NCOR1 Deficiency in Macrophages Aggravates Aortic Aneurysm Formation via the ANGPTL4-ALDOA-MMP2 Axis

doi:10.21203/rs.3.rs-5295423/v1

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NCOR1 Deficiency in Macrophages Aggravates Aortic Aneurysm Formation via the ANGPTL4-ALDOA-MMP2 Axis

https://doi.org/10.21203/rs.3.rs-5295423/v1

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An aortic aneurysm (AA) is a life-threatening cardiovascular condition characterized by progressive aortic dilation and potential rupture. In this study, we determined the function of nuclear receptor corepressor 1 (NCOR1) in macrophages in AA development. First, macrophage-specific NCOR1 knockout (MNKO) mice were generated, followed by treatment with β-aminopropionitrile to induce AA. AA formation was exacerbated in MNKO mice, with increased aortic dilation, elastin degradation, and inflammatory cell infiltration. Furthermore, NCOR1 deficiency promoted M1 macrophage polarization and upregulated the expression of proinflammatory cytokines and matrix metalloproteinases (MMPs). Mechanistically, NCOR1 regulates the ANGPTL4-ALDOA-MMP2 signaling axis in macrophages. Particularly, NCOR1 directly binds to the ANGPTL4 promoter, suppressing its transcription. ANGPTL4 knockdown attenuates NCOR1 deficiency-induced ALDOA and MMP2 upregulation. These findings suggest that macrophage-specific NCOR1 deficiency exacerbates AA formation via enhanced inflammatory responses and extracellular matrix degradation. The identification of the NCOR1-ANGPTL4-ALDOA-MMP2 axis provides new insights into the molecular mechanisms underlying AA development and highlights the essential role of epigenetic regulation in maintaining aortic wall integrity. This discovery may serve as therapeutic targets for preventing and treating AA.

Aortic aneurysm

Macrophages

NCOR1

Inflammation

Extracellular matrix degradation

An aortic aneurysm (AA) is a life-threatening cardiovascular condition characterized by progressive aortic dilatation; this can result in rupture and fatal consequences[1]. A combination of genetic factors, blood flow dynamics, and multiple disease processes such as inflammation, extracellular matrix (ECM) degradation, and changes in the behavior of vascular smooth muscle cells affect AA development[2, 3]. Chronic inflammation and ECM remodeling are key factors associated with initiating and progressing AA[4].

Macrophages play a key role in the inflammatory response associated with AA development[5]. After recruitment to the aortic wall, macrophages can be polarized into different phenotypes: M1 macrophages promote inflammation, whereas M2 macrophages contribute to tissue repair[6]. The balance between these macrophage subtypes is essential for maintaining vascular homeostasis. In AA, the proinflammatory M1 phenotype is often present in excess, increasing the levels of inflammatory cytokines and matrix-degrading enzymes such as matrix metalloproteinases (MMPs)[7].

As an evolutionarily conserved transcriptional corepressor, nuclear receptor corepressor 1 (NCOR1) regulates gene expression by interacting with various nuclear receptors and transcription factors[8]. NCOR1 recruits histone deacetylases and other chromatin-modifying enzymes to target gene promoters, thereby repressing transcription[9]. Recent studies have revealed the role of NCOR1 in regulating inflammatory responses and metabolism in various cell types, including macrophages[10]. However, the involvement of NCOR1 in AA progression, particularly in relation to macrophage function, remains unclear.

Angiopoietin-like protein 4 (ANGPTL4) is a secreted glycoprotein that participates in lipid metabolism, angiogenesis, and inflammation[11]. ANGPTL4 can modulate macrophage activation and lipid uptake in atherosclerosis[12], suggesting its role in vascular pathologies. Recently, aldolase A (ALDOA) has been implicated as a key glycolytic enzyme in non-metabolic functions such as transcriptional regulation and cell migration[13]. MMP2 is a well-established mediator of ECM degradation in AA, weakening the aortic wall[14]. Nevertheless, the interplay between these factors and their regulation by NCOR1 in AA remains unexplored.

In the present study, we explored the specific function of NCOR1 in macrophages in AA development and revealed the mechanisms that drive it. We hypothesized that in macrophages, NCOR1 deficiency exacerbates AA formation by promoting inflammatory responses and ECM degradation. To examine this theory, we specifically deleted NCOR1 in the macrophages of mice (MNKO) and exposed them to β-aminopropionitrile (BAPN) to induce AA. Then, we comprehensively analyzed AA progression, inflammatory responses, and ECM restoration, both in vivo and in vitro. Furthermore, we explored the downstream pathways regulated by NCOR1 in macrophages, with a focus on the ANGPTL4–ALDOA–MMP2 signaling axis.

2.1 Animals and AA Model

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine. The experiments were conducted according to the Guide for the Care and Use of Laboratory Animals.

