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.

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.

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.

Rationale: Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction, but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated. Objective: To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS. Methods and Results: Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared with those from non-MetS subjects. Rap1 was in GTP-associated active state in both types of lEVs, and Rap1-lEVs levels correlated with increased cardiovascular risks, including stenosis. MetS-lEVs, but not non-MetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of proinflammatory molecules and activation of ERK (extracellular signal-regulated kinase) 5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 completely prevented the effects of lEVs from MetS patients. High-fat diet-fed ApoE ^−/− mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from high-fat diet-fed ApoE ^−/− mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1. Conclusions: These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, proinflammatory profile, and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis. Graphical A graphical abstract is available for this article.