Abstract 3919: Macrophage Beta2 Integrin-mediated, HuR-dependent Stabilization of Angiogenic Factor-encoding mRNAs in Inflammatory Angiogenesis
Many angiogenic factors are encoded by labile transcripts bearing AU-rich elements (AREs) in their 3′-untranslated regions. These mRNA half-lives must be dynamically extended to allow significant protein production. We have demonstrated that engagement of the β2 integrin adhesion receptor, LFA-1, leads to stabilization of ARE-bearing mRNAs encoding TNF-α and IFN-γ in T lymphocytes through a required, rapid nuclear-to-cytoplasmic translocation of the RNA-binding protein HuR. We now address whether β2 integrin engagement stabilizes angiogenic factor mRNA in murine macrophages, and whether HuR is required for macrophage VEGF production at angiogenic sites in vivo. Raw 264.7 mouse macrophages and primary mouse bone marrow-derived macrophages were pretreated with PMA (1ng/ml) and allowed to adhere onto poly-L-lysine- or recombinant ICAM-1 (β2 integrin ligand)-coated dishes. RNA polymerase II (transcription) was inhibited at 3hr, after which RNA was harvested over 60 minutes for decay analysis. In both primary and cell line macrophages, angiogenic factor mRNA decayed to < 50% time 0 levels in the absence of integrin engagement, whereas the transcripts for VEGF, matrix metalloproteinase-9 and Angiopoietin-2 were all stable at 60 min in cells bound to ICAM-1. To study inflammatory angiogenesis in vivo, subcutaneous polyvinyl alcohol (PVA) sponges were implanted in wild-type and macrophage-specific HuR knockout (HuRflox/floxLysM-Cre) mice. FACS analysis on cells extracted from excised PVA sponges (1, 2 and 3 weeks) demonstrated a significant localization of F4/80+ macrophages. The recruitment and localization of monocyte/macrophages was identical in WT and macrophage HuR KO mice. Excised PVA sponge co-immunofluorescence confirmed equal macrophage localization but a dramatic reduction of macrophage VEGF production in macrophage HuR KOs. This correlated with a reduction in the number of CD31+ microvessels. This is the first report that macrophage β2 integrin (LFA-1, Mac-1) engagement results in stabilization of angiogenic factor mRNAs encoding VEGF, MMP-9 and Ang-2, and that macrophage HuR is required for neovascular responses mediated, in part, by macrophages. These findings are relevant in early angiogenic responses to tissue ischemia.