Pub Date : 2003-01-10DOI: 10.1161/01.RES.0000050921.53008.47
J. Martínez-González, J. Rius, A. Castelló, C. Cases-Langhoff, L. Badimón
Abstract— Vascular smooth muscle cells (VSMCs) migration and proliferation play a key role in the pathophysiology of cardiovascular disease. However, the transcription factors that regulate VSMC activation are not completely characterized. By a mRNA-differential display approach, we have identified neuron-derived orphan receptor-1 (NOR-1), a transcription factor within the NGFI-B subfamily of nuclear receptors, as a immediate-early gene in VSMCs. Two NOR-1 isoforms (&agr; and &bgr;) were identified and cloned from serum-induced porcine VSMC that shared high homology with the human isoforms. Northern blot analysis revealed a strong and transient (1 to 6 hours) upregulation of NOR-1 in both porcine and human coronary SMCs by growth factors (serum, platelet-derived growth factor-BB, and epidermal growth factor) and &agr;-thrombin but not by cytokines. NOR-1 upregulation is processed through G protein–coupled receptors and tyrosine kinase receptors, and involves Ca2+ mobilization, protein kinase C activation, and the mitogen-activated protein kinase pathway. This induction was closely dependent of the cAMP response elements present in NOR-1 promoter as transfection assays indicate. Human coronary atherosclerotic lesions overexpress NOR-1, and balloon angioplasty transiently induces NOR-1 in porcine coronary arteries with a pattern similar to that observed in VSMCs in culture. Antisense oligonucleotides against NOR-1 inhibited human coronary SMC proliferation (reduced de novo DNA synthesis, cell cycle progression, and VSMC wound repair) as efficiently as antisense against the protooncogene c-fos. These results show that NOR-1 modulates VSMC proliferation, and suggest that this transcription factor may play a role in both spontaneous and accelerated atherosclerosis.
{"title":"Neuron-Derived Orphan Receptor-1 (NOR-1) Modulates Vascular Smooth Muscle Cell Proliferation","authors":"J. Martínez-González, J. Rius, A. Castelló, C. Cases-Langhoff, L. Badimón","doi":"10.1161/01.RES.0000050921.53008.47","DOIUrl":"https://doi.org/10.1161/01.RES.0000050921.53008.47","url":null,"abstract":"Abstract— Vascular smooth muscle cells (VSMCs) migration and proliferation play a key role in the pathophysiology of cardiovascular disease. However, the transcription factors that regulate VSMC activation are not completely characterized. By a mRNA-differential display approach, we have identified neuron-derived orphan receptor-1 (NOR-1), a transcription factor within the NGFI-B subfamily of nuclear receptors, as a immediate-early gene in VSMCs. Two NOR-1 isoforms (&agr; and &bgr;) were identified and cloned from serum-induced porcine VSMC that shared high homology with the human isoforms. Northern blot analysis revealed a strong and transient (1 to 6 hours) upregulation of NOR-1 in both porcine and human coronary SMCs by growth factors (serum, platelet-derived growth factor-BB, and epidermal growth factor) and &agr;-thrombin but not by cytokines. NOR-1 upregulation is processed through G protein–coupled receptors and tyrosine kinase receptors, and involves Ca2+ mobilization, protein kinase C activation, and the mitogen-activated protein kinase pathway. This induction was closely dependent of the cAMP response elements present in NOR-1 promoter as transfection assays indicate. Human coronary atherosclerotic lesions overexpress NOR-1, and balloon angioplasty transiently induces NOR-1 in porcine coronary arteries with a pattern similar to that observed in VSMCs in culture. Antisense oligonucleotides against NOR-1 inhibited human coronary SMC proliferation (reduced de novo DNA synthesis, cell cycle progression, and VSMC wound repair) as efficiently as antisense against the protooncogene c-fos. These results show that NOR-1 modulates VSMC proliferation, and suggest that this transcription factor may play a role in both spontaneous and accelerated atherosclerosis.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2003-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77493961","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 : 2002-08-23DOI: 10.1161/01.RES.0000030861.13850.F1
C. Northcott, Matthew N. Poy, S. Najjar, S. Watts
Abstract— Arteries from deoxycorticosterone acetate (DOCA)-salt and N&ohgr;-nitro-l-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184±5 mm Hg] compared with sham rats (SBP=111±2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85&agr;, p110&agr;, p110&bgr;, and p110&dgr; but not p110&ggr; PI3-kinase subunits; p110&dgr; protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110&bgr; protein density, but neither L-NNA nor DOCA-salt had differences in p85&agr; and p110&agr;. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 &mgr;mol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110&dgr; density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.
摘要:来自醋酸脱氧皮质酮(DOCA)盐和N&ohgr;-硝基-l-精氨酸(L-NNA)高血压而非正常高血压大鼠的动脉出现自发性张力。LY294002和wortmannin，磷酸肌苷3-激酶(pi3 -激酶)抑制剂，消除自发张力。我们假设高血压患者pi3激酶蛋白和/或活性增加，并有助于观察到的收缩性增强。pi3激酶活性测定显示，与假手术大鼠(收缩压=111±2 mm Hg)相比，doca盐组(收缩压=184±5 mm Hg)胸主动脉pi3激酶活性提高了2倍。主动脉匀浆的Western分析显示存在p85&agr, p110&agr, p110&bgr，和p110&dgr;但不是p110&ggr;pi3激酶子单元;p110&dgr;与假手术组比较，高血压大鼠主动脉蛋白升高。L-NNA大鼠主动脉匀浆也有p110&bgr升高;蛋白密度，但L-NNA和DOCA-salt在p85&agr中均无差异;和p110&agr;。总Akt密度没有改变，但在doca盐大鼠的匀浆中，pAkt明显降低。LY294002 (20 μ mol/L)和硝苯地平(50 μ mol/L)可消除Ca2+诱导的doca盐大鼠主动脉自发张力。然而，LY294002没有改变bayk8644诱导的收缩，表明LY294002不直接抑制l型Ca2+通道。PTEN(磷酸酶和紧张素同源物)和pPTEN在doca盐和假手术大鼠主动脉中均有表达，但无显著差异。LY294002纠正了doca盐大鼠主动脉对KCl和去甲肾上腺素的收缩增强。这些数据支持pi3激酶活性和p110&dgr;L-NNA和doca盐大鼠主动脉密度变化。重要的是，这种增加有助于在两种高血压模型中观察到的收缩力增强。
{"title":"Phosphoinositide 3-Kinase Mediates Enhanced Spontaneous and Agonist-Induced Contraction in Aorta of Deoxycorticosterone Acetate-Salt Hypertensive Rats","authors":"C. Northcott, Matthew N. Poy, S. Najjar, S. Watts","doi":"10.1161/01.RES.0000030861.13850.F1","DOIUrl":"https://doi.org/10.1161/01.RES.0000030861.13850.F1","url":null,"abstract":"Abstract— Arteries from deoxycorticosterone acetate (DOCA)-salt and N&ohgr;-nitro-l-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184±5 mm Hg] compared with sham rats (SBP=111±2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85&agr;, p110&agr;, p110&bgr;, and p110&dgr; but not p110&ggr; PI3-kinase subunits; p110&dgr; protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110&bgr; protein density, but neither L-NNA nor DOCA-salt had differences in p85&agr; and p110&agr;. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 &mgr;mol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110&dgr; density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82436050","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 : 2002-08-23DOI: 10.1161/01.RES.0000030180.06028.23
Zhaokang Yang, C. Pascarel, Derek S. Steele, K. Komukai, F. Brette, C. H. Orchard
Abstract— Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.
{"title":"Na+-Ca2+ Exchange Activity Is Localized in the T-Tubules of Rat Ventricular Myocytes","authors":"Zhaokang Yang, C. Pascarel, Derek S. Steele, K. Komukai, F. Brette, C. H. Orchard","doi":"10.1161/01.RES.0000030180.06028.23","DOIUrl":"https://doi.org/10.1161/01.RES.0000030180.06028.23","url":null,"abstract":"Abstract— Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83478245","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 : 2002-08-23DOI: 10.1161/01.RES.0000031744.06353.D3
R. Kishore, I. Spyridopoulos, Corinne Luedemann, Douglas Losordo
Abstract— Local expression of tumor necrosis factor-&agr; (TNF-&agr;) at the sites of arterial injury after balloon angioplasty, suppresses endothelial cell (EC) proliferation and negatively affects reendothelialization of the injured vessel. We have previously reported that in vitro exposure of ECs to TNF-&agr; induced EC growth arrest and apoptosis. These effects were mediated, at least in part, by downregulation of cell cycle regulatory proteins. In the present study, we report potential mechanism(s) for TNF-&agr;–mediated suppression of cyclin A in ECs. TNF-&agr; exposure to ECs completely abrogated cyclin A mRNA expression via mechanisms involving both transcriptional and posttranscriptional modifications. TNF-&agr; inhibited de novo cyclin A mRNA synthesis and suppressed cyclin A promoter activity. Utilizing deletion mutants of human cyclin A promoter, we have identified CDE-CHR (C ell cycle–D ependent E lements–C ell cycle genes H omology R egion) region of cyclin A promoter as a target for TNF-&agr; suppressive action. Experiments to investigate CDE-CHR binding proteins/factors revealed a TNF-&agr;–mediated increase in specific DNA binding activity to the CHR elements. This increase in binding activity by TNF-&agr; was mediated via the induction of a functionally novel 84-kDa protein that binds specifically to CHR in Southwestern assays. UV cross-linking and SDS-PAGE analysis of proteins eluted from specific complex confirmed the presence of this 84-kDa protein. Moreover, induction of this protein by TNF-&agr; was protein synthesis dependent. Additionally, exposure of ECs to TNF-&agr; markedly reduced cyclin A mRNA stability. Targeted disruption of this protein could potentially be a therapeutic strategy to rescue EC proliferation in vivo.
{"title":"Functionally Novel Tumor Necrosis Factor-&agr;–Modulated CHR-Binding Protein Mediates Cyclin A Transcriptional Repression in Vascular Endothelial Cells","authors":"R. Kishore, I. Spyridopoulos, Corinne Luedemann, Douglas Losordo","doi":"10.1161/01.RES.0000031744.06353.D3","DOIUrl":"https://doi.org/10.1161/01.RES.0000031744.06353.D3","url":null,"abstract":"Abstract— Local expression of tumor necrosis factor-&agr; (TNF-&agr;) at the sites of arterial injury after balloon angioplasty, suppresses endothelial cell (EC) proliferation and negatively affects reendothelialization of the injured vessel. We have previously reported that in vitro exposure of ECs to TNF-&agr; induced EC growth arrest and apoptosis. These effects were mediated, at least in part, by downregulation of cell cycle regulatory proteins. In the present study, we report potential mechanism(s) for TNF-&agr;–mediated suppression of cyclin A in ECs. TNF-&agr; exposure to ECs completely abrogated cyclin A mRNA expression via mechanisms involving both transcriptional and posttranscriptional modifications. TNF-&agr; inhibited de novo cyclin A mRNA synthesis and suppressed cyclin A promoter activity. Utilizing deletion mutants of human cyclin A promoter, we have identified CDE-CHR (C ell cycle–D ependent E lements–C ell cycle genes H omology R egion) region of cyclin A promoter as a target for TNF-&agr; suppressive action. Experiments to investigate CDE-CHR binding proteins/factors revealed a TNF-&agr;–mediated increase in specific DNA binding activity to the CHR elements. This increase in binding activity by TNF-&agr; was mediated via the induction of a functionally novel 84-kDa protein that binds specifically to CHR in Southwestern assays. UV cross-linking and SDS-PAGE analysis of proteins eluted from specific complex confirmed the presence of this 84-kDa protein. Moreover, induction of this protein by TNF-&agr; was protein synthesis dependent. Additionally, exposure of ECs to TNF-&agr; markedly reduced cyclin A mRNA stability. Targeted disruption of this protein could potentially be a therapeutic strategy to rescue EC proliferation in vivo.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79190263","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 : 2002-08-23DOI: 10.1161/01.RES.0000031801.84308.F4
Bum-Rak Choi, Wonchul Nho, Tong Liu, G. Salama
Abstract— The nature and organization of electrical activity during ventricular fibrillation (VF) are important and controversial subjects dominated by 2 competing theories: the wavebreak and the dominant mother rotor hypothesis. To investigate spatiotemporal characteristics of ventricular fibrillation (VF), transmembrane potentials (Vm) were recorded from multiple sites of perfused rabbit hearts using a voltage-sensitive dye and a photodiode array or a CCD camera, and the time-frequency characteristics of Vm were analyzed by short-time fast Fourier transform (FFT) or generalized time-frequency representation with a cone-shaped kernel. The analysis was applied to all pixels to track VF frequencies in time and space. VF consisted of blobs, which are groups of contiguous pixels with a common frequency and an ill-defined shape. At any time t, several VF frequency blobs coexisted in the field of view, and the number of coexisting blobs was on average 5.9±2.1 (n=8 hearts) as they appeared and disappeared discontinuously with time and were not fixed in space. The life span of frequency blobs from birth to either annihilation or breakup to another frequency had a half-life of 0.39±0.13 second (n=4 hearts). The Ca2+ channel blocker nifedipine increased the stability of VF frequencies and reduced the number of frequency blobs progressing to a single frequency. In conclusion, VF consists of dynamically changing frequency blobs, which have a short life span and can be modified by pharmacological interventions, suggesting that VF is maintained by dynamically changing multiple wavelets.
{"title":"Life Span of Ventricular Fibrillation Frequencies","authors":"Bum-Rak Choi, Wonchul Nho, Tong Liu, G. Salama","doi":"10.1161/01.RES.0000031801.84308.F4","DOIUrl":"https://doi.org/10.1161/01.RES.0000031801.84308.F4","url":null,"abstract":"Abstract— The nature and organization of electrical activity during ventricular fibrillation (VF) are important and controversial subjects dominated by 2 competing theories: the wavebreak and the dominant mother rotor hypothesis. To investigate spatiotemporal characteristics of ventricular fibrillation (VF), transmembrane potentials (Vm) were recorded from multiple sites of perfused rabbit hearts using a voltage-sensitive dye and a photodiode array or a CCD camera, and the time-frequency characteristics of Vm were analyzed by short-time fast Fourier transform (FFT) or generalized time-frequency representation with a cone-shaped kernel. The analysis was applied to all pixels to track VF frequencies in time and space. VF consisted of blobs, which are groups of contiguous pixels with a common frequency and an ill-defined shape. At any time t, several VF frequency blobs coexisted in the field of view, and the number of coexisting blobs was on average 5.9±2.1 (n=8 hearts) as they appeared and disappeared discontinuously with time and were not fixed in space. The life span of frequency blobs from birth to either annihilation or breakup to another frequency had a half-life of 0.39±0.13 second (n=4 hearts). The Ca2+ channel blocker nifedipine increased the stability of VF frequencies and reduced the number of frequency blobs progressing to a single frequency. In conclusion, VF consists of dynamically changing frequency blobs, which have a short life span and can be modified by pharmacological interventions, suggesting that VF is maintained by dynamically changing multiple wavelets.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79956006","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 : 2002-08-23DOI: 10.1161/01.RES.0000031958.92781.9E
A. Milia, M. Salis, Tiziana Stacca, A. Pinna, P. Madeddu, M. Trevisani, P. Geppetti, C. Emanueli
Abstract— Proteinase-activated receptors (PAR-2) are expressed by the cardiovascular system and mediate vasodilation, plasma protein extravasation, and endothelial cell proliferation, all regarded as essential steps for neovascularization. We investigated the angiogenic action of PAR-2 signaling in vivo. The effect of the PAR-2 activating peptide (PAR-2AP, SLIGRL-NH2) was assessed in the absence of ischemia, and the therapeutic potential of PAR-2AP and the PAR-2 agonist trypsin (at 300 and 1.5 nmol IM daily for 21 days, respectively) was also tested in mice subjected to unilateral limb ischemia. PAR-2AP increased capillarity in normoperfused adductor skeletal muscles, whereas neither the vehicle of the PAR2-AP nor the PAR-2 reverse peptide (PAR-2RP, LRGILS-NH2) did produce any effect. In addition, both PAR-2AP and trypsin enhanced reparative angiogenic response to limb ischemia, an effect that was not produced by PAR-2RP or the vehicle of PAR-2 agonists. Potentiation of reparative angiogenesis by PAR-2AP or trypsin resulted in an accelerated hemodynamic recovery and enhanced limb salvage. In conclusions, our study is the first to demonstrate the angiogenic potential of PAR-2 stimulation in vivo. If similar effects occur in humans, PAR-2AP agonists could have some therapeutic potential for the treatment of tissue ischemia.
{"title":"Protease-Activated Receptor-2 Stimulates Angiogenesis and Accelerates Hemodynamic Recovery in a Mouse Model of Hindlimb Ischemia","authors":"A. Milia, M. Salis, Tiziana Stacca, A. Pinna, P. Madeddu, M. Trevisani, P. Geppetti, C. Emanueli","doi":"10.1161/01.RES.0000031958.92781.9E","DOIUrl":"https://doi.org/10.1161/01.RES.0000031958.92781.9E","url":null,"abstract":"Abstract— Proteinase-activated receptors (PAR-2) are expressed by the cardiovascular system and mediate vasodilation, plasma protein extravasation, and endothelial cell proliferation, all regarded as essential steps for neovascularization. We investigated the angiogenic action of PAR-2 signaling in vivo. The effect of the PAR-2 activating peptide (PAR-2AP, SLIGRL-NH2) was assessed in the absence of ischemia, and the therapeutic potential of PAR-2AP and the PAR-2 agonist trypsin (at 300 and 1.5 nmol IM daily for 21 days, respectively) was also tested in mice subjected to unilateral limb ischemia. PAR-2AP increased capillarity in normoperfused adductor skeletal muscles, whereas neither the vehicle of the PAR2-AP nor the PAR-2 reverse peptide (PAR-2RP, LRGILS-NH2) did produce any effect. In addition, both PAR-2AP and trypsin enhanced reparative angiogenic response to limb ischemia, an effect that was not produced by PAR-2RP or the vehicle of PAR-2 agonists. Potentiation of reparative angiogenesis by PAR-2AP or trypsin resulted in an accelerated hemodynamic recovery and enhanced limb salvage. In conclusions, our study is the first to demonstrate the angiogenic potential of PAR-2 stimulation in vivo. If similar effects occur in humans, PAR-2AP agonists could have some therapeutic potential for the treatment of tissue ischemia.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87460458","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 : 2002-08-23DOI: 10.1161/01.RES.0000031957.70034.89
D. Hooks, K. A. Tomlinson, S. Marsden, I. LeGrice, B. Smaill, A. Pullan, P. Hunter
Abstract— Our understanding of the electrophysiological properties of the heart is incomplete. We have investigated two issues that are fundamental to advancing that understanding. First, there has been widespread debate over the mechanisms by which an externally applied shock can influence a sufficient volume of heart tissue to terminate cardiac fibrillation. Second, it has been uncertain whether cardiac tissue should be viewed as an electrically orthotropic structure, or whether its electrical properties are, in fact, isotropic in the plane orthogonal to myofiber direction. In the present study, a computer model that incorporates a detailed three-dimensional representation of cardiac muscular architecture is used to investigate these issues. We describe a bidomain model of electrical propagation solved in a discontinuous domain that accurately represents the microstructure of a transmural block of rat left ventricle. From analysis of the model results, we conclude that (1) the laminar organization of myocytes determines unique electrical properties in three microstructurally defined directions at any point in the ventricular wall of the heart, and (2) interlaminar clefts between layers of cardiomyocytes provide a substrate for bulk activation of the ventricles during defibrillation.
{"title":"Cardiac Microstructure: Implications for Electrical Propagation and Defibrillation in the Heart","authors":"D. Hooks, K. A. Tomlinson, S. Marsden, I. LeGrice, B. Smaill, A. Pullan, P. Hunter","doi":"10.1161/01.RES.0000031957.70034.89","DOIUrl":"https://doi.org/10.1161/01.RES.0000031957.70034.89","url":null,"abstract":"Abstract— Our understanding of the electrophysiological properties of the heart is incomplete. We have investigated two issues that are fundamental to advancing that understanding. First, there has been widespread debate over the mechanisms by which an externally applied shock can influence a sufficient volume of heart tissue to terminate cardiac fibrillation. Second, it has been uncertain whether cardiac tissue should be viewed as an electrically orthotropic structure, or whether its electrical properties are, in fact, isotropic in the plane orthogonal to myofiber direction. In the present study, a computer model that incorporates a detailed three-dimensional representation of cardiac muscular architecture is used to investigate these issues. We describe a bidomain model of electrical propagation solved in a discontinuous domain that accurately represents the microstructure of a transmural block of rat left ventricle. From analysis of the model results, we conclude that (1) the laminar organization of myocytes determines unique electrical properties in three microstructurally defined directions at any point in the ventricular wall of the heart, and (2) interlaminar clefts between layers of cardiomyocytes provide a substrate for bulk activation of the ventricles during defibrillation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74239781","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 : 2002-08-23DOI: 10.1161/01.RES.0000030711.21521.AC
R. A. Kaiser, B. C. Oxhorn, Gracie L. Andrews, I. Buxton
Abstract— The presence of multiple receptors for disparate nucleotides on endothelial cells makes it unclear how the endothelium differentiates among these signals. We propose that endothelial P2Y receptors are organized into cholesterol-rich signaling domains, such as caveolae and respond to nucleotide agonists by mobilizing intracellular calcium. Treatment of endothelial cells with 5 mmol/L &bgr;-methyl-cyclodextrin prevents calcium release in response to the nucleotide receptor agonists 2-methylthio-ATP, ATP, ADP, and UTP, but not the kinin receptor agonist bradykinin, suggesting that depletion of membrane cholesterol disrupts signaling at P2Y receptors and that bradykinin receptors are not prelocalized to cholesterol microdomains in these cells. Direct measurement of cholesterol content after &bgr;-methyl-cyclodextrin treatment of aortic rings reveals a concentration-dependent depletion of cholesterol that parallels functional antagonism of P2Y-mediated relaxation. Nucleotide- and bradykinin-mediated relaxation is disrupted by 5 to 15 mmol/L &bgr; -methyl-cyclodextrin treatment or 1 to 10 &mgr;g/mL filipin III in a concentration-dependent fashion. Norepinephrine contracted aorta treated with A23187 relaxes in an endothelium-dependent fashion despite depletion of 84% of membrane-extractable cholesterol. These data indicate that in the basal state, P2Y receptors but not the kinin receptor may be compartmented to cholesterol-dependent signaling domains in guinea pig endothelium and that cholesterol-rich microdomains in these cells can respond to intracellular calcium in an agonist-specific manner. We suggest that the functional organization of cholesterol-rich signaling microdomains allows agonist-specific responses to increases in intracellular calcium and that this property may be a general phenomenon that permits cells to respond disparately to agonists that may signal through common calcium release pathways.
{"title":"Functional Compartmentation of Endothelial P2Y Receptor Signaling","authors":"R. A. Kaiser, B. C. Oxhorn, Gracie L. Andrews, I. Buxton","doi":"10.1161/01.RES.0000030711.21521.AC","DOIUrl":"https://doi.org/10.1161/01.RES.0000030711.21521.AC","url":null,"abstract":"Abstract— The presence of multiple receptors for disparate nucleotides on endothelial cells makes it unclear how the endothelium differentiates among these signals. We propose that endothelial P2Y receptors are organized into cholesterol-rich signaling domains, such as caveolae and respond to nucleotide agonists by mobilizing intracellular calcium. Treatment of endothelial cells with 5 mmol/L &bgr;-methyl-cyclodextrin prevents calcium release in response to the nucleotide receptor agonists 2-methylthio-ATP, ATP, ADP, and UTP, but not the kinin receptor agonist bradykinin, suggesting that depletion of membrane cholesterol disrupts signaling at P2Y receptors and that bradykinin receptors are not prelocalized to cholesterol microdomains in these cells. Direct measurement of cholesterol content after &bgr;-methyl-cyclodextrin treatment of aortic rings reveals a concentration-dependent depletion of cholesterol that parallels functional antagonism of P2Y-mediated relaxation. Nucleotide- and bradykinin-mediated relaxation is disrupted by 5 to 15 mmol/L &bgr; -methyl-cyclodextrin treatment or 1 to 10 &mgr;g/mL filipin III in a concentration-dependent fashion. Norepinephrine contracted aorta treated with A23187 relaxes in an endothelium-dependent fashion despite depletion of 84% of membrane-extractable cholesterol. These data indicate that in the basal state, P2Y receptors but not the kinin receptor may be compartmented to cholesterol-dependent signaling domains in guinea pig endothelium and that cholesterol-rich microdomains in these cells can respond to intracellular calcium in an agonist-specific manner. We suggest that the functional organization of cholesterol-rich signaling microdomains allows agonist-specific responses to increases in intracellular calcium and that this property may be a general phenomenon that permits cells to respond disparately to agonists that may signal through common calcium release pathways.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72672824","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 : 2002-08-23DOI: 10.1161/01.RES.0000031384.55006.DB
A. Gómez, B. Schwaller, H. Porzig, G. Vassort, E. Niggli, M. Egger
Abstract— Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca2+ handling associated with changes in the contractile function and arrhythmogenicity. The cardiac Na+-Ca2+ exchange (NCX) is an important mechanism for Ca2+ extrusion and cell relaxation. Its possible involvement in changes of excitation-contraction coupling (EC-coupling) with disease remains uncertain. We analyzed the NCX function in rat ventricular myocytes 5 to 6 months after experimental myocardial infarction (PMI) produced by left coronary artery ligation and from sham-operated (SO) hearts. Caged Ca2+ was dialyzed into the cytoplasm via a patch-clamp pipette and Ca2+ was released by flash photolysis to activate NCX and measure the associated currents (INaCa), whereas [Ca2+]i changes were simultaneously recorded with a confocal microscope. INaCa density normalized to the [Ca2+]i jumps was 2.6-fold higher in myocytes from PMI rats. The level of total NCX protein expression in PMI myocytes was also increased. Interestingly, although the INaCa density in PMI cells was larger, PMI and SO myocytes presented virtually identical Ca2+ transport via the NCX. This discrepancy was explained by a reduced surface/volume ratio (34.8%) observed in PMI cells. We conclude that the increase in NCX density may be a mechanism to maintain the required Ca2+ extrusion from a larger cell to allow adequate relaxation.
{"title":"Increased Exchange Current but Normal Ca2+ Transport via Na+-Ca2+ Exchange During Cardiac Hypertrophy After Myocardial Infarction","authors":"A. Gómez, B. Schwaller, H. Porzig, G. Vassort, E. Niggli, M. Egger","doi":"10.1161/01.RES.0000031384.55006.DB","DOIUrl":"https://doi.org/10.1161/01.RES.0000031384.55006.DB","url":null,"abstract":"Abstract— Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca2+ handling associated with changes in the contractile function and arrhythmogenicity. The cardiac Na+-Ca2+ exchange (NCX) is an important mechanism for Ca2+ extrusion and cell relaxation. Its possible involvement in changes of excitation-contraction coupling (EC-coupling) with disease remains uncertain. We analyzed the NCX function in rat ventricular myocytes 5 to 6 months after experimental myocardial infarction (PMI) produced by left coronary artery ligation and from sham-operated (SO) hearts. Caged Ca2+ was dialyzed into the cytoplasm via a patch-clamp pipette and Ca2+ was released by flash photolysis to activate NCX and measure the associated currents (INaCa), whereas [Ca2+]i changes were simultaneously recorded with a confocal microscope. INaCa density normalized to the [Ca2+]i jumps was 2.6-fold higher in myocytes from PMI rats. The level of total NCX protein expression in PMI myocytes was also increased. Interestingly, although the INaCa density in PMI cells was larger, PMI and SO myocytes presented virtually identical Ca2+ transport via the NCX. This discrepancy was explained by a reduced surface/volume ratio (34.8%) observed in PMI cells. We conclude that the increase in NCX density may be a mechanism to maintain the required Ca2+ extrusion from a larger cell to allow adequate relaxation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77437742","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 : 2002-08-23DOI: 10.1161/01.RES.0000031799.12850.1E
Yang Shi, J. Baker, Chenyang Zhang, J. Tweddell, Jidong Su, K. Pritchard
Abstract— Chronic hypoxia increases endothelial nitric oxide synthase (eNOS) production of nitric oxide (·NO) and cardioprotection in neonatal rabbit hearts. However, the mechanism by which this occurs remains unclear. Recent studies suggest that heat shock protein 90 (hsp90) alters eNOS function. In the present study, we examined the role of hsp90 in eNOS-dependent cardioprotection in neonatal rabbit hearts. Chronic hypoxia increased recovery of postischemic left ventricular developed pressure (LVDP). Geldanamycin (GA), which inhibits hsp90 and increases oxidative stress, decreased functional recovery in normoxic and hypoxic hearts. To determine if a loss in ·NO, afforded by GA, decreased recovery, GA-treated hearts were perfused with S-nitrosoglutathione (GSNO) as a source of ·NO. GSNO increased recovery of postischemic LVDP in GA-treated normoxic and hypoxic hearts to baseline levels. Although chronic hypoxia decreased phosphorylated eNOS (S1177) levels by ≈4- to 5-fold and total Akt and phosphorylated Akt by 4- and 5-fold, it also increased hsp90 association with eNOS by more than 3-fold. Using hydroethidine (HEt), a fluorescent probe for superoxide, we found that hypoxic hearts contained less ethidine (Et) staining than normoxic hearts. Normoxic hearts generated 3 times more superoxide by an N&ohgr;-nitro-l-arginine methyl ester (L-NAME)-inhibitable mechanism than hypoxic hearts. Taken together, these data indicate that the association of hsp90 with eNOS is important for increasing ·NO production and limiting eNOS-dependent superoxide anion generation. Such changes in eNOS function appear to play a critical role in protecting the myocardium against ischemic injury.
{"title":"Chronic Hypoxia Increases Endothelial Nitric Oxide Synthase Generation of Nitric Oxide by Increasing Heat Shock Protein 90 Association and Serine Phosphorylation","authors":"Yang Shi, J. Baker, Chenyang Zhang, J. Tweddell, Jidong Su, K. Pritchard","doi":"10.1161/01.RES.0000031799.12850.1E","DOIUrl":"https://doi.org/10.1161/01.RES.0000031799.12850.1E","url":null,"abstract":"Abstract— Chronic hypoxia increases endothelial nitric oxide synthase (eNOS) production of nitric oxide (·NO) and cardioprotection in neonatal rabbit hearts. However, the mechanism by which this occurs remains unclear. Recent studies suggest that heat shock protein 90 (hsp90) alters eNOS function. In the present study, we examined the role of hsp90 in eNOS-dependent cardioprotection in neonatal rabbit hearts. Chronic hypoxia increased recovery of postischemic left ventricular developed pressure (LVDP). Geldanamycin (GA), which inhibits hsp90 and increases oxidative stress, decreased functional recovery in normoxic and hypoxic hearts. To determine if a loss in ·NO, afforded by GA, decreased recovery, GA-treated hearts were perfused with S-nitrosoglutathione (GSNO) as a source of ·NO. GSNO increased recovery of postischemic LVDP in GA-treated normoxic and hypoxic hearts to baseline levels. Although chronic hypoxia decreased phosphorylated eNOS (S1177) levels by ≈4- to 5-fold and total Akt and phosphorylated Akt by 4- and 5-fold, it also increased hsp90 association with eNOS by more than 3-fold. Using hydroethidine (HEt), a fluorescent probe for superoxide, we found that hypoxic hearts contained less ethidine (Et) staining than normoxic hearts. Normoxic hearts generated 3 times more superoxide by an N&ohgr;-nitro-l-arginine methyl ester (L-NAME)-inhibitable mechanism than hypoxic hearts. Taken together, these data indicate that the association of hsp90 with eNOS is important for increasing ·NO production and limiting eNOS-dependent superoxide anion generation. Such changes in eNOS function appear to play a critical role in protecting the myocardium against ischemic injury.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88284063","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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