Pharmacologic Inhibition of Ferroptosis Attenuates Experimental Abdominal Aortic Aneurysm Formation

Abstract

Background:

The pathogenesis of abdominal aortic aneurysm (AAA) formation involves vascular inflammation, thrombosis formation and programmed cell death leading to aortic remodeling. In this study, we deciphered the role of ferroptosis, an excessive iron-mediated cell death in macrophages during aortic inflammation and vascular remodeling in AAA formation.

Methods:

Single cell RNA sequencing analysis was performed on human AAA tissue database. AAAs were induced in male and female C57BL/6 (WT) mice using two models with topical elastase or elastase+BAPN, with or without liproxstatin-1, a specific ferroptosis inhibitor, treatment. Aortic diameter, cytokine expression, histology, hallmarks of ferroptosis such as lipid peroxidation (MDA) and glutathione (GSH), and lipid analysis using mass spectrometry were measured in aortic tissue extracts. In vitro studies deciphered crosstalk of macrophages and smooth muscle cells (SMCs) and analyzed ferroptosis and matrix metalloproteinase (MMP) expressions.

Results:

Single cell-RNA sequencing analysis demonstrated significant differences in ferroptosis-related genes in macrophages from human AAAs compared to control aortic tissue. Using two established murine models of AAA and aortic rupture in WT mice, we observed that treatment with liproxstatin-1 significantly attenuated aortic diameter, pro-inflammatory cytokine production, immune cell infiltration (neutrophils and macrophages), elastic fiber disruption and increased smooth muscle cell α-actin expression compared to untreated mice.. Lipidomic analysis using mass spectrometry shows a significant increase in ceramides and a decrease in intact lipid species levels in murine AAA tissue compared to controls in the murine AAA model. Mechanistically, in vitro studies demonstrate that liproxstatin-1 treatment of macrophages mitigated ferroptosis and MMP9 expression, as well as the crosstalk with aortic smooth muscle cells (SMCs) by downregulating MMP2 secretion.

Conclusions:

Taken together, this study demonstrates that pharmacological inhibition by liproxstatin-1 mitigates macrophage-dependent ferroptosis contributing to inhibition of aortic inflammation and remodeling during AAA formation.

Graphical Abstract

INTRODUCTION

Abdominal aortic aneurysm (AAA) is a vascular disease that causes dilation of the aorta which can lead to aortic rupture and sudden death.^1–4 Aneurysms can occur at any location of the aorta, but the abdominal aorta is the most common site.⁵ The feared complication of AAA rupture carries a mortality rate between 65%−95% and accounts for 16,000 annual deaths.^{6, 7} One of the strongest predictors of rupture is maximum aortic diameter, with a five-year rupture rate of at least 25% for aneurysms larger than 5.0 cm.⁸ Currently, there are no directed medical therapies for AAA prevention or treatment. Therefore, there is an urgent need to develop novel targeted therapies that can attenuate aortic growth and prevent rupture.

The mechanism for AAA formation involves leukocyte recruitment and infiltration into the aortic wall media and adventitia with subsequent excessive production of pro-inflammatory cytokines and matrix degrading enzymes.^9–11 This activates programmed cell death pathways including apoptosis and non-apoptotic pathways like neutrophil extracellular traps (NETs) and pyroptosis, that cause progressive thinning of the aortic wall, increasing the likelihood of rupture.^12–14 The role of ferroptosis, a form of iron-dependent programmed cell death, in AAA formation has yet to be fully elucidated.¹⁵ Ferroptosis is triggered by oxidative damage and characterized by the accumulation of lipid peroxides in the context of increased reactive oxygen species (ROS) generation and inactivation of GSH peroxidase 4 (GPX4), a glutathione (GSH) dependent enzyme that prevents lipid peroxidation.^16–18 It is influenced by structurally diverse small molecules (e.g. erastin, sulfasalazine, and RSL3) and also prevented by lipophilic antioxidants (liproxstatins, ferrostatins, CoQ10, vitamin E) offering a source of therapeutic potential.^{19, 20} As excessive thrombus formation and iron deposition are hallmarks of chronic AAA, an excessive iron-mediated cell death is hypothesized to immunomodulate AAA formation with a potential for therapeutic interventions using specific inhibitors of ferroptosis ^{21, 22 23}.

We have previously shown that expression of key ferroptosis markers including increased iron content, lipid peroxidation (malondialdehyde; MDA), and depletion of GSH are present in aortic tissue of experimental murine AAAs.²⁴ Furthermore, in vitro studies have shown that additional markers of ferroptosis including ROS production and Nrf2 nuclear translocation are significantly increased in elastase treated macrophages.²⁴ Importantly, we have shown that proresolving lipid mediators such as Resolvin D1 can inhibit macrophage-dependent ferroptosis, supporting the notion that ferroptosis represents a suitable pathway for targeted pharmacologic therapy ²⁴.

One of the critical hallmarks of human AAA tissue is the formation of intraluminal thrombus that is a predominant feature of proteolysis resulting in the degradation and destabilization of the aortic wall ²⁵. Several components of the thrombus formation include erythrocyte trapping, hemagglutination in the thrombus, as well as ferrous iron (Fe2+), can contribute to the altered homeostasis of the vasculature. Accordingly, in this study, we evaluated the hallmarks of increased iron-mediated cell death and associated inflammatory signaling during ferroptosis, using human AAA tissue and elucidated the role of ferroptosis in murine models of chronic, thrombus forming AAA and preformed aneurysms. We hypothesize that accumulation of dead cell debris and thrombus formation leads to elevated iron and lipid peroxidation (ferroptosis), that triggers immune cell-parenchymal cell crosstalk leading to vascular remodeling, and that pharmacologic inhibition of ferroptosis attenuates ferroptosis-mediated AAA growth and prevents impending rupture.

MATERIALS AND METHODS

Data Availability Statement

Original data that support the findings of this study are available from the corresponding author upon reasonable request and approval of a research proposal. Single cell sequencing data was analyzed from published datasets available in the Gene Expression Omnibus under accession code GSE166676, as described below.

Human Single Cell RNA Sequencing

Single cell RNA-seq data set of human AAAs and controls was performed from Gene Expression Omnibus (GSE166676) and re-analyzed via Seurat²⁶. Cell cluster annotation was performed by extracting cluster-specific marker genes using the FindMarkers function in Seurat. Cell annotations were assigned by cross-referencing these marker gene sets and their expression in published human and mouse cell atlases via PanglaoDB²⁷. Cell annotations were verified via expression of putative cell markers for macrophage and SMC clusters as described in previously published analyses of scRNA datasets of the aorta^{28, 29}. Differential gene expression analysis in macrophage and SMC clusters between human AAA and controls were performed via the FindMarkers function in Seurat and statistical significance was assessed using the Wilcoxon Rank Sum Test. Differentially expressed genes (DEGs) were identified as genes with adjusted p-value < 0.05 and |fold change| > 2. A list of 1,317 ferroptosis related genes (FRGs) were curated via GeneCards search (query = ferroptosis) and cross-referenced with our list of DEGs to identify differentially expressed FRGs (DE FRGs)^{30, 31}. Average expression of each DE FRGs was calculated via Seurat within sample and condition³². Data was scaled using the ScaleData function and used as values for Expression Heatmap³³.

Animals

Adult male and female 8–12-week-old C57BL/6J WT mice were used in this study (Jackson Laboratory, Bar Harbor, ME). Since AAA formation is known to demonstrate sex specific differences with a significant male preponderance, majority of the experiments used male mice. A separate cohort of female mice were used to confirm key findings (Supplemental Figure S2). Mice were housed in a temperature-controlled room at 25°C in 12-hour light-dark cycles as per institutional animal protocols. Mice were provided drinking water and standard chow diet ad libitum. All Animal experiments followed protocol approved by the University of Florida’s Institutional Animal Care and Use committee (#20220000546).

Murine AAA Model Using Topical Elastase Treatment

C57BL/6 (wild-type; WT) mice were anesthetized using isoflurane and underwent exposure of the infrarenal abdominal aorta, as previously described ^{24, 34}. The aorta was dissected circumferentially away from the surrounding tissues and subjected to 3 minutes of either 5 µL of peri-adventitial elastase (0.4 U/mL type 1 porcine pancreatic elastase, Sigma Aldrich, St. Louis, MO) or heat-inactivated elastase as control. Separate groups of animals were intraperitoneally (i.p.) injected with either liproxstatin-1 (10mg/kg; Cayman Chemicals), erastin (10mg/kg, 50mg/kg, or 100mg/kg; Cayman Chemicals), or vehicle control (saline) from the day of the surgery (day 0) through post-operative day 7. The selection of liproxstatin-1 dosages were based on published literature ³⁵.

Aortas were harvested on day 14 after elastase application. Mice from each group were euthanized under anesthesia by overdose and exsanguination. The abdominal aorta, from below the left renal vein to the bifurcation was dissected. The external aortic adventitia diameter was measured at its maximum diameter and at the intact self-control portion just below the left renal vein using video microscopy with NIS-Elements D.5.10.01 software (Nikon SMZ-25, Melville, NY)²⁵. The aortic dilation percentage was determined using (maximal AAA diameter − self-control aortic diameter)/(maximal AAA diameter) × 100%. Aortic sections were harvested and preserved in formalin for immunohistochemistry or snap-frozen in liquid nitrogen and stored at −80 °C.

Chronic AAA and Aortic Rupture Model

In a second chronic AAA model linked to thrombus formation and aortic rupture³⁶, male mice were treated with 3 minutes of peri-adventitial application of either 5 µL of peri-adventitial elastase (0.4 U/mL type 1 porcine pancreatic elastase, Sigma Aldrich, St. Louis, MO) or heat-inactivated elastase as control. Mice were given drinking water containing 0.2% Beta-Aminopropionitrile (BAPN). Separate groups of mice were treated with liproxstatin-1 (10mg/kg administrated i.p.; Cayman Chemicals) or vehicle control (saline) daily from post-operative day 14 through post-operative day 27. Animals were euthanized on day 28 and aortic tissue was harvested as described above.

Lipidomic Analysis

Aortic samples were extracted using the Bligh-Dyer extraction method. Chromatographic separation was performed on an ultra-high-performance liquid chromatography (UHPLC) system (Dionex UHPLC; Thermo Scientific, San Jose, CA) using ACQUITY UPLC BEH C18 1.7 µm, 2.1mm x 50 mm column with ACQUITY UPLC BEH C18 VanGuard Pre-column 1.7 µm, 2.1 mm x 5 mm with a gradient program consisting of mobile phase A (60:40 acetonitrile/water) and mobile phase B (90:10 isopropanol/acetonitrile) with 10 mmol/L ammonium formation and 0.1% formic acid was used for sample analysis. The UHPLC system was coupled to a mass spectrometer (Q-Exactive Orbitrap; Thermo Scientific) to perform global lipidomic profiling in both positive and negative ionization modes through full-scan and iterative exclusion tandem mass spectrometry. For processing the data, LipidMatch Flow was utilized to manage peak identification, remove background noise, annotate, and merge results for both ion polarities. Semi-quantitative analysis was carried out using LipidMatch Normalizer.

Cytokine assay

Using isolated protein from murine abdominal aortas, cytokine panel assay (Bio-Rad Laboratories, Hercules, CA) was performed according to manufacturer instructions. High Mobility Group Box 1 (HMGB1) was measured in cell culture supernatants using an ELISA kit, following the manufacturer’s instructions (IBL International, Hamburg, Germany).

Histology

Aortic tissue was fixed in 4% buffered formaldehyde for 24 hours and embedded in paraffin and sectioned at 5µm. Immunostaining was performed for elastin (Verhoeff-van Gieson), neutrophils, macrophages, and smooth muscle α-actin, as previously described³⁶. Antibodies for immunohistochemical staining were anti-mouse Mac2 for macrophages (1:10,000, Cedarlane Laboratories, Burlington, ON, Canada; catalog no. CL8942AP), anti-mouse neutrophils for polymorphonuclear neutrophils (PMNs) (1:10,000, BioRad; catalog no. MCA771GA), anti-mouse α-smooth-muscle-actin (α-SMA, 1:1000, Sigma, St. Louis, MO; catalog no. A5691). Isotype IgG2a control antibody was used as negative control for immunostaining and murine spleen tissue or heat-inactivated elastase-treated aortic tissue was used as positive control. Histological analysis was performed on three different sections of aortic tissue from each animal and quantified using two independent observers. Representative images from different animals from each group are depicted for accuracy. Images were acquired with 20x magnification by an Olympus microscope equipped with a digital camera using NIS-Elements D.5.10.01 software (Nikon SMZ-25, Melville, NY). For grading, the positive staining area of the entire aortic tissue sample was selected and measured using integrated optical density of each section¹². The histological expression was quantified in aortic tissue sections using QuPath (v0.5) bioimage analysis software³⁷. Briefly, brightfield-stained images were imported, and the tissue was annotated manually to include the whole aortic tissue. Positive cell detection was designated using optimized threshold settings for optical density to identify positive regions. The percentage of positive staining area relative to the total annotated area was calculated and reported as positively stained % area. All analysis parameters were applied consistently across all samples.

In vitro experiments

Primary F4/80+ macrophages were isolated from WT male mice following the manufacturer’s instructions (Miltenyi Biotec, Germany). Primary aortic smooth muscle cells (SMCs) were purified from WT male mice as previously described.³⁸ Macrophages were subsequently exposed to 5 minutes of elastase treatment followed by washing the cells with PBS and replacing the media with/without liproxstatin-1 (10nM).²⁴ Lipid peroxidation (MDA; Millipore Sigma, St. Louis, MO) and glutathione (GSH; Cayman Chemicals, Ann Arbor, MI) were measured in tissue or cell culture extracts per the manufacturer’s instructions using colorimetric assay kits. Nrf2 transcription factor activation in nuclear extracts was measured using a colorimetric assay kit (Abcam, Cambridge, UK). Conditioned media transfer (CMT) experiments using macrophages and SMCs were also performed as previously described.²⁴ Macrophages from WT mice were grown to confluency in 6-well plates and exposed to transient elastase treatment with/without liproxstatin-1 treatment for quantifying matrix metalloproteinase-9 (MMP9) expression (Milliplex immunoassay kit; Millipore Sigma, Burlington, MA) . Additionally, in separate groups of elastase-treated macrophages, CMT was performed from macrophages to SMC cultures after 6 hrs and MMP2 activity was measured at 24 hours (Luminex bead array, Millipore Sigma).

Gelatin zymography for matrix metalloproteinase expression

Matrix metalloproteinases 2 (MMP2) and MMP9 activities were measured in aortic tissue from mice. Aortic samples were loaded onto a zymogram gel (Thermo Fisher Scientific) with 3 µg of isolated protein per lane. Protein samples were loaded individually, separated by gel electrophoresis, and then renatured with buffer (Thermo Fisher Scientific) for 30 minutes. The resulting gel was placed in a developing buffer and then stained with SimplyBlue SafeStain (Thermo Fisher Scientific), and bands were assessed by the Bio-Rad Image Lab 4.0 software (Bio-Rad).

Statistical Analysis

All experimental results derive from sample sizes that represent biological replicates and data are expressed as mean ± SEM. The parametric data were first analyzed using a Shapiro-Wilk normality test to determine data distribution. Multiple group comparisons were made using one-way ANOVA with Tukey’s posthoc analysis if they passed the normality test, or Kruskal-Wallis 1-way ANOVA if the normality test failed. Student t- test or the Mann-Whitney U test Rank-Sum test was used to compare data between two groups, depending on the result of normality tests. All data were analyzed using GraphPad Prism program (Version 10) and p≤0.05 was considered statistically significant.

RESULTS

Single-cell RNA-sequencing analysis reveals dysregulation of ferroptosis-related genes in AAA patients

To evaluate for evidence of ferroptosis dysregulation in human AAA, we analyzed single-cell RNA sequencing data from AAA patients and controls using a previously reported sequencing dataset (GSE1666676), and identified differences in ferroptosis-related gene (FRG) expression³⁹.Data was analyzed via Seurat and the macrophage or SMC clusters were identified for further analysis. Differential expression analysis comparing macrophages or SMCs from human AAAs (n=4) vs control aortas (n=2) was performed via FindMarkers calculated using Wilcoxon Rank Sum Testing. Genes with adjusted p-value < 0.05 and |fold-change| > 2 were considered significant (DEGs). A list of ferroptosis-related genes (FRGs) were generated using GeneCards and used to identify differentially expressed ferroptosis-related genes (DE FRGs). A total of 1530 genes were detected within the macrophage cluster and tested for differential expression (Figure 1A-B). Of the 1530 genes, 139 genes were downregulated, 147 were upregulated, and 1244 genes were unchanged (Figure 1C-D and Supplementary Table S1). Similarly, SMC cluster analysis demonstrated that 211 genes were downregulated, 164 were upregulated, and 1350 genes were unchanged between AAA and control tissue (Supplementary Figure S1 and Supplementary Table S2). These findings in human AAA tissue prompted the further exploration of ferroptosis and its role in preclinical models of AAA.

Figure 1. Ferroptosis-related genes are dysregulated in macrophages of human AAAs.

Bioinformatic analysis was performed on human AAA and control tissue and the macrophage cluster was isolated using Seurat. A, Uniform manifold projection (UMAP) plot of the annotated clusters from human AAAs and controls. The macrophage cluster was identified and subsetted for further analysis. B, Volcano plot displaying significantly downregulated (sky blue) and upregulated (orange) genes. DE FRGs are highlighted and colored (navy blue). C, Venn diagram displaying gene counts from this analysis. A total of 1530 genes were detected within the macrophage cluster and tested for differential expression. Of the 1530 genes, 139 genes were downregulated, 147 were upregulated, and 1244 genes were unchanged. 14/139 downregulated genes and 16/147 upregulated were related to ferroptosis. D, Heatmap of all 30 DE FRGs in macrophages displayed as average expression within each sample or aggregated between conditions (control vs AAA).

Pharmacologic inhibition of ferroptosis by Liproxstatin-1 decreases AAA formation

With this initial evidence that ferroptosis was involved in AAA, we sought to determine if pharmacologic inhibition could attenuate AAA formation in murine AAA models. First, we used the murine elastase AAA model in which aortic diameter was significantly increased in elastase-treated WT male mice compared to shams (121±12.2% vs. 1.99±3.3%; 0.74±0.04 vs. 0.34±0.01 mm; p<0.0001) (Figure 2A). Administration of liproxstatin-1 significantly attenuated aortic diameter compared to untreated mice on day 14 (78.4±12.9% vs. 121±12.2%; 0.57±0.03 vs. 0.74±0.17mm; p=0.02; Figure 2B-C). Similarly, liproxstatin-1 treated female mice also displayed a significant decrease in aortic diameter compared to untreated controls (Supplementary Figure S2A). There was no difference in aortic diameter of control (heat-inactivated elastase treated) male mice with or without liproxstatin-1 treatment (Supplementary Figure S2B). Conversely, mice were treated mice with erastin, a ferroptosis agonist, to evaluate the exacerbation of AAA formation. However, erastin treatment was not associated with increased aortic diameters on day 14 in the elastase-treated mice compared to elastase alone (Supplementary Figure S3).

Figure 2. Pharmacologic inhibition using Liproxstatin-1 mitigates AAA formation in the topical elastase murine AAA model.

A, Schematic description of the elastase treatment model of AAA. Male WT mice were treated with elastase or deactivated elastase (heat-inactivated; controls) with/without administration of liproxstatin-1 and aortic diameter was measured on day 14, followed by harvest of aortic tissue for further analysis. B, Elastase-treated mice administered with liproxstatin-1 demonstrated a significant decrease in aortic diameter compared with elastase-treated WT mice alone. *p<0.01; n=10–15/group; 1-way ANOVA C, Representative images of aortic phenotype in all groups. Scale bar=500μM.

Pharmacological inhibition of ferroptosis attenuates aortic inflammation and remodeling

Comparative histology and immunostaining of aortic tissue demonstrated distinct inflammatory cell infiltration profiles in liproxstatin-1 treated mice compared to untreated mice in the murine elastase AAA model on day 14 (Figure 3). On histological analysis elastase-treated mice demonstrated increased infiltration of neutrophils and macrophages, as well as elastin degradation, and decreased SMα-actin expression compared to sham controls. Importantly, liproxstatin-1-treated mice demonstrated preserved aortic morphology as demonstrated by mitigation of leukocyte infiltration and elastin degradation as well as increase in smooth muscle α-actin expression compared to elastase-treated mice alone (Figure 3A-B). Additionally, a significant reduction in pro-inflammatory cytokine and MMP9 expression was observed in the aortic tissue of liproxstatin-1-treated mice compared to untreated controls (Figure 4A and Supplementary Figure S4A). However, there was no significant difference in overall MMP2 expression in liproxtatin-1 treated mice compared to untreated mice (Supplementary Figure S4B). Moreover, aortic tissue of elastase-treated mice showed hallmarks of ferroptosis as demonstrated by a significant increase in lipid peroxidation (MDA) and decrease in GSH expression compared to sham controls, which were significantly altered in liproxstatin-1-treated mice as observed by decrease in MDA (0.11± 0.04 vs. 0.75± 0.1 nmol/mg; p<0.01) and increase in GSH expressions (12.1±1.2 vs. 4.7±0.7 µg/mg; p=0.04) (Figure 4B-C). Moreover, liproxstatin-1 treatment also inhibits total iron content in aortic tissue of elastase-treated mice compared to untreated controls (Supplementary Figure S5).

Figure 3. Liproxstatin-1 treatment preserves aortic morphology in the topical elastase murine AAA model.

A, In liproxstatin-1 treated WT male mice, comparative histology demonstrated increased SMα-actin expression, decreased elastin fiber disruption, and reduced immune cell infiltration (neutrophil and macrophage staining) compared to untreated mice. Arrows point to areas of immunostaining. Scale bar=100μM. B, Quantification of immunohistochemical staining demonstrates increased SMα-actin staining, and reduced elastin breaks, macrophage, and PMN staining in liproxstatin-1 treated tissue compared to mice treated with elastase alone. *p<0.05, **p<0.01; ns, not significant; n=4–7/group, 1-way ANOVA.

Figure 4. Liproxstatin-1 treatment reduces aortic inflammation, remodeling and ferroptosis during AAA formation.

A, Pro-inflammatory cytokine and MMP9 expressions in aortic tissue of male WT mice treated with liproxstatin-1 were significantly attenuated compared to elastase treatment alone. *p<0.01 vs. other groups; n=5/group, 1-way ANOVA.B, Glutathione depletion was significantly decreased in elastase-treated mice compared to controls. However, administration of liproxstatin-1 increased the GSH levels compared to untreated mice. *p<0.01 vs. other groups; n=5/group, 1-way ANOVA. C, Lipid peroxidation (MDA) expression in aortic tissue was significantly mitigated in liproxstatin-1 treated mice compared to elastase treatment alone. *p<0.01 vs. other groups; n=5/group, 1-way ANOVA.

Inhibition of ferroptosis decreases pre-formed aneurysm growth

To decipher the role of ferroptosis inhibition in a pre-formed AAA, we used a second, chronic inflammatory elastase+BAPN AAA and aortic rupture model that is associated with thrombus formation.^.25 To evaluate the protective effect of ferroptosis inhibition in a pre-formed AAA , mice were treated with liproxstatin-1 from postoperative days 14–27, after a small aneurysm formation (Figure 5A). Mice exposed to elastase+BAPN demonstrated a significant increase in aortic diameter on day 28 compared to sham controls (data not shown). The sham control mice on day 28 display similar characteristics for aortic diameter as the heat-inactivated controls as demonstrated above (data not shown). More importantly, liproxstatin-1-treated mice had significantly reduced aortic diameters compared to elastase+BAPN treated mice alone (246.7±21.3 vs. 459.7±52.5.; 1.15±0.08 vs. 1.77± 0.17mm; p<0.006) (Figure 5B-C). Importantly, liproxstatin-1 treated mice protected against aortic ruptures (incidence rate; 0/22 mice) compared to untreated controls (incidence rate; 2/20 mice) signifying the importance of ferroptosis inhibition in protection against AAA formation and aortic rupture.

Figure 5. Liproxstatin-1 administration mitigates pre-formed AAAs.

A, Schematic depicting the chronic AAA model with elastase+BAPN treatment in male WT mice. Liproxstatin-1 treatment was initiated on post-operative day 14 till day 27 and analysis was performed on day 28. B, Aortic diameter is significantly attenuated in liproxstain-1 treated mice compared to elastase+BAPN treated mice on day 28. *p=0.0007; n=20–21/group, Mann-Whitney U test. C, Comparative representative of aortic phenotype in each group. Scale bar=500μM. D, Liproxstatin-1 treatment preserves aortic morphology in the chronic murine AAA model. In liproxstatin-1 treated mice, comparative histology demonstrated increased SMα-actin expression, decreased elastin fiber disruption, and reduced immune cell infiltration (neutrophil and macrophage staining) compared to untreated mice. Arrows indicate areas of immunostaining. E, Quantification of immunohistochemical staining demonstrates increased SMα-actin staining, and reduced elastin breaks, macrophage, and PMN staining in liproxstatin-1 treated tissue compared to mice treated with elastase+BAPN alone. Scale bar=100μM.*p<0.05; n=5/group, unpaired t-test.

Liproxstatin-1 treatment of pre-formed AAA mitigates aortic inflammation and aortic remodeling

Comparative histology and immunostaining demonstrated marked decrease in inflammatory cell infiltration in mice treated with liproxstatin-1 compared to untreated mice after elastase+BAPN administration. Liproxstatin-1 treated mice demonstrated significant mitigation of neutrophil (7.12±1.2 vs. 13.2±1.9, p=0.03) and macrophage infiltrations (11.0±0.8 vs. 21.1±2.3, p=0.003), as well as increased SMα-actin expression (24.9±2.9 vs. 13.7±2.4, p=0.02) and attenuation of elastin breaks (12.8±1.6 vs. 19.0±1.8, p=0.04) compared to untreated mice (Figure 5D-E). Furthermore, aortic tissue of liproxstatin-1 treated mice demonstrated attenuation of pro-inflammatory cytokine and MMP9 expression compared to untreated mice (Figure 6A). Additionally, liproxstatin-1 treated mice demonstrated increased GSH (1.96±0.1 vs. 0.990±0.1 nmol/mg; p=0.002), and decreased MDA (3.03±0.8 vs. 11.0±1.4; p=0.001) expressions in aortic tissue compared to elastase+BAPN-treated mice (Figure 6B-C). These data demonstrate that liproxstatin-1 administration can effectively inhibit ferroptosis and decrease the growth of pre-formed AAAs.

Figure 6. Liproxstatin-1 treatment reduces aortic inflammation and hallmarks of ferroptosis in the chronic AAA model.

A, Aortic tissue from liproxstatin-1 administered male WT mice in the elastase+BAPN-treated group showed significant decrease in pro-inflammatory cytokine/chemokine production and MMP9 expressions compared to elastase+BAPN-treated WT mice. *p<0.03, n=5/group, unpaired t-test. B, Elastase+BAPN treatment significantly depletes GSH levels in aortic tissue which is mitigated in mice treated with liproxstatin-1. *p<0.01; n=5/group, unpaired t-test. C, MDA expression in aortic tissue is significantly reduced in aortic tissue treated with liproxstatin-1 compared to untreated controls. *p<0.01; n=5/group, unpaired t-test.

Ferroptosis-specific lipids are upregulated in experimental murine AAAs

We then sought to determine if there was evidence of ferroptosis-specific lipid breakdown products in a chronic, thrombus forming murine AAA and aortic rupture model (Figure 7A)³⁶. Lipidomic analysis of aortic tissue on day 28 demonstrates a significant association between ferroptosis and AAA growth. We observed significant decrease in the levels of intact lipid species in AAA mice compared to sham controls (Figure 7B). Specifically, we found decreased levels of intact lipid species such as triglycerides (TG), sphingomyelin (SM) and monogalactosyldiacylglycerol (MGDG) in aortic tissue of mice with AAAs compared to controls. Furthermore, there was significant increase in the levels of sphingosine (SO), a breakdown product of SM, as well as ceramide expression, that are activated lipids known to be associated with ferroptosis⁴⁰, in aortic tissue of AAAs. Additionally, phospholipids with higher degree of unsaturation that render the membrane more susceptible to lipid peroxidation, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were significantly increased in aortic tissue of mice with AAAs compared to controls (Figure 7B-C and Supplementary Table S3).

Figure 7. Liproxstatin-1 treatment protects against lipid oxidation during AAA formation.

A, Schematic description of the elastase+BAPN treatment in male WT mice model of AAA followed by harvest of aortic tissue for lipidomic analysis on day 28. B, Heatmap from untargeted lipidomic analysis depicting overview of lipid profiles between groups. n=6/group. C, Principal component analysis (PCA) to determine main differences in lipid profiles between experimental groups. D, Schematic description of the elastase+BAPN treatment model of AAA with or without liproxstatin-1 treatment followed by harvest of aortic tissue for lipidomic analysis on day 28. E, Heatmap from lipidomic analysis depicting overview of lipid profiles after liproxstatin-1 treatment. n=4–6/group. F, Principal component analysis to determine oxidized lipid perturbations after liproxstatin-1 treatment. Ox, oxidized; TG, triglycerides; MGDG, monogalactosyldiacylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; DG, diacylglycerols; MG, monoglyceride; SM, sphingomyelin; So, sphingosine; Cer, Ceramide.

Next, we tested the effect of pharmacological inhibition of ferroptosis on the lipidomic profile in AAA mice (Figure 7D). Our results showed that liproxstatin-1, a specific ferroptosis antagonist, offered protection against ferroptosis-related oxidized lipids in AAA. We observed a significant reduction in the levels of ceramides, PE and PC species with a higher degree of unsaturation, after liproxstatin-1 treatment compared to untreated controls (Figure 7E-F and Supplementary Table S4). Additionally, there was a significant downregulation in the levels of oxidized PE in liproxstatin-1 treated mice (Figure 7E). Collectively, these results indicate the involvement of ferroptosis in AAA and the efficacy of liproxstatin-1 to attenuate ferroptosis-specific targets in the aortic tissue of mice in the chronic AAA and rupture model.

Liproxstatin-1 treatment mitigates ferroptosis in macrophages to mitigate SMC activation

To evaluate the cell-specific role of ferroptosis in AAA formation, we focused on the role of macrophages and SMCs to delineate the crosstalk between immune cell-dependent inflammation and parenchymal cell-mediated vascular remodeling in the pathogenesis of AAAs. As we had previously shown that macrophages participate as mediators of ferroptosis and histological changes were most pronounced at the smooth muscle cell predominant media layer, we hypothesized that macrophage-SMC crosstalk was important for AAA formation²⁴. We first evaluated for hallmarks of ferroptosis including lipid peroxidation (MDA) and GSH depletion in macrophages exposed to transient elastase with and without liproxstatin-1 treatment. Liproxstatin-1 treated macrophages display decreased lipid peroxidation, restoration of glutathione levels, impediment of NRF2 translocation, and reduced expression of pro-inflammatory cytokine, HMGB1 as well as MMP9 compared to untreated controls (Figure 8A-E).

Figure 8. Liproxstatin-1 treatment mitigates macrophage mediated smooth muscle cell activation and MMP2 activation.

A-D, Hallmarks of ferroptosis including increased lipid peroxidation (MDA) and glutathione depletion, as well as increased nuclear translocation of Nrf2 in cell extracts and secretion of HMGB1 in culture supernatants were observed in macrophages treated with elastase. Liproxstatin-1 treated macrophages display mitigated lipid peroxidation, restoration of glutathione levels, impediment of Nrf2 translocation, and reduced HMGB1 expression. *p<0.01 vs. other groups; n=8/group, 1-way ANOVA. E, Liproxstatin-1 treated macrophages significantly attenuated elastase-treatment induced MMP9 expression compared to untreated controls. n=8/group, 1-way ANOVA. F-G, Conditioned media transfer (CMT) from elastase-exposed macrophages induced an increase in MMP2 expression which was mitigated by liproxstatin-1 treatment. *p<0.05 vs. other groups; n=8/group, 1-way ANOVA.

Next, we performed conditioned media transfer of elastase-exposed macrophages to SMCs with and without liproxstatin-1 treatment (Figure 8F). Mitigation of MMP2 activity and LDH release from SMCs was observed after CMT from liproxstatin-1 treated macrophages to SMCs compared to untreated controls (Figure 8G and Supplementary Figure S6). Conversely, CMT from elastase-treated SMCs to macrophages did not result in production of key inflammatory cytokines such as HMGB1, TNF-α and LDH release (data not shown). Moreover, induction of ferroptosis with erastin exacerbated elastase-exposed macrophage production of HMGB1 and TNF-α, which was significantly mitigated by liproxstatin-1 treatment (Supplementary Figure S7). Taken together, these results suggest that pro-inflammatory paracrine secretions from activated macrophages secondary to ferroptosis trigger SMC remodeling. This crosstalk can be significantly mitigated by inhibition of macrophage-dependent ferroptosis by treatment with liproxstatin-1, which then attenuates vascular remodeling and reduces the growth of AAAs.

DISCUSSION

This study demonstrates that excess iron-mediated cell death via ferroptosis is an important signaling event that triggers crosstalk between macrophages and SMCs to alter vascular inflammation and remodeling during AAA formation. Single cell RNA analysis of human AAA tissue demonstrated dysregulation of several ferroptosis associated-genes providing new avenues for targeted therapy. Importantly, in vivo and in vitro studies demonstrate the ability of liproxstatin-1 to pharmacologically modulate macrophage-mediated ferroptosis and mediate the balance of oxidized and intact lipid species that are the end-products of ferroptosis resulting in decreased growth of AAAs. Collectively, these results suggest an important role of ferroptosis in AAA and the efficacy of liproxstatin-1 to mitigate AAA formation and prevent aortic rupture.

The mechanism for AAA formation involves chronic damage to the aortic wall from increased production of pro-inflammatory cytokines and matrix degrading enzymes.^{9, 10} These increased inflammatory enzymes promote cell death pathways that weaken the aortic wall and contribute to AAA rupture.^{15, 24} Ferroptosis, an iron-mediated non-apoptotic cell death pathway, has recently generated significant interest in mediating the pathogenesis of cardiovascular diseases. The mechanistic role of ferroptosis may be particularly relevant to AAA due to the association between excess iron and intraluminal thrombus that often accompanies AAA.^{21, 22} In ferroptosis, intracellular transport of iron ultimately results in the formation of a oxidized lipids that increase cellular oxidative stress and generation of reactive oxygen species.^{13, 17, 18} These lipid hydroperoxides contribute to cell death via NADPH oxidase activation, a key enzyme in redox activation, which we have previously demonstrated to be involved in macrophage-dependent activation and HMGB1 secretion, thereby providing a link to pathogenesis and growth of aneurysms.^41–43

Recent studies have demonstrated the ability of pharmacological inhibition of ferroptosis using liproxstatin-1 to prevent BAPN-mediated aortic dissection in mice.⁴⁴ In the context of AAA, Ferrostatin-1, another pharmacologic inhibitor of ferroptosis, has been shown to reduce AAA size through GPX4 mediated vascular smooth muscle cell (VSMC) activation in the angiotensin II murine AAA model.⁴⁵ Liproxstatin-1 has been shown to be more effective in subverting ferroptosis due to its reaction stoichiometry to trap peroxyl radicals in lipid bilayers.⁴⁶ Using the topical elastase model, we had previously demonstrated phenotypic changes and alterations in expression of key ferroptosis markers like MDA excess and glutathione depletion in murine aortas.²⁴ Additional studies using the topical elastase model have demonstrated that PKM2-activated T lymphocyte-derived extracellular vesicles can mediate topic elastase induced AAA formation, which is then attenuated with liproxstatin-1.⁴⁷ However, few studies have studied the role of ferroptosis inhibition in a chronic or thrombus forming AAA model.

Induction of ferroptosis can affect glutathione peroxidase causing decreased intracellular antioxidant capacity and lipid ROS accumulation, that orchestrates redox imbalance and thrombus formation. These cellular events promote degradation of the extracellular matrix as well as smooth muscle cell remodeling during the aortic growth and progression of AAAs.⁴⁸ Glutathione metabolism can be altered by sphingolipids, a class of lipids, including ceramides, that can regulate cell proliferation, differentiation, apoptosis, and inflammation during aneurysm formation⁴⁹. Results from our study shows altered levels of ceramides and decreased PC and PE lipid species with higher degree of unsaturation, indicating a pivotal association of ferroptosis with lipid peroxidation in the tissue during vascular aortopathies. These oxidized lipids can lead to aortic wall injury and thrombus formation, resulting in the accumulation of immune cells and promoting the release of proinflammatory cytokines, which can aggravate vascular injury forming a self-amplified loop leading to uncontrolled growth and rupture. Key immune cells that are known to be involved in AAA formation include macrophages, neutrophils, and CD4+ T cells that orchestrate an inflammatory milieu affecting the aortic wall remodeling.⁵⁰ Since the contributory role of ferroptosis in SMCs has been previously reported, this study focused on delineating the mechanistic pathways preceding SMC activation by immune cells such as macrophages.⁵¹ Excess of iron-mediated sources of macrophages include phagocytosis of senescent RBCs to produce Fe²⁺, or extracellular Fe³⁺ that enters into macrophages through TFR and is reduced to Fe²⁺.⁵² This excessive iron deposition triggers ferroptosis during conditions of altered homeostasis, which then affects paracrine secretions and surrounding parenchymal cells in the aortic tissue.

Mechanistically, we provide evidence for ferroptosis-triggered macrophages to activate SMCs and treatment with liproxstatin-1 mitigating the crosstalk between macrophages and SMCs. Ferroptosis shares several commonalities between macrophages, and iron metabolism may contribute towards M1 polarization.^{53, 54 55} Studies have also shown that macrophages can activate SMCs to secrete MMPs and contribute to AAA progression.^{24, 56, 57} We first demonstrated that elastase-treated macrophages exhibit increased amounts of NRF2 nuclear translocation, which is upregulated in periods of oxidative stress. The transcription factor NRF2 mediates antioxidant response element (ARE)-related genes that regulate the expression of enzymes involved in glutathione synthesis ⁵⁸. Importantly, synthesis and function of GPx4, intracellular iron homeostasis, and lipid peroxidation can be mediated by NRF2 target genes.⁵⁹ Modulation of NRF2 in macrophages by liproxstatin, and subsequent mitigation of MMP9 in macrophages as well as MMP2 in SMCs indicates a significant correlation of these key processes during progression of AAA formation. Previous studies have elucidated that MMP2 and −9 work in concert during aortic aneurysm formation^{57, 60}. MMP-9 is the most abundant gelatinolytic MMP in AAA tissue, that is secreted in copious amounts in AAA explants indicating macrophage invasion while MMP2 is correlated with SMCs and fibroblasts. Our data indicates that liproxstatin-1 mitigates MMP9 activity from macrophages and can downregulate MMP2 activity from SMCs in the crosstalk between macrophages and SMCs. This data indicates that the important contribution of MMP2 to vascular remodeling during AAA formation that is largely from aortic intima and medial SMCs, and not from other parenchymal cells like adventitial fibroblasts.

The clinical relevance of ferroptosis mediated pathways is highlighted by the cell-specific analysis of aortic tissue from AAA patients. A prior study using single cell RNA sequencing of 14 AAAs and 8 transplant donor control aortas evaluated differential expression of 24 ferroptosis-related genes and found dysregulation of several of these genes in the AAA neck.⁶¹ We instead used a dataset containing aneurysmal aortic tissue, and deciphered a larger set of ferroptosis-related genes.³⁹ This single-cell RNA-sequencing analysis showed that patients with AAA disease have dysregulation of multiple ferroptosis-related genes including AIFM2, ATF3, PKM, and CASC9, which have been shown to be ferroptosis-related and associated with liproxstatin-1 mediated signaling in murine models.^{44, 47, 62} By interactions with liproxstatin-1, these genes can directly influence GPX4 activation and effect anti-oxidative activity in AAA development.

There are several limitations to consider in this study. The experimental models used to decipher the alterations in oxidized lipids were not performed in the presence of hypercholesteremia which can be an accompanying factor in clinical AAAs. Further studies deciphering the role of metabolic changes in the aortic wall relative to thrombus formation and ferroptosis in the presence of increased cholesterol using angiotensin II and ApoE^−/− and LdlR^−/− mice should help address the immune-parenchymal cell interactions for lipidomic perturbations in vascular pathologies. Additionally, translational efforts to expand these experiments to our previously described porcine AAA model may provide an opportunity to bridge the gap for the use of pharmacological inhibitors of ferroptosis for protection against AAA growth^{63, 64}. Also, although erastin treatment did not exacerbate AAA growth in the murine in vivo model, the increase of erastin-induced macrophage activation denotes the ferroptosis mediated enhancement of aortic inflammation. Additional limitations include detailed evaluation of the human sequencing datasets to perform subcluster analyses comparing differential gene expression in endothelial, macrophage, and smooth muscle cells. Ongoing studies with a robust database of cell-specific analysis of ferroptosis and lipidomic related pathways in murine and human AAA samples are postulated to expand our understanding of this critical signaling pathway. Finally, in addition to liproxstatin, several compounds that mediate iron metabolism, such as deferoxamine, ACSL4 (acyl-CoA synthetase long-chain family member 4) inhibitors, or lipid antioxidants like Vitamin E and selenium may have the potential to mediate inhibitory effects on ferroptosis and can be investigated in future studies using additional confirmatory murine models such as angiotensin II-induced AAA model in ApoE knockout mice.

In summary, we have shown that pharmacological inhibition by liproxstatin-1 mitigates macrophage-dependent ferroptosis and decreases SMC activation that contributes to inhibition of aortic inflammation and aortic remodeling during AAA formation. Our results highlight the dysregulation of several ferroptosis-related genes in human AAA, providing potential interventional molecular targets for future therapy. Further efforts should focus on elucidating the anti-ferroptotic effects of commercially available antioxidants or drugs that alter iron metabolism as targeted therapy for degenerative AAA.

Highlights.

• Human AAAs involve significant alterations in ferroptosis-related genes that are specifically associated with macrophages.

• Experimental murine models demonstrate that liproxstatin-1 attenuates ferroptosis-related oxidized lipids to mitigate aortic inflammation and vascular remodeling during AAA formation and aortic rupture.

• Pharmacological inhibition of macrophage-dependent ferroptosis correlates with preservation of smooth muscle cell remodeling and restoration of vascular inflammation and integrity.

Nonstandard Abbreviations and Acronyms

AAA

abdominal aortic aneurysms

SMC

smooth muscle cells

MDA

malondialdehyde

HMGB1

high mobility group box 1

BAPN

β-Aminopropionitrile

MMP

matrix metalloproteinase

GSH

glutathione

scRNA-seq

single-cell RNA sequencing

FRG

ferroptosis-related genes

Nrf2

nuclear factor erythroid 2-related factor 2

PMN

polymorphonuclear neutrophils

VVG

verhoeff-van Gieson

IL

interleukin

CMT

conditioned media transfer