An arginase-1 SNP that protects against the development of pulmonary hypertension in bronchopulmonary dysplasia enhances NO-mediated apoptosis in lymphocytes

Abstract

Arginase and nitric oxide synthase (NOS) share a common substrate, l‐arginine, and have opposing effects on vascular remodeling. Arginase is the first step in polyamine and proline synthesis necessary for cellular proliferation, while NO produced from NOS promotes apoptosis. Previously, we identified a single nucleotide polymorphism (SNP) in the arginase‐1 (ARG1) gene, rs2781666 (T‐allele) that was associated with a decreased risk for developing pulmonary hypertension (PH) in a cohort of infants with bronchopulmonary dysplasia (BPD). In this study, we utilized lymphocytes from neonates (the only readily available cells from these patients expressing the two genotypes of interest) with either the rs2781666 SNP (TT) or wild type (GG) to test the hypothesis that the protection of the ARG1 SNP against the development of PH in BPD would involve augmented NO production leading to more apoptosis. Lymphocytes were stimulated with IL‐4, IL‐13, and phorbol myristate acetate (PMA). We found that TT lymphocytes had similar levels of arginase I and arginase II expression, but there was a tendency for lower urea production (a surrogate marker of arginase activity), than in the GG lymphocytes. The TT lymphocytes also had significantly greater NO production than did GG lymphocytes despite no differences in iNOS expression between genotypes. Furthermore, the TT lymphocytes had lower numbers of viable cells, and higher levels of cleaved caspase‐3 than did GG lymphocytes. Inhibiting NOS activity using N ω‐Nitro‐l‐arginine methyl ester hydrochloride (l‐NAME) significantly decreased cleaved caspase‐3 levels in the TT lymphocytes. These data demonstrate that the TT genotype results in greater levels of NO production leading to more apoptosis, which is consistent with the concept that BPD patients with the TT genotype are protected against the development of PH by producing greater basal levels of endogenous NO.

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

“…; Trittmann et al. ). Briefly, 100 μ L of sample was placed in a reaction chamber containing a mixture of NaI in glacial acetic acid to reduce nitrite () to NO.…”

“…For cleaved caspase‐3, ‐8, and ‐9, the respective total caspase was used to control for protein loading as described previously (Trittmann et al. ).…”

“…Cell lysates were assayed for DDAH1, eNOS, cleaved caspase-3, total caspase-3, cleaved caspase-8, total caspase-8, cleaved caspase-9, total caspase-9, p21, PCNA, and bactin by western blot analysis as described previously (Nelin et al 2005;Toby et al 2010;Trittmann et al 2016b;White et al 2017). Cell lysate aliquots were diluted with SDS buffer and reducing agent, then heated to 80°C for 15 min, and centrifuged at 10,000g at room temperature for 2 min.…”

Arginase and α‐smooth muscle actin induction after hyperoxic exposure in a mouse model of bronchopulmonary dysplasia

“…Recently, our group demonstrated in infants with BPD, differential expression of a single nucleotide polymorphism (SNP) in the arginase‐1 gene between those patients with BPD and those with BPD and PH. Furthermore, lymphocytes harvested from patients with the arginase‐1 SNP had greater levels of NO production . We have also described that patients with BPD and PH have differential expression of a SNP in the dimethylarginine dimethylaminohydrolase‐1 (DDAH‐1) gene compared to patients with BPD alone .…”

“…Furthermore, lymphocytes harvested from patients with the arginase-1 SNP had greater levels of NO production. 12 We have also described that patients with BPD and PH have differential expression of a SNP in the dimethylarginine dimethylaminohydrolase-1 (DDAH-1) gene compared to patients with BPD alone. 13 Furthermore, we found higher plasma levels of asymmetric dimethylarginine (ADMA) in infants that went on to develop BPD with PH than in those with BPD alone.…”