Background-Nitric oxide (NO) plays an important part in lowering pulmonary vascular resistance after birth, and in persistent pulmonary hypertension of the newborn (PPHN), NO-mediated dilation is dysfunctional. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) circulates in plasma, and its concentrations are elevated in certain cardiovascular diseases, including pulmonary hypertension. ADMA is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), the activity of which regulates ADMA concentrations and provides a mechanism for modulating NO synthase in vivo. We investigated the changes in expression and activity of the 2 isoforms of DDAH in lungs from newborn piglets both during normal development and in PPHN. Methods and Results-Using Western blotting, we showed that DDAHI expression did not change in the normal developing lung; however, DDAHII increased after birth and reached a peak at 1 day. This was reflected in an increase in total DDAH activity according to an L-citrulline assay. With pulmonary hypertension, no changes in DDAHI expression were observed, but DDAHII expression was markedly decreased compared with age-matched controls. Total DDAH activity was similarly reduced. Conclusions-These

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and is metabolised by dimethylarginine dimethylaminohydrolase (DDAH).Elevated levels of circulating ADMA correlate with various cardiovascular pathologies but there is less known on the cellular effects of altered DDAH activity.We modified DDAH activity in cells and measured the changes in ADMA levels, morphological phenotypes on Matrigel and expression of vascular endothelial growth factor (VEGF). DDAH over-expressing ECV304 cells secreted less ADMA and when grown in Matrigel, had enhanced tube formation compared to untransfected cells. VEGF mRNA levels were 2.1-fold higher in both ECV304 and murine endothelial cells (sEnd.1) over-expressing DDAH. In addition the DDAH inhibitor, S-2-amino-4(3-methylguanidino)butanoic acid (4124W 1mM), markedly reduced human umbilical vein endothelial cell (HUVEC) tube formation in vitro. We have found that upregulating DDAH activity lowers ADMA levels, increases the levels of VEGF mRNA in endothelial cells and enhances tube formation in an in vitro model, whilst blockade of DDAH reduces tube formation.

Objective. Endothelial injury is central to the pathogenesis of vasculitis. The purpose of this study was to assess how indices of endothelial injury and repair change during different stages of disease activity in children with primary systemic vasculitis (PSV).Methods. Fifty children with PSV, 17 children with nonvasculitic inflammatory diseases (pediatric inflammatory disease controls), 35 healthy age-and sexmatched pediatric controls, and 27 healthy adult controls were included in the study. Biomarkers examined were endothelial microparticles (EMPs), circulating endothelial cells (CECs), angiogenic growth factors, and endothelial progenitor cells (EPCs). EMP binding to annexin V, EMPs expressing CD144 or E-selectin, and EPCs expressing vascular endothelial growth factor receptor 2 (VEGFR-2), CD133, and CD34 were examined by flow cytometry. CECs were enumerated using immunomagnetic bead extraction techniques, and VEGF and angiopoietin 2 (Ang-2) were measured by enzyme-linked immunosorbent assay.

Objective— Nitric oxide is a key to numerous physiological and pathophysiological processes. Nitric oxide production is regulated endogenously by 2 methylarginines, asymmetric dimethylarginine (ADMA) and monomethyl- l -arginine. The enzyme that specifically metabolizes asymmetric dimethylarginine and monomethyl- l -arginine is dimethylarginine dimethylaminohydrolase (DDAH). The first isoform dimethylarginine dimethylaminohydrolase 1 has previously been shown to be an important regulator of methylarginines in both health and disease. This study explores for the first time the role of endogenous dimethylarginine dimethylaminohydrolase 2 in regulating cardiovascular physiology and also determines the functional impact of dimethylarginine dimethylaminohydrolase 2 deletion on outcome and immune function in sepsis. Approach and Results— Mice, globally deficient in Ddah2, were compared with their wild-type littermates to determine the physiological role of Ddah2 using in vivo and ex vivo assessments of vascular function. We show that global knockout of Ddah2 results in elevated blood pressure during periods of activity (mean [SEM], 118.5 [1.3] versus 112.7 [1.1] mm Hg; P =0.025) and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue asymmetric dimethylarginine (SEM) was 0.89 (0.06) versus 0.67 (0.05) μmol/L ( P =0.02) and systemic nitric oxide concentrations. In a model of severe polymicrobial sepsis, Ddah2 knockout affects outcome (120-hour survival was 12% in Ddah2 knockouts versus 53% in wild-type animals; P <0.001). Monocyte-specific deletion of Ddah2 results in a similar pattern of increased severity to that seen in globally deficient animals. Conclusions— Ddah2 has a regulatory role both in normal physiology and in determining outcome of severe polymicrobial sepsis. Elucidation of this role identifies a mechanism for the observed relationship between Ddah2 polymorphisms, cardiovascular disease, and outcome in sepsis.