2.1.1 Generation of Macrophage-Specific NCOR1 Knockout Mice

MNKO mice with a macrophage-specific NCOR1 deletion were generated by crossing NCOR1^flox/flox mice (The Jackson Laboratory, USA) with Lysozyme2-Cre^+/+ mice (Nanjing University, China). The resulting Lysozyme2-Cre^+/−; NCOR1^flox/flox mice were designated MNKO, while Lysozyme2-Cre^−/−; NCOR1^flox/flox littermates served as controls (LC). Male NCOR1^flox/flox and Lysozyme2-Cre^+/+ mice on a C57BL/6J background, aged 8–10 weeks, were used. Mice were purchased from Jackson Laboratory, weighing 20-25g. All mice were specific pathogen-free (SPF) grade and had not undergone any other treatments prior to the experiment. Primer sequences for genotyping are listed in Table S1.

2.1.2 BAPN-Induced AA Model

This study employed a randomized controlled experimental design. The experimental unit was individual mice. Three-week-old male MNKO and LC mice were randomly divided into four groups: LC Control, LC BAPN, MNKO Control, and MNKO BAPN groups. Mice were randomly assigned to experimental and control groups using a random number table. The BAPN groups were administered 0.25% BAPN (Sigma) via drinking water for 4 weeks. The control groups were given standard drinking water. BAPN was added to drinking water at a concentration of 0.25%, replaced daily. Treatment lasted for 4 weeks. To minimize potential confounding factors, we randomized the order of treatments and measurements, and regularly rotated cage positions. Body weight and general condition of the mice were monitored weekly. In the 4th week, an ultrasound was conducted. Then, mice were sacrificed at the end of the same week to collect tissue samples. Treatment was conducted in the mouse facility, maintained at 22 ± 2℃, 50–60% relative humidity, with a 12-hour light/dark cycle.

The number of animals per group was: LC control group (n = 5), LC-BAPN group (n = 11), MNKO control group (n = 5), MNKO-BAPN group (n = 8). A total of 29 mice were used in the entire study. Sample size was determined based on a power analysis using G*Power software, with α = 0.05, power (1-β) = 0.8, and an effect size d = 1.2, which was estimated from preliminary experiments. This analysis ensured that the study had sufficient statistical power to detect biologically meaningful differences between groups while minimizing the use of animals in accordance with ethical guidelines. The operators were blinded to animal group assignments during experiment implementation and outcome assessment. Data analysis was performed by an independent researcher blinded to the group allocation.

Three-week-old male mice were selected based on previous studies[15, 16] indicating that this age group is most sensitive to BAPN-induced aortic aneurysm formation. While this may limit direct comparisons with adult human aortic aneurysms, it provides a sensitive model system for observing the effects of NCOR1 deficiency. The increased sensitivity at this age allows for a more pronounced demonstration of the role of NCOR1 in AA development, potentially revealing effects that might be subtler in older animals.

2.1.3 Anaesthesia, Analgesia, and Euthanasia

All animal procedures were performed under appropriate anaesthesia to minimize suffering. Anaesthesia was induced and maintained using isoflurane (initial dose: 4–5% for induction, maintenance dose: 1–2%) administered via inhalation. The adequacy of anaesthesia was monitored by checking for the absence of the pedal withdrawal reflex and observing the respiratory rate.

Analgesia was provided using buprenorphine (0.05-0.1mg/kg) administered subcutaneously every 12 hours as needed to alleviate pain. The animals were closely monitored for signs of pain and distress, including changes in behavior, posture, and food intake, throughout the study.

At the end of the experimental period, animals were euthanized using an overdose of pentobarbital sodium (200 mg/kg, intraperitoneally). The method of euthanasia was chosen to ensure rapid and humane termination of life, confirmed by the absence of heartbeat and respiratory movements.

2.2 Ultrasound Imaging and Analysis

The primary outcome measure of this study was the maximum aortic internal diameter measured by ultrasound[17]. Aortic imaging was performed using the Vevo 2100 small animal ultrasound system (FUJIFILM VisualSonics) with a 55 MHz probe. To perform imaging, the mice were anesthetized and kept in the supine position. The aortic root, ascending aorta, and aortic arch were imaged in two dimensions using the M-mode. The maximum internal diameter of the aorta was measured. The maximum internal diameter of the aorta was measured at the junction of the ascending aorta and the aortic arch. To ensure reproducibility, three independent measurements were taken for each mouse, and the average value was used for analysis. All measurements were performed by a trained operator blinded to the experimental groups. The coefficient of variation for repeated measurements was less than 5%, indicating high reproducibility. Images were acquired during diastole to minimize the influence of cardiac cycle on aortic dimensions. Furthermore, using the LC Control group as a reference, the relative changes in aortic diameter were calculated.

2.3 Histological and Immunohistochemical Analyses

2.3.1 Tissue Processing and Staining

Secondary outcome measures included elastin degradation score and degree of inflammatory cell infiltration. The aortic samples were treated with 4% paraformaldehyde, dehydrated, and then embedded in paraffin. Subsequently, 5-µm-thick sections were stained with Elastica van Gieson (EVG) to visualize elastin fibers. Antibodies against MAC-2 (1:100; Abcam, Cambridge, UK) were used to perform immunohistochemistry. An In Situ Cell Death Detection Kit (Roche) was used according to the manufacturer’s guidelines to perform the TUNEL assay.

2.3.2 Histological Evaluation

An elastin scoring system was used to assess elastin degradation in EVG-stained sections. Macrophage infiltration was assessed by counting MAC-2-positive cells. Apoptosis was assessed by quantifying TUNEL-positive cells. Image Pro Plus software was used to perform the quantifications.

The elastin scoring system used a scale from 0 to 4, where:

0 = No visible elastin fragmentation.

1 = Minimal fragmentation (< 25% of the vessel circumference affected).

2 = Moderate fragmentation (25–50% of the vessel circumference affected).

3 = Severe fragmentation (50–75% of the vessel circumference affected).

4 = Very severe fragmentation (> 75% of the vessel circumference affected or complete elastic lamina breakdown).

Scoring was performed independently by two blinded observers, and the average score was used for analysis. In cases of significant discrepancy (> 1 point difference), a third observer was consulted, and consensus was reached through discussion. The inter-observer reliability was assessed using Cohen's kappa coefficient, which was > 0.8, indicating strong agreement.

2.4 Flow Cytometry

Another secondary outcome measure was the quantification of inflammatory cell infiltration. Aortic tissues were digested with collagenase I (1 mg/mL) and DNase I (125 U/mL) at 37℃ for 45–60 min. Single-cell suspensions were stained with fluorochrome-conjugated antibodies against CD45, CD11b, F4/80, Ly6C, and Ly6G (BD Biosciences). BD FACSAria II was used to perform flow cytometry. Single-cell suspensions were first gated based on forward scatter (FSC) and side scatter (SSC) properties to exclude cell debris and aggregates. Live cells were then selected using a viability dye. From the live cell population, CD45 + cells were identified to focus on leukocytes. Within the CD45 + population, CD11b + cells were selected to identify myeloid cells. F4/80 and Ly-6C markers were then used to differentiate macrophages (F4/80high) and monocyte subsets (Ly-6Chigh and Ly-6Clow). For each population, fluorescence minus one (FMO) controls were used to set accurate gates. To ensure consistency, the same gating strategy was applied to all samples. FlowJo software was used to perform data analysis.

2.5 RNA Isolation and Quantitative Real-Time PCR

Gene expression levels of inflammatory markers and MMPs were assessed as secondary outcome measures. Total RNA was extracted from aortic tissues or cultured cells using TRIzol reagent (Invitrogen). The PrimeScript RT Reagent Kit (Takara) was used to reverse transcribe RNA into cDNA. qRT-PCR was performed using the ABI 7500 Real-Time PCR System) and SYBR Premix Ex Taq (Takara). Gene expression was adjusted by normalizing to GAPDH using the 2^−ΔΔCt method. Table S2 lists the primer sequences.

2.6 Western Blot Analysis

Protein expression levels of inflammatory markers and MMPs were also assessed as secondary outcome measures. SDS-PAGE was performed to separate protein samples. The separated proteins were transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies against NCOR1 (1:1000, Abcam), α-SMA (1:1000, Abcam), MMP2 (1:1000, Abcam), MMP9 (1:1000, Abcam), and GAPDH (1:5000, Cell Signaling Technology) overnight at 4℃. After washing, the membranes were incubated with horse radish peroxidase-conjugated secondary antibodies. An enhanced chemiluminescence system (Bio-Rad) was used to visualize protein bands. ImageJ software was used for quantification.

2.7 Cell Culture and Transfection

THP-1 cells were chosen for this study as they are a widely used human monocytic cell line that is easily cultured and manipulated. While derived from a leukemia patient and thus not fully representative of normal monocytes, they provide a valuable model for studying NCOR1 function in human macrophages. THP-1 cells have been extensively characterized and are known to differentiate into macrophage-like cells upon stimulation, making them suitable for our investigation[18–20]. However, we acknowledge that future studies using primary macrophages would be beneficial for validating our findings in a more physiologically relevant system.

In vitro experiments were conducted to assess the effect of NCOR1 knockdown on macrophage polarization and function as secondary outcome measures. THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. To differentiate macrophages, THP-1 cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 24 h, followed by incubation in PMA-free medium for 24 h. M1 polarization was induced by treatment with 100 ng/ml lipopolysaccharide and 20 ng/ml interferon-γ for 48 h.

To knock down NCOR1 and ANGPTL4, M1 macrophages were transfected with NCOR1-specific siRNA, ANGPTL4-specific siRNA or control siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. qRT-PCR and western blotting were performed to determine the knockdown efficiency. Primer sequences are listed in Table S3.

