Abstract
Endoglin (CD105), a transmembrane protein of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.
Highlights
Endoglin (CD105), a transmembrane protein of the transforming growth factor  superfamily, plays a crucial role in angiogenesis
Endoglin extracellular domain (ECD) Does Not Bind to Either Type I or Type II Receptors in Vitro—Previous studies performed on membrane-bound receptors have shown that endoglin can directly associate with type II receptor TGFRII [19] as well as type I receptors ALK5 and ALK1 [21, 27] without TGF family ligands present
To understand what role endoglin ECD plays in modulating interactions of TGF family ligands with their receptors, we tested binding of hEngECD 26 –586 to the ECD of 5 human type II and 7 type I TGF family receptors fused to hFc using surface plasmon resonance (SPR) technology (Biacore)
Summary
Materials—Biacore 3000, Biacore T100, research grade CM5 chips, amine coupling reagents, 10ϫ HBS-EP buffer, and rabbit anti-mouse IgG were purchased from Biacore (GE Healthcare). The filtered conditioned media material containing mEngECD-mFc 27–581, hEngECD-hFc 26 –586, or truncated variants of human endoglin was purified by affinity chromatography using mAb Select SuRe protein A chromatography. The following day a solution containing 12 g of pGL3 BRE-luciferase, 0.1 g of pRLCMV-luciferase, 30 l of FuGENE 6 (Roche Diagnostics), and 970 l of Opti-MEM (Invitrogen) was preincubated for 30 min at room temperature before adding to 24 ml of assay buffer (McCoy’s medium supplemented with 0.1% BSA). This mixture was applied to the plated cells (500 l/well) for incubation overnight at 37 °C. At the conclusion of the study, animals were euthanized by CO2 inhalation, and tumors were excised and frozen in liquid nitrogencooled isopentane
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