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C0418: miRNAs 16, 424 and 29C Regulate VEGF-A Expression in Endometrial Stromal Cells from Women with Endometriosis
ORAL COMMUNICATIONS / Thrombosis Research 133S3 (2014) S1–S34
C0418 MIRNAS 16, 424 AND 29C REGULATE VEGF-A EXPRESSION IN ENDOMETRIAL STROMAL CELLS FROM WOMEN WITH ENDOMETRIOSIS A. Braza-Boils1 , S. Salloum-Asfar2 , J. Mar´ı-Alexandre1 , A. Belen ´ 2 Arroyo2 , R. Gonzalez-Conejero ´ , V. Vicente2 , A. Estelles ´ 1, J. Gilabert-Estelles ´ 3 , C. Mart´ınez2 . 1 Grupo Hemostasia, Trombosis, Aterosclerosis y Biolog´ıa Vascular. Instituto de Investigaci´ on Sanitaria La Fe, Valencia, Spain; 2 Centro Regional de Hemodonaci´ on. Servicio de Hematolog´ıa y Oncolog´ıa M´edica. Hospital Universitario Morales ´ rea Maternoinfantil. Meseguer. Universidad de Murcia, IMIB, Spain; 3 A Hospital General Universitario, Valencia, Spain Background: Endometriosis is one of the most prevalent gynecological diseases that is characterized by the presence of endometrial tissue outside the uterus. These ectopic tissues are able to survive, to invade, and to proliferate into the peritoneal cavity. Moreover, previous results from our group indicated pathological alterations in angiogenesis and fibrinolysis, not only in the endometrial tissue but also in the peritoneal fluid from women with endometriosis. MicroRNAs (miRNAs) are non-coding RNAs that may regulate angiogenesis through protein translation modulation. In a recent study, we described a dysregulated miRNA expression profile in endometriosis, including miR-16, 29c and 424. In silico studies showed that these miRNAs may regulate VEGF-A expression. The aim of this study was to investigate regulation of VEGF-A by miR-16, 29c and 424 in primary cultures of endometrial stromal cells from women with and without endometriosis. Methods: Stromal primary cell cultures from endometrium from patients (n = 4), endometrium from control women (n = 4), and EAHY 926 cell line were employed to perform functional experiments. Transfections with miRNA were performed in triplicate using precursors (mimics) of miR-16, 29c, 424 and a scrambled mimic as negative control. Total RNA was extracted from cells and VEGF-A mRNA and protein levels were quantified by qRTPCR and western blot, respectively. Results: In EAHY 926, transfection of miR-16, 29c and 424 mimics induced a reduction of VEGF-A expression of 76%, 75%, and 82%, respectively. When the same transfections were performed in primary cell cultures from controls’ and patients’ endometrium, VEGF-A expression was reduced by 87%, 90%, and 90%, respectively, in endometrial cells from women without the disease, and 96%, 79%, and 78% in patients’ endometrial cells, respectively. In all cell types, VEGF-A mRNA expression after mimic transfection was not significantly modified. Conclusions: Functional studies employing mimics for miR-16, 29c and 424 suggested that these miRNAs regulate VEGF-A translation not only in EAHY 926 cell line but also in stromal primary cultures from endometrium from patients with endometriosis and control women. These promising results open up new therapeutic strategies for the treatment of endometriosis through the use of miRNAs. ISCIII and FEDER (PI011/00091, PI11/00566 and FI12/00012), RIC (RD12/0042/0029 and RD12/0042/0050), IIS La Fe 2011–211, Prometeo 2011/027, Fundacion ´ Dexeus (2011/0469), Contrato Sara Borrell CD13/0005.
S13
C0420 MICRORNA EXPRESSION PROFILE IN EPICARDIAL FAT IN SUDDEN CARDIAC DEATH FROM CORONARY ARTERY DISEASE A. Braza-Boils1 , J. Mar´ı-Alexandre1 , P. Molina2 , D. Domingo1 , ´ J. Sanz3 , J. Sancho2 , Y. Abellan ´ 2 , N. Castillo2 , M. Angel Arnau3 , 2 3 1 3 1 J. Giner , A. Montero , A. Estelles ´ , E. Zorio . Grupo de Hemostasia, Trombosis, Aterosclerosis y Biolog´ıa Vascular. Instituto de Investigaci´ on Sanitaria La Fe, Valencia, Spain; 2 Instituto de Medicina Legal, Valencia, Spain; 3 Servicio Cardiolog´ıa. Hospital Universitario y Polit´ecnico La Fe, Valencia, Spain Background: Coronary artery disease (CAD) is the leading cause of sudden cardiac death (SCD). The epicardial fat (EF) has been recently suggested to be a new cardiovascular risk factor. microRNAs (miRNAs) have been related to dyslipidemia and adipogenesis. To our knowledge no studies evaluating role of miRNAs in EF have been published. Aim: To identify a miRNA expression profile in EF associated to both generation and plaque destabilization responsible for ischemic SCD. Methods: 41 CAD-SCD and 15 non-CAD-sudden death (SD) cases who underwent forensic autopsy were enroled. miRNA arrays (Affymetrix platform) were performed in EF samples contiguous to coronary arteries with plaque in CAD-SCD patients and without plaque in non-CAD-SD subjects. To validate results from arrays, 5 miRNAs were quantified employing miRCURY LNA Universal RT microRNA PCR (EXIQON) in a larger cohort of samples including coronary arteries without plaque in CAD-SCD. Results: miRNA expression profile of EF from coronary arteries with plaque in patients and without plaque in non-CAD-SD clustered separately in the Principal Component Analysis. 35 mature miRNAs were differentially expressed (p < 0.05, 1.5 fold change) (17 upregulated and 18 down-regulated) in EF from arteries with plaque in CAD-SCD patients vs EF from arteries without plaque in nonCAD-SD individuals. Among them, in silico studies enabled us to choose 5 miRNAS with targets related to atherosclerosis (miR34a-3p, -34a-5p, -124-3p, -125a-5p and 628-5p). EF from coronary arteries with plaque in CAD-SCD patients presented significantly higher levels of miR-34a-3p and -5p (p < 0.05) than in those without plaque in non-CAD-SD individuals. miR-34a-3p and miR-34a-5p (p < 0.05) were also increased in EF from coronary arteries without plaque in CAD-SCD in comparison with those in non-CAD-SD. Moreover, miR-628-5p was significantly downregulated in paired EF of coronary arteries with plaque vs without plaque in CAD-SCD group (p < 0.05). Conclusions: This is the first study to describe a characteristic EF miRNA expression profile in EF in victims of CAD-SCD. An increased EF miR-34a levels seem to promote adjacent coronary atherosclerosis. Further studies are necessary to elucidate the role of miR-34a in CAD-SCD. (PI011/00091, IIS La Fe 2011–211, FI12/00012, RD12/0042/0029, Prometeo 2011/027, Premio Lopez ´ Borrasca-SETH, Contrato Sara Borrell CD13/0005, AstraZeneca). C0423 MICRORNAS IN SUDDEN CARDIAC DEATH FROM CORONARY ARTERY DISEASE. ITS RELATIONSHIP WITH DYSLIPIDEMIA AND NON-ALCOHOLIC FATTY LIVER DISEASE J. Mar´ı-Alexandre1 , A. Braza-Boils1 , P. Molina2 , D. Domingo1 , 4 ´ Y. Abellan ´ 2 , J. Sancho2 , P. Hevia2 , M. Angel Arnau3 , J. Gomez ´ , J. Giner2 , A. Salvador3 , A. Estelles ´ 1 , E. Zorio3 . 1 Grupo de Hemostasia, Trombosis, Aterosclerosis y Biolog´ıa Vascular. Instituto de Investigaci´ on Sanitaria La Fe, Valencia, Spain; 2 Instituto de Medicina Legal, Valencia, Spain; 3 Servicio Cardiolog´ıa. Hospital Universitario y Polit´ecnico La Fe, Valencia, Spain; 4 Hospital Universitario Dr. Peset, Valencia, Spain Background: Coronary artery disease (CAD) is the leading cause of sudden cardiac death (SCD). It has recently been pointed to
C0418 MIRNAS 16, 424 AND 29C REGULATE VEGF-A EXPRESSION IN ENDOMETRIAL STROMAL CELLS FROM WOMEN WITH ENDOMETRIOSIS A. Braza-Boils1 , S. Salloum-Asfar2 , J. Mar´ı-Alexandre1 , A. Belen ´ 2 Arroyo2 , R. Gonzalez-Conejero ´ , V. Vicente2 , A. Estelles ´ 1, J. Gilabert-Estelles ´ 3 , C. Mart´ınez2 . 1 Grupo Hemostasia, Trombosis, Aterosclerosis y Biolog´ıa Vascular. Instituto de Investigaci´ on Sanitaria La Fe, Valencia, Spain; 2 Centro Regional de Hemodonaci´ on. Servicio de Hematolog´ıa y Oncolog´ıa M´edica. Hospital Universitario Morales ´ rea Maternoinfantil. Meseguer. Universidad de Murcia, IMIB, Spain; 3 A Hospital General Universitario, Valencia, Spain Background: Endometriosis is one of the most prevalent gynecological diseases that is characterized by the presence of endometrial tissue outside the uterus. These ectopic tissues are able to survive, to invade, and to proliferate into the peritoneal cavity. Moreover, previous results from our group indicated pathological alterations in angiogenesis and fibrinolysis, not only in the endometrial tissue but also in the peritoneal fluid from women with endometriosis. MicroRNAs (miRNAs) are non-coding RNAs that may regulate angiogenesis through protein translation modulation. In a recent study, we described a dysregulated miRNA expression profile in endometriosis, including miR-16, 29c and 424. In silico studies showed that these miRNAs may regulate VEGF-A expression. The aim of this study was to investigate regulation of VEGF-A by miR-16, 29c and 424 in primary cultures of endometrial stromal cells from women with and without endometriosis. Methods: Stromal primary cell cultures from endometrium from patients (n = 4), endometrium from control women (n = 4), and EAHY 926 cell line were employed to perform functional experiments. Transfections with miRNA were performed in triplicate using precursors (mimics) of miR-16, 29c, 424 and a scrambled mimic as negative control. Total RNA was extracted from cells and VEGF-A mRNA and protein levels were quantified by qRTPCR and western blot, respectively. Results: In EAHY 926, transfection of miR-16, 29c and 424 mimics induced a reduction of VEGF-A expression of 76%, 75%, and 82%, respectively. When the same transfections were performed in primary cell cultures from controls’ and patients’ endometrium, VEGF-A expression was reduced by 87%, 90%, and 90%, respectively, in endometrial cells from women without the disease, and 96%, 79%, and 78% in patients’ endometrial cells, respectively. In all cell types, VEGF-A mRNA expression after mimic transfection was not significantly modified. Conclusions: Functional studies employing mimics for miR-16, 29c and 424 suggested that these miRNAs regulate VEGF-A translation not only in EAHY 926 cell line but also in stromal primary cultures from endometrium from patients with endometriosis and control women. These promising results open up new therapeutic strategies for the treatment of endometriosis through the use of miRNAs. ISCIII and FEDER (PI011/00091, PI11/00566 and FI12/00012), RIC (RD12/0042/0029 and RD12/0042/0050), IIS La Fe 2011–211, Prometeo 2011/027, Fundacion ´ Dexeus (2011/0469), Contrato Sara Borrell CD13/0005.
S13
C0420 MICRORNA EXPRESSION PROFILE IN EPICARDIAL FAT IN SUDDEN CARDIAC DEATH FROM CORONARY ARTERY DISEASE A. Braza-Boils1 , J. Mar´ı-Alexandre1 , P. Molina2 , D. Domingo1 , ´ J. Sanz3 , J. Sancho2 , Y. Abellan ´ 2 , N. Castillo2 , M. Angel Arnau3 , 2 3 1 3 1 J. Giner , A. Montero , A. Estelles ´ , E. Zorio . Grupo de Hemostasia, Trombosis, Aterosclerosis y Biolog´ıa Vascular. Instituto de Investigaci´ on Sanitaria La Fe, Valencia, Spain; 2 Instituto de Medicina Legal, Valencia, Spain; 3 Servicio Cardiolog´ıa. Hospital Universitario y Polit´ecnico La Fe, Valencia, Spain Background: Coronary artery disease (CAD) is the leading cause of sudden cardiac death (SCD). The epicardial fat (EF) has been recently suggested to be a new cardiovascular risk factor. microRNAs (miRNAs) have been related to dyslipidemia and adipogenesis. To our knowledge no studies evaluating role of miRNAs in EF have been published. Aim: To identify a miRNA expression profile in EF associated to both generation and plaque destabilization responsible for ischemic SCD. Methods: 41 CAD-SCD and 15 non-CAD-sudden death (SD) cases who underwent forensic autopsy were enroled. miRNA arrays (Affymetrix platform) were performed in EF samples contiguous to coronary arteries with plaque in CAD-SCD patients and without plaque in non-CAD-SD subjects. To validate results from arrays, 5 miRNAs were quantified employing miRCURY LNA Universal RT microRNA PCR (EXIQON) in a larger cohort of samples including coronary arteries without plaque in CAD-SCD. Results: miRNA expression profile of EF from coronary arteries with plaque in patients and without plaque in non-CAD-SD clustered separately in the Principal Component Analysis. 35 mature miRNAs were differentially expressed (p < 0.05, 1.5 fold change) (17 upregulated and 18 down-regulated) in EF from arteries with plaque in CAD-SCD patients vs EF from arteries without plaque in nonCAD-SD individuals. Among them, in silico studies enabled us to choose 5 miRNAS with targets related to atherosclerosis (miR34a-3p, -34a-5p, -124-3p, -125a-5p and 628-5p). EF from coronary arteries with plaque in CAD-SCD patients presented significantly higher levels of miR-34a-3p and -5p (p < 0.05) than in those without plaque in non-CAD-SD individuals. miR-34a-3p and miR-34a-5p (p < 0.05) were also increased in EF from coronary arteries without plaque in CAD-SCD in comparison with those in non-CAD-SD. Moreover, miR-628-5p was significantly downregulated in paired EF of coronary arteries with plaque vs without plaque in CAD-SCD group (p < 0.05). Conclusions: This is the first study to describe a characteristic EF miRNA expression profile in EF in victims of CAD-SCD. An increased EF miR-34a levels seem to promote adjacent coronary atherosclerosis. Further studies are necessary to elucidate the role of miR-34a in CAD-SCD. (PI011/00091, IIS La Fe 2011–211, FI12/00012, RD12/0042/0029, Prometeo 2011/027, Premio Lopez ´ Borrasca-SETH, Contrato Sara Borrell CD13/0005, AstraZeneca). C0423 MICRORNAS IN SUDDEN CARDIAC DEATH FROM CORONARY ARTERY DISEASE. ITS RELATIONSHIP WITH DYSLIPIDEMIA AND NON-ALCOHOLIC FATTY LIVER DISEASE J. Mar´ı-Alexandre1 , A. Braza-Boils1 , P. Molina2 , D. Domingo1 , 4 ´ Y. Abellan ´ 2 , J. Sancho2 , P. Hevia2 , M. Angel Arnau3 , J. Gomez ´ , J. Giner2 , A. Salvador3 , A. Estelles ´ 1 , E. Zorio3 . 1 Grupo de Hemostasia, Trombosis, Aterosclerosis y Biolog´ıa Vascular. Instituto de Investigaci´ on Sanitaria La Fe, Valencia, Spain; 2 Instituto de Medicina Legal, Valencia, Spain; 3 Servicio Cardiolog´ıa. Hospital Universitario y Polit´ecnico La Fe, Valencia, Spain; 4 Hospital Universitario Dr. Peset, Valencia, Spain Background: Coronary artery disease (CAD) is the leading cause of sudden cardiac death (SCD). It has recently been pointed to