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...

Transforming growth factor-beta 1 (TGF-beta 1) plays an important role in the modulation of cellular growth and differentiation and the production and degradation of the extracellular matrix. A number of experimental results suggest that TGF-beta 1 may be involved in cardiovascular physiopathology. In the present study, we assessed whether the TGF-beta 1 gene is a candidate gene for coronary heart disease or hypertension. We screened the coding region and 2181 bp upstream of the TGF-beta gene for polymorphisms and identified seven polymorphisms: 3 in the upstream region of the gene at positions -988, -800, and -509 from the first transcribed nucleotide; 1 in a nontranslated region at position +72; 2 in the signal peptide sequence Leu10-->Pro, Arg25-->Pro; and 1 in the region of the gene coding for the precursor part of the protein not present in the active form, Thr263-->Ile. We analyzed these TGF-beta 1 polymorphisms in 563 patients with myocardial infarction and 629 control subjects from four regions in Northern Ireland and France. The Pro25 allele was more frequent in patients than in control subjects in Belfast (P < .01) and Strasbourg (P < .05). The TGF-beta 1 polymorphisms were not associated with the degree of angiographically assessed coronary artery disease in patients. The presence of a Pro25 allele was associated with a lower systolic pressure in the four control groups (P < .002), and a history of hypertension was significantly less frequent in homozygotes or heterozygotes for Pro25 than in hormozygotes for Arg25 (odds ratio, 0.43, 95% confidence interval, 0.19 to 0.92; P < .03). Since the Pro25 allele was associated with an increased risk of myocardial infarction and a reduced risk of hypertension, we favor a cautious interpretation of these apparently inconsistent results. Other studies will need to verify whether these associations are real.

ALK1 is a type I receptor of the TGF-β family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.

Bone Morphogenetic Protein 9 (BMP9) has been recently found to be the physiological ligand for the activin receptor-like kinase 1 (ALK1), and to be a major circulating vascular quiescence factor. Moreover, a soluble chimeric ALK1 protein (ALK1-Fc) has recently been developed and showed powerful anti-tumor growth and anti-angiogenic effects. However, not much is known concerning BMP9. This prompted us to investigate the human endogenous sources of this cytokine and to further characterize its circulating form(s) and its function. Analysis of BMP9 expression reveals that BMP9 is produced by hepatocytes and intrahepatic biliary epithelial cells. Gel filtration analysis combined with ELISA and biological assays demonstrate that BMP9 circulates in plasma (1) as an unprocessed inactive form that can be further activated by furin a serine endoprotease, and (2) as a mature and fully active form (composed of the mature form associated with its prodomain). Analysis of BMP9 circulating levels during mouse development demonstrates that BMP9 peaks during the first 3 weeks after birth and then decreases to 2 ng/mL in adulthood. We also show that circulating BMP9 physiologically induces a constitutive Smad1/5/8 phosphorylation in endothelial cells. Taken together, our results argue for the role of BMP9 as a hepatocyte-derived factor, circulating in inactive (40%) and active (60%) forms, the latter constantly activating endothelial cells to maintain them in a resting state.

No abstract

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available -KO mice, we generated a conditional-KO mouse strain. The plasma from -KO mice, similarly to that of-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.

These results suggest that polymorphisms of the TNF-alpha gene are unlikely to contribute to CHD risk in an important way, but the TNF-alpha/-308 polymorphism should be investigated further in relation to obesity.