Veranstaltungsprogramm

Background: The abdominal aortic aneurysm (AAA) is a multifactorial disease with a high prevalence in individuals over 65 years of age, associated with significant mortality and morbidity. There is no specific pharmacological approach that directly targets AAA, and surgery remains the only treatment option. Matrix metalloproteinases (MMPs) constitute a family of enzymes capable of degrading nearly all components of the extracellular matrix (ECM). Among them, the secreted collagenase MMP13 is one of the few enzymes able to cleave the intact triple helix of collagen types I, II, and III. These collagens represent the most abundant structural proteins in the human body and collagenases play a pivotal role in maintaining matrix homeostasis. Owing to its markedly higher enzymatic activity compared to other MMPs, MMP13 is considered substantially more efficient and is thought to be a major contributor to the pathogenesis of AAA, thereby representing a potential therapeutic target. In this study, we explored MMP13 as a potential target for pharmacological inhibition for AAA in mice.

Methods: Four groups were established: An untreated wildtype (wt)-group with AAA; a wt-group receiving MMP13 inhibitor injections every second day starting three days before aneurysm induction (prophylactic); a wt-group receiving daily MMP13 inhibitor injections every second day starting three days after aneurysm induction (therapeutic); and a MMP13^-/-- group. AAA was induced in wt-mice as wells as in MMP13^-/--mice via surgery. The abdominal aorta was ligated, blood flow interrupted and filled with porcine-pancreas-elastase (PPE) for five minutes. Mice were harvested on day 6 after PPE to assess acute inflammation or on day 28 after PPE for tissue remodeling and fibrosis. Ultrasounds were performed to monitor surgery effects, aneurysm induction and growth. Snap-frozen aortas were embedded for histological analysis and stained for collagen, elastin, decorin, alpha-SMA, MMP activity and CD45/CD68.

Results: In both the prophylactic and therapeutic settings, the AAA diameter in mice with 28 days of MMP13-inhibiton was not different from MMP13^-/--mice (p=0.289; p=0.877). MMP13-inhibited groups and MMP13^-/--mice showed significantly smaller (p=0.039; p=0.009; p=0.007) AAA diameter compared to the control group. In the both settings as well as in MMP13^-/--mice, picro sirius staining revealed a higher amount of collagen as well as elastic fibers in aortic tissue when compared to the control group. Likewise, quantification of decorin and alpha-SMA showed increased levels of these structural proteins in both experimental settings and in MMP13^-/--mice relative to the control groups. Also, in both settings as well as in MMP13^-/--mice, we could observe less immune cell recruiting and less MMP-activity compared to the control group.

Conclusions: MMP13 as a potential target for pharmacological inhibition for AAA in mice shows a significant effect on diameter growth. Also, MMP13 inibition shows similar effectiveness as a knockout of MMP13 gene in mice. Stainings revealed that MMP13-Knockout as well as an inhibition stabilizes the aortic wall in AAA disease and lowers immune cell migration. Nevertheless, the pharmacological inhibition of MMP13 requires thorough characterization.

Atherosclerosis, the leading cause of cardiovascular disease, progresses from fatty streaks to unstable rupture-prone plaques that trigger thrombotic events. A hallmark of plaque instability is intraplaque angiogenesis (IPA), the growth of leaky neovessels from the adventitial vasa vasora, that promote intraplaque hemorrhage (IPH) and inflammation. While hypoxia is a known trigger, additional pro-angiogenic mechanisms remain unclear. As an age-related disease, atherosclerosis is characterized by the accumulation of senescent vascular smooth muscle cells (VSMCs), which are metabolically active and adopt a senescence-associated secretory phenotype (SASP) that promotes chronic inflammation and tissue remodeling.

Despite growing recognition of the detrimental role of senescent VSMCs in atherosclerosis and their spatial proximity to the vasa vasora, whether their SASP directly drives IPA and IPH has remained unexplored. Here, we investigated the link between VSMC senescence, SASP production, and angiogenesis, and assessed whether targeting SASP-related pathways could provide novel therapeutic opportunities.

Re-analysis of a scRNASeq dataset of human carotid atherosclerosis revealed a distinct population of senescent VSMCs within atherosclerotic lesions, identified by high expression of a 24-genes senescence signature. Moreover, gene ontology and gene set enrichment analyses further showed that these senescent VSMCs overexpress genes that are associated with angiogenesis. Importantly, immunohistochemical analysis confirmed that senescent VSMCs are significantly more abundant in unstable human carotid plaques than in stable ones.

To investigate their functional impact, we established two in vitro models of senescent VSMCs, replicative and doxorubicin-induced, and found that both models secreted significant higher levels of pro-angiogenic molecules, including VEGFA, compared to young control cells. Conditioned media from senescent VSMCs enhanced endothelial proliferation, migration, and tube formation in HUVECs.

Mechanistically, pharmacological inhibition of VEGFR2 in HUVECs abrogated these pro-angiogenic effects, identifying the VEGFA/VEGFR2 axis as a central mediator of VSMCs senescence-driven angiogenesis.

Our findings demonstrate that VSMC senescence actively contributes to plaque progression and instability through a strong pro-angiogenic SASP secretome. Targeting vascular senescence and the VEGFA/VEGFR2 pathway emerges as a promising strategy to inhibit intraplaque angiogenesis and stabilize high-risk atherosclerotic lesions.

Rationale: Aortic valve stenosis (AVS) and coronary artery disease (CAD) are highly prevalent comorbidity in elderly populations. Accumulation of atherosclerotic plaques triggers chronic inflammation and leads to progressive calcification in AVS. With transcatheter aortic valve replacement (TAVR) as the sole effective treatment, the urgent need for novel therapeutic targets is apparent. Long non-coding RNAs (lncRNAs) are crucial epigenetic regulators, yet their roles in AVS and CAD remain largely unexplored. Our preliminary transcriptomic data from calcified human aortic valves identified a significant upregulation of MIAT and MEG3, lncRNAs implicated in fibrosis and endothelial dysfunction. This study investigates their specific contribution to AVS and CAD pathogenesis to identify new RNA-based diagnostic and therapeutic strategies.

Methods and Results: Using primary human valvular interstitial (VICs) and endothelial cells (VECs) from calcified and non-calcified valves, as well as human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC), we performed siRNA-mediated knockdown of MIAT and MEG3. Silencing either lncRNA reduced cellular senescence and cytotoxicity without affecting viability. Functional assays revealed that knockdown attenuated endothelial cell proliferation and migration, while simultaneously affecting its angiogenic capacity. Upon oxLDL and TNFα stimulation on HCAEC, siMIAT and siMEG3 decreased the expression of pro-inflammatory genes. To model AVS pathogenesis, we induced endothelial-to-mesenchymal transition (EndMT) in VECs and calcification in VICs. siMIAT and siMEG3 potently suppressed key EndMT markers (ACTA2, TAGLN, COL1A1) in VECs and downregulated osteogenic drivers (BMP2, RUNX2, BGLAP) in VICs, indicating a profound amelioration of core disease phenotypes. Furthermore, intercellular communication studies via extracellular vesicles (EVs) and co-culture models demonstrated that silencing MIAT or MEG3 in donor VECs significantly reduced inflammatory markers (IL-6, TGF-β, ICAM-1, VCAM-1) in recipient VICs. An integrated in silico and proteomic approach identified caspase-3, p53, and HIF1α as potential downstream effectors mediating these lncRNA-driven inflammatory and calcification pathways.

Conclusion and Outlook: Our findings establish MIAT and MEG3 as critical promoters of calcification and EndMT in AVS, as well as drivers of cellular inflammation in CAD. Their silencing restores endothelial integrity and mitigates osteogenic differentiation, positioning them as promising therapeutic targets. Future work will employ RNA immunoprecipitation sequencing (RIP-seq) and pulldown assays to delineate the precise molecular mechanisms, supported by validation in valvular organoids and murine models to translate these findings towards clinical application.

Aim

Atherosclerosis is shaped by innate and adaptive immune responses against Apolipoprotein B-100 (ApoB) and low-density lipoprotein (LDL) cholesterol. A growing body of evidence has identified the microbiome as a fundamental modulator of immune cell maturation and -function. Here, we describe the generation of an ApoE-knockout-wildling mouse model with a C57BL/6 genetic background engrafted with a natural microbiome derived from wild mice (“wildling-mice”).

Methods

ApoE-/- mice with a wildling-microbiome (Wild-ApoE-/-) mice were generated by a fostering model, involving the transfer of newborn ApoE-/- mice from a specific-pathogen-free (SPF) facility to wildling mice. Wild-ApoE-/- and conventional SPF-ApoE-/- mice were subsequently fed a western diet for 16 weeks and assessed for their metabolic and inflammatory phenotypes by single-cell RNA Sequencing, histology, flow cytometry, and plasma metabolomics.

Results

The presence of a natural gut microbiome in Wild-ApoE-/- mice resulted in a more human-like immune phenotype with evidence of chronic immune activation compared to SPF-ApoE-/- controls. Wild-ApoE-/- mice gained significantly less weight compared to SPF-ApoE-/- mice, displayed reduced blood fasting glucose, and revealed a wide range of differences in circulating plasma metabolites. Moreover, a more natural gut microbiome resulted in a smaller atherosclerotic lesion size in the aortic root and features associated with a more stable plaque phenotype, including reduced plaque lipid deposition, an increased lesional collagen content, and smaller necrotic cores. Notably, immune and metabolic phenotypes remained stable across several generations of animals.

Conclusion

The natural microbiome potently modulates immune cell phenotypes and predisposition to cardiometabolic diseases, including atherosclerosis. The use of Wild-ApoE-/- mice may improve modeling of adult human immune responses and accuracy of therapeutic outcomes in preclinical atherosclerosis studies.

Medial smooth muscle cell depletion is a characteristic feature of aortic aneurysms, with necroptosis-mediated by RIPK3 and MLKL-potentially driving SMC death, the release of DAMPs, and inflammation. Even though the role of MLKL induced-necroptosis is well understood in several diseases, its role in development of aortic aneurysm remains largely uncertain. In this study, we monitored PPE-perfusion induced progression of AAA in C57BL/6N (WT) and MLKL knockout (Mlkl-/-) mice by ultrasound measurements, histological analyses and bulk mRNA sequencing to assess structural and molecular aortic changes. Additionally, we investigated the therapeutic potential of a new MLKL inhibitor for AAA. Ultrasound analysis showed that ~70% of the WT animals developed PPE induced-AAA with significant aortic structural alterations and enhanced myeloid cell infiltration. In contrast, Mlkl-/- mice were protected from AAA. This protection was associated with reduced adverse extracellular matrix (ECM) remodeling and leukocyte infiltration. MLKL deficiency was associated with a significant downregulation of genes involved in fibrinolysis, anti-inflammatory response, immune response and complement activation in aortic tissue in AAA. We also show that the WT animals treated with MLKL inhibitor were protected from the PPE induced aneurysm formation. Overall, these findings indicate that MLKL-induced necroptotic SMC death and subsequent proinflammatory cytokine and DAMP release might play a causative role in AAA development. We also show that pharmacological inhibition of MLKL represent a promising treatment strategy for AAA disease.

Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease characterized by chronic inflammation and immune cell infiltration. T cells are among the predominant immune cell types in AAA and play an important role in the inflammatory response. Moreover, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial and disease-causing immune response. This study aimed to provide a detailed analysis of T cell subsets and their functions in AAA formation and progression.

We performed single-cell RNA-sequencing and single-cell T cell receptor-sequencing of murine AAA at day 3, 7, 14 and 28 after elastase-perfusion and compared these to saline-perfused and non-perfused control aortae. Differentially-expressed genes were identified for each cluster to identify the different subpopulations and their distribution over AAA progression. Gene ontology, signaling pathway activities and cell-cell communication were analyzed to gain insights into the functional roles of the different lymphocyte subsets. In addition, clonality of T cell receptors was examined.

Natural killer (NK) cells, NKT cells, innate lymphoid cells (ILC) type 2 and 3, Cd4+ T cells, Cd8+ T cells, pro-inflammatory T cells and regulatory T cells (Treg) were identified in elastase-induced AAA. NK and NKT cells together constituted the majority of lymphocytes at all stages of AAA formation. While they peaked at day 3 and decreased with AAA progression, ILCs and Cd4+ T cells increased with AAA progression and peaked at day 28. Cd4+ T helper cells represented the largest T cell subset in AAA and were associated with T cell activation and differentiation as well as leukocyte cell-cell adhesion. They were predicted to strongly communicate with dendritic cells and macrophages via CD40-CD40L signaling, thus driving inflammation. Cd8+ T cells expressed genes associated with a memory phenotype and were mainly involved in lymphocyte differentiation. Tregs represented only a small subset in AAA and were relatively more abundant in controls. Furthermore, clonal-expanded T cells were detected in 11 of 16 AAA samples, but only in one control sample. The number of expanded clones ranged from 2 to 25 copies.

This study provides a detailed characterization of lymphocytes and particularly T cells in experimental AAA formation and progression and highlights a central role of T cells in the inflammatory response. Evidence of clonal expansion of T cells was found, supporting the notion that specific antigen-driven T cells facilitate AAA formation.

Background:

Regular physical activity (PA) and structured exercise are associated with a reduced incidence and mortality of cardiovascular disease (CVD). Accordingly, current guidelines recommend at least 150 minutes of moderate-intensity or 75 minutes of vigorous-intensity PA per week (or equivalent combinations). Nevertheless, approximately one-third of the global population fails to meet these recommendations, with physical inactivity continuing to rise. From a pathophysiological perspective, sedentary behavior promotes chronic low-grade systemic inflammation, thereby accelerating the development and progression of inflammatory diseases of the vessel wall, ultimately leading to atherosclerosis.

Methods:

To investigate immune adaptations induced by PA, C57BL/6 mice were subjected to six weeks of voluntary wheel running or maintained under sedentary conditions. Circulating leukocyte subsets and bone marrow hematopoiesis were assessed by flow cytometry. To determine whether PA induces leukocyte-intrinsic changes affecting vascular recruitment, adoptive transfer experiments were performed using FACS-sorted monocytes and neutrophils from physically active or sedentary donor mice transferred into ApoE⁻/⁻ recipient mice. Aortic recruitment and peripheral blood distribution were quantified 36 hours post-transfer.

Results:

Voluntary PA significantly reduced circulating leukocyte counts, including neutrophils, B cells, T cells, and Ly6C^hi monocytes, and induced distinct alterations in bone marrow leukocyte composition. In adoptive transfer experiments, monocytes and neutrophils derived from physically active donors exhibited markedly reduced recruitment to atherosclerotic vascular sites compared with cells from sedentary donors. Importantly, this effect was independent of the recipients’ activity status, indicating a leukocyte-intrinsic reprogramming induced by physical activity.

Translational Perspective:

To translate these findings to humans, the ISAR Inflex Trial evaluates the effects of different exercise intensities and durations on total leukocyte counts, subsets and systemic inflammation in a randomized cross-over design. Thirty-three sedentary, overweight but otherwise healthy adults undergo moderate-intensity continuous exercise (MICEx), vigorous-intensity continuous exercise (VICEx), and short maximal exercise bouts (SMEx). Comprehensive immunophenotyping, plasma proteomics, and transcriptomic profiling are performed before, during and after each intervention to delineate intensity- and duration-dependent immune responses.

Conclusions:

Physical activity induces systemic and leukocyte-intrinsic immune adaptations that attenuate inflammatory leukocyte recruitment in cardiovascular disease. Ongoing translational studies will clarify how exercise intensity and duration shape immune cell function in humans, supporting physical activity as a targeted, non-pharmacological immunomodulatory strategy in CVD.