siRNA-knockdown of ADAMTS-13 modulates endothelial cell angiogenesis.
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 this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA and the effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions. 15nM of ADAMTS-13 siRNA reduced HUVEC ADAMTS-13 protein levels by 90% after 24h incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Furthermore, this transfection did not affect the HUVEC endogenous protein level of ADAMTS-1, a related family member of ADAMTS-13 indicating the specificity of the siRNA. Transfection of HUVEC with 15nM of ADAMTS-13 siRNA resulted in a 21% decrease in proliferation after 24h incubation. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24h after transfection with control siRNA, there was increased cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased protein levels 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%. The protein level of vascular endothelial growth factor (VEGF), a well-known regulator of angiogenesis, was significantly decreased by 45% upon knockdown of ADAMTS-13. Moreover, activity of the AKT pathway, one of the VEGF angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through VEGF and AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.
['Tang|Huiyuan|H|', 'Lee|Manfai|M|', 'Kim|Eun Ho|EH|', 'Bishop|Daniel|D|', 'Rodgers|George M|GM|']
D000071120:ADAMTS13 Protein D002465:Cell Movement D049109:Cell Proliferation D002478:Cells, Cultured D015536:Down-Regulation D061307:Human Umbilical Vein Endothelial Cells D006801:Humans D018919:Neovascularization, Physiologic D051057:Proto-Oncogene Proteins c-akt D034622:RNA Interference D034741:RNA, Small Interfering D015398:Signal Transduction D013997:Time Factors D014162:Transfection D042461:Vascular Endothelial Growth Factor A D014841:von Willebrand Factor
ADAMTS-13 Endothelial cell Angiogenesis Von Willebrand factor siRNA Vascular endothelial growth factor