Abstract 20727: HuR-dependent Angiogenic Factor-Encoding mRNA Stabilization Mediated by β2 Integrin-Engagement in Macrophages during Ischemic and Inflammatory Angiogenesis
Expression of angiogenic factors and recruitment of macrophages play a crucial role in inflammatory angiogenesis. Furthermore, the regulation of stabilization of angiogenic factor-encoding mRNAs plays a large role in determining the magnitude of the mounted angiogenic response. Based on our previous studies that engagement of the β2 integrin leads to stabilization of ARE-bearing mRNAs in T lymphocytes through a required, rapid nuclear-to-cytoplasmic translocation of the RNA-binding protein HuR, here, we address whether HuR is required for macrophage VEGF mRNA stabilization mediated by β2-integrin engagement and for macrophage VEGF production at angiogenic sites in vivo. VEGF mRNA decay experiments in primary mouse bone marrow-derived macrophages (BMDMs) demonstrated a marked stabilization of the otherwise labile VEGF transcript upon cell adhesion to recombinant ICAM-1 (β2 integrin ligand), compared to poly-L-lysine control. This integrin-induced mRNA stabilization was absent in BMDMs obtained from macrophage-specific HuR gene-deleted mice (HuRflox/floxLysM-Cre+, designated KO, below) demonstrating that HuR is required for integrin-induced VEGF mRNA stabilization in macrophages. Correspondingly, ELISA-quantified VEGF protein accumulated over 24 hr in supernatants of ICAM-1-adhered wild-type (WT) but not KO BMDMs. In vivo, subcutaneous polyvinyl alcohol (PVA) sponges were implanted in WT and KO mice. FACS analysis on cells extracted from excised PVA sponges demonstrated identical numbers of F4/80+ macrophages but a marked reduction in macrophage-localized VEGF by co-IF, VEGF mRNA, and endothelial lectin-positive microvessels, in sponges obtained from KO vs. WT mice. Using the femoral artery ligation ischemia model, a significant attenuation of 2–4 week blood flow recovery was observed in macrophage-specific HuR KO, compared to WT mice, which correlated with more extensive hindlimb necrosis and greatly reduced neo-microvessel formation. This is the first report that macrophage β2 integrin engagement results in stabilization of VEGF mRNA, and that dynamic modulation of mRNA stability, in macrophages, is required for full amplitude angiogenic responses to ischemic and inflammatory stimuli, in vivo.
- © 2010 by American Heart Association, Inc.