Veranstaltungsprogramm

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.