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.
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.)
We used human arterial smooth muscle cells (SMCs) that had been reorganized three-dimensionally into aggregates, so-called spheroids, as a model system that might more closely correspond to arterial smooth muscle in vivo than do conventional monolayer cultures. After reaggregation the presence of serum in the culture medium strongly promoted the maintenance of spheroidal SMCs. With access to fresh serum, the spheroids developed into highly organized structures with an outer laminated shell of spindle
Excessive growth of the arterial smooth muscle is essential for the development of atherosclerosis and leads to arterial insufficiency in several other conditions. It is therefore important to elucidate the mechanisms that regulate the growth of the human arterial smooth-muscle cell, SMC. Like other untransformed cells, SMC require plasma for sustained growth in vitro. As found in an earlier study most of the material in plasma which stimulates SMC growth is related to the lipoproteins (LP), and is widespread among LP of different density classes. In the present study we investigated whether the growth-stimulating activity might be more specifically related to certain lipoproteins defined by criteria other than density or particle size. Activity was assayed using human SMC and human lung fibroblasts as both a change of culture size and DNA synthesis. The growth-stimulating activity was confined to apo B-containing LP, as defined by their strong affinity to heparin-Sepharose, electrophoretic beta-mobility, the presence of apo B and the absolute requirement of low density lipoprotein (LDL) receptors for the growth-stimulating effect to appear. It was strongly potentiated by PDGF-BB. A much higher level of LDL was required to initiate synthesis of DNA in SMC than in fibroblasts but at optimal LDL concentration the degree of activation was similar for both cell types. Apo B-containing LP are very powerfully related to atherosclerosis. As intimal thickening is a primary change in atherogenesis, the growth-stimulating effect of them may be of direct pathogenetic importance.
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