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.

Insolubilization of low density lipoprotein induces cholesterol accumulation in cultured subendothelial cells of human aorta.

Primary cultures of typical and modified smooth muscle cells isolated from the intima of human aorta were used to study the mechanism whereby low density lipoprotein (LDL) induces accumulation of intracellular cholesterol. Incubation of intimal cells with native LDL obtained from human plasma did not lead to deposition of total cholesterol. LDL added to the cultures simultaneously with hyaluronic acid, heparin, chondroitin sulfate, fibronectin, and mouse monoclonal antibody against LDL also failed to alter the cellular cholesterol. On the other hand, 24-h incubation of the cells with LDL in the presence of dextran sulfate, gelatin, particles of aortic elastin, particles of collagenase-resistant aortic matrix, goat polyclonal antibodies against LDL or latex beads caused a significant (1.5-7-fold) increase in total cholesterol. The compounds which stimulated cholesterol deposition are able to form precipitating complexes with LDL. On the contrary, the agents which failed to induce cholesterol accumulation were unable to insolubilize LDL. A direct correlation (r = 0.927) was found between the cholesterol content of the insoluble complex and the increment of cholesterol in the cultured cells. To find out whether LDL plays a specific role in the deposition of intracellular cholesterol, very low density lipoproteins and high density lipoproteins were used. These lipoproteins stimulated the accumulation of intracellular cholesterol in the presence of agents capable of forming insoluble associates with them. Our data suggest that insolubilization of lipoproteins is a key event in the LDL-mediated accumulation of intracellular cholesterol induced by various agents.

Stable analogues of prostacyclin and thromboxane A2 display contradictory influences on atherosclerotic properties of cells cultured from human aorta. The effect of calcium antagonists.

We examined the influence of stable prostacyclin analogues (carbacyclin) and thromboxane A2 (U46619) on atherosclerotic properties of cells: [3H]thymidine incorporation and intracellular cholesterol content. A primary culture of human aortic subendothelial cells derived from atherosclerotic plaques was used. Carbacyclin exerted a direct anti-atherosclerotic effect, significantly reducing atherosclerotic manifestations of cells, while agent U46619 stimulated proliferation and cholesterol accumulation, i.e. demonstrated atherogenic potency in culture. Calcium antagonists (verapamil and diltiazem) markedly enhanced anti-atherosclerotic properties of carbacyclin and restricted the atherogenicity of U46619.

Association of low-density lipoprotein with particulate connective tissue matrix components enhances cholesterol accumulation in cultured subendothelial cells of human aorta.

Low-density lipoproteins (LDL) were incubated with elastin particles, collagenase-resistant debris isolated from human aorta, and latex beads of 1.13 microns in diameter. As a result of incubation, insoluble LDL-associates were formed. These associates, as well as LDL-heparin-fibronectin-gelatin complexes described by other workers, were added to a 7-day primary culture of enzyme-isolated cells of human aortic subendothelial intima. The culture contained a mixed cell population made up mostly of typical and modified smooth muscle cells. 24 h later, total cholesterol, phospholipid, triacylglycerol, free cholesterol and cholesteryl ester levels were measured. Addition of insoluble LDL-complexes as well as LDL-associates to culture brought about a substantial accumulation of intracellular lipids; primarily, cholesteryl esters. The total cholesterol level in cultured cells was raised 3- to 8-fold. Addition of free LDL or LDL-free particles had no effect on the content of intracellular lipids. The results obtained allow the assumption that the occurrence of the LDL-mediated accumulation of intracellular lipids is due mainly to the LDL penetration inside the cell via 'nonspecific' phagocytosis and not through a regulated receptor-dependent pathway.

Low-density lipoprotein apheresis and regression of atherosclerotic plaque in vitro.

Short-term organ culture and primary cell culture of human aorta were used to study the effect of selective removal of low-density lipoproteins (LDLs) from the surrounding medium (LDL apheresis) on the lipid content of cultured tissue and cells, respectively. LDL apheresis was performed by passing the culture medium through immunosorbent containing agarose-bound goat antibodies against human LDL. LDL apheresis promoted the decrease of lipids of all classes in the cultured atherosclerotic plaque. However, only the cholesteryl ester level, but not free cholesterol, triglyceride, or phospholipid levels, was lowered in the cells cultured from the plaque. One may thus assume that LDL apheresis facilitates regression of lipoidosis by reducing the content of lipids in atherosclerotic lesions.

Atherogenicity of blood serum from patients with coronary heart disease.

The sera from 62 of 68 patients with coronary heart disease (CHD) caused a two to five fold elevation in the intracellular cholesterol in primary cultures of subendothelial cells derived from grossly normal intima of human aorta. The sera from 33 of 42 healthy subjects did not show atherogenic properties in culture. Atherogenic potential correlated directly with the serum apolipoprotein-B-apolipoprotein A1 ratio, but not with the level of total cholesterol, high-density-lipoprotein cholesterol, apo-B, or apo-A1. The sera from patients with CHD also facilitated deposition of lipids in the medial smooth muscle cells of human aorta and mononuclear blood cells, though to a lesser degree. They had no such effect on endothelial cells of human aorta and umbilical vein, or human embryo fibroblasts.