Pub Date : 2002-06-01DOI: 10.1161/01.ATV.0000020006.89055.11
I. Spyridopoulos, Corinne Luedemann, Donghui Chen, M. Kearney, Dongfen Chen, T. Murohara, N. Principe, J. Isner, Douglas Losordo
Vascular endothelial growth factor (VEGF) promotes angiogenesis by a variety of mechanisms including stimulation of endothelial cell proliferation and migration and increasing vascular permeability. Although its mitogenic activity is mediated primarily by the &bgr;2-isoforms of protein kinase C (PKC), little is known about the signaling pathways transducing its other physiological properties. Accordingly, we used a novel inhibitor molecule to examine the role of PKC isoforms &agr; and &bgr; in mediating VEGF-induced angiogenesis and vascular permeability. Because conventional inhibitors of PKC, such as staurosporine or calphostin C, also inhibit a variety of other protein kinases, we used a novel compound to specifically inhibit PKC. A myristoylated peptide, which mimics the pseudosubstrate motif of PKC-&agr; and -&bgr; subtypes, has been shown to be a highly selective and cell-permeable inhibitor of PKC. Blocking led, as expected, to abrogation of VEGF-induced endothelial cell proliferation in vitro. In vivo, VEGF-induced angiogenesis was impaired by myristoylated peptide. Surprisingly, selective inhibition of PKC induced vascular permeability in vivo via a NO-dependent mechanism. Moreover, PKC inhibition led to a 6.4-fold induction of NO synthase (NOS) activity in endothelial cells. Our findings demonstrate that activation of PKC is a major signaling pathway required for VEGF-induced proliferation and angiogenesis, whereas vascular permeability was enhanced by blocking PKC. Inhibition of calcium-dependent PKC by itself led to induction of NOS. Although NOS is a downstream target for VEGF-induced angiogenesis, its induction by PKC inhibition was not sufficient to promote neovascularization. These results reveal that angiogenesis and vascular permeability induced by VEGF are mediated by mechanisms which ultimately diverge.
{"title":"Divergence of Angiogenic and Vascular Permeability Signaling by VEGF: Inhibition of Protein Kinase C Suppresses VEGF-Induced Angiogenesis, but Promotes VEGF-Induced, NO-Dependent Vascular Permeability","authors":"I. Spyridopoulos, Corinne Luedemann, Donghui Chen, M. Kearney, Dongfen Chen, T. Murohara, N. Principe, J. Isner, Douglas Losordo","doi":"10.1161/01.ATV.0000020006.89055.11","DOIUrl":"https://doi.org/10.1161/01.ATV.0000020006.89055.11","url":null,"abstract":"Vascular endothelial growth factor (VEGF) promotes angiogenesis by a variety of mechanisms including stimulation of endothelial cell proliferation and migration and increasing vascular permeability. Although its mitogenic activity is mediated primarily by the &bgr;2-isoforms of protein kinase C (PKC), little is known about the signaling pathways transducing its other physiological properties. Accordingly, we used a novel inhibitor molecule to examine the role of PKC isoforms &agr; and &bgr; in mediating VEGF-induced angiogenesis and vascular permeability. Because conventional inhibitors of PKC, such as staurosporine or calphostin C, also inhibit a variety of other protein kinases, we used a novel compound to specifically inhibit PKC. A myristoylated peptide, which mimics the pseudosubstrate motif of PKC-&agr; and -&bgr; subtypes, has been shown to be a highly selective and cell-permeable inhibitor of PKC. Blocking led, as expected, to abrogation of VEGF-induced endothelial cell proliferation in vitro. In vivo, VEGF-induced angiogenesis was impaired by myristoylated peptide. Surprisingly, selective inhibition of PKC induced vascular permeability in vivo via a NO-dependent mechanism. Moreover, PKC inhibition led to a 6.4-fold induction of NO synthase (NOS) activity in endothelial cells. Our findings demonstrate that activation of PKC is a major signaling pathway required for VEGF-induced proliferation and angiogenesis, whereas vascular permeability was enhanced by blocking PKC. Inhibition of calcium-dependent PKC by itself led to induction of NOS. Although NOS is a downstream target for VEGF-induced angiogenesis, its induction by PKC inhibition was not sufficient to promote neovascularization. These results reveal that angiogenesis and vascular permeability induced by VEGF are mediated by mechanisms which ultimately diverge.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"90 9 1","pages":"901-906"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77980797","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 : 2002-06-01DOI: 10.1161/01.ATV.0000018305.95943.F7
N. Kaneider, P. Egger, S. Dunzendorfer, P. Noris, C. Balduini, D. Gritti, G. Ricevuti, C. Wiedermann
