Endothelial damage can occur in vivo in various conditions as a result of pathological disease entities such as chronic hypertension, atherosclerosis, or during invasive medical procedures. Wounding injuries to the vascular endothelium require a number of proteins to interact with the cell cytoskeleton for appropriate cellular movement. In addition to biochemical signaling, endothelial cells, interfaced between the flow of blood and the vascular wall, require biomechanical forces such as shear stress to direct their cellular movement. Biomechanical forces can directly influence endothelial cell structure and function, acutely and chronically, therefore constituting a novel paradigm of endothelial cell activation. However, the mechanisms by which shear forces affect adhesion proteins and growth factors, such as basic fibrobast growth factor (bFGF), for cellular movement remain elusive. We have preliminary data to suggest that laminar shear stress enhances human umbilical vein and coronary artery endothelial cells wound closure by mechanisms involving cell migration, spreading, and enhancement of specific adhesion proteins. In this proposal, the P.I. addresses the overall hypothesis that shear stress enhance the rate of endothelial cell wound closure by mechanisms involving expression of cell-cell (cadherin) and cell-matrix (integrin) adhesion proteins, and bFGF production.
Four specific aims are proposed.
In Specific Aim 1, we will characterize the relationship between shear stress and wound closure in endothelial cell monolayers. Measurements of wound width, cell area, and internuclear distances will indicate cell spreading and migration at the wound edge.
In Specific Aim 2, we will determine the functional significance of the relationship between shear stress and adhesion molecules during wound closure. Western blots of beta1 integrin chain and VE-cadherin protein expression and Northern blots of mRNA levels will be used to determine the adaptation response to shear stress during wound closure. Immunocytochemistry will be used to detect changes in the pattern of adhesion proteins during the wound healing process.
In Specific Aim 3, the mechanisms by which integrins and cadherins influence bFGF expression during wound closure will be investigated. Western blots for bFGF production, ribonuclease protection assays for bFGF mRNA will be used to determine the adaption response to shear stress. Effects of activating and blocking antibodies to beta1 integrin and VE-cadherin on bFGF expression will be assessed during wound closure.
In Specific Aim 4, we will examine the effect of shear stress on proteins related to actin-associated signaling. Tyrosine phosporylation and translocation of the p120 armadillo protein for enhancement of bFGF production will be assessed. Investigation of the mechanisms underlying endothelial repair during shear stress may lead to better strategies for the management of patients suffering from a variety of cardiovascular disorders.
