Barrier In Diabetic Retinopathy Research Articles

O-GlcNAcylation Inhibition Upregulates Connexin43 Expression in the Endothelium to Protect the Tight Junction Barrier in Diabetic Retinopathy.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on connexin43 (Cx43) expression and its subsequent effects on tight junction properties in diabetic retinopathy (DR). O-GlcNAcylation levels in primary human retinal vascular endothelial cells (HRVECs) and retinas from rats with diabetes were regulated by treatment with Thiamet G or alloxan. Immunoprecipitation was used to examine the relationship between O-GlcNAcylation and Cx43 expression. Stable overexpression and knockdown of Cx43 in HRVECs were achieved using lentivirus constructs; further, their effects on occludin and zonula occluden-1 (ZO-1) expression and tight junction barrier function were determined. O-GlcNAcylation level increased significantly, whereas Cx43 expression decreased in retinas from rats with diabetes and HRVECs cultured under high-glucose conditions. Immunoprecipitation revealed that Cx43 was modified by O-GlcNAcylation and phosphorylation simultaneously. O-GlcNAcylation inhibition negatively regulated both total Cx43 and phosphorylated Cx43 expression, subsequently disrupting tight junction properties. Conversely, Cx43 overexpression reversed the disruption of tight junction properties and downregulated vascular endothelial growth factor expression. Consistently, Cx43 overexpression increased transendothelial electrical resistance values in HRVEC layers. O-GlcNAcylation negatively regulated Cx43 expression, contributing to the disruption of the blood retinal barrier. However, O-GlcNAcylation inhibition and Cx43 overexpression could reverse the tight junction disruption. Therefore, O-GlcNAcylation inhibition is a potential target for avoiding tight junction disruption through the Cx43 pathway in DR.

Specialized Retinal Endothelial Cells Modulate Blood-Retina Barrier in Diabetic Retinopathy.

Endothelial cells (EC) play essential roles in retinal vascular homeostasis. This study aimed to characterize retinal EC heterogeneity and functional diversity using single-cell RNA sequencing. Systematic analysis of cellular compositions and cell-cell interaction networks identified a unique EC cluster with high inflammatory gene expression in diabetic retina; sphingolipid metabolism is a prominent aspect correlated with changes in retinal function. Among sphingolipid-related genes, alkaline ceramidase 2 (ACER2) showed the most significant increase. Plasma samples of patients with non-proliferative diabetic retinopathy (NPDR) with or without diabetic macular edema (DME) and active PDR were collected for mass spectrometry analysis. Metabolomic profiling revealed that the ceramide levels were significantly elevated in NPDR-NDME/DME and further increased in active PDR compared with control patients. In vitro analyses showed that ACER2 overexpression retarded endothelial barrier breakdown induced by ceramide, while silencing of ACER2 further disrupted the injury. Moreover, intravitreal injection of the recombinant ACER2 adeno-associated virus rescued diabetes-induced vessel leakiness, inflammatory response, and neurovascular disease in diabetic mouse models. Together, this study revealed a new diabetes-specific retinal EC population and a negative feedback regulation pathway that reduces ceramide content and endothelial dysfunction by upregulating ACER2 expression. These findings provide insights into cell-type targeted interventions for diabetic retinopathy.

Transcriptomic Profiling Reveals Chemokine CXCL1 as a Mediator for Neutrophil Recruitment Associated With Blood-Retinal Barrier Alteration in Diabetic Retinopathy.

Intravitreal CXCL1 injection and diabetes result in increased retinal vascular permeability with neutrophil and monocyte recruitment. Ly6G antibody treatment for neutrophil depletion in both animal models showed decreased retinal permeability and decreased cytokine expression. CXCL1 is produced by retinal endothelial cells, pericytes, and astrocytes. CXCL1 level is significantly increased in serum samples of patients with diabetic retinopathy. CXCL1, through neutrophil recruitment, alters the blood-retinal barrier in diabetic retinopathy and, thus, may be used as a therapeutic target.

Silencing Nogo-B improves the integrity of blood-retinal barrier in diabetic retinopathy via regulating Src, PI3K/Akt and ERK pathways

ObjectiveTo examine the mechanisms of Nogo-B (RTN4B) in the protection of blood-retinal barrier in experimental diabetic retinopathy. MethodsThe level of Nogo-B in vitreous and plasma samples was detected with ELISA. Diabetes was induced in Sprague-Dawley rats with intraperitoneal injection of streptozotocin. The rats were injected intravitreally with adeno-associated virus (AAV) for knockdown the expression of Nogo-B in retina or/and as AAV negative control. The permeability of blood-retinal barrier was detected with Rhodamine-B-dextran leakage assay. The expressions of Nogo-B, junctional proteins, inflammatory factors and signaling pathways were examined with Western blot and quantitative real-time PCR. ResultsNogo-B expression was significantly upregulated in clinical samples and experimental diabetic rat models. Under normal condition, Nogo-B knockdown resulted in the increased permeability of retinal blood vessels. In diabetic rat retinas, the vascular leakage was increased significantly, which was partially decreased by Nogo-B knockdown through increasing p/t-Src (Tyr529) and p/t-Akt (Ser473), and decreasing p/t-ERK1/2. ConclusionNogo-B was increased in diabetic retinopathy and silencing Nogo-B is a promising therapy for diabetic retinopathy.

Destruction of the blood-retina barrier in diabetic retinopathy depends on angiotensin-converting enzyme-mediated TGF-β1/Smad signaling pathway activation

Diabetes mellitus (DM) is a systemic, chronic metabolic disease that is related to heredity and autoimmunity and is often accompanied by complications of retinopathy. However, the causative mechanism involved in the pathological process remains unclear. In this research, treatment with fosinopril or LY2109761 was found to be responsible for the improvement of the pathological processes, serum biochemical indexes and retinopathy in rats with streptozotocin-induced diabetes. In addition, the upregulation of angiotensin-converting enzyme (ACE) in the serum and the increased expression of TGF-β1 in the pathological outer nuclear layer (ONL) and inner nuclear layer (INL) of the retina were also reduced. In vitro experiments demonstrated that ACE enhanced cell damage and TGF-β1/Smad signaling pathway activation in retinal capillary endothelial cells (RCECs) under high glucose conditions. In addition, the activity of ACE was also considered to be related to the increasing levels of activated TGF-β1 in both rat retinal Müller cells (RMCs) and RCECs, but ACE activity had no effect on the high glucose-mediated upregulation of total TGF-β1 in RMCs. Coculture experiments with RCECs and RMCs showed that the barrier that was established under normal conditions was significantly impaired when exposed to high glucose combined with ACE, and damage of barrier can be prevented by adding fosinopril or LY2109761. Finally, a similar auxiliary effect of ACE was also observed in the activated TGF-β1-mediated barrier damage in blood-retinal barrier model in vitro. In summary, ACE-mediated TGF-β1/Smad signaling pathway activation was found to be involved in the destruction of the blood-retina barrier during diabetic retinopathy in a model of streptozotocin-induced diabetes, and these data may provide evidence to guide the treatment of the complications of diabetes mellitus.

Interaction between microglia and retinal pigment epithelial cells determines the integrity of outer blood‐retinal barrier in diabetic retinopathy

Inner and outer blood-retinal barriers (BRBs), mainly composed of retinal endothelial cells and retinal pigment epithelial (RPE) cells, respectively, maintain the integrity of the retinal tissues. In this study, we aimed to investigate the mechanisms of the outer BRB disruption regarding the interaction between RPE and microglia. In mice with high-fat diet-induced obesity and streptozotocin-induced hyperglycemia, microglia accumulated on the RPE layer, as in those after intravitreal injection of interleukin (IL)-6, which is elevated in ocular fluids of patients with diabetic retinopathy. Although IL-6 did not directly affect the levels of zonula occludens (ZO)-1 and occludin in RPE cells, IL-6 increased VEGFA mRNA in RPE cells to recruit microglial cells. In microglial cells, IL-6 upregulated the mRNA levels of MCP1, MIP1A, and MIP1B, to amplify the recruitment of microglial cells. In this manner, IL-6 modulated RPE and microglial cells to attract microglial cells on RPE cells. Furthermore, IL-6-treated microglial cells produced and secreted tumor necrosis factor (TNF)-α, which activated NF-κB and decreased the levels of ZO-1 in RPE cells. As STAT3 inhibition reversed the effects of IL-6-treated microglial cells on the RPE monolayer in vitro, it reduced the recruitment of microglial cells and the production of TNF-α in RPE tissues in streptozotocin-treated mice. Taken together, IL-6-treated RPE and microglial cells amplified the recruitment of microglial cells and IL-6-treated microglial cells produced TNF-α to disrupt the outer BRB in diabetic retinopathy.

Cathepsin D plays a role in endothelial–pericyte interactions during alteration of the blood–retinal barrier in diabetic retinopathy

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-β, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca2+/calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 Mϕ-conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell-substrate impedance sensing (P= 0.007). The CM was fractioned, and the effective fraction (30-100 kDa) was analyzed by liquid chromatography-mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P= 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho-associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3-fold) and serum samples of patients with diabetic macular edema (1.6-fold) measured by Western blot and ELISA. These findings suggest an important role for Mϕ-derived CD in altering the blood-retinal barrier and reveal a potential therapeutic target in the treatment of DR.-Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Chemokine mediated monocyte trafficking into the retina: role of inflammation in alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2−/−) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.

Suppression of protein kinase C-ζ attenuates vascular leakage via prevention of tight junction protein decrease in diabetic retinopathy

To investigate the effect of protein kinase C (PKC)-ζ inhibition on vascular leakage in diabetic retinopathy, streptozotocin-induced diabetic mice were intravitreously injected with siPKC-ζ. According to the fluorescein angiography of the retinal vessels, suppression of PKC-ζ effectively attenuated vascular leakage in diabetic retina. Further evaluation on the retina with western blot analysis and immunohistochemistry revealed accompanying restoration of tight junction proteins on retinal vessels. As two major contributors to vascular leakage in diabetic retinopathy, vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) were investigated on the tight junction protein expression in endothelial cells. Inhibition of PKC-ζ attenuated VEGF-induced decrease of tight junction proteins and accompanying hyperpermeability in human retinal microvascular endothelial cells (HRMECs). PKC-ζ inhibition also attenuated AGE-induced decrease of tight junction proteins in HRMECs. Our findings suggest that inhibition of PKC-ζ could be an alternative treatment option for compromised blood-retinal barrier in diabetic retinopathy.

O-GlcNAcylation under hypoxic conditions and its effects on the blood-retinal barrier in diabetic retinopathy

An increase in O-linked N-acetylglucosamine (O-GlcNAc) protein modifications has been observerd in db/db mouse retinas. O-GlcNAc-modified proteins in the db/db mouse retina have been shown to be localized in the ganglion cell layer, the inner nuclear layer, the retina pigment epithelium (RPE) layer and the inner plexiform layer, in which hypoxia-inducible factor 1α (HIF1α) has also been shown to be localized. In the current study, we examined whether hypoxia increases O-GlcNAcylation in retinal vascular cells under high glucose conditions and whether HIF1α activation is consistent with the response to and activation of O-GlcNAcylation in retinal lesions in diabetic retinopathy. In addition, the effects of O-GlcNAcylation on the blood-retinal barrier were verified in vitro by the inhibition of O-GlcNAcylation. A time-dependent increase in the O-GlcNAcylation in bovine retinal vascular endothelial cells (BRVECs) was observed following incubation of the cells with high glucose medium (glucose 4.5 g/l) under hypoxic (1-3% O2) conditions. Hypoxia-induced BRVEC O-GlcNAcylation was not observed when the BRVECs were transfected with siRNA targeting O-GlcNAc transferase (OGT) or treated with alloxan (an OGT inhibitor) prior to exposure to high glucose. The increase in BRVEC O-GlcNAcylation induced by high glucose, as well as by thiamet G [an O-GlcNAcase (OGA) inhibitor] led to a reduction in occludin expression levels in vitro, which was prevented by treatment with OGT siRNA and alloxan. In conclusion, the current study demonstrates the relationship between O-GlcNAc glycosylation and hypoxia during diabetic retinopathy and that hyperglycemia induced O2 consumption activates HIF1α and O-GlcNAc modification protein in the same retinal layer. The reduced protein BRVEC O-GlcNAcylation levels exert protective effects on the blood-retinal barrier and thus represent a potential therapeutic target for the treatment of diabetic retinopathy.

RPE barrier breakdown in diabetic retinopathy: seeing is believing

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in working-age Americans. DR is traditionally regarded as a disorder of blood-retina barriers, and the leakage of blood content is a major pathological characteristic of the disease. While the breakdown of the endothelial barrier in DR has been investigated extensively, the vascular leakage through the retinal pigment epithelium (RPE) barrier in the disease has not been widely acknowledged. As the blood content leaked through the RPE barrier causes excessive water influx to the retina, the breakdown of the RPE barrier is likely to play a causative role in the development of some forms of diabetic macular edema, a major cause of vision loss in DR. In this article, we will discuss the clinical evidences of the diabetes-induced RPE barrier breakdown, the alteration of the RPE in diabetes, the molecular and cellular mechanism of RPE barrier breakdown, and the research tools for the analysis of RPE barrier leakage. Finally, we will discuss the methodology and potential applications of our recently developed fluorescent microscopic imaging for the diabetes- or ischemia-induced RPE barrier breakdown in rodents.

A Potential Role for Angiopoietin 2 in the Regulation of the Blood–Retinal Barrier in Diabetic Retinopathy

Although VEGF has been identified as an important mediator of the blood-retinal barrier alteration in diabetic retinopathy, the hypothesis for this study was that that other molecules, including the angiopoietins (Ang-1 and -2), may play a role. The expression of angiopoietins was analyzed in an animal model of diabetic retinopathy, and the role of Ang-2 in the regulation of diabetes-induced alterations of vascular permeability was characterized. Diabetes was induced in rats, and human retinal endothelial cells (HRECs) were grown in media with 5.5 or 30.5 mM glucose. Levels of Ang-1 and -2 mRNA and protein were analyzed. Fluorescence-based assays were used to assess the effect of Ang-2 on vascular permeability in vivo and in vitro. The effect of Ang-2 on VE-cadherin function was assessed by measuring the extent of tyrosine phosphorylation. Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.

Systemic administration of HMG-CoA reductase inhibitor protects the blood–retinal barrier and ameliorates retinal inflammation in type 2 diabetes

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are frequently used lipid-lowering drugs in type 2 diabetes. Recent emerging evidence suggests that statins protect cardiovascular function via lipid-independent mechanisms. However, the potential role of statins in diabetic retinopathy in type 2 diabetes is largely unclear. In the present study we have investigated the effect of lovastatin on blood–retinal barrier and inflammatory status in the retina of db/db mice and in cultured retinal cells. Male C57BL/KsJ db/db mice were randomly chosen to receive gastric gavage of lovastatin (10 mg/kg/day) or vehicle control for 6 weeks. Retinal vascular permeability, the tight junction and inflammation were determined. The results showed that db/db mice at the age of 19 weeks exhibited significantly increased retinal vascular leakage and decreased tight junction protein level in the retina. Moreover, the expression of pro-inflammatory factors, e.g. ICAM-1 and TNF-α, was drastically up-regulated in diabetic retina. Lovastatin treatment normalized all of these changes. In cultured bovine retinal capillary endothelial cells (RCECs) and human ARPE-19 cells, lovastatin attenuated the decrease of tight junction protein (occludin) and adherens junction protein (VE-cadherin) expression-induced by TNF-α, a major pro-inflammatory cytokine in diabetic retinopathy. Lovastatin also attenuated TNF-α expression in RCEC. Towards the mechanism, we showed that lovastatin ameliorated ICAM-1 expression-induced by hypoxia and TNF-α in both RCECs and ARPE-19 cells, in part through inhibition of NF-κB activation. Taken together, these findings indicate that lovastatin protects blood–retinal barrier in diabetic retinopathy, which is likely via its anti-inflammatory effects.

Reduction of retinal albumin leakage by the antioxidant calcium dobesilate in streptozotocin-diabetic rats

Calcium dobesilate stabilizes blood–retinal barrier in patients with diabetic retinopathy and possesses antioxidant properties in the retinas of rats with streptozotocin-induced diabetes, exposed ex vivo to ischemia–reperfusion. Here we investigated the action of calcium dobesilate on retinal albumin leakage in streptozotocin-diabetic rats, together with relevant in vivo retinal antioxidant and permeability markers, i.e., carboxymethyl-lysine-advanced glycation end product (CML-AGE) formation and vascular endothelial cell growth factor (VEGF) overexpression. Twenty days after streptozotocin administration, diabetic rats were treated for 10 days with calcium dobesilate (100 mg/kg/day per os) or vehicle. Retinal albumin leakage, CML-AGE formation, and VEGF overexpression were evaluated by immunohistochemistry of frozen eye sections. Diabetic rats exhibited dramatic increases in: (i) retinal albumin leakage (31% of positive vessels vs. 0.2% in nondiabetic rats, P<0.008), (ii) CML-AGE retinal occurrence (40±3% vs. undetectable positive vessels), and (iii) retinal VEGF protein expression (14.6±1.1 vs. 3.5±0.5 VEGF-positive spots/field, P<10 −4). Calcium dobesilate significantly reduced: (i) retinal albumin leakage (by 70%, P<0.008), (ii) retinal CML-AGEs contents (by 62%, P<0.008), and (iii) retinal VEGF expression (by 69.4%, P<0.008). In conclusion, calcium dobesilate orally given to diabetic rats markedly reduced retinal hyperpermeability, CML-AGE contents, and VEGF overexpression. These results strongly suggest that calcium dobesilate stabilizes blood–retinal barrier in diabetic retinopathy via an in situ antioxidant action. Further studies in patients are required to confirm such view.

High Glucose Down-regulates Intercellular Communication in Retinal Endothelial Cells by Enhancing Degradation of Connexin 43 by a Proteasome-dependent Mechanism

Intercellular communication through gap junctions (GJIC) is most likely relevant to maintaining the integrity of the blood-retinal barrier. In this study, we investigated the mechanism whereby high glucose enhances degradation of connexin 43 (Cx43), thus contributing to a decrease in GJIC. The levels of Cx43 in bovine retinal endothelial cells exposed to high glucose (25 mm) decreased about 50% as compared with controls (5.5 mm glucose). Consistently, the half-life of the protein decreased from 2.3 to 1.9 h. The proteasome inhibitors MG132 and lactacystin prevented the loss of Cx43 induced by high glucose and extended Cx43 half-life. The amount of phosphorylated Cx43 increased in high glucose and after proteasome inhibition. Scrape-loading dye transfer experiments show that high glucose is associated to a decrease of 40% in GJIC. Significantly, this reduction can be reversed by proteasome inhibitors. The decrease in GJIC in cells exposed to high glucose is associated with a loss of Cx43 from the plasma membrane, as demonstrated by immunofluorescence and biotinylation of cell-surface proteins. Results indicate that increased phosphorylation of Cx43 under high glucose is the mechanism targeting Cx43 for degradation by a proteasome-dependent mechanism. Increased degradation of Cx43 and reduction of GJIC in high glucose may be of physiological importance by contributing to endothelial cell dysfunction associated with the breakdown of the blood-retinal barrier in diabetic retinopathy.