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.

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.

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.

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 [...].

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.

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.

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.

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.

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.

Ursodeoxycholic Acid Halts Pathological Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy.

Retinopathy of prematurity (ROP) is the leading cause of blindness in infants. We have investigated the efficacy of the secondary bile acid ursodeoxycholic acid (UDCA) and its taurine and glycine conjugated derivatives tauroursodeoxycholic acid (TUDCA) and glycoursodeoxycholic acid (GUDCA) in preventing retinal neovascularization (RNV) in an experimental model of ROP. Seven-day-old mice pups (P7) were subjected to oxygen-induced retinopathy (OIR) and were treated with bile acids for various durations. Analysis of retinal vascular growth and distribution revealed that UDCA treatment (50 mg/kg, P7-P17) of OIR mice decreased the extension of neovascular and avascular areas, whereas treatments with TUDCA and GUDCA showed no changes. UDCA also prevented reactive gliosis, preserved ganglion cell survival, and ameliorated OIR-induced blood retinal barrier dysfunction. These effects were associated with decreased levels of oxidative stress markers, inflammatory cytokines, and normalization of the VEGF-STAT3 signaling axis. Furthermore, in vitro tube formation and permeability assays confirmed UDCA inhibitory activity toward VEGF-induced pro-angiogenic and pro-permeability effects on human retinal microvascular endothelial cells. Collectively, our results suggest that UDCA could represent a new effective therapy for ROP.

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.

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].

Carbon monoxide releasing molecule-A1 improves nonalcoholic steatohepatitis via Nrf2 activation mediated improvement in oxidative stress and mitochondrial function.

Nuclear factor-erythroid 2 related factor 2 (Nrf2)-mediated signaling plays a central role in maintaining cellular redox homeostasis of hepatic cells. Carbon monoxide releasing molecule-A1 (CORM-A1) has been reported to stimulate up-regulation and nuclear translocation of Nrf2 in hepatocytes. However, the role of CORM-A1 in improving lipid metabolism, antioxidant signaling and mitochondrial functions in nonalcoholic steatohepatitis (NASH) is unknown. In this study, we report that CORM-A1 prevents hepatic steatosis in high fat high fructose (HFHF) diet fed C57BL/6J mice, used as model of NASH. The beneficial effects of CORM-A1 in HFHF fed mice was associated with improved lipid homeostasis, Nrf2 activation, upregulation of antioxidant responsive (ARE) genes and increased ATP production. As, mitochondria are intracellular source of reactive oxygen species (ROS) and important sites of lipid metabolism, we further investigated the mechanisms of action of CORM-A1-mediated improvement in mitochondrial function in palmitic acid (PA) treated HepG2 cells. Cellular oxidative stress and cell viability were found to be improved in PA + CORM-A1 treated cells via Nrf2 translocation and activation of cytoprotective genes. Furthermore, in PA treated cells, CORM-A1 improved mitochondrial oxidative stress, membrane potential and rescued mitochondrial biogenesis thru upregulation of Drp1, TFAM, PGC-1α and NRF-1 genes. CORM-A1 treatment improved cellular status by lowering glycolytic respiration and maximizing OCR. Improvement in mitochondrial respiration and increment in ATP production in PA + CORM-A1 treated cells further corroborate our findings. In summary, our data demonstrate for the first time that CORM-A1 ameliorates tissue damage in steatotic liver via Nrf2 activation and improved mitochondrial function, thus, suggesting the anti-NASH potential of CORM-A1.

Inhibiting microRNA-144 potentiates Nrf2-dependent antioxidant signaling in RPE and protects against oxidative stress-induced outer retinal degeneration.

The retinal pigment epithelium (RPE) is consistently exposed to high levels of pro-oxidant and inflammatory stimuli. As such, under normal conditions the antioxidant machinery in the RPE cell is one of the most efficient in the entire body. However, antioxidant defense mechanisms are often impacted negatively by the process of aging and/or degenerative disease leaving RPE susceptible to damage which contributes to retinal dysfunction. Thus, understanding better the mechanisms governing antioxidant responses in RPE is critically important. Here, we evaluated the role of the redox sensitive microRNA miR-144 in regulation of antioxidant signaling in human and mouse RPE. In cultured human RPE, miR-144-3p and miR-144-5p expression was upregulated in response to pro-oxidant stimuli. Likewise, overexpression of miR-144-3p and -5p using targeted miR mimics was associated with reduced expression of Nrf2 and downstream antioxidant target genes (NQO1 and GCLC), reduced levels of glutathione and increased RPE cell death. Alternately, some protection was conferred against the above when miR-144-3p and miR-144-5p expression was suppressed using antagomirs. Expression analyses revealed a higher conservation of miR-144-3p expression across species and additionally, the presence of two potential Nrf2 binding sites in the 3p sequence compared to only one in the 5p sequence. Thus, we evaluated the impact of miR-144-3p expression in the retinas of mice in which a robust pro-oxidant environment was generated using sodium iodate (SI). Subretinal injection of miR-144-3p antagomir in SI mice preserved retinal integrity and function, decreased oxidative stress, limited apoptosis and enhanced antioxidant gene expression. Collectively, the present work establishes miR-144 as a potential target for preventing and treating degenerative retinal diseases in which oxidative stress is paramount and RPE is prominently affected (e.g., age-related macular degeneration and diabetic retinopathy).

M3 muscarinic receptor activation reduces hepatocyte lipid accumulation via CaMKKß/AMPK pathway.

Previously, we reported that hepatic muscarinic receptors modulate both acute and chronic liver injury, however, the role of muscarinic receptors in fatty liver disease is unclear. We observed in patients who underwent weight loss surgery, a decrease in hepatic expression of M3 muscarinic receptors (M3R). We also observed that fat loading of hepatocytes, increased M3R expression. Based on these observations, we tested the hypothesis that M3R regulate hepatocyte lipid accumulation. Incubation of AML12 hepatocytes with 1 mM oleic acid resulted in lipid accumulation that was significantly reduced by co-treatment with a muscarinic agonist (pilocarpine or carbachol), an effect blocked by atropine (a muscarinic antagonist). Similar treatment of Hepa 1-6 cells, a mouse hepatoblastoma cell line, showed comparable results. In both, control and fat-loaded AML12 cells, pilocarpine induced time-dependent AMPKα phosphorylation and significantly up-regulated lipolytic genes (ACOX1, CPT1, and PPARα). Compound C, a selective and reversible AMPK inhibitor, significantly blunted pilocarpine-mediated reduction of lipid accumulation and pilocarpine-mediated up-regulation of lipolytic genes. BAPTA-AM, a calcium chelator, and STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, attenuated agonist-induced AMPKα phosphorylation. Finally, M3R siRNA attenuated agonist-induced AMPKα phosphorylation as well as agonist-mediated reduction of hepatocyte steatosis. In conclusion, this proof-of-concept study demonstrates that M3R has protective effects against hepatocyte lipid accumulation by activating AMPK pathway and is a potential therapeutic target for non-alcoholic fatty liver disease.

MicroRNA-34a (miR-34a) Mediates Retinal Endothelial Cell Premature Senescence through Mitochondrial Dysfunction and Loss of Antioxidant Activities.

Stress-associated premature senescence (SAPS) is involved in retinal microvascular injury and diabetic retinopathy. We have investigated the role and mode of action of miR-34a in retinal endothelial cells senescence in response to glucidic stress. Human retinal microvascular endothelial cells (HuREC) were exposed to glucidic stress (high glucose (HG) = 25 mM d-glucose) and compared to cells exposed to normal glucose (NG = 5 mM) or the osmotic control l-glucose (LG = 25 mM). HG stimulation of HuREC increased the expression of miR-34a and induced cellular senescence. HG also increased the expression of p16ink4a and p21waf1, while decreasing the histone deacetylase SIRT1. These effects were associated with diminished mitochondrial function and loss of mitochondrial biogenesis factors (i.e., PGC-1α, NRF1, and TFAM). Transfection of the cells with miR-34a inhibitor (IB) halted HG-induced mitochondrial dysfunction and up-regulation of senescence-associated markers, whereas miR-34a mimic promoted cellular senescence and mitochondrial dysfunction. Moreover, HG lowered levels of the mitochondrial antioxidants TrxR2 and SOD2, an effect blunted by miR-34a IB, and promoted by miR-34a mimic. 3'-UTR (3'-untranslated region) reporter assay of both genes validated TrxR2 as a direct target of miR-34a, but not SOD2. Our results show that miR-34a is a key player of HG-induced SAPS in retinal endothelial cells via multiple pathways involved in mitochondrial function and biogenesis.