The clinical manifestations and complications of human atherosclerosis depend critically on the fine structure of the plaque and the integrity of its extracellular matrix. We and others have furnished evidence that cytokine-mediated inflammation in general, and alterations in extracellular matrix synthesis and degradation in particular regulate the stability of atheroma. Moreover, we have postulated that lipid lowering stabilizes atherosclerotic lesions (as shown by recent clinical trials) by reducing inflammation and ameliorating matrix weakening. This Project will test in vivo in transgenic mice three hypotheses related to the molecular regulation of plaque stability. (1) We will test the hypothesis that overexpression of an active form of a matrix-degrading proteinase in an evolving atheromatous lesion will alter features of its structure related to plaque stability. As an example of this strategy, we propose to drive an autoactivatable mutant form of stromelysin-1 with scavenger receptor promoter sequences in an atherosclerosis- susceptible strain of mice (e.g. those lacking LDL-receptors, LDL- R0). (2) using a similar strategy, we will test hypothesis that overexpression of the pro-inflammatory cytokine gamma interferon (IFN-gamma) in diet-induced lesions will alter the arterial matrix and activate atherogenic functions of lesional cells in LDL-Ro mice. Conversely, we will test whether local overexpression of the anti- inflammatory cytokine interleukin (IL)-10 modulates the structure of experimentally-induced atheroma or reduced endogenous proteinase expression and other indices of lesional cell activation. (3) We will test the hypothesis that on mechanism of 'lesion stabilization' by lipid lowering, a phenomenon disclosed by recent human studies, involves attenuation of the expression of genes encoding matrix- degrading pro-inflammatory cytokines and/or proteinases. For example, we will probe for expression (protein and mRNA) of cytokines (e.g. IFN-gamma) and metalloproteinases (e.g. MMP-9) in the arteries of atherosclerosis-susceptible mice to test whether modulation of plasma cholesterol levels (e.g. by diet) regulates the expression of these two model genes. In later studies, we will prepare LDL-R0 mice transgenic for constructs derived from the promoter regions of matrix metalloproteinase-9 (MMP-9) or genomic sequences of IFN-gamma linked to a luciferase reporter, and test the hypothesis that lipid lowering by diet decreases transcriptional activity of these genes in mouse atheroma. Together, these experiments should help to understand the molecular mechanisms that regulate the stability of atheroma in vivo, advance our understanding of the mechanisms of therapies suspected (without direct evidence) to owe their effectiveness in acute event reduction to an effect on the lesion itself, provide new tools for assessment of potential interventions, and furnish insight into lesion biology that may suggest new therapeutic avenues for atherosclerosis.
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