Efficient recovery of the immune system following bone marrow or stem cell transplantation requires optimal function of all obligatory supportive components of the marrow microenvironment. Work completed during previous periods of funding was focused on bone marrow stromal cells (BMSC) function subsequent to dose escalated chemotherapy. These studies demonstrated that BMSC VCAM-1 and CXCL-12 (SDF-1) proteins were diminished by VP-16 exposure. Treatment resulted in BMSC with reduced ability to support migration and survival of hematopoietic progenitor cells and dysregulation of expression and activity of BMSC derived matrix metalloproteinase-2 (MMP-2) and TGF-21. Experiments proposed in the current application extend these studies to osteoblasts as additional, essential constituents of the hematopoietic stem cell niche. Osteoblasts contribute to the "endosteal niche" as the unique anatomical location in which stem cells are maintained until recruited into the proliferative population available for subsequent differentiation. Pilot data relevant to the current application indicate that primary human osteoblasts exposed to melphalan or VP-16 have diminished CXCL12 expression and chemotactic support and altered support of CD34+ bone marrow cell differentiation. Furthermore, phosphorlyated-Smad-2 signaling in osteoblasts is enhanced by direct exposure to chemotherapy, suggesting autocrine effects of therapy-induced TGF-b. The influence of chemotherapy induced autocrine signaling via elevated TGF-2 was investigated by treatment of primary human osteoblasts to rTGF-b which resulted in elevated TGF-2 gene expression, suggesting potential for amplification of the effects of elevated TGF-2 signaling. Recombinant TGF-2 also reduced CXCL12 expression and blunted chemotactic support. Building on these preliminary data we will investigate the effects of dose escalated chemotherapy on osteoblasts with evaluation of the mechanisms that underlie changes in factors necessary for optimal chemotactic support of CD34+ human hematopoietic stem cell maintenance, with emphasis on the mechanisms that underlie diminished CXCL-12 gene expression and protein levels (Aim 1). We will extend our model to three dimensional matrices to include consideration of signaling that is influenced by architecture, an important aspect of models to investigate osteoblast function in a chemotherapy stressed bone marrow microenvironment (Aim 2) and will transition our studies to a murine model in which the osteoblast population can be specifically identified by virtue of constitutive expression of GFP (Col2.3-GFP mice) to allow isolation and characterization of ostoeblasts from mice exposed to chemotherapy in vivo. Successful completion of these aims we will test the hypothesis that TGF-b is a critical mediator of the effects of dose escalated chemotherapy on dysregulation of osteoblast function in the bone marrow microenvironment. Completion of this study will contribute to development of strategies in which neutralization of TGF-b can be optimized to enhance long-term hematopoietic recovery of bone marrow transplantation patients which relies, in part, on optimal function of the stem cell niche.
Long term hematopoietic recovery following bone marrow transplantation relies on a functional stem cell niche in the bone marrow to which transplanted cells must efficiently migrate and are impacted on by diverse developmental cues. Osteoblasts of the endosteal niche define one essential population that supports stem cell development. In the current proposal we investigate their vulnerability to dose escalated chemotherapy.
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