A. Estellés

MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors

STUDY QUESTION Could an aberrant microRNA (miRNA) expression profile be responsible for the changes in the angiogenic and fibrinolytic states observed in endometriotic lesions? SUMMARY ANSWER 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. WHAT IS ALREADY KNOWN?: 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. STUDY DESIGN, SIZE, DURATION Case-control study that involved 51 women with endometriosis and 32 women without the disease (controls). PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LIMITATIONS, REASONS FOR CAUTION Functional studies are needed to confirm the specific targets of the miRNAs differently expressed. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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.

Physiological coagulation inhibitors (protein S, protein C and antithrombin III) in severe preeclamptic states and in users of oral contraceptives

Protein C, protein S and antithrombin III were evaluated in normal pregnancy, severe preeclampsia and chronic hypertension with superimposed severe preeclampsia. The same study was performed on a group of 10 normal women using oral contraceptives. In normal pregnancy a significant decrease in the level of free and total PS was observed in the 2nd trimester of pregnancy and was sustained throughout the remaining months. No significant changes in the levels of protein C and antithrombin III were observed during normal pregnancy. In preeclamptic states a significant decrease in protein C was observed. It was more evident in severe preeclampsia when compared with the normal pregnancy group at similar gestational age. No statistically significant differences in protein S were found when the normal and pathological groups were compared. Antithrombin III decreased only slightly in the severe preeclamptic group. The decrease in protein C and antithrombin III levels in severe preeclampsia could be related with the microthrombotic state that these patients may present. However, the role played by protein S, which decreases during normal pregnancy and in preeclampsia, is not clear. A decrease in the level of total protein S was observed in the group of women using oral contraceptives. No significant changes in protein C and antithrombin III levels were observed in this group.

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.

Influence of peritoneal fluid on the expression of angiogenic and proteolytic factors in cultures of endometrial cells from women with endometriosis

BACKGROUND Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent benign gynaecological diseases. It has been suggested that both endometrial and peritoneal factors, related to angiogenesis and proteolysis, can be implicated in this disease. The aim of this study was to evaluate the influence of peritoneal fluid on the expression of angiogenic and proteolytic factors in cultures of endometrial cells from women with and without endometriosis. METHODS Endometrial cells were isolated, cultured and treated with endometriotic or normal peritoneal fluid. Vascular endothelial growth factor-A (VEGF-A), urokinase plasminogen activator (uPA), matrix metalloproteinase-3 (MMP-3) and their inhibitors including thrombospondin-1, plasminogen activator inhibitor-1 and MMP inhibitor type 1 (TIMP-1) mRNA levels were evaluated by quantitative RT-PCR, and protein levels were quantified by ELISA. RESULTS Peritoneal fluid from women with endometriosis induced an increase in VEGF-A and uPA protein and VEGF-A mRNA and uPA mRNA levels in endometrial cell culture from women with (P < 0.01) and without endometriosis (P < 0.05). The highest levels of VEGF-A and uPA were observed in endometrial cell cultures from patients with endometriosis and treated with peritoneal fluid from women with endometriosis. CONCLUSIONS Peritoneal fluid from women with endometriosis induced more VEGF and uPA expression in endometrial cell culture from women with endometriosis than did normal peritoneal fluid. Endometrial-peritoneal interactions increased angiogenic and proteolytic factors in endometrial cells, which could contribute to the development of endometriotic lesions.

Peritoneal fluid reduces angiogenesis-related microRNA expression in cell cultures of endometrial and endometriotic tissues from women with endometriosis.

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. Methods 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. Results 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.

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.

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.

O4 Vascular endothelial growth factor (VEGF) polymorphisms (−460C/T, +405G/C and 936C/T) and endometriosis. Their influence on VEGF expression

Endometriosis is a chronic gynecological disease defined as the presence of endometrial tissue outside the uterus. It is a multifactorial and polygenic disease in which angiogenesis may be implicated. Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis, is known to play a role in the development of endometriosis. However, the association of VEGF polymorphisms and endometriosis is unclear. The objective of this study was to analyse three functional VEGF polymorphisms ( 460C/T, +405G/C and 936C/T) in 186 women with endometriosis and in 180 controls, and their correlation with VEGF expression in endometrial tissue and peritoneal fluid. VEGF polymorphisms were determined using a PCRRFLP assay. Quantitative real-time RT-PCR assay was used to quantify VEGF-A mRNA. VEGF-A antigen levels were quantified by ELISA. Patients with endometriosis showed a higher VEGF 936T allele frequency than controls (P = 0.02). However, the distribution of genotypes and allele frequencies in the other two polymorphisms ( 460C/T, +405G/C) was similar in endometriosis and control groups. Endometrium and peritoneal fluid from women with endometriosis showed an increase in VEGF levels (P = 0.01 and P = 0.04), but no association was found between the VEGF polymorphisms studied and VEGF expression in endometrial tissue and peritoneal fluid. In conclusion, these findings suggest that the VEGF 936C/T polymorphism may be associated with the risk of endometriosis in a Caucasian population, but the increased VEGF levels observed in endometriosis do not appear to be associated with this polymorphism. Grants from Ministerio de Sanidad y Consumo, ISCIII (FIS PI050799, PI050844, Red RECAVA RD06/0014/0004), Beca Fundacion Salud 2000 (Serono), Spain.