bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanical load modulates the cargo of secreted extracellular vesicles (EVs) from living myocardial slices
Authors: Nicastro L , Catapano F , Nunez Toldra R , Floriano J , Oliveira C.C , Emanueli C , Terracciano C.M.N 
Published: November 2020

Abstract

Introduction
Mechanical overload plays an important role in the progression of chronic heart failure (CHF). Beside the numerous neurohormonal and chemical stimuli that contribute to structural and functional changes in the myocardium, intercellular communication mediated by extracellular vesicles (EVs), also plays a pivotal role in disease progression. Exosomes are a subset of EVs ranging between 30–150 nm; these mediators carry molecular cargoes such as miRNAs, mRNAs, DNA and several proteins. However, it is still unclear how specific pathological stimuli may induce a change in their release from tissues or a difference in their cargo content.
Living myocardial slices (LMS) are ultra-thin sections of heart tissue, ranging from 100–400 μm in thickness. They retain the multicellularity, electromechanical physiology, biochemistry and extracellular matrix of the adult heart.

Purpose
To investigate whether mechanical load influences both the released amount and content of LMS-derived exosomes after 24 hours culture.

Methods and results
LMS were prepared from the left ventricle of male Sprague Dawley rats using a high precision vibratome. LMS were stretched in the direction of the muscle fibers at a sarcomere length (SL) of 2.2 μm to recapitulate physiological preload, and at 2.4 μm SL to recapitulate a condition of volume overload. LMS were cultured for 24 hours under electromechanical stimulation. Media containing LMS-derived exosomes was harvested after culture and processed for exosome isolation by size exclusion chromatography columns. Particle size and concentration were assessed by nanoparticle tracking analysis and protein quantity by microBCA assay. There was no significant difference in the size of exosomes between overload and physiological conditions, with an average mean size of 113.475±8.35 nm for the physiological and 129.3±16.35 nm for the overload condition (p=0.61; n=4 physiological; n=3 overload). Although there was not significant difference in exosome amount between physiological and overload condition, (7.00E+10±2.53E+09 vs 1.04E+11±5.77E+09 particles/ml) (p=0.57; n=4 physiological; n=3 overload), exosomes released from overloaded LMS showed a significant increase in protein content compared to physiologically loaded LMS (150.57±25.682 vs 66.045±9.855 μg/ml) (p=0.04; n=3 overload; n=2 physiological).

Conclusions
Mechanical load influences the cargo content of EVs secreted from LMS after 24 hours culture under electromechanical stimulation. Understanding how mechanical load correlates with specific cargoes in EVs will reveal novel therapeutic targets for the treatment of CHF.

Funding Acknowledgement
Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation (BHF)
The crucial roles of coagulation factors in inducing brown adipose tissue dysfunction and systemic metabolic disorder in obesity
Authors: Hayashi Y , Shimizu I , Yoshida Y , Katsuumi G , Suda M , Fujiki S , Minamino T 
Published: November 2020

Abstract

The prevalence of obesity is increasing worldwide. Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Analyzing deposited microarray data, we found transcripts for coagulation factors including factor VII (F7), factor X (F10), and PAR1 receptor were increased in BAT from obese mice. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model.
Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. Fxa inhibition reduced reactive oxygen species (ROS) level in BAT. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. We generated BAT specific PAR1 KO mice. BAT-PAR1 KO mice exhibited re-browning of BAT along with reduced ROS level in this tissue. In BAT-PAR1 KO mice, glucose intolerance and thermogenic response under a metabolically stressed condition were ameliorated. In differentiated brown adipocytes, FXa markedly increased mitochondrial ROS and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes.

