This subproject will test hypotheses generated in our Program using transgenic and gene targeting technologies in mice. During the present grant period, our work has focused on aspects of oxidation (paraoxonase 1, myeloperoxidase, heme oxygenase-1, and secretory phospholipase A2) and macrophage function (macrophage colony stimulating factor, scavenger receptor CD68 and the nuclear receptor LXR). We now propose to extend some of these studies of paraoxonase-1 (PON1), heme oxygenase (HO-1) and macrophage colony stimulating factor (M-CSF) and to examine the functions of two additional proteins that have emerged as candidates from studies in this program: paraoxonase 3 (PON3) and 5-lipoxygenase (5-LO). PON1 and PON3 are members of a family of esterases, and both are carried on HDL. Using gene targeting, we provided strong evidence that PON1 protects against atherosclerosis and inhibits lipid oxidation. During the past two years, we and others have obtained evidence for a strong anti-oxidant function of PON3, and we will now test this using gene targeting. HO-1 has proved to be difficult to examine because HO-1 null mice breed poorly, although during the present grant period we did obtain evidence for its protective function in vivo using induction and inhibition with pharmacologic agents. We now propose to utilize transgenic approaches to pursue more detailed investigations of its role in atherosclerosis. M-CSF has been a long-term interest, since we originally cloned it in the mid-1980s. The expression of M-CSF is critical in the development of atherosclerosis in mice, and null mice have more than a 100-fold decrease in lesion development on an LDL receptor null background. But precisely how M-CSF contributes to the disease and which are the relevant sites of expression are unknown. These questions will be addressed using cell-specific knockouts of the M-CSF gene. Finally, 5-LO was identified in the subproject by Lusis as the gene controlling susceptibility to atherosclerosis in a genetic cross between inbred strains CAST and C57BL/6. 5-LO knockout were then shown to exhibit dramatically reduced atherosclerosis. We now propose to examine mechanistic questions pertaining to the role of 5-LO in atherosclerosis, including effects on monocyte proliferation and apoptosis.
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