LncRNA MALAT1-Targeting Antisense Oligonucleotide Ameliorates the AngII-Induced Vascular Smooth Muscle Cell Proliferation and Migration Through Nrf2/GPX4 Antioxidant Pathway

Abstract:

The high level of oxidative stress induced by angiotensin II (AngII) is the main pathophysiological process that promotes the proliferation and migration of vascular smooth muscle cells (VSMCs) and induces vascular remodeling. LncRNA metastasis-related lung adenocarcinoma transcript 1 (MALAT1) has been determined to play an important role in the modulation of oxidative stress and the development of cardiovascular diseases. Nevertheless, the function and underlying mechanism of MALAT1 in restenosis induced by hypertensive angioplasty remain unclear. AngII increased the expression of MALAT1 in VSMCs. We found that antisense oligonucleotide lncRNA MALAT1 (ASO-MALAT1) could inhibit AngII-induced reactive oxygen species production and VSMCs proliferation and migration by inducing the expression of glutathione peroxidase 4 (GPX4), which can be reversed by siRNA-GPX4. GPX4 overexpression can inhibit the proliferation and migration of VSMCs induced by AngII. In addition, we found that the process by which MALAT1 knockdown induces GPX4 expression involves nuclear factor erythrocyte 2–related factor 2 (Nrf2). Overexpression of Nrf2 can increase the expression of GPX4, and downregulation of GPX4 by ML385 (Nrf2 inhibitor) blocked the protective effect of ASO-MALAT1 on AngII-induced proliferation and migration of VSMCs. Ferrostatin-1 (Fer-1, ip 5 mg/kg per day for 2 weeks), a GPX4 agonist, significantly inhibited neointimal formation in spontaneously hypertensive rat by the inhibition of oxidative stress. In conclusion, these data imply that ASO-MALAT1 suppresses the AngII-induced oxidative stress, proliferation, and migration of VSMCs by activating Nrf2/GPX4 antioxidant signaling. GPX4 may be a potential target for the therapeutic intervention of hypertensive vascular restenosis.

INTRODUCTION

Hypertension is a chronic noncommunicable disease worldwide, one of the major diseases endangering human health, and the leading cause of global disease burden.¹ Hypertensive patients are prone to vascular stenosis and restenosis after percutaneous coronary intervention (PCI).^2,3 The proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in vascular remodeling and the formation of hypertension-related complications.⁴ As the main effector molecule of the renin-angiotensin system, AngII is one of the strongest active substances in the endogenous vasopressor system. It is not only involved in the formation of hypertension but also an important autocrine growth-promoting factor of VSMCs.⁵ In VSMCs, excessive production of reactive oxygen species (ROS) through angiotensin type 1 receptor (AT₁ receptor) is an important link leading to vascular stenosis and restenosis.^6,7 Therefore, despite the existence of anti-RAAS drugs, it is still necessary to explore the process of ROS generation at the molecular level to understand the pathogenesis of vascular restenosis in patients with hypertension and to identify potential targets to develop new treatment strategies for the disease.

The activation of ROS to promote VSMCs migration and proliferation is a key feature of hypertensive vascular restenosis.⁸ Most studies investigating the mechanism of vascular restenosis focus on protein coding genes.^9,10 Although recent genome-wide analyses have revealed an overwhelming number of transcriptions, there are still no translated genes. Therefore, transcription produces many long noncoding RNAs (lncRNAs), which can affect cellular processes, such as proliferation, migration, apoptosis, or cell senescence.¹¹ LncRNAs are associated with the progression of a variety of human cardiovascular diseases (heart failure, atherosclerosis, and hypertrophy).¹² LncRNA is a transcribed genomic region (longer than 200 nucleotides) and has no ability to encode proteins.¹³ MALAT1, an 8.7 kbp lncRNA, is a highly conserved lncRNA, usually in various tumor tissues, which regulate cardiovascular diseases.^14,15 Upregulation in blood vessels of MALAT1 in a spontaneously hypertensive rat (SHR) has been reported.¹⁶ More importantly, MALAT1 has been found to be overexpressed in patients with white coat hypertension.¹⁷ MALAT1 can regulate ROS/Nrf2 in diabetes and promote related complications.¹⁸ MALAT1 null mice show no overt phenotype. However, in transcriptome analysis of MALAT1 null mice, Chen et al¹⁹ found significant upregulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) regulated antioxidant genes, including Nqo1 and Cat, with significant reduction in ROS.

Nrf2, an important regulator of oxidative stress transcription, plays a key role in enhancing the ability of cells to resist damage caused by external oxidative stress mediators by interacting with antioxidant response elements to induce the expression of antioxidant stress protein genes. When the body is exposed to ROS, Nrf2 separates from Keap1 (Kelch-like ECH-related protein 1), translocates into nucleus and junctions with antioxidant response element, and initiates the transcription of downstream antioxidant genes HO-1 and GPX4.²⁰ However, a direct role of Nrf2/GPX4 signaling in VSMCs dysfunction during hypertensive vascular restenosis has not been demonstrated. Studying the relationship between the Nrf2/GPX4 pathway and the regulation of vascular restenosis in hypertension will help to identify new therapeutic targets and pharmacological options to prevent or reduce vascular restenosis.

