Aims: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia. Methods and results: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography. Conclusion: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium.

Aims: The most appropriate definition of perioperative myocardial infarction (pMI) after coronary artery bypass grafting (CABG) and its impact on clinically relevant long-term events is controversial. We aimed to (i) analyse the incidence of pMI depending on various current definitions in a ‘real-life’ setting of CABG surgery and (ii) determine the long-term prognosis of patients with pMI depending on current definitions. Methods and results: A consecutive cohort of 2829 coronary artery disease patients undergoing CABG from two tertiary university centres with the presence of serial perioperative cardiac biomarker measurements (cardiac troponin and creatine kinase-myocardial band) were retrospectively analysed. The incidence and prognostic impact of pMI were assessed according to (i) the 4th Universal Definition of Myocardial Infarction (4UD), (ii) the definition of the Society for Cardiovascular Angiography and Interventions (SCAI), and (iii) the Academic Research Consortium (ARC). The primary endpoint of this study was a composite of myocardial infarction, all-cause death, and repeat revascularization; secondary endpoints were mortality at 30 days and during 5-year follow-up. There was a significant difference in the occurrence of pMI (49.5% SCAI vs. 2.9% 4UD vs. 2.6% ARC). The 4th Universal Definition of Myocardial Infarction and ARC criteria remained strong independent predictors of all-cause mortality at 30 days [4UD: odds ratio (OR) 12.18; 95% confidence interval (CI) 5.00–29.67; P < 0.001; ARC: OR 13.16; 95% CI 5.41–32.00; P < 0.001] and 5 years [4UD: hazard ratio (HR) 2.13; 95% CI 1.19–3.81; P = 0.011; ARC: HR 2.23; 95% CI 1.21–4.09; P = 0.010]. Moreover, the occurrence of new perioperative electrocardiographic changes was prognostic of both primary and secondary endpoints. Conclusion: Incidence and prognosis of pMI differ markedly depending on the underlying definition of myocardial infarction for patients undergoing CABG. Isolated biomarker release-based definitions (such as troponin) were not associated with pMI relevant to prognosis. Additional signs of ischaemia detected by new electrocardiographic abnormalities, regional wall motion abnormalities, or coronary angiography should result in rapid action in everyday clinical practice.

Aims: Imbalances of iron metabolism have been linked to the development of atherosclerosis. However, subjects with hereditary haemochromatosis have a lower prevalence of cardiovascular disease. The aim of our study was to understand the underlying mechanisms by combining data from genome-wide association study analyses in humans, CRISPR/Cas9 genome editing, and loss-of-function studies in mice. Methods and results: Our analysis of the Global Lipids Genetics Consortium (GLGC) dataset revealed that single nucleotide polymorphisms (SNPs) in the haemochromatosis gene HFE associate with reduced low-density lipoprotein cholesterol (LDL-C) in human plasma. The LDL-C lowering effect could be phenocopied in dyslipidaemic ApoE−/− mice lacking Hfe, which translated into reduced atherosclerosis burden. Mechanistically, we identified HFE as a negative regulator of LDL receptor expression in hepatocytes. Moreover, we uncovered liver-resident Kupffer cells (KCs) as central players in cholesterol homeostasis as they were found to acquire and transfer LDL-derived cholesterol to hepatocytes in an Abca1-dependent fashion, which is controlled by iron availability. Conclusion: Our results disentangle novel regulatory interactions between iron metabolism, KC biology and cholesterol homeostasis which are promising targets for treating dyslipidaemia but also provide a mechanistic explanation for reduced cardiovascular morbidity in subjects with haemochromatosis.

Background: Mechanical stimulation of acute ischemic myocardium by shock wave therapy ( SWT ) is known to improve cardiac function by induction of angiogenesis. However, SWT in chronic heart failure is poorly understood. We aimed to study whether mechanical stimulation upon SWT improves heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis and to dissect underlying mechanisms. Methods and Results: SWT was applied in a mouse model of chronic myocardial ischemia. To study effects of SWT on postnatal vasculogenesis, wild‐type mice received bone marrow transplantation from green fluorescence protein donor mice. Underlying mechanisms were elucidated in vitro in endothelial cells and murine aortic rings. Echocardiography and pressure/volume measurements revealed improved left ventricular ejection fraction, myocardial contractility, and diastolic function and decreased myocardial fibrosis after treatment. Concomitantly, numbers of capillaries and arterioles were increased. SWT resulted in enhanced expression of the chemoattractant stromal cell–derived factor 1 in ischemic myocardium and serum. Treatment induced recruitment of bone marrow–derived endothelial cells to the site of injury. In vitro, SWT resulted in endothelial cell proliferation, enhanced survival, and capillary sprouting. The effects were vascular endothelial growth factor receptor 2 and heparan sulfate proteoglycan dependent. Conclusions: SWT positively affects heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis. SWT upregulated pivotal angiogenic and vasculogenic factors in the myocardium in vivo and induced proliferative and anti‐apoptotic effects on endothelial cells in vitro. Mechanistically, these effects depend on vascular endothelial growth factor signaling and heparan sulfate proteoglycans. SWT is a promising treatment option for regeneration of ischemic myocardium.

Background: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. Methods: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan ( Bgn ), Tlr3 , and IFN-α/β receptor alpha chain ( Ifnar1 )–deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. Results: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn ^−/− , Tlr3 ^−/− , and Ifnar1 ^−/− mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1–BGN–TLR3–interferon-α/β receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. Conclusions: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.