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The effect of oxidized LDL on macrophage cell growth : implications for the progression of early stage atherosclerosis

University of British Columbia

Early atherosclerotic lesions are characterized by the presence of cholesterol rich, macrophage-derived foam cells. It has recently been shown that macrophage proliferation occurs during the progression of early lesions and that oxidized low density lipoproteins (oxidized LDL) stimulates macrophage growth. Possible mechanisms by which oxidized LDL causes macrophage growth include potentiation of mitogenic signal transduction by a component of oxidized LDL following internalization, by interacting with integral plasma membrane proteins coupled to signalling pathways, or by direct or indirect activation of growth factor receptors on the cell surface. The present study was undertaken to further elucidate the factor(s) and cell surface receptor(s) that are involved in the growth-stimulating effect of oxidized LDL in macrophages, and to characterize some of the early intracellular signaling events by which oxidized LDL mediates macrophage cell growth. In this study, the growth stimulating effect of LDL was specific, in that neither native LDL nor acetyl LDL produced such an effect. Growth stimulation by oxidized LDL was shown to dependent on degree of LDL oxidation, requiring at least 15 hours of copper incubation to achieve significant levels of growth stimulation. Incubation of oxidized LDL with fatty acid-free BSA resulted in a 97% decrease in lysophosphatidylcholine (lysoPC) content, but only a 20% decrease in mitogenic activity. Similarly, treatment of native and acetyl LDL with phospholipase A₂ (PLA₂) led to a 90% increase in lysoPC formation, but only enhanced the ability of acetyl LDL to stimulate growth by 30% compared to that of oxidized LDL. When LDL was pretreated with PMSF to inhibit platelet activating factor-acetylhydrolase (PAF-AH) prior to oxidation, the hydrolysis of oxidized phospholipids to lysoPC was reduced by 70-80%, resulting in a 30% increase in growth stimulation over untreated oxidized LDL. Furthermore, oxidized LDL induced macrophage growth was reduced by approximately 60-70% with the PAF receptor antagonist L659,989, while growth of scavenger receptor type A class I/II (SR-AI/II) deficient cells was reduced by only 30% compared to control macrophages. In THP-1 macrophages, oxidized LDL was also found to increase protein-tyrosine phosphorylation and cause a 2-fold increase in phosphatidylinositol 3-kinase (PI3-kinase) activity compared to similar concentrations of native LDL. Finally, oxidized LDL induced macrophage growth was shown, in part, to require the activation of PI 3-kinase, as growth stimulating activity was reduced by 40-50% if cells were pretreated with PI 3-kinase inhibitors (100 nM wortmannin or 20 uM LY294002). The main findings of this study suggest that oxidized phospholipids, not lysoPC, account for the growth effect of oxidized LDL in macrophages, that PAP receptor activation may be required for the observed growth stimulating effect, and that at least part of the growth stimulating effect of oxidized LDL in macrophages is a result of PI 3-kinase activation.

Thesis/Dissertation

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2009-04-28

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http://hdl.handle.net/2429/7673

Experimental Medicine

University of British Columbia

UBCV

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