2.8 RNA Sequencing and Bioinformatics Analysis

Total RNA from aortic tissues was subjected to RNA sequencing using the Illumina HiSeq platform. HISAT2 was used to map clean reads to the mouse reference genome (mm10). DESeq2 was used to perform differential expression analysis. The criteria for significantly differentially expressed genes were as follows: |log₂FC| > 1 and p < 0.05. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database was used to identify the potential protein interaction network of NCOR1, with a focus on MMP-2-associated molecules. To ensure the accuracy and validity of our analysis, the reliability of the STRING predictions was evaluated by considering the sources, credibility, and biological significance of the interaction data. To explore potential regulatory networks associated with ANGPTL4, we performed bioinformatics analysis using Ingenuity Pathway Analysis (IPA) software. The input data consisted of differentially expressed genes (fold change > 1.5, p < 0.05) between ANGPTL4-treated and control groups. We focused on identifying upstream regulators and canonical pathways. Results were considered significant if p < 0.05 and |z-score| > 2.

2.9 Chromatin Immunoprecipitation (ChIP) Assay

The SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) was used according to the manufacturer’s instructions to perform ChIP. Briefly, cells were fixed with 1% formaldehyde, lysed, and sonicated to shear chromatin. Anti-NCOR1 antibody (Abcam) or IgG control was used to perform immunoprecipitation. Purified DNA was analyzed via qPCR using primers specific for the ANGPTL4 promoter region (Table S4).

2.10 Statistical Analysis

All statistical analyses were performed using GraphPad Prism 9.0 software. Data are expressed as mean ± standard deviation (SD). Normality of data was assessed using the Shapiro-Wilk test. Homogeneity of variance was evaluated using Levene's test. If data met parametric test assumptions, Student's t-test was used to compare two groups, whereas a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to compare multiple groups. If data did not meet parametric test assumptions, appropriate non-parametric test methods were employed: Mann-Whitney U test for two-group comparisons and Kruskal-Wallis test followed by Dunn's post hoc test for multiple group comparisons.

Survival curves were analyzed using the log-rank test. For the incidence of aortic aneurysm, Fisher's exact test was used. A p-value of less than 0.05 was considered statistically significant. Effect sizes with 95% confidence intervals were calculated for primary outcomes. For comparisons between two groups, Cohen's d was calculated. For ANOVA, partial eta squared (η2) was calculated as a measure of effect size. All experiments were performed at least three times independently. The exact number of biological replicates (n) for each experiment is indicated in the corresponding figure legend.

3.1 NCOR1 Deficiency in Macrophages Exacerbates BAPN-Induced AA Formation

To investigate the role of macrophage NCOR1 in AA progression, we generated macrophage-specific NCOR1 knockout (MNKO) mice by crossing NCOR1^flox/flox mice with Lysozyme2-Cre mice. The efficiency of NCOR1 deletion in macrophages was confirmed by quantitative RT-PCR and Western blot analyses, showing a significant reduction in NCOR1 mRNA and protein levels in MNKO macrophages compared to littermate control (LC) macrophages (Figure S1).

To assess the impact of macrophage NCOR1 deficiency on AA development, we subjected MNKO and LC mice to BAPN-induced AA (Fig. 1A). After 4 weeks of BAPN treatment, MNKO mice exhibited a significantly higher incidence of AA and increased mortality than LC mice (Fig. 1B-C). Specifically, the survival rate of MNKO-BAPN mice was significantly lower than that of LC-BAPN mice (p < 0.05), and the incidence of AA in MNKO-BAPN mice was markedly higher (p < 0.05). This indicates that NCOR1 deficiency in macrophages increases susceptibility to AA formation and worsens overall outcomes.

Ultrasound imaging revealed more severe aortic dilation in MNKO-BAPN mice than in LC-BAPN mice (Fig. 1D-E). The maximum internal diameter of the thoracic aorta was significantly larger in MNKO-BAPN mice (P < 0.01). Quantitative analysis showed that the relative increase in aortic diameter was substantially higher in MNKO-BAPN mice than in LC-BAPN mice.

Gross examination revealed a higher frequency of aortic rupture in MNKO-BAPN mice (Fig. 1F), highlighting the critical role of NCOR1 in maintaining aortic wall integrity. The aortic walls of MNKO-BAPN mice appeared more dilated and fragile, with visible signs of structural compromise, compared to LC-BAPN mice.

Histological analysis using Elastica van Gieson (EVG) staining demonstrated more extensive elastin degradation and disorganization in the aortic wall of MNKO-BAPN mice than in LC-BAPN mice (Fig. 2A). While LC-BAPN mice showed some degree of elastin fiber reduction and fragmentation, MNKO-BAPN mice exhibited more severe and widespread elastin degradation, with large areas of elastin loss. Quantification of elastin integrity using an elastin scoring system confirmed the increased severity of elastin degradation in MNKO-BAPN mice (Fig. 2B). This suggests that NCOR1 deficiency leads to enhanced extracellular matrix degradation, which is a hallmark of AA progression.

Analysis of α-smooth muscle actin (α-SMA) expression in aortic tissues using western blot and RT-PCR revealed a notable decrease in vascular smooth muscle cell (VSMC) content and markers of contractile phenotype in MNKO-BAPN mice when compared to LC-BAPN mice (Fig. 2C-D). Western blot analysis confirmed reduced α-SMA protein levels, while RT-PCR showed decreased α-SMA mRNA expression in MNKO-BAPN aortic tissues. This indicates that NCOR1 deficiency exacerbates the phenotypic switch of VSMCs from a contractile to a synthetic state, contributing to aortic wall weakening.

