Control of the thickness of the arterial wall is critical, as excessive overgrowth of constituent smooth muscle cells (SMCs) may interfere with blood flow. Effects on SMCs in vitro of several growth factors that are present in blood and/or that are produced endogenously in the arterial wall under certain conditions suggest that influences of endocrine, paracrine, and autocrine nature from stimulating and inhibiting factors may control the smooth muscle tissue mass in the artery. This possibility was explored further by investigating the degree of myodifferentiation in terms of the presence of differentiation-specific filamentous a-smooth muscle actin and growth, as measured by the synthesis of DNA and cell number, of SMCs as influenced by their exposure to the mitogens, platelet-derived growth factor and epidermal growth factor, and the bifunctional growth factor, transforming growth factor-01 (TGF-/31). Exposure to TGF-/31 markedly enhanced differentiation-specific filamentous o-smooth muscle actin. This effect did not require arrest of growth, which speaks against a direct causal relation between loss of myodifferentiation (modulation) and multiplication. When quiescent cultures were exposed to TGF-/31, <*-smooth muscle actin was further increased, indicating a more specific differentiation-promoting effect by TGF-/31 than mere inhibition of growth. Exposure to TGF-/31 also increased spreading, which occurred in parallel with increased filamentous tv-smooth muscle actin and appearance of stress fibers. Exposure to platelet-derived growth factor under serum-free conditions and to epidermal growth factor in cultures exposed to serum markedly decreased the number of cv-actin-positive SMCs, indicating a dedifferentiating effect by these mitogens. Exposure of SMCs to TGF-/31 under serum-free conditions had pronounced effects on growth, with a concentration-dependent inhibition of platelet-derived growth factor-induced DNA synthesis and cell multiplication. The basal synthesis of DNA in the absence of added growth factors was also greatly inhibited. With serum-free cultures, some loss of cells occurred even with very low concentrations of TGF-/31 (5 pg/ml), against which platelet-derived growth factor or a dense cultural state had a protective effect. Enhancement of cell multiplication was not detected for cultivated human SMCs exposed to TGF-/31, irrespective of culture density, in contrast to that reported for dense cultures of rat SMCs. TGF-/31 is present in and may be released from platelets in situations that promote platelet adherence such as endothelial injury; TGF-/31 may also be released from activated macrophages and T lymphocytes either during an immune reaction or inflammation or from the endothelium. The pronounced effects in vitro of this factor with promotion of myodifferentiation and inhibition of growth of SMCs could be consistent with a role for this factor of regulation of SMC growth and myodifferentiation in the arterial wall normally, during inflammation, and in atherosclerosis. (Arteriosclerosi...

The inhibition of experimental atherosclerosis by antioxidants and the presence of oxidized LDL (oxLDL) in atherosclerotic lesions indicate that oxLDL may play what is perhaps a primary role in atherogenesis. LDL promotes the growth of arterial smooth muscle cells (SMCs), and oxLDL has cytotoxic effects. Since excessive intimal growth alternating with necrosis is typical of atherosclerotic lesions, we wondered whether these extreme changes in the lesions could be related to the extreme effects of LDL and oxLDL on cells. We therefore examined the effects of increasing LDL oxidation on its capacity to induce cell growth or cell death and whether the latter could be due to apoptosis. Cells of the types present in the atherosclerotic artery used, ie, SMCs (human arterial), macrophages (human macrophage-like cell line THP-1), and human fibroblasts. Growth was evaluated by measuring the synthesis of DNA and culture size (MTT method) and apoptosis by using the in situ labeling of internucleosomally degraded DNA and, in the case of SMCs, the appearance of chromatin condensation. The oxidation of LDL was by UV or Fe ions. Shortly oxidized LDL had a markedly increased growth-promoting effect on all cell types. With prolonged exposure to UV, but not to Fe, LDL became increasingly cytotoxic, and this toxicity was paralleled by the appearance of apoptosis in all cell types. After prolonged UV treatment, low-molecular-weight material from the partially degraded LDL was responsible for the induction of apoptosis. The dual effect of oxLDL, ie, its strong growth-promoting effect or the induction of cell death by apoptosis, depending on the degree of change by oxidation, is compatible with the notion that oxLDL plays a role not only in atherogenesis but also more extensively in the development of the structure typical of the atherosclerotic lesion, with focal excessive growth alternating with necrosis.

Abstract-We recently reported on the immunolocalization of type II secretory nonpancreatic phospholipase A 2 (snpPLA 2 ) in human atherosclerotic lesions. In the present study, we present data on the distribution and ultrastructural localization of snpPLA 2 in adjacent nonatherosclerotic and atherosclerotic regions of human arteries. Electron microscopy (EM) of immunogold labeling techniques with a monoclonal antibody was used to analyze arterial tissue. The human specimens analyzed were obtained from autopsy and surgery cases. The results with EM showed a stronger snpPLA 2 immunoreactivity in regions of arteries with atherosclerotic lesions than in regions without lesions from the same individual. snpPLA 2 immunoreactivity was stronger in the arterial intima of atherosclerotic than of nonatherosclerotic tissue. EM-immunogold examination revealed that the majority of snpPLA 2 was localized along the extracellular matrix, associated with collagen fibers and other extracellular matrix structures. Intracellular snpPLA 2 was observed in electron-dense vesicles in intimal cells. snpPLA 2 was also found in contact with large, extracellular lipid droplets. These results support the hypothesis that extracellular snpPLA 2 is localized at sites where it may hydrolyze phospholipids from lipoproteins and lipid aggregates retained in the extracellular matrix of the arterial wall. This may be a mechanism for in situ release of proinflammatory lipids, free fatty acids, and lysophosphatidylcholine in regions of apolipoprotein B accumulation, which are abundant in atherosclerotic lesions. (Arterioscler Thromb Vasc Biol. 1998;18:519-525.)

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Smooth muscle proliferation leading to excessive intimal thickening is of prime importance in atherosclerosis. Human arterial smooth muscle cells (SMCs) and human lung fibroblasts are rather insensitive to mitogens under plasmafree conditions in vitro. This prompted us to study the distribution and nature of the growth-promoting material in human plasma. SMCs were obtained from explants of human aortic media. More than 80% of the growth-promoting activity of plasma was present in the lipoprotein (LP) fraction. The growth-promoting capacity of the different LPs was determined on fractions isolated with density gradient ultracentrifugation. Cytotoxic effects appeared if low-density lipoprotein (LDL) was not protected from oxidation and were aggravated with platelet-derived growth factor (PDGF)-BB. Very-lowdensity lipoprotein, LDL, and high-density lipoprotein (HDL) stimulated DNA replication and cell growth by themselves. The stimulation was considerable and equaled that obtained with PDGF-BB only. It was strongly increased in the presence of PDGF-BB. The effect on SMCs was not uniform for subfractions of HDL. A light portion inhibited growth in the absence but strongly stimulated it in the presence of PDGF-BB. For fibroblasts, HDL subfractions had a uniform effect, suggesting a cell type-dependent difference. Addition of cholesterol or essential fatty acids did not induce a growth response similar to that of LPs. This speaks strongly against mere nutritional supplementation as responsible for the mitogenic and growth-promoting effect of LPs and suggests that the effect may be more specific. Disordered LP metabolism is strongly related to atherosclerosis, and certain LPs have a potential role for the deposition of lipids. In addition to this, the distinct mitogenic and growth-stimulating effect of LPs by themselves, as demonstrated in the present report, suggests a mechanism by which intimal thickening, which is a prerequisite for atherosclerosis, may be induced. The pronounced amplification of this effect with PDGF-BB, a substance that also has been implicated in atherogenesis, might promote growth leading to the excessive intimal thickening in the atherosclerotic plaque. (Arterioscler Thromb. 1994;14:288-298.)