bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage-mediated interleukin-6 signaling drives ryanodine receptor-2 calcium leak in postoperative atrial fibrillation
Authors: Wehrens X , Keefe J A , Aguilar-Sanchez Y , Ong I , Navarro-Garcia J A , Paasche A , Abu-Taha I , Tekook M , Bruns F , Barazi D , Kamler M , Shen H Y , Dobrev D
Published: October 2024

Background

Postoperative atrial fibrillation (poAF) is self-limited AF that occurs within days after cardiac surgery in one-third of patients. Clinical and preclinical studies have demonstrated that the magnitude of the perioperative increase in systemic inflammation, in particular mediated by interleukin (IL)-6, correlates with poAF risk. However, no studies to-date have mechanistically linked IL-6 signaling to fundamental arrhythmia mechanisms or identified the specific cell types driving postoperative atrial IL-6 signaling.

Methods

We performed single-cell RNA sequencing (scRNAseq), followed by pseudotime trajectory and cell-cell communication analyses on atrial non-myocytes comparing mice with and without poAF. We followed up our scRNAseq findings with pharmacologic and genetic approaches in mice to validate pathways related to macrophage-mediated IL-6 signaling. We used confocal imaging to assess calcium-signaling mechanisms at the single atrial cardiomyocyte (ACM) level and performed parallel patch-clamp studies in isolated human ACMs as well as biochemical analyses of human atrial tissue to assess the translational relevance of our findings.

Results

Our scRNAseq results demonstrated macrophages to be the most prominently altered cell type in the atria of mice with poAF. Pseudotime trajectory analyses revealed IL-6 to be one of the top inflammatory pathways implicated in macrophage transcriptional state. Indeed, both macrophage depletion and conditional knockout of IL-6 receptors in macrophages were sufficient to prevent poAF. We found that inhibition of STAT3, which is downstream of the IL-6Ra, with FDA-approved phospho-STAT3 inhibitor TTI-101 prevented poAF in mice. Lastly, we demonstrate that enhanced STAT3 activity in poAF promotes arrhythmogenic Ca2+ sparks and waves through ryanodine receptor-2 dysfunction, and that CaMKII inhibition is sufficient to ameliorate Ca2+ mishandling at the single atrial myocyte level.

Conclusions

Taken together, we use an integrated approach incorporating mouse and human biochemical, functional, and single-cell sequencing data to demonstrate that infiltrating atrial macrophages are fundamentally required for poAF. IL-6 was found to be a critical pathway upregulated in poAF leading to downstream ryanodine receptor-2 dysfunction and atrial arrhythmogenesis. Our findings portend significant therapeutic utility by providing robust mechanistic evidence that targeting the IL-6 axis may be used for poAF prevention and treatment in a clinically amenable timeframe.
Calcitonin signalling system regulates function and arhythmogenicity of atrial cardiomyocytes
Authors: Moreira L , Robinson P , Sparrow A J , Mehta N , Agon K A , Steeples V , Akoumianakis I , Sayeed R , Krasopoulos G , Watkins H , Topfer C , Antoniades C , Nattel S , Redwood C , Reilly S
Published: October 2024

Background

Atrial fibrillation (AF) poses a significant therapeutic challenge due to myocardial remodelling, involving both structural and electrical alterations. Recent investigations have shed light on the role of atrial cardiomyocytes (CMs) in secreting Calcitonin (CT), a factor crucial for maintaining atrial tissue integrity. Dysregulated CT secretion in AF showed to contribute to fibrotic tissue production by atrial fibroblasts, exacerbating arrhythmogenesis [1]. However, the direct impact of CT on CM function and arrhythmogenicity remains unclear, driving the objectives of our study.

Methods/Results

Serum samples from 20 cardiac surgery patients revealed a noteworthy association between higher pre-operative CT levels and a significant ~2.8-fold reduction in the incidence of post-operative AF (poAF).

Using freshly isolated atrial guinea pig CMs, confirmed (by qPCR and immunofluorescence) that CMs express CT-receptor (CTR) to enable CT actions in the cell. Functional studies found that CT administration inhibits spontaneous calcium (Ca2+)-release events induced by pacing and decreases the Ca2+ transients amplitude (at 2 Hz; IonOptix μstep system) in fura2-loaded CMs (n=22 cells) in a concentration-dependent manner. Atrial iPSC-CMs transduced with global RGECO sensor (Genetically encoded Ca2+ indicator) and treated with 15 pM CT showed a reduction in the beat rate when paced at 2Hz, and a significantly increased the time to 50% baseline.

Evaluation of the expression and phosphorylation of selected Ca2+ handling proteins, which may potentially account for the observed changes in Ca2+ transients, showed no differences in total or phospho-(ser2808) ryanodine receptor, and SERCA2α in response to CT but an increase in phospho-(Ser16) Phospholamban.

Conclusion

Our findings highlight the effects of CT on atrial CM function. Maintaining physiological CT levels offer a promising approach for managing AF clinically, potentially through the use of already in clinical use CT-analogues.
Reduced Na current in dystrophin-deficient ventricular cardiomyocytes is rescued by tenascin-C inhibition
Authors: Marksteiner J , Sauer J , Koenig X , Todt H , Dostal C , Szabo P L , Podesser B K , Kiss A , Hilber K
Published: October 2024

Background

Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease triggered by the deficiency of the structural protein dystrophin, which leads to muscular degeneration mainly affecting young males. Major contributors to early death in DMD patients are cardiac arrhythmias and dystrophic cardiomyopathy. One cause for arrhythmias is impaired ventricular impulse conduction, which leads to ventricular asynchrony and reentrant mechanisms. We recently showed that the disruption of dystrophin results in a significant reduction of Na current in ventricular cardiomyocytes (vCMs) of the dystrophin-deficient mdx mouse model for human DMD. Na current reduction provides a mechanistic explanation for the impaired ventricular conduction and accompanying arrhythmias in the dystrophic heart.