Adenosine triphosphate and diphosphate that activate platelet, leukocyte, and endothelium functions are hydrolyzed by endothelial CD39/ATPDase. Because CD39/ATPDase is downregulated in endothelial cells by inflammation and this may be affected by HMG-CoA reductase inhibitors, we examined the role of cerivastatin and simvastatin in regulation of endothelial CD39/ATPDase expression, metabolism of ATP/ADP, and function in platelets. Thrombin-stimulated endothelial cells in vitro were treated with the statins, and hydrolysis of exogenous ADP and ATP was assessed by high-performance liquid chromatography and malachite green assay. Platelet aggregation studies were performed with endothelial cell supernatants as triggers. CD39/ATPDase surface expression by endothelial cells was determined immunologically by fluorescence-activated cell sorter, mRNA expression by RT-PCR, and thrombin-induced dissociation of Rho-GTPases by Western blotting. Treatment by simvastatin or cerivastatin restored impaired metabolism of exogenous ATP and ADP in thrombin-activated endothelial cells by preventing thrombin-induced downregulation of CD39/ATPDase. In platelet aggregation studies, ATP and ADP supernatants of thrombin-activated endothelial cells were less stimulatory in the presence of statins than in their absence. Data show that statins preserve CD39/ATPDase activity in thrombin-treated endothelial cells involving alterations by statins of Rho-GTPase function and CD39/ATPDase expression. Preservation of adenine nucleotide metabolism may directly contribute to the observed anti-thrombotic and anti-inflammatory actions of statins.
{"title":"Reversal of Thrombin-Induced Deactivation of CD39/ATPDase in Endothelial Cells by HMG-CoA Reductase Inhibition: Effects on Rho-GTPase and Adenosine Nucleotide Metabolism","authors":"N. Kaneider, P. Egger, S. Dunzendorfer, P. Noris, C. Balduini, D. Gritti, G. Ricevuti, C. Wiedermann","doi":"10.1161/01.ATV.0000018305.95943.F7","DOIUrl":"https://doi.org/10.1161/01.ATV.0000018305.95943.F7","url":null,"abstract":"Adenosine triphosphate and diphosphate that activate platelet, leukocyte, and endothelium functions are hydrolyzed by endothelial CD39/ATPDase. Because CD39/ATPDase is downregulated in endothelial cells by inflammation and this may be affected by HMG-CoA reductase inhibitors, we examined the role of cerivastatin and simvastatin in regulation of endothelial CD39/ATPDase expression, metabolism of ATP/ADP, and function in platelets. Thrombin-stimulated endothelial cells in vitro were treated with the statins, and hydrolysis of exogenous ADP and ATP was assessed by high-performance liquid chromatography and malachite green assay. Platelet aggregation studies were performed with endothelial cell supernatants as triggers. CD39/ATPDase surface expression by endothelial cells was determined immunologically by fluorescence-activated cell sorter, mRNA expression by RT-PCR, and thrombin-induced dissociation of Rho-GTPases by Western blotting. Treatment by simvastatin or cerivastatin restored impaired metabolism of exogenous ATP and ADP in thrombin-activated endothelial cells by preventing thrombin-induced downregulation of CD39/ATPDase. In platelet aggregation studies, ATP and ADP supernatants of thrombin-activated endothelial cells were less stimulatory in the presence of statins than in their absence. Data show that statins preserve CD39/ATPDase activity in thrombin-treated endothelial cells involving alterations by statins of Rho-GTPase function and CD39/ATPDase expression. Preservation of adenine nucleotide metabolism may directly contribute to the observed anti-thrombotic and anti-inflammatory actions of statins.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"50 1","pages":"894-900"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73145703","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 : 2002-06-01DOI: 10.1161/01.ATV.0000018306.68268.86
N. Kaneider, P. Egger, S. Dunzendorfer, C. Wiedermann
Platelet activation and aggregation is considered a crucial step in the initiation and aggravation of arterial thrombosis. ADP from activated platelets is recognized as major factor in thrombus formation and is a potent stimulator of oxygen-free radical release from neutrophils. The aim of the present investigation was to determine in vitro the direct effects of statins on ATP and ADP secretion by platelets and its impact on subsequent oxidative burst activity in neutrophils. Human neutrophils and platelets were isolated from peripheral blood. Levels of platelet-derived ATP and ADP were measured by high-performance liquid chromatography, oxygen-free radical release of neutrophils was measured fluorometrically, and chemotaxis experiments were performed. Rho-GTPases were studied by Western blot analysis. Thrombin-activated platelets primed neutrophils for enhanced oxygen-free radical release on triggering with formyl-Met-Leu-Phe, reduced by cerivastatin and simvastatin treatment of platelets. The two statins decreased the amount of adenosine-derivative release in these cells. Rho-GTPases, required for the thrombin signaling in platelets and neutrophils, were decreased after coincubation with statins. Data demonstrate that inhibition of Rho-GTPases by statins inhibit platelet ADP and ATP release and the consecutive augmentation of neutrophil oxygen-free radical release. Statins affect platelet-neutrophil interactions by altering Rho-GTPase–dependent adenosine nucleotide function.
{"title":"Rho-GTPase–Dependent Platelet-Neutrophil Interaction Affected by HMG-CoA Reductase Inhibition With Altered Adenosine Nucleotide Release and Function","authors":"N. Kaneider, P. Egger, S. Dunzendorfer, C. Wiedermann","doi":"10.1161/01.ATV.0000018306.68268.86","DOIUrl":"https://doi.org/10.1161/01.ATV.0000018306.68268.86","url":null,"abstract":"Platelet activation and aggregation is considered a crucial step in the initiation and aggravation of arterial thrombosis. ADP from activated platelets is recognized as major factor in thrombus formation and is a potent stimulator of oxygen-free radical release from neutrophils. The aim of the present investigation was to determine in vitro the direct effects of statins on ATP and ADP secretion by platelets and its impact on subsequent oxidative burst activity in neutrophils. Human neutrophils and platelets were isolated from peripheral blood. Levels of platelet-derived ATP and ADP were measured by high-performance liquid chromatography, oxygen-free radical release of neutrophils was measured fluorometrically, and chemotaxis experiments were performed. Rho-GTPases were studied by Western blot analysis. Thrombin-activated platelets primed neutrophils for enhanced oxygen-free radical release on triggering with formyl-Met-Leu-Phe, reduced by cerivastatin and simvastatin treatment of platelets. The two statins decreased the amount of adenosine-derivative release in these cells. Rho-GTPases, required for the thrombin signaling in platelets and neutrophils, were decreased after coincubation with statins. Data demonstrate that inhibition of Rho-GTPases by statins inhibit platelet ADP and ATP release and the consecutive augmentation of neutrophil oxygen-free radical release. Statins affect platelet-neutrophil interactions by altering Rho-GTPase–dependent adenosine nucleotide function.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"29 16","pages":"1029-1035"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91434120","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 : 2002-06-01DOI: 10.1161/01.ATV.0000019404.65403.71
C. Bernal‐Mizrachi, Sherry Weng, Bing Li, L. Nolte, Chu Feng, T. Coleman, J. Holloszy, C. Semenkovich
Insulin resistance is commonly associated with hypertension, a condition that causes vascular disease in people with obesity and type 2 diabetes. The mechanisms linking hypertension and insulin resistance are poorly understood. To determine whether respiratory uncoupling can prevent insulin resistance-related hypertension, we crossed transgenic mice expressing uncoupling protein 1 (UCP1) in skeletal muscle with lethal yellow (Ay/a) mice, genetically obese animals known to have elevated blood pressure. Despite increased food intake, UCP-Ay/a mice weighed less than their Ay/a littermates. The metabolic rate was higher in UCP-Ay/a mice than in Ay/a mice and did not impair their ability to alter oxygen consumption in response to temperature changes, an adaptation involving sympathetic nervous system activity. Compared with their nontransgenic littermates, UCP-Ay/a mice had lower fasting insulin, glucose, triglyceride, and cholesterol levels and were more insulin sensitive. Blood pressure, serum leptin, and urinary catecholamine levels were also lower in uncoupled mice. Independent of sympathetic nervous system activity, low-dose peripheral leptin infusion increased blood pressure in UCP-Ay/a mice but not in their Ay/a littermates. These data indicate that skeletal muscle respiratory uncoupling reverses insulin resistance and lowers blood pressure in genetic obesity without affecting thermoregulation. The data also suggest that uncoupling could decrease the risk of atherosclerosis in type 2 diabetes.
{"title":"Respiratory Uncoupling Lowers Blood Pressure Through a Leptin-Dependent Mechanism in Genetically Obese Mice","authors":"C. Bernal‐Mizrachi, Sherry Weng, Bing Li, L. Nolte, Chu Feng, T. Coleman, J. Holloszy, C. Semenkovich","doi":"10.1161/01.ATV.0000019404.65403.71","DOIUrl":"https://doi.org/10.1161/01.ATV.0000019404.65403.71","url":null,"abstract":"Insulin resistance is commonly associated with hypertension, a condition that causes vascular disease in people with obesity and type 2 diabetes. The mechanisms linking hypertension and insulin resistance are poorly understood. To determine whether respiratory uncoupling can prevent insulin resistance-related hypertension, we crossed transgenic mice expressing uncoupling protein 1 (UCP1) in skeletal muscle with lethal yellow (Ay/a) mice, genetically obese animals known to have elevated blood pressure. Despite increased food intake, UCP-Ay/a mice weighed less than their Ay/a littermates. The metabolic rate was higher in UCP-Ay/a mice than in Ay/a mice and did not impair their ability to alter oxygen consumption in response to temperature changes, an adaptation involving sympathetic nervous system activity. Compared with their nontransgenic littermates, UCP-Ay/a mice had lower fasting insulin, glucose, triglyceride, and cholesterol levels and were more insulin sensitive. Blood pressure, serum leptin, and urinary catecholamine levels were also lower in uncoupled mice. Independent of sympathetic nervous system activity, low-dose peripheral leptin infusion increased blood pressure in UCP-Ay/a mice but not in their Ay/a littermates. These data indicate that skeletal muscle respiratory uncoupling reverses insulin resistance and lowers blood pressure in genetic obesity without affecting thermoregulation. The data also suggest that uncoupling could decrease the risk of atherosclerosis in type 2 diabetes.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"22 1","pages":"961-968"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82778458","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 : 2002-06-01DOI: 10.1161/01.ATV.0000019051.88366.9C
T. Yamashita, S. Kawashima, M. Ozaki, M. Namiki, N. Inoue, K. Hirata, M. Yokoyama
Monocyte chemoattractant protein-1 (MCP-1), which binds to C-C chemokine receptor 2, has been implicated as the primary source of monocyte chemoattractant function in the early stages of atherosclerosis. Recently, propagermanium, a drug used clinically for the treatment of chronic hepatitis in Japan, has been shown to inhibit C-C chemokine receptor 2 function and suppress monocyte/macrophage infiltration in vitro and in vivo. Given the importance of monocyte infiltration in atherogenesis, the inhibition of it by propagermanium might prevent atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were fed an atherogenic high cholesterol diet with or without 0.005% propagermanium for 8 or 12 weeks. Although the plasma lipid levels were unchanged by the drug treatment, atherosclerotic lesion area in the aortic root was reduced by 50% in the drug-treated apoE-KO mice compared with the nontreated apoE-KO mice after 8 weeks of cholesterol feeding (0.62±0.12 versus 1.27±0.07 mm2, respectively;P <0.01). Moreover, the accumulation of macrophages in the lesions was markedly reduced in the drug-treated group (macrophage positive area, 0.23±0.06 mm2 [drug-treated group] versus 0.67±0.07 mm2 [control group];P <0.01). After 12 weeks of cholesterol feeding, atherosclerotic lesion formation in the aortic root and in the descending thoracic aorta was significantly reduced in the drug-treated group. Inhibition of macrophage infiltration by propagermanium prevented the formation of atherosclerotic lesions in apoE-KO mice. This drug may serve as a therapeutic tool for the treatment of atherosclerosis.
单核细胞趋化蛋白-1 (MCP-1)与C-C趋化因子受体2结合，被认为是动脉粥样硬化早期单核细胞趋化功能的主要来源。最近，日本临床用于治疗慢性肝炎的药物繁殖素在体外和体内被证明具有抑制C-C趋化因子受体2功能和抑制单核细胞/巨噬细胞浸润的作用。考虑到单核细胞浸润在动脉粥样硬化中的重要性，繁殖体对其的抑制可能会预防动脉粥样硬化。载脂蛋白E敲除(apoE-KO)小鼠分别饲喂含或不含0.005%繁殖苗的致动脉粥样硬化高胆固醇饮食8或12周。虽然药物治疗后血脂水平没有变化，但8周胆固醇喂养后，药物治疗的apoE-KO小鼠主动脉根部动脉粥样硬化病变面积比未治疗的apoE-KO小鼠减少了50%(分别为0.62±0.12 mm2比1.27±0.07 mm2, P <0.01)。药物治疗组病变内巨噬细胞的聚集明显减少(巨噬细胞阳性面积，药物治疗组为0.23±0.06 mm2，对照组为0.67±0.07 mm2, P <0.01)。胆固醇喂养12周后，药物治疗组主动脉根部和胸降主动脉动脉粥样硬化病变形成明显减少。通过繁殖体抑制巨噬细胞浸润可阻止apoE-KO小鼠动脉粥样硬化病变的形成。这种药物可以作为治疗动脉粥样硬化的一种治疗工具。
{"title":"Propagermanium Reduces Atherosclerosis in Apolipoprotein E Knockout Mice via Inhibition of Macrophage Infiltration","authors":"T. Yamashita, S. Kawashima, M. Ozaki, M. Namiki, N. Inoue, K. Hirata, M. Yokoyama","doi":"10.1161/01.ATV.0000019051.88366.9C","DOIUrl":"https://doi.org/10.1161/01.ATV.0000019051.88366.9C","url":null,"abstract":"Monocyte chemoattractant protein-1 (MCP-1), which binds to C-C chemokine receptor 2, has been implicated as the primary source of monocyte chemoattractant function in the early stages of atherosclerosis. Recently, propagermanium, a drug used clinically for the treatment of chronic hepatitis in Japan, has been shown to inhibit C-C chemokine receptor 2 function and suppress monocyte/macrophage infiltration in vitro and in vivo. Given the importance of monocyte infiltration in atherogenesis, the inhibition of it by propagermanium might prevent atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were fed an atherogenic high cholesterol diet with or without 0.005% propagermanium for 8 or 12 weeks. Although the plasma lipid levels were unchanged by the drug treatment, atherosclerotic lesion area in the aortic root was reduced by 50% in the drug-treated apoE-KO mice compared with the nontreated apoE-KO mice after 8 weeks of cholesterol feeding (0.62±0.12 versus 1.27±0.07 mm2, respectively;P <0.01). Moreover, the accumulation of macrophages in the lesions was markedly reduced in the drug-treated group (macrophage positive area, 0.23±0.06 mm2 [drug-treated group] versus 0.67±0.07 mm2 [control group];P <0.01). After 12 weeks of cholesterol feeding, atherosclerotic lesion formation in the aortic root and in the descending thoracic aorta was significantly reduced in the drug-treated group. Inhibition of macrophage infiltration by propagermanium prevented the formation of atherosclerotic lesions in apoE-KO mice. This drug may serve as a therapeutic tool for the treatment of atherosclerosis.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"62 1","pages":"969-974"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86251611","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 : 2002-06-01DOI: 10.1161/01.ATV.0000019734.89917.35
Layton H Smith, O. Boutaud, M. Breyer, J. Morrow, J. Oates, D. Vaughan
Reduction of plasma low density lipoprotein (LDL) levels is associated with a reduced risk of myocardial infarction, stroke, and death. Some of this clinical benefit may be derived from an improvement in endothelium-dependent vasodilation. In the present study, we examined the effects of LDL reduction on cyclooxygenase (COX) activity and prostacyclin (PGI2) production. Human umbilical vein endothelial cells exposed to reduced concentrations of LDL demonstrated increased PGI2 production in a dose-dependent manner (from 0.75±0.2 to 2.6±0.2 ng/mL, P <0.0001). This alteration in PGI2 production did not result from LDL-induced changes in PGI2 synthase expression. However, selective inhibition of COX-2, but not COX-1, blocked PGI2 production under low cholesterol conditions. Addition of exogenous cholesterol induces dose-dependent reductions in endothelial COX-2 expression as measured by reverse transcription–polymerase chain reaction and by Western blotting. Pretreatment of cells with actinomycin D, a transcription inhibitor, reduced COX-2–derived PGI2 production by 45.9% (from 0.55±0.09 to 0.25±0.08 ng/mL). Taken together, these observations indicate that endothelial PGI2 production is regulated by cholesterol at the transcriptional level and that cholesterol-sensitive transcriptional pathways that regulate COX-2 expression are present in vascular tissue.
血浆低密度脂蛋白(LDL)水平的降低与心肌梗死、中风和死亡风险的降低有关。一些临床益处可能来自于内皮依赖性血管舒张的改善。在本研究中，我们检测了LDL降低对环氧化酶(COX)活性和前列环素(PGI2)产生的影响。暴露于低浓度LDL的人脐静脉内皮细胞显示PGI2的产生以剂量依赖性的方式增加(从0.75±0.2到2.6±0.2 ng/mL, P <0.0001)。这种PGI2生成的改变不是由ldl诱导的PGI2合成酶表达的改变引起的。然而，选择性抑制COX-2，而不是COX-1，在低胆固醇条件下阻断PGI2的产生。通过逆转录聚合酶链反应和Western blotting检测，外源性胆固醇的添加诱导内皮细胞COX-2表达的剂量依赖性降低。用转录抑制剂放线菌素D预处理细胞，使cox -2来源的PGI2产量降低45.9%(从0.55±0.09 ng/mL降至0.25±0.08 ng/mL)。综上所述，这些观察结果表明内皮细胞PGI2的产生在转录水平上受到胆固醇的调节，并且血管组织中存在调节COX-2表达的胆固醇敏感转录途径。
{"title":"Cyclooxygenase-2–Dependent Prostacyclin Formation Is Regulated by Low Density Lipoprotein Cholesterol In Vitro","authors":"Layton H Smith, O. Boutaud, M. Breyer, J. Morrow, J. Oates, D. Vaughan","doi":"10.1161/01.ATV.0000019734.89917.35","DOIUrl":"https://doi.org/10.1161/01.ATV.0000019734.89917.35","url":null,"abstract":"Reduction of plasma low density lipoprotein (LDL) levels is associated with a reduced risk of myocardial infarction, stroke, and death. Some of this clinical benefit may be derived from an improvement in endothelium-dependent vasodilation. In the present study, we examined the effects of LDL reduction on cyclooxygenase (COX) activity and prostacyclin (PGI2) production. Human umbilical vein endothelial cells exposed to reduced concentrations of LDL demonstrated increased PGI2 production in a dose-dependent manner (from 0.75±0.2 to 2.6±0.2 ng/mL, P <0.0001). This alteration in PGI2 production did not result from LDL-induced changes in PGI2 synthase expression. However, selective inhibition of COX-2, but not COX-1, blocked PGI2 production under low cholesterol conditions. Addition of exogenous cholesterol induces dose-dependent reductions in endothelial COX-2 expression as measured by reverse transcription–polymerase chain reaction and by Western blotting. Pretreatment of cells with actinomycin D, a transcription inhibitor, reduced COX-2–derived PGI2 production by 45.9% (from 0.55±0.09 to 0.25±0.08 ng/mL). Taken together, these observations indicate that endothelial PGI2 production is regulated by cholesterol at the transcriptional level and that cholesterol-sensitive transcriptional pathways that regulate COX-2 expression are present in vascular tissue.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"14 1","pages":"983-988"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83550738","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 : 2002-06-01DOI: 10.1161/01.ATV.0000019360.14554.53
Lin Peng, Nitin Bhatia, Andrew C. Parker, Yanhong Zhu, W. Fay
We examined the roles of vitronectin and plasminogen activator inhibitor-1 (PAI-1) in neointima development. Neointima formation after carotid artery ligation or chemical injury was significantly greater in wild-type mice than in vitronectin-deficient (Vn−/−) mice. Vascular smooth muscle cell (VSMC) proliferation did not differ between groups, suggesting that vitronectin promoted neointima development by enhancing VSMC migration. Neointima formation was significantly attenuated in PAI-1–deficient (PAI-1−/−) mice compared with control mice. Because intravascular fibrin may function as a provisional matrix for invading VSMCs, we examined potential mechanisms by which vitronectin and PAI-1 regulate fibrin stability and fibrin-VSMC interactions. Inhibition of activated protein C by PAI-1 was markedly attenuated in vitronectin-deficient plasma. The capacity of PAI-1 to inhibit clot lysis was significantly attenuated in vitronectin-deficient plasma, and this effect was not explained simply by the PAI-1–stabilizing properties of vitronectin. The adhesion and spreading of VSMCs were significantly greater on wild-type plasma clots and PAI-1–deficient plasma clots than on vitronectin-deficient plasma clots. We conclude that endogenous levels of vitronectin and PAI-1 enhance neointima formation in response to vascular occlusion or injury. Their effects may be mediated to a significant extent by their capacity to promote intravascular fibrin deposition and by the capacity of vitronectin to enhance VSMC-fibrin interactions.
{"title":"Endogenous Vitronectin and Plasminogen Activator Inhibitor-1 Promote Neointima Formation in Murine Carotid Arteries","authors":"Lin Peng, Nitin Bhatia, Andrew C. Parker, Yanhong Zhu, W. Fay","doi":"10.1161/01.ATV.0000019360.14554.53","DOIUrl":"https://doi.org/10.1161/01.ATV.0000019360.14554.53","url":null,"abstract":"We examined the roles of vitronectin and plasminogen activator inhibitor-1 (PAI-1) in neointima development. Neointima formation after carotid artery ligation or chemical injury was significantly greater in wild-type mice than in vitronectin-deficient (Vn−/−) mice. Vascular smooth muscle cell (VSMC) proliferation did not differ between groups, suggesting that vitronectin promoted neointima development by enhancing VSMC migration. Neointima formation was significantly attenuated in PAI-1–deficient (PAI-1−/−) mice compared with control mice. Because intravascular fibrin may function as a provisional matrix for invading VSMCs, we examined potential mechanisms by which vitronectin and PAI-1 regulate fibrin stability and fibrin-VSMC interactions. Inhibition of activated protein C by PAI-1 was markedly attenuated in vitronectin-deficient plasma. The capacity of PAI-1 to inhibit clot lysis was significantly attenuated in vitronectin-deficient plasma, and this effect was not explained simply by the PAI-1–stabilizing properties of vitronectin. The adhesion and spreading of VSMCs were significantly greater on wild-type plasma clots and PAI-1–deficient plasma clots than on vitronectin-deficient plasma clots. We conclude that endogenous levels of vitronectin and PAI-1 enhance neointima formation in response to vascular occlusion or injury. Their effects may be mediated to a significant extent by their capacity to promote intravascular fibrin deposition and by the capacity of vitronectin to enhance VSMC-fibrin interactions.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"3 1","pages":"934-939"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75951721","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 : 2002-06-01DOI: 10.1161/01.ATV.0000017063.36768.87
S. Haberichter, M. A. Jozwiak, J. Rosenberg, P. Christopherson, R. Montgomery
The von Willebrand factor (VWF) propeptide (VWFpp) is critical for the targeting of VWF multimers to storage granules. VWFpp alone efficiently navigates the storage pathway in AtT-20 and endothelial cells and chaperones mature VWF multimers to storage granules when the two proteins are expressed in cis or in trans. To further define the role of VWFpp in granular sorting, we examined its ability to sort an unrelated protein, C3&agr; into the regulated secretory pathway. Chimeric constructs of VWFpp and the &agr;-chain of C3 were developed. The C3&agr; protein expressed alone did not sort to granules in AtT-20 cells. The trans expression of C3&agr; and VWFpp resulted in granular storage of VWFpp but no corresponding storage of C3&agr;. When C3&agr; is expressed as a single chain molecule with VWFpp that was rendered uncleavable by furin, C3&agr; is re-routed to storage and is colocalized with VWFpp. The uncleavable protein was expressed in bovine aortic endothelial cells where it sorted to Weibel-Palade bodies, colocalized with bovine VWF, and was released when agonist stimulated. We now demonstrate that VWFpp re-routes a constitutively secreted protein to the regulated storage pathway. Furthermore, our studies suggest that the VWFpp storage signal is contained within amino acids 201 to 741.
{"title":"The Von Willebrand Factor Propeptide (VWFpp) Traffics an Unrelated Protein to Storage","authors":"S. Haberichter, M. A. Jozwiak, J. Rosenberg, P. Christopherson, R. Montgomery","doi":"10.1161/01.ATV.0000017063.36768.87","DOIUrl":"https://doi.org/10.1161/01.ATV.0000017063.36768.87","url":null,"abstract":"The von Willebrand factor (VWF) propeptide (VWFpp) is critical for the targeting of VWF multimers to storage granules. VWFpp alone efficiently navigates the storage pathway in AtT-20 and endothelial cells and chaperones mature VWF multimers to storage granules when the two proteins are expressed in cis or in trans. To further define the role of VWFpp in granular sorting, we examined its ability to sort an unrelated protein, C3&agr; into the regulated secretory pathway. Chimeric constructs of VWFpp and the &agr;-chain of C3 were developed. The C3&agr; protein expressed alone did not sort to granules in AtT-20 cells. The trans expression of C3&agr; and VWFpp resulted in granular storage of VWFpp but no corresponding storage of C3&agr;. When C3&agr; is expressed as a single chain molecule with VWFpp that was rendered uncleavable by furin, C3&agr; is re-routed to storage and is colocalized with VWFpp. The uncleavable protein was expressed in bovine aortic endothelial cells where it sorted to Weibel-Palade bodies, colocalized with bovine VWF, and was released when agonist stimulated. We now demonstrate that VWFpp re-routes a constitutively secreted protein to the regulated storage pathway. Furthermore, our studies suggest that the VWFpp storage signal is contained within amino acids 201 to 741.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"22 1","pages":"921-926"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81797743","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 : 2002-06-01DOI: 10.1161/01.ATV.0000018300.43492.83
T. Abumiya, T. Sasaguri, Y. Taba, Y. Miwa, Megumi Miyagi
Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by ≈3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5′-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress–responsive element resided in the sequence between −94 and −31 bp, which contained putative nuclear factor-&kgr;B, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.
{"title":"Shear Stress Induces Expression of Vascular Endothelial Growth Factor Receptor Flk-1/KDR Through the CT-Rich Sp1 Binding Site","authors":"T. Abumiya, T. Sasaguri, Y. Taba, Y. Miwa, Megumi Miyagi","doi":"10.1161/01.ATV.0000018300.43492.83","DOIUrl":"https://doi.org/10.1161/01.ATV.0000018300.43492.83","url":null,"abstract":"Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by ≈3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5′-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress–responsive element resided in the sequence between −94 and −31 bp, which contained putative nuclear factor-&kgr;B, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"79 1","pages":"907-913"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88180059","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 : 2002-06-01DOI: 10.1161/01.ATV.0000017198.16727.27
Ute Kelkenberg, A. Wagner, Jasmin Sarhaddar, M. Hecker, H. E. von der Leyen
Many cytokine genes, including those encoding acute-phase proteins and immunoglobulins, share binding sites for the CCAAT/enhancer-binding protein (C/EBP) in their 5′-flanking regions, and C/EBP-related transcription factors regulate cell proliferation during terminal differentiation. Therefore, C/EBP represents an attractive target for inhibiting restenosis after balloon angioplasty. In a rabbit model of restenosis that combines balloon injury of the carotid artery with cholesterol-mediated chronic inflammation, a decoy oligodeoxynucleotide (ODN) capable of neutralizing C/EBP was administered to the site of injury for 30 minutes. Electrophoretic mobility shift analysis confirmed that C/EBP activity in decoy ODN–treated segments was virtually absent after 2 days. Morphometric analysis after 28 days revealed significant reduction (up to 50%) of neointimal formation and intravascular inflammation in decoy ODN–treated segments compared with mutant control ODN or vehicle-treated segments. In addition, de novo synthesis of endothelin-1 and the number of proliferating cell nuclear antigen–positive smooth muscle cells in the vessel wall were markedly attenuated at day 3. These findings suggest that decoy ODN–based neutralization of C/EBP may be a feasible and effective method to limit restenosis after angioplasty brought about, at least in part, by inhibiting the de novo synthesis of endothelin-1.
{"title":"CCAAT/Enhancer-Binding Protein Decoy Oligodeoxynucleotide Inhibition of Macrophage-Rich Vascular Lesion Formation in Hypercholesterolemic Rabbits","authors":"Ute Kelkenberg, A. Wagner, Jasmin Sarhaddar, M. Hecker, H. E. von der Leyen","doi":"10.1161/01.ATV.0000017198.16727.27","DOIUrl":"https://doi.org/10.1161/01.ATV.0000017198.16727.27","url":null,"abstract":"Many cytokine genes, including those encoding acute-phase proteins and immunoglobulins, share binding sites for the CCAAT/enhancer-binding protein (C/EBP) in their 5′-flanking regions, and C/EBP-related transcription factors regulate cell proliferation during terminal differentiation. Therefore, C/EBP represents an attractive target for inhibiting restenosis after balloon angioplasty. In a rabbit model of restenosis that combines balloon injury of the carotid artery with cholesterol-mediated chronic inflammation, a decoy oligodeoxynucleotide (ODN) capable of neutralizing C/EBP was administered to the site of injury for 30 minutes. Electrophoretic mobility shift analysis confirmed that C/EBP activity in decoy ODN–treated segments was virtually absent after 2 days. Morphometric analysis after 28 days revealed significant reduction (up to 50%) of neointimal formation and intravascular inflammation in decoy ODN–treated segments compared with mutant control ODN or vehicle-treated segments. In addition, de novo synthesis of endothelin-1 and the number of proliferating cell nuclear antigen–positive smooth muscle cells in the vessel wall were markedly attenuated at day 3. These findings suggest that decoy ODN–based neutralization of C/EBP may be a feasible and effective method to limit restenosis after angioplasty brought about, at least in part, by inhibiting the de novo synthesis of endothelin-1.","PeriodicalId":8418,"journal":{"name":"Arteriosclerosis, Thrombosis, and Vascular Biology: Journal of the American Heart Association","volume":"21 1","pages":"949-954"},"PeriodicalIF":0.0,"publicationDate":"2002-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78326086","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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