eNOS function is developmentally regulated: uncoupling of eNOS occurs postnatally

At birth, the transition to gas breathing requires the function of endothelial vasoactive agents. We investigated the function of endothelial nitric oxide synthase (eNOS) in pulmonary artery (PA) vessels and endothelial cells isolated from fetal and young (4-wk) sheep. We found greater relaxations to the NOS activator A-23187 in 4-wk-old compared with fetal vessels and that the NOS inhibitor nitro-L-arginine blocked relaxations in both groups. Relaxations in 4-wk vessels were not blocked by an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, but were partially blocked by catalase. We therefore hypothesized that activation of eNOS produced reactive oxygen species in 4-wk but not fetal PA. To address this question, we studied NO and superoxide production by endothelial cells at baseline and following NOS stimulation with A-23187, VEGF, and laminar shear stress. Stimulation of NOS induced phosphorylation at serine 1177, and this event correlated with an increase in NO production in both ages. Upon stimulation of eNOS, fetal PA endothelial cells (PAEC) produced only NO. In contrast 4-wk-old PAEC produced superoxide in addition to NO. Superoxide production was blocked by L-NAME but not by apocynin (an NADPH oxidase inhibitor). L-Arginine increased NO production in both cell types but did not block superoxide production. Heat shock protein 90/eNOS association increased upon stimulation and did not change with developmental age. Cellular levels of total and reduced biopterin were higher in fetal vs. 4-wk cells. Sepiapterin [a tetrahydrobiopterin (BH4) precursor] increased basal and stimulated NO levels and completely blocked superoxide production. We conclude that the normal function of eNOS becomes uncoupled after birth, leading to a developmental adaptation of the pulmonary vascular system to produce oxygen species other than NO. We speculate this may be related to cellular production and/or maintenance of BH4 levels.     

Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells

We previously reported association of eNOS with actin increases eNOS activity. In the present study, regulation of activity of eNOS by actin cytoskeleton during endothelial growth was studied. We found eNOS activity in PAEC increased when cells grew from preconfluence to confluence. eNOS activity was much greater in PAEC in higher density than those in lower density, suggesting increase in eNOS activity during cell growth is caused by increase in cell density. Although eNOS protein contents were also increased when endothelial cells grew from preconfluence to confluence, magnitude of increase in eNOS activity was much higher than increase in eNOS protein content, suggesting posttranslational mechanisms played an important role in regulation of eNOS activity during endothelial growth. Confocal fluorescence microscopy revealed eNOS was colocalized with G-actin in preconfluent cells in perinuclear region, with both G-actin in perinuclear area and cortical F-actin in plasma membrane in confluent cells. There was more beta-actin coimmunoprecipitated with eNOS in Triton X-100-soluble fraction in confluent cells in later growth phase and in high density. Decrease in eNOS association with beta-actin by silencing beta-actin expression using beta-actin siRNA causes inhibition of eNOS activity, NO production, and endothelial monolayer wound repair in PAEC. Moreover, PAEC incubation with cytochalasin D and jasplakinolide resulted in increases in eNOS/actin association and in eNOS activity without changes in eNOS protein content. Yeast two-hybrid experiments suggested strong association between eNOS oxygenase domain and beta-actin. These results indicate increase in eNOS association with actin is responsible for greater eNOS activity in confluent PAEC.     

Intrauterine growth restriction decreases pulmonary alveolar and vessel growth and causes pulmonary artery endothelial cell dysfunction in vitro in fetal sheep

Intrauterine growth restriction (IUGR) increases the risk for bronchopulmonary dysplasia (BPD). Abnormal lung structure has been noted in animal models of IUGR, but whether IUGR adversely impacts fetal pulmonary vascular development and pulmonary artery endothelial cell (PAEC) function is unknown. We hypothesized that IUGR would decrease fetal pulmonary alveolarization, vascular growth, and in vitro PAEC function. Studies were performed in an established model of severe placental insufficiency and IUGR induced by exposing pregnant sheep to elevated temperatures. Alveolarization, quantified by radial alveolar counts, was decreased 20% (P < 0.005) in IUGR fetuses. Pulmonary vessel density was decreased 44% (P < 0.01) in IUGR fetuses. In vitro, insulin increased control PAEC migration, tube formation, and nitric oxide (NO) production. This response was absent in IUGR PAECs. VEGFA stimulated tube formation, and NO production also was absent. In control PAECs, insulin increased cell growth by 68% (P < 0.0001). Cell growth was reduced in IUGR PAECs by 29% at baseline (P < 0.01), and the response to insulin was attenuated (P < 0.005). Despite increased basal and insulin-stimulated Akt phosphorylation in IUGR PAECs, endothelial NO synthase (eNOS) protein expression as well as basal and insulin-stimulated eNOS phosphorylation were decreased in IUGR PAECs. Both VEGFA and VEGFR2 also were decreased in IUGR PAECs. We conclude that fetuses with IUGR are characterized by decreased alveolar and vascular growth and PAEC dysfunction in vitro. This may contribute to the increased risk for adverse respiratory outcomes and BPD in infants with IUGR.     

Nitric oxide augments fetal pulmonary artery endothelial cell angiogenesis in vitro

Growth and development of the lung normally occur in the low oxygen environment of the fetus. The role of this low oxygen environment on fetal lung endothelial cell growth and function is unknown. We hypothesized that low oxygen tension during fetal life enhances pulmonary artery endothelial cell (PAEC) growth and function and that nitric oxide (NO) production modulates fetal PAEC responses to low oxygen tension. To test this hypothesis, we compared the effects of fetal (3%) and room air (RA) oxygen tension on fetal PAEC growth, proliferation, tube formation, and migration in the presence and absence of the NO synthase (NOS) inhibitor N(omega)-nitro-l-arginine (LNA), and an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). Compared with fetal PAEC grown in RA, 3% O(2) increased tube formation by over twofold (P < 0.01). LNA treatment reduced tube formation in 3% O(2) but had no affect on tube formation in RA. Treatment with SNAP increased tube formation during RA exposure to levels observed in 3% O(2). Exposure to 3% O(2) for 48 h attenuated cell number (by 56%), and treatment with LNA reduced PAEC growth by 44% in both RA and 3% O(2). We conclude that low oxygen tension enhances fetal PAEC tube formation and that NO is essential for normal PAEC growth, migration, and tube formation. Furthermore, we conclude that in fetal cells exposed to the relative hyperoxia of RA, 21% O(2), NO overcomes the inhibitory effects of the increased oxygen, allowing normal PAEC angiogenesis and branching. We speculate that NO production maintains intrauterine lung vascular growth and development during exposure to low O(2) in the normal fetus. We further speculate that NO is essential for pulmonary angiogenesis in fetal animal exposed to increased oxygen tension of RA and that impaired endothelial NO production may contribute to the abnormalities of angiogenesis see in infants with bronchopulmonary dysplasia.     

Developmental changes in nitric oxide synthase isoform expression and nitric oxide production in fetal baboon lung

Nitric oxide (NO), produced by NO synthase (NOS), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term = 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160-175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.     

Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O(2)(*-) scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O(2)(*-) in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O(2)(*-) in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.     

Asymmetric dimethylarginine inhibits HSP90 activity in pulmonary arterial endothelial cells: role of mitochondrial dysfunction

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.     

Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells

The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2-4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 microM) for 2-4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 microM) for 2-4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.     

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.     

Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.     

Bone morphogenetic protein receptor II is a novel mediator of endothelial nitric-oxide synthase activation

Activation of bone morphogenetic protein (BMP) receptor II (BMPRII) promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. Mutations to BMPRII are associated with the development of pulmonary arterial hypertension (PAH). Endothelial dysfunction, including decreased endothelial nitric-oxide synthase (eNOS) activity and loss of bioactive nitric oxide (NO), plays a prominent role in the development of PAH. We hypothesized that stimulation of BMPRII promotes normal PAEC function by activating eNOS. We report that BMPRII ligands, BMP2 and BMP4, (i) stimulate eNOS phosphorylation at a critical regulatory site, (ii) increase eNOS activity, and (iii) result in canonical changes in eNOS protein-protein interactions. The stimulation of eNOS activity by BMPRII ligands was largely dependent on protein kinase A (PKA) activation, as demonstrated using the PKA inhibitors H89 and myristoylated PKI(6-22) amide. PAEC migration stimulated by BMP2 and BMP4 was inhibited by the NOS inhibitor l-nitroarginine methyl ester, providing functional evidence of eNOS activation. Furthermore, BMP2 and BMP4 failed to stimulate eNOS phosphorylation when BMPRII was knocked down by siRNA. Most important to the pathophysiology of the disease, BMP2 and BMP4 failed to stimulate eNOS phosphorylation in PAECs isolated from patients with mutations in the BMPR2 gene. These data demonstrate a new action of BMPs/BMPRII in the pulmonary endothelium and provide novel mechanistic insight into the pathogenesis of PAH.     

Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.     

Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

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 microg/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 < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 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 NO synthase expression is attenuated in a fetal baboon model of chronic lung disease

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (VeNO(STP)) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups. VeNO(STP) remained stable in the range of 0.5-1.0 nl x kg(-1) x min(-1) from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.     

Substrate Inhibition of Nitric Oxide Synthase in Pulmonary Artery Endothelial Cells in Culture

The effects of arginine on nitric oxide synthase (NOS) activity and NO production were studied in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 0–100 μM arginine increased NO production, detected as nitrite in the culture medium, in a dose-dependent manner. In contrast, incubation with concentrations of arginine in excess of 100 μM resulted in a reversible dose-dependent inhibition of NO production, even though intracellular arginine content increased in these cells. The NOS enzyme kinetics were studied in a total membrane preparation and in purified NOS protein and revealed that theK_mof arginine as a substrate for NOS is 3–5 μM, theV_maxoccurred at 100 μM arginine, and substrate inhibition occurred at >100 μM arginine. Oxyhemoglobin, carboxy-PTIO, catalase, SOD, citrulline, hydroxyarginine, and -arginine did not change NOS kinetics. These results indicate that substrate inhibition of eNOS exists in porcine PAEC in vitro.     

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity -- role for iron-dependent oxidative stress

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H(2)O(2)). Here we examined the implications of H(2)O(2) for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H(2)O(2) exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H(2)O(2) had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H(2)O(2) enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H(2)O(2) impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H(2)O(2) treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H(2)O(2) increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H(2)O(2)-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H(2)O(2) impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.     

VEGF increases the proliferative capacity and eNOS/NO levels of endothelial progenitor cells through the calcineurin/NFAT signalling pathway

We have investigated whether VEGF (vascular endothelial growth factor) regulates the proliferative capacity and eNOS (endothelial nitric oxide synthase)/NO (nitric oxide) pathway of EPCs (endothelial progenitor cells) by activating CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) signalling. EPCs were obtained from cultured mononuclear cells isolated from the peripheral blood of healthy adults. Treatment with VEGF (50 ng/ml) potently promoted CaN enzymatic activity, activation of NFAT2, cell proliferation, eNOS protein expression and NO production. Pretreatment with cyclosporin A (10 μg/ml), a pharmacological inhibitor of CaN or 11R-VIVIT, a special inhibitor of NFAT, completely abrogated the aforementioned effects of VEGF treatment and increased apoptosis. The results indicate that VEGF treatment promotes the proliferative capacity of human EPCs by activating CaN/NFAT signalling leading to increased eNOS protein expression and NO production.     

Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung

Endothelial nitric oxide (NO) synthase (eNOS) produces NO, which contributes to vascular reactivity in the fetal lung. Pulmonary vasoreactivity develops during late gestation in the ovine fetal lung, during the period of rapid capillary and alveolar growth. Although eNOS expression peaks near birth in the fetal rat, lung capillary and distal air space development occur much later than in the fetal lamb. To determine whether lung eNOS expression in the lamb differs from the timing and pattern reported in the rat, we measured eNOS mRNA and protein by Northern and Western blot analyses and NOS activity by the arginine-to-citrulline conversion assay in lung tissue from fetal, newborn, and maternal sheep. Cellular localization of eNOS expression was determined by immunohistochemistry. eNOS mRNA, protein, and activity were detected in samples from all ages, and eNOS was expressed predominantly in the vascular endothelium. Lung eNOS mRNA expression increases from low levels at 70 days gestation to peak at 113 days and remains high for the rest of fetal life. Newborn eNOS mRNA expression does not change from fetal levels but is lower in the adult ewe. Lung eNOS protein expression in the fetus rises and peaks at 118 days gestation but decreases before birth. eNOS protein expression rises in the newborn period but is lower in the adult. Lung NOS activity also peaks at 118 days gestation in the fetus before falling in late gestation and remaining low in the newborn and adult. We conclude that the pattern of lung eNOS expression in the sheep differs from that in the rat and may reflect species-related differences in lung development. We speculate that the rise in fetal lung eNOS may contribute to the marked lung growth and angiogenesis that occurs during the same period of time.