The extracellular matrix protein tenascin-C (TN-C) is a significant remodeling factor in the injured and diseased heart and is strongly upregulated in dystrophic cardiomyopathy. To this date, it is unknown how the upregulation of TN-C in DMD patients affects dystrophic cardiomyopathy.

Purpose

In this study, we examined the effect of TN-C inhibition on diminished Na currents in dystrophin-deficient vCMs.

Methods

We compared four different mouse genotypes with each other, namely wild-type, dystrophin-deficient mdx, TN-C-deficient and dystrophin- plus TN-C-deficient mice. Furthermore, a cohort of mdx mice was injected with TN-C siRNA twice a week for 9 weeks to investigate the effect of TN-C knockdown. Hearts from adult male mice were enzymatically digested using a Langendorff system to isolate single vCMs. Na currents were then measured with the whole cell patch clamp technique.

Results

Na current densities were increased in TN-C deficient vCMs compared to wild-type vCMs. Accordingly, 24-hour incubation of wild-type vCMs with human recombinant TN-C resulted in a significant decrease in Na current. Na currents of vCMs from mdx mice were reduced, but restored to the wild-type level in vCMs from TN-C-deficient mdx mice. Moreover, vCMs of TN-C siRNA-treated mdx mice had significantly increased Na currents compared to control mdx vCMs.

Conclusion

Upregulation of TN-C in dystrophin-deficient vCMs reduces Na currents, whereas inhibition of TN-C prevents this reduction. Therefore, inhibition of TN-C in DMD patients may be considered as a potential new therapeutic strategy to improve ventricular conduction and reduce arrhythmia vulnerability.
Local ventricular activation at the distal great cardiac vein determines right- and left-sided approach for catheter ablation of outflow tract arrhythmias: a simple and rapid discriminator
Authors: Miyoshi M , Hasegawa K , Tsuji T , Nomura R , Mukai M , Tada H
Published: October 2024

Background

When the precordial transition zone (TZ) of idiopathic outflow tract arrhythmias (OTAs) is present around lead V3, it may be difficult to precisely determine which outflow tract is responsible for the origin of the OTAs from the ECG characteristics.

Purpose

We hypothesized that mapping around the distal portion of the great cardiac vein (dGCV) would be useful for identifying which outflow tract is the origin of OTAs during radiofrequency catheter ablation (RFA).

Methods

Among 28 OTA patients who underwent dGCV mapping and successful RFA at our institution from June 2021 to February 2024, 16 whose TZ during the OTAs was ＞lead V2 and ＜lead V4 were included in this study. In all patients, activation mapping around the dGCV with the novel 2.7 Fr over-the-wire (OTW)-type decapolar catheter was performed during the OTAs.

Results

In all patients, the mapping catheter could be easily placed in the dGCV. In patients whose OTAs were successfully ablated from the left-sided OT (n=7), the local ventricular activation time in the dGCV during OTAs preceded the QRS onset (LAT-dGCV) by -26.1±7.0 msec, which was significantly earlier than that of OTAs successfully ablated from the right-sided OT (n=9; -4.0±5.7 msec; p<0.001). An LAT-dGCV >-18 msec indicated a left-sided OTA, and right-sided and left-sided OTAs were completely distinct. Patients with an LAT-dGCV >-18 msec and mapping initiated first in the left-sided OT had shorter procedure times than those with mapping initiated first in the right-sided OT (p<0.001).

Conclusions

Mapping the dGCV with this OTW-type catheter is easy and safe. The earliness of the local ventricular activation in the dGCV during OTAs could easily and precisely differentiate between right-sided and left-sided OTAs, resulting in a successful ablation with a shorter procedure time.
Anatomical characteristics of the phrenic nerve and the spontaneous conduction block between the right atrium and the superior vena cava
Authors: Hojo R , Miura M , Suzuki M , Komine S , Sunagwa M , Tsuno W , Sasaki T , Kujiraoka H , Yamaoka K , Arai T , Yoshida K , Takahashi M , Tuchiyama T , Fukamizu S
Published: October 2024

Introduction

Although the superior vena cava isolation (SVCI) with minimal radiofrequency application using a spontaneous right atrium – superior vena cava (RA-SVC) conduction block has been reported, the anatomical characteristics of the spontaneous RA-SVC conduction block is not clear.

Purpose

The aim of this study to evaluate the anatomical relationship between the spontaneous RA-SVC conduction block and the phrenic nerve (PN).

Methods

This study consisted of 73 patients who attempted to undergo SVCI using a RA-SVC conduction block. The RA-SVC conduction block was visualized using the extended early meets late tool with the CARTO3 system. We set the first lower threshold at 1800/total local activation time to optimize the visualization of spontaneous RA-SVC conduction block. If the SVC was not isolated by ablation to the gap of the visualized conduction block line, the lower threshold was increased by 5%, and the new gap was ablated each time. The course of the right PN was detected by pacing with 20 mA /2 ms. The RA-SVC conduction block and the course of the PN were measured with reference to the height of the left atrial roof. The extend of the RA-SVC conduction block and PN were measured in angles with respect to the ventral side (Figure 1). These anatomical parameters were compared between the RA-SVC block line and the PN.

Results

Sixty-one patients (83.6%) had spontaneous RA-SVC conduction block, and the remaining 12 patients (16.4%) did not. Anatomical characteristics were evaluated in 61 patients with spontaneous RA-SVC conduction block. Compared with the course of the PN, RA-SVC conduction block extended more posteriorly (110 degrees vs 128 degrees, P = 0.013) and more caudally (9.5 mm vs 16.1 mm, P<0.0001) (Figure 2). In the patients who underwent SVCI using a RA-SVC conduction block, any patients did not undergo radiofrequency application on the course of the PN.