Additionally, TUNEL staining revealed increased apoptosis in the aortic wall of MNKO-BAPN mice (Fig. 2E-F). Quantification of TUNEL-positive cells showed a significantly higher percentage of apoptotic cells in MNKO-BAPN aortic tissues than in LC-BAPN tissues (P < 0.05), suggesting that NCOR1 plays a role in cell survival within the aortic tissue.

Collectively, these results demonstrate that macrophage-specific NCOR1 deficiency exacerbates BAPN-induced AA formation, which is characterized by increased aortic dilation, elastin degradation, VSMC phenotypic modulation, and enhanced apoptosis.

3.2 NCOR1 Deficiency Promotes Inflammatory Cell Infiltration and Cytokine Production in AA

To investigate the impact of NCOR1 deficiency on inflammatory responses in AA, we performed a flow cytometric analysis of aortic tissues. MNKO-BAPN mice exhibited a significantly higher percentage of CD11b⁺F4/80⁺ macrophages and CD11b⁺Ly6G⁺ neutrophils than LC-BAPN mice (Fig. 3A-B). The proportion of CD11b⁺F4/80⁺ macrophages in MNKO-BAPN aortic tissues was approximately twice that in LC-BAPN tissues. Similarly, the percentage of CD11b⁺Ly6G⁺ neutrophils was markedly elevated in MNKO-BAPN aortic tissues.

Immunofluorescence staining further confirmed the increased infiltration of MAC-2⁺ macrophages and Ly6G⁺ neutrophils in the aortic wall of MNKO-BAPN mice (Fig. 3C-D). Quantitative analysis of immunofluorescence images revealed a significantly higher number and density of MAC-2⁺ macrophages and Ly6G⁺ neutrophils in MNKO-BAPN aortic tissues than in LC-BAPN tissues. These findings indicate that NCOR1 deficiency enhances the recruitment of inflammatory cells to the site of AA formation.

RNA sequencing analysis was performed to assess the inflammatory profile of AA tissues. Heatmap visualization revealed distinct gene expression patterns between the MNKO-BAPN and LC-BAPN groups, with a significant upregulation of inflammatory genes in MNKO-BAPN mice (Fig. 4A). The heatmaps showed clusters of inflammatory genes that were consistently overexpressed in MNKO-BAPN aortic tissues compared to LC-BAPN tissues.

Quantitative RT-PCR analysis confirmed the increased expression of pro-inflammatory cytokines, including IL-1β, IL-6, and CD11b, in MNKO-BAPN aortic tissues (Fig. 4B). The mRNA levels of these inflammatory markers were significantly higher in MNKO-BAPN mice than in LC-BAPN mice.

Furthermore, we observed significant upregulation of MMPs, particularly MMP2, MMP9, and MMP12, in MNKO-BAPN aortic tissues compared with LC-BAPN tissues (Fig. 4C-D). In MNKO-BAPN mice, the mRNA levels of these MMPs were significantly higher, with MMP12 exhibiting the greatest increase.

Immunofluorescence staining for MMP12 showed an increased number of MMP12-positive cells in the aortic wall of MNKO-BAPN mice (Fig. 4E-F). Quantification of MMP12-positive cells revealed a significantly higher density of these cells in MNKO-BAPN aortic tissues than in LC-BAPN tissues.

ELISA analysis of serum MMP12 levels further confirmed elevated MMP12 production in MNKO-BAPN mice (Fig. 4G). Serum concentrations of MMP12 were significantly higher in MNKO-BAPN mice than in LC-BAPN mice, corroborating the tissue-level findings.

These results suggest that a lack of NCOR1 in macrophages encourages the influx of inflammatory cells and boosts the release of inflammatory cytokines and enzymes that break down tissues in AA, leading to the worsening of AA development.

3.3 NCOR1 Deficiency Enhances M1 Macrophage Polarization and Function

To further elucidate the role of NCOR1 in macrophage function, we performed in vitro experiments using THP-1 derived macrophages. THP-1 cells were differentiated into macrophages and polarized towards the M1 phenotype (Figure S2). Morphological observation under a microscope revealed that THP-1 cells changed from a round shape to an irregular form, with numerous vacuole-like structures in the cytoplasm after induction treatment, indicating successful polarization to M1 macrophages.

NCOR1 knockdown was achieved using siRNA transfection confirmed by qRT-PCR and western blotting (Fig. 5A-C). qPCR findings indicated a notable decrease in NCOR1 mRNA levels in cells transfected with siRNA compared to control cells. Western blot analysis further confirmed the substantial decrease in NCOR1 protein expression in knockdown cells.

NCOR1-deficient M1 macrophages exhibited enhanced expression of M1 markers, including IL-1β, IL-6, and Cd11b, at both the mRNA and protein levels compared to control M1 macrophages. qPCR analysis revealed significantly higher mRNA expression levels of these inflammatory markers in NCOR1-knockdown cells. And western blot analysis verified elevated protein levels of TNF-α, IL-1β, and IL-6 in M1 macrophages lacking NCOR1 (Fig. 5D-F).

