RESUMEN
Ischemia/reperfusion (I/R)-induced rapid inflammation involving activation of leukocyte-endothelial adhesive interactions and leukocyte infiltration into tissues is a major contributor to postischemic tissue injury. However, the molecular mediators involved in this pathological process are not fully known. We have previously reported that caveolin-2 (Cav-2), a protein component of plasma membrane caveolae, regulated leukocyte infiltration in mouse lung carcinoma tumors. The goal of the current study was to examine if Cav-2 plays a role in I/R injury and associated acute leukocyte-mediated inflammation. Using a mouse small intestinal I/R model, we demonstrated that I/R downregulates Cav-2 protein levels in the small bowel. Further study using Cav-2-deficient mice revealed aggravated postischemic tissue injury determined by scoring of villi length in H&E-stained tissue sections, which correlated with increased numbers of MPO-positive tissue-infiltrating leukocytes determined by IHC staining. Intravital microscopic analysis of upstream events relative to leukocyte transmigration and tissue infiltration revealed that leukocyte-endothelial cell adhesive interactions in postcapillary venules, namely leukocyte rolling and adhesion were also enhanced in Cav-2-deficient mice. Mechanistically, Cav-2 deficiency increased plasminogen activator inhibitor-1 (PAI-1) protein levels in the intestinal tissue and a pharmacological inhibition of PAI-1 had overall greater inhibitory effect on both aggravated I/R tissue injury and enhanced leukocyte-endothelial interactions in postcapillary venules in Cav-2-deficient mice. In conclusion, our data suggest that Cav-2 protein alleviates tissue injury in response to I/R by dampening PAI-1 protein levels and thereby reducing leukocyte-endothelial adhesive interactions.NEW & NOTEWORTHY The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.
Asunto(s)
Caveolina 2/deficiencia , Adhesión Celular , Células Endoteliales/metabolismo , Enfermedades del Yeyuno/metabolismo , Yeyuno/irrigación sanguínea , Rodamiento de Leucocito , Leucocitos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Daño por Reperfusión/metabolismo , Migración Transendotelial y Transepitelial , Vénulas/metabolismo , Animales , Caveolina 2/genética , Modelos Animales de Enfermedad , Células Endoteliales/patología , Enfermedades del Yeyuno/genética , Enfermedades del Yeyuno/patología , Leucocitos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Transducción de Señal , Vénulas/patologíaRESUMEN
Males have a higher risk for cardiovascular diseases (CVDs) than females. Ambient fine particulate matter (PM) exposure increases CVD risk with increased reactive oxygen species (ROS) production and oxidative stress. Endothelial progenitor cells (EPCs) are important to vascular structure and function and can contribute to the development of CVDs. The aims of the present study were to determine if sex differences exist in the effect of PM exposure on circulating EPCs in mice and, if so, whether oxidative stress plays a role. Male and female C57BL/6 mice (8-10 weeks old) were exposed to PM or a vehicle control for six weeks. ELISA analysis showed that PM exposure substantially increased the serum levels of IL-6 and IL-1ß in both males and females, but the concentrations were significantly higher in males. PM exposure only increased the serum levels of TNF-α in males. Flow cytometry analysis demonstrated that ROS production was significantly increased by PM treatment in males but not in females. Similarly, the level of circulating EPCs (CD34+/CD133+ and Sca-1+/Flk-1+) was significantly decreased by PM treatment in males but not in females. Antioxidants N-acetylcysteine (NAC) effectively prevented PM exposure-induced ROS and inflammatory cytokine production and restored circulating EPC levels in male mice. In sharp contrast, circulating EPC levels remained unchanged in female mice with PM exposure, an effect that was not altered by ovariectomy. In conclusion, PM exposure selectively decreased the circulating EPC population in male mice via increased oxidative stress without a significant impact on circulating EPCs in females independent of estrogen.
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Células Progenitoras Endoteliales/efectos de los fármacos , Células Progenitoras Endoteliales/metabolismo , Material Particulado/toxicidad , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Citocinas/sangre , Células Progenitoras Endoteliales/patología , Estrógenos/metabolismo , Femenino , Mediadores de Inflamación/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ovariectomía , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factores SexualesRESUMEN
Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.
Asunto(s)
Ciego/microbiología , Ácidos Grasos/metabolismo , Ácido Hipocloroso/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/enzimología , Peroxidasa/metabolismo , Sepsis/enzimología , Aldehídos/metabolismo , Animales , Antiinflamatorios/farmacología , Ciego/cirugía , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inflamación/inmunología , Inflamación/microbiología , Inflamación/prevención & control , Mediadores de Inflamación/antagonistas & inhibidores , Intestino Delgado/enzimología , Intestino Delgado/inmunología , Ligadura , Pulmón/enzimología , Pulmón/inmunología , Mastocitos/enzimología , Mastocitos/inmunología , Mesenterio/enzimología , Mesenterio/inmunología , Peroxidasa/antagonistas & inhibidores , Inhibidor 1 de Activador Plasminogénico/metabolismo , Punciones , Ratas Sprague-Dawley , Sepsis/inmunología , Sepsis/microbiología , Sepsis/prevención & control , Transducción de SeñalRESUMEN
Insulin-like growth factor-1 (IGF-1) decreases atherosclerosis in apolipoprotein E (Apoe)-deficient mice when administered systemically. However, mechanisms for its atheroprotective effect are not fully understood. We generated endothelium-specific IGF-1 receptor (IGF1R)-deficient mice on an Apoe-deficient background to assess effects of IGF-1 on the endothelium in the context of hyperlipidemia-induced atherosclerosis. Endothelial deficiency of IGF1R promoted atherosclerotic burden, when animals were fed on a high-fat diet for 12 wk or normal chow for 12 mo. Under the normal chow feeding condition, the vascular relaxation response to acetylcholine was increased in the endothelial IGF1R-deficient aorta; however, feeding of a high-fat diet substantially attenuated the relaxation response, and there was no difference between endothelial IGF1R-deficient and control mice. The endothelium and its intercellular junctions provide a barrier function to the vasculature. In human aortic endothelial cells, IGF-1 upregulated occludin, claudin 5, VE-cadherin, JAM-A, and CD31 expression levels, and vice versa, specific IGF1R inhibitor, picropodophyllin, an IGF1R-neutralizing antibody (αIR3), or siRNA to IGF1R abolished the IGF-1 effects on junction and adherens proteins, suggesting that IGF-1 promoted endothelial barrier function. Moreover, endothelial transwell permeability assays indicated that inhibition of IGF-1 signaling elevated solute permeability through the monolayer of human aortic endothelial cells. In summary, endothelial IGF1R deficiency increases atherosclerosis, and IGF-1 positively regulates tight junction protein and adherens junction protein levels and endothelial barrier function. Our findings suggest that the elevation of the endothelial junction protein level is, at least in part, the mechanism for antiatherogenic effects of IGF-1.NEW & NOTEWORTHY Endothelial insulin-like growth factor-1 (IGF-1) receptor deficiency significantly elevated atherosclerotic burden in apolipoprotein E-deficient mice, mediated at least in part by downregulation of intercellular junction proteins and, thus, elevated endothelial permeability. This study revealed a novel role for IGF-1 in supporting endothelial barrier function. These findings suggest that IGF-1's ability to promote endothelial barrier function may offer a novel therapeutic strategy for vascular diseases such as atherosclerosis.
Asunto(s)
Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Permeabilidad Capilar , Células Endoteliales/metabolismo , Receptor IGF Tipo 1/deficiencia , Animales , Antígenos CD/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Cadherinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/patología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Placa Aterosclerótica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Células THP-1 , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismoRESUMEN
Binge alcohol consumption is a health problem, but differences between the sexes remain poorly defined. We have examined the in vivo effects of three acute, repeat binge alcohol administration on the liver in male and female rats. Sprague-Dawley rats were gavaged with alcohol (5 g/kg body weight) three times at 12-hour intervals. Blood and liver tissues were collected 4 hours after the last binge ethanol. Subsequently, several variables were analyzed. Compared with male rats, females had higher levels of blood alcohol, alanine aminotransferase, and triglycerides. Liver histology showed increased lipid vesicles that were larger in females. Protein levels of liver cytochrome P4502E1 were higher in the liver of females than in the liver of males after binge. Hepatic phospho-extracellular signal-regulated kinase 1/2 and phosph-p38 mitogen-activated protein kinase levels were lower in females compared with males after binge alcohol, but no differences were found in the phospho-C-jun N-terminal kinase levels. Peroxisome proliferator-activated receptor γ-coactivator 1α and cyclic AMP response element binding (CREB) protein levels increased more in female than in male livers; however, increases in phospho-CREB levels were lower in females. Remarkably, c-fos was reduced substantially in the livers of females, but no differences in c-myc protein were found. Binge ethanol caused elevation in acetylated (H3AcK9) and phosphoacetylated (H3AcK9PS10) histone H3 in both sexes but without any difference. Binge alcohol caused differential alterations in the levels of various species of phosphatidylethanol and a larger increase in the diacylglycerol kinase-α protein levels in the liver of female rats compared with male rats. These data demonstrate, for the first time, similarities and differences in the sex-specific responses to repeat binge alcohol leading to an increased susceptibility of female rats to have liver injury in vivo. SIGNIFICANCE STATEMENT: This study examines the molecular responses of male and female rat livers to acute binge alcohol in vivo and demonstrates significant differences in the susceptibility between sexes.
