Pathogenesis Of Abdominal Aortic Aneurysm Research Articles

Abstract 1000: Platelets Act As Circulating Reservoir Of Tgfβ1: Role In Abdominal Aortic Aneurysms

Introduction: Transforming Growth Factor-Beta1 (TGFβ1) signaling plays a crucial role in the development and maintenance of the vasculature by orchestrating vascular remodeling signaling cascades. Aberrant TGFβ1 leads to aortic aneurysms, yet there is a persistent conflict in its role in driving or protecting against the degradation of the extracellular matrix (ECM) in abdominal aortic aneurysms (AAA). Notably, limited studies have delved into its cell-specific actions. This ambiguity highlights the need for an improved understanding of the TGFβ1 cell-specific role in AAA. Aims: To elucidate the role of platelet-derived TGFβ1 in AAA development thereon expanding our knowledge on the role of TGFβ1 in vascular remodeling. Methods and Results: Single-cell epitope and transcriptome analysis by using cellular-indexing of transcriptomes and epitopes by sequencing (CITE-seq) of the blood of patients with AAA revealed that platelets were enriched with TGFβ1 compared to healthy individuals. Comparative pathway analysis of the platelet populations showed the association of this cluster with ROS, platelet activation and chemokine signaling pathways that promote AAA pathogenesis. These results were validated in the Apolipoprotein E deficient (ApoE -/- ) mice treated with Angiotensin II for 28 days. To delve into the direct role of platelet-derived TGFβ1 in AAA, we generated mice with conditional knock-down of TGFβ1 in platelets (Tgfb1 flox/flox Pf4 Cre+ ) and subjected these mice to AAA. Remarkably, these mice exhibited reduced aneurysm development and preserved elastin microstructure compared to controls. The expression of phosphorylated smad2/3, one of the downstream TGFβ1-canonical signaling pathways, as well as matrix metalloproteinase, were repressed in the aorta of Tgfb1 flox/flox Pf4 Cre+ mice, suggesting the platelets could supplement Tgfb1 to the aortic tissue sufficient to fuel matrix degradation. These results were recapitulated when the platelets were depleted from the circulation using an anti-GP1b antibody. Conclusion: Our findings reveal that platelets represent an important reservoir of Tgfb1, clarify its cell-specific role in ECM remodeling and underscore its detrimental role in sustaining matrix degradation during AAA.

Can Biomarkers and PET Imaging Predict Abdominal Aortic Aneurysm Growth Rate?

Background: Abdominal aortic aneurysm (AAA) is a life-threatening condition due to the risk of aneurysm growth and rupture. Biomarkers linked to AAA pathogenesis are attractive candidates for AAA diagnosis and prognosis. The aim of this study was to assess circulating biomarkers levels relationship with PET imaging positivity and their predictive value in AAA growth rate. Methods: A total of 164 patients with AAA had whole body [18F]FDG PET/CT examination and blood drawn for biomarkers analysis at inclusion. Of these, 121 patients had at least one follow-up imaging assessment for AAA progression. Median (quartiles) imaging follow-up period was 32.8 months (15.2-69.6 months). Results: At baseline, PET was visually positive in 28 (17%) patients. Among PET+ patients, female proportion was higher compared to PET-patients (respectively, n = 6, 21.4% vs. n = 11, 8.1%, p = 0.046). Biomarkers of inflammation (CRP, CCL18), of proteolytic activity (MMP9), of extracellular matrix, and calcification regulation (OPN, OPG) were all significantly increased in PET+ patients (p < 0.05). During follow-up, rapid AAA growth (increase in size ≥ 1 cm per year) was observed in 36 (29.8%) patients and several biomarkers (CRP, MMP9, OPN, and OPG) were increased in those patients compared to patients without rapid growth (p < 0.05). Conclusions: Although PET positivity at baseline was not associated with rapid growth, CRP levels showed a significant association.

Unravelling CD4+ T cell diversity and tissue adaptation of Tregs in abdominal aortic aneurysms through single-cell sequencing.

Immune cell infiltration is a significant pathological process in abdominal aortic aneurysms (AAA). T cells, particularly CD4+ T cells, are essential immune cells responsible for substantial infiltration of the aorta. Regulatory T cells (Tregs) in AAA have been identified as tissue-specific; however, the time, location, and mechanism of acquiring the tissue-specific phenotype are still unknown. Using single-cell RNA sequencing (scRNA-seq) on CD4+ T cells from the AAA aorta and spleen, we discovered heterogeneity among CD4+ T cells and identified activated, proliferating and developed aorta Tregs. These Tregs originate in the peripheral tissues and acquire the tissue-specific phenotype in the aorta. The identification of precursors for Tregs in AAA provides new insight into the pathogenesis of AAA.

Neutrophil Extracellular Traps in Cardiovascular and Aortic Disease: A Narrative Review on Molecular Mechanisms and Therapeutic Targeting.

Neutrophil extracellular traps (NETs), composed of DNA, histones, and antimicrobial proteins, are released by neutrophils in response to pathogens but are also recognized for their involvement in a range of pathological processes, including autoimmune diseases, cancer, and cardiovascular diseases. This review explores the intricate roles of NETs in different cardiovascular conditions such as thrombosis, atherosclerosis, myocardial infarction, COVID-19, and particularly in the pathogenesis of abdominal aortic aneurysms. We elucidate the mechanisms underlying NET formation and function, provide a foundational understanding of their biological significance, and highlight the contribution of NETs to inflammation, thrombosis, and tissue remodeling in vascular disease. Therapeutic strategies for preventing NET release are compared with approaches targeting components of formed NETs in cardiovascular disease. Current limitations and potential avenues for clinical translation of anti-NET treatments are discussed.

N6-methyladenosine-associated genetic variants in NECTIN2 and HPCAL1 are risk factors for abdominal aortic aneurysm

Although N6-methyladenosine (m6A) modification has been implicated in the pathogenesis of abdominal aortic aneurysm (AAA), the relationship between m6A-associated single nucleotide polymorphisms (m6A-SNPs) and AAA remains unknown. This study used integrative multi-omics analysis and clinical validation approaches to systematically identify potential m6A-SNPs connected with AAA risk. We found that rs6859 and rs10198139 could modulate the expression of local genes, NECTIN2 and HPCAL1, respectively, which exhibited upregulation in AAA tissues, and their risk variants were significantly correlated with an increased susceptibility to AAA. Incorporating rs6859 and rs10198139 improved the efficiency of AAA risk prediction compared to the model considering only conventional risk factors. Additionally, these two SNPs were predicted to be located within the regulatory sequences, and rs6859 showed a substantial impact on m6A modification levels. Our findings suggest that m6A-SNPs rs6859 and rs10198139 confer an elevated risk of AAA, possibly by promoting local gene expression through an m6A-mediated manner.

SIRT6-mediated vascular smooth muscle cells senescence participates in the pathogenesis of abdominal aortic aneurysm

Background and aimsIn this study, we carried out a clinical sample study, and in vivo and in vitro studies to evaluate the effect of SIRT6 and SIRT6-mediated vascular smooth muscle senescence on the development of abdominal aortic aneurysm (AAA). Method and ResultsAAA specimen showed an increased P16, P21 level and a decreased SIRT6 level compared with control aorta. Time curve study of Ang II infusion AAA model showed similar P16, P21 and SIRT6 changes at the early phase of AAA induction. The in vivo overexpression of SIRT6 significantly prevented AAA formation in Ang II infusion model. The expression of P16 and P21 was significantly reduced after SIRT6 overexpression. SIRT6 overexpression also attenuated chronic inflammation and neo-angiogenesis in Ang II infusion model. The overexpression of SIRT6 could attenuate premature senescence, inflammatory response and neo-angiogenesis in human aortic smooth muscle cells (HASMC) under Ang II stimulation. ConclusionsSIRT6 overexpression could limit AAA formation via attenuation of vascular smooth muscle senescence, chronic inflammation and neovascularity.

