No abstract

Bovine aortic endothelial c~lls (BAEC) pulsed for 5 h with mildly oxidized low density lipoproteins (LDL), exhibit~ a broad, sustained and high peak of [Ca:*]l occurring several hours after the end of the pulse and reachilll~ very high [Ca"+]t values (around 2500--3000 nmol/I) and a concomitant drop of eytosolic pH (around 0.2-0.3 pH units) without any loss of cell viability. When BAEC were continuously pulsed with oxidized LDL, the peak of [Ca2*]~ was more sustained than in short pulse experiments and was associated with irreversible morphological ~hanges usually associat~l with cytotoxi¢ events (blebbin$) and with a marked loss of viability. The potential involvement of thes, biochemical and morphological changes in atherogenesis are discussed.

Oxidized low density lipoprotein (LDL) is thought to play a major role in atherogenesis. Atherosclerotic arteries exhibit structural changes associated with profound alterations in vascular tone that are potentially involved in arterial spasm and ischemic heart disease. We report here the role of oxidized LDL in the retraction of vascular smooth muscle cells. Mildly oxidized LDL elicited a broad and sustained peak in cytosolic calcium concentration ([Ca2+]i) in cultured arterial smooth muscle cells. Concomitant with the [Ca2+]i rise, oxidized LDL evoked a sustained and intense retraction of smooth muscle cells, as shown by the changes in cross-sectional area of single cells. Cell retraction was dependent on time, the concentration of oxidized LDL, and the level of LDL oxidation (native LDL induced neither a significant [Ca2+]i rise nor cell retraction). Oxidized LDL but not native LDL also elicited a delayed (12 +/- 2 hours) and sustained (14 +/- 2 hours) increase in isometric tension in deendothelialized arterial rings only, thus suggesting a protective role of intact endothelium. When triggered by nontoxic doses of oxidized LDL, retraction of cultured cells and the contractile response of aortic rings was reversible, whereas with higher (toxic) doses (> or = 200 micrograms apoB/mL), cell retraction was irreversible and led progressively to detachment and cell death. Cell retraction can be prevented in three ways: (1) by inhibiting LDL oxidation with supplements of antioxidants (indirect inhibition); (2) by blocking the pathogenic intracellular signaling elicited by oxidized LDL (direct inhibition), eg, by inhibiting calcium influx with EGTA or the calcium channel blocker nisoldipine or by blocking intracellular signaling (at a still-unknown step) by the lipophilic antioxidant alpha-tocopherol; and (3) by directly inhibiting myosin light chain kinase by 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1, 4-diazepine. In conclusion, oxidized LDL evoked a sustained and intense calcium-dependent retraction of cultured smooth muscle cell, which can be prevented by inhibiting LDL oxidation or by blocking the intracellular signaling induced by oxidized LDL.

Wolman disease in an inherited metabolic disease, characterized by a severe deficiency of the acid lipase and a massive lysosomal storage of triacylglycerols and cholesteryl esters, associated with hepatosplenomegaly, adrenal calcification and nearly always fatal in the first year of life. Cultured human lymphoblastoid cells and human adrenal cells are able to promote the formation of mildly oxidized low-density lipoproteins (LDL), which in turn exhibit a non-negligible cytotoxic effect on these cells. In contrast, fibroblasts induce only very low levels of LDL oxidation. Comparative experiments have shown that the cytotoxic effect of oxidized LDL was higher to Wolman-disease cells than to controls. The oxidative ability of Wolman cells was similar to that of normal ones. The over-cytotoxicity of mildly oxidized LDL to Wolman cells resulted from the higher uptake of mildly oxidized LDL through the LDL-receptor pathway, which is only poorly down-regulated in Wolman cells subsequently to the block of the lysosomal degradation of LDL-cholesteryl esters. In cultured adrenal cells, oxidized LDL induced a sustained rise in intracellular [Ca2+] which is directly involved in the cellular damage and cell death induced by oxidized LDL [Nègre-Salvayre and Salvayre (1992) Biochim. Biophys. Acta 1123, 207-215]. This Ca2+ peak is followed by a dramatic deposition of calcium in damaged or/and dead cultured adrenal cells, quite similar to that observed in Wolman-disease adrenal cortex. The cell-induced LDL oxidation and the subsequent cytotoxic effect can be prevented, at least in part, by antioxidants such as alpha-tocopherol and nordihydroguaiaretic acid. These findings support the hypothesis that the Wolman-disease adrenal damage (necrosis and calcification) could result from the association of the following events: mild oxidation of LDL by adrenal cells, over-uptake of mildly oxidized LDL by Wolman cells (resulting from the block of the lysosomal degradation of cholesteryl esters in Wolman cells), and cytotoxicity related to the amount of mildly oxidized LDL internalized by cells. The reported data also suggest that LDL oxidation induced by adrenal cells and their subsequent cytotoxicity can be prevented (in part) by antioxidants, and the potential therapeutic use of antioxidants in Wolman disease is discussed.