The regulatory roles of apoptosis-inducing factor in the formation and regression processes of ocular neovascularization

Abstract

The role of apoptosis in the formation and regression of neovascularization is largely hypothesized, although the detailed mechanism remains unclear. Inflammatory cells and endothelial cells both participate and interact during neovascularization. During the early stage, these cells may migrate into an angiogenic site and form a pro-angiogenic microenvironment. Some angiogenic vessels appear to regress, whereas some vessels mature and remain. The control mechanisms of these processes, however, remain unknown. Previously, we reported that the prevention of mitochondrial apoptosis contributed to cellular survival via the prevention of the release of proapoptotic factors, such as apoptosis-inducing factor (AIF) and cytochrome c. In this study, we investigated the regulatory role of cellular apoptosis in angiogenesis using two models of ocular neovascularization: laser injury choroidal neovascularization and VEGF-induced corneal neovascularization in AIF-deficient mice. Averting apoptosis in AIF-deficient mice decreased apoptosis of leukocytes and endothelial cells compared to wild-type mice and resulted in the persistence of these cells at angiogenic sites in vitro and in vivo. Consequently, AIF deficiency expanded neovascularization and diminished vessel regression in these two models. We also observed that peritoneal macrophages from AIF-deficient mice showed anti-apoptotic survival compared to wild-type mice under conditions of starvation. Our data suggest that AIF-related apoptosis plays an important role in neovascularization and that mitochondria-regulated apoptosis could offer a new target for the treatment of pathological angiogenesis.

Figure 1

A: Apoptotic cell death and mitochondrio-nuclear translocation of AIF in choroidal neovascularization (CNV). The CNV developed from choroidal tissue into the subretinal space in the WT mouse. B: Abundant TUNEL⁺ cells are observed in the CNV (green), and these cells also showed nuclear accumulation of AIF (red). In contrast, diffuse weak staining of AIF is observed in the cytosol of the nonapoptotic normal cells. C–F: Confocal microscopy also confirms co-localization of TUNEL (C) and AIF (D) in high resolution images. Nuclei are stained blue (E); merge is also shown (F). G and H: The electron microscopy shows the morphological characteristics of cellular apoptosis, namely chromatin condensation, cellular shrinkage, and apoptotic body formation in the CNV. Original magnification: ×200 (A); ×400 (B–F); ×750 (G) and ×2500 (H). ONL, outer nuclear layer.

Figure 2

Choroidal neovascularization observed by choroidal flat mount. CNV is visualized with fluorescent injection and analyzed with analysis software. AIF-deficient mice (B) induce larger CNV formation than normal WT mice (A). C: Even after 12 weeks AIF-deficient mice (black box) show persistent CNV, whereas WT mice (white box) show regression of formed CNV. D: The EGFP-positive WT macrophages reconstructed from transplanted bone marrow migrate in the CNV stained with rhodamine conjugated concanavalin A. E: The CNV size increased in AIF-deficient mice compared to WT mice; conversely, CNV size was decreased in AIF-deficient mice with WT bone marrow transplantation. *P < 0.05, **P < 0.01. Hq, Harlequin.

Figure 3

Immunohistochemistry for inflammatory and endothelial cells in CNV, which is observed at the laser injury site. A larger number of myeloid lineage cells (CD45) and macrophages (CD11b) migrate into CNV in apoptosis-inducing factor (AIF)-deficient mice compared to WT mice (A). TUNEL⁺ inflammatory and endothelial cells are abundant in WT mice compared to AIF-deficient mice (A, B). C: Immunohistochemistry of CD11b and four major angiogenic or inflammatory factors (VEGF, TNF-α, bFGF, and IL-1β) were examined in the CNV. These four factors are expressed in CD11b-positive macrophages. VEGF is strongly expressed in neovascular tissue; however, it is especially expressed in the macrophages. AIF-deficiency enhances macrophage accumulation and angiogenic factor expression in the CNV. bFGF, basic fibroblast growth factor; Hq/Y, harlequin hemizygous male mice.

Figure 4

Fluorescein angiography of the laser-induced CNV. Compared to WT mice (A), AIF-deficient mice (B) developed larger CNV formation and more severe leakage. C: Lesions were graded on an ordinal scale based on the spatial and temporal evolution of fluorescein leakage as follows: grade 0 (nonleaky): no leakage, faint hyperfluorescence, or mottled fluorescence without leakage; grade 1 (questionable leakage): hyperfluorescent lesion without progressive increase in size or intensity; grade 2A (leaky): hyperfluorescence increasing in intensity but not in size; no definite leakage; grade 2B (pathologically significant leakage): hyperfluorescence increasing in intensity and in size; definite leakage. Hq/Y, harlequin hemizygous male mice.

Figure 5

A: VEGF-induced corneal neovascularization by corneal micropocket assay. B: Pellets were implanted in the cornea, and the quantitative analysis of neovascularization in the mouse corneas was performed using image software. AIF-deficient mice (black box) develop larger neovascularization than WT mice (white box). The regression of the formed neovascularization is retarded in AIF-deficient mice compared to WT mice (A, B). Hq/Y, harlequin hemizygous male mice.

Figure 6

Immunohistochemistry for inflammatory and endothelial cells in the corneal micropocket assay. A: Immunostaining shows abundant infiltrated inflammatory cells (CD11b) and migrated neovascular endothelial cells (CD31) in AIF-deficient mice compared to WT mice. B: More TUNEL⁺ apoptotic cells are observed in WT mice than in AIF-deficient mice. TUNEL⁺ apoptotic cells had a trend to decrease on day 14 (late) in WT mice. In contrast, apoptotic cells had a trend to increase on day 14 (late) in AIF-deficient mice. Hq/Y, harlequin hemizygous male mice.

Figure 7

Peritoneal macrophage culture under starvation. Macrophages were collected from the peritoneal space and cultured using serum-free medium. A: WT mice show cytosolic (normal) and nuclear (apoptotic) staining for AIF, whereas AIF-deficient mice show no staining. B: TUNEL⁺ apoptotic macrophages increase after 24 hours of starvation in WT mice. In contrast, macrophages from AIF-deficient mice are resistant to starvation. *P < 0.05. Hq/Y, harlequin hemizygous male mice.