Distinct Mechanisms of Human Retinal Endothelial Barrier Modulation In Vitro by Mediators of Diabetes and Uveitis

Ocular diseases such as diabetic retinopathy (DR) and uveitis are associated with injury to the blood–retinal barrier (BRB). Whereas high glucose (HG) and advanced glycation end products (AGE) contribute to DR, bacterial infections causing uveitis are triggered by endotoxins such as lipopolysaccharide (LPS). It is unclear how HG, AGE, and LPS affect human retinal endothelial cell (HREC) junctions. Moreover, tumor necrosis factor-α (TNFα) is elevated in both DR and ocular infections. In the current study, we determined the direct effects of HG, AGE, TNFα, and LPS on the expression and intracellular distribution of claudin-5, VE-cadherin, and β-catenin in HRECs and how these mediators affect Akt and P38 MAP kinase that have been implicated in ocular pathologies. In our results, whereas HG, AGE, and TNFα activated both Akt and P38 MAPK, LPS treatment suppressed Akt but increased P38 MAPK phosphorylation. Furthermore, while treatment with AGE and HG increased cell-junction protein expression in HRECs, LPS elicited a paradoxical effect. By contrast, when HG treatment increased HREC-barrier resistance, AGE and LPS stimulation compromised it, and TNFα had no effect. Together, our results demonstrated the differential effects of the mediators of diabetes and infection on HREC-barrier modulation leading to BRB injury.

Hyperoxia Inhibits Proliferation of Retinal Endothelial Cells in a Myc-Dependent Manner

Oxygen supplementation is necessary to prevent mortality in severely premature infants. However, the supraphysiological concentration of oxygen utilized in these infants simultaneously creates retinovascular growth attenuation and vasoobliteration that induces the retinopathy of prematurity. Here, we report that hyperoxia regulates the cell cycle and retinal endothelial cell proliferation in a previously unknown Myc-dependent manner, which contributes to oxygen-induced retinopathy.

MiR-10b alleviates high glucose-induced human retinal endothelial cell injury by regulating TIAM1 signaling

Purpose: To investigate the effects of microRNA (miR)-10b on high glucose (HG)-induced human retinal endothelial cell (HREC) injury and the mechanisms involved.Methods: Levels of miR-10b were measured in HRECs using quantitative reverse transcriptasepolymerase chain reaction (qRT-PCR) after the addition of glucose (5.5 and 30 mM). Cell viability was measured using Cell Counting Kit-8 assay, while levels of reactive oxygen species (ROS) weredetermined using fluorimetry. An enzyme-linked immunosorbent assay (ELISA) was used to measure cellular apoptosis. Luciferase reporter assay was used to validate the miR-10b-binding sites of target genes. The levels of T-cell lymphoma invasion and metastasis (TIAM1) and NADPH oxidase-2 (NOX2) were determined using qRT-PCR. Ras-related C3 botulinum toxin substrate 1 (Rac1) activation was evaluated using a pull-down assay. The protein levels of TIAM1 and Rac1 were assayed by western blotting.Results: After HG stimulation, miR-10b expression was downregulated. Viability of HRECs decreased, whereas ROS production increased. However, the overexpression of miR-10b inhibited apoptosis and ROS production in HG-treated HRECs (p < 0.05), while luciferase reporter analysis revealed a possible binding site for miR-10b to target the 3'-untranslated region (UTR) of TIAM1. In addition, the overexpression of miR-10b distinctly reduced the expression levels of TIAM1 and NOX2, but decreased the activation of Rac1 in HG-treated HRECs (p < 0.05); these inhibitory effects of miR-10b were significantly reversed after TIAM1 application.Conclusion: MiR-10b alleviates HG-induced HREC injury by regulating TIAM1 signaling. MiR-10b therapy is a potential therapeutic strategy for patients suffering from diabetic retinopathy. Keywords: MicroRNA-10b, Human retinal endothelial cells, High glucose, TIAM1-Rac1 axis

Hyperoxia inhibits proliferation of retinal endothelial cells in Myc dependent manner

Oxygen supplementation is necessary to prevent mortality of severely premature infants. However, the supraphysiological concentration of oxygen utilized in these infants simultaneously creates retinovascular growth attenuation and vasoobliteration that induces retinopathy of prematurity. Here, we report that hyperoxia regulates the cell cycle and retinal endothelial cell proliferation in a previously unknown Myc dependent manner which contributes to oxygen-induced retinopathy.

Vitamin D Protects against Oxidative Stress and Inflammation in Human Retinal Cells

Diabetic retinopathy is a vision-threatening microvascular complication of diabetes and is one of the leading causes of blindness. Oxidative stress and inflammation play a major role in its pathogenesis, and new therapies counteracting these contributors could be of great interest. In the current study, we investigated the role of vitamin D against oxidative stress and inflammation in human retinal pigment epithelium (RPE) and human retinal endothelial cell lines. We demonstrate that vitamin D effectively counteracts the oxidative stress induced by hydrogen peroxide (H2O2). In addition, the increased levels of proinflammatory proteins such as Interleukin (IL)-6, IL-8, Monocyte chemoattractant protein (MCP)-1, Interferon (IFN)-γ, and tumor necrosis factor (TNF)-α triggered by lipopolysaccharide (LPS) exposure were significantly decreased by vitamin D addition. Interestingly, the increased IL-18 only decreased by vitamin D addition in endothelial cells but not in RPE cells, suggesting a main antiangiogenic role under inflammatory conditions. Moreover, H2O2 and LPS induced the alteration and morphological damage of tight junctions in adult retinal pigment epithelium (ARPE-19) cells that were restored under oxidative and inflammatory conditions by the addition of vitamin D to the media. In conclusion, our data suggest that vitamin D could protect the retina by enhancing antioxidant defense and through exhibiting anti-inflammatory properties.

Long noncoding RNA Hotair facilitates retinal endothelial cell dysfunction in diabetic retinopathy

Background: Retinal endothelial cell (REC) dysfunction induced by diabetes mellitus (DM) is an important pathological step of diabetic retinopathy (DR). Long noncoding RNAs (lncRNAs) have emerged as novel modulators in DR. The present study aimed to investigate the role and mechanism of lncRNA Hotair in regulating DM-induced REC dysfunction. Methods: The retinal vascular preparations and immunohistochemical staining assays were conducted to assess the role of Hotair in retinal vessel impairment in vivo. The EdU, transwell, cell permeability, CHIP, luciferase activity, RIP, RNA pull-down, and Co-IP assays were employed to investigate the underlying mechanism of Hotair-mediated REC dysfunction in vitro. Results: Hotair expression was significantly increased in diabetic retinas and high glucose (HG)-stimulated REC. Hotair knockdown inhibited the proliferation, invasion, migration, and permeability of HG-stimulated REC in vitro and reduced the retinal acellular capillaries and vascular leakage in vivo. Mechanistically, Hotair bound to LSD1 to inhibit VE-cadherin transcription by reducing the H3K4me3 level on its promoter and to facilitate transcription factor HIF1α-mediated transcriptional activation of VEGFA. Furthermore, LSD1 mediated the effects of Hotair on REC function under HG condition. Conclusion: The Hotair exerts its role in DR by binding to LSD1, decreasing VE-cadherin transcription, and increasing VEGFA transcription, leading to REC dysfunction. These findings revealed that Hotair is a potential therapeutic target of DR.