Evidence for enhanced eNOS function in coronary microvessels during the second window of protection

Nitric oxide (NO) derived from endothelial NO synthase (NOS) (eNOS) has been identified as a trigger for the second window of protection (SWOP), but its role as a mediator during the SWOP is a matter of debate. Eighteen mongrel dogs were chronically instrumented to measure left ventricular function, coronary blood flow, and wall thickening. Myocardial preconditioning was induced by 10 min coronary artery occlusion. After 24 h of reperfusion (during the SWOP), the hearts were excised. Coronary microvessels were isolated and incubated in presence of 1) the endothelium-dependent agonists carbachol and bradykinin, 2) the calcium ionophore A23187, and 3) the angiotensin-converting enzyme (ACE) inhibitors enalaprilat and ramiprilat. Nitrite, a metabolite of NO, was measured. Under baseline conditions, nitrite production in microvessels from SWOP was 30% higher than that from normal (96 +/- 4 vs. 74 +/- 3 pmol/mg, P < 0.01, respectively). Nitrite production in response to carbachol, bradykinin, and A23187 was also enhanced in microvessels from SWOP (P < 0.05). These enhanced responses were abolished by N(G)-nitro-l-arginine methyl ester (l-NAME) or the endothelial receptor-specific antagonists atropine and HOE-140. The level of eNOS protein in the SWOP myocardium was twofold higher than that in the non-SWOP myocardium. Nitrite production in response to the ACE inhibitors was greater in microvessels from SWOP. These effects were blocked by l-NAME, HOE-140, or dichloroisocoumarin (which inhibits kinin formation). We found that a brief ischemic episode induced delayed, enhanced NO production in coronary microvessels and an upregulation of eNOS protein. These findings suggest that eNOS is a mediator during the SWOP. The ability of ACE inhibitors to enhance NO release during the SWOP points to an additional clinical application for these drugs.     

Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs

Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg. kg(-1). day(-1)) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control (P < 0.05) and veratrine- (activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced (P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NO(x)) levels increased from 5.22 +/- 1.2 to 7. 79 +/- 1.3 microM (P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented (P < 0.05); resting in vivo myocardial oxygen consumption (MVO(2)) decreased from 6.8 +/- 0.6 to 5.9 +/- 0.4 ml/min (P < 0.05); NO-dependent regulation of MVO(2) in response to NO agonists was augmented in isolated myocardial segments (P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MVO(2).     

Hypoxic conditioning suppresses nitric oxide production upon myocardial reperfusion

Physiologically modulated concentrations of nitric oxide (NO) are generally beneficial, but excessive NO can injure myocardium by producing cytotoxic peroxynitrite. Recently we reported that intermittent, normobaric hypoxia conditioning (IHC) produced robust cardioprotection against infarction and lethal arrhythmias in a canine model of coronary occlusion-reperfusion. This study tested the hypothesis that IHC suppresses myocardial nitric oxide synthase (NOS) activity and thereby dampens explosive, excessive NO formation upon reperfusion of occluded coronary arteries. Mongrel dogs were conditioned by a 20 d program of IHC (FIO(2) 9.5-10%; 5-10 min hypoxia/cycle, 5-8 cycles/d with intervening 4 min normoxia). One day later, ventricular myocardium was sampled for NOS activity assays, and immunoblot detection of the endothelial NOS isoform (eNOS). In separate experiments, myocardial nitrite (NO(2)(-)) release, an index of NO formation, was measured at baseline and during reperfusion following 1 h occlusion of the left anterior descending coronary artery (LAD). Values in IHC dogs were compared with respective values in non-conditioned, control dogs. IHC lowered left and right ventricular NOS activities by 60%, from 100-115 to 40-45 mU/g protein (P < 0.01), and decreased eNOS content by 30% (P < 0.05). IHC dampened cumulative NO(2)(-) release during the first 5 min reperfusion from 32 +/- 7 to 14 +/- 2 mumol/g (P < 0.05), but did not alter hyperemic LAD flow (15 +/- 2 vs. 13 +/- 2 ml/g). Thus, IHC suppressed myocardial NOS activity, eNOS content, and excessive NO formation upon reperfusion without compromising reactive hyperemia. Attenuation of the NOS/NO system may contribute to IHC-induced protection of myocardium from ischemia-reperfusion injury.     

Effects of pulsatile shear stress on nitric oxide production and endothelial cell nitric oxide synthase expression by ovine fetoplacental artery endothelial cells

Placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression increase during pregnancy. Shear stress, the frictional force exerted on endothelial cells by blood flow, stimulates vessel dilation, endothelial NO production, and eNOS expression. In order to study the effects of pulsatile flow/shear stress, we adapted Cellco CELLMAX artificial capillary modules to study ovine fetoplacental artery endothelial (OFPAE) cells for NO production and eNOS expression. OFPAE cells were grown in the artificial capillary modules at 3 dynes/cm2. Confluent cells were then exposed to 10, 15, or 25 dynes/cm2 for up to 24 h. NO production by OFPAE cells exposed to pulsatile shear stress was inhibited to nondetectable levels by the NOS inhibitor l-NMMA and reversed by excess NOS substrate l-arginine. NO production and expression of eNOS mRNA and protein by OFPAE cells were elevated by shear stress in a graded fashion (P < 0.05). The rise in NO production with 25 dynes/cm2 shear stress (8-fold) was greater (P < 0.05) than that observed for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). The acute shear stress-induced rise in NO production by OFPAE cells was via eNOS activation, whereas the prolonged NO rise occurred by elevations in both eNOS expression and enzyme activation. Thus, elevations of placental blood flow and physiologic shear stress may be partly responsible for the increases in placental arterial endothelial eNOS expression and NO production during pregnancy.     

Restoration of impaired endothelium-dependent coronary vasodilation in failing heart: role of eNOS phosphorylation and CGMP/cGK-I signaling

In congestive heart failure (CHF), coronary vascular relaxation is associated with endothelial dysfunction and nitric oxide (NO) deficiency. This study explored the reversibility of this process in hearts recovering from CHF and its related mechanisms. Dogs were chronically instrumented to measure cardiac function and coronary blood flow (CBF). Heart failure was induced by right ventricular pacing at 240 beats/min for 3-4 wk, and cardiac recovery (CR) was allowed by the termination of cardiac pacing for 3-4 wk after the development of CHF, in which left ventricular contractile function was restored by 80-90%. The endothelium-dependent CBF response to bradykinin and acetylcholine was depressed in CHF and fully restored in CR. Myocardial NOx (nitrate/nitrite), endothelial NO synthase (eNOS) mRNA expression, total protein, and phosphorylated eNOS decreased significantly in failing hearts. However, myocardial NOx recovered to 78% of control and phosphorylated eNOS was fully restored in CR, despite the fact that eNOS mRNA expression and protein levels remained lower than control. Furthermore, the endothelium-independent CBF response to nitroglycerin did not change in CHF; however, it increased by 75% in CR, in conjunction with a near threefold increase in the phosphorylation of vasodilation-stimulated phosphoprotein (VASP) at Ser(239) in recovering hearts. Thus the complete restoration of endothelium-dependent coronary vascular relaxation during cardiac recovery from CHF was mediated by 1) a restoration of phosphorylated eNOS for partial recovery of the NO production and 2) an increase in cGMP/cGMP-dependent protein kinase-I pathway signaling activity for the enhancement of coronary vascular smooth muscle relaxation in response to NO.     

Growth-related oncogene-alpha induces endothelial dysfunction through oxidative stress and downregulation of eNOS in porcine coronary arteries

Growth-related oncogene-alpha (GRO-alpha) is a member of the CXC chemokine family, which is involved in the inflammatory process including atherosclerosis. We hypothesized that GRO-alpha may affect endothelial functions in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs). Vasomotor function was analyzed in response to thromboxane A2 analog U-46619 for contraction, bradykinin for endothelium-dependent vasorelaxation, and sodium nitroprusside (SNP) for endothelium-independent vasorelaxation. In response to 10(-6) M bradykinin, GRO-alpha (50 and 100 ng/ml) significantly reduced endothelium-dependent vasorelaxation by 34.73 and 48.8%, respectively, compared with controls (P < 0.05). There were no changes in response to U-46619 or SNP between treated and control groups. With the lucigenin-enhanced chemiluminescence assay, superoxide anion production in GRO-alpha-treated vessels (50 and 100 ng/ml) was significantly increased by 50 and 86%, respectively, compared with controls (P < 0.05). With real-time PCR analysis, endothelial nitric oxide synthase (eNOS) mRNA levels in porcine coronary arteries and HCAECs after GRO-alpha treatment were significantly decreased compared with controls (P < 0.05). The eNOS protein levels by both immunohistochemistry and Western blot analyses were also decreased in GRO-alpha-treated vessels. Antioxidant seleno-l-methionine and anti-GRO-alpha antibody effectively blocked these effects of GRO-alpha on both porcine coronary arteries and HCAECs. In addition, GRO-alpha immunoreactivity was substantially increased in the atherosclerotic regions compared with nonatherosclerotic regions in human coronary arteries. Thus GRO-alpha impairs endothelium-dependent vasorelaxation in porcine coronary arteries through a mechanism of overproduction of superoxide anion and downregulation of eNOS. GRO-alpha may contribute to human coronary artery disease.     

