Lymphocytic microparticles modulate macrophages function in experimental choroidal neovascularization

Abstract

Purpose: Pathological choroidal neovascularization (CNV) is the major cause of severe vision loss in patients with age-related macular degeneration (AMD). Inflammation is a key component in AMD, and macrophages play an important role in CNV generation. We have demonstrated that human T-lymphocyte-derived microparticles (LMPs) significantly inhibit angiogenesis in several models of ocular neovascularization. In this study, we investigated whether LMPs modulate angiogenic microenvironment by altering macrophages activities.

Methods: LMPs were produced from apoptotic human T lymphocytes after treated with actinomycin D. The effects of LMPs on cell viability and cell migration were studied in apoptosis assay and migration assay respectively. Gene expression in macrophages was evaluated using quantitative RT-PCR. The expression of IL-10 and IL-12 in macrophages (RAW264.7) were determined using FACS analysis. Cell growth of human retinal microvascular endothelial cells was assessed after cells were co-cultured with LMPs pre-treated macrophages. A laser-induced CNV model was used to determine the in vivo antiangiogenic effects of LMPs. CNV was analyzed with computer-assisted semi-quantitative assay using fluorescently labelled choroidal flat-mounts. Immunohistostaining was performed to reveal the infiltration of macrophages in CNV areas.

Results: LMPs dose-dependently inhibited macrophages cell growth without altering cell death. In addition, LMPs dramatically abrogated VEGF-induced macrophages migration. LMPs-pretreated macrophages exhibited strong inhibitory effect on endothelial cell growth and this effect was associated with the increased expression of IL-12, CD36 and HIF-1α. In vivo, intravitreal injection of LMPs significantly suppressed laser-induced CNV and reduced macrophages infiltration at the lesion sites.

Conclusions: These results suggest that LMPs are potent antiangiogenic therapeutic agent. In addition to the direct effects on endothelial cells, LMPs may interfere the proangiogenic environment through modulation of macrophages function during pathophysiological conditions.

Keywords: 412 age-related macular degeneration • 453 choroid: neovascularization • 609 neovascularization