The long-term objective of this proposal is to characterize the adhesion mechanisms mediating vaso-occlusion (VOC) in sickle cell disease. We have previously shown using intravital microscopy in sickle cell mice that sickle red blood cells (RBCs) interact with adherent leukocytes (WBCs) in inflamed venules in vivo. The interactions between RBCs and WBCs appear to be biologically important since they occur in both wild-type and sickle cell mice, and their numbers in sickle cell mouse venules correlate with reduced microvascular blood flow velocities and shorter survival during intravital microscopy. In addition, inhibition of RBC-WBC interactions protects sickle cell mice from VOC. Recent studies using a novel digital multichannel widefield fluorescence microscopy system has identified polymorphonuclear neutrophils (PMNs) as the major leukocyte subset capturing circulating RBCs. E-selectin-mediated signaling plays a critical role in enabling activated PMNs to capture RBCs. Other studies from the PI's laboratory have revealed that all E-selectin ligand (ESL) activity on PMN is conferred by three glycoproteins, PSGL-1, CD44 and ESL-1. Here, we propose to study further the cellular and molecular mechanisms mediating VOC in sickle cell mice.
In Specific Aim 1, we will identify which ESL induces leukocyte activation and the capture of circulating RBCs. We will use genetic deletion (Cd44 / , Selplg / , or mice lacking both) and RNA interference (short hairpin RNAi targeting ESL-1) introduced by lentiviral transduction of hematopoietic stem cells (HSCs).
In Specific Aim 2, we will define the microdomains and molecular determinants on leukocytes that mediate the capture of circulating sickle RBCs using high-speed digital videomicroscopy. Preliminary experiments in C57BL/6 mice suggest that receptors that cluster to the leading edge of adherent leukocytes mediate the capture of RBCs, and that expression of the b2 integrin Mac-1 is critical.
In Specific Aim 3, we will investigate the role of the leukocyte fucosyltransferase (Fut7), required for the synthesis of all selectin ligands, as therapeutic target for sickle cell VOC using RNAi downregulation of Fut7 expression in HSCs followed by generation of radiation chimeras, and using sickle cell mice deficient in Fut7. In the last Specific Aim, we will use the animal models of leukocyte adhesion defects that we have generated to evaluate the long-term impact of leukocyte adhesion deficits on chronic hemolysis, and on the function and pathology of target organs. The proposed studies will provide new mechanistic insights into the role of leukocytes in sickle cell vascular occlusion and may lead to new therapeutic options to prevent or treat this debilitating illness.
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