Pub Date : 2002-05-03DOI: 10.1161/01.RES.0000016963.43856.B1
Z. Paroo, J. Haist, M. Karmazyn, E. Noble
Exercise is a physiological inducer of the cardioprotective heat shock protein, Hsp70. The putative biological events involved in signaling this response exhibit sexual dimorphism. Thus, it was hypothesized that exercise-mediated induction of Hsp70 would demonstrate sex specificity. After treadmill running, male rats exhibited 2-fold greater levels of cardiac Hsp70 relative to the levels in gonadally intact female rats (P <0.001). Ovariectomized female rats exhibited exercise-mediated induction of Hsp70 similar to that observed for male rats, and estrogen treatment in these female rats reversed this effect (P <0.001). Attenuation of Hsp70 signaling by estrogen was non–receptor-mediated, possibly involving a cellular membrane–stabilizing mechanism of action. The physiological importance of this sex-specific hormone-mediated stress response is underscored by the disparity in functional adaptation in response to exercise between male rats and female rats. Exercise markedly improved postischemic left ventricular developed pressure, the maximal rate of contraction, and maximal rate of relaxation, and it reduced left ventricular end-diastolic pressure in male rats (P <0.001). No such benefit of exercise was observed in intact female rats. A causal role for Hsp70 in this sex-specific cardioprotective adaptation was indicated, inasmuch as ablation of Hsp70 induction with antisense oligonucleotides designed against Hsp70 transcripts attenuated improvement in the recovery of cardiac function in exercised male rats (P <0.05). Thus, the sex-specific hormone-mediated Hsp70 response to exercise results in cardioprotective adaptation, preferentially in male rats relative to female rats. These findings suggest that exercise may be more important for males than for females in defending against the effects of heart disease and offer a novel manner by which males may reduce the sex gap in susceptibility to adverse cardiac events.
{"title":"Exercise Improves Postischemic Cardiac Function in Males but Not Females: Consequences of a Novel Sex-Specific Heat Shock Protein 70 Response","authors":"Z. Paroo, J. Haist, M. Karmazyn, E. Noble","doi":"10.1161/01.RES.0000016963.43856.B1","DOIUrl":"https://doi.org/10.1161/01.RES.0000016963.43856.B1","url":null,"abstract":"Exercise is a physiological inducer of the cardioprotective heat shock protein, Hsp70. The putative biological events involved in signaling this response exhibit sexual dimorphism. Thus, it was hypothesized that exercise-mediated induction of Hsp70 would demonstrate sex specificity. After treadmill running, male rats exhibited 2-fold greater levels of cardiac Hsp70 relative to the levels in gonadally intact female rats (P <0.001). Ovariectomized female rats exhibited exercise-mediated induction of Hsp70 similar to that observed for male rats, and estrogen treatment in these female rats reversed this effect (P <0.001). Attenuation of Hsp70 signaling by estrogen was non–receptor-mediated, possibly involving a cellular membrane–stabilizing mechanism of action. The physiological importance of this sex-specific hormone-mediated stress response is underscored by the disparity in functional adaptation in response to exercise between male rats and female rats. Exercise markedly improved postischemic left ventricular developed pressure, the maximal rate of contraction, and maximal rate of relaxation, and it reduced left ventricular end-diastolic pressure in male rats (P <0.001). No such benefit of exercise was observed in intact female rats. A causal role for Hsp70 in this sex-specific cardioprotective adaptation was indicated, inasmuch as ablation of Hsp70 induction with antisense oligonucleotides designed against Hsp70 transcripts attenuated improvement in the recovery of cardiac function in exercised male rats (P <0.05). Thus, the sex-specific hormone-mediated Hsp70 response to exercise results in cardioprotective adaptation, preferentially in male rats relative to female rats. These findings suggest that exercise may be more important for males than for females in defending against the effects of heart disease and offer a novel manner by which males may reduce the sex gap in susceptibility to adverse cardiac events.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"51 1","pages":"911-917"},"PeriodicalIF":0.0,"publicationDate":"2002-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90930045","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 : 2002-05-03DOI: 10.1161/01.RES.0000016837.26733.BE
J. Fontana, D. Fulton, Yan Chen, Todd A. Fairchild, T. Mccabe, N. Fujita, T. Tsuruo, W. Sessa
Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.
{"title":"Domain Mapping Studies Reveal That the M Domain of hsp90 Serves as a Molecular Scaffold to Regulate Akt-Dependent Phosphorylation of Endothelial Nitric Oxide Synthase and NO Release","authors":"J. Fontana, D. Fulton, Yan Chen, Todd A. Fairchild, T. Mccabe, N. Fujita, T. Tsuruo, W. Sessa","doi":"10.1161/01.RES.0000016837.26733.BE","DOIUrl":"https://doi.org/10.1161/01.RES.0000016837.26733.BE","url":null,"abstract":"Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"115 1","pages":"866-873"},"PeriodicalIF":0.0,"publicationDate":"2002-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79166462","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 : 2002-05-03DOI: 10.1161/01.RES.0000016506.04193.96
R. Scotland, M. Morales-Ruiz, Yan Chen, Jun Yu, R. Rudic, David Fulton, J. Gratton, W. Sessa
Phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1179 can activate the enzyme, leading to NO release. Because eNOS is important in regulating vascular tone, we investigated whether phosphorylation of this residue is involved in vasomotion. Adenoviral transduction of endothelial cells (ECs) with the phosphomimetic S1179DeNOS markedly increased basal and vascular endothelial cell growth factor (VEGF)–stimulated NO release compared with cells transduced with wild-type virus. Conversely, adenoviral transduction of ECs with the non-phosphorylatable S1179AeNOS suppressed basal and stimulated NO release. Using a novel method for luminal delivery of adenovirus, transduction of the endothelium of carotid arteries from eNOS knockout mice with S1179DeNOS completely restored NO-mediated dilatation to acetylcholine (ACh), whereas vasomotor responses in arteries transduced with S1179AeNOS were significantly attenuated. Basal NO release was also significantly reduced in arteries transduced with S1179AeNOS, compared with S1179DeNOS. Thus, our data directly demonstrate that phosphorylation of eNOS at serine 1179 is an important regulator of basal and stimulated NO release in ECs and in intact blood vessels.
{"title":"Functional Reconstitution of Endothelial Nitric Oxide Synthase Reveals the Importance of Serine 1179 in Endothelium-Dependent Vasomotion","authors":"R. Scotland, M. Morales-Ruiz, Yan Chen, Jun Yu, R. Rudic, David Fulton, J. Gratton, W. Sessa","doi":"10.1161/01.RES.0000016506.04193.96","DOIUrl":"https://doi.org/10.1161/01.RES.0000016506.04193.96","url":null,"abstract":"Phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1179 can activate the enzyme, leading to NO release. Because eNOS is important in regulating vascular tone, we investigated whether phosphorylation of this residue is involved in vasomotion. Adenoviral transduction of endothelial cells (ECs) with the phosphomimetic S1179DeNOS markedly increased basal and vascular endothelial cell growth factor (VEGF)–stimulated NO release compared with cells transduced with wild-type virus. Conversely, adenoviral transduction of ECs with the non-phosphorylatable S1179AeNOS suppressed basal and stimulated NO release. Using a novel method for luminal delivery of adenovirus, transduction of the endothelium of carotid arteries from eNOS knockout mice with S1179DeNOS completely restored NO-mediated dilatation to acetylcholine (ACh), whereas vasomotor responses in arteries transduced with S1179AeNOS were significantly attenuated. Basal NO release was also significantly reduced in arteries transduced with S1179AeNOS, compared with S1179DeNOS. Thus, our data directly demonstrate that phosphorylation of eNOS at serine 1179 is an important regulator of basal and stimulated NO release in ECs and in intact blood vessels.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"13 1","pages":"904-910"},"PeriodicalIF":0.0,"publicationDate":"2002-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87270163","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 : 2002-05-03DOI: 10.1161/01.RES.0000016962.36404.04
B. Wolska, Grace M. Arteaga, J. Peña, G. Nowak, Ronald M. Phillips, Shalini Sahai, Pieter P. de Tombe, Anne F. Martin, Evangelia G. Kranias, R. J. Solaro
&bgr;-Adrenergic stimulation of the heart results in an enhanced relaxation rate in association with phosphorylation of both cardiac troponin I (cTnI) and phospholamban (PLB). We studied new lines of mice generated by crossbreeding mice that express slow skeletal troponin I (ssTnI) with PLB knockout (PLBKO) mice. This crossbreeding resulted in the generation of PLB/cTnI, PLB/ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. Perfusion with isoproterenol (ISO) significantly increased the peak amplitude of fura-2 ratio in PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI groups of mice. However, in the presence of ISO, there were no differences in the peak amplitude of fura-2 ratio among cells isolated from hearts of PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. In cells from PLB/cTnI mice, the extent of shortening was increased and the time of relaxation was significantly decreased during &bgr;-adrenergic stimulation. In PLBKO/cTnI cells, stimulation with ISO resulted in an increased extent of shortening and no change in time of relaxation. However, stimulation with ISO in cells isolated from PLBKO/ssTnI mice not only significantly increased the extent of cell shortening but also increased the time of relaxation. We also determined the kinetics of relaxation of papillary muscles isolated from all four groups of animals in the presence and absence of ISO. Perfusion with ISO increased the rate of relaxation only in PLB/cTnI, PLB/ssTnI, and PLBKO/cTnI muscles. During ISO stimulation, the time of relaxation was unchanged in PLBKO/ssTnI muscles. Our data directly demonstrate that phosphorylation of both PLB and cTnI contributes to increased rate of relaxation during &bgr;-adrenergic stimulation.
{"title":"Expression of Slow Skeletal Troponin I in Hearts of Phospholamban Knockout Mice Alters the Relaxant Effect of &bgr;-Adrenergic Stimulation","authors":"B. Wolska, Grace M. Arteaga, J. Peña, G. Nowak, Ronald M. Phillips, Shalini Sahai, Pieter P. de Tombe, Anne F. Martin, Evangelia G. Kranias, R. J. Solaro","doi":"10.1161/01.RES.0000016962.36404.04","DOIUrl":"https://doi.org/10.1161/01.RES.0000016962.36404.04","url":null,"abstract":"&bgr;-Adrenergic stimulation of the heart results in an enhanced relaxation rate in association with phosphorylation of both cardiac troponin I (cTnI) and phospholamban (PLB). We studied new lines of mice generated by crossbreeding mice that express slow skeletal troponin I (ssTnI) with PLB knockout (PLBKO) mice. This crossbreeding resulted in the generation of PLB/cTnI, PLB/ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. Perfusion with isoproterenol (ISO) significantly increased the peak amplitude of fura-2 ratio in PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI groups of mice. However, in the presence of ISO, there were no differences in the peak amplitude of fura-2 ratio among cells isolated from hearts of PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. In cells from PLB/cTnI mice, the extent of shortening was increased and the time of relaxation was significantly decreased during &bgr;-adrenergic stimulation. In PLBKO/cTnI cells, stimulation with ISO resulted in an increased extent of shortening and no change in time of relaxation. However, stimulation with ISO in cells isolated from PLBKO/ssTnI mice not only significantly increased the extent of cell shortening but also increased the time of relaxation. We also determined the kinetics of relaxation of papillary muscles isolated from all four groups of animals in the presence and absence of ISO. Perfusion with ISO increased the rate of relaxation only in PLB/cTnI, PLB/ssTnI, and PLBKO/cTnI muscles. During ISO stimulation, the time of relaxation was unchanged in PLBKO/ssTnI muscles. Our data directly demonstrate that phosphorylation of both PLB and cTnI contributes to increased rate of relaxation during &bgr;-adrenergic stimulation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"17 1","pages":"882-888"},"PeriodicalIF":0.0,"publicationDate":"2002-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75657647","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 : 2002-05-03DOI: 10.1161/01.RES.0000016504.08608.B9
P. Qiu, Li Li
Chromatin acetylation and deacetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) are closely related to eukaryotic gene transcription. Although the binding of serum response factor (SRF) to the CArG boxes in the promoter region is necessary for SM22 expression, it has never been examined whether the local chromatin modification is involved in SM22 gene regulation. In this study, we used the SM22 gene as a model to address whether transcriptional activation of the gene can be manipulated through adjusting histone acetylation of the chromatin template and whether SRF- and HAT-containing coactivators can be recruited onto the SM22 promoter region during gene activation. Here, we showed that the stimulation of the SM22 promoter by the coactivator CREB-binding protein (CBP) was dependent on HAT activity. Overexpression of HDACs decreased SM22 promoter activity, whereas trichostatin A, an HDAC inhibitor, stimulated SM22 promoter activity in a CArG box-dependent manner and induced endogenous SM22 gene expression. Chromatin immunoprecipitation assays showed that trichostatin A treatment in 10T1/2 cells induces chromatin hyperacetylation in the SM22 gene. Although histone hyperacetylation of the SM22 gene occurred during SM22 gene expression and SRF and CBP immunocomplexes possess HAT activities in smooth muscle cells, both SRF and CBP were recruited to the CArG box-containing region of the promoter. This study provides evidence that chromatin acetylation is involved in smooth muscle cell-specific gene regulation.
{"title":"Histone Acetylation and Recruitment of Serum Responsive Factor and CREB-Binding Protein Onto SM22 Promoter During SM22 Gene Expression","authors":"P. Qiu, Li Li","doi":"10.1161/01.RES.0000016504.08608.B9","DOIUrl":"https://doi.org/10.1161/01.RES.0000016504.08608.B9","url":null,"abstract":"Chromatin acetylation and deacetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) are closely related to eukaryotic gene transcription. Although the binding of serum response factor (SRF) to the CArG boxes in the promoter region is necessary for SM22 expression, it has never been examined whether the local chromatin modification is involved in SM22 gene regulation. In this study, we used the SM22 gene as a model to address whether transcriptional activation of the gene can be manipulated through adjusting histone acetylation of the chromatin template and whether SRF- and HAT-containing coactivators can be recruited onto the SM22 promoter region during gene activation. Here, we showed that the stimulation of the SM22 promoter by the coactivator CREB-binding protein (CBP) was dependent on HAT activity. Overexpression of HDACs decreased SM22 promoter activity, whereas trichostatin A, an HDAC inhibitor, stimulated SM22 promoter activity in a CArG box-dependent manner and induced endogenous SM22 gene expression. Chromatin immunoprecipitation assays showed that trichostatin A treatment in 10T1/2 cells induces chromatin hyperacetylation in the SM22 gene. Although histone hyperacetylation of the SM22 gene occurred during SM22 gene expression and SRF and CBP immunocomplexes possess HAT activities in smooth muscle cells, both SRF and CBP were recruited to the CArG box-containing region of the promoter. This study provides evidence that chromatin acetylation is involved in smooth muscle cell-specific gene regulation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"38 1","pages":"858-865"},"PeriodicalIF":0.0,"publicationDate":"2002-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85048327","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 : 2002-05-03DOI: 10.1161/01.RES.0000015885.27134.F0
H. Allayee, K. Krass, P. Pajukanta, R. Cantor, C. V. D. van der Kallen, R. Mar, J. Rotter, T. D. de Bruin, L. Peltonen, A. Lusis
Familial combined hyperlipidemia (FCH), a common cause of premature coronary artery disease, is genetically complex and poorly understood. Recently, a major locus on chromosome 1q21-23 exhibiting highly significant linkage was identified in Finnish FCH families by use of a parametric analysis. We now report highly significant evidence of linkage (maximum LOD score 3.8, recombination fraction 0) of an important FCH phenotype, elevated apolipoprotein B (apoB) levels, to a distinctly separate locus on chromosome 1p31 in Dutch pedigrees. ApoB is the major protein on very low density and low density lipoproteins, and elevated apoB levels have been used as a surrogate trait for FCH. Additional microsatellite markers in the 1p31 region were genotyped, and evidence of linkage improved (maximum LOD score 4.7) in a multipoint analysis of two markers in the peak region. The leptin receptor gene resides within this locus and is involved in obesity and insulin/glucose homeostasis. However, there was no evidence of an association between leptin receptor and apoB levels, raising the possibility that another gene on this chromosomal region contributes to elevated apoB levels in this Dutch population. This is one of the first loci identified for apoB levels in humans and is the second major locus implicated in the genetic etiology of FCH.
{"title":"Locus for Elevated Apolipoprotein B Levels on Chromosome 1p31 in Families With Familial Combined Hyperlipidemia","authors":"H. Allayee, K. Krass, P. Pajukanta, R. Cantor, C. V. D. van der Kallen, R. Mar, J. Rotter, T. D. de Bruin, L. Peltonen, A. Lusis","doi":"10.1161/01.RES.0000015885.27134.F0","DOIUrl":"https://doi.org/10.1161/01.RES.0000015885.27134.F0","url":null,"abstract":"Familial combined hyperlipidemia (FCH), a common cause of premature coronary artery disease, is genetically complex and poorly understood. Recently, a major locus on chromosome 1q21-23 exhibiting highly significant linkage was identified in Finnish FCH families by use of a parametric analysis. We now report highly significant evidence of linkage (maximum LOD score 3.8, recombination fraction 0) of an important FCH phenotype, elevated apolipoprotein B (apoB) levels, to a distinctly separate locus on chromosome 1p31 in Dutch pedigrees. ApoB is the major protein on very low density and low density lipoproteins, and elevated apoB levels have been used as a surrogate trait for FCH. Additional microsatellite markers in the 1p31 region were genotyped, and evidence of linkage improved (maximum LOD score 4.7) in a multipoint analysis of two markers in the peak region. The leptin receptor gene resides within this locus and is involved in obesity and insulin/glucose homeostasis. However, there was no evidence of an association between leptin receptor and apoB levels, raising the possibility that another gene on this chromosomal region contributes to elevated apoB levels in this Dutch population. This is one of the first loci identified for apoB levels in humans and is the second major locus implicated in the genetic etiology of FCH.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"166 2 1","pages":"926-931"},"PeriodicalIF":0.0,"publicationDate":"2002-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78132626","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}
Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300→His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by ≈600 &mgr;g/kg M5 versus ≈1200 &mgr;g/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.
{"title":"Prourokinase Mutant That Induces Highly Effective Clot Lysis Without Interfering With Hemostasis","authors":"Jian-ning Liu, Jianxia Liu, Bei-fang Liu, Ziyong Sun, Jian Zuo, Pei-xiang Zhang, Jing Zhang, Yu-hong Chen, V. Gurewich","doi":"10.1161/01.RES.0000014825.71092.BD","DOIUrl":"https://doi.org/10.1161/01.RES.0000014825.71092.BD","url":null,"abstract":"Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300→His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by ≈600 &mgr;g/kg M5 versus ≈1200 &mgr;g/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"1 1","pages":"757-763"},"PeriodicalIF":0.0,"publicationDate":"2002-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88569089","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 : 2002-04-19DOI: 10.1161/01.RES.0000014434.48463.35
C. Krämer, J. Sunkomat, J. Witte, M. Luchtefeld, M. Walden, B. Schmidt, R. Böger, W. Forssmann, H. Drexler, B. Schieffer
Angiotensin II (Ang II) type 1 receptor (AT1) antagonists such as losartan (LOS) are widely used for the treatment of hypertension and elicit antiinflammatory and antiaggregatory in vitro and in patients, although the underlying mechanism are unclear. Following computer-based molecule similarity, we proposed that on cytochrome-P450 degradation, the LOS metabolite EXP3179 is generated, which shows molecule homology to indomethacin, a cyclooxygenase inhibitor with antiinflammatory and antiaggregatory properties. Subsequently, serum-levels of EXP3179 were determined for 8 hours in patients receiving a single oral dose of 100 mg LOS. High-performance liquid chromatography followed by liquid chromatography–mass spectrometry (LC-MS) from serum samples revealed a maximum of 10−7 mol/L for EXP3179 peaking between 3 to 4 hours. The increase in serum-EXP3179 levels was associated with a significant reduction in platelet aggregation in vivo (−35±4%, P <0.001 versus control). EXP3179 generation was investigated in a chemical reaction mimicking the liver cytochrome-P450–dependent LOS-degradation and human endothelial cells were exposed to Ang II or lipopolysaccharides (LPS) in the presence of EXP3179 (10−7 mol/L). LPS- and Ang II–induced COX-2 transcription was abolished by EXP3179. Moreover, EXP3179 significantly reduced Ang II– and LPS-induced formation of prostaglandin F2&agr; as determined by LC-MS. Thus, antiinflammatory properties of LOS are mediated via its EXP3179 metabolite by abolishing COX-2 mRNA upregulation and COX-dependent TXA2 and PGF2&agr; generation. Serum levels of EXP3179 are detectable in patients in concentrations that exhibit antiinflammatory and antiaggregatory properties in vitro.
{"title":"Angiotensin II Receptor–Independent Antiinflammatory and Antiaggregatory Properties of Losartan: Role of the Active Metabolite EXP3179","authors":"C. Krämer, J. Sunkomat, J. Witte, M. Luchtefeld, M. Walden, B. Schmidt, R. Böger, W. Forssmann, H. Drexler, B. Schieffer","doi":"10.1161/01.RES.0000014434.48463.35","DOIUrl":"https://doi.org/10.1161/01.RES.0000014434.48463.35","url":null,"abstract":"Angiotensin II (Ang II) type 1 receptor (AT1) antagonists such as losartan (LOS) are widely used for the treatment of hypertension and elicit antiinflammatory and antiaggregatory in vitro and in patients, although the underlying mechanism are unclear. Following computer-based molecule similarity, we proposed that on cytochrome-P450 degradation, the LOS metabolite EXP3179 is generated, which shows molecule homology to indomethacin, a cyclooxygenase inhibitor with antiinflammatory and antiaggregatory properties. Subsequently, serum-levels of EXP3179 were determined for 8 hours in patients receiving a single oral dose of 100 mg LOS. High-performance liquid chromatography followed by liquid chromatography–mass spectrometry (LC-MS) from serum samples revealed a maximum of 10−7 mol/L for EXP3179 peaking between 3 to 4 hours. The increase in serum-EXP3179 levels was associated with a significant reduction in platelet aggregation in vivo (−35±4%, P <0.001 versus control). EXP3179 generation was investigated in a chemical reaction mimicking the liver cytochrome-P450–dependent LOS-degradation and human endothelial cells were exposed to Ang II or lipopolysaccharides (LPS) in the presence of EXP3179 (10−7 mol/L). LPS- and Ang II–induced COX-2 transcription was abolished by EXP3179. Moreover, EXP3179 significantly reduced Ang II– and LPS-induced formation of prostaglandin F2&agr; as determined by LC-MS. Thus, antiinflammatory properties of LOS are mediated via its EXP3179 metabolite by abolishing COX-2 mRNA upregulation and COX-dependent TXA2 and PGF2&agr; generation. Serum levels of EXP3179 are detectable in patients in concentrations that exhibit antiinflammatory and antiaggregatory properties in vitro.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"55 1","pages":"770-776"},"PeriodicalIF":0.0,"publicationDate":"2002-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88981343","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 : 2002-04-19DOI: 10.1161/01.RES.0000014966.97486.C0
S. Engelhardt, L. Hein, U. Keller, Kerstin Klämbt, M. Lohse
Chronic stimulation of the &bgr;1-adrenergic receptor leads to hypertrophy and heart failure in &bgr;1-adrenergic receptor transgenic mice and contributes to disease progression in heart failure patients. The cellular mechanisms underlying these detrimental effects are largely unknown. In this study, we have identified the cardiac Na+-H+ exchanger (NHE1) as a novel mediator of adrenergically induced heart failure. &bgr;1-Adrenergic receptor transgenic mice showed upregulation of both NHE1 mRNA (+140±6%) and protein (+42±19%). In order to test whether increased NHE1 is causally related to &bgr;1-adrenergic–induced hypertrophy, fibrosis, and heart failure, &bgr;1-adrenergic receptor transgenic (TG) and wild-type (WT) littermates were treated with a diet containing 6000 ppm of the NHE1 inhibitor cariporide or control chow for 8 months. There was significant hypertrophy of cardiac myocytes in &bgr;1-adrenergic receptor transgenic mice (2.3-fold increase in myocyte cross-sectional area), which was virtually absent in cariporide-fed animals. Interstitial fibrosis was prominent throughout the left ventricular wall in nontreated &bgr;1-adrenergic receptor transgenic mice (4.8-fold increase in collagen volume fraction); cariporide treatment completely prevented this development of fibrosis. Left ventricular catheterization showed that cariporide also prevented the loss of contractile function in &bgr;1-adrenergic receptor transgenic mice: whereas untreated transgenic mice showed a significant decrease in left ventricular contractility (5250±570 mm Hg/s TG versus 7360±540 mm Hg/s WT, dp/dtmax), this decrease was completely prevented by cariporide (8150±520 mm Hg/s TG cariporide). Inhibition of NHE1 prevented the development of heart failure in &bgr;1-receptor transgenic mice. We conclude that the cardiac Na+-H+ exchanger 1 is essential for the detrimental cardiac effects of chronic &bgr;1-receptor stimulation in the heart.
慢性刺激&bgr;1-肾上腺素能受体导致&bgr;1-肾上腺素能受体转基因小鼠肥厚和心力衰竭，并有助于心力衰竭患者的疾病进展。这些有害影响背后的细胞机制在很大程度上是未知的。在这项研究中，我们已经确定了心脏Na+-H+交换器(NHE1)作为肾上腺素能诱导心力衰竭的新介质。1-肾上腺素能受体转基因小鼠NHE1 mRNA表达上调(+140±6%)，蛋白表达上调(+42±19%)。为了测试NHE1升高是否与1-肾上腺素能诱导的肥大、纤维化和心力衰竭有因果关系，我们将转基因(TG)和野生型(WT)幼崽分别喂食含有6000 ppm NHE1抑制剂cariporide或对照饲料8个月。1-肾上腺素能受体转基因小鼠心肌细胞明显肥大(心肌细胞横截面积增加2.3倍)，而在cariporde喂养的动物中几乎没有这种现象。未处理的&bgr;1-肾上腺素能受体转基因小鼠左室壁间质纤维化明显(胶原体积分数增加4.8倍);Cariporide治疗完全阻止了纤维化的发展。左心室导管检查显示，卡利维亚还能防止1-肾上腺素能受体转基因小鼠的收缩功能丧失:未经治疗的转基因小鼠左心室收缩功能显著下降(TG为5250±570 mm Hg/s, WT为7360±540 mm Hg/s, dp/dtmax)，卡利维亚(TG为8150±520 mm Hg/s)完全阻止了这种下降。抑制NHE1可防止&bgr;1受体转基因小鼠心力衰竭的发生。我们得出结论，心脏Na+-H+交换器1对心脏慢性&bgr;1受体刺激的有害心脏效应至关重要。
{"title":"Inhibition of Na+-H+ Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in &bgr;1-Adrenergic Receptor Transgenic Mice","authors":"S. Engelhardt, L. Hein, U. Keller, Kerstin Klämbt, M. Lohse","doi":"10.1161/01.RES.0000014966.97486.C0","DOIUrl":"https://doi.org/10.1161/01.RES.0000014966.97486.C0","url":null,"abstract":"Chronic stimulation of the &bgr;1-adrenergic receptor leads to hypertrophy and heart failure in &bgr;1-adrenergic receptor transgenic mice and contributes to disease progression in heart failure patients. The cellular mechanisms underlying these detrimental effects are largely unknown. In this study, we have identified the cardiac Na+-H+ exchanger (NHE1) as a novel mediator of adrenergically induced heart failure. &bgr;1-Adrenergic receptor transgenic mice showed upregulation of both NHE1 mRNA (+140±6%) and protein (+42±19%). In order to test whether increased NHE1 is causally related to &bgr;1-adrenergic–induced hypertrophy, fibrosis, and heart failure, &bgr;1-adrenergic receptor transgenic (TG) and wild-type (WT) littermates were treated with a diet containing 6000 ppm of the NHE1 inhibitor cariporide or control chow for 8 months. There was significant hypertrophy of cardiac myocytes in &bgr;1-adrenergic receptor transgenic mice (2.3-fold increase in myocyte cross-sectional area), which was virtually absent in cariporide-fed animals. Interstitial fibrosis was prominent throughout the left ventricular wall in nontreated &bgr;1-adrenergic receptor transgenic mice (4.8-fold increase in collagen volume fraction); cariporide treatment completely prevented this development of fibrosis. Left ventricular catheterization showed that cariporide also prevented the loss of contractile function in &bgr;1-adrenergic receptor transgenic mice: whereas untreated transgenic mice showed a significant decrease in left ventricular contractility (5250±570 mm Hg/s TG versus 7360±540 mm Hg/s WT, dp/dtmax), this decrease was completely prevented by cariporide (8150±520 mm Hg/s TG cariporide). Inhibition of NHE1 prevented the development of heart failure in &bgr;1-receptor transgenic mice. We conclude that the cardiac Na+-H+ exchanger 1 is essential for the detrimental cardiac effects of chronic &bgr;1-receptor stimulation in the heart.