bluuebunny/crossref_metadata_2025 huggingface dataset:
Human calcified aortic valves release procoagulant microparticles inducing endothelial dysfunction and thrombogenicity: Role of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway
Authors: Hmadeh S , Trimaille A , Matsushita K , Marchandot B , Zobairi F , Sato C H , Kindo M , Minh Hoang T , Toti F , Pieper M , Schini Kerth V , Kauffenstein G , Morel O
Published: November 2023

Background

Aortic stenosis (AS) pathogenesis is a dynamic process with interplay between the hemostatic system and the valve leading to inflammation, oxidative stress, endothelial dysfunction, and calcification. Within the pathological valve, mechanical strain induces the release of cell-derived microparticles (MPs) that promote mineralization and carry a pro-oxidant activator signal. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) showed cardiovascular benefit and anti-inflammatory responses in patients with heart failure. Oxidative stress has been shown to trigger SGLT2 expression in endothelial cells (ECs) to induce endothelial dysfunction. Therefore, this study evaluated calcified AS valves and the effect of AS-derived MPs on ECs and, if so, the role of the SGLT2.

Methods

Pathological valves were collected from 90 AS and 15 aortic insufficiency (AI) patients. MPs were extracted from human pathological valves, and quantified through their prothrombinase activity. Primary cultures of porcine valvular endothelial cells (VECs) were treated with MPs (10 nM) or thrombin (1 U/ml). Protein expression levels were assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-qPCR, oxidative stress using dihydroethidium, and nitric oxide (NO) generation by DAF-FM diacetate. THP1 cells were used to evaluate the adhesiveness of VECs, and the generation of thrombin using a specific chromogenic substrate.

Results

Calcified AS valves showed increased levels of thrombogenic (TF, PAI-1), adhesive (VCAM-1, ICAM-1) and inflammatory markers (CD68, p-p65 NF-κB) and SGLT2 compared to non-calcified valves. These responses were associated with a higher level of oxidative stress that was reduced by inhibitors of the angiotensin system (ACEi, AT1R) and SGLT2, and an increased release of procoagulant MPs. Exposure of VECs to AS-MPs increased the expression of markers of the angiotensin system (ACE, p67 phox), inflammation (CXCL10, CCL11, CXCL8), thrombogenicity (TF, PAI-1), angiogenesis (VEGFR2, ANGPTL4), matrix remodeling (BMP-2, MMP-1), and SGLT2 and caused the down-regulation of eNOS. These responses were associated with a sustained pro-oxidant response, the nuclear translocation of NF-kB and blunted bradykinin-induced NO formation, which were prevented by inhibition of AT1R, NADPH oxidases, SGLT2, and NF-kB. AS-MPs-treated VECs showed also increased adhesion of THP-1 cells, and generation of thrombin.

Conclusions

Calcified AS valve is a potent reservoir of MPs, acting as a pro-thrombogenic source per se and prompting valvular endothelial cells dysfunction through activation of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. The observed protective effect of empagliflozin suggests SGLT2 inhibitors as a novel therapeutic option to curb valve dysfunction.
Aortic valve calcification is promoted by clonal hematopoiesis associated with DNMT3A and TET2 gene driver mutations
Authors: Abplanalp W , Schuhmacher B , Mas-Peiro S , Zuriaga M A , Matesanz N , Fuster J J , John D , Muhly-Reinholz M , Vasa-Nicotera M , Merten M , Hoffmann J , Dimmeler S , Zeiher A M
Published: November 2023

Background

Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations is associated with coronary heart disease and worse prognosis among patients with aortic valve stenosis (AVS) and inflammation. However, it is unknown what role CH plays in the pathogenesis of AVS. AVS is the most common age-related heart valve disease, with no medical therapy to halt progression.

Methods and Results

Single-cell RNA-sequencing of immune cells of CAVD patients (n=13) with and without the most prevalent CH-mutations (DNMT3A and TET2), with mean CH-driver gene variant allele frequency was 6.5% for DNMT3A and 17.9% for TET2. Monocytes of patients harboring DNMT3A and TET2 mutations shared 836 upregulated genes, many associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) and paracrine pro-calcific factors (oncostatin M (OSM), S100A9), suggesting that CH might be causally involved in AVS. Indeed, silencing of TET2 or DNMT3A in macrophages promotes M1-like polarization and increases release of pro-calcific mediators, particularly S100A9 and OSM. To assess whether CH-gene silenced macrophages may stimulate mesenchymal cells to secrete calcium, secreted factors from TET2 or DNMT3A-silenced macrophages were applied mesenchymal cells. Indeed, CH-gene silenced secretomes induced osteoblastic differentiation of mesenchymal cells, evidenced by increased Alizarin red calcium staining and elevated levels of ALP and RUNX2. This CH-mediated enhancement in osteoblastic conversion could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving TET2-/- bone marrow transplants demonstrated increased total calcified area (1.49-fold) along with increased numbers of calcification deposits (2.33-fold) as evidenced by von Kossa staining, along with enhanced OSM and S100A9 levels.

Conclusion

This is the first study to demonstrate that somatic CH-driver mutations in monocytes may accelerate AVS. Moreover, calcification was ablated via silencing of OSM in CH-gene silenced macrophages. These data suggest Oncostatin M may be a therapeutically promising target in halting the progression of AVS in patients harboring CH mutations.
The senescence-associated secretome for prediction of mortality in patients with aortic valve stenosis undergoing TAVR
Authors: Mas-Peiro S , Rasper T , Berkowitsch A , Dimmeler S , Zeiher A M
Published: November 2023

Background

Cellular senescence is an essential mechanism in the process of aging. The senescence-associated secretory phenotype (SASP) includes mainly pro-inflammatory cytokines, proteases, soluble signalling factors, and growth modulators. Degenerative aortic valve stenosis (AVS) is the prototypical inflammatory age-related cardiovascular disease.

Purpose

Our aim was to assess whether circulating concentrations of SASP factors are associated with prognosis in elderly patients with AVS undergoing transcatheter aortic valve replacement (TAVR), the current standard therapy for severe cases.

Methods

A total of 24 SASPs were measured using the Luminex Multiplex ELISA Assay (Biotechene, GmbH) in 120 AVS patients undergoing TAVR between February 2017 and February 2020 with a prospective follow-up of up to 4 years. Receiver operating characteristic (ROC) curves were created for each SASP protein in order to identify predictive markers. SASPs were considered positive if their baseline value was above the cut-off value derived from ROC-curves. The only 3 independent predictive factors were selected to develop an easy-to-calculate score based on the number of positive SASP proteins (0 to 3). Primary endpoint was long-term all-cause mortality.

Results

Median (IQR) age of the population was 83.2 (78.9-87.0) years, 56.7% were women. According to ROC curves, 9 out of 24 SASPs showed a significant area under the curve (AUC), with values above 0.623 (see Table). Only 3 of the markers reflecting potentially different underlying mechanisms remained independent predictors, after including all SASP factors in a Cox Regression analysis: GDF-15, ICAM-1 and Osteoprotegerin (all, p<0.03). A score based on the number of baseline factors being above the AUC-derived threshold level showed an excellent discrimination ability to predict survival, with long-term all-cause mortality substantially increasing for each additional positive marker (see Kaplan-Meier curves).

Conclusion

In conclusion, the presence of elevated baseline levels for 0, 1, 2, or 3 of the independent SASP factors reflecting different underlying pathophysiological mechanisms predicts increased long-term all-cause mortality with profoundly improved discrimination power in AVS patients undergoing TAVR. If confirmed in further prospective studies, a combination of these 3 SASP markers could serve as a highly useful risk stratification tool in the elderly population with AVS.
Changes in the immune cell landscape in the infarcted mouse heart
Authors: Mitre L , Burkard A , Hansen S , Li X , Ehret F , Heitlinger S , Mwinyella T , Marchini T , Olawale T , Gissler M , Horstmann H , Wolf D , Westermann D
Published: November 2023

Background

Myocardial Infarction (MI) and Ischemic Heart Failure represent the leading causes of death globally. Myocardial remodeling after MI is accompanied and driven by a strong immune response with an accumulation of several, partially opposing immune cell types. The temporal and spatial dynamics of this cardiac specific immune cell infiltration, however, have not been systematically evaluated.

Purpose

We characterized the dynamics of the immune cell repertoire in murine hearts following myocardial infarction by multi-color flow cytometry using a novel 12-marker panel allowing us to characterize major principal leukocyte lineages with a total of 10 distinct leukocyte populations. In parallel, we studied the immune response after MI in secondary lymph organs such as heart´s draining lymph nodes and the spleen. In addition, we performed transcriptional analysis of the healthy and infarcted hearts by bulk RNA at selected time points.

Methods

Permanent ligation of the left descending coronary artery (LAD), ischemic/reperfusion injury (I/R) and sham surgery was performed on C57BL6/J, 10 weeks old male mice. Leukocytes dynamics and T cell responses were screened in infarcted and remote areas of the heart, as well as in blood, spleen, and heat´s draining lymph nodes after 1, 3, 7, 14 and 28 days by multi-color flow cytometry. Also, immune activation and exhaustion transcription analysis by bulk RNA of healthy and infarcted hearts was performed at day 3,7 and 28 days after MI.

Results

We describe a sequential shift in the resident heart leukocyte population, a change in T cell activation states and identified potential new targets such as NK and γ/δ T cells. While macrophages area the most frequent leukocytes under steady state, making up to 75% of the total relative leukocyte population, at day 28 Lymphocytes represent 50%, among which B cells represent the most of them, followed by T helper cells. At early stages, CD4+ T cells secret pro inflammatory cytokines in the heart and in the heart´s draining lymph nodes, however, as stable scar in the heart is formed at day 7 immunomodulatory cytokines secretion such as IL-10 was observed. This is consistent with the build-up of a relevant adaptive immune response involving antigen presenting DCs and effector T and B cells in the healing heart.

Conclusions

Our findings contribute to identify novel cellular and transcriptional targets in the fight against ischemic heart failure.
Quantitative assessment of myocardial fibrosis by digital image analysis: an adjunctive tool for pathologist ground truth
Authors: Maltes S , Abecasis J , Dias N C , Pinto D G , Santos R R , Lopes P M D , Madeira M , Cardim N , Ramos S , Felix A
Published: November 2023

Aims

Myocardial fibrosis (MF) is a common pathological process in a wide range of cardiovascular diseases. Its quantity renders both current myocardial condition and previous accumulated damage. We aimed to assess if the use of an automated artificial intelligence software for the quantification of MF on endomyocardial biopsies (EMB) might improve individual pathologist´s semi-quantification and agreement, in terms of precision.

Methods and Results

EMB samples were studied from intraoperative biopsies of the interventricular septum of 30 patients with severe aortic stenosis submitted to surgical aortic valve replacement. Tissue sections were stained with Masson´s trichrome for collagen/fibrosis and whole slide images (WSI) from the experimental glass slides were obtained at a resolution of 0.5mm using a digital microscopic scanner. Three experienced pathologists made a first quantification of MF excluding the subendocardium. An automatic algorithm, developed on QuPath software for Masson`s trichrome brightfield WSI, was applied and MF quantification ensued (Figure 1), being revealed to the pathologists. A new blinded quantification was performed by each of the pathologists after two weeks. The agreement of MF quantification and the impact of the automatic quantification were tested with Bland-Altman type methodology.

Median values of MF on EMB were 8.33% [IQR 5.00-12.08%] and 13.60% [IQR 7.32-21.2%], respectively for the first pathologist´s and automatic algorithm quantification, being highly correlated (R2: 0.79; p<0.001) (Figure 2). Inter-observer discordance increased at first observation for higher percentages of MF, but the knowledge of the automatic quantification improved the overall pathologist´s agreement, which became unbiased for distinct grades of MF (Figure 3).

Conclusions

The use of an automatic digital pathology algorithm for the quantification of MF on EMB samples significantly improves the reproducibility of measurements by experienced pathologists. This may prove to be a useful adjunctive tool to both improve the reliability of the quantification of myocardial tissue components and imaging-pathology correlation studies.
Cardiac-specific deletion of myosin phosphatase targeting subunit 2 alleviates cardiac fibrosis in mineralocorticoid receptor-related hypertension
Authors: Ye Z , Okamoto R , Ito H , Moriwaki K , Ito R , Ida M , Ito M , Dohi K
Published: November 2023

Introduction

Myosin phosphatase targeting subunit 2 (MYPT2) is a crucial subunit of cardiac myosin light chain (MLC) phosphatase. The phosphorylation of MLC in the ventricle plays a pivotal role in cardiac hypertrophy and function. Our previous study demonstrated that overexpression of MYPT2 activated MLC phosphatase leading to cardiac dysfunction. Mineralocorticoid receptor (MR)-related hypertension is associated with extensive cardiac fibrosis. It remains unknown whether MYPT2 may play a role in the development of cardiac fibrosis in MR-related hypertension.

Purpose

Our purpose is to investigate the effect of MYPT2 on cardiac fibrosis in MR-related hypertension.

Methods

After the knockdown of MYPT2 using MYPT2-siRNA, HL-1 cells (murine cardiomyocytes) were incubated with aldosterone (ALDO) for 24h. Cardiac-­specific MYPT2 knockout (c-MYPT2-/-) mice were generated using the Cre-lox system (αMHC-­Cre; MYPT2f/f). MR-related hypertension mice were induced in c-MYPT2-/- and wild-type (MYPT2+/+) mice with the subcutanouse infusion of ALDO and 8% NaCl food for 4 weeks after uninephrectomy. Systolic blood pressure (SBP) was measured by tail cuffs every week. WB, qPCR, cardiac echo, and histological examinations were performed.

Results

In HL-1 cells, ALDO increased the expression of connective tissue growth factor (CTGF), MYPT2, and the catalytic subunit of the type-1 phosphatase δ isoform (PP1cδ) in a concentration-dependent manner. MYPT2 protein levels significantly decreased by 80% in HL-1 cells transfected with MYPT2 siRNA compared with cells transfected with unspecific siRNA. Knockdown of MYPT2 significantly decreased the protein level of CTGF. In the mice model, the MYPT2 expression in the heart from MYPT2-/- mice was decreased to less than 30% compared with MYPT2+/+ mice. Heart rate, blood pressure, and cardiac systolic function were normal in c-MYPT2-/- mice. PP1cδ was decreased in c-MYPT2-/- mice compared with MYPT2+/+ mice. The expression of cardiac MLC kinase was not changed. The level of phosphorylation of MLC in c-MYPT2-/- mice was significantly higher than MYPT2+/+. Blood pressure elevation and left ventricular hypertrophy were observed in both MR-related hypertension mice. No significant difference in heart size and the nuclear localization of MR in cardiomyocytes. However, ejection fraction and fractional shortening in echocardiography were higher in c-MYPT2-/- than in MYPT2+/+ in MR-related hypertension mice. Histopathological examinations revealed the degree of fibrosis in c-MYPT2-/- mice was lower than in MYPT2+/+ mice with the decreased expression of CTGF.

Conclusions

Cardiac-specific deletion of MYPT2 resulted in a decrease of MLC phosphatase and an increase of phosphorylation in MLC in vivo. MYPT2 deletion can inhibit cardiac fibrosis in a MR-related hypertension model.
High FABP4, associated with low-voltage areas, increases the epicardial fibrosis and its glucose metabolism
Authors: Vazquez-Abuin X , Fu X , Couselo-Seijas M , Lopez-Canoa J N , Martinez-Cereijo J M , Reija-Lopez L , Fernandez-Gonzalez A L , Gonzalez-Juanatey J R , Rodriguez-Manero M , Eiras S
Published: November 2023

Background

High plasma levels of FABP4 were associated with epicardial fat volume on persistent AF patients. FABP4 can be released by differentiated adipocytes and macrophages, which can be involved in fibrosis.

Purpose

Our main objective was to study the association between FABP4 and epicardial fibrosis, which can explain low-voltage areas (LVA) and atrial fibrillation (AF) recurrence.

Material and methods

Peripheral levels of FABP4 and Galectin-3 were analysed on AF patients undergoing pulmonary vein isolation (PVI) (n=299) and signed informed consent. During the intervention, LVA was evaluated by invasive electroanatomic voltage mapping (EAM). Epicardial (EAT) (n=6) and subcutaneous adipose tissue (SAT) (n=5) were obtained from undergoing open heart surgery patients. Stromal cells were isolated by collagenase digestion, cultured and treated with and without FABP4 100 ng/mL and/or galectin-3 30 ng/mL for 24 hours. The proliferation and migration rate were measured by wound healing assay; mitochondrial activity was studied by MTT assay; and glucose and lactate consumption were detected by colorimetric and fluorescence enzymatic assays, respectively. Gene expression levels were assessed by real-time PCR.

Results

Regression analysis determined that the main independent predictors of LVA were age, left atrial area and FABP4 levels. Preclinical "in vitro" studies have shown that FABP4 100 ng/mL induced fibrosis in epicardial stromal cells (p=0.0039) and subcutaneous stromal cells (p=0.0009), evidenced by wound healing assay and by the increment of associated fibroblast genes (COL1A2, VIM, TGF-β1). Moreover, the high FABP4 concentration modulates the protein levels and metabolic genes expression (CPT1, FASN, ATGL, related to transport, synthesis and degradation of fatty acids, respectively) on EAT stromal cells. Glucose metabolism without lactate production was also incremented after FABP4 treatment (p<0.0001). Even, on SAT cells, released lactate was reduced after FABP4 treatment (p=0.0218).

Conclusions

Plasma FABP4 was a predictor of LVA. Our results suggest that this association might be explained by the fibrotic and metabolic role of FABP4 in epicardial cells. Future studies will shed light on the benefits on atrial remodelling with FABP4 modulators.Figure 1Figure 2
ERBB4 stimulation mitigates atrial fibrillation and remodeling in a porcine DOCA model
Authors: Tubeeckx M R L , Goovaerts B , Van Fraeyenhove J , Murphy S L , De Meyer G R Y , De Keulenaer G W , Heidbuchel H , Segers V F M
Published: November 2023

Background

Before patients have spontaneous AF, pathologic remodeling is often already extensively present in the atria, creating a substrate for AF. Currently, no therapies exist that target this atrial remodeling, that can be divided into electrical (ion channel alterations) and structural (fibrosis, dilatation) remodeling. JK07 is a long-acting neuregulin-fusion protein with preventive effects towards ventricular fibrosis in models of heart failure through selective stimulation of the ERBB4 receptor.

Purpose

To test the ability of JK07 to reduce atrial fibrosis and AF inducibility in the minipig model of deoxycorticosterone acetate (DOCA, an aldosterone agonist) induced hypertension.

Methods

18 Aachener minipigs were randomized into 3 groups: control (CTRL), DOCA + Vehicle (DOCA) and DOCA+JK07. The control group did not undergo disease induction or therapeutic intervention. To induce hypertension and atrial fibrosis, pellets releasing 100 mg/kg DOCA over a period of 60 days were implanted in minipigs in the DOCA+VEH and DOCA+JK07 groups. The DOCA-implanted animals underwent weekly treatment with JK07 (0.3 mg/kg; DOCA + JK07) or vehicle control (DOCA), starting on the day of implantation (total of 9 administrations). After 60 days, arterial blood pressure was measured invasively and a decapolar catheter was placed in the right atrium. AF inducibility was tested by performing 50 burst pacing episodes and quantified as the percentage of "successful" episodes where an AF run ≥ 5 sec could be induced after the burst, out of the total attempts per group. Atrial fibrosis was quantified using ImageJ software on Masson trichrome staining of atrial specimens.

Results

Mean arterial pressure was significantly higher in the DOCA+VEH (142 ± 10 mmHg) and DOCA+JK07 (132 ± 15 mmHg) groups than in the CTRL group (105 ± 8 mmHg, p<0.01), without an effect of JK07.

AF inducibility in the DOCA+VEH group was significantly higher than in the CTRL group (66/250 vs. 9/300, p<0.001), and the DOCA + JK07 group (66/250 vs. 8/300, p<0.001).

The degree of fibrosis in the left atrial free wall was significantly higher in the DOCA group compared to CTRL (12.16 ± 5.06 vs. 5.14 ± 1.57, p=0.0089) and compared to DOCA+JK07 (12.16 ± 5.06 vs. 5.47 ± 3.69, p=0.026). Similar results were also found in the right atrial free wall and left and right atrial appendage.

Conclusions

ERBB4 stimulation prevents atrial fibrotic remodeling and AF inducibility in a porcine DOCA model. The effect of JK07 is unrelated to effects on blood pressure, but probably related to reduced atrial fibrogenesis.ERBB4 stimulation prevents AF inductionERBB4 stimulation prevents fibrosis
Interleukin 37 attenuates platelet activation and thrombosis formation
Authors: Gao W , Chen Y F , Luo X P
Published: November 2023

Aims

Interleukin 37 (IL-37) emerges as a natural suppressor of innate inflammatory and immune responses. Plasma IL-37 is abnormally increased in cardiovascular disease. Pleiotropic effects of IL-37 beyond anti-inflammation have been shown. However, the direct effects of IL-37 on platelet activation and thrombosis remain unclear. This study explored the direct effects of IL-37 on platelet activation and thrombosis.

Methods and results

A negative correlation between plasma IL-37 concentration and platelet aggregation was observed in patients with myocardial infarction. IL-37 directly inhibited ADP, thrombin and collagen induced platelet aggregation and dense granule release (Figure 1A, B). Platelet spreading in immobilized fibrinogen and clot retraction driven by integrin αIIbβ3 mediated out-side in signaling were inhibited by IL-37 concentration-dependently. In vivo thrombosis formation and stability in a FeCl3-injured mesenteric arteriole thrombosis mouse model was also severely suppressed by IL-37. Using co-immunoprecipitation, confocal microscopy and platelet-specific IL-1R8-deficient mice, we found that IL-37 bound to platelet IL-1R8 and IL-18Rα, and silencing of IL-1R8 impaired inhibitory effects of IL-37 on platelet activation. Mechanistic studies revealed that IL-37 combined with IL-1R8 to enhance PTEN activity and inhibited Akt, mitogen-activated protein kinase pathways. Platelet-specific IL-1R8-deficient mice presented impaired thrombosis and hemostasis but had improved cardiac function, reduced infarct size, decreased inflammatory response, and microthrombus after permanent ligation of the left anterior descending coronary. While control mice treated with IL-37 were protected from myocardial injury, IL-1R8-/- mice were not (Figure 1C, D).

Conclusions

IL-37 directly attenuated platelet activation and thrombosis formation by binding to platelet IL-18Rα and IL-1R8 and regulating the downstream signaling pathways, suggesting the potential role of exogenous IL-37 protein to prevent platelet hyperactivity thrombotic complications in patients with low plasma IL-37 levels.resultsmode picture
Incidentally induced atrial fibrillation during programmed electrical stimulation in patients with depressed left ventricular systolic function after an acute myocardial infarction: a CARISMA substudy
Authors: Sakthivel T , Thomsen A F , Jacobsen P K , Koeber L , Joergensen R M , Jacobsen U G , Huikuri H V , Bloch Thomsen P E , Joens C
Published: November 2023

Background

The aim of this study was to investigate the clinically significant value of incidentally induced atrial fibrillation (AF) during programmed electrical stimulation (PES) in patients with left ventricular systolic dysfunction (≤40%) after an acute myocardial infarction (MI).

Methods

In this study we included 231 patients from the Cardiac Arrhythmias and RIsk Stratification after Myocardial InfArction (CARISMA) study. These patients were identified 3-21 days after being diagnosed with an MI, had left ventricular ejection fraction ≤40% and had no prior history of AF. They all received an implantable cardiac monitor (ICM) and were continuously monitored for cardiac arrhythmias for 2 years. A PES was performed 6 weeks post-MI with pacing from the right atrium and coronary sinus for induction of SVT and from the ventricular apex and outflow tract with up to three extra stimuli for induction of VT. Induction of AF was unwanted but reported if this incidentally occurred.

Results

AF was induced in 34 patients (15%) during the PES of which 10 (29%) later developed AF. A total of 61 patients (26%) developed AF within 2-years follow-up and the overall risk of AF was not significantly increased in patients with PES induced AF compared to patients without inducible AF (HR 1.6, P=0.14), however, the risk of AF ≤188 days post-MI was slightly increased (HR 3.0, P=0.01). The risk of bradyarrhythmia (HR=0.2, P=0.07), ventricular arrhythmias (HR=0.7, P=0.77) and MACE (HR 0.5, P=0.28) was reduced in patients with inducible AF compared to patients without inducible AF.

Conclusions

Incidentally induced AF during an electrophysiological study in post-MI patients was relatively frequent (15%) but was not associated with a higher risk of atrial fibrillation, other cardiac brady- or tachy-arrhythmias or major cardiac events. Even though the short-term risk for AF slightly increased, this does not seem to justify anticoagulation treatment or intensified follow-up in these patients.
Histone Mono-methylation by SETD7 Orchestrates Angiogenic Response in Diabetic Limb Ischemia
Authors: Mohammed S A , Gorica E , Karsay G , Albiero M , Ambrosini S , Fadini G P , Ruschitzka F , Costantino S , Paneni F
Published: November 2023

Background

Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose: To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology: Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes.

Results

RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G, (Figure 1). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle (Figure 2). Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls (Figure 2) Conclusion: Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.
Increased levels of platelet function-related miR-16-5p predicts increased severity of ischemic stroke: in silico validation analysis
Authors: Eyileten C , Nowak A , Wicik Z , Keshwani D , Marszalek M , Domitrz I , Wierzchowska-Ciok A , Mirowska-Guzel D , Czlonkowska A , Postula M
Published: November 2023

Background

Ischemic stroke (IS) is one of the most frequent causes of death. Since miRNAs have been illustrated to play an important role in various processes through regulation of multiple genes, and platelet function, their utility as novel biomarkers should be determined. We aimed to identify and validate circulating miRNAs, modulated in acute ischemic stroke (IS) patients in order to distinguish the most specific as biomarkers to facilitate diagnosis and prognosis.

Method

Sixty patients with acute IS (24-h and 7-days after IS) and 30 age- and gender-matched individuals free of stroke with multiple risk factors for cardiovascular disease. Studied miRNAs were selected based on bioinformatic analysis that was previously published by our team (1-2-3). PlasmaRNA was extracted; quality RNA was assessed: fluorometric assay; RT-PCR for miRNAs expression measurement p<0.05.

Results

Let-7e expression 7-day of IS was significantly lower than both controls and first day of IS (p=0.007, p=0.001, res). miR-125a-3p was lower both at day-1 and day-7 of IS compared to controls (p=0.008, p=0.020, res). miR-125a-5p on the other hand, had significantly higher expression at day-1 compared to controls (p=0.002). Both miR-125a-3p and -5p showed diagnostic value in ROC curve analysis (AUC= 0.705, AUC= 0.709, res). Patients with moderate stroke (based on NIHSS) had significantly higher miR-16-5p expression levels compared to patients with minor stroke at the first day of IS (AUC: 0.718, (95% CI, 0.59-0.85) p = 0.004). High baseline expression of miR-16-5p and diabetes mellitus are independent predictors of increased severity of stroke to a multivariate analysis (OR: 4.341; 95% CI, 1.15–16.42; p = 0.031 and OR: 4.653; 95% CI, 1.07–20.19; p = 0.040, res).

Conclusion

In this study we validated miRNAs based on our previous bioinformatic analysis which could serve as novel, platelet-related predictive biomarkers of the IS severity, and can be used for early stratification of patients and prediction of severity of disease development in an individual.
A selective mineralocorticoid receptor blocker, esaxerenone, attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein e-deficient mice
Authors: Munkhjargal U , Daiju F , Masataka S
Published: November 2023

Introduction

Pharmacological blockade of mineralocorticoid receptors (MR) is a potential therapeutic approach to reduce cardiovascular complications because MR play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vascular function. We investigated the effects of a novel non-steroidal selective MR blocker, esaxerenone (ESAX), recently approved for hypertension treatment in Japan, on vascular function and the development of atherosclerosis.

Methods and results

ESAX (3mg/kg/day) or vehicle was orally administered to western type diet-fed apolipoprotein E-deficient mice for 20 weeks or for 8 weeks to investigate the effects on atherogenesis or vascular dysfunction. Administration of ESAX for 20 weeks suppressed the development of atherosclerotic plaques in the aortic arch compared with the vehicle group (p<0.001) without alteration of blood pressure. In addition, ESAX reduced the expression of intercellular cell adhesion molecule-1 (p<0.05), macrophage infiltration (p<0.01), lipid deposition (p<0.05) and tended to decrease the expression of vascular cell adhesion molecule-1 as determined by immunohistochemical analysis. Moreover, phosphorylation of eNOSSer1177 (p<0.05) and Akt (p<0.05) were increased in the aorta of ESAX-treated mice. Administration of ESAX for 8 weeks attenuated western type diet-induced endothelial dysfunction (p<0.05) accompanied with the decrease in expression of ICAM1 (p<0.05) and phosphorylation of JNK (p<0.05) in the descending aorta. In in vitro experiment, aldosterone decreased the phosphorylation of eNOSSer1177 and increased the phosphorylation of eNOSThr495 in a dose dependent manner, which were attenuated by ESAX in HUVECs. In ex vivo vascular reactivity assay, ESAX diminished the aldosterone-induced endothelial dysfunction.

Conclusion

ESAX ameliorated hyperlipidemia-induced atherosclerosis and vascular dysfunction in mice, suggesting that ESAX is a promising therapeutic approach for cardiovascular complications.
Ischemic stroke is associated with reduced sclerostin expression in human atherosclerotic plaques
Authors: Miteva K , Burger F , Roth A , Thouverey C , Ferrari S L , Mach F
Published: November 2023

Objectives

Vascular calcification (VC) is a hallmark of atherosclerosis and commonly found in aging population, increasing the risk of myocardial infarction (MI), stroke and mortality. Our data shows that Wnt/β-catenin signaling pathway and its main inhibitor, sclerostin (Scl, Sost) could be central to this process. However, Scl inhibition with romosozumab has been associated with a higher incidence of major adverse cardiovascular events (MACE). This increases in MACE call into question the safety of romosozumab use, particularly in patients with cerebrovascular and cardiovascular history. However, whether Scl expression levels in atherosclerotic plaques are associated with clinical ischemic events remains unknown. Here we explored the association between Scl expression in human atherosclerosis plaques and the extend of VC, VSMCs phenotypic switch to osteoblast-like cells (OBL), and its correlation to clinical cerebrovascular events.

Methods

Carotid plaque specimens of symptomatic (ischemic stroke) and asymptomatic patients undergoing endarterectomy for severe carotid stenosis were co-stained with VSMCs specific marker (Myh11), OBL marker (RUNX2), microcalcification sensitive fluorescent imaging probes (OsteoSense) and Scl and used for quantification analyses.

Results

scRNA seq data showed that Sost is upregulated by VSMCs in the aortic region protected against VC, suggesting an inhibitory role of Sost on VSMCs differentiation into OBL. Carotid plaque specimens from asymptomatic patients exhibited a significantly higher percentage of VSMCs expressing Scl (Myh11+Scl+) in parallel with a greater Scl+ area versus symptomatic patients. A negative correlation was observed between the percentage of Myh11+Scl+ cells and the extent of microcalcification (OsteoSense) as well as between the percentage of total Scl+ cells and induction of VSMCs phenotype switch to osteoblast-like cells (quantified as RUNX positive area).

Conclusion

In atherosclerotic plaques of patients with ischemic stroke, total and VSMCs -specific Scl expression levels were reduced as compared to asymptomatic patients. Furthermore, reduction in Scl levels was positively associated with the extent of microcalcification, and VMSCs phenotypic switch to OBL. The presented findings suggest that sclerostin expression in human atherosclerotic plaques could contribute to plaque stabilization and be protective against ischemic stroke, explaining why Scl inhibition with romosozumab resulted in a higher incidence of MACE.Figure 1Figure 2
Increased 12/15-lipoxygenase by disturbed flow promotes endothelial dysfunction and the development of atherosclerosis
Authors: Wang X Q , Chen J W , Lu L , Yang C D
Published: November 2023

Introduction

Atherosclerosis is predisposed at bifurcations, branch points and arterial curvatures where the blood flow is disturbed. Deposition and oxidative modification of low-density lipoprotein (LDL) in the subendothelial intima is an early event in the pathogenesis of atherosclerosis. However, the effect of disturbed flow (d-flow) on LDL oxidation and the underlying mechanisms remain unclear.

Purpose

In this study, we investigated the role of 12/15-lipoxygenase (LOX) in LDL oxidation in endothelial cells (EC) and the development of atherosclerosis at d-flow sites.

Methods

En face immunofluorescence was performed to analyze the expression of 12/15-LOX in EC under exposure to different flow patterns. Protein expression and LDL oxidation was studied in vitro by using 2 different flow apparatuses. Animal model of d-flow was established by partial ligation of the carotid artery.

Results

En face staining exhibited a significant increase in 12/15-LOX protein expression in EC in areas exposed to d-flow than those exposed to steady flow (s-flow). Carotid partial ligation in ApoE knockout mice led to substantially increased deposition of oxidized LDL in the subendothelial intima and formation of atherosclerotic plaques in the carotid artery, whereas these detrimental effects by d-flow were markedly attenuated in ApoE/12/15-LOX double knockout mice. In cultured EC, d-flow generated by a reversal flow pump markedly promoted the expression of 12/15-LOX and translocation of the protein onto the cell membrane. Inhibition of 12/15-LOX in EC, either by knockdown with its specific siRNA or a pharmacological inhibitor, suppressed production of 15s-HETE and LDL oxidation in response to d-flow. Mechanically, we found d-flow induced the expression of 12/15-LOX by activating a specific responsive element in the 12/15-LOX promoter through recruiting a shear stress-sensitive transcriptional factor SREBP2. Chromatin immunoprecipitation further confirmed the interaction of SREBP2 with the promoter of 12/15-LOX upon d-flow exposure.

Conclusions

These data define an essential role of 12/15-LOX in promoting the pathogenesis of atherosclerosis under d-flow by increasing LDL oxidation in EC through SREBP2 signaling.
Quantification of local heterogeneity from activation maps of omnipolar multielectrode recordings
Authors: Ruiperez-Campillo S , Pancorbo L , Castells F , Crespo M , Chorro F J , Merino J L , Casado-Arroyo R , Millet J
Published: November 2023

Background and Purpose

The study of cardiac arrhythmias requires understanding the heart's electrical conduction patterns. Using multielectrode catheters and other mapping systems have allowed a futher understanding of the wavefront. In particular, novel high-density (HD) grid catheters, enable the recording of omnipolar electrograms, which are purportedly not influenced by catheter orientation, allowing real-time determination of signal propagation direction. We propose a novel heterogeneity metric to quantify the degree of disorganisation in the vector maps from reconstructed omnipolar electrograms, which could potentially be utilised to detect and assess the degree of fibrosis in cardiac tissue.

Methods

We utilised basal and stimulated recordings from 5 Langendorff-perfused rabbit hearts, where signals were obtained using a multielectrode mapping catheter with 128 electrodes spaced at 1mm. Three recordings were taken for each series, basal at 37°C, plus stimulation performed using a bipolar electrode at 4 Hz and 6 Hz . An angle propagation matrix is computed for the local area of recording, by means of the orthogonal bipolar loops of each clique. A local heterogeneity metric is derived from the summation of angular variations of neighbouring elements. An illustration of the heterogeneity map, and heterogeneity metric for the recording area (H) is illustrated in figure 1. A statistical analysis is performed then to assess the distribution of heterogeneity values.

Results

Heterogeneity values for each experimental heart, and the distributions of H values obtained according to the stimulation type are shown in figure 2A. The ROC curve obtained from the values of H has an AUC of 0.996, with an optimal threshold value of 0.113, resulting in a confusion matrix presented in 12 TP, 30 TN, 3 FP and 0 FN. The study shows that the proposed heterogeneity metric can quantify the degree of disorganization in the vector maps and can be used to differentiate between basal and stimulated groups with p<0.001.

Conclusion

This study proposes a novel heterogeneity metric to quantify the degree of disorganization in the vector maps from reconstructed omnipolar electrograms. The methodology was assessed on basal and stimulated epicardial rabbit recordings and found to be effective in differentiating between the two groups with high accuracy. Future studies should focus on the efficiency of assessing fibrotic tissue using the proposed metric.Figure 1Figure 2
Direct oral anticoagulants versus vitamin K antagonists for left ventricular thrombus: a systematic review and meta-analysis
Authors: Pasqualotto E , Gewehr D M , Ferreira R , Silva C H A , Choque J W L , Cruz S A , Park A , Kubrusly L F
Published: November 2023

Introduction

Left ventricular thrombi (LVTs) increase the risk of stroke and systemic embolism and are commonly seen after acute myocardial infarction. While vitamin K antagonists (VKAs) are the recommended first-line treatment for LVT, direct oral anticoagulants (DOACs) are being used as an alternative. However, the comparative efficacy and safety of DOACs and VKAs in LVT treatment are unclear.

Purpose

We conducted a systematic review with meta-analysis to evaluate the impact of DOACs compared with VKAs therapy in patients with LVT.

Methods

PubMed, Embase, and Cochrane were systematically searched for studies that compared DOACs versus VKAs and reported efficacy and safety endpoints. We performed meta-analyses with fixed or random effect models based on the level of heterogeneity determined by the Cochrane Q-test and I2 statistics.

Results

Three randomised controlled trials (RCTs) and 29 cohort studies were included, with 4,400 patients assigned to either DOACs (1,307 patients) or VKAs (3,093 patients). Our findings indicate that DOACs therapy did not significantly reduce stroke/systemic embolic events (OR 0.90; 95% CI 0.65-1.26; p=0.54; I2=33%), stroke (OR 0.90; 95% CI 0.59-1.36; p=0.60; I2=7%), systemic embolic events (OR 0.65; 95% CI 0.39-1.06; p=0.08; I2=0%), thrombus resolution (OR 1.11; 95% CI 0.92-1.33; p=0.27; I2=0%), minor bleeding (OR 0.68; 95% CI 0.39-1.18; p=0.17; I2=0%), major bleeding (OR 0.84; 95% CI 0.47-1.49; p=0.54; I2=16%), or all-cause mortality (OR 1.01; 95% CI 0.75-1.36; p=0.97; I2=0%) compared to VKAs. However, DOACs therapy was associated with a statistically significant reduction in any bleeding (OR 0.72; 95% CI 0.55-0.96; p=0.02; I2=0%) and clinically relevant bleeding (OR 0.62; 95% CI 0.41-0.92; p=0.02; I2=0%) compared to VKAs therapy. Our subgroup analysis of patients treated with apixaban revealed no significant differences between DOACs and VKAs therapies for stroke, clinically relevant bleeding, and all-cause mortality. Conversely, rivaroxaban therapy was associated with a statistically significant reduction in systemic embolic events and clinically relevant bleeding compared to VKAs therapy.

Conclusion

DOACs therapy was not associated with reduced efficacy and safety endpoints compared to VKAs therapy, except for fewer haemorrhagic events. Rivaroxaban therapy had a significant reduction in systemic embolic and hemorrhagic events. These findings support the superiority of DOACs over VKAs therapy for LVT.PRISMA Flow-ChartForest plot of odds ratios (OR)
Efficacy and safety of antiarrhythmic drugs for pharmacological cardioversion of atrial fibrillation depending on gender: a subanalysis of CANT II Study
Authors: Wybraniec M , Maciag A , Miskowiec D , Balsam P , Wojcik M , Wrobel W , Farkowski M , Cwiek-Rebowska E , Szolkiewicz M , Bula K , Krzowski B , Kozinski M , Kasprzak J , Szwed H , Mizia-Stec K
Published: November 2023

Background/introduction

The data on gender differences concerning antiarrhythmic drugs (AAD) applied in atrial fibrillation (AF) patients is scarce.

Purpose

The aim of the study was to evaluate gender differences in terms of clinical profile of AF patients, and efficacy and safety of AAD used for pharmacological cardioversion (PCV) of AF.

Methods

This research was a sub-analysis of retrospective multicenter Cardioversion with ANTazoline II (CANT) registry, which comprised 1365 patients with short-duration AF referred for urgent PCV with the use of AAD. Patients were categorized in terms of gender and compared in terms of clinical parameters and outcome of PCV. The primary endpoint was return of sinus rhythm within 12 hours after drug infusion and the composite safety endpoint involved bradycardia <45 bpm, hypotension, syncope or death.

Results

The majority of patients qualified for PCV were men (n=725, 53.1%). Females were older and more symptomatic, had higher CHA2DS2-VASc score, greater prevalence of tachyarrhythmia and higher utilization of chronic anticoagulation. The overall efficacy (71.4% vs. 70.1%,P=0.588) and safety (5.2% vs. 4.6%,P=0.603) of PCV was comparable in men and women. Amiodarone (68.3% vs. 65.9%, P=0.664) and antazoline (77.1% vs. 80.0%, P=0.19) had similar efficacy in men and women, but propafenone had lower rate of rhythm conversion in men (64.7% vs. 79.3%, P=0.046; Figure). None of the assessed AAD differed in terms of safety profile in both genders. Logistic regression showed that, among other predictors, in women the use of amiodarone vs. other AAD (odds ratio [OR] 0.569, P=0.033), while in men administration of a amiodarone (OR 0.379, P=0.001) or propafenone (OR 0.331, P=0.002) vs. other AAD were associated with lower chance of successful rhythm conversion.

Conclusions

Female patients with AF share different clinical profile and similar efficacy and safety of AAD used for PCV as compared to male participants. Propafenone has significantly lower efficacy in men, which requires further prospective investigation.