ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease 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 endothelial cell (Lee et al. Microvascular Res 2012; 84:109) and glioblastoma cell (Lee et al. Blood 2013; 122:#2306) migration and proliferation. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA. 30pmols of ADAMTS-13 siRNA in a six-well plate inhibited HUVEC ADAMTS-13 expression levels by 90% after 24hr incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. The knockdown effect was decreased over time: 72hr after the initial knockdown, ADAMTS-13 expression was reduced by only 50%. The effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions were studied. Transfection of HUVEC with 10pmols of ADAMTS-13 siRNA in a 24-well plate resulted in a 21% and 22% decrease in proliferation after 24hr and 48hr incubation, respectively. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24hr after transfection with control siRNA, there was robust cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased expression of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. 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 in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through the AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.
Citation Format: Huiyuan Tang, Manfai Lee, Eun Ho Kim, Daniel Bishop, George M. Rodgers. Endogenous ADAMTS-13 regulates angiogenesis in cultured human endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1816. doi:10.1158/1538-7445.AM2017-1816