摘要:

Our previous studies have demonstrated that the introduction of angiotensin II type I receptor antisense (AT1R-AS) cDNA by a retrovirally mediated delivery system prevents the development of hypertension in the spontaneously hypertensive rat (SHR), an animal model for primary hypertension in humans. These results have led us to propose the hypothesis that an interruption of the renin-angiotensin system (RAS) activity at a genetic level would prevent hypertension on a permanent basis. F1and F2generations of offspring from a retroviral vector, LNSV- and LNSV-AT1R-AS–treated SHR, were generated, and various physiological parameters indicative of hypertension were studied and compared with those of their parents to investigate this hypothesis. Both F1and F2generations of LNSV-AT1R-AS–treated SHR expressed a persistently lower blood pressure, decreased cardiac hypertrophy and fibrosis, decreased medial thickness, and normalization of renal artery excitation-contraction coupling, Ca2+current, and [Ca2+]iwhen compared with offspring derived from the LNSV-treated SHR. In fact, the magnitude of the prevention of these pathophysiological alterations was similar to that observed in the LNSV-AT1R-AS–treated SHR parent. The prevention of cardiovascular pathophysiology and expression of normotensive phenotypes are, at least in part, a result of integration and subsequent transmission of AT1R-AS from the SHR parents to offspring. These data demonstrate that a single intracardiac injection of LNSV-AT1R-AS causes a permanent cardiovascular protection against hypertension as a result of a genomic integration and germ line transmission of the AT1R-AS in the SHR offspring. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The NH2-terminal decapeptide of &agr;-skeletal actin that contains a primary sequence specific for this isoform was used to raise a polyclonal antibody in rabbits. Using sequential affinity chromatography, we recovered from serum antibodies reacting exclusively with &agr;-skeletal actin when tested by immunoblotting and immunofluorescence. Epitope mapping by means of competition assays with synthetic peptides indicated that the acetyl group and the first 9 amino acids are essential for specificity. The monospecific antibody was then used to investigate the distribution of &agr;-skeletal actin in the myocardium of newborn and normal or hypertensive (with or without fibrotic areas) adult rats. Immunostaining of normal heart revealed that &agr;-skeletal actin is diffusely distributed within practically all myocardial fibers of the newborn rat, whereas it is restricted to a small proportion of adult rat cardiomyocytes, which appear intensely stained. A correlation, albeit not complete, was found between the distribution of &agr;-skeletal actin and &bgr;-myosin heavy chain. During cardiac hypertrophy induced by aortic ligature between the renal arteries, the expressions of &agr;-skeletal actin mRNA and protein were increased. The distribution of immunostaining had a focal pattern similar to that of normal adult rats, reactive fibers being more numerous and more intensely stained compared with normal myocardium. Positive fibers were particularly abundant at the periphery of fibrotic areas. Using this antibody, we have demonstrated for the first time the differential distribution of &agr;-skeletal actin in heart tissues. Changes in the distribution of this isoform in hypertrophic heart provide new insight into the mechanisms by which the heart adapts to work overload. This antibody will prove useful in exploring the mechanisms of expression of &agr;-skeletal actin and in defining its role in physiological and pathological situations. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

We have examined how different degrees (0.5%, 1.0%, 2.5%, 5.0%, and 10.0%) and directions of stretch regulate the turnover and accumulation of contractile proteins in cultured neonatal cardiac myocytes (NCMs). In pulse-chase experiments, stellate-shaped NCMs with random arrays of myofibrils (MFs) exhibited a threshold response to stretch. With respect to unstretched controls, the turnover of the contractile protein pool was suppressed 50% to 100% in stellate NCMs stretched 1.0% to 5.0% and was unaltered in stellate NCMs stretched 0.5% or 10.0%. The posttranslational metabolism of myosin heavy chain (MHC) and actin was regulated in parallel with the total contractile protein pool. The turnover of the cytoplasmic protein pool remained unchanged in response to stretch. NCMs plated onto an aligned matrix of type I collagen expressed an elongated, rod-like cell shape. The MFs of these cells were distributed in parallel with one another along a single unique axis. The tissue-like pattern of organization of these cultures made it possible to assay how specific directions of stretch affected cardiac protein turnover and MF organization. In pulse-chase