Decrease in high density lipoprotein binding sites is associated with decrease in intracellular cholesterol efflux in dedifferentiated aortic smooth muscle cells.

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.

ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet.

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.

Cell attachment and fibrinogen binding properties of platelet and endothelial cell thrombospondin are not affected by structural differences in the 70 and 18 kDa protease-resistant domains.

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.

Histological arguments for collagen and elastin synthesis by primary cultures of rat aortic media cells.

Using the histological staining methods of Weigert and of Masson on primary cultures of rat aortic media cells, we obtained additional proofs of the smooth muscle cell's ability to secrete collagen and elastin in vitro: the percentage of positive flasks with aorta rings was the same throughout the follow-up, but increased gradually for the new tissue growing around the rings.

Selection of a strain of rats with spontaneous high cholesterolemia.

By means of an inbred selection procedure utilizing positive assortative mating, high (SHC: spontaneous high cholesterolemic) and low (SLC: spontaneous low cholesterolemic) blood cholesterol strains of rats were developed. This procedure was shown to be much more efficient in increasing than in lowering the blood cholesterol level. The diet used throughout selection was normal laboratory chow; therefore the high and low blood cholesterol levels occurred spontaneously. Since the 6th generation there has been no overlap between the blood cholesterol values of the animals of the two strains. The cholesterol increase with ageing was found to be strongly related to sex, but weakly to the genes governing the differences between SHC and SLC rats. Cholesterol enhancement following a hyperlipidic diet did not differ between strains or sexes. It appears that the SHC rat strain could be an interesting model, particularly in pharmacological research.

High density lipoprotein alterations induced by bezafibrate in healthy male volunteers.

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.

Lipid biosynthesis in cultured arterial smooth muscle cells is related to their phenotype.

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.

Omega-3 fatty acids in smooth muscle cell phospholipids increase membrane cholesterol efflux.

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.

Enzyme cytochemical expression of aortic smooth muscle cell modulation in primary and secondary cultures.

"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.

[Experimental models of atherosclerosis. Contribution, limits and trends]. / Modèles expérimentaux d'athérosclérose. Apports, limites et perspectives.

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.

[Natural history of the regression of atherosclerosis: from animal models to men]. / Histoire naturelle de la régression de l'athérosclérose: des modèles animaux à l'homme.

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.

[Tricuspid valve replacement by Hancock's prosthesis]. / Le remplacement valvulaire tricuspidien par la prothèse de Hancock.

The medium term results of tricuspid valve replacement with the Hancock bioprosthesis are reported. Twenty eight patients underwent tricuspid valve replacement with this prosthesis between December 1974 and January 1978: mitral valve replacement with a Starr-Edwards or Cooley-Cutter prosthesis was associated in all cases and aortic valve replacement with a Björk-Shiley prosthesis in 11 cases. Follow-up at an average of 36,2 months after operation examined functional status, cardiac size and haemodynamics (in 12 patients). Three patients died in the immediate postoperative period and four others died later: the number of survivors was greater in the triple valve replacement (9/11) than in the double valve replacement group (12/17) but the difference was not statistically significant. Of the 23 surviving patients (average follow-up of 36,2 +/- 9,6 months), 17 were classified in functional Classes I or II of the NYHA classification. All patients had been Class III or IV before operation. The cardiothoracic ratio did not decrease significantly in patients undergoing triple valve replacement. Control cardiac catheterisation showed a significant increase in cardiac index (2,53 +/- 0,11 1/mn/m2, compared to 1,87 +/- 0,35 1/mn/m2 before operation; p less than 0,001) without significant reduction in pulmonary artery or right atrial pressures. The resting gradient across the Hancock bioprosthesis was not related to the size of the prosthesis (No 29-30: 2,17 +/- 2,57 mm Hg; No 31-33: 2,78 +/- 3,53 mm Hg) or with the quality of the functional result. However, on exercise, the gradient across the prosthesis was high, reaching an average of 10,3 +/- 5,2 mm Hg). The operative mortality of tricuspid valve replacement is relatively bioprosthesis associated with mitral and/or aortic valve replacement is relatively bioprosthesis associated with mitral and/or aortic valve replacement is relatively low (about 10%) and could be an argument in favour of broadening the indications for tricuspid valve replacement as resting tricuspid function with a bioprosthesis is satisfactory. However, the stenotic effects on exercise and the uncertainty over the long-term outcome of bioprostheses suggest that surgery should be limited to severe tricuspid stenoses and/or major tricuspid regurgitation organic or functional uncontrolled by digitalis and diuretic therapy.

[Modulation of arterial smooth muscle cells in culture and cholesterol exchange]. / Modulation des cellules musculaires lisses artérielles en culture et mouvements de cholestérol.

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.

[Establishment of a transformed line of arterial smooth muscle cells]. / Etablissement d'une lignée transformée de cellules musculaires lisses artérielles.

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.

High-density lipoprotein 3 stimulates phosphatidylcholine breakdown and sterol translocation in rat aortic smooth muscle cells by a phospholipase C/protein kinase C-dependent process.

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.

Enzyme histochemical expressions of smooth muscle cell modulation in arterial development, hypertension and remodeling.

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.

Proliferation of primary cultures from rat aortic media. Effects of hyperlipemic serum.

Tissue cultures have been made from explants of thoracic aortas to study the growth pattern of rat aortic mediacytes. Three parameters were measured weekly: the surface of the culture, the relative increase of this surface and the number of 3H-thymidine labelled cells per unit surface. The primary cultures showed two distinctive phases: a first phase with continuous growth followed by a plateau phase. We studied the growth effect of homologous hypercholesterolemic serum added to the cultures. The cell proliferation was affected by the cholesterol level in the medium and the stage of the culture at which serum incubation was initiated. An enhancing effect occurred in the rat resistant to such treatment in vivo.

[Formation of xanthomatous cells in vitro from the aortic media of the rat]. / Formation de cellules xanthomateuses in vitro à partir de media aortique de rat

The foam cell is viewed as a specific component of the atherosclerotic plaque found in human or experimentally induced in the animal. A study using light microscopy (staining with Sudan III) and electron microscopy was performed on cell cultures derived from rat aortic media. Sudanophilic and electron transparent vacuoles were observed in vitro in 11 week cultures. The sudanophilic cells were either scattered or crowded in clusters; some of them were found in a mitotic phase. Different serums were applied to the cultures starting from the 6th week: either calf serum (continuing the previous treatment), or normocholesterolemic rat serum (NCRS) or hypercholesterolemic rat serum (HCRS). Sudanophilic cells were observed more frequently in the cultures on exposure to HCRS than to NCRS (p less than 0.05). Thus it was possible to induce the formation of foam cells in vitro in cultures of arterial tissue derived from the rat, which is known to be resistant to atherosclerosis.

Isolation and partial characterization of an elastase-like protease from rat aorta smooth muscle cells. Possible role in the regulation of elastin biosynthesis.

An elastase-like protease was isolated from rat aorta smooth muscle cells and partially characterized. It appears to behave as an intracellular enzyme and may be involved in the regulation of elastin biosynthesis.