Conclusion

Since the caudal end of the RA-SVC conduction block is posterior to the caudal end of the phrenic nerve, SVCI using the RA-SVC conduction block may be able to avoid ablation near the PN.Figure1Figure2
Role of vitamin K2 and vitamin K cycle enzymes in aortic valvular interstitial cell calcification
Authors: Repges E , Al Zaidi M , Yousif A , Czogalla-Nitsche K , Oldenburg J , Tiyerili V , Nickenig G , Zimmer S , Aksoy A
Published: October 2024

Introduction

Calcific aortic valve disease (CAVD) is the most common valve disease. Currently, there is no medical treatment available for CAVD. Vitamin K antagonists promote CAVD while population-based studies suggest that dietary intake of vitamin K reduces the incidence of CAVD in adults. However, randomized trials have so far not proven a clear benefit of vitamin K supplementation on the progression of AVS. Consequently, there is a need for a deeper understanding of the role of vitamin K in valvular calcification.

This study aims to further investigate mechanisms of action of vitamin K during valvular calcification.

Methods and Results

Aortic valve samples were obtained from patients undergoing surgical aortic valve replacement for CAVD and valvular interstitial cells (VIC) were isolated by collagenase II digestion (Fig. 1). VIC expressed typical markers like Vimentin and α-SMA and were cultured in a control medium (CM) or further calcified in a pro-calcifying medium (PCM) containing sodium dihydrogen phosphate and ascorbic acid. PCM led to upregulation of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) expression, and ALP-activity after 7 and 21 days of culture. Moreover, alizarin red staining visualized significant calcium mineral deposition after 21 days of culture (Fig. 2).

The addition of the most potent vitamin K2 derivative (Menaquinone-7, MK-7) improved cell viability under both control and calcifying conditions (Fig. 3). Moreover, MK-7 protected VIC from ferroptotic cell death (Fig. 4) and increased expression of the vitamin K-dependent Matrix-Gla-protein (MGP), a major inhibitor of ectopic calcification (Fig. 5).

To fulfill its function as an activator of vitamin K-dependent proteins like MGP, vitamin K needs to be reduced to vitamin K hydroquinone by the vitamin K epoxide reductase enzymes VKORC1 and VKORC1L1. In VIC, using our calcification protocol, treatment with PCM led to the downregulation of VKORC1, but not of VKORC1L1, an enzyme with known anti-oxidative properties (Fig. 6). We next performed siRNA-mediated knockdown of VKORC1 and VKORC1L1 in VIC and observed calcification after 7 and 21 days of culture by measuring expression of ALP, RUNX2, quantifying ALP activity, and staining for calcium minerals (alizarin red). Intriguingly, VKORC1L1-knockdown sharply increased the expression of ALP.

To further elucidate these findings, we will perform overexpression experiments to analyze, if VKORC1L1 can alleviate PCM-induced calcification

Conclusion: Vitamin K2 inhibits in vitro calcification of valvular interstitial cells and upregulates the calcification inhibitor Matrix-Gla-Protein. Our results suggest the involvement of the vitamin K cycle enzyme VKORC1L1, which is known to exhibit anti-oxidative and cytoprotective effects. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding.
Peroxisome proliferator-activated receptor gamma regulates interleukin-6 induced lipoprotein(a) gene expression in human HepG2 cells
Authors: Harb T , Ziogos E , Amat-Alarcon N , Gerstenblith G , Arvanitis M , Leucker T M
Published: October 2024

Background

Lipoprotein(a)[Lp(a)] is an independent risk factor for atherosclerotic cardiovascular disease (ASCVD) and outcomes. The LPA gene promoter contains an interleukin 6 (IL-6) responsive binding site. IL-6 is a pro-inflammatory cytokine and data from patients with inflammatory diseases indicate an impact of the inflammatory milieu on LPA gene regulation and Lp(a) production. In a clinical study, the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone was associated with decreased Lp(a) levels. Publicly accessible ChIP-sequencing of human hepatocytes and measures of Lp(a) production by HepG2 cells provide an opportunity to explore PPARγ regulation of hepatic Lp(a) production.

Purpose

To investigate the role of PPARγ as a regulator of hepatic Lp(a) production.

Methods

Public ChIP-sequencing of human hepatocytes was analyzed. Commercially available HepG2 cells were incubated with one of the following: (1) scrambled small interfering RNA (siRNA) alone (control); (2) IL-6 (20ng/mL) and scrambled siRNA ; (3) IL-6 and PPARγ siRNA ; and (4) IL-6 and pioglitazone (25uM). LPA transcription was measured using the quantitative polymerase chain reaction.

Results

Analysis of the ChIP-sequencing data demonstrated that the LPA locus variant rs56393506, which is associated with increased Lp(a) levels and ASCVD risk in the general population, was within the location of the PPARγ binding site in the LPA promoter (Figure 1). IL-6 significantly increased LPA transcription in the HepG2 cells, compared to control (1.65±0.44 vs. 0.66±0.45, p=0.0027). In the presence of siRNA-mediated knock-down of PPARγ (58±17% PPARγ reduction, p<0.0001 vs. scrambled control), the effect of IL-6 on LPA transcription was significantly amplified (6.78±2.4 vs. 1.65±0.44, p=0.0009). IL-6 did not increase LPA transcription in the presence of the PPARγ agonist pioglitazone compared to control (0.16±0.03 vs. 0.66+0.45, p=0.11, Figure 2).

Conclusion

PPARγ is a negative regulator of IL-6 induced hepatic Lp(a) production and may represent a new therapeutic target in patients with inflammatory conditions.Figure 1Figure 2
Single-cell mass cytometry analysis unveils prothrombotic expression and heterogeneity in essential thrombocythemia
Authors: Kuehne S G , Dill V , Kirmes K , Han J , Klug M , Bloxham C J , Raake P W J , Bernlochner I , Bongiovanni D
Published: October 2024

Background/Introduction

Essential thrombocythemia (ET) is recognized for its hallmark elevated platelet count and a consequent increased risk of thromboembolic events including coronary events. Despite its clinical significance, the pathophysiological mechanisms predisposing ET patients to thrombosis remain largely undefined.

Purpose

The aim of this pioneering study was to shed light on the increased thrombotic risk in ET by conducting a detailed phenotypical characterization of platelet expression using single-cell mass cytometry, comparing ET patients to age- and sex-matched healthy controls.

Methods

Platelet samples from six patients diagnosed with ET and six healthy individuals were analyzed. This analysis utilized an extensive panel of 18 transmembrane regulators pivotal for platelet function and activation. Measurements were taken both at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). Advanced clustering analysis via the FlowSOM algorithm helped identify specific subclusters of platelets with a prothrombotic expression profile.

Results

Our investigation revealed a significant overexpression of the activation marker CD62P (P-Selectin, p=0.049) and of the collagen receptor GPVI (p=0.044) in non-stimulated ET platelets (Figure 1A-C). Conversely, a notable decrease in expression was observed for the fibrinogen receptor GPIIb/IIIa units CD41 (p=0.036) and CD61 (p=0.044), as well as for the von Willebrand factor receptor CD42b (p=0.044). The FlowSOM analysis distinguished two prothrombotic subclusters of ET platelets, particularly highlighting one cluster, which was significantly overrepresented in ET (22.13% of total ET platelets versus 2.94% in controls, p=0.035, Figure 1D-E).

Conclusion

This study presents compelling evidence of a distinct prothrombotic phenotype in ET, marked by significant alterations in platelet expression profiles. The overexpression of CD62P and GPVI, alongside the underexpression of CD41, CD61, and CD42b, underscores the complex pathophysiological landscape of ET. Importantly, we identified a specific subcluster, particularly overrepresented in ET, with a specific prothrombic signature. Our findings, while only hypothesis generating, propose GPVI as a potential therapeutic target to mitigate thrombosis in ET patients, paving the way for targeted interventions in this high-risk population.
"Honey pot"-like lesion formation: impact of catheter contact angle on lesion formation by novel temperature controlled ablation catheter in a porcine experimental model
Authors: Ogiso S , Fukaya H , Ogawa E , Mori H , Masuda Y , Yazaki Y , Murayama Y , Saito D , Kobayashi S , Nakamura H , Ishizue N , Kishihara J , Niwano S , Oikawa J , Ako J
Published: October 2024

Background

The DiamondTemp(DT) catheter enables precise temperature-controlled ablation. However, the effects of the DT catheter's contact angle on lesion formation are poorly understood.

Purpose

This study evaluated lesion formation using the DT catheter at different angles in a porcine experimental model.

Methods

We used freshly sacrificed porcine hearts, and radiofrequency catheter ablation with DT was performed at 50 watts for 15 seconds at an upper-temperature setting of 60°C. The contact force (5, 10, and 30 grams) and catheter contact angles (30°, 45°, and 90°) were changed in each set (n = 13 each). The surface width (SW), maximum lesion width (MW), lesion depth (LD), surface area (SA), and distance from the distal edge to the widest area (DW) were evaluated. In addition, the generator impedance drop during radiofrequency application was analyzed.

Results

Figure 1 shows the summarized data in this study. SW and MW were longer at 30° than at 90° (SW 5 g: 6.34 ± 0.91 vs. 5.07 ± 0.87 mm; SW 10 g: 7.46 ± 0.55. vs. 5.60 ± 0.84 mm; SW 30 g: 7.42 ± 0.74. vs. 6.14 ± 0.77 mm; MW 10 g: 8.81 ± 0.55. vs. 7.89 ± 0.84 mm; MW 30 g: 9.24 ± 0.67. vs. 8.03 ± 1.04 mm, p <0.05, respectively). There were no significant differences in LD by catheter angle except at 30 g. SA was larger at 30° than at 90° (SA 5 g: 30.03 ± 0.91. vs. 23.61 ± 0.87; SA 10 g: 29.24 ± 0.55. vs. 21.27 ± 0.84; SA 30 g: 35.86 ± 0.74. vs. 31.74 ± 0.77, p <0.05, respectively). Besides, DW was longer at 30° than at 90° (DW 5 g: 4.28 ± 1.17. vs. 2.50 ± 0.32 mm; DW 10 g: 4.94 ± 0.52. vs. 2.47 ± 0.38 mm; DW 30 g: 5.34 ± 0.26. vs. 2.96 ± 0.40 mm, p <0.05, respectively). There were no significant differences in the impedance drop according to the catheter angle.

Conclusion

With the DT catheters, the lesion width increased at a shallower contact angle, whereas the LD did not. SA also increased at a shallower contact angle. The DT catheter created a large ablation lesion on the proximal side of the catheter, which looked like a "honey pot." (Figure 2)summarized dataImage of honey pot-like lesion formation
Linking antibody against apolipoprotein A-1 to metabolic dysfunction-associated steatohepatitis in mice
Authors: Pagano S , Somm E , Juillard C , Ino F , Ferrari J , Braunersreuther V , Jornayvaz F , Vuilleumier N
Published: October 2024

Background

Metabolic dysfunction-associated fatty liver disease (MASLD) is a common liver condition associated with increased cardiovascular disease (CVD) risk. Cytokeratin 18 (CK-18) is indicative of liver injury and used as a surrogate biomarker covering MASLD to cirrhosis phenotypes. Autoantibodies against apolipoprotein A-1 (AAA-1) are known CVD risk factors inducing MASLD in vitro, but their role in modulating steatohepatitis and fibrosis in vivo is still elusive. We investigated AAA-1's contribution to systemic low-grade inflammation, liver steatosis, and fibrosis using a MASLD mouse model and a validated passive immunization protocol (PIP).

Methods

10 weeks-old male C57BL/6J mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) and immunized with AAA-1 (200ug) or control antibodies for ten days. At sacrifice, plasma samples were collected, and CK-18 levels, and proinflammatory cytokines were measured using the Mesoscale Discovery platform. Hematoxylin and Eosin and Sirius Red Fast Green staining were used to reveal liver steatosis and fibrosis, respectively. RNA were extracted from mouse liver samples and mRNA were then analyzed using a commercial fibrosis-specific genes panel (nCounter Human Fibrosis V2 Panel) and the nCounter Analysis System.

Results

Compared to control IgG recipients, CDAHFD mice injected with AAA-1 exhibited significantly elevated plasma levels of CK-18 (5.3 vs 2.1, p=0.031), IL-6 (13 vs 6.9, p=0.035), IL-10 (27.3 vs 9.8, p=0.007) and TNF-α (32.1 vs 24.2, p=0.032), and liver steatosis (93.4% vs 73.8%, p=0.007). Nanostring technology experiments revealed upregulation of several pro-fibrotic m-RNAs in liver tissue from AAA-1 immunized mice compared to the IgG immunized control group. These included Serum Amyloid A (Fold Change (FC) AAA-1 vs IgG : 2.5, p=0.02) , G-protein coupled receptors 65 (FC: 2.7, p=0.01), Periostin (FC: 2.6, p=0.001), Phospholipid transfer protein (FC: 2.2, p=0.0004), Vascular Cell Adhesion Molecule 1 (FC: 2.5, p=0.02) and Angiopoietin-like 4 (FC: 2.8, p=0.01). Histologically, the amount of liver fibrosis remained unaffected, probably due to the short time of the study.

Conclusions

Short AAA-1 PIP induced enhanced liver steatosis and inflammation accompanied by an elevation of the expression of several pro-fibrotic genes in CDAHFD mice, suggesting that AAA-1 may contribute to the transition from MASLD to MASH. Knowing whether increased exposure time to AAA-1 could lead to end stage disease (including cirrhosis and HCC), and if this could apply to humans as well warrant further investigations.
C-C motif chemokine ligand 3 is associated with atherosclerotic plaque vulnerability and increased risk of mortality
Authors: Li Y , Sun J , Persson A , Nitulescu M , Sundius L , Nilsson J , Edsfeldt A , Goncalves I
Published: October 2024

Background and Purpose

Chemokines play a crucial role in the inflammatory process underlying atherosclerotic plaque formation, progression and subsequent cardiovascular events. However, the specific role of C-C motif chemokine ligand 3 (CCL3) in atherosclerosis remains largely unexplored. This study aims to explore associations between intra-plaque levels of CCL3 and plaque vulnerability as well as to investigate the prognostic role of circulating CCL3 levels in patients undergoing carotid endarterectomy.

Methods

The CCL3 protein expression was measured using a proximity extension assay in 207 human carotid plaques and 558 plasma samples from patients undergoing carotid endarterectomy, all enrolled in the Carotid Plaque Imaging Project (CPIP) cohort. Protein expression is quantified as normalized protein expression (NPX), an arbitrary unit on a log2-scale. Plaque components and extracellular matrix including smooth muscle cells, macrophages, neutral lipids, intraplaque hemorrhage, collagen (by histology), and matrix metalloproteinases (assessed biochemically), were quantified. Plaque vulnerability index was calculated as the sum percentage of plaque areas stained for lipids, macrophages and hemorrhage, divided by the sum percentage of smooth muscle cells and collagen. Patients were followed up to 10.2 years to record cardiovascular events and mortality.

Results

Plaque and circulating levels of CCL3 were higher in symptomatic patients compared to asymptomatic patients (plaque, median NPX (interquartile range, IQR): 18.2 (17.4–19.1) vs 17.2 (16.4–18.0), P<0.001; plasma, median NPX (IQR): 1.78 (1.42–2.21) vs 1.56 (1.28–1.97), P<0.001). Plaque levels of CCL3 showed positive correlations with neutral lipids, macrophages, intraplaque hemorrhage, plaque vulnerability index and matrix metalloproteinases including MMP1, MMP2, MMP9, MMP10 and TIMP1, but inversely with smooth muscle cells. Among 511 patients with complete follow-up and circulating CCL3 measures, 144 (28.2%) patients developed cardiovascular events and 123 (24.1%) patients died. Elevated circulating CCL3 levels were associated with increased risk of cardiovascular events (hazard ratio (HR) per standard deviation (SD) = 1.40, 95% CI 1.06–1.84) and mortality (HR per SD=1.77, 95% CI 1.34–2.33), respectively. Higher levels of circulating CCL3 remained independently associated with an increased risk of mortality (HR per SD=1.50, 95% CI 1.09–2.04) even after adjustment for potential confounders.

Conclusions

Plaque levels of CCL3 are associated with plaque vulnerability features. Circulating levels of CCL3 predict higher risk of cardiovascular events and mortality among carotid endarterectomy patients.
Pericardial adipose tissue (PAT) as a cell model to assess patient responsiveness to docosahexaenoic acid (DHA) and eicopentaenoic acid (EPA)
Authors: Quarta S , Santarpino G , Cardetta F , Carluccio M A , Calabriso N , Madonna R , De Caterina R , Massaro M
Published: October 2024

Background

The abundance of epicardial (EAT) and pericardial adipose tissue (PAT) is associated with several cardiac risk factors and a higher carotid intima-media thickness, with a potential contributory role to the development of atherosclerosis and coronary artery disease (CAD). Under conditions promoting obesity, both epicardial and pericardial adipose tissue undergo a metabolic shift, acquiring an inflammatory and pro-atherogenic phenotype. Addressing dysmetabolism and inflammation associated with EAT and PAT are a promising and innovative therapeutic approach for preventing coronary atherosclerosis.

Recent data have highlighted a noteworthy association between the abundance of adipose tissue n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as well as their ratio to the omega-6 FA arachidonic acid (AA), with a lower risk of myocardial infarction.

Purpose

To develop an efficient method for isolation of adipocytes from human PAT and evaluation of adipocyte response to DHA and EPA.

Methods

PAT samples were collected from 9 CAD patients, undergoing coronary artery bypass graft surgery and immediately processed before to assess more than 50 experimental conditions. Isolated adipocytes were morphologically and molecularly characterized. Cell responsiveness was evaluated by cell exposure to inflammatory stimuli, with or without variable amounts of DHA, EPA, or AA assessing a spectrum of gene expressions and pro-inflammatory functional activities.

Results

Pure cultures of pericardial adipocytes exhibited prolonged viability (beyond 72 hours). and retained responsiveness to pro-inflammatory and lipolytic stimuli, while also showing the capacity to activate the insulin signalling pathway. Cell exposure to DHA or EPA resulted in a downregulation of messenger (m)RNA expression for monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6 and metalloproteinase (MMP)-9 (p<0.05) and in upregulation of uncoupling protein-(UCP)-1, UCP-2 and peroxisome proliferator-activated receptor (PPAR)γ (p< 0.05) (Figure 1).

In line with mRNA expression, a significant reduction in both cytokines and MMP-9 protein release was observed (p<0.05) (Figure 2). These effects paralleled a significant incorporation of DHA and EPA into total cell lipids (p<0.05). Functionally, the cell supernatant from DHA- or EPA-conditioned cells displayed diminished ability to attract monocytes in chemotaxis assays. AA exhibited no such activities.

Conclusion(s)

In a novel cellular model of isolated and cultured adipocytes from PAT, DHA and EPA attenuated the inflammatory and dysmetabolic characteristics of this cardiac adipose tissue.
Bidirectional roles of extracellular and intracellular HMGB2 in the development of atherosclerosis
Authors: Wang X Q , Chen J W , Chen C D , Lu L
Published: October 2024

Rationale: HMGB family plays an important role in homeostasis of immunity and regulation of inflammation. However, the role of HMGB2 in atherosclerosis remains uncharacterized.

Objective

The current study aimed to ascertain the relation of HMGB2 levels in macrophages of atherosclerotic plaques and in circulatory monocytes/macrophages with coronary artery disease (CAD), and to investigate the influence of HMGB2 on atherogenesis in mice and inflammatory reaction in vitro.

Methods/Results

We evaluated HMGB2 levels in macrophages of atherosclerotic plaques in coronary endarterectomy tissue from patients with angiographically documented severe coronary artery disease (CAD)(n=23), and in coronary artery specimens from autopsy cases with no or mild atherosclerosis (n=11) by immunohistochemistry. HMGB2 levels were significantly decreased in macrophages of progressive atherosclerotic plaques, as compared with those from mild plaques. Moreover, we also analyzed HMGB2 levels in peripheral blood monocytes from patients with CAD (n=44) or age- and sex-matched non-CAD controls (n=44). The results showed that decreased intracellular HMGB2 protein levels in monocytes were associated with CAD. In experiments, HMGB2 deficiency significantly promoted atherosclerosis in ApoE-/- mice, whereas such increment of atherosclerosis was markedly attenuated after transplantation of bone marrow derived macrophages from ApoE-/-mice. Mechanistically, intracellular HMGB2 repressed IL-1 transcription and induced autophagic degradation of NLRP3, thus suppressing NLRP3 inflammasome priming and activation. Extracellular HMGB2 levels increased in progressive atherosclerotic plaques with macrophage apoptosis. Recombinant HMGB2 protein promoted inflammation in vitro via RAGE and intraperitoneal administration of this protein exacerbated atherosclerosis in mice.

Conclusion

This study indicates that decreased HMGB2 level in macrophage is associated with inflammation and atherosclerosis in patients with CAD. Intracellular HMGB2 inhibits NLRP3 inflammasome and atherosclerosis in mice, whereas released HMGB2 promotes inflammation and atherosclerosis in mice.
Parathyroid hormone exacerbates pulmonary hypertension via parathyroid hormone receptors on pulmonary artery smooth muscle cells
Authors: Joki Y , Konishi H , Takasu K , Katsuumi G , Yoshida Y , Furihata T , Minamino T
Published: October 2024

Background

Pulmonary hypertension (PH) is a right heart failure disease and can be fatal. Parathyroid hormone (PTH) is a bone metabolism hormone and regulates calcium and phosphorus homeostasis. Previous studies have suggested that PTH affects the cardiovascular system and influences cardiovascular diseases. We hypothesized that PTH may play a role in the pathogenesis of PH.

Purpose

This study aimed to investigate whether PTH has a critical role in pulmonary hemodynamics.

Method

Serum PTH levels were measured in patients with PH or suspected PH undergoing right heart catheterization. We tested whether the regulation of PTH and the PTH receptor (PTH1R) affected PH in hypoxia (Hx)-induced PH model mice and Sugen/hypoxia (SuHx)-induced PH model ras. Hx mice were treated with PTH daily for 3 weeks or with an inhaled AAV-shRNA system to knockdown PTH1R expression in the lung. SuHx rats underwent parathyroidectomy (PTx). We measured physical data and right ventricular systolic pressure (RVSP) in these models. Human pulmonary artery smooth muscle cells (PASMCs) were cultured and treated with PTH to examine the direct effects of PTH.

Result

In the clinical study, we enrolled 30 participants and found serum PTH levels correlated with mean pulmonary artery pressure (r = 0.67) and pulmonary vascular resistance (r = 0.62). The ROC curve showed a cut-off PTH level of 46.0 pg/mL (68.2% sensitivity, 100% specificity) to predict PH. In animal models, PTH treatment exacerbated right ventricular hypertrophy and increased right ventricular systolic pressure (RVSP) in Hx mice compared with non-treated Hx mice (Figure 1). In contrast, knockdown of PTH1R in the lungs improved PH in Hx mice, and PTx significantly suppressed PH in SuHx rats (Figure 2). PTH promoted migration and proliferation through PTH receptor-ERK signaling in PASMCs.

Conclusion

Our clinical and experimental data showed that PTH exacerbates PH and suggest that PTH/PTH1R signaling would be a new target for PH treatment.
Suppression of Fibulin 5 Attenuated Pulmonary Arterial Hypertension Associated with Congenital Heart Disease via Mitigating Pulmonary Arterial Smooth Muscle Cell Dysfunction
Authors: Yang Y , Shu S R , Wang P Z , Zhou J J
Published: October 2024

Backgrounds: The specific molecular pathogenesis of pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) and the reversal still remain elusive. Fibulin 5 was involved in the regulation of cardiovascular system while little is known about the expression and functions of fibulin 5 in the development of CHD-PAH. This study aimed to investigate the role of fibulin 5 in the pathogenesis of the disease.

Methods

Lung samples were obtained both from patients with CHD-PAH and aortocaval shunt-associated PAH rat models. TGF-β1was used to induce human pulmonary artery smooth muscle cells (hPASMCs) dysfunction and lentiviruses were responsible for alteration of fibulin 5 expression. In an effort to explore the specific role of fibulin 5, an inhibitor of phosphatidylinositol-3-kinases (PI3K) /protein-serine-threonine kinase (AKT) which was defined as the TGF-β1-related downstream signaling was applied for further exploration.

Results

Fibulin 5 was pronouncedly elevated in the PASMCs of the remodeled pulmonary arterioles from patients with CHD-PAH and shunt-associated PAH rats. We found that over-expression of fibulin 5 could promote hPASMCs dysfunction induced by TGF-β1 and it could be reversed by the PI3K/AKT inhibitor LY294002 suggesting the essential role of TGF-β1/PI3K/AKT signaling activation in phenotypic switch, proliferation and migration of PASMCs. Moreover, an adeno-associated virus vector encoding fibulin 5 by intratracheally instillation with rats improve the hemodynamic parameters in shunt-associated PAH rats and its related pulmonary artery remodeling.

Conclusion

Over-expression of fibulin 5 could significantly promote hPASMCs dysfunction and pulmonary artery remodeling via reinforcing TGF-β1/PI3K/AKT signaling activation, which highlighted the potential valuable pharmacological target for the treatment of CHD-PAH.Fibulin 5 was over-expressed in PAHInhibition of fibulin 5 attenuated PAH
High glucose enhanced lipoprotein(a) [Lp(a)] oxidation in a manner inhibited by eicosapentaenoic acid (EPA) in vitro
Authors: Sherratt S , Libby P , Dunbar R L , Bhatt D L , Mason R P
Published: October 2024

Background

Elevated lipoprotein(a) (Lp(a)) levels are causally and independently associated with increased cardiovascular (CV) risk. Targeted molecular therapies are in development. Levels of oxidized Lp(a) were more predictive of CV events and endothelial dysfunction in type 2 diabetes than non-oxidized forms. EPA administered as icosapent ethyl (IPE) reduced CV events in statin-treated patients (REDUCE-IT) with diabetes and high CV risk. As a fatty acid with multiple conjugated double bonds, EPA inhibits lipoprotein oxidation by a potent lipid-centric scavenging mechanism. We therefore tested the effects of EPA on oxidation of Lp(a) and non-modified LDL under conditions of high glucose to mimic aspects of hyperglycemia in vivo.

Methods

Lp(a) was enriched to 41% of total ApoB-containing particles from patients with elevated levels by isopycnic centrifugation. Samples of Lp(a) and non-modified LDL were incubated under high glucose (200 mg/dL) conditions at 37°C for 30 min with EPA (50 µM). Samples were then subjected to copper sulfate-induced oxidation (20 µM) monitored by formation of malondialdehyde (MDA) via a colorimetric assay.

Results

Lp(a) enriched plasma underwent increased oxidation, peaking at 4 hr by 32-fold compared to baseline (0.37 ± 0.04 vs 12.27 ± 0.61 µM, p<0.001) under conditions of high glucose. Non-modified LDL also underwent oxidation in the presence of high glucose with a distinct rate compared to Lp(a), peaking after 3 h (0.28 ± 0.09 vs 10.91 ± 0.93 µM, p<0.001). EPA significantly inhibited both Lp(a) and LDL oxidation in a time-dependent manner. After peak oxidation was achieved in Lp(a) (4 h) and LDL (3 h), EPA inhibited oxidation by 57% (5.29 ± 0.25 µM, p<0.001) and 81% (2.12 ± 0.09 µM, p<0.001), respectively.

Conclusions

Under hyperglycemic conditions, EPA inhibited oxidation of Lp(a)-enriched plasma and non-modified LDL in a time-dependent manner. The activity of EPA may arise from its lipophilic properties and highly conjugated structure that scavenges free radicals in lipid-concentrated environments. The potent antioxidant activity of EPA may contribute to reduced CV risk in patients with diabetes and elevated Lp(a).
PI3 kinase delta governs regulatory T-cell biology and thus confers protection against atherosclerosis progression in mice
Authors: Zierden M , Berghausen E M , Gnatzy-Feik L , Millarg C , Picard F S R , Kiljan M , Geissen S , Polykratis A , Zimmermann L , Pasparakis M , Baldus S , Winkels H , Vantler M , Rosenkranz S
Published: October 2024

Introduction and Purpose

Atherosclerosis is a chronic inflammatory disease of arteries, critically involving innate and adaptive immune cells like macrophages as well as T and B lymphocytes. Macrophages are major drivers of disease through the ingestion of lipoproteins, foam cell formation, and secretion of inflammatory mediators. Although macrophages outnumber other leukocytes in atherosclerotic plaques, T and B cells can shape the course of disease by promoting or mitigating inflammatory responses. The phosphoinositide 3-kinase delta (PI3Kd) exerts a key role in the regulation of immune responses including activation, proliferation, differentiation, and effector function of lymphocytes. Therefore, PI3Kd may represent a promising target for the modulation of inflammatory diseases. Consequently, we aimed to analyse the role of PI3Kd during atherogenesis.

Methods and Results

To investigate the role of haematopoietic PI3Kd in atherosclerosis, bone marrow from PI3Kd-/- or PI3Kd+/+ mice was transplanted into Ldlr-/- mice. After a 6-weeks-challenge by high fat diet, PI3Kd-/- recipient Ldlr-/- mice displayed profoundly impaired CD4+ and CD8+ T-cell numbers, CD4+ T-cell activation, CD4+ effector T-cell differentiation, and proatherogenic CD4+ T-helper (Th) 1 responses in para-aortic lymph nodes and spleen compared with PI3Kd+/+ transplanted controls. Surprisingly, the net effect of PI3Kd deficiency was a substantial increase of aortic inflammation and atherosclerosis in Ldlr-/- mice. Whereas plaque content and functions of macrophages including foam cell formation, efferocytosis, and cytokine secretion remained largely unaffected, haematopoietic PI3Kd ablation strongly reduced mature B cells and serum immunoglobulins in Ldlr-/- mice. Importantly, PI3Kd deficiency severely impaired numbers, proliferation, immunosuppressive functions, and stability of regulatory CD4+ T cells (Tregs). Consequently, adoptive transfer of PI3Kd+/+ Tregs fully restrained the atherosclerotic plaque burden in PI3Kd-/- transplanted Ldlr-/- mice without affecting B-cell numbers and serum immunoglobulins, whereas transferred PI3Kd-/- Tregs failed to relieve atherosclerosis progression.

Conclusions

Here, we demonstrate that PI3Kd plays a crucial role in Tregs, Th1 cells, and B cells during atherogenesis. Lack of PI3Kd signalling specifically in atheroprotective Treg responses outplays its impact on B-cell and proatherogenic Th1 responses, thus leading to aggravated atherosclerosis. Hence, PI3Kd is a key regulator of Treg biology and thereby protects against atherosclerosis, suggesting that fine-tuning PI3Kd signalling may represent a promising target for Treg-directed therapy.
Inhibition of fractalkine signalling in patients with acute myocardial infarction - First results from the FRACTAL trial
Authors: Loh S , Ekinci Y , Bennaceur K , Ogundimu E , Bigirumurame T , Maier R , Jeyalan V , Egred M , Mcdiarmid A , Maredia N , Plein S , Olin T , Richardson G , Austin D , Spyridopoulos I
Published: October 2024

Background

We have previously demonstrated a strong association between expression of the fractalkine receptor CX3CR1 on lymphocytes and microvascular obstruction (MVO) in patients undergoing primary percutaneous coronary intervention (PPCI) for ST-elevation myocardial infarction. Our pre-clinical studies also found an improvement in infarct size, inflammation and intramyocardial haemorrhage (IMH) when rats were pre-treated with the fractalkine inhibitor KAND567.

Purpose

FRACTAL (FRACTalkine inhibition in Acute myocardial infarction) study was a phase IIa, pilot, randomized, 2-arm parallel group, placebo-controlled, double blinded, multi-centre trial. Its primary aim was to assess the safety and tolerability of intravenous and oral administration of the fractalkine inhibitor KAND567 as well as reducing signs of reperfusion injury in STEMI patients undergoing PPCI.

Methods

Patients with a new diagnosis of anterior STEMI, who had index chest pain within 5 hours and angiographic occlusion of the LAD, were randomised on a 1:1 basis to receive either KAND567 or placebo over a period of 72 hours. All patients then underwent a baseline MRI scan at 3 days with a follow-up scan at 3 months, paralleled by longitudinal deep immuno­phenotyping over 9 time points.

Results

A total of 71 patients were enrolled into the study with 64 (90.1%) being male patients. The mean age of patients was 59.2 years. 37 patients received KAND567, whilst 34 received placebo over 72-hour period. Throughout the 3-month follow-up period, 23 (62%) patients from the KAND567 arm as compared to 24 (71%) patients in the placebo arm had reported adverse events. There was no statistical difference in serious adverse events (SAEs) between both arms with 9 documented in the placebo arm and 12 in the KAND567 arm. Safety bloods also demonstrated no significant differences between the two groups across all time points. There was a higher incidence of left ventricular (LV) thrombus in the placebo arm as compared to the KAND567 arm (17.6% vs. 2.7%; p=0.049). Additionally, the study also demonstrated a lower occurrence of IMH in the KAND567 arm compared to the placebo arm (35.5% vs. 55.2%; p=0.19). Deep immunephenotyping suggested clear target engagement in the KAND567 arm in natural killer cells as well as in cytotoxic T-lymphocytes.

Conclusion

This first in-man study demonstrates that inhibition of the fractalkine pathway in STEMI patients appears safe and well-tolerated, while demonstrating a promising potential in preventing LV thrombus and IMH. The results suggest a new mechanism and class of drug to target reperfusion injury, clearly warranting a larger phase III study.LV thrombus and IMH