摘要:

To define the role of specific gene deletions and mutations in the development of transplant arteriosclerosis, we generated an accelerated model of the disease in mice. Carotid arteries were transplanted between B.10A(2R) (H-2h2) donor mice and C57BL/6J (H-2b) recipients and compared with arteries isografted between H-2bmice. Immunosuppressive drugs were not used. Within 7 days, the allografted carotid artery formed a neointima composed of mononuclear leukocytes (CD45+) that were predominantly monocytes or macrophages (ie, CD11b+ cells with single-lobed nuclei). CD4+ and CD8+ cells were present as well. By 30 days, the neointima became exuberant, and mononuclear leukocytes were largely replaced by smooth muscle cells. Cells staining for proliferating-cell nuclear antigen were abundantly present in the intima at both early and late time points, indicating the proliferation of mononuclear leukocytes and smooth muscle cells. The area of the intima increased from day 7 to day 30 (P<.0005), as did the number of nuclei (P=.0005), but the density of the nuclei decreased (P=.02), suggesting the formation of extracellular matrix. Six of the eight isografts formed no neointima, and in samples from the remaining two, a single layer of smooth muscle neointimal cells covered just a portion of the vessel circumference. This model, which reproduces many of the features of human transplant arteriosclerosis but at an accelerated pace, should prove useful for determining the roles in transplant arteriosclerosis of genes that code for components of immunologic and inflammatory responses.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The present study was designed to evaluate the effect of 8-methoxypsoralen (8-MOP) activated with visible light (419 nm) on the suppression of smooth muscle cell (SMC) proliferation in vitro. We hypothesize that if visible light (VL) instead of UVA is used to photoactivate 8-MOP, cytotoxic 8-MOP-DNA cross-link formation can be minimized. Bovine aorta SMCs (2 × 104/cm2) were incubated with 8-MOP (1 ×g/mL) for 30 minutes (in the dark) and exposed to a range of VL (2 to 69 J/cm2) to determine the dose of VL that inhibits SMC proliferation with minimal toxicity. The results show that 8-MOP in combination with 2 to 12 J/cm2VL reversibly inhibited SMC proliferation for up to 5 days after treatment. SMC viability was confirmed by trypan blue exclusion. 8-MOP in combination with 23- or 69-J/cm2VL irreversibly inhibited SMC proliferation. In cell cycle studies, 12-J/cm2VL was used to activate 8-MOP. A phase-specific G2blockade that correlated temporally with recovery of SMC replication was observed. Photoadduct repair studies showed that cell proliferation rates recovered when 60% of the adducts had been removed. These results demonstrate for the first time the possibility of using VL to activate 8-MOP to inhibit cell proliferation and suggest that 8-MOP/VL photochemotherapy can be used to control SMC growth.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The susceptibility of medial and neointimal arterial smooth muscle cells (SMCs) to acute cytomegalovirus (CMV) infection was investigated in immunocompetent and immunocompromised rats. The left common carotid artery of all the rats was injured by balloon catheterization. On days 14 and 17 after injury, rats were either intravenously infected with a rat CMV (RCMV) or mock-infected. Active RCMV infection was shown in the neointima of the injured arteries of immunosuppressed rats, characterized by specific cytopathological changes in hematoxylin-eosin-stained sections and by the presence of early viral antigens and the presence of the virus at different stages of maturation at the electron-microscopic level. Viral genome was shown as well by use of in situ hybridization procedures. However, medial cells were hardly ever infected, and RCMV did not infect neointimal parts that were recovered by endothelial cells. No infection was seen in control right carotid arteries or in the injured arteries of immunocompetent rats. Acute intimal RCMV infection was accompanied by infiltration of inflammatory cells, predominantly mononuclear cells that stained positive with an ED-1 monoclonal antibody. The majority of infected neointimal cells were SMCs containing smooth muscle actin. No changes were found in medial or neointimal cross-sectional areas of the infected arteries. In the present study, an active RCMV infection of arterial SMCs was established in vivo, and it was concluded that neointimal SMCs are far more susceptible than are medial SMCs and that the absence of endothelium and immunosuppression are necessary conditions for arterial RCMV infection.