The overall goal of this project is to dissect the marrow microenvironment (ME) for the purpose of identifying gene products that work in concert to regulate early events in hematopoiesis. Functionally distinct stromal cell lines have been generated to address this goal: one line supports immature progenitors (HS-27a) and one line drives differentiation (HS-5). Microarray technology has been used to generate gene expression profiles from these lines to identify differentially expressed genes (Data from this comparative analysis is available at htpp://parma.fhcrc.org/M Iwata).
In Aim 1, the array data will be confirmed for select genes by Northern and Western blotting and immune cytochemistry. Once their relevance has been established by detection in primary marrow cultures and marrow biopsies, the role of candidate gene products will be investigated by determining how gain and/or loss of function effects the ability of the ME to support hematopoiesis.
Aim 2 will address how monocytes/macrophages, which are an integral part of the ME, effect stromal function. Gene expression profiles have been generated for both normal monocytes and for HS-27a cells cultured alone. These have been compared to profiles from the two cell types cultured together to identify significant changes in both monocyte-derived and stroma-derived gene products. Several gene products with significantly altered expression and potential relevance to ME function have been selected for functional studies. These include: osteopontin (OPN) which down regulates Notch on CD34 cells; CXCL7 which may influence fibroblast growth; bone morphogenetic protein antagonist -1(BMPA-1); Cadherin-6 (CDH6) which is expressed by stroma and CD34 cells and mediates homophilic, yet heterotypic cell-cell interactions; and stromal derived factor-1 (SDF-1). The knowledge gained from Aims 1 and 2 will determine how normal monocytes interact with stromal cells and contribute to ME function. This information will be used as a platform to determine in Aim 3 how abnormal monocytes may compromise the ME. Specifically, we hypothesize that abnormal monocytes in patients with myelodysplastic syndrome (MDS) adversely affect the function of an intrinsically normal stroma, giving rise to the dysplasias characteristic of this disease. Additionally, we propose that MDS monocytes, through their retention after nonmyeloablative conditioning, cause the marrow graft failure and consequent disease progression seen in a significant proportion of MDS patients given stem cell transplants. This research plan is based on historical as well as new preliminary studies indicating that monocytes, normal or abnormal, interact with stromal cells to modulate ME function. Hypothetically this can advantageously or adversely influence hematopoiesis in general and stem cell engraftment in particular. A better understanding of these effects should facilitate the development of new therapies to correct dysplasias and optimize stem cell transplantation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062923-19
Application #
7011251
Study Section
Hematopoiesis Study Section (HP)
Program Officer
Thomas, John
Project Start
1984-08-01
Project End
2008-11-30
Budget Start
2005-12-01
Budget End
2006-11-30
Support Year
19
Fiscal Year
2006
Total Cost
$422,336
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Pillai, Manoj M; Yang, Xiaodong; Balakrishnan, Ilango et al. (2010) MiR-886-3p down regulates CXCL12 (SDF1) expression in human marrow stromal cells. PLoS One 5:e14304
Pillai, Manoj M; Hayes, Brian; Torok-Storb, Beverly (2009) Inducible transgenes under the control of the hCD68 promoter identifies mouse macrophages with a distribution that differs from the F4/80 - and CSF-1R-expressing populations. Exp Hematol 37:1387-92
Pillai, Manoj M; Venkataraman, Gopalakrishnan M; Kosak, Steven et al. (2008) Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple-integrant founder lines and determining zygosity. Transgenic Res 17:749-54
Iwata, Mineo; Pillai, Manoj; Ramakrishnan, Aravind et al. (2007) Reduced expression of inducible gelatinase B/matrix metalloproteinase-9 in monocytes from patients with myelodysplastic syndrome: Correlation of inducible levels with the percentage of cytogenetically marked cells and with marrow cellularity. Blood 109:85-92
Xu, Chang; Graf, Lynn F; Fazli, Ladan et al. (2007) Regulation of global gene expression in the bone marrow microenvironment by androgen: androgen ablation increases insulin-like growth factor binding protein-5 expression. Prostate 67:1621-9
Ramakrishnan, Aravind; Awaya, Norihiro; Bryant, Eileen et al. (2006) The stromal component of the marrow microenvironment is not derived from the malignant clone in MDS. Blood 108:772-3
Knudsen, Beatrice (2006) Migrating with myosin VI. Am J Pathol 169:1523-6
Pillai, Manoj M; Iwata, Mineo; Awaya, Norihiro et al. (2006) Monocyte-derived CXCL7 peptides in the marrow microenvironment. Blood 107:3520-6
Burroughs, Lauri; Mielcarek, Marco; Little, Marie-Terese et al. (2005) Durable engraftment of AMD3100-mobilized autologous and allogeneic peripheral-blood mononuclear cells in a canine transplantation model. Blood 106:4002-8
Iwata, Mineo; Awaya, Norihiro; Graf, Lynn et al. (2004) Human marrow stromal cells activate monocytes to secrete osteopontin, which down-regulates Notch1 gene expression in CD34+ cells. Blood 103:4496-502

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