Laura V. Gonzalez Bosc

NFATc3 Mediates Chronic Hypoxia-induced Pulmonary Arterial Remodeling with α-Actin Up-regulation

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-α-actin and -myosin heavy chain. NFATc3 (nuclear factor of activated T cells isoform c3), which is aCa2+-dependent transcription factor, has been recently linked to smooth muscle phenotypic maintenance through the regulation of the expression of α-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 α-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 α-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 α-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.

Intermittent Hypoxia in Rats Increases Myogenic Tone Through Loss of Hydrogen Sulfide Activation of Large-Conductance Ca 2+ -Activated Potassium Channels

Rationale: Myogenic tone, an important regulator of vascular resistance, is dependent on vascular smooth muscle (VSM) depolarization, can be modulated by endothelial factors, and is increased in several models of hypertension. Intermittent hypoxia (IH) elevates blood pressure and causes endothelial dysfunction. Hydrogen sulfide (H2S), a recently described endothelium-derived vasodilator, is produced by the enzyme cystathionine &ggr;-lyase (CSE) and acts by hyperpolarizing VSM. Objective: Determine whether IH decreases endothelial H2S production to increase myogenic tone in small mesenteric arteries. Methods and Results: Myogenic tone was greater in mesenteric arteries from IH than sham control rat arteries, and VSM membrane potential was depolarized in IH in comparison with sham arteries. Endothelium inactivation or scavenging of H2S enhanced myogenic tone in sham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in sham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H2S dilated and hyperpolarized sham and IH arteries, and this dilation was blocked by iberiotoxin, paxilline, and KCl preconstriction but not glibenclamide or 3-isobutyl-1-methylxanthine. Iberiotoxin enhanced myogenic tone in both groups but more in sham than IH. CSE immunofluorescence was less in the endothelium of IH than in sham mesenteric arteries. Endogenouse H2S dilation was reduced in IH arteries. Conclusions: IH appears to decrease endothelial CSE expression to reduce H2S production, depolarize VSM, and enhance myogenic tone. H2S dilatation and hyperpolarization of VSM in small mesenteric arteries requires BKCa channels.

Impairment of coronary endothelial cell ETB receptor function after short-term inhalation exposure to whole diesel emissions

Air pollutant levels positively correlate with increases in both acute and chronic cardiovascular disease. The pollutant diesel exhaust (DE) increases endothelin (ET) levels, suggesting that this peptide may contribute to DE-induced cardiovascular disease. We hypothesized that acute exposure to DE also enhances ET-1-mediated coronary artery constrictor sensitivity. Constrictor responses to KCl, U-46619, and ET-1 were recorded by videomicroscopy in pressurized intraseptal coronary arteries from rats exposed for 5 h to DE (300 microg/m(3)) or filtered air (Air). ET-1 constriction was augmented in arteries from DE-exposed rats. Nitric oxide synthase (NOS) inhibition [N(omega)-nitro-L-arginine (L-NNA), 100 microM] and endothelium inactivation augmented ET-1 responses in arteries from Air but not DE rats so that after either treatment responses were not different between groups. DE exposure did not affect KCl and U-46619 constrictor responses, while NOS inhibition augmented KCl constriction equally in both groups. Thus basal NOS activity does not appear to be affected by DE exposure. The endothelin type B (ET(B)) receptor antagonist BQ-788 (10 microM) inhibited ET-1 constriction in DE but not Air arteries, and constriction in the presence of the antagonist was not different between groups. Cytokine levels were not different in plasma from DE and AIR rats, suggesting that acute exposure to DE does not cause an immediate inflammatory response. In summary, a 5-h DE exposure selectively increases constrictor sensitivity to ET-1. This augmentation is endothelium-, NOS-, and ET(B) receptor dependent. These data suggest that DE exposure diminishes ET(B) receptor activation of endothelial NOS and augments ET(B)-dependent vasoconstriction. This augmented coronary vasoreactivity to ET-1 after DE, coupled with previous reports that DE induces production of ET-1, suggests that ET-1 may contribute to the increased incidence of cardiac events during acute increases in air pollution levels.

Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca2+-activated K+ channels and smooth muscle Ca2+ sparks

We 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(2+)-activated potassium channels (BKCa). Ca(2+) sparks are ryanodine receptor (RyR)-mediated Ca(2+)-release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H₂S activates Ca(2+) sparks in small mesenteric arteries. Ca(2+) sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca(2+) 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 μmol/l) increased Ca(2+) sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H2S with β-cyano-l-alanine (BCA) also reduced VSMC Ca(2+) spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC Em 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(+) currents in freshly dispersed mesenteric ECs. These results suggest that H₂S increases Ca(2+) spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

Nuclear Factor of Activated T Cells Regulates Osteopontin Expression in Arterial Smooth Muscle in Response to Diabetes-Induced Hyperglycemia

Objective—Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca2+/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. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3−/− mice. Conclusions—These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Mechanisms of intermittent hypoxia induced hypertension

•  Introduction •  Mechanisms of IH‐induced systemic hypertension ‐  Contribution of the nervous system ‐  Contribution of circulating and vascular factors ‐  Role of transcription factors in the inflammatory and cardiovascular consequences of IH •  NF‐κB •  NFAT •  HIF‐1 •  Intermittent hypoxia induced pulmonary hypertension •  Conclusions

Effect of chronic angiotensin II inhibition on the nitric oxide synthase in the normal rat during aging

Objective To assess the effect on the cardiovascular system, of enalapril (E) or losartan (L) given since weaning during 6 or 18 months to normal rats. Methods Animals were divided in three groups: control (C), E-treated and L-treated; treated rats received 10 mg/kg per day of drug. Systolic blood pressure (SBP), body weight, water and food intake (WI, FI), cardiac, left ventricular and aortic weight as well as the length of the tail were recorded. NADPH-diaphorase activity was determined as a marker of nitric oxide synthase (NOS) activity in aorta, arterioles of small intestine, heart and kidney of normal rats. NOS activity was measured as optical density (OD) in the stained tissue. Nitrate + nitrite urinary excretion was measured in 24 h urine. Only significant differences (P < 0.05) are reported. Results SBP, absolute cardiac, left ventricular and aortic weight increased with age. Both treatments delayed these increments. At 6 and 18 months, NOS activity was higher in aortic endothelium (Em) of L- and E-treated animals. Losartan treatment during 6 months also increased NOS activity in aortic smooth muscle (SM). Aortic Em NOS activity fell in the 18 months-treated and untreated animals. E increased NOS activity in the SM of intestinal arterioles at 6 months but reduced it at 18 months. Conclusions The fact that both E and L delayed cardiac hypertrophy/hyperplasia and aortic growth and raised aortic endothelium NOS activity indicates a protective effect on cardiovascular damage due to aging, exerted through inhibition of angiotensin II.

Endothelin-1 contributes to increased NFATc3 activation by chronic hypoxia in pulmonary arteries

Chronic hypoxia (CH) activates the Ca(2+)-dependent transcription factor nuclear factor of activated T cells isoform c3 (NFATc3) in mouse pulmonary arteries. However, the mechanism of this response has not been explored. Since we have demonstrated that NFATc3 is required for CH-induced pulmonary arterial remodeling, establishing how CH activates NFATc3 is physiologically significant. The goal of this study was to test the hypothesis that endothelin-1 (ET-1) contributes to CH-induced NFATc3 activation. We propose that this mechanism requires increased pulmonary arterial smooth muscle cell (PASMC) intracellular Ca(2+) concentration ([Ca(2+)](i)) and stimulation of RhoA/Rho kinase (ROK), leading to calcineurin activation and actin cytoskeleton polymerization, respectively. We found that: 1) CH increases pulmonary arterial pre-pro-ET-1 mRNA expression and lung RhoA activity; 2) inhibition of ET receptors, calcineurin, L-type Ca(2+) channels, and ROK blunts CH-induced NFATc3 activation in isolated intrapulmonary arteries from NFAT-luciferase reporter mice; and 3) both ET-1-induced NFATc3 activation in isolated mouse pulmonary arteries ex vivo and ET-1-induced NFATc3-green fluorescence protein nuclear import in human PASMC depend on ROK and actin polymerization. This study suggests that CH increases ET-1 expression, thereby elevating PASMC [Ca(2+)](i) and RhoA/ROK activity. As previously demonstrated, elevated [Ca(2+)](i) is required to activate calcineurin, which dephosphorylates NFATc3, allowing its nuclear import. Here, we demonstrate that ROK increases actin polymerization, thus providing structural support for NFATc3 nuclear transport.

NFATc3 is required for intermittent hypoxia-induced hypertension

Sleep apnea, defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension and peripheral vascular disease. Exposure of rodents to brief periods of intermittent hypercarbia/hypoxia (H-IH) during sleep mimics the cyclical hypoxia-normoxia of sleep apnea. Endothelin-1, an upstream activator of nuclear factor of activated T cells (NFAT), is increased during H-IH. Therefore, we hypothesized that NFATc3 is activated by H-IH and is required for H-IH-induced hypertension. Consistent with this hypothesis, we found that H-IH (20 brief exposures per hour to 5% O(2)-5% CO(2) for 7 h/day) induces systemic hypertension in mice [mean arterial pressure (MAP) = 97 +/- 2 vs. 124 +/- 2 mmHg, P < 0.05, n = 5] and increases NFATc3 transcriptional activity in aorta and mesenteric arteries. Cyclosporin A, an NFAT inhibitor, and genetic ablation of NFATc3 [NFATc3 knockout (KO)] prevented NFAT activation. More importantly, H-IH-induced hypertension was attenuated in cyclosporin A-treated mice and prevented in NFATc3 KO mice. MAP was significantly elevated in wild-type mice (Delta = 23.5 +/- 6.1 mmHg), but not in KO mice (Delta = -3.9 +/- 5.7). These results indicate that H-IH-induced increases in MAP require NFATc3 and that NFATc3 may contribute to the vascular changes associated with H-IH-induced hypertension.

Effect of chronic angiotensin II inhibition on the cardiovascular system of the normal rat

Previous studies have demonstrated in normal rats that chronic treatment, from weaning to 30 days, with either enalapril or losartan, induced significant changes in cardiovascular structure and function. The present study was performed to assess the effect of either enalapril or losartan on the structure and function of the heart and arteries given to normal rats from weaning until 6 months of age. Animals (n = 48) were divided into three groups: control, enalapril treated, and losartan treated; treated rats received 10 mg/kg/day of drug. Blood pressure, body weight, and water intake were recorded for that time period. DNA, cGMP, collagen, degree of fibrosis, and nitric oxide synthase-NADPH-diaphorase-dependent activity in the heart and arteries were determined. Only significant differences (P < .05) are reported. Blood pressure increased only in control rats (13 +/- 1 mm Hg), enalapril treatment enhanced water intake and reduced the rate of body growth (control, 672.9 +/- 15.4 g; losartan, 692.4 +/- 21.8 g; enalapril, 541.8 +/- 13.8 g). In the heart, DNA (control, 120 +/- 5; losartan, 99 +/- 4; enalapril, 93 +/- 6 microg/100 mg), collagen (control, 2.5 +/- 0.2; enalapril, 1.85 +/- 0.08 microg/100 mg), and fibrosis (control, 3.5 +/- 0.4%; losartan, 2.2 +/- 0.3%; enalapril, 2.1 +/- 0.4%) were reduced by treatment. In the aorta, cGMP (control, 0.15 +/- 0.01; losartan, 0.24 +/- 0.02 pmol/mg), and NADPH-diaphorase (control, 0.114 +/- 0.003; losartan, 0.148 +/- 0.006; enalapril, 0.169 +/- 0.003 as optical density) were enhanced. The enzyme was also higher in the aortic endothelium of treated animals (control, 0.193 +/- 0.010; losartan, 0.228 +/- 0.009; enalapril, 0.278 +/- 0.005). The lower rate of body weight increase, the enhanced water intake, and the reduced cardiac and left ventricular weight attributable to enalapril treatment do not seem to be related to inhibition of the renin-angiotensin system. On the other hand, renin-angiotensin system inhibition induces a protective effect on the heart and aorta through structural and functional changes. Most of this action seems to be exerted through angiotensin II type 1 receptors.