bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ventricular hyperinnervation in a mouse model of catecholaminergic polymorphic ventricular tachycardia
Authors: O'reilly M , De Waal T , Casini S , Remme C A 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): European Society of Cardiology Basic Research Fellowship.
Dutch Heart Foundation Dekker Grant

Background
Inherited arrhythmia syndromes are a leading cause of sudden cardiac death in young and otherwise healthy patients. They are often caused by mutations in genes that encode ion channels or transporters, leading to conditions such as Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). CPVT is caused by mutations in RYR2 (encoding ryanodine receptor 2, RyR2). In arrhythmia research, investigation into the effects of these mutations have exclusively been performed in cardiac cells. However, RyR2 is also present in neuronal tissue, including intracardiac neurons [1] (which modulate cardiac function). Moreover, patients often present with clinical signs of autonomic nervous system (ANS) dysfunction, and treatment strategies frequently target the ANS.

Aim
Our novel research line aims to investigate the neuronal phenotype induced by RyR2 mutations, using a CPVT mouse model to assess functional alterations as well as broader autonomic remodelling.

Results
Using immunocytochemistry, we reveal for the first time that RyR2 is also expressed in mouse stellate ganglia (crucial autonomic modulators of cardiac function). Through analysis of published RNA sequencing datasets, we find that RYR2 is more abundantly expressed in both stellate ganglion and intracardiac neurons than characteristic neuronal markers such as CHAT, SYN1 and SCN8A [2,3].
Functional patch clamp investigation of isolated stellate ganglion neurons have revealed two distinct populations based on their patterns of action potential (AP) firing, identifying both phasic-firing and tonic-firing type neurons. Investigation of an established CPVT mouse model (RyR2-R2474S) has thus far not revealed any differences in the AP firing frequency of isolated stellate ganglion neurons during sustained depolarising stimuli of various amplitudes. Assessment of calcium homeostasis and neurotransmitter release is currently ongoing.
We have furthermore investigated broader autonomic remodelling using immunohistochemistry in mouse heart cryosections. Neuronal innervation density was assessed by staining 10 µm sections of mid-ventricular myocardium for the sympathetic neuron marker tyrosine hydroxylase (TH). Quantification of the TH-positive area revealed a significant increase in the percentage of sympathetic neuronal innervation in ventricular tissue of CPVT mice (5.7% wild-type vs 7.2% RyR2-R2474S, p=0.0253, N=6).

Conclusions
The neurons that modulate heart function (stellate ganglion and intracardiac neurons) abundantly express RyR2. The RyR2-R2474S mutation leads to significant sympathetic hyper-innervation of ventricular myocardium, which may have important consequences for neurotransmitter release and hence cardiac (electrical) function and arrhythmogenesis.
A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes
Authors: Celeghin R , Risato G , Beffagna G , Cason M , Bueno Marinas M , Della Barbera M , Facchinello N , Branas Casas R , Rizzo S , Dalla Valle L , Argenton F , Thiene G , Tiso N , Pilichou K , Basso C 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Associazione Italiana per la Ricerca sul Cancro [AIRC grant IG-2017-19928], Italian Ministry of University and Research [grant PRIN 20173ZWACS]

Introduction
Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp).

Methods
We generated the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease.

Results
Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, edema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/β-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-β pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/β-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm.

Conclusion
Our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/β-catenin signalling cascade.
Exploring the regulation of circulating factors involved in aortic valve sclerosis onset
Authors: Moschetta D , Chiesa M , Massaiu I , Valerio V , Rusconi V , Bertolini F , Myasoedova V A , Poggio P 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): Ministero della Salute - Ricerca Finalizzata

Background
Aortic valve sclerosis (AVSc), the first stage of calcific aortic valve stenosis (CAVS), has a prevalence of 25-30% in subjects older than 65 years. Being a clinically silent phase, it is characterized only by non-uniform leaflet thickening due to fibrosis and microcalcifications, without the obstruction of the blood flow with an increased local oxidative stress, inflammation, and endothelial-to-mesenchymal transition (EndMT). However, to date, AVSc is not considered a pathology but 10% of cases evolve a full-blown CAVS.

Aim
Our aim is to investigate which systemic process(es) are activated in AVSc, in order to better define these subjects. These evaluations will allow us to identify potential druggable targets.

Methods
We consecutively enrolled 157 healthy subjects and detected AVSc presence by echocardiography. Plasma and serum samples were collected to evaluate circulating microRNA (RNA-sequencing) and cytokines (Luminex), respectively. Functional analysis was performed to reveal systemic pathway regulation. Healthy human valve endothelial cells were employed to evaluate viability (MTT assay) and EndMT (morphological analysis and quantitative PCR)

Results
AVSc was present in 77 patients (49%) and the differential expression analysis revealed that 59 microRNAs were down-regulated, while 93 were up-regulated in these subjects compared to healthy ones (adjusted-p&lt;0.05). The functional analysis revealed that the major enriched pathways in AVSc subjects were related to inflammation. Indeed, serum GROα and IL1β levels were higher in AVSc vs healthy (p&lt;0.05). In vitro, chronic GROα treatment resulted in lower cell viability (100.0±17.4 vs 61.8±16.5% of vital cells; p&lt;0.0001), while IL1β treatment induced a decrease in cell roundness and an increase in total area (0.40±0.18 vs 0.47±0.19AU, p&lt;0.0001; and 0.75±0.73 vs 0.45±0.48AU, p&lt;0.0001; respectively). Quantitative PCR revealed EndMT activation: SNAI1 (1.87±0.57 log2(foldchange), p&lt;0.0001) and SNAI2 (2.19±0.46 log2(foldchange), p&lt;0.0001) only after IL1β treatment.

Conclusions
Inflammation is the most regulated systemic pathway in AVSc subjects and its master regulator, IL1β, seems to be involved in the EndMT progression of valve endothelial cells, while GROα contributes to their death. These effects could be related to the early triggers of AVSc onset, leading to fibrosis and eventually calcification of aortic leaflets.
EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis
Authors: Kempkes R W M , Rief L C M , Roomen C P A A , Griffith G R , Vos W G , Bosmans L A , Gijbels M J J , Hoeksema M A , Prange K H M , De Winther M P J , Neele A E 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation (Hartstichting)

Aim
Epigenetic processes are essential modulators of macrophage inflammatory responses. We postulate that interference in the epigenetic machinery of macrophages might offer novel approaches to combat atherosclerosis. Here, we investigate the repressive histone modification H3K27Me3 deposited by the polycomb repressive complex 2 (PRC2) with its catalytic component EZH2. We studied the therapeutic potential of macrophage EZH2 inhibition in the context of atherosclerosis.

Methods
Human monocyte-derived macrophages and murine peritoneal and bone-marrow derived macrophages were treated with the EZH2-specific inhibitor GSK126 and subsequently activated with LPS to mimic TLR4-inflammatory responses. The impact of Ezh2 inhibition on macrophage differentiation and activation compared to vehicle (DMSO) was assessed by RNA-seq, ChIP-seq, flow cytometry, western blot, and ELISA. To study its impact on atherosclerosis, female Ldlr-/- mice on a high-cholesterol diet (9weeks) were treated the last four weeks with GSK126 or control.

Results
Ezh2 inhibition by GSK126 in vitro lowered global H3K27Me3 levels without altering macrophage viability and differentiation, showcasing effective EZH2 inhibition. RNA-seq revealed that of more than one-third of the LPS-induced genes were significantly downregulated by EZH2 inhibition. Subsequent pathway analysis identified cytokine and interferon signaling, co-stimulation, and cell migration as the top down-regulated pathways (padj&lt;0.05). Furthermore, ChIP-seq revealed considerably altered regulation of H3K27Me3 deposition. Indeed, we confirmed that gene and cytokine expression of the inflammatory mediators IL-6, IL-12, and TNF were reduced. Furthermore, membrane marker expression of co-stimulatory CD40, CD80, and CD86 were significantly decreased. In murine atherosclerosis, we observed a reduction in the Virmani plaque severity score and are currently assessing lesion size and composition.

Conclusion
Overall, we show that EZH2 inhibition reduces inflammatory responses in human and murine macrophages in vitro. We are currently assessing the impact of EZH2 inhibition on lesion size and composition in murine atherosclerosis. Additionally, we are performing ex vivo experiments on human endarterectomy plaques to assess the therapeutic potential of EZH2 inhibition on human atherosclerosis.
Aging related changes in cardiovascular system in healthy female population: photoplethysmography method and DFA analysis
Authors: Djuric B , Zikic K , Milosevic N , Nestorovic Z , Zikic D 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science Technological Development and Innovation

Background
The cardiovascular system undergoes dynamic changes during the biological aging in humans, leading to chronic cardiovascular and cerebrovascular diseases.

Purpose
Developing the novel method for non-invasive monitoring of age induced changes in arterial vessels.

Methodology
The pilot study was performed on 58 women, in good health, non-smokers, between the ages of 20 and 70, divided into three age groups. All subjects underwent arterial blood pressure measurement using a photoplethysmography sensor. Recordings were performed in left carotid artery and the left index finger area. Arterial blood flow waveform was then analyzed using DFA analysis, calculating the values of the scalar coefficients α1 and α2. Based on the waveform time interval shift between the standing and supine positions, the pulse wave velocity (PWV) ratio was determined.

Results
Systolic and diastolic arterial pressure showed a slight linear increase with the age, but without significant difference between the three age categories (p &gt; 0.5). Also, the PWV ratio between the supine and standing positions did not show a statistically significant increase between the age groups. Only scalar coefficients ratio α1/α2 demonstrated a significant difference in value between all age categories.

Conclusion
In subjects included in this pilot study, only the scalar coefficients ratio varied significantly with the age, indicating the occurrence of structural changes in the cardiovascular system caused by biological aging.
OPA1 affects lipid metabolism and atherosclerosis progression
Authors: Da Dalt L , Moregola A , Svecla M , Pedretti S , Fantini F , Donetti E , Mitro N , Scorrano L , Norata G D 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): European Atherosclerosis Society

Introduction
Mitochondria are involved in cellular metabolism, energy generation, calcium homeostasis, sterol and bile acids (BAs) production. Mitochondria continuously undergo biogenesis, fusion, fission and mitophagy, maintaining a continuous balance between all forms.

Purpose
On these premises, we test the impact of OPA1, an inner mitochondria membrane fusion protein, on mitochondrial tethering on lipid metabolism in the liver and the atherosclerotic plaque.

Methods
OPA1HepKO and OPA1 TG on LDLR KO background were fed with a Western-type diet (WTD) respectively for 12 weeks. Inverse calorimetry, GTT, and LipidToleranceTest (LTT) were performed. Paraffin-embedded tissues were used for histological analysis, frozen tissues were used for OMICs analysis. VSMCs were isolated and cultured with VLDL (50 µg/ml).

Results
While OPA1HepKO mice display altered systemic metabolism with reduced body weight and reduced circulating lipid levels OPA1 overexpression on LDLR KO background showed significantly high cholesterol levels. OPA1 deficiency impair hepatic bile acid conjugation with significant accumulation of primary unconjugated bile acids in the liver. On the contrary OPA1 overexpression leads to a reduction of unconjugated bile acids and higher percentage of conjugated bile acids. OPA1 reduction leads to a reduced atherosclerotic plaque while, on the contrary, its overexpression doesn’t affect atherosclerosis despite an increase in circulating lipid and lipoprotein levels. OPA1 overexpression is therefore promoting plaque stabilization with VSMCs that are less prone to change their metabolism to synthetic.

Conclusion
Hepatic Opa1 deficiency alters bile acids production and therefore circulating lipid profile with a consequent reduction in atherosclerotic plaque formation. OPA1 overexpression in the aorta affects plaque stability despite the highest levels of circulating lipids.
Circulating LIGHT/TNFSF14 levels are negatively associated with cardiovascular mortality, while intra-plaque levels are associated with symptomatic cerebrovascular disease
Authors: Shami A , Edsfeldt A , Nilsson J , Goncalves I 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): The Swedish Heart and Lung Foundation, Åke Wiberg's Foundation

Background
Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it is associated with a high-risk plaque phenotype and cardiovascular events is not known.

Purpose
This study investigates the association between intra-plaque and circulating LIGHT levels and cardiovascular disease.

Method
Plaque levels of LIGHT/TNFSF14 were measured in plasma and homogenised carotid endarterectomy plaques by Proximity Extension Assay in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=557 and 201, respectively). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through immunohistochemistry. Multivariate Cox proportional hazards analysis was performed on normalized (logarithmically transformed) values.

Results
Plaque, but not circulating, levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, intra-plaque levels of LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, fibromodulin, ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7) and matrix metalloproteinases 1, 2, 9 and 10. While plaque LIGHT levels were not predictive of cardiovascular death, circulating LIGHT levels were negatively associated with risk of cardiovascular death (after 6 ½ years) after correcting for the possible confounders age, sex, cerebrovascular symptoms, diabetes, high-sensitivity C-reactive protein, inflammatory disease, hypertension and smoking (hazard ratio=0.032, confidence interval=0.420-0.951, p=0.028).

Conclusion
Though intra-plaque LIGHT levels are associated with a rupture-prone plaque phenotype and pre-operative symptoms, circulating levels are associated with a lower risk of future cardiovascular mortality. This suggest that upregulation of LIGHT in atherosclerotic plaques may occur as part of a reparative rupture response and, systemically, reflect efficient wound-healing capacity.
The calcium-sensing-receptor (CaSR) in adipocytes contributes to sex-differences in the susceptibility to high fat diet induced obesity and atherosclerosis
Authors: Maas S L , Adam S , Huchzermeier R , Abschlag K , Hohl M , Santovito D , Jankowski J , Biessen E A L , Van Der Vorst E P C 
Published: May 2024

Abstract
Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): IZKF
Female mice are more resistant to the obesogenic effects of a high-fat diet (HFD), compared to male mice. Although the underlying mechanisms are poorly understood, sex hormones seem to play an important role. Interestingly, the activity of the estrogen receptor-α (ERα) is affected by the calcium-sensing-receptor (CaSR). Therefore, we investigated sex-differences upon HFD feeding and the role of adipocyte-specific CaSR herein.
Adipocyte-specific Casr deficient mice (AdipoqCre+Casrflox) and control mice (Casrflox) were injected with AAV8-PCSK9 and fed a HFD for 12 weeks.
Female mice have a lower visceral white adipose tissue (vWAT) mass then male mice, while this sex-difference is abolished upon adipocyte-specific Casr deficiency. Furthermore, while females showed an increase of IL-6, TNF-α and CCL2 in vWAT, compared to males, adipocyte-specific Casr deficiency rescued this sex-phenotype and demonstrated an inhibition of inflammatory signaling pathways. The expression of Erα, as well as associated genes involved in adipocyte differentiation, was increased in female mice in a mostly adipocyte-specific Casr dependent manner. Interestingly, circulating lipid levels were reduced in female compared to male mice, which coincided and correlated with decreased atherosclerotic plaque formation. These systemic effects were abrogated upon adipocyte-specific Casr deficiency.
Our findings indicate that female mice show a more pronounced obesity associated vWAT dysfunction compared to males. This sex effect is abolished in mice with an adipocyte-specific Casr deficiency. In contrast, females show a diminished hyperlipidemic response to HFD feeding and less atherosclerotic plaque formation compared to males, an effect that was rescued by adipocyte-specific Casr deficiency.
Incomplete microvascular reperfusion in a swine model of recanalized acute ischemic stroke
Authors: Wang M , Taha A , Raben J D , Noort R , Stam M , Duncker D J , Dippel D W J , Bobi J , Beusekom H M M 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CONTRAST CONSORTIUM (CVON, Dutch Heart Foundation)

Background
Incomplete microvascular reperfusion (IMR) could be a detrimental factor impairing regained functionality in stroke patients despite early recanalization. However, the presence of this phenomenon remains to be ascertained. Gyrencephalic large-animal models of acute ischemic stroke (AIS) such as swine are gaining attention as a next step in translation of preclinical to clinical stroke research, with brain anatomy and hemostasis more similar to humans. However, the occurrence and relevance of IMR in swine models of AIS is not well described yet, since most models do not include recanalization.

Purpose
We aimed to study the presence of IMR after brain ischemia in a translational swine model of AIS.

Methods
Anesthetized female Yorkshire-Landrace pigs (n=23) underwent craniotomy to occlude the right-sided middle cerebral arteries with aneurysm clips. Clips were released after 1 (n=5), 2 (n=5), and 4 (n=5) hours of occlusion to allow recanalization for 4 hours (recanalized AIS, n=15) or left in place for 8 hours (non-recanalized AIS, n=5). Three animals underwent the craniotomy without vessel clipping (Sham, n=3). 3D angiography was used to confirm occlusion and recanalization. Brains were collected after sacrifice, stained with TTC and then processed for histologic analysis. Infarct size was determined by TTC staining and expressed as percentage of the infarcted hemisphere. Histologic sections of the upper quadrant of the left and right hemispheres were immuno-stained with CD61 to visualize platelet aggregates in the microvasculature. Intravascular platelet aggregates (Figure 2A) were quantified using automated software analysis (Orbit) (CD61+ aggregates/mm2).

Results
All animals survived until the end of the procedure. Figure 1 shows differences in infarct size depending on the duration of occlusion (p=0.006). Figure 2 depicts an overall higher presence of platelet (CD61+) aggregates in the right hemisphere (infarct) compared to the left hemisphere (intact) (Figure 2B). There was a significant difference in platelet aggregates between left and right hemisphere but groupwise comparison showed only a trend (p=0.075) for more aggregates in the right hemisphere, compared to left, after 4 hours occlusion. Linear regression model could not identify whether infarct size and duration of occlusion were independent predictors of the number of platelet aggregates in the right hemisphere (p=0.191).

Conclusions
IMR occurs after brain ischemia in a swine model of AIS as a result of microvascular platelet aggregates. Further analysis and experiments are needed to elucidate which predictors can explain intravascular platelet aggregation.Infarct SizePlatelet aggregates