Rationale: Pathological angiogenesis is a critical component of diseases, such as ocular disorders, cancers, and atherosclerosis. It is usually caused by the abnormal activity of biological processes, such as cell proliferation, cell motility, immune, or inflammation response. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of these biological processes. However, the role of lncRNA in diabetes mellitus–induced microvascular dysfunction is largely unknown. Objective: To elucidate whether lncRNA-myocardial infarction–associated transcript (MIAT) is involved in diabetes mellitus–induced microvascular dysfunction. Methods and Results: Using quantitative polymerase chain reaction, we demonstrated increased expression of lncRNA-MIAT in diabetic retinas and endothelial cells cultured in high glucose medium. Visual electrophysiology examination, TUNEL staining, retinal trypsin digestion, vascular permeability assay, and in vitro studies revealed that MIAT knockdown obviously ameliorated diabetes mellitus–induced retinal microvascular dysfunction in vivo, and inhibited endothelial cell proliferation, migration, and tube formation in vitro. Bioinformatics analysis, luciferase assay, RNA immunoprecipitation, and in vitro studies revealed that MIAT functioned as a competing endogenous RNA, and formed a feedback loop with vascular endothelial growth factor and miR-150-5p to regulate endothelial cell function. Conclusions: This study highlights the involvement of lncRNA-MIAT in pathological angiogenesis and facilitates the development of lncRNA-directed diagnostics and therapeutics against neovascular diseases.

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Vascular dysfunction is a hallmark of ischemic, cancer, and inflammatory diseases, contributing to disease progression. Circular RNAs (circRNAs) are endogenous non-coding RNAs, which have been reported to be abnormally expressed in many human diseases. In this study, we used retinal vasculature to determine the role of circular RNA in vascular dysfunction. We revealed that cZNF609 was significantly up-regulated upon high glucose and hypoxia stress in vivo and in vitro. cZNF609 silencing decreased retinal vessel loss and suppressed pathological angiogenesis in vivo. cZNF609 silencing increased endothelial cell migration and tube formation, and protected endothelial cell against oxidative stress and hypoxia stress in vitro. By contrast, transgenic overexpression of cZNF609 showed an opposite effects. cZNF609 acted as an endogenous miR-615-5p sponge to sequester and inhibit miR-615-5p activity, which led to increased MEF2A expression. MEF2A overexpression could rescue cZNF609 silencing-mediated effects on endothelial cell migration, tube formation, and apoptosis. Moreover, dysregulated cZNF609 expression was detected in the clinical samples of the patients with diabetes, hypertension, and coronary artery disease. Intervention of cZNF609 expression is promising therapy for vascular dysfunction.

VEGF binding to VEGFR2 leads to VEGFR2 endocytosis and downstream signaling activation to promote angiogenesis.Methods: Using genetic strategies, we tested the requirement of α subunits of heterotrimeric G proteins (Gαi1/3) in the process.Results: Gαi1/3 are located in the VEGFR2 endocytosis complex (VEGFR2-Ephrin-B2-Dab2-PAR-3), where they are required for VEGFR2 endocytosis and downstream signaling transduction. Gαi1/3 knockdown, knockout or dominant negative mutation inhibited VEGF-induced VEGFR2 endocytosis, and downstream Akt-mTOR and Erk-MAPK activation. Functional studies show that Gαi1/3 shRNA inhibited VEGF-induced proliferation, invasion, migration and vessel-like tube formation of HUVECs. In vivo, Gαi1/3 shRNA lentivirus inhibited alkali burn-induced neovascularization in mouse cornea. Further, oxygen-induced retinopathy (OIR)-induced retinal neovascularization was inhibited by intravitreal injection of Gαi1/3 shRNA lentivirus. Moreover, in vivo angiogenesis by alkali burn and OIR was significantly attenuated in Gαi1/3 double knockout mice. Significantly, Gαi1/3 proteins are upregulated in proliferative retinal tissues of proliferative diabetic retinopathy (PDR) patients.Conclusion: These results provide mechanistic insights into the critical role played by Gαi1/3 proteins in VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis.

Atherosclerosis is one of the most common vascular disorders. Endothelial cell (EC) dysfunction and vascular smooth muscle cell (VSMC) proliferation contributes to the development of atherosclerosis. Long non-coding RNAs (lncRNAs) have been implicated in several biological processes and human diseases. Here we show that lncRNA-RNCR3 is expressed in ECs and VSMCs. RNCR3 expression is significantly upregulated in mouse and human aortic atherosclerotic lesions, and cultured ECs and VSMCs upon ox-LDL treatment in vitro. RNCR3 knockdown accelerates the development of atherosclerosis, aggravates hypercholesterolemia and inflammatory factor releases, and decreases EC and VSMC proliferation in vivo. RNCR3 knockdown also reduces the proliferation and migration, and accelerates apoptosis development of EC and VSMC in vitro. RNCR3 acts as a ceRNA, and forms a feedback loop with Kruppel-like factor 2 and miR-185-5p to regulate cell function. This study reveals that RNCR3 has an atheroprotective role in atherosclerosis, and its intervention is a promising strategy for treating atherosclerosis-related vascular dysfunction.

lncRNAs are involved in the pathogenesis of DR through the modulation of multiple pathogenetic pathways. MALAT1, a conserved lncRNA, may become a potential therapeutic target for the prognosis, diagnosis, and treatment of DR.

The crosstalk between vascular pericytes and endothelial cells (ECs) is critical for microvascular stabilization and remodeling; however, the crosstalk is often disrupted by diabetes, leading to severe and even lethal vascular damage. Circular RNAs are a class of endogenous RNAs that regulate several important physiological and pathological processes. Here we show that diabetes-related stress up-regulates cPWWP2A expression in pericytes but not in ECs. In vitro studies show that cPWWP2A directly regulates pericyte biology but indirectly regulates EC biology via exosomes carrying cPWWP2A. cPWWP2A acts as an endogenous miR-579 sponge to sequester and inhibit miR-579 activity, leading to increased expression of angiopoietin 1, occludin, and SIRT1. In vivo studies show that cPWWP2A overexpression or miR-579 inhibition alleviates diabetes mellitus-induced retinal vascular dysfunction. By contrast, inhibition of cPWWP2A-mediated signaling by silencing cPWWP2A or overexpressing miR-579 aggravates retinal vascular dysfunction. Collectively, this study unveils a mechanism by which pericytes and ECs communicate. Intervention of cPWWP2A or miR-579 expression may offer opportunities for treating diabetic microvascular complications.circular RNA | endothelial cell | pericyte | retinal vascular dysfunction | microRNA sponge

Excessive UV radiation and reactive oxygen species (ROS) cause retinal pigment epithelium (RPE) cell injuries. Nrf2 regulates transcriptional activation of many anti-oxidant genes. Here, we tested the potential role of 3H-1,2-dithiole-3-thione (D3T) against UV or ROS damages in cultured RPE cells (both primary cells and ARPE-19 line). We showed that D3T significantly inhibited UV-/H2O2-induced RPE cell death and apoptosis. UV-stimulated ROS production was dramatically inhibited by D3T pretreatment. D3T induced Nrf2 phosphorylation in cultured RPE cells, causing Nrf2 disassociation with KEAP1 and its subsequent nuclear accumulation. This led to expression of antioxidant response elements (ARE)-dependent gene heme oxygenase-1 (HO-1). Nrf2-HO-1 activation was required for D3T-mediated cytoprotective effect. Nrf2 shRNA knockdown or S40T dominant negative mutation as well as the HO-1 inhibitor Zinc protoporphyrin (ZnPP) largely inhibited D3T’s RPE cytoprotective effects against UV radiation. Yet, exogenous overexpression Nrf2 enhanced D3T’s activity in RPE cells. Further studies showed that D3T activated Akt/mTORC1 in cultured RPE cells. Akt-mTORC1 inhibitors, or Akt1 knockdown by shRNA, not only inhibited D3T-induced Nrf2-HO-1 activation, but also abolished the RPE cytoprotective effects. In vivo, D3T intravitreal injection protected from light-induced retinal dysfunctions in mice. Thus, D3T protects RPE cells from UV-induced damages via activation of Akt-mTORC1-Nrf2-HO-1 signaling axis.