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Tropomodulin is a 40.6-kD protein that colocalizes with actin filament pointed ends in skeletal muscle. We report the sequence of two partial-length complementary DNA (cDNA) clones of rat cardiac tropomodulin that cover 90% of the coding region. The cDNA sequence is 90% conserved between human and rat, with the predicted amino acid sequence similarity even higher at 95%. Anti-tropomodulin antibodies label a single polypeptide with an apparent mobility of 43 000 in Western blot analysis of rat cardiac muscle. Immunofluorescence experiments using this anti-tropomodulin antibody result in labeling that is coincident with thin filament ends, as demonstrated by double localization with α-actinin antibody. Tropomodulin protein is organized into a sarcomeric staining pattern with the earliest appearance of myofibrils in rat cardiocytes. The localization of tropomodulin protein at or near thin filament ends led us to examine the distribution of tropomodulin messenger RNA (mRNA) during myofibrillar development in vitro. Fluorescent in situ hybridization experiments using tropomodulin cDNA probe in cardiocytes that have been cultured for 3 to 5 days show a distribution of large mRNA patches. The cytoplasmic location of tropomodulin mRNA at this time, which bears no relation to the developed myofibrils, suggests that tropomodulin protein is targeted to thin filament ends rather than using localized translational machinery. However, the distribution of tropomodulin mRNA in cultured cardiocytes changes over the next 2 weeks from large perinu-clear patches to small concentrations arranged along myofibrils throughout the cell. The reorganization of tropomodulin mRNA throughout the cardiocyte appears to be distinct from the pattern of glyceraldehyde-3-phosphate dehydrogenase mRNA within the same time period. Increasing intracellular density of myofibrils within developing cardiocytes may lead to redistribution of selected mRNAs for localized translation.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+current sensitive to 4-aminopyridine and an inward Ca2+current but no Na+current. The Ca2+current showed all features of L-type Ca2+current, being highly sensitive to 1,4-dihydropyridines but not to ω-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+current, the delayed K+current, and Ifcurrent. Terminally differentiated cardiomyocytes expressed a Ca2+channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+currents (IK1and IK,Ach) and the ATP-modulated K+current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of α- and β-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+channels. The selective expression of α-car-diac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of theLacZconstruct driven by the α-cardiac MHC promotor region followed by ES cell differentiation and β-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The aim of the present study was to determine the changes in phospholamban protein levels and their regulatory effect on sarcoplasmic reticulum (SR) Ca2+uptake and left ventricular function in hypothyroid and hyperthyroid rat hearts. Hypothyroidism was associated with decreases in basal left ventricular function (+dP/dt and -dP/dt), whereas in hyperthyroidism these parameters were elevated compared with values for euthyroid hearts. The maximal SR Ca2+uptake rates were 12.8±1.1, 15.5±1.2, and 21.4±1.4 nmol Ca2+per milligram per minute, and the EC50values for Ca2+were 0.76±0.09, 0.41±0.07, and 0.30±0.05 μmol/L assayed in homogenates from hypothyroid, euthyroid, and hyperthyroid hearts, respectively. The relative tissue level of phospholamban was increased (135%) in hypothyroidism and decreased (75%) in hyperthyroidism compared with euthyroidism (100%). An opposite trend was observed for the SR Ca2+-ATPase, which was depressed (74%) in hypothyroid hearts but increased (134%) in hyperthyroid hearts. Consequently, the relative ratio of phospholamban to Ca2+-ATPase was highest in hypothyroid and lowest in hyperthyroid hearts, and these changes correlated with changes in the EC50of the SR Ca2+uptake for Ca2+. Stimulation of hearts with 0.1 μmol/L isoproterenol revealed that the relaxant effects were lower in hyperthyroid hearts and higher in hypothyroid hearts compared with euthyroid hearts, consistent with the alterations in the phospholamban levels. The maximal increases in the speed of relaxation, elicited by isoproterenol stimulation, correlated with the changes in the relative ratio of phospholamban to Ca2+-ATPase in these hearts. These findings indicate that alterations in phospholamban levels associated with various thyroid states may reflect alterations in (1) SR Ca2+uptake rates, (2) the affinity of the SR Ca2+pump for Ca2+, (3) the speed of relaxation of the myocardium, and (4) stimulatory effects by (β-adrenergic agonists.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The expression of 15 different potassium channel genes in rat atrial and ventricular muscle was quantitatively compared by use of an RNase protection assay. Of these genes, only five, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, were expressed at significant levels in cardiac muscle. In comparisons of atrial and ventricular RNA samples, transcripts from the Kvl.2 and Kv4.2 genes showed the largest differences in relative abundance. There was an approximately twofold decrease in total Kv4 subfamily mRNA expression in atrial muscle relative to ventricular muscle and a 70% increase in total Kvl subfamily mRNA. Variation of potassium channel mRNA expression within the left ventricular wall was also examined. There was a large gradient of Kv4.2 expression across the ventricular wall, and Kv4.2 expression in epicardial muscle was more than eight times higher than in papillary muscle. Other potassium channel genes were expressed at relatively uniform levels across the ventricular wall. The results suggest that transcriptional regulation makes a significant contribution to the control of potassium channel expression in cardiac muscle and to the variation of the electrophysiological phenotype of myocytes from different regions of the myocardium.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) proteins are potent vascular smooth muscle cell (SMC) mitogens that are expressed by endothelial cells and SMCs in vivo. Overexpression of these proteins in transfected cell lines can result in auto-crine transformation; therefore, the precise control of fibroblast growth factor gene expression in the vessel wall may be an important mechanism regulating vascular cell growth. In the present study, we demonstrate that bFGF can induce bFGF mRNA expression, but not aFGF mRNA expression, in serum-starved rat aortic SMCs. bFGF autoinduction is maximal at 4 hours, requires de novo RNA and protein synthesis, and is mediated predominantly by a protein kinase C-depen-dent signaling pathway. Furthermore, aFGF treatment of rat SMCs also increases bFGF mRNA and protein expression; however, aFGF mRNA levels are only slightly modulated. These results suggest that the local release of aFGF or bFGF within the vessel wall could promote a prolonged period of elevated bFGF synthesis. This, in turn, could be of importance in the SMC hyperplasia that occurs in response to vascular injury and during atherosclerotic plaque formation.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The deposition of platelets at the site of balloon angioplasty is thought to play a major role in the pathogenesis of restenosis. The antibody AZ-1, which binds to the rabbit platelet glycoprotein IIb/IIIa receptor and inhibits platelet function both in vitro and in vivo, was produced and tested in an experimental model of angioplasty. Atherosclerosis was induced by desiccation injury of the femoral artery, followed by a 28-day diet with 2% cholesterol and 6% peanut oil. Rabbits were randomized to receive an infusion of saline, a single infusion of 0.5 mg/kg of AZ-1, or an infusion of 0.6 mg/kg AZ-1 before angioplasty. The latter group received a second infusion of 0.6 mg/kg 72 hours later. Functional platelet inhibition was demonstrated by prolongation of the bleeding time in all treated animals. Angiography was performed at baseline, immediately after a standardized angioplasty, and again 28 days after angioplasty on a total of 42 vessels. There were no significant differences between the antibody-treated group and the control group in the mean angiographic minimum luminal diameter at any of the time points. There was also no difference in the initial improvement after angioplasty (acute gain), in the decrease in luminal diameter from immediately after angioplasty to 28 days after angioplasty (late loss), or in the overall improvement from before angioplasty to 28 days after angioplasty. Quantitative histological analysis confirmed the lack of a beneficial effect of AZ-1. There were no significant differences in the area of the intima, the media, or the combined intima and media between the antibody-treated groups and the control group. Thus, potent platelet inhibition for up to 6 days after balloon angioplasty using a monoclonal antibody that inhibits platelet aggregation did not reduce the response to vascular injury after balloon angioplasty in this rabbit model of experimental atherosclerosis.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

One of the possible causes of dilated cardiomyopathy is considered to be a sequel to myocarditis. Two mechanisms have been proposed in the process of progression of myocarditis into dilated cardiomyopathy: one is a persistent viral infection, and the other is an autoimmune myocardial injury. To clarify the possible part played by the autoimmune mechanism in the process, using an animal model, we investigated whether autoimmune myocarditis, exclusively not related to viral infection, might develop into dilated cardiomyopathy. Experimental autoimmune myocarditis was elicited in Lewis rats by immunization with cardiac myosin fraction. Rats of the control group were immunized with ovalbumin. The clinical course was observed over 4 months. Six rats from the myosin-immunized group died during the acute phase and the healing phase, and all those rats had severe myocarditis. All rats that survived until the end of the study showed enlarged and discolored hearts. Aneurysmal changes were observed in the right ventricle during thoracotomy. The ratio of heart weight to body weight of the myosin-immunized group was significantly higher than that of the control group (3.36±0.49 versus 2.69±0.06 g/kg, respectively;P<.005). The lengths of the anterior interventricular fissure and the posterior interventricular fissure of the hearts of the myosin-immunized group were significantly longer than those of the control group. The external diameter of the left ventricle of the myosin-immunized group was also significantly larger than that of the control group. Diffuse myocardial muscle loss and replacement fibrosis were the prominent histological findings of the rats of the myosin-immunized group. Inflammatory cell infiltrations disappeared from the majority of the lesions, but focal accumulations of mononuclear cells were rarely detected in the periphery of the lesions. When rats were immunized again 4 months after the initial immunization, severe myocarditis recurred in all these rats. Autoimmune giant cell myocarditis in the rat was demonstrated to develop into recurrent forms of myocarditis and to lead to dilated cardiomyopathy. This is a new model for postmyocarditis dilated cardiomyopathy not related to viral infection.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID