Structure of cerebral arterioles in mice deficient in expression of the gene for endothelial nitric oxide synthase

We examined effects of pharmacological inhibition of nitric oxide synthase (NOS) and genetic deficiency of the endothelial isoform of NOS (eNOS) on structure and mechanics of cerebral arterioles. We measured pressure, diameter, and cross-sectional area (CSA) of the vessel wall (histologically) in maximally dilated cerebral arterioles in mice that were untreated or treated for 3 months with the NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg per day in drinking water). Treatment with L-NAME increased systemic arterial mean pressure (SAP; 143+/-4 versus 121+/-4 mm Hg, P<0.05) and CSA (437+/-27 versus 310+/-34 microm2, P<0.05). These findings suggest that hypertension induced in mice by NOS inhibition is accompanied by hypertrophy of cerebral arterioles. To determine the role of the eNOS isoform in regulation of cerebral vascular growth, we examined mice with targeted disruption of one (heterozygous) or both (homozygous) genes encoding eNOS. Wild-type littermates served as controls. SAP and CSA were significantly increased in homozygous (SAP, 141+/-5 versus 122+/-3 mm Hg in wild-type mice, P<0.05; CSA, 410+/-18 versus 306+/-15 microm2 in wild-type mice, P<0.05), but not in heterozygous (SAP, 135+/-4 mm Hg; CSA, 316+/-32 microm2) eNOS-deficient mice. Carotid ligation normalized cerebral arteriolar pulse pressure did not prevent increases in CSA in homozygous eNOS-deficient mice. Thus, cerebral arterioles undergo hypertrophy in homozygous eNOS-deficient mice, even in the absence of increases in arteriolar pulse pressure. These findings suggest that eNOS plays a major role in regulation of cerebral vascular growth.     

Gastric stasis in neuronal nitric oxide synthase-deficient knockout mice

BACKGROUND & AIMS: Nitric oxide (NO) is a major inhibitory neurotransmitter in the gut. This study aimed to identify the effect of chronic deprivation of NO derived from neuronal (nNOS) or endothelial (eNOS) nitric oxide synthase on gastric emptying. METHODS: nNOS-deficient (knockout) mice were compared with wild-type mice for gastric size, fluoroscopic appearance after gavage of contrast, and histology of the pyloric sphincter. Wild-type mice treated with the NOS inhibitor N(omega)-nitro L-arginine (L-NA) and eNOS-deficient mice were also compared with wild-type and nNOS-deficient mice for liquid and solid gastric emptying. RESULTS: nNOS-deficient mice showed gastric dilation. Fluoroscopy showed delayed gastric emptying of radiologic contrast. There was no marked localized hypertrophy or luminal narrowing at the pyloric sphincter by histology of relaxed wild-type, nNOS-deficient, and eNOS-deficient tissues. Gastric emptying of both solids (28% +/- 27%) and liquids (22% +/- 18%) was significantly delayed in nNOS-deficient mice compared with control wild-type mice (82% +/- 22% for solids; 48% +/- 17% for liquids). eNOS-deficient mice showed no significant difference from wild-type mice (74% +/- 28% for solids; 47% +/- 23% for liquids). Wild-type mice treated acutely with L-NA showed delay in emptying of solids (43% +/- 31%) but not liquids (39% +/- 15%). CONCLUSIONS: Chronic depletion of NO from nNOS, but not eNOS, results in delayed gastric emptying of solids and liquids.     

In Vivo Electrophysiologic Studies in Endothelial Nitric Oxide Synthase (eNOS)-Deficient Mice

INTRODUCTION: Endothelial nitric oxide synthase (eNOS) mediates attenuation of the L-type calcium channel and modulates myocyte contractility. Arrhythmogenic afterdepolarizations are seen in vitro in ouabain-treated isolated myocytes from eNOS-deficient mice. The aim of these studies was to characterize the baseline electrophysiologic (EP) phenotype of eNOS-deficient mice and their potential susceptibility to cardiac conduction abnormalities and inducible arrhythmias. METHODS AND RESULTS: Surface ECG and in vivo intracardiac EP studies were performed in 27 mice lacking the eNOS gene and 21 wild-type littermate control mice. Baseline studies were performed in 10 eNOS-deficient mice and 10 wild-type controls. Subsequently, 17 eNOS-deficient mice and 11 wild-type controls were pretreated with digoxin, and ECG and EP testing were repeated. Data analysis revealed no significant differences in ECG intervals or cardiac conduction parameters, except sinus cycle length was higher in eNOS-deficient mice than wild-type mice (P < 0.01). After digoxin pretreatment, 7 of 17 eNOS-deficient mice had inducible ventricular tachycardia and 2 others had frequent ventricular premature beats, compared with only 3 of 11 wild-type mice with inducible ventricular tachycardia. In addition, 2 digoxin-treated eNOS-deficient mice and 1 wild-type mouse had inducible nonsustained atrial fibrillation. CONCLUSION: Mice with a homozygous targeted disruption of the eNOS gene have slower heart rates but no other distinguishable EP characteristics under basal sedated conditions. Partial inhibition of the Na+/K+ ATPase pump with digoxin administration increases ventricular ectopic activity in eNOS-/- mice, a phenotype analogous to afterdepolarizations seen in vitro in this eNOS-deficient mouse model.     

Endothelial nitric oxide gene knockout mice: cardiac phenotypes and the effect of angiotensin-converting enzyme inhibitor on myocardial ischemia/reperfusion injury.

We tested the hypothesis that nitric oxide (NO) released by endothelial NO synthase (eNOS) is not only important in blood pressure regulation but also involved in cardiac function and remodeling and in the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEi). With the use of a 2D Doppler echocardiography system equipped with a 15-MHz linear transducer, we evaluated left ventricular (LV) morphology and function in conscious eNOS knockout mice (eNOS(-/-); n=15) and their wild-type littermates (eNOS(+/+); n=16). We also studied whether in eNOS(-/-) mice (1) myocardial ischemia/reperfusion injury is more severe and (2) the cardioprotective effect of ACEi is diminished or absent. In comparison with the wild type, eNOS(-/-) mice had significantly increased systolic blood pressure (128+/-3 versus 108+/-5 mm Hg; P<0.001) and decreased heart rate (531+/-22 versus 629+/-18 bpm; P<0.001) associated with increased LV posterior wall thickness (0.80+/-0.04 versus 0.64+/-0.02 mm; P<0.001) and LV mass (18.3+/-0.9 versus 13.1+/-0.5 mg/10 g body weight; P<0.01). Despite hypertension and LV hypertrophy, LV chamber dimension, shortening fraction and ejection fraction (indicators of LV contractility), and cardiac output did not differ between the 2 strains, which indicates that LV function in eNOS(-/-) mice is well compensated. We also found that in eNOS(+/+) mice, ACEi decreased the ratio of myocardial infarct size to area at risk from 62.7+/-3.9% to 36.3+/-1.6% (P<0. 001), whereas in eNOS(-/-) mice this effect of ACEi was almost abolished: the ratio of myocardial infarct size to area at risk was 67.2+/-2.9% in the vehicle-treated group and 62.7+/-3.9% in mice treated with ACEi. Moreover, infarct size in vehicle-treated eNOS(-/-) mice was not significantly different from eNOS(+/+) mice given the same treatment. We concluded that (1) endothelium-derived NO plays an important role in the regulation of blood pressure homeostasis; (2) NO released under basal conditions has no significant impact on cardiac function; and (3) ACEi protect the heart against ischemia/reperfusion injury in mice and that this effect is mediated in part by endothelium-derived NO.     

Role of sex differences and effects of endothelial NO synthase deficiency in responses of carotid arteries to serotonin

We examined the hypothesis that contraction of the carotid arteries to serotonin is normally inhibited by endothelial NO synthase (eNOS) and is enhanced in mice lacking the gene for eNOS. Because the influence of eNOS may vary with the sex of the mouse, we also tested whether responses to serotonin were dependent on sex. We studied carotid arteries in vitro from littermate control (eNOS(+/+)) mice, heterozygous (eNOS(+/-)) mice, and homozygous eNOS-deficient (eNOS(-/-)) mice (male and female). Contraction to serotonin was greater in male eNOS(+/+) mice than in female eNOS(+/+) mice. In male mice, contraction to serotonin increased by approximately 40% and 2.5-fold in male eNOS(+/-) and eNOS(-/-) mice, respectively. Contraction to serotonin was more than doubled in female eNOS(+/-) mice and increased >5-fold in arteries from eNOS(-/-) mice. In contrast, maximum vasoconstriction to U46619 was similar in male and female eNOS(+/+), eNOS(+/-), and eNOS(-/-) mice. Relaxation to acetylcholine was not different in male and female eNOS(+/+) or eNOS(+/-) mice but was absent in eNOS(-/-) mice. These findings suggest that the contraction of carotid arteries to serotonin is influenced by the sex of the animal. eNOS deficiency in gene-targeted mice is associated with enhanced contraction to serotonin, particularly in female mice, providing direct evidence that eNOS is a major determinant of vascular effects of serotonin. The results with eNOS(+/-) mice suggest a "gene-dosing" effect for vascular responses to serotonin.     

Assessment of the hemostatic effectiveness of human platelets treated with aminomethyltrimethyl psoralen and UV A light using a rabbit ear bleeding time technique

The photochemical aminomethyltrimethyl psoralen (AMT), in conjunction with UV A light (UVA), has been shown to inactivate human immunodeficiency virus-1 and model viruses in platelet suspensions under conditions that have only a minimal effect on in vitro platelet properties. A rabbit ear bleeding time technique was used to assess the hemostatic effectiveness of human platelet suspensions treated with AMT/UVA. New Zealand White rabbits were made thrombocytopenic by a combination of irradiation and heterologous antirabbit platelet antiserum. Reticuloendothelial function in these rabbits was suppressed by the intravenous administration of ethyl palmitate. The hemostatic function of 1- and 5-day-old human platelet suspensions (14.5% plasma) that had been treated on day 1 with 40 micrograms/mL AMT and 24 kJ/m2 UVA (1 x UVA) was evaluated by measuring microvascular bleeding times after a standard incision. Comparable bleeding times were observed after infusion with both control and AMT/UVA-treated platelets stored for either 1 or 5 days. With the transfusion of AMT/1 x UVA-treated platelets stored for 5 days, the mean (+/- SD) bleeding time was 156.3 +/- 39.2 seconds (n = 10). With untreated platelets (no AMT/no UVA), stored for 5 days, the mean bleeding time was 189.2 +/- 36.4 seconds (n = 10). Neither AMT nor 1 x UVA treatment alone influenced the observed bleeding times. In contrast, the hemostatic effectiveness of human platelet suspensions was diminished if they were exposed to three times the standard UVA dose (72 kJ/m2) on day 1 and stored for 4 more days, regardless of whether AMT was present, with the mean bleeding time increasing to 442.2 +/- 122.6 seconds (n = 15, AMT present) or 396.0 +/- 45.9 seconds (n = 10, AMT absent). These results are consistent with data obtained from in vitro studies and indicate that virucidal AMT/1 x UVA treatment does not influence platelet hemostatic function. However, the final conditions to achieve these results must be carefully controlled.     

The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice

BACKGROUND & AIMS: Considerable debate exists concerning which isoform of nitric oxide synthase (NOS) is responsible for the increased production of NO in PHT. We used the portal vein ligation model of PHT in wild-type and eNOS- or iNOS-knockout mice to definitively determine the contribution of these isoforms in the development of PHT. METHODS: The portal vein of wild-type mice, or those with targeted mutations in the nos2 gene (iNOS) or the nos3 gene (eNOS), was ligated and portal venous pressure (Ppv), abdominal aortic blood flow (Qao), and portosystemic shunt determined 2 weeks later. RESULTS: In wild-type mice, as compared with sham-operated controls, portal vein ligation (PVL) resulted in a time-dependent increase in Ppv (7.72 +/- 0.37 vs 17.57 +/- 0.51 cmH(2)O, at 14 days) concomitant with a significant increase in Qao (0.12 +/- 0.003 vs 0.227 +/- 0.005 mL/min/g) and portosystemic shunt (0.47% +/- 0.01% vs 84.13% +/- 0.09% shunt). Likewise, PVL in iNOS-deficient mice resulted in similar increases in Ppv, Qao, and shunt development. In contrast, after PVL in eNOS-deficient animals, there was no significant change in Ppv (7.52 +/- 0.22 vs 8.07 +/- 0.4 cmH(2)0) or Qao (0.111 +/- 0.01 vs 0.14 +/-.023 mL/min/g). However, eNOS (-/-) mice did develop a substantial portosystemic shunt (0.33% +/- 0.005% vs 84.53% +/- 0.19% shunt), comparable to that seen in wild-type animals after PVL. CONCLUSIONS: These data support a key role for eNOS, rather than iNOS, in the pathogenesis of PHT.     

Essential role of endothelial nitric oxide synthase in vascular effects of erythropoietin

Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO has been recognized as a tissue-protective cytokine. In this study, we tested the hypothesis that endothelial NO synthase (eNOS) plays a key role in the vascular protective effect of EPO. A murine model of wire-induced injury of carotid artery was used to examine the effect of EPO on endothelial repair and arterial wall architecture. Recombinant human EPO (1000 U/kg, SC, biweekly) was administered for 2 weeks in wild-type and eNOS-deficient mice after which reactivity of isolated carotid arteries was studied in vitro, and the vasculature was histologically assessed. Injured arteries exhibited impairment of endothelium-dependent relaxations to acetylcholine (P<0.05). This was associated with increased medial cross-sectional area (P<0.05). EPO upregulated expression of phosphorylated Ser1177-eNOS and normalized the vasodilator response to acetylcholine (P<0.05). Furthermore, EPO prevented the injury-induced increase in medial cross-sectional area (P<0.05). The vascular protective effects of EPO were abolished in eNOS-deficient mice. Most notably, EPO significantly increased systolic blood pressure and enhanced medial thickening of injured carotid arteries in eNOS-deficient mice (P<0.05). Our results demonstrate that EPO prevents aberrant remodeling of the injured carotid artery. The protective effects of EPO are critically dependent on activation of eNOS.     

Receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice.

Receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) is emerging as an important regulator of vascular pathophysiology. Here, we demonstrate a novel role of RANKL as a vascular permeability factor and a critical role of endothelial nitric oxide synthase (eNOS) in RANKL-induced endothelial function. RANKL increased the vascular permeability and leukocyte infiltration in vivo and caused the breakdown of the blood-retinal barrier in wild-type mice but not in eNOS-deficient mice. In vitro, it increased endothelial permeability and reduced VE-cadherin-facilitated endothelial cell-cell junctions in a NO-dependent manner. RANKL also led to the activation of Akt and eNOS and to NO production in endothelial cells (ECs). These effects were suppressed by the inhibition of TRAF6, phosphoinositide 3'-kinase (PI3K), Akt, or NOS by genetic or pharmacologic means. Inhibition of the TRAF6-mediated NO pathway reduced EC migration and capillary-like tube formation in response to RANKL. Moreover, the effects of RANKL on ECs sprouting from the aorta, and neovessel formation in both the mouse Matrigel plug assay and corneal micropocket assay, were impaired in eNOS-deficient mice. These results demonstrate that RANKL promotes vascular permeability and angiogenesis by stimulating eNOS by a TRAF6-PI3K-Akt-dependent mechanism. These properties may be relevant to the pathogenesis of angiogenesis-dependent and inflammatory vascular diseases.     

Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 +/- 377 seconds) and arterioles (858 +/- 289 seconds) than in WT vessels (559 +/- 241 seconds, P < .001; 443 +/- 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively.     

Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption

Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O2 consumption and to further examine the role of nitric oxide (NO) in the control of mitochondrial respiration. Left ventricle O2 consumption in mice with defects in the expression of eNOS [eNOS (-/-)] and inducible NOS [iNOS (-/-)] was measured with a Clark-type O2 electrode. The rate of decreases in O2 concentration was expressed as a percentage of the baseline. Baseline O2 consumption was not significantly different between groups of mice. Bradykinin (10(-4) mol/L) induced significant decreases in O2 consumption in tissues taken from iNOS (-/-) (-28+/-4%), wild-type eNOS (+/+) (-22+/-4%), and heterozygous eNOS(+/-) (-22+/-5%) but not homozygous eNOS (-/-) (-3+/-4%) mice. Responses to bradykinin in iNOS (-/-) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with N-nitro-L-arginine methyl ester (L-NAME) (-2+/-5%, -3+/-2%, and -6+/-5%, respectively, P<0.05). In contrast, S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4) mol/L), which releases NO spontaneously, induced decreases in myocardial O2 consumption in all groups of mice, and such responses were not affected by L-NAME. In addition, pretreatment with bacterial endotoxin elicited a reduction in basal O2 consumption in tissues taken from normal but not iNOS (-/-)-deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O2 consumption and modulation of mitochondrial respiration by NO may have an important role in pathological conditions such as endotoxemia in which the production of NO is altered.     

Repeated thermal therapy up-regulates endothelial nitric oxide synthase and augments angiogenesis in a mouse model of hindlimb ischemia.

BACKGROUND: Nitric oxide (NO), constitutively produced by endothelial NO synthase (eNOS), plays roles in angiogenesis. Having reported that thermal therapy up-regulated the expression of arterial eNOS in hamsters, we investigated whether this therapy increased angiogenesis in mice with hindlimb ischemia. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced in apolipoprotein E-deficient mice, which were divided into control and thermal therapy groups. The latter mice were placed in a far-infrared dry sauna at 41 degrees C for 15 min and then at 34 degrees C for 20 min once daily for 5 weeks. Laser Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio in the thermal therapy group was significantly increased beyond that in controls (0.79+/-0.04 vs 0.54+/-0.08, p<0.001). Significantly greater capillary density was seen in thermal therapy group (757+/-123 /mm2 vs 416+/-20 /mm2, p<0.01). Western blotting showed thermal therapy markedly increased hindlimb eNOS expression. To study possible involvement of eNOS in thermally induced angiogenesis, thermal therapy was given to mice with hindlimb ischemia with or without N(G)-nitro-L-arginine methyl ester (L-NAME) administration for 5 weeks. L-NAME treatment eliminated angiogenesis induced using thermal therapy. Thermal therapy did not increase angiogenesis in eNOS-deficient mice. CONCLUSION: Angiogenesis was induced via eNOS using thermal therapy in mice with hindlimb ischemia.     

Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice

It has been shown that mice deficient in the gene coding for endothelial nitric-oxide synthase (eNOS) have increased pulmonary arterial pressure and pulmonary vascular resistance. In the present study, the effect of transfer to the lung of an adenoviral vector encoding the eNOS gene (AdCMVeNOS) on pulmonary arterial pressure and pulmonary vascular resistance was investigated in eNOS-deficient mice. One day after intratracheal administration of AdCMVeNOS to eNOS(-/-) mice, there was an increase in eNOS protein, cGMP levels, and calcium-dependent conversion of l-arginine to l-citrulline in the lung. The increase in eNOS protein and activity in eNOS(-/-) mice was associated with a reduction in mean pulmonary arterial pressure and pulmonary vascular resistance when compared with values in eNOS-deficient mice treated with vehicle or a control adenoviral vector coding for beta-galactosidase, AdCMVbetagal. These data suggest that in vivo gene transfer of eNOS to the lung in eNOS(-/-) mice can increase eNOS staining, eNOS protein, calcium-dependent NOS activity, and cGMP levels and partially restore pulmonary arterial pressure and pulmonary vascular resistance to near levels measured in eNOS(+/+) mice. Thus, the major finding in this study is that in vivo gene transfer of eNOS to the lung in large part corrects a genetic deficiency resulting from eNOS deletion and may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders in which eNOS activity is reduced.     

Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [l-N(6)-(1-iminoethyl) lysine, l-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) > or = WT with l-NIL or iNOS(-/-) > eNOS(-/-) > or = eNOS(-/-) with l-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS(-/-) mice but not in eNOS(-/-) mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.     

Sexual dimorphism in reduced-size liver ischemia and reperfusion injury in mice: role of endothelial cell nitric oxide synthase

We have recently reported that female mice are protected to a much greater extent from the injurious effects of reduced-size liver ischemia and reperfusion (RSL+I/R) than are males by an estrogen-dependent mechanism. The objective of this study was to examine the possibility that the protective effect observed in female mice depends on the up-regulation and/or activation of endothelial cell NO synthase (eNOS). Anesthetized female and male wild-type or eNOS-deficient C57BL/6 mice were subjected to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of ischemic tissue. Survival was monitored daily, whereas liver injury was quantified by using serum alanine aminotransferase determinations and histopathology. Hepatic eNOS mRNA, protein, and enzymatic activity were determined in male and female mice subjected to RSL+I/R. We found that liver injury was reduced and survival increased in female mice compared with males. This protective effect correlated with significant increases in hepatic eNOS message levels and enzyme activity but not protein expression compared with males subjected to the surgery. Furthermore, N(omega)-nitro-L-arginine methyl ester-treated or eNOS-deficient female mice responded to RSL+I/R with dramatic increases in liver injury and 100% mortality within 2 days of surgery. Finally, we found that pravastatin pretreatment significantly attenuated hepatocellular injury and increased survival of male mice, which was associated with enhanced expression of eNOS message. We conclude that the protective effect afforded female mice is due to the activation of hepatic eNOS activity and enhanced NO production.     

Erythropoietin protects cardiomyocytes from apoptosis via up-regulation of endothelial nitric oxide synthase

OBJECTIVE: Erythropoietin (EPO), a cytokine best known for its ability to increase red blood cell mass, has recently been shown to protect cardiomyocytes from apoptotic cell death. The objective of the present study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the anti-apoptotic effects of EPO in cardiomyocytes. METHODS AND RESULTS: Neonatal mouse ventricular cardiomyocytes were isolated and cultured from wild-type and eNOS(-/-) mice. Treatment with EPO significantly reduced apoptosis induced by norepinephrine (NE) in the wild-type cardiomyocytes. The reduction of apoptosis was associated with significant increases in eNOS expression, phosphorylation and NO production. However, the anti-apoptotic effects of EPO were significantly decreased in wild-type cardiomyocytes treated with L-NAME, which inhibits nitric oxide synthase activity. The results were further confirmed using eNOS(-/-) cardiomyocytes. To investigate the in vivo significance of eNOS in mediating the anti-apoptotic effects of EPO, wild-type and eNOS(-/-) mice were subjected to myocardial ischemia and reperfusion. EPO decreased myocardial apoptosis and infarct size in wild-type mice. However, the protective effects of EPO were significantly diminished in eNOS(-/-) mice. CONCLUSIONS: EPO increases eNOS expression and NO production in cardiomyocytes. The anti-apoptotic effects of EPO in cardiomyocytes are mediated by eNOS-derived NO production.     

Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model

OBJECTIVE: Angiogenesis is a complex multistep process that involves endothelial cell (EC) migration, proliferation and differentiation into vascular tubes. NO has been reported to be a downstream mediator in the angiogenic response to a variety of growth factors, but the mechanisms by which NO promotes neovessel formation is not clear. We hypothesized that NO directly contributes to EC migration and capillary tube formation. METHODS: Since previous studies have noted important biological differences between NO produced pharmacologically by NO-donor compounds compared to that from NO synthase (NOS), we used a cell-based gene transfer approach to increase NO production in a co-culture model of in vitro angiogenesis. Rat smooth muscle cells (SMCs) were transfected with plasmids containing VEGF(121), VEGF(165) (SMC(VEGF)), endothelial NOS (SMC(eNOS)) or the empty vector (SMC(Cont)). Expression of the eNOS in SMC(eNOS) was confirmed by Northern analysis, NADPH-diaphorase activity, and nitrite/nitrate levels, whereas VEGF production was confirmed using ELISA. Calf pulmonary artery ECs (CPAECs) were cultured on the fibrin matrix with (co-culture) or without underlying SMCs (monoculture). RESULTS: Co-culture of ECs with SMC(Cont) had no effect on EC differentiation compared with EC in monoculture (differentiation index, DI=2.8+/-3.4 vs. 2.1+/-2.8, respectively, NS). In contrast, co-culture with SMC(eNOS) resulted in the formation of extensive capillary-like structures within 48 h (DI=17.2+/-5.9, P<0.001 versus SMC(Cont)), which was significantly inhibited using a NOS inhibitor, L-NAME (3 mM) (DI=4.5+/-3.04, P<0.001 versus SMC(eNOS)). Similarly, SMC(VEGF121) induced an angiogenic response (DI=14.2+/-3.8), which was also significantly inhibited by L-NAME (DI=5.9+/-1.8, P<0.05). In using the Boyden chamber model, SMC(eNOS), but not SMC(Cont) increased EC migration to a similar extent as SMC(VEGF121), and both were significantly inhibited with L-NAME. CONCLUSIONS: These data support an important paracrine role for endogenously produced NO in EC migration and differentiation in vitro, and suggest that the cell-based eNOS gene transfer may be a useful approach to increase new blood vessel formation in vivo.     

Proangiogenic effect of angiotensin-converting enzyme inhibition is mediated by the bradykinin B(2) receptor pathway

Recent studies have suggested a proangiogenic effect of angiotensin-converting enzyme (ACE) inhibition. We hypothesized that such a proangiogenic effect of ACE inhibition may be mediated, in part, by bradykinin (BK) B(2)-receptor pathway. This study therefore examined the neovascularization induced by ACE inhibitor treatment in B(2) receptor-deficient mice (B(2)(-/-)) in a model of surgically induced hindlimb ischemia. After artery femoral occlusion, wild-type and B(2)(-/-) mice were treated with or without ACE inhibitor (perindopril, 3 mg/kg/d) for 28 days. Angiogenesis was then quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. The protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were determined by Western blot. In wild-type animals, vessel density and capillary number in the ischemic leg were raised by 1.8- and 1.4-fold, respectively, in mice treated with ACE inhibitor when compared with the nontreated animals (P<0.01). This corresponded to an improved ischemic/nonischemic leg perfusion ratio by 1.5-fold in ACE inhibitor-treated animals when compared with the untreated ones (0.87+/-0.07 versus 0.59+/-0.05, respectively, P<0.01). Activation of the angiogenic process was also associated with a 1.7-fold increase in tissue eNOS protein level in mice treated with ACE inhibitor (P<0.05 versus control) but not with changes in VEGF protein level. Conversely, ACE inhibition did not affect vessel density, blood flow, and eNOS protein level in ischemic hindlimb of B(2)(-/-) mice. Therefore, proangiogenic effect of ACE inhibition is mediated by B(2)-receptor signaling and was associated with upregulation of eNOS content, independently of VEGF expression.     

Stoichiometric relationships between endothelial tetrahydrobiopterin, endothelial NO synthase (eNOS) activity, and eNOS coupling in vivo: insights from transgenic mice with endothelial-targeted GTP cyclohydrolase 1 and eNOS overexpression

Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O2*-) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2*- rather than NO, possibly because of a mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS coupling in vivo remains undefined because no studies have investigated the regulation of eNOS by BH4 in the absence of vascular disease states that cause pathological oxidative stress through multiple mechanisms. We investigated the stoichiometry of BH4-eNOS interactions in vivo by crossing endothelial-targeted eNOS transgenic (eNOS-Tg) mice with mice overexpressing endothelial GTP cyclohydrolase 1 (GCH-Tg), the rate-limiting enzyme in BH4 synthesis. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with wild type. The ratio of eNOS dimer:monomer was significantly reduced in aortas from eNOS-Tg mice compared with wild-type mice but restored to normal in eNOS/GCH-Tg mice. NO synthesis was elevated by 2-fold in GCH-Tg and eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with wild type. Aortic BH4 levels were elevated in GCH-Tg and maintained in eNOS/GCH-Tg mice but depleted in eNOS-Tg mice compared with wild type. Aortic and cardiac O2*- production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME), suggesting O2*- production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O2*- production was similar to wild type, and L-NAME had no effect, indicating preserved eNOS coupling. These data indicate that eNOS coupling is directly related to eNOS-BH4 stoichiometry even in the absence of a vascular disease state. Endothelial BH4 availability is a pivotal regulator of eNOS activity and enzymatic coupling in vivo.     

Increased eNO and pulmonary iNOS expression in eNOS null mice.

Nitric oxide (NO) is a major regulatory molecule of the cardiovascular system; however, measurement of vascular NO synthesis in vivo represents a major challenge. NO stemming from the lower respiratory tract has been used as a marker of vascular endothelial function. Experimental evidence for this concept is lacking. Therefore, the aim of the present study was to investigate this relationship. Lower respiratory tract exhaled NO concentration, together with systemic and pulmonary artery pressure, was measured in endothelial nitric oxide synthase (NOS) (eNOS) null mice (eNOS-/-). Similar studies were performed in inducible NOS (iNOS) null mice (iNOS-/-). Defective endothelial NO synthesis in eNOS-/- mice (evidenced by systemic and pulmonary hypertension) was associated with augmented exhaled NO levels (12.5 +/- 1.9 versus 9.8 +/- 1.2 parts per billion (ppb), eNOS-/- versus wild type), whereas normal endothelial NO synthesis in iNOS-/- mice was associated with decreased exhaled NO levels (4.3 +/- 1.5 ppb). Augmented exhaled NO levels in eNOS-/- mice were associated with upregulation of iNOS expression in the lung. These results indicate that inducible nitric oxide synthase is a major determinant of gaseous nitric oxide production in the lung, and lower respiratory tract exhaled nitric oxide does not always represent a marker of vascular endothelial nitric oxide synthesis.