Mouse Model Of Abdominal Aortic Aneurysm Research Articles

Biogenesis of circRBM33 mediated by N6-methyladenosine and its function in abdominal aortic aneurysm

ABSTRACT This study aimed to explore whether m6A modification affects the biogenesis of circRBM33, which is involved in the progression of abdominal aortic aneurysm (AAA). For in vitro experiments, vascular smooth muscle cells (VSMCs) were treated with Ang II. MeRIP‒PCR was used to assess m6A modification of circRBM33. Gene expression was measured using RT‒qPCR and Western blotting. For in vivo experiments, a mouse model of AAA was established via Ang II infusion. HE, Sirius Red and TUNEL staining was performed to evaluate pathological changes and cell apoptosis in aortic vessels. The results showed that the m6A level of circRBM33 was abnormally increased in Ang II-induced VSMCs. In addition, METTL3 positively regulated circRBM33 expression. YTHDC1 deficiency decreased circRBM33 expression but had no effect on RBM33 mRNA expression. Notably, neither METTL3 nor YTHDC1 influenced the stability of circRBM33 or RBM33 mRNA. The interaction between circRBM33 and METTL3/YTHDC1 was verified by RIP analysis. Moreover, the Ang II-induced increase in circRBM33 expression was reversed by cycloleucine (an inhibitor of m6A methylation). Importantly, the m6A modification and expression of circRBM33 in the circRBM33-m6A-mut2-expressing VSMCs were not altered by METTL3 silencing. Mechanistically, METTL3/YTHDC1 modulates the biogenesis of circRBM33 in an m6A-dependent manner. In addition, circRBM33 knockdown alleviated AAA by reducing ECM degradation in the Ang II-infused mice. In conclusion, this study demonstrated that METTL3/YTHDC1-mediated m6A modification modulates the biogenesis of circRBM33 from exons of the RBM33 gene. Moreover, knockdown of circRBM33 alleviated AAA by reducing ECM degradation, which may provide a novel therapeutic strategy for treating AAA.

Mesenchymal stromal cell-derived exosomes protect against abdominal aortic aneurysm formation through CD74 modulation of macrophage polarization in mice

BackgroundMesenchymal stromal cell (MSC)-derived exosomes (MSC-Exo) have been recognized for their significant role in regulating macrophage polarization, a process crucial to the pathogenesis of abdominal aortic aneurysm (AAA). However, the therapeutic effects of MSC-Exo on AAA remain largely unexplored. Therefore, this study aimed to investigate the functional and mechanistic aspects of MSC-Exo in the progression of AAA.MethodsThe MSC-derived exosomes were characterized using Transmission Electron Microscopy, Nanoparticle Tracking Analysis, and Western blotting. An experimental mouse model of AAA was established through the administration of angiotensin II (Ang II) in male apoe−/− mice and calcium chloride (CaCl2) in male C57/B6 mice, with subsequent tail vein injection of exosomes to evaluate their efficacy against AAA. Macrophage polarization was assessed using immunofluorescence staining and WB analysis. Mechanistic analysis was performed using 4D Label-free Proteomics analysis.ResultsWe found that intravenous administration of MSC-Exo induced M2 polarization of macrophages within an inflammatory environment, effectively impeding AAA development in Ang II or CaCl2-induced AAA model. The therapeutic efficacy of MSC-Exo treatment was dependent on the presence of macrophages. Mechanistically, MSC-Exo suppressed the levels of cluster of differentiation 74 (CD74), modulating macrophage polarization through the TSC2-mTOR-AKT pathway. These findings highlight the potential of MSC-Exo as a therapeutic strategy for AAA by modulating macrophage polarization.

ATF4 Contributes to Abdominal Aortic Aneurysm Formation via Modulating M1 Macrophage Polarization and Inflammation.

Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular disease primarily in the male elderly population, but there is a lack of approved medical therapies to prevent the progression and rupture of AAA. Activating Transcription Factor 4 (ATF4) has been established to be involved in cardiovascular diseases, such as heart failure and calcific aortic valve disease. However, the role of ATF4 in the pathogenesis of AAA remains unclear. We found that ATF4 expression was significantly increased in patients with AAA and mouse models of AAA and was mainly confined to macrophages in arteries. ATF4 knockdown significantly attenuated aneurysm formation in experimental mouse model of AAA, while ATF4 overexpression promoted the development of AAA. RNA sequencing suggested that ATF4 was strongly related to the biological function of acute inflammatory response. Macrophages-specific ATF4 knockout significantly reduced the incidence and development of AAA, and decreased M1 polarization of macrophages in mice. Sphingomyelin phosphodiesterase 3 (SMPD3), a regulator of inflammatory responses in monocytes/macrophages, has been identified as a target gene of ATF4 through RNA sequencing, ChIP sequencing, and standard ChIP analyses. ATF4 induces M1 polarization of macrophages through the activation of SMPD3, thereby promoting inflammatory responses. Together, these results suggest that ATF4 mediated macrophage M1 polarization by regulating the expression of target genes SMPD3, leading to an increased inflammatory response, which further promotes the formation and development of AAA. These findings suggest ATF4 may be a new therapeutic target for AAA.

Neutrophil extracellular trap-induced ferroptosis promotes abdominal aortic aneurysm formation via SLC25A11-mediated depletion of mitochondrial glutathione

BackgroundNeutrophil extracellular traps (NETs) induce oxidative stress, which may initiate ferroptosis, an iron-dependent programmed cell death, during abdominal aortic aneurysm (AAA) formation. Mitochondria regulate the progression of ferroptosis, which is characterized by the depletion of mitochondrial glutathione (mitoGSH) levels. However, the mechanisms are poorly understood. This study examined the role of mitoGSH in regulating NET-induced ferroptosis of smooth muscle cells (SMCs) during AAA formation. MethodsConcentrations of NET markers were tested in plasma samples. Western blotting and immunofluorescent staining were performed to detect the expression and localization of NET and ferroptosis markers in tissue samples. The role of NETs and SMC ferroptosis during AAA formation was investigated using peptidyl arginine deiminase 4 gene (Padi4) knockout or treatment with a PAD4 inhibitor, ferroptosis inhibitor or activator in an angiotensin II-induced AAA mouse model. The regulatory effect of SLC25A11, a mitochondrial glutathione transporter, on mitoGSH and NET-induced ferroptosis of SMCs was investigated using in vitro and in vivo experiments. Transmission electron microscopy was used to detect mitochondrial damage. Blue native polyacrylamide gel electrophoresis was used to analyze the dimeric and monomeric forms of the protein. ResultsSignificantly elevated levels of NETosis and ferroptosis markers in aortic tissue samples were observed during AAA formation. Specifically, NETs promoted AAA formation by inducing ferroptosis of SMCs. Subsequently, SLC25A11 was identified as a potential biomarker for evaluating the clinical prognosis of patients with AAA. Furthermore, NETs decreased the stability and dimerization of SLC25A11, leading to the depletion of mitoGSH. This depletion induced the ferroptosis of SMCs and promoted AAA formation. ConclusionDuring AAA formation, NETs regulate the stability of the mitochondrial carrier protein SLC25A11, leading to the depletion of mitoGSH and subsequent activation of NET-induced ferroptosis of SMCs. Preventing mitoGSH depletion and ferroptosis in SMCs is a potential strategy for treating AAA.

Abstract 144: Pyruvate Dehydrogenase Kinase 4 Contributes To Abdominal Aortic Aneurysm Through Accelerating Smooth Muscle Cell Metabolic Reprogramming And Nlrp3 Inflammasome-dependent Pyroptosis

Background: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate upon rupture. Understanding its molecular mechanisms of progression is crucial for therapeutic strategery. Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular metabolism. This study explored the role of PDK4 in AAA development, focusing on its impact on vascular smooth muscle cells (VSMCs). Methods: The expression of PDK4 in human and mice AAA tissues was measured. The role of PDK4 in AAA development was evaluated in VSMC-specific PDK4 knockout mice using a PCSK9/Ang II-induced AAA model. Seahorse analysis was used to assess oxidative phosphorylation and glycolysis levels. RNA-sequencing analysis and mitochondrial functional analysis were used to explore the molecular mechanisms. Results: We observed an increased expression of VSMC-PDK4 in human AAA tissues and AAA tissues from a PCSK9/AngII-induced AAA mouse model. PDK4 deficiency in VSMCs significantly inhibited AAA formation, whereas PDK4 overexpression aggravated AAA development. PDK4 promoted the metabolic reprogramming of VSMCs and VSMC-specific deletion of PDK4 ameliorated the contractile-to-synthetic phenotype switch in VSMCs. PDK4 activated NLRP3 inflammasome by impairing mitochondrial function, which contributed to the pyroptosis of VSMCs and instigated an inflammatory response. Mitochondria-targeted ROS scavenging and NLRP3 inhibitor MCC950 effectively attenuated NLRP3 inflammasome activation and pyroptosis mediated by PDK4. Supplementation with MCC950 was beneficial to ameliorating AAA in a VSMC PDK4-dependent manner in mouse models. Conclusion: PDK4 promotes smooth muscle cell metabolic reprogramming and NLRP3 inflammasome-dependent pyroptosis in AAA development, suggesting that targeting PDK4 may be a promising therapeutic strategy for the prevention and treatment of AAA.

Extravascular administration of IGF1R antagonists protects against aortic aneurysm in rodent and porcine models.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. We identified plasma insulin-like growth factor 1 (IGF1) as an independent risk factor in patients with AAA by correlating plasma IGF1 with risk. Smooth muscle cell- or fibroblast-specific knockout of Igf1r, the gene encoding the IGF1 receptor (IGF1R), attenuated AAA formation in two mouse models of AAA induced by angiotensin II infusion or CaCl2 treatment. IGF1R was activated in aortic aneurysm samples from human patients and mice with AAA. Systemic administration of IGF1C, a peptide fragment of IGF1, 2 weeks after disease development inhibited AAA progression in mice. Decreased AAA formation was linked to competitive inhibition of IGF1 binding to its receptor by IGF1C and modulation of downstream alpha serine/threonine protein kinase (AKT)/mammalian target of rapamycin signaling. Localized application of an IGF1C-loaded hydrogel was developed to reduce the side effects observed after systemic administration of IGF1C or IGF1R antagonists in the CaCl2-induced AAA mouse model. The inhibitory effect of the IGF1C-loaded hydrogel administered at disease onset on AAA formation was further evaluated in a guinea pig-to-rat xenograft model and in a sheep-to-minipig xenograft model of AAA formation. The therapeutic efficacy of IGF1C for treating AAA was tested through extravascular delivery in the sheep-to-minipig model with AAA established for 2 weeks. Percutaneous injection of the IGF1C-loaded hydrogel around the AAA resulted in improved vessel flow dynamics in the minipig aorta. These findings suggest that extravascular administration of IGF1R antagonists may have translational potential for treating AAA.

LncRNA CARMN inhibits abdominal aortic aneurysm formation and vascular smooth muscle cell phenotypic transformation by interacting with SRF

Phenotypic transformation of vascular smooth muscle cells (VSMCs) plays a crucial role in abdominal aortic aneurysm (AAA) formation. CARMN, a highly conserved, VSMC-enriched long noncoding RNA (lncRNA), is integral in orchestrating various vascular pathologies by modulating the phenotypic dynamics of VSMCs. The influence of CARMN on AAA formation, particularly its mechanisms, remains enigmatic. Our research, employing single-cell and bulk RNA sequencing, has uncovered a significant suppression of CARMN in AAA specimens, which correlates strongly with the contractile function of VSMCs. This reduced expression of CARMN was consistent in both 7- and 14-day porcine pancreatic elastase (PPE)-induced mouse models of AAA and in human clinical cases. Functional analyses disclosed that the diminution of CARMN exacerbated PPE-precipitated AAA formation, whereas its augmentation conferred protection against such formation. Mechanistically, we found CARMN's capacity to bind with SRF, thereby amplifying its role in driving the transcription of VSMC marker genes. In addition, our findings indicate an enhancement in CAMRN transcription, facilitated by the binding of NRF2 to its promoter region. Our study indicated that CARMN plays a protective role in preventing AAA formation and restrains the phenotypic transformation of VSMC through its interaction with SRF. Additionally, we observed that the expression of CARMN is augmented by NRF2 binding to its promoter region. These findings suggest the potential of CARMN as a viable therapeutic target in the treatment of AAA.Graphical abstract

Targeting the smooth muscle cell Keap1-Nrf2-GSDMD-pyroptosis axis by cryptotanshinone prevents abdominal aortic aneurysm formation.

Rationale: Abdominal aortic aneurysm (AAA) is an inflammatory, fatal aortic disease that currently lacks any effective drugs. Cryptotanshinone (CTS) is a prominent and inexpensive bioactive substance derived from Salvia miltiorrhiza Bunge, a well-known medicinal herb for treating cardiovascular diseases through its potent anti-inflammatory properties. Nevertheless, the therapeutic effect of CTS on AAA formation remains unknown. Methods: To investigate the therapeutic effect of CTS in AAA, variety of experimental approaches were employed, majorly including AAA mouse model establishment, real-time polymerase chain reaction (PCR), RNA sequencing, western blot, co-immunoprecipitation, scanning/transmission electron microscopy (SEM/TEM), enzyme-linked immunosorbent assay (ELISA), seahorse analysis, immunohistochemistry, and confocal imaging. Results: In this study, we demonstrated that CTS suppressed the formation of AAA in apolipoprotein E knock-out (ApoE-/-) mice infused with Ang II. A combination of network pharmacology and whole transcriptome sequencing analysis indicated that activation of the Keap1-Nrf2 pathway and regulation of programmed cell death in vascular smooth muscle cells (VSMCs) are closely linked to the anti-AAA effect of CTS. Mechanistically, CTS promoted the transcription of Nrf2 target genes, particularly Hmox-1, which prevented the activation of NLRP3 and GSDMD-initiated pyroptosis in VSMCs, thereby mitigating VSMC inflammation and maintaining the VSMC contractile phenotype. Subsequently, by utilizing molecular docking, together with the cellular thermal shift assay (CETSA) and isothermal titration calorimetry (ITC), a particular binding site was established between CTS and Keap1 at Arg415. To confirm the binding site, site-directed mutagenesis was performed, which intriguingly showed that the Arg415 mutation eliminated the binding between CTS and the Keap1-Nrf2 protein and abrogated the antioxidant and anti-pyroptosis effects of CTS. Furthermore, VSMC-specific Nrf2 knockdown in mice dramatically reversed the protective action of CTS in AAA and the inhibitory effect of CTS on VSMC pyroptosis. Conclusion: Naturally derived CTS exhibits promising efficacy as a treatment drug for AAA through its targeting of the Keap1-Nrf2-GSDMD-pyroptosis axis in VSMCs.

Transcriptome analysis reveals therapeutic potential of NAMPT in protecting against abdominal aortic aneurysm in human and mouse

Abdominal Aortic Aneurysm (AAA) is a life-threatening vascular disease characterized by the weakening and ballooning of the abdominal aorta, which has no effective therapeutic approaches due to unclear molecular mechanisms. Using single-cell RNA sequencing, we analyzed the molecular profile of individual cells within control and AAA abdominal aortas. We found cellular heterogeneity, with increased plasmacytoid dendritic cells and reduced endothelial cells and vascular smooth muscle cells (VSMCs) in AAA. Up-regulated genes in AAA were associated with muscle tissue development and apoptosis. Genes controlling VSMCs aberrant switch from contractile to synthetic phenotype were significantly enriched in AAA. Additionally, VSMCs in AAA exhibited cell senescence and impaired oxidative phosphorylation. Similar observations were made in a mouse model of AAA induced by Angiotensin II, further affirming the relevance of our findings to human AAA. The concurrence of gene expression changes between human and mouse highlighted the impairment of oxidative phosphorylation as a potential target for intervention. Nicotinamide phosphoribosyltransferase (NAMPT, also named VISFATIN) signaling emerged as a signature event in AAA. NAMPT was significantly downregulated in AAA. NAMPT-extracellular vesicles (EVs) derived from mesenchymal stem cells restored NAMPT levels, and offered protection against AAA. Furthermore, NAMPT-EVs not only repressed injuries, such as cell senescence and DNA damage, but also rescued impairments of oxidative phosphorylation in both mouse and human AAA models, suggesting NAMPT supplementation as a potential therapeutic approach for AAA treatment. These findings shed light on the cellular heterogeneity and injuries in AAA, and offered promising therapeutic intervention for AAA treatment.

PANoptosis in vascular smooth muscle cells regulated by TNF-α/IL-1β can be a new target for alleviating the progression of abdominal aortic aneurysm.

PANoptosis is an inflammatory programmed cell death (PCD) regulated by multifaceted PANoptosome complexes with major features of pyroptosis, apoptosis, and/or necroptosis that cannot be accounted for by any of these PCD pathways alone. The aim of this study was to investigate the role of PANoptosis on the occurrence and development of abdominal aortic aneurysm (AAA). Clinical samples of patients with AAA, angiotensin II (ANG II)-induced AAA mouse model, and ANG II-induced vascular smooth muscle cells (VSMCs) in vitro model were used for investigation on PANoptosis features. The expressions of ZBP1, AIM2, and other markers related to pyroptosis, apoptosis, and necroptosis elevated obviously in aortic wall tissues of patients with AAA, mice with AAA, and ANG II-treated VSMCs. ANG II treatment increased inflammatory cytokines levels in VSMCs. The stimulation of tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) alone promoted VSMCs death, and the effect of TNF-α combined with IL-1β is more obvious. The expressions of ZBP1, AIM2, and related markers of pyroptosis, apoptosis, and necroptosis were increased by TNF-α and IL-1β combined treatment. Inhibition of TNF-α and/or IL-1β in mice with AAA improved the AAA pathology, reduced the loss of VSMCs, decreased the expression of ZBP1 and AIM2, and markers associated with pyroptosis, apoptosis, and necroptosis. PANoptosis features were observed in aortic wall tissues of patients with AAA, mice with AAA, and ANG II-treated VSMCs. The inhibition of TNF-α and IL-1β can alleviate PANoptosis in mice with AAA, which provides a new strategy for the prevention and treatment of AAA.NEW & NOTEWORTHY Early detection, diagnosis, and treatment are very important to improve the quality of life and prognosis of patients with abdominal aortic aneurysm (AAA). Based on the findings of apoptosis, necroptosis, and pyroptosis (PANoptosis) in AAA clinical samples, this study further explored the molecular mechanism in vivo and in vitro. Specifically, inhibition of tumor necrosis factor-α and interleukin-1β can reduce PANoptosis in vascular smooth muscle cell and thus alleviate the process of AAA.

Ganglioside GM3 Protects Against Abdominal Aortic Aneurysm by Suppressing Ferroptosis.

Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular condition, but approved medical therapies to prevent AAA progression and rupture are currently lacking. Sphingolipid metabolism disorders are associated with the occurrence and development of AAA. It has been discovered that ganglioside GM3, a sialic acid-containing type of glycosphingolipid, plays a protective role in atherosclerosis, which is an important risk factor for AAA; however, the potential contribution of GM3 to AAA development has not been investigated. We performed a metabolomics study to evaluated GM3 level in plasma of human patients with AAA. We profiled GM3 synthase (ST3GAL5) expression in the mouse model of aneurysm and human AAA tissues through Western blotting and immunofluorescence staining. RNA sequencing, affinity purification and mass spectrometry, proteomic analysis, surface plasmon resonance analysis, and functional studies were used to dissect the molecular mechanism of GM3-regulating ferroptosis. We conditionally deleted and overexpressed St3gal5 in smooth muscle cells (SMCs) in vivo to investigate its role in AAA. We found significantly reduced plasma levels of GM3 in human patients with AAA. GM3 content and ST3GAL5 expression were decreased in abdominal aortic vascular SMCs in patients with AAA and an AAA mouse model. RNA sequencing analysis showed that ST3GAL5 silencing in human aortic SMCs induced ferroptosis. We showed that GM3 interacted directly with the extracellular domain of TFR1 (transferrin receptor 1), a cell membrane protein critical for cellular iron uptake, and disrupted its interaction with holo-transferrin. SMC-specific St3gal5 knockout exacerbated iron accumulation at lesion sites and significantly promoted AAA development in mice, whereas GM3 supplementation suppressed lipid peroxidation, reduced iron deposition in aortic vascular SMCs, and markedly decreased AAA incidence. Together, these results suggest that GM3 dysregulation promotes ferroptosis of vascular SMCs in AAA. Furthermore, GM3 may constitute a new therapeutic target for AAA.

Higher matrix stiffness promotes VSMC senescence by affecting mitochondria-ER contact sites and mitochondria/ER dysfunction.

Abdominal aortic aneurysm (AAA) is a prevalent condition characterized by the weakening and bulging of the abdominal aorta. This study aimed to investigate the impact of a stiff matrix on vascular smooth muscle cells (VSMCs) in AAA development. Bioinformatics analysis revealed that differentially expressed genes (DEGs) in VSMCs of an AAA mouse model were enriched in cellular senescence and related pathways. To simulate aging-related changes, VSMCs were cultured on stiff matrices, and compared to those on soft matrices, the VSMCs cultured on stiff matrices exhibited cellular senescence. Furthermore, the mutual distance between mitochondria and endoplasmic reticulum (ER) in VSMCs was increased, indicating altered mitochondria-endoplasmic reticulum contacts (MERCs). The observed upregulation of reactive oxygen species (ROS) levels, antioxidant gene expression, and decreased mitochondrial membrane potential suggested the presence of mitochondrial dysfunction in VSMCs cultured on a stiff matrix. Additionally, the induction of ER stress-related genes indicated ER dysfunction. These findings collectively indicated impaired functionality of both mitochondria and ER in VSMCs cultured on a stiff matrix. Moreover, our data revealed that high lipid levels exacerbated the effects of high matrix stiffness on VSMCs senescence, MERC sites, and mitochondria/ER dysfunction. Importantly, treatment with the antilipemic agent CI-981 effectively reversed these detrimental effects. These findings provide insights into the role of matrix stiffness, mitochondrial dysfunction, ER stress, and lipid metabolism in AAA development, suggesting potential therapeutic targets for intervention.

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.

Vascular Ultrasound for In Vivo Assessment of Arterial Pathologies in a Murine Model of Atherosclerosis and Aortic Aneurysm.

Vascular diseases like atherosclerosis and abdominal aortic aneurysm (AAA) are common pathologies in the western world, promoting various potentially fatal conditions. Here, we evaluate high-resolution (HR) ultrasound in mouse models of atherosclerosis and AAA as a useful tool for noninvasive monitoring of early vascular changes in vivo. We used Apolipoprotein E-deficient (ApoE-/-) mice as an atherosclerosis model and induced AAA development by the implementation of Angiotensin II-releasing osmotic minipumps. HR ultrasound of the carotid artery or the abdominal aorta was performed to monitor vascular remodeling in vivo. Images were analyzed by speckle tracking algorithms and correlated to histological analyses and subsequent automated collagen quantification. Consistent changes were observed via ultrasound in both models: Global radial strain (GRS) was notably reduced in the AAA model (23.8 ± 2.8% vs. 12.5 ± 2.5%, p = 0.01) and in the atherosclerotic mice (20.6 ± 1.3% vs. 15.8 ± 0.9%, p = 0.02). In mice with AAA, vessel distensibility was significantly reduced, whereas intima-media thickness was increased in atherosclerotic mice. The area and collagen content of the tunica media were increased in diseased arteries of both models as measured by automated image analysis of Picrosirius Red-stained aortic sections. Correlation analysis revealed a strong correlation of multiple parameters, predicting early vascular damage in HR ultrasound and histological examinations. In conclusion, our findings underscore the potential of HR ultrasound in effectively tracing early alterations in arterial wall properties in murine models of atherosclerosis and AAA.

Increased plasma renin by vasodilators promotes the progression of abdominal aortic aneurysm.

Background: It is well-accepted that antihypertensive therapy is the cornerstone of treatment for abdominal aortic aneurysm (AAA) patients with hypertension. Direct-acting vasodilators were used in the treatment of hypertension by directly relaxing vascular smooth muscle but may have destructive effects on the aortic wall by activating the renin-angiotensin system axis. Their roles in AAA disease remain to be elucidated. In this study, we used hydralazine and minoxidil, two classical direct-acting vasodilators, to investigate their influence and potential mechanisms on AAA disease. Methods and results: In this study, we investigated the plasma renin level and plasma renin activity in AAA patients. Simultaneously, age and gender ratio-matched patients diagnosed with peripheral artery disease and varicose veins were selected as the control group using a ratio of 1:1:1. Our regression analysis suggested both the plasma renin level and plasma renin activity are positively associated with AAA development. In view of the well-established relationship between direct-acting vasodilators and increased plasma renin concentration, we established a porcine pancreatic elastase-infused AAA mouse model, followed by oral administration of hydralazine (250mg/L) and minoxidil (120mg/L) to investigate effects of direct-acting vasodilators on AAA disease. Our results suggested both hydralazine and minoxidil promoted the progression of AAA with increased aortic degeneration. Mechanistically, the vasodilators aggravated aortic inflammation by increased leukocyte infiltration and inflammatory cytokine secretion. Conclusion and relevance: The plasma renin level and plasma renin activity are positively associated with AAA development. Direct vasodilators aggravated experimental AAA progression, which raised cautionary concerns about their applications in AAA disease.

