DDAH1 siRNA Knockdown in a Human Pulmonary Vascular Co‐Culture Cell Model

Abstract

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of premature infants. Pulmonary hypertension (PH) develops in 25–40% of BPD patients, contributing to morbidity and mortality. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made in the pulmonary endothelium by nitric oxide synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous competitive NOS inhibitor. Dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA and DDAH1 is the most important for ADMA metabolism in vivo. Translational studies from our group have identified DDAH1 single nucleotide polymorphisms that are differentially expressed in BPD‐PH patients compared to BPD patients. We hypothesized that inhibiting DDAH1 in human pulmonary microvascular endothelial cells (hPMVECs) will lead to decreased apoptosis and increased proliferation of human pulmonary artery smooth muscle cells (hPASMCs) via effects in hPMVECs. We utilized a co‐culture model where hPMVECs were transfected with DDAH1 siRNA or scramble for 24h and then incubated in co‐culture with hPASMCs for 24h. Analysis included 1) trypan blue exclusion for viable cell numbers and 2) western blot analysis for cleaved caspase‐3 and 8, total caspase‐3 and 8, PCNA, and β‐actin. After DDAH1 siRNA transfection of hPMVECs and co‐culture with hPASMCs, we found greater hPASMC viable cell numbers compared to scramble hPASMC controls (N=3, p<0.05). After DDAH1 siRNA transfection of hPMVECs and co‐culture with hPASMCs, we found lower hPASMC levels of cleaved caspase‐3 protein (N=3, p<0.05) and cleaved caspase‐8 (N=3, p<0.05), with greater hPASMC protein levels of PCNA (N=3, p<0.05) compared to scramble. DDAH1 siRNA knockdown in hPMVEC results in greater hPASMC proliferation and less cleaved caspase‐3 mediated apoptosis. We speculate that DDAH1 is a potential therapeutic target for BPD‐PH. We speculate that these findings support the concept that DDAH1 regulates vascular remodeling by minimizing ADMA‐inhibition of eNOS, resulting in maintained NO production which would support angiogenesis and prevent/attenuate vascular remodeling. Future studies would include in vivo experiments in transgenic neonatal mice exposed to a BPD‐like lung injury.Support or Funding InformationThis work was funded by NIH‐K08 HL129080This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.