Date of Award
Open Access Thesis
Medical Doctor (MD)
Activated macrophages, via the secretion of pro-angiogenic factors, modulate the angiogenic response to inflammation. Several of these factors, including vascular endothelial growth factor (VEGF), are encoded by inherently unstable mRNA transcripts containing AU-rich elements (AREs) in their 3' untranslated region. During stress events, the half-lives of these mRNAs must be prolonged to allow for significant protein production and an adequate angiogenic response. This laboratory has demonstrated that engagement of the β-integrin receptor, LFA-1, leads to stabilization of ARE-bearing mRNAs in T lymphocytes through a nuclear-to-cytoplasmic translocation of the RNA-binding protein, HuR. Here, we address whether β-integrin engagement stabilizes VEGF mRNA in macrophages in vitro and at sites of angiogenesis via a HuR-dependent mechanism. To determine whether β-integrin engagement regulates VEGF mRNA stability, we adhered primary mouse bone marrow-derived macrophages to ICAM-1 β-integrin ligand) for 3 hours. VEGF mRNA degradation was measured by quantitative RT-PCR after transcriptional inhibition. In the cells bound to ICAM-1, the VEGF mRNA remained stable, whereas, in non-integrin engaged control cells, VEGF mRNA degraded to less than 50% of initial levels within 60 minutes. Furthermore, in BMDMs deficient in HuR expression, VEGF mRNA from integrin-engaged cells degraded to 50% within 60 minutes, thus supporting the essential role of HuR in this stabilization event. To study inflammatory angiogenesis in vivo, we used a subcutaneous polyvinyl alcohol (PVA) sponge model to assess differences in wild-type and macrophage-specific conditional HuR knockout (HuRflox/floxLysM-Cre) mice. Flow cytometry analyses of cells extracted from PVA sponges at 1 to 4 weeks showed equal recruitment of F4/80+ cells in wild-type and HuR knockout mice. Immunofluorescence staining of excised PVA sponges confirmed equal macrophage recruitment and localization but revealed a reduction of VEGF production. Preliminary results indicate that formation of CD31+ microvessels in HuR knockout mice is also decreased. We conclude that macrophage â2-integrin engagement results in stabilization of VEGF and that expression of HuR in macrophages is required for neovascular responses. These findings are relevant to understanding the post-transcriptional regulatory mechanisms of inflammatory angiogenesis.
Modi, Yasha, "Macrophage Integrin-Mediated, HuR-Dependent Angiogenic Factor Gene Expression" (2010). Yale Medicine Thesis Digital Library. 152.