Ana B. Arroyo

Peritoneal fluid modifies the microRNA expression profile in endometrial and endometriotic cells from women with endometriosis

STUDY QUESTION Could peritoneal fluid (PF) from patients with endometriosis alter the microRNA (miRNA) expression profile in endometrial and endometriotic cells from patients? SUMMARY ANSWER PF from patients with endometriosis modifies the miRNA expression profile in endometrial cells from patients. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTERESTS 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.

Regulation of Coagulation Factor XI Expression by MicroRNAs in the Human Liver

High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3′untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

Prognostic role of MIR146A polymorphisms for cardiovascular events in atrial fibrillation

There are few biomarkers able to forecast new thrombotic events in patients with AF. In this framework, microRNAs have emerged as critical players in cardiovascular biology. In particular, miR-146a-5p is recognised as an important negative regulator of inflammation. This study aims to evaluate the prognostic role and biological effect of functional MIR146A polymorphisms, rs2431697 and rs2910164, in non-valvular atrial fibrillation (AF) patients under oral anticoagulation.We studied 901 patients with permanent/paroxysmal AF stabilized for at least six months. Patients were followed-up for two years and adverse cardiovascular events (ACE) were recorded. In vitro studies were performed in monocytes from healthy homozygous for the two genotypes of rs2431697. Rs2910164 had no association with ACE. However, multivariate analysis (adjusted by CHA2DS2-VASc score) revealed that rs2431697TT was associated with adverse cardiovascular events [HR: 1.64 (1.09-2.47); p=0.017]. The predictive value of usefulness of the CHA2DS2-VASc+IL6+rs2431697 for predicting ACE, was statistically better than that predicted by CHA2DS2-VASc+IL6. Functional studies showed that after 24 hours incubation, monocytes from CC individuals showed a 65 % increase in miR-146a-5p levels, while TT individuals only showed a 28 % increase. Indeed, after 24 hours of LPS activation, TT monocytes showed a higher increase in IL6 mRNA expression than CC (52 % vs 26 %). Our study established MIR146A rs2431697 as a prognostic biomarker for ACE in anticoagulated AF patients. These data suggest that TT individuals, when submitted to an inflammatory stress, may be prone to a highest pro-inflammatory state due, in part, to lower levels of miR-146a-5p.

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a

BackgroundDevelopmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant.ResultsIn this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control.ConclusionsThese data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.

Effect of VKORC1, CYP2C9 and CYP4F2 genetic variants in early outcomes during acenocoumarol treatment

AIM To analyze VKORC1, CYP2C9 and CYP4F2 polymorphisms in relation to the main outcomes in the first stages of acenocoumarol therapy. PATIENTS & METHODS Nine hundred and forty one patients who had started therapy and in whom time to stable dosage, time to over-anticoagulation and adverse events occurred during 3 first months were retrospectively analyzed. RESULTS VKORC1 AA patients needed fewer days to reach stable dosage (p = 0.017). International normalized ratio [INR] at 72 h, and VKORC1 and CYP2C9 genotypes conditioned INR values >2.5 (p < 0.001, p = 0.002 and p < 0.001, respectively), whereas CYP4F2 T carriers had a low risk of the same outcome (p = 0.009). In regards to combined genotypes, CYP4F2 had a significant effect on over-anticoagulation at the beginning of therapy except for the VKORC1 AA and CYP2C9*3 combination. CONCLUSION In addition to VKORC1 and CYP2C9, CYP4F2 gene has a slight but significant role in reaching INR >2.5 during the first weeks of acenocoumarol therapy.

High on-treatment platelet reactivity in patients with ischemic cerebrovascular disease: assessment of prevalence and stability over time using four platelet function tests.

High on-treatment platelet reactivity (HTPR), referred to as a higher than expected platelet reactivity in patients under antiplatelet therapy, could influence outcome in cerebrovascular disease (CVD), but its prevalence and its stability over time is uncertain. Platelet reactivity was assessed in 18 patients with ischemic stroke/transient ischemic attack (TIA) 7 days (D7) and 90 days (D90) after prescription of clopidogrel, using four methods: light transmission aggregometry with 5 &mgr;mol/l ADP (LTA-ADP), vasodilator-stimulated phosphoprotein (VASP), Verify Now P2Y12 and platelet function analyzer (PFA) P2Y. HTPR was defined as LTA-ADP more than 46%; PFA-100-P2Y closure time less than 106 s; VerifyNow P2Y12, PRU greater than 235, VASP, PRI greater than 50%. Patients displayed, both at D7 and D90, a marked inhibition of platelet reactivity towards ADP in all tests as compared with reference levels. Correlations between the results obtained with all the tests at D7 and D90 and between measurements on each day in each test were low-to-moderate. The prevalence of HTPR for all the tests was 40% at D7 and 42% at D90. There was a moderate degree of agreement (k statistic < 0.5) between tests with regard to categorizing patients as HTPR/No-HTPR (D7 and D90). The on-clopidogrel platelet reactivity phenotype, HTPR/No-HTPR, remained stable in 55–72% of patients, depending on the test. A high prevalence of HTPR is found among CVD patients treated with clopidogrel and this platelet reactivity phenotype remains over time. There is poor agreement between the different platelet function tests for categorizing the platelet reactivity phenotype in these patients. The new PFA-100 P2Y equals other platelet function assays for evaluating HTPR in CVD.

microRNAs in the haemostatic system: More than witnesses of thromboembolic diseases?

MicroRNAs (miRNAs) are small endogenous RNAs that post-transcriptionally regulate gene expression. In the last few years, these molecules have been implicated in the regulation of haemostasis, and an increasing number of studies have investigated their relationship with the development of thrombosis. In this review, we discuss the latest developments regarding the role of miRNAs in the regulation of platelet function and secondary haemostasis. We also discuss the genetic and environmental factors that regulate miRNAs. Finally, we address the potential use of miRNAs as prognostic and diagnostic tools in thrombosis.

MiR-146a Regulates Neutrophil Extracellular Trap Formation That Predicts Adverse Cardiovascular Events in Patients With Atrial Fibrillation

Objective— Atrial fibrillation (AF) patients experience adverse cardiovascular events (ACEs) despite anticoagulant therapy. We reported that rs2431697 of miR-146a, a negative regulator of inflammation, predicts ACEs in patients with AF. The relationship between neutrophil extracellular traps and thrombogenesis is known. Thus, our aim was to evaluate the role of neutrophil extracellular trap compounds as prognostic markers of ACEs in AF and to study whether miR-146a affects NETosis. Approach and Results— We included 336 steadily anticoagulated AF patients with a median follow-up of 7.9 years (interquartile range, 7.3–8.1) and 127 healthy subjects. The reviewed ACEs included stroke (ischemic/embolic), acute coronary syndrome, acute heart failure, and global or vascular death. We quantified cell-free DNA and NE (neutrophil elastase) at diagnosis. Rs2431697 was genotyped. Neutrophils from human and mice were seeded to analyze shed cell-free DNA and H3cit (citrullinated histone 3) after activation. In human plasmas, higher NE levels (>55.29 ng/mL), but not cell-free DNA, were independently associated with higher risk of all-cause mortality (hazard ratio, 2.24; 95% CI, 1.36–3.68), cardiovascular mortality (hazard ratio, 4.77; 95% CI, 1.11–20.47), and composite cardiovascular events (hazard ratio, 1.84; 95% CI, 1.01–3.76). In patients, NE levels were associated with rs2431697 (TT: 51.82±2.73 versus CC: 40.01±3.05 ng/mL; P=0.040). In vitro, both human (TT for rs2431697) and miR-146a−/− mice neutrophils yielded higher levels of cell-free DNA and H3cit than CC or wild-type cells, respectively. Conclusions— NE activity can provide new ACE prognostic information in AF patients. These findings provide evidence of a potential role of miR-146a in neutrophil extracellular trap generation and cardiovascular risk in AF.

MiRNA-Based Regulation of Hemostatic Factors through Hepatic Nuclear Factor-4 Alpha.

MiRNAs have been reported as CIS-acting elements of several hemostatic factors, however, their mechanism as TRANS-acting elements mediated by a transcription factor is little known and could have important effects. HNF4α has a direct and important role in the regulation of multiple hepatic coagulation genes. Previous in vitro studies have demonstrated that miR-24-3p and miR-34a-5p regulate HNF4A expression. Here we aimed to investigate the molecular mechanisms of miR-24 and miR-34a on coagulation through HNF4A. Transfections with miR-24 and miR-34a in HepG2 cells decreased not only HNF4A but also F10, F12, SERPINC1, PROS1, PROC, and PROZ transcripts levels. Positive and significant correlations were observed between levels of HNF4A and several hemostatic factors (F5, F8, F9, F11, F12, SERPINC1, PROC, and PROS1) in human liver samples (N = 104). However, miR-24 and miR-34a levels of the low (10th) and high (90th) percentiles of those liver samples were inversely correlated with HNF4A and almost all hemostatic factors expression levels. These outcomes suggest that miR-24 and miR-34a might be two indirect elements of regulation of several hemostatic factors. Additionally, variations in miRNA expression profiles could justify, at least in part, changes in HNF4A expression levels and its downstream targets of coagulation.

MicroRNAs as potential regulators of platelet function and bleeding diatheses

Abstract Although a growing number of studies suggest that microRNAs (miRNAs) play a relevant role in platelet biology, their implications in bleeding diatheses are starting to be investigated. Indeed, several studies have shown that alterations in the intracellular levels of highly expressed platelet miRNAs provoke a thrombotic phenotype. On the other hand, primary immune thrombocytopenia (ITP), which is considered the hallmark of acquired bleeding disorders, has been recently associated with altered levels of miRNAs in peripheral blood mononuclear cells, plasma, and platelets. In this review, we will focus on miRNAs that may affect the hemostatic and thrombotic functions of platelets, and we will discuss the different studies that have attempted to associate miRNAs with regulatory mechanisms of ITP.