VWF and ADAMTS13 behavior in estradiol-treated HUVEC.
OBJECTIVE
In this study, the role of 17β-estradiol (E2) in the regulation of von Willebrand factor (VWF) and ADAMTS13 synthesis, storage, and secretion was investigated in cultured human umbilical vein endothelial cells (HUVEC).


METHODS
HUVEC were grown to 80-90% confluence and replaced with fresh medium containing E2 (1 nm) or vehicle for 24 h, after which the supernatant medium and cell lysates were collected to measure VWF and ADAMTS13. VWF was evaluated by VWF:Ag and multimeric analysis. ADAMTS13 was evaluated by SDS-PAGE. VWF and ADAMTS13 mRNA were quantified by real-time PCR after E2 or vehicle exposure for 18 h. A functional effect of ADAMTS13 on HUVEC VWF protein synthesis was further evaluated using a short hairpin RNA (shRNA) to knockdown the expression of endogenous ADAMTS13.


RESULTS
E2 did not increase the release or intracellular VWF levels in HUVEC. However, E2 increased the production of intracellular ADAMTS13, although there was no evidence of significant effects of their release into culture medium. Incubation of HUVEC with E2 resulted in a significantly increased expression of VWF and ADAMTS13 mRNA. ADAMTS13 gene inactivation upregulates release and intracellular VWF levels in E2-treated HUVEC.


CONCLUSIONS
The results demonstrated that E2 may play a role in the regulation of VWF and ADAMTS13 gene expression and in its production in human endothelial cells. The mechanism of the protective effects of E2 on the cardiovascular system could be explained by the intracellular regulation of VWF produced by ADAMTS13.
Powazniak
Yanina
Y
FONCyT/CONICET, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Buenos Aires, Argentina. yaninapowazniak@gmail.com
Kempfer
Ana Catalina
AC
Pereyra
Julio César Calderazzo
JC
Palomino
Juvenal Paiva
JP
Lazzari
Maria Angela
MA
D051722:ADAM Proteins / Q000096:biosynthesis*; D000071120:ADAMTS13 Protein; D002478:Cells, Cultured; D003470:Culture Media; D042783:Endothelial Cells / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014841:von Willebrand Factor / Q000096:biosynthesis*
D003470:Culture Media; D012333:RNA, Messenger; D014841:von Willebrand Factor; D004958:Estradiol; D012313:RNA; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human