Human pulmonary microvascular endothelial cell DDAH1-mediated nitric oxide production promotes pulmonary smooth muscle cell apoptosis in co-culture.

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in preterm infants, and pulmonary hypertension (PH) develops in 25%-40% of patients with BPD, 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), whereas DDAH1 overexpression will have the opposite effect. hPMVECs were transfected with small interfering RNA targeting DDAH1 (siDDAH1)/scramble or adenoviral vector containing DDAH1 (AdDDAH1)/AdGFP for 24 h and co-cultured for 24 h with hPASMC. Analyses included Western blot for cleaved and total caspase-3, caspase-8, caspase-9, ß-actin; trypan blue exclusion for viable cell numbers; terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL); and BrdU incorporation. Small interfering RNA targeting DDAH1 (siDDAH1) transfected into hPMVEC resulted in lower media nitrites, cleaved caspase-3 and caspase-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 caspase-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 were treated with hemoglobin to sequester NO. In conclusion, hPMVEC-DDAH1-mediated NO production positively regulates hPASMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.NEW & NOTEWORTHY BPD-PH is characterized by vascular remodeling. NO is an apoptotic mediator made in the pulmonary endothelium by eNOS. ADMA is an endogenous eNOS inhibitor metabolized by DDAH1. EC-DDAH1 overexpression resulted in greater cleaved caspase-3 and caspase-8 protein expression and lower viable cell numbers in co-cultured SMC. After NO sequestration, SMC viable cell numbers partially recovered despite EC-DDAH1 overexpression. EC-DDAH1-mediated NO production positively regulates SMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.

DDAH1 SNP rs480414 that protects against the development of pulmonary hypertension in bronchopulmonary dysplasia results in lower nitric oxide production in neonatal cord blood-derived lymphoblastoid cell lines.

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 ß-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.

PATET ratio by Doppler echocardiography: noninvasive detection of pediatric pulmonary arterial hypertension.

Pulmonary artery acceleration time (PAT) and PAT: ejection time (PATET) ratio are echocardiographic measurements of pulmonary arterial hypertension (PAH). These noninvasive quantitative measurements are ideal to follow longitudinally through the clinical course of PAH, especially as it relates to the 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 the shortening of the PATET ratio in PAH. At the same time, further development of PATET as an outcome measure for PAH in preclinical 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 preclinical 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. IMPACT: PATET ratio is a quantitative measurement by a noninvasive technique, Doppler echocardiography, providing clinicians a more precise/accurate, safe, and longitudinal assessment of pediatric PAH. We present a brief history/state of the art of PATET ratio to predict PAH in adults, children, infants, and fetuses, as well as in small animal models of PAH. In a preliminary study, PATET shortened by 18% during acute hypoxic exposure compared to pre-hypoxia. Studies are needed to establish PATET, especially in mouse models of disease, such as bronchopulmonary, as a routine measure of PAH.

Differential effects of the Src family tyrosine kinases Yes and Fyn on lipopolysaccharide-induced lung injury in mice.

Endothelial cell apoptosis is an early event in the development of acute lung injury (ALI). We have previously found that the Src family tyrosine kinase (STK) Yes activates caspase-3, whereas the STK Fyn inhibits caspase-3 activation in cultured pulmonary endothelial cells. We hypothesized that deficiency in Yes or Fyn in mice would have differential effects on lipopolysaccharide (LPS)-induced ALI. Mice were treated with LPS (10 mg/kg ip) for 24 h. Histological evidence of lung injury was greater in LPS-treated wild-type mice than in vehicle-treated wild-type mice, and the LPS-induced histological evidence of lung injury was attenuated in yes-/- mice and enhanced in fyn-/- mice. In wild-type or fyn-/- mice, LPS resulted in greater lung wet-to-dry weight ratios than in controls, whereas in yes-/- mice lung, wet-to-dry weight was similar between LPS and controls. LPS-exposed fyn-/- mice had greater respiratory system resistance and lower respiratory system compliance than did LPS-exposed wild-type mice. TUNEL positive cells in the lung following LPS treatment were greater in the fyn-/- mice and lower in the yes-/- mice compared with that in the wild-type mice. Following LPS treatment lung protein levels of PECAM-1 were lower in fyn-/- mice than in controls or yes-/- mice. LPS treatment increased cleaved caspase-3 protein levels in wild-type mice, whereas LPS-induced caspase-3 activation was attenuated in yes-/- mice and enhanced in fyn-/- mice. These results indicate that LPS-induced ALI is positively mediated via Yes-related mechanisms and negatively mediated by Fyn-related mechanisms.

Dual-specificity phosphatase (DUSP) genetic variants predict pulmonary hypertension in patients with bronchopulmonary dysplasia.

BACKGROUND: Pulmonary hypertension (PH) in patients with bronchopulmonary dysplasia (BPD) results from vasoconstriction and/or vascular remodeling, which can be regulated by mitogen-activated protein kinases (MAPKs). MAPKs are deactivated by dual-specificity phosphatases (DUSPs). We hypothesized that single-nucleotide polymorphisms (SNPs) in DUSP genes could be used to predict PH in BPD. METHODS: Preterm infants diagnosed with BPD (n = 188) were studied. PH was defined by echocardiographic criteria. Genomic DNA isolated from patient blood samples was analyzed for 31 SNPs in DUSP genes. Clinical characteristics and minor allele frequencies were compared between BPD-PH (cases) and BPD-without PH (control) groups. Biomarker models to predict PH in BPD using clinical and SNP data were tested by calculations of area under the ROC curve. RESULTS: In our BPD cohort, 32% (n = 61) had PH. Of the DUSP SNPs evaluated, DUSP1 SNP rs322351 was less common, and DUSP5 SNPs rs1042606 and rs3793892 were more common in cases than in controls. The best fit biomarker model combines clinical and DUSP genetic data with an area under the ROC curve of 0.76. CONCLUSION: We identified three DUSP SNPs as potential BPD-PH biomarkers. Combining clinical and DUSP genetic data yields the most robust predictor for PH in BPD.

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.

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

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% O2 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.

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

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 Nω-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.

The Src family tyrosine kinases src and yes have differential effects on inflammation-induced apoptosis in human pulmonary microvascular endothelial cells.

Endothelial cells are essential for normal lung function: they sense and respond to circulating factors and hemodynamic alterations. In inflammatory lung diseases such as acute respiratory distress syndrome, endothelial cell apoptosis is an inciting event in pathogenesis and a prominent pathological feature. Endothelial cell apoptosis is mediated by circulating inflammatory factors, which bind to receptors on the cell surface, activating signal transduction pathways, leading to caspase-3-mediated apoptosis. We hypothesized that yes and src have differential effects on caspase-3 activation in human pulmonary microvascular endothelial cells (hPMVEC) due to differential downstream signaling effects. To test this hypothesis, hPMVEC were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls. After recovery, the hPMVEC were treated with cytomix (LPS, IL-1ß, TNF-α, and IFN-γ). Treatment with cytomix induced activation of the extracellular signal-regulated kinase (ERK) pathway and caspase-3-mediated apoptosis. Treatment with siRNAsrc blunted cytomix-induced ERK activation and enhanced cleaved caspase-3 levels, while treatment with siRNAyes enhanced cytomix-induced ERK activation and attenuated levels of cleaved caspase-3. Inhibition of the ERK pathway using U0126 enhanced cytomix-induced caspase-3 activity. Treatment of hPMVEC with cytomix induced Akt activation, which was inhibited by siRNAsrc. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway using LY294002 prevented cytomix-induced ERK activation and augmented cytomix-induced caspase-3 cleavage. Together, our data demonstrate that, in hPMVEC, yes activation blunts the ERK cascade in response to cytomix, resulting in greater apoptosis, while cytomix-induced src activation induces the phosphatidylinositol 3-kinase pathway, which leads to activation of Akt and ERK and attenuation of apoptosis.

A single nucleotide polymorphism in the dimethylarginine dimethylaminohydrolase gene is associated with lower risk of pulmonary hypertension in bronchopulmonary dysplasia.

AIM: Pulmonary hypertension (PH) develops in 25-40% of bronchopulmonary dysplasia (BPD) patients, substantially increasing mortality. We have previously found that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) production, is elevated in patients with BPD-associated PH. ADMA is metabolised by N(á´³) ,N(á´³) -dimethylarginine dimethylaminohydrolase (DDAH). Presently, we test the hypothesis that there are single nucleotide polymorphisms (SNPs) in DDAH1 and/or DDAH2 associated with the development of PH in BPD patients. METHODS: BPD patients were enrolled (n = 98) at Nationwide Children's Hospital. Clinical characteristics and 36 SNPs in DDAH1 and DDAH2 were compared between BPD-associated PH patients (cases) and BPD-alone patients (controls). RESULTS: In BPD patients, 25 (26%) had echocardiographic evidence of PH (cases). In this cohort, DDAH1 wild-type rs480414 was 92% sensitive and 53% specific for PH in BPD, and the DDAH1 SNP rs480414 decreased the risk of PH in an additive model of inheritance (OR = 0.39; 95% CI [0.18-0.88], p = 0.01). CONCLUSION: The rs480414 SNP in DDAH1 may be protective against the development of PH in patients with BPD. Furthermore, the DDAH1 rs480414 may be a useful biomarker in developing predictive models for PH in patients with BPD.

Plasma asymmetric dimethylarginine levels are increased in neonates with bronchopulmonary dysplasia-associated pulmonary hypertension.

OBJECTIVE: To test the hypothesis that levels of the endogenous inhibitor of nitric oxide production, asymmetric dimethylarginine (ADMA), would be greater in preterm infants with bronchopulmonary dysplasia (BPD)-associated pulmonary hypertension (PH) than in infants with BPD alone. STUDY DESIGN: A case-control study of 23 patients with both BPD and PH (cases) and 95 patients with BPD but no evidence of PH (controls). Levels of ADMA were compared between cases and controls by t test. RESULTS: Patients with both BPD and PH had greater plasma levels of ADMA than patients with BPD alone (P = .04). In samples drawn before 28 days of life, greater levels of ADMA were again found in cases compared with controls (P = .02). The plasma arginine-to-ADMA ratio was lower in cases than in controls (P = .03), suggesting a greater likelihood of inhibition of nitric oxide production in patients with both BPD and PH than in patients with BPD alone. CONCLUSION: In this neonatal BPD cohort, ADMA levels are increased in patients with BPD who develop PH. We speculate that ADMA may be both a biomarker and a potential therapeutic target for preterm infants with BPD-associated PH.

Fellows as teachers: a model to enhance pediatric resident education.

OBJECTIVE: Pressures on academic faculty to perform beyond their role as educators has stimulated interest in complementary approaches in resident medical education. While fellows are often believed to detract from resident learning and experience, we describe our preliminary investigations utilizing clinical fellows as a positive force in pediatric resident education. Our objectives were to implement a practical approach to engage fellows in resident education, evaluate the impact of a fellow-led education program on pediatric resident and fellow experience, and investigate if growth of a fellowship program detracts from resident procedural experience. METHODS: This study was conducted in a neonatal intensive care unit (NICU) where fellows designed and implemented an education program consisting of daily didactic teaching sessions before morning clinical rounds. The impact of a fellow-led education program on resident satisfaction with their NICU experience was assessed via anonymous student evaluations. The potential value of the program for participating fellows was also evaluated using an anonymous survey. RESULTS: The online evaluation was completed by 105 residents. Scores were markedly higher after the program was implemented in areas of teaching excellence (4.44 out of 5 versus 4.67, p<0.05) and overall resident learning (3.60 out of 5 versus 4.61, p<0.001). Fellows rated the acquisition of teaching skills and enhanced knowledge of neonatal pathophysiology as the most valuable aspects of their participation in the education program. The anonymous survey revealed that 87.5% of participating residents believed that NICU fellows were very important to their overall training and education. CONCLUSIONS: While fellows are often believed to be a detracting factor to residency training, we found that pediatric resident attitudes toward the fellows were generally positive. In our experience, in the specialty of neonatology a fellow-led education program can positively contribute to both resident and fellow learning and satisfaction. Further investigation into the value of utilizing fellows as a positive force in resident education in other medical specialties appears warranted.