Date of Award

9-27-2010

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Jeffrey Bender

Second Advisor

Richard Bucala

Third Advisor

Michael Cleman

Abstract

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.

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