The first but not the second thrombospondin type 1 repeat of ADAMTS5 functions as an angiogenesis inhibitor
Section snippets
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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Cited by (31)
ADAMTS proteases in vascular biology
2015, Matrix BiologyCitation Excerpt :In this case, xenograft assays indicated that the anti-angiogenic properties of this protease were associated with the downregulation of several growth factors such as VEGF, PlGF, and PD-ECGF [18]. This same group also showed that the in vitro inhibitory activity was exclusive to the first, but not the second TSR [19]. A more complex function for the C-terminal ancillary domain was reported for ADAMTS12 [20].
ADAMTS proteases and cancer
2015, Matrix BiologyCitation Excerpt :Nevertheless, different mechanisms seem to underlie these angio-inhibitory capacities. For instance and similarly to ADAMTS-1, ADAMTS-2 [28], ADAMTS-5 [30,31] and ADAMTS-12 [36] do not require their catalytic activity to block angiogenesis, an effect most likely related to the TS1 motifs. By contrast, proteolytic activity of ADAMTS-9 [34] and ADAMTS-15 [37] are essential to promote angio-inhibitory capacity.
ADAMTS5: A new player in the vascular field
2012, American Journal of PathologyADAMTS5 functions as an anti-angiogenic and anti-tumorigenic protein independent of its proteoglycanase activity
2012, American Journal of PathologyCitation Excerpt :A P value of less than 0.05 was considered significant. Based on our previous result that the recombinant TSR1 of ADAMTS5 (TS5-TSR1) is anti-angiogenic in vitro,30 we analyzed whether the full-length ADAMTS5 would also influence angiogenesis. An equal amount (10 μg) of total protein of conditioned medium from TS5-FL (containing 23 nmol/L of TS5-FL) and empty vector transfected B16 melanoma cells (B16/Vec) were preincubated with HUVECs before plating the cells onto preformed Matrigel.
ADAMTS13 promotes angiogenesis and modulates VEGF-induced angiogenesis
2012, Microvascular ResearchCitation Excerpt :The thrombospondin motif has been shown to inhibit angiogenesis by modulating VEGF activity (Good et al., 1990; Luque et al., 2003; Tolsma et al., 1993; Rodriguez-Manzaneque et al., 2001). In the case of ADAMTS5, the first but not the second TSP1 repeat of ADAMTS5 was found to act as an angiogenic inhibitor (Sharghi-Namini et al., 2008). However, no reports have connected ADAMTS13 to angiogenesis at present.