Abstract 1960: NADPH oxidase Nox2 in Bone Marrow Vascular Niche Following Hindlimb Ischemia: Role in Stem/progenitor Cell Differentiation, Survival and Mobilization
Bone marrow (BM) microenvironment (niche) and mobilization of stem/progenitor cells from BM are important for post-ischemic neovascularization. We demonstrated that reactive oxygen species (ROS) derived from NADPH oxidase Nox2 play an essential role in reparative neovascularization induced by hindlimb ischemia. However, role of Nox2 in BM niche and stem/progenitor cell mobilization from BM remains unknown. Here we show that Nox2 is the most highly expressed Nox enzymes in BM mononuclear cells (BMCs) and BM c-Kit+/Lin-progenitor (BM-KL) cells. Nox2-dependent ROS production in both cells are significantly increased after hindlimb ischemia with peak at day 3 (7.2 and 1.6 fold, respectively). Mice lacking Nox2 show impaired ischemia-induced flow recovery (68%), which is associated with decrease of ROS production in BMCs and BM-KL cells (37% and 39%, respectively) as well as number of c-Kit+/Flk-1+ cells in peripheral blood (33%). Defective neovascularization in Nox2-deficient mice is rescued by transplantation of BM from wild-type mice. BM immuno-histological analysis with newly-developed Cys-SOH trapping probe reveals that Nox2+/c-Kit+ cells and oxidized proteins are accumulated in the BM vascular niche at 3 days after hindlimb ischemia. Furthermore, hindlimb ischemia increases the number of hematopoietic stem/ progenitors (c-Kit+/Sca-1+/Lin-(KSL) 1.8 fold) and myeloid progenitors (c-Kit+/Sca-1-/Lin−, 1.2 fold) as well as polyploid megakaryocyte and sinusoidal vessel formation in BM in WT mice, which are significantly inhibited in Nox2-deficient mice. This defective response is due to the increase in apoptosis (annexin V+/7AAD−, 28% in KSL, 71% in Lin−) but not due to the inhibition of proliferation (BrdU+). Mechanistically, SDF-1-induced cell survival kinase Akt phosphorylation (100%), chemotaxis (40%) and invasion (61%) are significantly inhibited in Nox2-deficient BM-KL cells. In summary, in response to hindlimb ischemia, Nox2-derived ROS is increased in BM microenvironment, which in turn regulates the differentiation and survival of BM progenitor cells by stimulating Akt phosphorylation, thereby facilitating to egress from the niche and to mobilize stem/progenitor cells leading to reparative neovascularization.
This research has received full or partial funding support from the American Heart Association, AHA National Center.