Veranstaltungsprogramm

Luis Berkan Quintana Selek1, Maximilian Sausen1, Nadim Tabaza1, Naresh Ganesh1, Maximillian Neuhaus1, Robert Mertens1, Paul Idel1, Susanne Just1, Maria C. Arrivas1, Julia Moellmann1, Nikolaus Marx1, Michael Lehrke1, Florian Kahles1

Background: Glucagon-like peptide 1 (GLP-1) is a gut incretin hormone secreted by intestinal L-cells, which induces post-prandial glucose-dependent insulin secretion. GLP-1 receptor agonists (GLP-1RA) are clinically used for the treatment of patients with diabetes and were found to improve cardiovascular outcomes in patients with diabetes at high cardiovascular risk and in patients with obesity. GLP-RA improved cardiovascular prognosis most likely by the reduction of vascular inflammation and atherosclerosis-associated events (stroke, myocardial infarction, revascularization). However, the exact underlying mechanisms are incompletely understood. The aim of our project was to decipher how GLP-1RA modulate inflammation in acute and chronic disease.

Methods and results: To investigate the effects of GLP-1RA on systemic inflammation we used 2 models of acute and chronic inflammation in mice. In the chronic inflammation model we induced atherosclerosis by injecting C57BL6J mice with PCSK9 virus and fed a diet high in cholesterol (HCD) for 12 weeks and administered liraglutide (GLP-1RA) vs. saline via osmotic minipumps. Liraglutide treatment strongly reduced atherosclerotic lesion size and plaque immune cell infiltration. Furthermore, liraglutide decreased circulating and intestinal immune cell numbers. To study the effects of GLP-1RA on acute inflammation we induced sepsis by injecting LPS (12.5 mg/kg) in C57BL6J mice treated with liraglutide vs. saline. Activation of the GLP-1RA reduced circulating and hepatic proinflammatory cytokines (TNF-a, IL-12, MCP-1) and significantly downregulated sepsis-induced influx of liver leukocytes (neutrophils, myeloid cells). Moreover, liraglutide prevented mobilization of hematopoetic stem and progenitor cells (HPSC) from the bone marrow in mice with sepsis.

Conclusion: GLP-1 RA directly reduce tissue immune cell infiltration and systemic inflammation in acute (sepsis) and chronic (atherosclerosis) disease models in mice without diabetes. Future work is needed to identify the responsible cell types and molecular pathways.

Clinical studies are warranted to investigate whether GLP-1 RA could be a novel therapeutic target for chronic and acute inflammatory diseases.

Introduction:

Resident macrophages account for 5% of the cells in the healthy heart. In response to cardiac injury, monocytes infiltrate and differentiate into recruited macrophages complementing the original resident macrophage population and their progeny. Overall macrophages decisively orchestrate cardiac remodeling. In this work, we aim to identify the extent to which macrophage origin, tissue location and type of cardiac injury determine macrophage phenotypes in ischemic and non-ischemic cardiac injuries over time.

Results and methods:

To this end, we use a tamoxifen inducible CX3CR1Yfp CreER/+:R26tdT/+ mouse line to visualize and quantify fluxes of resident and recruited macrophages and their respective localizations within the heart following ischemia and reperfusion (I/R) injury and pressure overload after transversal aortic constriction (TAC), respectively. While I/R represents an acute and local cardiac injury, TAC increases cardiac afterload continuously and affects the heart globally. Interestingly, macrophage numbers peaked during the first week post-surgery in both the local/acute and the global/continuous injury models. Initially, recruited macrophages outnumbered the resident macrophage pool within the infarct area but ultimately reached a near 1:1 equilibrium at 4 weeks post I/R injury. In the remote myocardium the 1:1 equilibrium established already during the first week. In TAC-injured hearts, the 1:1 ratio of recruited and resident hearts also established within the first week post-surgery and persisted up to 8 weeks of follow-up even as cardiac macrophage numbers declined and cardiac function deteriorated.

In the infarct, recruited macrophages showed increased cytokine synthesis, defensive response, and cell adhesion pathways during the first week after injury. Many inflammatory pathways remained activated up to day 28 post MI despite simultaneous activation of the anti-inflammatory response. Formerly resident macrophages and their progeny also mounted an immune response throughout the healing process. Unexpectedly, by day 28 post MI when a stable scar had formed, resident and recruited macrophage population transcriptoms had converted onto a common, pro-inflammatory steady state phenotype, that differed from their originally divergent gene expression profiles in the healthy heart by over one thousand genes. In the remote zone, however, resident macrophages maintained cellular homeostasis function, unlike recruited macrophages that retained an inflammatory program. Following TAC surgery, transcriptional profiles remained differentially regulated between recruited and resident cardiac macrophages at all time points tested, where recruited subset induced immune response and resident macrophages upregulated tissue remodeling genes. Yet again, gene expression profiles differed between macrophages located in the TAC heart and the remote myocardium.

Conclusion:

Ischemic and non-ischemic, focal and global, acute and chronic cardiac injuries induce a transient peak of monocyte recruitment and macrophage differentiation in and around cardiac lesions which lead to a permanent integration of recruited monocyte-derived macrophages into the pool of tissue resident macrophages – albeit at different paces. The type of injury and macrophage localization within the changing tissue microenvironment determine partial or complete override of ontogenic cell programs.

Background

Clonal hematopoiesis of indeterminate potential (CHIP) is an age associated expansion of blood cells that carry leucemia-associated somatic mutations. CHIP mutations increase the risk of cardiovascular disease and are associated with cardiovascular driven increase of mortality. Our study aimed to understand the relevance of CHIP in patients of cardiovascular risk, how CHIP mutations affect myeloid cell biology and their impact on cardiovascular tissue.

Methods and Results

We conducted a prospective, observational study to screen CHIP-driver mutations in patients who were admitted for percutaneous coronary intervention. 815 patients admitted for left heart coronary angiography were screened. Of 178 included patients 30% (n=53) were identified as CHIP carriers. The prevalence of CHIP in our cohort was at least 2 times higher than that in the general population. Gensini Score was used to quantify coronary artery disease severity, which was significantly worse in middle-aged (40-70 years) CHIP carriers than non-carriers. Within CHIP-carriers, the variant allele frequency (VAF) showed a strong performance as a distinguisher between coronary artery disease (CAD)- and non-CAD-patients (AUC 0.82 in middle aged patients).

We collected tissue and blood samples from CHIP-carriers undergoing carotid endarterectomy and heart surgery (n=9, n=4) to measure the accumulation of CHIP carrying myeloid cells in different tissues and to investigate their inflammatory profile. Droplet digital polymerase chain reaction (ddPCR) detected similar sizes of CHIP clones in circulating monocytes and tissue-bound macrophages even among CCR2– (resident) cardiac macrophages. Bulk RNA sequencing and GO-Term-Analysis revealed a pro-inflammatory phenotype of myeloid cells of CHIP-carriers compared to matched Non-carriers.

Conclusion

CHIP prevalence is at least doubled in CAD patients compared to the general public. Carrying a CHIP-mutation is associated with a higher disease severity in middle aged patients. In those middle aged CHIP carriers the VAF proves to be a strong predictor of CAD. When investigating cardiovascular (CV) tissue, mutated myeloid cells did not preferably accumulate in CV tissues but bulk RNA sequencing of tissue macrophages showed a significantly higher inflammation score in carriers than in their counterparts.

Objective: Cereals are an essential part of the human diet. All gluten-containing grains contain alpha-amylase-trypsin inhibitors (ATI), a group of 19 heat-stable peptides present in all gluten-containing cereals. Due to their stability, ATIs are present in their active form even in processed and heated foods like bread, pizza or cake. Since ATIs activate auto-inflammatory pathways via the Toll-like receptor 4 (TLR4) in the gut, they are suspected to promote inflammation in remote organ systems as it has been shown for non-alcoholic liver disease, multiple sclerosis and Morbus Alzheimer. This study aims to investigate the cardiovascular effects of an ATI-containing diet regarding vascular function and inflammatory hallmarks in a murine model.

Methods and results: Male C57BL/6J mice were fed an ATI-free, gluten-free diet for two weeks in advance to the experiment as a wash-out phase since standard diet in the breeding facility contains wheat and thus ATIs in an unknown concentration. Animals were divided in two groups. The control group received ATI-free, gluten-free diet for further four weeks while the ATI group was changed to a diet containing an ATI isolate (0,5 % w/w) from wheat flour. Blood pressure and body weight was monitored during the treatment. Vascular function (endothelium-dependent and -independent relaxation) was measured upon stimulation with acetylcholine and nitroglycerine. While blood pressure and weight gain remained unaltered by the ATI diet, a mild endothelial-dependent dysfunction occurred in the ATI fed mice. qPCR results revealed that CD11b and CD68 were significantly elevated in aortic tissue and perivascular adipose tissue indicating infiltration of inflammatory cells.

Conclusion: As most human diets contain cereals on a daily basis, almost all humans are exposed to significant amounts of ATIs. This study revealed for the first time that dietary ATIs have inflammatory properties on the cardiovascular system of healthy subjects. The measured endothelial dysfunction in combination with increased leukocyte markers indicates an inflammatory response to ATI exposure in remote organs. Further studies are necessary to explore the cardiovascular effects of ATI-containing diets in patients with cardiovascular disease or relevant cardiovascular risk factors.

Background

Jena auf Ziel – JaZ” is a prospective cohort study in which early combination therapy with atorvastatin 80 mg and ezetimibe 10 mg was initiated on admission in patients with ST – elevation myocardial infarction (STEMI) and lipid – lowering therapy (LLT) was escalated during follow – up with bempedoic acid (BA) and PCSK9 – inhibitors (PCSK9 – I). During follow – up 12 months after the index event in our outpatient lipid clinic LDL – C targets were attained in all patients.

Methods

After 12 months patients could decide to continue either with regular follow – ups in the outpatient lipid clinic or get routine standard care by general physicians (GP). 53 patients (65.4%) continued to visit the outpatient lipid clinic and 32 (33.6%) preferred standard care treatment. After 24 months differences between these two patient cohorts in regard to changes in lipid – lowering medications, LDL – C target attainment, LDL – C time on target during follow – up and major adverse cardiac events (MACE = nonfatal ischemic cardiovascular events, admission for heart failure, nonfatal stroke) were analyzed.

Results

A total of 85 consecutive patients were included in this study. 73 (85.9%) were male, 12 (14.1%) were female, the average age was 64.4 ± 13.1 years. All 85 patients were followed – up for 24 months. During the follow – up, 4 patients died due to non – cardiac causes. LLT after 24 months: 78 (96%) patients were on statins (atorvastatin / rosuvastatin), 71 (87.7%) patients were on ezetimibe, 14 (17.3%) patients were on BA, and 5 (6.2%) patients were on PCSK9 – I. The average LDL – C after 24 months was 1.38 ± 0.69 mmol/L in the total study population. 51 patients (63%) of the entire cohort were still on LDL – C target of 1.4 mmol/L or below (outpatient lipid clinic group: 72.5% vs. GP group: 27.5%; p 0.037). The average LDL – C in patients followed – up in the outpatient lipid clinic was significantly lower compared to patients who were treated by their GPs (1.3 ± 0.7 mmol/L vs. 2.33 ± 1.04 mmol/L; p < 0.01). Moreover, patients in the outpatient lipid clinic had a longer time on LDL – C targets compared to patients treated by GPs (82.4 ± 29.5% vs. 62.4 ± 36.6%; p < 0.01). The main cause of missed LDL – C targets was alterations of LLT by local GPs, surpassing non – adherence (2.33 ± 1.04 mmol/L vs. LDL – C: 1.52 ± 0.53 mmol/L; p < 0.01) in the overall study population. Patients with MACE during follow – up were characterized by a shorter time on LDL – C targets compared to patients without MACE (58.1 ± 29.9% vs. 79.1 ± 28.1%; p = 0.048) and higher LDL – C levels at 24 months (2.04 ± 1.26 mmol/L vs. 1.27 ± 0.47 mmol/L; p < 0.01). Altogether, male patients with three – vessel coronary artery disease and with reduced ejection fraction were significantly more affected by MACE.

Conclusion: Follow – up in specialized outpatient lipid clinics optimizes lipid – lowering therapies and improves LDL – C target attainment compared to standard care by general physicians.

Background:

Bruton's tyrosine kinase (BTK) is a key downstream mediator of B cell receptor (BCR) signalling in B cells. Upon antigen-BCR recognition and cross-linking, phosphorylated BTK drives B cell activation, proliferation, and differentiation. Ibrutinib is clinically used in B-cell malignancies including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) as a BTK inhibitor. Mechanistically, Ibrutinib irreversibly binds to BTK kinase domain and blunts downstream signaling by reducing BTK phosphorylating state. Here, we aimed to monitor B cell dynamics and activation in myocardial infarction (MI) in mice and test the effect of B cell inhibition by Ibrutinib in cardiac function.

Method:

MI was induced in male 10-week-old C57BL/6 mice by permanent LAD ligation and Ibrutinib (25 mg/kg/day) was given in the drinking water. After 3, 7, 14, and 28 days, single-cell suspensions were prepared for flow cytometry from peripheral blood, spleen, mediastinal lymph nodes (MLNs), and bone marrow, as well as from remote and infarcted myocardium. Total B cells and B cell subsets were quantified in the different locations by flow cytometry. B cell activation was evaluated by BTK551 phosphorylation. After 28 days, cardiac function was evaluated by echocardiography.

Results:

We found a decrease in total B cell numbers in blood, spleen, and MLN, together with increased plasma cells and plasma blasts in the infarcted heart on day 7 and 14 after MI. In the bone marrow, hematopoietic stem cells (HSC) increased after MI, while common lymphoid progenitors (CLP) and early-stage B cell progenitors (pre-pro and pro B cells) peaked at day 7 after MI. The later stage of B cell progenitors (pre and immature B cells) showed a biphasic response, with an early decrease after 7 days and a subsequent increase on day 14. Compared to the control group (vehicle), Ibrutinib-treated mice showed decreased B cell numbers in MLN and spleen by 27% and 14%, respectively, as well as decreased CLP and mature B cells in the bone marrow on day 28 after MI. Both BTK expression and phosphorylation in B cell progenitors were inhibited by Ibrutinib, with a mean fluorescence intensity (MFI) decreased by 8% and 15%, respectively. The inhibition of BTK phosphorylation was also observed in MLN and spleen. Notably, Ibrutinib-treated mice showed an improved cardiac function on day 28 after MI, with stroke volume (SV) elevated by 40%, and both left ventricular ejection fraction (LVEF) and fractional shortening (FS) elevated by almost 80%.

Conclusion:

Ibrutinib treatment in experimental MI hinders the development and activation of B cells in the bone marrow, as well as in peripheral lymphoid organs. Furthermore, B cell inhibition of improved cardiac function. Our results provide a conceptual basis for potential clinical treatment of patients suffering from myocardial infarction.

Background

Jena auf Ziel – “JaZ” is a prospective cohort study in which early combination therapy with atorvastatin 80 mg and ezetimibe 10 mg was initiated on admission in patients with ST – elevation myocardial infarction (STEMI). Lipid – lowering therapy (LLT) was escalated during follow – up with bempedoic (BA) acid and PCSK9 – inhibitors (PCSK9 – I) if low – density lipoprotein cholesterol (LDL – C) was not within the ESC / EAS goal of 1.4mmol/L or less. During follow – up 12 months after the index event in our outpatient lipid clinic LDL – C targets were attained in all patients.

Methods and objectives

We collected economic and clinical data, including in – hospital costs and length of stay. Costs related to LLT were derived from a pharmaceutical directory for Germany (Gelbe Liste Pharmindex), published by Medizinische Medien Informations GmbH (Langen, Germany). Follow – ups were conducted either by the outpatient lipid clinic through scheduled appointments or through routine standard care provided by general physicians (GPs), according to the patients' preferences. In this secondary analysis of “JaZ” we analyzed cost – effectiveness of intensive LLT therapy compared to a less effective LLT approach within the first 24 months after the index event.

Results

A total of 85 (73 men (85.9%), 12 women (14.1%), age: 64.4 ± 13.1 years) consecutive patients were included in this study. Mean and median length of the index hospital stay were: 7.37 ± 5.27 days or 6 (5 - 8) days, respectively. Overall, 631 hospital days accumulated for these 85 STEMI – patients including time spend in high – care monitoring units with average / median total costs of 9549.82 ± 5670.8 EUR / 3585.2 (3461.4 – 10167.2) EUR per patient. Total cumulative costs of these stays were 802.185,13 EUR .

24 months after discharge, 96.3% of the patients were on statins (atorvastatin: 55 (67.9%); rosuvastatin: 21 (25.9%)), 87.7% on ezetimibe, 17.3% on BA and 6.2% on PCSK9 – I. Mean LDL – C serum concentrations at baseline and after two years were 3.2 ± 1.2 mmol/L and 1.38 ± 0.69 mmol/L respectively, reflecting a mean LDL – C reduction of 1.78 ± 1.42 mmol/L.

During the 24 months of follow – up, the total costs for LLT amounted to: 35.495,78 EUR (average per case: 417,59 ± 100,49 EUR) for statins and ezetimibe; 19.983,87 EUR (average per case: 1.492,09 ± 480,97 EUR) for BA and 59.308,32 EUR (average per case: 11.861,66 ± 1.535,62 EUR) for PCSK9 – I.

Four patients died due to non – cardiac causes. MACE occurred in 10 patients (12.3%) that required a second hospital treatment and generated additional costs of 73.411,17 EUR.

Patients experiencing MACE during follow – up exhibited a shorter duration within the target LDL – C range compared to those without MACE (58.1 ± 29.9% vs. 79.1 ± 28.1%; p < 0.05). Additionally, their LDL – C levels after 24 – months were higher (2.04 ± 1.26 mmol/L vs. 1.27 ± 0.47 mmol/L; p < 0.001).

Patients with MACE had lower associated costs for LLT compared to patients without MACE (547.04 ± 475.93 EUR vs. 2.562,29 ± 3.170,20 EUR; p < 0.05). Incorporating the additional costs associated with MACE for the subsequent hospital stay, the overall costs for a more expensive LLT however proved to be less and thus highly cost – effective (2.562,29 ± 3.170,20 EUR vs. 7.921,25 ± 4.140,53 EUR; p < 0.01).

Conclusion

Intensive lipid – lowering therapy within the first 24 months after STEMI is highly cost effective compared to less effective lipid – lowering approach.

BACKGROUND: Previous work showed that atypical chemokine receptor 3 (ACKR3) expression on endothelial cells mediated CXCL12-driven atherosclerosis while ACKR3-deficient haematopoietic cells decrease atherosclerosis in Apolipoprotein E-deficient (Apoe-/-) mice. Notably, ACKR3 is highly expressed on B cells of the spleen and is essential in their differentiation into antibody-producing cells and their exit from the follicle of the spleen. While IgM-producing B1 B cells are well known to be atheroprotective, opposing data has been reported for the role of the larger population of B2 cells, which include plasma cells, in atheroprogression. Here, we aim to unravel the role of ACKR3 on B cells in atherosclerosis and hypothesised that its cell-specific loss of function will reduce the development of the disease.

METHODS: The role of ACKR3 expression is studied on an atherosclerotic mouse model (Apoe-/-) with cell-specific ACKR3 loss of function on B cells (CD19Cre+ ACKR3fl/fl Apoe-/-). These mice were fed either 4 (B cell-ACKR3 KO n=15, control n=20) or 12 weeks (B cell-ACKR3 KO n=17, control n=20) of Western Diet (WD) and atherosclerotic lesion characteristics, differential B cell subpopulations and plasma antibody titers were analysed. Statistical analysis was done using unpaired, two-sided t-test.

RESULTS: After 12 weeks of WD, mice lacking ACKR3 showed a significant decrease of aortic (p≥ 0,05) and root (p≥ 0,05) lesions. In addition, foam cells number (p≥ 0,05) and necrotic core size (p≥ 0,001) were significantly decreased after 12 weeks WD. The atheroprotective IgM content (p≥ 0,01) in the plaque was increased both after 4 and 12 weeks WD. Loss of B cell ACKR3 lead also to an increase of atheroprotective B1 cells in the blood (p≥ 0,001) and bone marrow (BM)(p≥ 0,01) compared with controls (ACKR3fl/fl Apoe-/-). On the other hand, atherogenic B2 cell numbers in the blood (p≥ 0,001) and BM (p≥ 0,01) as well as pro-atherogenic plasma cells in the BM (p≥ 0,05), lymph node (p≥ 0,01) and spleen (p≥ 0,001) were significantly reduced. Additionally, atherogenic plasma titers of IgG subclasses such as IgG1 (p≥ 0,001), IgG2a (p≥ 0,001) and IgG2b (p≥ 0,5) are decreased.

CONCLUSION: Our data suggest that cell-specific loss of ACKR3 on B cells has an atheroprotective role in atherosclerosis by decreasing pro-atherogenic B2 cells as well as plasma cells and pro-atherogenic antibody production while increasing atheropretective B1 cells and IgM antibodies leading to a reduction of foam cells and necrotic core area in the lesions. Further in vitro, we will investigate the role of B cell specific ACKR3 in both T cell-indepedente and T cell-dependent B cell activation as well as B cell migration towards chemoattractants. In addition, splenic B cell-ACKR3 deficiency is under investigation at a single cell level to unravel differential transcriptomics between the WT and KO mice under Western type Diet.

Rationale: Although constituting a relatively small population of cells in the heart, macro- phages are still important for maintaining cardiac health, for example through efferocytosis of cell debris / exosomes and via electromechanical coupling to cardiomyocytes. Apart from these heterocellular direct interactions, macrophages are exposed to constantly changing me- chanical forces in the beating heart. In this project we ask whether and how the stretch-acti- vated ion channel Piezo1 can influence cardiac macrophage phenotypes and myocardial ho- meostasis.

Methods and results: Macrophage-specific Piezo1 deficient mice (CX3CR1Cre:Piezo1fl/fl, KO) presented no apparent cardiac abnormalities at two months of age. At 6 and 12 months of age, however, KO mice had sponteanously developed an age-related cardiomyopathy with significantly reduced ejection fraction (52.73% WT vs. 45,31% KO; p<0,0001) and lower frac- tional shortening (27,9% WT vs. 22,89% KO; p<0,0001). Electron microscopic imaging of the murine KO hearts depicted pathological restructuring of cardiomyocytes with mitochondrial dysfunction. In search of the underlying mechanism, we performed high-dimensional single- cell RNA sequencing of sorted CD45+ leukocytes and single-nuclear RNA sequencing of stromal cells in WT and KO mice at 2 and 12 months of age (n=8 per group). Macrophage subclusters with resident cell and foamy cell phenotypes featured the largest number of differentially reg- ulated genes between the groups preceding the development of cardiomyopathy. Genes as- sociated with efferocytosis and regenerative growth factor signalling were reduced in Piezo1 deficient macrophages.

Conclusion: Our preliminary results indicate that mechanosensing by cardiac macrophages is essential to preserve cadiac tissue homeostasis and function during ageing.

Background: Post-myocardial infarction sterile inflammation results in the depletion of innate immune cells circulating in the body. Emergency hematopoiesis (EH), originating from the bone marrow, compensatorily activates to sustain early inflammatory monocyte recruitment, crucial for macrophage (Mφ)-mediated efferocytosis. However, unregulated myelopoiesis can exacerbate cardiac inflammation, contributing to maladaptive remodeling. Targeted reduction of hematopoietic activation may offer a novel strategy to preserve cardiac function. All-trans retinoic acid (ATRA), known for its pleiotropic modulation of EH and innate immunity, protects hematopoietic stem cells from activation while promoting survival and differentiation of myeloid cells.

Purpose: This study aimed to explore the impact of ATRA on EH and its implications in cardiac wound healing.

Methods: Myocardial infarction (MI) was induced through permanent coronary ligation in C57BL/6 mice, treated with daily ATRA injections (30mg/kg) or DMSO as a vehicle control for up to five days, starting 24 hours post-ligation. Flow cytometry (FACS) assessed cell cycle changes in hematopoietic stem cells (HSCs) and immunophenotyping of leukocytes in bone marrow (BM), blood, and heart. Cytokine expression was analyzed via qPCR in bulk infarct and sorted immune cells from bone marrow and cardiac origin. Echocardiography, Immunohistochemistry (IH), and Masson trichrome (MT) staining evaluated functional and histological infarct development.

Results: On the second day post-MI, ATRA-treated mice exhibited significantly reduced EH compared to vehicle controls, evident in cell cycle activity, single-cell proliferation potential of HSCs, and downstream myeloid cells in BM, blood, and heart. Consequently, early-phase infarct tissue showed diminished mRNA expression of key inflammatory cytokines, IL-1β, and TNFα. ATRA did not affect BM mobilization of leukocytes or the expression of recruitment factors on cardiac endothelial cells. However, despite these early anti-inflammatory changes, LV-ventricular remodeling was not prevented, and cardiac function was not preserved 21 days after MI. IH revealed a twofold increase in CD11b-positive myeloid cells in remote myocardium (RZ) alongside elevated expression of IL-6, TNFα, and TGFβ. MT-staining at day 21 demonstrated an almost threefold increase in collagen deposition in the RZ of ATRA-treated mice compared to controls. RNA-sequencing revealed dense expression of retinoic acid receptors on murine cardiac monocytes and Mφ. qPCR confirmed non-canonical activation of ATRA-primed monocyte-derived Mφ, promoting a pro-inflammatory phenotype with higher MMP2 and MMP9 expression. Additionally, prominent IL-1β and IL-6 expression in M2-polarized BM-derived Mφ indicated an impaired anti-inflammatory phenotype following ATRA treatment.

Conclusion: Despite a beneficial reduction in EH after MI, short-term ATRA treatment induced profound and persistent changes in cytokine expression of monocytes and Mφ from both BM and cardiac origin, leading to increased inflammatory and fibrotic activity. This hindered improvements in cardiac function due to myeloid and fibrotic expansion in remote myocardium. The data highlight the importance of the individual inflammatory behavior of immune cells in cardiac wound healing for enhancing scar care and improving cardiac outcomes post-MI.

Introduction

Optical coherence tomography angiography (OCT-A) has emerged as a non-invasive imaging technique for visualizing the microvasculature of both the retina and the choroid. Its application extends across ophthalmology, neuro-ophthalmology, neurology, and vascular medicine, proving invaluable in the diagnosis and assessment of various diseases. The quantification and interpretability of retinal vessel density relies heavily on the quality of scans. Therefore, effective quality management of OCT-A is essential in clinical studies.

Objective

To address the essential role of scan quality in OCT-A studies, we aimed to develop and implement an OCT-A quality management platform. This platform was applied in the Berlin Long Term Observation of Vascular Events study (BeLOVE), where individuals with various cardiovascular conditions and risk factors underwent examinations conducted by operators with varying levels of experience.

Methods

We refined published OCT-A quality criteria (OSCAR-MP) by conducting a comprehensive literature review, incorporating our own experiences, and consulting with peers. To capture and quantify the quality of each OCT-A scan, we designed an electronic case record form in the Research Electronic Data Capture System. The criteria were then applied to OCT-A scans obtained in the BeLOVE study, which included data from individuals with stroke, heart failure, heart attack, and individuals with risk factors for cardiovascular disease, i.e. diabetes mellitus. OCT-A was performed using the Cirrus HD-OCT 5000 (Carl Zeiss Meditec, Dublin, CA, USA) with scan protocols angiography 6x6 for the macula and ONH angiography 4.5x4.5 for the optic nerve head (ONH). Quality control was performed by one experienced grader.

Results

The quality criteria, represented by the acronym OMSCAR, encompass five categories. The first criterion, Obvious, aims to assess clearly identifiable quality problems such as incorrect scan regions or reversed tomograms. The next criterion covers Motion artifacts, including banding or blinking. The third criterion addresses Signal problems, such as signal strength, shadows, or focus. The fourth criterion focuses on Centration problems, ensuring that the ONH or macula is ideally centered within the scan. The criterion Algorithm evaluated the segmentation algorithm and A-scan registration of the manufacturer. Lastly, the criterion Retinal pathology involves identifying and assessing retinal pathologies that could affect the accuracy and interpretation of OCT-A parameters.

OMSCAR was assessed in 852 macular and 831 ONH OCT-A scans from 373 BeLOVE participants. Analysis of quality control showed Signal with 86% as the most prominent quality problem in macula scans followed by Motion with 74%. In contrast, ONH scans showed most quality issues in Motion (84%) followed by 77% of centration errors. Overall, the least quality problems were found in Algorithm and Retinal Pathology.

Conclusion

The OMSCAR quality management platform provides a standardized framework for assessing OCT-A image quality, facilitating consistent assessment and interpretation of retinal vascular alterations. The substantial number of scans with impaired quality, with Motion, Signal, and Centration being common quality issues, emphasizes the importance of rigorous quality management in multi-operator settings.

Specifically, in this study, there is an increased occurrence of artifacts due to the unique setting and characteristics of the cohort. In cohorts where challenging scans are anticipated, quality control becomes even more crucial. In order to improve the quality of OCT-A scans, it is recommended to provide targeted training and raise awareness among users about potential problems and their solutions. OMSCAR serves as a valuable resource and guide for future studies utilizing OCT-A, which stands out as a highly promising non-invasive tool in vascular research and medicine. Offering detailed angiographic information without the need for contrast agents, OCT-A facilitates precise evaluation and diagnosis of vascular conditions.

Background – Obesity, which is frequently associated with type 2 diabetes mellitus (T2DM), is a major contributor to ventricular arrhythmias (VA), which represent a considerable danger to cardiovascular health. Both obesity and T2DM increase the presence of senescent cells in the heart, which increases the susceptibility to VA. Application of senolytics compounds that selective eliminate senescent cells is a feasible treatment option with potential to renew heart tissue and restore organ performance, therefore positively altering the aging process. Targeting cellular senescence may represent a therapeutic approach for lowering ventricular arrhythmias in aged hearts.

Methods and Results – Male C57Bl/6J mice, aged 10-12 weeks, were subjected to a 12-week high-fat diet to induce obesity (DIO). Within one week, the DIO mice received either the senolytics dasatinib (5 mg/kg) and quercetin (50 mg/kg) (DQ) or a control treatment orally. To assess myocardial senescence, we measured Cdkn1a mRNA expression (a common senescence marker) using qPCR. DQ treatment of DIO mice resulted in a significant reduction in myocardial senescence compared to the control group. Susceptibility to ventricular arrhythmias was tested in isolated hearts through ventricular stimulation and epicardial fluorescence imaging. Cardiac contractility was determined by measuring sarcomere length and contraction speed in individual cardiomyocytes during pacing. As a result of DQ treatment, the amount of inducible VAs in DIO mice decreased, while the contractility of individual cardiomyocytes increased.

Western blot analyses were conducted to evaluate calcium signaling and cardiac innervation. Moreover, a quantitative enzyme-linked Immunosorbent Assay (ELISA) assessed cyclic adenosine monophosphate (cAMP) levels. DQ therapy significantly increased myocardial expression of tyrosine hydroxylase, β1-adrenoreceptor and cAMP.

Conclusions – Senescent cells are important in the development of age-related diseases. The senolytics dasatinib and quercetin successfully reduce cardiac tissue senescence and minimize the incidence of VA in obese and prediabetic patients. This work contributes to our understanding of arrhythmia formation and shows potential for targeted therapeutics in diabetes and senescence-related cardiovascular complications.

Background: Atherosclerosis is the most frequent pathology that causes coronary artery disease (CAD). It is characterized by chronic inflammation of the arterial wall driven by LDL-cholesterol (LDL-C) accumulation in the intima layer. Atherosclerosis is accompanied by an auto-immune response that involves autoreactive CD4+ T cells recognizing peptides from ApoB-100, the core protein of LDL-C. Regulatory T-cells (Tregs) can counteract this response by secreting anti-inflammatory cytokines such as IL-10. However, in mice, Tregs lose their protective properties over time, start expressing pro-inflammatory cytokines, including IFN-g, and ultimately promote disease development. Here, we aimed to systematically characterize circulating Tregs in human CAD.

Methods: Patients at high cardiovascular risk or with a history of CAD were included in the Adaptive Immunity in Atherosclerosis (ANIMATE) exploratory biomarker trial at the University Heart Centre Freiburg-Bad Krozingen, Germany. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a total of 359 patients. In a case-control design, we selected 80 patients that were matched according to their clinical parameters and divided into 4 groups: younger ≤ 55-year-old patients without (n=19) or with CAD (n=21), and older ≥ 70-year-old patients without (n=18) or with CAD (n=22). We studied the abundance, extracellular marker expression, and cytokine secretion in Tregs by flow cytometry with and without a stimulation with PMA at a concentration of 1:500.

Results: Younger patients without or with CAD did not show significant differences in the percentage of circulating Tregs that were defined as CD127-/CD25+/FoxP3+ CD4+ T cells (% of all CD4+ T cells). Surprisingly, the relative abundance of Tregs in older patients with CAD was significantly higher (increased by 2-fold) than in patients without CAD (p=0.0036), raising the question of age-dependent changes in Tregs homeostasis that might affect CAD progression. Accordingly, Tregs from older patients with CAD showed a lower baseline expression of the atheroprotective cytokine IL-10 (70% reduction), as well as higher IL-17, TNF-a, and INF-g protein expression levels in intracellular flow cytometry. While in younger patients Tregs sustained their atheroprotective function, these findings indicate a shift towards a proinflammatory phenotype in older patients with CAD. Interestingly, Treg numbers were significantly increased in patients with arterial hypertension, severe CAD (3-vessel-disease), and in patients with previous myocardial infarction. Moreover, Treg frequencies positively correlated with plasma levels of anti-ApoB IgG autoantibodies. Conversely, Treg frequencies negatively correlated with total cholesterol and LDL-C levels.

Conclusion: A switch of Tregs into pro-inflammatory T cells (exTregs) has been suggested from experimental atherosclerosis in mice. Here, we show that in older patients with CAD, Tregs are unexpectedly more frequent but show a more proinflammatory phenotype than in the control group without CAD or in younger individuals. These data support the concept of human Treg plasticity in CAD. This study provides insights into the immunological mechanisms underlying CAD development and progression and suggests potential therapeutic targets involving Treg modulation.

Objective: Most atherosclerosis-prone blood vessels are surrounded by perivascular adipose tissue (PVAT) which is contiguous with adventitial layer of arteries. PVAT is a physiologically and metabolically active endocrine tissue secreting various biologically active factors. Morphological, structural, and functional alternations of PVAT have been observed in obesity and other cardiovascular disorders. The dysfunction of this tissue is affecting the pathogenesis of atherosclerosis due to the secretion of pro-inflammatory adipokines and infiltration of inflammatory cells. The focus here is on the adipose tissue macrophages (ATMs) in PVAT which are central players in obesity-associated inflammation and metabolic stress due to their capability of switching their phenotype from alternatively activated anti-inflammatory M2 to classically activated pro-inflammatory M1. The receptor ChemR23 which is expressed in a number of immune cells including myeloid DCs, natural killer (NK) cells as well as macrophages and its ligand chemerin whose expression and secretion increases dramatically with adipogenesis seem to play a crucial role in the phenotype switching of ATMs and thus in the crosstalk between PVAT and arteries during atherosclerosis.

Methods: To study the role of ChemR23 expression, we use an atherosclerotic mouse model with a systemic knockout of ChemR23 expressing eGFP (enhanced green fluorescent protein) (Apoe-/- ChemR23-knockout/knockin mice). These mice are fed either a 4 or 12 weeks Western Diet (WD) and analysed for ATMs via FACS. PVAT will be analysed immunohistochemically and on transcriptomic level employing single nuclei RNA sequencing. Organs like spleen and lymph nodes will be analysed either histologically or at gene expression level. Using two Cre-lox mouse models with a cell-specific knockout of ChemR23 either on adipocytes or myeloid cells with an Apoe-/- deficient background will enable us to examine the impact of ChemR23 on those cells and to better understand the adipocyte-macrophage interaction in the absence of the receptor.

Results: We found that the systemic knockout of ChemR23 decreased the number of macrophages in various adipose tissues including the pericardial and epididymal adipose tissues as well as in periaortic adipose tissue (PVAT) after 4 weeks of Western Diet. Furthermore, the number of classically activated M1 macrophages increased in adipose tissues of mice lacking the ChemR23 receptor fed a WD for 4 weeks.

Conclusion: Based on the previous data, we hypothesize that the loss of ChemR23 expression has an adverse effect on the phenotypic switching of adipose tissue macrophages in perivascular adipose tissue in hyperlipidaemic mice during atherosclerosis. Further investigations are planned for unravelling the transcriptome of the ATMs using the single-cell RNA sequencing.

Background: The cannabinoid receptor CB1 modulates blood pressure and atherogenesis, although the underlying mechanisms regulating CB1 activation are not fully understood. Endothelial cells respond to shear stress, altering gene expression and cell morphology. Here, we addressed whether endothelial CB1 expression is affected by shear stress, thereby modulating vital cellular functions in a flow-dependent manner.

Methods: Murine samples were collected from Apoe-/- mice and mice with endothelial CB1 deficiency (Apoe-/-BmxCreERTCnr1flox/flox). In-situ hybridization for CB1 encoding gene Cnr1 expression, immunostainings of ICAM1 and VE-Cadherin were performed with aortic sections and whole mounted arteries. The inner curvature of the aortic arch exposed to oscillatory shear stress (OSS) was analyzed as an atheroprone area, the descending aorta, exposed to laminar shear stress (LSS) as an atheroprotective area. In vitro experiments were performed to study OSS (4dyn/cm2) and LSS (10dyn/cm2) responses in human aortic endothelial cells (HAECs). Gene expression levels were determined by qPCR. LDL uptake and monocyte adhesion assay were performed with and without CB1 activation.

Results: Endothelial Cnr1 expression was higher in atheroprone compared to atheroprotective areas of Apoe-/- aortas. En-face-stained aortas of mice with endothelial CB1 deficiency revealed decreased ICAM1 expression in atheroprone regions, suggesting a less inflammatory phenotype (n=4, p=0.0008). In HAECs, CNR1 was upregulated by OSS compared to static conditions (n=3, p=0.0005). HAECs treatment with CB1 agonist under LSS, but not static condition, resulted in enhanced expression of markers for lipid transcytosis (Caveolin-1) (n=4, p=0.0023), inflammation (ICAM, VCAM1), and glycolysis (PFKFB3), while anti-inflammatory markers were decreased. Upon pharmacological CB1 stimulation, reduced junctional adhesion protein VE-cadherin expression (n=3, p=0.0046), increased LDL uptake (n=3, p<0.0001) and monocyte adhesion (n=3, p=0.003) were observed. Conversely, antagonism of CB1 decreased LDL uptake (n=4, p<0.0001) and Caveolin-1 protein levels (n=4, p=0.0095).

Conclusion: Our results indicate that CB1 expression is upregulated by atheroprone flow conditions in vivo and in vitro, translating into a pro-inflammatory phenotype. How the mechanical changes translate into altered CB1 expression, and whether CB1 itself may act as a mechanosensor, remains to be investigated.

Background and Aims:

To assess the efficacy and safety of lomitapide in paediatric patients with homozygous familial hypercholesterolaemia (HoFH).

Methods:

APH-19 (NCT04681170) is an ongoing phase 3, open-label, single-arm trial of lomitapide in paediatric patients with HoFH receiving standard-of-care lipid-lowering therapy. The study consisted of a run-in period, followed by a 24-week efficacy phase, 80-week safety phase and follow-up period. Patients were stratified by age into three dose escalation groups: 5–10, 11– 15 and 16–17 years, where maximum daily doses were 20, 40 and 60 mg, respectively. Patients were titrated to maximum tolerated doses from a starting dose of 2 mg (patients 5–15 years) or 5 mg (16–17 years).

Results:

Forty-three patients were treated (Female: 55.8%; mean age: 10.7 years). APH-19 met its primary endpoint; mean change from baseline LDL-C at Week 24 was -53.5% (95% CI -61.6 – -45.4, p<0.0001), with results similar between patients aged 5–10 and 11–17. Mean reductions were also observed at Week 24 for non-high-density lipoprotein C (-53.9%; 95% CI -61.7 – -46.1, p<0.0001), total cholesterol (-50.1%; 95% CI -57.6 – -42.5, p<0.0001) and very-low-density lipoprotein cholesterol (-50.2%; 95% CI -59.1 – -41.2, p<0.0001). Patients reported mild (48.8%), moderate (30.2%) and severe (11.6%) adverse events (AEs). One patient experienced a life-threatening AE (MACE; treatment unrelated). No serious AEs led to discontinuation.

Conclusions:

In this study, lomitapide significantly reduced LDL-C levels in paediatric HoFH patients. Safety was consistent with the known profile of lomitapide, with no new signals identified.

Aims: Obesity is an epidemic that is a critical contributor to hypertension and other cardiovascular diseases. Current paradigms suggest that endothelial nitric oxide synthase (eNOS/NOS3) in the vessel wall is the primary regulator of vascular function and blood pressure. However, recent studies have revealed the presence of eNOS/NOS3 in the adipocytes of white adipose tissues and perivascular adipose tissues (PVATs). The current understanding of the role of adipocyte NOS3 is based mainly on studies using global knockout models. The present study aimed to elucidate the functional significance of adipocyte NOS3 for vascular function and blood pressure control. Methods and Results: We generated an adipocyte-specific NOS3 knockout mouse line using adiponectin promotor-specific Cre-induced gene inactivation. Control and adipocyte-specific NOS3 knockout (A-NOS3 KO) mice were fed a high-fat diet (HFD). Despite less weight gain, A-NOS3 KO mice exhibited a significant increase in blood pressure after HFD feeding, associated with exacerbated vascular dysfunction and remodeling. A-NOS3 KO mice also showed increased expression of signature markers of inflammation and hypoxia in the PVATs. Among the differentially expressed adipokines, we have observed an upregulation of a novel adipokine, chemerin, in A-NOS3 KO mice. Chemerin was recently reported to link obesity and vascular dysfunction. Treatment with chemerin neutralizing antibody normalized the expression of remodeling markers in the aorta segments cultured in serum from HFD-fed A-NOS3 KO mice ex vivo. Conclusions: These data suggest that NOS3 in adipocytes is vital in maintaining vascular homeostasis; dysfunction of adipocyte NOS3 contributes to obesity-induced vascular remodeling and hypertension.

Background and Aims: Microplastics are known to accumulate in our environment and represent a novel risk factor – not only but also – for humans. Microplastics trigger pro-inflammatory effects in many cells and tissues, which are also involved in atherogenesis. To investigate this aspect further we here analyzed the influence of polystyrene particles (PS), one of the most common plastic polymers, on pro-atherosclerotic processes and on high-fat diet (HFD)-induced experimental early atherosclerosis in Apolipoprotein E-deficient (Apoe^−/−) mice.

Methods and Results: PS particles (200, 500, 1000 nm, 10⁷/ml) penetrated epithelial and endothelial monolayers, increased their permeability to smaller substances (transwell assay), and impaired their regenerative capacity (scratch assay). Reactive oxygen species production (H₂DCFDA/DCF fluorescence) and oxidative stress-related genes (qPCR) were found to be upregulated in monocytes and endothelial cells after PS stimulation.

Apoe^−/− mice receiving an HFD for 6 weeks and during this time a gavage with PS particles (200, 500, 1000 nm = 500 µg) did not show changes in body weight, glucose metabolism (glucose tolerance test), and plasma cholesterol/triglyceride levels (enzymatic method). The extent of atherosclerosis in the aortic root (oil red O staining), the amount of inflammatory cells (MOMA-2 staining) in the plaques, and the vascular expression of inflammatory genes (qPCR) were not altered by the administration of PS particles in the animals. However, we observed significant alterations in the gut microbiome by 3^rd generation sequencing (Oxford Nanopore Technologies MinION) as a result of PS particle administration.

Conclusions: Our data show that PS particles are able to induce numerous effects associated with the initiation and progression of atherosclerosis. However, short-term administration of PS particles did not lead to an increase in experimental atherosclerosis in the mouse model, but to alterations in the composition of the gut microbiome.

Pharmacological anti-inflammatory treatments have showed promise in increasing left ventricular (LV) function recovery following an acute myocardial infarction (AMI). CD40, a critical immune modulatory molecule, is important in the inflammatory processes that accompany, among others, cardiovascular disorders. In this context, inhibiting CD40 signaling, specifically its interaction with tumor necrosis factor receptor-associated factor 6 (TRAF6), with small molecule inhibitors known as TRAF-STOPs, has shown potent anti-inflammatory effects, effectively reducing chronic inflammatory conditions such as atherosclerosis. However, the potential benefit of CD40 inhibition in the setting of acute myocardial infarction (AMI) are unclear. In this work, we aimed to determine the effect of CD40 inhibition via TRAF-STOP within a certain inflammatory phase following following AMI. CD40 suppression with TRAF-STOP starting 5 days after AMI leads to better LV function on days 14 and 28. This improvement is related to a TRAF-STOP-dependent reduction in the number of pro-fibrotic CD9+ macrophages, as well as a considerable attenuation of fibroblast activation, which leads to less cardiac scarring. This work highlights the therapeutic potential of targeted CD40 inhibition as a method of reducing inflammation and improving post-AMI cardiac recovery. The defined temporal modulation of CD40 inhibition with TRAF-STOP emerges as a promising approach to reduce myocardial damage, opening up new options for intervention in the treatment of AMI.

Background

Determining infarct and scar size is crucial for myocardial infarction (MI) research. These factors are often used to test the effectiveness of potential cardioprotective treatments. Currently, the most widely used method for measuring infarct size is 2,3,5-triphenyltetrazolium chloride (TTC) and Evans Blue staining. Although TTC staining is well established, it presents unique pitfalls that we aim to circumvent. Determining the infarct area and adjacent area poses challenges for the analyst due to demanding color nuances. A clear disadvantage is the limited option for simultaneous histological analysis such as co-stainings on the already TTC-stained tissue. A heart therefore cannot be used for any other analyses after it has been used for TTC.

Here, we compare classic histological methods with fluorescently-labeled agents like phalloidin and WGA to prove their specificity and offer experimenters an at least comparable alternative that allows additional and combined analyses including multicolor immunofluorescence.

Methods and Results

Mice underwent surgery to induce experimental MI by ligation the left anterior descending (LAD) coronary artery for 45 minutes followed by reperfusion. After 24 hours, we compared infarct size visualized with TTC/Evans Blue and fluorescence-labelled phalloidin staining. Phalloidin stains selective F-Actin from microfilaments in the cytoskeleton of cells. A strong correlation between these different staining methods was detected. Usually, TTC and Evans blue are used to visualize infarct area (non-viable tissue), area at risk (AAR; infarct + viable tissue devoid of blood supply), and tissue unaffected by the operation. To define these regions of interest in phalloidin-stained tissue, we applied Hoechst as fluorescent dye to explanted hearts by retrograde infusion while re-ligating the LAD. We could show different clearly defined areas of infarct, AAR and remote myocardium, which can be readily quantified using semi-automatic imaging analyses. Building on this technique, we examined invading immune cells in the infarct area using different cell type-specific markers 24h after MI.

For later time points (7d, 21d, 28d), we quantified scar size using collagen-based stainings and compared them with fluorescent WGA staining, a marker of glycoproteins in the cell membrane, that provide information about the integrity of tissue. We showed a strong correlation (Sirius Red, r=0.9752; Masson´s Trichrom, r=0.9920; Gomori r=0.8082) between WGA and these respective methods.

A comparison of phalloidin and WGA to define infarction or scar development results in the clear advantage of phalloidin for analyses until 7d post MI. A combination of both stainings is recommended for the quantification of later timepoints, due to the increase of fibrotic tissue.

Conclusion

In this work, we showed that fluorescent stainings were as reliable and impactful as their classical counterparts (e.g., TTC and collagen-based stainings) for infarct or scar size analyses after MI. Paramount benefits of these fluorescent dyes are their flexibility, as they are compatible with both paraffin and cryo-sections and allow for multicolor co-stainings for various specific research question. In addition, this technological advancement serves the 3R paradigm, as multiple analyses can be performed on a single heart.

Aim: Although the association between Lipoprotein (a) (Lp(a)) and incident/prevalent atherosclerotic cardiovascular disease (ASCVD) is well established, the evidence on the role of Lp(a) in subclinical carotid atherosclerosis (CA) remains controversial. Therefore, we aimed to investigate the association between Lp(a) and the presence of subclinical CA among asymptomatic subjects from the general population.

Methods: Lp(a) was measured in 7,482 participants of the population-based Hamburg City Health Study by a particle-enhanced quantitative turbidimetric immunoassay (molar-based; Roche Diagnostics). All subjects (median age: 62 years, 52.6% women) were free of prevalent ASCVD at time of enrollment. Subclinical CA was assessed by carotid ultrasound and defined as peak cIMT ≥ 1 mm or presence of plaques. Multivariable binary logistic regressions were performed after stratification of the study population according to Lp(a) risk-related cutoffs of < 75, 75-< 125 and ≥ 125 nmol/L.

Results: Median Lp(a) was 17.70 [IQR 7.80;59.48] nmol/L and 36.3% of study participants had subclinical CA on imaging. Subclinical CA was more prevalent in higher Lp(a) categories, being 35.1%, 39.3% and 41.5% in those with Lp(a) < 75, 75-< 125 and ≥ 125 nmol/L, respectively (p<0.001). High Lp(a) concentrations (i.e. ≥ 125 nmol/L) were associated with an elevated risk for subclinical CA (OR 1.20 (95%CI: 1.02-1.41); p=0.023) compared to individuals with low (i.e. < 75 nmol/L) Lp(a) values, independently of traditional cardiovascular risk factors and lipid-lowering medication. In individuals with Lp(a) concentration between 75 and < 125 nmol/L, corresponding OR was found to be 1.17 (95%CI: 0.95-1.43); p=0.14, fully-adjusted model).

Conclusion: High Lp(a) values were independently associated with presence of subclinical CA in asymptomatic subjects, thereby suggesting Lp(a) as an early indicator of atherosclerotic burden in the general population.

Background: Bioactive adrenomedullin 1-52 (bio-ADM) is a dynamic blood biomarker for real-time assessment of endothelial function. Bio-ADM was recently shown to be a prognostic marker in patients with acute heart failure and cardiogenic shock. The SMART (Second Manifestations of Arterial Disease) score is a validated tool for risk assessment in patients with established atherosclerotic cardiovascular disease (ASCVD). The aim of this study was to assess whether measurement of bio-ADM adds incremental value to the SMART score in stable patients with ASCVD.

Methods: Circulating bio-ADM levels were assessed in n=695 stable patients with ASCVD hospitalized at the Department of Cardiology at University Hospital Aachen, Germany (all-comer cohort). Endpoints evaluated were all-cause mortality and cardiovascular mortality; follow up was 3 years.

Results: Bio-ADM was higher in non-survivors (all-cause death: n=54, median 33.1 pg/mL) compared to survivors (n=641, median 17.9 pg/mL; p<0.0001). Univariable Cox regression analyses showed bio-ADM to be associated with adverse outcome [standardized hazard ratio (HR) of bio-ADM values: All-cause death: 2.4, 95% confidence interval (CI): 2.0-2.9; p<0.001, cardiovascular death: 2.5, 95% CI: 1.9-3.3; p<0.001]. This association remained significant in various multivariable Cox regression models. Bio-ADM was found to be a strong marker for mortality (c-index: 0.80, Chi²: 54.1) and proved to be superior to other markers including hs-Troponin T (c-index: 0.61, Chi²: 2.0) and eGFR CKD-EPI 2021 (c-index: 0.687, Chi²: 33.5). Addition of bio-ADM to the SMART score significantly improved model performance in predicting mortality (SMART score: c-index: 0.717, Chi²: 24.73; SMART score + bio-ADM: c-index: 0.832, Chi²: 63.24; Delta c-index: 0.115; Delta Chi²: 38.51; all p<0.001).

Conclusion:

Bio-ADM provides incremental added value (improved discrimination and calibration) on top of the SMART risk score in patients with ASCVD.

Objective

To investigate the association between body mass index (BMI) at acute myocardial infarction (AMI) and all-cause as well as cause-specific long-term mortality.

Methods

The analysis was based on 10,651 hospitalized AMI patients (age 25-84 years) recorded by the population-based Myocardial Infarction Registry Augsburg between 2000 and 2017. The median follow-up time was 6.7 years [IQR: 3.5-10.0)]. Cause-specific mortality was obtained by evaluating the death certificates as well as standardized questionnaires send out to the former treating physician or coroner. In multivariable adjusted COX regression models using cubic splines for the variable BMI, the association between BMI and cause specific mortality (all-cause, cardio-vascular, ischemic heart diseases, cancer) was investigated. Additionally, a subgroup analysis for 3 different age groups was performed for all-cause mortality.

Results

Overall, there was a significant U-shaped association between BMI at AMI and long-term mortality with the lowest hazard ratios (HR) found for BMI values between 25 and 30 kg/m². For cancer mortality, higher BMI values > 30 kg/m² were not associated with higher mortality. In younger patients, there was a significant association between high BMI values > 35 kg/m² and increased all-cause mortality; this association was missing in elderly patients. For all groups and for each specific mortality, low BMI values were significantly associated with higher mortality.

Conclusions

Overall, a low BMI – and also a high BMI in younger patients - are risk factors for increased mortality after AMI. Therefore, after AMI a BMI in a mid-range may be associated with favorable outcome.

Chemokine receptors play a pivotal role in sustaining chronic vascular inflammation. Our previously published research demonstrated that hematopoietic deficiency of the chemokine-like receptor ChemR23 led to an increased proportion of anti-inflammatory M2 macrophages and a reduction in plasmacytoid dendritic cell (pDC) recruitment to lymphatic organs and atherosclerotic lesions in murine models. This deficiency consequently slowed the progression of atherosclerotic lesions. However, observations from studies using systemic ChemR23-deficient animals suggest that ChemR23 may perform a cell-specific function within the context of atherosclerosis. To explore this hypothesis, we examined the specific role of ChemR23 in vascular cells.

We employed a knockout/knock-in reporter mouse model and conducted a bone marrow transplantation study. Apolipoprotein E-deficient (Apoe-/-) bone marrow was transplanted into both Apoe-/- and Apoe-/-ChemR23eGFP/eGFP mice, rendering these mice deficient in ChemR23 in non-hematopoietic (somatic) cells. When these mice were fed a Western diet for either 6 or 12 weeks, we observed that the absence of ChemR23 in somatic cells led to significantly larger atherosclerotic lesions and increased lipid accumulation. Histological analysis revealed that this loss of ChemR23 resulted in enhanced migration of smooth muscle cells (SMCs) into the plaque, as well as an increase in the formation of SMC-derived foam cells (SMFCs).

Bulk RNA sequencing of the aorta indicated that the lack of vascular ChemR23 promotes atherosclerotic pathways and upregulates genes associated with athero-progressive synthetic SMC phenotypes. Further in vitro studies using human aortic smooth muscle cells (HASMCs) treated with α-NETA, a small molecule antagonist of ChemR23, showed a rise in cholesterol uptake and a decrease in cholesterol efflux, suggesting that inhibiting ChemR23 encourages SMFC formation. Gene expression analysis in HASMCs revealed that α-NETA treatment upregulates synthetic athero-progressive genes such as KLF4, CD36, and MAC2. Additionally, α-NETA treatment increased HASMC migration, especially when compared to treatment with chemerin 9 (a ChemR23 ligand) in cells pre-treated with INF-γ.

As a therapeutic investigation, Apoe-/- mice were administered α-NETA, chemerin 9, or a vehicle control through an osmotic pump over a 4-week Western diet regimen. Both α-NETA and chemerin 9 treatments resulted in reduced lesion sizes but exhibited distinct plaque characteristics and underlying mechanisms, warranting further study.

In summary, our findings suggest that ChemR23 plays a crucial role in regulating the phenotypic switching of vascular smooth muscle cells (VSMCs), highlighting its potential as a therapeutic target in atherosclerosis.

Cardiovascular diseases (CVDs) encompass a broad spectrum of conditions that affect the heart and blood vessels, and are the leading cause of death worldwide. Inflammation plays a crucial role in the development and progression of CVDs. The aim of this study was to compare the composition of immune cells in different experimental mouse models of CVD. We generated a single-cell RNA sequencing (scRNA-seq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq; using a commercial 120 marker panel (TotalSeq™-C Mouse Universal Cocktail, V1.0, Biolegend)) dataset containing isolated immune cells from aortas and hearts from mouse models of atherosclerosis, abdominal aortic aneurysm (AAA) and myocardial infarction (MI). Specifically, the dataset contained immune cells from atherosclerotic aortas of old Apoe-/- mice (2847 cells), cells from aneurysmal tissue 3, 7 and 14 days after AAA induction via elastase perfusion (4061 cells) and cells from hearts 1 and 5 days after MI induction via transient occlusion of the left anterior coronary artery (2405 cells). In total, 28 distinct immune cell clusters were identified, all of which were detected – albeit in varying proportions - in all three CVD model tissues. Some T cell subpopulations accounted for a larger proportion of all immune cells in AAA and atherosclerosis than in MI. Other lymphocyte clusters like CD8+ T cells and B2 cells were most prevalent in atherosclerosis. We also found profound differences in the proportion of neutrophil and macrophage subpopulations between AAA and MI. Furthermore, we used the CITE-seq data to interrogate the expression of surface proteins at the single-cell level. We confirmed the immune cell types identified by RNA expression profiles by the expression of surface molecules and examined the correlation between RNA and protein expression in these cells on a cluster-restricted level. In summary, we present for the first time a pan-disease dataset that is devoid of any batch and other processing artifacts. In addition, parallel mRNA and surface protein analysis allows to transfer and interrogate long-established surface expression information to novel scRNASeq datasets. Thus, CITE-seq is suitable for the characterization of rare, previously unexplored subpopulations at the protein level, enabling further investigation of these cells with antibody-based methods such as flow cytometry.

Background:

The gut incretin hormone glucagon-like peptide 1 (GLP-1) induces post-prandial glucose-dependent insulin secretion. Clinical trials showed that GLP-1 receptor agonists improve cardiovascular outcomes in patients with diabetes at high cardiovascular risk. We found elevated GLP-1 levels to be associated with cardiovascular mortality in patients with acute myocardial infarction. The aim of this study was to analyze whether adjustment of the SMART (Second Manifestations of Arterial Disease) risk score by GLP-1 provides significant added value in the prediction of cardiovascular mortality in patients with coronary artery disease.

Methods:

We measured circulating GLP-1 levels in 2326 patients with coronary artery disease who underwent coronary angiography at baseline (1997-2000) and are part of the Ludwigshafen Risk and Cardiovascular Health Study. The primary endpoint of our study was cardiovascular mortality.

Results:

Multivariable Cox regression analysis found GLP-1 levels to be independently associated with cardiovascular mortality in patients with coronary artery disease (logarithmized GLP-1 tertile HR: 1.31; 95%-CI: 1.08–1.60; p=0.007; Harrell’s C-index: 0.76; multivariable model adjusted for age, sex, diabetes, smoking, hypertension, previous cardiovascular disease, eGFR CKD-EPI, hsCRP, LDL cholesterol, hs-TroponinT and NT-proBNP). Variable’s importance in the multivariable model was analysed and illustrated, proving the substantial higher influence of GLP-1 compared to other cardiovascular risk predictors like hs-TroponinT, LDL cholesterol, hsCRP, eGFR CKD-EPI, smoking, age and sex. The SMART risk score is a european guideline recommended tool for 10-year cardiovascular risk assessment in patients with clinical manifest atherosclerotic vascular disease. GLP-1 provides significant added value on top of the SMART risk score in risk prediction of cardiovascular mortality (ΔChi2: 12.29; logarithmized GLP-1 tertile HR: 1.32; 95%-CI: 1.09–1.61; p=0.004; Harrell’s C-index: 0.72). Furthermore, addition of GLP-1 to the SMART risk score leads to a better reclassification for medium-term cardiovascular risk prediction (continuous NRI: events 35.1%, non-events -7.9%).

Conclusion:

GLP-1 is a strong cardiovascular risk marker providing significant added value (improvement in risk discrimination, calibration, and reclassification) on top of the SMART risk score in patients with coronary artery disease.

Background Cardiovascular disease continues to be the main cause of death worldwide, despite improved risk management and modern pharmacological therapies. Thus, novel therapeutic approaches have to be identified to reduce cardiovascular morbidity and mortality. Data from the last 2 decades have shown that inflammation plays an important role in cardiovascular disease. Next to the spleen and the bone marrow, the gut contains one of the largest immune cell reservoirs of our bodies. We could recently show that gut immune cells regulate energy metabolism and play an important role in atherosclerosis. However, the role of intestinal immune cells in heart failure is unknown.

Methods and Results To investigate the role of gut immune cells in heart failure we performed sham procedure or transaortic constriction (TAC) in C57BL/6J mice to induce pressure-overload induced cardiac hypertrophy and analyzed intestinal leukocytes by spectral flow cytometry (FACS). Mice with established heart failure (5 weeks after TAC surgery) had higher numbers of intestinal immune cells compared to mice without heart failure (intraepithelial space: 2.5-fold increase of αβ and γδ T cells, p<0.05; lamina propria: 10-fold increase of T cells, p=0.036, 2.6-fold increase of macrophages, p<0.001). To explore the functional role of activated gut immune cells in heart failure we performed TAC surgery in wild type (n=10) or Integrin-β7^–/– mice (n=11), which are selectively deficient for intestinal immune cells with normal leukocyte counts in other organs. Interestingly gut immune cell deficient Integrin-β7^–/– mice were protected against left ventricular dysfunction induced by TAC surgery [LV ejection fraction (EF-SAX): 58.9 ± 4.4% in sham WT, 35.3 ± 7.0% in TAC WT, 45.0 ± 9.4% in TAC β7^–/–, p<0.05; LV fractional shortening: 30.7 ± 3.1% in sham WT, 17.9 ± 4.6% in TAC WT, 23.1 ± 5.9% in TAC β7^–/–, p<0.05, analyzed by echocardiography]. We validated this finding in the UK Biobank proteomics subset (ID 71300) and found in 53030 patients a positive association of circulating Integrin-β7 with lifetime prevalence of heart failure (ICD code I50, p=0.016).

Conclusion Here we identified an interorgan-crosstalk network between the intestinal immune system and the myocardium in mice with heart failure. Future work is needed to investigate the underlying mechanisms. These findings suggest that gut immune cells might be a novel therapeutical target for heart failure.

BACKGROUND: Previous work showed that atypical chemokine receptor 3 (ACKR3) expression on endothelial cells mediated CXCL12-driven atherosclerosis while ACKR3-deficient haematopoietic cells decrease atherosclerosis in Apolipoprotein E-deficient (Apoe^-/-) mice. Notably, ACKR3 is highly expressed on B cells of the spleen and is essential in their differentiation into antibody-producing cells and their exit from the follicle of the spleen. While IgM-producing B1 B cells are well known to be atheroprotective, opposing data has been reported for the role of the larger population of B2 cells, which include plasma cells, in atheroprogression. Here, we aim to unravel the role of ACKR3 on B cells in atherosclerosis and hypothesised that its cell-specific loss of function will reduce the development of the disease.

METHODS: The role of ACKR3 expression is studied on an atherosclerotic mouse model (Apoe^-/-) with cell-specific ACKR3 loss of function on B cells (CD19^Cre+ ACKR3^fl/fl Apoe^-/-). These mice were fed either 4 (B cell-ACKR3 KO n=15, control n=20) or 12 weeks (B cell-ACKR3 KO n=17, control n=20) of Western Diet (WD) and atherosclerotic lesion characteristics, differential B cell subpopulations and plasma antibody titers were analysed. Statistical analysis was done using unpaired, two-sided t-test.

RESULTS: After 12 weeks of WD, mice lacking ACKR3 showed a significant decrease of aortic (p≥ 0,05) and root (p≥ 0,05) lesions. In addition, foam cells number (p≥ 0,05) and necrotic core size (p≥ 0,001) were significantly decreased after 12 weeks WD. The atheroprotective IgM content (p≥ 0,01) in the plaque was increased both after 4 and 12 weeks WD. Loss of B cell ACKR3 lead also to an increase of atheroprotective B1 cells in the blood (p≥ 0,001) and bone marrow (BM)(p≥ 0,01) compared with controls (ACKR3^fl/fl Apoe^-/-). On the other hand, atherogenic B2 cell numbers in the blood (p≥ 0,001) and BM (p≥ 0,01) as well as pro-atherogenic plasma cells in the BM (p≥ 0,05), lymph node (p≥ 0,01) and spleen (p≥ 0,001) were significantly reduced. Additionally, atherogenic plasma titers of IgG subclasses such as IgG1 (p≥ 0,001), IgG2a (p≥ 0,001) and IgG2b (p≥ 0,5) are decreased.

CONCLUSION: Our data suggest that cell-specific loss of ACKR3 on B cells has an atheroprotective role in atherosclerosis by decreasing pro-atherogenic B2 cells as well as plasma cells and pro-atherogenic antibody production while increasing atheropretective B1 cells and IgM antibodies leading to a reduction of foam cells and necrotic core area in the lesions. Further in vitro, we will investigate the role of B cell specific ACKR3 in both T cell-indepedente and T cell-dependent B cell activation as well as B cell migration towards chemoattractants. In addition, splenic B cell-ACKR3 deficiency is under investigation at a single cell level to unravel differential transcriptomics between the WT and KO mice under Western type Diet.

Background and Aims

Accumulation of oxLDL within the vessel wall represents the central driver of atherogenesis and concomitant vascular inflammation. CD36 is an immunoregulatory scavenger receptor at the interface of lipid metabolism and inflammation that has been implicated in macrophage lipid uptake and foam cell formation in vitro. However, due to the lack of cell-specific in vivo models the exact role of CD36 on macrophages in atherosclerosis remains not sufficiently defined to date. In this context we here aimed to clarify the role of macrophage expressed CD36 in atherogenesis.

Methods

To screen for a potential role of macrophage expressed CD36 in atherosclerosis we assessed CD36 gene expression across different leukocyte populations in scRNAseq data from atherosclerotic LDLR-/- mice. To evaluate the role of macrophage expressed CD36 in atherogenesis, we generated tamoxifen inducible CX3CR1-specific CD36-KO mice on LDLR-/- background. Finally, potential clinical significance of CD36 in human atherosclerosis was assessed in complementing clinical study cohorts.

Results

Analysis of murine scRNAseq data identified macrophages as the main CD36 expressing cell type. Notably, Macrophage-specific CD36 deficiency substantially reduced lesional necrotic core area indicating ameliorated plaque vulnerability. In vitro, macrophage specific CD36 deficiency reduced ROS production and oxLDL uptake. Similar to our observations in mice, cellular CD36 expression was highest in human macrophages/monocytes. Pathway analysis revealed that CD36-expressing macrophages exhibited a significantly more pro-inflammatory gene signature than CD36-negative macrophages. Notably, unstable carotid plaques displayed a substantially higher CD36 expression compared to stable lesions. Finally, lesional CD36 expression was significantly associated with a high severity of symptoms and the occurrence of major adverse cardiovascular events in patients undergoing carotid endarterectomy.

Conclusions

All in all, our data indicate a pro-inlammatory role of macrophage expressed CD36 in murine and human atherosclerotic lesions. Our findings suggest that targeting CD36 on macrophages may represent a potential target against lipid-driven inflammation in atherosclerosis.

Aim:
This study investigates the cardiovascular effects of celiac disease (CeD) in a humanized mouse model, focusing on vascular inflammation, endothelial dysfunction, and oxidative stress. This study aims to highlight CeD as a non-traditional risk factor for cardiovascular complications.

Methods:
Employing the NOD.DQ8 mouse model susceptible to CeD, we induced CeD characteristics through a gluten-enriched diet and oral gavage, followed by a 14-day gluten-free diet to evaluate recovery. Assessments included duodenal histology, cardiovascular function (echocardiography, blood pressure), vascular reactivity, and markers of systemic inflammation and oxidative stress in various tissues, including plasma proteome profiling.

Results:
Gluten exposure successfully induced CeD, evidenced by intestinal inflammation. The gluten group exhibited increased blood pressure, impaired vascular relaxation, upregulated pro-inflammatory genes, increased CD11b+ myeloid cell infiltration, and heightened oxidative stress in aortic and heart tissues. Heart function remained unaffected. Plasma proteomics suggested interleukin-17A (IL-17A) as a potential mediator between gut and vascular inflammation.

The gluten-free diet phase normalized inflammatory parameters and significantly improved vascular function. Notably, oxidative stress markers in the vascular endothelium decreased, indicating a reversal of vascular health.

Conclusion:
Our study underscores celiac disease as a non-traditional risk factor for cardiovascular disease, revealing a gut-to-cardiovascular inflammatory axis potentially mediated by IL-17A and immune cell infiltration. These findings augment our understanding of CeD's link to cardiovascular disease and highlight the critical role of dietary interventions in managing cardiovascular risks associated with CeD.

Background: Aging and a consequential sedentary lifestyle, as well as slowed metabolism, can result in obesity and the development of diabetes mellitus (T2DM). Around 7 million Germans currently have T2DM, with 35% of those patients suffering from cardiovascular disease. Diabetes-associated accumulation of cellular senescence promotes a chronically inflamed environment resulting in activation and recruitment of immune cells like neutrophils. One promising approach to minimize the senescent burden are senolytics, which inhibit selective survival pathways of senescent cells, driving them to apoptosis. Here we study the effects of chronic inflammation attenuated by diabetes and cellular senescence on cardiovascular disease. We hope our findings can eventually minimize the risk of major cardiovascular events in diabetic patients. Methods and Results: Mice aged 10-12 weeks were fed a high-caloric diet for 10 weeks to induce diet-related obesity. We then treated them with senolytics (a combination of dasatinib and quercetin) 3 times within a week to induce senolysis. To gauge the effects of senolytics within the heart, we performed qPCR of Cdkn1a levels (codes for p21, a common senescent cell marker) in lysed heart tissue. The results showed a significant reduction in p21-positive senescent cells within the heart. Additionally, we performed a Senescence-Associated-ß-Galactosidase staining (also a common marker for senescent cells) of frozen heart sections where we could corroborate the qPCR results. We focused on investigating the immune system and inflammation following senolytics-treatment. Hearts were perfused in an ex vivo Langendorff system and effluates collected, followed by determination of cytokine levels produced by cardiac cells using a multiplex immune assay. This showed a significant reduction in pro-inflammatory cytokine levels directly connected to neutrophil activation and migration. We also excised hearts of mice treated with senolytics or the control treatment and performed tissue digestion to define the resident and circulatory immune phenotype, where we showed significantly reduced levels of resident neutrophils with lower levels of reactive oxygen species. After performing single cell sequencing analyses, we saw that the neutrophils within senolytics-treated hearts were seemingly less inflammatory. Conclusions: The senolytics combination dasatinib and quercetin effectively removes senescent cells in the pre-diabetic heart. Reduced senescence leads to a significant downregulation of pro-inflammatory cytokines associated with neutrophil recruitment. Thus, we can assume that senolytics may have cardioprotective qualities by lessening chronic inflammation in diabetic patients.

Objective: Most atherosclerosis-prone blood vessels are surrounded by perivascular adipose tissue (PVAT) which is contiguous with adventitial layer of arteries. PVAT is a physiologically and metabolically active endocrine tissue secreting various biologically active factors. Morphological, structural, and functional alternations of PVAT have been observed in obesity and other cardiovascular disorders. The dysfunction of this tissue is affecting the pathogenesis of atherosclerosis due to the secretion of pro-inflammatory adipokines and infiltration of inflammatory cells. The focus here is on the adipose tissue macrophages (ATMs) in PVAT which are central players in obesity-associated inflammation and metabolic stress due to their capability of switching their phenotype from alternatively activated anti-inflammatory M2 to classically activated pro-inflammatory M1. The receptor ChemR23 which is expressed in a number of immune cells including myeloid DCs, natural killer (NK) cells as well as macrophages and its ligand chemerin whose expression and secretion increases dramatically with adipogenesis seem to play a crucial role in the phenotype switching of ATMs and thus in the crosstalk between PVAT and arteries during atherosclerosis.

Methods: To study the role of ChemR23 expression, we use an atherosclerotic mouse model with a systemic knockout of ChemR23 expressing eGFP (enhanced green fluorescent protein) (Apoe^-/- ChemR23-knockout/knockin mice). These mice are fed either a 4 or 12 weeks Western Diet (WD) and analysed for ATMs via FACS. PVAT will be analysed immunohistochemically and on transcriptomic level employing single nuclei RNA sequencing. Organs like spleen and lymph nodes will be analysed either histologically or at gene expression level. Using two Cre-lox mouse models with a cell-specific knockout of ChemR23 either on adipocytes or myeloid cells with an Apoe^-/- deficient background will enable us to examine the impact of ChemR23 on those cells and to better understand the adipocyte-macrophage interaction in the absence of the receptor.

Results: We found that the systemic knockout of ChemR23 decreased the number of macrophages in various adipose tissues including the pericardial and epididymal adipose tissues as well as in periaortic adipose tissue (PVAT) after 4 weeks of Western Diet. Furthermore, the number of classically activated M1 macrophages increased in adipose tissues of mice lacking the ChemR23 receptor fed a WD for 4 weeks.

Conclusion: Based on the previous data, we hypothesize that the loss of ChemR23 expression has an adverse effect on the phenotypic switching of adipose tissue macrophages in perivascular adipose tissue in hyperlipidaemic mice during atherosclerosis. Further investigations are planned for unravelling the transcriptome of the ATMs using the single-cell RNA sequencing.