Amazingly, (the gene encoding ALK1) and in (the gene encoding its co-receptor endoglin) cause Rendu-Osler disease, also known as HHT (hereditary hemorrhagic telangiectasia).2 HHT is a rare genetic vascular disease characterized by numerous epistaxes, cutaneous telangiectasias and arteriovenous malformations (AVM) in the lungs, liver and central nervous system. malformations (AVM) in the lungs, liver and central nervous system. ALK1 inactivation in mice leads to embryonic lethality at E11 because of major angiogenesis defects.3,4 These data clearly demonstrate that ALK1 is an important player in angiogenesis, but its molecular role is still not completely clear. The retina of newborn mice is avascular, and development of retinal blood vessels progressively occurs during the first week after birth to form a highly organized vascular network composed of arteries, veins and capillaries. 5 Retinal vascularization in newborn mice is therefore a very interesting model to study physiologic angiogenesis. The roles of endoglin and ALK1 in the 5(6)-TAMRA vascularization of the retina DKFZp781B0869 have been recently demonstrated.6,7 Using endoglin-inducible KO in endothelial cells (Eng-iKOe), it was shown that absence of endoglin delayed remodeling of the capillary plexus, increased endothelial proliferation and induced localized AVMs in retinas.6 It was also published that injection of the extracellular domain of ALK1 (ALK1ecd) strongly affected retinal vascularization further supporting the importance of ALK1 and its ligands in retinal angiogenesis.7 5(6)-TAMRA In 2007, we identified bone morphogenetic protein 9 (BMP9) and BMP10 as specific ligands for ALK1.8 BMP9 was shown to be present in adult blood of rodents and humans and to circulate in both an active and an inactive form.9,10 On the other hand, BMP10 has been shown to be mainly expressed in the embryo and to be involved in heart development.11 We further showed that addition of serum to endothelial cells induced a phospho-Smad1/5 response that could be completely inhibited by the addition of a neutralizing anti-BMP9 antibody, supporting a major role for BMP9 in adult angiogenesis, while BMP10 function would mainly be restricted to embryogenesis.9,10 Therefore many studies have focused on the role of BMP9 on angiogenesis. The in vitro effects of BMP9 on endothelial cell migration and proliferation are still under debate, as some groups have found an inhibition,8,12 while another group, using endothelial cells from a different tissue origin, has described an induction.13 BMP9 was also shown to inhibit ex vivo endothelial sprouting from metatarsals12 and to inhibit FGF-2 induced angiogenesis in vivo in the mouse angiogenesis model of subcutaneously implanted sponges,10 while it increased angiogenesis in a Matrigel plug assay and in a xenograft model of human pancreatic cancer.13 Taken together these data demonstrate that BMP9 is involved in angiogenesis, although its precise cellular functions are still under debate. All of these prior studies have addressed 5(6)-TAMRA the role of BMP9 by supplementing BMP9 in vitro or in vivo. To date, nobody has addressed the effect of blocking BMP9 in vivo on angiogenesis. To address this issue, we investigated the role of endogenous BMP9 on retinal angiogenesis using anti-BMP9 antibodies and values of .05 or less. Results Anti-BMP9 treatment increases vascular density of the retina of WT mice It was previously described that injection of ALK1ecd to newborn pups increased postnatal retinal vascular density.7 This indicated that the ALK1 pathway controls postnatal angiogenesis. However, in this prior study, the nature of the ligand(s) blocked by the addition of ALK1ecd was not characterized. We have previously shown that BMP9 binds to ALK1 with strong affinity (EC50 = 2pM)8 and that BMP9 circulates in a biologically active form in human and mouse blood and is present at higher levels around birth than during adulthood (6 ng/mL in newborn vs 2 ng/mL in adult mice).9,10 We therefore asked whether circulating BMP9 triggered the biologic effects blocked by ALK1ecd. Analysis of mouse retinas at postnatal day 6 (P6) after a systemic treatment of pups (OF1 background) with a monoclonal anti-BMP9 antibody (5 mg/kg, at P1 and P3) revealed vascular patterning defects, with vessels forming a hyperbranched plexus (Figure 1A-B). We quantified the number of branching points both at the vascular front and at the capillary plexus and found that anti-BMP9 treatment significantly increased vascular branching (Figure 1D). We observed a similar effect with ALK1ecd treatment (5 mg/kg; Figure 1C-D). On the other hand, we did not observe any differences on radial vascular expansion.