevation of the IOP, simulating an angle closure glaucoma. In this case the effect was related to the containment of the upregulation of matrix metalloproteinase-9, heme oxygenase-1 (HO-1), and inducible nitric oxide synthase [87]. Mitochondrial protection by resveratrol treatment was shown in hypertensive model systems in vitro with RGC5 neuronal cells, and in vivo in rats fed with a resveratrol supplement before artificial clogging of the TM. In both systems neuronal cells improved mitochondrial function and survival was dependent on the activation of the AMPK/SIRT1/ PGC-1α signaling pathway, which is a critical pathway regulating mitochondrial biogenesis and function [88,89]. Finally, a critical process in the development of glaucoma is neuroinflammation, which is sustained by activated astrocytes producing cytotoxic mediators triggering RGC apoptosis. Resveratrol given in vitro to astrocytes under oxidative stress blunted their activation and improved their survival by decreasing caspases activation, toxic Tau processing, and neurofibrillar tangles formation [90]. Beside its direct neuroprotective activity on RGC, resveratrol may also work to decrease IOP. Topical administration of resveratrol eye drops in a rat model system of steroid-induced hypertensive glaucoma [91] could decrease by 25% the IOP via a mechanism involving adenosine receptors [92]. This hypotonizing effect was correlated with higher levels of metalloproteinase-2 in the aqueous humour (AH) and a decreased TM thickness beside a restoration of the retinal morphology and its redox status [92]. In the clinics, it has shown some degree of efficacy in neurologic pathologies such as Alzheimer disease and Friedreich ataxia [93], but no clinical data are available for glaucoma patients.

Melatonin is a very ancient molecule widely distributed in nature, in the vegetal and the animal world [94]. Melatonin is best known as a circadian regulator of sleep and other cyclical bodily functions [95]. Accordingly, melatonin is not produced only by the pineal gland, but also by several other organs, thus influencing through paracrine signaling the physiological functions of the whole organism [94] by differential binding to its three receptors MT1-3 [96]. In the eye, it is made by the retina [97], the ciliary body [98] and the lachrymal gland [99]. Melatonin and its metabolites have antioxidant and free radical scavenger activities including both reactive oxygen and nitrogen species [100,101], thus protecting from oxidative damage photoreceptors and other ocular tissues [102]. In preclinical studies melatonin showed clear neuroprotective effects. In a mouse model of ischemia/reperfusion of the retina after sudden IOP elevation, intraperitoneally injected melatonin prevented the toxicity due to the increased expression of the hypoxia inducible factor (HIF-1α) and Muller cells gliosis, thus improving RGC survival [103]. In a rat model of hypertensive glaucoma obtained by clogging of the TM, subcutaneous implantation of a pellet slowly releasing melatonin improved the antioxidant potential in the retina, decreased glutamate excitotoxicity with the final results of improving RGC survival and rescuing the retinal function as evidenced by the ERG [104]. In a rabbit model of intravitreal glutamate toxicity, simultaneous melatonin injection could blunt the oxidative stress and improve survival of RGC by decreasing their apoptotic cell death [105]. In a recent work with a rat model of hypertensive glaucoma obtained by episcleral injection of hypersaline solution, the intravitreal administration of Poly lactic-co-glycolic acid microspheres loaded with melatonin, CoQ10 and dexamethasone releasing the drugs for over 30 days, could preserve RGC from apoptotic cell death [106]. Interestingly, not only melatonin, but also agomelatine, the novel MT1/MT2 receptor agonist antidepressant, has shown significant neuroprotective features [107]. Melatonin not only shields RGC from apoptotic death, but also shows a direct effect on the IOP. Circadian changes of the IOP appear to be linked to melatonin [108], which has shown hypotensive effects on IOP [109], apparently mediated by the MT3 receptor [110,111]. Most recently, it has been shown that a nanomicellar formulation of melatonin and agomelatin given topically as eye drops is able to decrease by over 30% the IOP of normotensive rats, and by over 50% the IOP of hypertensive rats, after clogging of the TM [112]. Clinically, it has been reported that oral treatment with agomelatine could further decrease by 30% on average the IOP of 10 glaucomatous patients under maximum tolerated medical therapy (MTMT) with different hypotonizing drugs [113].

Cannabinoids are naturally present in our organism – including eye tissues and the retina – as endocannabinoids: anandamide, 2-arachidonoylglycerol and palmitoylethanolamide [114,115]. Phytocannabinoids (pCBs: agonists of the endocannabinoid receptors CB1 and CB2) are the active molecules found in cannabis (Cannabis sativa), and are mainly represented by Δ-9-tetrahydrocannabinol (THC), cannabinol and cannabidiol. THC is the molecule mainly responsible for the psychoactive effect of marijuana, while cannabinol is much less a psychotropic agent, and cannabidiol is not psychoactive at all [116]. Phytocannabinoids (much of all THC) have shown efficacy both as modulators of the IOP [117,118] and as neuroprotective agents [119]. It is likely the interaction of pCBs with the CB1 receptor that can lower the IOP by modulating production and outflow of AH [120,121]. The neuroprotective activity possibly depends on the antioxidant and antinflammatory effects of pCBs [122]. However, their psychotropic activity did not allow their free systemic use in ophthalmic diseases [123]. A valid alternative could be a topical formulation given as eye drops. Indeed, THC in nanotechnological formulations topically administered to normotensive rabbit’s eyes could reduce the IOP by 30% of the baseline value [124]. In a case report observation, oily based galenic ophthalmic formulations of pCBs with different proportions of THC and cannabidiol were given to patients unresponsive to drug treatments, however with little effect on the IOP. Only one patient with open angle glaucoma secondary to Fuchs heterochromic iridocyclitis had a partial response, probably due to the antinflammatory effect of the formulation with a higher content of THC [125].

Forskolin is a labdane diterpene extracted from the roots of the plant Indian Plectranthus barbatum (Coleus forskohlii). It is a lipid- soluble molecule that can cross cell membranes, thus working as a receptor independent activator of adenylate cyclase, finally resulting in the intracellular increase of cyclic adenosine monophosphate (cAMP) and cAMP-mediated functions [126]. Forskolin lowers IOP [127] by two independent and original mechanisms, both triggered by cAMP elevation: it decreases net secretion of AH by the ciliary body by a mechanism that involves the chloride channels and a reabsorption of AH from the posterior chamber into the stroma [128,129]; and by enhancing AH outflow through the TM by a mechanism that involves cell actin cytoskeleton disassembly through PKA activation and Rho kinase inhibition [130]. None of the existing glaucoma drugs (carbonic anhydrase inhibitors, beta-blockers, alfa-agonists, prostaglandin analogs) has similar mechanisms of action, therefore it might be expected that forskolin effects add upon those of such drugs. In fact, a clinical study in which POAG patients under MTMT were treated with a food supplement containing forskolin and rutin – an antioxidant and vasoactive ingredient [131] – showed that the food supplement resulted in a further decrease of the IOP, independent of the drug that the patients were taking [132,133]. Moreover, forskolin is also endowed with direct neuroprotective activities, mediated by the activation of paracrine signaling, due to the induction of the neurotrophin BDNF (brain derived neurotrophic factor) expression by astrocytes and vascular endothelial cells [134,135] and to the translocation of its receptor TrkB to the neuron cell membrane [136]. Preclinical data with a rat model system of hypertensive glaucoma obtained by clogging of the TM confirmed both the IOP control and the neuroprotective efficacy of oral forskolin [137]. Clinical data further corroborated the double efficacy of forskolin-based oral supplements. In a randomized, controlled clinical trial on 45 patients affected by POAG compensated by topical drugs, the 30 patients taking the food supplement with forskolin and rutin experienced during the 6 months of the trial a progressive decrease of the IOP and a concomitant increase of the PERG amplitude, suggesting an improvement of RGC survival [138]. Forskolin is also the basis of another association, with homotaurine – a neuroprotective compound used in Alzheimer [139] and Parkinson’s [140] disease – and carnosine, a wide spectrum antioxidant neuroprotector [141,142]. Preclinical evidence on a rat model of retinal ischemia/reperfusion obtained by sudden barometric increase of the IOP has shown a clear neuroprotective synergistic effect of the association of the three compounds intravitreally injected [143]. Improved RGC survival was reported, correlating with the induction of PI3K/Akt, the inhibition of GSK-3b and the reduction of calpain activity, all known to be linked to neurodegenerative events [143]. Results from a randomized, controlled clinical trial on 22 patients affected by POAG with IOP compensated by topical drugs, showed that patients taking the oral supplement containing the three components had a signiﬁcant further decrease of their IOP, an improvement of PERG amplitude at 6, 9, and 12 months, and of foveal sensitivity at 12 months, whilst all the above values remained stable in untreated control patients [144]. Most recently, a new association of forskolin with homotaurine and spearmint extract – rich in polyphenols and rosmarinic acid with antinflammatory and anti-oxidative effects, likely preventing neuron degeneration [145] – has been tested in a mouse model of ON crush, simulating the sudden compression at the ONH during acute glaucoma. The oral supplementation of forskolin, homotaurine, spearmint extract, and vitamins of the B group resulted in a significant improvement of RGC survival, correlating with a preservation of the ERG photopic negative response, which depends on the RGC health status, and with a decrease of inflammatory cytokines and pro-apoptotic markers [146]. Similar results were also obtained in a mouse model in which RGC degeneration depends on progressive IOP elevation, obtained by intracameral injection of polyetylen-glycol, which clogs the TM and decreases AH outflow [147].

Vitamins have been suggested as potential neuroprotective agents, and a recent metanalysis found relevant associations with vitamins A and C [148]. Deficiency of vitamins of the B complex has been reported in patients with glaucoma [148,149], which specifically showed significant lower serum levels of vitamins B1 and B12 [150,151]. Insufficient amounts of vitamins B6, B9 and B12 may lead to increased levels of homocysteine [152,153], known to be toxic to RGC [154], and found in relevant amounts in the AH and plasma of POAG patients [155]. A recent meta-analysis showed a high prevalence of hyperhomocysteinemia in patients with pseudoexfoliative glaucoma [156], which another study associated with decreased levels of vitamin B6, B9 and B12 [157]. Accordingly, increasing vitamin B9 intake appeared to lower the risk of pseudoexfoliative glaucoma probably by reducing homocysteine levels [158]. Vitamin C is an antioxidant, and an important factor involved in tissue repair [159]. Biochemical analyses have shown that the glycosaminoglycan content of TM in healthy subject is different from that of POAG patients, which contains less hyaluronic acid (HA) and more chondroitin sulfate [160]. Vitamin C stimulates the synthesis and secretion of HA in TM cells, more in those cells derived from a glaucomatous eye than from a normal eye, thus re-equilibrating the production of extracellular matrix (ECM) glycosaminoglycans [161]. Addition of vitamin C supplements was found to be associated in a dose-dependent way with decreased prevalence of glaucoma in a population sample in the United States [162]. In a large cross-sectional study on Afro-American women, a correlation has been described between higher consumption of food rich in vitamins A and C and carotenoids and a decreased incidence of glaucoma [163]. In a different epidemiologic evaluation, NTG patients were found to have lower blood levels of vitamin C and increased levels of uric acid [164]. Vitamin C has a role in maintaining lysosomal function and correct protein turnover in TM cells; therefore, an insufficient concentration of vitamin C in plasma and AH may alter the physiological function of TM cells, resulting in a decreased outflow capability and an increase of the IOP [165]. Vitamin E is a strong antioxidant [166]. Several studies on glaucoma patients have reported low levels of vitamin E in their blood [162,167] or AH [168]. A low intake of Vitamin A as retinol equivalents appears to be associated with an increased risk of POAG [150]. Vitamin D plays a role in the signaling pathways related to bone and mineral metabolism, cellular proliferation, immune modulation, and oxidative stress [169,170]. A high prevalence of vitamin D deficiency is observed in patients with glaucoma, confirmed by low circulating amounts of vitamin D in these subjects [171,172]. Metabolomic analysis on plasma samples from 72 POAG patients compared to 72 healthy controls revealed alterations of vitamin D metabolic pathways correlating with vitamin D insufficiency in POAG patients [173]; moreover, the presence of specific vitamin D receptor polymorphisms were found to be relevant risk factors in the development of glaucoma [174]. Accordingly, other studies have reported low serum levels of vitamin D in POAG patients [175].

