Physiological responses to chronic hypoxia include polycythemia, pulmonary arterial remodeling, and vasoconstriction. Chronic hypoxia causes pulmonary arterial hypertension leading to right ventricular hypertrophy and heart failure. During pulmonary hypertension, pulmonary arteries exhibit increased expression of smooth muscle-␣-actin and -myosin heavy chain. NFATc3 (nuclear factor of activated T cells isoform c3), which is a Ca 2؉ -dependent transcription factor, has been recently linked to smooth muscle phenotypic maintenance through the regulation of the expression of ␣-actin. The aim of this study was to determine if: (a) NFATc3 is expressed in murine pulmonary arteries, (b) hypoxia induces NFAT activation, (c) NFATc3 mediates the up-regulation of ␣-actin during chronic hypoxia, and (d) NFATc3 is involved in chronic hypoxia-induced pulmonary vascular remodeling. NFATc3 transcript and protein were found in pulmonary arteries. NFAT-luciferase reporter mice were exposed to normoxia (630 torr) or hypoxia (380 torr) for 2, 7, or 21 days. Exposure to hypoxia elicited a significant increase in luciferase activity and pulmonary arterial smooth muscle nuclear NFATc3 localization, demonstrating NFAT activation. Hypoxia induced up-regulation of ␣-actin and was prevented by the calcineurin/NFAT inhibitor, cyclosporin A (25 mg/kg/day s.c.). In addition, NFATc3 knock-out mice did not showed increased ␣-actin levels and arterial wall thickness after hypoxia. These results strongly suggest that NFATc3 plays a role in the chronic hypoxia-induced vascular changes that underlie pulmonary hypertension.As altitude increases, the barometric pressure and atmospheric oxygen partial pressure decrease. This decrease in barometric pressure is the basic cause of all hypoxia-related problems in high altitude pathophysiology. Similar levels of hypoxia are present in patients with chronic bronchitis, emphysema, cystic fibrosis, asthma, and severe restrictive lung diseases (1). Chronic hypoxia (CH) 2 causes pulmonary hypertension due to pulmonary vasoconstriction, arterial remodeling, and polycythemia, which ultimately results in right ventricular (RV) hypertrophy and often heart failure (2). Pulmonary vasoconstriction is thought to be caused by elevated vascular tone through increased pulmonary arterial smooth muscle cell (PASMC) intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (3-8) and increased sensitivity of the contractile apparatus to Ca 2ϩ (9 -12).Regardless of the cause of pulmonary hypertension, the structural change that is thought to underlie the increased vascular resistance is remodeling of small pulmonary arteries. A prominent feature of this vascular remodeling is medial thickening. In proximal pulmonary vessels, medial enlargement is caused by hypertrophy and hyperplasia of the pre-existing smooth muscle cells (reviewed in Ref.

Jackson-Weaver O, Osmond JM, Riddle MA, Naik JS, Gonzalez Bosc LV, Walker BR, Kanagy NL. Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca 2ϩ -activated K ϩ channels and smooth muscle Ca 2ϩ sparks. Am J Physiol Heart Circ Physiol 304: H1446 -H1454, 2013. First published March 22, 2013 doi:10.1152/ajpheart.00506.2012We have previously shown that hydrogen sulfide (H2S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H2S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca 2ϩ -activated potassium channels (BKCa). Ca 2ϩ sparks are ryanodine receptor (RyR)-mediated Ca 2ϩ -release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H2S activates Ca 2ϩ sparks in small mesenteric arteries. Ca 2ϩ sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca 2ϩ indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H2S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H2S donor NaHS (10 mol/l) increased Ca 2ϩ sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine ␥-lyase (CSE)-derived H2S with ␤-cyano-L-alanine (BCA) also reduced VSMC Ca 2ϩ spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC E m in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K ϩ currents in freshly dispersed mesenteric ECs. These results suggest that H2S increases Ca 2ϩ spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.cytochrome P-450 epoxygenase; sodium hydrosulfide; membrane potential HYDROGEN SULFIDE (H 2 S) is a recently described vasodilator produced in the vasculature by the enzymes cystathionine ␥-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). H 2 S has been proposed to cause vasodilation through a variety of mechanisms (1,3,26,33) and genetic knockout of the CSE gene causes hypertension (32). We previously reported that inhibiting CSE or disrupting the endothelium enhances myogenic tone in small mesenteric arteries from rats and that exogenous H 2 S hyperpolarizes vascular smooth muscle cell (VSMC) membrane potential (E m ) and dilates arteries through activation of large-conductance Ca 2ϩ -activated K (4) and ...

Objective-Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca 2ϩ /calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. Methods and Results-An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Key Words: NFAT Ⅲ diabetes Ⅲ hyperglycemia Ⅲ UTP Ⅲ vascular complications Ⅲ inflammation T he matrix cytokine osteopontin (OPN) is emerging as a key regulator of chronic inflammatory diseases, including vascular disease. Plasma OPN levels are associated with the presence and extent of coronary artery disease, 1 restenosis after balloon angioplasty, 2 and human abdominal aortic aneurysm. 3 OPN is highly expressed in human atherosclerotic lesions and is not only a marker of inflammation but also an active player in the progression of atherosclerosis and restenosis. 4 While OPN deficiency has been shown to result in reduced atherosclerotic lesion areas, 5,6 OPN overexpression is associated with enhanced aortic lesion size. 7 Atherosclerotic vascular disease is a major complication in diabetic patients, and the levels of OPN in vivo have been clinically associated with the progression of vascular complications. Plasma levels of OPN significantly correlate to the progression of diabetic nephropathy, 8 and OPN levels in the vitreous are enhanced in patients with diabetic retinopathy. 9 Furthermore, OPN expression is increased in the media of diabetic arteries. 10,11 Thus, mapping the signaling pathway leading to changes in OPN expression may reveal novel pharmacological targets for prevention of vascular disease.In the context of vascular remodeling, soluble factors, cytokines, hormones, and extracellular nucleotides have been shown to induce OPN expression. 12,13 In particular, UTP has been demonstrated to effectively increase OPN protein production by enhanced transcription and stabilization of OPN mRNA. 14,15 We and others have shown that stimulation of G-protein-coupled receptors effectively activates the Ca 2ϩ /calcineurin-dependent transcription factor NFAT in arterial smooth muscle. 16,17 More recently, we have shown that high glucose activates NFAT in intact arteries ex vivo by a mechanism involving the release of extracellular nucleotides (ie, UTP, UDP) acting on P2...