Regulation of the L-Arginine-ADMA-NO pathway in coronary and pulmonary endothelial and alveolar epithelial cells during hypoxia

Abstract

One important promoter of cardiovascular and especially pulmonary diseases is hypoxia, the insufficient supply with oxygen. It leads to hypoxic coronary vasodilation and hypoxic pulmonary vasoconstriction (HPV). Hypoxic coronary vasodilation is thought to be HIF-mediated; the mechanisms regulating HPV are less well understood. As lasting HPV might lead to vascular remodelling and the emergence of severe diseases, a deeper understanding of the mechanisms regulating HPV might help to gain insight into the emergence of those diseases and thereby provide putative therapeutic targets against hypoxia-related disorders. The L-arginine-ADMA-NO pathway plays an important role in the regional adaption of the blood flow. A differential hypoxic regulation of this pathway within coronary and pulmonary circuit might explain hypoxic coronary vasodilation and HPV. We therefore aimed to compare gene expression, protein expression and protein activity between normoxic and hypoxic human coronary and pulmonary artery endothelial cells. We found hypoxic regulation of genes involved in the L-arginine-ADMA-NO pathway, e.g. decreased NOS3 mRNA expression and increased DDAH2 mRNA expression in both cell lines after exposure to hypoxia. Interestingly, DDAH1 mRNA expression in HPAEC changed after short periods of hypoxia while it remained on normoxic level in HCAEC. However, those changes did not result in altered protein expression and ADMA concentrations remained unchanged. We further evaluated gene expression, protein expression and protein activity between normoxic and hypoxic alveolar epithelial A549 cells. Alveolar epithelial cells are the first layer of cells in contact to inhaled air, suggesting a putative role in oxygen sensing. Interestingly, we found decreased DDAH1 mRNA expression after exposure to hypoxia accompanied by increased SDMA and ADMA concentrations, the latter one being a well-known inhibitor of NO-synthesis. Thus, we propose a cellular cross talk between pulmonary endothelial and epithelial cells responsible for the mechanism of HPV. We further aimed to evaluate whether HIF is the responsible transcription factor regulating expression of genes involved in the L-arginine-ADMA-NO pathway within coronary and pulmonary artery endothelial as well as alveolar epithelial cell. We were able to show that hypoxic regulation of gene expression of the L-arginine-ADMA-NO pathway within those cells is not strictly HIF-dependent and that other transcription factors appear to be involved in hypoxic regulation of gene expression.