Asunto(s)
Consumo Excesivo de Bebidas Alcohólicas/genética , Consumo Excesivo de Bebidas Alcohólicas/fisiopatología , Epigénesis Genética , Etanol/efectos adversos , Hígado/efectos de los fármacos , Hígado/patología , Factores Sexuales , Animales , Consumo Excesivo de Bebidas Alcohólicas/metabolismo , Consumo Excesivo de Bebidas Alcohólicas/patología , Citocromo P-450 CYP2E1/metabolismo , Diacilglicerol Quinasa/metabolismo , Femenino , Glicerofosfolípidos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacosRESUMEN
Activation of large-conductance Ca2+-activated K+ (BKCa) channels evokes cell survival programs that mitigate intestinal ischemia and reperfusion (I/R) inflammation and injury 24 h later. The goal of the present study was to determine the roles of reactive oxygen species (ROS) and heme oxygenase (HO)-1 in delayed acquisition of tolerance to I/R induced by pretreatment with the BKCa channel opener NS-1619. Superior mesentery arteries were occluded for 45 min followed by reperfusion for 70 min in wild-type (WT) or HO-1-null (HO-1-/-) mice that were pretreated with NS-1619 or saline vehicle 24 h earlier. Intravital microscopy was used to quantify the numbers of rolling and adherent leukocytes. Mucosal permeability, tumor necrosis factor-α (TNF-α) levels, and HO-1 activity and expression in jejunum were also determined. I/R induced leukocyte rolling and adhesion, increased intestinal TNF-α levels, and enhanced mucosal permeability in WT mice, effects that were largely abolished by pretreatment with NS-1619. The anti-inflammatory and mucosal permeability-sparing effects of NS-1619 were prevented by coincident treatment with the HO-1 inhibitor tin protoporphyrin-IX or a cell-permeant SOD mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), in WT mice. NS-1619 also increased jejunal HO-1 activity in WT animals, an effect that was attenuated by treatment with the BKCa channel antagonist paxilline or MnTBAP. I/R also increased postischemic leukocyte rolling and adhesion and intestinal TNF-α levels in HO-1-/- mice to levels comparable to those noted in WT animals. However, NS-1619 was ineffective in preventing these effects in HO-1-deficient mice. In summary, our data indicate that NS-1619 induces the development of an anti-inflammatory phenotype and mitigates postischemic mucosal barrier disruption in the small intestine by a mechanism that may involve ROS-dependent HO-1 activity.NEW & NOTEWORTHY Antecedent treatment with the large-conductance Ca2+-activated K+ channel opener NS-1619 24 h before ischemia-reperfusion limits postischemic tissue injury by an oxidant-dependent mechanism. The present study shows that NS-1619-induced oxidant production prevents ischemia-reperfusion-induced inflammation and mucosal barrier disruption in the small intestine by provoking increases in heme oxygenase-1 activity.
Asunto(s)
Bencimidazoles/farmacología , Hemo-Oxigenasa 1/efectos de los fármacos , Inflamación/prevención & control , Canales de Potasio de Gran Conductancia Activados por el Calcio/agonistas , Proteínas de la Membrana/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/prevención & control , Animales , Hemo-Oxigenasa 1/genética , Inflamación/etiología , Precondicionamiento Isquémico , Leucocitos/efectos de los fármacos , Leucocitos/enzimología , Leucocitos/metabolismo , Activación de Macrófagos , Masculino , Proteínas de la Membrana/genética , Arteria Mesentérica Superior/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Membrana Mucosa/patología , Daño por Reperfusión/complicaciones , Daño por Reperfusión/fisiopatología , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The aim was to determine whether treatment with BAY 60-2770, a selective activator of oxidized soluble guanylate cyclase (sGC), near the end of an ischemic event would prevent postischemic inflammation and mitochondrial dysfunction in wild-type (WT) and heme oxygenase-1 KO (HO-1(-/-)) mice. This protocol prevented increases in leukocyte rolling (LR) and adhesion (LA) to intestinal venules along with elevated TNFα and circulating neutrophil levels that accompany ischemia-reperfusion (I/R) in both animal models. We further hypothesized that a component of BAY 60-2770 treatment involves maintenance of mitochondrial membrane integrity during I/R. Measurements on isolated enterocytes of calcein fluorescence (mitochondrial permeability) and JC-1 fluorescence ratio (mitochondrial membrane potential) were reduced by I/R, indicating formation of mitochondrial permeability transition pores (mPTP). These effects were abrogated by BAY 60-2770 as well as cyclosporin A and SB-216763, which prevented mPTP opening and inhibited glycogen synthase kinase-3ß (GSK-3ß), respectively. Western blots of WT and HO-1(-/-) enterocytes indicated that GSK-3ß phosphorylation on Ser(9) (inhibitory site) was reduced by half following I/R alone (increased GSK-3ß activity) and increased by one-third (reduced GSK-3ß activity) following BAY 60-2770. Other investigators have associated phosphorylation of the GSK-3ß substrate cyclophilin D (pCyPD) with mPTP formation. We observed a 60% increase in pCyPD after I/R, whereas BAY 60-2770 treatment of sham and I/R groups reduced pCyPD by about 20%. In conclusion, selective activation of oxidized sGC of WT and HO-1(-/-) during ischemia protects against I/R-induced inflammation and preserves mucosal integrity in part by reducing pCyPD production and mPTP formation.
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Enterocitos/metabolismo , Isquemia/metabolismo , Mitocondrias/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Células Cultivadas , Peptidil-Prolil Isomerasa F , Ciclofilinas/metabolismo , Enterocitos/efectos de los fármacos , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Hidrocarburos Fluorados/farmacología , Intestinos/irrigación sanguínea , Intestinos/citología , Potencial de la Membrana Mitocondrial , Ratones , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad MitocondrialAsunto(s)
Infiltración Neutrófila , Inhibidor 1 de Activador Plasminogénico , Humanos , Isquemia , ReperfusiónRESUMEN
OBJECTIVE: Integrins contribute to vascular morphogenesis through regulation of adhesion and assembly of the extracellular matrix. However, the role of ß1-integrin in the mature vascular wall is less clear. APPROACH AND RESULTS: We sought to determine the function of ß1-integrin in mature smooth muscle cells in vivo using a loss of function approach by crossing a tamoxifen-inducible sm22αCre line to a floxed ß1-integrin transgenic line. Adult mice lacking smooth muscle ß1-integrin survived only 10 weeks post induction. The deletion of ß1-integrin resulted in profound loss of vasomotor control. Histological analysis revealed progressive fibrosis in arteries with associated apoptosis of smooth muscle cells, which was not rescued by adventitial stem cells. Smooth muscle cell apoptosis was detected in arteries with dead cells replaced primarily by collagen. Despite the catastrophic effects on vascular smooth muscle, the deleted visceral smooth muscle remained viable with the exception of a short portion of the colon, indicating that vascular but not visceral smooth muscle is particularly sensitive to changes in ß1-integrin. CONCLUSIONS: This study reveals an essential function of ß1-integrin in the maintenance of vasomotor control and highlights a critical role for ß1-integrin in vascular, but not visceral, smooth muscle survival.
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Integrina beta1/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Vasoconstricción , Vasodilatación , Adaptación Fisiológica , Animales , Apoptosis , Supervivencia Celular , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Fibrosis , Integrina beta1/genética , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Factores de Tiempo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Abstract ß1-Subunits enhance the gating properties of large-conductance Ca(2+)-activated K(+) channels (BKCa) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), ß1-subunits are vital in coupling SR-generated Ca(2+) sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BKCa activity due to apparent differences in the functional ß1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. ß1 modulation of the α-subunit Ca(2+) sensitivity was studied using patch-clamp techniques. BKCa channel normalized open probability (NPo) versus membrane potential (Vm) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca(2+) was raised from 0.5 to 100 µm. Calculated V1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 µm Ca(2+)i to -89 ± 9 mV at 100 µm Ca(2+)i in cerebral compared with 101 ± 10 to -63 ± 7 mV in cremaster VSMCs. Cremaster BKCa channels thus demonstrated an â¼2.5-fold weaker apparent Ca(2+) sensitivity such that at a value of Vm of -30 mV, a mean value of [Ca(2+)]i of 39 µm was required to open half of the channels in cremaster versus 16 µm [Ca(2+)]i in cerebral VSMCs. Further, shortened mean open and longer mean closed times were evident in BKCa channel events from cremaster VSMCs at either -30 or 30 mV at any given [Ca(2+)]. ß1-Subunit-directed siRNA decreased both the apparent Ca(2+) sensitivity of BKCa in cerebral VSMCs and the appearance of spontaneous transient outward currents. The data are consistent with a higher ratio of ß1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this leads both to higher Ca(2+) sensitivity and NPo for BKCa channels in the cerebral vasculature relative to that of skeletal muscle.
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Encéfalo/irrigación sanguínea , Calcio/metabolismo , Activación del Canal Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Arteriolas/metabolismo , Circulación Cerebrovascular , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Masculino , Potenciales de la Membrana , Técnicas de Placa-Clamp , Fenotipo , Subunidades de Proteína , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional , Factores de Tiempo , Técnicas de Cultivo de Tejidos , TransfecciónRESUMEN
Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²âº. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function.
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Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Activadores de Enzimas/farmacología , Hemo-Oxigenasa 1/deficiencia , Hidrocarburos Fluorados/farmacología , Inflamación/prevención & control , Intestino Delgado/irrigación sanguínea , Isquemia/tratamiento farmacológico , Proteínas de la Membrana/deficiencia , Oclusión Vascular Mesentérica/tratamiento farmacológico , Pirazoles/farmacología , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Daño por Reperfusión/prevención & control , Enfermedades Vasculares/tratamiento farmacológico , Animales , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Activación Enzimática , Guanilato Ciclasa/metabolismo , Hemo-Oxigenasa 1/genética , Sulfuro de Hidrógeno/metabolismo , Inflamación/enzimología , Inflamación/genética , Inflamación/fisiopatología , Mediadores de Inflamación/metabolismo , Isquemia/enzimología , Isquemia/genética , Isquemia/fisiopatología , Rodamiento de Leucocito/efectos de los fármacos , Proteínas de la Membrana/genética , Arteria Mesentérica Superior/efectos de los fármacos , Arteria Mesentérica Superior/enzimología , Arteria Mesentérica Superior/cirugía , Isquemia Mesentérica , Oclusión Vascular Mesentérica/enzimología , Oclusión Vascular Mesentérica/genética , Oclusión Vascular Mesentérica/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Daño por Reperfusión/enzimología , Daño por Reperfusión/genética , Daño por Reperfusión/fisiopatología , Transducción de Señal/efectos de los fármacos , Guanilil Ciclasa Soluble , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Enfermedades Vasculares/enzimología , Enfermedades Vasculares/genética , Enfermedades Vasculares/fisiopatología , Vasodilatación/efectos de los fármacos , Vénulas/efectos de los fármacos , Vénulas/enzimologíaRESUMEN
While ethanol intake at high levels (3-4 or more drinks), either in acute (occasional binge drinking) or chronic (daily) settings, increases the risk for myocardial infarction and stroke, an inverse relationship between regular consumption of alcoholic beverages at light to moderate levels (1-2 drinks per day) and cardiovascular risk has been consistently noted in a large number of epidemiologic studies. Although initially attributed to polyphenolic antioxidants in red wine, subsequent work has established that the ethanol component contributes to the beneficial effects associated with moderate intake of alcoholic beverages regardless of type (red versus white wine, beer, spirits). Concerns have been raised with regard to interpretation of epidemiologic evidence for this association including heterogeneity of the reference groups examined in many studies, different lifestyles of moderate drinkers versus abstainers, and favorable risk profiles in moderate drinkers. However, better controlled epidemiologic studies and especially work conducted in animal models and cell culture systems have substantiated this association and clearly established a cause and effect relationship between alcohol consumption and reductions in tissue injury induced by ischemia/reperfusion (I/R), respectively. The aims of this review are to summarize the epidemiologic evidence supporting the effectiveness of ethanol ingestion in reducing the likelihood of adverse cardiovascular events such as myocardial infarction and ischemic stroke, even in patients with co-existing risk factors, to discuss the ideal quantities, drinking patterns, and types of alcoholic beverages that confer protective effects in the cardiovascular system, and to review the findings of recent experimental studies directed at uncovering the mechanisms that underlie the cardiovascular protective effects of antecedent ethanol ingestion. Mechanistic interrogation of the signaling pathways invoked by antecedent ethanol ingestion may point the way towards development of new therapeutic approaches that mimic the powerful protective effects of socially relevant alcohol intake to limit I/R injury, but minimize the negative psychosocial impact and pathologic outcomes that also accompany consumption of ethanol.
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Consumo de Bebidas Alcohólicas/epidemiología , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/prevención & control , Adaptación Fisiológica/efectos de los fármacos , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Enfermedades Cardiovasculares/metabolismo , Etanol/metabolismo , Etanol/farmacología , Etanol/uso terapéutico , Humanos , Precondicionamiento Isquémico Miocárdico , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/prevención & control , Transducción de Señal/efectos de los fármacos , Accidente Cerebrovascular/epidemiología , Accidente Cerebrovascular/prevención & controlRESUMEN
The objectives of this study were to determine whether neutrophil depletion with anti-neutrophil serum (ANS) or preconditioning with the hydrogen sulfide (H(2)S) donor NaHS (NaHS-PC) 24 h prior to ischemia-reperfusion (I/R) would prevent postischemic mitochondrial dysfunction in rat intestinal mucosa and, if so, whether calcium-activated, large conductance potassium (BK(Ca)) channels were involved in this protective effect. I/R was induced by 45-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in rats preconditioned with NaHS (NaHS-PC) or a BK(Ca) channel activator (NS-1619-PC) 24 h earlier or treated with ANS. Mitochondrial function was assessed by measuring mitochondrial membrane potential, mitochondrial dehydrogenase function, and cytochrome c release. Mucosal myeloperoxidase (MPO) and TNF-α levels were also determined, as measures of postischemic inflammation. BK(Ca) expression in intestinal mucosa was detected by immunohistochemistry and Western blotting. I/R induced mitochondrial dysfunction and increased tissue MPO and TNF-α levels. Although mitochondrial dysfunction was attenuated by NaHS-PC or NS-1619-PC, the postischemic increases in mucosal MPO and TNF-α levels were not. The protective effect of NaHS-PC or NS-1619-PC on postischemic mitochondrial function was abolished by coincident treatment with BK(Ca) channel inhibitors. ANS prevented the I/R-induced increase in tissue MPO levels and reversed mitochondrial dysfunction. These data indicate that neutrophils play an essential role in I/R-induced mucosal mitochondrial dysfunction. In addition, NaHS-PC prevents postischemic mitochondrial dysfunction (but not inflammation) by a BK(Ca) channel-dependent mechanism.
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Enfermedades Intestinales/prevención & control , Intestino Delgado/irrigación sanguínea , Precondicionamiento Isquémico/métodos , Procedimientos de Reducción del Leucocitos , Enfermedades Mitocondriales/prevención & control , Neutrófilos , Daño por Reperfusión/complicaciones , Sulfuros/administración & dosificación , Animales , Bencimidazoles/administración & dosificación , Citocromos c/metabolismo , Sulfuro de Hidrógeno/metabolismo , Enfermedades Intestinales/etiología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Enfermedades Mitocondriales/etiología , Peroxidasa/análisis , Canales de Potasio Calcio-Activados/agonistas , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/análisisRESUMEN
We recently demonstrated that preconditioning with an exogenous hydrogen sulfide donor (NaHS-PC) 24 h before ischemia and reperfusion (I/R) causes postcapillary venules to shift to an anti-inflammatory phenotype in C57BL/6J wild-type (WT) mice such that these vessels fail to support increases in postischemic leukocyte rolling (LR) and leukocyte adhesion (LA). The objective of the present study was to determine whether heme oxygenase-1 (HO-1) is a mediator of these anti-inflammatory effects noted during I/R in mice preconditioned with NaHS. Intravital fluorescence microscopy was used to visualize LR and LA in single postcapillary venules of the murine small intestine. I/R induced marked increases in LR and LA, effects that were prevented by NaHS-PC. Treatment with the HO inhibitor tin protoporphyrin IX, but not the inactive protoporphyrin CuPPIX, just before reperfusion prevented the anti-inflammatory effects of antecedent NaHS. The anti-inflammatory effects of NaHS-PC were mimicked by preconditioning with hemin, an agent that induces HO-1 expression. We then evaluated the effect of NaHS as a preconditioning stimulus in mice that were genetically deficient in HO-1 (HO-1(-/-) on an H129 background with appropriate WT strain controls). NaHS-PC was ineffective in HO-1(-/-) mice. Our work indicates that HO-1 serves as an effector of the anti-inflammatory effects of NaHS-PC during I/R 24 h later.
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Antiinflamatorios/farmacología , Células Endoteliales/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Sulfuro de Hidrógeno/metabolismo , Intestino Delgado/irrigación sanguínea , Proteínas de la Membrana/metabolismo , Daño por Reperfusión/prevención & control , Sulfuros/farmacología , Análisis de Varianza , Animales , Antiinflamatorios/metabolismo , Adhesión Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Endoteliales/enzimología , Células Endoteliales/inmunología , Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/deficiencia , Hemo-Oxigenasa 1/genética , Rodamiento de Leucocito/efectos de los fármacos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Metaloporfirinas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Fenotipo , Protoporfirinas/farmacología , Daño por Reperfusión/enzimología , Daño por Reperfusión/genética , Daño por Reperfusión/inmunología , Sulfuros/metabolismo , Factores de Tiempo , Vénulas/efectos de los fármacos , Vénulas/enzimología , Vénulas/inmunologíaRESUMEN
The present study determined whether AMP-activated protein kinase (AMPK) regulates heme oxygenase (HO)-1 gene expression in endothelial cells (ECs) and if HO-1 contributes to the biological actions of this kinase. Treatment of human ECs with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) stimulated a concentration- and time-dependent increase in HO-1 protein and mRNA expression that was associated with a prominent increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) protein. Induction of HO-1 was also observed in rat carotid arteries after the in vivo application of AICAR. Induction of HO-1 by AICAR was blocked by the AMPK inhibitor compound C, the adenosine kinase inhibitor 5'-iodotubercidin, and by silencing AMPK-α(1/2) and was mimicked by the AMPK activator A-769662 and by infecting ECs with an adenovirus expressing constitutively active AMPK-α(1). AICAR also induced a significant rise in HO-1 promoter activity that was abolished by mutating the antioxidant responsive elements of the HO-1 promoter or by the overexpression of dominant negative Nrf2. Finally, activation of AMPK inhibited cytokine-mediated EC death, and this was prevented by the HO inhibitor tin protoporphyrin-IX or by silencing HO-1 expression. In conclusion, AMPK stimulates HO-1 gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress.
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Adenilato Quinasa/metabolismo , Células Endoteliales/fisiología , Hemo-Oxigenasa 1/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Análisis de Varianza , Animales , Northern Blotting , Western Blotting , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Humanos , Regiones Promotoras Genéticas , Pirazoles/farmacología , Pirimidinas/farmacología , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Ribonucleótidos/farmacología , Factores de TiempoRESUMEN
We previously demonstrated that preconditioning induced by ethanol consumption at low levels [ethanol preconditioning (EPC)] or with 5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR-PC) 24 h before ischemia-reperfusion prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism that is initiated by nitric oxide formed by endothelial nitric oxide synthase. Recent work indicates that 1) ethanol increases the activity of AMP-activated protein kinase (AMPK) and 2) AMPK phosphorylates endothelial nitric oxide synthase at the same activation site seen following EPC (Ser1177). In light of these observations, we postulated that the heterotrimeric serine/threonine kinase, AMPK, may play a role in triggering the development of the anti-inflammatory phenotype induced by EPC. Ethanol was administered to C57BL/6J mice by gavage in the presence or absence of AMPK inhibition. Twenty-four hours later, the numbers of rolling and adherent leukocytes in postcapillary venules of the small intestine were recorded using an intravital microscopic approach. Following 45 min of ischemia, LEI were recorded after 30 and 60 min of reperfusion or at equivalent time points in control animals. Ischemia-reperfusion induced a marked increase in LEI relative to sham-operated control mice. The increase in LEI was prevented by EPC, an effect that was lost with AMPK inhibition during the period of ethanol exposure. Studies conducted in AMPK α(1)- and α(2)-knockout mice suggest that the anti-inflammatory effects of AICAR are not dependent on which isoform of the catalytic α-subunit is present because a deficiency of either isoform results in a loss of protection. In sharp contrast, EPC appears to be triggered by an AMPK α(2)-isoform-dependent mechanism.
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Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Endotelio Vascular/efectos de los fármacos , Precondicionamiento Isquémico/métodos , Leucocitos/efectos de los fármacos , Daño por Reperfusión/prevención & control , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Etanol/farmacología , Intestino Delgado/irrigación sanguínea , Intestino Delgado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión/tratamiento farmacológico , Ribonucleótidos/farmacología , Vénulas/efectos de los fármacosRESUMEN
Mast cells are best known for their role in allergic reactions but are also now recognized for their important contributions to a number of disparate inflammatory conditions through the release of inflammatory mediators, serglycin and other proteoglycans, and proteases. Because these tissue resident inflammatory cells express proteases in such great abundance and their enzymatic activity results in cleavage of a multitude of proteins and peptides, which in turn modify tissue function, their substrate specificity, tissue distribution, and mode of action have become the subjects of great interest. Although mast cell protease-dependent proteolysis is critical to host defense against invading pathogens, regulation of these hydrolytic enzymes is essential to limiting self-induced damage as well. Indeed, dysregulated release of mast cell proteases is now recognized to contribute to the pathogenesis of a number of inflammatory conditions including asthma, abdominal aortic aneurysm formation, vessel damage in atherosclerosis and hypertension, arthritis, and ischemia/reperfusion injury. Understanding how mast cell proteases contribute to inflammation will thus help unravel molecular mechanisms that underlie such immunologic disorders and will help identify new therapeutic targets for drug development.
RESUMEN
The objectives of this study were to determine the role of calcium-activated, small (SK), intermediate (IK), and large (BK) conductance potassium channels in initiating the development of an anti-inflammatory phenotype elicited by preconditioning with an exogenous hydrogen sulfide (H(2)S) donor, sodium hydrosulfide (NaHS). Intravital microscopy was used to visualize rolling and firmly adherent leukocytes in vessels of the small intestine of mice preconditioned with NaHS (in the absence and presence of SK, IK, and BK channel inhibitors, apamin, TRAM-34, and paxilline, respectively) or SK/IK (NS-309) or BK channel activators (NS-1619) 24 h before ischemia-reperfusion (I/R). I/R induced marked increases in leukocyte rolling and adhesion, effects that were largely abolished by preconditioning with NaHS, NS-309, or NS-1619. The postischemic anti-inflammatory effects of NaHS-induced preconditioning were mitigated by BKB channel inhibitor treatment coincident with NaHS, but not by apamin or TRAM-34, 24 h before I/R. Confocal imaging and immunohistochemistry were used to demonstrate the presence of BKα subunit staining in both endothelial and vascular smooth muscle cells of isolated, pressurized mesenteric venules. Using patch-clamp techniques, we found that BK channels in cultured endothelial cells were activated after exposure to NaHS. Bath application of the same concentration of NaHS used in preconditioning protocols led to a rapid increase in a whole cell K(+) current; specifically, the component of K(+) current blocked by the selective BK channel antagonist iberiotoxin. The activation of BK current by NaHS could also be demonstrated in single channel recording mode where it was independent of a change in intracellular Ca(+) concentration. Our data are consistent with the concept that H(2)S induces the development of an anti-adhesive state in I/R in part mediated by a BK channel-dependent mechanism.
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Sulfuro de Hidrógeno/uso terapéutico , Inflamación/prevención & control , Intestino Delgado/irrigación sanguínea , Isquemia/complicaciones , Precondicionamiento Isquémico , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Fenotipo , Animales , Apamina/farmacología , Células Cultivadas , Fenómenos Electrofisiológicos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Humanos , Sulfuro de Hidrógeno/farmacología , Inflamación/fisiopatología , Isquemia/fisiopatología , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Péptidos/farmacología , Pirazoles/farmacologíaRESUMEN
EtOH-PC reduces postischemic neuronal injury in response to cerebral (I/R). We examined the mechanism underlying this protective effect by determining (i) whether it was associated with a decrease in I/R-induced leukocyte-endothelial adhesive interactions in postcapillary venules, and (ii) whether the protective effects were mediated by activation of large conductance, calcium-activated potassium (BK(Ca)) channels. Mice were administered ethanol by gavage or treated with the BK(Ca) channel opener, NS1619, 24 hours prior to I/R with or without prior treatment with the BK(Ca) channel blocker, PX. Both CCA were occluded for 20 minutes followed by two and three hours of reperfusion, and rolling (LR) and adherent (LA) leukocytes were quantified in pial venules using intravital microscopy. The extent of DND, apoptosis and glial activation in hippocampus were assessed four days after I/R. Compared with sham, I/R elicited increases in LR and LA in pial venules and DND and apoptosis as well as glial activation in the hippocampus. These effects were attenuated by EtOH-PC or antecedent NS1619 administration, and this protection was reversed by prior treatment with PX. Our results support a role for BK(Ca) channel activation in the neuroprotective effects of EtOH-PC in cerebral I/R.
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Isquemia Encefálica/tratamiento farmacológico , Etanol/administración & dosificación , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Leucocitos/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Daño por Reperfusión/tratamiento farmacológico , Animales , Bencimidazoles/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Isquemia Encefálica/sangre , Isquemia Encefálica/patología , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Indoles/farmacología , Precondicionamiento Isquémico/métodos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/patología , Prosencéfalo/irrigación sanguínea , Prosencéfalo/efectos de los fármacos , Prosencéfalo/lesiones , Daño por Reperfusión/sangre , Daño por Reperfusión/patologíaRESUMEN
Heme oxygenase-1 knockout, H(mox)1(-/-), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in H(mox)1(-/-) mice. The contractile responses [superior mesenteric arteries (SMA), from female H(mox)1(-/-) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in H(mox)1(-/-) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in H(mox)1(-/-) than H(mox)1(+/+) mice (p < 0.001). Branches from H(mox)1(-/-) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. H(mox)1(-/-) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were H(mox)1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of H(mox)1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of H(mox)1(-/-) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both α1 and ß1 subunit levels were reduced to <50% of H(mox)1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of H(mox)1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.