Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000026056.81424.DA
M. Costell, R. Carmona, E. Gustafsson, M. González-Iriarte, R. Fässler, R. Muñoz-Chápuli
Perlecan is a heparan-sulfate proteoglycan abundantly expressed in pericellular matrices and basement membranes during development. Inactivation of the perlecan gene in mice is lethal at two developmental stages: around E10 and around birth. We report a high incidence of malformations of the cardiac outflow tract in perlecan-deficient embryos. Complete transposition of great arteries was diagnosed in 11 out of 15 late embryos studied (73%). Three of these 11 embryos also showed malformations of semilunar valves. Mesenchymal cells in the outflow tract were abnormally abundant in mutant embryos by E9.5, when the endocardial-mesenchymal transformation starts in wild-type embryos. At E10.5, mutant embryos lacked well-defined spiral endocardial ridges, and the excess of mesenchymal cells obstructed sometimes the outflow tract lumen. Most of this anomalous mesenchyme expressed the smooth muscle cell-specific &agr;-actin isoform, a marker of the neural crest in the outflow tract of the mouse. In wild-type embryos, perlecan is present in the basal surface of myocardium and endocardium, as well as surrounding presumptive neural crest cells. We suggest that the excess of mesenchyme at the earlier stages of conotruncal development precludes the formation of the spiral ridges and the rotation of the septation complex in order to achieve a concordant ventriculoarterial connection. The observed mesenchymal overpopulation might be due to an uncontrolled migration of neural crest cells, which would arrive prematurely to the heart. Thus, perlecan is involved in the control of the outflow tract mesenchymal population size, underscoring the importance of the extracellular matrix in cardiac morphogenesis.
{"title":"Hyperplastic Conotruncal Endocardial Cushions and Transposition of Great Arteries in Perlecan-Null Mice","authors":"M. Costell, R. Carmona, E. Gustafsson, M. González-Iriarte, R. Fässler, R. Muñoz-Chápuli","doi":"10.1161/01.RES.0000026056.81424.DA","DOIUrl":"https://doi.org/10.1161/01.RES.0000026056.81424.DA","url":null,"abstract":"Perlecan is a heparan-sulfate proteoglycan abundantly expressed in pericellular matrices and basement membranes during development. Inactivation of the perlecan gene in mice is lethal at two developmental stages: around E10 and around birth. We report a high incidence of malformations of the cardiac outflow tract in perlecan-deficient embryos. Complete transposition of great arteries was diagnosed in 11 out of 15 late embryos studied (73%). Three of these 11 embryos also showed malformations of semilunar valves. Mesenchymal cells in the outflow tract were abnormally abundant in mutant embryos by E9.5, when the endocardial-mesenchymal transformation starts in wild-type embryos. At E10.5, mutant embryos lacked well-defined spiral endocardial ridges, and the excess of mesenchymal cells obstructed sometimes the outflow tract lumen. Most of this anomalous mesenchyme expressed the smooth muscle cell-specific &agr;-actin isoform, a marker of the neural crest in the outflow tract of the mouse. In wild-type embryos, perlecan is present in the basal surface of myocardium and endocardium, as well as surrounding presumptive neural crest cells. We suggest that the excess of mesenchyme at the earlier stages of conotruncal development precludes the formation of the spiral ridges and the rotation of the septation complex in order to achieve a concordant ventriculoarterial connection. The observed mesenchymal overpopulation might be due to an uncontrolled migration of neural crest cells, which would arrive prematurely to the heart. Thus, perlecan is involved in the control of the outflow tract mesenchymal population size, underscoring the importance of the extracellular matrix in cardiac morphogenesis.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89134167","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-26DOI: 10.1161/01.RES.0000027813.55750.E7
P. Casanello, L. Sobrevia
Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetal development. The effect of IUGR on l-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced l-arginine transport (Vmax= 5.8±0.2 versus 3.3±0.1 pmol/&mgr;g protein per minute), with no significant changes in transport affinity (Km=159±15 versus 137±14 &mgr;mol/L). l-Arginine transport was trans-stimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of l-[3H]citrulline from l-[3H] arginine, lower nitrite and intracellular l-arginine, l-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of l-arginine transport and NO synthesis, and membrane depolarization were reversed by the NO donor S-nitroso-N-acetyl-l,d-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered l-arginine transport and NO synthesis (l-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in l-arginine/NO pathway could be critical for the physiological processes involved in the etiology of IUGR in human pregnancies.
宫内生长迟缓(IUGR)与血管并发症导致缺氧和胎儿发育异常有关。在人脐静脉内皮细胞(HUVECs)培养中，研究了IUGR对l-精氨酸转运和一氧化氮(NO)合成的影响。IUGR与膜去极化和L -精氨酸转运减少有关(Vmax= 5.8±0.2 vs 3.3±0.1 pmol/&mgr;g蛋白每分钟)，运输亲和力无显著变化(Km=159±15 vs 137±14 &mgr; L)。正常妊娠和IUGR妊娠细胞的l-精氨酸转运受到反式刺激(8- 9倍)。IUGR与l-[3H]精氨酸、低亚硝酸盐和细胞内l-精氨酸、l-瓜氨酸和cGMP产生的l-[3H]瓜氨酸减少有关。IUGR降低hCAT-1和hCAT-2B mRNA水平，升高eNOS mRNA和蛋白水平。一氧化氮供体s -亚硝基-n -乙酰- 1,d-青霉胺逆转了iugr相关的l-精氨酸运输和NO合成抑制以及膜去极化。综上所述，IUGR胎儿的内皮细胞表现出l-精氨酸运输和NO合成(l-精氨酸/NO通路)的改变，hCAT-1和hCAT-2B的表达和活性降低，eNOS活性降低。l-精氨酸/NO通路的改变可能对人类妊娠IUGR病因学中涉及的生理过程至关重要。
{"title":"Intrauterine Growth Retardation Is Associated With Reduced Activity and Expression of the Cationic Amino Acid Transport Systems y+/hCAT-1 and y+/hCAT-2B and Lower Activity of Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells","authors":"P. Casanello, L. Sobrevia","doi":"10.1161/01.RES.0000027813.55750.E7","DOIUrl":"https://doi.org/10.1161/01.RES.0000027813.55750.E7","url":null,"abstract":"Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetal development. The effect of IUGR on l-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced l-arginine transport (Vmax= 5.8±0.2 versus 3.3±0.1 pmol/&mgr;g protein per minute), with no significant changes in transport affinity (Km=159±15 versus 137±14 &mgr;mol/L). l-Arginine transport was trans-stimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of l-[3H]citrulline from l-[3H] arginine, lower nitrite and intracellular l-arginine, l-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of l-arginine transport and NO synthesis, and membrane depolarization were reversed by the NO donor S-nitroso-N-acetyl-l,d-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered l-arginine transport and NO synthesis (l-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in l-arginine/NO pathway could be critical for the physiological processes involved in the etiology of IUGR in human pregnancies.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91333725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000026421.61398.F2
A. Shioi, M. Katagi, Y. Okuno, K. Mori, S. Jono, H. Koyama, Y. Nishizawà
Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-&ggr; (IFN-&ggr;) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1&agr;,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of &bgr;-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-&agr; (TNF-&agr;) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-&agr; and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-&ggr; and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-&agr; and OSM.
{"title":"Induction of Bone-Type Alkaline Phosphatase in Human Vascular Smooth Muscle Cells: Roles of Tumor Necrosis Factor-&agr; and Oncostatin M Derived From Macrophages","authors":"A. Shioi, M. Katagi, Y. Okuno, K. Mori, S. Jono, H. Koyama, Y. Nishizawà","doi":"10.1161/01.RES.0000026421.61398.F2","DOIUrl":"https://doi.org/10.1161/01.RES.0000026421.61398.F2","url":null,"abstract":"Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-&ggr; (IFN-&ggr;) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1&agr;,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of &bgr;-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-&agr; (TNF-&agr;) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-&agr; and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-&ggr; and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-&agr; and OSM.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90359166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000026502.79063.66
Ho-Jin Park, Ulrike Begley, Dequan Kong, Haiyan Yu, L. Yin, F. Hillgartner, T. Osborne, J. Galper
We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M2 muscarinic receptor; the &agr;-subunit of the heterotrimeric G protein, G&agr;i2; and the inward rectifying K+ channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of G&agr;i2 promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the G&agr;i2 promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the G&agr;i2 promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative G&agr;i2 SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the G&agr;i2 SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of G&agr;i2 promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of G&agr;i2 promoter activity, suggesting that SREBP1 may play a role in the regulation of G&agr; i2 expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.
{"title":"Role of Sterol Regulatory Element Binding Proteins in the Regulation of G&agr;i2 Expression in Cultured Atrial Cells","authors":"Ho-Jin Park, Ulrike Begley, Dequan Kong, Haiyan Yu, L. Yin, F. Hillgartner, T. Osborne, J. Galper","doi":"10.1161/01.RES.0000026502.79063.66","DOIUrl":"https://doi.org/10.1161/01.RES.0000026502.79063.66","url":null,"abstract":"We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M2 muscarinic receptor; the &agr;-subunit of the heterotrimeric G protein, G&agr;i2; and the inward rectifying K+ channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of G&agr;i2 promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the G&agr;i2 promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the G&agr;i2 promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative G&agr;i2 SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the G&agr;i2 SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of G&agr;i2 promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of G&agr;i2 promoter activity, suggesting that SREBP1 may play a role in the regulation of G&agr; i2 expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75215004","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000026420.22406.79
E. Wijelath, Jacqueline Murray, S. Rahman, Y. Patel, A. Ishida, K. Strand, S. Aziz, Carlos Cardona, W. Hammond, G. Savidge, S. Rafii, M. Sobel
Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, &agr;5&bgr;1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to &agr;5&bgr;1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.
{"title":"Novel Vascular Endothelial Growth Factor Binding Domains of Fibronectin Enhance Vascular Endothelial Growth Factor Biological Activity","authors":"E. Wijelath, Jacqueline Murray, S. Rahman, Y. Patel, A. Ishida, K. Strand, S. Aziz, Carlos Cardona, W. Hammond, G. Savidge, S. Rafii, M. Sobel","doi":"10.1161/01.RES.0000026420.22406.79","DOIUrl":"https://doi.org/10.1161/01.RES.0000026420.22406.79","url":null,"abstract":"Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, &agr;5&bgr;1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to &agr;5&bgr;1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82983569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000024417.79275.23
M. Si, Tony J.‐F. Lee
It has been suggested in isolated porcine cerebral arteries that stimulation by nicotine of &agr;7-nicotinic acetylcholine receptors (&agr;7-nAChRs) on sympathetic nerves, but not direct stimulation of parasympathetic nitrergic nerves, caused nitrergic neurogenic dilation. Direct evidence supporting this hypothesis has not been presented. The present study, which used in vitro tissue bath and confocal microscopy techniques, was designed to determine whether choline, a selective agonist for &agr;7-nAChRs, induced sympathetic-dependent nitrergic dilation of porcine basilar arterial rings. Choline and several nAChR agonists induced exclusive relaxation of basilar arterial rings without endothelium. The relaxation was blocked by tetrodotoxin, nitro-l-arginine, guanethidine, and &bgr;2-adrenoceptor antagonists. Furthermore, the relaxation was blocked by methyllycaconitine and &agr;-bungarotoxin (preferential &agr;7-nAChR antagonists) and mecamylamine but was not affected by dihydro-&bgr;-erythroidine (a preferential &agr;4-nAChR antagonist). Confocal microscopic study demonstrated that choline and nicotine induced significant calcium influx in cultured porcine superior cervical ganglionic cells but failed to affect calcium influx in cultured sphenopalatine ganglionic cells, providing direct evidence that choline and nicotine did not act directly on the parasympathetic nitrergic neurons. The increased calcium influx in superior cervical ganglionic cells was attenuated by &agr;-bungarotoxin and methyllycaconitine but not by dihydro-&bgr;-erythroidine. These results support our hypothesis that activation of &agr;7-nAChRs on cerebral perivascular sympathetic nerves causes calcium influx and the release of norepinephrine, which then act on presynaptic &bgr;2-adrenoceptors located on the neighboring nitrergic nerve terminals, resulting in NO release and vasodilation. Endogenous choline may play an important role in regulating cerebral sympathetic activity and vascular tone.
{"title":"&agr;7-Nicotinic Acetylcholine Receptors on Cerebral Perivascular Sympathetic Nerves Mediate Choline-Induced Nitrergic Neurogenic Vasodilation","authors":"M. Si, Tony J.‐F. Lee","doi":"10.1161/01.RES.0000024417.79275.23","DOIUrl":"https://doi.org/10.1161/01.RES.0000024417.79275.23","url":null,"abstract":"It has been suggested in isolated porcine cerebral arteries that stimulation by nicotine of &agr;7-nicotinic acetylcholine receptors (&agr;7-nAChRs) on sympathetic nerves, but not direct stimulation of parasympathetic nitrergic nerves, caused nitrergic neurogenic dilation. Direct evidence supporting this hypothesis has not been presented. The present study, which used in vitro tissue bath and confocal microscopy techniques, was designed to determine whether choline, a selective agonist for &agr;7-nAChRs, induced sympathetic-dependent nitrergic dilation of porcine basilar arterial rings. Choline and several nAChR agonists induced exclusive relaxation of basilar arterial rings without endothelium. The relaxation was blocked by tetrodotoxin, nitro-l-arginine, guanethidine, and &bgr;2-adrenoceptor antagonists. Furthermore, the relaxation was blocked by methyllycaconitine and &agr;-bungarotoxin (preferential &agr;7-nAChR antagonists) and mecamylamine but was not affected by dihydro-&bgr;-erythroidine (a preferential &agr;4-nAChR antagonist). Confocal microscopic study demonstrated that choline and nicotine induced significant calcium influx in cultured porcine superior cervical ganglionic cells but failed to affect calcium influx in cultured sphenopalatine ganglionic cells, providing direct evidence that choline and nicotine did not act directly on the parasympathetic nitrergic neurons. The increased calcium influx in superior cervical ganglionic cells was attenuated by &agr;-bungarotoxin and methyllycaconitine but not by dihydro-&bgr;-erythroidine. These results support our hypothesis that activation of &agr;7-nAChRs on cerebral perivascular sympathetic nerves causes calcium influx and the release of norepinephrine, which then act on presynaptic &bgr;2-adrenoceptors located on the neighboring nitrergic nerve terminals, resulting in NO release and vasodilation. Endogenous choline may play an important role in regulating cerebral sympathetic activity and vascular tone.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74104471","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000024115.67561.54
S. Jo, V. Leblais, Ping H. Wang, M. Crow, R. Xiao
Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of &bgr;2-adrenoceptor (&bgr;2-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of &bgr;2-AR-coupled Gi proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the &bgr;2-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the &bgr;2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables &bgr;2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in &bgr;2-AR-induced cAMP formation. Blocking Gi or G&bgr;&ggr; signaling with pertussis toxin or &bgr;ARK-ct, a peptide inhibitor of G&bgr;&ggr;, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of &bgr;2-AR-PKA signaling sequentially involves Gi, G&bgr;&ggr;, and PI3K. Thus, PI3K constitutes a key downstream event of &bgr;2-AR-Gi signaling, which confines and negates the concurrent &bgr;2-AR/Gs-mediated PKA signaling.
{"title":"Phosphatidylinositol 3-Kinase Functionally Compartmentalizes the Concurrent Gs Signaling During &bgr;2-Adrenergic Stimulation","authors":"S. Jo, V. Leblais, Ping H. Wang, M. Crow, R. Xiao","doi":"10.1161/01.RES.0000024115.67561.54","DOIUrl":"https://doi.org/10.1161/01.RES.0000024115.67561.54","url":null,"abstract":"Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of &bgr;2-adrenoceptor (&bgr;2-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of &bgr;2-AR-coupled Gi proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the &bgr;2-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the &bgr;2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables &bgr;2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in &bgr;2-AR-induced cAMP formation. Blocking Gi or G&bgr;&ggr; signaling with pertussis toxin or &bgr;ARK-ct, a peptide inhibitor of G&bgr;&ggr;, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of &bgr;2-AR-PKA signaling sequentially involves Gi, G&bgr;&ggr;, and PI3K. Thus, PI3K constitutes a key downstream event of &bgr;2-AR-Gi signaling, which confines and negates the concurrent &bgr;2-AR/Gs-mediated PKA signaling.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74748730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000025268.10302.0C
K. Isoda, Kenichiro Nishikawa, Y. Kamezawa, Mikoto Yoshida, M. Kusuhara, M. Moroi, N. Tada, F. Ohsuzu
Osteopontin (OPN) is a soluble secreted phosphoprotein that binds with high affinity to several integrins and it has been found at the site of atherosclerotic lesions. However, the role of OPN expression in vivo is still poorly understood. To investigate the physiological role of OPN in detail, we generated transgenic mice (Tg) overexpressing the OPN gene under control of the cytomegalovirus enhancer/chicken &bgr;-actin promoter. We detected OPN mRNAs in almost all tissues of 3 lines of Tg mice by Northern blotting. The serum levels of OPN were significantly higher in Tg than in non-Tg mice (782±107 versus 182±44 ng/mL;P <0.001). Compared with non-Tg mice, a 73% (88±6 versus 51±7 &mgr;m;P <0.001) and 94% (126±15 versus 73±11 &mgr;m;P <0.0001) increase in the medial thickness of the aorta was determined in Tg mice at 16 and 32 weeks after birth. However, we found no evidence of inflammatory cells adhering to endothelial cells, intimal hyperplasia, or calcification in any region of Tg mice without artery injury. We then investigated the effect of cuff-induced injury to the femoral artery. The intimal thickening in Tg mice increased 2.9-fold more than that in non-Tg mice (4.9±1.9 versus 1.7±0.4 &mgr;m;P =0.022). The expression of OPN induces both medial thickening without injury and neointimal formation after injury, thus suggesting that OPN plays a role in the development of atherosclerosis, vascular remodeling, and restenosis after angioplasty in vivo.
骨桥蛋白(Osteopontin, OPN)是一种可溶性分泌磷蛋白，与几种整合素具有高亲和力，并且在动脉粥样硬化病变部位被发现。然而，OPN在体内表达的作用仍然知之甚少。为了详细研究OPN的生理作用，我们在巨细胞病毒增强子/鸡&bgr;-肌动蛋白启动子的控制下，培养了过表达OPN基因的转基因小鼠(Tg)。我们用Northern印迹法在3种Tg小鼠的几乎所有组织中检测到OPN mrna。Tg组血清OPN水平显著高于非Tg组(782±107 vs 182±44 ng/mL;P <0.001)。与非Tg小鼠相比，Tg小鼠在出生后16周和32周的主动脉内侧厚度增加了73%(88±6对51±7)和94%(126±15对73±11)P <0.0001)。然而，在没有动脉损伤的Tg小鼠的任何区域，我们没有发现炎症细胞粘附内皮细胞、内膜增生或钙化的证据。然后我们研究了袖带对股动脉损伤的影响。Tg小鼠的内膜增厚比非Tg小鼠增加了2.9倍(4.9±1.9比1.7±0.4;P =0.022)。OPN的表达可诱导未损伤时的内侧增厚和损伤后的内膜形成，提示OPN在体内动脉粥样硬化、血管重塑和血管成形术后再狭窄的发生中发挥作用。
{"title":"Osteopontin Plays an Important Role in the Development of Medial Thickening and Neointimal Formation","authors":"K. Isoda, Kenichiro Nishikawa, Y. Kamezawa, Mikoto Yoshida, M. Kusuhara, M. Moroi, N. Tada, F. Ohsuzu","doi":"10.1161/01.RES.0000025268.10302.0C","DOIUrl":"https://doi.org/10.1161/01.RES.0000025268.10302.0C","url":null,"abstract":"Osteopontin (OPN) is a soluble secreted phosphoprotein that binds with high affinity to several integrins and it has been found at the site of atherosclerotic lesions. However, the role of OPN expression in vivo is still poorly understood. To investigate the physiological role of OPN in detail, we generated transgenic mice (Tg) overexpressing the OPN gene under control of the cytomegalovirus enhancer/chicken &bgr;-actin promoter. We detected OPN mRNAs in almost all tissues of 3 lines of Tg mice by Northern blotting. The serum levels of OPN were significantly higher in Tg than in non-Tg mice (782±107 versus 182±44 ng/mL;P <0.001). Compared with non-Tg mice, a 73% (88±6 versus 51±7 &mgr;m;P <0.001) and 94% (126±15 versus 73±11 &mgr;m;P <0.0001) increase in the medial thickness of the aorta was determined in Tg mice at 16 and 32 weeks after birth. However, we found no evidence of inflammatory cells adhering to endothelial cells, intimal hyperplasia, or calcification in any region of Tg mice without artery injury. We then investigated the effect of cuff-induced injury to the femoral artery. The intimal thickening in Tg mice increased 2.9-fold more than that in non-Tg mice (4.9±1.9 versus 1.7±0.4 &mgr;m;P =0.022). The expression of OPN induces both medial thickening without injury and neointimal formation after injury, thus suggesting that OPN plays a role in the development of atherosclerosis, vascular remodeling, and restenosis after angioplasty in vivo.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79180719","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000024412.24491.CA
F. Jung, J. Haendeler, J. Hoffmann, Agnes Reissner, Elisabeth Dernbach, A. Zeiher, S. Dimmeler
Tyrosine kinase cascades may play a role in the hypoxic regulation of hypoxia-inducible factor (HIF)-1. We investigated the role of tyrosine kinase phosphorylation and of the Shc/Ras cascade on hypoxic HIF-1 stabilization. Exposure of human umbilical vein endothelial cells to hypoxia results in HIF protein stabilization as early as 10 minutes, with a maximum at 3 hours, and also in Shc tyrosine phosphorylation, with a maximum at 10 minutes. To test whether Shc directly mediates hypoxia-induced HIF stabilization, human umbilical vein endothelial cells were transfected with a dominant-negative Shc mutant (dnShc), resulting in significantly reduced HIF protein levels compared with control. Similar results were obtained with cells transfected with dominant-negative Ras, a known downstream effector of Shc. Hypoxia-induced Ras activity was significantly reduced in cells transfected with dnShc compared with control levels, indicating that Ras indeed acts downstream from Shc. Moreover, cells pretreated with a specific Raf-1 kinase inhibitor, a known downstream effector of Ras, exhibited reduced HIF protein levels. To examine the functional consequences of Shc in hypoxic signaling, HIF-1 ubiquitination, protein stabilization, and endothelial cell migration were assessed. Overexpression of dnShc increased ubiquitination of HIF-1 and reduced the half-life of the protein. Moreover, dnShc, dominant-negative Ras, or the Raf-1 kinase inhibitor significantly inhibited migration under hypoxia. Thus, Shc in concert with Ras and Raf-1 contributes to hypoxia-induced HIF-1&agr; protein stabilization and endothelial cell migration.
