摘要:

Endothelial cells (ECs) are constantly exposed to blood pressure-induced mechanical strain. We have previously demonstrated that cyclic strain can induce gene expression of monocyte chemotactic protein-1 (MCP-1). The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence indicates that intracellular reactive oxygen species (ROS) can act as a second messenger for signal transduction and thus affect gene expression. The potential role of ROS in strain-induced MCP-1 expression was investigated. ECs under cyclic strain induced a sustained elevated production of intracellular superoxide. ECs under strain or pretreated with either H2O2or xanthine oxidase/hypoxanthine induced MCP-1 expression. Strain- or oxidant-induced MCP-1 mRNA levels could be inhibited by treating ECs with catalase or antioxidant N-acetyl-cysteine (NAC). Functional analysis of MCP-1 promoter and site-specific mutations indicates that the proximal tissue plasminogen activator-responsive element (TRE) in the -60-bp promoter region is sufficient for strain or H2O sub 2 inducibility. Electrophoretic mobility shift assays demonstrated an increase of nuclear proteins binding to TRE sequences from ECs subsequent to strain or H2O2treatment. NAC or catalase pretreatment of ECs inhibited the strain- or H2O2-induced AP-1 binding. These results clearly indicate that cyclic strain inducibility of MCP-1 in ECs uses the interaction of AP-1 proteins with TRE sites via the elevation of intracellular ROS levels in strained ECs. These findings emphasize the importance of intracellular ROS in the modulation of hemodynamic force-induced gene expression in vascular ECs. (Circ Res. 1997;81:1-7.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Cytomegalovirus (CMV) infection and its periodic reactivation from latency may contribute to atherogenesis and restenosis. It is unknown how CMV is delivered to the vessel wall and is reactivated. We examined the following hypothesis: CMV, present in monocytes recruited to sites of vascular injury, is activated by endothelial cell (EC) or smooth muscle cell (SMC) contact and by oxidized low-density lipoproteins (oxLDLs). The CMV major immediate-early promoter (MIEP) controls immediate-early (IE) gene expression, and thereby viral replication. To determine whether elements of the vessel wall can activate CMV present in monocytes, we transiently transfected the promonocytic cell line HL-60 with a chloramphenicol acetyltransferase reporter gene construct driven by MIEP. MIEP activity increased 1.7 +/- 0.5-fold (P=.02) when the transfected HL-60 cells were cocultured with ECs, 4.5 +/- 1.5-fold when cocultured with SMCs (P=.03), and 2.0 +/- 0.5-fold (P=.01) when exposed to oxLDL. The combination of oxLDL and EC coculture increased MIEP activity over 7-fold. We also found that freshly isolated human monocytes, infected with endothelium-passaged CMV, were capable of transmitting infectious virus to cocultured ECs or SMCs. CMV-related progression of atherosclerosis or restenosis may, at least in part, involve monocyte delivery of the virus to the site of vascular injury, where the vascular milieu, ie, contact with ECs, SMCs, and oxLDL, can contribute to viral reactivation and/or replication by enhancing CMV IE gene expression. The virus may then infect neighboring ECs or SMCs, initiating a cascade of events predisposing to the development of atherogenesis-related processes. (Circ Res. 1997;81:8-16.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Proliferation of vascular smooth muscle cells (SMCs) is implicated in pathological events, including atherosclerosis and intimal hyperplasia following angioplasty. The glycosaminoglycan heparin is a growth inhibitor of SMCs in vitro and in vivo. The underlying mechanism, however, is still poorly understood. In the present study, we report that heparin inhibited the activation of the mitogen-activated protein kinase (MAPK) in baboon SMCs by serum but not by platelet-derived growth factor (PDGF). When fibroblast growth factor was used, heparin had a stimulatory effect on MAPK. The only MAPK-activating kinase found in SMCs was MAPK kinase (MAPKK)-1, although MAPKK-2 was present in comparable amounts. Activation of MAPKK-1 and DNA synthesis were affected by heparin in a similar fashion. Heparin does not appear to exert its effects through members of the protein kinase C family, which are downregulated by phorbol esters, because it was still capable of inhibiting MAPK/MAPKK-1 stimulation by FCS in phorbol ester-pretreated cells. The present findings support the conclusions that the effects of heparin depend on the nature of the mitogen and that heparin inhibits SMC proliferation by preventing activation of MAPKK-1. (Circ Res. 1997;81:17-23.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H2O2. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 micro mol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 micro mol/L H2O2. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 micro mol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries. (Circ Res. 1997;81:24-33.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

NO, synthesized in endothelial cells by endothelial NO synthase (NOS 3), is believed to be an important endogenous pulmonary vasodilator substance that contributes to the normal low pulmonary vascular resistance. To selectively investigate the role of NOS 3 in the pulmonary circulation, mice with targeted disruption of the NOS 3 gene were studied. Pulmonary hemodynamics were studied by measuring pulmonary artery pressure, left ventricular end-diastolic pressure, and lower thoracic aortic flow by using a novel open-chest technique. Transient partial occlusion of the inferior vena cava was used to assess the pulmonary artery pressure-flow relationship. Tension developed by isolated pulmonary artery segments after acetylcholine stimulation was measured in vitro. The histological appearance of NOS 3-deficient and wild-type murine lungs was compared. NOS 3-deficient mice (n=27), when compared with wild-type mice (n=32), had pulmonary hypertension (pulmonary artery pressure, 19.0 +/- 0.8 versus 16.4 +/- 0.6 mm Hg [mean +/- SE]; P<.05) that was due to an increased total pulmonary resistance (62 +/- 6 versus 33 +/- 2 mm Hg [centered dot] min [centered dot] g [centered dot] mL sup -1; P<.001). In vitro, acetylcholine induced vasodilation in the main pulmonary arteries of wild-type but not NOS 3-deficient mice. The morphology of the lungs of NOS 3-deficient mice did not differ from that of wild-type mice. We conclude that NOS 3 is a key enzyme responsible for providing basal pulmonary NO release. Congenital NOS 3 deficiency produces mild pulmonary hypertension in mice. (Circ Res. 1997;81:34-41.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Recent studies in conscious pigs and rabbits have demonstrated that a series of brief coronary occlusions renders the heart relatively resistant to myocardial "stunning" 24 hours later (late preconditioning [PC] against stunning). The mechanism of this powerful cardioprotective response is unknown. The goal of the present study was to test the hypothesis that the development of late PC against stunning is triggered by increased generation of NO during the first ischemic challenge. Conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). On day 1, rabbits received either an intravenous infusion of the NO synthase inhibitor Nomega-nitro-L-arginine (L-NA, 13 mg/kg before the first occlusion) (group II, n=10) or vehicle (group I [control], n=10). In the control group, on day 1 systolic wall thickening (WTh) in the ischemic/reperfused region remained significantly depressed for 4 hours after the sixth reperfusion, indicating myocardial stunning. On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 60% on day 2 and 55% on day 3 compared with day 1 (P<.01). In the L-NA-treated group, the total deficit of WTh on day 1 was similar to that observed in the control group. On day 2, however, the total deficit of WTh was not significantly different from that observed on day 1 and was 132% greater than that observed in control rabbits on day 2 (P<.01). On day 3, the total deficit of WTh was 66% less than that noted on day 2 (P<.01). Thus, in L-NA-treated rabbits the sequence of six coronary occlusions and reperfusions performed on day 1 failed to precondition against stunning on day 2, but the same sequence performed on day 2 did precondition against stunning on day 3. Another group of rabbits (group III, n=6) received L-NA on day 1 in the absence of ischemia and was subjected to the occlusion/reperfusion sequence on days 2 and 3. In these animals, the total deficit of WTh on day 2 did not differ from that observed in control rabbits on day 1, indicating that administration of L-NA did not exacerbate the severity of myocardial stunning 24 hours later; therefore, the absence of late PC against stunning on day 2 in group II cannot be ascribed to a delayed deleterious action of L-NA on WTh. In conclusion, these results demonstrate that the NO synthase inhibitor L-NA completely blocks the development of late PC against myocardial stunning in conscious rabbits, indicating that NO generated as a result of the PC ischemia triggers the development of the cardioprotective response observed 24 hours later. NO is known to exert numerous biological actions resulting in rapid but transient physiological responses. The present observations support a novel pathophysiological paradigm in which NO also plays a key role in the delayed myocardial adaptations to ischemic stress, acting as a signaling step in the transduction pathway that leads to increased resistance to subsequent ischemic injury. (Circ Res. 1997;81:42-52.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The goal of this study was to determine both the early and delayed effects of a brief (10-minute) coronary artery occlusion (CAO) and prolonged (5-day) reperfusion (CAR) on coronary endothelial function. Fourteen mongrel dogs were chronically instrumented to measure aortic and left ventricular pressures, wall thickness, and left circumflex coronary blood flow (CBF). Before CAO and during CAR, coronary vascular reactivity was investigated by 15-second CAO and subsequent reactive hyperemia (RH) and by the selective intracoronary infusion of acetylcholine (ACh, 10 micro g/min) and bradykinin (BK, 2.5 micro g/min), endothelium-dependent vasodilators, and sodium nitroprusside (SNP, 40 micro g/min), an endothelium-independent vasodilator. CBF responses to ACh and BK began to increase after 6 hours of CAR, reached a peak after 1 to 2 days of CAR, and then subsided over the subsequent 4 days. After 1 day of CAR, compared with before CAO, enhanced CBF responses (P<.05), associated with increased coronary sinus oxygen content, were observed for ACh (+66 +/- 20%), BK (+74 +/- 24%), and RH (+24 +/- 5%) but not SNP (-2 +/- 10%). Production of NO metabolites (nitrate and nitrite), measured as their coronary arteriovenous differencexCBF, was significantly increased after 1 to 2 days of CAR, both at baseline (153 +/- 56%) and during BK infusion (220 +/- 76%) (P<.05). Holding CBF at pre-CAO levels during the initial CAR period did not attenuate the delayed enhanced endothelial vasodilation to ACh and BK. However, NO blockade with intracoronary NG-nitro-L-arginine blocked the enhanced coronary vasodilation to ACh and BK. Thus, in contrast to previous studies, these data indicate that brief ischemic episodes induce delayed enhanced coronary endothelial function, which is delayed in onset and prolonged in duration. This can be explained by an upregulation of coronary vascular NO production, potentially involved in the mechanism of the delayed window of preconditioning. (Circ Res. 1997;81:53-59.