Introduction
Abdominal aortic aneurysm (AAA) represents a degenerative disorder that irreversibly affects human health, and it is associated with a high mortality once aortic rupture [
1]. AAA has the feature of progressive local dilatation of abdominal aorta with the diameter of more than 30 mm [
2]. Its risk factors are dyslipidaemia, hypertension, smoking, male gender, family history and atherosclerosis [
3]. At present, AAA can be mainly treated by invasive endovascular stent graft therapy or open surgery, while there have been no safe and effective medication available for disease management [
2].
AAA is an inflammatory vascular disease. Vascular smooth muscle cell (VSMC) apoptosis, maladaptive aortic wall remodeling and oxidative stress (OS) are pathological features of the progression of AAA formation [
4]. Chronic inflammation and excessive reactive oxygen species have long been recognized to be the main causes of AAA. In the initial phases of AAA diseases, infiltration of inflammatory cells into aortic wall can be observed, along with increased production of proinflammatory mediators. Then, infiltrating cells trigger the inflammatory response and promote VSMCs phenotypic switching. Furthermore, inflammatory cells secrete extracellular matrix (ECM) degrading enzymes to destroy aortic wall matrix during vascular remodeling [
5‐
7]. In addition, vascular oxidative damage is among the risk factors for early AAA. Genetic and pharmacological inhibition of excessive ROS production significantly reduced AAA diseases [
8].
Alpha-ketoglutarate (αKG) accounts for a critical metabolic intermediate produced from tricarboxylic acid (TCA) cycle for maintaining energy homeostasis [
9]. AKG has previously suggested to have an essential effect on anti-aging [
10], anti-inflammatory response [
11] and anti-tumor effects [
12]. Not only that, as an antioxidant, AKG is also important for diverse oxidative reactions [
9,
13]. These pleiotropic functions of AKG have a beneficial effect on the treatment of cardiovascular disease including enhancing myocardial energy production, reducing the incidence of myocardial ischemia and preventing cardiac remodeling [
9,
14]. According to the results, AKG possibly has certain impact on inhibiting AAA through anti-inflammation and antioxidant. Consequently, this work analyzed AKG’s role and mechanisms in mice with AAA.
This work focused on determining how AKG affected elastase-mediated AAA occurrence within mice. Here, we demonstrate that AKG treatment significantly inhibited AAA formation in this model. Apart from that, AKG supplement significantly reduced oxidative stress, macrophage infiltration in the aortic walls and inflammatory cytokines expression. Besides, we also show that AKG ameliorates AAA though PXDN/HCLO/ERK signaling pathways in vitro and in vivo.
Methods
Animals and mouse model
Our study protocols gained approval from the Animal Care and Use Committee of the Affiliated Hospital of Qingdao University. The C57BL/6 male mice aged 8–10 weeks were utilized for all experiments. To induce AAA, each male mouse was intraperitoneally injected with sodium pentobarbital for anesthesia, afterwards, pancreatic elastase (1.5 U) was applied locally into abdominal aorta. For exploring AKG’s efficacy in treating elastase-mediated AAA, all animals were randomized as sham (Sham), AAA, low-(0.1%, 100 mg/kg/d) and high-dose AKG 0.5% (500 mg/kg/d) groups. Accordingly, each mouse was given AKG (Macklin, China) dissolved in water and raised under the environmentally-controlled conditions for 28 consecutive days.
Cell culture, treatment and transfection
This work acquired mouse aortic smooth muscle cells (MOVAS) in American Type Culture Collection (ATCC). For establishing the aneurysmal microenvironment within MOVAS, this work adopted TNF-α for stimulating MOVAS [
15,
16]. The MOVAS cell line was grown in DMEM media that contained 1% penicillin/streptomycin and 10% fetal bovine serum (FBS), followed by incubation under 5% CO2 and 37 °C conditions. Before experiment, this work rinsed the MOVAS thrice with PBS, followed by pretreatment using AKG (5, 10, 20 mmol/L) in serum-free medium for two hour before TNF-α stimulation (100 ng/mL). Then, this work inoculated MOVAS at 50–70% confluency into the 6-well plates, followed by 6-h infection using the lentivirus expressing PXDN or lentiviral vector (MOI = 50) in serum-free medium. Afterwards, MOVAS were further cultured for a 24-h period in serum-depleted medium before TNF-α stimulation for the indicated times.
Elisa assay
The MOVAS cells were homogenized with PBS at 4 °C and centrifuged at 13,000 rpm. Then, the levels of MCP-1, IL-1β, and IL-6 were measured using ELISA kits (abcam) strictly following instructions.
MOVAS migration assay
For confirming cell migration, this work conducted Transwell assays. Briefly, the present work seeded MOVAS into the serum-free DMEM added into the upper Transwell chamber in the 24-well plates; whereas medium that contained 10% FBS was added into bottom chamber. At 24-h later, PBS was used to rinse cells on upper membrane surface, while those penetrating bottom chamber were subject to 4% formalin fixation as well as 1% crystal violet staining. The migrating cells were then counted microscopically for quantification. Also, cell migration was analyzed through scratch assay. In brief, MOVAS were inoculated in the 6-well plates and they attained 70–80% confluency at 24-h later. Cells were cultured for another 2-h period in serum-depleted medium prior to experiment, followed by stimulation with TNF-α and treatment with AKG for another 24 h in 0.1% FBS medium. Afterwards, one sterile micropipette tip was utilized to make scratch wounds on cell layer in every plate. Then, those detached cells were eliminated by PBS flushing, and cell proliferation was suppressed by replacing the original medium by serum-free medium, followed by TNF-α treatment of cells. After acquisition of images under the microscope at 0/24 h, the Image-Pro Plus (version 6.0) was adopted for evaluating recovered area proportion.
Detection of ROS generation in MOVAS and in mice
This study adopted fluorescence probes DCFH-DA and DHE for measuring intracellular ROS levels within abdominal aortic tissues or MOVAS. In brief, this work cultured tissues or cells for a 40-min period using 10 μM and DCFH-DA or 10 μM DHE within the humid incubator in dark. After obtaining the ROS fluorescence intensity, this study adopted Image-Pro Plus software for quantification.
Apoptosis assay in vivo and vitro
The present work adopted CellEvent Caspase-3/7 Green etection Reagent (Roche Diagnostics) for measuring caspase-3/7 activities in line with specific protocols. MOVAS were seeded into 48-well plates. Before experiment, MOVAS were cultured in serum-depleted medium for a 2-h period, followed by TNF-α stimulation and followed treated with AKG for another 24 h in 0.1% FBS medium. After images were captured microscopically, the Image-Pro Plus (version 6.0) was utilized for analysis.
Apoptosis of vascular cells in aortic wall was detected by TUNEL assay in line with specific instructions. Briefly, 7-μm aortic tissue sections were subject to 10-min permeabilization using 20 μg/mL Proteinase K solution under ambient temperature, followed by 10-min incubation using equilibration buffer under ambient temperature. Thereafter, aortic tissues were further incubated with terminal deoxynucleotidyl transferase (TdT) reaction mixture under 37 °C for a 60-min period, then the reaction was terminated using 2 × SSC buffer. Each slide was mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific, P36935). Finally, green fluorescence was measured.
Histology, immunohistochemistry (IHC) and immunofluorescence (IF) staining
Hematoxylin–Eosin (H&E) staining was conducted for analyzing cell morphology. The dilated aortic samples within infra-renal area of mice of diverse groups were treated by Tissue-Tek O.C.T. Compound embedding within liquid nitrogen, followed by slicing in the 5-μm consecutive sections. Afterwards, these sections were subject to deparaffinage, rehydration and staining using the Verhoeff-van Gieson staining kit to assess elastin in line with specific protocols.
Immunohistochemistry (IHC) staining of CD45, CD68, and MMP-9 was performed for observing changes in aortic wall morphology as well as inflammation. Briefly, the aortic slices (5 μm thickness) were later subject to 4% paraformaldehyde (PFA) fixation along with paraffin embedding. CD45, CD68 and MMP-9 were used for analysis. This study subsequently measured positive cell infiltration rate through the mean nucleus quantity surrounded by positively-stained cells under the microscope.
After blocking PFA-fixed tissue sections using 10% normal donkey serum, they were further probed under 4 °C overnight with primary ntibodies anti-α-SMA. Normal goat or rabbit IgG was applied as negative controls. After several washes with PBS, secondary antibodies were further used to probe sections under ambient temperature for a 1-h period. Nuclei were stained by DAPI. After acquisition of images under the microscope, Image-Pro Plus (version 6.0) was utilized for analysis.
RNA sequencing (RNA-seq)
To investigate the molecular mechanism underlying the regulatory effect of AKG on the development of AAA, total RNA was extracted from the aortic tissues of AAA-treated (n = 4) and AAA + AKG-treated (n = 4) mice. The mRNA library was constructed according to the manufacturer’s protocol (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina Kit (Vazyme, NR604-02). The complementary DNA libraries were sequenced using Illumina NovaSeq6000 with 2X150 running circles. In brief, 1 μg of total RNA from each sample was used for library construction. After capture and purification, mRNA was fragmented(85 °C, 6 min) into 250–450 bp and reverse transcribed. With end repair and adaptor ligation, the library was purified and underwent size selection. A total of 13 PCR cycles were used for the upcoming library amplification. Agencourt AMPure XPTM Beads (Beckman Coulter) were used for purification and sequencing was carried out on a NovaSeqTM(PE150, Illumina). Genes with expression changes of twofold compared with control samples were considered to be significantly up- or downregulated. The GO and KEGG was analysised using TopGO.
Transduction of mice aortic segments in vivo
We examined the role of PXDN in AKG inhibition of the development of AAA by transfection with adenovirus. The experimental procedure was performed as follows [
17]. In brief, the infrarenal segment of the abdominal aorta was exposed under anesthesia (2.5% isoflurane) via a midline incision, clamped just below the renal arteries, a vascular cannula introduced into the isolated vascular segment, and the aorta flushed with normal saline. For each mouse, the segment was transduced with HBAD-EGFP (50 μl, 2.51 × 10
11 PFU/mL) or HBAD-Adeasy-m-Pxdn-Null-EGFP (50 μl, 1.26 × 10
11 PFU/mL). After 20 min of incubation, the cannula was removed and the incision was sutured. After 2 weeks, the C57BL/6 male mice was intraperitoneally injected with sodium pentobarbital for anesthesia, afterwards, pancreatic elastase (1.5 U) was applied locally into abdominal aorta.
Western blotting (WB) assay
This work isolated total proteins in abdominal aorta and MOVAS. Thereafter, BCA Protein Assay Kit (Beyotime) was utilized to measure protein content. Subsequently, this study separated proteins through SDS-PAGE, followed by transfer onto PVDF membranes (pore size, 0.45 μm). Primary antibodies for MMP-2 (ABclonal), MMP-9 (Abclonal), a-SMA (Abcam), SM-22α (Abcam), PXDN (Abclonal), 3-Cl-tyr (Cell Science), BAX (Cell Signaling Technology), BCL2 (Cell Signaling Technology, USA) and ERK1/2/p-ERK1/2 (Cell Signaling Technology) were used. The present study adopted the ECL protocol for detecting the antigen–antibody complex by adopting goat anti-mouse or anti-rabbit IgG secondary antibody, with GAPDH being the endogenous reference.
RNA isolation and real-time PCR
This work utilized TRIzol reagent for isolating total RNA in MOVAS and abdominal aortic tissue. SYBR Green was used in the quantitative real-time PCR (Takara, Japan). By normalizing the samples to GAPDH, an internal reference, the samples were relatively measured.
Statistical analysis
GraphPad Prism 8.0 was adopted for statistical analysis. Results were displayed in a form of mean ± SEM. A Student t-test was used for statistical comparison between two groups. One-way analysis of variance was performed for analysis of three or more groups. P < 0.05 stood for statistical significance.
Discussion
AKG is an important intermediate produced during Krebs cycle, and it was originally identified as an antioxidant [
21]. It was involved in different metabolic and cellular pathways, including the oxidation of fatty acids, nitrogen and ammonia balance and energy metabolism [
22]. Thereafter, AKG is identified with several therapeutic activities, such as enhancing lifespan, protecting age-related osteoporosis, enhancing muscle strength and endurance, and managing non-alcoholic fatty liver disease (NAFLD) [
11,
23,
24]. AKG was beneficial for cell apoptosis, OS and inflammation, which indicates that it may be applied in treating cardiovascular diseases (CVDs) too [
25]. Nonetheless, the roles of AKG in AAA was previously unknown. In this study, AKG significantly suppressed the aneurysmal dilation by attenuating the oxidative stress, macrophage infiltration, elastin degradation and collagen fibers remodeling.
Previous studies have shown that AAA represents the degenerative vascular complication, with the pathological characteristics of depleted VSMCs, dysregulated ROS, infiltration of inflammatory cells and ECM degradation [
7,
8]. The production of ROS and oxidative stress is thought to be a critical mechanism implicated in AAA formation because these have been shown to promote inflammation, matrix degradation, and apoptosis of VSMCs in AAA formation [
26]. ROS, including hydroxyl radical (•OH), superoxide (O2
−) along with hydrogen peroxide (H2O2), are the highly reactive chemical molecules derived from oxygen [
27]. Excessive ROS levels implicated in VSMCs dysfunction, DNA damage, and lipid/protein peroxidation may result in cellular injury and death irreversibly [
28]. In the development of AAA, the large numbers of infiltrating macrophages can produce large amounts of O2
− and H
2O
2 through membrane-bound NADPH oxidase. In addition, endothelial cells, VMSCs, and fibroblasts are capable of forming O2
− via several pathways [
7]. As an antioxidant, AKG is adopted for treating some disorders, including inhibiting cardiac remodeling, suppressing colorectal cancer and reducing chronic renal failure. These may be related to regulating the activity of hypoxia, AMPK/mTOR and Wnt pathways [
9,
12,
21]. As far as we know, the present work is the first to analyze AKG’s role in AAA occurrence. As a result, AKG dramatically inhibited inflammatory factor levels within vascular walls (like TNF-α, IL-6, IL-1β) and triggered a dramatic decrease of ROS compared with sham group. In vitro, AKG treatment markedly suppressed TNF-α-induced VSMCs apoptosis and the production of superoxide. For investigating the mechanism by which AKG affected AAA occurrence, RNA-seq was conducted to comprehensively analyze whole-genome gene expression profiles within the abdominal aorta in AAA group and AKG-treated group. Consistent with previous studies, numerous related BPs, such as inflammatory response, cell response to OS, immune regulation and cellular energy and metabolism, were found. Finally, cells were treated with NAC for inhibiting AAA in vitro, aiming to investigate the necessity of ROS for AKG’s protection, and AKG could not further augment its improvements.
As a result, PXDN showed marked down-regulation within AKG group compared with AAA group by RNA-seq analysis. PXDN, which belongs to hemecontaining peroxidase family, shows high expression within cardiovascular system. Previous studies have shown that PXDN promoted the cardiovascular oxidative damage, including cardiac fibrosis after MI, myocardial ischemia–reperfusion injury and hypertresion [
29,
30]. A study by Huihui Peng showed that PXDN expression increased within aneurysmal tissues in mice and humans relative to healthy counterparts. PXDN catalyzed hypochlorous acid formation via H
2O
2, while markedly promoting the production of ROS [
20]. We confirmed that AKG inhibited PXDN expression in the vascular tissues and VMSCs. Collectively, we found that PXDN modulates MOVAS phenotypic switch whereas PXDN up-regulation reduced AKG’s protection in vitro. This work also discovered that AKG dramatically inhibited -Cl-tyr level in vitro and in vivo (which was produced by the reaction between HOCl and tyrosine residues). HOCl accounts for a potent ROS family oxidant and aggravates oxidative stress in AAA formation. PXDN overexpression blocked the role of AKG on inhibiting the production of HOCl. These results demonstrated that PXDN regulates the phenotype change of VSMCs via VPO1/HOCl pathway, and it possibly has an important function during AAA occurrence.
As a key biological compound, AKG exerted critical roles in different metabolic and cellular pathways, such as AKT /mTOR signaling, NF-kB pathway, Wnt signaling and AMPK signaling. However, whether other signal pathway is regulated by AKG on AAA formation remains unknown. It has been shown recently that PXDN promoted ROS production through ERK1/2 and AKT signaling in cardiovascular system [
18,
20]. Considering that PXDN is an important target for AKG to inhibit AAA, it was speculated that the AKG-regulated phenotype change of VSMCs was possibly associated with AKT and ERK1/2 pathways. According to our results, AKG inhibited the expression of p-ERK1/2 but not p-AKT whereas ERK activator blocked the effects. Moreover, ERK activator disrupted the protective of AKG on TNF-α-induced cells apoptosis and oxidative stress.
ROS levels elevate within AAA, and this enhances the inflammation, apoptosis and ECM degradation of VSMCs. Decreasing ROS within VSMCs alleviates AAA. In this work, we found that AKG supplementation exerted an anti-oxidative stress in the development of AAA formation. AKG inhibited ROS production and prevented the progression of AAA. Mechanistically, AKG reduced ROS generation by suppressing PXDN within MOVAS depending on HOCL/ERK1/2 pathway. According to our results, AKG alleviated AAA occurrence by its antioxidation within MOVAS. The findings will provide certain foundation for developing new drugs to treat AAA, and contribute to applying AKG clinically to prevent and treat AAA.
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