Murine Abdominal Aortic Aneurysm Research Articles

Abstract 4137500: The Histone Methyltransferase MLL1 Regulates Notch Signaling and T Cell Phenotype During Pathologic Abdominal Aortic Aneurysm Development

Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T H cells (Tregs) and pro-inflammatory T H 17 cells within the aortic wall. The mechanisms regulating CD4 + T H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T H cell phenotype by directly altering key transcription factors for T H 17 (RORγ) and Treg (FOXp3) differentiation in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T H 17 activity within AAA development and to determine key T H 17 cell signaling pathways that are altered Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3). Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4+ cells compared to non-aneurysmal control samples. Congruently, CD4 + T H cells isolated from the murine AngII-induced AAA model displayed a predominance of T H 17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4+ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4 + T H cell Notch signaling ( Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p<0.05) with decreased CD4+ T H 17 cell activity and reciprocal increase in Treg expression. Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.

Activation of nuclear receptor pregnane-X-receptor protects against abdominal aortic aneurysm by inhibiting oxidative stress

Abdominal aortic aneurysm (AAA) is a life-threatening condition, but effective medications to prevent its progression and rupture are currently lacking. The nuclear receptor pregnane-X-receptor (PXR) plays a crucial role in vascular homeostasis. However, the role of PXR in AAA development remains unknown. We first detected the PXR expression in human and murine AAA tissues by RT-qPCR and Western blot. To investigate the potential role of PXR in the development of AAA, we used adeno-associated virus-mediated overexpression of PXR and pharmacological activation of PXR by ginkgolide A (GA) in mouse AAA models induced by both angiotensin II (AngII) and calcium phosphate [Ca3(PO4)2]. The underlying mechanism was further explored using RNA-sequencing and molecular biological analyses. We found a significant decrease in both mRNA and protein levels of PXR in both human and murine aortic smooth muscle cells from AAA tissues, accompanied with phenotypic switching of vascular smooth muscle cell and increased oxidative stress. PXR overexpression in abdominal aortas and GA treatment successfully suppressed AAA formation in both mouse AAA models. RNA-sequencing data revealed that PXR activation inhibited gamma-aminobutyric acid type A receptor subunit alpha3 (GABRA3) expression. Additional mechanistic studies identified that PXR suppressed AAA through mitigating GABRA3-induced reactive oxygen species (ROS) generation and subsequent phosphorylation of c-Jun N-terminal kinase (JNK). Interestingly, p-JNK was found to induce ubiquitin-proteasome degradation of PXR. In summary, our data unveiled, for the first time, the protective role of PXR against AAA pathogenesis by inhibiting oxidative stress. These findings suggested PXR as a promising therapeutic target for AAA.

Single-Cell RNA Sequencing Deconstructs the Distribution of Immune Cells Within Abdominal Aortic Aneurysms in Mice.

Inflammation is a key component in the development of abdominal aortic aneurysm (AAA), yet insights into the roles of immune cells and their interactions in this process are limited. Using single-cell RNA transcriptomic analysis, we deconstructed the CD45+ cell population in elastase-induced murine AAA at the single-cell level. We isolated each group of immune cells from murine AAA tissue at different time points and divided them into several subtypes, listed the remarkable differentially expressed genes, explored the developmental trajectories of immune cells, and demonstrated the interactions among them. Our findings reveal significant differences in several immune cell subsets, including macrophages, dendritic cells, and T cells, within the AAA microenvironment compared with the normal aorta. Especially, conventional dendritic cell type 1 exclusively existed in the AAA tissue rather than the normal aortas. Via CellChat analysis, we identified several intercellular communication pathways like visfatin, which targets monocyte differentiation and neutrophil extracellular trap-mediated interaction between neutrophils and dendritic cells, which might contribute to AAA development. Some of these pathways were validated in human AAA. Despite the absence of external pathogenic stimuli, AAA tissues develop a complex inflammatory microenvironment involving numerous immune cells. In-depth studies of the inflammatory network shall provide new strategies for patients with AAA.

NINJ1 Facilitates Abdominal Aortic Aneurysm Formation via Blocking TLR4-ANXA2 Interaction and Enhancing Macrophage Infiltration.

Abdominal aortic aneurysm (AAA) is a common and potentially life-threatening condition. Chronic aortic inflammation is closely associated with the pathogenesis of AAA. Nerve injury-induced protein 1 (NINJ1) is increasingly acknowledged as a significant regulator of the inflammatory process. However, the precise involvement of NINJ1 in AAA formation remains largely unexplored. The present study finds that the expression level of NINJ1 is elevated, along with the specific expression level in macrophages within human and angiotensin II (Ang II)-induced murine AAA lesions. Furthermore, Ninj1flox/flox and Ninj1flox/floxLyz2-Cre mice on an ApoE-/- background are generated, and macrophage NINJ1 deficiency inhibits AAA formation and reduces macrophage infiltration in mice infused with Ang II. Consistently, in vitro suppressing the expression level of NINJ1 in macrophages significantly restricts macrophage adhesion and migration, while attenuating macrophage pro-inflammatory responses. Bulk RNA-sequencing and pathway analysis uncover that NINJ1 can modulate macrophage infiltration through the TLR4/NF-κB/CCR2 signaling pathway. Protein-protein interaction analysis indicates that NINJ1 can activate TLR4 by competitively binding with ANXA2, an inhibitory interacting protein of TLR4. These findings reveal that NINJ1 can modulate AAA formation by promoting macrophage infiltration and pro-inflammatory responses, highlighting the potential of NINJ1 as a therapeutic target for AAA.

Abstract 3002: Pcsk9 In Abdominal Aortic Aneurysm - Beyond Hypercholesterolemia

Background: Our lab has identified a role for proprotein convertase subtilisin/kexin type9 (Pcsk9) in murine abdominal aortic aneurysm (AAA). Beyond its pathological effects on degradation of LDL-receptors, and enhancement of tissue inflammation via the NF-kB pathway, reports suggest that it alters vascular smooth muscle cell (VSMC) senescence and metabolism. However, the specific cellular mechanisms connecting Pcsk9 to AAA disease (particularly SMC-derived Pcsk9) are still unclear. Hypothesis: Intercellular communication between VSMCs and macrophages via enhanced local Pcsk9 expression drives macrophages towards pro-inflammatory polarization and promotes AAA growth by upregulated aortic wall inflammation. Pcsk9 also enhances mitochondrial metabolism in VSMCs, contributing to cell senescence. Pcsk9 knockdown inhibits these effects. Aims: We explored the impacts of SMC-derived Pcsk9 on aortic inflammation and VSMC behavior, seeking to further understand AAA pathophysiology and provide a basis for re-purposing Pcsk9 inhibitors as a treatment for non-surgical AAA. Methods: Single-cell RNASeq of murine AAA tissue (porcine pancreatic elastase model) utilizing control and Pcsk9-knockout mice (systemic) was performed to examine local changes in aortic wall cell-subtype gene expression. Pcsk9 silenced VSMCs (with siRNA) were co-cultured with macrophages to assess polarization towards pro-inflammatory phenotype and pro-inflammatory gene/protein expression was identified. VSMCs were also treated either with exogenous Pcsk9 or Pcsk9 siRNA in vitro to assess mitochondrial metabolism via Seahorse assay. Results: Pcsk9 knock-out AAA tissue exhibited reductions in pro-inflammatory gene expression profiles in vascular cell sub-populations (compared to wildtype). In vitro, Pcsk9-silenced VSMC-cocultured macrophages presented decreased inflammatory profile assessed by flow cytometry. Further, exogenous Pcsk9-treated-VSMCs displayed metabolic changes compared to control (Mann-Whitney Test). Conclusions: Our data suggest significant mechanistic involvement of Pcsk9 in AAA disease at the cellular level. Absence of Pcsk9 appears to mitigate vascular inflammation and alter VSMC metabolism in ways that may decelerate AAA.

