摘要:

&NA;Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP‐dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG‐I alpha or the constitutively active catalytic domain of PKG‐I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin‐1 protein compared with control‐transfected PKG‐deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO‐cGMP signaling to inhibit VSMC migration and phenotypic modulation. (Circ Res. 1998;82:139‐146.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Wild‐type p53 (wt‐p53), a key protein in cell cycle regulation, inactivates the G1cyclins through direct activation of p21Waf‐1/Cip‐1/Sdi‐1. Persistent vascular smooth muscle cell (VSMC) proliferation following vascular interventions hinders the benefits of these therapeutics. Using the hemagglutinating virus of Japan/liposome‐mediated gene transfer method, we examined the inhibitory effect of overexpression of exogenous wt‐p53 on VSMC proliferation in vitro and in vivo. We assessed the proliferative activity of human p53 cDNA‐transduced bovine VSMCs by DNA synthesis assay, flow cytometry, and cell proliferation assay. p53 gene transfer reduced thymidine incorporation of VSMCs stimulated by platelet‐derived growth factor‐BB (P<.001). The p53‐transduced VSMCs underwent synthetic phase depletion (mean, 8.02% versus 33.7% of control; P<.001) and transient G2/M accumulation 2 days after gene transfection, and in almost all cells, G1arrest occurred (mean, 92.6% versus 79.3% of control; P<.001) 5 days later. The wt‐p53 gene transfection also inhibited the VSMC proliferation (P<.001) with no detectable induction of apoptosis. Cell death of p53‐transduced VSMCs was induced only by additional treatment with an apoptosis‐stimulating reagent, doxorubicin. The verification of apoptosis was made by DNA ladder, flow cytometry, and electron microscopy. In vivo transfection of p53 cDNA inhibited neointimal formation after balloon injury in rabbit carotid arteries, without apoptotic stimuli (P<.01). Thus, overexpression of the p53 gene in the injured arterial wall inhibits the proliferation of VSMCs in vitro and in vivo. This novel concept, including not only exogenous but also endogenous p53 overexpression in the vessel wall, may be one approach worth exploring in the treatment of patients with restenosis occurring after vascular interventions. (Circ Res. 1998;82:147‐156.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intracellular signaling mechanisms are still incompletely understood. We investigated the hypothesis that VSMCs binding to fibronectin activates the protein kinase C (PKC) pathway, causes differential intracellular PKC isoform translocation, and mediates cell spreading. VSMCs binding to poly‐L‐lysine or preincubated with Arg‐Gly‐Asp (RGD) peptides were used as controls. Diacylglycerol (DAG) and phospholipase D (PLD) activity were measured by thin‐layer chromatography. Intracellular distribution of PKC isoforms was assessed by confocal microscopy. VSMCs binding to fibronectin induced focal adhesions and cell spreading within 30 minutes. Fibronectin induced a rapid increase in DAG content, peaking at 10 minutes with a sustained response for <1 hour. In contrast, PLD activity was not influenced by specific binding to fibronectin. PKC isoforms alpha, delta, epsilon, and zeta were assessed by confocal microscopy. Fibronectin induced a PKC isoform translocation to the cell nucleus and to focal adhesions within minutes. The nuclear PKC alpha immunoreactivity was transiently increased. PKC isoforms alpha and epsilon were both translocated to focal adhesions. The intracellular distributions of other PKC isoforms were not influenced by fibronectin. The effects of fibronectin on DAG generation, the translocation of PKC alpha and PKC epsilon, and cell spreading were all abolished by the incubation with RGD peptides. Downregulation of PKC isoforms alpha and epsilon with specific antisense oligodinucleotides resulted in a significant inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform alpha is translocated to the nucleus, whereas PKC isoforms alpha and epsilon are translocated to focal adhesions. Both isoforms seem to play a role in inside‐out integrin signaling and cell spreading. (Circ Res. 1998;82:157‐165.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Carvedilol, a new vasodilating beta‐adrenoceptor antagonist and a potent antioxidant, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Recent clinical studies in patients with heart failure have demonstrated that carvedilol reduces morbidity and mortality and inhibits cardiac remodeling. The present study was designed to explore whether the protective effects of carvedilol on the ischemic myocardium include inhibition of apoptosis of cardiomyocytes and, if so, to determine its mechanism of action. Anesthetized rabbits were subjected to 30 minutes of coronary artery occlusion followed by 4 hours of reperfusion. Detection of apoptosis of cardiomyocytes was based on the presence of nucleosomal DNA fragments on agarose gels (DNA ladder) and in situ nick end labeling. Carvedilol (1 mg/kg IV), administered 5 minutes before reperfusion, reduced the number of apoptotic myocytes in the ischemic area from 14.7 +/‐ 0.4% to 3.4 +/‐ 1.8% (77% reduction, P<.001). Propranolol, administered at equipotent beta‐blocking dosage, reduced the number of apoptotic myocytes to 8.9 +/‐ 2.1% (39% reduction, P<.05). DNA ladders were observed in the hearts of all six vehicle‐treated rabbits but only one of six carvedilol‐treated rabbits (P<.01). Immunocytochemical analysis of rabbit hearts demonstrated an upregulation of Fas protein in ischemic cardiomyocytes, and treatment with carvedilol reduced both the intensity of staining as well as the area stained. Myocardial ischemia/reperfusion led to a rapid activation of stress‐activated protein kinase (SAPK) in the ischemic area but not in nonischemic regions. SAPK activity was increased from 2.1 +/‐ 0.3 mU/mg (basal) to 8.9 +/‐ 0.8 mU/mg after 30 minutes of ischemia followed by 20 minutes of reperfusion. Carvedilol inhibited the activation of SAPK by 53.4 +/‐ 6.5% (P<.05). Under the same conditions, propranolol (1 mg/kg) had no effect on SAPK activation. Taken together, these results suggest that carvedilol prevents myocardial ischemia/reperfusion‐induced apoptosis in cardiomyocytes possibly by downregulation of the SAPK signaling pathway, by inhibition of Fas receptor expression, and by beta‐adrenergic blockade. The former two actions represent novel and important mechanisms that may contribute to the cardioprotective effects of carvedilol. (Circ Res. 1998;82:166‐174.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti‐RyR antibodies showed a patchy network‐like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide‐stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti‐RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR‐Ca2+‐releasechannels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca2+content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5‐trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist‐induced Ca2+‐releasechannels and origins of Ca2+sparks, Ca2+oscillations, and Ca2+waves. (Circ Res. 1998;82:175‐185.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOS‐/‐). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOS‐/‐ mice. Aortic endothelial cells derived from eNOS‐/‐ mice displayed only background levels of NOxformation compared with wild‐type (WT) cells (88 versus 1990 pmol NOx[center dot] h‐1/mg protein‐1). eNOS‐/‐ mice were hypertensive (mean arterial pressure, 135 +/‐ 15 versus 107 +/‐ 8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff‐perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with Nomega‐nitro‐L‐argininemethyl ester (L‐NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, ‐35%) in WT hearts was followed by sustained vasodilation (+190%). L‐NAME significantly reduced vasodilation in WT hearts (+125%) but did not alter the initial vasoconstriction. In eNOS‐/‐ hearts, the initial vasoconstriction was augmented (‐70%), whereas the ACh‐induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh‐induced vasodilation into vasoconstriction (‐49% decrease of basal flow). This effect was even more pronounced in eNOS‐/‐ hearts (‐71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOS‐/‐ mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) prostaglandins are the main mediators of the ACh‐induced vasodilation in both WT and eNOS‐/‐ mice. (Circ Res. 1998;82:186‐194.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Endocardial endothelium and endothelium of coronary vessels produce NO. Histochemical methods have suggested that coronary arterial endothelial cells contain more endothelial constitutive NO synthase (ecNOS) than does coronary venous endothelium. We have further investigated the distribution of ecNOS in cardiac endothelium using immunofluorescence and en face confocal microscopy of rat heart. In endocardial endothelium, confocal microscopy revealed distinct ecNOS labeling of peripheral cell borders, cytoplasmic labeling, and labeling of the Golgi complexes. Labeling of the cell borders and of the Golgi complexes was confirmed by double staining for ecNOS and for platelet and endothelial cell adhesion molecule or Golgi 58k protein, respectively. Cytoplasmic labeling was strongest in coronary arterial endothelium. The size of the ecNOS‐labeled Golgi complexes decreased from coronary arterial endothelial cells (8.63 +/‐ 0.39 [micro sign]m2, mean +/‐ SE of 5 rats) to endocardial endothelium (7.07 +/‐ 0.61 [micro sign]m2) and to coronary venous endothelium (3.65 +/‐ 0.20 [micro sign]m2). In addition, pixel intensity of ecNOS labeling was higher in arterial endothelial cells than in venous endothelial cells. Endothelium of myocardial capillaries also contained small ecNOS‐labeled Golgi complexes. No correlation was observed between endothelial cell surface area and Golgi complex size. Caveolin‐1 labeling was strongest in capillaries and did not coincide completely with ecNOS labeling in endocardial and venous endothelium. These results suggest that endocardial and coronary arterial endothelium in the rat have a higher synthetic activity and might express more ecNOS than is expressed by cardiac venous and capillary endothelium. The observed heterogeneity in ecNOS distribution might be related to the specific mechanochemical environment and function of each endothelial compartment. (Circ Res. 1998;82:195‐203.