摘要:

The early response to vascular injury is characterized by migration of inflammatory cells, including monocytes, and platelets to the damaged vessel wall. These inflammatory cells may serve as a source of growth factors and cytokines that stimulate vascular smooth muscle cell (VSMC) migration and proliferation associated with intimal hyperplasia.JEis a platelet-derived growth factor (PDGF)-inducible “early” gene that encodes a monocyte chemoattractant and, as such, could play an important role in inflammation. We now report thatJEmRNA levels are increased in intact aorta after balloon injury. The time course of this increase, with maximal levels at 4 hours, is similar to that seen in PDGF-treated cultured rat aortic VSMCs. The accumulation ofJEmRNA in cultured VSMCs is accompanied by a marked increase in the secretion ofJEprotein. The elevation ofJEmRNA levels in VSMCs shows specificity for PDGF, because angiotensin II, α-thrombin, and epidermal growth factor fail to increaseJEmRNA levels. In contrast to 3T3 fibroblasts, the accumulation ofJEmRNA in VSMCs in response to PDGF is predominantly due to an increase inJEmRNA stability. The accumulation ofJEmRNA in VSMCs stimulated by PDGF appears to occur via a novel pathway(s) independent of Ca2+mobilization, Na+-H+exchange, protein kinase C activation, or elevation in cAMP levels. These findings suggest that VSMCs may take part in the early inflammatory response after injury through the production ofJE, a potent monocyte chemoattractant. Finally, our data suggest thatJEmay be a marker for PDGF-specific effects on VSMCs, both in vitro and in vivo. Thus, in addition to direct effects on VSMC growth and migration, PDGF may play a role in the early inflammatory response after vascular injury by inducing chemoattractants, such as that encoded byJE.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The effects of hydrogen peroxide (H2O2) on the action of basally produced endothelium-derived relaxing factor (EDRF) were investigated by measuring cGMP accumulation in single and cocultures of calf pulmonary artery endothelial cells (CPAEs) and rabbit pulmonary artery smooth muscle cells (RPASMs) as a model for determining the contribution of EDRF dysfunction to altered vascular tone and reactivity frequently associated with oxidant-induced vascular injury. Higher cGMP levels in long-term cocultures (20.4±1.8 pmol/mg protein/15 min) than in single-cell cultures (CPAE, 9.6±0.9 pmollmg protein/15 min; RPASM, 3.7±0.2 pmol/mg protein/15 min), and CPAE-induced increases (fivefold) in intracellular RPASM cGMP content in short-term cocultures suggest basal release of EDRF. Basal generation and release of an l-arginine-derived endothelial labile factor accounted for the increases in cGMP, since the response was completely blocked by pretreatment of CPAEs withNGmonomethyl l-arginine. Pretreatment of long-term cocultures with H2O2for 30 minutes resulted in a dose-dependent (0.5–2 mM) decrease in cGMP formation (49–79%o). To determine the effects of H2O2on EDRF synthesis, transport, and RPASM responsiveness, CPAEs or RPASMs were selectively pretreated with H2O2before establishment of short-term cocultures. In cocultures of H2O2-pretreated CPAEs with untreated RPASMs, RPASM cGMP levels were reduced, suggesting a decrease in EDRF production rather than deterioration of EDRF during transport, because cGMP levels were unaffected by posttreatment with oxygen radical scavengers during coculture. Pretreatment of RPASMs with H2O2attenuated the untreated CPAE-induced, the putative EDRF S-nitroso-l-cysteine-induced, or the nitroprusside-induced increases in RPASM cGMP levels. This attenuation was prevented by pretreatment with either dimethylthiourea, deferoxamine, or dithiothreitol, suggesting a mechanism of H2O2action involving iron-catalyzed formation of intracellular hydroxyl radicals and their attack on cellular thiols. H2O2diminution of cGMP accumulation was not associated with lytic cell injury in the experimental time frame, because morphology and51Cr release from prelabeled RPASMs and CPAEs were unchanged.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Cigarette smoking is associated with an increased incidence of a number of diseases. Minimal information is available at the molecular level concerning the mechanism of action of cigarette smoke. Platelet-activating factor (PAF) is one of the most potent proinflammatory agents described. PAF concentration may be regulated by the degradation of PAF as catalyzed by the plasma enzyme, PAF acetylhydrolase (PAF-AH). This enzyme is associated with the lipoprotein fraction. The exposure of low density lipoprotein to a cigarette smoke extract (CSE) has been shown to alter the charge of low density lipoprotein and its uptake by macrophages. The activity of PAF-AH in the lipoprotein fraction has been assayed after exposure to CSE. The activity of PAF-AH was inhibited by the CSE in a dose-dependent manner. The inhibition of PAF-AH by the CSE was not altered by superoxide dismutase or catalase addition. Sulfihydryl compounds prevented and reversed the inhibition of PAF-AH caused by CSE. The inhibitor present in CSE was not nicotine, its major metabolic product, (-)-cotinine, or several compounds known to be present in the extract. The charge alteration reaction(s) and PAF-AH inhibition appear to be localized at different sites of the lipoprotein molecule. The observed inhibition may account for the increase in the plasma PAF concentration that is known to occur in smokers. The increase of PAF may contribute to the increased incidence of cardiovascular and lung diseases known to be present in smokers.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Intravascular stents, currently in experimental human use for recurrent arterial stenosis, are plagued by subacute thrombosis. As a therapeutic approach to stent-related thrombosis, we and others have suggested coating stents with endothelial cells before implantation. In a previous study we demonstrated the feasibility of coating stents with endothelial cells that were genetically modified to secrete large amounts of human tissue plasminogen activator. In the present study we attempted both to develop a clinically applicable protocol for stent seeding and to test whether seeded cells would remain adherent to stents after exposure to pulsatile flow. Endothelial cells were harvested from the saphenous veins of sheep with survival of the donor animals. Harvested cells were transduced with a retroviral vector containing a marker gene and seeded onto catheter-mounted stents under sterile conditions. Scanning electron microscopy revealed complete coverage of the stent surfaces by seeded cells. Stents were expanded and exposed to pulsatile flow in vitro. Substantial cell retention was observed on the lateral stent surfaces by light microscopy and scanning electron microscopy; fewer cells were seen on the luminal and abluminal surfaces. Removal of seeded cells from flow-exposed stents by trypsin digestion resulted in the recovery of approximately 70% of the seeded cells. These cells were viable and healthy as judged by their ability to proliferate to confluence with the same kinetics as control (non-flow-exposed) cells. Autologous genetically modified endothelial cells can be seeded onto catheter-mounted stents in a sterile manner, and stent deployment under flow conditions results in substantial retention of viable cells.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The influence of platelet-vessel wall interactions on leukocyte rolling was investigated in rabbit mesenteric venules (diameter, 21–40 μm) using intravital videomicroscopy. Puncture of the wall with glass micropipettes (tip, 6–8 μm) evoked the formation of a thrombus in all venules. In most vessels, emboli were produced as well. The rolling of leukocytes (i.e., their movement along the vessel wall at a velocity clearly lower than that of the other blood cells) was quantitated simultaneously in vessel segments upstream and downstream from a thrombus up to 10 minutes after puncture. During embolization the number of rolling leukocytes decreased significantly from the upstream to the downstream vessel segment (median decrease, 45%;p≤ 0.001). It was still decreased by ∼50% after embolization had stopped, indicating that the decrease in leukocyte rolling was not caused by inclusion of leukocytes in the emboli. In venules without embolization, leukocyte rolling did not change systematically, indicating that fluid dynamic changes induced by the thrombus do not influence leukocyte rolling. Inhibition of prostaglandin formation with aspirin (100 mg/kg) almost completely abolished the influence of the thromboembolic reaction on leukocyte rolling, but blockade of thromboxane A2receptors with sulotroban (30 mg/kg) had no effect. In conclusion, this is the first report on a functional interaction in vivo, at a site of vessel wall injury, between platelets, vascular cells, and leukocytes. The findings suggest that substances produced by activated platelets and/or damaged vascular cells diminish leukocyte rolling. The identity of these substances is not yet clear, but the present study indicates that prostaglandins other than thromboxane A2are involved.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The effect ofNω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of endothelium-derived relaxing factor production, on the vasodilator response to efferent vagal stimulation was investigated in the pulmonary vascular bed of the intact-chest cat under conditions of controlled blood flow and constant left atrial pressure. When pulmonary vascular tone was increased with U46619, efferent vagal stimulation decreased lobar arterial pressure in a stimulus-frequency-dependent manner. The decreases in lobar arterial pressure were enhanced by pretreatment with reserpine, were blocked by atropine, and were not altered by propranolol, indicating that the neurogenic vasodilator response was cholinergic in nature. The decreases in lobar arterial pressure in response to vagal stimulation and to exogenously administered acetylcholine were reduced after administration of L-NAME (100 mg/kg i.v.). Although l-NAME decreased pulmonary vasodilator responses to vagal stimulation and to acetylcholine, responses to adenosine, nicorandil, lemakalim, isoproterenol, prostaglandin E1, sodium nitroprusside, and 8-bromo-cGMP, agents that act by a variety of mechanisms, were not decreased. These results are consistent with the hypothesis that efferent vagal stimulation releases acetylcholine, which dilates the pulmonary vascular bed by stimulating the production of nitric oxide or a labile nitroso compound from l-arginine.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Bovine heart 67-kd protein (p67) was coisolated with calpactin I complex by cycles of Ca2+-dependent precipitation followed by solubilization with EGTA-containing buffer. Using affinity-purified anti-p67 antibody and anti-p36 (36-kd subunit of calpactin 1) antibody, we examined the localization of the two proteins in secretory atrial myocytes and other endocrine tissues of adult rats. Immunofluorescence microscopy showed that p67 was expressed both in the atrial myocytes in situ and in cultured atrial myocytes in which we failed to detect p36 and that p67 appeared to be closely associated with the cell surface. We also found that p67 was colocalized with p36 in the thyroid follicle epithelium and zona reticularis of the adrenal gland. On the other hand, neither p67 nor p36 was detectable in pancreas islet cells. Immunoelectron microscopy revealed that p67 was localized at the sarcolemma in the atrial myocytes in situ. The p67, which was shown to be a globular molecule with a diameter of 18–25 nm by a low-angle rotary shadowing method, bound radioactive Ca2+on a nitrocellulose membrane. The results suggest that Ca2+-binding proteins expressed in endocrine cells seem to vary from tissue to tissue and that p67 may function in Ca2+-mediated events at the plasma membrane of secretory atrial myocytes and some types of endocrine cells expressing this protein.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Microvascular functions have been shown to be sensitive to agents associated with changes in cyclic nucleotide levels. The central hypothesis of the current study was that one measure of capillary exchange capacity, hydraulic conductivity (Lp), would be elevated by agents shown to elevate cellular levels of cGMP. To evaluate the hypothesis, frog mesenteric capillary Lpwas measured during luminal exposure to 1) atrial natriuretic peptide (ANP), 2) the truncated atriopeptins ANP-I and ANP-III, 3) the nitrovasodilator sodium nitroprusside (SNP), 4) the cGMP phosphodiesterase inhibitor M&B 22948, and 5) methylene blue dye, both alone and in combination with ANP or SNP. ANP (100 nM) elevated Lpby 2.3±0.2-fold from control levels (n=15); 10 nM ANP induced a 2.1±0.3-fold change (n=8), while 10 nM ANP-III elicited a 1.7±0.4-fold change (n=8). In contrast, Lpdid not change from basal levels during 10 nM ANP-I infusion (LpANP-1/Lpcontrol=1.2±0.2;n=14). SNP (1 μM) induced a reversible, 2.6±0.5-fold increase in Lp(n=30) that was inhibitable by methylene blue dye (LpSNP+MetB/Lpcntrol=1.1±0.1;n=8). Methylene blue did not mask the response to 100 nM ANP (LpANP+MetB/Lpcontrol=2.1±0.5;n=7). M&B 22948 (30 μM) increased Lpby 2.8±0.6-fold (n=9). These data constitute strong inference that agents demonstrated to elevate cGMP also mediate an increase in capillary Lpin in situ, perfused exchange vessels.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Although the tricarboxylic acid (TCA) cycle is the prime means of carbon metabolism for energy generation in normal myocardium, the noninvasive quantification of TCA cycle flux in intact cardiac tissues is difficult. A novel approach for estimating citric acid cycle flux using13C nuclear magnetic resonance (NMR) is presented and evaluated experimentally by comparison with measured myocardial oxygen consumption over a wide range of cardiac contractile function in intact, beating rat hearts. Continuous series of13C NMR spectra, obtained after the introduction of [2-13C] acetate as substrate, quantified the time course of13C appearance in the carbon positions of myocardial glutamate, which are sequentially enriched via citric acid cycle metabolism. A TCA cycle flux parameter was calculated using the premise that TCA cycle flux is inversely proportional to the time difference between13C appearance in the C-4 and C-2 positions of glutamate (glutamate δt50[minutes]), which are enriched in subsequent “turns” of the TCA cycle. This TCA cycle flux parameter, termed KT, correlated strongly with myocardial oxygen consumption over a range of developed pressures in hearts perfused with 5 mM acetate (r=0.98,p<0.001), as well as in separate studies in hearts perfused with 5 mM glucose and 0.5–0.8 mM acetate (r=0.94,p<0.001). Results of numerical modeling of13C glutamate kinetics suggest that this TCA cycle flux parameter, KT, is relatively insensitive to changes in metabolite pool sizes that could occur during metabolism of other substrates or during conditions of altered oxygen availability. Additional studies in separate hearts indicated that the time course of13C appearance in citrate, which is predominantly mitochondrial in the rat heart, is similar to that in glutamate, further supporting the premise that the described13C NMR parameters reflect mitochondrial citric acid cycle activity in intact cardiac tissues.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The role of local accumulation and diffusion of CO2to modify cellular loss and extracellular accumulation of K+during the initial, reversible phase of myocardial ischemia was investigated in isolated, cylindrical papillary muscles of the rabbit. The muscles were blood-perfused through their vascular tree and placed in a (permanently flowing) humidified gas mixture with predetermined partial pressures of N2, O2, and CO2. Ischemia was produced by total arrest of perfusion and O2withdrawal from the gas mixture. With surface Pco2kept constant during ischemia, [K+]ovaried markedly with muscle geometry. After 10 minutes of ischemia, K+accumulation was ∼2.5 mM in muscles with a radius of 0.35 mm and ∼14 mM in muscles with a radius of 0.9 mm, indicating that a large fraction of K+accumulation was dependent on diffusion of a volatile metabolite. Computer simulation of CO2accumulation and diffusion within a tissue cylinder suggested a close phenomenological relation between Pco2and [K+]oin ischemia. This was confirmed by the finding that an increase of tissue Pco2in small cylinders before or during ischemia by externally applied CO2produced an increase in K+accumulation. The importance of CO2diffusion for local inhomogeneities in K+within the same preparation was demonstrated by showing [K+]ogradients with simultaneous or consecutive measurements between the papillary muscle cylinders and the adjacent septum and within 300 μm from the surface of the papillary muscle cylinders. These gradients predict an inhomogeneity of impulse conduction that might contribute to the genesis of ventricular arrhythmias. Besides the demonstration that accumnulation and diffusion introduce inhomogeneities of [K+]oin ischemia, our results suggest that a significant component of cellular ischemic K+loss is associated with production and extrusion of metabolic acid. On the basis of previous measurements of pHoand pH4in identical conditions, possible mechanisms of ischemic cellular K+loss are discussed.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID