bluuebunny/crossref_metadata_2025 huggingface dataset:
Deeper insights into heart failure with preserved ejection fraction through rna-sequencing of circulating endothelial cells - advanced protocol establishment enables complete transcriptome analysis
Authors: Wurmbrand L , Kalies K , Knoepp K , Dutzmann J , Sedding D
Published: May 2024

Funding Acknowledgements 

None.

Heart failure with preserved ejection fraction (HFpEF) continues to pose a significant challenge with a bleak prognosis and a rising incidence, particularly among the aging population in Western countries. An in-depth comprehension of cellular-level pathogenesis, with a specific emphasis on the pivotal role of cellular senescence in endothelial dysfunction, is imperative for the evolution of innovative therapeutic interventions. Many previous approaches to investigate the genesis of HFpEF are based only on HFpEF-mouse models, which cannot directly be transferred to humans. The isolation of circulating endothelial cells from peripheral blood samples represents a more translatable, feasible, less invasive way to study endothelial dysfunction, a central component of HFpEF-pathogenesis, in human material for the identification of new therapeutical targets.

This study focuses on establishing a new protocol for isolating circulating endothelial cells (CEC) in HFpEF patients to address endothelial dysfunction and pinpoint novel therapeutic targets through a comprehensive whole transcriptome analysis. Methodologically, CEC isolation from whole blood samples was achieved using a newly established Fluorescence-Activated Cell Sorting (FACS) panel. HFpEF patients were recruited based on their HFA-PEFF score, with non-diseased controls comprising both young and old subjects meticulously matched for sex and age. Subsequently, the isolated CECs were subjected to transcriptome analysis.

Our new protocol is based on red blood cell lysis followed by magnetic cell sorting to deplete CD45+ cells. This is followed by staining with fluorescent-conjugated monoclonal antibodies. This FACS panel includes known endothelial-specific markers including CD11b- and CD45-, CD146+, CD34+ and CD31+ as well as DAPI- as a nucleus marker. The prepared CECs were sorted using FACS and a specially developed gating. We isolated the RNA in preparation for sequencing. Our protocol was validated by immunofluorescence staining of isolated CECs (markers: CD31, vWF, CD146, Phalloidin) and PCR analysis (markers: CD31, CD146 and vWF).

In summary, we have successfully established a new, robust and highly targeted workflow for consistently isolating circulating endothelial cells from blood and performing RNA sequencing on the collected samples. This is a key advance towards a deeper understanding of the pathophysiology of HFpEF. The application of our methodology may enable the characterization of patients with HFpEF at the transcriptome level. The whole transcriptome analyses provide potential starting points for further in vitro and in vivo studies. As we extend our investigations to larger patient cohorts in the future, there is potential for the identification of novel therapeutic targets.
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

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

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

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<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<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<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<0.0001; and 0.75±0.73 vs 0.45±0.48AU, p<0.0001; respectively). Quantitative PCR revealed EndMT activation: SNAI1 (1.87±0.57 log2(foldchange), p<0.0001) and SNAI2 (2.19±0.46 log2(foldchange), p<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

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<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

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 > 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

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

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

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

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
Proteomic profiling reveals higher presence of glycolytic enzymes in vulnerable atherosclerotic lesions
Authors: Palm K C A , Theofilatos K , Van Der Laan S , Diez Benavente E , De Borst G J , De Kleijn D P V , Wotja J , Mayr M , Den Ruijter H , Mokry M , Pasterkamp G
Published: May 2024

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Leducq PlaqOmics

Background

Our definition of vulnerable plaques is predominantly driven by histological analysis. The molecular mechanisms behind the vulnerable histological characteristics are not fully understood. Here we use mass spectrometry-based proteomics data, to identify molecular mechanisms linked to vulnerable plaques.

Methods

Atherosclerotic lesions from two hundred patients undergoing endarterectomy surgery were collected (149 male and 51 female plaques). From a total of two hundred plaque samples, extracellular matrix proteins were extracted and processed for untargeted proteomics analysis. The resulting protein levels were linked to existing histological characteristics, transcriptomic, and clinical data.

