Macrophage Promotes Vasculogenesis In Experimental Choroidal Neovascularization Via Upregulating SDF-1 Expression By RPE

Backgrounds Choroidal neovascularization (CNV)is new vessel membrane which often implicates the macular, leading to exudation or hemorrhage and finally scar and thus cause blindness. Both angiogenesis and vasculogenesis are involved in CNV formation which happens in many kinds of ocular diseases in the posterior of eye, especially the age-related macular degeneration (AMD) which is the most common cause of blindness in the west. The incidence of the disease in China is higher than the average of the world wild. And along with the social aging acceleration, the problem may become a social one. As CNV contributed to 90% blindness in AMD, it has been an imperatitve issue to find ways to prevent and treat the disease. To date, the etiology and pathology of AMD and CNV was still not figured out. Recent studies have implied immunology-inflammation may be the inducer and promoter of AMD. In the past, macrophages (Mφ) had often been seen in CNV and might be playing important roles but with obscure detail. Considering that Mφplayed central role in immunol and inflammatory response, we believe that to probe the exact role of Mφin CNV as well as the mechanism would help understanding the pathology of CNV. Moreover, it may get us an acceess to novel therapeutic strategy for CNV.Aim To investigate the role of monocyte-macrophage (MC/Mφ) in experimental CNV especially the vasculogenesis aspect and its mechanism. Methods Evaluation of pharmacal depletion effectiveness: clodronate-lipsome was administrated intravenously to deplete MC/Mφ. Immunohistochemistry against F4/80 in spleen was done to detect the number of Mφ. Pharmacal toxicity evaluation: DiO labeled liposome was prepared and injected intravenously into mice. The relationships of liposomes and retinal epithelium (RPE) cells, endothelial cells (EC) and Mφwere examined by Immunoinflurorescence. Besides, RPE cells and Rhesus macaque choroid-retinal endothelial (RF/6A) cells were cultured with liposomes in vitro. Then proliferation ability of the cells was assessed by MTT. The expression level of vascular endothelial growth factor (VEGF) and stromal derived factor-1α(SDF-1α) was detected by ELISA; Effect of depletion on CNV: Green fluorescent protein (GFP) chimeric mice were developed by transplanting unpurified bone marrow cells from GFP +/+ transgenic mice (6 ~ 8 wk) into wild type C57BL/6J (6 ~ 8 wk). The qualified chimeric mice underwent laser photocoagulation to induce CNV. Chimeric were injected with clodronate-liposome injection via tail vein to systemically deplete monocyte-macrophage. Another two groups of control mice were injected with PBS-liposome or nothing. GFP expressing cells were calculated at the site of laser spots and CNV area was measured at 3 days and 2wk after lasering by choroidal flatmounts. Tissue section of the eye balls underwent immunofluorecence staining to assess VEGF and SDF-1 expression at the laser spots at 3 days after lasering and endothelial cells marker CD31 and smooth muscle cell markerα-SMA were detected in CNV at 2wk to see the differentiation status. Beseides, young wild type C57 mice which received old GFP transgenic mice ( > 18m) bone marrow cells were also depleted monocyte and then the GFP expressing cells and CNV area were evaluated. Human monocyte was isolated form blood. SDF-1 and VEGF mRNA in RPE and SDF-1αand VEGF in the supernatant were detected by PCR and ELISA after human monocyte and RPE were co-cultured in a contact pattern or indirect contact for 4h and 24h.Results clodronate-liposome were effective in depleting MC/Mφin spleen and also innoxious inflected by in vivo no obviously phagocytized by RPE and EC and in vitro unchanged proliferation ability and undiminished expression of VEGF and SDF-1 in the supernatant; Chemeric rate were higher than 90% in chemeric mice. After monocyte depletion, GFP expression cells dramatically decreased at laser spots at day 3 and 2wk. CNV area at 2wk attenuated markedly. The two control group were almost the same. Meanwhile, compared with PBS-lip group, most GFP cells were beneath the CNV membrane instead of participating in the CNV membrane. The GFP cells which were CD31 orα-SMA positive were also reduced. VEGF and SDF-1 expression also decreased at day 3 after lasering. And the mice transplanted with bone marrow from old GFP transgenic mice also presented apparente attenuation in GFP cells and CNV area. More SDF-1 mRNA and SDF-1αprotein were detected in RPE cocultured with monocytes in the contact pattern than RPE alone or cocultured with monocytes in Millicell. However the VEGF protein and mRNA expression levels were not upregulated after co-culture compared with cultured alone.Conclusion Systemically depleting monocytes could attenuate CNV and reduce the recruitment and incorporation of bone marrow derived cells into CNV. The descent of VEGF and SDF-1 may be attributed to the inhibition status of CNV development. Contact of MC/Mφwith RPE may potently stimulate SDF-1 in RPE to promote the vasculogenesis in CNV. The lack of alteration of VEGF in vitro implied downregulation of VEGF in vivo a sequential or accompanying event.