Previous studies have suggested that the NG2 proteoglycan interacts with type VI collagen. We have further characterized this interaction using a solid phase binding assay in which purified NG2 was shown to bind to pepsin-solubilized type VI collagen. In addition, NG2 bound a recombinant α2 (VI) collagen chain but did not appreciably bind to the recombinant α1 (VI) chain or the N-terminal domain of α3 (VI) (N9-N2). Binding of NG2 to type VI collagen was shown to be concentration-dependent and saturable and to depend mainly on the NG2 core protein, since chondroitinase-treated NG2 bound the collagen as well as undigested samples. In addition, the binding studies revealed several other possible ligands for NG2, including type II collagen, type V collagen, tenascin, and laminin. Binding of the proteoglycan to these molecules was also shown to be mediated by domains contained within the NG2 core protein. The ability of NG2 to bind to these extracellular matrix molecules was compared with that of the chondroitin sulfate proteoglycan decorin, revealing an almost identical binding pattern of the two proteoglycans to the different collagen types. In addition, decorin was found to effectively inhibit the ability of NG2 to bind to collagen, thus suggesting that the two proteoglycans may bind to some of the same regions on the collagen substrates. In contrast, decorin did not bind tenascin and was ineffective in inhibiting the binding of NG2 to tenascin or laminin, indicating that NG2 may bind these two molecules using a separate domain that is distinct from its collagen binding region.

NG2 is a membrane-spanning proteoglycan with a primary structure unique among cell surface or extracellular matrix proteins. To characterize the interaction between NG2 and extracellular matrix proteins, we have used a eukaryotic expression system to produce and purify several recombinant fragments covering not only the entire ectodomain of NG2 but also distinct subdomains of the molecule. Using a solid phase binding assay with various extracellular matrix proteins, we have identified two main ligands for NG2, namely, collagens V and VI. Consistent with previous models of glycosaminoglycan attachment, roughly 50% of the recombinant NG2 fragments containing the central domain have chondroitin sulfate chains attached to the protein core. These glycosaminoglycan chains are not directly involved in collagen binding, since chondroitinase-treated fragments exhibit an unimpaired ability to bind to both collagens. Using more restricted recombinant fragments of NG2, we mapped the binding site for both collagens to the central domain of NG2. Electron microscopy after rotary shadowing of native NG2 molecules indicates that this extended nonglobular domain provides a flexible connection joining the two N- and C-terminal globular regions of NG2. Rotary shadowing of mixtures of NG2 and collagen V or VI confirms a direct interaction between the molecules and indicates that the collagens align with the central region of NG2, giving the appearance of a rod between the N- and C-terminal globules.

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 in vivo. First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro 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 Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad 1–5 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 of most of the biological BMP activity found in plasma.

Rendu-Osler-Weber syndrome, also known as hereditary hemorrhagic telangiectasia (HHT), is an autosomal dominant vascular disorder. Three genes are causally related to HHT: the ENG gene encoding endoglin, a co-receptor of the TGFß family (HHT1), the ACVRL1 gene encoding ALK1 (activin receptor-like kinase 1), a type I receptor of the TGFß family (HHT2), and the SMAD4 gene, encoding a transcription factor critical for this signaling pathway. Bone morphogenetic proteins (BMPs) are growth factors of the TGFß family. Among them, BMP9 and BMP10 have been shown to bind directly with high affinity to ALK1 and endoglin, and BMP9 mutations have recently been linked to a vascular-anomaly syndrome that has phenotypic overlap with HHT. BMP9 and BMP10 are both circulating cytokines in blood, and the current working model is that BMP9 and BMP10 maintain a quiescent endothelial state that is dependent on the level of ALK1/endoglin activation on endothelial cells. In accordance with this model, to explain the etiology of HHT we hypothesize that a deficient BMP9/BMP10/ALK1/endoglin pathway may lead to re-activation of angiogenesis or a greater sensitivity to an angiogenic stimulus. Resulting endothelial hyperproliferation and hypermigration may lead to vasodilatation and formation of arteriovenous malformation (AVM). HHT would thus result from a defect in the angiogenic balance. This review will focus on the emerging role played by BMP9 and BMP10 in the development of this disease and the therapeutic approaches that this opens.