The L-arginine/NO pathway is an important regulator of pulmonary hypertension, the leading cause of mortality in patients with the chronic lung disease of prematurity, bronchopulmonary dysplasia. L-arginine can be metabolized by NO synthase (NOS) to form L-citrulline and NO, a potent vasodilator. Alternatively, L-arginine can be metabolized by arginase to form urea and L-ornithine, a precursor to collagen and proline formation important in vascular remodelling. In the current study, we hypothesized that C3H/HeN mice exposed to prolonged hyperoxia would have increased arginase expression and pulmonary vascular wall cell proliferation. C3H/HeN mice were exposed to 14 days of 85% O or room air and lung homogenates analyzed by western blot for protein levels of arginase I, arginase II, endothelial NOS (eNOS), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), and α-smooth muscle actin (α-SMA). Hyperoxia did not change arginase I or eNOS protein levels. However, arginase II protein levels were 15-fold greater after hyperoxia exposure than in lungs exposed to room air. Greater protein levels of ODC and OAT were found in lungs following hyperoxic exposure than in room air animals. α-SMA protein levels were found to be 7-fold greater in the hyperoxia exposed lungs than in room air lungs. In the hyperoxia exposed lungs there was evidence of greater pulmonary vascular wall cell proliferation by α-SMA immunohistochemistry than in room air lungs. Taken together, these data are consistent with a more proliferative vascular phenotype, and may explain the propensity of patients with bronchopulmonary dysplasia to develop pulmonary hypertension.

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 ( DDAH 1). We tested the hypothesis that DDAH 1 expression is essential for maintaining NO production in human fetal pulmonary microvascular endothelial cells (hf PMVEC ), such that knockdown of DDAH 1 expression will lead to decreased NO production resulting in less caspase‐3 activation and less tube formation. We found that hf PMVEC transfected with DDAH 1 si RNA had lower NO production than control, with no difference in eNOS protein levels between groups. hf PMVEC transfected with DDAH 1 si RNA had lower protein levels of cleaved caspase‐3 and ‐8 than control. Both DDAH 1 si RNA ‐ and ADMA ‐treated hf PMVEC had greater numbers of viable cells than controls. Angiogenesis was assessed using tube formation assays in matrigel, and tube formation was lower after either DDAH 1 si RNA transfection or ADMA treatment than controls. Addition of an NO donor restored cleaved caspase‐3 and ‐8 protein levels after DDAH 1 si RNA transfection in hf PMVEC to essentially the levels seen in scramble control. Addition of a putative caspase‐3 inhibitor to DDAH 1 si RNA 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 hf PMVEC , DDAH 1 is central to the regulation of NO ‐mediated caspase‐3 activation and the resultant apoptosis and angiogenesis. Our findings suggest that DDAH 1 may be a potential therapeutic target in pulmonary hypertensive disorders.

Pulmonary artery acceleration time (PAT) and PAT: ejection time (PATET) ratio are echocardiographic measurements of pulmonary arterial hypertension (PAH). These non-invasive quantitative measurements are ideal to follow longitudinally through the clinical course of PAH, especially as it relates to need for and/or response to treatment. This review article focuses on the current literature of PATET measurement for infants and children as it relates to shortening of the PATET ratio in PAH. At the same time, further development of PATET as an outcome measure for PAH in pre-clinical models, particularly mice, such that the field can move forward to human clinical studies that are both safe and effective. Here we present what is known about PATET in infants and children and discuss what is known in pre-clinical models with particular emphasis on neonatal mouse models. In both animal models and human disease, PATET allows for longitudinal measurements in the same individual, leading to more precise determinations of disease/model progression and/or response to therapy.

Bronchopulmonary dysplasia (BPD) is chronic lung disease in preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD and is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made by nitric oxide synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA). Dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Translational studies from our group identified a single nucleotide polymorphism (SNP) in the DDAH1 gene, rs480414, that was associated with a decreased risk for PH in BPD patients. Therefore, we aim to determine if the DDAH1 SNP rs480414 affects DDAH1 function in cell culture. Neonatal cord blood specimens were treated with Epstein‐Barr virus to select for B‐lymphocytes and transform into lymphoblastoid cell lines (LCLs) that were genotyped (SNP or wild‐type) at the rs480414 locus. We tested the hypothesis that in LCLs the SNP rs480414 results in a gain of function mutation in DDAH1, which would lead to greater NO‐mediated apoptosis as compared to DDAH1 wild‐type (WT). LCLs (WT, n=3 and SNP, n=3) were stimulated with phorbol myristate (PMA), IL‐4, and IL‐13 for 48 hours, protein was analyzed by western blot analysis for DDAH1, cleaved and total caspase‐3 and ‐8, and β‐actin. RT‐PCR was performed and expression of DDAH1 and iNOS mRNA was evaluated. Cell media was assayed for concentrations of nitrite using a chemiluminescence NO analyzer. Spectrophotometry was used to measure the conversion of ADMA to L‐citrulline. A standard L‐citrulline curve was used to calculate DDAH activity after normalization to protein concentration. LCLs with the DDAH1 SNP had similar levels of DDAH1 protein expression (n=3, p=0.58), similar mRNA expression (n=3, p=0.1), and similar DDAH activity (p=0.35) compared to DDAH1 WT. We found that LCLs with the DDAH1 SNP had similar iNOS mRNA expression (p=0.6), lower nitrite levels (p=0.04), lower cleaved caspase‐3 levels (p=0.04), and similar cleaved caspase‐8 levels (p=0.2) than DDAH1 WT LCLs. Contrary to our hypothesis, these finding suggests that the DDAH1 SNP rs480414 may result in a loss of function mutation, resulting in lower NO production and lower apoptosis in LCLs. This is the first evidence that the DDAH1 SNP rs480414 has an effect on cell function, however we speculate that pulmonary vascular endothelial cells will likely have a different physiology. Support or Funding Information This work was supported by NIH‐K08 HL129080.

Objective Pulmonary hypertension (PH) predicts mortality in patients with bronchopulmonary dysplasia (BPD), and chronic hyperoxia exposure contributes to BPD‐PH disease progression. BPD‐PH is characterized by aberrant pulmonary vascular wall proliferation/remodeling, which occludes the microvascular lumen, increasing pulmonary vascular resistance. Endogenous nitric oxide (NO) production by pulmonary vascular endothelial cells promotes apoptosis of pulmonary vascular endothelial and smooth muscle cells. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase‐1 (DDAH1) promotes NO‐mediated apoptosis. DDAH1 metabolizes the majority of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial NO synthase. Therefore, our hypothesis is that pharmacologically enhancing DDAH1 expression in hPMVEC will restore endogenous endothelial NO production. Methods hPMVEC were grown at 37 ̊C to ~70% confluence. Farnesoid X receptor (FXR) agonist, GW4064 (1.25 µM) was used to bind FXR element (FXRE) of the DDAH1 promoter to enhance transcription. hPMVEC were pre‐treated with FXR agonist for 24 hours and then exposed to 85%O2 or 21%O2 for an additional 24 hours. DDAH1 protein expression was analyzed by Western blot. Groups were compared by 1‐way ANOVA. Results In hPMVEC, hyperoxia exposure resulted in lower DDAH1 protein levels compared to hPMVEC grown in normoxia (N=6 in each group, p=0.003). Treatment with GW4064 prior to exposing to hyperoxia resulted in 4.3‐fold greater DDAH1 protein levels than in vehicle/85%O2 hPMVEC (N=6, p=0.044), and restored DDAH1 protein levels to 72% of that seen in the vehicle/21%O2exposed hPMVEC. Conclusions Chronic hyperoxia suppresses DDAH1 levels, and FXR agonist promotes DDAH1 recovery in hPMVEC. We speculate that promoting endogenous DDAH1 expression may prevent aberrant proliferation in the pulmonary vascular wall and thereby attenuate or prevent the vascular remodeling characteristic of pulmonary hypertension in BPD.