{"title":"Hypoxic Induction of the Hypoxia-Inducible Factor Is Mediated via the Adaptor Protein Shc in Endothelial Cells","authors":"F. Jung, J. Haendeler, J. Hoffmann, Agnes Reissner, Elisabeth Dernbach, A. Zeiher, S. Dimmeler","doi":"10.1161/01.RES.0000024412.24491.CA","DOIUrl":"https://doi.org/10.1161/01.RES.0000024412.24491.CA","url":null,"abstract":"Tyrosine kinase cascades may play a role in the hypoxic regulation of hypoxia-inducible factor (HIF)-1. We investigated the role of tyrosine kinase phosphorylation and of the Shc/Ras cascade on hypoxic HIF-1 stabilization. Exposure of human umbilical vein endothelial cells to hypoxia results in HIF protein stabilization as early as 10 minutes, with a maximum at 3 hours, and also in Shc tyrosine phosphorylation, with a maximum at 10 minutes. To test whether Shc directly mediates hypoxia-induced HIF stabilization, human umbilical vein endothelial cells were transfected with a dominant-negative Shc mutant (dnShc), resulting in significantly reduced HIF protein levels compared with control. Similar results were obtained with cells transfected with dominant-negative Ras, a known downstream effector of Shc. Hypoxia-induced Ras activity was significantly reduced in cells transfected with dnShc compared with control levels, indicating that Ras indeed acts downstream from Shc. Moreover, cells pretreated with a specific Raf-1 kinase inhibitor, a known downstream effector of Ras, exhibited reduced HIF protein levels. To examine the functional consequences of Shc in hypoxic signaling, HIF-1 ubiquitination, protein stabilization, and endothelial cell migration were assessed. Overexpression of dnShc increased ubiquitination of HIF-1 and reduced the half-life of the protein. Moreover, dnShc, dominant-negative Ras, or the Raf-1 kinase inhibitor significantly inhibited migration under hypoxia. Thus, Shc in concert with Ras and Raf-1 contributes to hypoxia-induced HIF-1&agr; protein stabilization and endothelial cell migration.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85137372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-07-12DOI: 10.1161/01.RES.0000023391.40106.A8
C. Tiruppathi, M. Freichel, S. Vogel, B. Paria, D. Mehta, V. Flockerzi, A. Malik
We investigated the possibility that the TRPC gene family of putative store-operated Ca2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4−/−) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4−/− LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+ transient secondary to influx of Ca2+. Ca2+ influx activated by thrombin was blocked by La3+ (1 &mgr;mol/L). In TRPC4−/− LECs, thrombin or TFLLRNPNDK-NH2 produced a similar initial increase of intracellular Ca2+ secondary to Ca2+ store depletion, but Ca2+ influx induced by these agonists was drastically reduced. The defect in Ca2+ influx in TRPC4−/− endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (Kf,c), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4−/−; La3+ (1 &mgr;mol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4−/−. These results show that TRPC4-dependent Ca2+ entry in mouse LECs is a key determinant of increased microvascular permeability.
{"title":"Impairment of Store-Operated Ca2+ Entry in TRPC4−/− Mice Interferes With Increase in Lung Microvascular Permeability","authors":"C. Tiruppathi, M. Freichel, S. Vogel, B. Paria, D. Mehta, V. Flockerzi, A. Malik","doi":"10.1161/01.RES.0000023391.40106.A8","DOIUrl":"https://doi.org/10.1161/01.RES.0000023391.40106.A8","url":null,"abstract":"We investigated the possibility that the TRPC gene family of putative store-operated Ca2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4−/−) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4−/− LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+ transient secondary to influx of Ca2+. Ca2+ influx activated by thrombin was blocked by La3+ (1 &mgr;mol/L). In TRPC4−/− LECs, thrombin or TFLLRNPNDK-NH2 produced a similar initial increase of intracellular Ca2+ secondary to Ca2+ store depletion, but Ca2+ influx induced by these agonists was drastically reduced. The defect in Ca2+ influx in TRPC4−/− endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (Kf,c), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4−/−; La3+ (1 &mgr;mol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4−/−. These results show that TRPC4-dependent Ca2+ entry in mouse LECs is a key determinant of increased microvascular permeability.","PeriodicalId":10314,"journal":{"name":"Circulation Research: Journal of the American Heart Association","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83069554","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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