The early lesions of atherosclerosis, in human subjects and experimental animals, consistently develop in a non-random pattern in the arterial vasculature, and the geometry of these ~lesion-prone areas~ correlates with branch points, curvatures and other regions of altered blood flow. Recent studies have demonstrated direct effects of biomechanical forces on the expression of pathophysiologically relevant genes by the endothelial cell (EC). This leads us to hypothesize that hemodynamic forces, in particular wall shear stresses generated by laminar and disturbed laminar flow, can function as both positive and negative stimuli in atherogenesis, via effects on EC gene expression. To test this hypothesis, we propose a series of interrelated in vitro and in vivo experiments, utilizing a combination of cell biological, molecular biological and experimental pathological techniques, under three Specific Aims: In the First Specific Aim, RT-PCR-based differential display will be used to define the molecular genetic programs elicited in cultured EC by exposure to the steady laminar shear stresses that characteristically occur in 'lesion-protected' areas, versus the altered shear stresses found in 'lesion-prone' areas. These differential patterns of gene expression then will be validated in vitro in a specially designed model system that generates a disturbed flow field over a cultured EC monolayer, and 'athero-protective' and 'athero-pathogenic' candidate genes will be selected, based on structural and functional data. In the Second Specific Aim, the potential synergism or antagonism of known biochemical risk factors (e.g., constituents of oxidized lipoproteins, such as lysophosphatidylcholine and elevated levels of homocysteine), and anti-atherogenic substances (e.g., antioxidants) with altered shear stress stimuli will be examined in vitro models. In the Third Specific Aim, the patterns of expression of candidate athero-protective and athero-pathogenic genes in EC will be examined in vivo in the lesion-protected and lesion-prone areas of the aorta of atherosclerosis-susceptible (ApoE-deficient and LDL receptor-deficient) mouse strains, and human arterial specimens. A final test of pathophysiological relevance will be attempted by examining the effects of transgenic overexpression of selected candidate genes on lesion localization/progression/regression. The results of these studies should increase our understanding of the interplay of biomechanical and biochemical 'risk factors' in atherogenesis, and may lead to the definition of the critical EC genes involved.
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