OCULAR SURFACE DYSFUNCTION(Dry Eye)

The most recent definition of the dry eye syndrome by “The Tear Film and Ocular Surface Society Dry Eye Workshop Report”, defines the key points and possible therapeutic targets of the disease. Dry eye is “a multifactorial disease of the ocular surface” leading to tear film instability in which “hyperosmolarity, ocular surface inflammation, and neurosensory abnormalities play etiological roles” [176]. The etiology of dry eye is complex, and no cure for it is available. Artificial tears provide a way to blunt signs and symptoms of the disease, but with limited efficacy, both in terms of intensity and duration. Therefore, there is an increasing interest in the use of nutritional supplements as a complementary approach for the prevention and management of the dry eye syndrome [177]. Many scientific reports have highlighted an association between incidence and severity of dry eye disease and nutritional deficiencies [178-183]. The identification and appropriate use of nutraceuticals able to work on dry eye may provide insights into dry eye pathogenesis and increase the efficacy of conventional therapies.

Polyunsaturated Fatty Acids

Among dietary supplements, there is a growing interest in the use of polyunsaturated fatty acids (PUFAs) to prevent or alleviate signs and symptoms of dry eye [184,185]. PUFAs are involved in the synthesis of photoreceptor outer segment disks, and in ocular surface physiology; therefore, they are essential for proper visual development and retinal function [186]. PUFAs can be grouped into two main families: omega-3 and omega-6 [187]. The omega-3 include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and alpha-linolenic acid (ALA), while omega-6 include linoleic acid (LA), gamma-linolenic acid (GLA) and arachidonic acid (AA). Multiple clinical trials looked at the effect of omega-3 and omega-6 supplements in the treatment of dry eye syndrome. Miljanović et al., evaluated the association between the dietary intake of different ratios of omega-3 and omega-6 and dry eye syndrome occurrence in a large cross sectional study on women. Results showed that women with a higher dietary intake of omega-3 had a lower prevalence of dry eye symptoms and that a high ratio of omega-6/omega-3 (>15:1) was associated with a greater prevalence (more than 2-folds) of dry eye syndrome [188]. A meta-analysis of 9 randomized controlled trials (collected between 2002 and 2011) on PUFAs dietary supplements reported an improvement of the burning sensation and reflex lacrimation in dry eye patients assuming PUFAs [189]. A similar meta-analysis of other 7 randomized controlled trials collected from 2007 to 2013 reported not only the improvement of the above symptoms, but also an improvement of signs like Schirmer Test and Tear Break Up Time [190].

Meibomian gland dysfunction (MGD) is a chronic disease of the homonymous glands [191-193], leading to altered secretion of the tear film lipid layer, finally resulting in increased tear evaporation and tear osmolarity, a trigger for dry eye signs and symptoms [191-193]. One key factor of the potential benefits of systemic supplementation with PUFAs is the evidence that dietary fatty acids can be incorporated in lacrimal gland phospholipids [194-196]. Wojtowicz et al., explored the potential effect of dietary supplementation with omega-3 on lipid composition in a case-control trial, where treated patients received for 90 days fish oil, containing 450 mg of EPA, 300 mg of DHA, and 1000 mg of flaxseed oil (rich in ALA). At the end of the study, most of the patients (about 70%) became asymptomatic with an increment of tear secretion [197]. Another study recruiting a large sample of postmenopausal women, showed that high omega-3 consumption and moderate omega-6 consumption were protective against MGD [198]. Tear film hyperosmolarity is a focal event in dry eye syndrome [199]. In a double-blind, randomized, parallel trial, consumption of sea buckthorn oil (high in omega-3 and omega-6 fatty acids) for 3 months attenuated the increase in tear film osmolarity during the cold season and positively affected symptoms in dry eye patients [200]. Furthermore, a moderate daily dose of long-chain omega-3 PUFAs given over three months reduced tear osmolarity and increased tear stability in subjects with dry-eye disease [201]. In a different clinical study on patients with contact lens associated dry eye, 6 months of oral treatment with evening primrose oil rich in the omega-6 GLA resulted in improvements in dryness symptoms at 3 and 6 months and in overall lens wearing comfort at 6 months [202]. Oleñik et al., evaluated efficacy and tolerability of a food supplement containing a combination of omega-3 and antioxidants (a mixture of DHA 1050 mg, EPA 127.5 mg, DPA 90 mg + vitamins C/E and GSH + trace elements) on dry eye related symptoms in patients under treatment with artificial tears [203]. After 12 weeks, treated patients showed a significant improvement of symptoms with a lesser need of using artificial tears and a better tolerance to contact lens wearing. Comparable outcomes were achieved with treatment administered for shorter periods of time [204] or with lower concentrations of omega-3 PUFAs [205]. In another recent study, dietary omega-3 fatty acids administration showed efficacy in alleviating computer work- related dry-eye symptoms, associated with altered tear evaporation rate (Figure 2) [206].

Figure 2. Eye dryness and ocular surface dysfuntion. The malfunctioning of one ore more components of the ocular surface functional unit may lead to pathologic alterations of the ocular surface eventually causing eye dryness, discomfort and pain (upper panel). Some food supplements have been shown to prevent and/or alleviate symptoms and signs of dry eye (bottom panel).

Chronic inflammatory processes associated to immunologic activation have a pivotal role in dry eye pathogenesis [207,208] and PUFAs have been shown to have significant anti-inflammatory activity [209], which might turn beneficial in avoiding the excessive damage caused by chronic inflammation. In a mouse dry eye model induced by scopolamine injection in a dry environment, treatment with topical ALA for 9 days resulted in a significant decrease of inflammatory cytokines like TNF-α, and a reduction of corneal fluorescein staining in comparison to untreated controls [210]. Barabino and colleagues investigated the efficacy and anti-inflammatory activity of oral supplements of systemic ω-6 (LA and GLA) given twice per day for 45 days, on chronic ocular inflammation in patients with dry eye. They observed an improvement in symptoms and ocular surface inflammation in the study group patients when compared to controls [211]. A recent prospective case-control study showed that patients receiving an oral combination of antioxidants and omega-3 PUFAs for 3 months displayed reduced tear levels of inflammatory mediators such as the interleukins: IL-1b, IL-6, and IL-10 [212]. Brignole-Baudouin et al., in a multicentre trial, demonstrated that supplementation with omega-3 and omega-6 fatty acids can reduce the ectopic expression of the HLA-DR inflammatory marker and may help to improve dry eye symptoms [213]. Prostaglandins are well-known inflammatory mediators, playing a key role in the modulation of the inflammatory response in dry eye disease [214]. More specifically, tear levels of prostaglandin-E2 are known to be positively correlated with dry eye symptoms [215,216]. Black currant seed oil, rich in GLA and ALA, has been shown to modulate cell membrane lipid composition and eicosanoid production, finally reducing prostaglandin-E2 production [217]. Cermak et al. highlighted that in women with primary or secondary Sjögren’s syndrome (a systemic autoimmune inflammatory disease of the exocrine glands, including the lacrimal gland, thus resulting in a serious dry eye syndrome [218]), the intake of LA, and other specific PUFAs was significantly lower with respect to gender matched controls [219]. In another clinical trial involving 20 patients with primary Sjögren’s syndrome, an oral mixture of omega-6 induced a significant increase in anti-inflammatory PGE-1 content in tears, improving the symptoms of ocular discomfort and the signs derived from corneal epithelial defects [220]. The drug cyclosporine, present in eye drops approved for the treatment of dry eye disease [221], prevents T-cell activation and production of inflammatory cytokines, therefore breaking the inflammatory cycle of autoimmune dry eye disease and increasing the production of tears and mucin-producing Goblet cells in the conjunctiva [222]. However, the use of cyclosporine is not free from side effects which depend on the dose used; therefore it is important to maximise its effects in order to use lower doses. To this purpose, the effects of topical cyclosporine on dry eye signs and symptoms has been evaluated either alone, or in association with a prescription-only medical food supplement containing omega-3 and omega-6 PUFAs acids. It has been reported that a proper balance of omega-3 and omega-6 essential fatty acids could indeed improve the efficacy of cyclosporine on tear break up time and patients’ symptoms [223]. PUFAs have also proven to be effective in dry eye syndrome further to autoimmune rosacea disease. In fact, in rosacea patients which develop dry eye, diet supplementation with omega-3 for 6 months ameliorated dry eye parameters such as tear breakup time and Schirmer score [224].

Vitamins D and A

A nutritional deficiency, often associated to dry eye, is the lack of vitamin D [177,178,180,182,183]. Patients with vitamin D deficiency have a higher risk to develop dry eye disease [225]. The correlation of Vitamin D levels and inadequate sunlight exposure with severity of dry eye syndrome was highlighted in a recent study on Korean adults, concluding that sufficient sunlight exposure or vitamin D supplementation may attenuate signs and symptoms of dry eye [183]. Furthermore, an interesting correlation has been shown between single nucleotide polymorphisms in the vitamin D receptor gene and the incidence of dry eye disease in the Chinese Han population [226]. Vitamin D deficiency is relatively frequent in patients with primary Sjögren syndrome and also in this case its serum levels correlate with the severity of dry eye parameters [182,227,228]. In a retrospective observational study, in which subjects were divided into 3 groups according to serum Vitamin D levels (sufficient, inadequate or deficient group), tear break-up time and tear secretion were lower in the vitamin D-deficient group compared to the sufficient group [229]. In a recent work Dikci et al. observed that vitamin D deficiency may cause conjunctival squamous metaplasia and loss of goblet cells on the ocular surface in the eyes of 36 patients with mild to severe dry eye [230]. Inflammatory reactions and oxidative damage are common findings in dry eye, and in fact a study run on 217 patients (397 eyes) either normal or with various severity of dry eye has shown a correlation between its severity and the amount of oxidative tissue damage and polymorphonuclear leucocytes infiltration [231]. Within this frame, Eksioglu et al. evaluated the antioxidant effects of active vitamin D against high-dose radioiodine therapy-associated oxidative damage of the lacrimal gland in Wistar albino rats. Radioiodine caused significant oxidative stress and inflammation in lacrimal glands and vitamin D could blunt these effects, thus showing anti-inflammatory, antioxidant and radio-protective effects [232]. As mentioned before, chronic inflammatory events further to immunologic activation have a pivotal role in dry eye pathogenesis, and vitamin D is known to have immunomodulatory properties [179]. Further research on experimental animal models has provided evidence about the antinflammatory and immunomodulatory roles of vitamin D on the ocular surface, particularly in the cornea [233,234], and clinical data confirmed that a low serum vitamin D level is associated with different pathological states, such as autoimmune diseases, lymphoma, or neuropathy [218]. Reins et al. highlighted that vitamin D modulates the expression of inflammatory cytokines and protects corneal epithelial barrier function [180,235,236]. Antigen presenting Langerhans cells resident in the ocular surface epithelia have a role in aqueous tear-deficient dry eye pathogenesis [237], and topical administration of vitamin D can suppress ocular surface inflammation via inhibition of the increased Langerhans cells migration into mouse corneas [234]. The activation of inflammatory ROS-NLRP3-IL1β signaling axis induced by hyperosmotic stress has been recognized as a key priming stage of epithelial inflammation in dry eye pathogenesis [238,239]. Vitamin D has shown the ability to inhibit this signaling axis in human corneal epithelial cells under hyperosmotic stress via the activation of a competitive antioxidant signaling cascade dependent on the activation of Nrf2, a transcription factor regulating the expression of genes coding for antioxidant proteins [240]. Shetty et al. investigated the correlation between levels of serum vitamin D and tear-inflammatory proteins. They found that decreased serum vitamin D was significantly associated with higher levels of inflammatory interleukin IL-17A/F, interferon-γ, monocyte chemotactic protein 1, intercellular adhesion molecule 1, IL-4, IL-10, and decreased levels of antinflammatory IL-2 in tears of dry eye patients in comparison to healthy controls [241]. Altered tear fluid soluble factors with potential to modulate nociception exhibited a distinct association with the ocular surface discomfort status [242]. Vitamin D deficiency was found to be associated with neuralgia and chronic pain. In a case report it was observed that a vitamin D-deficient patient with corneal neuralgia had relief from burning sensation and pain by vitamin D supplementation, 1,000 IU/day, while topical therapies and lubricants were not effective [243]. It is thus probable that low vitamin D could contribute to the severity of ocular surface symptoms either by directly influencing nociceptive mechanisms or by affecting the presence of inflammatory cytokines [235,236,241,242]. Khamar et al., reported the dysregulation of tear fluid nociception-associated factors, corneal dendritic cell density, and vitamin D levels in dry eye patients. The use of lubricants (artificial tears or biological tear substitutes) is one of the strategies to manage dry eye symptoms [244,245], since tear film instability can lead to ocular surface damage [246]. Hwang et al., showed that Vitamin D enhanced the efficacy of topical carbomer- based lipid-containing artificial tears and Hyaluronic Acid (HA) in patients with dry eye and it may be a useful adjuvant therapy for patients with dry eye syndrome refractory to topical lubricants [247].

Vitamin A deficiency has also been suggested to be a contributory factor in the development of dry eye disease [248-250]. Vitamin A is essential for multiple functions in mammals including vision [251]; in fact it is a precursor in the formation of the rod photopigment rhodopsin [252]. An adequate consumption of vitamin A avoids night blindness, exophthalmia [253,254] and its deficiency is the main cause for blindness among children in third world Countries [255]. Apoptotic death of corneal epithelial cells is part of the pathological process of dry eye [256,257]. Vitamin A has been found to be able to prevent this event in a mice model of dry eye induced by benzalkonium chloride by decreasing the ratio between the pro-apoptotic gene Bax and the anti-apoptotic gene Bcl-2 [258]. In a recent clinical study on dry eye patients, tear ferning grades and tear osmolarity values were improved after vitamin A supplementation at a daily dose of 1,500 mg for 3 days [259].

lactoferrin

Lactoferrin deficiency in tears is a frequent finding in dry eye disease, especially in the elderlies [260]. Lactoferrin is a glycoprotein with several functions, including anti-inflammatory and antimicrobial, as well as promotion of cell growth, antiangiogenic and antitumoral [261]. Lactoferrin represents around 25% of tear proteins, with an average concentration in healthy subjects of 1.42 mg/ mL [262]. Its concentration decreases with prolonged closure of the eyelids, as it happens during sleep [263]. Lactoferrin concentration is also decreasing with age [264], and the decrease of its concentration in tear fluid correlates with the severity of dry eye disease [265-268]. Shimmura et al. reported a protective effect of lactoferrin against oxidative cellular damage in cultured corneal epithelial cells [269]. Accordingly, Fujihara et al. showed that lactoferrin eye drops may suppress the loss of corneal epithelial integrity produced in rabbits’ eyes by an ocular speculum blocking blinking [270]. Patients affected by Sjogren’s syndrome with dry eye symptoms and receiving 270 mg/day oral lactoferrin supplementation, showed improvements of both symptoms and tear film stability. This improvement reverted on cessation of treatment [271]. Moreover, oral lactoferrin administration preserves lacrimal gland function in aged mice by attenuating oxidative damage and suppressing subsequent gland inflammation [272]. Small incisions during cataract surgery may induce post-surgical dry eye, in which case oral lactoferrin may improve such condition [273]. Higuchi et al. showed the efficacy of Selenium-containing lactoferrin eye drops in a tobacco smoke exposure rat dry eye model and a short- term rabbit dry eye model of blinking block [274]. Rusciano et al. described the potential use of lactobionic acid, a functional mimetic of lactoferrin, in the management of age-related dry eye [260], and its efficacy is supported by the results of a recent study showing that lactobionic acid —with or without hyaluronic acid—favored wound healing both in vitro and in vivo, through an improvement of re- epithelization and the reduction of inflammatory markers [275].

Amino Acids

Amino acids are the building blocks of proteins and are essential for the construction and the functioning of the entire organism [276,277]. Amino acids are naturally present in human tears and their imbalance or deficiency could contribute to the development of the pathologies affecting the ocular surface [278]. Two different studies have reported that the concentration of amino acids is different in human tears and in serum [279,280] and that tear concentration changes in patients affected by ocular dryness [280,281]. As mentioned before, we know that chronic inflammatory events have a pivotal role in the pathogenesis of dry eye [207,208], and several studies have indicated that amino acids are involved in antinflammatory processes [282-284]. Dietary supplementation of arginine can enhance wound healing, regulate endocrine activity and potentiate immunity [285]. Glutamine may improve immunocompetence, thus reducing the susceptibility to infections and favoring the recovery of the seriously ill, therefore minimizing their hospital stay [286-292]. Branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, play critical roles in the regulation of energy homeostasis, nutrition metabolism, gut health, immunity and disease in humans and animals [293]. BCAAs have shown anti-inflammatory and anti-genotoxic activity in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages [294]. In a randomized, double-blind, placebo-controlled study, Dunn- Lewis et al. evaluated the antinflammatory properties and the capacity of improving physical performance of a multi-nutrient supplement containing leucine, isoleucine, valine, taurine plus anti-inflammatory plant extracts, and B vitamins. They reported that the multi-nutrient supplement was effective in improving the inflammatory status in both men and women, ameliorating the markers of pain, joints’ pain, strength, and power in men only, and both anxiety and balance (a risk factor for hip fracture) in women [295]. Refractive surgery may alter ocular surface homeostasis and Meibomian gland function, thus leading to the development of dry eye signs and symptoms [296-299]. Two different studies reported that oral administration of an amino acids mixture promotes the recovery and maintenance of corneal stroma health after refractive surgery [300,301]. In the first, Vinciguerra et al., demonstrated that oral supplementation of amino acids improved the healing rate of patients subjected to photorefractive keratectomy (PRK) and had shown re-epithelization defects, by favoring keratocyte growth and repopulation of the corneal stroma [300]. In the second study, Torres Munoz et al. reported that oral supplementation with a mixture of 13 different amino acids favored an active healing process and the remodelling of the stroma in patients operated of cataract surgery, by inducing growth and migration of stromal cells [301]. The identical oral supplement with 13 amino acids, used in the previous study, was also used by Roszkowska (Roszkowska, 2006, personal communication). The analyses of the corneal epithelium removed from patients during the procedure of PRK showed that amino acid treatment resulted in decreased EGF expression, suggesting a low proliferation rate of epithelial cells and keratocytes (hence, a decreased risk of scar formation); an increased expression of TGFβ, correlating again with decreased keratocyte proliferation and increased motility of epithelial cells (hence, faster re-epithelization); and a significant increase in the expression of polyamines, suggesting regulated differentiation and proliferation. Meduri et al. gave to patients undergoing PRK a cysteine oral supplement at a daily dose of 200 mg, then reporting shorter times for re-epithelization in comparison with eyes of untreated controls [302]. Observations at the confocal microscopy of the cornea of dry eye patients treated with eye drops containing HA and amino acids showed less hyper- reflecting cells in the epithelial layers (a sign of metabolic damage) in comparison to those receiving HA alone; nerve tortuosity in the subepithelial layer was also improving faster and better in the presence of topic amino acids [303]. The administration as eye drops of the amino acid taurine changed the tear proteome of contact lens wearers and dry eye patients shifting it towards the profile of healthy control subjects [304]. Taurine has then been included in a contact lens cleaning solution because of the regenerative effect on the tear film of contact lens wearers [305]. Taurine in combination with sodium hyaluronate has shown antioxidant and osmoprotective activity in in vitro and in vivo models of dry eye [306]. In albino rabbits, taurine effectively protected ocular surface tissues from chemical damage induced by hypochlorous acid, and arrested the progression of tissue damage (as measured by the level of lactate dehydrogenase activity) that had already been initiated by hypochlorous acid [307]. A mixture of taurine, alpha-ketoglutarate, pyruvate and pantothenate prevented corneal damage induced by 2-chloroethyl-ethyl sulfide [308]. Taurine could protect corneal stroma and epithelium from lactic acidosis caused by contact lens wearing [309], because of its acid buffering ability against lactic acidosis [310].

