Stromal Microenvironment Regulates Progenitor Survival through Nitric Oxide Production.

Abstract 1440

Poster Board I-463

Nitric oxide (NO) is a small gaseous molecule with diverse roles including the regulation of cell proliferation, differentiation, apoptosis, adhesion and migration. NO is derived from L-arginine by the nitric oxide synthase (NOS) family of enzymes. At least three distinct NOS isoforms have been identified in mammalian cells including the endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). Recently, we have shown that NO donors induce CXCR4 expression in human CD34 positive cells suggesting that NO production may regulate the migration and adhesion of hematopoietic progenitors. To determine the in vivo relevance of these findings and define the role of NO in the biology of hematopoietic progenitor cells, we first investigated NOS expression in hematopoietic and non-hematopoietic cells. Using quantitative reverse transcription-PCR analysis, we demonstrate that nNOS, and eNOS isoforms are highly expressed in Human Umbilical Vein Endothelial Cells (HUVEC) and osteoblastic cell lines, but there was no or low expression of NOS in hematopoietic cells including cell lines and mature blood cells except macrophages that express high level of iNOS. In agreement with RNA analysis, we found large amounts of nitrite (a stable derivative of NO) in the culture medium of stromal (MS-5) and osteoblasic (MG-63) cell lines. To determine the effects of NO on hematopoietic progenitor cells survival, cord blood CD34+ were cultured on MS-5 cells in the presence/absence of NOS inhibitor (L-NAME) for three days, then hematopoietic progenitor numbers were determined by culture in a semi-solid medium and colonies were quantified 14 days later. Treatment with L-NAME reduced colony number by 33, 42 and 54% with 10, 100 and 500 μM L-NAME concentrations, respectively. This effect is NO specific since the diminution of progenitor number was reverted by adding NO donor in the culture medium.

These results indicate that NO is produced by bone marrow stromal cells. This production regulates the survival of hematopoietic progenitors in vitro. Further experiments are needed to determine if these effects are dependent on the regulation of CXCR4/SDF-1 signaling.