Results

We detected 1499 proteins in atherosclerotic lesions, which were mostly affiliated with biological processes including extracellular matrix organization, coagulation, and peptidase activity. Linear regression identified 240 proteins associated with plaque vulnerability index (a semi-quantitative score summarizing the presence of SMC cells, macrophages, plaque hemorrhage, and fat in the plaque). Among the proteins associated with high plaque vulnerability index we identified an overrepresentation of enzymes involved in glycolysis – including the rate limiting enzymes HK3 (beta =0.40 p =0.003) and PKM (beta =0.19 p =0.009). This was driven by macrophage numbers and fat content in the plaque. Furthermore, data from a validation cohort of 120 patients from the Medical University of Vienna, Austria, revealed that HK3 and PKM were associated with plaque progression (p =<0.001, p =0.017) and clinical symptoms (p = <0.001, p = <0.001).

Conclusion

Enzymes involved in the glycolysis process show higher presence in vulnerable plaques. This suggests an important role for energy metabolism in plaque vulnerability.
Increased bone morphogenetic protein 10 in precapillary pulmonary hypertension patients
Authors: Llucia-Valldeperas A , Van Wezenbeek J , Groeneveldt J A , Smal R , Sanchez-Duffhues G , Becher C , Handoko M L , Meijboom L , Marcus J T , Symersky P , Niessen H W M , Vonk-Noordegraaf A , Bogaard H J , Goumans M J , De Man F S
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organization for Scientific Research: NWO-VICI num. 918.16.610 and NWO-VIDI num. 917.18.338.

Dutch Heart Foundation Dekker senior post-doc grant num. 2018T059 and the Netherlands CardioVascular Research Initiative: CVON-2017-10 DOLPHIN-GENESIS and CVON-2018-29 PHAEDRA-IMPACT.

Fundació La Marató de TV3 num.202038.

The Spanish Ministry of Science through the Ramón y Cajal grant num.RYC2021-030866-I.

Introduction

Precapillary pulmonary hypertension (PH) results in increased right atrial (RA) stretch and pressure. The right atrium is the major source of bone morphogenetic protein (BMP) 10 (BMP10) in adults, mostly produced by RA cardiomyocytes. The major predisposing risk factor for PH involves loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene, through which BMP10 and BMP9 transduce signals.

Purpose

The aim of this study was to investigate if BMP10 expression is altered in PH, and whether BMP10 signaling activity associates with right ventricular (RV) adaptation, RA function and disease progression.

Methods

We investigated BMP10 gene and protein expressions in RA tissue. We determined BMP10 and BMP9 plasma levels by ELISA. We studied BMP10 signaling activity in PH and CTEPH patients before and after pulmonary endarterectomy (PEA).

Results

BMP10 mRNA, protein and activity were significantly increased in the PH right atrium. While circulating levels of BMP10 and BMP9 proteins were increased, no significant changes were observed in BMP10 signaling activity between PH and controls. In a cohort of CTEPH patients, BMP10 signaling activity was reduced after PEA which coincided with improved hemodynamics and RV function. Interestingly, high BMP10 activity was associated with reduced RV function and adaptation, increased RA dilation and decreased RA function. No associations were observed with hemodynamics or general disease markers.

Conclusions

RA BMP10 expression and plasma levels are increased in precapillary PH. High BMP10 transcriptional activity was associated with worse RV function, RV adaptation, RA dilation and RA function. Future studies are needed to further understand the mechanism underlying BMP10 secretion and function in PH.
Modulation of mitochondrial dynamism in kupffer cells impacts systemic metabolism
Authors: Fantini F , Da Dalt L , Moregola A , Vingiani G B , Svecla M , Terenghi O , Nour J , Bellini R , Bonacina F , Norata G D
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): University of Milan

Background

Kupffer Cells (KC) are hepatic resident macrophages that are essential for liver physiology and contribute to the development of Non-Alcoholic Fatty Liver Disease (NAFLD). The liver of patients with NAFLD shows different expression of some key regulators of inner mitochondrial membrane fusion compared with healthy subjects, including OPA1 protein, which is a mitochondrial protein whose activity promotes mitochondrial fusion and positive modulation of oxidative phosphorylation.

Aim

Given that Kupffer Cells have both immune and metabolic roles, their mitochondria are critical for their and energy requirements and their functionality. This project aims to study how the inhibition of OPA1-driven mitochondrial fusion affects immune-metabolic response and progression of NAFLD by exploring the role of OPA1 in both a Standard and High-fat diet context.

Methods

mice selectively lacking OPA1 in Kupffer Cells were fed with either a Standard diet or a High-fat diet for 20 weeks. The metabolic phenotype was assessed by in vivo indirect calorimetry, measurement of plasma and tissue lipid profile, glucose and insulin sensitivity assays, and complete immunophenotype characterization. Single cell RNA sequencing was also performed to profile with higher resolution the impact of OPA1 deficiency on Kupffer Cell function and cross talk with other liver cells types within the hepatic niche.

Results

Under Standard diet conditions mice selectively lacking OPA1 in Kupffer Cells show a significant reduction in the KC1 pro-inflammatory subset, while the pro-resolutive KC2 increases significantly. In addition, mice lacking OPA1 in KCs show a metabolic preference towards carbohydrates, despite comparable energy expenditure, O2 consumption and CO2 production. The systemic immune profile was comparable between the two groups, with an expected reduction in KC total number under High-fat diet conditions, where no significant differences were observed between the two groups in plasma cholesterol and triglyceride levels, while a significant reduction in liver fibrosis was observed with liver histology.

Conclusions

Taken together these preliminary data suggest that OPA1-mediated inhibition of mitochondrial fusion in Kupffer Cells affects the systemic metabolic response under Standard diet with a reflection also on High-fat diet conditions, where systemic metabolism is comparable between the two groups, but the loss of OPA1 appears to influence the progression of NAFLD through a reduction in total liver fibrosis. Ongoing studies aim to understand the molecular mechanisms underlying these different immune-metabolic responses.
Unraveling the metabolic pattern of angiopoietin-like 3 knock-out mouse models under fasting and fed conditions
Authors: Terenghi O , Da Dalt L , Fantini F , Moregola A , Bonacina F , Norata G D
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): University of Milan

Background

Angiopoietin-like 3 (ANGPTL3) is a hepatokine and a regulator of lipid and lipoprotein metabolism through lipoprotein lipase and endothelial lipase inhibition, preventing the hydrolyzation of lipoprotein-derived triglycerides.

A lack of ANGPTL3 results into a hypolipidemic phenotype, with a lower profile of lipids and lipoproteins, and this is evident in humans carrying a loss of function mutation, or in cellular or in vivo models.

ANGPTL3 action, from the liver, is finely regulated between oxidative and storage tissues in the fasting and postprandial phases; from the current pharmacological treatments a possible ANGPTL3 intrahepatic role besides the lipases’ inhibition is emerging, thus possibly affecting the systemic metabolism with different implications.

Purpose

An axis between ANGPTL3 and other Angiopoietin-like proteins is known to regulate the triglyceride partitioning after a meal or during fasting, however the effect on tissues and the following metabolic and pathologic implications are still not known.

Therefore, we aim to describe the impact of ANGPTL3 deficiency in fasting or in a postprandial state, and in different compartments of the organism.

Methods

As models, we utilized Angptl3 Knock-out (KO) mice (C57BL6/J background) and their littermate controls (wild-type, WT) fed a chow diet for 16 weeks. We assessed the metabolic phenotype with indirect calorimetry and with lipids, glucose, and insulin tolerance tests; we evaluated changes in plasma lipids under fast, fed, and fast-refeed settings and we investigated the histology of key metabolic organs.

Results

ANGPTL3 KO mice fed ad libitum a chow diet are hypolipidemic (plasma triglycerides levels: 42,42±8,80 mg/dL in ANGPTL3 KO mice vs. 122,02±55,09 mg/dL in WT mice; plasma cholesterol levels: 44,00±9,11 mg/dL in ANGPTL3 KO mice vs. 76,51±15,87 mg/dL in WT mice). The oral charge lipid challenge suggests that KO mice have lower lipid levels at all time points.

Conclusions

This preliminary profiling of ANGPTL3 KO mice under fasting and fed conditions highlights the hypolipidemia of these models, and the potentially beneficial impact of ANGPTL3 deficiency on their metabolism compared to controls.
Endothelial cellular response to oxidative stress key to proteasome inhibitor cardiovascular toxicity profile
Authors: Mansson Broberg A G N E T A , Wardell E V A , Sjoborg Alpman M , Lund J L , Sylven C
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Karolinska Institutet

Background/Introduction

Proteasome inhibitors (PI) have contributed to longer survival in multiple myeloma although PI related cardiovascular adverse events are common. Associated to carfilzomib hypertension, coronary ischemic events and heart failure (both with reduced and preserved left ventricular ejection fraction). Here we explore the effect of PIs on endothelial gene expression.

Methods

Human aortic endothelial cells (HAEC) were incubated for 24 h with carfilzomib or bortezomib at different concentrations. Gene expression determined using using bcl2fastq (v2.20.0), and read quality was assessed using FastQC (v0.11.8). For each group the 100 genes with highest ratio were used for analysis of gene ontology (GO), biological processes using ClusterProfiler. The carfilzomib / bortezomib >1.5 was determined in the relevant GO groups.

Results

PIs induce gene expression related to inflammatory response and oxidative stress, more pronounced for carfilzomib. 44 / 31 genes related to "cytokine-mediated signaling pathway" and "response to oxidative stress pathway. NR4A3 was 15.5 fold above control while its increase with bortezomib was modest.

Discussion

Both carfilzomib and bortezomib induce genes related in inflammation and oxidative stress. Carfilzomib induced more pronounced upregulations in several genes, that stimulates expression of increased cellular respiration. Our result show a differentiated effect on endothelial cell gene expression by carfilzomib and bortezomib -a possible explanation to the differences in cardiovascular toxicity profile.

Figure 1.

Selected GO, Biological function characteristics calculated on the top 100 fold-change genes in each group. C2 high dose carfilzomib, B1 and B2 low and high dose bortezomib. C1 did not converge to any GO characteristics.
Impact of the 2022 pulmonary hypertension definition on haemodynamic classification and mortality in patients with aortic stenosis undergoing valve replacement
Authors: Maeder Micha T , Weber Lukas , Pohle Susanne , Chronis Joannis , Baty Florent , Rigger Johannes , Brutsche Martin , Haager Philipp , Rickli Hans , Brenner Roman
Published: May 2024

Aims

With the 2022 pulmonary hypertension (PH) definition, the mean pulmonary artery pressure (mPAP) threshold for any PH was lowered from ≥25 to >20 mmHg, and the pulmonary vascular resistance (PVR) value to differentiate between isolated post-capillary PH (IpcPH) and combined pre- and post-capillary PH (CpcPH) was reduced from >3 Wood units (WU) to >2 WU. We assessed the impact of this change in the PH definition in aortic stenosis (AS) patients undergoing aortic valve replacement (AVR).

Methods and results

Severe AS patients (n = 503) undergoing pre-AVR cardiac heart catheterization were classified according to both the 2015 and 2022 definitions. The post-AVR mortality [median follow-up 1348 (interquartile range 948–1885) days] was assessed. According to the 2015 definition, 219 (44% of the entire population) patients had PH: 63 (29%) CpcPH, 125 (57%) IpcPH, and 31 (14%) pre-capillary PH. According to the 2022 definition, 321 (+47%) patients were diagnosed with PH, and 156 patients (31%) were re-classified: 26 patients from no PH to IpcPH, 38 from no PH to pre-capillary PH, 38 from no PH to unclassified PH, 4 from pre-capillary PH to unclassified PH, and 50 from IpcPH to CpcPH (CpcPH: +79%). With both definitions, only the CpcPH patients displayed increased mortality (hazard ratios ≈ 4). Among the PH-defining haemodynamic components, PVR was the strongest predictor of death.

Conclusion

In severe AS, the application of the 2022 PH definition results in a substantially higher number of patients with any PH as well as CpcPH. With either definition, CpcPH patients have a significantly increased post-AVR mortality.
Leprosy reactions: Unraveling immunological mechanisms underlying tissue damage in leprosy patients
Authors: Serrano-Coll Héctor , Wan Eric L , Restrepo-Rivera Lina , Cardona-Castro Nora
Published: May 2024

Leprosy is a chronic granulomatous infectious and disabling disease caused by two mycobacteria, Mycobacterium leprae and Mycobacterium lepromatosis. Acute inflammatory responses, known as leprosy reactions, are significant contributors to disabilities. Three types of leprosy reactions have been identified based on excessive cytokine release (e.g. type 1) or the accumulation of immune complexes in tissues inducing multiorgan damage (e.g. types 2 and 3). The type of leprosy reaction has implications on treatment and management strategies, yet are not well understood by health workers caring for leprosy patients. We attempt to describe the immunologic mechanisms behind the different leprosy reactions and the rationale for tailoring clinical treatment and management to the particular type of leprosy reaction based on the underlying immunologic situation.
Longitudinal Trajectories of Stress and Positive Aspects of Dementia Caregiving: Findings From the IDEAL Programme
Authors: Quinn Catherine , Gamble Laura D , Morris Robin G , Pentecost Claire , Rusted Jennifer M , Clare Linda
Published: May 2024

Objectives

Understanding what influences changes over time in caregiver well-being is important for the development of effective support. This study explores differences in trajectories of caregiver stress and positive aspects of caregiving (PAC).

Methods

Caregivers of community-dwelling individuals with mild-to-moderate dementia at baseline from the IDEAL cohort were interviewed at baseline (n = 1,203), 12 months (n = 917), and 24 months (n = 699). Growth mixture models identified multiple growth trajectories of caregiver stress and PAC in the caregiver population. Associations between study measures and trajectory classes were examined using multinomial logistic regression and mixed-effects models.

Results

Mean stress scores increased over time. A 4-class solution was identified: a “high” stable class (8.3%) with high levels of stress, a “middle” class (46.1%) with slightly increasing levels of stress, a “low” class (39.5%) with initial low levels of stress which slightly increased over time, and a small “increasing” class (6.1%) where stress level started low but increased at a steeper rate. Mean PAC scores remained stable over time. A 5-class solution was identified: 3 stable classes (“high,” 15.2%; “middle,” 67.6%; “low” 9.3%), a small “increasing” (3.4%) class, and 1 “decreasing” class (4.5%). For stable classes, positive ratings on study measures tended to be associated with lower stress or higher PAC trajectories and vice versa. Those with “increasing” stress also had worsening trajectories of several study measures including depression, relationship quality, competence, and ability to cope.

Discussion

The findings highlight the importance of identifying caregivers at risk of increased stress and declining PAC and offering them targeted support.
Loneliness Trajectories and Chronic Loneliness Around the World
Authors: Sheftel Mara Getz , Margolis Rachel , Verdery Ashton M
Published: May 2024

Objectives

We examine cross-national variation in (a) loneliness trajectories and (b) the association between common social risk factors and chronic loneliness in middle and older adulthood.

Methods

Using longitudinal data, we assess the country-level prevalence of loneliness trajectories (chronic, transitory, and no loneliness) and the extent of common social risk factors for loneliness (living alone, widowhood, divorce, no grandchildren, having chronic conditions, and never working) among adults 50 and older in 20 countries covering 47% of the global population in this age bracket. Additionally, we compare how the associations between social risk factors and chronic loneliness vary across countries.

Results

We find considerable variation in the prevalence of chronic loneliness cross-nationally, ranging between 4% (Denmark) and 15% (Greece) of adults 50 and older. Living arrangements have the most consistent association with the likelihood of chronic loneliness across countries, with those ever living alone having an 8% higher likelihood of chronic loneliness on average across countries, with a range of 2%–25%. Additionally, those who never report working and those with chronic conditions have a higher likelihood of chronic loneliness across more than a third of the countries.

Discussion

These results suggest that policies and interventions targeted to middle age and older adults living alone and with limited work histories or with chronic conditions are critical in reducing the public health challenges of chronic loneliness.
Practical Guidance and Workflows for Identifying Fast Evolving Non-Coding Genomic Elements Using PhyloAcc
Authors: Thomas Gregg W C , Gemmell Patrick , Shakya Subir B , Hu Zhirui , Liu Jun S , Sackton Timothy B , Edwards Scott V
Published: May 2024

Synopsis

Comparative genomics provides ample ways to study genome evolution and its relationship to phenotypic traits. By developing and testing alternate models of evolution throughout a phylogeny, one can estimate rates of molecular evolution along different lineages in a phylogeny and link these rates with observations in extant species, such as convergent phenotypes. Pipelines for such work can help identify when and where genomic changes may be associated with, or possibly influence, phenotypic traits. We recently developed a set of models called PhyloAcc, using a Bayesian framework to estimate rates of nucleotide substitution on different branches of a phylogenetic tree and evaluate their association with pre-defined or estimated phenotypic traits. PhyloAcc-ST and PhyloAcc-GT both allow users to define a priori a set of target lineages and then compare different models to identify loci accelerating in one or more target lineages. Whereas ST considers only one species tree across all input loci, GT considers alternate topologies for every locus. PhyloAcc-C simultaneously models molecular rates and rates of continuous trait evolution, allowing the user to ask whether the two are associated. Here, we describe these models and provide tips and workflows on how to prepare the input data and run PhyloAcc.