The objectives of this work are to observe and quantify sickle erythrocyte adhesion to cultured endothelial cell monolayers exposed to blood under controlled levels of arteriole-like shear stress, to assess related endothelial cell injury by downstream measurement of production of prostacyclin (PGI2) and intracellular enzymes (lactic dehydrogenase, LDH), and to relate findings, among children and young adults, to clinical severity of homozygous sickle cell disease and of sickle-thalassemia. Key to the first of these aims is an in vitro flow chamber a bounding surface of which is a monolayer of cultured endothelial cells. Thickness of the flow path (254MuM) and rate fo flow from a well-stirred, constant temperature blood reservoir by means of a roller pump allow for control of shear stress at the blood monolayer interface. The chamber also accomodates a video microscope whereby red cells adherent to endothelial cells can be imaged by EPI-fluorescent microscopy. Endothelial cell function and injury will be monitored by means of a special further chamber feature: inclusion of a magnetically stirred flow region immediately downstream from the monolayer. In this flow region, blood samples for PGI2 (measured by RIA for 6-keto PGF1Alpha) or LDH permit calculation (concentration x flow rate) of rate of productionof PGI 2 or LDH by the monolayer as a whole. Direct observation by phase contrast microscopy (after displacing blood by buffer, but before chamber disassembly) yields complementary information concerning endothelial cell detachment and morphologic change. Relationships will be sought between erythrocyte adherence and monolayer dysfunction and injury, on the one hand, and clinical severity of disease, on the other. Clinical severity will be evaluated according to a scoring system which places emphasis on manifestations (presence and frequency) of microvascular occlusion. Before study, bloods from patients and normal subjects will be characterized with respect to whole blood viscosity and deformability. The proposed experiments are intended to place the well-documented phenomenon of sickle erythrocyte adherence to endothelium on a quantitative basis with respect to levels of physiologic shear stress, provide a quantitative assessment of endothelial cell function and morphological changes in response to such adherence, examine the role of platelets by injury (cold, hypoxia, trauma) of monolayers grown to confluence on collagen-coated substrate, and study the effects of low-dose aspirin and selected anti-sickling agents.
