ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Gene transfer to blood vessels is a promising new approach to the treatment of the vascular diseases, but the feasibility of gene transfer to adult human vessels has not been explored. We introduced an adenovirus vector encoding a marker gene human placental alkaline phosphatase into normal and atherosclerotic human vessels in organ culture. In the normal vessels, recombinant gene was expressed preferentially in the endothelial cells ([approximate]100%), intimal smooth muscle cells (1.3 +/- 0.4%, 1.4 +/- 1.0%, and 3.8 +/- 0.8% in the internal mammary arteries, saphenous veins, and normal coronary arteries, respectively), and various adventitial cells. Advanced, complicated atherosclerotic plaques demonstrated a similar efficiency of recombinant gene expression (3.1 +/- 0.5% and 3.8 +/- 0.3% of nonendothelial intimal cells in the coronary artery and carotid artery plaques, respectively). Of these intimal cells, macrophages and smooth muscle cells expressed a transgene, identifying them as targets for gene transfer. Areas of plaque rupture and thrombus are sites of predilection for expression of recombinant genes. Collagenase and elastase treatment increased the percentage of transgenic alkaline phosphatase-positive cells 7 times (P<0.001), suggesting that the pattern of gene expression was affected by the amount of surrounding extracellular matrix. These studies demonstrate the feasibility of gene transfer to human blood vessels. However, these studies also highlight important barriers to adenoviral gene delivery to the actual normal and atherosclerotic human vessels of clinical interest. (Circ Res. 1998;82:1243-1252.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

or=to4x109plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in beta-galactosidase expression through 2x1010pfu/mL, whereas in in vivo arteries, titers above 4x109pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time- and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1x1011pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury. (Circ Res. 1998;82:1253-1262.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Inhibition of NO synthesis has recently been shown to increase oxygen extraction in vivo, and NO has been proposed to play a significant role in the regulation of oxygen consumption by both skeletal and cardiac muscle in vivo and in vitro. It was our aim to determine whether NO also has such a role in the kidney, a tissue with a relatively low basal oxygen extraction. In chronically instrumented conscious dogs, administration of an inhibitor of NO synthase, nitro-L-arginine (NLA, 30 mg/kg IV), caused a maintained increase in mean arterial pressure and renal vascular resistance and a decrease in heart rate (all P<0.05). At 60 minutes, urine flow rate and glomerular flow rate decreased by 44 +/- 12% and 45 +/- 7%, respectively; moreover, the amount of sodium reabsorbed fell from 16 +/- 1.7 to 8.5 +/- 1.1 mmol/min (all P<0.05). At this time, oxygen uptake and extraction increased markedly by 115 +/- 37% and 102 +/- 34%, respectively (P<0.05). Oxygen consumption also significantly increased from 4.5 +/- 0.6 to 7.1 +/- 0.9 mL O2/min. Most important, the ratio of oxygen consumption to sodium reabsorbed increased dramatically from 0.33 +/- 0.07 to 0.75 +/- 0.11 mL O2/mmolNa+(P<0.05), suggesting a reduction in renal efficiency for transporting sodium. In vitro, both a NO-donating agent and the NO synthase-stimulating agonist bradykinin significantly decreased both cortical and medullary renal oxygen consumption. In conclusion, NO plays a role in maintaining a balance between oxygen consumption and sodium reabsorption, the major ATP-consuming process in the kidney, in conscious dogs, and NO can inhibit mitochondrial oxygen consumption in canine renal slices in vitro. (Circ Res. 1998;82:1263-1271.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Angiotensin II (Ang II) has been previously shown to stimulate the extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase family members. Little is known regarding the upstream signaling molecules involved in Ang II-mediated JNK activation. Ang II has been shown to activate the Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT) pathway, suggesting similarities to cytokine signaling. In response to cytokines such as interleukin-1 and tumor necrosis factor-alpha, the p21-activated kinase (PAK) has been identified as an upstream component in JNK activation. Therefore, we hypothesized that PAK may be involved in JNK activation by Ang II in vascular smooth muscle cells (VSMCs). alpha PAK activity was measured by myelin basic protein phosphorylation in rat aortic VSMCs. In response to Ang II, alpha PAK was rapidly stimulated within 1 minute, with a peak (5-fold increase) at 30 minutes. alpha PAK stimulation preceded activation of JNK in VSMCs. Ang II-mediated activation of both alpha PAK and JNK was Ca2+dependent and inhibited by downregulation of phorbol ester-sensitive protein kinase C isoforms (by pretreatment with phorbol 12,13-dibutyrate) but not by pretreatment with GF109203X. Activation of both PAK and JNK was partially inhibited by tyrosine kinase inhibitors but not by specific Src inhibitors, suggesting regulation by a tyrosine kinase other than c-Src. Finally, introduction of dominant negative PAK markedly reduced the JNK activation by Ang II in both Chinese hamster ovary and COS cells stably expressing the Ang II type 1 receptor (AT1R). Our data provide evidence for alpha PAK as an upstream mediator of JNK in Ang II signaling and extend the role of Ang II as a proinflammatory mediator for VSMCs. (Circ Res. 1998;82:1272-1278.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

p130Casis a signaling molecule that was initially found to be tyrosine-phosphorylated in v-Crk and v-Src transformed cells. We characterized the regulation of p130Castyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130 (Cas) tyrosine phosphorylation, which was sensitive to the actin polymerization inhibitor cytochalasin D and to the intracellular Ca2+chelator BAPTA-AM but not the Ca2+channel blocker verapamil. The Ang II-induced tyrosine phosphorylation of p130Caswas also dependent on an active Src family tyrosine kinase, since it could be blocked by the Src kinase inhibitors geldanamycin and PP1. Ang II treatment resulted in the ability of p130Casto bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase C alpha and the cell adhesion signaling molecule pp120 formed temporal associations with p130Casin response to Ang II. c-Src was found to associate with p130Casin a manner that was independent of Ang II treatment. Inhibition of protein kinase C by either calphostin C or phorbol 12-myristate 13-acetate downregulation inhibited the Ang II-induced tyrosine phosphorylation of p130Cas. These results are the first to demonstrate that the tyrosine phosphorylation of p130Casby Ang II is transduced by the Src, intracellular Ca2+, protein kinase C signaling pathway. (Circ Res. 1998;82:1279-1288.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O (2) sup [center dot] -) and oxidize LDL in vitro; however, the role of O2sup [center dot] - in endothelial cell-induced LDL oxidation is unclear. Incubation of human LDL (200 [micro sign]g/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5 +/- 1.1 versus 6.3 +/- 0.2 [mean +/- SEM] nmol malondialdehyde/mg LDL protein, respectively; P<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 [micro sign]g/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O2sup [center dot] - release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O2sup [center dot] - contributes to endothelial cell-induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells. (Circ Res. 1998;82:1289-1297.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The endothelium is a source of reactive oxygen species in short-term models of hypercholesterolemia and atherosclerosis. We examined a chronic model of atherosclerosis for increased vascular production of superoxide (O2-[center dot]) and determined whether endothelial overexpression of superoxide dismutase (SOD) would improve endothelium-dependent relaxation. Superoxide generation was 3 times higher in isolated aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits (2 to 4 years old) compared with aortas from New Zealand White (NZ) rabbits (43 +/- 10 versus 14 +/- 2 relative light units [center dot] min-1[center dot] mm-2, n=9, P<0.05). After in vitro transduction with adenovirus containing the gene for CuZn-SOD (AdCMVCuZn-SOD) or extracellular SOD (AdCMVEC-SOD), endothelial O2-[center dot] levels in WHHL aortas were significantly reduced. Gene transfer of SOD to WHHL aortas, however, failed to improve the impaired relaxation to acetylcholine or calcium ionophore. By use of the oxidative fluorescent dye hydroethidine, an in situ assay indicated markedly increased generation of O2-[center dot] throughout the wall of WHHL aorta, especially within layers of smooth muscle. This finding was confirmed by demonstrating increased O2-[center dot] levels in smooth muscle cells cultured from WHHL aorta. We conclude that elevated O2-[center dot] levels in atherosclerotic vessels are not confined to the endothelium but occur throughout the vascular wall, including smooth muscle cells. Reduction in endothelial O2-[center dot] levels is not sufficient to improve endothelium-dependent relaxation. Generation of reactive oxygen species within the media may contribute to vasomotor dysfunction in atherosclerosis. (Circ Res. 1998;82:1298-1305.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Protease-activated receptors (PARs) are a family of G protein-coupled receptors activated by a tethered ligand sequence within the amino terminal that are revealed by site-specific proteolysis. The thrombin-sensitive PAR-1 and trypsin-activated PAR-2 mediate endothelium-dependent vascular relaxation in a number of species. Because both thrombin and trypsin-like enzymes have been implicated in coronary artery disease, the purpose of this study was to investigate whether similar receptors are present in human coronary arteries. Thrombin (0.001 to 0.1 U/mL) and trypsin (0.001 to 1 U/mL) caused concentration- and endothelium-dependent relaxations of human coronary artery ring segments suspended in organ chambers for isometric tension recording and contracted with the thromboxane A2mimetic U46619. These relaxations were dependent on the catalytic activity of each enzyme and were inhibited by the NO synthase inhibitor NG-nitro-L-arginine(100 [micro sign]mol/L) and the NO scavenger oxyhemoglobin (20 [micro sign]mol/L). The synthetic PAR-1 tethered ligand sequence SFLLRN-NH2(0.01 to 10 [micro sign]mol/L) also caused endothelium-dependent relaxation of U46619-contracted human coronary arteries; however, the equivalent PAR-2 ligand SLIGKV-NH (2) caused almost no relaxation. In addition, desensitization to either thrombin or trypsin resulted in cross-desensitization to the other enzyme but had only a minimal affect on the response to SFLLRN-NH2. Therefore, we conclude that human coronary artery endothelial cells possess a PAR-1-like receptor that is potently activated by thrombin, trypsin, and SFLLRN-NH2to cause NO-mediated vascular relaxation. Once cleaved, this receptor is recycled in a truncated form, able to respond to exogenous application of only its tethered ligand sequence, suggesting the presence of another endogenous activator possibly acting independently of receptor cleavage. (Circ Res. 1998;82:1306-1311.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID