Susceptibility towards Irradiation-Induced Bone Marrow (BM) Dysplasia in Vivo Is Determined by the BM Vasculogenic Phenotype: Correlation with MDS Patients BM Samples

Bone marrow (BM) endothelial cells regulate hematopoiesis by promoting mobilization, survival and proliferation of hematopoietic progenitors. Interfering with BM endothelium function conditions BM recovery following myelosuppression. In the present study, we established a mouse model of BM dysfunction to study the importance of the endothelial compartment in regulating the incidence and the onset of BM disease. For this purpose, mice were submitted to a cycle of 3 whole body sub-lethal (250 rad) irradiations (one month apart), after which the BM phenotype and incidence of BM disease were characterized. Under these conditions, common mouse strains (FVB) approximately 40% of irradiated mice develop BM dysfunction followed by malignant transformation and demise. FACS analysis of BM cells revealed that sick mice developed thrombocytopenia, accompanied by a significant reduction in hematopoietic (sca1+) endothelial (flk1+) cells and B lymphocyte precursor (CD19+) cells. Microssatelite study of BM flk1+ endothelial cells from irradiated mice demonstrated these contained characteristic chromosome 2 alterations. Since BM endothelial cells and endothelial precursors were decreased and presented cytogenetic alterations in (irradiated) diseased mice, next we tested whether interfering with BM endothelial cells content in our mouse model of BM dysfunction might increase disease onset and worsen disease outcome. First, we studied the incidence of the BM dysfunction phenotype described above in high (Balb/c) versus low (C57/BL6) vasculogenic mouse strains (as published by Shaked Y et al, 2005). For this purpose, 30 mice of each strain, sex and age matched, were sub-lethally irradiated, and BM disease phenotype and incidence were characterized as above. The incidence of BM-dysfunction and leukemia-related deaths differed significantly between the 2 strains: 25% Balb/c versus 50% C57/BL6 mice. These differences correlated with reduced endothelial-differentiation capacity (Colony Forming Units-Endothelial Cells) and increased hematopoietic CFU differentiation (in methylcellulose cultures) from C57/BL6 BM flk1+ or sca1+ BM cells, respectively. BM flk1+ from C57BL/6 were also significantly more sensitive to the apoptotic effects of irradiation (1200 rad) in vitro. These data indicated that BM endothelial cells and endothelial precursors from C57BL/6 are more sensitive to the effects of irradiation. Second, we followed a different approach to interfere with BM endothelial cells content. Normal FVB mice (which had intermediate sensitivity in our irradiation-induced model of BM dysfunction) were treated with DC101 (neutralizing antibody to mouse VEGFR-2, previously shown to reduce BM endothelial cells, Zhou et al 2007) every 3 days for 3 weeks after the last irradiation, after which BM disease was characterized, as above. Under these conditions, BM disease incidence doubled, and was accompanied by a significant reduction in BM flk1+ cells. The results obtained from our mouse model suggested the irradiation-induced BM dysfunction was similar to the BM phenotype of Myelodisplastic syndrome (MDS) patients. Next, we validated our in vivo observations in MDS BM patient samples. First we showed that AC133⁺VEGFR2+ cells from MDS patients with different chromosomal alterations (one with 20q-, one with del(5q-) and one with monossomy 7) were shown to harbour the same cytogenetic alterations. Next, besides confirming the presence of several cytopenias, FACS analysis of BM samples from 21 MDS patients (low, intermediate and high risk) revealed a significantly higher number of CD117⁺ hematopoietic precursors and a significant decrease in BM endothelial cells (VEGFR-2+) and endothelial progenitors (AC133+VEGFR-2+) in high-risk patients (those most likely to progress into acute leukemias). Several reports have demonstrated the BM endothelial content is reduced in high risk MDS patients, suggesting such a reduction might somehow correlate with greater likelihood of these patients of developing acute leukemias. On the other hand, the incidence of cytogenetic alterations in BM endothelial cells from such patients suggests the process of malignant transformation involved different cell types. In the present study we reveal the BM vasculogenic phenotype strongly correlates with BM disease onset and progression, in a mouse model of irradiation-induced BM dysfunction and also in high risk MDS patients.