High Glucose Aggravates Retinal Endothelial Cell Dysfunction by Activating the RhoA/ROCK1/pMLC/ Connexin43 Signaling Pathway

PURPOSE. This research aims to explore the mechanism underlying the relationship between RhoA/ROCK signaling and Connexin43 (Cx43) in retinal endothelial cell dysfunction and to evaluate the protective effect of ROCK inhibitors against retinal endothelial cell dysfunction in diabetic retinopathy (DR) models. METHODS. TUNEL staining, hematoxylin and eosin staining, a retinal digestion assay, and Evans blue assay were conducted to explore the effect of fasudil in alleviating retinal dysfunction induced by DR. ELISA, the CCK-8 assay, and flow cytometry were conducted to study inflammation, viability, and apoptosis of mouse retinal microvascular endothe-lial cells treated with high glucose and ROCK inhibitors. The qRT-PCR and Western blotting were used to evaluate the expression of RhoA, ROCK1, ROCK2, MLC, pMLC, and Cx43. Co-immunoprecipitation was used to verify the interaction between pMLC and Cx43. Immunofluorescence and scrape-loading and dye transfer were used to evaluate the expression and function of Cx43. RESULTS. Marked endothelial cell dysfunction resulting from the activation of RhoA/ROCK1 signaling was found in in vivo and in vitro models of DR. Via interaction with pMLC, which is downstream of RhoA/ROCK1, a significant downregulation of Cx43 was observed in retinal endothelial cells. Treatment with ROCK inhibitors amelio-rated retinal endothelial dysfunction in vitro. The ROCK inhibitor, fasudil, significantly alleviated retinal dysfunction as shown by a decrease of retinal acellular capillaries, an improvement of vascular permeability, and a reduction of cell apoptosis in vivo. CONCLUSIONS. Our study highlights a novel mechanism that high glucose could activate RhoA/ROCK1/pMLC signaling, which targets the expression and localization of Cx43 and is responsible for cell viability, apoptosis, and inflammation, resulting in retinal endothelial cell injury. ROCK inhibitors markedly ameliorate endothelial cell dysfunction, suggesting their therapeutic potential for diabetic retinopathy.