Anti-angiogenic properties of ADAMTS-4 in vitro

Abstract

Angiogenesis is an indispensable mechanism in development and in many pathologies, including cancer, synovitis and aberrant wound healing. Many angiogenic stimulators and inhibitors have been investigated, and some have progressed to the clinic. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a group of multifunctional proteinases. ADAMTS-1 and ADAMTS-8 have been reported to be anti-angiogenic. Here, we provide evidence that ADAMTS-4, like ADAMTS-1, is expressed by endothelial cells and binds to vascular endothelial groth factor (VEGF). Moreover, ADAMTS-4 inhibited human dermal microvascular endothelial cells (HuDMEC) VEGF-stimulated VEGF receptor (R) R2 phosphorylation, differentiation and migration, suggesting that ADAMTS-4 may be a novel anti-angiogenic molecule.

Figure 1

Relative mRNA expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1, -4, -5, -8 and -9 in human umbilical vein endothelial cells (HUVECs) and human dermal microvescular endothelial cells (HuDMECs) as determined by real-time RT-PCR. ADAMTS expression is shown compared with GAPDH expression (normalized to 1) using the formula 2^−ΔCT, and data are displayed as median and range of repeat analyses.

Figure 2

Binding of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and Vascular endothelial growth factor (VEGF) as detected by co-IP. Lane 1: Recombinant ADAMTS-4 and VEGF were mixed, and antibody against VEGF and protein A/G beads were added to the protein mixture to precipitate VEGF. Lane 2: As in lane 1, except an isotype control antibody was used. Lane 3: As in lane 1, but antibody was omitted. The densitometric result (OD/mm²) is shown under each band. Detection in all lanes was with an anti-His tag to identify the His-tagged ADAMTS-4.

Figure 3

Effects of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 and ADAMTS-4 on VEGFR2 phosphorylation. (A) VEGFR2 phosphorylation was reduced by ADAMTS-4 in a dose-dependent manner. The amount of phosphorylated VEGFR2 compared with total VEGFR2 was calculated and plotted against the concentration of ADAMTS-4. (B) A representative Western blot of VEGFR2 phosphorylation in the presence or absence of Vascular endothelial growth factor (VEGF) with or without ADAMTS-4 and -1. Lane 1: control cells (starving medium only); lane 2: 30 nM ADAMTS-1; lane 3: 30 nM ADAMTS-4; lane 4: 20 ng/ml VEGF; lane 5: ADAMTS-1 + VEGF; lane 6: ADAMTS-4 + VEGF; and lane 7: recombinant VEGFR2 control. (C) Densitometric analysis normalized to VEGF-treated cells. Data are mean ± SEM of five repeats. *P = 0.004.

Figure 4

Effects of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 and ADAMTS-4 on human dermal microvescular endothelial cells (HuDMEC) differentiation on Matrigel in vitro. (A) Representative images of Matrigel assay; (a) control cells; (b) 20 ng/ml Vascular endothelial growth factor (VEGF); (c) ADAMTS-1 + VEGF; (d) ADAMTS-4 + VEGF. (B) The relative numbers of cord-like structures per well are presented as mean ± SEM of the data normalized to the relevant control. *P < 0.05 with respect to the relevant control. N = 3.

Figure 5

Effects of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 and ADAMTS-4 on human dermal microvescular endothelial cells (HuDMEC) migration. (A) A set of representative images of scratch closure at time 0 (left) and 18 h later (right) in the absence of added Vascular endothelial growth factor (VEGF) and ADAMTS-4 (control, top), in the presence of added VEGF (middle) and in the presence of both added VEGF and ADAMTS-4 (bottom). The two migrating fronts of cells are highlighted with dotted lines. (B) The degree of scratch closure normalized to control or VEGF treatment, respectively. N = 4. *P < 0.05 with respect to relevant control.

Figure 6

The structure of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 and ADAMTS-4. The structures of full-length ADAMTS-1 and -4 and recombinant (rh)ADAMTS-1 and -4 are shown. Names of each domain are also identified.