Aitana Braza-Boïls

Background Acute thoracic aortic dissections and ruptures, the main life-threatening complications of the corresponding aneurysms, are an important cause of sudden cardiac death. Despite the usefulness of the molecular diagnosis of these conditions in the clinical setting, the corresponding forensic field remains largely unexplored. The main goal of this study was to explore and validate a new massive parallel sequencing candidate gene​ assay as a diagnostic tool for acute thoracic aortic dissection autopsy cases. Materials and methods Massive parallel sequencing of 22 thoracic aortic disease candidate genes performed in 17 cases of thoracic aortic dissection using AmpliSeq and Ion Proton technologies. Genetic variants were filtered by location, type, and frequency at the Exome Aggregation Consortium and an internal database and further classified based on the American College of Medical Genetics and Genomics (ACMG) recommendations published in 2015. All prioritized results were confirmed by traditional sequencing. Results From the total of 10 potentially pathogenic genetic variants identified in 7 out of the 17 initial samples, 2 of them were further classified as pathogenic, 2 as likely pathogenic, 1 as possibly benign, and the remaining 5 as variants of uncertain significance, reaching a molecular autopsy yield of 23%, approximately. Conclusions This massive parallel sequencing candidate gene approach proved useful for the molecular autopsy of aortic dissection sudden cardiac death cases and should therefore be progressively incorporated into the forensic field, being especially beneficial for the anticipated diagnosis and risk stratification of any other family member at risk of developing the same condition.

Background & Aims: Non‐alcoholic fatty liver disease (NAFLD) appears to be a new risk factor for the development of coronary artery disease (CAD). Members of a class of non‐coding RNAs, termed microRNAs (miRNAs), have been identified as post‐transcriptional regulators of cholesterol homoeostasis and can contribute to the development of NAFLD. The aims of this study were to (i) to assess the relationship between NAFLD and sudden cardiac death (SCD) from severe CAD in forensic autopsies and (ii) to quantify several hepatic miRNAs previously associated with lipid metabolism and NAFLD to correlate their expression with the presence of NAFLD, CAD, obesity parameters and postmortem lipid profile.Methods: A total of 133 cases of autopsies with SCD and established CAD (patient group, CAD‐SCD) and 106 cases of non‐CAD sudden death (control group, non‐CAD‐SD) were included. miRNAs were quantified in frozen liver tissues.Results: Males predominated in both groups. Patients more frequently exhibited NAFLD and necroinflammatory steatohepatitis (NASH) than controls (62% vs 26%, P = 0.001 and 42% vs 26%, P = 0.001 respectively). In both groups, the presence of NAFLD correlated with body mass index and abdominal circumference (P < 0.05). An increase in miR‐34a‐5p and a decrease in miR‐122‐5p and ‐29c‐3p in patients with NASH vs controls without NAFLD were observed (P < 0.05). Finally, significant correlations between miR‐122‐5p and unfavourable lipid profile and also hs‐CRP and miR‐34a‐5p were noted.Conclusions: CAD is associated with NAFLD and NASH. The hepatic miRNAs studied appear to be associated with NAFLD severity and may promote CAD through lipid metabolism alteration and/or promotion of the systemic inflammation.

MicroRNAs (miRNAs) are small non-coding RNAs (18–22 nt) that function as modulators of gene expression. Since their discovery in 1993 in C. elegans, our knowledge about their biogenesis, function, and mechanism of action has increased enormously, especially in recent years, with the development of deep-sequencing technologies. New biogenesis pathways and sources of miRNAs are changing our concept about these molecules. The study of the miRNA contribution to pathological states is a field of great interest in research. Different groups have reported the implication of miRNAs in pathologies such as cancer, diabetes, cardiovascular, and gynecological diseases. It is also well-known that miRNAs are present in biofluids (plasma, serum, urine, semen, and menstrual blood) and have been proposed as ideal candidates as disease biomarkers. The goal of this review is to highlight the current knowledge in the field of miRNAs with a special emphasis to their role in endometriosis and the newest investigations addressing the use of miRNAs as biomarkers for this gynecological disease.

Could peritoneal fluid (PF) from patients with endometriosis alter the microRNA (miRNA) expression profile in endometrial and endometriotic cells from patients? PF from patients with endometriosis modifies the miRNA expression profile in endometrial cells from patients. Angiogenesis is a pivotal system in the development of endometriosis, and dysregulated miRNA expression in this disease has been reported. However, to our knowledge, the effect of PF from patients on the miRNA expression profile of patient endometrial cells has not been reported. Moreover, an effect of three miRNAs (miR-16-5p, miR-29c-3p and miR-424-5p) on the regulation of vascular endothelial growth factor (VEGF)-A mRNA translation in endometrial cells from patients with endometriosis has not been demonstrated. Primary cultures of stromal cells from endometrium from 8 control women (control cells) and 11 patients with endometriosis (eutopic cells) and ovarian endometriomas (ectopic cells) were treated with PF from control women (CPF) and patients (EPF) or not treated (0PF) in order to evaluate the effect of PF on miRNA expression in these cells. MiRNA expression arrays (Affymetrix platform) were prepared from cells (control, eutopic, ectopic) treated with CPF, EPF or 0PF. Results from arrays were validated by quantitative reverse transcription–polymerase chain reaction in cultures from 8 control endometrium, 11 eutopic endometrium and 11 ovarian endometriomas. Functional experiments were performed in primary cell cultures using mimics for miRNAs miR-16-5p, miR-29c-3p and miR-424-5p to assess their effect as VEGF-A expression regulators. To confirm a repressive action of miR-29c-3p through forming miRNA:VEGFA duplexes, we performed luciferase expression assays. EPF modified the miRNA expression profile in eutopic cells. A total of 267 miRNAs were modified in response to EPF compared with 0PF in eutopic cells. Nine miRNAs (miR-16-5p, miR-21-5p, miR-29c-3p, miR-106b-5p, miR-130a-5p, miR-149-5p, miR-185-5p, miR-195-5p, miR-424-5p) that were differently expressed in response to EPF, and which were potential targets involved in angiogenesis, proteolysis or endometriosis, were validated in further experiments (control = 8, eutopic = 11, ectopic = 11). Except for miR-149-5p, all validated miRNAs showed significantly lower levels (miR-16-5p, miR-106b-5p, miR-130a-5p; miR-195-5p and miR-424-5p, P < 0.05; miR-21-5p, miR-29c-3p and miR-185-5p, P < 0.01) after EPF treatment in primary cell cultures from eutopic endometrium from patients in comparison with 0PF. Transfection of stromal cells with mimics of miRNAs miR-16-5p, miR-29c-3p and miR-424-5p showed a significant down-regulation of VEGF-A protein expression. However, VEGFA mRNA expression after mimic transfection was not significantly modified, indicating the miRNAs inhibited VEGF-A mRNA translation rather than degrading VEGFA mRNA. Luciferase experiments also corroborated VEGF-A as a target gene of miR-29c-3p. The study was performed in an in vitro model of endometriosis using stromal cells. This model is just a representation to try to elucidate the molecular mechanisms involved in the development of endometriosis. Further studies to identify the pathways involved in this miRNA expression modification in response to PF from patients are needed. This is the first study describing a modified miRNA expression profile in eutopic cells from patients in response to PF from patients. These promising results improve the body of knowledge on endometriosis pathogenesis and could open up new therapeutic strategies for the treatment of endometriosis through the use of miRNAs. This work was supported by research grants by ISCIII and FEDER (PI11/00091, PI11/00566, PI14/01309, PI14/00253 and FI12/00012), RIC (RD12/0042/0029 and RD12/0042/0050), IIS La Fe 2011-211, Prometeo 2011/027 and Contrato Sara Borrell CD13/0005. There are no conflicts of interest to declare.

Could an aberrant microRNA (miRNA) expression profile be responsible for the changes in the angiogenic and fibrinolytic states observed in endometriotic lesions? This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient and their correlation with the most important angiogenic and fibrinolytic factors. An important role for dysregulated miRNA expression in the pathogenesis of endometriosis is well documented. However, to the best of our knowledge, there are no reports of the relationship between angiogenic and fibrinolytic factors and miRNAs when endometrial tissue and different types of endometriotic lesions from the same patient are compared. Case–control study that involved 51 women with endometriosis and 32 women without the disease (controls). The miRNA expression profiles were determined using the GeneChip miRNA 2.0 Affymetrix array platform, and the results were analysed using Partek Genomic Suite software. To validate the obtained results, 12 miRNAs differentially expressed were quantified by using miRCURY LNA™ Universal RT microRNA PCR. Levels of vascular endothelial growth factor (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) proteins were quantified by ELISA. Patient endometrial tissue showed significantly lower levels of miR-202-3p, miR-424-5p, miR-449b-3p and miR-556-3p, and higher levels of VEGF-A and uPA than healthy (control) endometrium. However, tissue affected by ovarian endometrioma showed significantly lower expression of miR-449b-3p than endometrium from both controls and patients, and higher levels of PAI-1 and the angiogenic inhibitor TSP-1. A significant inverse correlation between miR-424-5p and VEGF-A protein levels was observed in patient endometrium, and an inverse correlation between miR-449b-3p and TSP-1 protein levels was observed in ovarian endometrioma. Peritoneal implants had significantly higher levels of VEGF-A than ovarian endometrioma samples. Functional studies are needed to confirm the specific targets of the miRNAs differently expressed. Differences in miRNA levels could modulate the expression of VEGF-A and TSP-1, which may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in eutopic endometrium from patients might facilitate the implantation of endometrial cells at ectopic sites. This work was supported by research grants from ISCIII-FEDER (PI11/0091, Red RIC RD12/0042/0029), Consellería de Educación-Generalitat Valenciana (PROMETEO/2011/027), Beca de Investigación Fundación Dexeus para la Salud de la Mujer (2011/0469), and by Fundación Investigación Hospital La Fe (2011/211). A.B-B. has a Contrato Posdoctoral de Perfeccionamiento Sara Borrell-ISCIII (CD13/00005). J.M-A. has a predoctoral grant PFIS-ISCIII (FI12/00012). The authors have no conflicts of interest to declare.

Thrombosis and Haemostasis publishes original basic research and clinical studies in vascular biology and medicine, serving as an international platform for dissemination and debate of scientific information at the cutting edge.

Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis. Endometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222) by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively. Control and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion, this “in vitro” study indicates that peritoneal fluid from women with endometriosis modulates the expression of miRNAs that could contribute to the angiogenic and proteolytic disequilibrium observed in this disease.

Which is the role of microRNAs (miRNAs) related to several angiogenesis regulators such as VEGF-A (Vascular endothelial growth factor-A) and TSP-1 (Thrombospondin-1) in endometrial cancer? A dysregulated expression of miRNAs related to angiogenesis and an increase in the VEGF-A levels were observed in endometrial cancer in comparison with control. The different expression of miRNAs could modulate the expression of angiogenic and antiangiogenic factors, which may play an important role in the pathogenesis of endometrial cancer. Dysregulated miRNA expression has been previously evaluated in endometrial adenocarcinoma. To the best of our knowledge, there are no studies on the relationship between angiogenic factors and miRNAs in endometrial cancer. Case–control study: 41 patients with histologically proven endometrioid endometrial cancer and 56 women without endometrial cancer. RNAs isolated from tissue samples were analyzed using the GeneChip miRNA 2.0 Array platform (Affymetrix). TaqMan qRT–PCR was used to assess the expression of the selected miRNAs related to angiogenesis (miR-15b, -16, -17-5p, -20a, -21, -125a, -200b, -210, -214*, -221, -222 and -424), and VEGF-A and TSP-1 mRNAs were assessed by qRT–PCR using SYBR Green. Protein levels were quantified by ELISAs. Compared with the miRNAs in the control endometrium, eight miRNAs (miR-15b, -17-5p, -20a, -125a, -214*, -221, -222 and -424) were significantly down-regulated and two miRNAs (miR-200b and -210) were significantly up-regulated in the cancerous endometrium. A significant increase in VEGF-A mRNA and protein expression and in TSP-1 protein levels (P <0.01) was observed in endometrial cancer. Moreover, significant inverse correlations between VEGF-A protein levels and miR-20a, -125a, -214*, -221, -222 and -424 were detected. In contrast, a positive correlation was observed between VEGF-A and miR-200b and -210. Furthermore, stage IB endometrial cancer was associated with a higher VEGF-A protein/mRNA ratio and lower miR-214*, -221 and -222 expression in comparison with stage IA. Future functional studies (e.g. miRNA inhibition or ectopic overexpression) in cell culture models are needed to confirm the VEGF targeting by the miRNAs found in the present study. The findings of the present study have potential implications for diagnostics and therapeutics of endometrial carcinoma. This work was supported by research grants from the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, PI080185, PI0110091) and Red RECAVA (RD06/0014/0004), by Consellería de Sanidad (AP-141/11) and Consellería de Educación (PROMETEO/2011/027), Generalitat Valenciana, by Beca Fibrinolisis 2009 and Becario 2010, 2011 from Fundación Española de Trombosis y Hemostasia and by the Fundación Investigación Hospital La Fe, Spain. None of the authors have any conflicts of interest.