The Functional Analysis of Thrombospondin Type I Domain Containing 7A in Vascular Endothelial Cell Migration during Angiogenesis

• Main Authors: Wang, Chieh-Huei, 王捷暉
• Other Authors: Chuang, Yung-Jen
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/23064932237633465063

博士 === 國立清華大學 === 生物資訊與結構生物研究所 === 99 === Angiogenesis is a highly organized process under the control of guidance cues that direct endothelial cell (EC) migration, proliferation and differentiation. Recently, many molecules that were initially described as regulators of neural guidance were subsequently shown to also direct EC migration during angiogenesis. Here we report a novel protein, Thrombospondin-typeⅠDomain-containing Protein 7A (THSD7A), which is required for EC migration and involved in the vascular patterning during development. Identified by SAGE (serial analysis of gene expression) database mining and immunohistochemistry, THSD7A is highly expressed in human placenta vasculatures. To determine the function of THSD7A, we altered endogenous THSD7A expression in human umbilical vein endothelial cells (HUVECs) for subsequent angiogenesis assays. Our data indicated that downregulation of THSD7A in HUVECs enhanced cell migration and promoted tube formation, while overexpression of a THSD7A carboxyl-terminal fragment inhibited HUVEC migration and disrupted tube formation. Immunohistological analysis revealed that THSD7A was expressed at the leading edge of migrating HUVECs, and it co-localized with alpha-V-beta-3 integrin and paxillin. This distribution was dispersed from focal adhesions after disruption of the actin cytoskeleton, suggesting the involvement of THSD7A in alpha-V-beta-3 integrin and paxillin that mediates cytoskeletal reorganization during directed EC migration. To characterize THSD7A in vivo, we performed whole-mount in-situ hybridization to reveal the spatiotemporal expression of THSD7A orthologue during zebrafish embryonic development, by which we detected zebrafish thsd7a transcripts in the central nervous system. Notably, this expression exhibited a unique pattern along the ventral edge of neural tube, correlating with the growth path of angiogenic intersegmental vessels (ISVs). Antisense oligonucleotide-mediated gene knockdown of Thsd7a caused a lateral deviation of angiogenic ECs below the thsd7a-expressing sites, resulting in aberrant ISV patterning. Collectively, our study revealed that THSD7A mediates angiogenic EC migration via cytoskeletal reorganization, and that zebrafish Thsd7a is a neural protein required for ISV angiogenesis during development. Future analysis on this novel protein shall provide a new perspective on the underlying mechanisms of directed EC migration, and shed light on the complex communication network between the nervous and vascular systems.