Rat Retinal Endothelial Cells Research Articles

The Role of the Inflammatory Mediators TNF‐α and IFN‐γ in the Hyperglycemia‐induced PECAM‐1 Ubiquitination and Degradation in Diabetic Retinopathy

ObjectiveThe aim of this study was to investigate the role of TNF‐α and IFN‐γ in PECAM‐1 ubiquitination and subsequent degradation under hyperglycemic conditions in the diabetic retina.IntroductionInflammation is a major contributor to the development of diabetic retinopathy (DR). Two of the key mediators of inflammation, tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), have been shown to contribute to the pathophysiological changes leading to the progression of DR. However, their role in the hyperglycemia‐induced loss of retinal PECAM‐1 is not known. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has an important role in the maintenance of the endothelium and its functions. We have previously found a significant decrease in PECAM‐1 levels in the diabetic retina and cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions. Thus, an investigation into the possible mechanism/s of PECAM‐1 loss may help in understanding the molecular processes leading to DR, and provide a possible therapeutic target.Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with TNF‐α and/or IFN‐γ (20 ng/ml and 30 ng/ml, respectively) added 24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. Retinas and plasma were collected eight weeks post‐STZ injection for analysis. PECAM‐1, TNF‐α, IFN‐γ, and ubiquitin levels were assessed using western blotting, ELISA, Immunofluorescence, and immunoprecipitation assays.ResultsTotal ubiquitination levels were significantly increased in the diabetic retina and in RRMECs grown under HG conditions (35% and 40% increase, respectively, p<0.05). Moreover, PECAM‐1 ubiquitination levels were significantly increased in RRMECs grown under hyperglycemic conditions (3‐fold, p<0.05). TNF‐α levels were significantly increased in diabetic STZ plasma and RRMECs grown under HG conditions (31%, 74.4% increase, respectively, p<0.05). Additionally, IFN‐γ levels were significantly increased in the diabetic retina (35% increase, p<0.05). PECAM‐1 levels were significantly decreased in RRMECs when treated with TNF‐α and/or IFN‐γ (60–75% decrease, p<0.05), with a significant increase in total ubiquitination levels (20–28% increase, p<0.05).ConclusionThese results indicate a possible role for TNF‐α and IFN‐γ in the hyperglycemia‐induced loss of PECAM‐1 in retinal endothelial cells, possibly by upregulating PECAM‐1 ubiquitination and subsequent degradation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health (NIH) EY025632This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-204-5p promotes diabetic retinopathy development via downregulation of microtubule-associated protein 1 light chain 3.

Diabetic retinopathy (DR) is a chronic and progressive complication of diabetes mellitus. DR impairs sight due to neuronal and vascular dysfunction in the retina. It is critical to investigate the pathogenesis of DR to develop effective treatment. In the present study, a streptozotocin (STZ)-induced diabetic rat model was constructed and the expression of microRNA (miR)-204-5p and vascular endothelial growth factor (VEGF) were determined. Immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were employed to detect the effects of miR-204-5p on the expression of microtubule-associated protein 1 light chain 3 (LC3B). RT-qPCR analysis demonstrated that miR-204-5p and VEGF were significantly upregulated in the retina tissue of diabetic rats compared with the control group (P<0.01). Immunohistochemistry and western blotting revealed that the protein expression levels of LC3B-II and the ratio of LC3B-II/LC3B-I were significantly suppressed in the diabetes group compared with the control (P<0.01). In retinal tissues, anti-miR-204-5p treatment significantly enhanced the protein expression levels of LC3B-II and the ratio of LC3B-II/LC3B-I and these levels were significantly reduced in response to miR-204-5p mimic treatment compared with the negative miR control (P<0.01). In rat retinal endothelial cells isolated from diabetic rats, anti-miR-204-5p treatment increased the number of autophagic vacuoles, and significantly promoted LC3B-II expression and the LC3B-II/LC3B-I ratio compared with the negative control (P<0.01). The results of the present study revealed that miR-204-5p downregulated the expression of LC3B-II to inhibit autophagy in DR. Therefore, miR-204-5p may be considered as a novel effective therapeutic target during the development of DR.

Loss of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) in the Diabetic Retina: Role of Matrix Metalloproteinases.

PurposeTo test the hypothesis that high glucose and matrix metalloproteinases (MMPs) contribute to the diabetes-induced loss of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the retinal microvasculature.MethodsPECAM-1 and MMP protein, activity, and interactions with PECAM-1 were assessed using western blotting, zymography, immunofluorescence, or coimmunoprecipitation assays. These assays were conducted using primary rat retinal microvascular endothelial cells (RRMECs) grown either in normal glucose (5 mM) or high glucose (25 mM) conditions and using retinas collected from streptozotocin-induced diabetic or control rats. The broad-spectrum MMP inhibitor GM6001 was administered in vivo and in vitro to ascertain the role of MMPs in the hyperglycemia-induced loss of PECAM-1.ResultsA dramatic decrease in PECAM-1 (western blotting, immunofluorescence) was observed in both the diabetic retina and in hyperglycemic RRMECs. The decrease in PECAM-1 was accompanied by a significant increase in the presence and activity of matrix metalloproteinase-2 (MMP-2) (but not matrix metalloproteinase-9 [MMP-9]) in the diabetic plasma (P < 0.05) and in hyperglycemic RRMECs (P < 0.05). Moreover, RRMEC PECAM-1 significantly decreased when treated with plasma collected from diabetic rats. Several MMP-2 cleavage sites on PECAM-1 were identified using in silico analysis. Moreover, PECAM-1/MMP-2 interactions were confirmed using coimmunoprecipitation. PECAM-1 was significantly decreased in RRMECs treated with MMP-2 (P < 0.05), but became comparable to controls with the MMP inhibitor GM6001 in both the diabetic retina and hyperglycemic RRMECs.ConclusionsThese results indicate a possible role of MMP-2 in hyperglycemia-induced PECAM-1 loss in retinal endothelial cells.

Resveratrol exhibits an effect on attenuating retina inflammatory condition and damage of diabetic retinopathy via PON1

Diabetic retinopathy (DR), an obstacle of the visual microvascular system, is a serious complication of diabetic patients. Paraoxonase 1 (PON1) has been extensively evaluated as a genetic candidate for diabetic microvascular complications, and PON1 is associated with DR. In this study, the biological functions of PON1 and its related proteins were determined via gene ontology (GO) enrichment analysis; we demonstrated that treatment with resveratrol alleviated retinal inflammatory activities to evaluate its protective effects on streptozotocin (STZ)-induced diabetic rats and high-glucose (HG) stimulated rat retinal endothelial cells (RRECs). The GO enrichment analysis suggested that PON1 may regulate inflammatory responses and microvascular complications in DR. In an in vivo study, resveratrol significantly recovered the insulin level and PON1 expression and activity, as well as clearly reduced the retinal vascular permeability, retinal AGEs, LDL, Ox-LDL, caspase3 activity, retinal damage, IL-1β, IL-6, TNFα, VEGF, IFNγ and MCP-1 in STZ-diabetic rats. Moreover, resveratrol reduced the caspase3 activity and Ox-LDL expression in HG stimulated RRECs. However, its protective effect was a deficiency in PON1-silenced RRECs. PON1 is a pivotal modulator in the role of resveratrol in reversing the RREC damage induced by HG. Furthermore, we found that resveratrol exhibits an effect on attenuating the retinal inflammatory condition and damage of DR via PON1. Our study suggests that resveratrol-induced PON1 in the retina may be a promising therapeutic strategy to prevent diabetes-related retinopathy.

Inhibition of Cx43 gap junction uncoupling prevents high glucose-induced apoptosis and reduces excess cell monolayer permeability in retinal vascular endothelial cells

The aim of this study was to investigate whether inhibition of connexin 43 gap junction-uncoupling is sufficient to prevent retinal vascular cell loss under high glucose condition and reduce cell monolayer permeability. Rat retinal endothelial cells were grown for 3, 5, and 7 days in normal (5 mM) or high glucose (30 mM) medium; in parallel, cells grown in high glucose medium were exposed for 3, 5, and 7 days to 100 nM danegaptide, which stabilizes connexin 43-mediated cell coupling. Additionally, cells grown in normal medium were treated with a connexin 43 blocker as a negative control. To determine gap junction intercellular communication, scrape load dye transfer assay was performed at the three time points. Cells were assessed for apoptosis and cell monolayer permeability by differential dye staining and in vitro permeability assays, respectively. Cells treated with danegaptide preserved gap junction intercellular communication, decreased cell death, and reduced cell monolayer permeability. Scrape load dye transfer assay indicated that cells exposed to danegaptide for 3, 5, and 7 days under high glucose condition maintained gap junction intercellular communication. Importantly, danegaptide significantly prevented high glucose-induced apoptosis at all three time points, and inhibited cell monolayer permeability by day 5. Cells exposed to a connexin 43 blocker, which decreased cell coupling, showed excess apoptosis and cell monolayer permeability. These findings suggest that prevention of high glucose-induced compromised cell-cell coupling may be a useful strategy for inhibiting apoptosis and excess vascular permeability associated with diabetic retinopathy.

