Elastase-induced Abdominal Aortic Aneurysm Research Articles

Inhibiting YAP1 reduced abdominal aortic aneurysm formation by suppressing adventitial fibroblast phenotype transformation and migration.

The adventitial fibroblast (AF) is the most abundant cell in the vascular adventitia, a few studies had confirmed that AF contributed to abdominal aortic aneurysm (AAA) development; YAP1 involved in several vascular diseases by promoting AF transformed to myofibroblast, the role of YAP1 in AAA is not clear yet. This study aims to determine whether YAP1 play a role in AAA process by regulating AF function. We found the expression of YAP1was significantly increased in aneurysm tissues of AAA patients compared to normal adjacent vascular tissues and mainly in adventitia. YAP1 also upregulated in elastase-induced and CaCl2-induced mice AAA model. Suppressed YAP1 function with YAP1 inhibitor-Verteporfin declined AAA incident rate remarkably in mice, and the collagen deposition in the adventitia was alleviated obviously. Afterwards, we studied the effect of YAP1 on the function of AF, Verteporfin was used to block YAP1 invitro, the process of AF transforming to myofibroblast and migration were almost completely eliminated after inhibiting YAP1 expression. This study demonstrated that YAP1 may play a key role in AAA development, inhibiting YAP1 significantly reduced AAA formation through suppressed the process of AF transformed to myofibroblast and migration.

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.

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration.

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

Abstract 3013: Indoleamine 2,3-dioxygenase-mediated Tryptophan Catabolism Limits Experimental Abdominal Aortic Aneurysms

Background: Indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression. Genetic IDO deficiency limits angiotensin II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic mice. We investigated the influence of IDO inhibition or IDO-catabolized tryptophan catabolite on elastase-induced AAAs in normolipidemic mice. Methods: AAAs were induced in male C57B/6J mice via intra-aortic porcine pancreatic elastase (PPE) infusion. Mice were treated with IDO inhibitor 1-methyl-DL-tryptophan (MT, 2 mg/ml in drinking water), tryptophan metabolite 3-hydroxyanthranilic acid (HAA, 200 mg/kg, oral gavage), or vehicle, starting 3 days prior to PPE infusion for 17 days. AAAs were assessed via ultrasonography and histopathology at sacrifice. Results: The Figure summarizes aortic diameters and histopathology in differentially treated PPE-infused mice. Rapid aortic enlargement was noted in MT-, as compared to vehicle-treated mice following PPE infusion. AAA rupture, unusual for this model, was noted in MT-treated AAA mice. No difference was noted between groups in medial elastin degradation, smooth muscle cell depletion, or macrophage accumulation. However, mural neoangiogenesis and lymphocyte accumulation were remarkably augmented in MT-treated mice. HAA Treatment attenuated aortic enlargement in conjunction with improved medial elastin and smooth muscle cell retention and reduced aortic leukocyte accumulation and neovessels. Conclusion: IDO activity and its tryptophan metabolite HAA limit experimental AAA progression in the PPE model. These results provide further support for investigating tryptophan metabolites in clinical AAA disease suppression.

The incidence rate and histological characteristics of intimal hyperplasia in elastase-induced experimental abdominal aortic aneurysms in mice.

Intimal hyperplasia (IH) is a negative vascular remodeling after arterial injury. IH occasionally occurs in elastase-induced abdominal aortic aneurysm (AAA) mouse models. This study aims to clarify the incidence and histological characteristics of IH in aneurysmal mice. A retrospective study was conducted by including 42 male elastase-induced mouse AAA models. The IH incidence, aortic diameters with or without IH, and hyperplasia lesional features of mice were analyzed. Among 42 elastase-induced AAA mouse models, 10 mice developed mild IH (24%) and severe IH was found in only 2 mice (5%). The outer diameters of the AAA segments in mice with and without IH did not show significant difference. Both mild and severe IH lesions show strong smooth muscle cell positive staining, but endothelial cells were occasionally observed in severe IH lesions. There was obvious macrophage infiltration in the IH lesions of the AAA mouse models, especially in mice with severe IH. However, only a lower numbers of T cells and B cells were found in the IH lesion. Local cell-secreted matrix metalloproteinases (MMP) 2 was highly expressed in all IH lesions, but MMP9 was only overexpressed in severe lesions. In conclusion, this study is the first to demonstrate the occurrence of aneurysmal IH and its histological characteristics in an elastase-induced mouse AAA model. This will help researchers better understand this model, and optimize it for use in AAA-related research.

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 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.

