Expression and activation of the ketone body receptor HCAR2/GPR109A promotes preservation of retinal endothelial cell barrier function.

Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.

Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neuronal and Vascular Damage.

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia.

Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction.

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease.

Deoxycholylglycine, a conjugated secondary bile acid, reduces vascular tone by attenuating Ca2+ sensitivity via rho kinase pathway.

Patients with cirrhosis have reduced systemic vascular resistance and elevated circulating bile acids (BAs). Previously, we showed that secondary conjugated BAs impair vascular tone by reducing vascular smooth muscle cell (VSMC) Ca2+ influx. In this study, we investigated the effect of deoxycholylglycine (DCG), on Ca2+ sensitivity in reducing vascular tone. First, we evaluated the effects of DCG on U46619- and phorbol-myristate-acetate (PMA)-induced vasoconstriction. DCG reduced U46619-induced vascular tone but failed to reduce PMA-induced vasoconstriction. Then, by utilizing varied combinations of diltiazem (voltage-dependent Ca2+ channel [VDCC] inhibitor), Y27632 (RhoA kinase [ROCK] inhibitor) and chelerythrine (PKC inhibitor) for the effect of DCG on U46619-induced vasoconstriction, we ascertained that DCG inhibits VDCC and ROCK pathway with no effect on PKC. We further assessed the effect of DCG on ROCK pathway. In ß-escin-permeabilized arteries, DCG reduced high-dose Ca2+- and GTPγS (a ROCK activator)-induced vasoconstriction. In rat vascular smooth muscle cells (VSMCs), DCG reduced U46619-induced phosphorylation of myosin light chain subunit (MLC20) and myosin phosphatase target subunit-1 (MYPT1). In permeabilized VSMCs, DCG reduced Ca2+- and GTPγS-mediated MLC20 and MYPT1 phosphorylation, and further, reduced GTPγS-mediated membrane translocation of RhoA. In VSMCs, long-term treatment with DCG had no effect on ROCK2 and RhoA expression. In conclusion, DCG attenuates vascular Ca2+ sensitivity and tone via inhibiting ROCK pathway. These results enhance our understanding of BAs-mediated regulation of vascular tone and provide a platform to develop new treatment strategies to reduce arterial dysfunction in cirrhosis.

Carbon monoxide releasing molecule A-1 attenuates acetaminophen-mediated hepatotoxicity and improves survival of mice by induction of Nrf2 and related genes.

Acute liver injury is frequently associated with oxidative stress. Here, we investigated the therapeutic potential of carbon monoxide releasing molecule A-1 (CORM A-1) in oxidative stress-mediated liver injury. Overnight-fasted mice were injected with acetaminophen (APAP; 300 mg/kg; intraperitoneally) and were sacrificed at 4 and 12 h. They showed elevated levels of serum transaminases, depleted hepatic glutathione (GSH) and hepatocyte necrosis. Mice injected with CORM A-1 (20 mg/kg) 1 h after APAP administration, had reduced serum transaminases, preserved hepatic GSH and reduced hepatocyte necrosis. Mice that received a lethal dose of APAP (600 mg/kg), died by 10 h; but those co-treated with CORM A-1 showed a 50% survival. Compared to APAP-treated mice, livers from those co-treated with CORM A-1, had upregulation of Nrf2 and ARE genes (HO-1, GCLM and NQO-1). APAP-treated mice had elevated hepatic mRNA levels of inflammatory genes (Nf-κB, TNF-α, IL1-ß and IL-6), an effect blunted in those co-treated with CORM A-1. In tert-butyl hydroperoxide (t-BHP)-treated HepG2 cells, CORM A-1 augmented cell viability, reduced oxidative stress, activated the nuclear factor erythroid 2-related factor 2 (Nrf2) and anti-oxidant response element (ARE) genes. The molecular docking profile of CO in the kelch domain of Keap1 protein suggested that CO released from CORM A-1 mediated Nrf2 activation. Collectively, these data indicate that CORM A-1 reduces oxidative stress by upregulating Nrf2 and related genes, and restoring hepatic GSH, to reduce hepatocyte necrosis and thus minimize liver injury that contributes to an overall improved survival rate.

Unveiling the gut-eye axis: how microbial metabolites influence ocular health and disease.

The intricate interplay between the gut microbiota and ocular health has surpassed conventional medical beliefs, fundamentally reshaping our understanding of organ interconnectivity. This review investigates into the intricate relationship between gut microbiota-derived metabolites and their consequential impact on ocular health and disease pathogenesis. By examining the role of specific metabolites, such as short-chain fatty acids (SCFAs) like butyrate and bile acids (BAs), herein we elucidate their significant contributions to ocular pathologies, thought-provoking the traditional belief of organ sterility, particularly in the field of ophthalmology. Highlighting the dynamic nature of the gut microbiota and its profound influence on ocular health, this review underlines the necessity of comprehending the complex workings of the gut-eye axis, an emerging field of science ready for further exploration and scrutiny. While acknowledging the therapeutic promise in manipulating the gut microbiome and its metabolites, the available literature advocates for a targeted, precise approach. Instead of broad interventions, it emphasizes the potential of exploiting specific microbiome-related metabolites as a focused strategy. This targeted approach compared to a precision tool rather than a broad-spectrum solution, aims to explore the therapeutic applications of microbiome-related metabolites in the context of various retinal diseases. By proposing a nuanced strategy targeted at specific microbial metabolites, this review suggests that addressing specific deficiencies or imbalances through microbiome-related metabolites might yield expedited and pronounced outcomes in systemic health, extending to the eye. This focused strategy holds the potential in bypassing the irregularity associated with manipulating microbes themselves, paving a more efficient pathway toward desired outcomes in optimizing gut health and its implications for retinal diseases.

CORM-A1 Alleviates Pro-Atherogenic Manifestations via miR-34a-5p Downregulation and an Improved Mitochondrial Function.

Atherogenesis involves multiple cell types undergoing robust metabolic processes resulting in mitochondrial dysfunction, elevated reactive oxygen species (ROS), and consequent oxidative stress. Carbon monoxide (CO) has been recently explored for its anti-atherogenic potency; however, the effects of CO on ROS generation and mitochondrial dysfunction in atherosclerosis remain unexplored. Herein, we describe the anti-atherogenic efficacy of CORM-A1, a CO donor, in in vitro (ox-LDL-treated HUVEC and MDMs) and in vivo (atherogenic diet-fed SD rats) experimental models. In agreement with previous data, we observed elevated miR-34a-5p levels in all our atherogenic model systems. Administration of CO via CORM-A1 accounted for positive alterations in the expression of miR-34a-5p and transcription factors/inhibitors (P53, NF-κB, ZEB1, SNAI1, and STAT3) and DNA methylation pattern, thereby lowering its countenance in atherogenic milieu. Inhibition of miR-34a-5p expression resulted in restoration of SIRT-1 levels and of mitochondrial biogenesis. CORM-A1 supplementation further accounted for improvement in cellular and mitochondrial antioxidant capacity and subsequent reduction in ROS. Further and most importantly, CORM-A1 restored cellular energetics by improving overall cellular respiration in HUVECs, as evidenced by restored OCR and ECAR rates, whereas a shift from non-mitochondrial to mitochondrial respiration was observed in atherogenic MDMs, evidenced by unaltered glycolytic respiration and maximizing OCR. In agreement with these results, CORM-A1 treatment also accounted for elevated ATP production in both in vivo and in vitro experimental models. Cumulatively, our studies demonstrate for the first time the mechanism of CORM-A1-mediated amelioration of pro-atherogenic manifestations through inhibition of miR-34a-5p expression in the atherogenic milieu and consequential rescue of SIRT1-mediated mitochondrial biogenesis and respiration.

Clerodendron glandulosum.Coleb leaf extract attenuates in vitro macrophage differentiation and expression of VCAM-1 and P-selectin in thoracic aorta of atherogenic diet fed rats.

Present inventory evaluates the anti-atherogenic potential of C. glandulosum.Coleb leaf extract (CG) using in vivo and in vitro experimental models. Serum markers of low density lipoprotein (LDL-C) oxidation, cholesterol, triglycerides, lipoproteins, auto-antibody titer, ex vivo LDL-C oxidation, LDL-C aggregation, aortic lipids, histopathological evaluations and immunolocalization of macrophage surface marker (F4/80), vascular cell adhesion molecule-1 (VCAM-1) and P-selectin were performed in CON [rats treated with single dose of saline (i.p.) and fed with laboratory chow], ATH [rats treated with single dose of vitamin D3 (600,000 IU, i.p) and fed with atherogenic diet] and ATH+CG [rats treated with single dose of vitamin D3 (600,000 IU, i.p.) and fed with atherogenic diet and simultaneously treated with 200 mg/kg CG extract, p.o.] for 8 weeks. CG extract supplementation to atherogenic diet fed rats significantly prevented increment in serum cholesterol, triglycerides, and lipoproteins, markers of LDL-C oxidation, auto-antibody titer and aortic lipids. Also, LDL-C isolated from ATH+CG rats recorded mimimal aggregation and susceptibility to undergo ex vivo LDL-C oxidation. Microscopic evaluation of thoracic aorta of ATH+CG rats reveled prevention of atheromatous plaque formation, accumulation of lipid laden macrophages, calcium deposition, distortion/defragmentation of elastin, accumulation of macrophages and, down regulation of cell adhesion molecules (VCAM-1 and P-selectin) expression. Further, in vitro monocyte to macrophage differentiation was significantly attenuated in presence of CG extract (200 µg/mL). It can be concluded from the present study that, CG extract is capable of controlling induction of experimental atherosclerosis and warrants further scrutiny at the clinical level as a possible therapeutic agent.

Sida rhomboidea.Roxb aqueous extract down-regulates in vivo expression of vascular cell adhesion molecules in atherogenic rats and inhibits in vitro macrophage differentiation and foam cell formation.

The present study evaluates efficacy of Sida rhomboidea.Roxb (SR) leaves extract in ameliorating experimental atherosclerosis using in vitro and in vivo experimental models. Atherogenic (ATH) diet fed rats recorded significant increment in the serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very LDL (VLDL), autoantibody against oxidized LDL (Ox-LDL), markers of LDL oxidation and decrement in high-density lipoprotein (HDL) along with increment in aortic TC and TG. The ex vivo LDL oxidation assay revealed an increased susceptibility of LDL isolated from ATH rats to undergo copper mediated oxidation. These set of changes were minimized by simultaneous co-supplementation of SR extract to ATH diet fed rats. Histopathology of aorta and immunolocalization studies recorded pronounced atheromatous plaque formation, vascular calcification, significant elastin derangements and higher expression of macrophage surface marker (F4/80), vascular cell adhesion molecule-1 (VCAM-1) and p-selectin in ATH rats. Whereas, ATH+SR rats depicted minimal evidence of atheromatous plaque formation, calcium deposition, distortion/defragmentation of elastin and accumulation of macrophages along with lowered expression of VCAM-1 and P-selectin compared to ATH rats. Further, monocyte to macrophage differentiation and in vitro foam cell formation were significantly attenuated in presence of SR extract. In conclusion, SR extract has the potency of controlling experimental atherosclerosis and can be used as promising herbal supplement in combating atherosclerosis.

Anthocyanin-rich red cabbage (Brassica oleracea L.) extract attenuates cardiac and hepatic oxidative stress in rats fed an atherogenic diet.

BACKGROUND: Oxidative stress induced by reactive oxygen species plays an important role in the aetiology of several diseases including atherosclerosis and coronary heart disease. Anthocyanin-rich extracts have been shown to possess a variety of therapeutic roles, including antioxidant, cardioprotective and hepatoprotective properties. The present inventory was undertaken to evaluate the protective role of anthocyanin-rich red cabbage extract (ARCE) on an atherogenic (ATH) diet-induced hypercholesterolaemia and related cardiac and, hepatic oxidative stress in rats. RESULTS: ARCE (100 mg kg(-1) body weight) treatment of rats fed the ATH diet significantly prevented elevation in serum and tissue lipids, circulating levels of cardiac and hepatic damage markers, and resulted in excretion of lipids through faeces. Also, the ARCE extract significantly attenuated alterations in the cardiac and hepatic antioxidants and lipid peroxidation, and histopathological changes in cardiac and hepatic tissue. CONCLUSION: Thus, the present study provides the first scientific evidence for a protective role of ARCE against ATH diet-induced hypercholesterolaemia and cardiac and hepatic oxidative stress.

Autonomous regulation of retinal insulin biosynthesis in diabetes.

Diabetic retinopathy (DR) is a neurodegenerative disease that results as a complication of dysregulated glucose metabolism, or diabetes. The signaling of insulin is lost or dampened in diabetes, but this hormone has also been shown to be an important neurotrophic factor which supports neurons of the brain. The role of local insulin synthesis and secretion in the retina, however, is unclear. We have investigated whether changes in local insulin synthesis occur in the diabetic retina and in response to stressors known to initiate retinal neurodegenerative processes. The expression of insulin and its cleavage product, c-peptide, were examined in retinas of a Type I diabetes animal model and human postmortem donors with DR. We detected mRNAs for insulin I (Ins1), insulin II (Ins2) and human insulin (Ins) by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Using an ex-vivo system, isolated neuroretinas and retinal pigmented epithelium (RPE) layers were exposed to glycemic, oxidative and inflammatory environments to measure insulin gene transcripts produced de novo in the retina under disease-relevant conditions. The expression of insulin in the retina was altered with the progression of diabetes in STZ mice and donors with DR. Transcription factors for insulin, were simultaneously expressed in a pattern matching insulin genes. Furthermore, de novo insulin mRNA in isolated retinas was induced by acute stress. RPE explants displayed the most pronounced changes in Ins1 and Ins2. This data reveals that the retina, like the brain, is an organ capable of producing local insulin and this synthesis is altered in diabetes.

Sida rhomboidea. Roxb leaf extract down-regulates expression of PPARγ2 and leptin genes in high fat diet fed C57BL/6J Mice and retards in vitro 3T3L1 pre-adipocyte differentiation.

Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity.

Oxidative Stress and Inflammation in Retinal Degeneration.

Inflammation and oxidative stress play prominent roles in the pathogenesis of many degenerative diseases of the retina, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), retinal vein occlusion, and retinitis pigmentosa [...].

A Comprehensive Review Evaluating the Impact of Protein Source (Vegetarian vs. Meat Based) in Hepatic Encephalopathy.

Hepatic encephalopathy (HE) is a common neurological consequence in patients with cirrhosis and has a healthcare burden of USD 5370 to 50,120 per patient annually. HE significantly hampers the quality of life and is a major cause of morbidity and mortality. Patients with cirrhosis are at a high risk for protein-calorie malnutrition due to altered metabolism. Current evidence has changed the old belief of protein restriction in patients with cirrhosis and now 1.2 to 1.5 g/kg/day protein intake is recommended. Case series and studies with small numbers of participants showed that a vegetarian protein diet decreases the symptoms of HE when compared to a meat-based diet, but the evidence is limited and requires further larger randomized controlled trials. However, vegetable or milk-based protein diets are good substitutes for patients averse to meat intake. Branch chain amino acids (BCAA) (leucine, isoleucine and valine) have also been shown to be effective in alleviating symptoms of HE and are recommended as an alternative therapy in patients with cirrhosis for the treatment of HE. In this review, we provide an overview of current literature evaluating the role of protein intake in the management of HE in cirrhosis.

Pharmacological and Metabolic Significance of Bile Acids in Retinal Diseases.

Bile acids (BAs) are amphipathic sterols primarily synthesized from cholesterol in the liver and released in the intestinal lumen upon food intake. BAs play important roles in micellination of dietary lipids, stimulating bile flow, promoting biliary phospholipid secretion, and regulating cholesterol synthesis and elimination. Emerging evidence, however, suggests that, aside from their conventional biological function, BAs are also important signaling molecules and therapeutic tools. In the last decade, the therapeutic applications of BAs in the treatment of ocular diseases have gained great interest. Despite the identification of BA synthesis, metabolism, and recycling in ocular tissues, much remains unknown with regards to their biological significance in the eye. Additionally, as gut microbiota directly affects the quality of circulating BAs, their analysis could derive important information on changes occurring in this microenvironment. This review aims at providing an overview of BA metabolism and biological function with a focus on their potential therapeutic and diagnostic use for retinal diseases.

Protective effect of Clerodendron glandulosum extract against experimentally induced metabolic syndrome in rats.

CONTEXT: Metabolic syndrome (MetS) has become one of the major health burdens worldwide. To date, no single pharmacological agent has been developed to correct metabolic abnormalities associated with MetS. Use of indigenous medicinal plants as alternative medicines against MetS could be beneficial due to multiple therapeutic usage, easy availability, and relatively few side effects. OBJECTIVE: To investigate the protective effect of Clerodendron glandulosum Coleb. (Verbenaceae) aqueous leaf extract (CgE) against experimentally induced MetS in rats. METHODS: Changes in body weight, food and fluid intake, plasma glucose, insulin, fasting insulin resistance index (FIRI), plasma total lipid profile, free fatty acids (FFA), oral glucose tolerance test (OGTT), blood pressure and vascular reactivity have been investigated in various experimental groups. RESULTS: Fructose+CgE groups recorded significant decrement (P <0.05) in plasma glucose, insulin, FIRI, total cholesterol, triglycerides, LDL, VLDL and FFA, whereas plasma HDL level was significantly increased (P <0.05) along with an efficient clearance of glucose during OGTT and lowered area under curve values. FRU+CgE groups also showed significantly decreased (P <0.05) mean arterial blood pressure along with decreased vasoconstriction and increased vasorelaxation in response to administration of various pharmacological agents. These results were comparable with metformin treated rats. DISCUSSION: C. glandulosum leaf extract ameliorates experimentally induced MetS by improving dyslipidemia and insulin resistance. CONCLUSION: This study provides the first pharmacological evidence for the protective role of C. glandulosum leaves against experimentally induced MetS. Thus, therapeutic use of C. glandulosum in controlling MetS is indicated.

Data on the role of miR-144 in regulating fetal hemoglobin production in retinal pigmented epithelial cells.

The data presented in this article are connected to our related article entitled "Inhibiting microRNA-144 potentiates Nrf2-dependent antioxidant signaling in retinal pigmented epithelial cells (RPE) and protects against oxidative stress-induced outer retinal degeneration" [1] where, we have shown that miR-144 induces oxidative stress in RPE cells by targeting Nrf2 expression. Previous studies from our laboratory have shown that like erythroid cells, RPE cells express α, ß and γ-globin and produce hemoglobin locally in retina. Further, the ability to therapeutically reactivate fetal hemoglobin production in these cells, a strategy of high potential benefit in the treatment of complications of sickle cell disease, including retinopathy, is impacted by Nrf2-mediated signaling [2,3]. Studies by others [4,5] provide compelling evidence of a regulatory role for miR-144 and Nrf2 in fetal hemoglobin production in erythroid cells. Our current work confirms this finding in human RPE. We additionally show that miR-144-mediated regulation of fetal hemoglobin production in RPE cells is independent of kruppel like factor 1 (KLF-1). This supports the plausibility that in RPE, hemoglobin, particularly fetal hemoglobin, may be important for functions other than oxygen transport (e.g., antioxidant defense). Indeed, our new data on miR-144 in RPE supports strongly the potential mechanistic between fetal hemoglobin production and the regulation of oxidative stress in this cell type [1].

Implications of NAD+ Metabolism in the Aging Retina and Retinal Degeneration.

Nicotinamide adenine dinucleotide (NAD+) plays an important role in various key biological processes including energy metabolism, DNA repair, and gene expression. Accumulating clinical and experimental evidence highlights an age-dependent decline in NAD+ levels and its association with the development and progression of several age-related diseases. This supports the establishment of NAD+ as a critical regulator of aging and longevity and, relatedly, a promising therapeutic target to counter adverse events associated with the normal process of aging and/or the development and progression of age-related disease. Relative to the above, the metabolism of NAD+ has been the subject of numerous investigations in various cells, tissues, and organ systems; however, interestingly, studies of NAD+ metabolism in the retina and its relevance to the regulation of visual health and function are comparatively few. This is surprising given the critical causative impact of mitochondrial oxidative damage and bioenergetic crises on the development and progression of degenerative disease of the retina. Hence, the role of NAD+ in this tissue, normally and aging and/or disease, should not be ignored. Herein, we discuss important findings in the field of NAD+ metabolism, with particular emphasis on the importance of the NAD+ biosynthesizing enzyme NAMPT, the related metabolism of NAD+ in the retina, and the consequences of NAMPT and NAD+ deficiency or depletion in this tissue in aging and disease. We discuss also the implications of potential therapeutic strategies that augment NAD+ levels on the preservation of retinal health and function in the above conditions. The overarching goal of this review is to emphasize the importance of NAD+ metabolism in normal, aging, and/or diseased retina and, by so doing, highlight the necessity of additional clinical studies dedicated to evaluating the therapeutic utility of strategies that enhance NAD+ levels in improving vision.

Inhibition of HDAC6 Attenuates Diabetes-Induced Retinal Redox Imbalance and Microangiopathy.

We investigated the contributing role of the histone deacetylase 6 (HDAC6) to the early stages of diabetic retinopathy (DR). Furthermore, we examined the mechanism of action of HDAC6 in human retinal endothelial cells (HuREC) exposed to glucidic stress. Streptozotocin-induced diabetic rats (STZ-rats), a rat model of type 1 diabetes, were used as model of DR. HDAC6 expression and activity were increased in human diabetic postmortem donors and STZ-rat retinas and were augmented in HuREC exposed to glucidic stress (25 mM glucose). Administration of the HDAC6 specific inhibitor Tubastatin A (TS) (10 mg/kg) prevented retinal microvascular hyperpermeability and up-regulation of inflammatory markers. Furthermore, in STZ-rats, TS decreased the levels of senescence markers and rescued the expression and activity of the histone deacetylase sirtuin 1 (SIRT1), while downregulating the levels of free radicals and of the redox stress markers 4-hydroxynonenal (4-HNE) and nitrotyrosine (NT). The antioxidant effects of TS, consequent to HDAC6 inhibition, were associated with preservation of Nrf2-dependent gene expression and up-regulation of thioredoxin-1 activity. In vitro data, obtained from HuREC, exposed to glucidic stress, largely replicated the in vivo results further confirming the antioxidant effects of HDAC6 inhibition by TS in the diabetic rat retina. In summary, our data implicate HDAC6 activation in mediating hyperglycemia-induced retinal oxidative/nitrative stress leading to retinal microangiopathy and, potentially, DR.

Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina.

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.