Abstract 18601: The Role of ADMA Metabolism in the Regulation of Human Pulmonary Endothelial Cell Function

Abstract

Objective: Asymmetric dimethylarginine (ADMA), a nitric oxide synthase inhibitor, is increased in patients with idiopathic pulmonary hypertension (IPAH). We hypothesized that ADMA abrogates gap junctional communication required for the coordinated regulation of pulmonary endothelial permeability and angiogenesis, contributing to the disease. Methods and Results. The effects of ADMA on expression and function of gap junctional proteins were studied in human pulmonary artery endothelial cells (HPAECs). ADMA inhibited protein expression and cell surface localization of connexin 43 (Cx43) in HPAECs (2-fold decrease, P<0.01), resulting in impaired gap junctional communication, as shown by Fluorescence Recovery After Photobleaching (FRAP). ADMA regulated Cx43 expression and function via the nitric oxide signalling pathway, as effects could be reversed by the nitric oxide donor SNAP, and nitric oxide secondary messengers, cAMP and cGMP. Defective gap junction communication in HPAECs led to endothelial dysfunction, including increased trans-endothelial permeability (1.7-fold increase, P<0.001), and impaired angiogenesis(1.7-fold decrease, P<0.001). The effects of ADMA were prevented by over expressing dimethylarginine dimethylaminohydrolase I (DDAHI), an enzyme that metabolised ADMA, or treatment with the Cx43-gap junction activating drug, rotigaptide.Reduced Cx43 expression was also found in lungs of mice deficient in DDAHI. In vivo lung permeability was increased in DDAHI homozygous knockout mice (1.2-fold increase, P<0.05), and was alleviated by application of rotigaptide. Endothelial colony forming cells from IPAH patients showed reduced expression of DDAHI and Cx43 (2-fold decrease, P<0.05), resulting in impaired endothelial barrier function and abnormal angiogenesis. Conclusions. ADMA is critical in the regulation of gap junctional communication in pulmonary endothelium. Endothelial cells from IPAH patients show reduced ADMA metabolism resulting in defective gap junctional communication and abnormal angiogenic responses. Enhancing Cx43 function may have endothelium-protective effects in conditions associated with reduced nitric oxide signalling, such as pulmonary hypertension.