IMEX   05356
INSTITUTO DE MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Modification of ADAMTS13 isoforms by estradiol and histamine treatment
Autor/es:
ALBERTO MF; WOODS AI; PAIVA J; POWASNIAK Y; DOS SANTOS C; KEMPFER AC; SANCHEZ-LUCEROS A; LAZZARI MA
Lugar:
Montpeliere
Reunión:
Congreso; Congress of ISTH-SSC; 2016
Resumen:
Modificationof ADAMTS13 isoforms by estradiol and histamine treatment Juvenal Paiva, Ana Catalina Kempfer,María Fabiana Alberto, Yanina Powazniak, Analía Sánchez-Luceros, Adriana InésWoods, Célia Dos Santos,  María A. LazzariIntroduction:ADAMTS13 is a physiological von Willebrand factor (VWF) cleavingprotease; it is synthesized by hepatic stellate cells, megakaryocytes, endothelial cells and other celllines. The expression of mRNA of ADAMTS13 in several organs,peripheral blood leukocytes and human platelets was demonstrated. In HEP3B was found ADAMTS13 isoforms. We described previously mRNA of isoform 1 (Iso1) and isoform 2 (Iso2)in HUVEC, platelets and two breast cancer lines (MDA-MB23, MCF7). IntracellularmRNA levels of ADAMTS13 were increased significantly in HUVEC after treatmentswith estradiol (E2) and Histamine (H).Aim: Determinethe effect of E2 and H on levels of ADAMTS13 Iso1 and Iso2 in HUVEC, MDA-MB23 and MCF7.Methods:Reagents:1nM E2, 100μM H, 1mM H inhibitors (I) (HRH1: Ketotifen, HRH2:Cimetidine, HRH3: Clobenpropit).Treatments:a)vehicle, b)H, c)E2, d)H+E2, e)H+I, f)E2+I, g)H+E2+I, h)I.mRNA wasextracted from cells using TRIzol reagent followed by precipitation withphenol/chloroform. The integrity was verified by 260/280 optical density ratio.The mRNA was reverse transcribed into cDNA using random primers. We designedprimers to amplify the sequence of ADAMTS13 to differentiate Iso1 (610 bp) andIso2 (442bp) at the same time. β-actin was used as control. The PCR productswere analyzed on agarose gel containing SYBR Safe. Results:The HUVECstreated with b) showed normal level of Iso1 and with c), d), e), f), h) slightdecrease of Iso1. Treatments e), f), h), also decreased Iso2; on the otherhand, the HUVECs treated with b), c), d), g) promoted increase of the Iso2. Thesamples were compared with a)In MDA-MB23and MCF7 (with initial high levels of Iso1 and Iso2 compared with HUVEC) we didnot observe important differences between levels of Iso1 and Iso2 treated or notConclusion:Our resultsshow that treatment with b) and c) influence directly or indirectly in splicingmechanism modifying the levels of Iso1 and Iso2 in HUVEC, but not in MDA-MB23and MCF7 at the concentration used.