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Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in preterm infants, and pulmonary hypertension (PH) develops in 25-40% of BPD patients, increasing morbidity and mortality. BPD-PH is characterized by vasoconstriction and vascular remodeling. Nitric oxide (NO) is a pulmonary vasodilator and apoptotic mediator made in the pulmonary endothelium by NO synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous eNOS inhibitor, primarily metabolized by dimethylarginine dimethylaminohydrolase-1 (DDAH1). Our hypothesis is that DDAH1 knockdown in human pulmonary microvascular endothelial cells (hPMVEC) will result in lower NO production, decreased apoptosis, and greater proliferation of human pulmonary arterial smooth muscle cells (hPASMC), while DDAH1 overexpression will have the opposite effect. hPMVEC were transfected with siDDAH1/scramble or AdDDAH1/AdGFP for 24h and co-cultured for 24h with hPASMC. Analyses included Western blot for cleaved and total caspase-3, -8, -9, β-actin, trypan blue exclusion for viable cell numbers, TUNEL, and BrdU incorporation. Small interfering RNA targeting DDAH1 (siDDAH1) transfected into hPMVEC resulted in lower media nitrites, cleaved caspase-3 and -8 protein expression, and TUNEL staining; and greater viable cell numbers and BrdU incorporation in co-cultured hPASMC. Adenoviral mediated transfection of the DDAH1 gene (AdDDAH1) into hPMVEC resulted in greater cleaved-caspase-3 and -8 protein expression and lower viable cell numbers in co-cultured hPASMC. Partial recovery of hPASMC viable cell numbers after AdDDAH1-hPMVEC transfection was observed when media was treated with hemoglobin to sequester NO. In conclusion, hPMVEC DDAH1-regulated NO production positively regulates hPASMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is chronic lung disease of prematurity and pulmonary hypertension (PH) is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a vasodilator and apoptotic mediator made by NO synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA), and dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Previously, in a BPD patient cohort, we identified single nucleotide polymorphism (SNP) DDAH1 rs480414 (G >  A) that was protective against developing PH. This study aims to determine functional consequences of the DDAH1 SNP in lymphoblastoid cell lines (LCLs) derived from neonatal cord blood. We tested the hypothesis that DDAH1 SNP (AA) results in DDAH1 gain of function, leading to greater NO-mediated apoptosis compared to DDAH1 wild-type (GG) in LCLs. METHODS: LCLs were analyzed by Western blot (DDAH1, cleaved and total caspase-3 and -8, and β-actin), and RT-PCR (DDAH1, iNOS). Cell media assayed for nitrites with chemiluminescence NO analyzer, and conversion of ADMA to L-citrulline was measured by spectrophotometry. RESULTS: LCLs with DDAH1 SNP had similar levels of DDAH1 protein and mRNA expression, as well as DDAH activity, compared to DDAH1 WT LCLs. There were also no changes in cleaved caspase-3 and -8 protein levels. LCLs with DDAH1 SNP had similar iNOS mRNA expression. Nitrite levels in media were lower for DDAH1 SNP LCLs compared to DDAH1 WT LCLs (p <  0.05). CONCLUSION: Contrary to our hypothesis, we found that NO production was lower in DDAH1 SNP LCLs, indicative of a loss of function phenotype.