In the current study, we have explored the possibility that ADM exerts angiogenic effects in a laser-induced CNV model. The ADM receptor was expressed in choroidal endothelial cells and ADM was upregulated in the choroid/RPE complex during CNV formation. Moreover, an ADM pharmaceutical antagonist and a mAb to ADM efficiently suppressed CNV formation and mediated synergistic effects together with a potent VEGF-A inhibitor. These findings suggest that ADM could represent a therapeutic target and be an attractive option for the treatment of CNV in AMD.
The biological functions of ADM are well- recognized to be dilatation of resistance vessels 
, increases of cardiac output 
, regulation of vascular permeability 
and contribution to mobilization, adhesion and differentiation into endothelial progenitor cells of bone marrow-derived cells 
. Additionally, several lines of evidence have suggested that inhibiting the ADM pathway with antibodies or antagonists directed against ADM or ADM receptors can reduce angiogenesis and tumor cell proliferation in mouse cancer models 
. Although ADM is reported to affect not only angiogenesis but also tumor cell proliferation via its autocrine and paracrine mechanisms in cancer, there is little data showing the involvement of ADM in a disease model whose central pathogenesis depends on sprouting angiogenesis 
ADM immunoreactivity was reported in cardiac myocytes, vascular smooth muscle cells, ECs, renal distal and collecting tubules, mucosal and glandular epithelia of the digestive, respiratory and reproductive system, the endocrine and neuroendocrine system, as well as in the central nervous system 
. In the current model, upregulation of ADM was not detected in the retina after laser treatment (data not shown), although Blom et al. reported the expression of ADM in neural retina. However, we saw ADM in the RPE/choroid. Secreted inflammatory cytokines after laser burn might be enhancing ADM expression and further accumulation of inflammatory cells.
This was confirmed using a primary RPE/choroid culture model in the present study. However, ADM expression 24 hr after TNF-α stimulation was lower than at 12 hr, in contrast to findings with different cell lines such as RAW 264.7, RPE-19 and bEnd.3. We hypothesize that this difference can be attributed to the use of transformed cell lines which could display different expression patterns compared to that of primary cultures. Local upregulation of ADM at each photocoagulated site could induce direct ADM effects, i.e. EC proliferation, migration and tube formation. Currently, anti-VEGF therapy has become the major treatment modality for neovascular AMD. However, numerous injections of the anti-VEGF drug may be required to maintain clinical benefit. Moreover, after treatment with anti-VEGF drugs, it can be hypothesized that a hypoxic response could occur and subsequently upregulate ADM, because this has been observed in ECs 
. Chen et al. reported reciprocal regulation of ADM and HIF-1α expression exerted synergistic effects on proliferation of ECs in vitro 
. Induction and nuclear translocation of HIF-1 α controls the expression of several angiogenic factors 
. Therefore, the requirement for additional drugs together with anti-VEGF therapy is rational even if anti-ADM treatment partially suppresses CNV formation.
Although we expected that ADM injection would enhance the angiogenic response, exogenous ADM did not alter the size of CNV. We hypothesize that ADM was already saturated in the lesion and therefore additional factor would not be able to further activate the ADM receptor and exert any greater effects.
Chen et al. reported that tumor-associated macrophages (TAM) express both ADM and ADM receptor components. They also reported that ADM from TAM stimulated ECs and furthered angiogenesis via a paracrine pathway; moreover, it also potentiated the differentiation of TAM from the M1 to M2 state in an autocrine manner 
. In the current study, infiltrating macrophages in CNV eyes expressed more ADM; therefore we cannot completely exclude the possibility that upregulation of ADM in macrophages could cause them to change their characteristics and to secrete other angiogenic factors such as VEGF. However, the finding of an inhibitory effect on EC proliferation of ADM antagonists in supernatant from macrophage cultures implies that ADM originating from macrophages must be at least partly responsible for CNV formation.
Udono et al. reported that hypoxia and inflammatory cytokines induced the expression of ADM in human RPE cells and that ADM could enhance RPE proliferation 
. Therefore, in this CNV model, it is also possible that ADM expression in RPE was upregulated and that ADM secreted by RPE could promote angiogenesis. However, Huang et al. reported that ADM inhibited the migration of RPE cells in association with reductions in [Ca2+
. Although there is some controversy about the function of ADM in RPE cells, we could demonstrate that inflammatory stimulation up-regulated the expression of ADM in RPE in vitro. Indeed, it is technically difficult to sort the RPE cells by flow cytometry using specific surface markers and we were unable to determine their ADM expression level. Although we have to carefully evaluate the major source of ADM in the CNV model, the observed paracrine and autocrine effects of ADM could induce CNV.