Pub Date : 2002-08-09DOI: 10.1161/01.RES.0000027562.91075.56
M. V. Alvarez-Arroyo, S. Yagüe, R. Wenger, D. Pereira, S. Jiménez, F. R. González-Pacheco, M. A. Castilla, J. Deudero, C. Caramelo
Abstract— The relative importance of cyclophilin (CyP) versus calcineurin (Cn)-mediated mechanisms in the effect of cyclosporin A (CsA) on endothelial cells (ECs) is largely unknown. In cultured ECs, CsA was cytotoxic/proapoptotic or cytoprotective/antiapoptotic at high or low concentrations, respectively. CsA analogs (MeVal-4-CsA and MeIle-4-CsA), which bind to CyP but do not inhibit Cn, closely reproduced the CsA effects. Based on our previous data, the role of vascular endothelial growth factor (VEGF) as a mediator of CsA-induced cytoprotection was further analyzed. The actions of CsA and CsA analogs were shifted from a protective to a cell-damaging pattern in the presence of a specific anti-VEGF monoclonal antibody (mAb). This positive interaction was further supported by a transient increase in cytosolic free calcium concentration ([Ca2+]i) by VEGF after pretreatment with either CsA or MeVal-4-CsA and an increase in the expression and synthesis of VEGF receptor 2 (VEGFR2). Of functional importance, blockade of the interaction between VEGF and VEGFR2 by a VEGFR2 mAb abolished the cytoprotective effect of CsA. In addition, preconditioning with low concentrations of CsA or CsA analogs increased both cytoprotection and VEGFR2 mRNA expression when EC were exposed to higher concentrations of CsA. In summary, our results reveal that (1) the biphasic responses to CsA in EC are related to the interaction of CsA with CyP rather than with Cn and (2) VEGF is a critical factor in the cytoprotective effect of CsA, by a mechanism that involves VEGFR2.
{"title":"Cyclophilin-Mediated Pathways in the Effect of Cyclosporin A on Endothelial Cells: Role of Vascular Endothelial Growth Factor","authors":"M. V. Alvarez-Arroyo, S. Yagüe, R. Wenger, D. Pereira, S. Jiménez, F. R. González-Pacheco, M. A. Castilla, J. Deudero, C. Caramelo","doi":"10.1161/01.RES.0000027562.91075.56","DOIUrl":"https://doi.org/10.1161/01.RES.0000027562.91075.56","url":null,"abstract":"Abstract— The relative importance of cyclophilin (CyP) versus calcineurin (Cn)-mediated mechanisms in the effect of cyclosporin A (CsA) on endothelial cells (ECs) is largely unknown. In cultured ECs, CsA was cytotoxic/proapoptotic or cytoprotective/antiapoptotic at high or low concentrations, respectively. CsA analogs (MeVal-4-CsA and MeIle-4-CsA), which bind to CyP but do not inhibit Cn, closely reproduced the CsA effects. Based on our previous data, the role of vascular endothelial growth factor (VEGF) as a mediator of CsA-induced cytoprotection was further analyzed. The actions of CsA and CsA analogs were shifted from a protective to a cell-damaging pattern in the presence of a specific anti-VEGF monoclonal antibody (mAb). This positive interaction was further supported by a transient increase in cytosolic free calcium concentration ([Ca2+]i) by VEGF after pretreatment with either CsA or MeVal-4-CsA and an increase in the expression and synthesis of VEGF receptor 2 (VEGFR2). Of functional importance, blockade of the interaction between VEGF and VEGFR2 by a VEGFR2 mAb abolished the cytoprotective effect of CsA. In addition, preconditioning with low concentrations of CsA or CsA analogs increased both cytoprotection and VEGFR2 mRNA expression when EC were exposed to higher concentrations of CsA. In summary, our results reveal that (1) the biphasic responses to CsA in EC are related to the interaction of CsA with CyP rather than with Cn and (2) VEGF is a critical factor in the cytoprotective effect of CsA, by a mechanism that involves VEGFR2.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79159072","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-08-09DOI: 10.1161/01.RES.0000029232.42227.16
K. Regula, K. Ens, L. Kirshenbaum
Abstract— In this study, we provide evidence for the operation of BNIP3 as a key regulator of mitochondrial function and cell death of ventricular myocytes during hypoxia. In contrast to normoxic cells, a 5.6-fold increase (P <0.05) in myocyte death was observed in cells subjected to hypoxia. Moreover, a significant increase in BNIP3 expression was detected in postnatal ventricular myocytes and adult rat hearts subjected to hypoxia. An increase in BNIP3 expression was detected in adult rat hearts in vivo with chronic heart failure. Subcellular fractionation experiments indicated that endogenous BNIP3 was integrated into the mitochondrial membranes during hypoxia. Adenovirus-mediated delivery of full-length BNIP3 to myocytes was toxic and provoked an 8.3-fold increase (P <0.05) in myocyte death with features typical of apoptosis. Mitochondrial defects consistent with opening of the permeability transition pore (PT pore) were observed in cells expressing BNIP3 but not in cells expressing BNIP3 missing the carboxyl-terminal transmembrane domain (BNIP3&Dgr;TM), necessary for mitochondrial insertion. The pan-caspase inhibitor z-VAD-fmk (25 to 100 &mgr;mol/L) suppressed BNIP3-induced cell death of ventricular myocytes in a dose-dependent manner. Bongkrekic acid (50 &mgr;mol/L), an inhibitor of the PT pore, prevented BNIP3-induced mitochondrial defects and cell death. Expression of BNIP3&Dgr;TM suppressed the hypoxia-induced integration of the endogenous BNIP3 protein and cell death of ventricular myocytes. To our knowledge, the data provide the first evidence for the involvement of BNIP3 as an inducible factor that provokes mitochondrial defects and cell death of ventricular myocytes during hypoxia.
{"title":"Inducible Expression of BNIP3 Provokes Mitochondrial Defects and Hypoxia-Mediated Cell Death of Ventricular Myocytes","authors":"K. Regula, K. Ens, L. Kirshenbaum","doi":"10.1161/01.RES.0000029232.42227.16","DOIUrl":"https://doi.org/10.1161/01.RES.0000029232.42227.16","url":null,"abstract":"Abstract— In this study, we provide evidence for the operation of BNIP3 as a key regulator of mitochondrial function and cell death of ventricular myocytes during hypoxia. In contrast to normoxic cells, a 5.6-fold increase (P <0.05) in myocyte death was observed in cells subjected to hypoxia. Moreover, a significant increase in BNIP3 expression was detected in postnatal ventricular myocytes and adult rat hearts subjected to hypoxia. An increase in BNIP3 expression was detected in adult rat hearts in vivo with chronic heart failure. Subcellular fractionation experiments indicated that endogenous BNIP3 was integrated into the mitochondrial membranes during hypoxia. Adenovirus-mediated delivery of full-length BNIP3 to myocytes was toxic and provoked an 8.3-fold increase (P <0.05) in myocyte death with features typical of apoptosis. Mitochondrial defects consistent with opening of the permeability transition pore (PT pore) were observed in cells expressing BNIP3 but not in cells expressing BNIP3 missing the carboxyl-terminal transmembrane domain (BNIP3&Dgr;TM), necessary for mitochondrial insertion. The pan-caspase inhibitor z-VAD-fmk (25 to 100 &mgr;mol/L) suppressed BNIP3-induced cell death of ventricular myocytes in a dose-dependent manner. Bongkrekic acid (50 &mgr;mol/L), an inhibitor of the PT pore, prevented BNIP3-induced mitochondrial defects and cell death. Expression of BNIP3&Dgr;TM suppressed the hypoxia-induced integration of the endogenous BNIP3 protein and cell death of ventricular myocytes. To our knowledge, the data provide the first evidence for the involvement of BNIP3 as an inducible factor that provokes mitochondrial defects and cell death of ventricular myocytes during hypoxia.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75723600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000027529.37429.38
H. Reuter, S. Henderson, T. Han, T. Matsuda, A. Baba, R. Ross, J. Goldhaber, K. Philipson
The role of the Na+-Ca2+ exchanger as a major determinant of cell Ca2+ is well defined in cardiac tissue, and there has been much effort to develop specific inhibitors of the exchanger. We use a novel system to test the specificity of two putative specific inhibitors, KB-R7943 and SEA0400. The drugs are applied to electrically stimulated heart tubes from control mouse embryos or embryos with the Na+-Ca2+ exchanger knocked out. We monitored effects of the drugs on Ca2+ transients. Both drugs depress the Ca2+ transients at low concentrations even in the absence of any Na+-Ca2+ exchanger. KB-R7943 and SEA0400 are not completely specific and should be used with caution as Na+-Ca2+ exchange inhibitors.
{"title":"Knockout Mice for Pharmacological Screening: Testing the Specificity of Na+-Ca2+ Exchange Inhibitors","authors":"H. Reuter, S. Henderson, T. Han, T. Matsuda, A. Baba, R. Ross, J. Goldhaber, K. Philipson","doi":"10.1161/01.RES.0000027529.37429.38","DOIUrl":"https://doi.org/10.1161/01.RES.0000027529.37429.38","url":null,"abstract":"The role of the Na+-Ca2+ exchanger as a major determinant of cell Ca2+ is well defined in cardiac tissue, and there has been much effort to develop specific inhibitors of the exchanger. We use a novel system to test the specificity of two putative specific inhibitors, KB-R7943 and SEA0400. The drugs are applied to electrically stimulated heart tubes from control mouse embryos or embryos with the Na+-Ca2+ exchanger knocked out. We monitored effects of the drugs on Ca2+ transients. Both drugs depress the Ca2+ transients at low concentrations even in the absence of any Na+-Ca2+ exchanger. KB-R7943 and SEA0400 are not completely specific and should be used with caution as Na+-Ca2+ exchange inhibitors.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82168091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000028149.15986.4C
Ho-Jin Park, Dequan Kong, L. Iruela‐Arispe, Ulrike Begley, Dongjiang Tang, J. Galper
Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor–stimulated chick chorioallantoic membranes and basic fibroblast growth factor–stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.
{"title":"3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Interfere With Angiogenesis by Inhibiting the Geranylgeranylation of RhoA","authors":"Ho-Jin Park, Dequan Kong, L. Iruela‐Arispe, Ulrike Begley, Dongjiang Tang, J. Galper","doi":"10.1161/01.RES.0000028149.15986.4C","DOIUrl":"https://doi.org/10.1161/01.RES.0000028149.15986.4C","url":null,"abstract":"Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor–stimulated chick chorioallantoic membranes and basic fibroblast growth factor–stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86598994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000028150.51130.36
F. Coussin, R. H. Scott, A. Wise, G. Nixon
Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein–coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160ROCK (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate–dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44mapk in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P1/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.
{"title":"Comparison of Sphingosine 1-Phosphate–Induced Intracellular Signaling Pathways in Vascular Smooth Muscles: Differential Role in Vasoconstriction","authors":"F. Coussin, R. H. Scott, A. Wise, G. Nixon","doi":"10.1161/01.RES.0000028150.51130.36","DOIUrl":"https://doi.org/10.1161/01.RES.0000028150.51130.36","url":null,"abstract":"Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein–coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160ROCK (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate–dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44mapk in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P1/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87154936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000028008.99774.7F
M. Mehrabian, H. Allayee, Jack Wong, W. Shih, Xuping Wang, Z. Shaposhnik, C. Funk, A. Lusis
We previously reported the identification of a locus on mouse chromosome 6 that confers almost total resistance to atherogenesis, even on a hypercholesterolemic (LDL receptor–null) background. 5-Lipoxygenase (5-LO) is the rate-limiting enzyme in leukotriene synthesis and was among the chromosome 6 locus candidate genes that we examined. The levels of 5-LO mRNA were reduced about 5-fold in a congenic strain, designated CON6, containing the resistant chromosome 6 region derived from the CAST/Ei strain (CAST), as compared with the background C57BL/6J (B6) strain. 5-LO protein levels were similarly reduced in the CON6 mice. Sequencing of the 5-LO cDNA revealed several differences between CON6 and the B6 strain. To test the whether 5-LO is responsible for the resistant phenotype, we bred a 5-LO knockout allele onto an LDL receptor–null (LDLR−/−) background. On this background, the mice bred poorly and only heterozygous 5-LO knockout mice were obtained. These mice showed a dramatic decrease (>26-fold;P <0.0005) in aortic lesion development, similar to the CON6 mice. Immunohistochemistry revealed that 5-LO was abundantly expressed in atherosclerotic lesions of apoE− /− and LDLR−/− deficient mice, appearing to colocalize with a subset of macrophages but not with all macrophage-staining regions. When bone marrow from 5-LO+/− mice was transplanted into LDLR−/−, there was a significant reduction in atherogenesis, suggesting that macrophage 5-LO is responsible, at least in part, for the effect on atherosclerosis. These results indicate that 5-LO contributes importantly to the atherogenic process and they provide strong presumptive evidence that reduced 5-LO expression is partly responsible for the resistance to atherosclerosis in CON6 mice.
{"title":"Identification of 5-Lipoxygenase as a Major Gene Contributing to Atherosclerosis Susceptibility in Mice","authors":"M. Mehrabian, H. Allayee, Jack Wong, W. Shih, Xuping Wang, Z. Shaposhnik, C. Funk, A. Lusis","doi":"10.1161/01.RES.0000028008.99774.7F","DOIUrl":"https://doi.org/10.1161/01.RES.0000028008.99774.7F","url":null,"abstract":"We previously reported the identification of a locus on mouse chromosome 6 that confers almost total resistance to atherogenesis, even on a hypercholesterolemic (LDL receptor–null) background. 5-Lipoxygenase (5-LO) is the rate-limiting enzyme in leukotriene synthesis and was among the chromosome 6 locus candidate genes that we examined. The levels of 5-LO mRNA were reduced about 5-fold in a congenic strain, designated CON6, containing the resistant chromosome 6 region derived from the CAST/Ei strain (CAST), as compared with the background C57BL/6J (B6) strain. 5-LO protein levels were similarly reduced in the CON6 mice. Sequencing of the 5-LO cDNA revealed several differences between CON6 and the B6 strain. To test the whether 5-LO is responsible for the resistant phenotype, we bred a 5-LO knockout allele onto an LDL receptor–null (LDLR−/−) background. On this background, the mice bred poorly and only heterozygous 5-LO knockout mice were obtained. These mice showed a dramatic decrease (>26-fold;P <0.0005) in aortic lesion development, similar to the CON6 mice. Immunohistochemistry revealed that 5-LO was abundantly expressed in atherosclerotic lesions of apoE− /− and LDLR−/− deficient mice, appearing to colocalize with a subset of macrophages but not with all macrophage-staining regions. When bone marrow from 5-LO+/− mice was transplanted into LDLR−/−, there was a significant reduction in atherogenesis, suggesting that macrophage 5-LO is responsible, at least in part, for the effect on atherosclerosis. These results indicate that 5-LO contributes importantly to the atherogenic process and they provide strong presumptive evidence that reduced 5-LO expression is partly responsible for the resistance to atherosclerosis in CON6 mice.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82888962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000028007.91385.EE
R. Black, Yiru Guo, Z. Ge, S. Murphree, S. Prabhu, W. Jones, R. Bolli, J. Auchampach
We used a genetic approach to determine whether increasing the level of A3 adenosine receptors (A3ARs) expressed in the heart confers protection against ischemia without causing cardiac pathology. We generated mice carrying one (A3tg.1) or six (A3tg.6) copies of a transgene consisting of the cardiomyocyte-specific &agr;-myosin heavy chain gene promoter and the A3AR cDNA. A3tg.1 and A3tg.6 mice expressed 12.7±3.15 and 66.3±9.4 fmol/mg of the high-affinity G protein–coupled form of the A3AR in the myocardium, respectively. Extensive morphological, histological, and functional analyses demonstrated that there were no apparent abnormalities in A3tg.1 transgenic mice compared with nontransgenic mice. In contrast, A3tg.6 mice exhibited dilated hearts, expression of markers of hypertrophy, bradycardia, hypotension, and systolic dysfunction. When A3tg mice were subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion, infarct size was reduced ≈30% in A3tg.1 mice and ≈40% in A3tg.6 mice compared with nontransgenic littermates. The reduction in infarct size in the transgenic mice was not related to differences in risk region size, systemic hemodynamics, or body temperature, indicating that the cardioprotection was a result of increased A3AR signaling in the ischemic myocardium. The results demonstrate that low-level expression of A3ARs in the heart provides effective protection against ischemic injury without detectable adverse effects, whereas higher levels of A3AR expression lead to the development of a dilated cardiomyopathy.
{"title":"Gene Dosage-Dependent Effects of Cardiac-Specific Overexpression of the A3 Adenosine Receptor","authors":"R. Black, Yiru Guo, Z. Ge, S. Murphree, S. Prabhu, W. Jones, R. Bolli, J. Auchampach","doi":"10.1161/01.RES.0000028007.91385.EE","DOIUrl":"https://doi.org/10.1161/01.RES.0000028007.91385.EE","url":null,"abstract":"We used a genetic approach to determine whether increasing the level of A3 adenosine receptors (A3ARs) expressed in the heart confers protection against ischemia without causing cardiac pathology. We generated mice carrying one (A3tg.1) or six (A3tg.6) copies of a transgene consisting of the cardiomyocyte-specific &agr;-myosin heavy chain gene promoter and the A3AR cDNA. A3tg.1 and A3tg.6 mice expressed 12.7±3.15 and 66.3±9.4 fmol/mg of the high-affinity G protein–coupled form of the A3AR in the myocardium, respectively. Extensive morphological, histological, and functional analyses demonstrated that there were no apparent abnormalities in A3tg.1 transgenic mice compared with nontransgenic mice. In contrast, A3tg.6 mice exhibited dilated hearts, expression of markers of hypertrophy, bradycardia, hypotension, and systolic dysfunction. When A3tg mice were subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion, infarct size was reduced ≈30% in A3tg.1 mice and ≈40% in A3tg.6 mice compared with nontransgenic littermates. The reduction in infarct size in the transgenic mice was not related to differences in risk region size, systemic hemodynamics, or body temperature, indicating that the cardioprotection was a result of increased A3AR signaling in the ischemic myocardium. The results demonstrate that low-level expression of A3ARs in the heart provides effective protection against ischemic injury without detectable adverse effects, whereas higher levels of A3AR expression lead to the development of a dilated cardiomyopathy.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91508305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000026057.13161.42
S. Shirao, S. Kashiwagi, Masafumi Sato, Saori Miwa, F. Nakao, T. Kurokawa, N. Todoroki-Ikeda, K. Mogami, Y. Mizukami, S. Kuriyama, K. Haze, Michiyasu Suzuki, Sei Kobayashi
Although recent investigations have suggested that a Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated. In addition, the involvement of protein kinase C (PKC) may also be related to cerebral vasospasm. We recently reported that sphingosylphosphorylcholine (SPC), a sphingolipid, induces Rho-kinase–mediated Ca2+ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca2+ sensitization of the bovine middle cerebral artery (MCA) and the relation to signal transduction pathways mediated by Rho-kinase and PKC. In intact MCA, SPC induced a concentration-dependent (EC50=3.0 &mgr;mol/L) contraction, without [Ca2+]i elevation. In membrane-permeabilized MCA, SPC induced Ca2+ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca2+ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominant-negative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca2+-independent contraction induced by phorbol ester. In contrast, phorbol ester–induced Ca2+ sensitization was resistant to a Rho-kinase inhibitor and a dominant-negative Rho-kinase. In primary cultured vascular smooth muscle cells, SPC induced the translocation of cytosolic Rho-kinase to the cell membrane. We propose that SPC is a novel messenger for Rho-kinase–mediated Ca2+ sensitization of cerebral arterial smooth muscle and, therefore, may play a pivotal role in the pathogenesis of abnormal contraction of the cerebral artery such as vasospasm. The SPC/Rho-kinase pathway functions independently of the PKC pathway.
{"title":"Sphingosylphosphorylcholine Is a Novel Messenger for Rho-Kinase–Mediated Ca2+ Sensitization in the Bovine Cerebral Artery: Unimportant Role for Protein Kinase C","authors":"S. Shirao, S. Kashiwagi, Masafumi Sato, Saori Miwa, F. Nakao, T. Kurokawa, N. Todoroki-Ikeda, K. Mogami, Y. Mizukami, S. Kuriyama, K. Haze, Michiyasu Suzuki, Sei Kobayashi","doi":"10.1161/01.RES.0000026057.13161.42","DOIUrl":"https://doi.org/10.1161/01.RES.0000026057.13161.42","url":null,"abstract":"Although recent investigations have suggested that a Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated. In addition, the involvement of protein kinase C (PKC) may also be related to cerebral vasospasm. We recently reported that sphingosylphosphorylcholine (SPC), a sphingolipid, induces Rho-kinase–mediated Ca2+ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca2+ sensitization of the bovine middle cerebral artery (MCA) and the relation to signal transduction pathways mediated by Rho-kinase and PKC. In intact MCA, SPC induced a concentration-dependent (EC50=3.0 &mgr;mol/L) contraction, without [Ca2+]i elevation. In membrane-permeabilized MCA, SPC induced Ca2+ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca2+ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominant-negative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca2+-independent contraction induced by phorbol ester. In contrast, phorbol ester–induced Ca2+ sensitization was resistant to a Rho-kinase inhibitor and a dominant-negative Rho-kinase. In primary cultured vascular smooth muscle cells, SPC induced the translocation of cytosolic Rho-kinase to the cell membrane. We propose that SPC is a novel messenger for Rho-kinase–mediated Ca2+ sensitization of cerebral arterial smooth muscle and, therefore, may play a pivotal role in the pathogenesis of abnormal contraction of the cerebral artery such as vasospasm. The SPC/Rho-kinase pathway functions independently of the PKC pathway.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85666797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000028004.76218.B8
C. Vecchione, R. Brandes
3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) improve endothelial function. We determined whether withdrawal of statin therapy affects endothelium-dependent relaxation in mice and studied the underlying mechanism. Mice were treated with daily injections of cerivastatin (2 mg/kg per day SC), atorvastatin (1 and 10 mg/kg per day SC), or placebo. Vascular reactivity was studied in aortic rings from these mice after 10 days of treatment and after cessation of therapy for several days. Both statins improved endothelium-dependent relaxation to acetylcholine. Compared with control, withdrawal of statin treatment transiently (from day 4 to 7) attenuated endothelium-dependent relaxation. In vessels from animals subjected to atorvastatin withdrawal, the antioxidant tiron restored relaxations. Vascular superoxide anion generation was unaffected by statin therapy but was increased during withdrawal. In mice lacking the gp91phox subunit of the NADPH oxidase, no attenuation of acetylcholine-induced relaxation and no increase in superoxide generation were observed after withdrawal of atorvastatin. In human umbilical vein endothelial cells, statins, which decrease the membrane association of NADPH oxidase–activating Rac-1, increased the activity of this GTPase in whole-cell lysates. Withdrawal of statins induced a translocation of Rac-1 from the cytosol to the membrane and transiently increased NADPH-induced lucigenin chemiluminescence in membrane preparations. Rac-1 inactivation by Clostridium difficile toxin B inhibited the cerivastatin-induced oxygen radical production in human umbilical vein endothelial cells. These observations indicate that the withdrawal of statins induces endothelial dysfunction. The underlying mechanism involves activation of a gp91phox-containing NADPH oxidase by Rac-1 and the subsequent scavenging of endothelium-derived NO by superoxide anions generated from this enzyme.
{"title":"Withdrawal of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Elicits Oxidative Stress and Induces Endothelial Dysfunction in Mice","authors":"C. Vecchione, R. Brandes","doi":"10.1161/01.RES.0000028004.76218.B8","DOIUrl":"https://doi.org/10.1161/01.RES.0000028004.76218.B8","url":null,"abstract":"3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) improve endothelial function. We determined whether withdrawal of statin therapy affects endothelium-dependent relaxation in mice and studied the underlying mechanism. Mice were treated with daily injections of cerivastatin (2 mg/kg per day SC), atorvastatin (1 and 10 mg/kg per day SC), or placebo. Vascular reactivity was studied in aortic rings from these mice after 10 days of treatment and after cessation of therapy for several days. Both statins improved endothelium-dependent relaxation to acetylcholine. Compared with control, withdrawal of statin treatment transiently (from day 4 to 7) attenuated endothelium-dependent relaxation. In vessels from animals subjected to atorvastatin withdrawal, the antioxidant tiron restored relaxations. Vascular superoxide anion generation was unaffected by statin therapy but was increased during withdrawal. In mice lacking the gp91phox subunit of the NADPH oxidase, no attenuation of acetylcholine-induced relaxation and no increase in superoxide generation were observed after withdrawal of atorvastatin. In human umbilical vein endothelial cells, statins, which decrease the membrane association of NADPH oxidase–activating Rac-1, increased the activity of this GTPase in whole-cell lysates. Withdrawal of statins induced a translocation of Rac-1 from the cytosol to the membrane and transiently increased NADPH-induced lucigenin chemiluminescence in membrane preparations. Rac-1 inactivation by Clostridium difficile toxin B inhibited the cerivastatin-induced oxygen radical production in human umbilical vein endothelial cells. These observations indicate that the withdrawal of statins induces endothelial dysfunction. The underlying mechanism involves activation of a gp91phox-containing NADPH oxidase by Rac-1 and the subsequent scavenging of endothelium-derived NO by superoxide anions generated from this enzyme.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88887921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000027815.75000.33
M. Okada, Catherine Y. Wang, Daniel W Hwang, Taichi Sakaguchi, K. E. Olson, Yasushi Yoshikawa, K. Minamoto, S. Mazer, S. Yan, D. Pinsky
Expression of the zinc finger transcription factor early growth response gene-1 (Egr-1) is triggered rapidly after mechanical vascular injury or after a precipitous drop in ambient oxygen, whereupon it induces the expression of diverse gene families to elicit a pathological response. Initially characterized as an early response transcriptional activator, the role of Egr-1 in more chronic forms of vascular injury remains to be defined. Studies were designed to examine whether Egr-1 induction may serve as a causal link between early preservation injury and delayed vascular consequences, such as coronary allograft vasculopathy (CAV). The preservation and transplantation of heterotopic murine cardiac allografts strongly induce Egr-1 expression, leading to increased expression of its downstream target genes, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and platelet-derived growth factor A chain. Expression of these Egr-1–inducible gene targets is virtually obliterated in homozygous Egr-1–null donor allografts, which also exhibit attenuated parenchymal rejection and reduced CAV as long as 60 days. Congruous data are observed by treating donor hearts with a phosphorothioate antisense oligodeoxyribonucleotide directed against Egr-1 before organ harvest, which blocks subsequent expression of Egr-1 mRNA and protein and suppresses the late development of CAV. These data indicate that Egr-1 induction represents a central effector mechanism in the development of chronic rejection characterized by CAV. Blocking the expression of this proximal transcription factor solely at the time of organ harvest elicits beneficial delayed consequences for the cardiac allograft.
{"title":"Transcriptional Control of Cardiac Allograft Vasculopathy by Early Growth Response Gene-1 (Egr-1)","authors":"M. Okada, Catherine Y. Wang, Daniel W Hwang, Taichi Sakaguchi, K. E. Olson, Yasushi Yoshikawa, K. Minamoto, S. Mazer, S. Yan, D. Pinsky","doi":"10.1161/01.RES.0000027815.75000.33","DOIUrl":"https://doi.org/10.1161/01.RES.0000027815.75000.33","url":null,"abstract":"Expression of the zinc finger transcription factor early growth response gene-1 (Egr-1) is triggered rapidly after mechanical vascular injury or after a precipitous drop in ambient oxygen, whereupon it induces the expression of diverse gene families to elicit a pathological response. Initially characterized as an early response transcriptional activator, the role of Egr-1 in more chronic forms of vascular injury remains to be defined. Studies were designed to examine whether Egr-1 induction may serve as a causal link between early preservation injury and delayed vascular consequences, such as coronary allograft vasculopathy (CAV). The preservation and transplantation of heterotopic murine cardiac allografts strongly induce Egr-1 expression, leading to increased expression of its downstream target genes, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and platelet-derived growth factor A chain. Expression of these Egr-1–inducible gene targets is virtually obliterated in homozygous Egr-1–null donor allografts, which also exhibit attenuated parenchymal rejection and reduced CAV as long as 60 days. Congruous data are observed by treating donor hearts with a phosphorothioate antisense oligodeoxyribonucleotide directed against Egr-1 before organ harvest, which blocks subsequent expression of Egr-1 mRNA and protein and suppresses the late development of CAV. These data indicate that Egr-1 induction represents a central effector mechanism in the development of chronic rejection characterized by CAV. Blocking the expression of this proximal transcription factor solely at the time of organ harvest elicits beneficial delayed consequences for the cardiac allograft.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90790956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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