Impaired flow-induced dilation in mesenteric resistance arteries from receptor protein tyrosine phosphatase-mu-deficient mice

The transmembrane receptor-like protein tyrosine phosphatase-mu (RPTPmu) is thought to play an important role in cell-cell adhesion-mediated processes. We recently showed that RPTPmu is predominantly expressed in the endothelium of arteries and not in veins. Its involvement in the regulation of endothelial adherens junctions and its specific arterial expression suggest that RPTPmu plays a role in controlling arterial endothelial cell function and vascular tone. To test this hypothesis, we analyzed myogenic responsiveness, flow-induced dilation, and functional integrity of mesenteric resistance arteries from RPTPmu-deficient (RPTPmu(-/-)) mice and from wild-type littermates. Here, we show that cannulated mesenteric arteries from RPTPmu(-/-) mice display significantly decreased flow-induced dilation. In contrast, mechanical properties, myogenic responsiveness, responsiveness to the vasoconstrictors phenylephrine or U-46619, and responsiveness to the endothelium-dependent vasodilators methacholine or bradykinin were similar in both groups. Our results imply that RPTPmu is involved in the mechanotransduction or accessory signaling pathways that control shear stress responses in mesenteric resistance arteries.     

Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction

Matrix metalloproteinase-2 (MMP-2) is prominently overexpressed both after myocardial infarction (MI) and in heart failure. However, its pathophysiological significance in these conditions is still unclear. We thus examined the effects of targeted deletion of MMP-2 on post-MI left ventricular (LV) remodeling and failure. Anterior MI was produced in 10- to 12-wk-old male MMP-2 knockout (KO) and sibling wild-type (WT) mice by ligating the left coronary artery. By day 28, MI resulted in a significant increase in mortality in association with LV cavity dilatation and dysfunction. The MMP-2 KO mice had a significantly better survival rate than WT mice (56% vs. 85%, P < 0.05), despite a comparable infarct size (50 +/- 3% vs. 51 +/- 3%, P = not significant), heart rate, and arterial blood pressure. The KO mice had a significantly lower incidence of LV rupture (10% vs. 39%, P < 0.05), which occurred within 7 days of MI. The KO mice exerted less LV cavity dilatation and improved fractional shortening after MI by echocardiography. The LV zymographic MMP-2 level significantly increased in WT mice after coronary artery ligation; however, this was completely prevented in KO mice. In contrast, the increase in the LV zymographic MMP-9 level after MI was similar between KO and WT mice. MMP-2 activation is therefore considered to contribute to an early cardiac rupture as well as late LV remodeling after MI. The inhibition of MMP-2 activation may therefore be a potentially useful therapeutic strategy to manage post-MI hearts.     

Heart rate-associated mechanical stress impairs carotid but not cerebral artery compliance in dyslipidemic atherosclerotic mice

The cardiac cycle imposes a mechanical stress that dilates elastic carotid arteries, while shear stress largely contributes to the endothelium-dependent dilation of downstream cerebral arteries. In the presence of dyslipidemia, carotid arteries stiffen while the endothelial function declines. We reasoned that stiffening of carotid arteries would be prevented by reducing resting heart rate (HR), while improving the endothelial function would regulate cerebral artery compliance and function. Thus we treated or not 3-mo-old male atherosclerotic mice (ATX; LDLr(-/-):hApoB(+/+)) for 3 mo with the sinoatrial pacemaker current inhibitor ivabradine (IVA), the β-blocker metoprolol (METO), or subjected mice to voluntary physical training (PT). Arterial (carotid and cerebral artery) compliance and endothelium-dependent flow-mediated cerebral dilation were measured in isolated pressurized arteries. IVA and METO similarly reduced (P < 0.05) 24-h HR by ≈15%, while PT had no impact. As expected, carotid artery stiffness increased (P < 0.05) in ATX mice compared with wild-type mice, while cerebral artery stiffness decreased (P < 0.05); this paradoxical increase in cerebrovascular compliance was associated with endothelial dysfunction and an augmented metalloproteinase-9 (MMP-9) activity (P < 0.05), without changing the lipid composition of the wall. Reducing HR (IVA and METO) limited carotid artery stiffening, but plaque progression was prevented by IVA only. In contrast, IVA maintained and PT improved cerebral endothelial nitric oxide synthase-dependent flow-mediated dilation and wall compliance, and both interventions reduced MMP-9 activity (P < 0.05); METO worsened endothelial dysfunction and compliance and did not reduce MMP-9 activity. In conclusion, HR-dependent mechanical stress contributes to carotid artery wall stiffening in severely dyslipidemic mice while cerebrovascular compliance is mostly regulated by the endothelium.     

Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.     

Arterial remodeling and plasma volume expansion in caveolin-1-deficient mice

Caveolin-1 (Cav-1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide (NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin-1 may thus be expected to cause arterial dilatation and increased vessel wall mass (remodeling). This was tested in Cav-1 knockout (KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media-to-lumen ratio in KO. Pressure-induced myogenic tone and flow-induced dilatation were decreased in KO arteries, but both were increased toward wild-type (WT) levels following NO synthase (NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length-force relationships in KO, and the force response to alpha(1)-adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO (38 +/- 6%) compared with WT (17 +/- 3%). Tracer-dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav-1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav-1 KO mice.     

Functional characterization and sex differences in small mesenteric arteries of the estrogen receptor-beta knockout mouse

The role of the estrogen receptor (ER) subtypes in the modulation of vascular function is poorly understood. The aim of this study was to characterize ex vivo the functional properties of small arteries and their response to estrogens in the mesenteric circulation of female and male ER-beta knockout mice (beta-ERKO) and their wild-type (WT) littermates. Responses to changes in intraluminal flow and pressure were obtained before and after incubation with 17beta-estradiol or ER-alpha agonist propyl-pyrazole-triol (3 h; 10 nM). Cumulative concentration-response curves to acetylcholine, norepinephrine, and passive distensibility were compared with respect to sex and genotype. The collagen and elastin content within the vascular wall and ER expression were also determined. Endothelial morphology was visualized by scanning electron microscopy. 17beta-Estradiol and propyl-pyrazole-triol-treated arteries from female beta-ERKO and WT mice showed enhanced flow-mediated dilation, but this was not evident in males. Distensibility was decreased in arteries from beta-ERKO females. Sex differences in myogenic tone were observed in 17beta-estradiol-treated arteries, but were similar between beta-ERKO and WT mice. Acetylcholine- and norepinephrine-induced responses were similar between groups and sexes. ER-alpha was similarly expressed in the endothelium and media of arteries from all groups studied, as well as ER-beta in WT animals. Endothelial morphology was similar in arteries from animals of both sexes and genotype; however, arterial elastin content was decreased, and collagen content was increased in beta-ERKO male compared with WT male and with beta-ERKO female. We suggest that ERs play a sex-specific role in estrogen-mediated flow responses and distensibility, and that deletion of ER-beta affects artery structure but only in male animals. Further studies in beta-ERKO mice with established hypertension and in alpha-ERKO mice are warranted.     

Calcitonin gene-related peptide released from endothelial progenitor cells inhibits the proliferation of rat vascular smooth muscle cells induced by angiotensin II

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.     

Key role of alpha(1)beta(1)-integrin in the activation of PI3-kinase-Akt by flow (shear stress) in resistance arteries

Resistance arteries are the site of the earliest manifestations of many cardiovascular and metabolic diseases. Flow (shear stress) is the main physiological stimulus for the endothelium through the activation of vasodilatory pathways generating flow-mediated dilation (FMD). The role of FMD in local blood flow control and angiogenesis is well established, and alterations in FMD are early markers of cardiovascular disorders. alpha(1)-Integrin, which has a role in angiogenesis, could be involved in FMD. FMD was studied in mesenteric resistance arteries (MRA) isolated in arteriographs. The role of alpha(1)-integrins in FMD was tested with selective antibodies and mice lacking the gene encoding for alpha(1)-integrins. Both anti-alpha(1) blocking antibodies and genetic deficiency in alpha(1)-integrin in mice (alpha(1)(-/-)) inhibited FMD without affecting receptor-mediated (acetylcholine) endothelium-dependent dilation or endothelium-independent dilation (sodium nitroprusside). Similarly, vasoconstrictor tone (myogenic tone and phenylephrine-induced contraction) was not affected. In MRA phosphorylated Akt and phosphatidylinositol 3-kinase (PI3-kinase) were significantly lower in alpha(1)(-/-) mice than in alpha(1)(+/+) mice, although total Akt and endothelial nitric oxide synthase (eNOS) were not affected. Pharmacological blockade of PI3-kinase-Akt pathway with LY-294002 inhibited FMD. This inhibitory effect of LY-294002 was significantly lower in alpha(1)(-/-) mice than in alpha(1)(+/+) mice. Thus alpha(1)-integrin has a key role in flow (shear stress)-dependent vasodilation in resistance arteries by transmitting the signal to eNOS through activation of PI3-kinase and Akt. Because of the central role of flow (shear stress) activation of the endothelium in vascular disorders, this finding opens new perspectives in the pathophysiology of the microcirculation and provides new therapeutic targets.     

Coronary artery myogenic response in a genetic model of hypertrophic cardiomyopathy

Hypertrophic cardiac myopathy (HCM) is the leading cause of mortality in young athletes. Abnormalities in small intramural coronary arteries have been observed at autopsy in such subjects. The walls of these intramural vessels, especially in the ventricular septum, are thickened, and the lumen frequently appears narrowed. Whether these morphological characteristics have functional correlates is unknown. We studied coronary myogenic tone in a transgenic mouse model of HCM that has mutations in the cardiac alpha-myosin heavy chain gene. This transgenic mouse has a cardiac phenotype that resembles that occurring in humans. We examined the possible vascular contributions to the pathology of HCM. Septal arteries from 3- and 11-mo-old wild-type (WT) and transgenic (TG) mice were studied on a pressure myograph. The myogenic response to increased intravascular pressure in older animals was significantly reduced [maximal constriction: 32 +/- 4% (TG) and 46 +/- 4% (WT), P < 0.05]. After inhibition of endothelin receptors with bosentan, both WT and TG mice had similar increases in myogenic constriction. The sensitivity to exogenous endothelin was significantly reduced in TG mice, suggesting that the reduced myogenic constriction in HCM was due to reduced receptor sensitivity. In conclusion, we show for the first time that 1) myogenic tone in the coronary septal artery of the mouse is regulated by a basal release of endothelin, and 2) pressure-induced myogenic activation is attenuated in HCM, possibly consequent to a reduction in endothelin responsiveness. The associated reduction in coronary vasodilatory reserve may increase susceptibility to ischemia and arrhythmias.     

Gonadal hormones affect diameter of male rat cerebral arteries through endothelium-dependent mechanisms

Gender is known to influence the incidence and severity of cerebrovascular disease. In the present study, luminal diameter was measured in vitro in pressurized middle cerebral artery segments from male rats that were either untreated, orchiectomized (ORX), ORX with testosterone treatment (ORX+TEST), or ORX with estrogen treatment (ORX+EST). The maximal passive diameters (0 Ca(2+) + 3 mM EDTA) of arteries from all four groups were similar. In endothelium-intact arteries, myogenic tone was significantly greater in arteries from untreated and ORX+TEST compared with arteries from either ORX or ORX+EST. During exposure to N(G)-nitro-L-arginine-methyl ester (L-NAME), an NO synthase (NOS) inhibitor, myogenic tone significantly increased in all groups. The effect of L-NAME was significantly greater in arteries from untreated and ORX+EST compared with arteries from ORX and ORX+TEST rats. Differences in myogenic tone between ORX and ORX+TEST persisted after inhibition of NOS. After endothelium removal or inhibition of the cyclooxygenase pathway combined with K(+) channel blockers, myogenic tone differences between ORX and ORX+TEST were abolished. Wall thickness and forced dilation were not significantly different between arteries from ORX and ORX+TEST. Our data show that gonadal hormones affect myogenic tone in male rat cerebral arteries through NOS- and/or endothelium-dependent mechanisms.     

Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice

The role of mitochondrial manganese-superoxide dismutase (Mn-SOD) in the maintenance of vascular function has not yet been studied. Thus we examined flow- and agonist-induced dilations in isolated mesenteric arteries (approximately 90 microm in diameter) of Mn-SOD heterozygous (Mn-SOD+/-) and wild-type (WT) mice. Increases in flow elicited dilations in all vessels, but the magnitude of the dilation was significantly less in vessels of Mn-SOD+/- mice than in those of WT mice (64 vs. 74% of passive diameter). N(omega)-nitro-L-arginine methyl ester inhibited the dilation in vessels of WT mice but had no effect on vessels of Mn-SOD+/- mice. Tempol or tiron (superoxide scavengers) increased flow-induced dilation in vessels of Mn-SOD+/- mice. Acetylcholine- and sodium nitroprusside-induced, but not adenosine-induced, dilations were also decreased in arteries of Mn-SOD+/- mice. Superoxide levels in the arteries of Mn-SOD+/- mice were significantly increased. Western blot analysis confirmed a 50% reduction of Mn-SOD protein in the vessels of Mn-SOD+/- mice. A 41% reduction in endothelial nitric oxide synthase (eNOS) protein and a 37% reduction in eNOS activity were also found in the vessels of Mn-SOD+/- mice. Whereas there was no difference in eNOS protein in kidney homogenates of WT and Mn-SOD+/- mice, a significant reduction of nitric oxide synthase activity was found in Mn-SOD+/- mice, which could be restored by the administration of tiron. We conclude that an increased concentration of superoxide due to reduced activity of Mn-SOD, which inactivates nitric oxide and inhibits eNOS activity, contributes to the impaired vasodilator function of isolated mesenteric arteries of Mn-SOD+/- mice. These results suggest that Mn-SOD contributes significantly to the regulation of vascular function.     

EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice

Vasodilation to increases in flow was studied in isolated gracilis muscle arterioles of female endothelial nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT) mice. Dilation to flow (0-10 microl/min) was similar in the two groups, yet calculated wall shear stress was significantly greater in arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin, which inhibited flow-induced dilation in vessels of WT mice by approximately 40%, did not affect the responses of eNOS-KO mice, whereas miconazole and 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses. Basal release of epoxyeicosatrienonic acids from arterioles was inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in arterioles of eNOS-KO mice but had no effect on arterioles of WT mice. In WT mice, neither N(omega)-nitro-L-arginine methyl ester nor miconazole alone affected flow-induced dilation. Combination of both inhibitors inhibited the responses by approximately 50%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone inhibited flow-induced dilation by approximately 49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT mice, nitric oxide and prostaglandins mediate flow-induced dilation. When eNOS is inhibited, endothelium-derived hyperpolarizing factor substitutes for nitric oxide. In female eNOS-KO mice, metabolites of cytochrome P-450, via activation of large-conductance Ca2+-activated K+ channels of smooth muscle, mediate entirely the arteriolar dilation to flow.     

Effects of cardiac hormones on arterial pressure and sodium excretion in NPRA knockout mice

These studies were designed to determine if the atria contains natriuretic substances that act through a non-natriuretic peptide type A (NPRA) receptor mechanism. C57BL/6 mice, either wild-type NPRA++ (WT) or NPRA-- knockout (KO), were anesthetized with pentobarbital. Catheters were placed in the trachea, carotid artery, jugular vein, and bladder. Urine was collected for six 30-min periods. Both groups received an iv injection of 100 ng of rat atrial natriuretic peptide (rANP) in 200 microl of saline after the first period (30 mins) and 200 microl of rat atrial extract after the fourth period (120 mins). ANP injection increased urine flow (UF) to 2.7 +/- 0.5 microl/min in the WT versus 1.9 +/- 0.2 in KO. Extract increased UF to 7.9 +/- 1.5 microl/min in WT versus 2.7 +/- 0.4 in KO (P < 0.01). ANP increased sodium excretion (ENa) to 0.47 +/- 0.10 micromoles/min in WT versus 0.27 +/- 0.04 in KO (P < 0.05). Extract increased ENa to 1.44 +/- 0.47 micromoles/min in WT versus 0.26 +/- 0.06 in KO (P < 0.05). Extract decreased mean arterial pressure (MAP) to 62 +/- 3 mm Hg in the WT versus 81 +/- 5 in KO (P < 0.01). ENa and MAP responses to extract in KO were not different from responses to 200 microl of saline. A constant 150-min infusion of rat atrial extract increased urine flow by 3-fold and ENa by 5-fold (both P < 0.05) in the WT mice but had no significant effect in the KO mice. Thus, acute renal and MAP responses to atrial extracts require the NPRA receptor.     

Inhibition of tumor necrosis factor receptor-1-mediated pathways has beneficial effects in a murine model of postischemic remodeling

The aim of the present study was to investigate the importance of tumor necrosis factor (TNF)-alpha receptor-1 (TNFR1)-mediated pathways in a murine model of myocardial infarction and remodeling. One hundred and ninety-four wild-type (WT) and TNFR1 gene-deleted (TNFR1KO) mice underwent left coronary artery ligation to induce myocardial infarction. On days 1, 3, 7, and 42, mice underwent transesophageal echocardiography. Hearts were weighed, and the left ventricle (LV) was assayed for matrix metalloproteinase (MMP)-2 and -9 activity and for tissue inhibitor of MMP (TIMP)-1 and -2 expression. Deletion of the TNFR1 gene substantially improved survival because no deaths were observed in TNFR1KO mice versus 56.4% and 18.2% in WT males and females, respectively (P < 0.002). At 42 days, LV remodeling, assessed by LV function (fractional area change of 31.9 +/- 7.9%, 32.2 +/- 7.7%, and 21.6 +/- 7.1% in TNFR1KO males, TNFR1KO females, and WT females, respectively, P < 0.04), and hypertrophy (heart weight-to-body weight ratios of 5.435 +/- 0.986, 5.485 +/- 0.677, and 6.726 +/- 0.704 mg/g, P < 0.04) were ameliorated in TNFR1KO mice. MMP-9 activity was highest at 3 days postinfarction and was highest in WT males (1.9 +/- 0.4 4, 3.6 +/- 0.24, 1.15 +/- 0.28, and 1.3 +/- 1.2 ng/100 microg protein, respectively, in TNFR1KO males, WT males, TNFR1KO females, and WT females, respectively, P < 0.002), whereas at 3 days TIMP-1 mRNA fold upregulation compared with type- and sex-matched controls was lowest in WT males (138.32 +/- 13.05, 46.74 +/- 5.43, 186.09 +/- 28.07, and 101.76 +/- 22.48, respectively, P < 0.002). MMP-2 and TIMP-2 increased similarly in all infarcted groups. These findings suggest that the benefits of TNFR1 ablation might be attributable at least in part to the attenuation of cytokine-mediated imbalances in MMP-TIMP activity.     

Mechanisms of endothelial dysfunction in resistance arteries from patients with end-stage renal disease

The study focuses on the mechanisms of endothelial dysfunction in the uremic milieu. Subcutaneous resistance arteries from 35 end-stage renal disease (ESRD) patients and 28 matched controls were studied ex-vivo. Basal and receptor-dependent effects of endothelium-derived factors, expression of endothelial NO synthase (eNOS), prerequisites for myoendothelial gap junctions (MEGJ), and associations between endothelium-dependent responses and plasma levels of endothelial dysfunction markers were assessed. The contribution of endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxation was impaired in uremic arteries after stimulation with bradykinin, but not acetylcholine, reflecting the agonist-specific differences. Diminished vasodilator influences of the endothelium on basal tone and enhanced plasma levels of asymmetrical dimethyl L-arginine (ADMA) suggest impairment in NO-mediated regulation of uremic arteries. eNOS expression and contribution of MEGJs to EDHF type responses were unaltered. Plasma levels of ADMA were negatively associated with endothelium-dependent responses in uremic arteries. Preserved responses of smooth muscle to pinacidil and NO-donor indicate alterations within the endothelium and tolerance of vasodilator mechanisms to the uremic retention products at the level of smooth muscle. We conclude that both EDHF and NO pathways that control resistance artery tone are impaired in the uremic milieu. For the first time, we validate the alterations in EDHF type responses linked to kinin receptors in ESRD patients. The association between plasma ADMA concentrations and endothelial function in uremic resistance vasculature may have diagnostic and future therapeutic implications.     

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.     

Vascular reactivity, 5-HT uptake, and blood pressure in the serotonin transporter knockout rat

The handling of serotonin [5-hydroxytryptamine (5-HT)] depends on the serotonin transporter (SERT). A SERT knockout (KO) rat is a useful model to test the hypothesis that SERT is the primary mechanism for arterial 5-HT uptake and to investigate the impact of SERT removal on blood pressure. Wild-type (WT) and KO rats were used to measure 5-HT content (plasma, raphe, aorta, carotid, and mesenteric artery), aortic isometric contraction, and blood pressure. HPLC supported the lack of circulating 5-HT in plasma (ng/ml plasma, WT, 310 +/- 96; and KO, 1.0 +/- 0.5; P < 0.05). Immunohistochemistry and Western blot analyses validated the presence of the SERT protein in the WT rats and a lesser expression in the KO rat. The aorta isolated from KO rats had a normal contraction to phenylephrine and norepinephrine and a normal relaxation to the endothelium-dependent agonist acetylcholine compared with the aorta from WT. In contrast, the potency of 5-HT was increased in the aorta from KO rats compared with WT rats [-log EC(50) (M); WT, 5.71 +/- 0.08; and KO, 6.7 +/- 0.18] and maximum contraction was reduced [%phenylephrine (10 muM) contraction, WT, 113 +/- 6%; and KO, 52 +/- 12%]. 5-HT uptake was reduced but not abolished in arteries of the KO compared with the WT rats. Diurnal mean arterial blood pressure, heart rate, and locomotor activity level of the KO rats were similar to the WT rats. These data suggest that there are other mechanisms of 5-HT uptake in the arteries of the rat and that although the absence of circulating 5-HT and/or SERT function sensitizes arteries to 5-HT, SERT dysfunction does not impair normal blood pressure.     

Acetylcholine causes endothelium-dependent contraction of mouse arteries

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).     

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.