Pathogenesis-Guided Rational Engineering of Nanotherapies for the Targeted Treatment of Abdominal Aortic Aneurysm by Inhibiting Neutrophilic Inflammation.

Abdominal aortic aneurysm (AAA) remains a fatal disease in the elderly. Currently, no drugs can be clinically used for AAA therapy. Considering the pivotal role of neutrophils in the pathogenesis of AAA, herein we propose the targeted therapy of AAA by site-specifically regulating neutrophilic inflammation. Based on a luminol-conjugated α-cyclodextrin material (LaCD), intrinsically anti-inflammatory nanoparticles (NPs) were engineered by simple nanoprecipitation, which were examined as a nanotherapy (defined as LaCD NP). After efficient accumulation in the aneurysmal aorta and localization in pathologically relevant inflammatory cells in rats with CaCl2-induced AAA, LaCD NP significantly alleviated AAA progression, as implicated by the decreased aortic expansion, suppressed elastin degradation, inhibited calcification, and improved structural integrity of the abdominal aorta. By functionalizing LaCD NP with alendronate, a calcification-targeting moiety, the in vivo aneurysmal targeting capability of LaCD NP was considerably enhanced, thereby affording significantly potentiated therapeutic outcomes in AAA rats. Mechanistically, LaCD NP can effectively inhibit neutrophil-mediated inflammatory responses in the aneurysmal aorta. Particularly, LaCD NP potently attenuated the formation of neutrophil extracellular traps (NETs), thereby suppressing NETs-mediated pro-inflammatory events and NETosis-associated negative effects responsible for AAA progression. Consequently, we demonstrated the effectiveness and underlying mechanisms of anti-NETosis nanotherapies for the targeted treatment of AAA. Our findings provide promising insights into discovering precision therapies for AAA and other inflammatory vascular diseases.

Cellular, Molecular and Clinical Aspects of Aortic Aneurysm-Vascular Physiology and Pathophysiology.

A disturbance of the structure of the aortic wall results in the formation of aortic aneurysm, which is characterized by a significant bulge on the vessel surface that may have consequences, such as distention and finally rupture. Abdominal aortic aneurysm (AAA) is a major pathological condition because it affects approximately 8% of elderly men and 1.5% of elderly women. The pathogenesis of AAA involves multiple interlocking mechanisms, including inflammation, immune cell activation, protein degradation and cellular malalignments. The expression of inflammatory factors, such as cytokines and chemokines, induce the infiltration of inflammatory cells into the wall of the aorta, including macrophages, natural killer cells (NK cells) and T and B lymphocytes. Protein degradation occurs with a high expression not only of matrix metalloproteinases (MMPs) but also of neutrophil gelatinase-associated lipocalin (NGAL), interferon gamma (IFN-γ) and chymases. The loss of extracellular matrix (ECM) due to cell apoptosis and phenotype switching reduces tissue density and may contribute to AAA. It is important to consider the key mechanisms of initiating and promoting AAA to achieve better preventative and therapeutic outcomes.

The future for the therapeutics of abdominal aortic aneurysm: engineered nanoparticles drug delivery for abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a severe cardiovascular disease with a high mortality rate. Several screening and diagnostic methods have been developed for AAA early diagnosis. Open surgery and endovascular aortic repair (EVAR) are clinically available for patients who meet the indications for surgery. However, for non-surgical patients, limited drugs exist to inhibit or reverse the progression of aneurysms due to the complex pathogenesis and biological structure of AAA, failing to accumulate precisely on the lesion to achieve sufficient concentrations. The recently developed nanotechnology offers a new strategy to address this problem by developing drug-carrying nanoparticles with enhanced water solubility and targeting capacity, prolonged duration, and reduced side effects. Despite the rising popularity, limited literature is available to highlight the progression of the field. Herein, in this review, we first discuss the pathogenesis of AAA, the methods of diagnosis and treatment that have been applied clinically, followed by the review of research progressions of constructing different drug-loaded nanoparticles for AAA treatment using engineered nanoparticles. In addition, the feasibility of extracellular vesicles (EVs) and EVs-based nanotechnology for AAA treatment in recent years are highlighted, together with the future perspective. We hope this review will provide a clear picture for the scientists and clinicians to find a new solution for AAA clinical management.

