The first but not the second thrombospondin type 1 repeat of ADAMTS5 functions as an angiogenesis inhibitor

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Abstract

Angiogenesis is critical for tumour growth and metastasis where factors that regulate this process are potential targets for development of anti-cancer drugs. In this study, we show that the first TSR domain of the extracellular matrix protease ADAMTS5, unlike the second TSR, has anti-angiogenic activities where it inhibits endothelial cell tube formation on Matrigel, reduces cell proliferation and attachment, while promoting cell apoptosis and migration, all in a dose-dependent manner. Furthermore, it influences the architecture of endothelial cells by disrupting actin stress fibres and reducing focal adhesions, likely via suppressing RhoA activation. TSR1 of ADAMTS5 is therefore a novel anti-angiogenic peptide and could serve as a prototype for future development into anti-cancer drugs.

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Materials and methods

Expression and purification of TS5-TSR1 protein. TSR1 was cloned into pET-M vector (modified from pET32, Novagen), expressed in Escherichia coli (BL21DE3), and purified using Ni–NTA affinity chromatography in 6 M urea according to the manufacturers’ instructions (Qiagen, USA). The protein was then dialysed against PBS and its concentration was determined using BioRad Bradford assay reagent. The concentrations of TS5-TSRs in all assays were between 1 and 1000 nM unless specified. Synthetic

TS5-TSR1 but not TS5-TSR2 inhibits EC tube-like structure formation on Matrigel

There are two TSRs in ADAMTS5, a centrally located TSR1 and a C-terminal TSR2 (Fig. 1A). Sequence comparison and structural analysis indicated that TS5-TSR1 is highly similar to the anti-angiogenic TSR2 and TSR3 of TSP-1, suggesting TS5-TSR1 could potentially be anti-angiogenic. Synthetic TS5-TSRs were mixed with ECs and plated onto pre-formed Matrigel. Results indicated that TS5-TSR1, but not TS5-TSR2, inhibited tube-like structure formation in a dose-dependent manner (Fig. 1B). A recombinant

Conclusions

This study demonstrates that TSR1, but not TSR2 of ADAMTS5, is a novel anti-angiogenic peptide. It prevents EC tube-like structure formation in vitro, possibly through multiple mechanisms including inhibition of EC attachment to matrix, stimulation of EC migration, and induction of apoptosis. These functions are likely the results of this peptide’s ability to mitigate VEGF-induced formation of actin stress fibres and focal adhesions, through suppressing RhoA activation. TS5-TSR1 can therefore

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