Growing evidence suggests that ceramides play an important role in the development of atherosclerotic and valvular heart disease. Ceramides are biologically active sphingolipids that are produced by a complex network of enzymes. Lowering cellular and tissue levels of ceramide by inhibiting the ceramide-producing enzymes counteracts atherosclerotic and valvular heart disease development in animal models. In vascular tissues, ceramides are produced in response to hyperglycemia and TNF (tumor necrosis factor)-α signaling and are involved in NO-signaling and inflammation. In humans, elevated blood ceramide levels are associated with cardiovascular events. Furthermore, important cardiovascular risk factors, such as obesity and diabetes, have been linked to ceramide accumulation. This review summarizes the basic mechanisms of how ceramides drive cardiovascular disease locally and links these findings to the intriguing results of human studies on ceramides as biomarkers for cardiovascular events. Moreover, we discuss the current state of interventions to therapeutically influence vascular ceramide metabolism, both locally and systemically.

The transfer of microRNAs (miRs) via extracellular vesicles (EVs) is a functionally relevant mechanism of intercellular communication that regulates both organ homoeostasis and disease development. Little is known about the packaging of miRs into EVs. Previous studies have shown that certain miRs are exported by RNA‐binding proteins into small EVs, while for other miRs and for large EVs, in general, the export mechanisms remain unclear. Therefore, a proteomic analysis of endothelial cell‐derived large EVs was performed, which revealed that heterogeneous nuclear ribonucleoprotein U (hnRNPU) is abundantly present in EVs. EVs were characterized by electron microscopy, immunoblotting and nanoparticle tracking analysis. Taqman microRNA array and single qPCR experiments identified specific miR patterns to be exported into EVs in an hnRNPU‐dependent way. The specific role of hnRNPU for vesicular miR‐sorting was confirmed independently by gain‐ and loss‐of‐function experiments. In our study, miR‐30c‐5p was the miR whose export was most significantly regulated by hnRNPU. Mechanistically, in silico binding analysis showed that the export of miRs into EVs depends on the binding efficiency of the respective miRs to hnRNPU. Among the exported miRs, a significant enrichment of the sequence motif AAMRUGCU was detected as a potential sorting signal. Experimentally, binding of miR‐30c‐5p to hnRNPU was confirmed independently by RNA‐immunoprecipitation, electrophoretic mobility shift assay and reciprocally by miR‐pulldown. Nuclear binding of miR‐30c‐5p to hnRNPU and subsequent stabilization was associated with a lower cytoplasmatic abundance and consequently reduced availability for vesicular export. hnRNPU‐dependent miR‐30c‐5p export reduced cellular migration as well as pro‐angiogenic gene expression in EV‐recipient cells. In summary, hnRNPU retains miR‐30c‐5p and other miRs and thereby prevents their export into large EVs. The data presented provide a novel and functionally relevant mechanism of vesicular miR export.

Background: Transcatheter aortic valve replacement (TAVR) is a well-established treatment option for high- and intermediate-risk patients with severe symptomatic aortic valve stenosis. A majority of patients exhibit improvements in left ventricular ejection fraction (LVEF) after TAVR in response to TAVR-associated afterload reduction. However, a specific role for circulating microRNAs (miRNAs) in the improvement of cardiac function for patients after TAVR has not yet been investigated. Here, we profiled the differential expression of miRNAs in circulating extracellular vesicles (EVs) in patients after TAVR and, in particular, the novel role of circulating miR-122-5p in cardiomyocytes. Methods: Circulating EV-associated miRNAs were investigated by use of an unbiased Taqman-based human miRNA array. Several EV miRNAs (miR-122-5p, miR-26a, miR-192, miR-483-5p, miR-720, miR-885-5p, and miR-1274) were significantly deregulated in patients with aortic valve stenosis at day 7 after TAVR compared with the preprocedural levels in patients without LVEF improvement. The higher levels of miR-122-5p were negatively correlated with LVEF improvement at both day 7 ( r =−0.264 and P =0.015) and 6 months ( r =−0.328 and P =0.0018) after TAVR. Results: Using of patient-derived samples and a murine aortic valve stenosis model, we observed that the expression of miR-122-5p correlates negatively with cardiac function, which is associated with LVEF. Mice with graded wire injury–induced aortic valve stenosis demonstrated a higher level of miR-122-5p, which was related to cardiomyocyte dysfunction. Murine ex vivo experiments revealed that miR-122-5p is highly enriched in endothelial cells compared with cardiomyocytes. Coculture experiments, copy-number analysis, and fluorescence microscopy with Cy3-labeled miR-122-5p demonstrated that miR-122-5p can be shuttled through large EVs from endothelial cells into cardiomyocytes. Gain- and loss-of-function experiments suggested that EV-mediated shuttling of miR-122-5p increases the level of miR-122-5p in recipient cardiomyocytes. Mechanistically, mass spectrometry, miRNA pulldown, electrophoretic mobility shift assay, and RNA immunoprecipitation experiments confirmed that miR-122-5p interacts with the RNA-binding protein hnRNPU (heterogeneous nuclear ribonucleoprotein U) in a sequence-specific manner to encapsulate miR-122-5p into large EVs. On shuttling, miR-122-5p reduces the expression of the antiapoptotic gene BCL2 by binding to its 3′ untranslated region to inhibit its translation, thereby decreasing the viability of target cardiomyocytes. Conclusions: Increased levels of circulating proapoptotic EV-incorporated miR-122-5p are associated with reduced LVEF after TAVR. EV shuttling of miR-122-5p regulates the viability and apoptosis of cardiomyocytes in a BCL2-dependent manner.

Aims: Chronic kidney disease (CKD) is an independent risk factor for the development of coronary artery disease (CAD). For both, CKD and CAD, the intercellular transfer of microRNAs (miRs) through extracellular vesicles (EVs) is an important factor of disease development. Whether the combination of CAD and CKD affects endothelial function through cellular crosstalk of EV-incorporated miRs is still unknown. Methods and results: Out of 172 screened CAD patients, 31 patients with CAD + CKD were identified and matched with 31 CAD patients without CKD. Additionally, 13 controls without CAD and CKD were included. Large EVs from CAD + CKD patients contained significantly lower levels of the vasculo-protective miR-130a-3p and miR-126-3p compared to CAD patients and controls. Flow cytometric analysis of plasma-derived EVs revealed significantly higher numbers of endothelial cell-derived EVs in CAD and CAD + CKD patients compared to controls. EVs from CAD + CKD patients impaired target human coronary artery endothelial cell (HCAEC) proliferation upon incubation in vitro. Consistent with the clinical data, treatment with the uraemia toxin indoxyl sulfate (IS)-reduced miR-130a-3p levels in HCAEC-derived EVs. EVs from IS-treated donor HCAECs-reduced proliferation and re-endothelialization in EV-recipient cells and induced an anti-angiogenic gene expression profile. In a mouse-experiment, intravenous treatment with EVs from IS-treated endothelial cells significantly impaired endothelial regeneration. On the molecular level, we found that IS leads to an up-regulation of the heterogenous nuclear ribonucleoprotein U (hnRNPU), which retains miR-130a-3p in the cell leading to reduced vesicular miR-130a-3p export and impaired EV-recipient cell proliferation. Conclusion: Our findings suggest that EV-miR-mediated vascular intercellular communication is altered in patients with CAD and CKD, promoting CKD-induced endothelial dysfunction.

Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular disease. However, the expression pattern of circulating extracellular vesicle (EV)-incorporated lncRNAs in patients with coronary artery disease (CAD) is still poorly investigated. A human lncRNA array revealed that certain EV-lncRNAs are significantly dysregulated in CAD patients. Circulating small EVs (sEVs) from patients with (n = 30) or without (n = 30) CAD were used to quantify PUNISHER (also known as AGAP2-antisense RNA 1 [AS1]), GAS5, MALAT1, and H19 RNA levels. PUNISHER (p = 0.002) and GAS5 (p = 0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Fluorescent labeling and quantitative real-time PCR of sEVs demonstrated that functional PUNISHER was transported into the recipient cells. Mechanistically, the RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK), interacts with PUNISHER, regulating its loading into sEVs. Knockdown of PUNISHER abrogated the EV-mediated effects on endothelial cell (EC) migration, proliferation, tube formation, and sprouting. Angiogenesis-related gene profiling showed that the expression of vascular endothelial growth factor A (VEGFA) RNA was significantly increased in EV recipient cells. Protein stability and RNA immunoprecipitation indicated that the PUNISHER-hnRNPK axis regulates the stability and binding of VEGFA mRNA to hnRNPK. Loss of PUNISHER in EVs abolished the EV-mediated promotion of VEGFA gene and protein expression. Intercellular transfer of EV-incorporated PUNISHER promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.

Background: Cardiovascular disease (CVD) remains the leading cause of death worldwide. The main driving force behind this association is coronary artery disease (CAD), the manifestation of atherosclerosis in the coronary circulation. Cornerstones in the development of CAD are pathologies in lipid metabolism. In recent years, ongoing research has identified ceramides, a subclass of sphingolipids to be mediators of CVD. The aim of this study is to investigate the influence of type II diabetes mellitus (DM) on circulating ceramides and hexosylceramides (HexCers) in CAD patients. Methods: 24 patients aged 40–90 years with CAD confirmed by angiography were included into a pilot study. Patients with DM were identified by analysis of discharge letters or other medical documents available at the study center. During coronary angiography, arterial blood samples were collected and quantification of sphingolipids in patient serum was performed by mass spectrometry. Results: Statistical analysis showed nine significantly different HexCers in CAD patients with DM compared to patients without DM. Among the nine significantly regulated HexCers, we identified seven d18:1 HexCers. This group contributes to the fourth most abundant subgroup of total ceramides and HexCers in this dataset. HexCer-d18:1–23:1(2-OH) showed the strongest downregulation in the patient group with DM. Conclusion: This study suggests that levels of circulating HexCers are downregulated in patients with CAD and concomitant DM compared to patients without DM. Further research is needed to investigate the underlying mechanisms and the suitability of HexCers as possible mediators and/or prognostic markers in CAD.