Pub Date : 2002-06-14DOI: 10.1161/01.RES.0000020017.84398.61
G. D. De Meyer, D. D. De Cleen, S. Cooper, M. Knaapen, Dominique M. Jans, W. Martinet, A. Herman, H. Bult, M. Kockx
In human occluded saphenous vein grafts, we previously demonstrated cytotoxic foam cells, presumably derived from macrophages engulfing platelets. In the present study, we investigated whether platelet phagocytosis occurs in human atherosclerotic plaques, whether this activates macrophages, and whether the platelet constituent, amyloid precursor protein (APP), was involved. Immunohistochemistry documented the presence of APP, &bgr;-amyloid peptide (A&bgr;, cleaved from APP), and platelets (CD9), along with inducible NO synthase (iNOS) and cyclooxygenase-2, two markers of macrophage activation, around microvessels in advanced human carotid artery plaques (n=18). A&bgr; colocalized with iNOS-expressing macrophages that were often surrounded by platelets. In vitro, murine J774 and human THP-1 macrophages were incubated with or without washed human platelets. Coincubation of macrophages and platelets led to platelet phagocytosis (electron and confocal microscopy) and formation of lipid-, APP-, and A&bgr;-containing foam cells. These expressed iNOS mRNA (reverse transcription–polymerase chain reaction) and protein and produced nitrite and tumor necrosis factor-&agr; (ELISA). Macrophage pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, a protease inhibitor, reduced APP processing and inhibited NO biosynthesis induced by platelet phagocytosis but not by lipopolysaccharides. Human atherosclerotic plaques and J774 and THP-1 macrophages contained mRNA of the APP-cleaving enzyme &bgr;-secretase. This is the first demonstration of A&bgr;, a peptide extensively studied in Alzheimer’s disease, in human atherosclerotic plaques. It was present in activated iNOS-expressing perivascular macrophages that had phagocytized platelets. In vitro studies indicate that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to A&bgr;, resulting in iNOS induction. This represents a novel mechanism for macrophage activation in atherosclerosis.
{"title":"Platelet Phagocytosis and Processing of &bgr;-Amyloid Precursor Protein as a Mechanism of Macrophage Activation in Atherosclerosis","authors":"G. D. De Meyer, D. D. De Cleen, S. Cooper, M. Knaapen, Dominique M. Jans, W. Martinet, A. Herman, H. Bult, M. Kockx","doi":"10.1161/01.RES.0000020017.84398.61","DOIUrl":"https://doi.org/10.1161/01.RES.0000020017.84398.61","url":null,"abstract":"In human occluded saphenous vein grafts, we previously demonstrated cytotoxic foam cells, presumably derived from macrophages engulfing platelets. In the present study, we investigated whether platelet phagocytosis occurs in human atherosclerotic plaques, whether this activates macrophages, and whether the platelet constituent, amyloid precursor protein (APP), was involved. Immunohistochemistry documented the presence of APP, &bgr;-amyloid peptide (A&bgr;, cleaved from APP), and platelets (CD9), along with inducible NO synthase (iNOS) and cyclooxygenase-2, two markers of macrophage activation, around microvessels in advanced human carotid artery plaques (n=18). A&bgr; colocalized with iNOS-expressing macrophages that were often surrounded by platelets. In vitro, murine J774 and human THP-1 macrophages were incubated with or without washed human platelets. Coincubation of macrophages and platelets led to platelet phagocytosis (electron and confocal microscopy) and formation of lipid-, APP-, and A&bgr;-containing foam cells. These expressed iNOS mRNA (reverse transcription–polymerase chain reaction) and protein and produced nitrite and tumor necrosis factor-&agr; (ELISA). Macrophage pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, a protease inhibitor, reduced APP processing and inhibited NO biosynthesis induced by platelet phagocytosis but not by lipopolysaccharides. Human atherosclerotic plaques and J774 and THP-1 macrophages contained mRNA of the APP-cleaving enzyme &bgr;-secretase. This is the first demonstration of A&bgr;, a peptide extensively studied in Alzheimer’s disease, in human atherosclerotic plaques. It was present in activated iNOS-expressing perivascular macrophages that had phagocytized platelets. In vitro studies indicate that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to A&bgr;, resulting in iNOS induction. This represents a novel mechanism for macrophage activation in atherosclerosis.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77756579","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 : 2002-06-14DOI: 10.1161/01.RES.0000021432.70309.28
M. Frid, Vishakha A Kale, K. Stenmark
Though in the past believed to be a rare phenomenon, endothelial-mesenchymal transdifferentiation has been described with increasing frequency in recent years. It is believed to be important in embryonic vascular development, yet less is known regarding its role in the adult vasculature. Using FACS and immunomagnetic (Dynabeads) purification techniques (based on uptake of DiI-acetylated low-density lipoproteins and/or PECAM-1 expression) and double-label indirect immunostaining (for endothelial and smooth muscle [SM] markers), we demonstrate that mature bovine vascular endothelium contains cells of an endothelial phenotype (defined by VE-cadherin, von Willebrand factor, PECAM-1, and elevated uptake of acetylated low-density lipoproteins) that can undergo endothelial-mesenchymal transdifferentiation and further differentiate into SM cells (as defined by expression of &agr;-SM-actin, SM22&agr;, calponin, and SM-myosin). “Transitional” cells, coexpressing both endothelial markers and &agr;-SM-actin, were consistently observed. The percentage of cells capable of endothelial-mesenchymal transdifferentiation within primary endothelial cultures was estimated as 0.01% to 0.03%. Acquisition of a SM phenotype occurred even in the absence of proliferation, in &ggr;-irradiated (30 Gy) and/or mitomycin C–treated primary cell cultures. Initiation of transdifferentiation correlated with disruption of cell-cell contacts (marked by loss of VE-cadherin expression) within endothelial monolayers, as well as with the action of transforming growth factor-&bgr;1. In conclusion, our in vitro data show that mature bovine systemic and pulmonary endothelium contains cells that can acquire a SM phenotype via a transdifferentiation process that is transforming growth factor-&bgr;1– and cell-cell contact–dependent, but proliferation-independent.
