ALK1 is an endothelial-specific type I receptor of the TGF␤ receptor family whose heterozygous mutations cause hereditary hemorrhagic telangiectasia type 2. Although TGF␤1 and TGF␤3 have been shown to bind ALK1 under specific experimental conditions, they may not represent the physiological ligands for this receptor. In the present study, we demonstrate that BMP9 induces the phosphorylation of Smad1/5/8 in microvascular endothelial cells, and this phosphorylation lasts over a period of 24 hours. BMP9 also activates the ID1 promoter-derived BMP response element (BRE) in a dosedependent manner (EC 50 ‫؍‬ 45 ؎ 27 pg/ mL), and this activation is abolished by silencing ALK1 expression or addition of ALK1 extracellular domain. Overexpression of endoglin increases the BMP9 response, whereas silencing of both BMP-RII and ActRIIA expressions completely abolishes it. BMP10, which is structurally close to BMP9, is also a potent ALK1 ligand. Finally, we demonstrate that BMP9 and BMP10 potently inhibit endothelial cell migration and growth, and stimulate endothelial expression of a panel of genes that was previously reported to be activated by the constitutively active form of ALK1. Taken IntroductionActivin receptor-like kinase 1 (ALK1) is an endothelial-specific type I receptor of the TGF␤ receptor family that is implicated in the pathogenesis of the Rendu-Osler disease also known as hereditary hemorrhagic telangiectasia (HHT). 1 The disease is an autosomal dominant vascular dysplasia affecting 1 in 10 000 people. The clinical abnormalities in HHT are caused by direct arteriovenous connections without an intervening capillary bed. The resulting telangiectases occur in the oral cavity (lips and tongue), in the nose, and on the fingertips. Larger arteriovenous malformations (AVMs) can be encountered in the lung, brain, and liver. 2 There is wide variation in the penetrance and severity of these symptoms in patients even within the same family, suggesting that environmental or other genetic factors influence the phenotype. The majority of cases are caused by mutations in either Endoglin (ENG) or ALK1 (ACVRL1) genes, thus defining HHT1 and HHT2, respectively. Recently, mutations in SMAD4 have also been described in a few cases with combined juvenile polyposis and HHT syndromes. 3 Each of the 3 genes implicated in HHT (ENG, ACVRL1, and SMAD4) encode receptors or signaling molecules from the TGF␤ family. Most TGF␤ family ligands bind to heteromeric complexes of type I and type II serine/threonine kinase receptors (for review, see Shi and Massague 4 ). In addition, the type III receptors (betaglycan and endoglin) act as coreceptors that can potentiate the signaling cascade. Upon ligand binding, the type II receptor phosphorylates and activates the type I receptor, also known as activin receptor-like kinase (ALK), which in turn phosphorylates a receptor-regulated Smad protein (Smad1, Smad2, Smad3, Smad5, or Smad8). This phosphorylated Smad dimerizes with a common partner, Smad4, and this complex translocates to the nucleus where ...

Abstract-Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-␤ receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC 50 (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence. Key Words: BMP9 Ⅲ ALK1 Ⅲ HHT Ⅲ angiogenesis B one morphogenetic proteins (BMPs), which belong to the transforming growth factor (TGF)␤ superfamily, were originally identified as inducers of ectopic bone growth and cartilage formation. Since then, there has been substantial progress in our knowledge of the multiple functions of these growth factors. 1 BMPs regulate cell growth, differentiation, and apoptosis of various cell types, and they are critically important in the morphogenesis and differentiation of tissues and organs. BMP9, also known as growth differentiation factor-2, is expressed in the adult liver by nonparenchymal cells (ie, endothelial, stellate, and Kupffer cells) 2 and in the septum and spinal cord of mouse embryos. 3 BMP9 has been described as a hematopoietic, hepatogenic, osteogenic, and chondrogenic factor. It has also been identified as a regulator of glucose metabolism, capable of reducing glycemia in diabetic mice and as a differentiation factor for cholinergic neurons in the central nervous system. 3 More recently, it was shown to induce the expression of hepcidin, a hormone that plays a key role in iron homeostasis. 4 ALK1 (activin receptor like-kinase 1) is an endothelialspecific type I receptor of the TGF␤ receptor family that is implicated in the pathogenesis of hereditary hemorrhagic telangiectasia type 2 (HHT2), also known as the Rendu-Osler...