Ayuko Sawada

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 &agr;-catalytic subunit of AMPK, either &agr;1 (eAMPK&agr;1−/−&agr;2+/+) or &agr;2 (eAMPK&agr;1+/+&agr;2−/−) alone or both of them (eAMPK&agr;1−/−&agr;2−/−). 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 eAMPK&agr;1−/−&agr;2−/− and eAMPK&agr;1−/−&agr;2+/+ but not in eAMPK&agr;1+/+&agr;2−/− mice when compared with respective control. Importantly, endothelium-dependent relaxations, EDH, and coronary flow increase were all significantly reduced in eAMPK&agr;1−/−&agr;2−/− and eAMPK&agr;1−/−&agr;2+/+ but not in eAMPK&agr;1+/+&agr;2−/− mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (a NO donor), NS-1619 (a Ca2+-activated K+ channel opener), and exogenous H2O2 were almost comparable among the groups. In eAMPK&agr;1−/−&agr;2−/− 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 &agr;1 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 (H2O2) 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 H2O2. 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.

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.