Although oxidative stress is known to contribute to endothelial dysfunction-associated systemic vascular disorders, its role in pulmonary vascular disorders is less clear. Our previous studies, using isolated pulmonary arteries taken from lambs with surgically created heart defect and increased pulmonary blood flow (Shunt), have suggested a role for reactive oxygen species (ROS) in the endothelial dysfunction of pulmonary hypertension, but in vivo data are lacking. Thus the initial objective of this study was to determine whether Shunt lambs had elevated levels of ROS generation and whether this was associated with alterations in antioxidant capacity. Our results indicate that superoxide, but not hydrogen peroxide, levels were significantly elevated in Shunt lambs. In addition, we found that the increase in superoxide generation was not associated with alterations in antioxidant enzyme expression or activity. These data suggested that there is an increase in superoxide generation rather than a decrease in scavenging capacity in the lung. Thus we next examined the expression of various subunits of the NADPH oxidase complex as a potential source of the superoxide production. Results indicated that the expression of Rac1 and p47(phox) is increased in Shunt lambs. We also found that the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduced dihydroethidium (DHE) oxidation in lung sections prepared from Shunt but not Control lambs. As DPI can also inhibit endothelial nitric oxide synthase (eNOS) superoxide generation, we repeated this experiment using a more specific NADPH oxidase inhibitor (apocynin) and an inhibitor of NOS (3-ethylisothiourea). Our results indicated that both inhibitors significantly reduced DHE oxidation in lung sections prepared from Shunt but not Control lambs. To further investigate the mechanism by which eNOS becomes uncoupled in Shunt lambs, we evaluated the levels of dihydrobiopterin (BH(2)) and tetrahydrobiopterin (BH(4)) in lung tissues of Shunt and Control lambs. Our data indicated that although BH(4) levels were unchanged, BH(2) levels were significantly increased. Finally, we demonstrated that the addition of BH(2) produced an increase in superoxide generation from purified, recombinant eNOS. In conclusion our data demonstrate that the development of pulmonary hypertension in Shunt lambs is associated with increases in oxidative stress that are not explained by decreases in antioxidant expression or activity. Rather, the observed increase in oxidative stress is due, at least in part, to increased expression and activity of the NADPH oxidase complex and uncoupled eNOS due to elevated levels of BH(2).

SM. Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 294: L46-L56, 2008. First published November 16, 2007 doi:10.1152/ajplung.00247.2007.-Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P Ͻ 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P Ͻ 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow. carnitine metabolism; oxidative stress THE DEVELOPMENT OF PULMONARY HYPERTENSION and its associated increased vascular reactivity are common accompaniments of congenital heart disease with increased pulmonary blood flow (3). Endothelial dysfunction is thought to be an early hallmark of pulmonary hypertension (4). There is increasing histological and physiological evidence that endothelial injury and the resulting aberration in the balance of its regulatory mechanisms play an important role in the development of pulmonary hypertension (4). Children with pulmonary hypertension have evidence of endothelial dysfunction as indicated by impaired endothelium-dependent relaxation in early disease and decreased endothelial nitric oxide synthase (eNOS) protein levels in late disease (11,23). However, data delineating the role of endotheliumderived NO in the disease have been less clear. For example, we (5) and others (72) have shown that pulmonary expression of NOS can be paradoxically inc...

Objective To determine the association between preoperative nutritional status and postoperative outcomes in children undergoing surgery for congenital heart defects (CHD). Methods Seventy-one patients with CHD were enrolled in a prospective, two-center cohort study. We adjusted for baseline risk differences using a standardized risk adjustment score for surgery for CHD. We assigned a World Health Organization Z-score for each subjects’ preoperative triceps skinfold measurement, an assessment of total body fat mass. We obtained preoperative plasma concentrations of markers of nutritional status (prealbumin, albumin) and myocardial stress (B-type natriuretic peptide, BNP). Associations between indices of preoperative nutritional status and clinical outcomes were sought. Results Subjects had a median (IQR) age of 10.2 (33) months. In the UCSF cohort, duration of mechanical ventilation (median 19 hours, IQR 29), length of ICU stay (median 5 days, IQR 5), duration of any continuous inotropic infusion (median 66 hours, IQR 72) and preoperative BNP levels (median 30 pg/mL, IQR 75) were associated with a lower preoperative triceps skinfold Z-score (p<0.05). Longer duration of any continuous inotropic infusion and higher preoperative BNP levels were also associated with lower preoperative prealbumin (12.1 ± 0.5 mg/dL) and albumin (3.2 ± 0.1) (p<0.05). Conclusions Lower total body fat mass and acute and chronic malnourishment are associated with worse clinical outcomes in children undergoing surgery for CHD at UCSF, a resource-abundant institution. There is an inverse correlation between total body fat mass and BNP levels. Duration of inotropic support and BNP increase concomitantly as measures of nutritional status decrease, supporting the hypothesis that malnourishment is associated with decreased myocardial function.

