摘要:

Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules and engages diverse ligands relevant to distinct pathological processes. One class of RAGE ligands includes glycoxidation products, termed advanced glycation end products, which occur in diabetes, at sites of oxidant stress in tissues, and in renal failure and amyloidoses. RAGE also functions as a signal transduction receptor for amyloid beta peptide, known to accumulate in Alzheimer disease in both affected brain parenchyma and cerebral vasculature. Interaction of RAGE with these ligands enhances receptor expression and initiates a positive feedback loop whereby receptor occupancy triggers increased RAGE expression, thereby perpetuating another wave of cellular activation. Sustained expression of RAGE by critical target cells, including endothelium, smooth muscle cells, mononuclear phagocytes, and neurons, in proximity to these ligands, sets the stage for chronic cellular activation and tissue damage. In a model of accelerated atherosclerosis associated with diabetes in genetically manipulated mice, blockade of cell surface RAGE by infusion of a soluble, truncated form of the receptor completely suppressed enhanced formation of vascular lesions. Amelioration of atherosclerosis in these diabetic/atherosclerotic animals by soluble RAGE occurred in the absence of changes in plasma lipids or glycemia, emphasizing the contribution of a lipid- and glycemia-independent mechanism(s) to atherogenesis, which we postulate to be interaction of RAGE with its ligands. Future studies using mice in which RAGE expression has been genetically manipulated and with selective low molecular weight RAGE inhibitors will be required to definitively assign a critical role for RAGE activation in diabetic vasculopathy. However, sustained receptor expression in a microenvironment with a plethora of ligand makes possible prolonged receptor stimulation, suggesting that interaction of cellular RAGE with its ligands could be a factor contributing to a range of important chronic disorders. (Circ Res. 1999;84:489-497.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The role of basement membrane-degrading matrix metalloproteinases (MMPs) in enabling vascular smooth muscle cell migration after vascular injury has been established in several animal models. In contrast, the role of their native inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), has remained unproven despite frequent coregulation of MMPs and TIMPs in other disease states. We have investigated the time course of expression and localization of TIMP-4 in rat carotid arteries 6 hours, 24 hours, 3 days, 7 days, and 14 days after balloon injury by in situ hybridization, immunohistochemistry, and Western blot analysis. TIMP-4 protein was present in the adventitia of injured carotid arteries from 24 hours after injury. At 7 and 14 days after injury, widespread immunostaining for TIMP-4 was observed throughout the neointima, media, and adventitia of injured arteries. Western blot analysis confirmed the quantitative increase in TIMP-4 protein at 7 and 14 days. In situ hybridization detected increased expression of TIMP-4 as early as 24 hours after injury and a marked induction in neointimal cells 7 days after injury. We then studied the effect of TIMP-4 protein on the migration of smooth muscle cells through a matrix-coated membrane in vitro and demonstrated a 53% reduction in invasion of rat vascular smooth muscle cells. These data and the temporal relationship between the upregulation of TIMP-4, its accumulation, and the onset of collagen deposition suggest an important role for TIMP-4 in the proteolytic balance of the vasculature controlling both smooth muscle migration and collagen accumulation in the injured arterial wall. (Circ Res. 1999;84:498-504.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

This study investigates the role of extracellular signal-regulated kinases (ERKs) in angiotensin II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+concentration, ie, [Ca2+]i, and pHi) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (KWY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+]i, pHi, and contractile responses to Ang II (10-12to 10-6mol/L) in the absence and presence of PD98059 (10-5mol/L). [Ca2+]iand pHiwere measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II-stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca2+]iand pHiand contracted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (41.9 +/- 5.1% reduction in cell length relative to basal length) than in WKY (28.1 +/- 3.0% reduction in cell length relative to basal length). Basal [Ca2+]i, but not basal pHi, was higher in SHR compared with WKY. [Ca2+]iand pHieffects of Ang II were enhanced (P<0.05) in SHR compared with WKY (maximum Ang II-induced response [Emax] of [Ca2+]i, 576 +/- 24 versus 413 +/- 43 nmol/L; Emaxof pHi, 7.33 +/- 0.01 versus 7.27 +/- 0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II-elicited contractile responses in SHR (Emax, 19.3 +/- 3% reduction in cell length relative to basal length). Ang II-stimulated [Ca2+]i(Emax, 294 +/- 55 nmol/L) and pHi(Emax, 7.27 +/- 0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In conclusion, Ang II-stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR. (Circ Res. 1999;84:505-515.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-kappa B and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO-induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2). N-Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin-specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti-E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides that contained nuclear factor-kappa B or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil-endothelial adhesion response. (Circ Res. 1999;84:516-524.