Abstract
Directed migration of endothelial cell (EC) plays a critical role in vascular growth and patterning processes. It is tightly regulated by various guidance cues and molecules. Previously, we identified a novel protein, Thrombospondin Type-1 Domain Containing 7A (THSD7A), which may involved in this process, but not much is known regarding its structural-functional mechanism. By bioinformatic analysis, THSD7A was predicted as a membrane glycoprotein which contains eleven thrombospondin-type-1 repeats (TSR) and one RGD motif. These characters imply THSD7A may be involved in cell migration and cell-to-ECM interactions. In our previous study, we found out that the down-regulation of zTHSD7A (THSD7A homolog in zebrafish) gene expression resulted in abnormal branching and stalled of intersegmental vessel (ISV). On the other hand, the endothelial tip cell at the leading front of growing vessel showed excessive filopodia formation which was thought to guide its direction. We also discovered that Thsd7a transcript was expressed in zebrafish nervous system. Based on these findings, we hypothesize that THSD7A may be a vessel guidance protein secreted by neural tissue and act as an exogenous negative regulator of the directed migration of endothelial tip cell during vascular growth. The specific aim of this thesis study is focusing on analyzing the functional domain of THSD7A in vitro. First, recombinant full-length human THSD7A was expressed by mammalian cells to reveal the isoforms of THSD7A and modification. Baculoviral vectors containing truncated fragment 2 (TF2) of THSD7A was then constructed and used to express the target protein region by insect cells system. The resulting TF2 were then tested with two in vitro angiogenic assays, which are the transwell migration assay and tube-like formation assay. These results demonstrated that there was a secreted form of THSD7A which was glycosylated. His-tagged TF2 (TF2-His) can indeed be expressed by High5 insect cells in Express 5 medium successfully, and an optimal purification procedure was established. However, we found TF2 has no activity on regulating the migration and tube-formation of HUVEC. This suggests that the functional domain of THSD7A may not locate within the TF2 region.