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Cholesteryl esters (CE) comprise the principal lipid class that accumulates within macrophages and smooth muscle cells of the atherosclerotic lesion. Acyl-CoA cholesterol acyl-transferase (ACAT) is the major enzyme responsible for esterification of intracellular cholesterol. We evaluated the ability of E5324 (n-butyl-N'-[-2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methyl-phenyl]urea), a novel, orally absorbable ACAT inhibitor, to inhibit esterification of fatty acids to cholesterol and CE accumulation in macrophages and in smooth muscle cells. E5324 significantly inhibited cholesterol esterification in rat aortic smooth muscle cells and in macrophages. In addition, E5324 reduced the cellular mass of CE, the significant measure of the efficacy of drugs designed to modulate cholesterol metabolism. E5324 treatment of macrophages exposed to acetylated low-density lipoprotein reduced CE mass by 97%, and treatment of lipid-loaded smooth muscle cells reduced CE mass by 29%. Although free cholesterol increased approximately twofold, this free cholesterol would presumably be accessible to the membrane for efflux in vivo (reverse cholesterol transport). These results demonstrate that E5324 can inhibit cholesterol esterification and CE mass in atherosclerotic foam cells, derived from either macrophages or arterial smooth muscle cells.

Previous studies of age-related susceptibility to viral infection have focused largely on the effects of aging on the immune response. Little attention has been given to age-related changes in the infectivity of target cells. We show here a fourfold greater plating efficiency of herpes simplex virus type 1 (HSV-1) for cultured vascular smooth muscle cells derived from adult rats compared with cells from genetically identical pup rats. The difference in plating efficiency appeared to be due to differences in initial attachment of the virion to the cell surface. There were no differences in the rate of viral entry or the efficiency of viral replication at high multiplicities of infection and no resistant "subpopulation" of pup cells. The pup cells did not release a soluble inhibitor of infection. Infection in both cell types was inhibited similarly by basic fibroblast growth factor (bFGF). Although adult cells exhibited a more vigorous mitogenic response to bFGF than did pup cells, binding studies did not demonstrate significant differences in the binding of bFGF to the cell surface, suggesting that differential expression of high-affinity FGF receptors could not be correlated with the difference in infectivity. We speculate that differences in the distribution of heparan sulfate in the cell surface, which serves as the initial attachment site for HSV-1, may explain the observed differences in plating efficiency. Since age is a risk factor for the development of atherosclerosis, these results have potential implications for susceptibility of the vasculature to herpesviral infections as a function of the development of the vessel wall.

The effects of transforming growth factor-beta (TGF-beta) on low density lipoprotein (LDL) receptor-mediated cholesterol metabolism were evaluated in vascular smooth muscle cells. TGF-beta significantly increased the binding, uptake, and degradation of 125I-LDL. This increase was paralleled by an increase in LDL receptor mRNA steady state levels and an increase in cholesterol esterification. The increase in LDL cholesterol metabolism was independent of proliferation. LDL receptor expression in response to TGF-beta was not affected by coincubation with an antibody against platelet-derived growth factor or by cyclooxygenase inhibitors in arterial smooth muscle cells, suggesting that TGF-beta's effect was not mediated through platelet-derived growth factor or prostaglandins, as demonstrated in other cell systems. However, coincubation with pertussis toxin abrogated the effect of TGF-beta on LDL receptor expression, suggesting that a pertussis toxin-sensitive G-protein may be involved in the signal transduction pathway. These results are discussed in terms of their potential effects on cellular cholesterol trafficking.

Recent clinical studies have shown that calcium channel blockers can retard and possibly reduce the angiographic progression of coronary artery disease. Calcium channel blockers also inhibit dietary-induced atherosclerosis in animal models of this disease. In this study, we delineate potential cellular and molecular mechanisms by which nicardipine, a dihydropyridine calcium antagonist, may alter lipoprotein and cholesterol trafficking, affect the regulatory signal transduction pathways involved in accelerating cholesteryl ester (CE) catabolism in vascular smooth muscle cells, and modulate cell-cell interactions of vascular and inflammatory cells. We demonstrate in arterial smooth muscle cells that nicardipine increases 1) LDL binding, uptake, and degradation, 2) RNA transcript levels for the LDL receptor, 3) CE catabolic activity, 4) PGI2 release, and 5) RNA transcript levels for cyclooxygenase. Furthermore, nicardipine blocked cytokine-induced monocyte adhesion to endothelial cells and smooth muscle cells. Taken together, these findings support the hypothesis that nicardipine may function as an anti-atherosclerotic agent by promoting CE catabolism and cholesterol clearance and by reducing monocyte adhesion to the activated endothelium.

In vivo studies have suggested that the aorta from an old animal responds to injury with an exaggerated proliferation of smooth muscle cells (SMCs) compared with the response of this aorta from a young animal. In this study we compared proliferation of SMCs derived from uninjured old (less than 19 mo) and young (3-4 mo) rat aortas. Old SMCs grew more rapidly than young SMCs in the presence of medium containing competence factors (10% FCS or platelet-derived growth factor [PDGF]) as well as in their absence (2% PDS or serum-free media) as determined both by a short-term thymidine incorporation assay and by cell counts. Lysates prepared from old SMCs that had been grown in the absence of serum or PDGF stimulated proliferation of target cells more than lysates prepared from young SMCs; the effect was inversely related to cell density of the SMCs. This stimulatory effect of lysates was completely blocked by antibody to PDGF. After the growth-promoting activity of lysates was eliminated by anti-PDGF, growth-inhibiting activity was revealed. Lysates prepared from old SMCs had significantly less capacity to inhibit target cell growth. In the presence of exogenous heparin both the serum- or PDGF-stimulated proliferation and serum-free proliferation of old SMCs were decreased to the level of proliferation of young SMCs. These results suggest that the balance between growth-promoting and growth-inhibiting factors is altered in SMCs from old rats. This may contribute to the increased proliferative capacity of these cells in culture and may facilitate the development of atherosclerosis with age.

Herpes simplex viruses (HSVs) contain a function that can cause the degradation of host mRNA and mediate the shutoff of host protein synthesis. Previously, we observed that HSV infection causes a 40-fold increase in cholesteryl ester (CE) accretion in arterial smooth muscle cells due, in part, to a substantial decrease in CE hydrolysis. In studies reported herein, we found that HSV infection leads to reduced immunoprecipitable lysosomal (acid) CE hydrolase (ACEH) and beta-galactosidase, another lysosomal enzyme in vascular smooth muscle cells. The HSV-induced reduction was greater with respect to ACEH than beta-galactosidase. To determine whether degradation of host cellular mRNA or inhibition of cellular translation was responsible for decreased CE hydrolysis in HSV-infected smooth muscle cells, we utilized an in vitro translation system that permitted us to compensate for any mRNA degradation during viral infection. Reduced ACEH activity was observed in the total cellular RNA translation products of HSV-infected smooth muscle cells compared to uninfected cells owing to posttranscriptional modification. We conclude that the decrease in CE hydrolysis in HSV-infected smooth muscle cells is caused primarily by decreased ACEH synthesis and activity, which can contribute to CE accretion in these vascular cells.