DIABETIC RETINOPATHY

Diabetic retinopathy (DR) is a multifactorial microvascular complication of diabetes mellitus caused by damage to the blood vessels of the retina, the light-sensitive tissue located at the back of the eye. DR has been included by the World Health Organization in the priority list of eye diseases which can be partly prevented, but not cured yet. In its early stages, DR may cause only mild vision problems but, over time, persistent high blood sugar levels can lead to the obstruction of the tiny blood vessels that nourish the retina, cutting off its blood supply. As a result, the eye reacts by triggering an abnormal growth of new retinal vessels, causing micro-hemorrhages and edemas in the macular region, thus leading to severe visual impairment and eventually blindness. These intra-retinal microvascular changes are used to classify DR into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). NPDR is characterized by a complex array of vasodegenerative lesions within the retinal microvascular bed, such as thickening of capillary basement membranes (BMs), loss of pericytes and vascular smooth muscle cells, capillary occlusion and microaneurysms [311]. PDR is caused by an abnormal growth of new blood vessels (retinal neovascularization) in response to inflammation and/or ischemic damage and hypoxia, eventually giving rise to vitreous hemorrhages and tractional retinal detachment. A direct consequence of inner blood–retinal barrier (iBRB) breakdown is the development of macular edema. Retinal neovascularization and macular edema are the result of increasing secretion of pro-inflammatory cytokines, and pro-angiogenic growth factors, among which predominates the vascular endothelial growth factor (VEGF) [312]. The retina is a highly metabolic active tissue, and high-glucose concentrations are particularly detrimental to its functioning. As reported by Sayin and collegues, chronic hyperglycemia is the main risk factor for DR because of its toxic effects on pericytes, to which vascular endothelial dysfunction may follow, also involving the retina [313]. A chronic hyperglycemic status will activate alternative energetic and metabolic pathways besides glycolysis, generating a toxic oxidative stress leading pericytes to apoptotic death, and causing vascular degeneration in the retina [314-316]. The physiopathology of DR could be represented by a pyramidal scheme in which the hyperglycemic state is at the bottom of the pyramid, immediately topped by oxidative stress, lipid peroxidation, apoptosis, mytochondrial alterations, leading to endothelial dysfunction and finally to retinal damage and neurodegeneration [317-319]. Under normal conditions, a certain amount of reactive oxygen species (ROS) can be tolerated, because they derive from the metabolic processes necessary to sustain cellular proliferation, differentiation and their physiological activities; however, if the production of free radicals overwhelms the capacity of the antioxidant defenses, the normal cellular metabolism will be disrupted by oxidative stress, degrading nucleic acids, proteins and enzymes, eventually leading to organ and tissue pathologies, such as DR [320]. Kowluru et al. [321] and Kaštelan et al. [322] have demonstrated that oxidative stress not only is involved in the physiopathology of DR, but also hinders its remission when glycemic values return within the normal interval. Typically, metabolic pathways altered in DR (polyol, hexosamine and protein kinase C pathways) lead to ROS imbalance, or result from oxidative stress, giving rise to advanced glycation end products (AGE). It is well known that visual impairment is already manifest in the early stages of DR, but currently no efficient treatment has been found to arrest its progression. Treatments have been developed for the late stages, and PDR can be treated or at least contained by pan-retinal laser photocoagulation, vitreoretinal surgery, or intravitreal injections of anti-inflammatory and anti-angiogenic drugs, even though retinal and visual function integrity cannot be preserved. Therefore, effective treatments for all stages of DR are needed to help in preventing or delaying the development and progression of this diabetes-induced visual dysfunction [323]. Most recently [324], it has been suggested to implement the use of nutraceuticals, which may act upstream the disease, reducing neuronal stress and favoring neuroprotection, in order to slow down its progression. Considering that antioxidants can act as free radical scavengers providing protection against oxidative stress, a good strategy could be to use nutrients with antioxidant and/ or anti-inflammatory activity and a good safety profile. In fact, in the last years the optimization of the glycemic and lipidemic control by a correct dietetic regimen has been shown to be of great relevance in controlling diabetes progression and its complications [325,326]. Thanks to their low toxicity in comparison to drugs, the use of food- derived bioactive molecules can be considered a good strategy to contrast the risk factors contributing to DR. In this paragraph, we will sum up the effects of nutritional strategies used as adjuvant therapies, on the different pathways involved in the physiopathology of DR (Figure 3).

Figure 3. Diabetic retinopathy. Diabetic retinopathy is a frequent complication of diabetes, and mainly results from the oxidative stress and the inflammatory events triggered by the continuous hyperglycemic state of the organism and the retinal vasculature. Initially, local injuries are visible (NPDR), eventually leading to the proliferation of new vessels in the macular area (PDR) and the loss of sight (upper panel). The food supplements that have been shown some efficacy in slowing down the progression of DR have antioxidant, antinflammatory and vasoprotective activities (bottom panel).

Polyunsaturated Fatty Acids (PUFAs)

A healthy diet widely known and appreciated is the Mediterranean diet, rich in vegetables, fish and extra virgin olive oil containing antioxidant and anti-inflammatory factors. Among these, we can find phenolic antioxidants; gamma- and delta-tocopherols and tocotrienols; long chain PUFAs: omega-6 and mostly omega-3, from which DHA and EPA are derived; several carotenoids of which lycopene may be the most active; isothiocyanates from cruciferous vegetables; sulfur compounds from allium vegetables; terpenoids. Interestingly, most of these health-promoting nutrients function, at least in part, by raising the nuclear factor Nrf2 (nuclear erythroid factor 2-related factor 2), responsible for the transcription of over 500 genes in the human genome, most of which have cytoprotective functions [327]. The role of PUFAs in the retina has been intensely studied for their involvement in regulating vascular function and angiogenesis. Tikhonenko et al. have demonstrated, by transcriptomic analysis in diabetic rats, that diabetes induced a downregulation of retinal enzymes known as elongases, necessary to metabolize PUFAs. This event resulted in a decreased content of retinal DHA, as well as decreased incorporation of very long-chain poly unsaturated fatty acids, particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with the presence of an inflammatory status in diabetic retina, reflected by an increase in gene expression of the proinflammatory markers IL6, VEGF, and ICAM-1 [328]. Similar results were obtained by a lipidomic analysis showing that the omega-3 long-chain poly unsaturated fatty acids metabolites derived from the activity of cyclooxygenases and lipoxygenases, inhibit inflammation and angiogenesis [329]. Connor and colleagues showed that in mouse models of oxygen-induced retinopathy the dietary intake of omega-3- and omega-6 PUFAs decreased the avascular area of the retina and increased vessel regrowth after injury, thereby reducing the hypoxia- induced pathological neovascularization. Moreover, while the intake of omega-6 PUFAs increased the microglial production of the pro- inflammatory and pro-angiogenic cytokine TNF-α, such increase was prevented by the intake of omega-3 PUFAs, which are the precursors of the mediators neuroprotectin-D1, resolvin-D1 and resolvin-E1, known to be potent inhibitors of neovascularization [330]. This anti- angiogenic effect induced by omega-3 PUFAs on retinopathy in the mouse eye is comparable in magnitude to treatment with an inhibitor of VEGF [331]. Therefore, increased dietary intake of omega-3 PUFAs could be a useful addition to anti-VEGF therapy to control pathological retinal angiogenesis. Another important function of omega-3 PUFAs is correlated with the attenuation of apoptotic events induced by mytochondrial and endoplasmic reticulum oxidative stress by a reduction of caspase-3 activity in adipocytes via AMPK activation, and increased production of resolvins, a family of neuroprotector molecules derived from EPA and DHA [332]. Overall, omega-3 fatty acids act on different pathophysiological pathways of DR such as inflammation, oxidative stress and neoangiogenesis, showing a remarkable potential in the prevention of retinal disease progression.

Polyphenols

Polyphenols belong to a family of natural compounds characterized by one or more hydroxyl groups (OH) linked to a benzene ring. These natural substances are present in a variety of foods including grapes, berries, dark chocolate, coffee and tea to mention a few, and are characterized by strong antioxidant and anti-inflammatory power, so that they are widely used in the prevention and/or treatment of different pathologies [316,324]. Several studies have shown that dietary polyphenols exert a protective effect against progression of age-related ocular abnormalities such as cataract, glaucoma, DR and macular degeneration [333]. Based on their structure, polyphenols are classified into non-flavonoid and flavonoid compounds. Flavonoids consist of two phenolic rings linked to a pyranosic ring and are highly represented in fruits, vegetables, roots, and wine.

Anthocyanins (anthos = flower and kyanos = blue) are a subgroup of flavonoids, water-soluble pigments very common in the plant kingdom and in many typical foods of the Mediterranean diet such as blueberries, aubergines, peaches, oranges, figs, cherries and olives. Consumption of foods rich in anthocyanins has been associated with a reduced risk of cardiovascular diseases. As reported by Fang and colleagues [334] anthocyanins can be absorbed as such through the gastrointestinal wall, where they undergo an extensive first- pass metabolism and the metabolites so formed enter the systemic circulation. In fact, anthocyanin metabolites were detected in the blood stream in much higher concentrations than their parent compounds. Several studies reported that dietary intake of anthocyanins may confer benefits in different brain functions, including vision. For example, in blueberry-fed pigs, which are a suitable model to mimic human digestive absorption, anthocyanins were detected in all examined tissues, including brain and eyes, thus indicating that anthocyanins can accumulate also in tissues beyond the blood-brain barrier [335]. Other studies have shown the role of anthocyanins as antioxidants in the retinal pigmented epithelium (RPE) where they can neutralize the ROS formed by the metabolic activity of photoreceptors thus playing a role in neuroprotection as also shown by their protective effect in a model system of rat retinal neurons damaged by N-methyl- N-nitrosourea [336]. Song and colleagues [337] addressed the effects of blueberry anthocyanins on retinas of diabetic rats subjected to oxidative stress and inflammation, demonstrating their antioxidant potency resulting in increased expression of HO-1 and nuclear localization of the transcription factor Nrf2 (which controls the expression of antioxidant response element-dependent genes) finally resulting in increased expression of GSH and glutathione peroxidase, and reduced levels of malondialdehyde (MDA) and ROS. Moreover, cyanidins and the orthodihydroxy group of anthocyanins can inhibit lipid peroxidation by chelation of metal ions [338].

Another flavonoid with antioxidant and anti-inflammatory properties in the retina is quercetin. It has been reported that in mouse retinal photoreceptor cells, quercetin inactivated the pro-inflammatory transcription factor NF-κB through inhibition of both mitogen- activated protein kinases (MAPK) and Akt, reducing in this way VEGF-induced inﬂammation [339]. Kumar et al. [340] demonstrated the retinal neuroprotective effects of quercetin. In streptozotocin- induced diabetic rats, quercetin blunted inflammation and glyosis decreasing the retinal expression level of glial fibrillary acidic protein (GFAP) and NF-κB in speciﬁc retinal layers, such as the nerve ﬁber layer, the inner plexiform layer, and the inner nuclear layer. The effect of quercetin on NF-κB was also associated with decreased levels of TNFα and IL-1β. Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a non-flavonoid polyphenol, and a phytoalexin found in red wine and grape skin. Many studies demonstrated its antioxidative and anti- inﬂammatory properties, so that it has been often used to mitigate those eye diseases with a prevalence of oxidative stress, such as DR. In vitro studies have shown that resveratrol prevents ROS-induced apoptosis in retinal capillary endothelial cells stressed by high glucose, through the activation of the AMPK/Sirt1/PGC-1α pathway [341]. Other in vitro studies on the human pigmented epithelium ARPE-19 cell line put under chemically mimicked hypoxia by cobalt chloride have demonstrated that resveratrol could reduce oxidative stress and the production of pro-angiogenic and pro-fibrotic factors [342]. In the same model system, hower subjected to high glucose oxidative stress, resveratrol inhibited the stimulated secretion of inﬂammatory cytokines such as IL-6 and IL-8, and prevented the downregulation of gap junction associated connexin 43 while inducing the activation of TGFβ, PKCβ, and COX-2 [343]. In vivo studies by Chen and colleagues with streptozotocin (STZ)-induced diabetic rats, have revealed the effects of paraoxonase-1 (PON1) as an intermediary in the protective effects exerted by resveratrol. In fact, oral resveratrol treatment – via induction of PON1 in the retina – blunted the increased retinal vascular permeability, pro-apoptotic caspase-3 activity, retinal damage, as well as clearly showed an inhibitory effect on inflammatory markers, such as IL-1β, IL-6, TNFα, VEGF, IFN-γ and monocyte chemoattractant protein-1. Therefore, these data suggest that PON1 induction by resveratrol in the retina may be a promising therapeutic strategy to prevent the progression of diabetes-related retinopathy [344]. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)- 1,6-heptadiene-3,5-dione) is another non-flavonoid polyphenol extracted from the root of Curcuma longa and exhibits a wide range of pharmacological properties, which include antioxidant, anti- inflammatory, antimutagenic, antimicrobial and anticancer activities. For its anti-inﬂammatory and antioxidant properties, curcumin is exploited as oral supplementation therapy for retinal degenerative diseases, including DR [345]. The World Health Organization stated that an acceptable daily intake of curcumin can be up to 3mg/kg as a food additive [346]. However, this “golden spice” despite a good efficacy has a limited bioavailability due to poor absorption and rapid metabolism and elimination. Clinical trials have shown that curcumin is safe in humans and well tolerated up to doses of 12g/day [347]. The antioxidant and anti-inﬂammatory activities of curcumin (10 µM) have been tested on human retinal pigmented epithelial cells, human retinal endothelial cells and human retinal pericytes exposed to oxidative stress with high glucose. Results demonstrated a signiﬁcant decrease of ROS concentration in retinal pigmented epithelial cells and of TNF-α release in retinal endothelial cells, while protecting retinal pericytes from high-glucose damage [348-349]. Human retinal endothelial cells exposed to high glucose and treated with curcumin (10 µM) showed an increase of HO-1 expression [350], a stress response protein that is highly inducible under various conditions, such as oxidative or heat stress. This observation suggests that curcumin may have both a direct and indirect antioxidant activity, due to the activation of Nrf2. Once activated, Nrf2 translocates into the nucleus and promotes the transcription of genes that encode antioxidant enzymes including HO-1 [351,352]. In vivo studies have been done in rats with STZ-induced diabetes and treated with curcumin given by different routes. Intraperitoneal injections of curcumin (80mg/ kg, once a day), prevented the retinal increase of malondialdehyde (MDA: a marker of oxidative stress) and the decrease of antioxidant GSH levels [353]. In the same model, Gupta et al. [354] have shown that oral curcumin (1 gr/Kg) showed hypoglycemic activity, decreased significantly the superoxide dismutase and catalase activities and prevented the increase of the proinflammatory cytokines TNF-α and VEGF in diabetic retinae. Moreover, they observed increased antioxidant levels of superoxide dismutase (SOD), catalase, and GSH and a lesser increase of retinal nitrotyrosine, a marker of oxidative protein damage, and of 8-hydroxy-20-deoxyguanosine, a marker of oxidative DNA damage [355].

Carotenoids

Carotenoids are naturally occurring pigments found in fruits and vegetables, but also in plants, algae, and photosynthetic bacteria. Carotenoids can be divided into two main classes: carotenes (precursors of vitamin A) and xanthophylls (non precursors of vitamin A). Carotenes are non-polar molecules, containing only carbon and hydrogen atoms, while xanthophylls are polar carotenoids, containing at least one oxygen atom [356]. Several studies demonstrated the beneficial power of carotenoids on human health and more specifically on eye health, exerting primarily antioxidant effects against AMD, age-related cataract (ARC), uveitis and DR [357]. Humans cannot synthesize carotenoids which must be supplemented by foods in order to be distributed to various tissues, especially to the retina. Lutein and zeaxanthin are non-provitamin A carotenoids from the xanthophyll family. These carotenoids are characterized by OH groups on each end. Their amphipathic character allows them to be inserted into the lipid bilayer of cell membranes and in the outer monolayer of lipoproteins. These xanthophylls have been found in the human retina, particularly in the macular region [358]. The highest concentration is found in the fovea, and in primates they are responsible for the characteristic central yellow coloration known as the macula lutea. The fovea contains approximately 13ng/ mm2 of carotenoids, compared with approximately 0.05ng/mm2 in the peripheral retina [359]. The main mechanism by which lutein and zeaxanthin are involved in the prevention of retinal diseases appears to be due to their local antioxidant activity and their capacity to filter the blue light, responsible of photo-oxidative damage [360]. Elimination of lutein from the diet of experimental animals resulted in early degenerative signs in the retina, and patients with an acquired condition of macular pigment loss (Macular Telangiectasia) showed a serious visual handicap, highlighting the importance of macular pigment. Accordingly, a recent clinical study reported that thanks to lutein capacity of filtering short-wavelenght light, its administration to patients with NPDR led to some improvement in glare sensitivity [361]. Another study on diabetic rats treated with zeaxanthin has shown its ability to bring the retinal concentration levels of VEGF and intercellular adhesion molecule-1 (ICAM-1: related to the inflammatory state of the tissue) to values comparable to healthy controls. Moreover, zeaxanthin supplementation prevented diabetes-associated retinal damage by decreasing nitrotyrosine levels, DNA oxidative damage, lipid peroxidation and the decrease of retinal SOD [362]. Similarly, lutein administration to diabetic mice resulted to prevent retinal oxidative stress and restored normal retinal ROS levels [363]. Hu et al. addressed the potential benefits of supplementation with lutein and zeaxanthin in type 2 diabetic patients with diagnosed NPDR [364]. In this study, plasma concentration of these carotenoids was substantially lower in the diabetic group than in normal subjects at baseline. After 3 months treated patients presented higher plasma concentrations in comparison to untreated controls, correlating with reduction of the macular edema and improvement of visual acuity. Overall, these data indicate that lutein and zeaxanthin are main actors on the photo-oxidative stage, by quenching oxygen singlets, scavenging free radicals and protecting retinal cells from oxidative damage. Their dietary intake may be used to ameliorate or even to reverse vision loss in DR patients. Astaxanthin is a third member of the xanthophyll family, a ketocarotenoid generated by the oxidation of β-carotene, and is a common pigment in algae, fishes, and birds. One of the most important properties of astaxanthin is a potent antioxidant activity, almost 10 times higher than other known compounds such as polyphenols or β-carotene [365]. Because of this property, astaxanthin has been studied, mainly in animal and cell models, for its possible role in the treatment of chronic diseases involving oxidative stress, such as in diabetes and its complications. Yeh et al. [366] addressed the effect of orally administered astaxanthin on STZ-induced diabetic rats. Ocular tissues from astaxanthin and lutein treated rats showed a significant reduction of oxidative stress and inflammatory mediators depending on NF-κB transcription activity, and increased levels of antioxidant enzymes, thus preserving retinal architecture and function. Zhou and colleagues further demonstrated that astaxanthin can regulate the glycemic state and reduce insulin resistance, and can also exert an anti-inﬂammatory and anti-angiogenic effect by decreasing the expression of NF-κβ and TNF-α, thus inhibiting the expression of proinﬂammatory molecules such as ICAM-1, the monocyte chemoattractant protein-1 and VEGF [367]. In conclusion, several studies indicate that astaxanthin, zeaxanthin and lutein may blunt the generation of oxidation products in the retina, so that their oral uptake could be a good strategy to control the development of DR by reducing the oxidative damage to DNA, proteins, and lipids.

Vitamin c

Vitamin C is a water-soluble vitamin that exists in two main forms, ascorbic and dehydroascorbic acid. It is an important nutraceutical used as a support to therapy to treat oxidative-stress-induced diseases. Vitamin C scavenges ROS such as superoxide and peroxynitrite in plasma and cells (thus avoiding damage to proteins, lipids and DNA), prevents occludin dephosphorylation and the loosening of tight junctions. Several studies have reported that serum vitamin C levels were lower in diabetic patients in comparison to healthy subjects [368], and these levels were much lower in diabetic patients with DR [369]. The vitreous level of vitamin C is responsible for the regulation of oxygen tension and oxidative stress in the eye, which are implicated in retinal ischemia and the development of PDR [370]. Duarte et al. have demonstrated that in healthy individuals the concentration of vitamin C in vitreous humor is remarkably higher as compared to that in serum [371]. In patients with PDR, the vitreous level of vitamin C was decreased up to tenfold compared to healthy controls and was associated with the degree of macular ischemia, suggesting that vitreous vitamin C depletion may contribute to macular ischemia in PDR patients [370]. Ascorbic acid (AsA) has anti-inﬂammatory, immunostimulant, and antibacterial properties, and is used as adjuvant therapy in many inﬂammatory disorders [372]. In a double- blind cross-over trial [373], 8 diabetic patients were treated with AsA tablets showing that the supplementation of 1000 mg AsA/day for 2 weeks reduced the activity of the enzyme aldose reductase thus inhibiting the polyol pathway, which is activated in DR. Diabetic hyperglycemia triggers apoptosis of vascular pericytes thus impairing vascular regulation and weakening vessels, especially in brain and retina. May et al. demonstrated that vitamin C prevented high glucose- induced perycytes apoptosis, that was largely due to the activation of the receptor for advanced glycation end products (RAGE) [374]. AsA may also have a role in angiogenesis. A recent study conducted on human umbilical vein endothelial cells (HUVECs) has shown that AsA prevented the increase of endothelial barrier permeability induced by VEGF. This effect may contribute to reduce the macular edema in DR [375].

Alpha-lipoic Acid

Alpha-Lipoic Acid (1,2 dithiolane-3-pentanoic acid) (aLA), also known as thioctic acid, is a natural thiol antioxidant. After oral supplementation, it is quickly transported to intracellular compartments and reduced by enzymes to dihydrolipoic acid. This reduced form has many biochemical functions. It acts as biological antioxidant, metal chelator, and regenerates by reducing their oxidized forms other antioxidant agents such as vitamin C, E and GSH. It also functions as modulator of the signaling transduction of pathways involving insulin or NF-kB [376]. aLA plays an essential role in mitochondrial bioenergetic reactions, which makes it useful in managing diabetic vascular complications such as retinopathy and peripheral neuropathy. In an in vivo study [377], the mitochondrial functional integrity, biogenesis and DNA copy number were analyzed in the retina of STZ-diabetic rats. Animals under good glycemic control and fed with aLA have shown a lesser dysregulation of retinal mitochondrial biogenesis as measured by citrase synthase (a marker of mitochondria functional integrity) and the presence of fewer acellular capillaries (a marker of DR). In another experimental study [378], Wistar rats were treated with aLA (60 mg/kg bodyweight) i.p. daily for 30 weeks. Results demonstrated that aLA treatment reduced oxidative stress, NF-kB activation and angiopoietin-2 expression, and reduced the amount of VEGF in the diabetic retina by 43%. In a randomized, double-blind placebo-controlled study, 32 patients with preretinopathic diabetes were treated with aLA (400 mg/daily) in combination with other antioxidants (genistein and vitamins). Oxidative stress levels in plasma and changes in the full field ERG were evaluated. Results revealed that the oral treatment with antioxidants in preretinopathic diabetes subjects may have a protective effect on retinal cells, as detected by ERG analyses observed after 30 days of treatment [379]. In conclusion, several studies show that nutritional supplementation of natural molecules, as discussed above, protects neuronal and vascular cells from inflammatory damage and mitochondrial degeneration, thus slowing down DR progression and helping diabetic patients to spare their vision for longer times [380].

AGE-RELATED-MACULAR DEGENERATION

AMD is a progressive degenerative disease of the retina principally affecting the macula, a highly specialized region of the central retina responsible for fine and colour vision. It is currently considered the leading cause of visual impairment and blindness among patients over 60 years [381]. AMD can occur in two distinct forms: the most frequent (roughly 90% of the cases) is the dry (atrophic, non- exudative) form, characterized by diffuse insoluble debris (known as drusen) causing the death of RPE cells and photoreceptors [381]; the wet (neovascular, exudative) form is less frequent (roughly 10% of the cases) but still responsible of the majority of blindness cases, and is characterized by the development of abnormal new vessels inside the macular region (choroidal neovascularisation) [382]. Oxidative stress and inflammation along with genetic risk factors are all involved in AMD pathogenesis [383]. An important and characteristic clinical hallmark of AMD is the presence of drusen – small yellow or whitish accumulations of extracellular debris (made of proteins and lipids) located between the basal lamina of the RPE and the inner collagenous layer of Bruch’s membrane [382,384]. In particular, these protein/ lipid deposits are associated with an inefficient metabolism of RPE cells, which cannot get rid of catabolic products which then tend to accumulate in the extracellular spaces giving rise to drusen bodies [385]. Another early molecular event of AMD is the accumulation within RPE cells of lipofuscin, a remnant of the recycling of photoreceptor outer segments, that contributes to RPE degeneration [386]. Oxidative stress is a leading mechanism in the generation and progression of AMD, therefore antioxidant self-defense mechanisms are crucial for the mainteinance of retinal health. Two main targets of oxidative stress in RPE cells are the elements involved in autophagy and the transcription factor Nrf2. Dysregulation of both autophagy and the induction of antioxidant enzymes regulated by Nrf2 may trigger the cascade of events finally leading to lipofuscin and drusen generation, and start the inflammatory process causing AMD [387]. Inflammation processes thus induced occur through recruitment of macrophages and microglia, and complement activation [388]. Allelic variations of genes coding for complement factors, beside those participating in lipid metabolism or angiogenesis, constitute risk factors for AMD development [389]. Other risk factors include age, genetic predisposition, exposure to light, race, smoking habit, altered body/mass index, and diet [390-392]. Currently, the only therapy approved for the treatment of the dangerous wet form of AMD involves the inhibition of the pro-angiogenic factor VEGF in the retina. However, the search for more effective treatments of dry AMD is actively ongoing (Figure 4) [387].

Figure 4. Age related macular degeneration. Oxidative stress and inflammation further to ageing, a wrong lifestyle and hyper reactivity of the natural immunity (mainly the complement system) may lead to the accumulation of catabolic debris (drusen) between the RPE and the Bruch’s membrane (dry or atrophic MD), and eventually to the rupture of the Bruch’s membrane and the invasion of new blood vessels into the macular area (wet or neovascular MD) (upper panel). Food supplements that may be useful to blunt these events have antioxidant, antinflammatory, antiapoptotic and antiangiogenic effects (bottom panel).

Food Supplements

In recent years, epidemiological and clinical studies have suggested the importance of nutrients and food supplements to prevent or slow down the progression of AMD in the elderly. In particular, since free radicals and ROS are known to be the principal factors behind this pathology, most of the nutrients studied have antioxidant and free radical scavenger characteristics [382,393]. They belong to several classes: polyphenols (anthocyanins, tannic acid, quercetin, curcumin, EGCG, resveratrol and herb rosemary), carotenoids (lutein, zeaxanthin, and β-carotene), vitamins (A, C, D and E) and minerals (zinc, selenium and copper). Other molecules with relevant antiaging and antioxidant properties used for AMD are aLA, omega-3 fatty acids and Lactobacillus paracasei KW3110 [382,394-396]. The first important trial evaluating food supplements for AMD prevention has been the Age-Related Eye Disease Study (AREDS) started in 1992. Results have shown that a formulation with vitamin C (500 mg), vitamin E (400 IU), β-carotene (15 mg) and zinc (zinc oxide 80 mg and cupric oxide 2 mg) showed a 25% risk reduction of the progression to advanced AMD over 5 years in patients with intermediate or advanced AMD [397]. A second trial, designated AREDS2, conducted between 2006 and 2012, was carried out to explore the added benefits of lutein, zeaxanthin and omega-3 fatty acids to the AREDS1 diet [398]. This study provided an opportunity to further refine the original AREDS formulation by eliminating ß-carotene (because of increasing risk of lung cancer in smokers) and lowering the dose of zinc (because of the potential health risks of taking large quantities of zinc). AREDS2 found that lutein and zeaxanthin reduced the risk of developing advanced AMD by a further 10 per cent. For participants who had a poor dietary intake of lutein and zeaxanthin a further decrease of 20% of the risk of progression to advanced AMD was observed; omega-3 fatty acids did not appear to further improve the efficacy of the AREDS1 diet, and the reduced amount of zinc conserved the same effects of the original higher amount [398,399].

Polyphenols

Anthocyanins (a subgroup of flavonoids, with cyanidin and delphinidin among the most common) are red–purple pigments found in plants [400]. Berries, such as blueberry, blackcurrant, strawberry, and wolfberry (better known as ‘goji berry’), are all rich in anthocyanins [401]. They have shown favorable effects on AMD for their antioxidant and anti-inflammatory properties, as demonstrated by both in vitro and in vivo models. Anthocyanins such as cyanidin 3-glucoside (C3G) promote regeneration and new synthesis of rhodopsin (the photosensible pigment of rod photoreceptor cells) and protect the retina from overexposure to visible and UV light [402-404]. A clinical study on 72 post-menopausal women showed that combining anthocyanin supplements with lutein and zeaxanthin did not influence their uptake in serum and macular pigment, so that an additive protection effect could be expected [405]. Tannic acid (a tannin derivative) contained in the polyphenolic extract of some plants, has been shown to protect human RPE cells from UVB damage, by inhibiting the activation of the transcription factor STAT3 and the downstream induction of the pro-inflammatory cytokine IL-6 and downregulating the expression of the complement factor B, also involved in the inflammatory cascade leading to AMD [406]. Quercetin is an another natural polyphenol with known antioxidant and anti-apoptotic properties, the protective efficacy of which has been demonstrated both in vitro and in vivo models. RPE exposed to cytotoxic and inflammatory effects induced by 4- hydroxynonenal [407] or by blue light [408] were prevented by quercetin. In a different model of peroxide-induced oxidative stress model in RPE cells [409] quercetin treatment influenced the transcription of genes involved in apoptosis regulation, including the anti-apoptotic Bcl-2 and the pro- apoptotic BAX and FADD, and the effectors of apoptosis Caspase-3 and Caspase-9. Intraperitoneal administration of quercetin blunted the photo-oxidative damage of rat retinas’ photoreceptors exposed to intense light [410]. Curcumin, a polyphenol from Curcuma longa (turmeric plant) belonging to the ginger family, is also able to modulate apoptosis, inflammation and oxidative stress defense proteins [411]; these effects follow the upregulation of the anti-apoptotic protein Bcl- 2, the downregulation of the pro-inflammatory transcription factor NF-kB, and the upregulation of the antioxidant proteins SOD, GR and HO-1, as shown in human retinal cell models exposed to oxidative damage [350,412]. An active phenolic component of green tea, EGCG, protected human ARPE19 cells from UVA-induced damage by decreasing both the production of hydrogen peroxide and the activation of the pro-inflammatory MAPK and COX-2 [413]; moreover, it has shown a protective effect in RPE cells against oxidative stress-induced damage by modulating the expression of the transcription factor Nrf2 [414]. Resveratrol (3,4,5-trihydroxystilbene), a polyphenolic antioxidant belonging to the stilbene family and commonly found in grape skin and seeds, has been shown to be protective against UVA-induced damage in ARPE19 cells [415] and to prevent the development of choroidal neovascularization by modulating protein kinase A (PKA) in macrophages [416]. A recent study has suggested that resveratrol could mitigate the adverse effects induced by repeated injections of bevacizumab (anti-VEGF antibody) in wet-AMD therapy [417]. One of the major limitations of anti-VEGF therapy is an excessive reduction of the available secreted VEGF, which is also important to maintain the homeostatic function of the RPE. In ARPE-19 cells, various combinations of resveratrol and bevacizumab prevented VEGF reduction observed after bevacizumab alone treatment [417]. A prospective, randomized clinical trial (NCT02625376) started on August 2015, the results of which have not been released yet, aimed to evaluate the safety and efficacy of resveratrol to reduce the progression of exudative AMD between two groups of patients, either receiving 250mg of resveratrol or placebo, after 24 months of follow-up. The common herb rosemary (Rosmarinus officinalis) contains a variety of polyphenolic compounds which, combined with zinc, decreased the amount of CEP-proteins (carboxyethyl-pyrrole modified proteins, found in drusen and considered a significant risk factor for the development of AMD), and improved photoreceptor cell survival in rat retinas exposed to photo-oxidative damage [418]. Chronic supplementation with rosemary antioxidants has been suggested to be a useful addition to the therapeutic benefits of AREDS supplements in slowing down the progression of AMD [419,420].

carotenoids

Lutein, zeaxanthin and meso-zeaxanthin are carotenoids present in high concentration in the macula where they form the macular pigment protecting the retina from the oxidative stress constantly induced by light [421]. They are not synthesized by mammalian cells but found in egg yolk and vegetables such as fruits, spices, lettuce, broccoli, and spinach [422]. In an oxidative stress model obtained in manganese superoxide dismutase knockout mice, zeaxanthin supplementation blunted the oxidative stress and preserved the structure of the RPE [423]. Human clinical studies demonstrated that lutein and zeaxanthin, as well as β–carotene supplementations have protective effects on AMD, improving visual performance measured as contrast sensitivity, glare tolerance, and photo-stress recovery [396,424]. Several other studies have shown that high blood levels of lutein and zeaxanthin correlate with a significant lower risk of AMD [425,426].

