Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are known to play an important role in the proliferation and viability of vascular smooth muscle cells. In this study, we determined the effects of increased superoxide dismutase and catalase activity on fetal pulmonary arterial smooth muscle cell (FPASMC) proliferation and viability using EUK-134, a superoxide dismutase/catalase mimetic. Treatment of FPASMC with EUK-134 or with a combination of superoxide dismutase and catalase enzymes decreased superoxide and hydrogen peroxide levels as detected by the fluorescent dyes dihydroethidium and dichlorodihydrofluorescein diacetate, respectively. EUK-134 (5 microM) attenuated serum-induced FPASMC proliferation, whereas 50 microM EUK-134 decreased the number of viable cells, suggesting cell death. Conversely, combined superoxide dismutase and catalase enzyme activity equivalent to 50 microM EUK-134 prevented proliferation but did not reduce the number of viable FPASMC. The loss of mitochondrial membrane potential after 18 h, an increase in caspase-9 and caspase-3 activity after 24 h, and the subsequent appearance of TdT-mediated dUTP nick end labeling-positive nuclei were detected in FPASMC after treatment with 50 microM EUK-134. This indicates an induction of programmed rather than necrotic cell death and suggests that prolonged removal of ROS is required to stimulate apoptosis. Compounds such as EUK-134 may, therefore, prove more effective than enzymic antioxidants over longer periods, especially when the aim is to decrease the number of smooth muscle cells in diseases resulting from excessive muscularization.     

Endothelin-1 decreases endothelial NOS expression and activity through ETA receptor-mediated generation of hydrogen peroxide

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased endothelial nitric oxide synthase (eNOS) gene expression in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are elevated in the pulmonary arteries from PPHN lambs and that ET-1 increases ROS production in pulmonary arterial smooth muscle cells (PASMC) in culture. Thus the objective of this study was to determine whether there was a feedback mechanism between the ET-1-mediated increase in ROS in fetal PASMC (FPASMC) and a decrease in eNOS gene expression in fetal pulmonary arterial endothelial cells (FPAEC). Our results indicate that ET-1 increased H2O2 levels in FPASMC in an endothelin A receptor-dependent fashion. This was observed in both FPASMC monoculture and in cocultures of FPASMC and FPAEC. Conversely, ET-1 decreased H2O2 levels in FPAEC monoculture in an endothelin B receptor-dependent fashion. Furthermore, ET-1 decreased eNOS promoter activity by 40% in FPAEC in coculture with FPASMC. Promoter activity was restored in the presence of catalase. In FPAEC in monoculture treated with 0-100 microM H2O2, 12 microM had no effect on eNOS promoter activity, but it increased eNOS protein levels by 50%. However, at 100 microM, H2O2 decreased eNOS promoter activity and protein levels in FPAEC by 79 and 40%, respectively. These data suggest a role for smooth muscle cell-derived H2O2 in ET-1-mediated downregulation of eNOS expression in children born with PPHN.     

Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling

Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca(2+)](i)). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca(2+) desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca(2+)-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca(2+)](i). We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca(2+) sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca(2+) desensitization through inhibition of RhoA/ROK.     

NO inhibits signal transduction pathway for ATP release from erythrocytes via its action on heterotrimeric G protein Gi

The release of ATP from erythrocytes involves a signal transduction pathway of which cystic fibrosis transmembrane conductance regulator, PKA, adenylyl cyclase, and the heterotrimeric G proteins G(s) and G(i) are components. In the pulmonary circulation, ATP released from the erythrocyte stimulates nitric oxide (NO) synthesis, thereby regulating vascular resistance. We reported that NO liberated from an NO donor inhibited ATP release from erythrocytes in response to decreased Po(2) or mechanical deformation. Here, we investigated the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of G(i). Washed rabbit erythrocytes were incubated in the presence or absence of the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate; 100 nM, 20 min), followed by treatment with agents that activate specific components of the signal transduction pathway promoting ATP release. Neither ATP release nor cAMP accumulation induced by either forskolin (100 microM, n = 7) or iloprost (100 nM, n = 6) was inhibited by spermine NONOate. These experiments suggest that the inhibitory action of NO is not the result of inactivation of adenylyl cyclase or G(s), respectively. However, spermine NONOate completely inhibited ATP release in response to mastoparan (10 microm, P < 0.05, n = 5), a specific activator of G(i). Spermine (100 nM, 20 min), the polyamine remaining after liberation of NO from spermine NONOate, had no affect on mastoparan-induced ATP release (n = 4). These results support the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein G(i).     

A role for platelet-derived growth factor beta-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-alpha and -beta receptors (PDGF-alphaR and -betaR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-betaR, but not PDGF-alphaR, on arterial smooth muscle. PDGF-BB, a major PDGF-betaR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-betaR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-betaR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-betaR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.     

Release of intracellular Zn<Superscript>2+</Superscript> in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn²⁺ release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn²⁺ release and subsequent cell death. Cortical neurons were loaded with the Zn²⁺ selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn²⁺ levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn²⁺. Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn²⁺ induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn²⁺ release appeared to peak at 1 μM spermine NONOate (123.8 ± 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn²⁺ levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn²⁺ in cortical neurons. The possibility that release of intracellular Zn²⁺ in response to NO could play a role in intracellular signaling is discussed.     

Impaired NO-dependent inhibition of store- and receptor-operated calcium entry in pulmonary vascular smooth muscle after chronic hypoxia

We have recently demonstrated that chronic hypoxia (CH) attenuates nitric oxide (NO)-mediated decreases in pulmonary vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca2+]i) and promotes NO-dependent VSM Ca2+ desensitization. The objective of the current study was to identify potential mechanisms by which CH interferes with regulation of [Ca2+]i by NO. We hypothesized that CH impairs NO-mediated inhibition of store-operated (capacitative) Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) in pulmonary VSM. To test this hypothesis, we examined effects of the NO donor, spermine NONOate, on SOCE resulting from depletion of intracellular Ca2+ stores with cyclopiazonic acid, and on UTP-induced ROCE in isolated, endothelium-denuded, pressurized pulmonary arteries (213 +/- 8 microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We found that the change in [Ca2+]i associated with SOCE and ROCE was significantly reduced in vessels from CH animals. Furthermore, spermine NONOate diminished SOCE and ROCE in vessels from control, but not CH animals. We conclude that NO-mediated inhibition of SOCE and ROCE is impaired after CH-induced pulmonary hypertension.     

Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation

We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.     

Shear stress inhibits smooth muscle cell migration via nitric oxide-mediated downregulation of matrix metalloproteinase-2 activity

Vascular smooth muscle cell (SMC) migration is a hallmark of intimal hyperplasia (IH), the progression of which is affected by hemodynamic conditions at the diseased site. The realization that SMCs are exposed to blood flow in both denuded vessels (direct blood flow) and intact vessels (interstitial blood flow) motivated this study of the effects of fluid flow shear stress (SS) on SMC migration. Rat aortic SMCs were seeded onto Matrigel-coated cell culture inserts, and their migratory activity toward PDGF-BB when exposed to SS in a rotating disk apparatus was quantified. Four hours of either 10 or 20 dyn/cm2 SS significantly inhibited SMC migration to the bottom side of the insert. This inhibition was associated with downregulation of SMC matrix metalloproteinase (MMP)-2 activation. Four hours of 10 dyn/cm2 SS also drastically increased SMC production of NO. A NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester; 100 microM) abolished the shear-induced increase in SMC NO production as well as the inhibition of migration and MMP-2 activity. A NO donor (S-nitroso-N-acetyl-penicillamine; 500 microM) suppressed SMC migration via the reduction of both total and active MMP-2 levels. Addition of 10 microM MMP-2 inhibitor I to inserts significantly reduced SMC migration. Western blots showed no effect of 4 h of 20 dyn/cm2 SS on SMC production of PDGF-AA, another chemical known to suppress SMC migration. Thus it appears that SS acts to suppress SMC migration by upregulating the cellular production of NO, which in turn inhibits MMP-2 activity.     

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.     

Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells

Pulmonary hypertension is characterized by vascular remodeling involving smooth muscle cell proliferation and migration. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators, and the inhibition of aortic smooth muscle cell (ASMC) proliferation by NO has been documented, but less is known about the effects of CGRP. The mechanism by which overexpression of CGRP inhibits proliferation in pulmonary artery smooth muscle cells (PASMC) and ASMC following in vitro transfection by the gene coding for prepro-CGRP was investigated. Increased expression of p53 is known to stimulate p21, which inhibits G(1) cyclin/cdk complexes, thereby inhibiting cell proliferation. We hypothesize that p53 and p21 are involved in the growth inhibitory effect of CGRP. In this study, CGRP was shown to inhibit ASMC and PASMC proliferation. In PASMC transfected with CGRP and exposed to a PKA inhibitor (PKAi), cell proliferation was restored. p53 and p21 expression increased in CGRP-treated cells but decreased in cells treated with CGRP and PKAi. PASMC treated with CGRP and a PKG inhibitor (PKGi) recovered from inhibition of proliferation induced by CGRP. ASMC treated with CGRP and then PKAi or PKGi recovered only when exposed to the PKAi and not PKGi. Although CGRP is thought to act through a cAMP-dependent pathway, cGMP involvement in the response to CGRP has been reported. It is concluded that p53 plays a role in CGRP-induced inhibition of cell proliferation and cAMP/PKA appears to mediate this effect in ASMC and PASMC, whereas cGMP appears to be involved in PASMC proliferation.     

Mechanism by which avenanthramide-c, a polyphenol of oats, blocks cell cycle progression in vascular smooth muscle cells

Previously, we reported that avenanthramide-c (Avn-c), one of the major avenanthramides, polyphenols of oats, inhibited the serum-induced proliferation of vascular smooth muscle cells (SMC), which is an important process in the initiation and development of atherosclerosis. In the present study, we further investigated its cell cycle inhibitory mechanism. Rat embryonic aortic smooth muscle cell line A10 was used in this study. Flow cytometry analysis revealed that treatment of A10 cells with 80 muM Avn-c arrested the cell cycle in G1 phase as indicated by an increase in the number of cells in G1 phase and a decrease in the number of cells in S phase. This cell cycle arrest was associated with a decrease in the phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1 to S transition in the cell cycle. The inhibition of pRb phosphorylation with Avn-c was accompanied by a decrease in cyclin D1 expression and an increase in cyclin-dependent kinase inhibitor p21cip1 expression, without significant changes in p27kip1 expression. Furthermore, Avn-c treatment increased the expression level and stability of p53 protein, which could account for the increase of p21cip1 expression. Our results demonstrate for the first time that Avn-c, which is a unique polyphenol found in oats, arrests SMC proliferation at G1 phase by upregulating the p53-p21cip1 pathway and inhibiting pRB phosphorylation. This inhibitory effect of Avn-c on SMC proliferation is an additional indication for the potential health benefit of oat consumption in the prevention of coronary heart disease beyond its known effect through lowering blood cholesterol.     

Interactions of arterial cells: III. Stathmokinetic analyses of smooth muscle cells cocultured with endothelial cells

Arterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell-cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum-free ECCM with antiplatelet-derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF-like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.     

Characteristics of spontaneously transformed human endothelial ECV304 cell line: II. Functional responses of ECV304 cells

The functional peculiarities of spontaneously transformed human endothelial ECV304 cell line were studied to estimate its adequacy as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of the proliferative activity of this line on the presence of growth factors was shown. The absence of serum in the nutrition medium leads to the blockage of cells in the G₁ phase of cell cycle, which is not characteristic of tumor cell lines. Low doses of beta particles emitted during the decay of the [³H]-thymidine blocked, dose-dependent proliferation of these cells in the G₂/M phase. The incubation of the cells in medium with another source of β particles, ³H₂O, resulted in the predominant accumulation of cells in the S phase under conditions of equal specific tritium activities. The different efficiency of β particles of tritium as a part of the H₂O molecule or thymidine demonstrates that different mechanisms are responsible for different checkpoints. The checkpoint of G₁/S is absent, which agrees with the presence of the deletion of chromosome 9 at locus p21. The level of NO produced by the constitutive form of NO synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that the ECV304 line cells retained properties of the initial spontaneously transformed cell line obtained from the human umbilical vein (HUVEC) and can be used as a model system for further studies of properties of the vascular endothelium.     

Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia

Vasodilatory responses to exogenous nitric oxide (NO) are diminished following exposure to chronic hypoxia (CH) in isolated, perfused rat lungs. We hypothesized that both endothelium-derived reactive oxygen species (ROS) and endothelin-1 (ET-1) mediate this attenuated NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined vasodilatory and vascular smooth muscle (VSM) Ca2+ responses to the NO donor spermine NONOate in UTP-constricted, isolated pressurized small pulmonary arteries from control and CH rats. Consistent with our previous findings in perfused lungs, we observed attenuated NO-dependent vasodilation following CH in endothelium-intact vessels. However, in endothelium-denuded vessels, responses to spermine NONOate were augmented in CH rats compared with controls, thus demonstrating an inhibitory influence of the endothelium on NO-dependent reactivity following CH. Whereas both the ROS scavenger tiron and the ETA receptor antagonist BQ-123 augmented NO-dependent reactivity in endothelium-intact vessels from CH rats, neither fully restored vasodilatory responses to those observed following endothelium denudation in vessels from CH rats. In contrast, the combination of tiron and BQ-123 or the nonselective ET receptor antagonist PD-145065 enhanced NO responsiveness in endothelium-intact vessels from CH rats similar to that observed following endothelium denudation. We conclude that both endothelium-derived ROS and ET-1 attenuate NO-dependent pulmonary vasodilation following CH. Furthermore, CH augments pulmonary VSM reactivity to NO.     

Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression

Arterial remodeling in response to pathological insult is a complex process that depends in part on the balance between vascular cell apoptosis and proliferation. Studies in experimental models suggest that HO-1 mediates neointimal formation while limiting lumen stenosing, indicating a differential effect on vascular endothelial (EC) and smooth muscle cells (SMC). We investigated the effect of HO-1 expression on cell cycle progression in EC and SMC. The addition of SnMP (10 microM), an inhibitor of HO activity, to EC or SMC for 24h, resulted in significant abnormalities in DNA distribution and cell cycle progression compared to cells treated with the HO-1 inducers, heme (10 microM) or SnCl(2) (10 microM). SnMP increased G(1) phase and decreased S and G(2)/M phases in EC while heme or SnCl(2) decreased G(1) phase, but increased S and G(2)/M phases (p<0.05). Opposite effects were obtained in SMC. SnMP decreased G(1) phase and increased S and G(2)/M phases while heme or SnCl(2) increased G(1) phase but decreased S and G(2)/M phases (p<0.05). Our data demonstrate that HO-1 regulates the cell cycle in a cell-specific manner; it increases EC but decreases SMC cycle progression. The mechanisms underlying the HO-1 cell-specific effect on cell cycle progression within the vascular wall are yet to be explored. Nevertheless, these findings suggest that cell-specific targeting of HO-1 expression may provide a novel therapeutic strategy for the treatment of cardiovascular diseases.     

Expression and roles of Cl- channel ClC-5 in cell cycles of myeloid cells

This study investigated the effect of exogenous nitric oxide (NO) on endothelial glucocorticoid receptor (GR) function. The NO donor diethylenetriamine NONOate (DETA, 50-500microM) caused concentration dependent nuclear localization of transfected chimeric green fluorescent protein GFP-GR and elevated expression of secreted alkaline phosphatase (SEAP) from a glucocorticoid response element (GRE) promoter construct in bovine aortic endothelial cells. Other weaker NO donors (S-nitroso-N-acetylpenicillamine and spermine NONOate) failed to induce GFP-GR nuclear localization, but all the NO donors activated GRE-SEAP expression, a response unaffected by the antioxidant N-acetyl-L-cysteine. Overall, exogenous NO from high concentration donors can directly activate GR, suggesting a potential feedback mechanism for NO to regulate endothelial inducible nitric oxide synthase (iNOS) expression.     

YC-1-mediated vascular protection through inhibition of smooth muscle cell proliferation and platelet function

YC-1, a synthetic benzyl indazole derivative, is capable of stimulating endogenous vessel wall cyclic guanosine monophosphate (cGMP) production and attenuating the remodeling response to experimental arterial angioplasty. In an effort to investigate the mechanisms of this YC-1-mediated vasoprotection, we examined the influence of soluble YC-1 or YC-1 incorporated in a polyethylene glycol (PEG) hydrogel on cultured rat vascular smooth muscle cell (SMC) cGMP synthesis, SMC proliferation, and platelet function. Results demonstrate that soluble YC-1 stimulated SMC cGMP production in a dose-dependent fashion, while both soluble and hydrogel-released YC-1 inhibited vascular SMC proliferation in a dose-dependent fashion without effects on cell viability. Platelet aggregation and adherence to collagen were both significantly inhibited in a dose-dependent fashion by soluble and hydrogel-released YC-1. Arterial neointima formation following experimental balloon injury was significantly attenuated by perivascular hydrogel-released YC-1. These results suggest that YC-1 is a potent, physiologically active agent with major anti-proliferative and anti-platelet properties that may provide protection against vascular injury through cGMP-dependent mechanisms.     

Specificity of antioxidant enzyme inhibition in skeletal muscle to reactive nitrogen species donors

Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.