s. A probability value of less than 0.05 was considered statistically significant. 5 Adrenomedullin in CNV Results To elucidate the involvement of ADM signaling in CNV, we first investigated the expression of the ADM receptor in choroidal vascular ECs in the CNV model. After we confirmed that CNV did not invade retinal tissue, CD31+CD452 ECs in the RPE-choroid complex were analyzed by flow cytometry and their number was calculated as a percentage of a total of 56104 choroid/RPE complex cells 7 days after laser induction. We confirmed that the number of ECs increases after induction of CNV in laser-treated eyes compared to control eyes . There was no change in the number of CD31+ CD45+ or CD312CD45+ cells; however, CD11b cells Aglafoline site increased after laser-induction of CNV . Because ADM signaling is known to be mediated through CRLR/RAMP2 and CRLR/RAMP3 receptors, we analyzed the expression of CRLR, RAMP2 and RAMP3 mRNA in choroid/RPE complexes after induction of CNV. CRLR, RAMP2 and RAMP3 were highly expressed in the CD31+CD452 ECs compared to CD312CD452 non-ECs. However, the level of expression of these ADM receptor components in ECs was not altered after CNV induction compared to sham-operated control groups. Moreover, we confirmed the expression of CRLR in the ECs of CNV by immunostaining. Next, we assessed the expression of ADM during CNV formation. In the 9726632 laser-induced CNV model, the presence of different angiogenic factors in RPE-Choroid complexes has already been reported. We analyzed the expression of ADM mRNA and found that it increased in a time-dependent fashion and peaked 4 days after laser treatment. Because it is reported that ADM promotes angiogenesis in both an autocrine and a paracrine manner, we focused on two cell populations which could be isolated by flow cytometry: CD31+ ECs and CD11b+ monocytes/macrophages. We confirmed that both ECs and macrophages express more ADM mRNA compared to CD312CD11b2 cells. Furthermore, after CNV induction, ADM expression was significantly upregulated in both ECs and macrophages compared to the same cells in shamoperated mice. Therefore, these data suggest that ADM is involved in this laser-induced CNV model. It is well known that inflammatory cytokines upregulate ADM expression in various cells. We confirmed this by using macrophage, EC, and retinal pigment epithelial 
cell lines stimulated with TNF-a and LPS. It has been reported that ADM induces EC proliferation, migration and tube formation through phosphatidylinositol 39 kinase /Akt, extracellular signal-regulated kinase, and tyrosine phosphorylation of focal adhesion kinase . Thus, we tested whether culture supernatant from RAW264.7 cells after TNF-a stimulation promotes EC proliferation and whether this can be inhibited by a widelyaccepted ADM antagonist, ADM . Although the inhibitory effect was weak compared to a potent VEGF-A signaling inhibitor, SU1498, ADM did significantly suppress proliferation of EC cultured not only in supernatant from TNF-a-stimulated 8114006 macrophages but also to some extent in medium containing TNF-a without macrophages. Additionally, we confirmed the absence of the potential toxicity of ADM using an in vitro dose-response model. These data suggest that ADM signaling affects EC proliferation in an autocrine and a paracrine manner. Moreover, we evaluated the expression of ADM in 24 hour-cultured primary RPE/choroid complexes obtained from wild-type mice. TNF-a stimulation of cultured primary RPE/choroid complexes sign