Trimethylamine N-oxide promotes abdominal aortic aneurysm by inducing vascular inflammation and vascular smooth muscle cell phenotypic switching

ObjectiveInflammation and vascular smooth muscle cell (VSMC) phenotypic switching are implicated in the pathogenesis of abdominal aortic aneurysm (AAA). Trimethylamine N-oxide (TMAO) has emerged as a crucial risk factor in cardiovascular diseases, inducing vascular inflammation and calcification. We aimed to evaluate the effect of TMAO on the formation of AAA. Approach and resultsHere, we showed that TMAO was elevated in plasma from AAA patients compared with nonaneurysmal subjects by liquid chromatography‒mass spectrometry (LC‒MS) detection. Functional studies revealed that increased TMAO induced by feeding a choline-supplemented diet promoted Ang II-induced AAA formation. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot analyses revealed that TMAO induced macrophage infiltration and inflammatory factor release. Conversely, inhibition of TMAO by supplementation with DMB suppressed AAA formation and the inflammatory response. Molecular studies revealed that TMAO regulated VSMC phenotypic switching. Flow cytometry analyses showed that TMAO induces macrophage M1-type polarization. Furthermore, pharmacological intervention experiments suggested that the nuclear factor-κB (NF-κB) signaling pathway was critical for TMAO to trigger AAA formation. ConclusionsTMAO promotes AAA formation by inducing vascular inflammation and VSMC phenotypic switching through activation of the NF-κB signaling pathway. Thus, TMAO is a prospective therapeutic AAA target.

Integrated analysis of bulk RNA-seq and single-cell RNA-seq reveals the function of pyrocytosis in the pathogenesis of abdominal aortic aneurysm.

Pyrocytosis is involved in the development of abdominal aortic aneurysm (AAA), we explored the pyrocytosis-related hub genes in AAA and conducted a diagnostic model based on the pyrocytosis-related genes score (PRGs). A total of 2 bulk RNA-seq (GSE57691 and GSE47472) datasets and pyrocytosis-related genes were integrated to obtain 24 pyrocytosis-related different expression genes (DEGs). The LASSO Cox regression analysis was conducted to filter out 7 genes and further establish the nomogram signature based on the PRGs that exhibited a good diagnosis value. Weighted gene co-expression network analysis (WGCNA) established 14 gene modules and further identified 6 hub genes which were involved in the regulatory process of pyrocytosis in AAA. At the single cell level, we further identified 3 immune cells were highly associated with the pyrocytosis process in AAA. Finally, the cell-cell communication demonstrated that fibroblasts and endothelial cells and myeloid cells maintained close communications. Here, we identified the dysfunctional expressed pyrocytosis-related genes and immune cells in AAA, which provide a comprehensive understanding of the pathogenesis of AAA.

Exploring the impact of gut microbiota on abdominal aortic aneurysm risk through a bidirectional Mendelian randomization analysis

ObjectiveThe abdominal aortic aneurysm (AAA) is associated with alterations in the composition of the gut microbiota; however, the precise causal relationship remains unclear. Elucidating this complex interplay could provide new insights into the pathogenesis of AAA. MethodsA bidirectional two-sample Mendelian randomization analysis was conducted using genome-wide association study summary data on the gut microbiota (n = 18,340) and AAA (n = 353,087). A total of 196 gut microbial taxa across taxonomic levels were examined for their potential causal effects on AAA risk. Conversely, the effect of AAA on these microbial taxa was also analyzed. ResultsNine microbial taxa were identified as having a causal influence on AAA risk. Specifically, increased risk were associated with genus Bilophila (odds ratio [OR], 1.359; P = .0119), genus Catenibacterium (OR, 1.348; P = .0058), genus family XIII AD3011 group (OR, 1.507; P = .004), genus Oxalobacter (OR, 1.157; P = .0449), and genus Prevotella 7 (OR, 1.194; P = .0306), whereas decreased risks were linked to class Lentisphaeria (OR, 0.829; P = .0361), order Victivallales (OR, 0.829; P = .0361), family Victivallaceae (OR, 0.814; P = .0057), and genus Anaerotruncus (OR, 0.773; P = .0497). Furthermore, AAA was found to influence the abundance of 14 microbial taxa across various taxonomic levels. Notably, bidirectional associations were observed with the class Lentisphaeria and the order Victivallales. ConclusionsThis study provides novel evidence for a reciprocal causal relationship between gut microbiota and AAA risk, thereby offering new insights into the pathogenesis of AAA. These findings also suggest promising avenues for microbiome-based therapeutic interventions.

