Aitana Braza-Boïls

microRNAs expression in endometriosis and their relation to angiogenic factors

BACKGROUND Endometriosis is a common, multifactorial disease in which angiogenesis may be involved in the growth of endometrium outside the uterus. microRNAs (miRNAs) are 21-22 nucleotide non-coding RNAs that regulate gene expression and play fundamental roles in biological processes. The objective of this study was to analyze several miRNAs related to angiogenesis and the angiogenic factors, vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1), in endometriotic lesions (ovarian endometrioma, peritoneal lesion and rectovaginal nodule) and eutopic endometrium from women with endometriosis. METHODS TaqMan real-time PCR was used to assess the expression of the miRNAs (miR-15b, -16, -17-5p, -20a, -21, -125a, -221 and -222), while VEGF-A and TSP-1 mRNA were assessed by real-time PCR, with SYBR Green I and VEGF-A and TSP-1 protein levels were quantified by ELISA. Included in the study were 58 women with endometriosis and 38 control women. RESULTS In paired samples, ovarian endometrioma showed significantly lower VEGF-A mRNA (P = 0.02) and protein (P = 0.002) expression than eutopic endometrium and higher expression of miR-125a (P = 0.003) and miR-222 (P <0.001). However, ovarian endometrioma had significantly higher expression of the angiogenic inhibitor TSP-1 and lower expression of miR-17-5p than eutopic endometrium (P < 0.001). Moreover, a significant inverse correlations between miR-222 and VEGF-A protein levels (-0.267, P = 0.018) and between miR-17-5p and TSP-1 protein levels (-0.260, P=0.022) were observed. Peritoneal lesions showed a significant increase in VEGF-A in comparison with ovarian endometrioma (P < 0.01). CONCLUSIONS Expression levels of miRNAs related to angiogenesis were different in eutopic endometrium from that observed in ovarian endometrioma. This could influence the expression of angiogenic factors and play a role in the pathogenesis of endometriosis.

microRNAs related to angiogenesis are dysregulated in endometrioid endometrial cancer

STUDY QUESTION 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? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION Case-control study: 41 patients with histologically proven endometrioid endometrial cancer and 56 women without endometrial cancer. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS The findings of the present study have potential implications for diagnostics and therapeutics of endometrial carcinoma. STUDY FUNDING/COMPETING INTEREST(S) 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.

miRNAs Regulation and Its Role as Biomarkers in Endometriosis

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.

microRNAs and angiogenesis in endometriosis

miRNAs function as important regulators of a wide range of cellular processes, such as angiogenesis and fibrinolysis, by postranscriptional modulation of gene expression. We present a review on the role of miRNAs and angiogenesis in endometriosis. Endometriosis, defined as the implantation of endometrial tissue outside the uterine cavity, is one of the most frequent benign gynecological diseases and it has important consequences on the quality of life and fertility of patients. Similarly to tumor metastasis, the ectopic endometrium acquires the capability to adhere, proliferate and infiltrate the extracellular matrix. Endometriosis is a multifactorial and polygenic disease in which angiogenesis and proteolysis may be involved, and emerging data provide evidence that a dysregulation of miRNA expression may be implicated in these processes. The detection of circulating miRNAs in plasma and other body fluids and their relative stability has raised the possibility that they might serve as non-invasive biomarkers for the diagnosis of the disease. On the other hand, the development of therapies that might block the expression or mimic the functions of miRNAs could represent new therapeutic strategies for the treatment of endometriosis.

Deregulated hepatic microRNAs underlie the association between non-alcoholic fatty liver disease and coronary artery disease

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.

Plasminogen activator inhibitor-1 (PAI-1) 4 G/5 G polymorphism and endometrial cancer. Influence of PAI-1 polymorphism on tissue PAI-1 antigen and mRNA expression and tumor severity

Plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism may have significance for PAI-1 expression. High levels of PAI-1 in endometrial cancer patients are associated with a poor prognosis. The objective of this study was to evaluate the PAI-1 4G/5G polymorphism in women with and without endometrial cancer and to analyze the influence of this polymorphism on PAI-1 expression in endometrial tissue. In 423 women (212 patients with endometrial cancer and 211 controls) PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. Quantitative real-time RT-PCR assay was used to quantify PAI-1 mRNA and PAI-1 protein levels were quantified by ELISA in tissue extracts from 33 patients with endometrial cancer and from 70 endometrial tissues from control women. The frequency of PAI-1 4G/4G genotype (P=0.010) and the PAI-1 4G allele (P=0.009) was significantly higher in patients than in controls. The frequency of PAI-1 4G allele was significantly higher in patients with stage IB than in those with stage IA (P=0.03). Control women with the 4G/4G genotype had higher endometrial PAI-1 protein (P=0.018) and mRNA (P=0.004) levels than those with the 5G/5G genotype. A significant increase in PAI-1 protein and mRNA was observed in endometrial cancer tissue in comparison with the endometrial tissue from control women (P<0.01). In conclusion, frequencies of the PAI-1 4G allele and 4G/4G genotype were found significantly more often in women with endometrial cancer than in controls. PAI-1 levels in endometrial tissue seem to be associated with PAI-1 4G/5G polymorphism. These findings suggest that the PAI-1 4G/4G genotype may be associated with the risk of endometrial cancer in a Caucasian population. Further studies with a larger number of patients are needed to clarify the influence of this PAI-1 polymorphism in endometrial cancer.

Thickness and an Altered miRNA Expression in the Epicardial Adipose Tissue Is Associated With Coronary Heart Disease in Sudden Death Victims

INTRODUCTION AND OBJECTIVES An increased epicardial adipose tissue (EAT) thickness has become a new risk factor for coronary heart disease (CHD). We aimed to study the role of EAT dysfunction as a CHD marker by focusing on its thickness and microRNA (miRNA) expression profile, and the potential factors possibly influencing them. METHODS One hundred and fifty-five CHD sudden cardiac death victims and 84 non-CHD-sudden death controls were prospectively enrolled at autopsy. A representative subset underwent EAT thickness measurements and EAT miRNA expression profiling. RESULTS Epicardial adipose tissue thickness was increased and allowed an accurate diagnosis of patient status (among other measurements, EAT score area under the curve 0.718, P < .001). Epicardial adipose tissue from patients showed 14 up- and 14 down-regulated miRNAs and miR-34a-3p, -34a-5p, -124-3p, -125a-5p, 628-5p, -1303 and -4286 were validated by quantitative real-time polymerase chain reaction. Patients exhibited higher EAT levels of miR-34a-3p and -34a-5p than controls (with a positive trend considering EAT from coronaries without stenosis, with stable stenosis and complicated plaques) and correlated with age only in controls. The mild positive correlation between liver and EAT miR-34a-5p levels in patients (r = 0.295, P = .020) dramatically increased in EAT from complicated plaques (r = 0.799, P = .017). Similar correlations were observed for high-sensitivity-C-reactive protein levels and miR-34a-5p levels both in EAT and liver extracts. CONCLUSIONS Increased age-independent levels of miR-34a-3p and -34a-5p characterize the EAT miRNA expression profile of CHD regardless of EAT thickness, anthropometric parameters, and the presence of underlying atherosclerotic plaques.

Micro-RNA profile and proteins in peritoneal fluid from women with endometriosis: their relationship with sterility

OBJECTIVE To define the microRNA (miRNA) profile and its relationship with cytokines content in peritoneal fluid (PF) from endometriosis patients. DESIGN Case-control study. SETTING University hospital, research institute. PATIENT(S) One hundred twenty-six women with endometriosis (EPF) and 45 control women (CPF). MAIN OUTCOMES MEASURE(S) MiRNA arrays were prepared from six EPF and six CPF. Quantitative reverse transcription-polymerase chain reaction validation of nine selected miRNAs (miR-29c-3p, -106b-3p, -130a-3p, -150-5p, -185-5p, -195-5p, -451a, -486-5p, and -1343-5p) was performed. Vascular endothelial growth factor-A (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-3 (MMP3), tissue inhibitor of metalloproteinases type 1 (TIMP-1), interleukin (IL)-6, IL-8, IL-17A, macrophage inflammatory protein 1β (MIP1beta), platelet-derived growth factor α-polypeptide A, and regulated on activation, normal T cell expressed and secreted (RANTES) were quantified by ELISA and MILLIPLEX. RESULT(S) MiRNA arrays showed 126 miRNAs differentially expressed (fold change ±1.2) (78 down-regulated, 48 up-regulated) in EPF. Validation showed higher levels of miR-106b-3p, -451a, -486-5p, IL-6, IL-8, uPA, and TIMP-1 in EPF. In menstrual phase, EPF presented up-regulation of miR-106b-3p, -130a-3p, -150-5p, -185-5p, -451a, -486-5p, VEGF-A, IL-8, MIF 1β, uPA, and PAI-1 compared with other phases; however, CPF did not. MiRNA-486-5p was up-regulated in sterile EPF compared with sterile controls, and VEGF-A, IL-8, and TIMP-1 were increased in sterile and fertile EPF compared with fertile CPF. CONCLUSION(S) MiRNAs seem to be involved in the peritoneal alterations in endometriosis, suggesting new mechanisms by which ectopic lesions could implant in endometriosis patients; and to serve as biomarkers for fertility outcome prediction.

C0081 Tissue factor expression and micrornas −17-5p and –20a in endometrial cell cultures from women with endometriosis: Its modulation by peritoneal fluid

Background: Endothelial colonies forming cells (ECFC) are good candidates for cell-based therapy in cardiovascular diseases. Concerns have been raised about the potential risks of ECFC-based cell therapy, in terms of thrombogenicity. We recently published that cord blood ECFC (cb-ECFC) express Tissue Factor (TF). The objective of the present study is to detect TF in two different cell therapy products (CTP). As we previously reported a co-regulation of TF with proangiogenic MMPs, we also focused on the expression of MMP-2 and MMP-9 in CTP. Methods: ECFC were cultured from cord blood after isolation of CD34+ cells. CTP were obtained from an ongoing clinical trial evaluating cell therapy to improve vascularization in chronic critical limb ischemia (NIH NCT00533104). Two different CTP were analyzed: 1Peripheral blood mononuclear cells (PB-MNC) obtained by cytapheresis; 2Bone marrow mononuclear cells (BM-MNC). Results: Full length TF mRNA was detected in both CTP. The alternatively spliced form (as-TF) was also detected but represented a very minor part of total TF mRNA. TF mRNA was estimated to be a 2 fold higher in BM-MNC than in cb-ECFC. On the contrary, TF mRNA was a 5 fold lower in PB-MNC than in cb-ECFC. TF mRNA levels was also compared with CD34+ cells and found respectively 3 fold lower in BM-MNC and 27 fold lower in PB-MNC. The source of TF in CTP could be monocytes or CD34+ cells. Indeed, the percentage of CD14+ cells was respectively 17% and 26% in BM-MNC and in PBMNC whereas the percentage of CD34+ cells was 2.6% in BM-MNC and 0.1% in PB-MNC. TF protein was found in both CTP at a very low range. Still, in the clinical trial, one patient who had received PB-MNC developed a deep vein thrombosis at the site of injection. In BM-MNC, MMP-2 and MMP-9 were expressed at a very high level. In PB-MNC, MMP-2 and MMP-9 could be detected but at a very low level. In comparison, cb-ECFC strongly expressed MMP-2 whereas the expression of MMP-9 was weak. Comment: The profile of TF and MMPs is different in BM-MNC and in PB-MNC, suggesting a higher proangiogenic potential for BM-MNC. The different cell composition of CTP may explain these differences. However, the prothrombotic potential of CTP has to be considered.