Vitamins

Vitamins A, C and E appeared to reduce the risk of macular degeneration according to the AREDS studies I and II [397,398]. Positive outcomes concerning high dietary intakes of fruits and vegetables rich in pro vitamin A and vitamin C have been reported [394,427]. Vitamin C is known to be a potent antioxidant protecting proteins, lipids, carbohydrates and nucleic acids from free radicals and ROS damage [428]. Moreover, the AREDS Study Group has revealed that high vitamin C intake is associated with a reduced likelihood of neo-vascular AMD [429]. Vitamin E, similarly to vitamin C, is a powerful antioxidant. Its deprivation leads to lipofuscin accumulation [430], retinal damage [431] and loss of photoreceptors [432]. High dietary intakes of vitamin E have been clinically correlated with a slower progression of AMD [433]. Vitamin D may exert beneficial effects on AMD prevention by modulating the immune system and inhibiting inflammation and angiogenesis. The role of vitamin D in the progression of AMD has been addressed recently, because the cognate receptor is expressed by mammalian RPE [434]. However, among the many clinical observations and studies run on the efficacy of vitamins A, C, E, not all the results could confirm their efficacy on the decourse of AMD [396]. As to vitamin D, its role in the prevention of AMD seems to be ascertained, though the amount to be given as food supplement is still in need of better clarification [435].

Minerals

Zinc, copper and selenium play important roles in the normal functioning of antioxidant enzymes and in retinal cell survival [436,437]. Supplementation of zinc and copper decreases the risk of AMD progression suggesting that these metals play an important role in the homeostasis of AMD and in retinal health [438]. In particular, zinc supplementation effectively blunts the risk of AMD and visual loss by its antioxidant properties, and by intervening in the regulation of the light-rhodopsin reaction, in the modulation of synaptic transmission and of complement activation [438]. Selenium is a well-known anti-oxidant agent, and may reduce the risk of AMD by blunting the oxidative damage of membrane lipids and by participating in the activity of GSH antioxidant defense [437]. Moreover, a diet deficient in vitamin E and selenium in laboratory animals resulted in a consistent decrease of total PUFAs in RPE and in retinal rod outer segments, which could be recovered by an adequate diet supplementation [439]. However, more clinical evidence has to be raised in order to better understand the role, the efficacy and the dietary doses of these minerals in the prevention and treatment of AMD.

Alpha-Lipoic Acid

aLA is a naturally occurring fatty acid found in some foods such as yeast, spinach, broccoli and potatoes [376]. It works as a cofactor of mitochondrial dehydrogenase and as a free radical scavenger, regenerating endogenous antioxidant systems like vitamins C and E and the enzyme SOD [440]. aLA dietary supplementation has been shown to improve vision-related quality of life in dry AMD patients most likely by improving the antioxidant defense [441]. aLA entered a phase 2 clinical trial in May of 2016 sponsored by the University of Pennsylvania (NCT02613572), with the aim of assessing safety and tolerability of the 800 and 1200 mg daily doses in patients affected by geographic atrophy [417].

Omega-3 Fatty Acids

The deficiency of omega-3 fatty acids DHA and EPA in photoreceptor membrane lipids play an important role in the pathogenesis of AMD because their imbalance contribute to drusen formation in the RPE and sub-RPE layers [442]. Oral treatment with a complex mixture of fatty acids (commercially available as Macular- FAG®) has been used in a mouse model system of AMD induced by subretinal injection of polyethylene glycol (PEG-400). Results have shown a blunting of complement-mediated inflammation, a decrease of macrophage recruitment and modulation of the production of pro-inflammatory and angiogenic cytokines, finally resulting in a functional rescue of retinal electrophysiology [443].

Lactobacillus Paracasei KW3110

Anti-inflammatory agents appear to be optimal candidates for AMD prevention or slowing of its progression. Most recently, Morita et al. have demonstrated that Lactobaciullus paracasei KW3110 (a lactic acid bacterium) may activate M2 macrophages (macrophages associated with anti-inflammatory reactions), thus suppressing blue light-induced retinal inflammation both in vitro and in vivo. These results suggest that L. paracasei KW3110 may have a preventive effect against degenerative retinal diseases, including AMD [395].

MYOPIA

Aristotle is considered to be the first person who described myopia. The term myopia derives from the Greek’s verb μύειν, which means ‘to close’ and ωψ which means ‘eye’, and together describe a typical action of myopic individuals who squeeze their eyes to improve farsight vision. This action reduces the opening of the iris diaphragm, thus increasing the focal depth. Scientifically, myopia (near-sightdness) is a spherical refractive error that occurs when rays of light running parallel to the optic axis focus the image anterior to the retinal plane [444]. This disorder is mostly due to an over-growth of the eyeball, which brings the retina behind the focal plan [445]. According to the recent definition by the IMI (International Myopia Institute), myopia can be divided into low myopia and high myopia, in which the spherical equivalent refractive error is ≤ −0.50 diopters (D) and ≤ −6.00 D, respectively [445]. Myopia is among the most common ocular disorders causing visual loss. In 2010 a systematic analysis estimated that 1.9 billion of people in the world were myopic (about 27% of the world’s population), among which 70 million had high myopia [446]. Holden et al. estimated that in 2050 there will be almost 5 billion myopic people (50% of world’s population) among which almost 1 billion with high myopia [447]. However, myopia is not equally distributed worldwide; 90% of the youngsters are myopic in East Asian countries, while in the United States and Europe their proportion remains around 50% [448]. The prevalence of myopia also varies with age. In the Beaver Dam Eye Study, data suggested that the prevalence of myopics decreased from 42.9% among adults aged 43-54 years to 14% in individuals older than 75 years [449]. To date, hundreds of genes and around twenty chromosomal loci have been identified to be associated with the occurrence of refractive error and myopia, although they cannot fully explain it [450-452]. A seminal study published in 1969 concerning Inuit people living in North Alaska, showed that only 2 out of 131 adults, grown up in an isolated group, were myopic. On the contrary, about 50% of the next generation individuals were shortsighted [453], demonstrating that genes alone cannot explain such increase in just one generation, because genetic changes require longer times. However, genes can determine the individual susceptibility to environmental factors, and familiarity is evident within families and within populations [454-456]. Environmental factors including socio-economic and the time spent in outdoor activities have also been correlated with myopia development. While the time spent indoor represents an environmental risk, the concomitant outdoor time may be an important protective factor [457-458]. High level of outdoor activity is positively correlated with lower odds of myopia in different epidemiological studies [459-461]. The idea that outdoor activities reduce the onset of myopia, introduces the concept that the amount of exposition to bright light has a pivotal role in this prevention. In fact, light stimulates the release of dopamine and its accumulation in the retina, which starts a signaling cascade involving the retinal pigmented epithelium, finally slowing down the growth of the eye globe [462-465]. The molecular etiology of myopia is not clear yet. In a recent study, we observed that atropine, a molecule used to slow down myopia progression in children and teenagers, increased extra-cellular matrix production from scleral fibroblasts in vitro, thus contrasting the scleral thinning often observed during myopia progression; on the contrary, atropine effect on choroidal fibroblasts resulted in a decrease of ECM components production, suggesting an alternative mechanism, by which atropine might improve choroidal blood perfusion, known to be reduced in myopic eyes [466]. Further investigations are needed to better clarify the physio-pathological events leading to myopia. Current anti-myopic treatments include: use of eye-glasses or soft contact lenses, orthokeratology, topical administration of low dose atropine or pirenzepine, oral administration of 7-methylxanthine, corrective surgery. However, in many cases a “rebound effect” can be observed after interruption of treatment (Figure 5) [467-470].

Figure 5. Myopia. Myopia mostly results from the elongation of the eye globe, which brings the macular region of the retina out of focus, and puts a mechanical stress on this delicate tissue, increasing the risk of a retinal detachment and/or laceration (upper panel). Food supplements that might be useful are those reinforcing the matrix of the sclera, thus retarding its abnormal growth and elongation (bottom panel).

Nutrition and Food Supplements

The use of food supplements to correct nutrition deficiencies is a strategy widely accepted and used to improve the general health and try to prevent many different pathologies, also including eye diseases. Eating brightly colored foods like green vegetables and fresh fruits, loaded with vitamins, is highly recommended also to intervene on eye growth. Moreover, the consumption of fish like salmon, tuna and mackerel is very important for a sufficient intake of omega-3 fatty acids, needful to the brain and the eye to modulate neuronal cell membrane activity and their kinetics of transport systems [471]. In the study KNHANES VII (Korean National Health and Nutrition Examination Survey 2016-2017) the prevalence of high and low grade myopia among South Korean children 5-18 years of age and the associated risk factors were analyzed. In this study, a correlation was noted between high body mass index and high myopia [472]. In another study on Asian children a high dietary intake of cholesterol and fat was found to be associated with myopia progression [473]. Glycemic index can be regarded as a risk factor for myopia and refractive error is more frequent in young patients with newly diagnosed type I diabetes mellitus [474,475].

Microelements: Microelements are important during the entire life, since they are essential co-factors for many enzymes, and more specifically they have a critical role to support a normal growth in children. Fedor and coworkers [476] found an association between low serum concentration of zinc and selenium, and a high Cu/Zn ratio in a cohort of 83 Polish myopic children aged 7-17 years, which might suggest that a dietary supplement of Zinc and Selenium could be indicated in myopic children. However, the finding was not confirmed by another study on American children [477], and more clinical data are necessary to consider microelements as a necessary supplement in the control of myopia progression in children.

Vitamin D: Although there is no direct evidence on the effect of vitamins on myopia progression, some studies have shown a correlation between vitamin D and myopia. The biosynthesis of vitamin D is promoted by sunlight exposure, and might be related to the time that individuals spend outdoor. Intake of folate and calcium also correlate with the amount of circulating vitamin D. In a study on 22 young American students (13 to 25 years old) the 14 myopes showed lower blood levels of vitamin D (average of 3.4 ng/ml) compared with non-myopes when adjusted for age and dietary intakes, despite the fact that both groups spent similar amounts of time outdoor [478]. In a Western Australian Pregnancy Cohort (Raine) Study, Yzar and co-workers [479] analyzed 946 children during a follow-up of 20 years. Among these, 23.4% were myopic and had lower serum vitamin D3 concentrations compared to non-myopic individuals. Univariate analysis showed that lower serum vitamin D3 concentration was associated with higher risk of progressive myopia (with a cut-off value of 50 nmol/L the odds ratio for developing myopia with low vitamin D3 was 2.63). Two more studies investigated the correlation between low serum levels of vitamin D3 and myopia. A first study analyzed 2,666 European children aged 6 years [480], and the second study enrolled more than 15,000 Korean young adults (aged 20 years or more) [481]. Both studies found an association between low serum levels of vitamin D3 and presence of myopia, independently from the time spent outdoor.

7-Methylxanthine: 7-Methylxanthine (7-MX) is an intermediate metabolite in the synthesis of caffeine in plants, and a product of the metabolism of caffeine in humans [482]. Unpublished evidence by K. Trier had shown an effect of caffeine on the production of ECM components in the sclera of treated rabbits. Further evidence by the same author has shown that 7-MX – a non-selective adenosine receptor antagonist – may have a role in the retardation of myopia progression. 7-MX effects resemble those of atropine (a muscarinic receptor antagonist), both increasing ECM production and collagen fibril diameter, thus increasing thickness of the sclera [483,484]. A thicker sclera should be less prone to axial elongation, which is the main cause of myopia development. In a recent study, infant macaques with myopia induced by the use of corrective lenses were treated with 100 mg/kg of oral 7-MX twice daily, demonstrating the ability of 7-MX to retard myopia progression in primate puppets [483]. 7-MX is a common human dietary ingredient for coffee drinkers, which has shown no significant side effects during long term treatment. In a clinical study, it has been shown to limit eye elongation and myopia progression in childhood myopia [485].

crocetin: Another promising natural compound is crocetin, a carotenoid compound consisting of a C-20 carbon chain contained in saffron and used in traditional herbal medicine [486]. This powerful compound is able to delay the growth of cancer cells by inhibiting the synthesis of nucleic acids and the activation of defense anti-oxidative systems, interfering with growth factor signaling pathways and finally inducing cancer cells apoptosis [487]. In a murine model of lens-induced myopia, a dietary crocetin supplement delayed myopia progression. Compared to controls (n = 14), crocetin administration resulted in a significant smaller change of refractive errors and axial elongation [488]. In a randomized clinical trial, 69 myopic children aged 6 to 12 years received either a placebo or oral crocetin for 24 weeks. The spherical equivalent refractive error progressed during the 6 months of the study by -0.41± 0.05 Diopters in placebo controls, and by −0.33 ± 0.05 Diopters in crocetin treated (p < 0.05); the axial length increased by 0.21 ± 0.02 mm in placebo controls, and by 0.18 ± 0.02 mm in crocetin treated (p < 0.05), thus showing the potential effects of crocetin in slowing down myopia progression [489].

CATARACT

Cataract is the opacification of the normally transparent lens of the eye, which may occur centrally (nuclear and posterior subcapsular) thus impairing vision, or in periphery (cortical cataract) [490]. Oxidative stress in the lens, occurring as consequence of normal aging, or accelerated by genetic and environmental causes, plays an important role in the onset and progression of cataract [491,492]. Currently, the only treatment for cataract is surgical removal of the cloudy lens, which typically is then replaced with an intraocular lens (IOL) during cataract surgery (Figure 6).

Figure 6. Cataract. Oxidative damage is the main and only recognized factor for lens opacification (upper panel). Antioxidants like vitamin C, lutein and zeaxanthin are the only food supplements that have shown some efficacy in retarding the insurgence of cataract (bottom panel).

Food Supplements

Despite the evidence indicating oxidative damage to lens epithelial cells as a likely common cause for cataractogenesis, the efficacy of nutritional supplements and antioxidants in the management of age- related cataracts is still under debate. Several studies reported that nutritional supplements showed a long-term inhibitory effect on the development of sight threatening cataract. A randomized clinical trial enrolling 14,000 U.S. male physicians older than 50 years with a follow-up of 11 years indicated that long-term daily multivitamin use had a slight, though significant effect in decreasing the risk of cataract [493]. A nutrition survey including more than 30,000 women older than 49 years followed for over 7 years detected a 13% lesser risk of developing cataract among those indicating a higher dietary intake of antioxidants. The main contributors to dietary total antioxidant capacity in the study population were fruit and vegetables (44.3%), whole grains (17.0%) and coffee (15.1%) [494]. Another large study of adult women in the United States correlated the eating of foods rich in a variety of vitamins and minerals with the delay of cataracts development [495]. A 10-year study of more than 2,400 older adults in Australia found that participants with the highest quintile of total intake (diet + supplements) of vitamin C had a reduced risk of incident nuclear cataract. An above-median intake of combined antioxidants (vitamins C and E, beta-carotene, and zinc) was associated with a reduced risk of incident nuclear cataract. Antioxidant intake was not associated with incident cortical or posterior sub-capsular cataract [496]. A large study of female health professionals in the United States enrolled 1802 women aged 50 to 79 years with intakes of lutein and zeaxanthin above the 78th (high) and below the 28th (low) percentiles. Four to seven years later, it was found that diets rich in lutein and zeaxanthin are moderately associated with a decreased prevalence of nuclear cataract in older women [497]. However, a Cochrane metanalysis including 9 clinical trials involving more than 117,000 patients concluded that dietary supplements of beta-carotene, vitamin E, and vitamin C had no clear beneficial effects on the inhibition of ARC development [498]. It has to be considered though that clinical studies on cataract development and progression require several years of observation, during which time the compliance of patients to treatment indications can be less tight, and that the endpoint parameters of such studies can be highly subjective. Most recently, a randomized, prospective, double-blind, placebo controlled human clinical trial has been launched to test the efficacy of a 6 months treatment with a nutritional supplement in inhibiting nuclear cataract progression on 20 patients subjected to Pars Plana Vitrectomy surgery. The nutritional supplement contained 11 compounds with reported cataract inhibitory properties: Riboflavin, L-GSH, C-Phycocyanin (as Spirulina Algae Extract), LA, Pyruvate (as Calcium Pyruvate), aLA, Quercetin, Turmeric, Silybin (as Milk Thistle Extract), Lutein, Zeaxanthin, and Astaxanthin [499]. In this study, cataract formation and progression were monitored by the serial Pentacam Nuclear Staging measurements (Oculus, Arlington, Wash.), supposed to be an objective, quantitative, and reproducible measurement of nuclear cataract density. Results showed a marginal borderline effect of the nutritional supplement, which however might suggest some efficacy, given the short observation time and the paucity of enrolled patients [499]. All nutrients associated with cataract prevention in clinical studies can be found in eye vitamins and vision supplements. Many experts believe these substances should be acquired from a healthy diet rather than from nutritional supplements. However, very often it happens that common dietary habits may lack key nutrients because not containing enough fruits and vegetables. In such occurrence, it could be wise to consider taking one or more daily nutritional supplements to assure the organism the intake of all the nutrients needed for optimum eye health. Antioxidant vitamins and phytochemicals that are supposed to reduce the risk of cataracts include vitamins A, C and E, lutein and zeaxanthin; consumption of fish high in omega-3 fatty acids has also been linked to a potential reduction of the risk of cataracts or their progression [500]. Here follows a sample of recent research suggesting the role of such nutrients in the prevention of cataracts.

Antioxidants and Vitamins

Seminal preclinical studies performed in the 80’s of the past century on cataract induced in mice by hyperbaric oxygen exposure [501] or by photo-oxidative stress in isolated rat lenses [502] have highlighted the potential protective activity of antioxidants, also including vitamin C, against cataract. The level of AsA is known to be low or absent in cataractous patients, with a concomitant increase of di-hydro-AsA (the oxidized form of ascorbic acid) [503]. However, the concentration of vitamin C in the aqueous humor and lens of human subjects could be increased after supplementation of 2 gr per day for 2 to 4 weeks prior to lens removal [504]. Along the same line, the Second National Health and Nutrition Examination Survey reported an inverse association between serum AsA and self-reported cataract [505], confirmed by the findings of the Nutrition and Vision Project, in which the inverse association between cortical and posterior subcapsular cataract with the use of long-term vitamin C supplement was evidenced [506]. These results are also consistent with those from the Beaver Dam Eye study where the 5-year risk to develop nuclear or cortical cataract was 40% and 60% lower among individuals who reported the abitual use of multivitamins or any other supplement containing vitamin C [507]. Daily oral use for 3 years of a mixture of antioxidant micronutrients including vitamin C produced a small deceleration in the progression of ARC [508]. In a population-based cross-sectional analytic study, which included 5638 people (aged ≥60 years), a strong inverse association between vitamin C and cataract was described [509]. Finally, a recent prospective cohort study, has shown that vitamin C protected against nuclear cataract progression as evaluated approximately 10 years after the baseline [510].

lutein and Zeaxanthin

Lutein and zeaxanthin are the main carotenoids found in the eye and look like promising nutrients in the fight against cataracts. Recent preclinical and clinical works have addressed the risk of developing cataracts in relation to dietary assumption of these nutrients.

Diabetic cataract is a frequent eye complication in diabetes. Dietary lutein effects on the prevention of diabetic cataract have been addressed in a rat model of diabetes induced by streptozotocin [511]. An oral lutein supplement (0.5 mg/kg) did not significantly affect blood glycemic values or body weights in diabetic rats. Most (81%) of diabetic rats’ eyes developed lens opacity and 43% developed mature cataract. Diabetic rats treated with lutein, developed lens opacity in 38% of the eyes and no mature cataracts were observed. Diabetic rats’ lenses showed a parallel increase of MDA and a decrease of GSH levels. Lutein treatment blunted MDA levels, though not raising GSH, hence indicating that lutein effects on the prevention of diabetic cataract may work through the inhibition of lipid peroxidation, finally suggesting that a lutein dietary supplement, combined with glycemic control, might work in the prevention of diabetic cataract [511]. The Nurses’ Health Study enrolled over 77,000 US female nurses aged 45- 71 y, and observed for 12 years. Results showed that those subjects with a dietary intake of high amounts of lutein+zeaxanthin (more than the average amount of 6 mg/day registered in the study) had a reduced need for cataract surgery [511]. The POLA (Pathologies Oculaires Liées à l’Age) study evaluated the associations of plasma lutein and zeaxanthin and other carotenoids with the risk of age- related maculopathy and cataract in 899 residents in the south of France and aged > 60 years. Results were strongly suggestive of a protective role of the xanthophylls, in particular zeaxanthin, for the development and progression of age-related maculopathy and nuclear cataract [512]. The Melbourne Visual Impairment Project recruited 2322 permanent residents aged > 40 years, with a follow-up of 2 years. This study confirmed the inverse association between high dietary zeaxanthin intake and prevalence of nuclear cataract [513]. On the contrary, the AREDS2 study concluded that daily supplementation with lutein/zeaxanthin had no detectable effects on rates of cataract surgery or vision [514]. However, these results are biased by the fact that the enrolled population was relatively well nourished and given at baseline a multivitamin supplement which could already have a protective effect on cataract progression, thus masking the effects of lutein and zeaxanthin [515].

Conclusion

Most often pathologies result from the loss of homeostasis of the whole organism, and may involve one or more organs at a time. Pache and Flammer defined POAG as “a sick eye in a sick body” [516], and indeed this definition might be extended to more ophthalmic pathologies, which can be derived or aggravated by imbalances of the homeostatic equilibrium of the body. Food supplements aim at re-establishing such equilibrium, both at the organism level, and sometimes specifically at the organ level, in case an organ is in major need of some specific nutrients or defense molecules that can be delivered by the food supplement. In fact, medical treatments are specifically aimed at the diseased organ, and their effect might be potentiated by food supplements cooperating with the drug(s) both at the specific site, and more generally at the organism level. Several decades of preclinical and clinical research have substantiated this hypothesis, telling us that nutrition is the most important element in our lifestyle influencing our health and disease status. Food supplements can thus be seen as nutritional aids to prevent disease, but also to cooperate with the treatment of diseases, of the eyes, but of other organs as well.

Abbreviations

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