Abstract 155: Platelet-derived Transforming Growth Factor-β1 Ameliorates AAA in a Murine Model

Background: Platelets are rapidly recruited to the forming intraluminal thrombus in abdominal aortic aneurysm (AAA), stabilizing the aortic wall and preventing rupture. Adhesion and activation of platelets releases a mix of inflammatory mediators such as transforming growth factor β (TGFβ), which mediates cellular proliferation, inflammatory signaling, and fibrosis. Importantly, while TGFβ neutralization results in increased rupture and disease progression in mouse models of AAA, the source of protective TGFβ signaling remains controversial. We hypothesized that platelet-derived TGFβ1 attenuates aortic growth in the elastase AAA model. Methods and Results: Ldlr -/- mice with platelet-specific deletion of Tgfβ1 were produced by breeding hemizygous male mice expressing Cre under the control of the Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Mice were subjected to laparotomy and topical elastase application to the abdominal aorta (10 mg/mL porcine pancreatic elastase for 5 minutes) and sacrificed at day 28 to determine AAA progression via aortic diameter. Male TGFβ1-Pf4 Cre+ mice (n=8) had significantly increased abdominal aortic diameters compared to Male TGFβ1-Pf4 Cre- mice (n=7) ( Cre- : 1.70 ± 0.12 mm; Cre+ : 2.66 ± 0.25 mm; P < 0.004). Additionally, 25% of male TGFβ1-Pf4 Cre+ mice died of AAA rupture compared to 0% in the TGFβ1-Pf4 Cre- cohort. To determine if these actions were occurring via TGFβ1 activation of the TGFβ receptor 2 (βr 2 ), Ldlr -/- /βr2 Flox /PF4 Cre+ and Cre- mice were created, similarly to the previously described Tgfβ1 mice. Male βr2-Pf4 Cre+ (n=7) had increased aortic diameter compared to βr2-Pf4 Cre- (n=6) ( Cre- : 1.70± 0.20 mm; Cre+ : 2.45 ± 0.38 mm; P < 0.138). Additionally, female βr2-Pf4 Cre+ (n=9) also had significantly larger aortic diameters compared to βr2-Pf4 Cre- (n = 5) ( Cre- : 1.27± 0.04 mm; Cre+ : 1.90 ± 0.23 mm; P < 0.01). Conclusions: Our results further support TGFβ signaling playing a protective role in AAA. More specifically, platelet-derived TGFβ1 ligand and signaling via platelet TGFβr2 mitigates aortic diameter expansion. Further research into the pertinent TGFβ isoforms and the mechanism of TGFβr2 activation will ideally yield insights into therapeutic targets for an, as yet, intractable disease.

Mesenchymal stem cell-derived extracellular vesicles protect against abdominal aortic aneurysm formation by inhibiting NET-induced ferroptosis

Neutrophil extracellular traps (NETs) play an important role in abdominal aortic aneurysm (AAA) formation; however, the underlying molecular mechanisms remain unclear. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) may exert therapeutic effects on AAA through their immunomodulatory and regenerative abilities. This study aimed to examine the role and mechanism of MSC-EVs in regulating the development of NET-mediated AAA. Excessive release of NETs was observed in patients with AAA, and the levels of NET components were associated with the clinical outcomes of the patients. Datasets from the Gene Expression Omnibus database were analyzed and revealed that the PI3K/AKT pathway and ferroptosis were strongly associated with NETosis during AAA formation. Further experiments verified that NETs promoted AAA formation by inducing ferroptosis in smooth muscle cells (SMCs) by inhibiting the PI3K/AKT pathway. The PI3K agonist 740 Y-P, the ferroptosis inhibitor ferrostatin-1, and Padi4 deficiency significantly prevented AAA formation. MSC-EVs attenuated AAA formation by reducing NET release in an angiotensin II-induced AAA mouse model. In vitro experiments revealed that MSC-EVs reduced the release of NETs by shifting NETosis to apoptosis. Our study indicates an important role for NET-induced SMC ferroptosis in AAA formation and provides several potential targets for AAA treatment.

MicroRNA-19b-3p dysfunction of mesenchymal stem cell-derived exosomes from patients with abdominal aortic aneurysm impairs therapeutic efficacy

Senescence of vascular smooth muscle cells (VSMCs) contributes to the formation of abdominal aortic aneurysm (AAA). Although mesenchymal stem cell exosomes (MSC-EXO) have been confirmed to restrict the development of AAA, their biological activity depends largely on the physiological state of the MSCs. This study aimed to compare the effects of adipose-derived MSC-EXO from healthy donors (HMEXO) and AAA patients (AMEXO) on senescence of VSMCs in AAA and explore the underlying mechanisms. An ApoE-/- mouse model of AAA was used to investigate the therapeutic effects of HMEXO, AMEXO or miR-19b-3p-AMEXO on AAA development. This in vitro model of AAA was established by treating VSMCs with Ang II (Angiotensin II). The senescence of VSMCs was determined by senescence-associated β-galactosidase (SA-β-gal) staining. The morphology of mitochondria in VSMCs was examined by MitoTracker staining. HMEXO exhibited superior capacity compared with AMEXO to inhibit VSMC senescence and attenuate AAA formation in Ang II–treated ApoE-/- mice. In vitro, both AMEXO and HMEXO inhibited Ang II-induced VSMC senescence via downregulation of mitochondrial fission. Notably, compared with HMEXO, the ability of AMEXO to inhibit VSMC senescence was significantly decreased. miRNA sequencing and the expression of miR-19b-3p was significantly decreased in AMEXO compared with HMEXO. Luciferase assay suggested that MST4 (Mammalian sterile-20-like kinase 4) is a potential target of miR-19b-3p. Mechanistically, miR-19b-3p in HMEXO ameliorated VSMC senescence by inhibiting mitochondrial fission via regulation of the MST4/ERK/Drp1 signaling pathway. Overexpression of miR-19b-3p in AMEXO improved their beneficial effect on AAA formation. Our study reveals that MSC-exosomal miR-19b-3p exerts protective effects against Ang II-induced AAA and VSMC senescence via regulation of the MST4/ERK/Drp1 pathway. The pathological state of AAA patients alters the miRNA components of AMEXO and impairs their therapeutic benefits.

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.

Reactive oxygen species-induced long intergenic noncoding RNA p21 accelerates abdominal aortic aneurysm formation by promoting secretary smooth muscle cell phenotypes

Whether long noncoding RNAs participate in the formation of abdominal aortic aneurysms (AAAs) through the regulation of SMC phenotypic switching is unknown. lincRNA-p21 induced by reactive oxygen species (ROS) is likely functionally associated with SMC phenotypic switching. We thus investigated the role of lincRNA-p21 in SMC phenotypic switching-associated AAA formation and its underlying mechanisms. An analysis of human and mouse abdominal aortic samples revealed that the lincRNA-p21 levels were significantly higher in AAA tissue. Stimulation with hydrogen peroxide upregulated the expression of lincRNA-p21 in a dose-dependent manner and converted SMCs from a contractile phenotype to a synthetic, proteolytic, and proinflammatory phenotype in vitro. Moreover, lincRNA-p21 promoted fracture of elastic fibres, reconstruction of the vascular wall, and AAA formation in vivo by modulating SMC phenotypic switching in two mouse models of AAA induced by angiotensin II or porcine pancreatic elastase (PPE) perfusion. Using a bioinformatics prediction method and luciferase reporter gene assays, we further proved that lincRNA-p21 sponged miR-204-5p to release the transcriptional activity of Mekk3 and promoted the NF-κB pathway and thereby played a role in the SMC phenotypic switch and AAA formation. The ROS levels were positively correlated with the lincRNA-p21 levels in human and mouse AAA tissues. The knockdown of lincRNA-p21 in a PPE-induced mouse AAA model increased the miR-204-5p levels and reduced the expression of Mekk3, whereas lincRNA-p21 overexpression had the opposite effect. Collectively, the results indicated that ROS-induced lincRNA-p21 sponges miR-204-5p to accelerate synthetic and proinflammatory SMC phenotypes through the Mekk3/NF-κB pathway in AAA formation. Thus, lincRNA-p21 may have therapeutic potential for AAA formation.