The Role of Matrix Metalloproteinase 2 (MMP2) In Diabetes‐induced Loss of PECAM‐1 in The Retina: Direct and Indirect Mechanisms

ObjectiveMatrix metalloproteinases (MMPs) are calcium‐dependent, zinc‐containing endopeptidases that are responsible for degrading extracellular matrix proteins, and for cleavage of various substrates such as cell surface receptors, proteoglycans, chemokines and cytokines. The aim of this study was to investigate the molecular mechanisms by which MMP2 may lead to PECAM‐1 loss in the diabetic retina, either directly through cleavage, or indirectly by modulating tumor necrosis factor‐α (TNF‐α) levels and activity.BackgroundPlatelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has major roles in barrier function, endothelial cell‐endothelial cell adhesion, and cell signaling. We have previously found a significant decrease (50–90%, P&lt;0.05) in PECAM‐1 levels in the diabetic retina, and in cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions (30–40%, P&lt;0.05). The decrease in PECAM‐1 levels was accompanied by a significant increase in MMP2 levels and activity in the diabetic plasma (72% and 78.5% respectively, P&lt;0.05), and in RRMECs grown under high glucose conditions (12‐fold increase, P&lt;0.05).Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with treatments added 6–24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. GM6001, a broad spectrum MMP inhibitor, was started at week six post‐STZ injection for two weeks (4 mM, 25 μl/eye, in sterile eye drops), and retinas were collected eight weeks post‐STZ injection for analysis. PECAM‐1, MMP2, and TNF‐α levels and interactions were assessed using western blotting, zymography, immunofluorescence, immunoprecipitation, or ELISA assays. In silico analysis for possible protease cleavage sites was done using PROSPER, provided by Monash University, Australia.ResultsSeveral PECAM‐1 cleavage sites by MMP2 were identified using in silico analysis. Moreover, we confirmed PECAM‐1/MMP2 interactions using immunoprecipitation. PECAM‐1 levels were significantly decreased in RRMECs treated with MMP2 (81%, P&lt;0.05), with a significant increase in TNF‐α levels (118.2%, P&lt;0.05). Moreover, RRMECs treated with TNF‐α had a significant decrease in PECAM‐1 levels (34%, P&lt;0.05). Furthermore, TNF‐α levels were significantly increased in diabetic STZ plasma, and in RRMECs grown under HG conditions (31%, 74.4% respectively, P&lt;0.05). To investigate the protective effect of MMP2 inhibition on PECAM‐1 levels, we treated diabetic rats with GM6001 eye drops, and saw an increase of PECAM‐1 levels (77.2%, P&lt;0.05) when compared to untreated rats.ConclusionThese results indicate a possible role of MMP2 in the diabetes‐induced loss of PECAM‐1 in the retina, which can be via direct cleavage of PECAM‐1, or indirectly via upregulation of TNF‐α, with the two mechanisms evaluated in future studies.Support or Funding InformationNIH EY025632, and AHA Predoctoral Fellowship 16PRE30080003This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Matrix Metalloproteinases (MMPs) in Platelet Endothelial Cell Adhesion Molecule‐1 (PECAM‐1) Loss in Diabetic Retinopathy: In Vivo and in Vitro Studies

OBJECTIVEThe aim of this study is to understand the mechanisms involved in the diabetes‐induced loss of PECAM‐1 in the retina, and whether it can be attributed to PECAM‐1 cleavage by matrix metalloproteinases (MMPs).INTRODUCTIONPlatelet endothelial cell adhesion molecule‐1 (PECAM‐1) is expressed on the surface of endothelial cells of the vasculature, and has major roles in inflammation, permeability, and angiogenesis. In addition, PECAM‐1 functions in cell signaling, cell‐cell adhesion, and cell survival. However, its physiological role in the pathophysiology of diabetic retinopathy (DR), the leading cause of blindness among working age adults worldwide, has not yet been investigated. We have previously found a significant decrease (50–90%) in retinal PECAM‐1 levels in a diabetic model of type I diabetes in rats.DESIGN AND METHODSFor our animal studies, we injected age‐matched male Wistar rats with either vehicle (control) or streptozotocin (STZ, diabetic), with the latter at 30 mg/kg/day for three consecutive days to elicit a model of type I diabetes. For our in vitro studies, primary rat retinal microvascular endothelial cells (RRMECs) were grown in either normal glucose (NG, 5mM) or high glucose (HG, 25 mM) media, and treated for 6 days with 0 or 10% plasma collected from control or diabetic rats 8 weeks post‐injection. PECAM‐1, and MMP levels or activity, were assessed using immunofluorescence staining, western blotting, or zymography.RESULTSFor RRMECs grown in NG conditions, treatment with 10% diabetic plasma resulted in significantly lower levels of PECAM‐1 than treatment with 10% control plasma (27%, p&lt;0.05). PECAM‐1 loss was further exaggerated when cells were grown under HG conditions and treated with 10% diabetic plasma (74%, p&lt;0.01). In contrast, control plasma added to RRMECs in NG conditions increased the expression of PECAM‐1 in a dose‐dependent manner. These findings indicate that normal plasma contributes to the expression of PECAM‐1, but some factor(s) in diabetic plasma has the reverse effect. MMPs, which are calcium‐dependent zinc‐containing endopeptidases, are known to cleave PECAM‐1. Plasma samples from diabetic rats revealed a significant increase in MMP‐2 levels (2‐fold, p&lt;0.05), and a significant increase in MMP‐7 activity (p&lt;0.01). Moreover, RRMECs showed a significant increase in MMP2 levels (12‐fold, p&lt;0.01) when grown in HG media.CONCLUSIONThese results indicate a possible role for MMPs in the diabetes‐induced loss of PECAM‐1. Further studies will be conducted to investigate the potential benefits of protecting the retinal expression of PECAM‐1 by targeting the actions of MMPs.Support or Funding InformationNIH RO1 EY017599

NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.

To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.

Beneficial effects of fenofibric acid on overexpression of extracellular matrix components, COX-2, and impairment of endothelial permeability associated with diabetic retinopathy

In the Fenofibric Acid (FA) Intervention and Event Lowering in Diabetes (FIELD) study, FA, a lipid-lowering drug, has been shown to significantly reduce macular edema in diabetic patients. In the present study, we investigated whether FA reduces vascular permeability by inhibiting cyclooxygenase-2 (COX-2), a critical mediator of inflammation, and reducing overexpression of fibronectin (FN) and collagen IV (Coll IV), two basement membrane (BM) components upregulated in diabetic retinopathy. Rat retinal endothelial cells (RRECs) were grown in normal (N:5 mM glucose) or high glucose (HG:30 mM glucose) medium with or without FA for 7 days. Total protein isolated from these cells was assessed for FN, Coll IV, COX-2, and zonula occludens-1 (ZO-1), a tight junction protein, using Western blot analysis. In addition, the distribution and localization of ZO-1 was determined by immunofluorescence microscopy, and cell monolayer permeability was studied by in vitro permeability (IVP) assay. RRECs grown in HG medium showed significant increase in FN, Coll IV, and COX-2 expression (179%, 144%, 139% of N respectively), and a decrease in ZO-1 expression (48% of N) compared to those of N cells. Cells grown in HG medium supplemented with FA significantly reduced FN, Coll IV, and COX-2 expression by 47%, 32%, and 34% respectively, with concomitant increase in ZO-1 expression by 42%. In parallel studies, IVP assays showed a significant increase (139% of N) in cell monolayer permeability in RRECs grown in HG medium, which was significantly reduced with FA treatment. Additionally, immunostaining results indicated FA prevents HG-induced downregulation of ZO-1. The findings indicate that the beneficial effect of FA in reducing excess permeability is mediated, at least in part, by downregulating abnormal overexpression of BM components and inflammatory factors and preventing compromised tight junctions associated with diabetic retinopathy.

Ras-related C3 botulinum toxin substrate 1 activation is involved in the pathogenesis of diabetic retinopathy.

This study used a streptozotocin (STZ)-induced rat model of diabetes to investigate whether Ras-related C3 botulinum toxin substrate 1 (Rac1) was involved in the pathogenesis of diabetic retinopathy. The effects of Rac1 inhibition on vascular endothelial (VE)-cadherin and β-catenin expression in high glucose-induced rat retinal endothelial cells (RRECs) were additionally examined. Rac1 activation in the retinas from STZ-induced diabetic rats and in high glucose-induced RRECs was measured by reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemistry and western blot analysis. The expression levels of VE-cadherin and β-catenin were also examined with or without Rac1 inhibition through small interfering (si)RNA transfection. STZ-induced diabetes was associated with an increase in the vascular permeability of the retina. Furthermore, Rac1 activation was increased in the retina of STZ-induced diabetic rats and in high glucose-induced RRECs compared with that in the controls. Immunohistochemistry showed that immunostaining of Rac1 was localized in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers and in the retinal microvasculature of rats. The expression of β-catenin was increased in the retinas of the diabetic rats at four, eight and 12 weeks after the induction of diabetes compared with that in the controls. Additionally, Rac1 activation was required for the high glucose-induced VE-cadherin expression decrease and for β-catenin expression in high glucose-induced RRECs. Rac1 inhibition by Rac1-siRNA transfection effectively prevented hyperpermeability, β-catenin expression and the VE-cadherin expression decrease in high glucose-induced RRECs. In conclusion, diabetes affects the expression of Rac1 in the retina. Rac1 may be involved in the diabetes-induced damage and/or alterations to the blood-retinal barrier through changes in VE-cadherin and β-catenin expression.

Changes in the daily rhythm of lipid metabolism in the diabetic retina.

Disruption of circadian regulation was recently shown to cause diabetes and metabolic disease. We have previously demonstrated that retinal lipid metabolism contributed to the development of diabetic retinopathy. The goal of this study was to determine the effect of diabetes on circadian regulation of clock genes and lipid metabolism genes in the retina and retinal endothelial cells (REC). Diabetes had a pronounced inhibitory effect on the negative clock arm with lower amplitude of the period (per) 1 in the retina; lower amplitude and a phase shift of per2 in the liver; and a loss of cryptochrome (cry) 2 rhythmic pattern in suprachiasmatic nucleus (SCN). The positive clock arm was increased by diabetes with higher amplitude of circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 (bmal1) and phase shift in bmal1 rhythmic oscillations in the retina; and higher bmal1 amplitude in the SCN. Peroxisome proliferator-activated receptor (PPAR) α exhibited rhythmic oscillation in retina and liver; PPARγ had lower amplitude in diabetic liver; sterol regulatory element-binding protein (srebp) 1c had higher amplitude in the retina but lower in the liver in STZ- induced diabetic animals. Both of Elongase (Elovl) 2 and Elovl4 had a rhythmic oscillation pattern in the control retina. Diabetic retinas lost Elovl4 rhythmic oscillation and had lower amplitude of Elovl2 oscillations. In line with the in vivo data, circadian expression levels of CLOCK, bmal1 and srebp1c had higher amplitude in rat REC (rREC) isolated from diabetic rats compared with control rats, while PPARγ and Elovl2 had lower amplitude in diabetic rREC. In conclusion, diabetes causes dysregulation of circadian expression of clock genes and the genes controlling lipid metabolism in the retina with potential implications for the development of diabetic retinopathy.

Polyol effects on growth factors and MAPK signaling in rat retinal capillary cells.

Recent studies report that growth factor and signaling changes in rat lenses do not directly result from the presence of diabetes or sorbitol/galactitol (polyol) formation/accumulation, but from secondary osmotic changes associated with the aldose reductase (AR) catalyzed polyol formation. AR is also present in rat retinal pericyte and endothelial cells; however, significant polyol formation only occurs in pericytes and this does not appear to be linked to osmotic changes. The purpose of this study was to determine whether polyol formation and AR activity are similarly linked to growth factor and signaling changes in the rat capillary cells despite the apparent absence of osmotic stress. Conditionally immortalized rat retinal pericyte (TR-rPCT) and endothelial (TR-iBRB) cell lines were cultured on collagen type 1-coated dishes in the DMEM containing 5.5 mM glucose. After 24 h of initial culture, the medium was replaced with a serum-free medium containing 5.5, 25, or 50 mM glucose or galactose with/without the aldose reductase inhibitors (ARIs) AL1576 or tolrestat for periods of up to 48 h. Growth factors and transduction pathways were measured by Western blots using the antibodies against basic FGF, IGF-1, TGF-β, P-ERK1/2, P-SAPK/JNK, and P-Akt. Sorbitol accumulation was only observed in pericytes, while galactitol was present in both pericytes and endothelial cells. Pericytes cultured in high glucose showed increased expression of the growth factors basic FGF, IGF-1, TGF-β, and signaling in P-Akt, P-ERK1/2, and P-SAPK/JNK compared with those cultured in 5.5 mM glucose and these expressions were normalized by the presence of ARIs. Similar results were observed with galactose media. In contrast, endothelial cells cultured in high glucose media showed neither growth factor or signaling changes. In galactose media, endothelial cells showed increased expression of basic FGF, IGF-1, TGF-β, P-ERK1/2, and P-SAPK/JNK, which were only partially reduced by ARIs. Growth factor and MAPK signaling expression in pericytes are linked to the presence of polyols. Pericytes, which readily accumulate sorbitol/galactitol that is inhibited by ARIs, show expression changes similar to those observed in rat lenses. In contrast, endothelial cells only show partial expression changes that are linked to galactitol accumulation.

High Glucose–Induced Downregulation of Connexin 30.2 Promotes Retinal Vascular Lesions: Implications for Diabetic Retinopathy

To investigate whether high glucose (HG) alters expression of connexin 30.2 (Cx30.2) and influences gap junction intercellular communication (GJIC) in retinal endothelial cells and promotes vascular lesions characteristic of diabetic retinopathy (DR). Western blot analysis and immunostaining were performed to determine Cx30.2 protein expression and localization in rat retinal endothelial cells (RRECs) grown in normal (N; 5 mM) or HG (30 mM) medium for 7 days. Concurrently, GJIC was assessed in cells grown in N or HG medium and in cells transfected with Cx30.2 siRNA. Similarly, retinal Cx30.2 expression was assessed in nondiabetic and diabetic rats. Additionally, the effect of reduced Cx30.2 on development of acellular capillaries (ACs) and pericyte loss (PL) was studied in retinas of Cx30.2 knockout mice. Cx30.2 was identified in RRECs in vitro and in vascular cells of retinal capillaries. RRECs grown in HG exhibited significantly reduced Cx30.2 protein levels consistent with decreased Cx30.2 immunostaining compared with those grown in N medium. Cells grown in HG and cells transfected with Cx30.2 siRNA exhibited significantly diminished dye transfer compared with N or nontransfected cells. Importantly, Cx30.2 protein level and immunostaining were decreased in diabetic retinas compared with nondiabetic retinas. Retinal capillaries of Cx30.2 knockout mice exhibited increased numbers of ACs and PL compared with those of wild-type mice. These results indicate that HG- or diabetes-induced downregulation of Cx30.2 expression and decrease in GJIC activity play a critical role in the development of retinal vascular lesions in early DR.

Downregulation of Mitochondrial Connexin 43 by High Glucose Triggers Mitochondrial Shape Change and Cytochrome c Release in Retinal Endothelial Cells

To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression and whether altered mtCx43 channel activity is involved in promoting apoptosis in retinal endothelial cells. MtCx43 localization was determined using immunostaining, green fluorescent protein (GFP)-tagged Cx43 followed by confocal imaging, and Western blot analysis using protein isolated from mitochondria of rat retinal endothelial cells (RRECs). To assess HG effects on mtCx43 expression, RRECs were grown in normal (5 mM) or HG (30 mM) medium for 7 days, and mtCx43 protein level assessed by Western blot analysis. To determine if mtCx43 channel inhibition affected mitochondrial morphology, RRECs grown sparsely were left untreated or treated with β-glycerrhetinic acid (β-GA), an inhibitor of connexin channels, and imaged using confocal microscopy. Additionally, mitochondria isolated from RRECs were treated with β-GA, and cytochrome c release assessed by Western blot. Cx43 localization on the mitochondria of RRECs was confirmed with immunofluorescence staining using Cx43 antibody and GFP-tagged Cx43 imaged in live cells. Western blot analysis indicated that Cx43 was located primarily on the inner mitochondrial membrane, and mtCx43 protein level was significantly reduced in RRECs grown in HG condition. Treatment of RRECs with β-GA significantly decreased mtCx43 phosphorylation, induced mitochondrial fragmentation, and isolated mitochondria treated with β-GA showed increased cytochrome c release. HG-induced downregulation of mtCx43 protein resulting in decreased channel activity may promote mitochondrial morphology changes and cytochrome c release, suggesting a novel mechanism for hyperglycemia-induced apoptosis in diabetic retinopathy.

Application of Membrane Permeability Evaluated in In Vitro Analyses to Estimate Blood–Retinal Barrier Permeability

The relationship between the in vitro membrane permeability and systemic blood-retinal barrier (BRB) permeability of drugs was investigated. To determine membrane permeability trend lines in this relationship, the apparent permeability (P(app)) and initial uptake rate (V) of 23 compounds were evaluated in a parallel artificial membrane permeability assay and the uptake study with a rat retinal endothelial cell line (TR-iBRB2 cells) for comparison with their retinal uptake index (RUI). The RUI values of compounds undergoing passive diffusion across the BRB were correlated with a log of the P(app) [RUI = 7.93 × 10 × exp (0.994 × log P(app)), r(2) = 0.660] and a log of the V [RUI = 26.5 × exp (1.55 × log V), r(2) = 0.581]. The RUI values of compounds undergoing carrier-mediated transport across the BRB were correlated with a log of the V [RUI = 26.5 × exp (0.887 × log V), r(2) = 0.559]. These results showed that the membrane permeability trend lines derived from the RUI and V values reflect the transport of drugs at the BRB, suggesting that an in vitro analysis-based estimation of the BRB permeability can be obtained using TR-iBRB2 cells and membrane permeability trend lines.

High Glucose-induced Altered Basement Membrane Composition and Structure Increases Trans-endothelial Permeability: Implications for Diabetic Retinopathy

Purpose: The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) and affect vascular permeability in diabetic retinopathy.Materials and Methods: To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days to confluency in normal (N, 5 mM) or high glucose (HG, 30 mM) medium on transwell inserts and subjected to an in vitro cell monolayer permeability assay. Inserts were cut out and viewed under a transmission electron microscope to assess extracellular matrix (ECM) accumulation and cell morphology. In parallel cell cultures, fibronectin and collagen IV protein expression were determined using Western Blot analysis.Results: Electron microscopic analysis of cells exposed to short-term HG showed no difference in inter-endothelial cell tight junctions (TJs) or in the number of vesicles or coated pits compared to those of normal cells. However, ECM accumulation underlying HG cells was significantly increased compared to that of cells grown in N medium (139 ± 7% of control, p = 0.04), with areas of focal thickening. Western blot analysis showed increased fibronectin and collagen IV expression (152 ± 24% of control, p = 0.01; 146 ± 16% of control, p = 0.02, respectively) in cells grown in HG compared to those grown in N medium. Cell monolayers grown in HG exhibited increased permeability to FITC-dextran compared to cells grown in N medium (134 ± 15% of control, p = 0.02).Conclusions: High glucose-induced excess ECM accumulation and altered composition underlies structural and functional changes that allow increased permeability. This finding provides evidence for the first time that the thickened vascular basement membrane contributes to the development of excess permeability seen in diabetic retinopathy.

Response of Rat Retinal Capillary Pericytes and Endothelial Cells to Glucose

The purpose of this study was to investigate the effects of hyperglycemia, its fluctuations, and glucose starvation on the expression of glucose-regulated protein 78/binding immunoglobulin protein (GRP78/BiP), one of the most commonly used markers of endoplasmic reticulum stress, in rat capillary pericytes and endothelial cells cultured separately and together. Conditionally immortalized rat retinal pericyte and endothelial cell lines were cultured in dishes coated with collagen type I in Dulbecco's modified Eagle's medium containing 5.5 mM glucose. For cocultures, pericytes and endothelial cells were seeded together on rat tail collagen type I-coated cell culture plates. After 24 h of initial culture, the medium was replaced with serum-free medium containing 0-100 mM glucose for periods of up to 72 h. GRP78/BiP, caspase-3, and nuclear factor-κB expression were investigated using western blots. No significant increase in GRP78/BiP expression was observed when pericytes, endothelial cells, or cocultures were exposed to either 25, 50, or 100 mM glucose for 48 h compared with the control level of 5.5 mM glucose. Similarly, no change in expression of GRP78/BiP was observed when media glucose levels were reduced from either 5.5 or 25 to 1 mM. GRP78/BiP expression significantly increased when cells were cultured for 24 h in glucose-deprived medium. This was accompanied by a time-dependent increase in the expression of caspase-3 and nuclear factor-κB. In diabetic retinopathy, hyperglycemia has been reported to induce apoptosis in retinal capillary vascular cells, but these studies suggest that the apoptosis is not linked to the expression of GRP78/BiP, one of the most commonly used markers of endoplasmic reticulum stress. However, GRP78/BiP-linked apoptosis may play a role in vascular changes associated with retinal ischemia/reperfusion.

Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.

High Glucose Disrupts Mitochondrial Morphology in Retinal Endothelial Cells: Implications for Diabetic Retinopathy

Mitochondrial dysfunction has been implicated in diabetic complications; however, it is unknown whether hyperglycemia affects mitochondrial morphology and metabolic capacity during development of diabetic retinopathy. We investigated high glucose (HG) effects on mitochondrial morphology, membrane potential heterogeneity, cellular oxygen consumption, extracellular acidification, cytochrome c release, and apoptosis in retinal endothelial cells. Rat retinal endothelial cells grown in normal (5 mmol/L) or HG (30 mmol/L) medium and double-stained with MitoTracker Green and tetramethylrhodamine-ethyl-ester-perchlorate were examined live with confocal microscopy. Images were analyzed for mitochondrial shape change using Form Factor and Aspect Ratio values, and membrane potential heterogeneity, using deviation of fluorescence intensity values. Rat retinal endothelial cells grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification using an XF-24 flux analyzer, cytochrome c release by Western blot, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Rat retinal endothelial cells grown in HG medium exhibited increased mitochondrial fragmentation concurrent with membrane potential heterogeneity. Metabolic analysis showed increased extracellular acidification in HG with reduced steady state/maximal oxygen consumption. Cytochrome c and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were also increased in HG. Thus, HG-induced mitochondrial fragmentation with concomitant increase in membrane potential heterogeneity, reduced oxygen consumption, and cytochrome c release may underlie apoptosis of retinal endothelial cells as seen in diabetic retinopathy.

Involvement of system A in the retina-to-blood transport of l-proline across the inner blood–retinal barrier

The purpose of the present study was to elucidate the mechanisms of retina-to-blood transport of l-proline across the blood–retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The vitreous humor/retina-to-blood transport of [ 3H] l-proline across the BRB was evaluated by microdialysis. Transport mechanisms of [ 3H] l-proline were investigated by cellular uptake using an in vitro model of the inner BRB (TR-iBRB2 cells). The mRNA level of system A was determined by quantitative real-time PCR analysis with specific primers. [ 3H] l-Proline and [ 14C] d-mannitol, which is a bulk flow marker, were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [ 3H] l-proline during the terminal phase was 1.6-fold greater than that of [ 14C] d-mannitol. The terminal elimination rate constant difference between [ 3H] l-proline and [ 14C] d-mannitol was reduced in the retinal presence of 3 mM l-proline and 5 mM α-methylaminoisobutyric acid, suggesting that l-proline is transported via a carrier-mediated retina-to-blood transport process across the BRB. [ 3H] l-Proline uptake by TR-iBRB2 cells appeared to be mediated through a saturable and Na +-dependent process. The corresponding Michaelis–Menten constant was 392 μM. This process was reduced by substrates for system A, suggesting that system A is involved in l-proline uptake. Of the isoforms of system A, ATA1, ATA2, and ATA3, ATA2 mRNA is predominantly expressed in TR-iBRB2 cells and isolated rat retinal endothelial cells. In conclusion, system A, most likely ATA2, is responsible for the retina-to-blood transport of l-proline across the inner BRB and may play a role in maintaining the concentration of small neutral amino acids in the retina.