Regulation of serine palmitoyl-transferase and Rac1-Nox2 signaling in diabetic retinopathy.

Hyperlipidemia is considered as one of the major systemic factors associated with the development of diabetic retinopathy, and animal models have documented that its presence in a hyperglycemic environment exacerbates cytosolic ROS production (via activation of the Rac1-Nox2 axis) and mitochondrial damage. Hyperglycemia also accelerates Rac1 transcription via dynamic DNA methylation-hydroxymethylation of its promoter. In diabetes, ceramide metabolism in the retina is impaired and its accumulation is increased. Our aim was to investigate the effect of inhibition of the rate limiting enzyme of the de novo ceramide biosynthesis, serine palmitoyl-transferase (SPT), on Rac1 activation in diabetic retinopathy. Using human retinal endothelial cells, transfected with SPT-siRNA, and incubated in 20 mM D-glucose in the presence or absence of 50 µM palmitate (glucolipotoxic and glucotoxic, respectively), activities of Rac1 and Nox2, and ROS levels were quantified. For Rac1 transcriptional activation, 5 hydroxymethyl cytosine (5hmC) levels at its promoter were quantified. Key parameters were confirmed in retinal microvessels from streptozotocin-induced diabetic mice on a normal diet (type 1 diabetic model) or on a high-fat diet (45% kcal, type 2 diabetic model), injected intravitreally with SPT-siRNA. Compared to normal glucose, cells in high glucose, with or without palmitic acid, had increased Rac1-Nox2-ROS signaling, Rac1 transcripts and 5hmC levels at its promoter. Inhibition of SPT by SPT-siRNA or myriocin prevented glucotoxic- and glucolipotoxic-induced increase in Rac1-Nox2-ROS signaling and 5hmC at the Rac1 promoter. Similarly, in both type 1 and type 2 diabetic mouse models, SPT-siRNA attenuated the increase in the Rac1-Nox2-ROS axis and 5hmC at the Rac1 promoter. Thus, inhibition of the rate limiting enzyme of ceramide de novo biosynthesis, SPT, regulates activation of DNA methylation-hydroxymethylation machinery and prevents increased Rac1 transcription. This ameliorates the activation of Rac1-Nox2 signaling and protects the mitochondria from damaging cytosolic ROS, which prevents accelerated capillary cell loss. These results further raise the importance of regulating lipid levels in diabetic patients with dyslipidemia.

Epigenetic modifications in diabetes.

Diabetes is now considered as a 'silent epidemic' that claims over four million lives every year, and the disease knows no socioeconomic boundaries. Despite extensive efforts by the National and International organizations, and cutting-edge research, about 11% world's population is expected to suffer from diabetes (and its complications) by year 2045. This life-long disease damages both the microvasculature and the macrovasculature of the body, and affects many metabolic and molecular pathways, altering the expression of many genes. Recent research has shown that external factors, such as environmental factors, lifestyle and pollutants can also regulate gene expression, and contribute in the disease development and progression. Many epigenetic modifications are implicated in the development of micro- and macro- vascular complications including DNA methylation and histone modifications of several genes implicated in their development. Furthermore, several noncoding RNAs, such as micro RNAs and long noncoding RNAs, are also altered, affecting many biochemical pathways. Epigenetic modifications, however, have the advantage that they could be passed to the next generation, or can be erased. They are now being explored as therapeutical target(s) in the cancer field, which opens up the possibility to use them for treating diabetes and preventing/slowing down its complications.

Susceptibility to high-altitude pulmonary edema is associated with circulating miRNA levels under hypobaric hypoxia conditions.

Hypobaric hypoxia poses stress to sojourners traveling to high-altitude. A cascade of physiological changes occurs to cope with or adapt to hypobaric hypoxia. However, an insufficient physiological response to the hypoxic condition resulting from imbalanced vascular homeostasis pathways results in high-altitude pulmonary edema (HAPE). The present study aims to identify the implication of miRNAs associating with HAPE and adaptation. We analyzed the expression of 1,113 miRNAs in HAPE-patients (HAPE-p), HAPE-free controls (HAPE-f), and highland natives (HLs). Based on miRNA profiling and in silico analyses, miR-124-3p emerged relevantly. We observed a significant overexpression of miR-124-3p in HAPE-p. In silico analyses revealed a direct interaction of miR-124-3p with vascular homeostasis and hypoxia-associated genes NOS3 (endothelial nitric oxide synthase), Apelin, and ETS1 (V-Ets avian erythroblastosis virus E2 oncogene homolog 1). Moreover, the transcript and biolevel expression of these genes were significantly decreased in HAPE-p when compared with HAPE-f or HLs. Our in vitro analysis in human umbilical vein endothelial cells demonstrated a significant knockdown of these genes both at transcript and protein levels following miR-124-3p overexpression. Conclusively, our results showed that miR-124-3p might play a plausible role in HAPE pathophysiology by inhibiting the expression of NOS3, Apelin, and ETS1.

Epigenetics and Mitochondrial Stability in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy.

Retinopathy continues to progress even when diabetic patients try to control their blood sugar, but the molecular mechanism of this 'metabolic memory' phenomenon remains elusive. Retinal mitochondria remain damaged and vicious cycle of free radicals continues to self-propagate. DNA methylation suppresses gene expression, and diabetes activates DNA methylation machinery. Our aim was to investigate the role of DNA methylation in continued compromised mitochondrial dynamics and genomic stability in diabetic retinopathy. Using retinal endothelial cells, incubated in 20 mM glucose for four days, followed by 5 mM glucose for four days, and retinal microvessels from streptozotocin-induced diabetic rats in poor glycemia for four months, followed by normal glycemia for four additional months, DNA methylation of mitochondrial fusion and mismatch repair proteins, Mfn2 and Mlh1 respectively, was determined. Retinopathy was detected in trypsin-digested microvasculature. Re-institution of good glycemia had no beneficial effect on hypermethylation of Mfn2 and Mlh1 and retinal function (electroretinogram), and the  retinopathy continued to progress. However, intervention of good glycemia directly with DNA methylation inhibitors (Azacytidine or Dnmt1-siRNA), prevented Mfn2 and Mlh1 hypermethylation, and ameliorated retinal dysfunction and diabetic retinopathy. Thus, direct regulation of DNA methylation can prevent/reverse diabetic retinopathy by maintaining mitochondrial dynamics and DNA stability, and prevent retinal functional damage.

Homocysteine Disrupts Balance between MMP-9 and Its Tissue Inhibitor in Diabetic Retinopathy: The Role of DNA Methylation.

High homocysteine is routinely observed in diabetic patients, and this non-protein amino acid is considered as an independent risk factor for diabetic retinopathy. Homocysteine biosynthesis from methionine forms S-adenosyl methionine (SAM), which is a major methyl donor critical in DNA methylation. Hyperhomocysteinemia is implicated in increased oxidative stress and activation of MMP-9, and in diabetic retinopathy, the activation of MMP-9 facilitates capillary cell apoptosis. Our aim was to investigate the mechanism by which homocysteine activates MMP-9 in diabetic retinopathy. Human retinal endothelial cells, incubated with/without 100 µM homocysteine, were analyzed for MMP-9 and its tissue inhibitor Timp1 expressions and interactions, and ROS levels. Timp1 and MMP-9 promoters were analyzed for methylated and hydroxymethylated cytosine levels (5mC and 5hmC respectively) by the DNA capture method, and DNA- methylating (Dnmt1) and hydroxymethylating enzymes (Tet2) binding by chromatin immunoprecipitation. The results were confirmed in retinal microvessels from diabetic rats receiving homocysteine. Homocysteine supplementation exacerbated hyperglycaemia-induced MMP-9 and ROS levels and decreased Timp1 and its interactions with MMP-9. Homocysteine also aggravated Dnmts and Tets activation, increased 5mC at Timp1 promoter and 5hmC at MMP-9 promoter, and suppressed Timp1 transcription and activated MMP-9 transcription. Similar results were obtained from retinal microvessels from diabetic rats receiving homocysteine. Thus, hyperhomocysteinemia in diabetes activates MMP-9 functionally by reducing Timp1-MMP-9 interactions and transcriptionally by altering DNA methylation-hydroxymethylation of its promoter. The regulation of homocysteine could prevent/slow down the development of retinopathy and prevent their vision loss in diabetic patients.

Faulty homocysteine recycling in diabetic retinopathy.

BACKGROUND: Although hyperglycemia is the main instigator in the development of diabetic retinopathy, elevated circulating levels of a non-protein amino acid, homocysteine, are also associated with an increased risk of retinopathy. Homocysteine is recycled back to methionine by methylenetetrahydrofolate reductase (MTHFR) and/or transsulfurated by cystathionine ß-synthase (CBS) to form cysteine. CBS and other transsulfuration enzyme cystathionine-γ-lyase (CSE), through desulfuration, generates H2S. Methionine cycle also regulates DNA methylation, an epigenetic modification associated with the gene suppression. The aim of this study was to investigate homocysteine and its metabolism in diabetic retinopathy. METHODS: Homocysteine and H2S levels were analyzed in the retina, and CBS, CSE and MTHFR in the retinal microvasculature from human donors with established diabetic retinopathy. Mitochondrial damage was evaluated in retinal microvessels by quantifying enzymes responsible for maintaining mitochondrial dynamics (fission-fusion-mitophagy). DNA methylation status of CBS and MTHFR promoters was examined using methylated DNA immunoprecipitation technique. The direct effect of homocysteine on mitochondrial damage was confirmed in human retinal endothelial cells (HRECs) incubated with 100 µM L-homocysteine. RESULTS: Compared to age-matched nondiabetic control human donors, retina from donors with established diabetic retinopathy had ~ 3-fold higher homocysteine levels and ~ 50% lower H2S levels. The enzymes important for both transsulfuration and remethylation of homocysteine including CBS, CSE and MTHFR, were 40-60% lower in the retinal microvasculature from diabetic retinopathy donors. While the mitochondrial fission protein, dynamin related protein 1, and mitophagy markers optineurin and microtubule-associated protein 1A/1B-light chain 3 (LC3), were upregulated, the fusion protein mitofusin 2 was downregulated. In the same retinal microvessel preparations from donors with diabetic retinopathy, DNA at the promoters of CBS and MTHFR were hypermethylated. Incubation of HRECs with homocysteine increased reactive oxygen species and decreased transcripts of mtDNA-encoded CYTB. CONCLUSIONS: Compromised transsulfuration and remethylation processes play an important role in the poor removal of retinal homocysteine in diabetic patients. Thus, regulation of their homocysteine levels should ameliorate retinal mitochondrial damage, and by regulating DNA methylation status of the enzymes responsible for homocysteine transsulfuration and remethylation, should prevent excess accumulation of homocysteine.

Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy.

Purpose: Diabetes causes dysfunction in the retinal mitochondria and increases base mismatches in their DNA (mtDNA). The enzyme responsible for repairing the base mismatches, MutL homolog 1 (Mlh1), is compromised. Diabetes also favors many epigenetic modifications and activates DNA methylation machinery, and Mlh1 has a CpG-rich promoter. Our aim is to identify the molecular mechanism responsible for impaired mtDNA mismatch repair in the pathogenesis of diabetic retinopathy. Methods: Human retinal endothelial cells, incubated in 20 mM glucose, were analyzed for mitochondrial localization of Mlh1 by an immunofluorescence technique, Mlh1 promoter DNA methylation by the methylated DNA capture method, and the binding of Dnmt1 and transcriptional factor Sp1 by chromatin immunoprecipitation. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice, with or without Dnmt inhibitors, and from human donors with diabetic retinopathy. Results: Compared with cells in 5 mM glucose, high glucose decreased Mlh1 mitochondrial localization, and its promoter DNA was hypermethylated with increased Dnmt-1 binding and decreased Sp1 binding. Dnmt inhibitors attenuated Mlh1 promoter hypermethylation and prevented a decrease in its gene transcripts and an increase in mtDNA mismatches. The administration of Dnmt inhibitors in mice ameliorated a diabetes-induced increase in Mlh1 promoter hypermethylation and a decrease in its gene transcripts. Similar decreases in Mlh1 gene transcripts and its promoter DNA hypermethylation were observed in human donors. Conclusions: Thus, as a result of the epigenetic modifications of the Mlh1 promoter, its transcription is decreased, and decreased mitochondrial accumulation fails to repair mtDNA mismatches. Therapies targeted to halt DNA methylation have the potential to prevent/halt mtDNA damage and the development of diabetic retinopathy.

Functional Regulation of an Oxidative Stress Mediator, Rac1, in Diabetic Retinopathy.

PURPOSE: Early activation of cytosolic NADPH oxidase-2 (Nox2) in diabetes increases retinal ROS production, damaging their mitochondria. The assembly of Nox2 holoenzyme requires activation of a small molecular weight G protein Rac1. Rac1 activation is regulated by guanine exchange factors and guanine nucleotide-dissociation inhibitors, and post-translational modifications assist in its association with exchange factors and dissociation inhibitors. The goal of this study is to investigate the mechanisms of Rac1 activation in the development of diabetic retinopathy. METHODS: The levels of the dissociation inhibitor, prenylating enzyme (farnesyltransferase, FNTA), and exchange factor Vav2 were quantified in human retinal endothelial cells, incubated in normal or high glucose for 96 h. The roles of prenylation and Vav2 in Rac1-Nox2-ROS mitochondrial damage were confirmed in FNTA-siRNA-transfected cells and using the Vav2 inhibitor EHop, respectively. Retinal histopathology and functional changes associated with diabetic retinopathy were analyzed in diabetic mice receiving EHop for 6 months. Key parameters of Rac1 activation were confirmed in the retinal microvasculature from human donors with diabetic retinopathy. RESULTS: In HRECs, glucose increased FNTA and Vav2 and decreased the dissociation inhibitor. FNTA-siRNA and EHop inhibited glucose-induced activation of Rac1-Nox2-ROS signaling. In diabetic mice, EHop ameliorated the development of retinopathy and functional/structural abnormalities and attenuated Rac1-Nox2-mitochondrial damage. Similar alterations in Rac1 regulators were observed in retinal microvasculature from human donors with diabetic retinopathy. In diabetes, Rac1 prenylation and its interactions with Vav2 contribute to Nox2-ROS-mitochondrial damage, and the pharmacological inhibitors to attenuate Rac1 interactions with its regulators could have the potential to halt/inhibit the development of diabetic retinopathy. Graphical Abstract Activation of prenylating enzyme farnesyltransferase (FNTA) in diabetes, prenylates Rac1. The binding of Rac1 with guanine nucleotide-dissociation inhibitor (GDI) is decreased, but its association with the guanine exchange factor, Vav2, is increased, resulting in Rac1 activation. Active Rac1 helps in the assembly of Nox2 holoenzyme, and Nox2 activation increases cytosolic ROS production, damaging the mitochondria. Damaged mitochondria accelerate capillary cell apoptosis, and ultimately, results in the development of diabetic retinopathy.

Matrix metalloproteinase-14 is a biomarker of angiogenic activity in proliferative diabetic retinopathy.

Purpose: Matrix metalloproteinase-14 (MMP-14) is a transmembrane MMP that plays a critical role in promoting angiogenesis. We investigated the expression levels of MMP-14 and correlated the levels with clinical disease activity and with the levels of the angiogenic factors vascular endothelial growth factor (VEGF) and MMP-9 in proliferative diabetic retinopathy (PDR). To reinforce the findings at the functional level, we examined the expression of MMP-14 in the retinas of diabetic rats. Methods: Vitreous samples from 34 patients with PDR and 18 nondiabetic patients and epiretinal membranes from 13 patients with PDR and the retinas of rats were studied with enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and real-time reverse transcription PCR (RT-PCR). Results: The MMP-14, VEGF, and MMP-9 levels were statistically significantly higher in the vitreous samples from patients with PDR than in the samples from the nondiabetic controls (p<0.001 for all comparisons). The MMP-14 levels in patients with PDR with active neovascularization were statistically significantly higher than those in patients with inactive PDR (p<0.001). There were statistically significant positive correlations between levels of MMP-14 and levels of VEGF (r = 0.3; p = 0.032) and MMP-9 (r = 0.54; p<0.001). In the epiretinal membranes, MMP-14 was expressed in vascular endothelial cells, leukocytes, and myofibroblasts. Statistically significant positive correlations were detected between the numbers of blood vessels expressing CD31 and the numbers of blood vessels (r = 0.74; p = 0.004) and stromal cells (r = 0.72; p = 0.005) expressing MMP-14. Statistically significant increases of MMP-14 mRNA and protein were detected in rat retinas after induction of diabetes. Conclusions: These results suggest that MMP-14 is involved in PDR angiogenesis.

Rho-Associated Protein Kinase-1 Mediates the Regulation of Inflammatory Markers in Diabetic Retina and in Retinal Müller Cells.

GOAL: To investigate the effects of blocking Rho kinase pathway on the expression of inflammatory signaling pathways in the retina of diabetic mice and in human retinal Müller glial cells stimulated with high-glucose to replicate hyperglycemia. PROCEDURES: Retinas from diabetic mice and human retinal Müller glial cells (MIO-M1) were studied. Western blot analysis, immunofluorescence, and enzyme-linked immunosorbent assay were utilized to study the effect of the Rho kinase inhibitor fasudil on the expression of Rho-associated protein kinase-1 (ROCK-1), extracellular signal-regulated kinases1&2(ERK ½), phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB), inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1/CCL2). RESULTS: Treatment of human retinal Müller cells with high-glucose induced significant upregulation of ROCK-1, VEGF, and MCP-1/CCL2. Fasudil co-treatment normalized the high-glucose-induced upregulation of these mediators. Similarly, fasudil attenuated high-glucose-induced enhanced immunoreactivity for ROCK-1 and VEGF. Diabetes induced upregulation of ROCK-1, p-ERK ½, p-NF-κB and iNOS expression in retinas of mice. Constant fasudil intake from the onset of diabetes did not affect the metabolic status of diabetic mice but it attenuated diabetes-induced upregulation of these inflammatory signaling pathways. CONCLUSIONS: Our finding suggests that Rho-associated protein kinase-1 activation mediates regulation of inflammatory signaling pathways in diabetic retina.

Association of 150-kDa oxygen-regulated protein with vascular endothelial growth factor in proliferative diabetic retinopathy.

PURPOSE: 150-kDa oxygen-regulated protein (ORP150), a member of heat-shock protein family located in endoplasmic reticulum (ER), has a critical role in secretion of vascular endothelial growth factor (VEGF). We investigated expression levels of ORP150 and correlated these levels with VEGF and total vitreous antioxidant capacity (TAC) in proliferative diabetic retinopathy (PDR). We also examined expression of ORP150 in retinas of diabetic rats and in human retinal microvascular endothelial cells (HRMEC). METHODS: Vitreous samples from 40 PDR and 20 non-diabetic patients, epiretinal membranes from 14 patients with PDR, retinas of rats and HRMEC were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. RESULTS: We showed a significant increase in expression of VEGF and ORP150 in vitreous samples from PDR patients compared with controls (p < 0.0001 for both comparisons). Total vitreous antioxidant capacity (TAC) levels were significantly lower in patients with PDR than those in controls (p < 0.0001). Vascular endothelial growth factor (VEGF) and ORP150 levels in PDR with active neovascularization were significantly higher than that in inactive PDR (p = 0.016; p = 0.011, respectively). A significant positive correlation was observed between levels of ORP150 and levels of VEGF (r = 0.42; p = 0.001). In epiretinal membranes, ORP150 was expressed in vascular endothelial cells and stromal cells. We also demonstrated colocalization of the nuclear cell proliferation marker Ki67 and ORP150 in endothelial cells of pathologic new blood vessels. 150-kDa oxygen-regulated protein (ORP150) levels were significantly increased in rat retinas after induction of diabetes. Vascular endothelial growth factor (VEGF) and the pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) induced upregulation of ORP150 in HRMEC. CONCLUSION: These results suggest a role for ORP150 in PDR angiogenesis.

Differential expression and localization of human tissue inhibitors of metalloproteinases in proliferative diabetic retinopathy.

PURPOSE: Tissue inhibitors of metalloproteinases (TIMPs) block the catalysis by matrix metalloproteinases (MMPs) and have additional biologic activities, including regulation of cell growth and differentiation, apoptosis, angiogenesis and oncogenesis. We investigated the expression levels of all the four human TIMPs and correlated these levels with those of MMP-9 and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples from 38 PDR and 21 nondiabetic control patients and epiretinal membranes from 14 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR) were studied by enzyme-linked immunosorbent assay, Western blot analysis and immunohistochemistry. RESULTS: Tissue inhibitor of metalloproteinases-1, TIMP-4, MMP-9 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic controls (p < 0.0001 for all comparisons), whereas TIMP-2 and TIMP-3 levels did not differ significantly. TIMP-1, TIMP-4, MMP-9 and VEGF levels in PDR with active neovascularization were significantly higher than those in inactive PDR (p < 0.0001, 0.001, 0.013, 0.004, respectively). Significant positive correlations existed between levels of TIMP-1 and levels of TIMP-4 (r = 0.37; p = 0.004), MMP-9 (r = 0.65; p < 0.0001) and VEGF (r = 0.59; p < 0.0001), between levels of TIMP-4 and levels of MMP-9 (r = 0.61; p < 0.0001) and VEGF (r = 0.62; p < 0.0001) and between levels of MMP-9 and VEGF (r = 0.62; p < 0.0001). TIMP-1 and TIMP-3 were expressed in vascular endothelial cells in PDR epiretinal membranes and in myofibroblasts and leucocytes in PDR and PVR epiretinal membranes. CONCLUSION: The differential expression of TIMPs in PDR suggests that among the 4 TIMPs, TIMP-1 and TIMP-4 may be possible biomarkers of disease activity.

Unbalanced Vitreous Levels of Osteoprotegerin, RANKL, RANK, and TRAIL in Proliferative Diabetic Retinopathy.

PURPOSE: We investigated the expression of the proinflammatory and proangiogenic factor osteoprotegerin (OPG) and its ligands, receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and the receptor RANK in proliferative diabetic retinopathy (PDR). MATERIALS AND METHODS: Vitreous samples from PDR and nondiabetic control patients and epiretinal membranes from PDR patients were studied by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot analysis. RESULTS: Vascular endothelial growth factor, OPG, and soluble RANK levels in vitreous samples from PDR patients were significantly higher than that in nondiabetic controls. Soluble TRAIL levels were significantly lower in PDR patients than that in nondiabetic control, whereas soluble RANKL levels did not differ significantly. RANKL, RANK, and TRAIL were expressed in vascular endothelial cells, myofibroblasts, and CD45-expressing leukocytes in PDR epiretinal membranes. CONCLUSIONS: Dysregulated expression of OPG/RANKL/RANK pathway and TRAIL might be related to inflammation and angiogenesis in PDR.

Osteoprotegerin Is a New Regulator of Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

Purpose: Osteoprotegerin (OPG) is a novel regulator of endothelial cell function, angiogenesis, and vasculogenesis. We correlated expression levels of OPG with those of the angiogenic and inflammatory factors vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1/CCL2) in proliferative diabetic retinopathy (PDR). We also examined expression of OPG in retinas from diabetic rats and diabetic patients and measured production of OPG by human retinal microvascular endothelial cells (HRMEC) and investigated its angiogenic activity. Methods: Vitreous samples from 47 PDR and 28 nondiabetic patients, epiretinal membranes from 14 patients with PDR, human retinas (10 from diabetic patients and 10 from nondiabetic subjects), and rat retinas and HRMEC were studied by using enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, Western blot analysis, and RT-PCR. In vitro and in vivo angiogenesis assays were performed. Results: We showed a significant increase in the expression of OPG, VEGF, and MCP-1/CCL2 in a comparison between vitreous samples from PDR patients and those from nondiabetic controls. Significant positive correlations were found between levels of OPG and levels of VEGF and MCP-1/CCL2. In epiretinal membranes, OPG was expressed in vascular endothelial cells and stromal cells. Significant increases of OPG mRNA and protein were detected in the retinas from diabetic patients. The proinflammatory cytokines TNF-α and IL-1ß, but not VEGF, MCP-1/CCL2 or thrombin, induced upregulation of OPG in HRMEC. Osteoprotegerin induced ERK1/2 and Akt phosphorylation in HRMEC and stimulated their migration. Osteoprotegerin potentiated the angiogenic effect of VEGF in the in vivo protein gelatin plug assay. Conclusions: These results suggest that OPG is involved in PDR angiogenesis.

Elevated blood plasma levels of epinephrine, norepinephrine, tyrosine hydroxylase, TGFß1, and TNFα associated with high-altitude pulmonary edema in an Indian population.

Biomarkers are essential to unravel the locked pathophysiology of any disease. This study investigated the role of biomarkers and their interactions with each other and with the clinical parameters to study the physiology of high-altitude pulmonary edema (HAPE) in HAPE-patients (HAPE-p) against adapted highlanders (HLs) and healthy sojourners, HAPE-controls (HAPE-c). For this, seven circulatory biomarkers, namely, epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor beta 1, tumor necrosis factor alpha (TNFα), platelet-derived growth factor beta beta, and C-reactive protein (CRP), were measured in blood plasma of the three study groups. All the subjects were recruited at ~3,500 m, and clinical features such as arterial oxygen saturation (SaO2), body mass index, and mean arterial pressure were measured. Increased levels of epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor-beta 1, and TNFα were observed in HAPE-p against the healthy groups, HAPE-c, and HLs (P<0.0001). CRP levels were decreased in HAPE-p against HAPE-c and HLs (P<0.0001). There was no significant difference or very marginal difference in the levels of these biomarkers in HAPE-c and HLs (P>0.01). Correlation analysis revealed a negative correlation between epinephrine and norepinephrine (P=4.6E-06) in HAPE-p and positive correlation in HAPE-c (P=0.004) and HLs (P=9.78E-07). A positive correlation was observed between TNFα and CRP (P=0.004) in HAPE-p and a negative correlation in HAPE-c (P=4.6E-06). SaO2 correlated negatively with platelet-derived growth factor beta beta (HAPE-p; P=0.05), norepinephrine (P=0.01), and TNFα (P=0.005) and positively with CRP (HAPE-c; P=0.02) and norepinephrine (HLs; P=0.04). Body mass index correlated negatively with epinephrine (HAPE-p; P=0.001) and positively with norepinephrine and tyrosine hydroxylase in HAPE-c (P<0.05). Mean arterial pressure correlated positively with TNFα in HAPE-p and norepinephrine in HLs (P<0.05). Receiver operating characteristic curve analysis yielded a positive predictive value for these biomarkers with HAPE (area under the curve >0.70, P<0.05). The results clearly suggest that increased plasma levels of these circulatory biomarkers associated with HAPE.

Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

PURPOSE: Selective proteolytic activation of protease-activated receptor-1 (PAR1) by thrombin and matrix metalloproteinase-1 (MMP-1) plays a central role in enhancing angiogenesis. We investigated the expression levels of thrombin, MMP-1, and PAR1 and correlated these levels with vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of PAR1 and thrombin in the retinas of diabetic rats and PAR1 in human retinal microvascular endothelial cells (HRMEC) following exposure to high-glucose, the proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and the hypoxia mimetic agent cobalt chloride (CoCl2). METHODS: Vitreous samples from 32 PDR and 23 nondiabetic patients, epiretinal membranes from 10 patients with PDR, retinas of rats, and HRMEC were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and Western blot analysis. An assay for in vitro cell migration angiogenesis was performed in HRMEC. RESULTS: In epiretinal membranes, PAR1 was expressed in vascular endothelial cells, CD45-expressing leukocytes, and myofibroblasts. ELISA and Western blot assays revealed significant increases in the expression levels of thrombin, MMP-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between the levels of VEGF and the levels of thrombin (r = 0.41; p = 0.006) and MMP-1 (r = 0.66; p < 0.0001). Significant increases of cleaved PAR1 (approximately 50 kDa) and the proteolytically active thrombin (approximately 50 kDa) were detected in rat retinas after induction of diabetes. The proinflammatory cytokines IL-1ß and TNF-α, but not high-glucose and CoCl2, induced upregulation of cleaved PAR1 (approximately 30 kDa) in HRMEC. In addition, thrombin and MMP-1 induced VEGF in HRMEC and vorapaxar, a PAR1 inhibitor, inhibited thrombin-induced migration in HRMEC. CONCLUSIONS: Interactions among thrombin, MMP-1, PAR1, and VEGF might facilitate angiogenesis in PDR.

Mutual enhancement between high-mobility group box-1 and NADPH oxidase-derived reactive oxygen species mediates diabetes-induced upregulation of retinal apoptotic markers.

The expression of the proinflammatory cytokine high-mobility group box-1 (HMGB1) is upregulated in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and in the diabetic retina. We hypothesized that a novel mechanism exists where HMGB1 and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are mutually enhanced in the diabetic retina, which may be a novel mechanism for promoting upregulation of retinal apoptotic markers induced by diabetes. Vitreous samples from 48 PDR and 34 nondiabetic patients, retinas from 1-month diabetic rats and from normal rats intravitreally injected with HMGB1 and human retinal microvascular endothelial cells (HRMEC) stimulated with HMGB1 were studied by enzyme-linked immunosorbent and spectrophotometric assays, Western blot analysis, RT-PCR, and immunofluorescence. We also studied the effect of the HMGB1 inhibitor glycyrrhizin and apocynin on diabetes-induced biochemical changes in the retinas of rats (n = 5-7 in each groups). HMGB1 and the oxidative stress marker protein carbonyl content levels in the vitreous fluid from PDR patients were significantly higher than in controls (p = 0.021; p = 0.005, respectively). There was a significant positive correlation between vitreous fluid levels of HMGB1 and the levels of protein carbonyl content (r = 0.62, p = 0.001). HMGB1 enhanced interleukin-1ß, ROS, Nox2, poly (ADP-ribose) polymerase (PARP)-1, and cleaved caspase-3 production by HRMEC. Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of ROS, Nox2, PARP-1, and cleaved caspase-3 in the retina. Constant glycyrrhizin and apocynin intake from onset of diabetes did not affect the metabolic status of the diabetic rats, but restored these increased mediators to control values. The results of this study suggest that there is a mutual enhancement between HMGB1 and Nox-derived ROS in the diabetic retina, which may promote diabetes-induced upregulation of retinal apoptotic markers.

Tiam1-Rac1 Axis Promotes Activation of p38 MAP Kinase in the Development of Diabetic Retinopathy: Evidence for a Requisite Role for Protein Palmitoylation.

BACKGROUND/AIMS: Evidence in multiple tissues, including retina, suggests generation of reactive oxygen species (ROS) and the ensuing oxidative stress as triggers for mitochondrial defects and cell apoptosis. We recently reported novel roles for Tiam1-Rac1-Nox2 axis in retinal mitochondrial dysfunction and cell death leading to the development of diabetic retinopathy. Herein, we tested the hypothesis that activation of p38 MAP kinase, a stress kinase, represents the downstream signaling event to Rac1-Nox2 activation in diabetes-induced metabolic stress leading to capillary cell apoptosis. METHODS: Activation of p38 MAP kinase was quantified by Western blotting in retinal endothelial cells incubated with high glucose (20 mM) for up to 96 hours, a duration where mitochondrial dysfunction and capillary cell apoptosis can be observed. NSC23766 and 2-bromopalmitate (2-BP) were used to assess the roles of Tiam1-Rac1 and palmitoylation pathways, respectively. RESULTS: Activation of p38 MAP kinase was observed as early as 3 hours after high glucose exposure, and continued until 96 hours. Consistent with this, p38 MAP kinase activation was significantly higher in the retina from diabetic mice compared to age-matched normal mice. NSC23766 markedly attenuated hyperglycemia-induced activation of p38 MAP kinase. Lastly, 2-BP inhibited glucose-induced Rac1, Nox2 and p38 MAP kinase activation in endothelial cells. CONCLUSIONS: Tiam1-Rac1-mediated activation of Nox2 and p38 MAP kinase constitutes early signaling events leading to mitochondrial dysfunction and the development of diabetic retinopathy. Our findings also provide the first evidence to implicate novel roles for protein palmitoylation in this signaling cascade.

The Chemokine Platelet Factor-4 Variant (PF-4var)/CXCL4L1 Inhibits Diabetes-Induced Blood-Retinal Barrier Breakdown.

PURPOSE: To investigate the expression of platelet factor-4 variant (PF-4var/CXCL4L1) in epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) and the role of PF-4var/CXCL4L1 in the regulation of blood-retinal barrier (BRB) breakdown in diabetic rat retinas and human retinal microvascular endothelial cells (HRMEC). METHODS: Rats were treated intravitreally with PF-4var/CXCL4L1 or the anti-vascular endothelial growth factor (VEGF) agent bevacizumab on the first day after diabetes induction. Blood-retinal barrier breakdown was assessed in vivo with fluorescein isothiocyanate (FITC)-conjugated dextran and in vitro in HRMEC by transendothelial electrical resistance and FITC-conjugated dextran cell permeability assay. Occludin, vascular endothelial (VE)-cadherin, hypoxia-inducible factor (HIF)-1α, VEGF, tumor necrosis factor (TNF)-α, receptor for advanced glycation end products (RAGE), caspase-3 levels, and generation of reactive oxygen species (ROS) were assessed by Western blot, enzyme-linked immunosorbent assays, or spectrophotometry. RESULTS: In epiretinal membranes, vascular endothelial cells and stromal cells expressed PF-4var/CXCL4L1. In vitro, HRMEC produced PF-4var/CXCL4L1 after stimulation with a combination of interleukin (IL)-1ß and TNF-α, and PF-4var/CXCL4L1 inhibited VEGF-mediated hyperpermeability in HRMEC. In rats, PF-4var/CXCL4L1 was as potent as bevacizumab in attenuating diabetes-induced BRB breakdown. This effect was associated with upregulation of occludin and VE-cadherin and downregulation of HIF-1α, VEGF, TNF-α, RAGE, and caspase-3, whereas ROS generation was not altered. CONCLUSIONS: Our findings suggest that increasing the intraocular PF-4var/CXCL4L1 levels early after the onset of diabetes protects against diabetes-induced BRB breakdown.