Abstract 3010: Inhibition Of Endothelial Cell-Mediated Efferocytosis Promotes Abdominal Aortic Aneurysm Formation

Introduction: Aortic wall inflammation and vascular smooth muscle (VSMC) apoptosis can lead to accumulation of apoptotic cells and debris during abdominal aortic aneurysm (AAA) formation. We hypothesize that dysregulation of MerTK receptor-mediated efferocytosis (clearance of apoptotic cell debris) by endothelial cells (ECs) can significantly contribute to aortic inflammation and vascular remodeling in AAA pathogenesis. Methods: Single-cell RNA sequencing of human AAA tissue from a published dataset (PMID: 33779682) was evaluated for cells of the aortic wall (VSMC/EC) identified via SingleR and cell-specific marker s. Differentially expressed genes (DEGs) were identified via edgeR using the QL F-test and efferocytosis-related genes (ERGs) were recognized using a list of 141 ERGs curated from GeneCards. MerTK expression was assessed in human aortic tissue and ECs via western blot and immunofluorescence. PKH67-labelled apoptotic VSMCs were overlayed on ECs to assess efferocytosis activity and visualized via confocal microscopy. In a murine elastase AAA model, endothelial-specific MerTK knockout ( Cdh5 Cre-ERT2 / MerTK fl/fl ) and littermate male mice were analyzed on postoperative day 14 for aortic diameter. Statistical analyses were performed by Welch’s T-test and p<0.05 was considered statistically significant. Results: ERGs (ANXA2, PHACTR1, PPARG, ADAM9, CD14, IL6) were differentially expressed (p<0.05, log 2 FC > |1|) between aortic wall cells of human AAAs and control. Human AAA tissue displayed a significant increase in MerTK protein compared to controls (1±0.1 vs. 1.51±0.4, n=6/group, p=0.01). The expression of MerTK in aortic ECs was confirmed by western blot and immunofluorescence(n=3). Pharmacologic inhibition of MerTK using UNC5293, a specific MerTK inhibitor, attenuated EC-mediated efferocytosis of apoptotic VSMCs (qualitative data; 10 images/group n=4). Importantly, EC-specific MerTK -/- mice had a significant increase in aortic diameter compared to littermate controls (155±40.4 vs. 103±30.9%, n=5-6/group, p=0.04) on day 14. Conclusion: EC-mediated efferocytosis can be regulated by MerTK to mediate AAA formation. Ongoing studies will delineate mechanisms of EC-MerTK signaling in AAA formation.

Abstract 1006: Pathological PERK/ATF4 Activation In Vascular Smooth Muscle Cells Drives Abdominal Aortic Aneurysm Development

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). Mice with vascular smooth muscle cell-specific knockout of PERK (PERK SMKO ) underwent tamoxifen injection. AAA maximum diameters were quantified and VSMCs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. Both In vivo pharmacological inhibition of the PERK/eIF2A/ATF4 ER stress response via 4-PBA injection and vascular smooth muscle depletion of PERK (PERK SMKO ) inhibited AAA formation resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the PERK/eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

Abstract 3005: Transgenerational Impact Of E-cigarette Nicotine Vaping On Abdominal Aortic Aneurysm

Background: Smoking and family history are major risk factors for abdominal aortic aneurysm (AAA). Smoking is a powerful modulator of DNA methylation, and nicotine can cause heritable epigenetic alterations in animals and humans. We found that nicotine infusion and e-cigarette (e-cig) vaping augment murine AAA, and that parental nicotine infusion augments model AAA in offspring, altering tissue DNA methylation patterns. Hypothesis: We investigated the effects of maternal e-cig vaping in mice on their offspring’s experimental AAA risk, and transgenerational DNA methylation. Methods: Two treatment arms were employed: 1) pre-fertilization or 2) peri-natal. Female ApoE-/- or C57BL6 mice (F0) were exposed to e-cig nicotine (24 mg/ml) puffs, 9 sec/min, 1 hour/day (or room air). Arm 1: treated 28 days, then mated to untreated controls. Arm 2: mated after 1 week of e-cig, then maintained on treatment until gestation was complete. Aortic tissue of 10-week-old F1 offspring was evaluated using RRBS-Seq DNA methylation analysis. Parallel offspring underwent angiotensin-II infusion (1μg/kg/min; ApoE-/-) or elastase infusion (2U/ml for 5 min at 120 mmHg; C57) to induce AAA. Aneurysm growth was ultrasound tracked over 28 days. Results: Maternal vape exposure altered aortic DNA methylation in offspring, with numerous differentially methylated regions (DMRs; q<0.1). Hundreds of these DMRs were located in the same genes previously identified in offspring of nicotine-infused mice (see Background). Vaping altered offspring aortic methylation patterns in AAA-related genes, including miRNAs. DMR pathway analysis was enriched in transcription factors. Further, many DMRs were located in genes harboring risk loci for human AAA from our recent collaborative publication. Maternal e-cig exposure augmented AAA size in both models, arms, and genders (p<0.05). AAA incidence and mortality were increased in ApoE-/- in both genders and arms. Effects were most significant in males. Conclusions: E-cig nicotine exposure augments experimental AAA growth in multiple models across generations. These effects are accompanied by broad aortic epigenetic changes, including in key AAA modulator genes, with enrichment in transcription factors that target known AAA-related genes.

Glycoprotein VI Platelet Receptor Blockade Attenuates Progression of Established Abdominal Aortic Aneurysms in Murine Models

Objective: Abdominal aortic aneurysms (AAA) frequently feature the formation of a nonocclusive intraluminal thrombus (ILT) along the site of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation via the glycoprotein VI receptor pathway impacts the pathogenesis of AAA. Methods and Results: RNA sequencing was performed on age-matched control human aortic tissue, AAA aortic tissue, and AAA ILT. Platelet transcripts comprised 25% of significantly enriched genes in the comparison of AAA thrombus to AAA tissue and control aortic wall, with GPVI increased by 9.1 Log2-FC (P = 3.21E−08). Circulating GPVI was significantly elevated in platelets isolated from AAA patients versus age-matched controls. Soluble GPVI (sGPVI—indicator of platelet activation), was significantly increased in the plasma of patients with fast-growing AAAs compared to slow-growing AAAs and healthy control subjects. To determine if GPVI blockade was protective from AAA progression, mice underwent either the angiotensin II (AngII) infusion or the topical elastase models of aneurysm formation. Following model initiation, aortic diameter was monitored by weekly ultrasounds. After two weeks, mice which developed an aneurysm (defined as an aortic diameter of >1.2mm) were randomized into control (IgG—50μg) or treatment (JAQ1—50μg, monoclonal GPVI antibody) groups. As expected, platelets isolated from JAQ1 treated mice did not activate in response to the GPVI specific agonist convulxin. Furthermore, JAQ1 treatment attenuated aneurysm progression, increased type I collagen deposition, attenuated macrophage accumulation, and increased survival in both murine AAA models versus IgG control treated mice. Conclusions: Platelets play a critical role in AAA pathophysiology and specific blockade of the GPVI-mediated activation pathway attenuates progression of established aneurysms in two independent murine models. Thus, our work demonstrates that specific targeting of GPVI to blunt platelet activation in AAA could be a potential therapeutic for a pathology with no current pharmacological treatments. R01HL147171-04 and R01HL158801-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

Crosstalk of platelets with macrophages and fibroblasts aggravates inflammation, aortic wall stiffening, and osteopontin release in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases, but their role in AAA is poorly understood. The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the extracellular matrix (ECM). They are responsible for the up-regulation of SPP1 (osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodelling and also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity result in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity were also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodelling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall. In conclusion, our data strongly support the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients.

Application and challenges of TCR and BCR sequencing to investigate T- and B-cell clonality in elastase-induced experimental murine abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays autoimmune disease characteristics. Particularly, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial immune response. Single-cell RNA (scRNA) T cell receptor (TCR) and B cell receptor (BCR) sequencing is a powerful tool for investigating clonality. However, difficulties such as limited numbers of isolated cells must be considered during implementation and data analysis, making biological interpretation challenging. Here, we perform a representative single-cell immune repertoire analysis in experimental murine AAA and show a reliable bioinformatic processing pipeline highlighting opportunities and limitations of this approach. We performed scRNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aorta of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham-operated mice at days 3 and 28 and non-operated mice served as controls. Comparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed neither differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified several clones in 11 of 16 AAA samples and one of eight control samples. Immune receptor repertoire comparison indicated that only a few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2 + TRBV13-2. We found no clonal expansion of B cells but evidence for clonal expansion of T cells in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive number of lymphocytes to prevent undersampling and potentially detect rare clones. Thus, further experiments are necessary to confirm our findings. In summary, this paper examines TCR and BCR sequencing results, identifies limitations and pitfalls, and offers guidance for future studies.

