The overall objective of this proposal is to understand the adhesive mechanisms mediating vaso-occlusion in sickle cell disease. Although several studies have documented roles for a number of adhesion molecules in sickle cell adhesion in vitro, there is very little in vivo data. The recent development of a mouse strain that exclusively expresses human globins provides us with the opportunity to study the adhesive mechanisms in vivo using intravital microscopy. Our preliminary studies indicate that cytokine-induced inflammation produces severe vasoocclusion in post-capillary venules in the cremaster muscle of sickle cell mice. Our results indicate that adherent leukocytes play a key role in this process since they can bind circulating sickle cell erythrocytes (SS RBCs) and initiate venular occlusion. Endothelial selectin-deficient mice, which display defects in leukocyte recruitment in venules, show few SS RBC-leukocyte interactions and no vasooclusion. In this proposal, we wish to further investigate the hypothesis that vasooclusion in sickle cell disease is initiated by the adhesive interaction between SS RBCs and adherent leukocytes.
In specific aim I, we will identify the type of leukocyte (mononuclear vs polymorphonuclear) that interacts with SS RBCs in vivo, and we will further evaluate the roles of leukocyte and endothelial adhesion molecules in vasoocclusion using adhesion molecule knockout mice and inhibitory antibodies; we will also develop a model to investigate vasoocclusion in the bone marrow microvasculature, an important target in sickle cell disease.
In specific aim II, we will develop adhesion assays to dissect the molecular mechanisms mediating SS RBC-leukocyte interactions and, in parallel, we will conduct intravital experiments to confirm putative adhesion pathways.
In specific aim III, we propose to evaluate the in vivo functions of three adhesion molecules previously shown to participate in sickle cell adhesion (von Willebrand factor, beta3 integrins and thrombospondin), using adhesion molecule knockout mice and sickle cell animals. The proposed studies should shed new light on the in vivo mechanisms of sickle cell vasoocclusion and may lead to novel ways to treat sickle cell crises and other complications of this disease.
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