ur research interests are centered on studying the role of cell adhesion molecules in vascular biology. There are two ongoing projects in the laboratory which relate to molecular mechanisms of hematopoietic stem cell trafficking and of sickle cell-induced vascular occlusion.
Hematopoietic Stem Cell trafficking in vivo
The traffic between the blood and the bone marrow is a two-way street in which blood progenitor cells home to and egress from the bone marrow under various physiological or iatrogenic stimuli.
Homing to Bone Marrow
Homing of stem and progenitor cells ( HSCs/HPCs) to the bone marrow occurs naturally during development and can be dramatically recapitulated during clinical bone marrow transplantation. Unlike transplantation of solid organs, the bone marrow can be transplanted to another individual by simple intravenous injection. This and the fact that full reconstitution can occur by injection of very few cells, suggested that the phenomenon was highly specific. Accumulating evidence, however, indicates that HSCs in fact use the same adhesion molecules as mature blood cells to migrate to the bone marrow, but in different combinations. For example, E-selectin and their ligands closely cooperate with P-selectin / ligands for the recruitment of mature myeloid cells, while E-selectin / ligands instead cooperate with a4 integrins ( a4b1 and a4b7) and their counterreceptors to recruit HSCs/HPCs to the bone marrow .
We recently found that human progenitors themselves are heterogenous in their expression of functional selectin ligands . We have compared the ability of human progenitors, enriched by CD34 antigen purification, derived from three clinically relevant hematopoietic sources: bone marrow, mobilized peripheral blood (mPB), and cord blood (CB), to interact with the bone marrow microvessels using intravital microscopy in NOD/SCID mice. The still frame below depicts a representative example of the bone marrow microvasculature of a NOD/SCID mouse with a bright fluorescent CD34+ cell rolling in a collecting venule. We found that the initial interactions of neonatal-derived cells were significantly reduced compared to those obtained from adults.
You can click on the photograph to download a sample video.
Despite expression of normal levels of P-selectin glycoprotein ligand-1 (PSGL-1), a large subpopulation (30%) of human cord blood CD34+ cells do not bind P- or E-selectin. Preliminary data suggest that the abnormality results from reduced fucosyltransferase (FT) actvitity in this sub population. We are currently evaluating the biological significance of this abnormality and testing whether enhancing selectin binding by exogenous recombinant FT treatment improves homing and engraftment of cord blood cells in NOD/SCID mice.
Mobilization from the Bone Marrow
While much progress has been made on the molecular mechanisms leading to progenitor homing to the bone marrow, less is known about the traffic from the bone marrow to the blood compartment. Hematopoietic stem and progenitor cells can be mobilized following the stimulation with various agents including hematopoietic growth factors, inflammatory cytokines, chemokines, complement components etc. Recent studies have suggested that the downregulation of stromal derived factor-1 (SDF-1 or CXCL12) in the bone marrow played a critical role in the mobilization of stem/progenitor cells. While investigating the role of adhesion phenomena that mediate stem cell egress from the bone marrow, we discovered that a sulfated fucose polymer, fucoidan, could rapidly mobilize progenitors in a manner that did not require selectin binding. Searching for its mechanism of action, we found that fucoidan can bind to the leukocyte b2 integrin aMb2 (Mac-1). Our recent data indeed suggest that Mac-1 acts as a molecular anchor that retains progenitors in the bone marrow .
We were intrigued by the similar biological characteristics fucoidan and sulfated glycolipids. The latter is found in hematopoietic cells. We are currently evaluating the function of these galactolipids in stem cell mobilization and hematopoiesis using genetically engineered knockout mice and other molecular techniques. Our recent studies suggest that mice lacking galactocerebrosides (CGT-/- which are deficient in galactosylceramide and its sulfated form sulfatide) exhibited severe postnatal atrophy of all lymphoid organs, due to a maturational arrest before the pro-B / T cell stage . The defect was specific to the lymphoid lineage since the numbers of myeloid progenitors (CFU-GM) and stem cells (as assessed by long-term competitive assays) in the BM of CGT-/- mice were normal. The defect originated from the bone marrow stroma since it was not transplantable to irradiated wild-type recipients. Remarkably, CGT-/- long-term B lymphoid BM cultures displayed severe deficits in the number of CD45 negVCAM-1 pos stromal cells and fibronectin matrix assembly, and produced floating macrophages rather than B lymphocytes. The fibronectin network was also altered in the CGT-deficient bone marrow parenchyma. These results point to an essential role for galactolipids in the formation of fibronectin-enriched lymphoid-specific stromal niches in the bone marrow.
Mechanisms of Sickle Cell Vascular Occlusions
Sickle cell disease originates from a single mutation in the b-globin gene that leads to anemia, painful vasoocclusive episodes, and end-organ damage. Adhesion molecules are thought to play a role in this disease since the "young" (low density) sickle cells have been shown to adhere to endothelial cells. We have used intravital microcopy of the cremaster muscle - which allows us to directly visualize adhesion processes in the microvasculature - to evaluate the mechanisms of vasoocclusive episodes in a sickle mouse model mice expressing exclusively human globin genes. We have found that sickle red blood cells (RBCs) interact with leukocytes that were adherent to the venular wall . The interactions between sickle RBCs and leukocytes were enhanced by stimulation using an inflammatory cytokine (TNF-a), and produced complete vascular occlusions. Mice deficient in P-and E-selectins, which display a defect in the recruitment of adherent leukocytes, are protected from vascular occlusions. These data are the first demonstration of a direct role for adherent leukocytes in the pathophysiology of sickle cell disease in vivo. For a review article, see . We have recently found that intravenous human gammaglobulins (IVIG) can inhibit the interactions between RBCs and WBCs . We are currently investigating the molecular mechanisms mediating these interactions and the effect of IVIG using a new multichannel intravital microscopy system which allows us to dissect the cellular and molecular components of sickle occlusion in real time in live mice.