Conference Agenda

Patients with chronic kidney disease (CKD) show a highly increased cardiovascular risk. Beyond a higher risk of myocardial infarction (MI), CKD patients also suffer from a reduced survival following MI, but the underlying mechanisms remain largely unclear. Here, we examined the impact of CKD on cardiac remodeling and function post-MI using a mouse model of adenine-induced CKD, with validation of main identified mediators in patients.

After MI, CKD mice showed a stronger cardiac dysfunction compared to non-CKD controls. While immunohistochemical and immunofluorescence analyses did not reveal changes in cardiomyocyte apoptosis, infarction size or myofibroblast content, we uncovered a disturbed cardiac tissue metabolism with impaired glycolysis, a reduced glycerol-3-phosphate shuttle and a shortage of the cellular energy metabolite Coenzyme A in CKD vs. non-CKD mice post-MI by integrating metabolomics and RNAseq data. Furthermore, CKD mice exhibited an increased amount of circulating myeloid cells post-infarction and an increased neutrophil infiltration in the heart, as shown by flow cytometry. Combining RNAseq, untargeted kinome profiling, western blotting and mass spectrometry revealed that post-MI, CKD enhanced the accumulation of the acute stress protein complex S100A8/A9 in circulation and the heart, and enforced MAP-kinase p38 activation and NR4A1 phosphorylation in the myocardium as pathways underlying cardiomyocyte dysfunction. S100A8/A9 also exerted a direct detrimental impact on calcium flux and sarcomere shortening in primary cardiomyocytes ex vivo. Increased myeloid cell-derived S100A8/A9 expression was confirmed in the infarcted human heart based on single nuclear RNAseq data, and patients with CKD were shown to present with higher post-infarction S100A8/A9 levels compared to patients without kidney dysfunction.

In summary, our study reveals innate immune activation, inflammatory and metabolic alterations to underlie worsened cardiac dysfunction post-MI in CKD vs. non-CKD conditions. This could contribute to the worsened outcome of CKD patients post-MI.

Background: Patients with chronic kidney disease (CKD) present with an increased cardiovascular risk. Chemokines play an important regulatory role in inflammation and cardiovascular disease but have been understudied in CKD.

Methods and results: In a chemokine profiling, we identified CCL15 as the strongest upregulated chemokine in CKD patients compared to healthy controls. Blood CCL15 levels inversely correlated with kidney filtration function and were associated with kidney interstitial fibrosis and tubular atrophy in kidney biopsies. Furthermore, CCL15 predicted future cardiovascular events in the NEFRONA study and was an independent predictor of decompensated heart failure and death in the CARE for HOMe study. In the UK population biobank, CCL15 also independently predicted heart failure risk in the general population. Single-cell RNAseq of human kidney biopsies indicated that CCL15 is expressed in tubules and injured endothelium and is upregulated in CKD. Mechanistically, CCL15 was able to induce fibrosis in human cardiac organoids and kidney pericytes beyond its effect on inflammatory cell recruitment, may contributing to disease progression and increased cardiovascular risk.

Conclusion: Overall, we identify CCL15 as the first chemokine biomarker predicting heart failure risk and mortality in CKD. CCL15 was able to drive fibrosis, and could thereby contribute to CKD progression and increased cardiovascular risk.

Background: Mitral valve insufficiency (MI) is among the most prevalent valvular heart diseases worldwide and represents a major cause of morbidity and mortality. Despite its clinical relevance, the molecular and cellular mechanisms underlying MI pathogenesis remain poorly understood. Current therapeutic strategies are largely limited to symptomatic treatment or surgical intervention, underscoring the urgent need for a deeper mechanistic understanding and the development of targeted therapies. One major limitation to advancing research in this field is the lack of an appropriate mouse model that faithfully recapitulates MI. In this study, with the genetic knock-out mouse model dzip1^S14R/+ we established and characterized a new model of MI to investigate the underlying mechanisms contributing to disease onset and progression.

Methods: For our study, male and female dzip1^S14R/+-mice (n=16) were used as well a wildtype(wt)-mice (n=4) as control group. From 12 weeks of age onward, echocardiography was performed in parasternal long axis, parasternal short axis, as well as apical four chamber view and suprasternal view on a weekly basis to monitor methodological effects and the temporal onset of a MI. Right before harvest, we additionally performed MRIs and PET-CTs. After harvesting the mice, autoradiography and gamma counting was performed to visualize MI effects. Hearts were embedded in paraffin, sliced in 4 um slices and the heart as well as the mitral valves leaflets were stained for calcification via von kossa staining and for structural analysis via HE-staining.

Results: Female dzip1^S14R/+-mice showed a MI between 19-20-week-old, ultrasound revealed mild regurgitation. Interestingly, male dzip1^S14R/+-mice showed a MI between 23-24-week-old with a mild regurgitation. Ejection fraction was higher in dzip1^S14R/+-mice compared to wt-mice, calcification of the valves could not be detected using PET-CT, autoradiography, gamma counting or von Kossa staining. The model appears to have a primarily degenerative component, as the E-to-A wave ratio was not significantly altered. Stainings revealed an alteration of the mitral valve leaflets, which appear to undergo a measurable thickening. Suprisingly, the aortic valve showed no significant difference in the dzip1^S14R/+-mice which already showed an MI.

Conclusions: Our study enabled the establishment of a novel MI models (dzip1^S14R/+-model) that reflects distinct aspects of the pathology. Nevertheless, the models requires thorough characterization following its initial development before it can be considered for use as standard model.

Cardiovascular disease (CVD) and cancer, the leading causes of death worldwide, share risk factors and appear to also interact pathomechanistically. Patients with chronic lymphocytic leukemia (CLL) have a 9% higher risk of developing CVD – however concise pathomechanisms remain poorly understood (Larsson et al., Br J Haematol 2020).

We here aim to investigate whether myocardial infarction (MI) might influence growth of TCL1-tg B cells, a B cell lymphoma model.

TCL1-tg cells were adoptively transferred five days prior MI induction by permanent ligation of the left descending anterior artery (LAD). Cardiac function was assessed biweekly by echocardiography. In parallel, CLL burden (CD5+CD19+ cells) and immune cell changes in the blood were determined by flow cytometry (FACS), using a pan leukocyte panel and markers of exhaustion, memory and proliferation. Once mice reached termination criteria, bone marrow, spleen, lymph nodes, heart, and blood were collected for FACS and histological analysis.

Echocardiographic and histological analyses confirmed the successful induction of MI. By FACS, accelerated tumor growth was observed in mice with MI (week 6: 38.73 ± 17.78%) vs. sham (23.19 ± 17.73%). In addition we observed increased frequencies of Tregs and Ly6Chi monocytes in blood and spleen upon harvest (week 8). Both cell types are well-established contributors to tumor progression. We further observed more exhausted T and NKT cells, as well as a trend toward more Tregs in the tumor microenvironment (TME) of MI mice. Future directions will unravel how MI impacts on the observed immune cell changes in the TME.

Clinical data suggests that cardiovascular diseases, including myocardial infarction

(MI), increase the risk to develop different malignancies. Patients with MI have a 46%

increased Hazard Ratio to develop cancer compared to those without (Rinde, 2017).

First interfaces of cross communication in other tumor entities, like breast cancer,

point to immune cell reprogramming (Koelwyn, 2020), the mechanistic crosstalk

underlying MI-accelerated melanoma progression remains uncharacterized.

We here aim to investigate the impact of MI on melanoma growth in mice. C57BL/6J

mice (10 to 14-week-old, male and female) underwent LAD-Ligation to induce MI or

sham surgery (thoracotomy only). B16F10 melanoma cells were injected

subcutaneously at day -3 or day 7 of surgery. Tumor size was determined by

micrometer calliper daily, heart function was analysed by echocardiography once

weekly. At the end of the experiment (day 15 post B16F10 injection), immune cell

changes of tumor tissue, lymph nodes, blood and spleen were determined by

spectral flow cytometry and heart tissue was used for histological analysis.

Our results demonstrate that MI accelerates melanoma growth in mice. Flow

cytometry analysis of circulating immune cells in blood revealed a systemic

reprogramming, characterized by an increase in pro inflammatory Ly6Chigh CCR2+

monocytes and a decrease in CD4+ T cells and Tregs (FoxP3+CD25+) in MI affected

mice vs. controls. Future experiments will reveal the role of these altered immune cell

subsets for acceleration of tumor growth.

Myocardial infarction (MI) triggers a systemic inflammatory response, mainly sustained by emergency hematopoiesis in the bone marrow (BM). While it is well established that hematopoietic progenitors and precursors react to MI by increasing leukocyte production, it remains elusive whether immune cell phenotypes also change during the inflammatory response.

We performed single-cell RNA sequencing (scRNA-seq) on BM leukocytes from C57BL/6 mice at steady state and at 12-, 24-, 48-, and 72-hours post-MI (permanent ligation of the left ascending coronary artery). This analysis identified an expansion/the occurrence of a distinct subpopulation of Ly6Chigh monocytes in response to infarction. In particular, this population expressed genes typically associated with neutrophils, including S100a9, S100a8, Lcn2, and Camp. Thus, this subpopulation was classified as “neutrophil-like monocytes” (Neu-Mo). We validated Neu-Mo dynamics over time in the BM, blood, and heart of infarcted mice using flow cytometry and identified this population as S100A9highMHCIInegLy6Chigh monocytes. Our analysis indicates that Neu-Mo represent a small monocyte subpopulation (6.98±2.92% of circulating monocytes). Following MI, Neu-Mo numbers start to rise first in the BM, then in the blood and ultimately in the heart at 48 hours post-infarction (15.36±9.03 cells/mg). These Neu-Mo kinetics were further confirmed using S100a9EGFP/+ transgenic mice, which allow tracking of Neu-Mo post-MI.

We next investigated potential blood-borne factors driving Neu-Mo production. In-vitro experiments identified interleukin 6 (IL-6) as a primary inducer of Neu-Mo production. Wild-type mice injected with IL-6 confirmed Neu-Mo expansion in the BM (15,093±14,359 cells/femur vs. 98,025±57,775 cells/femur). Conversely, experiments in Il6-/- mice demonstrated a significant reduction in Neu-Mo accumulation in the ischemic area 48 hours after MI (21.66±10.29 cells/mg vs. 4.139±2.382 cells/mg). To explore whether MI induces phenotypic shifts in monocyte precursors, we performed RNA-seq on sorted BM monocyte precursors at steady state and 12 hours post-MI. Monocyte precursors upregulate neutrophil-related genes such as Lcn2 and S100a9 in response to MI, indicating that Neu-Mo expansion is a direct consequence of phenotypic changes in upstream monocyte precursors.

Our findings identify a previously uncharacterized monocyte subpopulation that significantly contributes to the inflammatory response post-MI, highlighting the active role of the BM in modulating leukocyte phenotypes. Rather than representing a simple acceleration of baseline hematopoiesis, emergency hematopoiesis following MI involves a more specialized and branched hematopoietic process. This process facilitates the expansion of distinct subpopulations, such as Neu-Mo cells, which do not arise during typical hematopoiesis. Additionally, we demonstrate the critical role of IL-6 in regulating the heart-BM axis to promote Neu-Mo production. Ongoing research aims to elucidate the specific functions of Neu-Mo in the context of MI.