This is a renewal application for a project entitled """"""""Animal Models for Atherosclerosis Research."""""""" The hypothesis that was being tested in the original application was that since 15-lipoxygenase (15-LO) had been documented to have the capacity to oxidatively modify low density lipoprotein (LDL), and oxidized LDL being much more atherogenic than native LDL, animals that overexpresses 15-LO would have greatly increased susceptibility to atherosclerosis development. The PI produced transgenic rabbits with a 15-LO transgene driven by a lysozyme (Lys) promoter. The enzyme was expressed in a macrophage-specific manner. Over the course of three years, he performed three sets of experiments, two in transgenic and control rabbits with a wild-type background and one in animals with a heterozygous Watanabe heritable hyperlipidemic (WHHL) background. In all 3 sets of experiments, the presence of the 15-LO transgene was associated with protection against diet-induced atherosclerosis. This was a surprise finding contrary to dogma. In this renewal application, he will further examine the role of 15-LO and macrophage in atherogenesis. Since rabbit experiments are time-consuming and expensive, and few genetic models are available, he will perform all experiments in this application in mice. The first part of the project (Specific Aims 1-3) deals with 15-LO transgenic mice, cross-bred into a 12/15-LO null background, and the second part (Specific Aim 4) deals with the role of macrophages in atherosclerosis development.
In Specific Aim 1, he will generate 15-LO transgenic mice with 2 different macrophage-specific promoters, the Lys promoter and the human scavenger receptor A (SR-A) promoter. Animals will be bred into a 12/15-LO null background, and into apoE and LDLR knockout mice. The susceptibility of these animals to an atherogenic diet feeding will be studied.
In specific Aim 2, he will generate 12/15-LO null transgenic mice expressing human 15-LO using a 130-kb PAC clone as transgene. In these animals, expression of 15-LO would be directed by its natural promoter and their susceptibility to diet-induced atherosclerosis will be examined.
In Specific aim 3, Dr. Chan will generate mice expressing 15-LO in a regulatable binary transactivation system. This system responds to exogenously administered ligand RU486 at a subphysiological dose by induction of 15-LO in a macrophage-specific manner. Similarly, genetic crosses with 12/15-LO, apoE and LDLR null backgrounds will be made before atherogenesis experiments. Macrophage plays a central role in atherogenesis, 15-LO production being only one facet of its many functions in vivo.
In Specific Aim 4, he will produce transgenes containing conditional pro-apoptotic alleles that are driven by Lys and SR-A promoters. By this strategy, he can ablate macrophages in transgenic mice at predetermined times of atherosclerosis development by treating them with FK1012. Use of these conditional pro-apoptotic mutants should allow examination of the role of macrophages in atherosclerosis development. The overall project represents an in-depth study on the role of an enzyme, 15-LO, and a particular cell type, macrophages, in atherosclerosis initiation and progression in animal models in vivo.
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