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Vascular smooth muscle cells (VSMCs) as well as macrophages have been shown to generate a substantial amount of NO in inflammatory vascular lesions. Prostaglandin (PG) D2(PGD2) is produced by inflammatory cells, including mast cells and macrophages. We investigated whether PGD2modulates NO metabolism in rat VSMCs. PGD2at a concentration of 10‐7mol/L or greater dose‐dependently inhibited nitrite accumulation in the medium of cultured VSMCs stimulated with interleukin 1 beta (IL‐1 beta). In a dose‐response analysis of IL‐1 beta and nitrite accumulation, PGD2was seen to decrease the maximal ability of VSMCs to generate NO, arguing against competition by PGD2at cytokine receptors. Northern analysis showed that PGD2suppresses induction of inducible NO synthase (iNOS) mRNA in IL‐1 beta‐stimulated VSMCs, with consequent inhibition of iNOS protein expression in Western analysis. A thromboxane A2(TXA2) analogue, U46619 (10‐5mol/L), produced less inhibition of NO generation than did PGD2. Neither the PGI2analog carbaprostacyclin nor PGE1showed any inhibition. PGD2dose‐dependently inhibited NO generation despite the addition of the TXA2antagonist SQ29548. PGJ2, Delta12‐PGJ2, and 15‐deoxy‐Delta12,14‐PGJ2, all metabolites of PGD2, were as potent as or slightly stronger than PGD2in the inhibition of NO generation. These data suggest that PGD2suppresses NO generation in VSMCs by inhibiting iNOS mRNA expression, most likely through the cascade of the PGJ2series rather than through the TX receptor or cAMP upregulation. Such action makes it likely that PGD2regulates NO metabolism in vascular lesions. (Circ Res. 1998;82:204‐209.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Pressure‐induced activation of vascular smooth muscle may involve electromechanical as well as nonelectrome‐chanical coupling mechanisms. We compared calcium‐tone relations of cannulated rat mesenteric small arteries during pressure‐induced activation, depolarization (16 to 46 mmol/L K+), and alpha1‐adrenergicstimulation (1 [micro sign]mol/L phenylephrine). The intracellular calcium concentration was expressed as the fura‐2 ratio, normalized to the maximal and minimal ratios. In order to compare activation levels at various pressures, tone was expressed as the ratio of active wall tension to the maximal active tension. The passive and maximal active pressure‐diameter relations needed for the calculation of tone were determined in a separate set of experiments, using isometric loading of cannulated vessels. Pressure steps from 20 to 60 and then to 100 mm Hg caused a modest rise of calcium. Nifedipine (1 [micro sign]mol/L) blocked both the calcium rise and the resulting myogenic responses. Electromechanical coupling could not fully account for the myogenic response: the calcium sensitivity, defined as the slope of the calcium‐tone relation, was five times higher during pressure‐induced activation compared with potassium stimulation and twice as high as the sensitivity during alpha1‐adrenergicstimulation. We therefore conclude that the myogenic response involves a small but necessary rise in calcium due to influx through L‐type calcium channels, as well as a nonelectromechanical coupling mechanism that greatly enhances the calcium sensitivity of the contractile machinery. (Circ Res. 1998;82:210‐220.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular morphogenesis. For this purpose, we performed long‐term lineage tracing using quail‐chick chimera techniques and premigratory NCC infection with a replication‐incompetent retrovirus containing the LacZ reporter gene in combination with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the anterior cardinal veins was also determined to be NC‐derived. NC‐derived elastic arteries differed from non‐NC elastic vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating a muscular or elastic artery. Boundaries between NC‐infested areas and mesodermal vessel structures were mostly very sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in the aortic and pulmonary trunks. (Circ Res. 1998;82:221‐231.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID