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.

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.

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.

The gap junction modifier ZP1609 decreases cardiomyocyte hypercontracture following ischaemia/reperfusion independent from mitochondrial connexin 43.

BACKGROUND AND PURPOSE: Dysregulation of gap junction-mediated cell coupling contributes to development of arrhythmias and myocardial damage after ischaemia/reperfusion (I/R). Connexin 43 (Cx43) is present at ventricular gap junctions and also in the mitochondria of cardiomyocytes. The dipeptide (2S, 4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid (ZP1609) has antiarrhythmic properties and reduces infarct size when given at reperfusion. However, it is unclear, whether ZP1609 targets Cx43-containing mitochondria and affects cardiomyocyte hypercontracture following I/R. EXPERIMENTAL APPROACH: We studied the effects of ZP1609 on the function of murine sub-sarcolemmal mitochondria (SSM, containing Cx43) and interfibrillar mitochondria (IFM, lacking Cx43). Murine isolated cardiomyocytes were subjected to simulated I/R without and with ZP1609 (applied during I/R or at the onset of reperfusion only), and the number of cardiomyocytes undergoing hypercontracture was quantified. Biochemical pathways targeted by ZP1609 in cardiomyocytes were analysed. KEY RESULTS: ZP1609 inhibited ADP-stimulated respiration and ATP production in SSM and IFM. ROS formation and calcium retention capacities in SSM and IFM were not affected by ZP1609, whereas potassium uptake was enhanced in IFM. The number of rod-shaped cardiomyocytes was increased by ZP1609 (10 µM) when administered either during I/R or reperfusion. ZP1609 altered the phosphorylation of proteins contributing to the protection against I/R injury. CONCLUSIONS AND IMPLICATIONS: ZP1609 reduced mitochondrial respiration and ATP production, but enhanced potassium uptake of IFM. Additionally, ZP1609 reduced the extent of cardiomyocytes undergoing hypercontracture following I/R. The protective effect was independent of mitochondrial Cx43, as ZP1609 exerts its effects in Cx43-containing SSM and Cx43-lacking IFM.

Escherichia coli in marine water: Comparison of methods for the assessment of recreational bathing water samples.

Bathing Water Directive (2006/7/EC) specifies two reference methods for Escherichia coli detection: ISO 9308-1 and 9308-3. The revised ISO 9308-1 is recommended only for waters with a low bacterial background flora. Considering the extended time needed for analysis and, generally, the lack of experience in using ISO 9308-3 in the Mediterranean, the suitability of ISO 9308-1 for the examination of E. coli in bathing water was evaluated. The present study was aimed at a comparison of data obtained by the reference method in seawater samples (110 beaches, N=477) with data received from six alternative methods. Results show that recently used TSA/TBA method may overestimate E. coli numbers in marine waters. The temperature modified ISO 9308-1 (44°C) did not significantly alter the results, but outperformed the antibiotic supplemented agar at reducing non-E. coli bacteria on the plates, allowing the use of the respective method for monitoring coastal water.