VASCULAR CELL INTERACTIONS AND FLOW IN VIVO

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This project focuses on the role of the terminal microvascular network in determining the variable behavior of blood cells (erythrocytes, RBC and leukocytes, WBC) and in determining blood flow distributions in the microvasculature. We view the heterogeneity in blood flow and blood cell behavior as a predictable result of local biophysical phenomena and their biological outcomes. Gen. Aim 1 centers on determinants of RBC distribution. Aim 1.1 tests the hypothesis that RBC distribution into, and flow through, capillaries is a function of RBC surface to volume ration. With Project 3, flow properties of modified RBCs will be determined in capillaries both acute and chronically; cell mechanical properties will be measured in vitro and RBC flux and velocity and capillary geometry in vivo. Aim 1.2 tests the hypothesis that bifurcation geometry contributes to asymmetries in distribution of RBCs, using RBC suspensions perfused through microchannel systems constructed to mimic terminal arteriolar bifurcation geometry. Gen. Aim 2 explores the relationship between endothelial cell (EC) cytoskeletal realignment with flow, and the geometry (curvature) of the EC growth surface. Aim 2.1 tests the hypothesis that the cytoskeletal responses of ECs to flow is different in cells growing on curved compared to flat surfaces. F-actin distribution will be measured in ECs of different origins grown under varying flow and curvature. Focal adhesion distribution will also be measured. Aim 2.2 tests the hypothesis that ECs in vivo can respond to flow changes with cytoskeletal realignment. Local perfusion of arterioles will be used to mix microvessels and stain F-actin, under differing flow of vasoactive stimuli. Gen. Aim 3 addresses the mechanisms underlying WBC-EC interactions in vivo. Aim 3.1 tests the hypothesis that heterogeneity in WBC distribution among venules is due to preferred flow channels for WBCs. WBC flow variables will be quantitated in related to the venular architecture, at differing flows, or without selected adhesion molecules. Local venular heterogeneity in adhesion molecules will be quantitated from the distribution of P-selectin and ICAM-1, and also by using a WBC stopped time analysis developed by Project 1. Aim 3.2 tests they hypothesis that EC-WBC interactions can be changed due to flow related changes in ECs. With Project 2 we will measure EC volume changes and WBC flows in vivo before and after tissue ischemia, and with amiloride to prevent EC changes. In microchannels we will explore that mechanism underlying the EC volume response to flow, and effects on WBC-EC interactions.