DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells.

Nitric Oxide (NO) is an endogenous pulmonary vasodilator produced by endothelial NO synthase (eNOS). Asymmetric dimethyl L-arginine (ADMA) is an endogenous inhibitor of eNOS activity. In endothelial cells, ADMA is hydrolyzed to L-citrulline primarily by dimethylarginine dimethyl-aminohydrolase-1 (DDAH1). We tested the hypothesis that DDAH1 expression is essential for maintaining NO production in human fetal pulmonary microvascular endothelial cells (hfPMVEC), such that knockdown of DDAH1 expression will lead to decreased NO production resulting in less caspase-3 activation and less tube formation. We found that hfPMVEC transfected with DDAH1 siRNA had lower NO production than control, with no difference in eNOS protein levels between groups. hfPMVEC transfected with DDAH1 siRNA had lower protein levels of cleaved caspase-3 and -8 than control. Both DDAH1 siRNA- and ADMA-treated hfPMVEC had greater numbers of viable cells than controls. Angiogenesis was assessed using tube formation assays in matrigel, and tube formation was lower after either DDAH1 siRNA transfection or ADMA treatment than controls. Addition of an NO donor restored cleaved caspase-3 and -8 protein levels after DDAH1 siRNA transfection in hfPMVEC to essentially the levels seen in scramble control. Addition of a putative caspase-3 inhibitor to DDAH1 siRNA transfected and NO-donor treated cells led to greater numbers of viable cells and far less angiogenesis than in any other group studied. We conclude that in hfPMVEC, DDAH1 is central to the regulation of NO-mediated caspase-3 activation and the resultant apoptosis and angiogenesis. Our findings suggest that DDAH1 may be a potential therapeutic target in pulmonary hypertensive disorders.