Funding Acknowledgement
Type of funding source: None
PCSK9 is associated with disease activity and implicated in immune activation and cardiovascular disease in Systemic Lupus Erythematosus
Authors: Frostegard J , Frostegard J , Rahman M , Hafstrom I , Ajeganova S , Liu A 
Published: November 2020

Abstract

Background
The risk of atherosclerosis and cardiovascular disease (CVD) is very high in SLE. This is both a clinical problem and of interest in studies of immunity in these conditions. LDL-levels are increased by Proprotein convertase subtilisin kexin 9 (PCSK9) which targets the LDL-receptor (LDLR). We recently reported that PCSK9 ameliorates dendritic cell (DC) activation by oxidized LDL (OxLDL), which is abundant in atherosclerotic plaques and also raised and associated with cardiovascular disease (CVD) in SLE. We here investigate the role of PCSK9 in SLE.

Methods
PCSK9-levels were determined by ELISA among SLE patients (n=109) and age- and sex-matched population-based controls (n=91). Common carotid intima-media thickness (IMT) and plaque occurrence were determined by B-mode ultrasound. Plaques were graded by echogenicity. Human peripheral blood monocytes from SLE patients or controls were differentiated into DCs. Effects of PCSK9 and its inhibition by silencing were studied.

Results
PCSK9-levels were non-significantly higher among SLE-patients as compared to controls but associated significantly with SLE disease activity, as determined by SLAM (0.020) or SLEDAI (0.0178). There was no association between PCSK9-levels and atherosclerosis as determined by IMT, prevalence of plaques or echolucent (potentially vulnerable) plaques. PCSK9 levels were significantly associated with CVD among SLE-patients.
OxLDL induced PCSK9 in DCs and DC-maturation with increased expression of CD86 and HLA-DR. The effects were significantly stronger in DC from SLE patients than from controls. Silencing of PCSK9 abolished OxLDL-induced DC-maturation.

Conclusions
PCSK9 is associated with disease activity in SLE. One underlying cause could be OxLDL, promoting DC-activation which depends on PCSK9. OxLDL induces PCSK9, an effect which is higher among SLE-patients. PCSK9 could play an unexpected immunological role in SLE.

Funding Acknowledgement
Type of funding source: Private company. Main funding source(s): Amgen; Swedish Heart and Lung Foundation
MicroRNA-146a regulates angiogenesis and functional regeneration after myocardial infarction
Authors: Knoepp K , Teske R , Korte L , Dutzmann J , Rieckmann M , Daniel J.M , Thum T , Bauersachs J , Sedding D.G 
Published: November 2020

Abstract

Background
The growth or regeneration of new blood vessels is critical for physiological angiogenesis and for tissue repair in response to injury, such as myocardial infarction. However, the mechanisms underlying the angiogenic processes and, specifically, the regulation of neo-vascularization by microRNAs are not well understood.

Purpose
To study the potential beneficial role of miRNA-146a in vessel regeneration after myocardial infarction.

Methods
Mice were subjected to myocardial infarction or hind limb ischemia and vascular development was followed in zebra fish. microRNA-146a was blocked or overexpressed in endothelial cells in vitro or in mice or zebra fish in vivo. Furthermore, Blood samples from patients with ST-elevation myocardial infarction (STEMI) were studied for miR-146a expression.

Results
MicroRNA-146a was upregulated in the ischemic hind limb following ligation of the femoral artery and in the ischemic myocardium in male C57BL/6 mice following ligation of the left anterior descending (LAD) artery, as well as in patients with STEMI. In vitro, overexpression of microRNA-146a significantly attenuated endothelial cell proliferation and migration, abolished endothelial capillary network formation, and inhibited cell sprouting from endothelial spheroids. In contrast, microRNA-146a silencing had opposite effects. Mechanistically, Nox4, Notch1, and nRas were identified by in silico prediction and microarray mRNA expression analysis, and validated as direct targets and effector molecules of microRNA-146a in endothelial cells according to mRNA and protein expression profiles, as well as luciferase gene reporter assays and respective rescue experiments. In zebrafish, overexpression of microRNA-146a inhibited the development of the trunk vasculature. Blocking the upregulation of endothelial microRNA-146a in mice,using specific inhibitors, resulted in significantly enhanced 1 angiogenesis and revascularization both in an ischemic hind limb model and in the infarcted myocardium. This was accompanied by a significantly reduced myocardial infarct size and preserved cardiac function post MI.