Mechanism of coronary vasodilation to insulin and insulin-like growth factor I is dependent on vessel size

Insulin and insulin-like growth factor I (IGF-I) influence numerous metabolic and mitogenic processes; these hormones also have vasoactive properties. This study examined mechanisms involved in insulin- and IGF-I-induced dilation in canine conduit and microvascular coronary segments. Tension of coronary artery segments was measured after constriction with PGF(2alpha). Internal diameter of coronary microvessels (resting diameter = 112.6+/-10.1 microm) was measured after endothelin constriction. Vessels were incubated in control (Krebs) solution and were treated with N(omega)-nitro-L-arginine (L-NA), indomethacin, or K(+) channel inhibitors. After constriction, cumulative doses of insulin or IGF-I (0.1-100 ng/ml) were administered. In conduit arteries, insulin produced modest maximal relaxation (32 +/- 5%) compared with IGF-I (66+/-12%). Vasodilation was attenuated by nitric oxide synthase (NOS) and cyclooxygenase inhibition and was blocked with KCl constriction. Coronary microvascular relaxation to insulin and IGF-I was not altered by L-NA, indomethacin, tetraethylammonium chloride, glibenclamide, charybdotoxin, and apamin; however, tetrabutylammonium chloride attenuated the response. In conclusion, insulin and IGF-I cause vasodilation in canine coronary conduit arteries and microvessels. In conduit vessels, NOS/cyclooxygenase pathways are involved in the vasodilation. In microvessels, relaxation to insulin and IGF-I is not mediated by NOS/cyclooxygenase pathways but rather through K(+)-dependent mechanisms.     

Signaling cascade that mediates endothelial nitric oxide synthase activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) induces activation of nitric oxide-synthase (NOS). Aims: to identify the isoform of NOS involved in ANP effects, to study whether ANP modifies NOS expression and to investigate the signaling pathways and receptors involved in NOS stimulation. NOS activation induced by ANP would be mediated by endothelial NOS (eNOS) since neuronal or inducible NOS inhibition did not alter ANP effect. The peptide induced no changes in eNOS protein expression. NOS activity stimulated by ANP, in the kidney, aorta and left ventricle, was partially abolished by the NPR-A/B antagonist, as well as PKG inhibition, but no difference in atria was observed. 8-Br-cGMP partially mimicked the effect of ANP on NOS in all tissues. NOS stimulation by ANP in atria disappeared when G protein was inhibited, but this effect was partial in the other tissues. Calmodulin antagonist abolished NOS stimulation via ANP. Inhibition of the PLC, PKC or PI3 kinase/Akt pathway failed to alter NOS activation induced by ANP. ANP would activate eNOS in the aorta, heart and kidney without modifying the expression of the enzyme. ANP would interact with NPR-C coupled via G proteins leading to the activation of Ca(2+)-calmodulin-dependent NOS in atria; while in ventricle, aorta and kidney, ANP could also interact with NPR-A/B, increasing cGMP, which in turns activates PKG to stimulate eNOS.     

Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat

Thisstudy examined mRNA and protein expressions of neuronal (nNOS),inducible (iNOS), and endothelial nitric oxide synthases (eNOS) inperipheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. Onesciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve werequantitatively measured by competitive PCR, and protein was determinedby Western blotting and immunohistochemical staining. The resultsshowed that, after ischemia (2 h), both nNOS and eNOS proteinexpressions decreased. After I/R (2 h of ischemia followed by3 h of reperfusion), expression of both nNOS and eNOS mRNA andprotein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal thedynamic expression of individual NOS isoforms during the course of I/Rinjury. An understanding of this modulation on a cellular and molecularlevel may lead to understanding the mechanisms of I/R injury and tomethods of ameliorating peripheral nerve injury.

     

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.     

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.     

Differential regulation of glomerular and interstitial endothelial nitric oxide synthase expression in the kidney of hibernating ground squirrel.

Hibernating animals transiently reduce renal function during their hypothermic periods (torpor), while completely restoring it during their periodical rewarming to euthermia (arousal). Moreover, structural integrity of the kidney is preserved throughout the hibernation. Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) is a crucial vasodilatory mediator and a protective factor in the kidney. We investigated renal NOS expression in hibernating European ground squirrels after 1 day and 7 days of torpor (torpor short, TS, and torpor long, TL, respectively), at 1.5 and at 10 h of rewarming (arousal short, AS, and arousal long, AL, respectively), and in continuously euthermic animals after hibernation (EU). For that purpose, we performed NOS activity assay, immunohistochemistry and real-time PCR analysis. Immunohistochemistry revealed a decreased glomerular eNOS expression in hibernating animals (TS, TL, AS, and AL) compared to non-hibernating animals (EU, p < 0.05), whereas no difference was found in the expression of interstitial eNOS. Expression of iNOS and nNOS did not differ between all groups. The reduced glomerular eNOS was associated with a significantly lower eNOS mRNA levels and NOS activity of whole kidney during torpor and arousal (TS, TL, AS, and AL) compared to EU. In all methods used, torpid and aroused squirrels did not differ. These results demonstrate differential regulation of eNOS in glomeruli and interstitium of hibernating animals, which is unaffected during arousal. The differential regulation of eNOS may serve to reduce ultrafiltration without jeopardizing tubular structures during hibernation.     

Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats.

Increased vascular nitric oxide (NO) production has been implicated in the pathogenesis of the hyperdynamic circulation in liver cirrhosis. This study investigated the expression of three isoforms of NO synthase (NOS) in rat cirrhotic livers. Cirrhosis was induced by chronic bile duct ligation (BDL). NOS enzyme activity was assessed by L-citrulline generation. Competitive RT-PCR was performed to detect the mRNA levels of NOS. In situ hybridization was done to localize NOS mRNA. Protein expression of NOS was evaluated by Western blotting and immunohistochemistry. The L-citrulline assay showed that constitutive NOS (cNOS) enzymatic activity was decreased, while inducible NOS (iNOS) activity was increased in BDL livers. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) mRNA were detected in BDL and sham rats, but with enhanced expression in BDL rats. eNOS protein was redistributed with less expression in sinusoidal endothelial cells, but the total levels in liver were not changed. nNOS was induced in hepatocytes of BDL rats, in contrast to only a weak signal observed around some blood vessels in sham livers. Intense mRNA and protein expression of iNOS was induced in livers of BDL rats and was localized in hepatocytes, with no or a negligible amount in control livers. In conclusion, iNOS was induced in cirrhotic liver with its activity increased. In contrast, cNOS activity was impaired, regardless of unchanged eNOS protein levels and enhanced nNOS expression. These results suggest that all three types of NOS have a role in cirrhosis, but their expression and regulation are different.     

Serum amyloid A induces endothelial dysfunction in porcine coronary arteries and human coronary artery endothelial cells

The objective of this study was to determine the effects and mechanisms of serum amyloid A (SAA) on coronary endothelial function. Porcine coronary arteries and human coronary arterial endothelial cells (HCAECs) were treated with SAA (0, 1, 10, or 25 microg/ml). Vasomotor reactivity was studied using a myograph tension system. SAA significantly reduced endothelium-dependent vasorelaxation of porcine coronary arteries in response to bradykinin in a concentration-dependent manner. SAA significantly decreased endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein levels as well as NO bioavailability, whereas it increased ROS in both artery rings and HCAECs. In addition, the activities of internal antioxidant enzymes catalase and SOD were decreased in SAA-treated HCAECs. Bio-plex immunoassay analysis showed the activation of JNK, ERK2, and IkappaB-alpha after SAA treatment. Consequently, the antioxidants seleno-l-methionine and Mn(III) tetrakis-(4-benzoic acid)porphyrin and specific inhibitors for JNK and ERK1/2 effectively blocked the SAA-induced eNOS mRNA decrease and SAA-induced decrease in endothelium-dependent vasorelaxation in porcine coronary arteries. Thus, SAA at clinically relevant concentrations causes endothelial dysfunction in both porcine coronary arteries and HCAECs through molecular mechanisms involving eNOS downregulation, oxidative stress, and activation of JNK and ERK1/2 as well as NF-kappaB. These findings suggest that SAA may contribute to the progress of coronary artery disease.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.