Targeted PERK inhibition with biomimetic nanoclusters confers preventative and interventional benefits to elastase-induced abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a progressive aortic dilatation, causing ∼80% mortality upon rupture. Currently, there is no approved drug therapy for AAA. Surgical repairs are invasive and risky and thus not recommended to patients with small AAAs which, however, account for ∼90% of the newly diagnosed cases. It is therefore a compelling unmet clinical need to discover effective non-invasive strategies to prevent or slow down AAA progression. We contend that the first AAA drug therapy will only arise through discoveries of both effective drug targets and innovative delivery methods. There is substantial evidence that degenerative smooth muscle cells (SMCs) orchestrate AAA pathogenesis and progression. In this study, we made an exciting finding that PERK, the endoplasmic reticulum (ER) stress Protein Kinase R-like ER Kinase, is a potent driver of SMC degeneration and hence a potential therapeutic target. Indeed, local knockdown of PERK in elastase-challenged aorta significantly attenuated AAA lesions in vivo. In parallel, we also conceived a biomimetic nanocluster (NC) design uniquely tailored to AAA-targeting drug delivery. This NC demonstrated excellent AAA homing via a platelet-derived biomembrane coating; and when loaded with a selective PERK inhibitor (PERKi, GSK2656157), the NC therapy conferred remarkable benefits in both preventing aneurysm development and halting the progression of pre-existing aneurysmal lesions in two distinct rodent models of AAA. In summary, our current study not only establishes a new intervention target for mitigating SMC degeneration and aneurysmal pathogenesis, but also provides a powerful tool to facilitate the development of effective drug therapy of AAA.

Nicotine Administration Augments Abdominal Aortic Aneurysm Progression in Rats.

Inflammation and elastin degradation are key hallmarks in the pathogenesis of abdominal aortic aneurysms (AAAs). It has been acknowledged that activation of alpha7 nicotinic acetylcholine receptors (α7nAChRs) attenuates inflammation, termed the cholinergic anti-inflammatory pathway (CAP). Thus, we hypothesize that low-dose nicotine impairs the progression of elastase-induced AAAs in rats by exerting anti-inflammatory and anti-oxidative stress properties. Male Sprague-Dawley rats underwent surgical AAA induction with intraluminal elastase infusion. We compared vehicle rats with rats treated with nicotine (1.25 mg/kg/day), and aneurysm progression was monitored by weekly ultrasound images for 28 days. Nicotine treatment significantly promoted AAA progression (p = 0.031). Additionally, gelatin zymography demonstrated that nicotine significantly reduced pro-matrix metalloproteinase (pro-MMP) 2 (p = 0.029) and MMP9 (p = 0.030) activity in aneurysmal tissue. No significant difference was found in the elastin content or the score of elastin degradation between the groups. Neither infiltrating neutrophils nor macrophages, nor aneurysmal messenger RNA (mRNA) levels of pro- or anti-inflammatory cytokines, differed between the vehicle and nicotine groups. Finally, no difference in mRNA levels of markers for anti-oxidative stress or the vascular smooth muscle cells' contractile phenotype was observed. However, proteomics analyses of non-aneurysmal abdominal aortas revealed that nicotine decreased myristoylated alanine-rich C-kinase substrate and proteins, in ontology terms, inflammatory response and reactive oxygen species, and in contradiction to augmented AAAs. In conclusion, nicotine at a dose of 1.25 mg/kg/day augments AAA expansion in this elastase AAA model. These results do not support the use of low-dose nicotine administration for the prevention of AAA progression.

DOCK2 Deficiency Attenuates Abdominal Aortic Aneurysm Formation-Brief Report.

Abdominal aortic aneurysm (AAA) is a potentially lethal disease that lacks pharmacological treatment. Degradation of extracellular matrix proteins, especially elastin laminae, is the hallmark for AAA development. DOCK2 (dedicator of cytokinesis 2) has shown proinflammatory effects in several inflammatory diseases and acts as a novel mediator for vascular remodeling. However, the role of DOCK2 in AAA formation remains unknown. Ang II (angiotensin II) infusion of ApoE-/- (apolipoprotein E deficient) mouse and topical elastase-induced AAA combined with DOCK2-/- (DOCK2 knockout) mouse models were used to study DOCK2 function in AAA formation/dissection. The relevance of DOCK2 to human AAA was examined using human aneurysm specimens. Elastin fragmentation in AAA lesion was observed by elastin staining. Elastin-degrading enzyme MMP (matrix metalloproteinase) activity was measured by in situ zymography. DOCK2 was robustly upregulated in AAA lesion of Ang II-infused ApoE-/- mice, elastase-treated mice, as well as human AAA lesions. DOCK2-/- significantly attenuated the Ang II-induced AAA formation/dissection or rupture in mice along with reduction of MCP-1 (monocyte chemoattractant protein-1) and MMP expression and activity. Accordingly, the elastin fragmentation observed in ApoE-/- mouse aorta infused with Ang II and elastase-treated aorta was significantly attenuated by DOCK2 deficiency. Moreover, DOCK2-/- decreased the prevalence and severity of aneurysm formation, as well as the elastin degradation observed in the topical elastase model. Our results indicate that DOCK2 is a novel regulator for AAA formation. DOCK2 regulates AAA development by promoting MCP-1 and MMP2 expression to incite vascular inflammation and elastin degradation.

Trehalose attenuates abdominal aortic aneurysm formation by inducing autophagy in smooth muscle cells.

Trehalose is a naturally occurring disaccharide, which has been identified as an autophagy inducer and exhibits protective effect in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of trehalose in abdominal aortic aneurysm (AAA) remains undefined. To study the effect of trehalose in AAA, trehalose (1 g/kg per day) were given for 14 continuous days in a mouse model of elastase-induced abdominal aortic aneurysm. On day 14, ultrasound was performed to measure aortic diameter before the abdominal aortas were harvested and processed for further analysis. Verhoeff-Van Gieson staining and TUNEL staining were performed on paraffin sections to evaluate vascular histology and apoptosis, immunofluorescence staining and Western-blot were performed to evaluate expression of autophagy markers. Echocardiography and in situ pictures demonstrated that trehalose attenuated infrarenal aorta dilation. Verhoeff-Van Gieson staining showed elastin degradation was improved in trehalose-treated group. Compared with vehicle-treated mice, trehalose treatment restored smooth muscle cell contractile phenotype with increased α-SMA, Calponin and Myh11 expression. Furthermore, trehalose also attenuated cell apoptosis and leukocytes infiltration. Importantly, trehalose induced autophagy with decrease SQSTM1/p62 accumulation, increased lamp2 expression and LC3B conversion. Trehalose attenuated AAA progression with decreased inflammation and restored SMC contractile phenotype by inducing autophagy. These results demonstrated the therapeutic potential of trehalose in AAA.

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

BAF60c prevents abdominal aortic aneurysm formation through epigenetic control of vascular smooth muscle cell homeostasis.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. BAF60c, a unique subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, is critical for cardiac and skeletal myogenesis, yet little is known about its function in the vasculature and, specifically, in AAA pathogenesis. Here, we found that BAF60c was downregulated in human and mouse AAA tissues, with primary staining to vascular smooth muscle cells (VSMCs), confirmed by single-cell RNA-sequencing. In vivo studies revealed that VSMC-specific knockout of Baf60c significantly aggravated both angiotensin II- (Ang II-) and elastase-induced AAA formation in mice, with a significant increase in elastin degradation, inflammatory cell infiltration, VSMC phenotypic switch, and apoptosis. In vitro studies showed that BAF60c knockdown in VSMCs resulted in loss of contractile phenotype, increased VSMC inflammation, and apoptosis. Mechanistically, we demonstrated that BAF60c preserved VSMC contractile phenotype by strengthening serum response factor (SRF) association with its coactivator P300 and the SWI/SNF complex and suppressing VSMC inflammation by promoting a repressive chromatin state of NF-κB target genes as well as preventing VSMC apoptosis through transcriptional activation of KLF5-dependent B cell lymphoma 2 (BCL2) expression. Our identification of the essential role of BAF60c in preserving VSMC homeostasis expands its therapeutic potential in preventing and treating AAA.

Peptide-siRNA nanoparticles targeting NF-κB p50 mitigate experimental abdominal aortic aneurysm progression and rupture.

Abdominal aortic aneurysm (AAA) is a progressive vascular condition associated with high risk of mortality if left untreated. AAA is an inflammatory process with excessive local production of extracellular matrix degrading enzymes, leading to dilatation and rupture of the abdominal aorta. We posit that targeting NF-κB, a signaling pathway that controls inflammation, will halt AAA progression and prevent rupture. In an elastase-induced AAA model we observed that NF-κB activation increased progressively post-elastase perfusion. Unexpectedly, we found that AAA progression was marked by predominant nuclear accumulation of the NF-κB p50 subunit at the exclusion of p65. Using the amphipathic peptide p5RHH to form nanocomplexes with siRNA, we sought to mitigate AAA progression by knocking down the expression of different NF-κB subunits. We found that the administration of NF-κB p65 siRNA was only beneficial when given early (day 3 post-elastase perfusion) while p50 siRNA was still effective in mitigating elastase-induced AAA even when delivery was delayed until day 5. Additionally, systemic delivery of p50 siRNA, but not p65 siRNA decreased the risk of aortic rupture and sudden death in the transforming growth factor-beta blockade model of AAA. In both murine models, knockdown of NF-κB was accompanied by a significant decrease in leukocyte infiltrates, inflammatory cytokine release, inducible nitric oxide synthase expression, and cell apoptosis. These results suggest that the NF-κB p50 and p65 subunits contribute differentially at different stages of disease and the timing of in vivo siRNA delivery was of critical importance. The results also provide a rationale for selective targeting of p50 for more specific therapeutic intervention in the medical treatment of small AAA.

Galectin-1 prevents pathological vascular remodeling in atherosclerosis and abdominal aortic aneurysm.

Pathological vascular remodeling is the underlying cause of atherosclerosis and abdominal aortic aneurysm (AAA). Here, we analyzed the role of galectin-1 (Gal-1), a β-galactoside–binding protein, as a therapeutic target for atherosclerosis and AAA. Mice lacking Gal-1 (Lgals1−/−) developed severe atherosclerosis induced by pAAV/D377Y-mPCSK9 adenovirus and displayed higher lipid levels and lower expression of contractile markers of vascular smooth muscle cells (VSMCs) in plaques than wild-type mice. Proteomic analysis of Lgals1−/− aortas showed changes in markers of VSMC phenotypic switch and altered composition of mitochondrial proteins. Mechanistically, Gal-1 silencing resulted in increased foam cell formation and mitochondrial dysfunction in VSMCs, while treatment with recombinant Gal-1 (rGal-1) prevented these effects. Furthermore, rGal-1 treatment attenuated atherosclerosis and elastase-induced AAA, leading to higher contractile VSMCs in aortic tissues. Gal-1 expression decreased in human atheroma and AAA compared to control tissue. Thus, Gal-1–driven circuits emerge as potential therapeutic strategies in atherosclerosis and AAA.

Hexarelin attenuates abdominal aortic aneurysm formation by inhibiting SMC phenotype switch and inflammasome activation.

Hexarelin, a synthetic growth hormone-releasing peptide, is shown to be protective in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of hexarelin in abdominal aortic aneurysm (AAA) remains undefined. The present study determined the effect of hexarelin administration (200μg/kg twice per day) in a mouse model of elastase-induced abdominal aortic aneurysm. Echocardiography and in situ pictures showed hexarelin decreased infrarenal aorta diameter. Histology staining showed elastin degradation was improved in hexarelin-treated group. Hexarelin rescued smooth muscle cell contractile phenotype with increased α-SMA and decreased MMP2. Furthermore, hexarelin inhibited inflammatory cell infiltration, NLRP3 inflammasome activation and IL-18 production. Particularly, hexarelin suppressed NF-κB signaling pathway which is a key initiator of inflammatory response. These results demonstrated that hexarelin attenuated AAA development by inhibiting SMC phenotype switch and NF-κB signaling mediated inflammatory response.

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

T lymphocyte-derived extracellular vesicles aggravate abdominal aortic aneurysm by promoting macrophage lipid peroxidation and migration via pyruvate kinase muscle isozyme 2

T lymphocyte and macrophage infiltration in the aortic wall is critical for abdominal aortic aneurysm (AAA). However, how T lymphocytes interact with macrophages in the pathogenesis of AAA remains largely uncharacterized. In an elastase-induced murine AAA model, we first found that the expression of pyruvate kinase muscle isozyme 2 (PKM2), the last rate-limiting enzyme in glycolysis, was increased in infiltrated T lymphocytes of vascular lesions. T lymphocyte-specific PKM2 deficiency in mice (LckCrePKM2fl/fl) or intraperitoneal administration of the sphingomyelinase inhibitor GW4869 caused a significant attenuation of the elastase-increased aortic diameter, AAA incidence, elastic fiber disruption, matrix metalloproteinases (MMPs) expression, and macrophage infiltration in the vascular adventitia compared with those in PKM2fl/fl mice. Mechanistically, extracellular vesicles (EVs) derived from PKM2-activated T lymphocytes elevated macrophage iron accumulation, lipid peroxidation, and migration in vitro, while macrophages treated with EVs from PKM2-null T lymphocytes or pretreated with the lipid peroxidation inhibitors ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), or the iron chelating agent deferoxamine mesylate (DFOM) reversed these effects. In vascular lesions of elastase-induced LckCrePKM2fl/fl mice with AAA, the oxidant system weakened, with downregulated 4-hydroxynonenal (4-HNE) levels and strengthened antioxidant defense systems with upregulated glutathione peroxidase 4 (GPX4) and cystine/glutamate antiporter solute carrier family 7 member 11 (Slc7a11) expressions in macrophages. High-throughput metabolomics showed that EVs derived from PKM2-activated T lymphocytes contained increased levels of polyunsaturated fatty acid (PUFA)-containing phospholipids, which may provide abundant substrates for lipid peroxidation in target macrophages. More importantly, upregulated T lymphocyte PKM2 expression was also found in clinical AAA subjects, and EVs isolated from AAA patient plasma enhanced macrophage iron accumulation, lipid peroxidation, and migration ex vivo. Therefore, from cell-cell crosstalk and metabolic perspectives, the present study shows that PKM2-activated T lymphocyte-derived EVs may drive AAA progression by promoting macrophage redox imbalance and migration, and targeting the T lymphocyte-EV-macrophage axis may be a potential strategy for early warning and treating AAA.

Kunming mouse strain is less susceptible to elastase-induced abdominal aortic aneurysms.

BackgroundPorcine pancreatic elastase (PPE) is successfully used to induce abdominal aortic aneurysm (AAA) in mice. However, differences between mouse strains in susceptibility to PPE induction have been reported. Kunming mouse is one of the most frequently used strains in China but whether it is suitable for induction of AAA by PPE application remains unclear.MethodsPPE infusion (1.5 units/ml) in temporary controlled aorta was performed to induce AAAs in both C57BL/6J and Kunming mice. Phosphate‐buffered saline (PBS) application was used as vehicle control. The aorta diameters of all mice were measured at days 0 and 14 after surgery to evaluate the AAA formation.ResultsAfter 14 days of PPE or PBS infusion, all mice were sacrificed and aorta tissues were collected for histological staining analysis. At the 14th day after infusion, PPE successfully induced aortic dilation in Kunming mice and typical AAA in C57BL/6J mice. The aorta diameter increased by 0.23 mm in Kunming mice after PPE infusion, while it was 0.72 mm in the C57BL/6J strain. PPE induced mild elastin degradation, smooth muscle cell (SMC) depletion and mural leucocyte infiltration in Kunming mice, but in PPE‐sensitive C57BL/6J mice, it induced total loss of SMCs, elastin disappearance and diffused infiltrated leucocytes in aortic aneurysmal segments. The effects of PPE in inducing angiogenesis and upregulating matrix metalloproteinase 2 and 9 expression in Kunming mice were also weaker than that in C57BL/6J mice.ConclusionAt the reported dose of PPE, Kunming mouse is not as susceptible to AAA formation as C57BL/6J mice. The failure of PPE to induce AAA formation in Kunming mice may be associated to its inability to boost a strong inflammatory response.

High-Frequency Three-Dimensional Lumen Volume Ultrasound Is a Sensitive Method to Detect Early Aneurysmal Change in Elastase-Induced Murine Abdominal Aortic Aneurysm.

Objective The aim of this study was to investigate the reproducibility of anterior–posterior diameter (APd max ) and three-dimensional lumen volume (3DLV) measurements of abdominal aortic aneurysms (AAA) in a classical murine AAA model. We also compared the magnitude of change in the aortic size detected with each method of assessment. Methods Periadventitial application of porcine pancreatic elastase (PPE AAA) or sham surgery was performed in two cohorts of mice. Cohort 1 was used to assess for observer variability with the APd max and 3DLV measurements. Cohort 2 highlighted the relationship between APd max and 3DLV and changes in AAA detected. Results There was no significant observer variability detected with APd max measurement. Similarly, no significant intraobserver variability was evident with 3DLV; however, a small but significant interobserver difference was present. APd max and 3DLV measurements of PPE AAA significantly correlated. However, changes in the AAA morphology were detected earlier with 3DLV. Conclusion APd max and 3DLV are both reliable methods for measuring an AAA. Both these methods correlate with each other. However, changes in AAA morphology were detected earlier with 3DLV, which is important to detect subtle but important changes to aortic geometry in a laboratory setting. 3DLV measurement of AAA is a simple, reproducible, and comprehensive method for assessing changes in disease morphology.