Increased expression of miR-224-5p in circulating extracellular vesicles of patients with reduced coronary flow reserve.

BACKGROUND: Endothelial and microvascular dysfunction are pivotal causes of major adverse cardiac events predicted by coronary flow reserve (CFR). Extracellular Vesicles (EVs) have been studied extensively in the pathophysiology of coronary artery disease. However, little is known on the impact of the non-coding RNA content of EVs with respect to CFR. METHODS: We carried out a study among 120 patients divided by high-CFR and low-CFR to profile the miRNA content of circulating EVs. RESULTS: A multiplex array profiling on circulating EVs revealed mir-224-5p (p-value ≤ 0.000001) as the most differentially expressed miRNA in the Low-CFR group and showed a significantly independent relationship to CFR. Literature survey indicated the origin of the miR from liver cells and not of platelet, leukocyte, smooth muscle or endothelial (EC) origin. A q-PCR panel of the conventional cell type-EVs along with hepatic EVs showed that EVs from liver cells showed higher expression of the miR-224-5p. FACS analysis demonstrated the presence of liver-specific (ASGPR-1+/CD14-) EVs in the plasma of our cohort with the presence of Vanin-1 required to enter the EC barrier. Hepatic EVs with and without the miR-224-5p were introduced to ECs in-vitro, but with no difference in effect on ICAM-1 or eNOS expression. However, hepatic EVs elevated endothelial ICAM-1 levels per se independent of the miR-224-5p. CONCLUSION: This indicated a role of hepatic EVs identified by the miR-224-5p in endothelial dysfunction in patients with Low CFR.

Microvesicles in plasma reflect coronary flow reserve in patients with cardiovascular disease.

High levels of microvesicles (MVs), a type of extracellular vesicles, are detected in several pathological conditions. We investigated the connection between coronary flow reserve (CFR), a prognostic clinical parameter that reflects blood flow in the heart, with levels of MVs and their cargo, from plasma of patients with cardiovascular disease. The PROFLOW study consists of 220 patients with prior myocardial infarction and measured CFR with transthoracic echocardiography. The patients were divided into high and low CFR groups. Plasma MVs were captured with acoustic trapping. Platelet- and endothelial-derived MVs were measured with flow cytometry, and vesicle lysates were analyzed with proteomic panels against cardiovascular biomarkers. Flow cytometry was further applied to identify cellular origin of biomarkers. Our data show a negative correlation between MV concentration and CFR values. Platelet and endothelial MV levels were significantly increased in plasma from the low CFR group. CFR negatively correlates with the levels of several proteomic biomarkers, and the low CFR group exhibited higher concentrations of these proteins in MVs. Focused analysis of one of the MV proteins, B cell activating factor (BAFF), revealed platelet and not leukocyte origin and release upon proinflammatory stimulus. Higher levels of MVs carrying an elevated concentration of proatherogenic proteins circulate in plasma in patients with low CFR, a marker of vascular dysfunction, reduced blood flow, and poor prognosis. Our findings demonstrate a potential clinical value of MVs as biomarkers and possible therapeutic targets against endothelial deterioration.NEW & NOTEWORTHY We investigated how microvesicles (MVs) from patients with cardiovascular diseases are related to coronary flow reserve (CFR), a clinical parameter reflecting blood flow in the heart. Our results show a negative relationship between CFR and levels of platelet and endothelial MVs. The pattern of MV-enriched cardiovascular biomarkers differs between patients with high and low CFR. Our findings suggest a potential clinical value of MVs as biomarkers of reduced blood flow and proatherogenic status, additional to CFR.

Bilberry Supplementation after Myocardial Infarction Decreases Microvesicles in Blood and Affects Endothelial Vesiculation.

SCOPE: Diet rich in bilberries is considered cardioprotective, but the mechanisms of action are poorly understood. Cardiovascular disease is characterized by increased proatherogenic status and high levels of circulating microvesicles (MVs). In an open-label study patients with myocardial infarction receive an 8 week dietary supplementation with bilberry extract (BE). The effect of BE on patient MV levels and its influence on endothelial vesiculation in vitro is investigated. METHODS AND RESULTS: MVs are captured with acoustic trapping and platelet-derived MVs (PMVs), as well as endothelial-derived MVs (EMVs) are quantified with flow cytometry. The in vitro effect of BE on endothelial extracellular vesicle (EV) release is examined using endothelial cells and calcein staining. The mechanisms of BE influence on vesiculation pathways are studied by Western blot and qRT-PCR. Supplementation with BE decreased both PMVs and EMVs. Furthermore, BE reduced endothelial EV release, Akt phosphorylation, and vesiculation-related gene transcription. It also protects the cells from P2X7 -induced EV release and increase in vesiculation-related gene expression. CONCLUSION: BE supplementation improves the MV profile in patient blood and reduces endothelial vesiculation through several molecular mechanisms related to the P2X7 receptor. The findings provide new insight into the cardioprotective effects of bilberries.

Effect of exercise on the plasma vesicular proteome: a methodological study comparing acoustic trapping and centrifugation.

Extracellular vesicles (EVs) are a heterogeneous group of actively released vesicles originating from a wide range of cell types. Characterization of these EVs and their proteomes in the human plasma provides a novel approach in clinical diagnostics, as they reflect physiological and pathological states. However, EV isolation is technically challenging with the current methods having several disadvantages, requiring large sample volumes, and resulting in loss of sample and EV integrity. Here, we use an alternative, non-contact method based on a microscale acoustic standing wave technology. Improved coupling of the acoustic resonator increased the EV recovery from 30% in earlier reports to 80%, also displaying long term stability between experiment days. We report a pilot study, with 20 subjects who underwent physical exercise. Plasma samples were obtained before and 1 h after the workout. Acoustic trapping was compared to a standard high-speed centrifugation protocol, and the method was validated by flow cytometry (FCM). To monitor the device stability, the pooled frozen plasma from volunteers was used as an internal control. A key finding from the FCM analysis was a decrease in CD62E+ (E-selectin) EVs 1 h after exercise that was consistent for both methods. Furthermore, we report the first data that analyse differential EV protein expression before and after physical exercise. Olink-based proteomic analysis showed 54 significantly changed proteins in the EV fraction in response to physical exercise, whereas the EV-free plasma proteome only displayed four differentially regulated proteins, thus underlining an important role of these vesicles in cellular communication, and their potential as plasma derived biomarkers. We conclude that acoustic trapping offers a fast and efficient method comparable with high-speed centrifugation protocols. Further, it has the advantage of using smaller sample volumes (12.5 µL) and rapid contact-free separation with higher yield, and can thus pave the way for future clinical EV-based diagnostics.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells.

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.

Comparative Proteomic Analysis of Extracellular Vesicles Isolated by Acoustic Trapping or Differential Centrifugation.

Extracellular vesicles (ECVs), including microparticles and exosomes, are submicrometer membrane vesicles released by diverse cell types upon activation or stress. Circulating ECVs are potential reservoirs of disease biomarkers, and the complexity of these vesicles is significantly lower compared to their source, blood plasma, which makes ECV-based biomarker studies more promising. Proteomic profiling of ECVs is important not only to discover new diagnostic or prognostic markers but also to understand their roles in biological function. In the current study, we investigated the protein composition of plasma-derived ECVs isolated by acoustic seed trapping. Additionally, the protein composition of ECVs isolated with acoustic trapping was compared to that isolated with a conventional differential centrifugation protocol. Finally, the proteome of ECVs originating from ST-elevation myocardial infarction patients was compared with that of healthy controls using label-free LC-MS quantification. The acoustic trapping platform allows rapid and automated preparation of ECVs from small sample volumes, which are therefore well-suited for biobank repositories. We found that the protein composition of trapped ECVs is very similar to that isolated by the conventional differential centrifugation method.