Background-Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Methods and Results-Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Conclusions-Endothelial

BackgroundCirculating 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 ResultsTen 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.ConclusionMVs 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.

High glucose condition increases NADPH oxidase activity in endothelial microparticles that amplify endothelial inflammation and impair endothelial function by promoting activation of the endothelium. These findings provide new insights into the pathogenesis of diabetes-associated atherosclerosis.

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Background: Obesity is a major risk factor for the development of heart failure. The pathophysiology of obesity cardiomyopathy is complex, and the exact mechanisms of disease remain poorly understood. The accumulation of lipids in the heart, caused by a mismatch between uptake and oxidation of fatty acids, is a powerful determinant of lipotoxic damage, apoptosis and impaired contractile function. The transcription factor JunD -a component of the Activator Protein-1 (AP-1) complex -has recently emerged as a pivotal modulator of triglyceride metabolism in the liver of obese mice. Purpose: To investigate whether JunD participates to cardiac lipid accumulation and myocardial damage. Methods: JunD transcriptional activity as well as the expression of genes involved in triglyceride uptake and storage were assessed in neonatal rat ventricular myocytes (NRVM) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to palmitic acid (PA, 200uM) and vehicle for 48 hours. Gene silencing of JunD was perfomed by siRNA technology. Western blot and real-time PCR were used to assess JunD expression and lipid-related genes. Binding of JunD to peroxisome proliferator-activated receptor gamma (PPARγ) promoter was investigated by chromatin immunoprecipitation (ChIP) assay. Apoptosis and oxidative stress were investigated by caspase-3 and 3-nitrotyrosine (3-NT) assays. The impact of JunD on cardiac lipid uptake and PPARγ signaling was also investigated in the heart of JunD knockout (JunD−/−) mice as well as in transgenic mice with cardiac-specific overexpression of JunD via the α-myosin heavy chain promoter (α-MHC-JunDtg). Myocardial triglyceride content was assessed on crude heart homogenates. Results: Exposure of NRVM and hiPSC-CMs to PA significantly increased JunD expression and transcriptional activity. ChIP assays in PA-treated cells showed that JunD binds PPARγ promoter, leading to its upregulation and subsequent overexpression of PPARγ-dependent genes, namely CD36, fatty acid synthase (FAS) and Perilipin 5 (Plin5). Interestingly, JunD knockdown abolished PA-induced PPARγ transcriptional programs and intracellular lipid uptake both in rat and human cardiomyocytes. Caspase-3 activity and 3-NT levels were also significantly reduced by JunD silencing. Consistently, PPARγ signaling and intramyocardial lipid content were blunted in the heart of JunD−/− mice as compared to wild-type littermates. By contrast, α-MHC-JunDtg mice displayed cardiac steatosis due to massive upreguation of PPARγ and its downstream genes CD36, FAS and Plin5. Lipid accumulation in α-MHC-JunDtg mice was associated with apoptosis and oxidative stress. Conclusions: JunD drives the expression of PPARγ-related genes, leading to cardiac lipid accumulation and damage. This study -which employs JunD gainand loss of function mouse models -provides insights into new anti-steatotic therapies for the prevention of obesity cardiomyopathy phenotype.

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Introduction Cardiac hypertrophy and heart failure are wide spread diseases of elderly patients in the industrialised world and drive a majority of healthcare costs in these countries. Therefore, there is a high urgency to understand the development and progress of heart failure to find new therapeutic strategies especially in early stages. Microvesicles are involved in the development and propagation of almost all cardiac diseases and increased levels of circulating microvesicles can be found in blood of patients with chronic heart failure. To point out the underlying mechanisms and to provide a new animal model based approach, we investigated microvesicle (MV)-release in mice that underwent Transverse Aortic Constriction (TAC). TAC is a common method to induce cardiac hypertrophy and heart failure in mice by inducing pressure overload. We hypothesized that TAC leads to upregulation of total MV and MV of specific origin. Methods and results Wildtype C57BL/6 mice underwent TAC to induce cardiac hypertrophy and heart failure. After TAC, mice developed cardiac hypertrophy as determined by altered heart weight/ bodyweight ratio, end-diastolic and end-systolic diameter and decreased fractional shortening. Total numbers of circulating microvesicles were detected 1, 4 and 12 weeks after TAC. We found that total numbers of circulating macrovesicles raised in a time dependent manner. Similar observations could be done with samples stained for annexin V, although results were not significant. Moreover, microvesicles were stained with specific surface markers for lymphocyte (CD3), monocyte (CD14), endothelial cells (CD31), thrombocytes (CD41), B-cells (CD45) and neutrophils (Lys6). One week after TAC increased numbers of specific microvesicles could be detected which in the course declined rapidly. Only microvesicles subgroup of lymphocyte origin showed significant increase one week after TAC-OP. Conclusion In this study, we show that total number of circulating microvesicles raise after TAC over an observation period of 12 weeks. Furthermore, we found that increased numbers of circulating microvesicles of specific origin like lymphocytes, monocytes, endothelial cells, thrombocytes, B-cells and neutrophils showed a trend towards increased levels one week after TAC with a rapid decline in subsequent detection. To our knowledge this is the first time that the impact of TAC on number of circulating microvesicles in mice was investigated. Future studies should characterize the content and effects of these MV on recipient cells to elucidate possible contributions to heart failure progression or protective effects. Detecting new harmful or protective effects of heart failure triggered by circulating microvesicles could offer new highly needed approaches to suppress heart failure development or deliver the possibility to develop new drugs for heart failure treatment. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Else Kröner-Fresenius

Sirtuin 4 (SIRT4) is a mitochondrial NAD+-dependent deacylase which inhibits the oxidation of glucose and fatty acids, and has been implicated in the regulation of oxidative stress. Given the importance of cardiac energy depletion and ROS during heart failure development, we aimed to define the role of SIRT4 in the development of heart failure. Mice with deletion (SIRT4−/−) or overexpression (SIRT4 TG) of SIRT4 were subjected to transverse aortic constriction (TAC) for 12 weeks or underwent sham procedures. Using echocardiography, ejection fraction (EF) was not different between SIRT4 TG and WT mice subjected to sham operations. In contrast, TAC induced a more pronounced decrease in EF (35% vs. 51%; p<0.05), and a more pronounced increase in LV endsystolic diameter (4.5mm vs. 3.6mm; p<0.05) and myocardial fibrosis (2.2-fold; p<0.05) in SIRT4 TG mice compared to WT mice. Myocardial levels of the lipid peroxidation product 4-hydroxynonenal were increased in WT mice following TAC and were synergistically increased in SIRT4 TG mice following TAC (+66% vs. WT TAC; p<0.05). Administration of the mitochondria-targeted antioxidant MitoQ normalized 4-hydroxynonenal levels, markedly attenuated the decline in EF and almost normalized endsystolic LV diameter in SIRT4 TG mice following TAC. Cardiac function and morphology were unaffected in SIRT4−/− mice during normal or increased workload conditions. Thus, while SIRT4 is not required to maintain cardiac function even in response to increased energy demands, increased expression of SIRT4 accelerates the development of heart failure following TAC, at least in part due to increased mitochondrial oxidative stress. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation