Intersitial collagen critically influences the structure of atherosclerotic arteries. Activated macrophages in human atheromata overexpress matrix-degrading enzymes including interstitial collagenases (MMP-l/collagenase-1, MMP- 13/collagenase-3, and MMP-8/collagenase-2). We recently showed that collagenase resistance due to mutation of collagen type I at the cleavage site for collagenases yields increased collagen accumulation in mouse atheromata. However, direct in vivo evidence of the importance of specific collagenolytic enzymes in arterial remodeling during atherogenesis remains scant. We therefore propose to study in vivo mechanisms of collagen remodeling by collagenases. This project will test three hypotheses in genetically-altered mice in vivo or with cells derived from such animals or with genetically-manipulated human macrophages in vitro.
Specific Aim 1 will test the hypothesis that specific collagenases regulate plaque structure during atherogenesis in mice with targeted deletions of the major murine interstitial collagenases. We will determine whether deficiency of MMP-8 or -13 or both collagenases increases collagen accumulation in atheromata and influences other variables of plaque structure in atherosclerosis-susceptible apoE mice.
Specific Aim 2 will explore the roles of interstitial collagenases (MMP-8, -13 and -14) from cells derived from bone-marrow (primarily macrophages) in collagen accumulation and arterial remodeling in atheromata. We hypothesize that selective restoration of MMP activity in apoE mice lacking MMP-13 or -8 by transfer of bone marrow from apoE-/- mice, wild-type for the respective MMP, will decrease collagen content, yield thinner fibrous caps in lesions and/or cause local ectasia. We will also determine whether selective deletion of MMP-14 in bone marrow-derived cells affects these variables (the MMP-14-/- mouse has early lethality).
Specific Aim 3 will test the hypothesis that MMP-8, -13 or -14 mediates matrix degradation and vascular cell and macrophage migration in vitro. We will test whether mouse macrophages deficient in these MMPs have altered ability to degrade collagen in matrices generated by SMC in culture. We will perform similar experiments with human macrophages with reduced expression of specific collagenases in siRNA """"""""knock-down"""""""" experiments. We will further test the hypothesis that SMC and macrophages deficient in the interstitial collagenases (MMP-13, -8, or -14) have reduced ability to migrate through extracellular matrix in vitro. These experiments will provide mechanistic insights into the phenotypes that we will likely obtain in the in vivo experiments of the first two specific aims. Together, this project should help in understanding the mechanisms of extracellular matrix remodeling during atherogenesis, a key determinant of the clinical expression of this disease.
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