eNOS Knockout Mice Research Articles

Ascorbic acid synthesis due to l-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding l-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting l-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.

COX-2 contributes to the maintenance of flow-induced dilation in arterioles of eNOS-knockout mice

Our previous studies demonstrated that, in gracilis muscle arterioles of male mice deficient in the gene for endothelial nitric oxide synthase (eNOS), flow-induced dilation (FID) is mediated by endothelial PGs. Thus the present study aimed to identify the specific isoform of cyclooxygenase (COX) responsible for the compensatory mediation of FID in arterioles of eNOS-knockout (KO) mice. Experiments were conducted on gracilis muscle arterioles of male eNOS-KO and wild-type (WT) mice. Basal tone and magnitude of FID of arterioles were comparable in the two strains of mice. A role for COX isoforms in the mediation of the responses was assessed by use of valeryl salicylate (3 mM) and NS-398 (10 microM), inhibitors of COX-1 and COX-2, respectively. In eNOS-KO arterioles, valeryl salicylate or NS-398 alone inhibited FID (at maximal flow rate) by approximately 51% and approximately 58%, respectively. Administration of both inhibitors eliminated the dilation. In WT arterioles, inhibition of COX-2 did not significantly affect FID, whereas inhibition of COX-1 decreased the dilation by approximately 57%. The residual portion of the response was abolished by additional administration of Nomega-nitro-L-arginine methyl ester. Western blot analysis indicated a comparable content of COX-1 protein in arterioles of WT and eNOS-KO mice. COX-2 protein, which was not detectable in arterioles of WT mice, was strongly expressed in arterioles of eNOS-KO mice, together with an upregulation of COX-2 gene expression. Immunohistochemical staining confirmed the presence of COX-2 in the endothelium of eNOS-KO arterioles. In conclusion, COX-2-derived PGs are the mediators responsible for maintenance of FID in arterioles of eNOS-deficient mice.

1007: Vasoreactivity Changes in Corporal Tissue of eNOS Knockout Mice May Induced by Apoptosis

1007: Vasoreactivity Changes in Corporal Tissue of eNOS Knockout Mice May Induced by Apoptosis

Endothelial Nitric Oxide Synthase is Essential for Postpneumonectomy Compensatory Vasodilation

After pneumonectomy, the remaining lung vasculature must vasodilate to compensate for increased blood volume. We hypothesized that endothelial nitric oxide synthase (eNOS) is essential for compensatory vasodilation after pneumonectomy. Adult, wild-type C57BL6 (WT) and eNOS knockout (eNOS-/-) mice underwent left pneumonectomy and recovered under normoxic conditions. Animals were lightly anesthetized at 1, 3, 7, or 14 days after pneumonectomy, and closed chest, systolic right ventricular pressure (RVP) was recorded using fine-needle cannulation. The right ventricle to left ventricle plus septum weight ratios were measured as an index of right ventricular hypertrophy. Two additional groups of mice (WT and eNOS-/-) were recovered after pneumonectomy in inhaled nitric oxide (iNO, 10 ppm), and RVP was measured on day 7. The eNOS-/- mice had significantly higher preoperative RVP than did WT (17.1 +/- 0.4 versus 14.2 +/- 0.2 cmH2O, p = 0.001). Both groups exhibited transient periods of pulmonary hypertension after pneumonectomy. On day 1, RVP was 80% above baseline in eNOS-/- mice (30.7 +/- 0.8 cmH2O) versus 42% in WT mice (20.2 +/- 0.7 cmH2O, p = 0.0001). The RVP returned to baseline in WT mice (16.3 +/- 0.2 cmH2O) but remained significantly elevated in eNOS-/- mice (28.6 +/- 0.9 cmH2O) at day 3 and at each time thereafter (p = 0.0001). The iNO significantly reduced RVP in eNOS-/- animals to 15.2 +/- 0.3 cmH2O (p = 0.0001) while having no effect in WT animals. Right ventricular hypertrophy was not observed in any group. Pneumonectomy results in a transient increase in RVP. Under normal circumstances, these pressures return to baseline within 3 days. The eNOS-/- mice failed to display compensatory vasodilation yet could be rescued with iNO. These results suggest that eNOS is essential for postpneumonectomy compensatory vasodilation.

Mechanisms of Vascular Remodeling in eNOS Knockout Mice

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone. It has been shown that mice deficient in eNOS have impaired endothelium, such as alterations in structure and function. However, the mechanism of this impairment is unclear. Previous studies have implicated reactive oxygen species (ROS) as playing an important role in the initiation and progression of endothelial damage. We hypothesize that a deficiency in endothelial derived NO initiates ROS production therefore promoting increased activation of matrix metalloproteinases (MMP) and subsequent vascular remodeling. The descending thoracic aorta of wild type (WT) and eNOS−/− mice were removed, cleaned, dissected and homogenates were prepared. Results: The assessment of aortic function by in vivo echocardiography showed an increase in wall thickness and decreased blood flow in the eNOS−/− aortas suggesting increased wall stiffness. Quantitative PCR analysis revealed a marked increase in NADPH oxidase (Nox-4) and in contrast a decrease in thioredoxin (Trx) as well as an increase in MMP -2 and -9 in the knockout animal. Gelatin zymography showed a selective increase in MMP -9 activity in the eNOS−/− mice. Western blot analysis in eNOS−/− indicated a robust decrease in elastin expression. The stretch-strain relationship of aortic rings was assessed in an isometric tissue myobath. WT aortas relaxed in response to 0.5g of tension while the eNOS−/− aortas remained constricted, indicating reduced vascular elastic compliance. These results show that eNOS deficiency leads to the degradation of vessel wall structural components initiated by oxidative stress and increased MMP activity that collectively result in aortic remodeling and eventual irregularities in the regulation of vascular tone.

NO deficiency enhances age‐dependent decline in telomerase activity in mesenteric arteries. A gender‐dependent response

The purpose of the present study was to investigate the effect of NO deficiency on telomerase activity (TA) and telomere length (TL) in mesenteric arteries of 6, 14 and 24-month-old male and female eNOS knockout (KO) and wild type (WT) mice. TA and TL were assessed by the Telo TAGGG Telomerase PCR ELISAPLUS kit and Telo TAGGG Telomere Length Assay kit (Roche). We found that aging continuously and significantly decreased TA. Compared to vessels of 6-month-old mice, TA in vessels of 24-month-old mice decreased by 54 to 75%. There were significant differences in TA between vessels of male and female mice. For example, the optical densities of TA in vessels of 6, 14 and 24-month female WT mice were 0.50, 0.36 and 0.22, whereas in corresponding vessels of male WT mice they were only 0.38, 0.21 and 0.12, respectively. NO deficiency had no significant effect on TA in female vessels, but it significantly decreased TA in male vessels. For example, TA in vessels of 6- and 24-month male KO mice was only 0.31 and 0.08, values which are significantly less than those in corresponding WT vessels. TL in mesenteric arteries was 64 – 80 Kb. There were no significant differences in TL among the groups. We conclude that telomerase activity is mainly influenced by the aging process. Female hormones and/or genetic encoding provide a significant protection of telomerase activity in female vessels. Nitric oxide, derived from eNOS, plays a significant role in the maintenance of telomerase activity in vessels of male mice. (Supported by NIH grants HL-43023, HL-68813 and HL-070653)

Sodium Sensitivity of Arterial Blood Pressure in L-NAME Hypertensive but not eNOS Knockout Mice

The present studies determined the sensitivity of mean arterial pressure (MAP) to sodium intake in endothelial nitric oxide synthase (eNOS) knockout mice, wild-type mice (C56BL/6J), and wild-type mice intravenously administered the nonspecific NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME, 8.6 mg/kg/d). Arterial blood pressure was measured from chronically implanted femoral arterial catheters in conscious, freely moving mice. The MAP was unaltered from the low sodium ( approximately 200 microEq/d) intake level of 106 +/- 3 mm Hg in wild-type mice when sodium intake was increased to approximately 1000 microEq/d (n = 12). Chronic administration of L-NAME to wild-type mice led to a sodium-dependent increase in MAP from 111 +/- 7 mm Hg to 124 +/- 5 mm Hg when the mice were placed on an elevated sodium intake (n = 7). In contrast to the L-NAME-treated mice, MAP was unaltered in eNOS knockout mice (n = 8) when sodium intake was increased (128 +/- 3 mm Hg v 129 +/- 5 mm Hg). These experiments demonstrate that eNOS knockout and L-NAME-treated wild-type mice are hypertensive relative to wild-type controls when sodium intake is elevated, but only L-NAME-treated mice exhibited a sodium-sensitive increase in MAP. Therefore, nitric oxide produced by eNOS does not appear to be important in the physiologic adaptation to elevated sodium chloride intake.

A central role of eNOS in the protective effect of wine against metabolic syndrome

The positive health effects derived from moderate wine consumption are pleiotropic. They appear as improvements in cardiovascular risk factors such as plasma lipids, haemostatic mechanisms, endothelial function and antioxidant defences. The active principles would be ethanol and mainly polyphenols. Results from our and other laboratories support the unifying hypothesis that the improvements in risk factors after red wine consumption are mediated by endothelial nitric oxide synthase (eNOS). Many genes are involved, but the participation of eNOS would be a constant feature. The metabolic syndrome is a cluster of metabolic risk factors associated with high risk of cardiovascular disease (CVD). The National Cholesterol Education Programmmes Adult Treatment Panel III (NCEPATP III) clinical definition of the metabolic syndrome requires the presence of at least three risk factors, from among abdominal obesity, high plasma triacylglycerols, low plasma HDL, high blood pressure and high fasting plasma glucose. The molecular mechanisms responsible for the metabolic syndrome are not known. Since metabolic syndrome apparently affects 10-30% of the population in the world, research on its pathogenesis and control is needed. The recent finding that eNOS knockout mice present a cluster of cardiovascular risk factors comparable to those of the metabolic syndrome suggests that defects in eNOS function may cause human metabolic syndrome. These mice are hypertensive, insulin resistant and dyslipidemic. Further support for a pathogenic role of eNOS comes from the finding in humans that eNOS polymorphisms associate with insulin resistance and diabetes, with hypertension, with inflammatory and oxidative stress markers and with albuminuria. So, the data sustain the hypothesis that eNOS enhancement should reduce metabolic syndrome incidence and its consequences. Therefore red wine, since it enhances eNOS function, should be considered as a potential tool for the control of metabolic syndrome. This hypothesis is supported by epidemiological observations and needs experimental validation in human intervention studies.

Nitric oxide in obstructive uropathy: role of endothelial nitric oxide synthase.

Nitric oxide (NO) ameliorates fibrosis in experimental obstructive uropathy. Previously, we demonstrated that renal fibrosis was decreased after 2 weeks of unilateral ureteral obstruction in inducible nitric oxide synthase (iNOS) knock-out mice given L-arginine supplemented drinking water. We proposed that the 2 constitutive isoforms of nitric oxide synthase (NOS) mediated down-regulation of renal fibrosis in response to prolonged ureteral obstruction. To determine the specific role of endothelial NOS (eNOS) versus neuronal NOS in modulating renal fibrosis due to obstructive uropathy, we evaluated renal injury following unilateral ureteral obstruction in C57BL/6J mice subjected to biochemical inhibition of the constitutive isoforms of NOS and in eNOS knockout mice. Four groups of C57BL/6J mice were studied. Complete unilateral ureteral obstruction was created by ligating the right ureter at age 8 weeks. A single daily intraperitoneal injection of 30 mg./kg. S-methyl-L-thiocitrulline (SMLT), a selective neuronal and endothelial NOS inhibitor was started 24 hours before ureteral obstruction and administered to half of the study animals. SMLT treated mice and control animals were further subdivided to receive either regular tap water or 1% L-arginine (weight/volume) supplemented water after unilateral ureteral obstruction. Animals were sacrificed on postoperative day 3, 7 or 14. In addition, eNOS knockout mice with unilateral ureteral obstruction were given tap water or L-arginine supplemented water to drink and sacrificed after 14 days. Urine specimens from the bladder and the obstructed renal pelvis along with serum were collected. Nitrite level in each fluid was determined. Renal morphology and cortical thickness were assessed in the normal and obstructed kidneys. Interstitial fibrosis was evaluated using trichrome stain. SMLT was well tolerated by C57BL/6J mice. Serum nitrite levels and nitrite excretion in bladder urine were similar in all SMLT treated groups throughout the duration of unilateral ureteral obstruction. A reduction of pelvic urine nitrite levels by 89%, 68% and 48% versus bladder urine nitrite levels was observed after 3, 7 and 14 days of unilateral ureteral obstruction (p <0.05). Administration of SMLT resulted in a significant increase in bladder urine nitrite level at 7 days and in pelvic urine nitrite levels at 14 days. No significant histological differences in the obstructed kidney were seen after 3, 7 or 14 days of unilateral ureteral obstruction in SMLT treated versus control mice regardless of whether they received tap water or L-arginine supplemented drinking water. In eNOS knockout mice with unilateral ureteral obstruction for 14 days L-arginine supplementation had no effect on pelvic urine nitrite levels and did not alter renal histopathology or cortical thickness. NO production is decreased in the obstructed kidney in mice with unilateral ureteral obstruction. Biochemical inhibition of constitutive NOS did not modulate renal injury after 14 days of unilateral ureteral obstruction. In contrast to previous findings with iNOS knockout mice, dietary supplementation with L-arginine had no effect on the degree of fibrosis in the obstructed kidney in SMLT treated C57BL/6J or eNOS knockout mice. We conclude that NO derived from eNOS within the kidney has a pivotal role in protecting against renal fibrosis in response to unilateral ureteral obstruction.

Inducible Nitric Oxide Synthase Plays a Stimulatory Role in Platelet Activation.

Platelets generate nitric oxide (NO) in response to agonist stimulation. Previous reports have shown that the endothelial nitric oxide synthase (eNOS) plays a role in agonist-stimulated platelet NO production and in platelet activation. Here we show that platelets from eNOS knockout mice (eNOS−/−) showed only partial reduction in thrombin-induced NO production compared to wild type platelets (50% reduction), indicating the presence of another NOS isoform in platelets. More importantly, we show that resting platelets express functional inducible nitric oxide synthase (iNOS), which participates in platelet activation. Compared to wild type platelets, thrombin-induced NO production was reduced by 54% in platelets isolated from iNOS knockout mice (iNOS−/−), indicating an iNOS-dependent NO production in platelets induced by thrombin. Since thrombin-induced NO production occurs during the first 3 min of thrombin stimulation, our findings provide the first evidence for a short-term regulation of iNOS activity independent of transcription regulation. In contrast, previous description of iNOS activation was primarily at the transcriptional level and required much longer time of induction. To determine the role of iNOS in platelet activation, platelets from wild type and iNOS−/− mice were stimulated with low concentrations of agonists. iNOS−/− platelets exhibited lower aggregation and secretion response compared to wild type control, indicating that iNOS plays a stimulatory role in platelet activation. We also examined the effect of iNOS inhibitors on platelet activation. Human and mouse platelets preincubated with iNOS specific inhibitors, 1400W and aminoguanidine, exhibited a dose-dependent inhibition of platelet secretion and aggregation induced by either low-dose thrombin or collagen. Furthermore, the inhibitory effect of iNOS-specific inhibitors was only shown in wild type mouse platelets, but was lacking in iNOS−/− platelets. Thus, activation of both iNOS and eNOS is important in agonist-induced NO production which stimulates platelet secretion and aggregation.

Effects of repeated in vivo inhalant nitrite exposure on gene expression in mouse liver and lungs

Exposure to inhalant organic nitrites (drugs of abuse commonly known as "poppers") has been reported to enhance tumor growth in mice, but the mechanism is not fully defined. This study examined the effect of repeated in vivo nitrite exposures on gene expression in the mouse liver and lungs using a gene array panel of 94 cancer- and angiogenesis-related genes. Using 2-fold change as a threshold criterion, repeated nitrite exposure was found to alter the expression of 65 and 23 genes in the liver and lungs, respectively. Six genes were significantly upregulated (p<or=0.05), viz., those encoding VEGF, VEGFD (vascular endothelial growth factor A and D, respectively) in the lungs and FGF1, FGF4 (fibroblast growth factor 1 and 4, respectively), Hsp70 (heat shock 70kDa protein 4), and PF4 (platelet factor 4) in the liver. mRNA encoding HO-1 (heme oxygenase-1) and Smad7 were marginally (p=0.057) stimulated in the liver. Follow-up studies in the liver revealed significant nitrite-induced expression of VEGF protein and mRNA. Immuno-staining of liver slices revealed that the increased hepatic VEGF expression resided mainly in hepatocytes. Stimulation of hepatic VEGF expression by ISBN was not different in endothelial nitric oxide synthase (eNOS) knockout vs. wild-type mice. In conclusion, multiple exposures to inhalant nitrite appeared to cause alteration in the expression of a number of genes relating to cancer and angiogenesis, including VEGF. eNOS presence did not appear to be essential for nitrite-induced VEGF expression. These studies demonstrate that in vivo exposure to inhalant nitrites results in changes in the angiogenesis cascade.

Alterations in ventricular myocyte contraction caused by C-type natriuretic peptide and nitric oxide in eNOS–/– mice

Lack of endothelial nitric oxide synthase (eNOS) may affect the sensitivity of cyclic GMP signaling through soluble guanylyl cyclase (sGC). We hypothesized that in eNOS knockout (eNOS–/–) mice, stimulation of guanylyl cyclase would have enhanced effects inhibiting cardiac contraction. We measured cell shortening and calcium transients in isolated ventricular myocytes from adult eNOS–/– and wild-type (WT) mice after stimulating particulate guanylyl cyclase (pGC) with C-type natriuretic peptide (CNP, 10 –8 and 10 –7 M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP, NO donor, 10 –6 and 10 –5 M). Although sGC activity was increased by +71% in eNOS–/–, SNAP had similar effects in the two groups (%shortening –39% control vs. –37% eNOS–/–), suggesting that the cyclic GMP pathway was desensitized in eNOS–/– myocytes. CNP had significantly smaller effects on cell contraction (%shortening –34% control vs. –14% eNOS–/–) and pGC activity was not changed in eNOS–/– myocytes. Similar effects were also produced by guanylin and carbon monoxide, stimulators of pGC and sGC. CNP's effects on Ca 2+ transients were also attenuated in eNOS–/– myocytes. SNAP did not alter Ca 2+ transients in eNOS–/– or control cells. In the eNOS–/– mice, cyclic GMP-dependent protein kinase and cyclic AMP phosphodiesterase activity were reduced. This study demonstrated that the downstream cyclic GMP pathway was attenuated in eNOS–/– mice and this was partially compensated for by increased sGC, but not pGC activity in ventricular myocytes.

The role of nitric oxide in mitochondria. Focus on “Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells”

nitric oxide (NO) plays an important role in intracellular signaling, and stole the limelight in 1992 by winning science's equivalent of an “Oscar”: Science magazine's “Molecule of the Year.” Subsequently, the importance and versatility of its known roles has expanded. Furthermore, this

Endothelial nitric oxide synthase is critical for ischemic remodeling, mural cell recruitment, and blood flow reserve.

The genetic loss of endothelial-derived nitric oxide synthase (eNOS) in mice impairs vascular endothelial growth factor (VEGF) and ischemia-initiated blood flow recovery resulting in critical limb ischemia. This result may occur through impaired arteriogenesis, angiogenesis, or mobilization of stem and progenitor cells. Here, we show that after ischemic challenge, eNOS knockout mice [eNOS (-/-)] have defects in arteriogenesis and functional blood flow reserve after muscle stimulation and pericyte recruitment, but no impairment in endothelial progenitor cell recruitment. More importantly, the defects in blood flow recovery, clinical manifestations of ischemia, ischemic reserve capacity, and pericyte recruitment into the growing neovasculature can be rescued by local intramuscular delivery of an adenovirus encoding a constitutively active allele of eNOS, eNOS S1179D, but not a control virus. Collectively, our data suggest that endogenous eNOS-derived NO exerts direct effects in preserving blood flow, thereby promoting arteriogenesis, angiogenesis, and mural cell recruitment to immature angiogenic sprouts.

Correction

HomeCirculation ResearchVol. 97, No. 1Correction Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection Originally published3 Apr 2018https://doi.org/10.1161/res.97.1.e6Circulation Research. 2005;97:e6This article corrects the followingHMG-CoA Reductase Inhibitors Inhibit Endothelial Exocytosis and Decrease Myocardial Infarct SizeIn an article published by Yamakuchi et al (Circ Res. 2005:96:1185–1192.), Figure 5 contained an errors in Panel B. The corrected figure is below: Download figureDownload PowerPointFigure 5. Simvastatin inhibited leukocyte rolling in vivo. A, Simvastatin decreased leukocyte adhesion to murine venules. Mice were treated with vehicle (PBS) or simvastatin (1 mg/kg) IP, and after 24 hours the mice were anesthetized and injected with rhodamine 6G to label leukocytes. The mesentery was exteriorized, superfused with 20 μL histamine 1 mmol/L, and rolling leukocytes were imaged with a fluorescent digital camera. B, Quantification of simvastatin inhibition of leukocyte adhesion in mice (n=3 to 5±SEM. *P<0.04 vs vehicle). C, Simvastatin increased S-nitrosylation of NSF in vivo. Wild-type or eNOS knockout mice were treated with simvastatin for 24 hours, and spleen lysates were immunoprecipitated with antibody to nitrosocysteine. Precipitants were fractionated and immunoblotted with antibody to NSF (upper) to detect nitrosylated NSF (NSF-NO). To confirm that simvastatin did not change total NSF expression, total cell lysates were also immunoblotted with antibody to NSF (lower). Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesHMG-CoA Reductase Inhibitors Inhibit Endothelial Exocytosis and Decrease Myocardial Infarct SizeMunekazu Yamakuchi, et al. Circulation Research. 2005;96:1185-1192 July 8, 2005Vol 97, Issue 1 Advertisement Article InformationMetrics https://doi.org/10.1161/res.97.1.e6 Originally publishedApril 3, 2018 PDF download Advertisement

Endothelium-Derived Nitric Oxide Regulates Postischemic Myocardial Oxygenation and Oxygen Consumption by Modulation of Mitochondrial Electron Transport

Nitric oxide (NO) production is increased in postischemic myocardium, and NO can control mitochondrial oxygen consumption in vitro. Therefore, we investigated the role of endothelial NO synthase (eNOS)-derived NO on in vivo regulation of oxygen consumption in the postischemic heart. Mice were subjected to 30 minutes of coronary ligation followed by 60 minutes of reperfusion. Myocardial oxygen tension (Po2) was monitored by electron paramagnetic resonance oximetry. In wild-type, N-nitro-L-arginine methyl ester (L-NAME)-treated (with 1 mg/mL in drinking water), and eNOS knockout (eNOS-/-) mice, no difference was observed among baseline myocardial Po2 values (8.6+/-0.7, 10.0+/-1.2, and 10.1+/-1.2 mm Hg, respectively) or those measured at 30 minutes of ischemia (1.4+/-0.6, 2.3+/-0.9, and 3.1+/-1.4 mm Hg, respectively). After reperfusion, myocardial Po2 increased markedly (P<0.001 versus baseline in each group) but was much lower in L-NAME-treated and eNOS-/- mice (17.4+/-1.6 and 20.4+/-1.9 mm Hg) than in wild-type mice (46.5+/-1.7 mm Hg; P<0.001). A transient peak of myocardial Po2 was observed at early reperfusion in wild-type mice. No reactive hyperemia was observed during early reperfusion. Endothelial NO decreased the rate-pressure product (P<0.05), upregulated cytochrome c oxidase (CcO) mRNA expression (P<0.01) with no change in CcO activity, and inhibited NADH dehydrogenase (NADH-DH) activity (P<0.01) without alteration of NADH-DH mRNA expression. Peroxynitrite-mediated tyrosine nitration was higher in hearts from wild-type mice than in eNOS-/- or L-NAME-treated hearts. eNOS-derived NO markedly suppresses in vivo O2 consumption in the postischemic heart through modulation of mitochondrial respiration based on alterations in enzyme activity and mRNA expression of NADH-DH and CcO. The marked myocardial hyperoxygenation in reperfused myocardium may be a critical factor that triggers postischemic remodeling.

Protective Effects of Statins Involving Both eNOS and tPA in Focal Cerebral Ischemia

Previous studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) protect the brain against ischemic injury by upregulating endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that statins provide additional beneficial effects by also upregulating endogenous tissue plasminogen activator (tPA) and enhancing clot lysis in a mouse model of embolic focal ischemia. Heterologous blood clots (0.2 mm) were injected into the distal internal carotid artery to occlude blood flow in the middle cerebral artery territory after long-term (14 days) simvastatin, atorvastatin or vehicle treatment. Ischemic lesion volume, neurologic deficits, as well as residual blood clots were measured at 22 h. Reverse transcription-polymerase chain reaction assessed mRNA levels of eNOS, tPA, and the endogenous plasminogen activator inhibitor PAI-1. Ischemic lesion volumes and neurologic deficits were significantly reduced in wild-type mice by both simvastatin and atorvastatin. Statins increased eNOS and tPA mRNA levels but did not change mRNA levels of PAI-1. In eNOS knockout mice, atorvastatin reduced the volume of ischemic tissue and improved neurologic outcomes after arterial occlusion by blood clot emboli. In contrast, statins did not have protective effects in tPA knockout mice after embolic focal ischemia, but only in a filament model where focal ischemia was achieved via mechanical occlusion. These results suggest that statins protect against stroke by multiple mechanisms involving both eNOS and tPA. The involvement of each pathway may be revealed depending on the choice of experimental stroke model.

HMG-CoA Reductase Inhibitors Inhibit Endothelial Exocytosis and Decrease Myocardial Infarct Size

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors protect the vasculature from inflammation and atherosclerosis by cholesterol dependent and cholesterol independent mechanisms. We hypothesized that HMG-CoA reductase inhibitors decrease exocytosis of Weibel-Palade bodies, endothelial cell granules whose contents promote thrombosis and vascular inflammation. We pretreated human aortic endothelial cells with simvastatin for 24 hours, then stimulated the cells with thrombin, and measured the amount of vWF released into the media. We then measured the effect of simvastatin on myocardial infarction in mice. Simvastatin decreased thrombin-stimulated Weibel-Palade body exocytosis by 89%. Simvastatin inhibited exocytosis in part by increasing synthesis of nitric oxide (NO), which S-nitrosylated N-ethylmaleimide sensitive factor (NSF), a critical regulator of exocytosis. Simvastatin treatment attenuated myocardial infarct size by 58% in wild-type but not eNOS knockout mice. Furthermore, simvastatin decreased endothelial exocytosis and neutrophil infiltration into ischemic-reperfused myocardium, which was mediated in part by P-selectin contained in Weibel-Palade bodies. However, simvastatin did not affect exocytosis and inflammation in myocardial infarcts of eNOS knockout mice. Inhibition of endothelial exocytosis is a novel mechanism by which HMG-CoA reductase inhibitors may reduce vascular inflammation, inhibit thrombosis, and protect the ischemic myocardium. These findings may explain part of the pleiotropic effects of statin therapy for patients with cardiovascular disease.

Transcriptional basis for exercise limitation in male eNOS-knockout mice with age: heart failure and the fetal phenotype

Endothelium-derived nitric oxide (NO) is pivotal in regulating mitochondrial O(2) consumption (Vo(2)) and glucose uptake in mice. The aim of this study was to investigate the mechanism of age- and genotype-related exercise limitation in male endothelial NO synthase (eNOS)-knockout (KO, n = 16) and wild-type (WT, n = 19) mice. Treadmill testing was performed at 12, 14, 16, 18, and 21 mo of age. Vo(2), CO(2) production, respiratory exchange ratio, and maximal running distance were determined during treadmill running. There were good linear correlations for increase of speed with increase of Vo(2). The difference between KO and WT mice was not significant at 12 mo but was significant at 18 mo. Linear regression showed that KO mice consumed more O(2) at the same absolute and relative workloads, suggesting that Vo(2) was not inhibited by NO in KO mice. KO mice performed 30-50% less work than WT mice at each age (work = vertical distance x weight). In contrast to WT mice, the work performed by KO mice significantly decreased from 17 +/- 1.4 m.kg at 12 mo to 9.4 +/- 1.7 m.kg at 21 mo. Running distance was significantly decreased from 334 +/- 27 m at 12 mo to 178 +/- 38 m at 21 mo, and maximal Vo(2), CO(2) production, and respiratory exchange ratio per work unit were significantly higher in KO than in WT mice. Gene arrays showed evidence of a fetal phenotype in KO mice at 21 mo. In conclusion, age- and genotype-related exercise limitations in maximal work performed and maximal running distance in male eNOS-KO mice indicated that fetal phenotype and age were related to onset of heart failure.

Superoxide scavenger tempol markedly improves angiotensin II induced renal injury and mortality in eNOS knockout mice

Superoxide scavenger tempol markedly improves angiotensin II induced renal injury and mortality in eNOS knockout mice