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Intracoronary thrombus formation is associated with epicardial vasoconstriction distal to the thrombotic site. To investigate the mechanisms of abnormal vasomotor function of the artery distal to the thrombotic site, we studied coronary vessels in dogs with cyclic flow variations (CFVs) of the left anterior descending coronary artery (LAD) stenosis with endothelial injury. Coronary rings isolated from the LAD (proximal, stenotic, and distal sites) and control circumflex coronary arteries were tested for responsiveness to endothelium-dependent (acetylcholine and A23187) and endothelium-independent vasodilators (NaNO2). Endothelium-independent relaxation was intact in all 4 sites. Endothelium-dependent relaxation was intact in the control and proximal sites and impaired in the stenotic sites. Relaxations not only to acetylcholine and A23187 but also to serotonin, ADP, and thrombin were impaired in the distal sites after observing CFVs for 80 minutes. Electron microscopy revealed the loss of endothelial integrity with leukocyte adherence to the endothelium in the distal sites. Immunohistochemical expression of P-selectin on the endothelial cells was more upregulated in the distal site than in the proximal site, and P-selectin mRNA expression was significantly greater in the ischemic region distal to the thrombotic site than in the proximal nonischemic region. PB1.3, a neutralizing monoclonal antibody against P-selectin, and sialyl LewisX(SLeX)-containing oligosaccharide SLeX, a carbohydrate analogue of selectin ligand, preserved endothelial function without affecting CFVs. SLeX-containingoligosaccharide preserved endothelial integrity of the distal site and inhibited P-selectin expression of the distal site. Thus, the adhesive interaction between endothelial P-selectin and leukocyte SLeXmay play an important role in endothelial injuries of the coronary artery distal to the thrombotic site. (Circ Res. 1999;84:525-535.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The recent discoveries of inositol 1,4,5-trisphosphate (IP3) receptor subtypes with different affinities for IP3and their potential involvement in development has important consequences for vascular smooth muscle. This study has examined the expression and distribution of the type 1 and type 3 IP3receptor subtypes in developing rat vascular smooth muscles. Immunoblotting of portal vein and aorta from neonatal (2 to 4 days) and fully developed (6 weeks) rats revealed significantly higher levels of the type 3 IP3receptor expression in neonatal, compared with developed, vascular smooth muscles. In contrast, expression of the type 1 IP3receptor in neonates was lower compared with developed vascular smooth muscles. Immunolocalization of the type 3 IP3receptors in neonatal tissues revealed that staining corresponded to the distribution of the sarcoplasmic reticulum (visualized by osmium ferricyanide staining of thin tissue sections), which suggested localization of the type 3 IP3receptor throughout the sarcoplasmic reticulum network. We conclude that type 3 IP3receptors are the predominant subtype in the development of vascular smooth muscle and are distributed throughout the sarcoplasmic reticulum in these cells. The switch in isoforms of the IP3receptor during development from the type 3 with low affinity for IP3to the higher-affinity type 1 receptor may play a role in calcium-mediated regulation of developing vascular smooth muscle. (Circ Res. 1999;84:536-542.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Tranilast, which is an antiallergic drug, has a potent effect on preventing postangioplasty restenosis. To elucidate this mechanism, we studied the effect of tranilast on the proliferation of vascular smooth muscle cells (SMCs) in vitro and in vivo. Tranilast decreased the growth rate of SMCs stimulated by either 10% FBS or platelet-derived growth factor. The IC50value, evaluated as cell number, was 100 [micro sign]mol/L. These inhibitory effects were associated with inhibition of the retinoblastoma gene product (pRb) phosphorylation. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDK), we investigated CDK2 and CDK4 activities and the expression of CDK inhibitor p21waf1/cip1/sdi1(p21). When SMCs were stimulated by 10% FBS or platelet-derived growth factor, CDK2 and CDK4 activities reached a maximum near the G1/S80% without reduction of CDK2/cyclin E and CDK4/cyclin D1 protein levels. These inhibitory effects were associated with enhanced expression of p21 and elevated complexing of p21 with CDK2/CDK4. Next, rat balloon-injured carotid artery was analyzed for intimal thickening and p21 expression. Tranilast-treated rats had a 70% (P<0.001) smaller neointima/media area ratio at 14 days after balloon injury compared with the controls. Immunohistochemical staining demonstrated that, in tranilast-treated rats, p21 was already present in the neointima at day 7 and strongly expressed throughout the neointima at day 14. In control rats, p21 was not observed in the neointima at day 7 but was sparsely expressed at day 14. These data demonstrate that inhibition of CDK2/CDK4 activities by the increased expression of p21 may be one mechanism by which tranilast inhibits SMC proliferation and prevents postangioplasty restenosis. (Circ Res. 1999;84:543-550.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The properties of the transient outward current (Ito) differ between rabbit and human atrial myocytes. In particular, rabbit Itois known to recover more slowly than its human counterpart and to show much more frequency dependence. To assess the possibility that these physiological differences may reflect differing expression of K+channel subunit gene products, we used a combination of whole-cell voltage-clamp, heterologous expression, pharmacological, antisense, and Western blot techniques. The inactivation of Itoin rabbit atrial myocytes was significantly slowed by hydrogen peroxide, with human Itobeing unaffected. Use-dependent unblocking with 4-aminopyridine was not seen for rabbit Itonor for Kv1.4 currents in Xenopus oocytes, whereas human Itoshowed strong use-dependent unblock (as did Kv4 currents). Western blots indicated the presence of Kv4 proteins in both human and rabbit atrial membranes, but Kv1.4 was only detected in the rabbit. Antisense oligodeoxynucleotides directed against Kv4.3, Kv4.2, or Kv1.4 subunit sequences significantly inhibited Itocurrent density in cultured rabbit atrial myocytes, whereas only Kv4.3 antisense significantly inhibited Itoin human cells. Neither mismatch oligodeoxynucleotides nor vehicle altered currents in either species. We conclude that, unlike human atrial myocytes, rabbit atrial myocytes express Kv1.4 channel subunits, which likely contribute to a number of important physiological differences in Itoproperties between the species. To our knowledge, these studies constitute the first demonstration of a functional role for Kv1.4 channels in cardiac membranes and provide insights into the molecular mechanisms of an important cardiac repolarizing current. (Circ Res. 1999;84:551-561.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Pacing-induced heart failure in the dog recapitulates many of the electrophysiological and hemodynamic abnormalities of the human disease; however, the mechanisms underlying altered Ca2+handling have not been investigated in this model. We now show that left ventricular midmyocardial myocytes isolated from dogs subjected to 3 to 4 weeks of rapid pacing have prolonged action potentials and Ca2+transients with reduced peaks, but durations [approximate]3-fold longer than controls. To discriminate between action potential effects on Ca2+kinetics and direct changes in Ca2+regulatory processes, voltage-clamp steps were used to examine the time constant for cytosolic Ca2+removal (tauCa). tauCawas prolonged by just 35% in myocytes from failing hearts after fixed voltage steps in physiological solutions (tauCacontrol, 216 +/- 25 ms, n = 17; tau (Ca) failing, 292 +/- 23 ms, n = 22; P<0.05), but this difference was markedly accentuated when Na+/Ca2+exchange was eliminated (tauCacontrol, 282 +/- 30 ms, n = 13; tauCafailing, 576 +/- 83 ms, n = 11; P<0.005). Impaired sarcoplasmic reticular (SR) Ca2+uptake and a greater dependence on Na+/Ca(2+) exchange for cytosolic Ca2+removal was confirmed by inhibiting SR Ca (2+) ATPase with cyclopiazonic acid, which slowed Ca2+removal more in control than in failing myocytes. beta-Adrenergic stimulation of SR Ca2+uptake in cells from failing hearts sufficed only to accelerate tauCato the range of unstimulated controls. Protein levels of SERCA2a, phospholamban, and Na+/Ca2+exchanger revealed a pattern of changes qualitatively similar to the functional measurements; SERCA2a and phospholamban were both reduced in failing hearts by 28%, and Na+/Ca2+exchange protein was increased 104% relative to controls. Thus, SR Ca (2+) uptake is markedly downregulated in failing hearts, but this defect is partially compensated by enhanced Na+/Ca2+exchange. The alterations are similar to those reported in human heart failure, which reinforces the utility of the pacing-induced dog model as a surrogate for the human disease. (Circ Res. 1999;84:562-570.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Ca2+transients measured in failing human ventricular myocytes exhibit reduced amplitude, slowed relaxation, and blunted frequency dependence. In the companion article (O'Rourke B, Kass DA, Tomaselli GF, Kaab S, Tunin R, Marban E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart, I: experimental studies. Circ Res. 1999;84:562-570), O'Rourke et al show that Ca2+transients recorded in myocytes isolated from canine hearts subjected to the tachycardia pacing protocol exhibit similar responses. Analyses of protein levels in these failing hearts reveal that both SR Ca2+ATPase and phospholamban are decreased on average by 28% and that Na+/Ca2+exchanger (NCX) protein is increased on average by 104%. In this article, we present a model of the canine midmyocardial ventricular action potential and Ca2+transient. The model is used to estimate the degree of functional upregulation and downregulation of NCX and SR Ca2+ATPase in heart failure using data obtained from 2 different experimental protocols. Model estimates of average SR Ca2+ATPase functional downregulation obtained using these experimental protocols are 49% and 62%. Model estimates of average NCX functional upregulation range are 38% and 75%. Simulation of voltage-clamp Ca2+transients indicates that such changes are sufficient to account for the reduced amplitude, altered shape, and slowed relaxation of Ca2+transients in the failing canine heart. Model analyses also suggest that altered expression of Ca2+handling proteins plays a significant role in prolongation of action potential duration in failing canine myocytes. (Circ Res. 1999;84:571-586.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID