Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). Previously, we reported on the development of an anti-AM antibody that potently inhibits tumor cell proliferation in vitro and tumor growth in vivo. Here, we report the effect of anti-AM receptor antibodies (alphaAMRs) on angiogenesis and tumor growth. We demonstrate that alphaAMRs decrease in a dose-dependent manner the growth of U87 glioblastoma cells and HT-29 colorectal cancer cells, but not A549 lung cancer cells, in vitro. In vivo, AM in Matrigel plugs induces angiogenesis by promoting recruitment of endothelial cells, pericytes, myeloid precursor cells, and macrophages and by promoting channel formation. Remarkably, systemic administration of alphaAMRs every 3 d markedly reduced neovascularization of Matrigel plugs in a dose-dependent fashion, as demonstrated by reduced numbers of the recruited cells and vessel structures. Several human tumor xenografts in athymic mice were used to examine the effect of alphaAMR treatment on tumor angiogenesis and growth. AlphaAMR treatment significantly suppressed the growth of glioblastoma, lung, and colon tumors. Histological examination of alphaAMR-treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial and pericyte cells, and increased tumor cell apoptosis. These findings support the conclusion that alphaAMR treatment inhibits tumor growth by suppression of angiogenesis and tumor growth and suggest that AMRs may be useful therapeutic targets.

CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs) . IntroductionCD146 is a component of the endothelial junction primarily expressed in endothelial cells. It is involved in the control of cell and tissue architecture, as demonstrated by the regulation of its expression during endothelium monolayer formation, its involvement in the control of paracellular permeability, and its colocalization with the actin cytoskeleton. 1 Besides its structural role, CD146 is also involved in cell signaling. 2,3 We have recently demonstrated that CD146 is involved in the regulation of monocyte transendothelial migration. 4 Recent findings indicate that CD146 displays angiogenic properties. In one study, the authors showed that an anti-CD146 antibody, mAb AA98, displayed antiangiogenic properties in chicken chorioallantoic membrane assays and inhibited tumor growth in different xenografted human tumor models in mice. In a model of human umbilical vein endothelial cells (HUVECs), it was also shown that silencing CD146 with specific siRNA inhibited the proliferation and migration of the cells. [5][6][7] Of interest, we have established that CD146 also exists in a soluble form (sCD146) as the result of metalloprotease-dependent shedding of membrane CD146. 4,8 sCD146 is detectable in the human serum, and its level is modulated in different pathologies, such as inflammatory bowel diseases, 9 pathologic pregnancies, 10 and chronic renal failure. 11 However, its exact role is still largely unknown.Postischemic neovascularization occurs as a result of 2 mechanisms: angiogenesis, which relies on mature endothelial cells already present at the ischemic site; and vasculogenesis, which involves the homing and endothelial differentiation of endothelial progenitor cells (EPCs) mobilized from the bone marrow. 12,13 Different angiogenic factors have been shown to trigger angiogenesis and/or vasculogenesis by directly or indirectly stimulating proliferation, differentiation, and migration of mature or precursor cells. Among these factors, the more effective are fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF), and angiopoietins (Ang). FGF-1 has been shown to stimulate the proliferation and differentiation of all cell types necessary for the constitution of an arterial vessel, including endothelial cells and smooth muscle cells. FGF-2 also promotes endothelial cell proliferation and organization of endothelial cells into capillary-like structures. 14 In vitro studies have clearly demonstrated that VEGF is a potent stimulator of angiogenesis, stimulating endothelial cell mitogenesis and migration, 15 and numerous clinical trials have been co...

Neuroendocrine (NE) differentiation in prostate cancer (CaP) has been reported to be an early marker associated with the development of androgen independence. The mechanisms by which CaP acquires NE properties are poorly understood. In this study, a putative role of adrenomedullin (AM) in the NE differentiation was investigated. The expression of AM and AM receptors (calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein-2 and -3 (RAMP2 and RAMP3) was evaluated after experimental manipulation of androgen status. Levels of AM mRNA and immunoreactive AM (ir-AM) increased four-to sevenfold in androgen-sensitive LNCaP cells after androgen withdrawal in vitro and in LNCaP xenografts in animals after castration. Treatment of LNCaP cells with androgen analogue (dihydrotestosterone; 10 À9 M) prevented the increase in AM mRNA and ir-AM levels. Interestingly, the expression of CRLR, RAMP2 and RAMP3 is not regulated by androgen status. We demonstrate that in the presence of serum, AM is able to induce an NE phenotype in LNCaP cells via CRLR/ RAMP2 and RAMP3, which includes extension of neuritic processes and expression of the neuron-specific enolase (NSE), producing cGMP in a dose-dependent manner, which is mediated by a pertussis toxin-sensitive GTP-binding protein. 8-bromo-cGMP mimicked the effects of AM on cell differentiation. We demonstrate that AM induces a G-kinase Ia translocation to the nucleus. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both AM and 8-bromocGMP. In noncastrated animals, administration of AM enhanced expression of NSE and chromogranin A in LNCaP xenografts with a significant increase of NSE levels in serum and no changes in tumor growth. In castrated animals, intraperitoneal injection of AM resulted in a 240718% (Po0.001) increase in tumor volume 36 days after treatment, indicating that the nature of effect of AM in CaP depends on the presence or absence of endogenous androgen. Together, these results demonstrate that AM may function as a mediator of NElike differentiation in culture as well as in vivo and indicate that its production may be important for tumor resurgence following androgen ablation.