Loss of ceramide synthase 5 inhibits the development of experimentally induced aortic valve stenosis.

AIM: Inflammation and calcification are hallmarks in the development of aortic valve stenosis (AVS). Ceramides mediate inflammation and calcification in the vascular tissue. The highly abundant d18:1,16:0 ceramide (C16) has been linked to increased cardiovascular mortality and obesity. In this study, we investigate the role of ceramide synthase 5 (CerS5), a critical enzyme for C16 ceramide synthesis, in the development of AVS, particularly in conjunction with a high-fat/high-cholesterol diet (Western diet, WD). METHODS: We used wild-type (WT) and CerS5-/- mice on WD or normal chow in a wire injury model. We measured the peak velocity to determine AVS development and performed histological analysis of the aortic valve area, immune cell infiltration (CD68 staining), and calcification (von Kossa). In vitro experiments involved measuring the calcification of human aortic valvular interstitial cells (VICs) and evaluating cytokine release from THP-1 cells, a human leukemia monocytic-like cell line, following CerS5 knockdown. RESULTS: CerS5-/- mice showed a reduced peak velocity compared to WT only in the experiment with WD. Likewise, we observed reduced immune cell infiltration and calcification in the aortic valve of CerS5-/- mice, but only on WD. In vitro, calcification was reduced after knockdown of CerS5 in VICs, while THP-1 cells exhibited a decreased inflammatory response following CerS5 knockdown. CONCLUSION: We conclude that CerS5 is an important mediator for the development of AVS in mice on WD and regulates critical pathophysiological hallmarks of AVS formation. CerS5 is therefore an interesting target for pharmacological therapy and merits further investigation.

Comparison of Arctic Front Advance Pro and POLARx cryoballoons for ablation therapy of atrial fibrillation: an intraprocedural analysis.

INTRODUCTION: Cryoballoon (CB) ablation has become a popular method for pulmonary vein isolation (PVI) in atrial fibrillation (AF) treatment. This study aimed to compare the intraprocedural ablation characteristics of two cryoballoons, Arctic Front Advance Pro™ (AFA-Pro, Medtronic) and POLARx™ (Boston Scientific). METHODS AND RESULTS: In this retrospective single-center study, 230 symptomatic paroxysmal or persistent AF patients underwent CB ablation with either AFA-Pro or POLARx. Propensity-score matching resulted in two cohorts of 114 patients each. Baseline and procedural characteristics were comparable between both CBs. POLARx achieved lower minimal temperatures (e.g., left superior pulmonary vein, LSPV: AFA-Pro - 49.0 °C vs. POLARx - 59.5 °C) and lower temperatures at time-to-isolation (TTI). Additionally, POLARx reached lower temperatures faster, as evidenced by lower temperatures after 40 and 60 s, and a larger mean temperature change between 20 and 40 s. POLARx also had a greater area under the curve below 0 °C and a longer thawing phase. Both CBs achieved comparable high rates of final PV-isolation. TTI, minimal esophagus temperature, and first-pass isolation rates were similar between groups. Periprocedural complications, including phrenic nerve injuries, were comparable. Troponin levels in the left atrium were elevated with both systems. Values and change in troponin were numerically higher in the POLARx group (delta troponin: AFA-Pro 36.3 (26.4, 125.4) ng/L vs. POLARx 104.9 (49.5, 122.2) ng/L), p = 0.077). CONCLUSION: AFA-Pro and POLARx are both highly effective and safe CB systems for PVI. POLARx exhibited significant faster and lower freezing characteristics, and numerically higher troponin levels might indicate greater myocardial injury. However, these differences did not translate into improved performance, procedural efficiency, or safety.

Analysis of circulating ceramides and hexosylceramides in patients with coronary artery disease and type II diabetes mellitus.

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.

A prospective, randomized, controlled, multicentre trial for secondary prevention in patients with chronic coronary syndrome using a smartphone application for digital therapy: the CHANGE study protocol.

Aims: Coronary artery disease (CAD) remains the leading cause of death worldwide. 'Stable' CAD is a chronic progressive condition, which recent European guidelines recommend referring to as 'chronic coronary syndrome' (CCS). Despite therapeutic advances, morbidity and mortality among patients with CCS remain high. Optimal secondary prevention in patients with CCS includes optimization of modifiable risk factors with behavioural changes and pharmacological therapy. The CHANGE study aims to provide evidence for optimization of secondary prevention in CCS patients by using a smartphone application (app). Methods and results: The CHANGE study is designed as a prospective, randomized, controlled trial with a 1:1 allocation ratio, which is currently performed in nine centres in Germany in a parallel group design. 210 patients with CCS will be randomly allocated either to the control group (standard-of-care) or to the intervention group, who will be provided the VantisTherapy* app in addition to standard-of-care to incorporate secondary prevention into their daily life. The study will be performed in an open design. Outcomes will be assessed using objective data from three in-person visits (0, 12, and 24 weeks). Primary outcomes will involve adherence to secondary prevention recommendations and quality of life (QoL). The recruitment process started in July 2022. Conclusion: The CHANGE study will investigate whether a smartphone-guided secondary prevention app, combined with a monitor function compared with standard-of-care, has beneficial effects on overall adherence to secondary prevention guidelines and QoL in patients with CCS. Trial registration: The study is listed at the German study registry (DRKS) under the registered number DRKS00028081.

Impact of transradial versus transfemoral access for preprocedural coronary angiography on TAVR-associated complications.

Background: Vascular injury and bleeding complications remain frequent after transcatheter aortic valve replacement (TAVR). Whether the access-site of preprocedural coronary angiography (CAG) affects TAVR-related complications is not known. The aim of this study was to evaluate the impact of transradial (TRA) versus transfemoral access (TFA) for preprocedural CAG on outcomes in patients undergoing subsequent TAVR. Methods: The study cohort included 1002 patients undergoing transfemoral TAVR, of whom 39.4% (395/1002) had undergone radial and 60.6% (607/1002) femoral access for pre-TAVR CAG. The primary endpoint was a composite of 30-day mortality and major vascular complications after TAVR. Key secondary endpoints included VARC-3-defined complications. Results: The primary endpoint occurred less frequently in patients with prior TRA (3.3%) as compared to patients with prior TFA (6.3%, p = 0.04), which was mainly driven by significantly lower rates of major vascular complications (0.8% vs 2.5%, p = 0.05). Moreover, incidences of periprocedural access-related vascular injury and unplanned endovascular interventions were lower in TRA patients (13.2% vs 18.0%, p = 0.05). The rate of major bleeding tended to be lower in the TRA (1.5%) as compared to the TFA group (3.5%) but was not significantly different (p = 0.07). Moreover, the rate of life-threatening bleeding was comparable between both groups (0.5% vs 0.8%, p = 0.71). Conclusion: Transradial access for preprocedural CAG was associated with significantly lower rates of vascular complications following subsequent TAVR as compared to transfemoral access. However, despite the tendency to lower major bleedings with transradial access, no significant association was detectable between the access-site of coronary angiography and TAVR-related bleeding complications.

Toll-like receptor-3 contributes to the development of aortic valve stenosis.

Aortic valve stenosis (AS) development is driven by distinct molecular and cellular mechanisms which include inflammatory pathways. Toll-like-receptor-3 (TLR3) is a lysosomal pattern-recognition receptor that binds double-stranded RNA and promotes pro-inflammatory cellular responses. In recent years, TLR3 has emerged as a major regulator of vascular inflammation. The exact role of TLR3 in the development of AS has not been investigated. Isolated human valvular interstitial cells (VICs) were stimulated with the TLR3-agonist polyIC and the resulting pro-inflammatory and pro-osteogenic response measured. Severe AS was induced in wildtype- and TLR3-/- mice via mechanical injury of the aortic valve with a coronary springwire. TLR3 activation was achieved by polyIC injection every 24 h after wire injury, while TLR3 inhibition was realized using Compound 4a (C4a) every 48 h after surgery. Endothelial mesenchymal transition (EndoMT) of human valvular endothelial cells (VECs) was assessed after polyIC stimulation. Stimulation of human VICs with polyIC promoted a strong inflammatory and pro-osteogenic reaction. Similarly, injection of polyIC marginally increased AS development in mice after wire injury. AS induction was significantly decreased in TLR3-/- mice, confirming the role of endogenous TLR3 ligands in AS pathology. Pharmacological inhibition of TLR3 with C4a not only prevented the upregulation of inflammatory cytokines and osteogenic markers in VICs, and EndoMT in VECs, but also significantly abolished the development of AS in vivo. Endogenous TLR3 activation significantly contributes to AS development in mice. Pharmacological inhibition of TLR3 with C4a prevented AS formation. Therefore, targeting TLR3 may be a viable treatment option.

Optimal protamine-to-heparin dosing ratio for the prevention of bleeding complications in patients undergoing TAVR-A multicenter experience.

BACKGROUND: Despite major advances, transcatheter aortic valve replacement (TAVR) is still associated with procedure-specific complications. Although previous studies reported lower bleeding rates in patients receiving protamine for heparin reversal, the optimal protamine-to-heparin dosing ratio is unknown. HYPOTHESIS: The aim of this study was a comparison of two different heparin antagonization regimens for the prevention of bleeding complications after TAVR. METHODS: The study included 1446 patients undergoing TAVR, of whom 623 received partial and 823 full heparin antagonization. The primary endpoint was a composite of 30-day mortality, life-threatening, and major bleeding. Safety endpoints included stroke and myocardial infarction at 30 days. RESULTS: Full antagonization of heparin resulted in lower rates of the primary endpoint as compared to partial heparin reversal (5.6% vs. 10.4%, p < .01), which was mainly driven by lower rates of life-threatening (0.5% vs. 1.6%, p = .05) and major bleeding (3.2% vs. 7.5%, p < .01). Moreover, the incidence of major vascular complications was significantly lower in patients with full heparin reversal (3.5% vs. 7.5%, p < .01). The need for red-blood-cell transfusion was lower in patients receiving full as compared to partial heparin antagonization (10.4% vs. 15.9%, p < .01). No differences were observed in the incidence of stroke and myocardial infarction between patients with full and partial heparin reversal (2.2% vs. 2.6%, p = .73 and 0.2% vs. 0.4%, p = .64, respectively). CONCLUSIONS: Full heparin antagonization resulted in significantly lower rates of life-threatening and major bleeding after TAVR as compared to partial heparin reversal. The occurrence of stroke and myocardial infarction was low and comparable between both groups.

Ceramide Metabolism in Cardiovascular Disease: A Network With High Therapeutic Potential.

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 lncRNA MRPL20-AS1 is associated with severe OSAS and downregulated upon hypoxic injury of endothelial cells.

INTRODUCTION: Obstructive sleep apnea syndrome (OSAS) is the most common sleep disorder in humans. Although OSAS is clearly related to arterial hypertension, coronary artery disease, and heart failure, it remains unknown through which pathomechanisms OSAS influences cardiovascular health. Recent research has pinpointed long non-coding RNAs (lncRNA) as important molecular mediators of various cardiovascular pathologies. In this study, we have identified the lncRNA MRPL20-AS1 to be affected by OSAS in patients as well as by hypoxia in vitro. METHODS AND RESULTS: A transcriptomic analysis was performed on peripheral blood from four patients with severe OSAS taken after one night of polygraphic assessment. We found that three lncRNAs were significantly dysregulated, of which MRPL20-AS1 was the most significant. In a larger cohort of 22 OSAS patients, MRPL20-AS1 was inversely correlated with the apnea-hypopnea index (AHI). This indicates that OSAS patients with higher AHI levels and therefore more severe OSAS had lower levels of MRPL20-AS1 in the blood. The results were recapitulated in vitro by subjecting endothelial cells to hypoxia. In these experiments, hypoxia led to a significant downregulation of MRPL20-AS1 in endothelial cells. CONCLUSION: MRPL20-AS1 may serve as a useful tool to identify patients suffering from severe OSAS and further research should be done to evaluate the therapeutic potential of MRPL20-AS1 as a target to counteract the cardiovascular effects of OSAS.

Circulating MicroRNA-122-5p Is Associated With a Lack of Improvement in Left Ventricular Function After Transcatheter Aortic Valve Replacement and Regulates Viability of Cardiomyocytes Through Extracellular Vesicles.

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.

Predictive value of the Fibrosis-4 index in patients with severe aortic stenosis undergoing transcatheter aortic valve replacement.

BACKGROUND: Liver dysfunction is associated with an increased risk of mortality after cardiac interventions. The Fibrosis-4 (FIB-4 index), a marker of hepatic fibrosis, has been associated with a worse prognosis in heart failure. The prognostic relevance of the index in patients undergoing transcatheter aortic valve replacement (TAVR) is unknown. The aim of this study was to evaluate the clinical implications associated with the FIB-4 index in patients undergoing TAVR. METHODS: Between May 2012 and June 2019, 941 patients undergoing TAVR were stratified into a low or high FIB-4 index group, based on a cutoff value that was determined according to a receiver operating characteristic curve predicting 1-year all-cause mortality. RESULTS: Patients with a high FIB-4 index (n = 480), based on the cutoff value of 1.82, showed higher rates of pulmonary hypertension (43.8% vs. 31.8%, p < 0.01), right-ventricular systolic dysfunction (29.5% vs. 19.2%, p < 0.01) and larger inferior vena cava diameter (1.6 ± 0.6 cm vs. 1.3 ± 0.6 cm, p < 0.01) than patients with a low FIB-4 index (n = 461). Furthermore, a high FIB-4 index was associated with a significantly higher cumulative 1-year all-cause mortality (17.5% vs. 10.2%, p < 0.01) and non-cardiovascular mortality (12.1% vs. 2.5%, p < 0.01), compared to a low FIB-4 index. Multivariable analysis revealed that a high FIB-4 index was independently associated with all-cause mortality (HR: 1.75 [95% CI: 1.18-2.59], p < 0.01). CONCLUSIONS: A high FIB-4 index is associated with right-sided heart overload and an increased risk of mortality in patients undergoing TAVR. The FIB-4 index may be useful as an additional predictor of outcomes in these patients.

Analysis of nocturnal, hypoxia-induced miRNAs in sleep apnea patients.

INTRODUCTION: Obstructive sleep apnea syndrome (OSAS) is associated with an increased cardiovascular risk. The underlying mechanisms are largely unclear. MicroRNAs (miRNAs) are RNAs circulating in the blood that can be released into the bloodstream during hypoxia. In the present study, we investigate if OSAS-induced hypoxia results in a release of miRNAs that may mediate OSAS-associated cardiovascular damage. METHODS: Blood was sampled from 23 OSAS patients before and after a polygraphically monitored night. Total circulating RNA was isolated from the plasma and quantified using real-time qPCR. Using a Taqman miRNA array, the levels of 384 different miRNAs were compared between evening and morning after polysomnography. The most highly upregulated miRNA (miRNA-505) and four additionally upregulated miRNAs (miRNA-127, miRNA-133a, miRNA-145, and miRNA-181a) were then quantified in a bigger patient cohort individually. RESULTS: Apnea/Hypopnea-Index (AHI) was evaluated and averaged at 26 per hour on nocturnal polygraphy. In an initial miRNA array, a total of 4 miRNAs were significantly regulated. A significant increase of miRNA-145 was observed in the larger patient cohort. No significant changes in concentration were detected for miRNA-127, miRNA-133a, miRNA-181a, and miRNA-505 in this larger cohort. CONCLUSION: OSAS results in the nocturnal release of miRNAs into the bloodstream. Our collected data may indicate a hypoxia-induced release of miRNAs into the bloodstream of OSAS-patients. In vitro experiments are needed to confirm the secretion of these miRNAs under hypoxia and evaluate the effect on the cardio vasculature.

MicroRNA-mediated vascular intercellular communication is altered in chronic kidney disease.

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.

Large extracellular vesicles in the left atrial appendage in patients with atrial fibrillation-the missing link?

Atrial fibrillation (AF) is the most frequent arrhythmic disease in humans, which leads to thrombus formation in the left atrial appendage and stroke through peripheral embolization. Depending on their origin, large extracellular vesicles (lEVs) can exert pro-coagulant functions. In the present study, we investigated how different types of AF influence the levels of large EV subtypes in three distinct atrial localizations. Blood samples were collected from the right and left atrium and the left atrial appendage of 58 patients. 49% of the patients had permanent AF, 34% had non-permanent AF, and 17% had no history of AF. Flow cytometric analysis of the origin of the lEVs showed that the proportion of platelet-derived lEVs in the left atrial appendage was significantly higher in permanent AF patients compared to non-permanent AF. When we grouped patients according to their current heart rhythm, we also detected significantly higher levels of platelet-derived lEVs in the left atrial appendage (LAA) in patients with atrial fibrillation. In vitro studies revealed, that platelet activation with lipopolysaccharide (LPS) leads to higher levels of miR-222-3p and miR-223-3p in platelet-derived lEVs. Treatment with lEVs from LPS- or thrombin-activated platelets reduces the migration of endothelial cells in vitro. These results suggest that permanent atrial fibrillation is associated with increased levels of platelet-derived lEVs in the LAA, which are potentially involved in LAA thrombus formation.

Transverse aortic constriction-induced heart failure leads to increased levels of circulating microparticles.

BACKGROUND: Circulating microparticles represent one type of signal transmission between cells. Previous studies revealed increased levels of circulating microparticles in patients with heart failure, while composition, temporal occurrence and biological effects are largely unknown. METHODS: Circulating microparticles were quantified by flow cytometry in mice following TAC. Microparticles were characterized by NTA and immunoblotting for Flotillin-1. Microparticle content was investigated by microRNA analyses. RESULTS: After TAC induction of heart failure could be demonstrated. Simultaneously we observed increased numbers of circulating microparticles in the first week after TAC with a rapid decline thereafter. The most relevant fraction of circulating EVs after TAC derived from lymphocytes containing has-miR-26a-5p and / -146b-5p known to be involved in inflammatory processes. CONCLUSION: This work provides a previously unknown timely limited occurrence of circulating microparticles after new onset of heart failure which might have important influence on disease development and progression and thereby are of probable therapeutic relevance.

Activation of neutral sphingomyelinase 2 through hyperglycemia contributes to endothelial apoptosis via vesicle-bound intercellular transfer of ceramides.

BACKGROUND: Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Local cellular ceramide accumulation mediates endothelial apoptosis, especially in type 2 diabetes mellitus, which is a major cardiovascular risk factor. In recent years, large extracellular vesicles (lEVs) have been identified as an important means of intercellular communication and as regulators of cardiovascular health and disease. A potential role for lEVs as vehicles for ceramide transfer and inductors of diabetes-associated endothelial apoptosis has never been investigated. METHODS AND RESULTS: A mass-spectrometric analysis of human coronary artery endothelial cells (HCAECs) and their lEVs revealed C16 ceramide (d18:1-16:0) to be the most abundant ceramide in lEVs and to be significantly increased in lEVs after hyperglycemic injury to HCAECs. The increased packaging of ceramide into lEVs after hyperglycemic injury was shown to be dependent on neutral sphingomyelinase 2 (nSMase2), which was upregulated in glucose-treated HCAECs. lEVs from hyperglycemic HCAECs induced apoptosis in the recipient HCAECs compared to native lEVs from untreated HCAECs. Similarly, lEVs from hyperglycemic mice after streptozotocin injection induced higher rates of apoptosis in murine endothelial cells compared to lEVs from normoglycemic mice. To generate lEVs with high levels of C16 ceramide, ceramide was applied exogenously and shown to be effectively packaged into the lEVs, which then induced apoptosis in lEV-recipient HCAECs via activation of caspase 3. Intercellular transfer of ceramide through lEVs was confirmed by use of a fluorescently labeled ceramide analogue. Treatment of HCAECs with a pharmacological inhibitor of nSMases (GW4869) or siRNA-mediated downregulation of nSMase2 abrogated the glucose-mediated effect on apoptosis in lEV-recipient cells. In contrast, for small EVs (sEVs), hyperglycemic injury or GW4869 treatment had no effect on apoptosis induction in sEV-recipient cells. CONCLUSION: lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides.

NcRNAs in Vascular and Valvular Intercellular Communication.

Non-coding RNAs have been shown to be important biomarkers and mediators of many different disease entities, including cardiovascular (CV) diseases like atherosclerosis, aneurysms, and valvulopathies. Growing evidence suggests a central role of ncRNAs as regulators of different pathological pathways involved in endothelial dysfunction, cardiovascular inflammation, cell differentiation, and calcification. This review will discuss the role of protein-bound and extracellular vesicular-bound ncRNAs as biomarkers of vascular and valvular diseases, their role as intercellular communicators, and regulators of disease pathways and also highlights possible treatment strategies.

CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling.

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.