Antiatherosclerotic effects of calcium antagonists. Study in human aortic cell culture.

To investigate the effects of calcium antagonists on atherosclerotic cellular indices, [3H]thymidine incorporation and intracellular cholesterol content, primary culture of cells isolated from subendothelial intima of human atherosclerotic aorta was used. Among tested drugs were: verapamil, nifedipine, diltiazem, papaverin, nicardipine, D-600, cinnarizine, PN 200 110 and PY 108 068. Verapamil proved to be the most effective. It significantly reduced the total intracellular cholesterol and sharply decreased the incorporation of [3H]thymidine. With respective efficacy verapamil was followed by nifedipine and PY 108 068. Neither beta-blocker (metoprolol) nor nitrate (nitroglycerin) modified antiatherosclerotic effects of calcium antagonist (nifedipine). Calcium agonist Bay K 8644 which facilitates the penetration of calcium into cells caused the accumulation of intracellular cholesterol and stimulated cell proliferation. Simultaneous addition of nifedipine and Bay 8644 led to the inhibition of the agonist's atherogenic effect. Thus, facilitation of calcium influx into cells causes atherosclerotic alterations in the arterial cells; atherogenic calcium effects are inhibited by calcium channel blockers. Furthermore, based on the results of application of blood plasma from patients treated with calcium antagonists or beta-blocker to primary cultures of atherosclerotic cells, it can be assumed that calcium antagonists affect an anti-atherosclerotic and beta-blockers an atherogenic action.

Triggerlike stimulation of cholesterol accumulation and DNA and extracellular matrix synthesis induced by atherogenic serum or low density lipoprotein in cultured cells.

A 72-hour incubation of cultured cells with blood sera or plasma of patients suffering from coronary heart disease (CHD) with angiographically assessed coronary atherosclerosis caused a threefold to fourfold elevation of intracellular cholesterol. An elevated cholesterol level in the cells precultured with patients' sera was retained several days after the removal of the examined serum from culture. The accumulation of intracellular cholesterol was accompanied by enhanced synthesis of DNA, total protein, collagen, sulfated glycosaminoglycans, and hyaluronic acid. Enhanced DNA and total protein synthesis was retained for at least 9 days after the serum had been removed from culture. The obtained results suggest that the sera of CHD patients possess an atherogenic potential that manifests itself at the arterial cell level in the stable stimulation of atherosclerotic cellular processes: proliferation, lipidosis, and fibrosis. The examined sera of healthy donors were devoid of such an atherogenic potential. The low density lipoprotein (LDL) fraction (density, 1.030-1.050 g/cm3) obtained from an atherogenic serum had the same atherogenic potential as a whole serum. Atherosclerotic alterations in cultured intimal cells caused by atherogenic LDL were retained for at least 3 days after the removal of the lipoprotein from culture. Preincubation of intimal cells with LDL obtained from healthy donors had no effect on the intracellular cholesterol level or the synthesis of DNA and extracellular matrix. One may assume that the atherogenic potential of CHD patients' sera is related to the presence of LDLs that are qualitatively different from the LDL of healthy subjects.

Cyclic nucleotides and atherosclerosis: studies in primary culture of human aortic cells.

A primary culture of cells derived from uninvolved and atherosclerotic intima of human aorta was used to elucidate the role of cyclic nucleotides in atherogenesis. The cells cultured from fatty streaks and atherosclerotic plaques had a 2- to 8-fold lower cyclic AMP level and a 1.5- to 2-fold higher level of cyclic GMP compared with those of a grossly normal intima. Medial cells cultured from nonlesioned and atherosclerotic aortic segments showed no differences in the cyclic nucleotide concentrations. Reduction of the intracellular cyclic AMP with 2'-deoxyadenosine or a cyclic GMP elevation with its dibutyryl derivative, or liposomes containing cyclic GMP stimulated the uptake of [3H]thymidine and protein synthesis in the cells cultured from unaffected intima. On the contrary, a rise of the intracellular cyclic AMP caused by adenylate cyclase activators, a phosphodiesterase inhibitor, dibutyryl cyclic AMP, and liposomes containing cyclic AMP inhibited cell proliferation and protein synthesis. Elevation of the intracellular cyclic AMP stimulated the hydrolysis of lipids which led to reduction of lipid levels in the cells cultured from atherosclerotic lesions. The results of this study corroborate the existence of a relationship between the alterations of intracellular cyclic nucleotide levels and the metabolic disorders occurring in atherosclerosis.

Trapidil derivatives as potential antiatherosclerotic drugs.

Trapidil, a triazolopyrimidine, and its derivatives are coronary vasodilating drugs. Trapidil reduces the serum level of low density lipoprotein- and very low density lipoprotein-cholesterol and increases the serum level of high density lipoprotein-cholesterol in hyperlipemic patients. The present study demonstrates that trapidil and five different trapidil derivatives inhibit the proliferation of cells cultured from grossly normal intima and fatty streaks of human aorta. The inhibiting effect of trapidil derivatives is about 60%, similar to the standard substance 3-isobutyl-1-methyl-xanthine (MIX). In cells cultured from atherosclerotic plaques trapidil and trapidil derivatives reduced the content of cholesteryl esters by 36% for trapidil and between 47% and 68% for 4 of 5 trapidil derivatives, respectively. The trapidil derivative AR 12463 (5-piperidino-7-[N-(n-amyl)-N-(beta-hydroxyethyl)amino]-s-triazolo[1,5- a]pyrimidine) reduces the free cholesterol content by 29%, but the other trapidil derivatives are without effect on this parameter. Four of five derivatives decrease the content of triglycerides by 53 to 70%. The synthesis of collagen is inhibited by the trapidil derivative AR 12463 (25%). Trapidil and other derivatives have a smaller or no effect on the synthesis of collagen. These effects of trapidil derivatives point to potential antiatherosclerotic properties. The possible mechanisms are discussed.

Disorders in the system of cyclic nucleotides in atherosclerosis: cyclic AMP and cyclic GMP content and activity of related enzymes in human aorta.

Cyclic AMP and cyclic GMP content and activities of cyclic nucleotide metabolic enzymes were determined in intima and media of atherosclerotic and unaffected human aorta obtained shortly after death due to myocardial infarction. Cyclic AMP content in fatty streaks and atherosclerotic plaques was lower by three- and five-fold, respectively, as compared with uninvolved intima. Cyclic GMP level in atherosclerotic lesions was estimated to be three-fold higher than in grossly normal area. Basal activity of adenylate cyclase in fatty streaks and plaques was two- to six-fold lower than in unaffected intima. Besides, the ability of adenylate cyclase to be stimulated by the stable analogue of prostacyclin, carbacyclin, was suppressed in plaques. Guanylate cyclase activity in fatty streaks was 1.5- to three-fold higher than in normal tissue. The thiol-reducing agent, dithiothreitol, decreased the enzyme activity to normal level, suggesting the oxidative nature of guanylate cyclase activation in the lesion zone. There were no significant changes in cyclic AMP phosphodiestease activity in the regions of the atherosclerotic lesion. Cyclic GMP phosphodiesterase activity in atherosclerotic plaques was two-fold lower than in the intima of unaffected areas. We did not find differences in the content of cyclic nucleotides or related enzyme activities in the media of uninvolved areas of human aorta nor in the media underlying atherosclerotic lesions. Our findings suggest that development of human atherosclerotic lesions is accompanied by dramatic changes in the cyclic nucleotide metabolism featuring gradual hormonal receptor uncoupling from adenylate cyclase, activation of guanylate cyclase in fatty streaks and inhibition of cyclic GMP phosphodiesterase in plaques.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary culture of human aortic intima cells as a model for testing antiatherosclerotic drugs. Effects of cyclic AMP, prostaglandins, calcium antagonists, antioxidants, and lipid-lowering agents.

Smooth muscle cells isolated from atherosclerotic lesions of human aorta retain in primary culture their intrinsic in vivo characteristics: namely, enhanced proliferative activity and high lipid levels. We have tested the effect of different compounds on [3H]thymidine uptake and on the levels of phospholipids, triglycerides, cholesterol, and cholesteryl esters in cultured aortic cells. Effects, such as the inhibition of cellular proliferation and/or lowering of the intracellular lipid levels which would be regarded as antiatherosclerotic if exerted in vivo, were observed in vitro by the following compounds: dibutyryl cyclic AMP, cholera toxin, forskolin, methylisobutylxanthine, stable prostacyclin analogues, prostaglandins E2 and D2, verapamil, reserpine, alpha-tocopherol, butylated hydroxytoluene, lipostabil, and high density lipoproteins. In this paper, we discuss the possibility of using a primary culture of smooth muscle cells from an atherosclerotic human aorta for testing drugs for possible antiatherosclerotic activity.

Regression of atherosclerotic manifestations in primary culture of human aortic cells: effects of prostaglandins.

The effect of different prostaglandins on cell proliferation and intracellular lipid content in primary culture of human aortic cells has been studied. It was demonstrated that prostacyclin, its stable analogues (carbacyclin and 6 beta-PGI1), and prostaglandins E1, E2, D2 taken in the concentration of 0.5 micrograms/ml and 10 micrograms/ml decreased the 3H-thymidine uptake into cultured intimal smooth muscle cells by 2-3-fold. Carbacyclin and 6 beta-PGI1 also lowered by 1.5-2-fold the triglyceride and cholesteryl ester levels in cells cultured from atherosclerotic lesions. Prostaglandins E1, E2, D2 in the concentration of 10 micrograms/ml decreased by 20-40% total cholesterol level in cultured cells.