Arteriolar Neovasculogenesis and Sinusoidal Collapse Are Pathologic Features of the Hematopoietic Stem Cell Niche in Sickle Cell Disease (SCD) Mice

Sickle cell disease is a worldwide distributed hereditary red cell disorder, caused by pathologic hemoglobin S (HbS). The main clinical manifestations of SCD are chronic hemolytic anemia and acute vaso-occlusion, leading to multiple end-organ complications. To what extent, vaso-occlusion may affect the bone marrow microenvironments that regulate hematopoiesis in SCD is poorly defined. Here, we first used quantitative 2-D and 3-D imaging analyses to study the bone marrow parenchyma. In 8-12 week-old SCD (SS) mice (humanized Townes model, SS), we observed a disorganized vasculature with both sinusoidal ( laminin⁺ CD105⁺ Sca-1^- alpha smooth muscle actin^- ) and arteriolar (laminin⁺ CD105^- Sca-1⁺ alpha smooth muscle actin⁺) structural abnormalities compared to healthy (AA) or HbS- carrier (SA) mice. The majority of sinusoids have either small or collapsed lumens, often filled with large Ter 119⁺ red blood cells (RBC) clumps, suggesting RBC sickling in localized areas. The RBC clumps involve significant numbers of Gr-1⁺/CD11b⁺ myeloid cells, which also are concentrated perisinusoidally consistent with a local inflammatory process. These features are amplified under hypoxia-reoxygenation conditions, used to mimick acute SCD vaso-occlusive crisis. Most strikingly, is the dramatic increase in small, tortuous artery/arteriole vessel formation, particularly in the central portions, i.e. non-endosteal regions, of the bone marrow cavity. Using laser scanning cytometry (LaSC) of whole femur sections, we determined a four fold increase in arterioles in the central bone marrow. In support of hypoxia induced pathways responsible for neovasculogenesis, we show that HIF-1alpha staining is dramatically increased in the bone marrow of SCD (SS) mice compared to healthy (AA) mice, and interestingly the distribution pattern also is concentrated to the central, non-endosteal portions of the marrow. Moreover the bone marrow supernatant, only in SS mice, exhibits high levels of vascular endothelial growth factor, VEGF in agreement with increased levels of HIF-1alpha. Because both the sinusoidal and arteriolar vascular structures are recognized components of the hematopoietic stem cell (HSC) niche, we explored the early stages of hematopoiesis by multiparameter flow cytometry. We found that the long term HSC population (Lin^- Sca-1⁺ c-Kit⁺ CD48^- CD150⁺) is reduced by 50% in SCD mice. Taken together, we propose a model for SCD bone marrow whereby vaso-occlusive events are accompanied by defects in both sinusoidal and arteriolar vessels and resultant severe tissue hypoxia. Finally, we speculate that the abnormal vascular niche structures in SS mice affect hematopoietic stem cell homeostasis, which requires further study. These findings may have significant implications for gene therapy protocols utilizing autologous hematopoietic stem cell transplantation.

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