虽然过去认为内皮-间充质转分化是一种罕见的现象，但近年来对内皮-间充质转分化的描述越来越频繁。它被认为在胚胎血管发育中很重要，但它在成人血管系统中的作用却鲜为人知。利用FACS和免疫磁(Dynabeads)纯化技术(基于摄取双乙酰化低密度脂蛋白和/或PECAM-1表达)和双标记间接免疫染色(用于内皮和平滑肌[SM]标记)，我们证明成熟的牛血管内皮含有内皮型细胞(由VE-cadherin, von Willebrand因子，PECAM-1，和乙酰化低密度脂蛋白的摄取升高)，可以经历内皮-间充质转分化并进一步分化为SM细胞(通过表达&agr;-SM-actin, SM22&agr; calponin和SM-myosin来定义)。“移行”细胞，共同表达内皮标志物和&agr;-SM-actin，一致被观察到。在原代内皮培养物中，能够内皮-间充质转分化的细胞百分比估计为0.01%至0.03%。在辐照(30 Gy)和/或丝裂霉素c处理的原代细胞培养中，即使在没有增殖的情况下，也能获得SM表型。转分化的启动与内皮单层内细胞-细胞接触的破坏(以VE-cadherin表达的缺失为标志)以及转化生长因子的作用相关1。总之，我们的体外数据表明，成熟的牛全身和肺内皮细胞可以通过转化生长因子- 1和细胞-细胞接触依赖的转分化过程获得SM表型，但不依赖于增殖。
{"title":"Mature Vascular Endothelium Can Give Rise to Smooth Muscle Cells via Endothelial-Mesenchymal Transdifferentiation: In Vitro Analysis","authors":"M. Frid, Vishakha A Kale, K. Stenmark","doi":"10.1161/01.RES.0000021432.70309.28","DOIUrl":"https://doi.org/10.1161/01.RES.0000021432.70309.28","url":null,"abstract":"Though in the past believed to be a rare phenomenon, endothelial-mesenchymal transdifferentiation has been described with increasing frequency in recent years. It is believed to be important in embryonic vascular development, yet less is known regarding its role in the adult vasculature. Using FACS and immunomagnetic (Dynabeads) purification techniques (based on uptake of DiI-acetylated low-density lipoproteins and/or PECAM-1 expression) and double-label indirect immunostaining (for endothelial and smooth muscle [SM] markers), we demonstrate that mature bovine vascular endothelium contains cells of an endothelial phenotype (defined by VE-cadherin, von Willebrand factor, PECAM-1, and elevated uptake of acetylated low-density lipoproteins) that can undergo endothelial-mesenchymal transdifferentiation and further differentiate into SM cells (as defined by expression of &agr;-SM-actin, SM22&agr;, calponin, and SM-myosin). “Transitional” cells, coexpressing both endothelial markers and &agr;-SM-actin, were consistently observed. The percentage of cells capable of endothelial-mesenchymal transdifferentiation within primary endothelial cultures was estimated as 0.01% to 0.03%. Acquisition of a SM phenotype occurred even in the absence of proliferation, in &ggr;-irradiated (30 Gy) and/or mitomycin C–treated primary cell cultures. Initiation of transdifferentiation correlated with disruption of cell-cell contacts (marked by loss of VE-cadherin expression) within endothelial monolayers, as well as with the action of transforming growth factor-&bgr;1. In conclusion, our in vitro data show that mature bovine systemic and pulmonary endothelium contains cells that can acquire a SM phenotype via a transdifferentiation process that is transforming growth factor-&bgr;1– and cell-cell contact–dependent, but proliferation-independent.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90273994","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 : 2002-06-14DOI: 10.1161/01.RES.0000020404.01971.2F
R. Touyz, Xin Chen, F. Tabet, Guoying Yao, G. He, M. Quinn, P. Pagano, E. Schiffrin
A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)–regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase–polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II–mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.
{"title":"Expression of a Functionally Active gp91phox-Containing Neutrophil-Type NAD(P)H Oxidase in Smooth Muscle Cells From Human Resistance Arteries: Regulation by Angiotensin II","authors":"R. Touyz, Xin Chen, F. Tabet, Guoying Yao, G. He, M. Quinn, P. Pagano, E. Schiffrin","doi":"10.1161/01.RES.0000020404.01971.2F","DOIUrl":"https://doi.org/10.1161/01.RES.0000020404.01971.2F","url":null,"abstract":"A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)–regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase–polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II–mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87734246","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 : 2002-05-31DOI: 10.1161/01.RES.0000019757.57344.D5
T. Adachi, R. Matsui, Shanqin Xu, M. Kirber, H. Lazar, V. Sharov, C. Schöneich, R. Cohen
Antioxidants improve endothelial function in hypercholesterolemia (HC); however, whether this includes improvement of the vascular smooth muscle response to NO is unknown. NO relaxes arteries, in part, by stimulating Ca2+ uptake via sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in aortic smooth muscle, and HC impairs SERCA function and the response to NO. HC induces oxidative stress, which could impair SERCA function. To study the effect of antioxidants, which are known to improve endothelium-dependent relaxation in HC, smooth muscle SERCA activity and NO-induced relaxation were studied in rabbits fed normal chow or a 0.5% cholesterol diet for 13 weeks. The antioxidant t-butylhydroxytoluene (BHT, 1%) was mixed with the HC diet in the last 3 weeks. HC impaired acetylcholine- and NO-induced relaxation, and these were restored by BHT. After inhibiting SERCA with thapsigargin, no difference existed in NO-induced relaxation among the three groups. Reduced aortic SERCA activity in HC was restored by BHT without changing SERCA protein expression. 3-Nitrotyrosine was notably increased in the media of the HC aorta, where it colocalized with SERCA. Tyrosine-nitrated SERCA protein was immunoprecipitated in the aortas of HC rabbits, where it was decreased by BHT, and it was also detected in the aortas of atherosclerotic humans. Thus, the antioxidant reverses impaired smooth muscle SERCA function in HC, and this is correlated with the improved relaxation to NO. These beneficial effects may depend on reducing the direct effects on SERCA of reactive oxygen species that are augmented in HC.
{"title":"Antioxidant Improves Smooth Muscle Sarco/Endoplasmic Reticulum Ca2+-ATPase Function and Lowers Tyrosine Nitration in Hypercholesterolemia and Improves Nitric Oxide–Induced Relaxation","authors":"T. Adachi, R. Matsui, Shanqin Xu, M. Kirber, H. Lazar, V. Sharov, C. Schöneich, R. Cohen","doi":"10.1161/01.RES.0000019757.57344.D5","DOIUrl":"https://doi.org/10.1161/01.RES.0000019757.57344.D5","url":null,"abstract":"Antioxidants improve endothelial function in hypercholesterolemia (HC); however, whether this includes improvement of the vascular smooth muscle response to NO is unknown. NO relaxes arteries, in part, by stimulating Ca2+ uptake via sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in aortic smooth muscle, and HC impairs SERCA function and the response to NO. HC induces oxidative stress, which could impair SERCA function. To study the effect of antioxidants, which are known to improve endothelium-dependent relaxation in HC, smooth muscle SERCA activity and NO-induced relaxation were studied in rabbits fed normal chow or a 0.5% cholesterol diet for 13 weeks. The antioxidant t-butylhydroxytoluene (BHT, 1%) was mixed with the HC diet in the last 3 weeks. HC impaired acetylcholine- and NO-induced relaxation, and these were restored by BHT. After inhibiting SERCA with thapsigargin, no difference existed in NO-induced relaxation among the three groups. Reduced aortic SERCA activity in HC was restored by BHT without changing SERCA protein expression. 3-Nitrotyrosine was notably increased in the media of the HC aorta, where it colocalized with SERCA. Tyrosine-nitrated SERCA protein was immunoprecipitated in the aortas of HC rabbits, where it was decreased by BHT, and it was also detected in the aortas of atherosclerotic humans. Thus, the antioxidant reverses impaired smooth muscle SERCA function in HC, and this is correlated with the improved relaxation to NO. These beneficial effects may depend on reducing the direct effects on SERCA of reactive oxygen species that are augmented in HC.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84047260","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 : 2002-05-31DOI: 10.1161/01.RES.0000019892.41157.24
J. Silvestre, R. Tamarat, T. Senbonmatsu, Toshihiro Icchiki, T. Ebrahimian, M. Iglarz, Sandrine Besnard, M. Duriez, T. Inagami, B. Lévy
This study examined the potential role of angiotensin type 2 (AT2) receptor on angiogenesis in a model of surgically induced hindlimb ischemia. Ischemia was produced by femoral artery ligature in both wild-type and AT2 gene–deleted mice (Agtr2−/Y). After 28 days, angiogenesis was quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. Protein levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), Bax, and Bcl-2 were determined by Western blot analysis in hindlimbs. The AT2 mRNA level (assessed by semiquantitative RT-PCR) was increased in the ischemic hindlimb of wild-type mice. Angiographic vessel density and laser Doppler perfusion data showed significant improvement in ischemic/nonischemic leg ratio, 1.9- and 1.7-fold, respectively, in Agtr2−/Y mice compared with controls. In ischemic leg of Agtr2−/Y mice, revascularization was associated with an increase in the antiapoptotic protein content, Bcl-2 (211% of basal), and a decrease (60% of basal) in the number of cell death, determined by TUNEL method. Angiotensin II treatment (0.3 mg/kg per day) raised angiogenic score, blood perfusion, and both VEGF and eNOS protein content in ischemic leg of wild-type control but did not modulate the enhanced angiogenic response observed in untreated Agtr2−/Y mice. Finally, immunohistochemistry analysis revealed that VEGF was mainly localized to myocyte, whereas eNOS-positive staining was mainly observed in the capillary of ischemic leg of both wild-type and AT2-deficient mice. This study demonstrates for the first time that the AT2 receptor subtype may negatively modulate ischemia-induced angiogenesis through an activation of the apoptotic process.
{"title":"Antiangiogenic Effect of Angiotensin II Type 2 Receptor in Ischemia-Induced Angiogenesis in Mice Hindlimb","authors":"J. Silvestre, R. Tamarat, T. Senbonmatsu, Toshihiro Icchiki, T. Ebrahimian, M. Iglarz, Sandrine Besnard, M. Duriez, T. Inagami, B. Lévy","doi":"10.1161/01.RES.0000019892.41157.24","DOIUrl":"https://doi.org/10.1161/01.RES.0000019892.41157.24","url":null,"abstract":"This study examined the potential role of angiotensin type 2 (AT2) receptor on angiogenesis in a model of surgically induced hindlimb ischemia. Ischemia was produced by femoral artery ligature in both wild-type and AT2 gene–deleted mice (Agtr2−/Y). After 28 days, angiogenesis was quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. Protein levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), Bax, and Bcl-2 were determined by Western blot analysis in hindlimbs. The AT2 mRNA level (assessed by semiquantitative RT-PCR) was increased in the ischemic hindlimb of wild-type mice. Angiographic vessel density and laser Doppler perfusion data showed significant improvement in ischemic/nonischemic leg ratio, 1.9- and 1.7-fold, respectively, in Agtr2−/Y mice compared with controls. In ischemic leg of Agtr2−/Y mice, revascularization was associated with an increase in the antiapoptotic protein content, Bcl-2 (211% of basal), and a decrease (60% of basal) in the number of cell death, determined by TUNEL method. Angiotensin II treatment (0.3 mg/kg per day) raised angiogenic score, blood perfusion, and both VEGF and eNOS protein content in ischemic leg of wild-type control but did not modulate the enhanced angiogenic response observed in untreated Agtr2−/Y mice. Finally, immunohistochemistry analysis revealed that VEGF was mainly localized to myocyte, whereas eNOS-positive staining was mainly observed in the capillary of ischemic leg of both wild-type and AT2-deficient mice. This study demonstrates for the first time that the AT2 receptor subtype may negatively modulate ischemia-induced angiogenesis through an activation of the apoptotic process.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86934426","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 : 2002-05-31DOI: 10.1161/01.RES.0000019580.64013.31
Agustín D. Martínez, V. Hayrapetyan, A. Moreno, E. Beyer
Two gap junction proteins, connexin43 (Cx43) and connexin45 (Cx45), are coexpressed in many cardiac and other cells. Homomeric channels formed by these proteins differ in unitary conductance, permeability, and regulation. We sought to determine the ability of Cx43 and Cx45 to oligomerize with each other to form heteromeric gap junction channels and to determine the functional and regulatory properties of these heteromeric channels. HeLa cells were transfected with Cx45 or (His)6-tagged Cx43 or sequentially transfected with both connexins. Immunoblots verified production of the transfected connexins, and immunofluorescence demonstrated that they were colocalized in the HeLa-Cx43(His)6/Cx45 cells. Connexons were solubilized from HeLa-Cx43(His)6/Cx45 cells by using Triton X-100 and were applied to a Ni2+-NTA column, which binds the His6 sequence. Cx45 was coeluted from the column with Cx43(His)6, demonstrating that some hemichannels contain both connexins. Single-channel recordings showed that the HeLa-Cx43(His)6/Cx45 cells exhibited single-channel conductances that were not observed in cells expressing either connexin alone. Dye-coupling experiments showed that HeLa-Cx43(His)6 cells readily passed Lucifer yellow and N-(2-aminoethyl)biotinamide hydrochloride (neurobiotin); in contrast, HeLa-Cx45 and HeLa-Cx43(His)6/Cx45 cells showed extensive intercellular passage of neurobiotin but little coupling with Lucifer yellow. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate reduced junctional conductance in cells expressing Cx43, Cx45, or both connexins, but it reduced the extent of neurobiotin transfer only in HeLa-Cx43(His)6 and HeLa-Cx43(His)6/Cx45 cells but not in the HeLa-Cx45 cells. Thus, biochemical and electrophysiological evidence suggests that Cx43 and Cx45 extensively mix to form heteromeric channels; however, individual connexin components dominate aspects of the physiological behavior of these channels.
两个间隙连接蛋白，connexin43 (Cx43)和connexin45 (Cx45)，在许多心脏和其他细胞中共表达。这些蛋白形成的同质通道在单一电导率、渗透性和调节方面存在差异。我们试图确定Cx43和Cx45相互寡聚形成异质间隙连接通道的能力，并确定这些异质通道的功能和调控特性。用Cx45或(His)6标记的Cx43转染HeLa细胞，或依次转染两种连接蛋白。免疫印迹证实了转染的连接蛋白的产生，免疫荧光显示它们在HeLa-Cx43(His)6/Cx45细胞中共定位。使用Triton X-100从HeLa-Cx43(His)6/Cx45细胞中溶解连接子，并将其应用于结合His6序列的Ni2+-NTA柱上。用Cx43(His)6从色谱柱中分离出Cx45，表明一些半通道包含两种连接蛋白。单通道记录显示，HeLa-Cx43(His)6/Cx45细胞表现出单通道电导，这在单独表达任何连接蛋白的细胞中都没有观察到。染料偶联实验表明，HeLa-Cx43(His)6细胞容易通过路西法黄和N-(2-氨基乙基)生物胺盐酸盐(神经生物素);相比之下，HeLa-Cx45和HeLa-Cx43(His)6/Cx45细胞显示神经生物素的广泛细胞间传代，但与路西弗黄的偶联很少。蛋白激酶C激活剂12- o -十四烷酰酚13-醋酸酯处理降低了表达Cx43、Cx45或两种连接蛋白的细胞的连接电导，但它只降低了HeLa-Cx43(His)6和HeLa-Cx43(His)6/Cx45细胞的神经生物素转移程度，而在HeLa-Cx45细胞中没有。因此，生化和电生理证据表明，Cx43和Cx45广泛混合形成异质通道;然而，单个连接蛋白成分主导着这些通道的生理行为。
{"title":"Connexin43 and Connexin45 Form Heteromeric Gap Junction Channels in Which Individual Components Determine Permeability and Regulation","authors":"Agustín D. Martínez, V. Hayrapetyan, A. Moreno, E. Beyer","doi":"10.1161/01.RES.0000019580.64013.31","DOIUrl":"https://doi.org/10.1161/01.RES.0000019580.64013.31","url":null,"abstract":"Two gap junction proteins, connexin43 (Cx43) and connexin45 (Cx45), are coexpressed in many cardiac and other cells. Homomeric channels formed by these proteins differ in unitary conductance, permeability, and regulation. We sought to determine the ability of Cx43 and Cx45 to oligomerize with each other to form heteromeric gap junction channels and to determine the functional and regulatory properties of these heteromeric channels. HeLa cells were transfected with Cx45 or (His)6-tagged Cx43 or sequentially transfected with both connexins. Immunoblots verified production of the transfected connexins, and immunofluorescence demonstrated that they were colocalized in the HeLa-Cx43(His)6/Cx45 cells. Connexons were solubilized from HeLa-Cx43(His)6/Cx45 cells by using Triton X-100 and were applied to a Ni2+-NTA column, which binds the His6 sequence. Cx45 was coeluted from the column with Cx43(His)6, demonstrating that some hemichannels contain both connexins. Single-channel recordings showed that the HeLa-Cx43(His)6/Cx45 cells exhibited single-channel conductances that were not observed in cells expressing either connexin alone. Dye-coupling experiments showed that HeLa-Cx43(His)6 cells readily passed Lucifer yellow and N-(2-aminoethyl)biotinamide hydrochloride (neurobiotin); in contrast, HeLa-Cx45 and HeLa-Cx43(His)6/Cx45 cells showed extensive intercellular passage of neurobiotin but little coupling with Lucifer yellow. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate reduced junctional conductance in cells expressing Cx43, Cx45, or both connexins, but it reduced the extent of neurobiotin transfer only in HeLa-Cx43(His)6 and HeLa-Cx43(His)6/Cx45 cells but not in the HeLa-Cx45 cells. Thus, biochemical and electrophysiological evidence suggests that Cx43 and Cx45 extensively mix to form heteromeric channels; however, individual connexin components dominate aspects of the physiological behavior of these channels.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90838233","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 : 2002-05-31DOI: 10.1161/01.res.0000019756.88731.83
S. Sandow, M. Tare, H. Coleman, C. Hill, H. Parkington
The nature of the vasodilator endothelium-derived hyperpolarizing factor (EDHF) is controversial, putatively involving diffusible factors and/or electrotonic spread of hyperpolarization generated in the endothelium via myoendothelial gap junctions (MEGJs). In this study, we investigated the relationship between the existence of MEGJs, endothelial cell (EC) hyperpolarization, and EDHF-attributed smooth muscle cell (SMC) hyperpolarization in two different arteries: the rat mesenteric artery, where EDHF-mediated vasodilation is prominent, and the femoral artery, where there is no EDHF-dependent relaxation. In the rat mesenteric artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization of both ECs and SMCs, and characteristic pentalaminar MEGJs were found connecting the two cell layers. In contrast, in the femoral artery, ACh evoked hyperpolarization in only ECs but not in SMCs, and no MEGJs were present. Selective hyperpolarization of ECs or SMCs evoked hyperpolarization in the other cell type in the mesenteric artery but not in the femoral artery. Disruption of gap junctional coupling using the peptide Gap 27 markedly reduced the ACh-induced hyperpolarization in SMCs, but not in ECs, of the mesenteric artery. These results show that transfer of EC hyperpolarization or of a small molecule to SMCs through MEGJs is essential and sufficient to explain EDHF.
{"title":"Involvement of Myoendothelial Gap Junctions in the Actions of Endothelium-Derived Hyperpolarizing Factor","authors":"S. Sandow, M. Tare, H. Coleman, C. Hill, H. Parkington","doi":"10.1161/01.res.0000019756.88731.83","DOIUrl":"https://doi.org/10.1161/01.res.0000019756.88731.83","url":null,"abstract":"The nature of the vasodilator endothelium-derived hyperpolarizing factor (EDHF) is controversial, putatively involving diffusible factors and/or electrotonic spread of hyperpolarization generated in the endothelium via myoendothelial gap junctions (MEGJs). In this study, we investigated the relationship between the existence of MEGJs, endothelial cell (EC) hyperpolarization, and EDHF-attributed smooth muscle cell (SMC) hyperpolarization in two different arteries: the rat mesenteric artery, where EDHF-mediated vasodilation is prominent, and the femoral artery, where there is no EDHF-dependent relaxation. In the rat mesenteric artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization of both ECs and SMCs, and characteristic pentalaminar MEGJs were found connecting the two cell layers. In contrast, in the femoral artery, ACh evoked hyperpolarization in only ECs but not in SMCs, and no MEGJs were present. Selective hyperpolarization of ECs or SMCs evoked hyperpolarization in the other cell type in the mesenteric artery but not in the femoral artery. Disruption of gap junctional coupling using the peptide Gap 27 markedly reduced the ACh-induced hyperpolarization in SMCs, but not in ECs, of the mesenteric artery. These results show that transfer of EC hyperpolarization or of a small molecule to SMCs through MEGJs is essential and sufficient to explain EDHF.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80884153","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 : 2002-05-31DOI: 10.1161/01.RES.0000019241.12929.EB
S. Galt, S. Lindemann, L. Allen, Donald Medd, Jeanne M Falk, T. McIntyre, S. Prescott, L. Kraiss, G. Zimmerman, A. Weyrich
Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix proteins. These enzymes are implicated in a variety of physiological and pathological events characterized by extracellular matrix remodeling. Recent studies suggest that MMPs may have a signaling capacity, but direct evidence supporting this concept is lacking. In the present study, we demonstrate that outside-in signals delivered by exogenous MMP-1 (interstitial collagenase) markedly increase the number of tyrosine-phosphorylated proteins in platelets. Active MMP-1 also targets &bgr;3 integrins to areas of cell contact and primes platelets for aggregation. Examination of the endogenous enzyme demonstrated that activated platelets process latent MMP-1 into its active form. Neutralization of MMP-1 activity with MMP inhibitors or specific blocking antibodies markedly attenuates agonist-induced phosphorylation of intracellular proteins, movement of &bgr;3 integrins to cell contact points, and intercellular aggregation. The finding that MMP-1 is rapidly activated in platelets and controls functional responses identifies a new role for this metalloproteinase as a signaling molecule that regulates thrombotic events.
{"title":"Outside-In Signals Delivered by Matrix Metalloproteinase-1 Regulate Platelet Function","authors":"S. Galt, S. Lindemann, L. Allen, Donald Medd, Jeanne M Falk, T. McIntyre, S. Prescott, L. Kraiss, G. Zimmerman, A. Weyrich","doi":"10.1161/01.RES.0000019241.12929.EB","DOIUrl":"https://doi.org/10.1161/01.RES.0000019241.12929.EB","url":null,"abstract":"Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix proteins. These enzymes are implicated in a variety of physiological and pathological events characterized by extracellular matrix remodeling. Recent studies suggest that MMPs may have a signaling capacity, but direct evidence supporting this concept is lacking. In the present study, we demonstrate that outside-in signals delivered by exogenous MMP-1 (interstitial collagenase) markedly increase the number of tyrosine-phosphorylated proteins in platelets. Active MMP-1 also targets &bgr;3 integrins to areas of cell contact and primes platelets for aggregation. Examination of the endogenous enzyme demonstrated that activated platelets process latent MMP-1 into its active form. Neutralization of MMP-1 activity with MMP inhibitors or specific blocking antibodies markedly attenuates agonist-induced phosphorylation of intracellular proteins, movement of &bgr;3 integrins to cell contact points, and intercellular aggregation. The finding that MMP-1 is rapidly activated in platelets and controls functional responses identifies a new role for this metalloproteinase as a signaling molecule that regulates thrombotic events.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83832154","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 : 2002-05-31DOI: 10.1161/01.RES.0000020861.20064.7E
J. Edelberg, Lilong Tang, K. Hattori, D. Lyden, S. Rafii
Delivery of young bone marrow–derived stem cells offers a novel approach for restoring the impaired senescent cardiac angiogenic function that may underlie the increased morbidity and mortality associated with ischemic heart disease in older individuals. Recently, we reported that alterations in endothelial cells of the aging heart lead to a dysregulation in the cardiac myocyte platelet-derived growth factor (PDGF)-B–induced paracrine pathway, which contributes to impaired cardiac angiogenic function. Based on these results, we hypothesized that cellular restoration of the PDGF pathway by bone marrow–derived endothelial precursor cells (EPCs) could reverse the aging-associated decline in angiogenic activity. In vitro studies revealed that young murine (3-month-old) bone marrow–derived EPCs recapitulated the cardiac myocyte–induced expression of PDGF-B, whereas EPCs from the bone marrow of aging mice (18-month-old) did not express PDGF-B when cultured in the presence of cardiac myocytes. Transplantation of young, but not old, genetically marked syngeneic bone marrow cells into intact, unirradiated aging mice that populated the endogenous senescent murine bone marrow incorporated into the neovasculature of subsequently transplanted syngeneic neonatal myocardium. Moreover, the young bone marrow–derived EPCs restored the senescent host angiogenic PDGF-B induction pathway and cardiac angiogenesis, with graft survival and myocardial activity in the aging murine host (cardiac allograft viability: 3-month-old controls, 8/8; 18-month-old controls, 1/8; 18-month-old donors receiving bone marrow from 3-month-old mice, 15/16; or 18-month-old mice, 0/6;P <0.05). These results may offer a foundation for the development of novel therapies for the prevention and treatment of cardiovascular disease associated with aging. The full text of this article is available at http://www.circresaha.org.
{"title":"Young Adult Bone Marrow–Derived Endothelial Precursor Cells Restore Aging-Impaired Cardiac Angiogenic Function","authors":"J. Edelberg, Lilong Tang, K. Hattori, D. Lyden, S. Rafii","doi":"10.1161/01.RES.0000020861.20064.7E","DOIUrl":"https://doi.org/10.1161/01.RES.0000020861.20064.7E","url":null,"abstract":"Delivery of young bone marrow–derived stem cells offers a novel approach for restoring the impaired senescent cardiac angiogenic function that may underlie the increased morbidity and mortality associated with ischemic heart disease in older individuals. Recently, we reported that alterations in endothelial cells of the aging heart lead to a dysregulation in the cardiac myocyte platelet-derived growth factor (PDGF)-B–induced paracrine pathway, which contributes to impaired cardiac angiogenic function. Based on these results, we hypothesized that cellular restoration of the PDGF pathway by bone marrow–derived endothelial precursor cells (EPCs) could reverse the aging-associated decline in angiogenic activity. In vitro studies revealed that young murine (3-month-old) bone marrow–derived EPCs recapitulated the cardiac myocyte–induced expression of PDGF-B, whereas EPCs from the bone marrow of aging mice (18-month-old) did not express PDGF-B when cultured in the presence of cardiac myocytes. Transplantation of young, but not old, genetically marked syngeneic bone marrow cells into intact, unirradiated aging mice that populated the endogenous senescent murine bone marrow incorporated into the neovasculature of subsequently transplanted syngeneic neonatal myocardium. Moreover, the young bone marrow–derived EPCs restored the senescent host angiogenic PDGF-B induction pathway and cardiac angiogenesis, with graft survival and myocardial activity in the aging murine host (cardiac allograft viability: 3-month-old controls, 8/8; 18-month-old controls, 1/8; 18-month-old donors receiving bone marrow from 3-month-old mice, 15/16; or 18-month-old mice, 0/6;P <0.05). These results may offer a foundation for the development of novel therapies for the prevention and treatment of cardiovascular disease associated with aging. The full text of this article is available at http://www.circresaha.org.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75959239","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 : 2002-05-31DOI: 10.1161/01.RES.0000021044.53156.F5
M. Engelse, J. Lardenoye, Jolanda M. Neele, J. Grimbergen, M. D. de Vries, M. Lamfers, H. Pannekoek, P. Quax, C. D. de Vries
Activin A alters the characteristics of human arterial smooth muscle cells (SMCs) toward a contractile, quiescent phenotype. We hypothesize that activin A may prevent SMC-rich neointimal hyperplasia. Here, we study the effect of adenovirus-mediated expression of activin A on neointima formation in vitro and in vivo. Human saphenous vein organ cultures, in which a neointima is formed spontaneously, were infected either with activin A- or lacZ-adenovirus. Activin A-overexpression reduces neointima formation by 78%, whereas no significant reduction was observed after control infection. In addition, the effect of activin A on neointima formation was assessed in vivo in mice with cuffed femoral arteries. In activin A adenovirus-infected mice (IV injection), neointimal hyperplasia is reduced by 77% compared with the SMC-rich neointima in mock-infected or in noninfected mice. Cultured human saphenous vein SMCs and murine aorta SMCs were incubated with activin A and an increased expression of SM22&agr; and SM &agr;-actin mRNA, and SM &agr;-actin protein was demonstrated. Laser-capture microdissection on sections of cuffed murine arteries and subsequent real-time RT-PCR established in vivo induction of SM &agr;-actin mRNA in the media of activin A–treated mice. In summary, activin A inhibits neointima formation in vitro and in vivo by preventing SMC dedifferentiation.
{"title":"Adenoviral Activin A Expression Prevents Intimal Hyperplasia in Human and Murine Blood Vessels by Maintaining the Contractile Smooth Muscle Cell Phenotype","authors":"M. Engelse, J. Lardenoye, Jolanda M. Neele, J. Grimbergen, M. D. de Vries, M. Lamfers, H. Pannekoek, P. Quax, C. D. de Vries","doi":"10.1161/01.RES.0000021044.53156.F5","DOIUrl":"https://doi.org/10.1161/01.RES.0000021044.53156.F5","url":null,"abstract":"Activin A alters the characteristics of human arterial smooth muscle cells (SMCs) toward a contractile, quiescent phenotype. We hypothesize that activin A may prevent SMC-rich neointimal hyperplasia. Here, we study the effect of adenovirus-mediated expression of activin A on neointima formation in vitro and in vivo. Human saphenous vein organ cultures, in which a neointima is formed spontaneously, were infected either with activin A- or lacZ-adenovirus. Activin A-overexpression reduces neointima formation by 78%, whereas no significant reduction was observed after control infection. In addition, the effect of activin A on neointima formation was assessed in vivo in mice with cuffed femoral arteries. In activin A adenovirus-infected mice (IV injection), neointimal hyperplasia is reduced by 77% compared with the SMC-rich neointima in mock-infected or in noninfected mice. Cultured human saphenous vein SMCs and murine aorta SMCs were incubated with activin A and an increased expression of SM22&agr; and SM &agr;-actin mRNA, and SM &agr;-actin protein was demonstrated. Laser-capture microdissection on sections of cuffed murine arteries and subsequent real-time RT-PCR established in vivo induction of SM &agr;-actin mRNA in the media of activin A–treated mice. In summary, activin A inhibits neointima formation in vitro and in vivo by preventing SMC dedifferentiation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83297205","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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