Thrombocytopenia, is a life threatening complication of treatment of many hematological and oncological malignancies. High dose chemotherapeutic regimens used for treatment of resistent lymphomas, leukemias, and bone marrow transplantation frequently result in profound refractory thrombocytopenia. This complication not only limits further treatment, but also may result in life threatening hemorrhagic complications. Even though many cytokines such as GM-CSF, IL-3, and erythropoietin, that enhance erythroid and myeloid progenitor cell proliferation and maturation are in clinical use, there is no known cytokine(s) that can selectively induce(s) platelet formation. Platelet formation is a complex process that involves sequential maturation of megakaryocytes, the formation of a pseudopod-like projection called the proplatelet, and fragmentation into platelets. The cellular events leading to proplatelet formation and platelet production are complex processes that are poorly understood. Although it is known that mature megakaryocytes are located adjacent to the subluminal surface of bone marrow endothelial cells (BMEC) and extend pseudopods into sinusoidal space, the manner by which pseudopods are formed and fragment into platelets and the role of the BMEC in this process is incompletely understood. The central premise of this proposal is that BMEC is a unique type of endothelium that regulates megakaryocytopoiesis, and thrombopoiesis. In preliminary studies we have developed a technique for isolation and cultivation of BMEC and megakaryocytes. We show that BMEC elaborates cytokine that induces megakaryocyte maturation and induce platelet formation.
The specific aim of this proposal is to characterize the cellular and molecular factor(s) mediated by BMEC that is (are) responsible for final stages of megakaryocyte maturation and platelet formation. The known cytokines elaborated by BMEC will be examined by ELISA, Northern analysis, and by functional studies. Platelet-like particle formation and maturation of GPIIb/Illa positive megakaryocytic precursors from pluripotent stem cells are in vitro models that will be used to characterize the functional activity of BMEC post-culture supernatant and will allow for the isolation and purification of putative thrombopoietins. Classical cloning approaches using reagents derived from these studies will lead to cloning of potentially useful cytokines. Functional expression cloning using mammalian (Cos) cells will provide an alternative approach, independent of successful purification, for defining and cloning BMEC elaborated cytokines. These studies may not only result in isolation, purification and cloning of a novel thrombopoietin/platelet maturation factor (PMF), but may also lay the foundation for the in vitro production of platelets.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08HL002926-01A1
Application #
2210795
Study Section
Special Emphasis Panel (ZHL1-CCT-M (M1))
Project Start
1994-07-01
Project End
1997-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
201373169
City
New York
State
NY
Country
United States
Zip Code
10065
Mohle, R; Bautz, F; Rafii, S et al. (1999) Regulation of transendothelial migration of hematopoietic progenitor cells. Ann N Y Acad Sci 872:176-85;discussion 185-6
Mohle, R; Rafii, S; Moore, M A (1998) The role of endothelium in the regulation of hematopoietic stem cell migration. Stem Cells 16 Suppl 1:159-65
Mohle, R; Bautz, F; Rafii, S et al. (1998) The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor-1. Blood 91:4523-30
Frey, B M; Hackett, N R; Bergelson, J M et al. (1998) High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectors. Blood 91:2781-92
Frey, B M; Rafii, S; Teterson, M et al. (1998) Adenovector-mediated expression of human thrombopoietin cDNA in immune-compromised mice: insights into the pathophysiology of osteomyelofibrosis. J Immunol 160:691-9
Song, W; Kong, H L; Carpenter, H et al. (1997) Dendritic cells genetically modified with an adenovirus vector encoding the cDNA for a model antigen induce protective and therapeutic antitumor immunity. J Exp Med 186:1247-56
Cannizzo, S J; Frey, B M; Raffi, S et al. (1997) Augmentation of blood platelet levels by intratracheal administration of an adenovirus vector encoding human thrombopoietin cDNA. Nat Biotechnol 15:570-3
Mohle, R; Moore, M A; Nachman, R L et al. (1997) Transendothelial migration of CD34+ and mature hematopoietic cells: an in vitro study using a human bone marrow endothelial cell line. Blood 89:72-80
Mohle, R; Green, D; Moore, M A et al. (1997) Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci U S A 94:663-8
Rafii, S; Mohle, R; Shapiro, F et al. (1997) Regulation of hematopoiesis by microvascular endothelium. Leuk Lymphoma 27:375-86

Showing the most recent 10 out of 15 publications