The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H 2 O 2 ) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H 2 O 2 to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K + channel opener levcromakalim. Exogenous H 2 O 2 elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H 2 O 2 upon stimulation by ACh and that the H 2 O 2 production was markedly reduced in eNOS-KO mice. These results indicate that H 2 O 2 is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.J. Clin. Invest. 106:1521-1530. cular smooth muscle (21,22). In this study, we thus tested our hypothesis that H 2 O 2 is an EDHF and also examined a possible role of eNOS as a major source of the reactive oxygen species, using control and eNOS knockout (eNOS-KO) mice (23). MethodsThis study was reviewed by the Committee on Ethics in Animal Experiments of the Kyushu University and was carried out according to the Guidelines for Animal Experiments of the Kyushu University and of the Japanese government.Animals and tissue preparation. Male eNOS-KO mice and C57BL/6 mice, 10-16 weeks of age, were used. The eNOS-KO mice were originally provided by P. Huang and M. Fishman (Harvard Medical School, Boston, Massachusetts, USA) (23) and maintained in the Laboratory of Animal Experiments in the Kyushu University. The eNOS-KO mice were derived from a cross between SV129J and C57BL/6 mice and were backcrossed to C57BL/6 mice over ten generations. Thus, C57BL/6 mice were used as a wild-type control; they were also maintained in the Laboratory of Animal Experiments in the Kyushu University. Some eNOS-KO mice were treated with hydralazine in order to examine the effect of mildly elevated blood pressure on the EDHF-mediated responses. The treatment with hydralazine hydrochloride (20 mg/kg/d) was performed for 6 weeks from 10-16 weeks of age. Systolic blood pressure was measured by tail-cuff method under conscious conditions before the animals were killed. Th...

Abstract. The Ca2+ signal is the primary determinant of the contraction of the vascular smooth muscle. However, the alteration of the Ca 2+ sensitivity of the contractile apparatus also plays an essential role. The regulation of the myosin light chain phosphatase (MLCP) activity is considered to be the most important mechanism underlying the regulation of Ca 2+ sensitivity. The investigations during the last 15 years have identified many proteins that participate in the regulation of the MLCP activity. Recently, the Ca 2+ signal has also been shown to cross-talk with the mechanisms regulating the Ca 2+ sensitivity. Consequently, Rho kinase, protein kinase C, CPI-17, and MYPT1 have all been suggested to play a physiologically important role in the regulation of the MLCP activity. We are now close to elucidating the major rules regulating the MLCP activity and the Ca 2+ sensitivity during vascular contractions. This article will give an overview of the current understanding of the biochemical basis for the regulation of the MLCP activity, while also discussing their functional roles from a physiological point of view. I hope this article will help to develop new pharmacological strategies for the prevention and treatment of the pathological vasoconstriction often seen in vascular diseases.

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Abstract-Proteinase-activated receptors (PARs) belong to a family of G protein-coupled receptors, thus mediating the cellular effects of proteinases. In the vascular system, thrombin and other proteinases in the coagulation-fibrinolysis system are considered to be the physiologically relevant agonists, whereas PARs are among the most important mechanisms mediating the interaction between the coagulation-fibrinolysis system and the vascular wall. Under physiological conditions, PARs are mainly expressed in endothelial cells, and participate in the regulation of vascular tone, mostly by inducing endothelium-dependent relaxation. PARs in endothelial cells are also suggested to contribute to a proinflammatory phenotypic conversion and an increase in the permeability of vascular lesions. In smooth muscle cells, PARs mediate contraction, migration, proliferation, hypertrophy, and production of the extracellular matrix, thereby contributing to the development of vascular lesions and the pathophysiology of such vascular diseases as atherosclerosis. However, the expression of PARs in the smooth muscle of normal arteries is limited. The upregulation of PARs in the smooth muscle is thus considered to be a key step for PARs to participate in the pathogenesis of vascular lesions. Elucidating the molecular mechanism regulating the PARs expression is therefore important to develop new strategies for the prevention and treatment of vascular diseases. (Arterioscler Thromb Vasc Biol. 2007;27:27-36.)

Background-Peroxisome proliferator-activated receptor ␥ (PPAR␥) activators, such as troglitazone (Tro), not only improve insulin resistance but also suppress the neointimal formation after balloon injury. However, the precise mechanisms have not been determined. Angiotensin II (Ang II) plays crucial roles in the pathogenesis of atherosclerosis, hypertension, and neointimal formation after angioplasty. We examined the effect of PPAR␥ activators on the expression of Ang II type 1 receptor (AT 1 -R) in cultured vascular smooth muscle cells (VSMCs). Methods and Results-AT 1 -R mRNA and AT 1 -R protein levels were determined by Northern blot analysis and radioligand binding assay, respectively. Natural PPAR␥ ligand 15-deoxy-⌬ 12,14 -prostaglandin J 2 , as well as Tro, reduced the AT 1 -R mRNA expression and the AT 1 -R protein level. The PPAR␥ activators also reduced the calcium response of VSMCs to Ang II. PPAR␥ activators suppressed the AT 1 -R promoter activity measured by luciferase assay but did not affect the AT 1 -R mRNA stability, suggesting that the suppression occurs at the transcriptional level. Conclusions-PPAR␥ activators reduced the AT 1 -R expression and calcium response to Ang II in VSMCs. Downregulation of AT 1 -R may contribute to the inhibition of neointimal formation by PPAR␥ activators. (Circulation.