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n=8) or 3-morpholinosydnonimine (SIN-1, n=6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n=6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 micro mol/L, n=6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 micro mol/L, n=6), and (3) mimicked by 8-bromo-cGMP (n=6) with no additional positive chronotropic effect of SIN-1 (n=5). The response to 10 micro mol/L SNP (n=28) or 50 micro mol/L SIN-1 (n=16) was unaffected by ICa-L antagonism with nifedipine (0.2 micro mol/L) but was abolished after blockade of the hyperpolarization-activated inward current (If) by Cs sup + (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 micro mol/L). The effect on Ifwas further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n=21), where we found that 5 micro mol/L SNP or SIN-1 caused a reversible Cs sup +-sensitive increase in this current (+130% at -70 mV and +250% at -100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of Ifvia the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO. (Circ Res. 1997;81:60-68.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The oxidative capacity of mitochondria isolated from myocardium is undiminished after myocardial stunning, which is remarkable because stunning affects many other cellular functions. The aim of the present study was to assess the mitochondrial oxidative response in intact rather than isolated myocardium. The mean response time of mitochondrial O2consumption to heart rate steps (tmito) was measured before and after 15-minute ischemia or high-flow hypoxia in isolated rabbit hearts. The tmitowas calculated from the time course of venous O2tension to steps in heart rate, with corrections made for diffusion and vascular transport delay. Isovolumic hearts were perfused with Tyrode's solution at 37 degrees C. Developed left ventricular pressure at 35 minutes of reperfusion was decreased significantly to 67 +/- 3% after ischemia (mean +/- SEM, n=8) and to 79 +/- 6% after hypoxia (n=8) relative to the control condition (n=8), without increased cellular creatine kinase release. Before ischemia or hypoxia, tmitowas 4.3 +/- 0.3 seconds. During reperfusion after ischemia or hypoxia, the increase in tmito(by 62 +/- 10% and 64 +/- 18%, respectively) was significantly larger than that in time controls (24 +/- 12% increase). The major determinant of decreased contractility and slower mitochondrial response appeared to be O2deprivation and/or reintroduction rather than other consequences of stopped flow. O2consumption at a given rate-pressure product was not increased after ischemia or hypoxia, indicating undiminished cardiac contractile economy. Brief ischemia or hypoxia, resulting in stunning, was associated with a slowing of the in vivo mitochondrial oxidative response, indicating that energy transfer and/or signaling between energy-consuming sites and mitochondria is affected in stunned myocardium. (Circ Res. 1997;81:69-75.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The mechanism(s) by which mutations in sarcomeric proteins cause hypertrophic cardiomyopathy (HCM) remains unknown. A leading hypothesis proposes that mutant sarcomeric proteins impair cardiac myocyte contractility, providing an impetus for compensatory hypertrophy. To test this hypothesis, we determined the impact of expression of a mutant (Arg92Gln) human cardiac troponin T (cTnT), known to cause HCM in humans, on adult cardiac myocyte contractility. A full-length human cTnT cDNA was cloned, and the Arg92Gln mutation was induced. Recombinant adenoviruses Ad5/CMV/cTnT-N and Ad5/CMV/cTnT-Arg92Gln were generated through homologous recombination. Adult feline cardiac myocytes were infected with recombinant adenoviruses or a control viral vector (Ad5 Delta E1) at a multiplicity of infection of 100. Expression levels of the full-length normal and mutant cTnT proteins were equal on Western blots. Expression of the exogenous cTnT proteins in cardiac myocytes was also shown by immunocytochemistry and immunofluorescence, and their incorporation into myofibrils was confirmed by Western blotting on myofibrillar extracts. Electron microscopy showed intact sarcomere structure in rod-shaped cardiac myocytes in all groups. Cell fractional shortening and the peak velocity of shortening were not significantly different among the groups 24 hours after transduction. However, 48 hours after transduction, both fractional shortening and the peak velocity of shortening were significantly reduced (24% [P<.001] and 26% [P<.001], respectively) in cardiac myocytes in the Ad5/CMV/cTnT-Arg92Gln compared with the Ad5/CMV/cTnT-N groups. The magnitude of the reductions was greater at 72 hours after transduction (45% and 39%, respectively; P<.001). Our results indicated that expression of the mutant (Arg92Gln) cTnT, known to cause HCM in humans, impaired intact adult cardiac myocyte contractility. Our data also show that both normal and mutant cTnT were incorporated into myofibrils. These results provide a potential mechanism by which mutations in sarcomeric proteins cause HCM. (Circ Res. 1997;81:76-85.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID