Introduction Obstructive sleep apnea syndrome (OSAS) is the most common sleep disorder and it is associated with arterial hypertension, heart failure, coronary artery disease as well as atrial fibrillation. The underlying pathomechanisms for this association are only incompletely understood. In recent years long non-coding RNAs (lncRNA) have been shown to be involved in various cardiovascular pathologies. The aim of this study is to identify lncRNAs which are associated with OSAS in order to provide potential therapeutic targets. Methods and results In order to tackle this issue, we included 23 Patients with a suspected OSAS in a pilot study and sampled blood on the evening before and on the morning after polysomnographic analysis. Citrate was used to inhibit clotting of the blood and cellular components were removed by centrifugation, before the plasma was stored at −80°C. One patient had no sign of OSAS as indicated by an Apnea/Hypopnea-Index (AHI) <5 per hour and was therefore excluded. The remaining 22 patients had a mean age of 44.45 years and a mean AHI of 28.38 per hour. Three of the patients were female and nineteen were male. In four of those patients with high AHI levels, we performed a RNA sequencing analysis from the citrate plasma and found MRPL20-AS1 to be the most significantly regulated lncRNA after the night of polysomnographic analysis (A). Low coding potential of MRPL20-AS1 was confirmed in silico via the Coding-Potential Assessment Tool (http://lilab.research.bcm.edu/) resulting in a coding probability of 0.06148. Then MRPL20-AS1 levels were measured via qPCR in the remaining patients. From 19 patients, we obtained evaluable results. We found that MRPL20-AS1 blood levels had the tendency to be lower after the night of polysomnographic assessment (B). Interestingly MRPL20-AS1 levels were inversely correlated with the AHI of the OSAS patients (C). This indicates that severe OSAS was associated with low levels of MRPL20-AS1 in our cohort (D). In order to further investigate these results in vitro, we subjected human coronary artery endothelial cells (HCAECs) to hypoxia (1% and 5% O2) for 24 h. We found that hypoxia leads to a significant downregulation of MRPL20-AS1 in HCAECs (E). Conclusion MRPL20-AS1 blood levels are lower in patients with severe OSAS after nocturnal hypoxia. In endothelial cells MRPL20-AS1 is downregulated after hypoxia. MRPL20-AS1 can therefore be useful to identify patients suffering from severe OSAS. Further investigations are needed to elucidate the biological function of MRPL20-AS1 in the context of OSAS and to investigate MRPL20-AS1 as a potential therapeutic target to counteract cardiovascular effects of OSAS Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Unviversity of BonnUniversity of Cologne
Background Ceramide synthase 5 (CerS5) is essential to maintain C:16 ceramide levels in mice. Ceramides contribute to inflammation and calcification, two hallmarks of aortic valve stenosis development. It is known, that loss of CerS5 function reduces diet-induced obesity. Purpose The aim of this study was to investigate the effect of ceramide synthase 5 and high-fat diet on the development of aortic valve stenosis with a mouse model. Methods Our experiment consists of four groups of mice: Wild type (WT) and CerS5 (−/−) mice, with normal and high fat diet. We induced the aortic valve stenosis development through a wire-injury. For this model, a coronary angiography wire is introduced into the left ventricle over the right carotid artery, followed by rotation on the aortic valve level to generate a defined injury. Development of aortic valve stenosis was determined by measuring the peak velocity 14, 28 and 42 days after the wire-injury. With histological analysis of the aortic valve, we measured macrophage infiltration with CD68 immunostaining and calcification using von Kossa staining. For statistical analysis ANOVA and Turkeys multiple comparisons were performed. Results Histological analysis of the aortic valve revealed, that in CerS5 (−/−) mice with high fat diet (HFD) immune cell infiltration is reduced, while there is no difference between CerS5 (−/−) mice and wild type, when fed with normal diet (A). Also calcification showed a trend towards a reduction in CerS5 mice with high fat diet, but was not statistically significant (B). Echocardiography could detect a reduced peak velocity of CerS5 mice with high fat diet, in comparison to the other groups, four and six weeks after wire injury (C). Conclusion Our experiments indicate that loss of CerS5 function reduces the development of aortic valve stenosis in mice with high fat diet, by decreasing immune cell infiltration and calcification. Interestingly, in the groups with normal chow, loss of CerS5 function had no effect. In addition, high-fat diet alone had no negative effect in our mouse model. Based on our results it can be assumed that upon high fat diet CerS5 mediates for pro-inflammatory effects in the aortic valve, which are absent on normal diet. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Ernst & Berta Grimmke Foundation (13/19)
Background Chronic kidney disease is closely associated with cardiovascular disease which contributes to a high mortality and morbidity in these patients. Calcific aortic valve stenosis is the most common valve disease among adults. CKD leads to a higher incidence of AS and is associated with impaired clinical outcomes after valve replacement. CKD leads to retention of uremic toxins, such as indoxyl sulfate (IS), which is known to induce inflammatory and pro-calcific processes. The influence of IS on AS pathophysiology is currently not known. Methods and results Human aortic valvular interstitial cells (VICs) were stimulated by 4 different conditions: Control medium (CM), CM and IS in a concentration of 50 mmol/l, pro-calcifying medium (PCM) which was produced by adding NaH2PO4 and L-ascorbic acid to CM and PCM+IS. After 7 days of incubation, VICs were fixed in formalin and calcification was evaluated through staining with alizarin red solution (B). Staining was quantified by photometric measurements at 540 nm. We observed a significantly higher degree of VIC calcification under the influence PCM+IS compared to PCM alone (C). VICs were stimulated for 7 days and next-generation RNA Sequencing (RNA Seq) was performed. Analysis of RNA Seq dataset identified naked cuticle homologue 2 (NKD2) to be strongly regulated by IS. This effect was confirmed by qPCR using VICs from a total of three different human donors and immunoblot analysis (D, E). We observed a more pronounced upregulation of NKD2 gene expression under PCM+IS vs. PCM alone (p<0.0001) (D). To investigate the influence of NKD2 on VIC calcification genetic knockdown of NKD2 was performed by transfecting VICs with NKD2 siRNA compared and scrambled siRNA as negative control (nc siRNA) (F). After 21 days of stimulation and subsequent alizarin staining, we observed a significant decrease in VIC calcification under NKD2 knockdown compared to negative control under PCM+IS conditions (G, H). To identify upstream mechanisms that lead to NKD2 upregulation by IS we investigated the influence of organic anion transporters (OAT) which are responsible for cellular uptake of IS. Probenecid, known as a potent inhibitor of OATs showed a dose dependent suppression of NKD2 upregulation under PCM+IS (I). Systematic analysis of RNA Seq data regarding SLCO genes coding for OAT and OAT-peptides revealed that only SLCO3A1, coding for OAT3A1 is highly abundant in VICs (J). SLCO3A1 knockdown showed a significant decrease of NKD2 expression after stimulation with PCM+IS for 7 days compared to negative control (K). Conclusion Our findings show that in vitro conditions of uremia increase calcification of aortic VICs. NKD2 expression is regulated by PCM and IS, and PCM+IS treated cells show the strongest upregulation of NKD2. Genetic knockdown of NKD2 attenuates VIC calcification suggesting a role of NKD2 in this process. Further, our findings suggest that OAT3A1 is involved in IS mediated regulation of NKD2 expression. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Medical faculty of the University of Bonn, German Cardiac Society (DGK), German Research Foundation (DFG), TRR259
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