Sickle cell bone marrow niche and regulation of platelet activation and erythropoiesis

Platelets in both murine and human Sickle Cell Disease (SCD) are increased in number and activation state, and likely contribute to the pathogenesis of SCD. Another complication of SCD is anemia, due to destruction of red blood cells and altered erythropoiesis, including reduced expansion of erythroid progenitor cells and increased mobilization of progenitors to the peripheral blood. The molecular mechanisms regulating platelet production, activation and erythropoiesis in the sickle bone marrow remain elusive. We previously identified Mesenchymal Stem Cells (MSCs) as key players in improving platelet formation and lowering baseline platelet activation. MSCs secrete CXCL12, a key factor for promoting megakaryocyte migration and for maintaining progenitor cells in the bone marrow. In our preliminary studies, we found MSCs also express Gas6, an important factor for erythropoiesis and can synergize with and enhance EPOR signaling. In SCD, we demonstrated that heme activation of Toll-like receptor 4 (TLR4) signaling in MSCs leads to reduced functionality, decreased cytokine production, and increased apoptosis. Furthermore, it was previously reported that erythroid progenitors are reduced in the SCD bone marrow and found at an increased presence in the peripheral blood, which we confirmed in our preliminary studies. Thus, our overall hypothesis is that heme driven alterations in SCD MSCs reduce Cx43, Gas6, and Cxcl12 expression and contribute to increased platelet activation, platelet production, and impaired early erythropoiesis in SCD. In our first aim, we plan to investigate the contribution of MSCs to elevated platelet formation and activation in SCD. Using a rigorous combination of human and murine samples, in vitro co-culture studies, chemical inhibitors/antibody blocking assays, and in vivo genetic mouse models, we will examine whether heme mediated activation of TLR4-NFB or ROS-Nrf2 in MSCs leads to reduced Cx43 expression and in turn promotes increased platelet production and baseline platelet activation in SCD. In our second aim we will investigate the mechanisms by which MSCs have impaired support of erythropoiesis in SCD. Using heme treated mouse models, heme scavengers, TLR4 inhibitors, and transfusions, we will examine MSC ability to maintain erythroid progenitors in the bone marrow along with their ability to promote the proliferation of early erythroid progenitor cells. These proposed studies will significantly contribute to the knowledge base of the bone marrow niche at steady state and in SCD, which could initiate development of novel anemia treatments or anti-platelet and anti-thrombotic drugs to reduce arterial thrombosis and benefit patients with SCD.