Structure-based Protein Engineering In Anti-angiogenesis Therapeutic Development

Angiogenesis plays an important role in the growth,development,wound repair and tissue regeneration.Dysregulation of angiogenesis can lead to many severe diseases,such as cardiovascular disease,cancer,inflammation and so on.BMP9(Bone Morphogenetic Protein 9),a member of TGFβ(Transforming Growth Factor β)superfamily,is an important signaling molecule that regulates osteogenesis,chondrogenesis,hepatogenic development,forebrain neuron development and angiogenesis.Studies in BMP9-knockout mice have shown significant roles in the development of blood vessels,lymphatic vessels and cardiac fibrosis.However,BMP9 is double-sided in blood vessels,and it has not yet been clearly to regard BMP9 as promotor or inhibitor for angiogenesis.In normal physiological environment,BMP9 is synthesized as prepropeptide precursor,and then cleaved by proteolytic enzymes to obtain active BMP9,so is there an interaction between the two forms of BMP9? Therefore,successfully accounting for the molecular mechanism of BMP9 signal necessitates a clear understanding of the molecular basis for angiogenesis and the development of novel target therapeutics.Herein,we designed,expressed and purified the pro-BMP9 and pro BMP9 to detect their effect on angiogenesis.Firstly,we demonstrated that pro BMP9 can inhibit pro-BMP9 signaling in vitro by C2C12 differentiation assay.Subsequently,we continued to explore the functions and interactions of the two BMP9 in vivo through the chicken embryo chorioallantoic membrane assay and mouse subcutaneous Matrigel assay,which pro-BMP9 strongly inhibited the angiogenesis,as well pro BMP9 was an important step in the antagonism of BMP9 signaling in vivo.This is a basis for rationalizing anti-vascular disease drug design.Moreover,we design other mutants to discover more available form to impact BMP9 signaling.The principles adopted in our structure-based design might be applicable to a more general scheme in therapeutic development.