JMBD

Introduction

AAA is a severe vascular disease with >85% mortality upon rupture and rising incidence, lacking effective therapies [1]. Oxidative stress and infiltration of inflammatory cells, predominantly macrophages, are significant pathological changes in AAA [2,3]. Macrophage apoptosis triggers a complex immune-inflammatory cascade and plays a critical role in the initiation and progression of AAA [4,5]. Current research reveals macrophage apoptosis plays a paradoxical role in AAA development, showing protective roles in early stages but contributing to disease progression in advanced phases [6,7]. Nevertheless, research on macrophage death in the context of AAA remains relatively limited. The detail of modulating macrophage apoptosis in AAA has not been fully elucidated.

Oxidative stress and inflammation are critical factors in AAA Pathogenesis. Reactive oxygen species (ROS) play essential roles in maintaining cellular homeostasis and regulating various physiological functions at physiological concentration. For example, ROS can stimulate the differentiation of osteoclast through NF-κB Signaling [8]. However, excessive ROS triggers oxidative stress and leads to aberrant cellular functions including cell migration, proliferation, and apoptosis. These pathological alterations exacerbate inflammatory responses and facilitate vascular remodeling, thereby promoting the development of many vascular diseases including AAA [9]. Nevertheless, the precise mechanisms by which ROS induces macrophage apoptosis and its role in AAA pathophysiology remain to be fully understood.

Gamma-glutamylcysteine (γ-GC), a direct precursor of glutathione (GSH), is a critical intracellular antioxidant and plays a pivotal role in maintaining cellular antioxidant capacity and modulating oxidative stress responses [10]. Studies have demonstrated that γ-GC improves endothelial function and alleviates inflammatory injury by modulating antioxidant and signal transduction [11]. Additionally, GSH has been found to reduce the risk of thrombosis by attenuating platelet aggregation [12]. A decrease in γ-GC levels significantly elevated intracellular oxidative stress, contributing to the pathogenesis of various diseases. Supplementation with exogenous γ-GC has been shown to significantly alleviate oxidative stress and mitigate cellular damage [13]. γ-GC is efficiently absorbed by various cell types, including macrophages, where it promotes GSH synthesis and exerts anti-inflammatory effects [14,15]. However, the connection between γ-GC and AAA remains underexplored, and its role in AAA-associated inflammation and macrophage apoptosis is not yet fully understood.

MiRNAs play critical roles in ROS-induced apoptosis, especially in the apoptosis of macrophage. For example, miR-21 targeted MKK3 and up-regulated pro-apoptotic p38-CHOP, thereby promoting macrophage apoptosis [16]. MiR-221-3p could target ADAM22 and suppress ox-LDL-induced macrophage apoptosis and foam cell formation [17]. Intronic miRNAs are located within the introns of protein-coding genes and usually share the same promoters with their host genes. MiR-483-3p is a typical intronic miRNA located in the second intron of IGF2 gene and participates in the development of various metabolic and malignant diseases. MiR-483-3p over-expression in murine 3T3-L1 cells could induce lipotoxicity and insulin resistance by impeding the lipid storage of adipocytes. MiR-483-3p inhibition increased the homing of endothelial progenitor cells in venous thrombosis rats [18]. In nephroblastoma, colon cancer, liver cancer, etc., miR-483-3p was up-regulated and inhibited tumor cell apoptosis, thereby promoting tumorigenesis [19]. However, a study also indicated that miR-4833p promoted apoptosis and inhibited tumor cell proliferation [20]. Nevertheless, the relationships among oxidative stress, miR-4833p, γ-GC, macrophage apoptosis, and AAA remain unclear.

Herein, we found significant increases in macrophage apoptosis, miR-483-3p and its host gene IGF2 expression in aneurysm tissues of AAA mice, accompanied by a pronounced downregulation of γ-glutamylcysteine synthetase (γ-GCS). H2O2 significantly upregulated the expression of miR-483-3p and its host gene IGF2 when inducing macrophage apoptosis, which were significantly ameliorated by γ-GC. MiR-483-3p upregulated p53/p21 by suppressing MED1, promoting macrophage apoptosis, while IGF2 enhances this process. γ-GC may be a potential therapeutic target for AAA.

Materials and Methods

Ethics Statement and Sample Collection

Blood and fecal samples were collected from 20 patients diagnosed with AAA and healthy individuals. AAA diagnosis was confirmed via CT angiography (CTA), with an abdominal aortic diameter exceeding 3 cm. Healthy individuals underwent abdominal CT scans to ensure the absence of AAA. Exclusion criteria for AAA patients included age above 80 years, the presence of severe hypertension, diabetes, or dyslipidemia, as well as other aneurysms, cardiovascular diseases, infectious diseases, gastrointestinal disorders, autoimmune conditions, hepatic or renal diseases, cancer, or a history of abdominal surgery. This study was approved by the Ethics Committee of Shandong University Qilu Hospital (2018-110) and verbal consent was obtained from the subjects. All procedures involving human participants adhered to the principles outlined in the Declaration of Helsinki and were conducted in accordance with applicable local laws and institutional guidelines. All animal procedures followed the US NIH Guide for the Care and Use of Laboratory Animals.

AAA Model Construction

ApoE-/- male specific pathogen-free mice, purchased from Beijing Vital River Laboratory Animal Technology Co, were housed in a controlled environment with 23-24°C, relative humidity of 50%, and a consistent 12:12-hour light-dark cycle. The mice were allowed free access to high-fat diet (containing 0.25% cholesterol and 15% cocoa butter) and water. The Ang II-induced AAA mice model was established according to previously published protocols [21]. Briefly, ApoE-/- mice were implanted subcutaneously with micro-osmotic pumps (Alzet, model 2004) to deliver either Ang II (1000 ng/min/kg) or PBS for 4 weeks. Upon completion of the experiment, mice were anesthetized, and tissue specimens were collected for other experiments.

Cell culture and treatment

HEK293T and RAW264.7 cells purchased from Shanghai Cell Bank of Chinese Academy of Sciences were cultured in DMEM (Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (Biological Industries, Israel) at 37 ºC with 5% CO2. RAW264.7 cells treated with 300 μM H2O2 were used as the experimental group, and RAW264.7 cells treated with H2O2 at physiological concentration (10 nM) were used as the control group. After treatment for 24 hours, cells were harvested for the following experiments. In some experiments, RAW264.7 cells were treated with recombinant IGF2 active protein (Abcam, USA) at 200 ng/ml for 48 hours.

Transfection

The expression plasmid of MED1 (pCMV6-MED1) was transfected into cells using Lipofectamine 3000 reagent (Life Technologies, USA) according to the manufacturer’s instructions. The mimic and inhibitor of miR-483-3p and the corresponding negative controls (Ribobio, China) respectively were transfected at a final concentration of 50 nM using Lipofectamine RNAiMAX reagent (Life Technologies, USA).

Target gene prediction and functional enrichment analysis

The target genes of miR-483-3p were predicted by TargetScan, miRWalk and miRDB. The target genes predicted by all three prediction tools were selected, and their functional enrichment analysis were performed by Enrichr (http://amp.pharm.mssm.edu/ Enrichr/) [22,23].

RNA isolation and Real-time quantitative PCR (qPCR)

Total RNA was extracted using TRIZOL reagent. Mir-X miRNA First-Strand Synthesis Kit (Clotech, Japan) and RT-PCR kit Mir-X miRNA qRT-PCR TB Green Kit (Clotech, Japan) were used for miRNA cDNA synthesis and PCR amplification. Human and murine RNU6B gene were used as endogenous standards. The mRNA expressions of MED1, IGF2, p53 and p21 were detected using PrimeScript RT Master Mix (Takara, Japan) and TB Green Premix Ex Taq II (Takara, Japan) in a CFX96 real-time PCR system (Bio Rad, USA). β-actin was used as an endogenous standard. The 2-ΔΔCt method was used for quantification of qPCR data. The primers in this study were listed in (Table S1).

Table S1: PCR Primers usedin this study

Western Blot

Total proteins were isolated using RIPA buffer (Beyotime, China). Protein concentrations were determined by bicinchoninic acid (BCA) method. Then, 20µg total protein was separated by 10% SDS-PAGE and transferred to PVDF membrane (Bio-Rad, USA). The membrane was incubated with primary antibodies at 4 °C overnight and subsequently incubated with second antibody (Proteintech Group, USA) at room temperature for 2 h. The antibodies used in this study were listed in (Table S2).

Table S2: Antibodies used in this study

Dual-Luciferase Reporter Assay

The 3’-UTR sequence of MED1 containing the wild-type (WT) or mutant (Mut) bases in the predicted binding site of miR-4833p was cloned into the SacI/XbaI restriction site of pmir-GLO (Promega, USA) to generate the plasmid pG-WT-MED1-3’-UTR or pG-Mut-MED1-3’-UTR, respectively. Then, HEK-293T cells were co-transfected, respectively, with the above vectors, miR-4833p mimics or miR-483-3p inhibitors and corresponding negative controls using Lipofectamine 3000 reagent and Lipofectamine RNAiMAX reagent (Life Technologies, USA). After 48h, the luciferase activity was determined using Dual-Luciferase Reporter Assay System (Promega, USA) on Centro LB 960 Microplate Luminometer (Berthold, Germany). Primers are listed in Table S1.

Flow Cytometry Analysis of Apoptosis

The apoptosis of RAW264.7 was detected using FITC Annexin V Apoptosis Detection Kit I (Becton Dickinson, USA) according to the manufacturer’s protocol. Cells with different treatments were resuspended in binding buffer to a concentration at 1×105/ ml. Next, the cells were stained with 5 µl FITC Annexin V and Propidium Iodide (PI), and incubated for 15 minutes in the dark at room temperature. The apoptotic rate was detected using the FACSCalibur (Becton Dickinson, USA) and data were analyzed using FlowJo X software (Flow Jo, USA).

Tunel Staining

AAA tissue sections were deparaffinized and washed with PBS, then immunolabeled with CD68 polyclonal antibody (Santa Cruz, USA) to identify macrophages. Macrophage apoptosis was subsequently detected using a TUNEL staining kit (Beyotime, China). Briefly, deparaffinized sections were incubated with TUNEL reaction mixture at room temperature for 30 min in a humidified chamber.

After three PBS washes, nuclei were counterstained with DAPI. Macrophage apoptosis was then observed and analyzed using fluorescence microscopy.

RNA Fish

The experiment was conducted in accordance with the manufacturer’s protocol for the RNA FISH paraffin section kit (GenePharma, China). The results were observed under a fluorescence microscope and RNA expression in the tissue was analyzed using Image pro plus 6.0 software (Media Cybernetics, USA).

Statistical analysis

Data are presented as mean ± SD. The normality was checked using Shapiro–Wilk test. T-test was used for significance analysis between two group. One-way ANOVA was used for statistical tests of multiple comparisons. Correlations were examined by the Spearman’s rank correlation coefficients with corresponding multiple comparison correction. All experiments were repeated at least three times independently and p < 0.05 was considered as statistically significant. Statistical analysis was performed using GraphPad Prism v8.0.2.

Results

Significant increases in levels of macrophage apoptosis, and the expression of miR-483-3p and its host gene IGF2 in AAA tissues

We found that compared to normal aortic tissues, the expression of γ-GCS, the key enzyme responsible for synthesizing the antioxidant γ-GC, was significantly reduced in the aneurysmal tissues of angiotensin II-induced AAA mice (Figure 1A). The co-localization area of CD68 and TUNEL staining was markedly enhanced in

AAA tissues compared to those in normal abdominal aorta tissues, indicating a significant increase in macrophage apoptosis (Figure 1B). RNA fluorescent in situ hybridization showed that miR-483-3p expression was significantly upregulated in AAA aneurysmal tissues (Figure 1C). The qPCR results further demonstrated the elevated levels of miR-483-3p and its host gene, IGF2, in macrophages isolated from aneurysmal tissues (Figure1D,1E). Moreover, correlation analyses revealed strong positive associations between macrophage apoptosis, miR-483-3p expression, and IGF2 expression levels (Figure 1F,1H). Additionally, targeted metabolomic analysis showed that serum γ-GC levels were significantly lower in AAA patients compared to healthy individuals (Figure1I). These findings suggest that oxidative stress is significantly elevated in AAA aneurysmal tissues and is closely associated with increased macrophage apoptosis and the upregulation of miR-483-3p and its host gene IGF2.

Figure 1: Significant changes in levels of γ-GCS, macrophage apoptosis, miR-483-3p expression and their relationships in AAA.

Comparison of expression levels of γ-glutamylcysteine synthetase (γ-GCS) mRNA in abdominal aorta tissues from normal mice (NC) and aneurysmal tissues from Ang II-induced AAA mice (AAA). (B) Representative immunofluorescence images of CD68 and TUNEL in abdominal aorta tissues of NC and AAA groups and their quantitative comparison. (C) Representative images of fluorescence in situ hybridization of miR-483-3p in abdominal aorta tissues of NC and AAA groups and their quantitative comparison. (D,E) Comparison of expression levels of miR-483-3p (D) and IGF2 (E) in abdominal aorta tissues of NC and AAA groups. (F,H) Correlation analyses between macrophage apoptosis, miR-483-3p and IGF2 expression levels. (I) Comparison of serum levels of γ-glutamylcysteine (γGC) in AAA patients (hAAA) and healthy individuals (healthy). Statistical analyses were performed using Student’s t-test for (A-E, I).

Spearman’s correlation analysis was used for (F,H). ***p < 0.001.

MiR-483-3p was predicted to be involved in multiple biological functions and significantly promoted macrophage

H2O2 is the main source of oxidative stress in cells. We found that high concentration of H₂O₂ induced the apoptosis of macrophages (Figure 2A). Compared to the macrophages treated by H₂O₂ at physiological concentration (10 nM), the expression of miR-483-3p was significantly up-regulated in macrophages treated with 300 μM H₂O₂ for 24 hours (Figure 2B). However, miR-483-3p mimic or inhibitor had no significant effects on the apoptosis of macrophages without H2O2 treatment (Figure 2C). Despite that, miR-483-3p mimic markedly enhanced the apoptosis of macrophages treated with 300 μM H₂O₂, while miR-483-3p inhibitor had no obvious effect on the H₂O₂-induced macrophage apoptosis (Figure 2D). To further unravel the mechanism by which miR-483-3p promoted H₂O₂-induced macrophage apoptosis, the target genes of miR-483-3p were predicted by three commonly used tools including TargetScan, miRWalk and miRDB. To reduce false positives in the predicted results, 45 target genes of the miR-483-3p predicted by all three tools were selected as the potential target genes of miR-483-3p (Figure 2E). The functional enrichment analysis of these 45 genes showed that miR-483-3p was involved in multiple important biological functions, including the regulation of apoptosis, intracellular lipid and cholesterol transport, LDL binding, cellular response to ROS and immunity (Figure 2F). Among them, the enrichment of regulation of apoptotic process was the most significant. Besides, these potential target genes were also significantly enriched in several important signaling pathways that regulate the cellular inflammation, proliferation, and apoptosis, such as NF-κB, Notch1, Wnt, P53 and IGF1 pathways (Figure 2G). These results suggested that miR-483-3p was associated with multiple biological functions and pathways including apoptosis, implying the importance of miR-483-3p in functional regulation.

Figure 2: Hydrogen peroxide-induced apoptosis, miR-483-3p expression, and their interplay in macrophages and the predicted target genes of miR-483-3p. (A,B) Comparison of apoptosis rates (A) or miR-483-3p expression levels (B) in RAW264.7 cells treated with 10 nM (Con) or 300 μM H2O2 (H2O2). (C,D) Comparison of apoptosis rates in RAW264.7 cells (C) or 300 μM H2O2-treated RAW264.7 cells (D) with various treatments. NC-mi: treatment with negative control for miR-483-3p; NC-IHT: treatment with negative control for miR-483-3p inhibitor. Inhibitor: treatment with miR-483-3p inhibitor. (E) Venn diagram of the potential target genes of miR-483-3p predicted by TargetScan, miRWalk and miRDB. (F,G) Significantly enriched biological processes (F) and KEGG signaling pathways (G) of the potential target genes of miR-483-3p. Student’s t was used for (A,B). ANOVA test was used for (C,D). *: p < 0.05, **: p < 0.01, ***: p < 0.001. ns: no significance.

MiR-483-3p targeted MED1 gene to inhibit its expression in macrophages

MED1 was predicted to be a target gene of miR-483-3p. The results of RNAhybrid also indicated a binding site of miR-483-3p seed sequence in the 3’-UTR sequence of MED1, and the binding energy was as low as -22.4 kcal/mol (Figure 3A). In addition, the highconcentration H₂O₂ significantly reduced MED1 expression at mRNA and protein level in macrophages (Figure 3B,3C). Moreover, MED1 expression levels were significantly negatively correlated with miR-483-3p expression levels (Figure 3D). At the mRNA level, miR-483-3p mimic markedly down-regulated MED1 expression in macrophages whereas the effect of miR-483-3p inhibitor was opposite (Figure 3E). At the protein level, miR-483-3p mimic also remarkably decreased MED1 expression, but miR-483-3p inhibitor had no obvious effect on MED1 expression (Figure 3F). Additionally, the pmir-GLO dual luciferase reporter vector containing wild-type (WT) or mutated (Mut) 3’-UTR sequence of MED1 were constructed and respectively co-transfected with miR-483-3p mimic or its negative control (NC) into 293T cells. The results showed that the co-transfection of WT dual luciferase reporter vector and miR-4833p mimic significantly decreased the luciferase activity whereas co-transfection of Mut dual luciferase reporter vector and miR-483-3p mimic had no influence on the luciferase activity (Figure 3G). These results demonstrated that MED1 is a target gene of miR-483-3p, and its expression is down-regulated by miR-483-3p.

Figure 3: MED1 was the target gene of miR-483-3p. (A) The predicted binding site and binding energy of miR-483-3p in 3’-UTR of MED1 gene. Green letters indicate the sequence in MED1 3’-UTR that complementarily binds to the miR-483-3p seed region. Red italic letters: the mutation of the binding sequence used to construct the dual luciferase reporter vector. (B,C) Comparison of MED1 expression at mRNA (B) and protein (C) level in RAW264.7 cells treated with 10 nM (Con) or 300 μM H2O2 (H2O2). (D) Spearman correlation between the expression levels of MED1 mRNA (left panel) or protein (right panel) and miR-483-3p levels. (E, F) Relative expression of MED1 mRNA (E) and protein (F) in RAW264.7 cells with various treatments. (G) Relative luciferase active (FL/RL) detected by dual luciferase reporter assay. H293T cells were respectively co-transfected with the dual luciferase reporter vector containing the widetype (WT) or mutant (Mut) sequence of the miR-483-3p binding site in MED1 3’-UTR and miR-483-3p mimic or its negative control. NC-mi: treatment with negative control for miR-483-3p; NC-IHT: treatment with negative control for miR-483-3p inhibitor. Inhibitor: treatment with miR-483-3p inhibitor. Statistical analysis was performed using student’s t. Spearman’s correlation analysis was used for (D). *: p < 0.05, ***: p < 0.001. ns: no significance.

miR-483-3p enhanced H2O2-induced macrophage apoptosis through MED1-p53/p21 pathway in concert with its host gene IGF2

MiR-483-3p is a typical intron miRNA located in the second intron of IGF2 gene (Figure 4A). Our results showed that high-concentration H2O2 significantly increased expression of IGF2 mRNA and protein in macrophages (Figure 4B). IGF2 expressions were positively correlated with levels of miR-483-3p expression (Figure 4C). These results indirectly implied that miR-483-3p and its host gene IGF2 might share the same transcriptional regulatory elements. Additionally, miR-483-3p mimic remarkably upregulated the expression of p53 and p21 in macrophages. However, the overexpression of MED1 significantly diminished the above effects of miR-483-3p mimic and reduced the apoptosis of macrophages induced by H₂O₂ (Figure 4D,4F). We found that as a secreted protein, while IGF2 treatment alone did not influence H₂O₂ -induced macrophage apoptosis, the combination of IGF2 and miR-483-3p could significantly increase the apoptosis of macrophage induced by H₂O₂. Moreover, MED1 overexpression also significantly abrogates the synergistic effect of IGF2 and miR-483-3p in promoting hydrogen peroxide-induced macrophage apoptosis. The results indicated a synergistic effect of miR-4833p and its host gene on regulating macrophage apoptosis (Figure 4G). The above results demonstrated that miR-483-3p could enhance H₂O₂-induced macrophage apoptosis through its target gene MED1 and downstream p53/p21 in concert with its host gene IGF2.