Conclusions
Our findings identify microRNA-146a as a critical regulator of angiogenesis and vascular regeneration. Inhibition of microRNA-146a may represent an attractive strategy for the angiogenic therapy of ischemic tissue diseases.

Funding Acknowledgement
Type of funding source: None
Implication of anti-angiogenic VEGF-A165b in angiogenesis and systolic function after reperfused myocardial infarction
Authors: Marcos Garces V , Rios-Navarro C , Hueso L , Diaz A , Bonanad C , Ruiz-Sauri A , Sanz M.J , Chorro F.J , Piqueras L , Bodi V 
Published: November 2020

Abstract

Background
Angiogenesis participates in re-establishing microcirculation after myocardial infarction (MI).

Purpose
In this study, we aim to further understand the role of the anti-angiogenic isoform vascular endothelial growth factor (VEGF)-A165b after MI and explore its potential as a co-adjuvant therapy to coronary reperfusion.

Methods
Two mice MI models were formed: 1) permanent coronary ligation (non-reperfused MI), 2) transient 45-min coronary occlusion followed by reperfusion (reperfused MI); in both models, animals underwent echocardiography before euthanasia at day 21 after MI induction. Serum and myocardial VEGF-A165b levels were determined. In both experimental MI models, functional and structural implication of VEGF-A165b blockade was assessed. In a cohort of 104 ST-segment elevation MI patients, circulating VEGF-A165b levels were correlated with cardiovascular magnetic resonance-derived left ventricular ejection fraction at 6-months and with the occurrence of adverse events (death, heart failure and/or re-infarction).

Results
In both models, circulating and myocardial VEGF-A165b presence was increased 21 days after MI induction. Serum VEGF-A165b levels inversely correlated with systolic function evaluated by echocardiography. VEGF-A165b blockage increased capillary density, reduced infarct size, and enhanced left ventricular function in reperfused, but not in non-reperfused MI experiments. In patients, higher VEGF-A165b levels correlated with depressed ejection fraction and worse outcomes.

Conclusions
In experimental and clinical studies, higher serum VEGF-A165b levels associates with a worse systolic function. Its blockage enhances neoangiogenesis, reduces infarct size, and increases ejection fraction in reperfused, but not in non-reperfused MI experiments. Therefore, VEGF-A165b neutralization represents a potential co-adjuvant therapy to coronary reperfusion.

Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): This study was funded by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (Exp. PIE15/00013, PI17/01836, PI18/00209 and CIBERCV16/11/00486).
Purification of human iPSC-derived cardiomyocytes by HDAC inhibition through inducing apoptosis and cell arrest in non-cardiomyocytes
Authors: Tanaka A , Watanabe K , Kondo S , Tamura N , Nishimoto T , Yoshida Y 
Published: November 2020

Abstract

Background
Cell therapy is one of the most promising strategies for treatment of heart failure. During preparation of iPSC-derived cardiomyocytes for cells therapy, it is important to eliminate the presence of residual proliferative non-target cells in the preparation as these non-target cells could present the risk of tumorgenicity. To minimize the risk, selective and more precise purification process is necessary.

Purpose
We hypothesized that the differences in proliferative activity between cardiomyocytes and non-target cells might result in different sensitivities to the drugs targeting cell growth/survival. The aim of this study is to obtain compounds that eliminate non-cardiomyocytes selectively and to study the mechanism of action of these compounds.

Methods and results
We screened 314 small compounds using both iPSCs and sorted hiPSC-derived cardiomyocytes (hiPSC-CMs). We identified several compounds, which markedly decreased the cell numbers of iPSCs, but showed minimum effects on those of hiPSC-CMs. Among them, HDAC inhibitors were selected as the most promising candidates. We examined whether HDAC inhibitors could purify hiPSC-CMs containing non-cardiomyocytes populations, which were differentiated using the classical embryoid body (EB) method. As a result, HDAC inhibitors increased the purity of cardiomyocytes (up to 98%) by decreasing non-target cells such as smooth muscle cells, endothelial cells, and endodermal lineage cells. Moreover, we confirmed HDAC inhibitors could be used for purification of cardiomyocytes in monolayer differentiation protocol using GSK3β inhibitor and Wnt inhibitor. We measured the expression levels of mRNA and protein in iPSCs and hiPSC-CMs (EB method) treated with HDAC inhibitors. mRNA levels of CDKN1A (p21) and BAX were upregulated in iPSCs. Western blotting analyses revealed that HDAC inhibitors also induced the expression of p21 and the cleavage of Caspase3 in iPSCs.

Conclusions
Our result suggests that the inhibition of HDAC enables an efficient purification of hiPSC-CMs in multiple differentiation methods. Furthermore, our data indicate that HDAC inhibitors induce the apoptosis and cell cycle arrest in iPSCs but not hiPSC-CMs.
Figure 1

Funding Acknowledgement
Type of funding source: Private company. Main funding source(s): Takeda Pharmaceutical Company Limited
Bioinspired cell-derived nanovesicles protect the heart from ischemia reperfusion injury
Authors: Wang J , Huang C.Y , Neupane Y.R , Wang X , Zharkova O , Chong S.Y , Lee C.K , Wacker M.G , Storm G , Pastorin G 
Published: November 2020

Abstract

Introduction
Exosomes have been proven to alleviate myocardial ischemia reperfusion (I/R) injury in preclinical studies. However, the laborious and low-yield production process of naturally secreted exosomes has been impeding their translation into clinical trials.

Purpose
We aim to develop a simple and cost-effective protocol to produce exosome mimetics, bioinspired Cell-Derived Nanovesicles (CDNs), and examine their intrinsic bioactivity in a mouse model of I/R injury.

Methods
CDNs were produced from human U937 monocytes using cell shearing approach and characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Lipid composition of cells and CDNs was analysed by lipidomics. I/R injury was induced by transient occlusion of left coronary artery. Data was analysed with Mann-Whitney U test. P&lt;0.05 was considered statically significant.

Results
We obtained 538 mg (protein content) of CDNs, or 3x109 CDNs, via cell shearing approach from 2×107 cells, approximately 15 folds of exosomes via natural secretion from the same number of cells. CDNs were 125±8 nm in diameter with negative surface charge (zeta potential −7.0±0.8) and presented as double-membranous vesicles under TEM. In vitro, CDNs showed strong antioxidant activity and could be taken up by bone marrow-derived macrophages. Following intravenous administration, as demonstrated by the IVIS Spectrum imaging system, CDNs accumulated specifically in the infarct area of the heart within 3 hours. Compared with saline treatment, CDNs reduced myocardial infarct size by 31.6% (p&lt;0.01) after 24 hours of I/R injury. Intriguingly, CDNs generated from human mesenchymal stem cells showed similar therapeutic efficacy. Mechanistically, CDNs inhibited infiltration of inflammatory cells (macrophages) and promoted upregulation of the anti-inflammatory cytokine interleukin 10 (IL10) in the I/R injured hearts. In the blood stream, CDNs increased IL10 protein level and exerted antioxidant activity. Furthermore, CDNs reduced I/R injury-induced cell apoptosis in the myocardium.

Conclusion
We have established a cost-effective approach to produce exosome mimetics, bioinspired CDNs, which protect the heart from I/R injury via inhibition of inflammation, oxidative stress and cardiac cell apoptosis. CDNs have intrinsic cardioprotective capability in heart injury, comparable to exosomes.

Funding Acknowledgement
Type of funding source: Public Institution(s). Main funding source(s): National University Health System.
Dapagliflozin protects Doxorubicin induced cardiotoxicity in patients with diabetes via suppressing ER stress
Authors: Wei-Ting C , Lin Y.W , Ho C.H , Chen Z.C 
Published: November 2020

Abstract

Objectives
Cancer patients with diabetes have an increasing risk of Doxorubicin (Dox)-induced cardiotoxicity. Despite previous studies reporting benefits of dapagliflozin on the cardiovascular system, it remains unknown whether dapagliflozin has a cardioprotective effect in cancer patients with diabetes receiving Dox therapy. The purpose of this study is to investigate the potentials of dapagliflozin in preventing Dox induced cardiotoxicity.

Methods
Using National Health Insurance Database, the incidence of heart failure of breast cancer patients with or without diabetes was investigated. Also, streptozotocin (STZ) induced diabetic rats were pretreated with oral dapagliflozin (10 mg/kg/day) for 6 weeks and received Dox (5 mg/kg/week) for 4 weeks via intraperitoneally injection. Sequential echocardiography was applied to assess the cardiac function. For in vitro analysis, H9C2 cardiomyocytes, cultured in high glucose (30 mM), were treated with dapagliflozin at 10 mM and subsequently exposed to Dox at 1 mM. The apoptosis and endoplasmic reticulum (ER) stress related protein expression were measured by immunohistochemistry and western blotting.

Results
Among the studied patients, those with diabetes have a higher risk of heart failure while patients prescribed with dapagliflozin lowered the risks. Correspondingly, dapagliflozin treatment effectively inhibited Dox-induced apoptosis and reactive oxygen species accumulation in cardiomyocytes. Also, the reduction of cardiac fibrosis as well as apoptosis in rats treated with dapagliflozin resulted in significantly improved cardiac function. Dapagliflozin also decreased the cardiac expression of Bax, cleaved caspase 3 and increased Bcl-2. Mechanistically, we showed that dapagliflozin significantly reduced ER stress-associated proteins including GRP78, PERK, eIF 2a, ATF-4, and CHOP.

Conclusions
Our study revealed for the first time that dapagliflozin mitigated Dox-induced cardiomyocyte apoptosis in diabetes via inhibiting ER stress. These results indicate that dapagliflozin could be useful in preventing cardiotoxicity in diabetic cancer patients receiving Dox treatment.

Funding Acknowledgement
Type of funding source: Private hospital(s). Main funding source(s): Chi-Mei Medical Center, Tainan, Taiwan
Genome-wide association study of patients with atrioventricular nodal reentry tachycardia
Authors: Andreasen L , Ahlberg G , Hartmann J , Paludan-Mueller C , Jensen H.K , Riahi S , Hansen J , Sandgaard N , Haunsoe S , Kanters J.K , Ellervik C , Bundgaard H , Svendsen J.H , Olesen M.S 
Published: November 2020

Abstract

Background
Supraventricular tachycardias (SVTs) originate from the atria or the area close to the AV node. AV nodal reentry tachycardia (AVNRT) is one of the tachyarrhytmias comprising the group of SVTs. The typical patient is female, young at disease onset, with a structurally normal heart. At present we do not know the etiology of AVNRT. We therefore hypothesized that AVNRT might be caused by changes in the DNA.

Methods
DNA from purified blood was obtained from patients with AVNRT verified by an invasive electrophysiological study. Patients were recruited from five ablation centers in Denmark and individuals from the general population of Denmark (the BEFUS cohort) served as controls. DNA was subjected to chip genotyping, imputation and analyses in a genome-wide association study (GWAS) setup.

Results
A GWAS on 1,143 AVNRT patients and 3,004 controls revealed one locus close to the gene MYH6 to reach genome-wide significance for association with AVNRT (P=4.8x10–8). MYH6 encodes the α-isoform of the protein myosin heavy chain important for the contractile units of the heart, the sarcomeres. The gene is predominantly expressed in the atria. Additional subthreshold loci located close to other plausible arrhythmia genes were identified.

Conclusion
We report the first genetic locus to be associated with AVNRT close to the sarcomere gene MYH6. This is, to our knowledge, the first gene ever associated with AVNRT.
Manhattan plot

Funding Acknowledgement
Type of funding source: Public hospital(s). Main funding source(s): Rigshospitalets Forskningspulje - 3 years PhD salary