Establishment and Validation of an Animal Model of Abdominal Aortic Aneurysm in Beagles Through Vascular Patch Angioplasty and Elastase Infusion

To evaluate the effect of an abdominal aortic aneurysm (AAA) animal model established in beagles by way of vascular patch angioplasty combined with elastase infusion. A total of 60 beagle dogs were included in this study. Among them, 10 beagles were assigned to a control group to obtain normal abdominal aortic wall tissue, while the other 50 underwent vascular patch angioplasty combined with elastase infusion in order to establish the AAA disease model. In order to evaluate the outcome of modeling, abdominal vascular ultrasonography was performed 14 days after the modeling surgery was performed and ultrasound and computed tomographic angiography (CTA) were performed 28 days after the modeling surgery. The criterion for evaluating modeling success is that the maximum diameter of the abdominal aortic aneurysm is 50% greater than the diameter of the normal abdominal aorta below the renal artery. A total of 20 beagles of the modelling group and 5 control beagles were sacrificed 35 days after the modeling surgery and infrarenal abdominal aortic wall tissues were harvested. Then, hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and elastic van Gieson (EVG) staining were conducted to observe the pathology features of abdominal aortic wall tissues. A total of 50 beagles underwent the AAA modeling procedures, with the average operative and anesthesia time being (119.4±18.9) and (137.4±15.8) minutes, respectively, the average blood loss volume being (43.6±7.7) mL, the average abdominal aorta block time being (39.7±5.3) minutes during the modeling surgery, and the average abdominal aorta diameter measured during the surgery being (6.5±0.4) mm. Intraoperative mortality was 0%. Mortality within 30 days after the surgery was 2% (1 out of the 50 beagles). Postoperative ultrasound and CTA results revealed that the success rate of AAA modeling was 100%. Pathology examination suggested that the animal model rather successfully simulated the pathophysiologic changes associated with human AAA in regard to the morphological and pathological changes. Vascular patch angioplasty combined with elastase infusion can be used to successfully establish AAA model in beagles. The AAA modeling method described in our report demonstrates stability and reliability in aneurysm formation effect and the surgical procedures are easy to replicate. The method integrates the advantages of previous animal modeling methods and can be used to study the pathogenesis of AAA.

B cells and immunoglobulins promotes aortic dissection in mice

Abstract Background Aortic dissection (AD) is a devastating aortic disease with high mortality, for which molecular pathogenesis is largely unknown. Recent studies have highlighted the importance of inflammatory response in AD. We have reported that B cells and immunoglobulins promote inflammatory response in the aorta and are involved in the pathogenesis of abdominal aortic aneurysm, another form of aortic disease. However, it is not known whether and how B cells participate in AD development or progression. Methods and results Immunohistochemical staining of human AD tissue revealed that B cells were clustered together with T cells, macrophages, and neutrophils at the entry site of AD with medial disruption. B cell cluster was also observed at the site of medial disruption in mouse model of AD that was induced by continuous infusion of beta-aminopropionitrile and angiotensin II (BAPN+AngII). In muMT mouse, which is genetically deficient for B cells and immunoglobulins, BAPN+AngII induced significantly less severe AD compared to that in wild type. The rate of aortic rupture and sudden death was approximately 42% in wild type mice, while that in muMT mouse was 12% (P&amp;lt;0.05). Administration of mouse normal polyclonal IgG to muMT mice resulted in dramatic increase in aortic rupture and sudden death, starting at day 7 of BAPN+AngII infusion, and reaching 69% of rupture rate, indicating the critical role of IgG in AD. Transcriptome analysis revealed that IgG administration enhanced the expression of inflammation-related genes in muMT mice before BAPN+AngII infusion, indicating that IgG increased susceptibility to AD. Immunohistochemistry of aorta confirmed exogenous IgG deposition, colocalized with C3, which is known to contribute to innate immunity. Conclusion These findings demonstrated B cells and IgG are critically involved in the destructive inflammation of AD pathogenesis. Further, the aortic deposition of IgG and C3, a part of the components of innate immunity, precedes the development of AD. These findings may provide the therapeutic opportunities to AD.

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 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 &gt;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.

Abstract 12098: Evaluation of Inflammatory Biomarkers and Causal Association With Abdominal Aortic Aneurysm Development

Introduction: Observational analyses have linked circulating inflammatory biomarkers with an increased risk of abdominal aortic aneurysms (AAA). Although observational studies implicate inflammatory biomarkers in AAA, whether these are a cause or consequence of the disease is unknown. A limited number of trials have explored agents that reduce inflammation in slowing the progression of AAA. Observational study quality may be limited by residual confounding and reverse causality. We sought to leverage genetic instruments and Mendelian Randomization (MR) to evaluate the human genetic evidence supporting inflammatory biomarkers in the pathogenesis of AAA. Methods: We leveraged a meta-analysis of 6 genome-wide association studies (GWAS) of 66 circulating inflammatory markers including up to 59,969 participants of European ancestry. We tested the association for each biomarker with AAA using summary statistics from a meta-analysis of 17 individual GWAS from 14 discovery cohorts in the AAAgen consortium comprising 39,221 individuals with AAA. We employed Wald ratio or inverse-variance weighted MR; weighted median and weighted mode methods were applied as sensitivity analyses. Results: After accounting for multiple testing by controlling the false discovery rate (q &lt; 0.05), we identified a total of 6/66 (9.1%) inflammatory biomarkers that had a significant association with AAA: sIL6R, CCL8, CCL7, ILRN, F2, and PIGF. These results were consistent across a range of linkage-disequilibrium thresholds to identify independent genetic instruments. Some of these biomarkers have been previously implicated in the development of AAA but have not been validated in human RCTs of aneurysm development, growth, or rupture. Conclusions: This analysis highlights the role of several circulating inflammatory markers in the development of AAA. These findings represent potential therapeutic targets for further investigation in mechanistic trials.

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.

Effect of Hydralazine on Angiotensin II-Induced Abdominal Aortic Aneurysm in Apolipoprotein E-Deficient Mice.

The rupture of an abdominal aortic aneurysm (AAA) causes about 200,000 deaths worldwide each year. However, there are currently no effective drug therapies to prevent AAA formation or, when present, to decrease progression and rupture, highlighting an urgent need for more research in this field. Increased vascular inflammation and enhanced apoptosis of vascular smooth muscle cells (VSMCs) are implicated in AAA formation. Here, we investigated whether hydralazine, which has anti-inflammatory and anti-apoptotic properties, inhibited AAA formation and pathological hallmarks. In cultured VSMCs, hydralazine (100 μM) inhibited the increase in inflammatory gene expression and apoptosis induced by acrolein and hydrogen peroxide, two oxidants that may play a role in AAA pathogenesis. The anti-apoptotic effect of hydralazine was associated with a decrease in caspase 8 gene expression. In a mouse model of AAA induced by subcutaneous angiotensin II infusion (1 µg/kg body weight/min) for 28 days in apolipoprotein E-deficient mice, hydralazine treatment (24 mg/kg/day) significantly decreased AAA incidence from 80% to 20% and suprarenal aortic diameter by 32% from 2.26 mm to 1.53 mm. Hydralazine treatment also significantly increased the survival rate from 60% to 100%. In conclusion, hydralazine inhibited AAA formation and rupture in a mouse model, which was associated with its anti-inflammatory and anti-apoptotic properties.