SM. Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress. Am J Physiol Lung Cell Mol Physiol 295: L756 -L766, 2008. First published August 29, 2008 doi:10.1152/ajplung.00146.2007.-Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 g/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO x), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P Ͻ 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P Ͻ 0.05). Conversely, NO x levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P Ͻ 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P Ͻ 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P Ͻ 0.05) and were greater in shunted lambs than controls at all ages (P Ͻ 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P Ͻ 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO. pulmonary circulation; oxidant stress; congenital heart disease; reactive oxygen species INFANTS AND CHILDREN with congenital cardiac defects that cause significantly increased pulmonary blood flow suffer morbidity due to early aberrations in pulmonary vascular function (17). In fact, this early pulmonary vascular dysfunction is often exacerbated in the immediate postoperative period, manifesting as increased vascular reactivity that may produce severe hypoxemia, acidosis, low cardiac output, and death if not treated immediately (7,11,31). A complete understanding of the mechanisms responsible for this pulmonary vascular dysfunction is lacking, but evidence suggests that aberrant nitric oxide (NO)-cGMP signaling and oxidative stress may participate (1,3,6,11,15,33,39,41).Basal NO production by the vascular endothelium is integral to the maintenance of the normal low resistance state of the pulmonary vasculature, and dynamic alterations in NO production modulate vascular relaxation and constriction in response to various stimuli. NO is produced in ...

B-type natriuretic peptide determinations might be a useful tool for clinicians caring for infants and children after surgical intervention for congenital heart disease.

erator-activated receptor type gamma (PPAR␥) is a subgroup of the PPAR transcription factor family. Recent studies indicate that loss of PPAR␥ is associated with the development of pulmonary hypertension (PH). We hypothesized that the endothelial dysfunction associated with PPAR␥ inhibition may play an important role in the disease process by altering cellular gene expression and signaling cascades. We utilized microarray analysis to determine if PPAR␥ inhibition induced changes in gene expression in pulmonary arterial endothelial cells (PAEC). We identified 100 genes and expressed sequence tags (ESTs) that were upregulated by Ͼ1.5-fold and 21 genes and ESTs that were downregulated by Ͼ1.3-fold (P Ͻ 0.05) by PPAR␥ inhibition. The upregulated genes can be broadly classified into four functional groups: cell cycle, angiogenesis, ubiquitin system, and zinc finger proteins. The genes with the highest fold change in expression: hyaluronan-mediated motility receptor (HMMR), VEGF receptor 2 (Flk-1), endothelial PAS domain protein 1 (EPAS1), basic fibroblast growth factor (FGF-2), and caveolin-1 in PAEC were validated by real time RT-PCR. We further validated the upregulation of HMMR, Flk-1, FGF2, and caveolin-1 by Western blot analysis. In keeping with the microarray results, PPAR␥ inhibition led to re-entry of cell cycle at G1/S phase and cyclin C upregulation. PPAR␥ inhibition also exacerbated VEGF-induced endothelial barrier disruption. Finally we confirmed the downregulation of PPAR␥ and the upregulation of HMMR, Flk-1, FGF2, and Cav-1 proteins in the peripheral lung tissues of an ovine model of PH. In conclusion, we have identified an array of endothelial genes modulated by attenuated PPAR␥ signaling that may play important roles in the development of PH. microarray; cell signaling; peroxisome proliferator-activated receptor PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-␥ (PPAR␥) was initially identified in adipose tissue and is a key regulator of lipid metabolism and glucose homeostasis (47, 50). Later, it was shown that PPAR␥ is also expressed in the vasculature including endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and monocytes/macrophages (13, 43). Upon activation by its ligands (fatty acids, arachidonic acid metabolites, and thiazolidinediones, etc), PPAR␥ forms heterodimers with the retinoid X receptor (RXR), and binds to specific PPAR response elements (PPRE) in the promoter region of its target genes, thereby regulating downstream gene expression (15,46). PPAR␥-mediated gene regulation can also be modulated by its interactions with specific co-activators and co-repressors (15, 26). PPAR␥ activation has been shown to alleviate atherosclerotic lesion formation and tumor progression (25, 30). The antiproliferative effects on VSMCs and antiangiogenic effects on tumor vasculogenesis by PPAR␥ activation seem to play important roles in these beneficial effects (9,31,53).Recently, it has been shown that loss of PPAR␥ is associated with pulmonary hypertension (PH) (35). PPAR␥ expression has been sho...

The perioperative changes in B-type natriuretic peptide levels and their ability to predict outcomes are lesion-specific. Characterization of these changes might be useful in caring for infants after congenital heart surgery.

Previous in vivo studies indicate that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity and that this decrease is associated with significant increases in pulmonary vascular resistance (PVR) upon the acute withdrawal of inhaled NO (rebound pulmonary hypertension). In vitro studies suggest that superoxide and peroxynitrite production during inhaled NO therapy may mediate these effects, but in vivo data are lacking. The objective of this study was to determine the role of superoxide in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy in vivo. In control lambs, 24 h of inhaled NO (40 ppm) decreased NOS activity by 40% (P<0.05) and increased endothelin-1 levels by 64% (P<0.05). Withdrawal of NO resulted in an acute increase in PVR (60.7%, P<0.05). Associated with these changes, superoxide and peroxynitrite levels increased more than twofold (P<0.05) following 24 h of inhaled NO therapy. However, in lambs treated with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) during inhaled NO therapy, there was no change in NOS activity, no increase in superoxide or peroxynitrite levels, and no increase in PVR upon the withdrawal of inhaled NO. In addition, endothelial NOS nitration was 18-fold higher (P<0.05) in control lambs than in PEG-SOD-treated lambs following 24 h of inhaled NO. These data suggest that superoxide and peroxynitrite participate in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy. Reactive oxygen species scavenging may be a useful therapeutic strategy to ameliorate alterations in endogenous NO signaling during inhaled NO therapy.