Characterization of Tie-2 involvement in modulation of vessel maturation

• Main Authors: Yi-An Chang, 張怡安
• Other Authors: Li-Wha Wu
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/22371539978953371153

碩士 === 國立成功大學 === 分子醫學研究所 === 92 === 　　The formation of endothelial cell (EC) tubes requires the recruitment of pericytes or smooth muscle cells (SMCs). ECs are believed to play an important role in this recruitment event. In the paper by Vikkula et al., venous malformation, a disease that leads to variable thickness of SMCs, was mapped to the receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie2) where an arginine-to-tryptophan substitution (R849W) results in ligand-independent activation of Tie2. By using VMs as a disease model, the specific aim of this study is to delineate how the Tie2 and its ligand, Ang1, regulate the SMCs surrounding the ECs and subsequently modulate vessel maturation. We utilized yeast two hybrid approach to investigate possible molecules that might be involved in this signaling pathway. Four putative interacting proteins were identified by screening an endothelial cDNA library. Among these four proteins, two of them were previously identified to be associated with Tie2, and the remaining two were confirmed to interact with Tie2 in vivo using co-immunoprecipitation. Ang1-expressing stable cell clones were established to produce Ang1-containing conditioned medium. Also, we have successfully established primary culture of SMCs from human umbilical arteries. SMCs were further co-cultured with either wild type or mutant Tie2-expressing ECs to elucidate whether signaling of Tie2 mutant (R849W) would influence the recruitment of SMCs to ECs by evaluating the migration ability of SMC. Under co-culturing condition, we found ECs became elongated and parallelly aligned, and expressed less VE-cadherin than ECs alone. The mRNA level of PDGF-BB and TGF-β of co-cultured ECs were both down-regulated. When we over-expressed normal and mutant Tie2 in the co-cultured ECs, the down-regulation of PDGF-BB and TGF-β appeared to require a functional kinase activity of Tie2. Moreover, we observed that the kinase activity of Tie2 played different roles in mediating the expression of PDGF-BB in the presence or absence of SMCs. However, when we compared the number of recruited SMCs by ECs overexpressing normal or mutated Tie2, there was no significant difference among them. Although more studies are needed to clarify how Tie2 modulate the cross-talk between ECs and SMCs, the present findings suggest that PDGF-BB might be a crucial molecule involved in Tie2 signaling in the vessel maturation.