Abstract 4930: NADPH Oxidase 2 Regulates Stem/Progenitor Cell Mobilization via Modulating Hypoxic Gradient and Proteolytic Enzymes in Bone Marrow Following Hindlimb Ischemia

Abstract

Bone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cells mobilization during postnatal neovascularization. We demonstrated that NADPH oxidase2 (Nox2)-derived reactive oxygen species (ROS) in BM play a role in ischemia-induced stem/progenitor mobilization and revascularization. However, role of Nox2 in regulating BM microenvironment remains unknown. Here we show that hindlimb ischemia of mice increases Nox2 expression (2.2-fold) and ROS production (7.2-fold) in BM cells with peak at day 3, which is cotemporaneous with increase in circulating Sca-1 + /Flk-1 + endothelial progenitor cells (2.0-fold). These increases are inhibited in Nox2-deficient mice. Using hypoxia bioprobe, BM section shows that endosteum region is hypoxic in basal steady-state. After contralateral hindlimb ischemia, hypoxic areas are expanded with an increase in HIF-1alpha and Nox2 expression throughout the BM peaking at day 3 in wild type (WT) mice, which are markedly reduced in Nox2-deficient mice. In parallel, hindlimb ischemia increases the expansion of cKit + Lin − cells (1.6-fold) and angiogenic CXCR4 + VEGFR1 + Lin low progenitor cells (31%) in WT mice, which are inhibited in Nox2-deficient mice. This is due to increase in apoptotic (annexin V+) Lin- cells (2.0-fold) and decrease in proliferative (BrdU+) primitive stem/progenitor cKit + /Sca1 + /Lin − cells (36%). Moreover, hindlimb ischemia increases Akt phosphorylation (2.2-fold), MT1-MMP (4.2-fold) expression, and decreases its membrane-anchored inhibitor, RECK (62%) in BM, which are inhibited in Nox2-deficient mice. In summary, Nox2-derived ROS in BM regulate expansion of stem/progenitors, thereby promoting local hypoxia that increases HIF-1alpha in response to hindlimb ischemia. They also activate proteolytic enzyme activity in BM, which may facilitate mobilizing stem/progenitor cells leading to reparative neovascularization. This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).