Endothelial (EC) and smooth muscle cells (SMC) form the inner lining of blood vessels that are continuously subjected to pulsatile hydrostatic pressures that operate rhythmically during a lifetime. Damage to this lining initiates a series of events, which as EC and SMC proliferation and secretion of substances, which ultimately result in intimal healing, media wall thickening or thrombus formation. Much of our knowledge of EC and SMC biology has come from studies of cells cultured in vitro. Compared to the in vivo millieu, however, these conditions are artificially static and may be sub-obtimal or inappropriate for the study of their behavior. The purpose of this proposal is to evaluate the immediate and long-term responses of EC and SMC to repetitive stress in vitro in order to determine the role of physical forces in modulating the response of EC and SMC to vessel injury by cell proliferation or production of anticoagulants prostacyclin, PGI2, plasminogen activator, PA) or procoagulants (von Willebrand factor, lagen). We hypothesize that the response to physical stress varies with different cell types but what even within the same cell type, the stretched cells will have different growth kinetics, morphology, orientation, and secretion of macromolecules and matrix elements compared to those of the static cells. This response will be dependent on inter-related physical factors, such as the amplitude of the force, its duration, frequency and shear rate. EC or SMC will be grown on flexible- bottomed culture dishes injected to varying cycles of tensional deformation in vitro by a vacuum-operated stress-providing unit. The kinetics of EC and SMC generation will be assessed by determining cell number and 3H-thymidine incorporation, EC and SMC morphology will be observed by light and immunofluorescent F-actin microscopy and by transmission and scanning electron microscopy. The acute, intrinsic response of EC will be assessed by the measurement of PGI2, vWF and PA factors involved in the early phase of thrombus formation. Chronic adaptive response, such as protein and collagen synthesis, will be evaluated with 2D gel electrophoresis. The effect of stretch on cyclic AMP generation and phosphoinositide metabolism will be examined to define the intracellular messengers producing the acute responses of the stretched cells. The significance of this study is the characterization of mechanical stress on EC and SMC biologiy to identify the role of physical forces in stimulating EC and SMC proliferation during vessel injury and enhancing the production and secretion of substances involved in thrombus formation.
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