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To develop an MRI method for the evaluation of contrast enhancement in early atherosclerotic plaque development in the abdominal aorta of a mouse model. Male apoE-/- mice from three groups, respectively 4 (n = 6), 8 (n = 11) and 16 (n = 4) weeks were included. Axial T1 spin echo images of the abdominal aorta were obtained above and below the renal arteries (90 microm spatial resolution) before and over 1 h after the injection of a macromolecular contrast agent. Signal enhancement was measured in the vessel wall and compared to histological features. Maximal arterial wall signal enhancement was obtained from 16 to 32 min post injection. During this time, the signal-to-noise ratio increased by a factor up to 1.7 in 16 week mice and 2.7 and 2.4 in 8 and 4 weeks mice, respectively. The enhancement of the arterial wall appeared less pronounced in the oldest mice, 16 weeks old, exhibiting more advanced lesions. Using a macromolecular gadolinium agent, contrast uptake in atherogenesis varies with lesion stage and may be related to vessel-wall permeability. Dynamic contrast-enhanced MRI may be useful to evaluate the atherosclerotic plaque activity in mice.

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To simulate diabetic conditions, the effects of high glucose concentration on collagen synthesis and cholesterol level in cultured aortic smooth muscle cells of Psammomys were investigated. For collagen biosynthesis, smooth muscle cells (SMCs) were incubated in synthetic proliferative phase and in postconfluent phase with 3H-proline. Cellular cholesterol was determined by enzymatic method. Under high glucose concentration, the results showed morphological modifications characterized by morphometric cellular, nuclear, and nucleolar changes. In biochemical studies, the authors observed an increase of free and esterified cellular cholesterol as well as of total proteins, collagen biosynthesis, and alpha1 (I+III) and alpha2 (I) chains of collagen contained in the SMCs and in the extracellular matrix. These results showed the sensitivity of Psammomys aortic SMCs to high glucose concentration and would constitute an interesting cellular model to study atherosclerosis pathogeny in experimental diabetes.

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In this report, we have shown that the standard laboratory diet administered to Psammomys obesus (sand rat) from Beni Abbes in Algeria, induced a non-insulin dependent diabetes, characterised by increase of body weight (p<0.001) as well as hyperinsulinemia, hyperglycemia and hypercholesterolemia. In cultured aortic smooth muscle cells (SMC) of sand rats, type I and type III collagen biosynthesis and insulin effects, at low dose, on these parameters were investigated. In all experimental conditions of cultured SMC study, The alpha chains of type I collagen were analysed by immunoblotting in media and cells. Metabolic radiolabelling and Immunochemical procedures revealed that, in diabetic state, synthetic SMC (SMCs) actively produce type I and III collagen which are synthesised in the cells and secreted in the medium; type I collagen was predominant as compared with type III collagen. Diabetes enhanced the collagen synthesis. Low dose of Insulin added to the medium, during 48 h of incubation, induced a marked reduction in the synthesis of collagen types, especially type I collagen.

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Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE(-/-) mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE(-/-)) or double-knockout (DKO; ApoE(-/-), ICAM-1(-/-)) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

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Smooth muscle cells (SMCs), before migration and proliferation in the intima of the vessel wall, change from a normal contractile to a pathological proliferating phenotype. The molecular regulatory mechanisms implicated in such phenotypic changes remain poorly understood. In this study, using differential display, we have isolated for the first time a new gene (2A3-2) that is overexpressed in a rapidly proliferating, but not synthetic, rat SMC line. This was further confirmed by northern blot performed on the 2 cell types. Moreover, balloon catheter injury of rat carotids showed, by a virtual northern technique, an upregulation of this new gene in hyperplasia vessels. This new gene (2A3-2, 1.2 kb) was present in skeletal muscle, heart, aorta, lung, liver, kidney, and spleen. In addition, 5' rapid amplification of cDNA ends (5' RACE) allowed the cloning and sequencing of this 1.2-kb gene. Comparison of this newly identified gene sequence with data banks showed a strong homology to human and bovine mitochondrial translational elongation factor. The 2A3-2 gene, identified in this study, may play a vital role in the cascade of events implicated in switching SMC phenotype from a quiescent to a proliferate one.

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The aim of our work was to determine whether fatty acid modifications in smooth muscle cell phospholipids affect cholesterol efflux and desorption. [3H]Cholesterol was used to label cholesterol pools in the whole cell or selectively in the plasma membrane. Cells were incubated for 12 h in order to increase oleate, linoleate, arachidonate, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in phospholipids. Cholesterol efflux was monitored using native or tetranitromethane modified high-density lipoprotein3 (HDL3). When all cholesterol pools were labeled, the efflux from cells treated with different fatty acids were not different. Plasma membrane cholesterol efflux remained unchanged after oleate, linoleate or arachidonate treatments, but was markedly increased after EPA and DHA enrichment, both with native HDL3 and with tetranitromethane-high-density lipoprotein. These results suggest that the positive effects of n-3 fatty acid consumption on the atherosclerotic process could be linked in part to an increase in plasma membrane cholesterol efflux from vascular smooth muscle cells.

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The aim of this study was to elucidate signal transduction pathways following high-density lipoprotein 3 (HDL3) fixation to HDL high-affinity binding sites and leading to translocation of newly synthesized cholesterol to the plasma membrane pool for efflux. First, membrane phosphatidylcholine (PC) breakdown and 1,2-diacylglycerol (DAG) production were investigated following HDL3 or tetranitromethane (TNM)-HDL incubation with smooth muscle cells in culture. Second, newly synthesized cholesterol was labeled using [3H] mevalonolactone. Phospholipase C (PLC) and protein kinase C (PKC) were stimulated using carbachol and phorbol 12-myristate 13-acetate. Translocation and efflux of newly synthesized cholesterol were monitored using the cholesterol oxidase method and TNM-HDL as cholesterol acceptor. Results showed that: (1) native HDL3 but not modified HDL was able to stimulate PC breakdown and DAG formation; and (2) PLC and PKC stimulation using specific agents induce cholesterol translocation from intracellular to plasma membrane pool. Taken together, these two sets of results suggest that native HDL3 could induce cholesterol translocation by a PLC/PKC process in smooth muscle cells.

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One of the key features of atherosclerosis formation and progression is 'dedifferentiation' of contractile arterial smooth muscle cells (SMC) in synthetic cells. In primary cultures and subcultures before 10 and after 200 passages, SMC exhibit contractile-like, synthetic and transformed phenotypes, respectively, providing a good model for studying dedifferentiation process in vitro: the rationale for comparing these phenotypes of SMC in vivo rests in similar changes in cytoenzymatic and cytoskeletal features. In vivo, dedifferentiated SMC are transformed into foam cells by accumulating lipids. Thus, the aim of this study was to determine whether cholesterol metabolism undergoes changes in dedifferentiated cells and the three cultured phenotypes were compared in regard to their cholesterol efflux mechanisms. Phenotypic changes were shown to be associated with decrease in intracellular cholesterol apoprotein mediated efflux and translocation but also with decrease in high affinity binding sites for native HDL. Thus, the dedifferentiation process triggers a need for increased supply of cholesterol for membrane synthesis and efflux down-regulation mechanisms are aimed at maximizing cholesterol availability to the cell. Plasma membrane cholesterol efflux, which seems to be apoprotein-independent, decrease slightly with cell dedifferentiation suggesting either modifications in the dedifferentiated cell membranes physical properties. Taken together, these different results showed that dedifferentiation of arterial SMC is associated with decrease in the different steps of the efflux process, which could constitute one of the early events in their foam cell transformation.

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During the atherogenic process in vivo, arterial smooth muscle cells (SMC) undergo changes in their phenotype. In the present study, rat SMC from primary cultures and from subcultures before 10 and after 200 passages, showing contractile-like, synthetic and transformed phenotypes, respectively, were compared in regard to their lipid content and biosynthesis. The rationale for comparing these phenotypes rests in the similar changes in phenotype of SMC that occur in the formation and progression of atherosclerotic lesions. Phenotype changes were shown to be associated with changes in the phospholipid content of SMC. Phospholipid levels increased, but not as significantly as did cholesterol levels when passing from contractile to synthetic and transformed cells (1.23 +/- 0.18, 2.28 +/- 0.26 and 3.25 +/- 0.23 micrograms/10(6) cells, respectively). Cholesterol normalized in respect to cell protein was increased to the same extent. Lipid synthesis as judged by [14C]acetate incorporation was increased 3- to 12-fold in the synthetic and transformed cells, respectively, compared to contractile cells. After thin-layer chromatography, radioactivity was shown to be markedly increased in most of the lipid fractions, but label in the cholesterol fraction of synthetic and transformed cells was increased by 7- and 21-fold, respectively. Thus, SMC in vitro were shown to drastically increase cholesterol biosynthesis associated with phenotype changes. Such changes are known to occur in vivo and might represent a critical step in the deposition of excess cholesterol within foam cells.

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"Contractile" arterial smooth muscle cells (SMC) return to a less differentiated "synthetic" state during adaptative and proliferative processes in vitro and in cell cultures. At present, the enzyme expression of the modulation of cultured SMC is partially unknown. In order to define metabolic events associated with cell modulation in vitro, we studied 16 enzyme activities in primary and secondary (P1-P3-P10) SMC cultures in comparison to in situ SMC in fetal and adult rat aorta. The "contractile" SMC in aorta of 2 months old rat showed very high nucleotide hydrolase activities (5'-nucleotidase, Mg-ATPase, Ca-ATPase), and naphthylesterase activities and weak lysosomal acid phosphatase activity; the glycolysis-linked dehydrogenases were expressed with higher activities than Krebs cycle-linked enzymes. In primary cultures, the SMC near the explant expressed a "contractile-like" enzyme behaviour, in opposite to cells in the peripheral part of growing area enzymatically similar to sub-cultured SMC. Proliferating SMC in secondary cultures were characterized by increased lysosomal activities and by the decrease or disappearance of Ca-ATPase, Mg-ATPase, 5'-nucleotidase, and butyrylcholinesterase activities like fetal SMC in vivo. These enzyme changes in subcultures might be related to a deficiency of nucleotide ester hydrolysis, abnormal adenosine and AMP levels, lowered lipolytic capability and increased lysosomal reactivity. In conclusion, subcultured "synthetic" SMC expressed enzyme cytochemical patterns different from those of "contractile" SMC and similar to those of fetal immature SMC. Their enzyme behaviour is unfavourable to contractile function and favourable to cell proliferation and lipid accumulation, two characteristic features of SMC in atherosclerotic plaques.

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Experimental approaches to the problem of atherosclerosis involve animal or cellular models and procedures of lesional induction. Relevant animal models are rare. The rat, the mouse and the dog are free of "natural" atherosclerosis and only develop diffuse lipidosis after high cholesterol diet and thyroid block. They are more appropriate models of experimental arteriosclerosis and intimal proliferation induced by different procedures. The rabbit, also free of spontaneous atherosclerosis, is extremely sensitive to lipid-rich diets, but the lesions induced resemble more a xanthomatosis than an atherosclerosis. Immunological procedures in this model result in a generalised immune arteriosclerotic arteriopathy. The monkey and pig, which are phylogenetically close to man, develop spontaneous atherosclerosis exacerbated by lipid-rich diets or other procedures: hormones, psychosocial stress. The cost and problems of upkeep make these two models inaccessible to most laboratories. Although the hen, turkey and pigeon are grain-eating, they develop natural atherosclerosis, are sensitive to atherogenic diets, and provide satisfactory replacement models, especially for research into the viral and tumoral theories of atherogenesis. The pigeon is particularly suitable for studying cellular, biochemical and genetic aspects of atherosclerosis: these spontaneous plaques, similar to those in man, are ontogenetically and topographically predictable. The species include genetic types both sensitive and resistant to the disease. Moderately lipid-rich diets induce lesions even in very young pigeons. They also lend themselves well to the study of the antiatherosclerotic effects of pharmacological agents. Endothelial, smooth muscle and macrophage cell cultures are widely used to study the factors influencing cellular modulation and proliferation, lipid metabolism and movement of cholesterol, cellular biosynthesis and cell-cell and cell-matrix interactions.

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Smooth muscle cell proliferation is an important feature of atherogenesis. Some works have hypothesized that a transformation of smooth muscle cells could arise during this pathological process. The present paper describes two spontaneously transformed cell lines of arterial smooth muscle cells (SMC) established from aortic media of adult rat. The cell lines have been designated V6 and V8; some of their morphologic, growth, and metabolic characteristics are described and compared to their parent cells. The two cell lines appeared distinct by their morphology and by their degree of transformation. V6 cells appeared as elongated spindle-shaped cells whereas V8 cells were spread cells with a cobblestone pattern. Karyotypes of both cell lines showed a high polyploidy level. V6 and V8 cell lines were immortalized and showed growth characteristics of transformed cells: low requirement of serum to grow, ability to form colonies in soft agar and tumorigenicity in nude mice; V8 cells presented a higher malignancy than V6 cells. Both V6 and V8 cells exhibited characteristics of cultured arterial SMC: ultrastructure, alpha actin expression at the protein and mRNA level, prostacyclin production. The remarkably different morphologies of the V6 and V8 lines and their transformed phenotype suggest that these cell lines could be useful models to study SMC differentiation and proliferation with respect to atherosclerotic or hypertensive vascular diseases.

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The phenotypic modulation and the enhanced proliferation of smooth muscle cells (SMC) as well as their foam transformation are major processes in arterial pathophysiology and during atherogenesis. Arterial SMC play a crucial role, in response to several stimuli: the SMC "activation" is an essential condition leading to the adult atherosclerotic plaque formation. Owing to the difficulty to study the SMC regulation in vivo, most of the literature in this field refers to in vitro models. Modulated SMC in culture, changing from a contractile to a synthetic state, share similar features with atherosclerotic plaques cells. The phenotypic modulation of SMC is expressed by morphological, biochemical, metabolic and functional modifications. The regulation of cholesterol movements might influence the foam transformation process of arterial SMC.

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In order to define metabolic profiles of smooth muscle cell (SMC) modulation, 16 enzyme activities linked to nucleotide hydrolysis, lipolysis, lysosomal reactivity and intermediate glucose catabolism were compared in four rat arterial models, exhibiting four metabolic phenotypes of modulated smooth muscle cells: (i) "primary synthetic" statein immature aorta; (ii) "contractile" state in adult aorta; (iii) "hypertensive" state in aorta of hypertensive rat, SHR; (iiii) "secondary synthetic" state in diffuse intimal thickening of ligated carotid artery. Contractile SMC presented strong activities of enzymes linked to nucleotide ester hydrolysis and contractility (ATP-A-Ca, ATP-A-Mg, ATP-A-Ca/Mg, 5'nucleotidase) and to lipolytic process (butyryl cholinesterase, acid esterase). These enzyme activities were more pronounced in "hypertensive SMC". Incontrast, the same enzymes were weakly active or not expressed in "synthetic SMC". Increased lysosomal enzyme reactivity was a particular expression of "secondary synthetic SMC". The observed enzyme abnormalities in reactively modulated SMC (proliferative-synthetic phenotype) might be related to the loss of contractility and to the enhanced cell proliferation and lipid accumulation, characteristic features of modulated SMC in atherogenesis.

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Numerous studies carried out on animal models (apes excepted) have given encouraging results as regards the regression of experimental atherosclerosis after return to a normal or hypocaloric diet combined or not with various drugs. Regression is more obvious when lesions are recent and less severe: lipid striae disappear in less than 12 months, whereas more advanced and complicated lesions take years to regress. Intracellular lipids and cell alterations vanish more readily than extracellular lipids and alterations of connective and matrical tissues. Excessive accumulation of collagen accounts for the irreversibility of complicated plaques. Lesions of the intima are less stubborn than those of the media. Involution does not take place at the same time in coronary vessels and in the aorta. In non human primates, however, no noticeable regression is observed before several months if not years. In these animals, the degree and rapidity of involution after return to the normal vegetarian diet depend on the severity of the lesions induced, on the degree of fibrosis, on the level of residual hypercholesterolaemia and on the adjunction to the diet of certain drugs such as cholestyramine or alpha-alpha. The results of therapeutic trials conducted in man have not been so good because the patients treated had old and severe atherosclerosis: after a few years' treatment with low-cholesterol diet and appropriate drugs less than 10 p. 100 of them showed a clear-cut angiographic improvement. It is therefore illusory to rely on spontaneous regression when tackling a case of clinically detectable atherosclerosis. A preventive treatment is more promising, since infraclinical lesions may regress.

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Structural differences between platelet and endothelial cell thrombospondin (TBSP) were found in two protease-resistant domains (70 and 18 kDa). The 70 kDa fragment is involved in the binding of TBSP to fibrinogen and the 18 kDa fragment in the attachment to various cultured cells. Despite these structural differences, platelet and endothelial cell TBSP bound with the same affinity to fibrinogen and mediated the attachment of smooth muscle cells but not of endothelial cells.

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Biology of the vascular cells is widely studied by means of cell culture techniques. In the present work the description of a transformed cell line of arterial smooth muscle cells is presented. The cell line, named V8, has been established from cells of adult rat aortic media. The cells presented proliferation characteristics in vitro, in soft agar, and in vivo in nude mice demonstrating a tumorigenic ability. This cell line provides an interesting model for the study of growth regulation of arterial smooth muscle cells specially in the areas of hypertension and atherosclerosis.

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The alterations of HDL structure and metabolism induced by bezafibrate administration were studied in healthy male volunteers. As usually observed in hyperlipaemic patients, bezafibrate induced a decrease of the plasma concentrations of apo B and LDL-cholesterol and an increase of that of HDL-cholesterol. Analysis of HDL by gradient polyacrylamide gel electrophoresis revealed that bezafibrate administration resulted in a change of the particle size distribution likely suggesting a drop of the HDL2/HDL3 ratio. This was accompanied by a 30% enhancement of the plasma concentration of apoprotein A-II, while that of apoprotein A-I remained unchanged. These data suggest an increase of the HDL concentration, preferentially in the HDL3 subfraction. In spite of these HDL alterations, there was no evidence of change in the three stages of the reverse pathway of cholesterol, since bezafibrate did not induce any significant alteration in the in vitro properties of plasma with respect to (a) cholesterol transport from cultured cells, (b) cholesterol esterification, and (c) transfer of cholesteryl esters from HDL to VLDL-LDL.

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