Endothelium-dependent hyperpolarization-mediated vasodilatation compensates nitric oxide-mediated endothelial dysfunction during ischemia in diabetes-induced canine coronary collateral microcirculation in vivo.

OBJECTIVES: It has been previously demonstrated that endothelial caveolin-1 plays crucial roles to produce an endothelium-derived hyperpolarizing factor in mouse mesenteric arteries. We examined whether this mechanism is involved in the endothelium-dependent hyperpolarization-mediated responses to compensate reduced NO-mediated responses in diabetes mellitus during coronary occlusion in dogs in vivo. METHODS: Canine subepicardial collateral coronary small arteries (≥100 µm) and arterioles (<100 µm) were observed by an intravital microscope. Experiments were performed during occlusion of the left anterior descending coronary artery (90 minutes) under the following conditions (n = 6 each); (i) control, (ii) diabetes mellitus, and (iii) diabetes mellitus+L-NMMA+KCa channel blockade. Vascular and myocardial levels of caveolin-1, eNOS, and caspase-3 were measured by ELISA. RESULTS: Caveolin-1 levels in the ischemic area were greater in coronary microvessels than in conduit arteries in the control group. NO-mediated coronary vasodilatations of small arteries to bradykinin did not increase in diabetes mellitus associated with decreased eNOS phosphorylation at Ser1177 compared with baseline of controls and were restored by compensation of endothelium-dependent hyperpolarization and were suppressed by KCa channel blockade. CONCLUSIONS: NO-mediated vasodilatations of small coronary arteries during coronary occlusion are impaired in diabetes mellitus and are compensated by endothelium-dependent hyperpolarization of arterioles in dogs in vivo.

Maintenance of endothelial guanosine triphosphate cyclohydrolase I ameliorates diabetic nephropathy.

In diabetes, endothelial nitric oxide synthase (eNOS) produces superoxide anion rather than nitric oxide, referred to as "eNOS uncoupling," which may contribute to endothelial dysfunction, albuminuria, and diabetic nephropathy. Reduced levels of endothelium-derived tetrahydrobiopterin (BH4), an essential cofactor for eNOS, promote eNOS uncoupling. Accelerated degradation of guanosine triphosphate cyclohydrolase I (GTPCH I), the rate-limiting enzyme in BH4 biosynthesis, also occurs in diabetes, suggesting that GTPCH I may have a role in diabetic microvascular disease. Here, we crossed endothelium-dominant GTPCH I transgenic mice with Ins2(+/Akita) diabetic mice and found that endothelial overexpression of GTPCH I led to higher levels of intrarenal BH4 and lower levels of urinary albumin and reactive oxygen species compared with diabetic control mice. Furthermore, GTPCH I overexpression attenuated the hyperpermeability of macromolecules observed in diabetic control mice. In addition, we treated Ins2(+/Akita) mice with metformin, which activates AMP-activated protein kinase (AMPK) and thereby slows the degradation of GTPCH I; despite blood glucose levels that were similar to untreated mice, those treated with metformin had significantly less albuminuria. Similarly, in vitro, treating human glomerular endothelial cells with AMPK activators attenuated glucose-induced reductions in phospho-AMPK, GTPCH I, and coupled eNOS. Taken together, these data suggest that maintenance of endothelial GTPCH I expression and the resulting improvement in BH4 biosynthesis ameliorate diabetic nephropathy.

Erythropoietin enhances hydrogen peroxide-mediated dilatation of canine coronary collateral arterioles during myocardial ischemia in dogs in vivo.

We have previously demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) plays an important role in the canine coronary microcirculation as an endothelium-derived hyperpolarizing factor in vivo. However, it remains to be examined whether endogenous H(2)O(2) is involved in the dilatation of coronary collaterals during myocardial ischemia in vivo and, if so, whether erythropoietin (EPO) enhances the responses. Canine subepicardial native collateral small arteries (CSA; ≥ 100 µm) and arterioles (CA; <100 µm) were observed using an intravital microscope. Experiments were performed after left anterior descending coronary artery ischemia (90 min) under the following eight conditions (n = 5 each): control, EPO, EPO+catalase, EPO+N-monomethyl-l-arginine (l-NMMA), EPO+l-NMMA+catalase, EPO+l-NMMA+iberiotoxin [Ca(2+)-activated K(+) (K(Ca)) channel blocker], EPO+l-NMMA+apamin+charybdotoxin (K(Ca) channel blocker), and EPO+wortmannin (phosphatidylinositol 3-kinase inhibitor). Myocardial ischemia caused significant vasodilatation in CA but not in CSA under control conditions, which was significantly decreased by catalase in CA. After EPO, the vasodilatation was significantly increased in both sizes of arteries and was significantly decreased by catalase. The enhancing effect of EPO was reduced by l-NMMA but not by catalase in CSA and was reduced by l-NMMA+catalase in CA, where the greater inhibitory effects were noted with l-NMMA+catalase, l-NMMA+iberiotoxin, L-NMMA+apamin+charybdotoxin, or wortmannin. EPO significantly ameliorated ischemia-induced impairment of myocardial Akt phosphorylation, which was abolished by l-NMMA+catalase or wortmannin. EPO also ameliorated oxidative stress and myocardial injury, as assessed by plasma 8-hydroxydeoxyguanosine and troponin-T, respectively. These results indicate that EPO enhances H(2)O(2)-mediated dilatation of coronary collateral arterioles during myocardial ischemia in dogs in vivo.

Angiotensin II type 1 receptor blocker ameliorates uncoupled endothelial nitric oxide synthase in rats with experimental diabetic nephropathy.

BACKGROUND: Recent studies showed that angiotensin II type 1 receptor blocker (ARB) slows progression of chronic renal disease in patients with type 2 diabetes, regardless of changes in blood pressure. We showed that the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) due to endothelial NO synthase (eNOS) uncoupling contributed to renal dysfunction in the diabetic nephropathy. The aim of this study was to determine the effects of ARB on uncoupled eNOS in rat diabetic nephropathy. METHODS: Diabetes was induced in Sprague-Dawley rats with streptozotocin (65 mg/ kg body weight). After 6 weeks, rats were divided into saline (DM; n = 11) and ARB, losartan groups (DM+Los; n = 11). After 2-week treatment, glomerular ROS production was assessed by 2',7'-dichlorofluorescin diacetate (DCFH-DA)-derived chemiluminescence. Renal NO and ROS production were imaged by confocal laser microscopy after renal perfusion with DCFH-DA and diaminorhodamine-4M acetoxymethyl ester with L-arginine. The dimeric form of eNOS was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Serum tetrahydrobiopterin (BH4) concentrations were determined by high-performance liquid chromatography. Protein and mRNA expression of GTP cyclohydrolase 1 (GTPCH1), key enzyme of BH4 synthesis, were examined. RESULTS: Losartan attenuated glomerular ROS production in DM. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in DM glomeruli. Losartan reversed the decreased GTPCH1 and decreased dimeric form of eNOS and glomerular NO production by increased BH4 bioavailability. CONCLUSIONS: ARB improved the NOS uncoupling in diabetic nephropathy by increasing BH4 bioavailability.

Oral administration of tetrahydrobiopterin attenuates testicular damage by reducing nitric oxide synthase activity in a cryptorchid mouse model.

Experimental cryptorchidism has been shown to induce germ cell apoptosis. Nitric oxide (NO), a ubiquitous free radical produced by NO synthases (NOSs), has been associated with apoptosis in a number of cell types. However, the regulation of NOSs in experimental cryptorchid testes remains unknown. Tetrahydrobiopterin (BH4), an essential cofactor of NOS, plays an important role in the generation of NO. It has been reported that activation of the immune system stimulates an increase in endogenous BH4 rate-limiting enzyme GTP cyclohydrolase I (GTPCH I) activity, resulting in an increase in intracellular BH4 levels and BH4-dependent NO synthesis in various cells. We examined the effect of dietary treatment with BH4 on GTPCH I, BH4 synthesis, NO production, and testicular damage in cryptorchid model mice. Male mice were treated with oral BH4 starting from age 4 weeks or received standard diet only, and right cryptorchid testes were created surgically at age 10 weeks. The testes were evaluated 0, 3, 5, 7, and 10 days after surgery by assays of testicular weight, BH4 and dihydrobiopterin (oxidized BH4) levels, GTPCH I mRNA levels, NOS protein expression levels, NO concentration, and nitrotyrosine (product of ONOO(-); determinant of NO-dependent damage) levels. In untreated mice, GTPCH I mRNA and BH4 levels increased and eNOS protein expression, NO concentration, and nitrotyrosine levels increased gradually. BH4 treatment decreased GTPCH I mRNA and BH4 levels, with concomitant reduction of eNOS protein levels, nitrotyrosine levels, and NO concentration, resulting in reduced testicular damage. Our findings demonstrate that supplementation with BH4 could provide a new therapeutic intervention for heat stress-based testicular dysfunction.

A specific role for eNOS-derived reactive oxygen species in atherosclerosis progression.

OBJECTIVE: When the availability of tetrahydrobiopterin (BH4) is deficient, endothelial nitric oxide synthase (eNOS) produces superoxide rather than NO (uncoupled eNOS). We have shown that the atherosclerotic lesion size was augmented in apolipoprotein E-deficient (ApoE-KO) mice overexpressing eNOS because of the enhanced superoxide production. In this study, we addressed the specific importance of uncoupled eNOS in atherosclerosis, and the potential mechanistic role for specific versus nonspecific antioxidant strategies in restoring eNOS coupling. METHODS AND RESULTS: We crossed mice overexpressing eNOS in the endothelium (eNOS-Tg) with mice overexpressing GTP-cyclohydrolase I (GCH), the rate-limiting enzyme in BH4 synthesis, to generate ApoE-KO/eNOS-Tg/GCH-Tg mice. As a comparison, ApoE-KO/eNOS-Tg mice were treated with vitamin C. Atherosclerotic lesion formation was increased in ApoE-KO/eNOS-Tg mice compared with ApoE-KO mice. GCH overexpression in ApoE-KO/eNOS-Tg/GCH-Tg mice increased vascular BH4 levels and reduced plaque area. This reduction was associated with decreased superoxide production from uncoupled eNOS. Vitamin C treatment failed to reduce atherosclerotic lesion size in ApoE-KO/eNOS-Tg mice, despite reducing overall vascular superoxide production. CONCLUSION: In contrast to vitamin C treatment, augmenting BH4 levels in the endothelium by GCH overexpression reduced the accelerated atherosclerotic lesion formation in ApoE-KO/eNOS-Tg mice, associated with a reduction of superoxide production from uncoupled eNOS.

Coronary microcirculation in the beating heart.

The phase opposition of velocity waveforms between coronary arteries (predominantly diastolic) and veins (systolic) is the most prominent characteristic of coronary hemodynamics. This unique arterial and venous flow patterns indicate the importance of intramyocardial capacitance vessels and variable resistance vessels during a cardiac cycle. It was shown that during diastole the intramyocardial capacitance vessels have two functional components, unstressed volume and ordinary capacitance. Unstressed volume is defined as the volume of blood in a vessel at zero transmural pressure. In vivo observation of systolic narrowing of arterioles in mid-wall and in subendocardium indicates the increase in resistance by cardiac contraction.

Changes of asymmetric dimethylarginine, nitric oxide, tetrahydrobiopterin, and oxidative stress in patients with acute myocardial infarction by medical treatments.

The relationship among the nitric oxide synthase (NOS) inhibitor [asymmetric dimethylarginine (ADMA)], NOS cofactor [tetrahydrobiopterin (BH(4))], and superoxide anion in the patients with acute myocardial infarction (AMI) is still unknown. This study sought to assess the NOS inhibitor and cofactor with oxidative stress in AMI patients (n=9) during initial administration and 4 weeks after medical treatments. We measured plasma NOS inhibitor and cofactor (ADMA and BH(4)) by HPLC and plasma oxidized-LDL by ELISA. Blood samples from age-matched healthy volunteers (n=9) were taken for comparison. In AMI, plasma ADMA, oxidized-BH(4) (BH(2)+biopterin) and oxidized-LDL significantly increased (P<0.0001, P<0.01 and P<0.05 vs. healthy volunteers) and plasma BH(4), plasma nitrate and L-arginine/ADMA significantly decreased compared with healthy volunteers (P<0.0001, P<0.05 and P<0.005 vs. healthy volunteers). Medical treatments improved plasma ADMA, nitrate, BH(4) and oxidized-LDL. In conclusion, ADMA increased, and NO and BH(4) decreased with oxidative stress in AMI, and these mediators improved in AMI patients with medical treatments. These findings indicated that inhibition of NOS with oxidative stress plays a crucial role in endothelial dysfunction in patients with AMI.

Beneficial effect of hydroxyfasudil, a specific Rho-kinase inhibitor, on ischemia/reperfusion injury in canine coronary microcirculation in vivo.

OBJECTIVES: We examined whether hydroxyfasudil, a specific Rho-kinase inhibitor, exerts cardioprotective effect on coronary ischemia/reperfusion (I/R) injury and, if so, whether nitric oxide (NO) is involved. BACKGROUND: Recent studies have demonstrated that Rho-kinase is substantially involved in the pathogenesis of cardiovascular diseases; however, it remains to be examined whether it is also involved in ischemia/reperfusion (I/R) injury. METHODS: Canine subepicardial small arteries (SA, >or=100 microm) and arterioles (A, <100 microm) were observed by a charge-coupled device intravital microscope during I/R. Coronary vascular responses to endothelium-dependent (acetylcholine, intracoronary [IC]) and -independent (papaverine, IC) vasodilators were examined after I/R under the following four conditions: control (n = 7), NO synthase inhibitor alone (N(G)-monomethl-L-arginine [L-NMMA], IC, n = 4), hydroxyfasudil alone (IC, n = 7), and hydroxyfasudil plus L-NMMA (n = 7). RESULTS: Hydroxyfasudil significantly attenuated serotonin (IC)-induced vasoconstriction of SA (-7 +/- 1% vs. 2 +/- 1%, p < 0.01). Coronary I/R significantly impaired coronary vasodilation to acetylcholine after I/R (SA, p < 0.05; and A, p < 0.01 vs. before I/R) and L-NMMA further reduced the vasodilation, whereas hydroxyfasudil completely preserved the responses. The vasoconstriction by L-NMMA after I/R was significantly improved by hydroxyfasudil in both-sized arteries (both p < 0.01). Expression of endothelial nitric oxide synthase (eNOS) protein in the ischemic endocardium of left anterior descending coronary artery area (as determined by Western blotting) significantly decreased (79 +/- 4%) compared with the nonischemic endocardium of LCX area (100 +/- 7%), which was improved by hydroxyfasudil (105 +/- 6%, p < 0.01). Hydroxyfasudil significantly reduced myocardial infarct size, and hydroxyfasudil with L-NMMA also reduced the infarct size compared with L-NMMA alone. CONCLUSIONS: Hydroxyfasudil exerts cardioprotective effects on coronary I/R injury in vivo, in which NO-mediated mechanism may be involved through preservation of eNOS expression.

Cardioprotective effect of hydroxyfasudil as a specific Rho-kinase inhibitor, on ischemia-reperfusion injury in canine coronary microvessels in vivo.

Rho-kinase modulates calcium sensitivity of the myosin light chain in smooth muscle cells and has been implicated as playing a pathogenetic role in cardiovascular disorders. This paper was aimed to determine whether hydroxyfasudil (a specific Rho-kinase inhibitor) exerts cardioprotective effect on coronary ischemia-reperfusion (I/R) injury, and if so, whether NO is involved. Canine subepicardial small arteries (diameter > or = 100 microm) and arterioles (diameter < 100 microm) were observed by a CCD intravital microscope during coronary I/R. Coronary vascular responses to endothelium-dependent (acetylcholine) and -independent (papaverine) vasodilators were examined after I/R under three conditions: control, preconditioning, and hydroxyfasudil. Coronary I/R significantly impaired coronary vasodilation to acetylcholine, whereas hydroxyfasudil completely preserved the responses, as did preconditioning. Hydroxyfasudil also significantly reduced myocardial infarct size. These results indicated that hydroxyfasudil exerts cardioprotective effects on coronary I/R injury in vivo, for which NO-mediated mechanism may be involved.

Hydrogen peroxide, an endogenous endothelium-derived hyperpolarizing factor, plays an important role in coronary autoregulation in vivo.

BACKGROUND: Recent studies in vitro have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans. The aim of this study was to evaluate our hypothesis that endothelium-derived H2O2 is an EDHF in vivo and plays an important role in coronary autoregulation. METHODS AND RESULTS: To test this hypothesis, we evaluated vasodilator responses of canine (n=41) subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) with an intravital microscope in response to acetylcholine and to a stepwise reduction in coronary perfusion pressure (from 100 to 30 mm Hg) before and after inhibition of NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). After L-NMMA, the coronary vasodilator responses were attenuated primarily in small arteries, whereas combined infusion of L-NMMA plus catalase (an enzyme that selectively dismutates H2O2 into water and oxygen) or tetraethylammonium (TEA, an inhibitor of large-conductance K(Ca) channels) attenuated the vasodilator responses of coronary arteries of both sizes. Residual arteriolar dilation after L-NMMA plus catalase or TEA was largely attenuated by 8-sulfophenyltheophylline, an adenosine receptor inhibitor. CONCLUSIONS: These results suggest that H2O2 is an endogenous EDHF in vivo and plays an important role in coronary autoregulation in cooperation with NO and adenosine.

Role of endogenous hydrogen peroxide during angiotensin type 1 receptor blockers administration in pacing-induced metabolic coronary vasodilatation in dogs in vivo.

BACKGROUND: We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in canine coronary microcirculation in vivo. However, the role of H2O2/EDHF during angiotensin type 1 receptor blockers (ARB) administration in metabolic coronary dilatation in vivo remains to be examined. We examined whether H2O2 during ARB administration is involved in pacing-induced metabolic coronary vasodilatation in dogs in vivo and if so, whether such beneficial effects of ARB administration acutely improve coronary vasodilatation in diabetes mellitus (DM). METHODS: Canine subepicardial coronary small arteries (CSA,≥ 100 µm) and arterioles (CA, <100 µm) in left anterior descending artery area were continuously observed by an intravital microscope under cyclooxygenase blockade(ibuprofen, 12.5 mg/kg, intravenous infusion, iv). Experiments were performed during paired right ventricular pacing under the following 4 conditions (n=5 each); (i) control, (ii) DM(alloxan 40 mg/ kg, iv, 1 week prior to study), (iii) DM+ARB(olmesartan, 10 µg/kg/min, 10 min, intracoronary infusion,ic)+L-NMMA (NOS inhibitor, 2 !mol/min, ic) and (iv)DM+ARB+catalase (H2O2 discomposer, 1000 U/ml, 5 min, ic). RESULTS: Cardiac tachypacing (60 to 120 bpm) caused coronary vasodilatation in both-sized arteries under control conditions. DM significantly decreased the vasodilatation compared with control in CSA and there was a residual vasodilatation for the loss of NO in CA, whereas DM+ARB+L-NMMA improved the vasodilatation compared with DM alone in CA and was significantly decreased by DM+ARB+catalase in CA. CONCLUSIONS: These results indicate that H2O2 during ARB administration is involved in pacing-induced metabolic coronary vasodilatation in DM in vivo and that there are substantial compensatory interactions between NO and H2O2.

Impaired endothelial function may be due to decreased aortic tetrahydrobiopterin, assessed by a new flow-mediated vasodilation in vivo in hypercholesterolemic/atherogenic mice.

Tetrahydrobiopterin (BH4) is an important cofactor for endothelial nitric oxide synthase activity. The relationship between endothelial function in vivo and aortic BH4 level is not fully understood, however. In the present study, we aimed to clarify whether reduction of aortic BH4 levels contributes to endothelial dysfunction in vivo using spontaneously hyperlipidemic mice. To estimate endothelial function in vivo and in real-time state, we developed a flow-mediated vasodilation (FMV) method in mice, which measured changes in the diameter of the femoral artery in response to increased blood flow. C57BL/6 mice and apoE/low-density lipoprotein receptor double knock-out mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 12 weeks from 6 weeks of age. HFD feeding impaired FMV in double knock-out mice, but not in C57BL/6 mice. Furthermore, HFD feeding reduced plasma NOx concentration and aortic BH4 level in double knock-out mice. Conversely, exogenous injection of BH4 (2 mg/kg) markedly increased aortic BH4 levels and restored endothelial function. In conclusion, we demonstrated that HFD feeding impaired nitric oxide-mediated endothelial function and reduced BH4 level in vivo, and that acute augmentation of aortic BH4 levels improved endothelial function. These findings indicate that BH4 is a critical determinant of nitric oxide-mediated endothelial function in hypercholesterolemia.

Role of Cu,Zn-SOD in the synthesis of endogenous vasodilator hydrogen peroxide during reactive hyperemia in mouse mesenteric microcirculation in vivo.

We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD(-/-) mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, N(G)-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-SOD(-/-) mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD(-/-) mice. In mesenteric arterioles of the Cu,Zn-SOD(-/-) mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H(2)O(2) production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD(-/-) mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H(2)O(2) that contributes to RH in mouse mesenteric smaller arterioles.

Crucial role of nitric oxide synthases system in endothelium-dependent hyperpolarization in mice.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several relaxing factors, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have previously demonstrated in animals and humans that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF that is produced in part by endothelial NO synthase (eNOS). In this study, we show that genetic disruption of all three NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]) abolishes EDHF responses in mice. The contribution of the NOS system to EDHF-mediated responses was examined in eNOS(-/-), n/eNOS(-/-), and n/i/eNOS(-/-) mice. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased, whereas vascular smooth muscle function was preserved. Loss of eNOS expression alone was compensated for by other NOS genes, and endothelial cell production of H(2)O(2) and EDHF-mediated responses were completely absent in n/i/eNOS(-/-) mice, even after antihypertensive treatment with hydralazine. NOS uncoupling was not involved, as modulation of tetrahydrobiopterin (BH(4)) synthesis had no effect on EDHF-mediated relaxation, and the BH(4)/dihydrobiopterin (BH(2)) ratio was comparable in mesenteric arteries and the aorta. These results provide the first evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries.

Fluvastatin reverses endothelial dysfunction and increased vascular oxidative stress in rat adjuvant-induced arthritis.

OBJECTIVE: To investigate the effect of statins on vascular dysfunction in rat adjuvant-induced arthritis (AIA). METHODS: Fluvastatin (5 mg/kg/day) was administered orally to rats with AIA, for 21 days after the onset of arthritis. The vasodilatory response to acetylcholine of aortic rings isolated from rats with AIA that were not treated or were treated with fluvastatin and from normal rats was determined. The amounts of 4-hydroxy-2-nonenal (HNE) and nitrotyrosine in aortas were measured by Western blotting. In vitro and in situ superoxide production in aortas was evaluated based on fluorogenic oxidation of dihydroethidium to ethidium. Expression of NAD(P)H components and endothelial nitric oxide synthase (eNOS) in aortas was examined by real-time reverse transcriptase-polymerase chain reaction and Western blotting. Serum levels of tetrahydrobiopterin, a critical eNOS cofactor, were determined by high-performance liquid chromatography. RESULTS: Fluvastatin reversed endothelial dysfunction in AIA without affecting the clinical severity of arthritis or serum cholesterol concentration. Fluvastatin reduced the amounts of HNE and nitrotyrosine in the aorta, and the levels of superoxide expressed in endothelial cells and smooth muscle cells in the tissue, in rats with AIA. NADH- or L-arginine-induced superoxide production was not observed in the aortic samples from fluvastatin-treated rats with AIA. Fluvastatin decreased the levels of expression of messenger RNA for p22phox, a NAD(P)H oxidase component, in the aortas of rats with AIA, but did not affect the expression of eNOS. Serum levels of tetrahydrobiopterin were significantly reduced in rats with AIA, and were increased by administration of fluvastatin. CONCLUSION: Our findings demonstrate that fluvastatin has potent vascular protective effects in AIA and provide additional scientific rationale for the use of statins to reduce cardiovascular mortality in patients with rheumatoid arthritis.

Important role of endogenous hydrogen peroxide in pacing-induced metabolic coronary vasodilation in dogs in vivo.

OBJECTIVES: We examined whether endogenous hydrogen peroxide (H2O2) is involved in pacing-induced metabolic vasodilation in vivo. BACKGROUND: We have previously demonstrated that endothelium-derived H2O2 is an endothelium-derived hyperpolarizing factor in canine coronary microcirculation in vivo. However, the role of endogenous H2O2 in metabolic coronary vasodilation in vivo remains to be examined. METHODS: Canine subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) were continuously observed by a microscope under cyclooxygenase blockade (ibuprofen, 12.5 mg/kg intravenous [IV]) (n = 60). Experiments were performed during paired right ventricular pacing under the following 7 conditions: control, nitric oxide (NO) synthase inhibitor (N(G)-monomethyl-L-arginine [L-NMMA], 2 micromol/min for 20 min intracoronary [IC]), catalase (a decomposer of H2O2, 40,000 U/kg IV and 240,000 U/kg/min for 10 min IC), 8-sulfophenyltheophylline (SPT) (an adenosine receptor blocker, 25 mug/kg/min for 5 min IC), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA) (K(Ca)-channel blocker, 10 microg/kg/min for 10 min IC), and L-NMMA+catalase+8-SPT. RESULTS: Cardiac tachypacing (60 to 120 beats/min) caused coronary vasodilation in both-sized arteries under control conditions in response to the increase in myocardial oxygen consumption. The metabolic coronary vasodilation was decreased after L-NMMA in subepicardial small arteries with an increased fluorescent H2O2 production compared with catalase group, whereas catalase decreased the vasodilation of arterioles with an increased fluorescent NO production compared with the L-NMMA group, and 8-SPT also decreased the vasodilation of arterioles. Furthermore, the metabolic coronary vasodilation was markedly attenuated after L-NMMA+catalase, L-NMMA+TEA, and L-NMMA+catalase+8-SPT in both-sized arteries. CONCLUSIONS: These results indicate that endogenous H2O2 plays an important role in pacing-induced metabolic coronary vasodilation in vivo.

Edaravone preserves coronary microvascular endothelial function after ischemia/reperfusion on the beating canine heart in vivo.

We examined whether edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, exerts its protective effect on coronary microvessels after ischemia/reperfusion (I/R) in vivo. Ninety-minute coronary occlusion followed by reperfusion was performed in 16 open-chest dogs with and without edaravone administration. Coronary small artery (> or = 100 microm in size) and arteriolar (< 100 microm) vasodilation, in the presence of endothelium-dependent (acetylcholine) or -independent (papaverine) vasodilators, was directly observed using intravital microscopy before and after I/R. I/R impaired microvascular vasodilation in response to acetylcholine, whereas administration of edaravone preserved the response in microvessels of both sizes, but to a greater extent in the coronary small arteries. No significant changes were noted with papaverine administration. In the edaravone group, the fluorescent intensity from reactive oxygen species (ROS) was lower, whereas nitric oxide (NO) intensity was higher relative to controls in the microvessels of the ischemic area. In conclusion, edaravone preserves coronary microvascular endothelial function after I/R in vivo. These effects, which were NO-mediated, were attributed to the ROS scavenging properties of edaravone.