Numerous observations support the connection between atherosclerotic lesion progression and disturbed blood flow. Changes that allow lesion development require the matrix-degrading action of matrix metalloproteinases (MMPs), which have emerged as key agents of vascular wall remodeling. Due to their destabilizing action, understanding MMP control and function may have important applications for managing acute cardiovascular events. However, little is known about the regulation of MMPs produced by endothelial cells (EC), the flow sensor of the arterial wall. We propose to investigate the mechanism by which disturbed flow mediates the action of EC MMPs. Previous in vitro studies indicate that oscillatory shear increases the oxidative stress in cultured EC and decreases cell viability. We previously demonstrated that reactive oxygen species (ROS) increase MMP expression and activity. We presently hypothesize that shear-induced oxidative stress increases EC MMP production and activity, specifically of the inducible MMP-9, in areas of disturbed flow. We further propose that increased MMP activity has functional consequences related to degradation of EC basement membrane, and decreased EC viability, which precipitates EC loss, and may contribute to plaque erosion, the major failure mode of otherwise stable plaques. To investigate this potential mechanism, which would connect disturbed blood flow, oxidative stress, and endothelial dysfunction via increased MMP activity, we propose the following specific aims: 1. To investigate whether EC MMP expression, especially of the inducible MMP-9, is modulated by shear or oxidative stress; 2. To identify regulatory elements involved in MMP-9 induction by shear or ROS; 3. To investigate whether effects of disturbed shear upon EC MMP-9 expression are mediated via oxidative stress;
AIM 4. To assess whether EC MMP mediate increased basement membrane degradation and cell loss under disturbed shear. Unidirectional and disturbed blood flow will be modeled in vitro and in vivo, and their effects analyzed in vitro, using cultured human and mouse EC, and in vivo, by creating a stenosis by banding of mouse aorta. We will investigate EC MMP-9 transcriptional regulation, mRNA stability, protein, and activity. Computed flow patterns of the aortic stenosis will be superimposed with the in vivo map of EC MMP-9 induction obtained from transgenic mouse overexpressing the MMP-9 promoter driving a LacZ reporter (MMP-9/LacZ), and with maps of EC dysfunction and loss. Specific contributions of MMP-9 and oxidative stress will be verified in vitro and in vivo using chemical inhibitors and relevant strains of genetically deficient mice. Our preliminary results support our hypothesis and demonstrate the feasibility of these studies which should help elucidate the contribution of EC-derived MMPs to endothelial dysfunction and progression of atherosclerosis towards acute clinical events.
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