Current notions regarding the etiology and inception of atherosclerosis suggest that loss and/or injury to the arterial endothelium represent primary cellular events in the disease process. Regulation of the endothelial cell (EC) motile response to injury may ultimately determine whether the vessel wall will be repaired or vascular disease will ensue. Morphological and biochemical experiments are planned which may reveal the molecular and structural mechanisms underlying the cellular basis of intimal integrity and the motile response to injury. Specifically, molecules of the extracellular matrix (ECM), which are present within basal laminae of blood vessels, will be used as test substrates upon which large and microvascular EC will spread and migrate following mechanical denudation in vitro. The rate and extent of these EC movements will be quantitated as a function of ECM composition using a computer-assisted, time-lapse video-micrography tape recording system. Blocking and digestion of ECM components with specific antibodies and matrix-degrading enzymes may reveal which basement membrane molecules influence the EC motile response to injury. To gain insight into the EC cytoskeletal mechanisms involved in wound repair and intimal restabilization, specific contractile protein markers will be used. Fluorescent and colloidal gold-labeled antibodies will reveal the light microscopic form and detailed ultrastructural arrangement of the major EC contractile elements. Staining patterns and molecular configurations of cytoplasmic actin, myosin and vinculin will be compared, contrasted and quantitated in normal and wounded populations of EC with documented motility as a function of ECM composition. These studies may ultimately reveal how matrix molecules recruit or reorganize the cytoskeleton into specific, supramolecular arrays capable of motile force production, tension generation and wound closure in situ. Biosynthetically-labeled cytoplasms of normal and wounded EC will be fractionated into soluble and insoluble cytoskeletal parts and immunoprecipitated with monospecific contractile protein antibodies. Electrophoretic and fluorographic analyses of resting vs. injured EC may reveal if the motile wound healing response requires new contractile protein synthesis or induces alterations in their physical form and distribution. If so, the ECM influence on this biochemical response to injury will be examined. Results of these basic experiments may ultimately reveal the molecular mechanisms utilized by EC 1) for the promotion of intimal integrity and 2) to effect wound repair following injury in vivo.
