Endogenous Adamts-13 Regulates Angiogenesis in Cultured Human Endothelial Cells

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease enzyme that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis; for example; addition of exogenous ADAMTS-13 enhances angiogenesis in cell migration and proliferation assays. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cell (HUVEC) via siRNA. 100pmols of ADAMTS-13 siRNA inhibited HUVEC ADAMTS-13 expression levels by 60% after 7hr incubation, whereas control siRNA did not reduce endogenous ADAMTS-13. The effects of reduced endogenous ADAMTS-13 on HUVEC function were studied in proliferation and scratch wound assays. Transfection of HUVEC with 100pmols of ADAMTS-13 siRNA resulted in a 47% decrease in proliferation after 19hr incubation. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also studied. 9hr after transfection with control siRNA, cells initiated migration across the scratch wound. This migration did not occur in ADAMTS-13 knockdown cells. The activity of the AKT pathway, one of the angiogenesis downstream signaling pathways was down regulated by ADAMTS-13 siRNA while ERK, a component of the MAP kinase pathway was not affected upon knockdown of ADAMTS-13. These data indicate that one role of endogenous ADAMTS-13 in HUVEC is regulation of angiogenesis, mediated through the AKT signaling pathway. Overall our data suggest an additional model of endogenousADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.