Effects of lipid oxidation on transcriptional regulation and cell death

Elevated levels of low density lipoprotein (LDL) in plasma are associated with an increased risk for atherosclerosis. Excessive accumulation of lipoproteins and lipids in the vascular wall is thought to lead to the development of advanced atherosclerotic lesions prone to rupture. In the present investigation, we have studied the cellular effects of oxidized lipids and lipoproteins, as well as lipid accumulation, on the regulation of gene expression. We have also characterized cell death induced by oxysterols, oxidized derivatives of cholesterol, in smooth muscle cells.

Oxidized LDL (ox-LDL) inhibited activation of NF-KB, a transcription factor which induces the expression of a large number of genes encoding proteins involved in immune and inflammatory responses. While this effect would seem to be anti-inflammatory, ox-LDL may have other, pro-inflammatory effects, such as activation of the transcription factor AP- 1, which was for the first time demonstrated in the present study. The biologically active component of ox-LDL responsible for AP-1 induction appears to be Iysophosphatidylcholine, while inhibition of NF-KB may be due to other lipid oxidation products such as aldehydes and oxysterols. The effects of ox-LDL on AP-I and NF-KB were similar in smooth muscle cells, endothelial cells and macrophages stimulated with lipopolysaccharide or TNF, whether LDL was oxidized with copper or UV light.

Activation of AP-I by ox-LDL was associated with increased TNF mRNA expression and protein secretion in adherent monocytes. Conditioned medium from monocytes treated with ox-LDL stimulated proliferation of smooth muscle cells. Antibodies against TNF abolished 45% of the ox-LDL-dependent mitogenicity of conditioned medium, indicating that TNF is a major smooth muscle cell mitogen secreted by monocytes in response to ox-LDL.

Cytotoxic oxysterols are formed during oxidation of LDL. We have shown that several oxysterols can induce apoptosis in smooth muscle cells. Side-chain hydroxylated oxysterols were most potent in inducing apoptosis, but the reasons for this remain to be explained. Both 7ß-hydroxycholesterol and 25-hydroxycholesterol induced intracellular Ca2+ oscillations and activated extracellular signal-regulated protein kinases within a few minutes after addition. Within a few hours, oxysterol treatment caused disorganization, but not swelling, of the ER and Golgi membranes, suggesting that Ca2+ is a critical mediator of oxysterol toxicity.

When macrophages were loaded with lipid by incubation in the presence of acetylated LDL, inducibility of AP-I and TNF expression were decreased, suggesting that lipid accumulation and foam cell formation decrease the inflammatory potential of macrophages. NF-KB was unaffected by lipid loading, but ox-LDL suppressed NF-KB activity, demonstrating that the effects of lipid loading on AP-I were not due to lipoprotein oxidation. Taken together, our results support a model for the pathogenesis of atherosclerosis where atherogenic lipoproteins initially stimulate but subsequently suppress cell proliferation and inflammation.