Abstract 1145
Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. To assess the possible role of ADAMTS13 in angiogenesis, cell proliferation and migration of human umbilical vein endothelial cells (HUVEC) were studied in vitro. ADAMTS13 was found to be a highly potent chemoattractant, and additionally was capable of neutralizing VEGF activity in two angiogenesis assays-cell proliferation and cell migration. In the Boyden chamber cell migration assay, treatment of endothelial cells with exogenous recombinant ADAMTS13 promoted cell migration in a dose-dependent manner, with 1 ng/mL increasing cell migration across a gelatinized polycarbonate membrane by 14-fold. In the same model, 5 ng/mL VEGF
165 (molar ratio of ADAMTS13:VEGF
165 = 1/19) only increased cell migration by 7 fold. A steady decrease in endothelial cell migration was observed when the concentration of ADAMTS13 exceeded 1 ng/mL (
Figure 1). Coincubation of 30 ng/mL ADAMTS13 with 6.16 ng/mL VEGF
165 (molar ratio of ADAMTS13/VEGF
165 = 1.3/1) inhibited endothelial cell migration by 45% compared to VEGF alone (
Figure 2). A second model using an in vitro scratch-wound assay confirmed the Boyden chamber data.
Substitution of ADAMTS13 with ADAM17, an analog of ADAMTS13 without the thrombospondin domain reversed the inhibition of VEGF-mediated cell migration, suggesting that the thrombospondin domain of ADAMTS13 is responsible for the inhibitory interaction with VEGF165. This finding was in agreement with our previously published co-immunoprecipitation assay data (Blood 2010, 116, 4307). Similar patterns of inhibition were observed with VEGF121 and VEGF189, indicating that other isoforms of VEGF may interact with the TSP domain of ADAMTS13. Using a manual proliferation assay method, HUVEC treated with 30 ng/mL ADAMTS13 and 6.16 ng/mL VEGF165 proliferated 40% slower than the control treated with VEGF alone. Combined with our findings on the inhibition of endothelial cell-tube formation in a Matrigel assay with ADAMTS13 and VEGF165 previously reported, our cumulative data suggest that 1) ADAMTS13 promotes angiogenesis by increasing cell migration and 2) ADAMTS13 can modulate VEGF-mediated angiogenic activities.
Disclosures:
No relevant conflicts of interest to declare.
