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Chronic inflammation within the bone marrow (BM) microenvironment has been proposed as a key driver of aging-associated hematopoietic defects including loss of hematopoietic stem cell (HSC) self-renewal, myeloid-biased HSC differentiation, and a predisposition towards development of myeloid neoplasms. In support of this idea, we recently discovered that constitutive activation of the NFKB signaling pathway within endothelial cells (ECs) of young mice leads to an inflammatory stress within the BM, resulting in loss of HSC self-renewal and a myeloid-biased output at the expense of lymphopoiesis. Since chronic vascular inflammation has emerged as a key component of the systemic aging process, we hypothesized that suppression of NFKB dependent vascular inflammation during physiological aging could temporize BM inflammation and preserve HSC health. To test this hypothesis, we utilized the Tie2.IkBss mouse model wherein the canonical NFKB signaling pathway is suppressed specifically within ECs of adult mice, and physiologically aged these mice to assess their hematopoietic function. Our findings demonstrate that aged Tie2.IkBss mice displayed a significant decrease in their peripheral blood myeloid biased output as compared to their aged littermate controls. HSC transplantation assays revealed that HSCs derived from aged Tie2.IkBss mice demonstrated a significant decrease in their myeloid-biased differentiation and displayed enhanced lymphopoiesis. Furthermore, serial transplantation revealed that HSCs from aged Tie2.IkBss mice displayed balanced multi-lineage reconstitution ability during both primary as well as secondary transplantation assays. Collectively, these results suggest that inflammatory cytokines derived from the aged vascular niche play a crucial role in promoting aging-associated myeloid bias of HSCs, and strategies directed towards suppression of chromic vascular inflammation will provide novel therapeutic interventions towards prevention of aging-associated myeloid malignancies and improving overall healthspan.

Drosophila Muscleblind (Mbl) proteins control terminal muscle and neural differentiation, but their molecular function has not been experimentally addressed. Such an analysis is relevant as the human Muscleblind-like homologs (MBNL1-3) are implicated in the pathogenesis of the inherited muscular developmental and degenerative disease myotonic dystrophy. The Drosophila muscleblind gene expresses four protein coding splice forms (mblA to mblD) that are differentially expressed during the Drosophila life cycle, and which vary markedly in their ability to rescue the embryonic lethal phenotype of muscleblind mutant flies. Analysis of muscleblind mutant embryos reveals misregulated alternative splicing of the transcripts encoding Z-band component alpha-Actinin, which can be replicated in human cells expressing a Drosophilaalpha-actinin minigene and epitope-tagged Muscleblind isoforms. MblC appreciably altered alpha-actinin splicing in this assay, whereas other isoforms had only a marginal or no effect, demonstrating functional specialization among Muscleblind proteins. To further analyze the molecular basis of these differences, we studied the subcellular localization of Muscleblind isoforms. Consistent with the splicing assay results, MblB and MblC were enriched in the nucleus while MblA was predominantly cytoplasmic. In myotonic dystrophy, transcripts bearing expanded non-coding CUG or CCUG repeats interfere with the function of human MBNL proteins. Co-expression of CUG repeat RNA with the alpha-actinin minigene altered splicing compared with that seen in muscleblind mutant embryos, indicating that CUG repeat expansion RNA also interferes with Drosophila muscleblind function. Moreover MblA, B, and C co-localize with CUG repeat RNA in nuclear foci in cell culture. Our observations indicate that Muscleblind isoforms perform different functions in vivo, that MblC controls muscleblind-dependent alternative splicing events, and establish the functional conservation between Muscleblind and MBNL proteins both over a physiological target (alpha-actinin) and a pathogenic one (CUG repeats).

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents.