Deletion Of ADAR1 Impairs Endothelial Cell Function And Its Mechanism

BackgroundAs the development of living condition, the incidences of cardiovascular diseases are on the rise. Vascular endothelium is a monocellular layer that covers the inner surface of the whole vascular system. It plays a major role in several physiological processes, including the control of vascular tone, the maintenance of anti-coagulant environment, the regulation of permeability, and the formation of new blood vessels. Many cardiovascular disease risk factors are associated with endothelial dysfunction. Therefore, understanding the mechanism of endothelial dysfunction improves the prevention and cure of cardiovascular diseases.Adenosine deaminases that act on RNA (ADARs) convert adenosines (A) to inosines (I) in structured and double-stranded RNA (dsRNA). Three mammalian ADAR genes are found in mammals, which encode two active deaminases (ADAR1 and ADAR2) and one inactive deaminase (ADAR3). Previously studies demonstrated that ADAR 1 is essential for embryonic development, hematopoietic stem cell differentiation, autoimmune disorders and virus infection. Knocking out ADAR1 resulted in cell death in the embryos at the early stage of organogenesis and the homozygous embryos were not able to survive beyond E11.5-E12.0. Previously study showed that ADAR1-/- embryos had pale, bloodless yolk sacs, which indicated embryonic circulatory defects. These demonstrate that ADAR1 has important functions in vascular development. However, the mechanisms underlying the impact of ADAR1 in endothelial cell function has not been addressed.ObjectiveFocused on the ADAR1, the aim of the present study was to examine the roles of ADAR1 in endothelial cell function. We also tested the mechanism underlying the effect of ADAR1 on endothelial cells.1. To investigate the effect of ADAR1 on endothelial cell function and its mechanism, endothelial cells tube formation and cell permeability were detected in vitro by using the ADAR1 siRNA.2. To investigate the role of ADAR1 on endothelial homeostasis and its mechanism, vascular permeability and angiogenesis were measured by using endothelium-specific ADAR1 knockout (ADAR1ECKO) mice.MethodsBriefly, wild-type (WT) mice, ADAR1-/- embryos, ADAR1ECKO mice, ADAR1flox/flox mice and Human umbilical vein endothelial cells (HUVECs) were used. Animal protocols received institutional review and committee approval by IACUC of Second millitary medical university. Knockdown of ADAR1 was performed by siRNA against human ADAR1 using Lipofectamine 2000. Tissues were collected for histological observation or transmission electron microscope detection. Vascular permeability was measured by Evans Blue-labeled albumin in vivo and in vitro. Hindlimb ischemia model and aorta ring assay were used in mice and tube formation assay was used in HUVECs. Expression protein was measured by Western blotting or Immunofluorescence.Results1. We investigated the effect of siRNA-mediated knockdown of ADAR1 in HUVECs tube formation and permeability. siRNA-mediated knockdown of ADAR1 suppresses basal endothelial cells proliferation, tube formation and increases cell permeability.2. We found that ADAR1 siRNA significantly inhibited Caveolin-1 expression, but not VEGF, Sprouty 2, p-eNOS, and MMP-9. Results also show that ADAR1 siRNA treatment reduced Cav-2 and VE-Cadherin protein expression in HUVECs.3. The cell-permeable peptide cavtratin, which encodes the Cav-1 scaffolding domain, significantly nullified, in part, the ADAR1 siRNA-induced impairment of angiogenesis and permeability which was not found in DMSO treated group. Delivery of cavtratin to siRNA-treated HUVECs reverses the ADAR1 siRNA-induced loss in VE-Cadherin.4. VE-Cadherin-Cre mice were cross-bred with ADARlflox/flox mice to generate ADAR1ECKO. Nearly 3/4 of the ADAR1ECKO pups identified at birth succumbed during the postnatal 4 weeks. The significant feature of lungs from ADAR1ECKO mice were constricted alveolar spaces, thickened septa, and hyper-cellularity. We also examined aorta, carotid, heart, liver and kidneys microscopically, but no abnormal histology was detected.5. Endothelium from ADAR1ECKO mice showed dilated interendothelial junctions, In ADAR1ECKO mice, pulmonary Evans Blue accumulation was significantly increased compared with ADAR1flox/flox mice. Immunofluorescent staining also shows that both Cav-1 and VE-Cadherin are diminished in the lung capillary endothelium of ADAR1ECKO mice.6. Hindlimb ischemia model and aorta ring asssay were used to evaluate the role of ADAR1 in angiogenesis in vivo. endothelial cell-specific deletion of ADAR1 decreased the number of microvessels sprouting from aortic rings compared with ADAR1flox/flox mice. In ADARlflox/flox mice, the blood flow of the ischemic leg recovered≈80% of the blood flow of the untreated leg by 4 weeks, but blood flow recovery was significantly impaired in ADAR1ECKO mice.Conclutions1. ADAR1 siRNA impaired angiogenesis and cell permeability in vitro.2. Decreased Caveolin-1/VE-Cadherin expression were associated with damage to endothelial cell function caused by ADAR1 siRNA. Cavtratin reverses effects of ADAR1 siRNA on VE-Cadherin expression, permeability and tube formation in HUVECs.3. Partial postnatal lethality and lung defects in ADAR1ECKO mice.4. Dilated interendothelial junctions, increased vascular permeability and impaired angiogenesis in ADAR1ECICO mice.5. Cav-1 and VE-cadherin expression were decreased in ADAR1ECK0 mice.This study demonstrates that ADAR1 is critical for endothelium integrity and angiogenesis. The loss of ADAR1 contributes to decreased Cav-1 expression and the subsequent suppression of VE-Cadherin, thereby adversely affecting vascular permeability and angiogenesis. These findings contribute significantly to our understanding of vasculopathy mechanisms and might lead to new targets for disease intervention or treatment.