Abstract-Flow-induced dilation (FID) is dependent largely on hyperpolarization of vascular smooth muscle cells (VSMCs) in human coronary arterioles (HCA) from patients with coronary disease. Animal studies show that shear stress induces endothelial generation of hydrogen peroxide (H 2 O 2 ), which is proposed as an endothelium-derived hyperpolarizing factor (EDHF). We tested the hypothesis that H 2 O 2 contributes to FID in HCA. Arterioles (135Ϯ7 m, nϭ71) were dissected from human right atrial appendages at the time of cardiac surgery and cannulated with glass micropipettes. Changes in internal diameter and membrane potential of VSMCs to shear stress, H 2 O 2 , or to papaverine were recorded with videomicroscopy. In some vessels, endothelial H 2 O 2 generation to shear stress was monitored directly using confocal microscopy with 2Ј,7Ј-dichlorofluorescin diacetate (DCFH) or using electron microscopy with cerium chloride. Catalase inhibited FID (%max dilation; 66Ϯ8 versus 25Ϯ7%; PϽ0.05, nϭ6), whereas dilation to papaverine was unchanged. Shear stress immediately increased DCFH fluorescence in the endothelial cell layer, whereas treatment with catalase abolished the increase in fluorescence. Electron microscopy with cerium chloride revealed shear stress-induced increase in cerium deposition in intimal area surrounding endothelial cells. Exogenous H 2 O 2 dilated (%max dilation; 97Ϯ1%, ED 50 ; 3.0Ϯ0.7ϫ10 Ϫ5 mol/L) and hyperpolarized HCA. Dilation to H 2 O 2 was reduced by catalase, 40 mmol/L KCl, or charybdotoxin plus apamin, whereas endothelial denudation, deferoxamine, 1H-1,2,4 -oxadiazole-[4,3-a]quinoxalin-1-one, or glibenclamide had no effect. These data provide evidence that shear stress induces endothelial release of H 2 O 2 and are consistent with the idea that H 2 O 2 is an EDHF that contributes to FID in HCA from patients with heart disease. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2003;92:e31-e40.)Key Words: human Ⅲ coronary microcirculation Ⅲ flow-induced dilation Ⅲ hydrogen peroxides P hysiologically, shear stress plays a critical role in the regulation of vascular tonus and vascular homeostasis, contributing to the maintenance of tissue perfusion and vascular integrity. Shear stress-induced release of nitric oxide (NO) from endothelial cells is widely recognized as one of the most important and common mechanisms for shear-induced vasomotion. For example, flow-induced release of NO is responsible for the mediation of flow-induced vasodilation (FID). [1][2][3] Animal studies have reported that the contribution of NO to FID is reduced as oxidative stress increases in the presence of risk factors for cardiovascular disease such as hypercholesterolemia 4 and hypertension. 5 In humans, in vivo and in vitro studies have demonstrated that relaxant factor(s) other than NO compensate to maintain FID when NO availability is reduced. 6,7 We recently reported that FID is mediated largely by endothelium-derived hyperpolarizing factor (EDHF) in human coronary arterioles (HCAs...

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe -/-mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1 -/-mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe -/-mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Background-Kϩ channel activation in vascular smooth muscle cells (VSMCs) plays a key role in regulating vascular tone. It has been proposed that endothelium-derived hyperpolarizing factor (EDHF) contributes to microvascular dilation more than nitric oxide (NO) does. Whether hyperpolarization is important for coronary arteriolar dilation in humans is not known. Bradykinin (BK), an endogenous vasoactive substance, is released from ischemic myocardium and regulates coronary resistance. Therefore, we tested the effects of inhibiting NO synthase, cyclooxygenase, and K ϩ channels on the changes in diameter and membrane potential (Em) in response to BK in isolated human coronary microvessels. Methods and Results-Arterioles (97Ϯ4 m; nϭ120) dissected from human right atrial appendages (nϭ78) were cannulated at a distending pressure of 60 mm Hg and zero flow. Changes in vessel diameter (video microscopy) and VSMC Em (glass microelectrodes) were measured simultaneously. In vessels constricted and depolarized (Em; Ϫ50Ϯ3 to Ϫ28Ϯ2 mV) with endothelin-1 (ET), dilation to BK was associated with greater membrane hyperpolarization (Ϫ48Ϯ3 mV at 10 Ϫ6 mol/L) than dilation to sodium nitroprusside (SNP) (Ϫ34Ϯ2 mV at 10 Ϫ4 mol/L) for similar degrees of dilation. Treatment with N

Abstract-ATP-sensitive K ϩ channels (K ATP ) contribute to vasomotor regulation in some species. It is not fully understood the extent to which K ATP participate in regulating vasomotor tone under physiological and pathophysiological conditions in the human heart. Arterioles dissected from right atrial appendage were studied with video microscopy, membrane potential recordings, reverse transcription-polymerase chain reaction, and immunohistochemistry. Hypoxia produced endothelium-independent vasodilation and membrane hyperpolarization of vascular smooth muscle cells, both of which were attenuated by glibenclamide. Aprikalim, a selective K ATP opener, also induced a potent endothelium-independent and glibenclamide-sensitive vasodilation with membrane hyperpolarization. Reverse transcription-polymerase chain reaction detected mRNA expression for K ATP subunits, and immunohistochemistry confirmed the localization of the inwardly rectifying Kir6.1 protein in the vasculature. In patients with type 1 or type 2 diabetes mellitus (DM), vasodilation was reduced to both aprikalim (maximum dilation, DM(ϩ) 90Ϯ2% versus DM(Ϫ) 96Ϯ1%, PϽ0.05) and hypoxia (maximum dilation, DM(ϩ) 56Ϯ8% versus DM(Ϫ) 85Ϯ5%, PϽ0.01) but was not altered to sodium nitroprusside or bradykinin. Baseline myogenic tone and resting membrane potential were not affected by DM. We conclude that DM impairs human coronary arteriolar dilation to K ATP opening, leading to reduced dilation to hypoxia. This reduction in K ATP function could contribute to the greater cardiovascular mortality and morbidity in DM.