Cardiovascular complications of diabetes: role of non-coding RNAs in the crosstalk between immune and cardiovascular systems.

Diabetes mellitus, a group of metabolic disorders characterized by high levels of blood glucose caused by insulin defect or impairment, is a major risk factor for cardiovascular diseases and related mortality. Patients with diabetes experience a state of chronic or intermittent hyperglycemia resulting in damage to the vasculature, leading to micro- and macro-vascular diseases. These conditions are associated with low-grade chronic inflammation and accelerated atherosclerosis. Several classes of leukocytes have been implicated in diabetic cardiovascular impairment. Although the molecular pathways through which diabetes elicits an inflammatory response have attracted significant attention, how they contribute to altering cardiovascular homeostasis is still incompletely understood. In this respect, non-coding RNAs (ncRNAs) are a still largely under-investigated class of transcripts that may play a fundamental role. This review article gathers the current knowledge on the function of ncRNAs in the crosstalk between immune and cardiovascular cells in the context of diabetic complications, highlighting the influence of biological sex in such mechanisms and exploring the potential role of ncRNAs as biomarkers and targets for treatments. The discussion closes by offering an overview of the ncRNAs involved in the increased cardiovascular risk suffered by patients with diabetes facing Sars-CoV-2 infection.

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 comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. METHODS: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17ß-estradiol (E2) (1 nmol/L, 24 h). miRNA-mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-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 according to miRNA target prediction databases. The analysis identified 588 miRNA-target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion 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.

Approaching Sex Differences in Cardiovascular Non-Coding RNA Research.

Cardiovascular disease (CVD) is the biggest cause of sickness and mortality worldwide in both males and females. Clinical statistics demonstrate clear sex differences in risk, prevalence, mortality rates, and response to treatment for different entities of CVD. The reason for this remains poorly understood. Non-coding RNAs (ncRNAs) are emerging as key mediators and biomarkers of CVD. Similarly, current knowledge on differential regulation, expression, and pathology-associated function of ncRNAs between sexes is minimal. Here, we provide a state-of-the-art overview of what is known on sex differences in ncRNA research in CVD as well as discussing the contributing biological factors to this sex dimorphism including genetic and epigenetic factors and sex hormone regulation of transcription. We then focus on the experimental models of CVD and their use in translational ncRNA research in the cardiovascular field. In particular, we want to highlight the importance of considering sex of the cellular and pre-clinical models in clinical studies in ncRNA research and to carefully consider the appropriate experimental models most applicable to human patient populations. Moreover, we aim to identify sex-specific targets for treatment and diagnosis for the biggest socioeconomic health problem globally.

Mechanisms underlying the influence of oestrogen on cardiovascular physiology in women.

Women show a lower incidence of cardiovascular diseases than age-matched men, but this benefit disappears after menopause. Oestrogen-mediated vascular actions are mainly attributed to oestradiol and exerted by oestrogen receptors (ERα, ERß and G protein-coupled oestrogen receptor), 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 oestrogen's 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 ERß 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. 17ß-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 endothelium-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.

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, ERß 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 ERß, 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.

miRNA as a 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.

Extracellular histones disarrange vasoactive mediators release through a 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.

Mobilization of endothelial progenitor cells in acute cardiovascular events in the PROCELL study: time-course after acute myocardial infarction and stroke.

The mobilization pattern and functionality of endothelial progenitor cells after an acute ischemic event remain largely unknown. The aim of our study was to characterize and compare the short- and long-term mobilization of endothelial progenitor cells and circulating endothelial cells after acute myocardial infarction or atherothrombotic stroke, and to determine the relationship between these cell counts and plasma concentrations of vascular cell adhesion molecule (VCAM-1) and Von Willebrand factor (VWF) as surrogate markers of endothelial damage and inflammation. In addition, we assessed whether endothelial progenitor cells behave like functional endothelial cells. We included 150 patients with acute myocardial infarction or atherothrombotic stroke and 145 controls. Endothelial progenitor cells [CD45-, CD34+, KDR+, CD133+], circulating endothelial cells [CD45-, CD146+, CD31+], VWF, and VCAM-1 levels were measured in controls (baseline only) and in patients within 24h (baseline) and at 7, 30, and 180 days after the event. Myocardial infarction patients had higher counts of endothelial progenitor cells and circulating endothelial cells than the controls (201.0/mL vs. 57.0/mL; p<0.01 and 181.0/mL vs. 62.0/mL; p<0.01). Endothelial progenitor cells peaked at 30 days post-infarction (201.0/mL vs. 369.5/mL; p<0.01), as did VCAM-1 (573.7 ng/mL vs. 701.8 ng/mL; p<0.01). At 180 days post-infarction, circulating endothelial cells and VWF decreased, compared to baseline. In stroke patients, the number of endothelial progenitor cells - but not circulating endothelial cells - was higher than in controls (90.0/mL vs. 37.0/mL; p=0.01; 105.0/mL vs. 71.0/mL; p=0.11). At 30 days after stroke, however, VCAM-1 peaked (628.1/mL vs. 869.1/mL; p<0.01) but there was no significant change in endothelial progenitor cells (90/mL vs. 78/mL; p<0.34). At 180 days after stroke, circulating endothelial cells and VWF decreased, compared to baseline. Cultured endothelial progenitor cells from controls and myocardial infarction patients had endothelial phenotype characteristics and exhibited functional differences in adhesion and Ca(2+) influx, but not in proliferation and vasculogenesis. In myocardial infarction patients, VCAM-1 levels and mobilization of endothelial progenitor cells peaked at 30 days after the ischemic event. Although a similar VCAM-1 kinetic was observed in stroke patients, endothelial progenitor cells did not increase. Endothelial progenitor cells had mature endothelial capabilities in vitro.

Dynamics of serum-induced endothelial cell apoptosis in patients with myocardial infarction.

BACKGROUND: In patients with ST-segment elevation myocardial infarction (STEMI) reperfused with primary coronary intervention (PCI), the dynamics of endothelial cell (EC) viability, apoptosis and necrosis and its relationship with the structural consequences on the left ventricle have not been addressed so far. DESIGN: In 20 STEMI patients, we incubated human umbilical vein endothelial cells (HUVECs) with serum drawn before reperfusion and subsequently afterwards (24, 96 h, 30 days). Viability, apoptosis and necrosis percentages were evaluated by flow cytometry. Values were compared with 12 age- and sex-matched control subjects with normal coronary arteries. Cardiac magnetic resonance (CMR) was performed during the first week after infarction. RESULTS: Serum from STEMI patients induced a progressive loss of EC viability, with a nadir of 67.7 ± 10.2% at 96 h (baseline: 75 ± 6% and controls: 80.2 ± 3.9%, P < 0.001 in both cases). This is due to an increase in apoptosis that peaked at 96 h after reperfusion (15.2 ± 7.1% vs. 11 ± 6 at baseline and 5.8 ± 1.6% in controls, P < 0.001 in both cases). However, no significant dynamic changes in EC necrosis were detected. Extensive myocardial oedema (> 30%, median of left ventricular mass) was the only CMR variable significantly associated with a higher percentage of EC apoptosis at 96 h (extensive vs. nonextensive oedema: 18.3 ± 6.8% vs. 12.1 ± 6.3%, P < 0.05). CONCLUSIONS: Dynamic changes in EC viability occur in the setting of STEMI patients reperfused with PCI, these changes peak late after reperfusion, they are mainly the result of an increase of apoptosis and are associated with the presence of extensive myocardial oedema.

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.

Complete blockade of the vasorelaxant effects of angiotensin-(1-7) and bradykinin in murine microvessels by antagonists of the receptor Mas.

The heptapeptide angiotensin-(1-7) is a biologically active metabolite of angiotensin II, the predominant peptide of the renin-angiotensin system. Recently, we have shown that the receptor Mas is associated with angiotensin-(1-7)-induced signalling and mediates, at least in part, the vasodilatory properties of angiotensin-(1-7). However, it remained controversial whether an additional receptor could account for angiotensin-(1-7)-induced vasorelaxation. Here, we used two different angiotensin-(1-7) antagonists, A779 and d-Pro-angiotensin-(1-7), to address this question and also to study their influence on the vasodilatation induced by bradykinin. Isolated mesenteric microvessels from both wild-type and Mas-deficient C57Bl/6 mice were precontracted with noradrenaline, and vascular reactivity to angiotensin-(1-7) and bradykinin was subsequently studied using a small-vessel myograph. Furthermore, mechanisms for Mas effects were investigated in primary human umbilical vein endothelial cells. Both angiotensin-(1-7) and bradykinin triggered a concentration-dependent vasodilatation in wild-type microvessels, which was absent in the presence of a nitric oxide synthase inhibitor. In these vessels, the pre-incubation with the Mas antagonists A779 or d-Pro-angiotensin-(1-7) totally abolished the vasodilatory capacity of both angiotensin-(1-7) and bradykinin, which was nitric oxide mediated. Accordingly, Mas-deficient microvessels lacked the capacity to relax in response to either angiotensin-(1-7) or bradykinin. Pre-incubation of human umbilical vein endothelial cells with A779 prevented bradykinin-mediated NO generation and NO synthase phosphorylation at serine 1177. The angiotensin-(1-7) antagonists A779 and d-Pro-angiotensin-(1-7) equally block Mas, which completely controls the angiotensin-(1-7)-induced vasodilatation in mesenteric microvessels. Importantly, Mas also appears to be a critical player in NO-mediated vasodilatation induced by renin-angiotensin system-independent agonists by altering phosphorylation of NO synthase.

Effects of estrogen on vascular inflammation: a matter of timing.

OBJECTIVE: Our study aims to determine the role of time of menopause on vascular inflammation biomarkers and how it affects their modulation by estrogen and raloxifene in postmenopausal women. METHODS AND RESULTS: Uterine arteries from 68 postmenopausal women were divided into 3 segments and cultured for 24 hours in tissue culture media containing 17ß-estradiol (100 nmol/L), raloxifene (100 nmol/L), or vehicle. Assessment of arterial concentration of 13 inflammatory biomarkers was performed by multiplex immunobead-based assay. Aging per se has a positive correlation with the generation of several proinflammatory markers. Although short-term estradiol exposure correlates with lower expression of tumor necrosis factor-α, vascular endothelial growth factor, and interleukin-1ß in all age groups, for most biomarkers aging was associated with a switch from a beneficial anti-inflammatory action by estrogen, at earlier stages of menopause, to a proinflammatory profile after 5 years past its onset. Raloxifene has no significant effect on the expression of all proinflammatory markers. Western blot analysis of estrogen receptor expression (estrogen receptor-α and estrogen receptor-ß) showed that estrogen receptor-ß increases with aging, and this increase has a positive correlation with the generation of several proinflammatory markers. CONCLUSIONS: Aging alters estrogen-mediated effects on the modulation of inflammatory biomarkers in women. How aging affects estrogen responses on vascular inflammation is not clear, but our data show a positive association between increased estrogen receptor-ß expression with aging and proinflammatory effects by estrogen.

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 µg/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.

Extracellular Histones Activate Endothelial NLRP3 Inflammasome and are Associated with a Severe Sepsis Phenotype.

Introduction: Circulating extracellular histones acquire relevance as cytotoxic mediators in sepsis. Extracellular histones act as damage-associated molecular patterns (DAMPs), which induce oxidative stress and NLRP3 inflammasome activation. Inflammasome mediates pyroptosis, a programmed cell death mechanism that produces inflammation. Despite evidence for inflammasome activation in immune cells during sepsis, it was unknown whether extracellular histones can produce endothelial inflammasomes activation. Methods: We used human umbilical vein endothelial cells (HUVEC) to explore the activation of pyroptosis, endothelial function and inflammation by extracellular histones. We evaluated pyroptosis by flow cytometry, caspase-1 activity assay, and gene and protein expression analysis by RT-qPCR and Western blot, respectively. The upstream molecular responses involved in pyroptosis activation by extracellular histones were validated by means of using antioxidant glutathione ethyl ester and NLRP3 inflammasome inhibitors. Finally, using mass spectrometry, we measured circulating histones in blood from critically-ill patients and demonstrated that circulating histone levels correlated with the expression of pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity. Results: We found that extracellular histones mediate the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how the hyperacetylation of extracellular histones or the use of antioxidants decreased pyroptosis. In addition, we showed that pyroptosis is a feasible process occurring in septic shock patients. Discussion: Circulating histone levels correlated with the expression of pro-inflammatory and pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity. We propose to block histone-mediated pyroptosis as a feasible therapeutic strategy in sepsis.

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-α, 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.

Histone Citrullination Mediates a Protective Role in Endothelium and Modulates Inflammation.

NETosis is a key host immune process against a pathogenic infection during innate immune activation, consisting of a neutrophil "explosion" and, consequently, NET formation, containing mainly DNA, histones, and other nuclear proteins. During sepsis, an exacerbated immune host response to an infection occurs, activating the innate immunity and NETosis events, which requires histone H3 citrullination. Our group compared the circulating histone levels with those citrullinated H3 levels in plasma samples of septic patients. In addition, we demonstrated that citrullinated histones were less cytotoxic for endothelial cells than histones without this post-translational modification. Citrullinated histones did not affect cell viability and did not activate oxidative stress. Nevertheless, citrullinated histones induced an inflammatory response, as well as regulatory endothelial mechanisms. Furthermore, septic patients showed elevated levels of circulating citrullinated histone H3, indicating that the histone citrullination is produced during the first stages of sepsis, probably due to the NETosis process.

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 profile and affect their results. This study determines if the blood starting material is a source of variance in miRNA profile 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 significant increase was observed in patients with Non-ST-elevation myocardial infarction (NSTEMI) compared to control volunteers. When normalized by the expression of miR-484, different patterns of miRNA expression between serum and plasma. Although NSTEMI modified 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, differences were only detected in serum (p = 0.0240), and conversely, miR-499a showed differences 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 different patterns of circulating miRNA expression in NSTEMI and suggest that results from studies with different starting material could not be comparable.

Microparticles harbouring Sonic hedgehog morphogen improve the vasculogenesis capacity of endothelial progenitor cells derived from myocardial infarction patients.

Aims: Endothelial progenitor cells (EPC) play a role in endothelium integrity maintenance and regeneration. Decreased numbers of EPC or their impaired function correlates with an increase in cardiovascular events. Thus, EPC are important predictors of cardiovascular mortality and morbidity. Microparticles carrying Sonic hedgehog (Shh) morphogen (MPShh+) trigger pro-angiogenic responses, both in endothelial cells and in ischaemic rodent models. Here, we propose that MPShh+ regulates EPC function, thus enhancing vasculogenesis, and correcting the defects in dysfunctional EPC obtained from acute myocardial infarction (AMI) patients. Methods and results: The mechanisms underlying Shh pathway function and nitric oxide (NO) production in EPC were evaluated. MPShh+ increased both the in vitro and in vivo vasculogenic capacity of EPC isolated from adult human peripheral blood samples. MPShh+ treatment significantly increased the expression of Shh signalling pathway genes (PTCH1, SMO, and GLI1) and masters of pro-angiogenic genes (NOS3, VEGFA, KDR, and KLF2) in EPC. Moreover, MPShh+ increased both the protein expression and activity of eNOS, resulting in increased NO production. Most importantly, MPShh+ improved the vasculogenic capacity of EPC from AMI patients to levels similar to that of EPC from healthy patients. All these effects were due to the activation of Shh pathway. Conclusion: MPShh+ increase both the vasculogenesis of EPC and their capacity to produce NO, including EPC from patients who have recently suffered an AMI. This study emphasizes MPShh+ and EPC as potential therapeutic tools for improving vascular regeneration as a treatment for cardiovascular ischaemic disease.

Catalyzing Transcriptomics Research in Cardiovascular Disease: The CardioRNA COST Action CA17129.

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field.COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).

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.