Moreover, NCOR1 knockdown resulted in increased expression of MMP2 and MMP9 in M1 macrophages (Fig. 5G-I). Both qPCR and western blot analyses showed significant upregulation of MMP2 and MMP9 at mRNA and protein levels, respectively, in NCOR1-deficient cells compared to control cells.

These results suggest that NCOR1 deficiency enhances M1 macrophage polarization and promotes the expression of proinflammatory mediators and matrix-degrading enzymes, providing a mechanistic link between NCOR1 deficiency and exacerbated AA formation.

3.4 NCOR1 Regulates the ANGPTL4-ALDOA-MMP2 Signaling Axis in Macrophages

To investigate the molecular mechanisms by which NCOR1 regulates macrophage function in AA, we conducted bioinformatics analysis. We identified ANGPTL4 and ALDOA as potential downstream targets of NCOR1 (Fig. 6A and S3). Bioinformatics analysis revealed a strong correlation between NCOR1 expression and the levels of ANGPTL4 and ALDOA, suggesting a potential regulatory relationship.

Quantitative RT-PCR analysis confirmed the upregulation of ANGPTL4, ALDOA, and MMP2 mRNA expression in NCOR1-deficient macrophages (Fig. 6B). The mRNA levels of ANGPTL4 were increased approximately 4-fold, ALDOA expression was elevated approximately 2.5-fold, and MMP2 mRNA levels were upregulated more than 5-fold in NCOR1-knockdown cells compared to those in control cells.

To investigate the relationship between NCOR1 and ANGPTL4, we performed chromatin immunoprecipitation (ChIP) assays. The results demonstrated the binding of NCOR1 to the ANGPTL4 promoter region, which was significantly reduced in NCOR1-deficient macrophages (Fig. 6C-D). ChIP-qPCR analysis showed a marked decrease in NCOR1 occupancy of the ANGPTL4 promoter in NCOR1-knockdown cells, suggesting that NCOR1 directly regulates ANGPTL4 transcription.

To further validate the NCOR1-ANGPTL4-ALDOA-MMP2 signaling axis, we conducted knockdown experiments targeting ANGPTL4 alone or in combination with NCOR1 in M1 macrophages. Single knockdown of ANGPTL4 (MAKO) and double knockdown of ANGPTL4 and NCOR1 (MAKO་MNKO) were confirmed by qRT-PCR and western blotting (Fig. 7A-F). The efficiency of ANGPTL4 knockdown was greater than 70%, as demonstrated by both the mRNA and protein analyses. In the double knockdown experiments, both NCOR1 and ANGPTL4 expression was significantly reduced.

Interestingly, ANGPTL4 knockdown attenuated NCOR1 deficiency-induced upregulation of ALDOA and MMP2 expression (Fig. 7G-K). In NCOR1-deficient cells, the additional knockdown of ANGPTL4 resulted in a significant reduction in ALDOA and MMP2 mRNA and protein levels, bringing their expression closer to that observed in control cells. This suggests that ANGPTL4 acts as an intermediate in NCOR1-mediated regulation of ALDOA and MMP2.

These findings suggest that NCOR1 regulates the ANGPTL4-ALDOA-MMP2 signaling axis in macrophages, potentially contributing to its protective role in AA development. The discovery of this communication pathway offers fresh perspectives on the molecular processes through which NCOR1 affects macrophage activity and AA development.

In summary, our results demonstrate that macrophage-specific NCOR1 deficiency exacerbates AA formation through enhanced inflammatory responses, increased matrix degradation, and activation of the ANGPTL4-ALDOA-MMP2 signaling axis. These findings highlight the critical role of NCOR1 in regulating macrophage function and maintaining aortic wall integrity, offering new perspectives on AA pathogenesis and potential therapeutic targets for its prevention and treatment.

In our comprehensive study, we revealed that macrophage-specific NCOR1 deficiency exacerbates AA formation in a BAPN-induced mouse model. Our findings suggest a novel protective role of NCOR1 in AA development by regulating macrophage function and the ANGPTL4–ALDOA–MMP2 signaling axis. This discovery not only advances our understanding of AA pathogenesis but also unfolds new avenues for potential therapeutic interventions.

Complex interactions between various cell types and molecular pathways are involved in AA pathogenesis[1]. In this study, we focused on macrophage function, which plays an essential role in inflammation associated with AA progression and growth[5]. We observed that NCOR1 deficiency in macrophages significantly increased AA incidence, enhanced aortic dilation, and caused more severe elastin degradation in BAPN-induced mice. These findings are consistent with those of previous studies, highlighting the importance of macrophage-mediated inflammation in AA pathogenesis[21, 22].

As a transcriptional corepressor, NCOR1 plays a role in regulating inflammatory responses across different biological contexts[23]. In this study, we significantly extended these observations to the field of vascular biology, particularly in the context of AA. We noted that NCOR1 deficiency promotes the infiltration of inflammatory cells, especially macrophages and neutrophils, into the aortic wall. This enhanced inflammatory cell recruitment is accompanied by a considerable increase in the expression of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and Cd11b. These findings are consistent with those of previous studies that have revealed that NCOR1 suppresses inflammatory gene expression in macrophages[10, 24].

The effect of NCOR1 deficiency on macrophage function extends beyond inflammatory cell recruitment. This shift toward the M1 phenotype, which is a more inflammatory phenotype, may contribute to the accelerated AA progression noted in our mouse model. The role of NCOR1 in macrophage polarization adds a new layer of complexity to our understanding of how this transcriptional corepressor affects vascular pathology.

One of the key mechanisms underlying AA progression is the degradation of ECM components, particularly elastin and collagen[25]. MMPs play a key role in this process[26]. We observed that NCOR1 deficiency significantly upregulated several MMPs, including MMP2, MMP9, and MMP12, in AA tissues. This increased MMP expression may have enhanced the elastin degradation and aortic wall weakening observed in NCOR1-deficient mice. MMP regulation by NCOR1 has been previously reported in other contexts, including cancer[27]. Our study extends these findings to the cardiovascular system.

To further elucidate the mechanisms by which NCOR1 regulates macrophage function in AA, in vitro experiments were conducted using THP-1-derived macrophages. We observed that NCOR1 deficiency enhances M1 macrophage polarization and promotes the expression of proinflammatory mediators and matrix-degrading enzymes. These results are consistent with those of previous studies that have revealed the significance of NCOR1 in regulating macrophage polarization and inflammatory reactions[28, 29].

The identification of the ANGPTL4-ALDOA-MMP2 signaling axis as a downstream target of NCOR1 in macrophages is a novel and particularly interesting aspect of our study. This novel finding highlights how NCOR1 regulates macrophage activity and affects atherosclerosis development. ANGPTL4 has been previously implicated in lipid metabolism and inflammation[30]. On the other hand, ALDOA plays a role in glycolysis. However, recent studies have revealed the non-metabolic functions of ALDOA, suggesting that this enzyme plays a more complex role in cellular processes[31].

In the present study, ChIP revealed that NCOR1 binds directly to the ANGPTL4 promoter, suggesting that NCOR1 directly regulates the transcription of this gene. The observation that ANGPTL4 knockdown attenuates NCOR1 deficiency-induced ALDOA and MMP2 upregulation provides strong evidence for the presence of a signaling axis connecting these molecules. This finding is particularly significant because it reveals a previously unknown regulatory pathway in the context of AA pathogenesis.

The role of ANGPTL4 in vascular biology is complex and context-dependent. While some studies have revealed the protective effects of ANGPTL4 in atherosclerosis[32], others have revealed that the proinflammatory and proangiogenic effects of this molecule[33]. In the present study, we hypothesized that increased ANGPTL4 expression owing to NCOR1 deficiency may contribute to AA progression. This apparent contradiction with the findings of previous studies highlights the importance of understanding the functions of multifaceted proteins, including ANGPTL4. Our findings on ANGPTL4's role in AA are further supported by previous research. Notably, Gabel et al. (2017) identified ANGPTL4 as a key molecular fingerprint in aortic disease[34]. Their work highlighted the potential importance of ANGPTL4 in aortic pathology, which our current study confirms and extends. The consistency between these findings underscores the significance of ANGPTL4 in vascular biology and suggests it may be a promising target for therapeutic interventions in AA.

The association between ANGPTL4 and ALDOA in vascular diseases is a novel finding of our study. Although ALDOA has been thoroughly investigated with respect to cancer metabolism[35], its role in cardiovascular diseases remains unclear. Our results suggest the potential non-glycolytic function of ALDOA in regulating MMP2 expression in macrophages, possibly contributing significantly to ECM degradation in AA. This unexpected connection between glycolytic enzymes and matrix degradation unfolds new avenues for investigations in the field of vascular biology.

The precise mechanisms by which ANGPTL4 regulates ALDOA and how ALDOA affects MMP2 expression in macrophages remain unclear. We hypothesized that ANGPTL4 activates signaling pathways, thereby increasing ALDOA expression or activity. This, in turn, can regulate MMP2 expression via transcriptional or post-transcriptional mechanisms. Alternatively, ALDOA may directly interact with the transcriptional regulators of MMP2 or affect its activity via metabolic changes in the cell. These possibilities represent exciting avenues for future studies.

The discovery of the NCOR1-ANGPTL4-ALDOA-MMP2 axis offers new perspectives on the molecular pathways involved in AA progression and suggests potential targets for treatment. The modulation of this pathway may offer novel strategies for preventing or treating AA. For example, targeting ANGPTL4 or ALDOA in macrophages may help alleviate the detrimental effects of NCOR1 deficiency in AA. In the future, studies should be undertaken to explore the development of small-molecule inhibitors or activators that can modulate this pathway, facilitating new treatment options for patients with AA.

In addition, our findings have implications for developing diagnostic and prognostic biomarkers for AA. The expression of NCOR1 and its downstream targets (ANGPTL4, ALDOA, and MMP2) in blood mononuclear cells may serve as indicators of AA risk and disease progression. In the future, researchers should focus on developing and validating blood-based tests to measure these markers in clinical settings. Such tests can revolutionize AA management by allowing earlier detection and a more accurate monitoring of disease progression.

Although our study findings provide valuable insights into the role of NCOR1 in AA development, they have several limitations that warrant further investigation. First, we primarily focused on a specific animal model and cell type. This may not fully represent the complexity of human AA. In the future, studies should be undertaken to explore these effects in other cell types and AA models and validate the findings using human clinical data. Second, in-depth examinations are warranted to elucidate the precise molecular mechanisms and signaling pathways involved. Longitudinal studies can offer insights into AA progression and regression. Lastly, the broader systemic effects of NCOR1 deficiency should be assessed to determine the specificity of its effects on vascular health. These limitations highlight the need for additional studies to fully elucidate the role of NCOR1 in AA and its potential as a therapeutic target.

In conclusion, we reported that macrophage-specific NCOR1 deficiency exacerbates AA formation by enhancing inflammatory responses and ECM degradation. The ANGPTL4–ALDOA–MMP2 signaling axis can serve as a new downstream target of NCOR1 in macrophages, offering new perspectives on the molecular pathways involved in AA development. Our findings not only improve our understanding of AA biology but also suggest therapeutic strategies for preventing or treating this life-threatening condition.

The clinical implications of this study are far-reaching, offering new prospects for improved diagnosis, risk stratification, and targeted treatment approaches for patients with AA. As we continue to discover the complex biology of AA, studies such as ours will pave the way for a new era of precision medicine for managing vascular disease, thereby promising better outcomes for patients suffering from this challenging condition. Future studies on these findings could transform mechanistic insights into practical advancements in patient care, possibly decreasing the morbidity and mortality rates of AAs.

Conflict of interest

The authors declared that they have no conflicts of interest in this work.

Funding

This research was supported by the National Natural Science Foundation of China under grant numbers 82070262 and 82270298. The funding organization had no involvement in the study's design, data collection, analysis, interpretation, or manuscript preparation.

Author Contribution

D.Z. and H.Z. designed and performed the experiments, analyzed the data, and wrote the main manuscript text. W.F. and Y.P. assisted in designing the experiments and provided critical revisions to the manuscript. R.L. supervised the project, provided funding acquisition, and reviewed the manuscript. All authors reviewed and approved the final version of the manuscript.

Acknowledgement

First and foremost, I would like to express my heartfelt gratitude to everyone who has assisted me in the completion of this paper.I extend my sincere thanks and appreciation to my supervisor, Mrs. Li Ruogu, whose invaluable suggestions and unwavering encouragement have provided me with profound insights into the field of translation studies. It has been an honor and a privilege to work under her guidance and supervision. I am deeply grateful for her remarkable personality and diligence, qualities that I will always cherish. My appreciation for her knows no bounds.Additionally, I received indispensable assistance and support from Zhu Hong and Fu Wenxia during the writing process of this article. Pan Yitong also provided numerous insightful opinions and suggestions, significantly enhancing the quality of this work.I am extremely grateful to all my friends and classmates who have kindly offered their assistance and companionship throughout the preparation of this paper. Furthermore, I would like to express my deep appreciation to my family for their unwavering love and support. Lastly, I extend my gratitude to all those who have taken the time to read this thesis and provide me with valuable advice, which will undoubtedly benefit me in my future studies.

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No competing interests reported.

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NCOR1 Deficiency in Macrophages Aggravates Aortic Aneurysm Formation via the ANGPTL4-ALDOA-MMP2 Axis

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Animals and AA Model

2.1.1 Generation of Macrophage-Specific NCOR1 Knockout Mice

2.1.2 BAPN-Induced AA Model

2.1.3 Anaesthesia, Analgesia, and Euthanasia

2.2 Ultrasound Imaging and Analysis

2.3 Histological and Immunohistochemical Analyses

2.3.1 Tissue Processing and Staining

2.3.2 Histological Evaluation

2.4 Flow Cytometry

2.5 RNA Isolation and Quantitative Real-Time PCR

2.6 Western Blot Analysis

2.7 Cell Culture and Transfection

2.8 RNA Sequencing and Bioinformatics Analysis

2.9 Chromatin Immunoprecipitation (ChIP) Assay

2.10 Statistical Analysis

3. Results

3.1 NCOR1 Deficiency in Macrophages Exacerbates BAPN-Induced AA Formation

3.2 NCOR1 Deficiency Promotes Inflammatory Cell Infiltration and Cytokine Production in AA

3.3 NCOR1 Deficiency Enhances M1 Macrophage Polarization and Function

3.4 NCOR1 Regulates the ANGPTL4-ALDOA-MMP2 Signaling Axis in Macrophages

4. Discussion

Declarations

Conflict of interest

Funding

Author Contribution

Acknowledgement

References

Additional Declarations

Supplementary Files

Status:

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