Disruption of Physiological Balance Between Nitric Oxide and Endothelium-Dependent Hyperpolarization Impairs Cardiovascular Homeostasis in Mice.

OBJECTIVE: Endothelium-derived nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) play important roles in modulating vascular tone in a distinct vessel size-dependent manner; NO plays a dominant role in conduit arteries and EDH in resistance vessels. We have recently demonstrated that endothelial NO synthase (eNOS) is functionally suppressed in resistance vessels through caveolin-1 (Cav-1)-dependent mechanism, switching its function from NO to EDH/hydrogen peroxide generation in mice. Here, we examined the possible importance of the physiological balance between NO and EDH in cardiovascular homeostasis. APPROACH AND RESULTS: We used 2 genotypes of mice in which eNOS activity is genetically upregulated; Cav-1-knockout (Cav-1-KO) and endothelium-specific eNOS transgenic (eNOS-Tg) mice. Isometric tension recordings and Langendorff experiments with isolated perfused hearts showed that NO-mediated relaxations were significantly enhanced, whereas EDH-mediated relaxations were markedly reduced in microcirculations. Importantly, impaired EDH-mediated relaxations of small mesenteric arteries from Cav-1-KO mice were completely rescued by crossing the mice with those with endothelium-specific overexpression of Cav-1. Furthermore, both genotypes showed altered cardiovascular phenotypes, including cardiac hypertrophy in Cav-1-KO mice and hypotension in eNOS-Tg mice. Finally, we examined cardiac responses to chronic pressure overload by transverse aortic constriction in vivo. When compared with wild-type mice, both Cav-1-KO and eNOS-Tg mice exhibited reduced survival after transverse aortic constriction associated with accelerated left ventricular systolic dysfunction, reduced coronary flow reserve, and enhanced myocardial hypoxia. CONCLUSIONS: These results indicate that excessive endothelium-derived NO with reduced EDH impairs cardiovascular homeostasis in mice in vivo.

Endothelial AMP-activated protein kinase regulates blood pressure and coronary flow responses through hyperpolarization mechanism in mice.

OBJECTIVE: Vascular endothelium plays an important role to maintain cardiovascular homeostasis through several mechanisms, including endothelium-dependent hyperpolarization (EDH). We have recently demonstrated that EDH is involved in endothelial metabolic regulation in mice. However, it remains to be examined whether AMP-activated protein kinase (AMPK), an important metabolic regulator, is involved in EDH and if so, whether endothelial AMPK (eAMPK) plays a role for circulatory regulation. APPROACH AND RESULTS: We examined the role of eAMPK in EDH, using mice with endothelium-specific deficiency of α-catalytic subunit of AMPK, either α1 (eAMPKα1 (-/-)α2 (+/+)) or α2 (eAMPKα1 (+/+)α2 (-/-)) alone or both of them (eAMPKα1 (-/-)α2 (-/-)). We performed telemetry, organ chamber, electrophysiological, and Langendorff experiments to examine blood pressure, vascular responses, hyperpolarization of membrane potential, and coronary flow responses, respectively. Hypertension was noted throughout the day in eAMPKα1 (-/-)α2 (-/-) and eAMPKα1 (-/-)α2 (+/+) but not in eAMPKα1 (+/+)α2 (-/-) mice when compared with respective control. Importantly, endothelium-dependent relaxations, EDH, and coronary flow increase were all significantly reduced in eAMPKα1 (-/-)α2 (-/-) and eAMPKα1 (-/-)α2 (+/+) but not in eAMPKα1 (+/+)α2 (-/-) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (a NO donor), NS-1619 (a Ca(2+)-activated K(+) channel opener), and exogenous H2O2 were almost comparable among the groups. In eAMPKα1 (-/-)α2 (-/-) mice, antihypertensive treatment with hydralazine or long-term treatment with metformin (a stimulator of AMPK) failed to restore EDH-mediated responses. CONCLUSIONS: These results provide the first direct evidence that α1 subunit of eAMPK substantially mediates EDH responses of microvessels and regulates blood pressure and coronary flow responses in mice in vivo, demonstrating the novel role of eAMPK in cardiovascular homeostasis.

Essential role of bone marrow for microvascular endothelial and metabolic functions in mice.

RATIONALE: We have previously demonstrated that the importance of endothelium-derived hyperpolarizing factor (EDHF) increases as the vessel size decreases and that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in animals and humans, for which endothelial nitric oxide synthase (eNOS) is the major source. Recent studies have suggested the important role of the bone marrow (BM) in modulating cardiovascular and metabolic functions. OBJECTIVE: We aimed to examine whether BM plays a role in modulating microvascular endothelial and metabolic functions in mice, and if so, to elucidate the mechanisms involved. METHODS AND RESULTS: Male eNOS(-/-) mice were transplanted with BM cells from wild-type (WT) or eNOS(-/-) mice and were maintained for 6 weeks. Endothelium-dependent relaxations and hyperpolarizations of mesenteric arteries to acetylcholine were reduced in eNOS(-/-) mice and were markedly improved when transplanted with WT-BM but not with eNOS(-/-)-BM. The enhanced component of endothelium-dependent relaxations was abolished by catalase, indicating that the improved responses were mediated by H(2)O(2). In contrast, no such beneficial effect was noted in the aorta. Reduced plasma adiponectin levels and impaired glucose tolerance in eNOS(-/-) mice were also improved by WT-BM transplantation. Neuronal nitric oxide synthase (nNOS) in mesenteric arteries of eNOS(-/-) mice was significantly upregulated only when transplanted with WT-BM. Importantly, the beneficial effects of WT-BM transplantation were absent in eNOS(-/-)/adiponectin(-/-) or eNOS(-/-)/nNOS(-/-) mice. CONCLUSIONS: These results provide the first evidence that BM plays an important role in modulating microvascular endothelial and metabolic functions, for which adiponectin and nNOS may be involved.

Mechanisms for enhanced endothelium-derived hyperpolarizing factor-mediated responses in microvessels in mice.

BACKGROUND: Endothelium-derived relaxing factors play an important role in cardiovascular homeostasis. Among them, endothelium-derived hyperpolarizing factor (EDHF) is important especially in microcirculation. It has previously been demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in animals and humans and that endothelial nitric oxide synthase (eNOS) plays diverse roles as a nitric oxide (NO) generating system in conduit arteries and as an EDHF/H(2)O(2) generating system in microvessels. As compared with NO-mediated responses, those by EDHF are resistant to atherosclerosis, contributing to the maintenance of cardiovascular homeostasis. The aim of this study is to elucidate the molecular mechanisms for enhanced EDHF-mediated responses in microvessels. METHODS AND RESULTS: This study used male wild-type mice and caveolin-1-deficient mice (caveolin-1(-/-) mice). In the endothelium, eNOS was functionally suppressed in mesenteric arteries (microvessels) compared with the aorta (conduit arteries), for which Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) and caveolin-1 are involved, as EDHF-mediated responses were inhibited by STO-609 (an inhibitor of CaMKKß) and in caveolin-1(-/-) mice, respectively. In vascular smooth muscle, relaxation responses to H(2)O(2) were enhanced through a protein kinase G1α (PKG1α)-mediated mechanism in mesenteric arteries compared with the aorta, as they were inhibited by Rp-8-Br-cGMPS (an inhibitor of PKG1α). CONCLUSIONS: These results indicate that CaMKKß, caveolin-1, and PKG1α are substantially involved in the mechanisms for the enhanced EDHF-mediated responses in microvessels in mice.

Combination therapy with olmesartan and azelnidipine improves EDHF-mediated responses in diabetic apolipoprotein E-deficient mice.

BACKGROUND: The endothelium modulates vascular tone by synthesizing and releasing several vasodilating factors, including vasodilator prostaglandins, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). In the present study, we examined whether an angiotensin-receptor blocker, a calcium-channel blocker or their combination improved EDHF-mediated responses in diabetic apolipoprotein E-deficient (ApoE(-/-)) mice. METHODS AND RESULTS: We used male C57BL/6N (control) and streptozocin-induced diabetic ApoE(-/-) mice. The diabetic ApoE(-/-) mice were administered oral vehicle (untreated), olmesartan (OLM, 30 mgxkg(-1)xday(-1)), azelnidipine (AZL, 10 mgxkg(-1)xday(-1)), their combination (OLM + AZL), or hydralazine (HYD 5 mgxkg(-1)xday(-1)) for 5 weeks. In the untreated group, systolic blood pressure was significantly higher and both EDHF-mediated relaxation and endothelium-dependent hyperpolarization were markedly reduced as compared with the control group. Although EDHF-mediated relaxation was not significantly improved in the HYD, OLM and AZL groups, it was significantly improved in the OLM + AZL group, as was also the case with phosphorylation of Akt and endothelial NO synthase (eNOS). In contrast, the endothelium-independent relaxation response to sodium nitroprusside or NS-1619 (a direct opener of K(Ca) channels) was unaltered in any group. CONCLUSIONS: OLM + AZL may improve the severely impaired EDHF-mediated responses in diabetic ApoE(-/-) mice, in which activation of the endothelial Akt - eNOS pathway may be involved.