Abstract
Purpose: :
Inflammatory angiogenesis has been implicated in the pathophysiology of age-related macular degeneration and diabetic retinopathy. Expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), and recruitment of macrophages play a crucial role in the disease progression. It is well known that induction of VEGF during hypoxia and inflammation is due in large part to stabilization of its mRNA, which is mediated primarily by an RNA-binding protein HuR. Expression of VEGF by macrophages in the choroidal neovascular membranes has also been described. Here, we address whether β2-integrin (a cell-adhesion molecule that mediates intracellular signals) engagement stabilizes VEGF mRNA in macrophages in vitro and at angiogenic sites in vivo via a HuR-dependent mechanism.
Methods: :
1. To determine whether in vitro engagement of β2-integrins regulates VEGF mRNA stability, we allowed primary mouse bone marrow-derived macrophages to adhere to recombinant ICAM-1 (β2 integrin ligand) or poly-L-lysine (as a negative control) for 3 hours and measured VEGF mRNA degradation by quantitative RT-PCR following inhibition of transcription. 2. To study inflammatory angiogenesis in vivo, we used a macrophage-dependent model of subcutaneous implantation of polyvinyl alcohol (PVA) sponges to assess differences in wild-type and macrophage-conditional HuR knockout (HuRflox/floxLysM-Cre) mice.
Results: :
1. In the cells bound to poly-L-lysine, more than half of the VEGF mRNA degraded within 60 min. However, VEGF mRNA remained stable when macrophages were engaged to ICAM-1.2. Flow cytometry analyses of cells extracted from PVA sponges at 1 to 4 weeks showed essentially equal recruitment of F4/80+ macrophages in wild-type and HuR knockout mice. Immunofluorescence staining of excised PVA sponges confirmed equal macrophage recruitment and localization but revealed a significant reduction of VEGF production and formation of CD31+ microvessels in HuR knockout mice.
Conclusions: :
This is the first report that macrophage β2-integrin engagement results in stabilization of VEGF and that expression of HuR in macrophages is required for neovascular responses. These findings provide evidence for a post-transcriptional regulatory mechanism of VEGF expression in macrophages. Clinically, these research findings are relevant to the mechanisms of inflammatory angiogenesis in age-related macular degeneration and diabetic retinopathy.
Keywords: vascular endothelial growth factor • inflammation • stress response