Mechanisms underlying the influence of oestrogen on cardiovascular physiology in women

Women show a lower incidence of cardiovascular diseases than aged-matched men, but this benefit disappears after menopause. Oestrogen-mediated vascular actions are mainly attributed to oestradiol and exerted by oestrogen receptors (ERα, ERβ, and GPER), through rapid and/or genomic mechanisms, but these effects depend on ageing and inflammation. A cardiovascular approach in women's health has arisen due to controversy regarding oestrogens' beneficial impact as reported in experimental and observational studies and large randomized trials. These can be explained, in part, by two mutually non-exclusive hypotheses. On the one hand, the timing hypothesis, which states that oestrogen-mediated benefits occur before the detrimental effects of ageing are established in the vasculature; on the other hand, ageing and/or hormonal-associated changes in ER expression that could lead to a deleterious imbalance in favour of ERβ over ERα, generally associated with higher inflammation and endothelial dysfunction. In experimental studies, oestradiol acting on ERα promotes the release of vasoactive compounds such as nitric oxide (NO) and prostacyclin, and shifts the angiotensin axis towards angiotensin 1-7 production. Mechanisms underlying oestradiol vascular function also include anti-inflammatory and epigenetic modifications. 17β-oestradiol changes the transcriptomic profile of endothelial cells, and the involvement of miRNA in the regulatory pathways of vascular function reinforces assumptions regarding the vascular actions of oestrogen. Thus, the present symposium review aims to postulate the role of ERα in oestrogen modulation of endothelial-derived mediators and vascular physiology, as well as its relationship with miRNA and inflammation, and elucidate how physiological changes in postmenopausal women counteract the observed effects

Role of miRNA in the Regulatory Mechanisms of Estrogens in Cardiovascular Ageing

Cardiovascular diseases are a worldwide health problem and are the leading cause of mortality in developed countries. Together with experimental data, the lower incidence of cardiovascular diseases in women than in men of reproductive age points to the influence of sex hormones at the cardiovascular level and suggests that estrogens play a protective role against cardiovascular disease and that this role is also modified by ageing. Estrogens affect cardiovascular function via their specific estrogen receptors to trigger gene expression changes at the transcriptional level. In addition, emerging studies have proposed a role for microRNAs in the vascular effects mediated by estrogens. miRNAs regulate gene expression by repressing translational processes and have been estimated to be involved in the regulation of approximately 30% of all protein-coding genes in mammals. In this review, we highlight the current knowledge of the role of estrogen-sensitive miRNAs, and their influence in regulating vascular ageing

miRNA as New Regulatory Mechanism of Estrogen Vascular Action

The beneficial effects of estrogen on the cardiovascular system have been reported extensively. In fact, the incidence of cardiovascular diseases in women is lower than in age-matched men during their fertile stage of life, a benefit that disappears after menopause. These sex-related differences point to sexual hormones, mainly estrogen, as possible cardiovascular protective factors. The regulation of vascular function by estrogen is mainly related to the maintenance of normal endothelial function and is mediated by both direct and indirect gene transcription through the activity of specific estrogen receptors. Some of these mechanisms are known, but many remain to be elucidated. In recent years, microRNAs have been established as non-coding RNAs that regulate the expression of a high percentage of protein-coding genes in mammals and are related to the correct function of human physiology. Moreover, within the cardiovascular system, miRNAs have been related to physiological and pathological conditions. In this review, we address what is known about the role of estrogen-regulated miRNAs and their emerging involvement in vascular biology

MicroRNA as crucial regulators of gene expression in estradiol-treated human endothelial cells.

Background/Aims: Estrogen signalling plays an important role in vascular biology as it modulates vasoactive and metabolic pathways in endothelial cells. Growing evidence has also established microRNA (miRNA) as key regulators of endothelial function. Nonetheless, the role of estrogen regulation on miRNA profile in endothelial cells is poorly understood. In this study, we aimed to determine how estrogen modulates miRNA profile in human endothelial cells and to explore the role of the different estrogen receptors (ERα, ERβ and GPER) in the regulation of miRNA expression by estrogen. Methods: We used miRNA microarrays to determine global miRNA expression in human umbilical vein endothelial cells (HUVEC) exposed to a physiological concentration of estradiol (E2; 1 nmol/L) for 24 hours. miRNA-gene interactions were computationally predicted using Ingenuity Pathway Analysis and changes in miRNA levels were validated by qRT-PCR. Role of ER in the E2-induced miRNA was additionally confirmed by using specific ER agonists and antagonists. Results: miRNA array revealed that expression of 114 miRNA were significantly modified after E2 exposition. Further biological pathway analysis revealed cell death and survival, lipid metabolism, reproductive system function, as the top functions regulated by E2. We validated changes in the most significantly increased (miR-30b-5p, miR-487a-5p, miR-4710, miR-501-3p) and decreased (miR-378h and miR-1244) miRNA and the role of ER in these E2-induced miRNA was determined. Results showed that both classical, ERα and ERβ, and membrane-bound ER, GPER, differentially regulated specific miRNA. In silico analysis of validated miRNA promoters identified specific ER binding sites. Conclusion: Our findings identify differentially expressed miRNA pathways linked to E2 in human endothelial cells through ER, and provide new insights by which estrogen can modulate endothelial function

Regulatory network analysis in estradiol-treated human endothelial cells.

Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a com-prehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miR-NA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17ß- Estradiol (E2) (1 nmol/lL, 24 h). miRNA--mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miR-NA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA--target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship accord-ing to miRNA target prediction databases. The analysis identified 588 miRNA--target interactions between 102 miRNAs and 588 targets. Specifically, 63 up-regregulated miRNAs interacted with 295 down-regregulated targets, while 39 down-regregulated miRNAs were paired with 293 up-regregulated mRNA targets. Functional characterization of miRNA/mRNA association analy-sis highlighted hypoxia signallignaling, integrin, ephrin receptor signaling, and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Tran-scription factors and downstream genes analysis revealed a total of eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhe-sion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells

An affordable method to obtain cultured endothelial cells from peripheral blood

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell-like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above-mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols

Extracellular histones disarrange vasoactive mediators reléase through COX-NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies

Extracellular histones trigger oxidative stress-dependent induction of the NF-kB/CAM pathway via TLR4 in endothelial cells.

Extracellular histones have been reported to aggravate different pathophysiological processes by increasing vascular permeability, coagulopathy, and inflammation. In the present study, we elucidate how extracellular histones (10-100 mu g/mL) concentration dependently increase cytosolic reactive oxygen species (ROS) production using human umbilical vein endothelial cells (HUVECs). Furthermore, we identify cyclooxygenase (COX) and NADPH oxidase (NOX) activity as sources of ROS production in extracellular histone-treated HUVEC. This COX/NOX-mediated ROS production is also involved in enhanced NF-kB activity and cell adhesion molecules (VCAM1 and ICAM1) expression in histone-treated HUVEC. Finally, by using different toll-like receptor (TLR) antagonists, we demonstrate the role of TLR4 in CAMs overexpression triggered by extracellular histones in endothelial cells. In conclusion, our data suggest that through TLR4 signaling, extracellular histones increase endothelial cell activation, a mechanism involving increased COX- and NOX-mediated ROS. These findings increase our understanding on how extracellular histones enhance systemic inflammatory responses in diseases in which histone release occurs as part of the pathological processes

Circulating miRNA Fingerprint and Endothelial Function in Myocardial Infarction : Comparison at Acute Event and One-Year Follow-Up

MicroRNAs (miRNA) are major regulators of intercellular communication and key players in the pathophysiology of cardiovascular disease. This study aimed to determine the miRNA fingerprint in a cohort of 53 patients with acute myocardial infarction (AMI) with non-ST-segment elevation (NSTEMI) relative to miRNA expression in healthy controls (n = 51). miRNA expression was initially profiled by miRNA array in the serum of patients undergoing cardiac catheterization during NSTEMI (n = 8) and 1 year past the event (follow-up, n = 8) and validated in the entire cohort. In total, 58 miRNAs were differentially expressed during AMI (p < 0.05), while 36 were modified at follow-up (Fisher's exact test: p = 0.0138). Enrichment analyses revealed differential regulation of biological processes by miRNA at each specific time point (AMI vs. follow-up). During AMI, the miRNA profile was associated mainly with processes involved in vascular development. However, 1 year after AMI, changes in miRNA expression were partially related to the regulation of cardiac tissue morphogenesis. Linear correlation analysis of miRNA with serum levels of cytokines and chemokines revealed that let-7g-5p, let-7e-5p, and miR-26a-5p expression was inversely associated with serum levels of pro-inflammatory cytokines TNF- , and the chemokines MCP-3 and MDC. Transient transfection of human endothelial cells (HUVEC) with let-7e-5p inhibitor or mimic demonstrated a key role for this miRNA in endothelial function regulation in terms of cell adhesion and angiogenesis capacity. HUVEC transfected with let-7e-5p mimic showed a 20% increase in adhesion capacity, whereas transfection with let-7e-5p inhibitor increased the number of tube-like structures. This study pinpoints circulating miRNA expression fingerprint in NSTEMI patients, specific to the acute event and changes at 1-year follow-up. Additionally, given its involvement in modulating endothelial cell function and vascularization, altered let-7e-5p expression may constitute a therapeutic biomarker and target for ischemic heart disease

Disparate miRNA expression in serum and plasma of patients with acute myocardial infarction: a systematic and paired comparative analysis

Despite the promising value of miRNAs in the diagnostic and prognostic of cardiovascular disease (CVD), recent meta-analyses did not support their potential. Methodological variances in studies may interfere with miRNA profle and afect their results. This study determines if the blood starting material is a source of variance in miRNA profle by performing a paired comparison in plasma and serum of the expression of primary miRNAs associated with CVD. Circulating miRNA yield was similar in both plasma and serum, although a signifcant increase was observed in patients with Non-ST-elevation myocardial infarction (NSTEMI) compared to control volunteers. When normalized by the expression of miR-484, diferent patterns of miRNA expression between serum and plasma. Although NSTEMI modifed the expression of miR-1 and miR-208 in both serum and plasma, plasma displayed a higher variance than serum (Levene's test p<0.01). For miR-133a and miR-26a, diferences were only detected in serum (p=0.0240), and conversely, miR-499a showed diferences only in plasma of NSTEMI (p=0.001). Interestingly, miR-21 showed an opposite pattern of expression, being increased in serum (2−ΔΔCt:5.7, p=0.0221) and decreased in plasma (2−ΔΔCt: 0.5, p=0.0107). Plasma and serum exhibit diferent patterns of circulating miRNA expression in NSTEMI and suggest that results from studies with diferent starting material could not be comparable