Felix Jansen

MicroRNA Expression in Circulating Microvesicles Predicts Cardiovascular Events in Patients With Coronary Artery Disease

Background Circulating microRNAs (miRNAs) are differentially regulated and selectively packaged in microvesicles (MVs). We evaluated whether circulating vascular and endothelial miRNAs in patients with stable coronary artery disease have prognostic value for the occurrence of cardiovascular (CV) events. Methods and Results Ten miRNAs involved in the regulation of vascular performance—miR‐126, miR‐222, miR‐let7d, miR‐21, miR‐20a, miR‐27a, miR‐92a, miR‐17, miR‐130, and miR‐199a—were quantified in plasma and circulating MVs by reverse transcription polymerase chain reaction in 181 patients with stable coronary artery disease. The median duration of follow‐up for major adverse CV event–free survival was 6.1 years (range: 6.0–6.4 years). Events occurred in 55 patients (31.3%). There was no significant association between CV events and plasma level of the selected miRNAs. In contrast, increased expression of miR‐126 and miR‐199a in circulating MVs was significantly associated with a lower major adverse CV event rate. In univariate analysis, above‐median levels of miR‐126 in circulating MVs were predictors of major adverse CV event–free survival (hazard ratio: 0.485 [95% CI: 0.278 to 0.846]; P=0.007) and percutaneous coronary interventions (hazard ratio: 0.458 [95% CI: 0.222 to 0.945]; P=0.03). Likewise, an increased level of miR‐199a in circulating MVs was associated with a reduced risk of major adverse CV events (hazard ratio: 0.518 [95% CI: 0.299 to 0.898]; P=0.01) and revascularization (hazard ratio: 0.439 [95% CI: 0.232 to 0.832]; P=0.01) in univariate analysis. miRNA expression analysis in plasma compartments revealed that miR‐126 and miR‐199a are present mainly in circulating MVs. MV‐sorting experiments showed that endothelial cells and platelets were found to be the major cell sources of MVs containing miR‐126 and miR‐199a, respectively. Conclusion MVs containing miR‐126 and miR‐199a but not freely circulating miRNA expression predict the occurrence of CV events in patients with stable coronary artery disease.

Endothelial Microparticle Uptake in Target Cells Is Annexin I/Phosphatidylserine Receptor Dependent and Prevents Apoptosis

Objective—Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. Methods and Results—EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I–downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. Conclusion—EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor–dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.

Vascular endothelial microparticles-incorporated microRNAs are altered in patients with diabetes mellitus

BackgroundCirculating microRNAs (miRs) are differentially regulated and selectively packaged into microparticles (MPs). We evaluated whether diabetes mellitus alters circulating vascular and endothelial MP-incorporated miRs expression levels.Methods and resultsCirculating MPs were isolated from 135 patients with or without diabetes mellitus type II and characterized using flow cytometer and electron microscope. Nine miRs involved in the regulation of vascular performance—miR-126, miR-222, miR-let7d, miR-21, miR-30, miR-92a, miR-139, miR-199a and miR-26a—were quantified in circulating MPs by reverse transcription polymerase chain reaction. Among those, miR-126 and miR-26a were significantly reduced in diabetic patients compared to non-diabetic patients. Patients with low miR-26a and miR-126 levels were at higher risk for a concomitant coronary artery disease. MP-sorting experiments showed that endothelial cells were the major cell sources of MPs containing miR-126 and miR-26a, respectively. Finally, in accordance with our clinical results, in vitro experiments revealed that hyperglycemia reduces the packaging of miR-126 and miR-26a into EMPs.ConclusionDiabetes mellitus significantly alters the expression of vascular endothelial miRs in circulating endothelial MPs with potential implications on vascular heath.

Effects of high intensity training and high volume training on endothelial microparticles and angiogenic growth factors.

Aims Endothelial microparticles (EMP) are complex vesicular structures shed from activated or apoptotic endothelial cells. As endurance exercise affects the endothelium, the objective of the study was to examine levels of EMP and angiogenic growth factors following different endurance exercise protocols. Methods 12 subjects performed 3 different endurance exercise protocols: 1. High volume training (HVT; 130 min at 55% peak power output (PPO); 2. 4×4 min at 95% PPO; 3. 4×30 sec all-out. EMPs were quantified using flow cytometry after staining platelet-poor-plasma. Events positive for Annexin-V and CD31, and negative for CD42b, were classified as EMPs. Vascular endothelial growth factor (VEGF), migratory inhibiting factor (MIF) and hepatocyte growth factor (HGF) were determined by ELISA technique. For all these measurements venous blood samples were taken pre, 0′, 30′, 60′ and 180′ after each intervention. Furthermore, in vitro experiments were performed to explore the effect of collected sera on target endothelial functions and MP uptake capacities. Results VEGF and HGF significantly increased after HIT interventions. All three interventions caused a significant decrease in EMP levels post exercise compared to pre values. The sera taken after exercise increased the uptake of EMP in target endothelial cells compared to sera taken under resting conditions, which was shown to be phosphatidylserin-dependent. Increased EMP uptake was associated with an improved protection of target cells against apoptosis. Sera taken prior and after exercise promoted target endothelial cell migration, which was abrogated after inhibition of VEGF. Conclusion Physical exercise leads to decreased EMP levels and promotes a phosphatidylserin-dependent uptake of EMP into target endothelial cells, which is associated with a protection of target cells against apoptosis.

Extracellular Vesicles in Cardiovascular Disease: Potential Applications in Diagnosis, Prognosis, and Epidemiology

Extracellular vesicles originate from diverse subcellular compartments and are released in the extracellular space. By transferring their cargoes into target cells and tissues, they now emerge as novel regulators of intercellular communication between adjacent and remote cells. Because vesicle composition and biological content are specific signatures of cellular activation and injury, their potential as diagnostic and prognostic biomarkers has raised significant interest in cardiovascular diseases. Characterization of circulating vesicles- or nonvesicles-bound nucleic acids represents a valuable tool for diagnosing and monitoring cardiovascular diseases, recently referred to as a liquid biopsy. Circulating extracellular vesicles offer a noninvasive and almost continuous access to circulating information on the disease state in epidemiological investigations. Finally, genetic engineering and cell-specific application of extracellular vesicles could display a novel therapeutic option for the treatment of cardiovascular diseases. In this review, we summarize the current knowledge about extracellular vesicles as diagnostic and prognostic biomarkers, as well as their potential applications for longitudinal epidemiological studies in cardiovascular diseases.

Acute Effects of Different Exercise Protocols on the Circulating Vascular microRNAs -16, -21, and -126 in Trained Subjects

Aim: mircoRNAs (miRNAs), small non-coding RNAs regulating gene expression, are stably secreted into the blood and circulating miRNAs (c-miRNAs) may play an important role in cell–cell communication. Furthermore, c-miRNAs might serve as novel biomarkers of the current vascular cell status. Here, we examined how the levels of three vascular c-miRNAs (c-miR-16, c-miR-21, c-miR-126) are acutely affected by different exercise intensities and volumes. Methods: 12 subjects performed 3 different endurance exercise protocols: 1. High-Volume Training (HVT; 130 min at 55% peak power output (PPO); 2. High-Intensity Training (HIT; 4 × 4 min at 95% PPO); 3. Sprint-Interval Training (SIT; 4 × 30 s all-out). c-miRNAs were quantified using quantitative real-time PCR with TaqMan probes at time points pre, 0′, 30′, 60′, and 180′ after each intervention. The expression of miR-126 and miR-21 was analyzed in vitro, in human coronary artery endothelial cells, human THP-1 monocytes, human platelets, human endothelial microparticles (EMPs) and human vascular smooth muscle cells (VSMCs). To investigate the transfer of miRNAs via EMPs, VSMCs were incubated with EMPs. Results: HVT and SIT revealed large increases on c-miR-21 [1.9-fold by HVT (cohens d = 0.85); 1.5-fold by SIT (cohens d = 0.85)] and c-miR-126 [2.2-fold by SIT (cohens d = 1.06); 1.9-fold by HVT (cohens d = 0.85)] post-exercise compared to pre-values, while HIT revealed only small to moderate changes on c-miRs-21 (cohens d = −0.28) and c-miR-126 (cohens d = 0.53). c-miR-16 was only slightly affected by SIT (1.4-fold; cohens d = 0.57), HVT (1.3-fold; cohens d = 0.61) or HIT (1.1-fold; cohens d = 0.2). Further in vitro experiments revealed that miR-126 and miR-21 are mainly of endothelial origin. Importantly, under conditions of endothelial apoptosis, miR-126 and miR-21 are packed from endothelial cells into endothelial microparticles, which were shown to transfer miR-126 into target vascular smooth muscle cells. Conclusion: Taken together, we found that HVT and SIT are associated with the release of endothelial miRNAs into the circulation, which can function as intercellular communication devices regulating vascular biology.

Role and Function of MicroRNAs in Extracellular Vesicles in Cardiovascular Biology

Intercellular communication mediated by extracellular vesicles is crucial for preserving vascular integrity and in the development of cardiovascular disease. Extracellular vesicles consist of apoptotic bodies, microvesicles, and exosomes that can be found in almost every fluid compartment of the body like blood, saliva, and urine. In the recent years, a lot of reports came up suggesting that major cardiovascular and metabolic pathologies like atherogenesis, heart failure, or diabetes are highly influenced by transfer of microRNAs via extracellular vesicles leading to altered protein expression and phenotypes of recipient cells. The following review will summarize the fast developing field of intercellular signaling in cardiovascular biology by microRNA-containing extracellular vesicles.

Intercellular transfer of miR-126-3p by endothelial microparticles reduces vascular smooth muscle cell proliferation and limits neointima formation by inhibiting LRP6

BACKGROUND Vascular smooth muscle cell (VSMC) proliferation is of importance in the pathogenesis of vascular diseases such as restenosis or atherosclerosis. Endothelial microparticles (EMPs) regulate function and phenotype of target endothelial cells (ECs), but their influence on VSMC biology is unknown. We aim to investigate the role of EMPs in the regulation of vascular smooth muscle cell (VSMC) proliferation and vascular remodeling. METHODS AND RESULTS Systemic treatment of mice with EMPs after vascular injury reduced neointima formation in vivo. In vitro, EMP uptake in VSMCs diminished VSMC proliferation and migration, both pivotal steps in neointima formation. To explore the underlying mechanisms, Taqman microRNA-array was performed and miR-126-3p was identified as the predominantly expressed miR in EMPs. Confocal microscopy revealed an EMP-mediated miR-126 transfer into recipient VSMCs. Expression of miR-126 target protein LRP6, regulating VSMC proliferation, was reduced in VSMCs after EMP treatment. Importantly, genetic regulation of miR-126 in EMPs showed a miR-126-dependent inhibition of LRP6 expression, VSMC proliferation and neointima formation in vitro and in vivo, suggesting a crucial role of miR-126 in EMP-mediated neointima formation reduction. Finally, analysis of miR-126 expression in circulating MPs in 176 patients with coronary artery disease revealed a reduced PCI rate in patients with high miR-126 expression level, supporting a central role for MP-incorporated miR-126 in vascular remodelling. CONCLUSION EMPs reduce VSMC proliferation, migration and subsequent neointima formation by delivering functional miR-126 into recipient VSMCs.

Kinetics of Circulating MicroRNAs in Response to Cardiac Stress in Patients With Coronary Artery Disease

Background Circulating microRNAs (miRNAs/miRs) are regulated in patients with coronary artery disease. The impact of transient coronary ischemia on circulating miRNA levels is unknown. We aimed to investigate circulating miRNA kinetics in response to cardiac stress in patients with or without significant coronary stenosis. Methods and Results Eighty of 105 screened patients with stable coronary artery disease underwent dobutamine stress echocardiography before coronary angiography. Nine circulating vascular miRNAs (miRNA‐21, miRNA‐26, miRNA‐27a, miRNA‐92a, miRNA‐126‐3p, miRNA‐133a, miRNA‐222, miRNA‐223, and miRNA‐199‐5p) were quantified in plasma by reverse transcription polymerase chain reaction before, immediately after, and 4 and 24 hours after dobutamine stress echocardiography. Quantitative polymerase chain reaction revealed increased miRNA‐21, miRNA‐126‐3p, and miRNA‐222 levels at 24 hours after dobutamine stress echocardiography in all patients. On coronary angiography, significant coronary artery stenoses (>80% diameter stenosis) were found in 41 patients. Stratifying patients according to the prevalence of significant stenoses, patients with stenosis showed an increase of circulating miRNA‐21, miRNA‐126‐3p, and miRNA‐222 in response to cardiac stress. In patients without significant stenoses (<50% diameter stenosis), miRNA‐92a levels gradually increased in response to cardiac stress. Conclusions miRNAs are distinctly released into the circulation in response to cardiac stress depending on the prevalence of significant coronary stenoses.

Endothelial- and Immune Cell-Derived Extracellular Vesicles in the Regulation of Cardiovascular Health and Disease

Summary Intercellular signaling by extracellular vesicles (EVs) is a route of cell-cell crosstalk that allows cells to deliver biological messages to specific recipient cells. EVs convey these messages through their distinct cargoes consisting of cytokines, proteins, nucleic acids, and lipids, which they transport from the donor cell to the recipient cell. In cardiovascular disease (CVD), endothelial- and immune cell-derived EVs are emerging as key players in different stages of disease development. EVs can contribute to atherosclerosis development and progression by promoting endothelial dysfunction, intravascular calcification, unstable plaque progression, and thrombus formation after rupture. In contrast, an increasing body of evidence highlights the beneficial effects of certain EVs on vascular function and endothelial regeneration. However, the effects of EVs in CVD are extremely complex and depend on the cellular origin, the functional state of the releasing cells, the biological content, and the diverse recipient cells. This paper summarizes recent progress in our understanding of EV signaling in cardiovascular health and disease and its emerging potential as a therapeutic agent.