Painful vascular occlusion is one of the cardinal features of sickle cell disease. It, more than any other complication, leads patients to seek medical attention. Several processes participate in vasoocclusion, including increased adherence of sickle RBC to vascular endothelial cells. Specific cytoadhesion receptors, ligands, and heterocellular mechanisms involved in adherence have been and are continuing to be discovered. We propose that endothelial cell receptors and changes in their expression that occur with cell activation are important variables in vasoocclusion. The dramatic changes in endothelial cell function effected by known antagonists include alterations in adhesivity, a shift in the repertoire displayed on cell surfaces, changes in receptor activity, cell contraction, and interendothelial cell gap formation. A wide variety of agonists that alter endothelial cells are germane to the pathophysiology of sickle cell vasoocclusion. We propose to use cultured endothelial cells to study agonists that induce sickle RBC adherence and the endothelial cell receptors that mediate enhanced adherence. The agonists we will study include thrombin, histamine, tumor necrosis factor-alpha, interleukin- 1beta, and ischemia/reperfusion. The blocking agents to be used to assess the receptors on cultured endothelial cells involved in baseline and induced adherence include a blocking monoclonal antibody against GP Ib, RGD-containing oligopeptides, and blocking monoclonal antibodies against integrin and integrin subunits. In addition we will investigate the role of yet unidentified adhesive proteins using immunologic methods. We will prepare rabbit monoclonal antibodies against activated endothelial cells and, as an alternative, use phage display technology to determine molecular changes in endothelial cells that result from activation. Affinity of monoclonal antibodies or, alternatively, recombinant bacteriophage expressing antibody fragments specific for surface antigens on activated endothelial cells will be used to identify molecules that have been induced by agonists and to assess their role in induced adherence. The bacteriophage library containing cloned human immunoglobulin variable region cDNA expresses 7 x 10 9 antibody specificities. We will also study the cell mechanisms responsible for increased activity of receptors on endothelial cells--quantitatively increased expression, changes in cell surface distribution, cell contraction exposing previously occupied receptors, and increased activation of receptors. This work complements that proposed by Drs. Sam Test and Frans Kuypers, with whom we propose collaborative studies. In the later phases of this work we will use New Zealand White rabbits to produce monoclonal antibodies. Our experiments are anticipated to enhance our knowledge of the molecular mechanisms responsible for sickle RBC- endothelial cell adherence, improve our grasp of the pathophysiology of sickle cell vasoocclusion, and expand our knowledge of endothelial cell biology.
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