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"195 1","pages":"814-819"},"PeriodicalIF":0.0,"publicationDate":"2002-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82700903","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 : 2002-04-19DOI: 10.1161/01.RES.0000015592.95986.03
T. Casey, Julian L. Pakay, M. Guppy, P. Arthur
The aim was to identify energy-consuming processes, other than contraction, downregulated during moderate hypoxia (≈5 &mgr;mol/L, 0.5% O2) and severe hypoxia (<0.5 &mgr;mol/L, <0.05% O2) in isolated neonatal cardiomyocytes. The metabolic response of cardiomyocytes to moderate and severe hypoxia was assessed by measuring rates of energy consumption and energetic status of cells maintained under these conditions. We found that the rates of energy production were decreased during both forms of hypoxia. Decreased rates of energy production under moderate hypoxia were associated with reduced energy wastage through a downregulation of proton leak in the mitochondria. Cellular protein synthesis and RNA synthesis, major energy-consuming pathways, were downregulated only during severe hypoxia, when oxygen concentrations were low enough to induce energetic stress (quantitatively defined as being any situation in which phosphocreatine concentrations had fallen by ≥40%). Our results suggest that energetic stress is the signal responsible for this downregulation.
{"title":"Hypoxia Causes Downregulation of Protein and RNA Synthesis in Noncontracting Mammalian Cardiomyocytes","authors":"T. Casey, Julian L. Pakay, M. Guppy, P. Arthur","doi":"10.1161/01.RES.0000015592.95986.03","DOIUrl":"https://doi.org/10.1161/01.RES.0000015592.95986.03","url":null,"abstract":"The aim was to identify energy-consuming processes, other than contraction, downregulated during moderate hypoxia (≈5 &mgr;mol/L, 0.5% O2) and severe hypoxia (<0.5 &mgr;mol/L, <0.05% O2) in isolated neonatal cardiomyocytes. The metabolic response of cardiomyocytes to moderate and severe hypoxia was assessed by measuring rates of energy consumption and energetic status of cells maintained under these conditions. We found that the rates of energy production were decreased during both forms of hypoxia. Decreased rates of energy production under moderate hypoxia were associated with reduced energy wastage through a downregulation of proton leak in the mitochondria. Cellular protein synthesis and RNA synthesis, major energy-consuming pathways, were downregulated only during severe hypoxia, when oxygen concentrations were low enough to induce energetic stress (quantitatively defined as being any situation in which phosphocreatine concentrations had fallen by ≥40%). Our results suggest that energetic stress is the signal responsible for this downregulation.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":"7 1","pages":"777-783"},"PeriodicalIF":0.0,"publicationDate":"2002-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78436726","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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