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We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20

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Bio Breeding (BB) rats develop a genetically determined insulin-dependent diabetes, because of the early destruction of pancreatic beta cells of Langerhans islets, massively infiltrated by inflammatory mononuclear cells. S 5682, registered as Daflon, 500 mg, is a purified micronized flavonoid fraction (90% diosmin, 10% hesperidin), which has been shown to possess antiinflammatory properties, including anti-free radical activity, effects on vascular permeability, venous tone, and perivenous inflammation. We studied the effect of S 5682 on the course of pancreatic insulitis in diabetic BB rats. All the diabetic BB rats were hyperglycemic, with an increase of plasma levels of fructosamine, alpha-1 acid glycoprotein, and fibrinogen, and a dramatic decrease of C-peptide level. These parameters were not modified by S 5682. Pancreas histologic studies showed that in S 5682-treated diabetic BB rats, lymphocytic infiltration of Langerhans islets was less important and frequent than in untreated diabetic BB rats. By quantitative analysis, a highly significant difference was observed for insulitis, as well as perivasculitis, between S 5682-treated and untreated diabetic BB rats. This inhibitory effect of S 5682 on pancreatic mononuclear cell infiltration may be useful for a complementary treatment to decrease the development of insulitis in human insulin-dependent diabetes mellitus.

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Benfluorex is a clinical lipid-lowering agent with antihyperglycemic properties. The effect of long-term oral treatment (10 mg/kg/day for 7.5 months) on carbohydrate and lipid metabolism and aortic morphology was investigated in 24 insulin-resistant sand rats receiving a standard laboratory diet supplemented with cholesterol (2%). Untreated controls (n=34) developed impaired glucose tolerance, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia and elevated plasma LDL- and VLDL-cholesterol, positively correlated with the proportion of the thoracic aorta displaying oil red O-positive atherosclerosis; ultrastructural examination showed intimal lipid deposits, foam cells, polymorph infiltrates and fibrosis. Benfluorex-treated animals showed significant decreases in glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and plasma LDL- and VLDL-cholesterol, with no evidence of aortic atheroma. The metabolic benefits of benfluorex may protect against the long-term development of atherosclerosis in the insulin-resistant dyslipidemic syndrome.

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OBJECTIVE: Elastic arteries were found to be less prone to intimal hyperplasia than muscular arteries. The internal mammary artery (IMA), which is elastic in its proximal segment, presents a gradual decrease of media elastic fibers along its downstream course. Metabolic and morphometric studies of the distal end of the IMA with regard to its local susceptibility to develop intimal changes were undertaken in order to evaluate the reliability of its use as an anastomotic site for bypass grafting. METHODS: Twenty distal segments of IMA were harvested from patients who had undergone myocardial revascularization. Histologic, enzyme-histochemical and morphometric studies were undertaken on these arterial segments. RESULTS: Histologic examinations indicated an elastomuscular structure in 13 patients, a muscular structure in 6 and an elastic structure in 1. Of the 20 IMAs, none was found to have intimal thickening of greater than 25% of the diameter of the lumen. The enzyme-histochemical profile of the proliferating cells found in the intimal thickening differed from normal contractile smooth muscle medial cells in the loss of myosin and mitochondrial ATPase, plasma membrane 5' nucleotidase, moderately decreased aerobic dehydrogenase and increased lactate dehydrogenase activity and ribonucleoprotein-linked pyroninophilia. Lysosomal beta-glucuronidase and sulfatase were strongly active. This enzyme behavior is unfavorable to contractile function and favorable to cell proliferation and lipid accumulation, two events strongly involved in the atherogenic process. CONCLUSION: Intimal proliferative changes were observed in the distal segment of the IMA. Although there was no histologic evidence of atherosclerotic plaque, the enzyme-histochemical profile of this intimal thickening was favorable to cell proliferation and lipid accumulation. These findings suggest that it may be beneficial to avoid coronary anastomoses with the distal end of the IMA and to use a more proximal/elastic segment.

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The ability of human internal mammary artery smooth muscle cells to maintain histoenzymatic activity and contractile response after various times of cold anoxia prior to and following cryostorage was evaluated. The results showed that the enzyme histochemical status of human mammary arteries was largely unchanged after both cold anoxia and cryopreservation. Neither in fresh nor in cryopreserved mammary arteries did cold anoxia for up to 24 h change maximal contractile responses to potassium depolarization and norepinephrine. However, compared to unfrozen controls, the contractile responses were significantly reduced in cryopreserved mammary arteries. In conclusion, after cryopreservation of human mammary arteries, the enzyme activities were globally maintained, whereas the contractile responses were reduced. For up to 24 h after harvesting cold anoxia at 4 degrees C is well tolerated and allows preservation of metabolic and functional properties of these arteries.

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Biologic or synthetic grafts have had limited success in small vessel applications. Studies were initiated to assess the potential use of cryopreserved (CP) arteries as coronary artery bypass conduits. Sheep carotid arteries (internal diameter: 4 mm; length: 10 cm) were cryopreserved in a nutrient media containing 10% DMSO and were stored in a nitrogen vapor at -150 degrees C. After thawing, histological, enzyme-histochemical and functional studies showed slight histological alterations, preservation of enzymal activities and an abolition of the contractile response. In a sheep model, arterial substitution of a 10 cm segment of carotid artery was realised by implantation of fresh autografts ( n = 4); fresh allografts (n = 9) and CP allografts (n = 9). After 3 months, all autografts were patent with slight histological alterations. Fresh and CP allografts showed similar modifications: patency rate was 7/9 in both groups. Intimal thickening with cell proliferation was seen in fresh (3/7) and CP (4/8) arteries; loss of smooth muscle medial cells was constant. Adventitia was always involved by a marked inflammatory reaction. One characteristic of CP allografts was the frequent presence of large dystrophic calcifications. In conclusion, morphologic and functional arterial changes occurred after freezing and thawing. In spite of vascular rejection, the patency rate of allografts after 3 months of implantation in arterial circulation remained high and does not seem influenced by cryopreservation.

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The effects of a 12-week endurance training programme (treadmill) upon the passive and the noradrenaline-activated properties of the aorta were studied in 15 trained and 24 sedentary rats. Aortic compliance was studied by measuring the length-tension curves of rings of the descending aorta without (passive properties) and with noradrenaline (noradrenaline activated) in a bubbling Krebs bath kept at a temperature of 37 degrees. The training effect on aortic volume compliance was studied by transforming the tension-length curves into a cross-sectional area-pressure curve according to Laplace's law. The noradrenaline responsiveness was studied by the dose-effect curve. The mechanical data were correlated with the results of a histomorphometric study which measured the aortic wall thickness and the percentages and amounts of elastic, connective and muscle components. Passive aortic compliance and volume compliance were higher in endurance-trained rats whose tunica media presented a lower percentage of collagen and a larger amount of elastic tissue. The dose-effect curve showed that the maximal aortic response to noradrenaline was stronger in trained rats but that the half maximal effective dose was not different. As a consequence, the length-tension curves of the noradrenaline fully activated aorta were similar in trained and sedentary rats except at the highest tensions where collagen is the main factor determining aortic stiffness. The increased noradrenaline response in trained rats was probably the result of the hypertrophy of the smooth muscle cells as maximal active strain (Newtons per square metre) was similar in trained and sedentary rats.

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Use of cryopreserved small-caliber arterial allografts for arterial bypass procedures has been suggested. In addition to immunological tolerance, long term in vivo success of these grafts may be dependent on the viability of arterial cells after cryopreservation. Metabolic and functional capabilities of arterial smooth muscle cells were evaluated by studying the enzyme histochemical expressions of sheep carotid arteries after various time of cryopreservation (7 days, 90 to 150 days) and their contractile responses after freezing. The results indicated that cryopreserved arteries exhibited 4 features: (a) the enzyme activities were globally maintained, (b) the spontaneous endogenous oxido-reduction (NitroBT test) was reduced, (c) contractile responses against KCl and norepinephrine were abolished, (d) metabolic status of frozen arteries was independent of the duration of cryopreservation. These data suggest that cryopreserved arterial muscle cells may be depleted in electron donors and/or energy-rich nucleotides substrates. This defect is present after short or long time of cryopreservation.

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Plasma levels of fibrinogen, alpha 1-acid glycoprotein (AG) and albumin, pancreatic insulitis quantitative scores, and erythrocyte velocity in the mesoappendix microvessels were measured in BB diabetic (BBD) and streptozotocin-diabetic rats (WSTZ) in order to answer the following questions: (a) Does hyperfibrinogenemia or increase in AG plasma level occur in BBD and WSTZ rats, and if so, are these alterations secondary to the hyperglycemia or to an inflammatory process such as insulitis? (b) Is there a decrease in microcirculatory flow in the BBD and WSTZ rats, and if so, is it secondary to the hyperfibrinogenemia and/or the hyperglycemia? Insulitis was present in the BBD rats after 5 weeks of disease (with a score of 2.9 +/- 0.1 vs. 1.4 +/- 0.6 in the normoglycemic controls), but absent in WSTZ rats after 5 months of disease (1.2 +/- 0.06 vs. 1.1 +/- 0.06). Increase in fibrinogen and AG plasma levels was observed in the BBD rats only and appears linked to the insulitis. The major acute phase protein AG level is increased in BBD rats already on the first day of appearance of glycosuria. In the WSTZ rats, without insulitis, chronic hyperglycemia alone did not induce an increase in fibrinogen and AG plasma levels. A decreased microcirculatory erythrocyte velocity has been found in both BBD and WSTZ rats. Thus an increase in fibrinogen or AG plasma levels is not necessary for inducing a decrease in erythrocyte velocity. Hyperglycemia is probably the main factor responsible for the decrease in microcirculatory flow in the BBD and WSTZ rats.

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Calcic ions play a role in the initial and chronic development of atherosclerosis lesions. Demonstration of anti atherogenic properties of calcium antagonists has open a new therapeutic approach of atherosclerosis and of its complications. Mechanism of protection remains unclear but it is essentially a preventive effect requiring very early administration before onset of lesions. Several experimental studies have shown that calcium antagonists could reduce cholesterol content of arterial wall without any modification of lipid plasma or profile. Several other mechanisms have been proposed but none of them can actually explain the observed preventive effect of these drugs. The clinical relevance in human of such a preventive effect of calcium antagonists requires further investigations.

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The effect of pravastatin, an inhibitor of HMG CoA reductase, on blood lipids and aortic lipidosis was studied in young cholesterol-fed White Carneau pigeons. The birds were fed with normal ('N group', n = 20) or atherogenic diet (grains + 0.4% cholesterol + 4% lard) alone ('C group', n = 20) and in association with pravastatin ('P group', n = 20). Plasma lipids and aortic intima lipidosis were studied after 3-5 and 8-12 months of the diet. Compared to the N group, pigeons from C group exhibited hypercholesterolemia (TC = 1000 mg/dl) and hyperlipoproteinemia of which level was independent of the duration of the diet. Total VLDL (VLDL+LDL)-cholesterol and apolipoprotein-B levels rose significantly 15, 8 and 4 times, respectively, whereas HDL were increased two times (P < 0.01) in females only. Macroscopically visible intima lipidosis areas covered 40% and 80% of aortic surface after 3-5 and 8-12 months of the diet. In P group, the increase in plasma lipid values was significantly lower than in WC from C group: -40% for total cholesterol (600 mg/dl) (P < 0.01), -71% for VLDL (P < 0.001), -53% for (VLDL+LDL)-cholesterol (P < 0.01) and -54% for apo-B (P < 0.05). HDL remained as high as in C group. Consequently TC/HDL-C ratio was improved and atherogenic risk of cholesterol was reduced by 41% (P < 0.05). Intima lipidosis areas were lowered by 35% (P < 0.01). We conclude that pravastatin treatment involves (1) a decrease in hypercholesterolemia and hyperlipoproteinemia and (2) a lowering in extensiveness and severity of macroscopically visible aortic lipidosis in cholesterol-fed White Carneau pigeon.

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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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The risk of developing macroangiopathy associated with diabetes led us to study in sand rats the long-term consequences of non-insulin-dependent diabetes on the development of arterial lesions promoted by feeding a high-cholesterol diet. Gliclazide, an agent whose preventive effect has previously been suggested in other experimental models of atheroma, was also investigated in these diabetic and hypercholesterolemic animals. Sand rats were fed a natural diet (ND group), a standard laboratory feed (StD group), or a high-cholesterol feed (HCD group) for 15 months. Biologic parameters were monitored throughout the period of the study, and histologic and histochemical examinations were conducted when the animals were killed (month 15). One StD group and one HCD group were treated with gliclazide from month 3 to month 15. The StD group developed a syndrome of obesity, hyperglycemia, hyperinsulinemia, and triglyceridemia. The high cholesterol feed further increased hypercholesterolemia. These biologic abnormalities were accompanied by arterial lesions (thickening of the intima, deposition of glycosaminoglycans). Foam cells were seen in the intima, and microthrombi were present in the lumen of the arteries of animals in the HCD group. Long-term gliclazide medication at doses that normalized serum glucose levels also reduced the obesity, hyperinsulinemia, lipid disorders, and it prevented or retarded the appearance of arterial lesions.

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The purpose of the study was to investigate the development of microangiopathic complications in North African sand rats with diabetes induced by a long-term standard laboratory diet. Hyperinsulinaemic rats, whether non-diabetic obese or diabetic, developed capillary basement membrane (CBM) thickening in the skin; in insulin-dependent animals, this change was diffuse. Many PAS positive areas were demonstrated in skeletal muscle and myocardium, together with evidence of microangiopathy; the primary myocardial lesion in insulin-dependent disease was ischaemic fibrosis. The kidney was also affected with marked basement membrane thickening in Bowman's capsule and glomerular capillaries; glomerulosclerosis and tubular changes were found in insulin-dependent disease. No evidence of diabetic retinopathy was found, and there was a high incidence of cataract.

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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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Kinetic studies of the histochemical and histoenzymatic behavior of rabbit pancreatic parenchymas were performed 5, 30 and 90 days after Wirsung duct ligation. In control pancreas, some enzyme activities (EA) were more prominent in Langerhans islets [glucose-6-phosphatase, glucose-6-phosphate dehydrogenase (DH), isocitrate DH, glycerol-3-phosphate DH, NADPH DH], others were strongly marked in acini and ducts (alkaline phosphatase, beta-glucuronidase, acid esterase aryl-sulfatase). Histochemical and enzyme abnormalities observed in experimental rabbits reflect the post-ligation degenerative and reactive processes in both exocrine and endocrine pancreas: (1) the decrease in Krebs cycle and pentose pathway linked EA and the increased lysosomal and acid phosphatase EA reflect early (day 5) degeneration and necrosis of islets and acini (day 30); (2) proliferative processes in developed ductal epithelia are shown by an increase in both glycolytic and lysosomal EA (days 30 and 90); (3) connective tissue neogenesis and interstitial fibrosis occurred as shown by activated beta-glucuronidase, aryl-sulfatase, alkaline phosphatase and increased ribonucleoproteins and glycoaminoglycans contents (day 30); (4) on day 90, the neoformed cell clusters presenting glucose-6-phosphatase positivity (B-cell marker) are seen in the pancreas remnant. At the same time, blood insulin level increases correlated with a decrease of hyperglycemia.

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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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Eighteen male Wistar rats weighing 230 g (9 wk old) were fed casein diets containing 10% protein (HC), 50% protein (HP) or 10% protein plus 2% DL-methionine (MET) for 2 yr. In HC rats, mean body weight was 570 g; the carcass contained 13.5% protein and 37% lipid. The HP-fed rats had a 100 g lower body weight than HC rats due solely to a smaller amount of body lipid. Liver urea concentration and kidney weight were higher in HP rats than in HC rats. The body weight of MET-fed rats was lower than the other two groups and body lipid was only 30% that of HC rats. Histologic examination showed a normal aspect of the thoracic aorta from HC rats, whereas in HP, moderate signs of vascular aging--thicker intima and media with hypertrophy of smooth muscular cells (smc) with collagen enrichment and diffuse fibrosis--were observed. Aortas from MET rats also exhibited thicker intima and media due to smc hypertrophy. Some smc presented degenerative aspects and necrosis; other smc were replaced by chondroid cells and foci of fibrosis, resulting in a loss of the distension capacity of the aorta. Such an advanced stage of vascular aging is not normally found in 2-yr-old rats.

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