Abstract 13582: Interferon Regulatory Factor 8 Promotes Abdominal Aortic Aneurysm via Inducing Type I Conventional Dendritic Cell Differentiation

Introduction: Abdominal aortic aneurysm (AAA) remains to be the most common true aneurysm worldwide. Previous studies identified the participation of dendritic cells (DC) in the pathogenesis of AAA. However, the detailed subtypes involved and mechanisms remain unknown. Our previous RNA-Seq data determined dramatically increased Irf8 in AAA samples, which drives DC differentiating to Type I conventional dendritic cells (cDC1). Aim: The aim of the work is to determine the role of interferon regulatory factor 8 (IRF8) in the development of AAA. Methods and Results: Murine AAA were induced via elastase infusion. The single-cell transcriptomic sequencing of CD45+ cells in murine AAA samples identified the dramatically increased cDC1. By flow cytometry, we found that the ratio of CD103 + cDC1s reached maximum at day 7. Compared with the control group, Irf8 CAG-LSL Itgax Cre mice ( Irf8 overexpression in DC) suffered significantly more severe aneurysm expansion, elastin degradation and apoptosis of smooth muscle cells, while the AAA development in Irf8 flox/flox Itgax Cre mice ( Irf8 deletion in DC) was less sharp. Batf3 -/- mice which were cDC1 depleted, showed similar characteristic in AAA expansion with Irf8 flox/flox Itgax Cre mice. The effective CD8 + T cells increased significantly in Irf8 CAG-LSL Itgax Cre mice than the control group, while decreased in Irf8 flox/flox Itgax Cre ones. Deletion of Clec9a which blocks cross-present dead-cell-associated antigens to the CD8 + T cells, also attenuated AAA development, in accordance with Irf8 flox/flox Itgax Cre mice in gross anatomy and pathology changes. Conclusion: Our findings suggest that the activation of IRF8 promotes cDC1 differentiation, which subsequently activate CD8 + T cells and contribute to destruction of the aortic-wall and AAA formation.

Abstract 17672: Implications of Pcsk9 on Abdominal Aortic Aneurysm

Objective: Recent genome-wide association studies revealed a potential role of proprotein convertase subtilisin/kexin type9(Pcsk9) in the human abdominal aortic aneurysm(AAA) disease.Altered Pcsk9 gene expression and smaller AAA diameter were observed in murine AAA using the porcine pancreatic elastase(PPE) model in our lab. Pcsk9 increases plasma LDL-cholesterol,enhances tissue inflammation and induces vascular smooth muscle cell (VSMC) senescence, hallmarks in AAA.However, the exact cellular mechanisms of Pcsk9 in AAA disease remain unclear.Despite the absence of a pharmacological treatment for AAA, monoclonal Pcsk9 antibody therapies have proven effective against statin-resistant hypercholesterolemia.This study aimed to explore the impacts of Pcsk9 on aortic vascular inflammation and VSMC dedifferentiation with a view to better understanding AAA pathophysiology and providing a basis for future investigations into Pcsk9 inhibitors. Hypothesis: We hypothesized that local vascular injury escalates Pcsk9 expression,amplifying vascular inflammation and driving VSMC towards dedifferentiation that further advances AAA. Conversely,Pcsk9 inhibition will mitigate these processes. Methods: AAA was induced by PPE model in wildtype C57Bl/6 mice and Pcsk9 knock-out (KO)mice.Sonography was used to evaluate the aneurysm growth. Single-cell RNA sequencing was employed to examine local changes in the aortic wall. Local vascular Pcsk9,pro-inflammatory and VSMC marker gene/protein expression were quantified using qPCR and immunohistochemistry.VSMCs were treated with exogenous Pcsk9 in vitro to assess metabolism via Seahorse. Results: Reduced AAA diameters were observed in Pcsk9 KO mice compared to wildtype controls. Significant upregulation of local Pcsk9 expression accompanied AAA induction. Single-cell RNA sequencing revealed altered gene expression profiles across vascular cell populations. In vitro,VSMCs displayed changes in marker gene/protein expression after treatment with exogenous Pcsk9, alongside metabolic changes (Mann-Whitney Test). Conclusions: The findings suggest a significant role of Pcsk9 in AAA pathogenesis. Its inhibition appears to mitigate AAA growth,indicating a promising treatment target for AAA.

Abstract 15171: Endothelium-Derived C-Type Natriuretic Peptide Prevents the Development and Progression of Abdominal Aortic Aneurysm

Background: Abdominal aortic aneurysm (AAA) is a life-threatening disease characterised by dilatation, inflammation and structural weakness of the abdominal aorta. Since the only effective existing intervention is surgery, development of pharmacological therapies is desperately needed. C-type natriuretic peptide (CNP) is a key endothelium-derived molecule that possesses vasodilator, anti-inflammatory and anti-atherogenic activity. Thus, we explored the role and therapeutic potential of CNP in AAA pathogenesis. Methods: Suprarenal aortic diameter was assessed by ultrasonography at baseline and following Angiotensin II (AngII, 1.44mg/kg/day) infusion in wild type (WT) or endothelium-restricted CNP deficient (ecCNP -/- ) mice infected with an AAV-expressing proprotein convertase subtilisin/kexin type 9 gain-of-function mutation (D377Y) or backcrossed on an apolipoprotein E deficient (ApoE -/- ) background. At 28 days, aortas were harvested for qRT-PCR, pharmacological assessment of vasorelaxant activity and histological analysis; plasma CNP concentrations were also determined. In some studies, CNP (0.2mg/kg/day) was infused to rescue any adverse phenotype. Results: ecCNP -/- exhibited an aggravated AAA phenotype compared to WT, exemplified by greater dilation, fibrosis and elastin degradation (without changes in blood pressure). Whilst plasma CNP concentrations were reduced in murine AAA, CNP mRNA abundance was elevated within aneurysmal tissue of ecCNP -/- , accompanied by increased abundance of mRNA encoding markers of inflammation ( IL-6, Adgre1, VCAM-1, CCL-2 ), extracellular matrix remodelling/calcification ( MMP-2, Notch-1, BMPR2, smoothelin ), fibrosis ( fibronectin ) and apoptosis ( Bax ) . CNP-induced relaxations were enhanced in aortic rings from aneurysmal tissue compared to control; an effect possibly underpinned by upregulation of natriuretic peptide receptor expression. Prophylactic administration of CNP protected against the severity of AngII-induced AAA. Conclusions: Endothelial CNP plays an important role in attenuating AAA formation by preserving aortic structure and function. Therapeutic strategies aimed at mimicking CNP bioactivity may prevent the clinical progression of AAA.

Abstract 12885: Pathological Perk/Atf4 Activation in Abdominal Aortic Aneurysm Formation

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). The elastase induced AAA model was used as a secondary murine model. AAA maximum diameters were quantified and VSCMs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. In vivo pharmacological inhibition of the eIF2A/ATF4 ER stress response via 4-PBA injection inhibited AAA formation in both the AngII-induced and elastase-induced AAA model resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

Abstract 18814: Glycoprotein VI is Critical for the Detection and Progression of Abdominal Aortic Aneurysms

Objective: Abdominal aortic aneurysms (AAA) frequently feature the formation of a nonocclusive intraluminal thrombus (ILT) along the site of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understand. Thus, we sought to investigate how platelet activation via the glycoprotein VI (GPVI) platelet receptor impacts the pathogenesis of AAA. Methods and Results: RNA sequencing of age-matched control human aortic tissue, AAA aortic tissue, and AAA ILT demonstrates that platelet transcripts comprised 25% of significantly enriched genes AAA thrombus compared to AAA tissue and control aortic wall, with GPVI increased by 9.1 Log2-FC (P = 3.21E -08 ). Examination of soluble GPVI (sGPVI), a specific marker of platelet activity, in 2 independent AAA patient cohorts is predictive of AAA case status, associates strongly with aneurysm growth rate, and better predicts the presence of a fast-growing AAA when compared to D-dimer. To determine if GPVI blockade ameliorates AAA progression, mice were subjected to either the angiotensin II (AngII) infusion or the topical elastase models of aneurysm formation. Following model initiation, aortic diameter was monitored by weekly ultrasounds. After two weeks, mice which developed an aneurysm (defined as an aortic diameter of >1.2mm) were randomized into control (IgG-50μg) or treatment (JAQ1-50μg, monoclonal GPVI antibody) groups. Treatment with JAQ1 blunted GPVI mediated platelet activation, attenuated aneurysm progression, and increased survival in both murine AAA models versus control. Conclusions: The highly specific platelet activation biomarker soluble glycoprotein VI strongly predicts both AAA case status and growth rate with potential use in identifying high risk patients. Specific blockade of GPVI-mediated platelet activation attenuates progression of established aneurysms in two independent murine models. Thus, our work demonstrates that specific targeting of GPVI to blunt platelet activation in AAA could be a potential therapeutic for a pathology with no current pharmacological treatments.

The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-β regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Gut Microbiota-Derived Trimethylamine N-Oxide Contributes to Abdominal Aortic Aneurysm Through Inflammatory and Apoptotic Mechanisms.

Large-scale human and mechanistic mouse studies indicate a strong relationship between the microbiome-dependent metabolite trimethylamine N-oxide (TMAO) and several cardiometabolic diseases. This study aims to investigate the role of TMAO in the pathogenesis of abdominal aortic aneurysm (AAA) and target its parent microbes as a potential pharmacological intervention. TMAO and choline metabolites were examined in plasma samples, with associated clinical data, from 2 independent patient cohorts (N=2129 total). Mice were fed a high-choline diet and underwent 2 murine AAA models, angiotensin II infusion in low-density lipoprotein receptor-deficient (Ldlr-/-) mice or topical porcine pancreatic elastase in C57BL/6J mice. Gut microbial production of TMAO was inhibited through broad-spectrum antibiotics, targeted inhibition of the gut microbial choline TMA lyase (CutC/D) with fluoromethylcholine, or the use of mice genetically deficient in flavin monooxygenase 3 (Fmo3-/-). Finally, RNA sequencing of in vitro human vascular smooth muscle cells and in vivo mouse aortas was used to investigate how TMAO affects AAA. Elevated TMAO was associated with increased AAA incidence and growth in both patient cohorts studied. Dietary choline supplementation augmented plasma TMAO and aortic diameter in both mouse models of AAA, which was suppressed with poorly absorbed oral broad-spectrum antibiotics. Treatment with fluoromethylcholine ablated TMAO production, attenuated choline-augmented aneurysm initiation, and halted progression of an established aneurysm model. In addition, Fmo3-/- mice had reduced plasma TMAO and aortic diameters and were protected from AAA rupture compared with wild-type mice. RNA sequencing and functional analyses revealed choline supplementation in mice or TMAO treatment of human vascular smooth muscle cells-augmented gene pathways associated with the endoplasmic reticulum stress response, specifically the endoplasmic reticulum stress kinase PERK. These results define a role for gut microbiota-generated TMAO in AAA formation through upregulation of endoplasmic reticulum stress-related pathways in the aortic wall. In addition, inhibition of microbiome-derived TMAO may serve as a novel therapeutic approach for AAA treatment where none currently exist.

Purinergic receptor P2X7 contributes to abdominal aortic aneurysm development via modulating macrophage pyroptosis and inflammation

The purinergic receptor P2X7 has been established as an important mediator of inflammation and participates in a variety of cardiovascular diseases including atherosclerosis, however, its role in abdominal aortic aneurysms (AAA) remains unclear. In this study, we demonstrate that P2X7 plays essential roles in AAA development via modulating macrophage pyroptosis and inflammation. P2X7 is highly expressed in human AAA specimen, as well as in experimental murine AAA lesions (both CaCl2- and Angiotensin II-induced AAA models), and it mainly confines in macrophages. Furthermore, P2X7 deficiency or pharmacological inhibition with its antagonist could significantly attenuate aneurysm formation in experimental murine AAA models, while P2X7 agonist could promote AAA development. The caspase-1 activity, matrix metalloproteinase (MMP) activity, reactive oxygen species (ROS) production and pro-inflammatory gene expression were significantly reduced in experimental AAA lesions in mice with P2X7 deficiency or inhibition. Mechanistically, macrophage P2X7 can mediate the activation of NLRP3 inflammasome and activate its downstream caspase-1 to initiate the pyroptosis pathway. After caspase-1 activation, it further cleaves pro-interleukin (IL)-1β and gasdermin D (GSDMD). Consequently, the N-terminal fragment of GSDMD forms pores on the cell membrane, leading to macrophage pyroptosis and release of the pro-inflammatory factor IL-1β. The resulting vascular inflammation further leads to the upregulation of MMP and ROS, thereby promoting AAA development. In summary, these data identify P2X7-mediated macrophage pyroptosis signaling pathway as a novel contributory mechanism of AAA formation.