The objectives of the research are to elucidate (1) the effects of mechanical shear conditions, especially secondary flow and flow separation, on gene expression in vascular endothelial cells, and (2) the molecular biomechanical basis for the rolling leukocytes (WBCs) on endothelial cells (ECs). These experiments will be performed in vivo on arterial vascular endothelial cells and in vitro with the aid of flow channel and micropipette-microaspiration techniques. The in vivo studies will focus on regional variations in the expression of genes related atherogenesis as a function of local variations in hemodynamic patterns, especially secondary flow and flow separation. The in vitro flow channel studies will be performed in order to allow a better definition of the relationship between flow pattern and gene expression. The devices to be used will include the newly developed step flow channels with step changes in height or width to create flow disturbances and the tapered flow channel in which the shear stress varies linearly along the axis of flow. Cultured human umbilical vein endothelial cells will be subjected to well- defined shearing conditions, and the effects on gene expression and the signal transduction mechanisms involved will be determined. With the use of immunohistochemical techniques, in situ hybridization, and Northern blotting, the expression of a number of immediate early genes which are important in transcriptional activation, e.g., c-fos and c-jun, and target genes coding for proteins that can induce monocyte adhesion and smooth muscle proliferation, e.g., selectins, ICAM-1, VCAM, PDGF, and monocyte chemoattractive protein-1 (MCP-1). Studies will also be made on some of the signaling molecules (e.g., Ca2+, cAMP, and tyrosine kinase) related to the gene expression in response to varying flow conditions. The functional expression of ICAM-1, VCAM and MCP-1 will be assessed by determining the force required to detach monocytes from EC monolayers. In order to understand the molecular mechanisms and micromechanics of the initial interactions (rolling) of WBCs (neutrophils or monocytes) on EC and their relevance to circulatory dynamics, the cell adhesion studies will be performed under various flow conditions with emphasis on the roles of the carbohydrate moiety (e.g., sialyl Lewis-x and sialyl Lewis-a) and the lectin-EGF-like domain of selectins. These experiments will be performed on WBCs rolling on selectin-coated slides and on EC monolayers treated with IL-1beta or fMLP which can modulate the EC surface adhesive molecules and the underlying cytoskeletal proteins involved in regulation these interactions. Flow channel and micropipette techniques will be used to determine the force required for detaching WBCs from the cultured ECs, and a combination of experimental and theoretical approaches will be employed to assess the adhesive energies. The proposed research has considerable significance in elucidating several fundamental biological processes in health and pathophysiological changes in diseases such as atherosclerosis and inflammation.
NIH ExPorter
NIH ExPorter