experiments, stretch in parallel with the MFs did not alter the turnover of the total contractile protein pool, the cytoplasmic protein pool, MHC, or actin. The total cellular concentration of MHC and actin remained constant, and MF alignment was not overtly affected. In contrast, even modest degrees of stretch across the short axis of the MFs suppressed total contractile protein turnover, the turnover of MHC and actin, and promoted the accumulation of these MF subunits. The parallel alignment of MFs deteriorated in myocytes stretched greater than 5%. The characteristic response of aligned myocytes to stretch was not affected by the contractile state of the cells. Isoproterenol (ISO) treatment in concert with stretch in parallel with the MFs modestly accelerated contractile protein turnover. Conversely, contractile protein turnover was suppressed in cells treated with ISO and stretched across the short axis of the MFs. Contractile arrest with nifedipine (NIFED) accelerated total myofibrillar protein turnover. Stretch across the short axis, but not in parallel with the MFs, suppressed protein turnover in cells treated with NIFED. The turnover of the cytosolic proteins remained constant under all conditions assayed. These data suggest that specific directions of stretch may play a crucial role in regulating MF organization and the metabolism of contractile proteins in the cardiac myocyte. The full text of this article is availabale at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

This study was designed to investigate whether insulin-like growth factor-1 (IGF-1) transduces signaling through the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and to assess the upstream signals of serine and tyrosine phosphorylation of STAT family proteins. Primary cultured neonatal rat cardiomyocytes were stimulated with IGF-1 (10−8mol/L). JAK1, but not JAK2 or Tyk2, was phosphorylated by IGF-1 as early as 2 minutes and peaked at 5 minutes. IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3. Tyrosine phosphorylation of STAT1 peaked at 15 minutes and correlated with that of JAK1, whereas that of STAT3 was sustained up to 120 minutes and was dissociated from the activation of JAK1. Tyrosine phosphorylation of STAT3 was unaffected by the preincubation with CV11974 (AT1blocker), TAK044 (endothelin-1 receptor blocker), RX435 (anti-gp130 blocking antibody), PD98058, wortmannin, EDTA, or KN62 but was significantly attenuated by BAPTA-AM and chelerythrine. The time course of a gel mobility shift of SIE (sis-inducing element) coincided with the phosphorylation of STAT3. Serine phosphorylation of STAT1 peaked at 30 minutes and that of STAT3 was observed from 5 to 60 minutes. These results indicated that (1) IGF-1 activated JAK1 but not JAK2 or Tyk2 in rat cardiomyocytes; (2) IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3; and (3) the tyrosine phosphorylation of STAT3 was not caused by JAK1 alone, and protein kinase C and intracellular Ca2+were required for phosphorylation.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Nonesterified fatty acids (NEFAs) are acutely liberated during lipolysis and are chronically elevated in pathological conditions, such as insulin resistance, hypertension, and obesity, which are known risk factors for atherosclerosis. The purpose of this study was to investigate the effect and mechanism of action of NEFAs on the epithelial growth factor (EGF) receptor (EGFR). In the ECV-304 endothelial cell line, unsaturated fatty acids triggered a time- and dose-dependent tyrosine phosphorylation of EGFR (polyunsaturated fatty acids [PUFAs] were the most active), whereas saturated FAs were inactive. Although less potent than PUFAs, oleic acid (OA) was used because it is prominent in the South European diet and is only slightly oxidizable (thus excluding oxidation derivatives). EGFR is activated by OA independent of any autocrine secretion of EGF or other related mediators. OA-induced EGFR autophosphorylation triggered EGFR signaling pathway activation (as assessed through coimmunoprecipitation of SH2 proteins such as SHC, GRB2, and SHP-2) and subsequent p42/p44 mitogen-activated protein kinase (as shown by the use of EGFR- deficient B82L and EGFR- transduced B82LK+cell lines). OA induced in vitro both autophosphorylation and activation of intrinsic tyrosine kinase of immunopurified EGFR, thus suggesting that EGFR is a primary target of OA. EGFR was also activated by mild surfactants, Tween-20 and Triton X-100, both in vitro (on immunopurified EGFR) and in intact living cells, thus indicating that EGFR is sensitive to amphiphilic molecules. These data suggest that EGFR is activated by OA and PUFAs, acts as a sensor for unsaturated fatty acids (and amphiphilic molecules), and is a potential transducer by which diet composition may influence vascular wall biology.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Through the genotype/phenotype cosegregation analysis of an F2intercross, from the crossbreeding of stroke-prone spontaneously hypertensive rats (SHRSP) and stroke-resistant spontaneously hypertensive rats (SHR), we previously identified a quantitative trait locus for stroke on rat chromosome 5 (STR2) that colocalized with the genes encoding atrial and brain natriuretic peptides (ANPandBNP) and conferred a stroke-delaying effect. To further characterizeANPandBNPas candidates for stroke, we performed additional studies. Comparative sequence analysis revealed point mutations in both the coding and regulatory regions ofANP, whereas no interstrain differences were found forBNP. In in vitro studies in COS-7 and AtT-20 cells that were performed to test the relevance of a G→A substitution at position 1125, a Gly→Ser transposition in the SHRSP pro-ANP peptide resulted in different posttranslational processing of the SHRSP ANP gene product that was also associated with higher cGMP production (P<0.05). Furthermore, an analysis of a 5′ end mutation affecting a PEA2 regulatory binding site in the 5′ untranslated regulatory sequence of SHRSPANPdemonstrated a significantly lowerANPpromoter activation in endothelial cells (P<0.05 versus the SHRANP). In addition, the expression ofANPwas significantly reduced in the brain, but not in the atria, of SHRSP compared with SHR (P<0.0001). No differences were detected with regard toBNPexpression. The present results reveal substantial differences inANP, but notBNP, structure and product among SHR and SHRSP, which supports a role ofANPin the pathogenesis of stroke in the SHRSP animal model.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and its tissue inhibitor (TIMP-2) are mainly known for their roles in the (patho)physiological remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. A mechanism of action of MMP-2 is the proteolytic breakdown of specific extracellular matrix proteins. The amino acid sequences in interstitial collagen (Gly-Leu/Ile) and laminin-5 (Ala-Leu) that are cleaved by MMP-2 are homologous to a region (Gly32-Leu33) within human big endothelin-1[1 to 38] (big ET-1). Big ET-1 requires cleavage to an active form to produce vasoconstriction. We tested the hypothesis that vascular MMP-2 can cleave big ET-1, thus generating a vasoconstrictor peptide. In perfused rat mesenteric arteries with an intact endothelium, inhibition of vascular MMP-2 with TIMP-2 reduced (by 16.2±4.2%) the vasoconstrictor effects of big ET-1 (50 pmol). However, when the endothelium was mechanically removed, TIMP-2 abolished (>90%) the vasoconstriction of big ET-1, and this effect was mimicked by an anti–MMP-2 antibody. Incubation of big ET-1 with recombinant human MMP-2 resulted in the specific cleavage of the Gly32-Leu33bond of big ET-1. Moreover, the resultant peptide ET-1[1 to 32] exerted greater vasoconstrictor effects than big ET-1. We conclude that vascular MMP-2 contributes to the vasoconstrictor effects of big ET-1 by cleaving big ET-1 to yield a novel and potent vasoconstrictor, ET-1[1 to 32]. These data implicate, for the first time, the endogenous MMP-2/TIMP-2 system in the regulation of vascular reactivity.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Moyamoya disease is a progressive cerebrovascular occlusive disease that primarily affects children. The cause is unknown. We examined the production of prostanoids and the expression of cyclooxygenase-2 (COX-2) in cultured arterial smooth muscle cells (SMCs) derived from patients with moyamoya disease. Twelve moyamoya and 8 control cell strains were examined. The steady-state levels of prostanoids in the culture medium did not differ between moyamoya and control SMCs. When the cells were stimulated with interleukin-1&bgr; (IL-1&bgr;), prostaglandin E2(PGE2) release into the medium was significantly greater from moyamoya SMCs than from control SMCs, whereas the amounts of prostacyclin and thromboxane B2did not differ. IL-1&bgr;–induced PGE2production by moyamoya SMCs was completely blocked by the addition of indomethacin or NS-398. IL-1&bgr; significantly stimulated cell migration and DNA synthesis in control SMCs but had an inhibitory effect on moyamoya SMCs. The inhibitory effects on the growth and migration of moyamoya SMCs were caused by excessive secretion of PGE2and was reversed with indomethacin treatment. Immunofluorescence studies and Western blot analysis showed greater amounts of COX-2 protein expression in IL-1&bgr;–stimulated moyamoya SMCs. These findings suggest that moyamoya SMCs respond to inflammatory stimuli to produce excess amounts of PGE2through the activation of COX-2, which increases vascular permeability and decreases vascular tone. This facilitates the exposure of vessels to blood constituents and promotes the development of intimal thickening in moyamoya disease.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID