Modulating Cholesterol Levels on Bone Marrow Cells Affects Endothelial and Hematopoietic Cell Mobilization and Differentiation.

Abstract

A decrease in cholesterol (CH) levels associated with hematological malignancies, such as acute leukemia, correlates with Hematopoietic suppression. Moreover, patients with mevalonate kinase deficiency (which results in hypocholesterolemia) have besides neurological defects, hepatosplenomegaly, thrombocytopenia, anemia and eventually progress into Myelodisplasic Syndromes/Leukemia. For this study, we hypothesized that CH levels might affect hematopoietic differentiation (acting directly on hematopoietic stem cells, HSC) or hematopoietic cell mobilization. For this purpose we started by studying the bone marrow (BM) expression pattern of CD36 and ApoE, 2 proteins involved in CH cellular transport, in BM samples of normal adult mice. Immunofluorescent staining showed that CD36 and ApoE are both expressed within the BM microenvironment, being particularly evident in BM sinusoids and also small subsets of hematopoietic cells. Next, we took advantage of CD36 and ApoEKO models to study their hematological parameters and BM cellular contents. To do so, CD36/ApoEKO mice and their counterparts were euthanized and BM and peripheral blood cells were collected, stained for CD11b (myeloid cells), B220 (B lymphocytes), Sca1 (progenitor cells), Flk1 (endothelial cells) and analyzed by FACS. FACS analysis of BM cells revealed that KO and WT mice had similar progenitor cells and B lymphocytes percentage, but substantially less myeloid cells. Peripheral blood analysis revealed a substantial increase in circulating B lymphocytes, and a decrease in circulating progenitor cells and myeloid cells. Regarding circulating endothelial cells, no differences were detected between KO and WT mice. However, ApoEKO mice had increased levels of BM endothelial cells and progenitor cells. As B lymphocytes are increased in the periphery and diminished in the BM, CD36 deficiency (which results in diminished CH uptake) seems to promote B lymphocyte exit from the BM. An opposing effect seems to occur in the progenitor cell populations, since less percentage in the peripheral blood might imply a failure in their exit from the BM. Alternatively, altered CH levels may affect selectively progenitor cell (subsets) differentiation. In order to investigate the role of CH modulation in hematopoietic differentiation/commitment, progenitor cells (Lin-Sca+) from CD36 WT mice were cultured in a methylcellulose hematopoietic differentiation assay and also cultured in endothelial differentiation conditions in the presence/absence of a CH lowering agent (Pravastatin). At day 9 of differentiation, cells were collected and expression of Sca1, CD11b and Flk1 was analyzed by flow cytometry. Reduced CH availability, as a result of Pravastatin treatment, resulted in substantial lower levels of differentiated endothelial cells, suggesting a blockade in this differentiation process, and in a reduced number of progenitor cells, suggesting progenitor cell death. Notably, the percentage of myeloid cells was not affected by Pravastatin treatment. Taken together, we suggest that CH influences not only trafficking between BM and peripheral blood, influences endothelial cell differentiation, and might also influence BM recovery to injury by diminishing progenitor cell number available for subsequent hematopoiesis. Current studies aim at comparing the BM hematopoietic recovery of CD36, ApoEKO vs control in response to sublethal irradiation, and the contribution of the BM endothelial compartment in this process.