In this study, we explored the protective effect of ASO-MALAT1 on AngII-induced VSMCs proliferation and migration through Nrf2/GPX4-dependent antioxidant pathway. We also investigated the potential impact of GPX4 agonist (Fer-1) on the prevention of intimal hyperplasia after angioplasty in hypertension.

MATERIALS AND METHODS

Materials

MTT Kit and AngII were purchased from Sigma Co (St. Louis, MO). GPX4 was purchased from Proteintech. GAPDH was purchased from Santa Cruz Biotechnology. H3 polyclonal antibodies and proliferating cell nuclear antigen (PCNA) antibody were purchased from Cell Signaling Technology (CST, Danvers, MA). Nrf2 inhibitor ML385 and Fer-1 were from MedChemExpress (Monmouth Junction, NJ). Futurepagetm protein prefabrication gel (10%) and Western blot–related apparatus were purchased from Nonin Biotech Co Ltd (Shanghai, China). All serum, cell media, and antibiotics were purchased from Procell Co Ltd (Wuhan, China). All organic solvents and nitrocellulose filter membrane (NC membrane) were obtained from Sangon Biotech Co Ltd (Shanghai, China). Transwell assays were obtained from Thermo Fisher Scientific (Waltham, MA).

Cell Culture and Cell Transfection

A10 cell line was obtained from ATCC (Hercules, CA). A10 cells were cultured in 10% FBS-DMEM which containing 1% penicillin–streptomycin Solution. Cells were cultured at 37°C in 95% air and 5% CO₂ atmosphere. Cells were allowed to grow to approximately 70%–80% confluence and then serum-deprived in 0.1% FBS-DMEM. Next, quiescent A10 cells were treated with AngII for 24 hours before MTT assay, RNA or protein extraction, or other performance of biochemical assays. For the transfection of cells, ASO targeting MALAT1, scramble nontargeting ASO, and GPX4 siRNA were obtained from RiboBio (Guangzhou, China); meanwhile, GPX4 plasmid (pLV3-CMV-Gpx4(rat)-3×FLAG-CopGFP-Puro) was purchased from Sangon Biotech Co Ltd. Procedures of cell transfection procedures refer to the manufacturer's manual protocol. Specifically, different ASO, plasmids (1 μg/mL), or siRNA (50 nM) were transfected into A10 cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Collect transfected cells for use after 48 hours. The siRNA sequence with the best interference effect was selected for further experiments. The siRNA sequences were as follows:

ASO1:5′-TCTTATGTTTCCGAACCGTT-3'.

ASO2:5′-TGCCTTTAGGATTCTAGACA-3'.

Cell Proliferation Assay

The abilities of A10 cells proliferation were detected by MTT assays, trypan blue rejection assays, EdU staining, and detection of PCNA. For MTT assays, A10 cells were seeded into 96-well culture plates (Corning, Lowell, MA) and cultured to 70% and then treated with different stimulus and cultured for 24 hours. MTT solution was added according to the kit instructions. Finally, the absorbance value (OD) at 490 nm was detected on a microplate reader (model 680; Bio-Rad, CA). For trypan blue rejection assays, A10 cells were seeded into 24-well cell culture plates at a concentration of 1 × 10⁸/L, 1 mL per well, and 6 duplicate wells were designed for each group. In addition, 48 hours after different treatment, the supernatant was discarded, and the adherent cells were digested with trypsin to prepare a single-cell suspension. The cell suspension was mixed with trypan blue solution (0.4%) at a ratio of 9:1. The viable cells, determined by the uptake of 0.4% trypan blue, were counted within 3 minutes using a hemocytometer. For Edu staining, A10 cells were seeded into a 6-well culture plate for 24 hours. According to the kit instructions, the following steps were performed in sequence: EdU-labeled staining, fixed cells, Apollo staining, DNA staining. Finally, use a confocal microscope (Olympus FV1200, Tokyo, Japan) to observe, photograph, and count cells. Immunofluorescence staining reflects the expression level of PCNA.²¹ Finally, the results of immunofluorescence staining were visualized by using a confocal microscope (Olympus FV1200).

Cell Migration Assay

Cell migration was determined by scratch wound assays and transwell assays. For transwell migration assay, 200 μL A10 cell suspension (1 × 10⁵ cells/well) was added to the upper chambers, serum-containing culture medium were added to the lower chambers, and then cultured for 24 hours. Finally, the cells were fixed in paraformaldehyde and stained with 0.1% crystal violet. Migrating cells were observed and counted under a microscope. For the scratch migration test, A10 cells were seeded in 6-well plates, approximately 1 × 10⁶ cells per well. When the cell density reaches 90%, scratch with a 200 μL pipette tip perpendicular to the bottom surface of the 6-well plate. After washing 3 times with phosphate-buffered saline (PBS), serum-free medium was added for continued incubation.²² Photographs were taken at 0 hours and 24 hours using an Olympus IX-70 inverted microscope (Olympus, Tokyo, Japan), and the scratch areas were measured using Image J 1.44 software (NIH, MD).

Cell ROS Detection

2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe (Beyotime) was used to detect the level of ROS. A10 cells were treated with AngII (10⁻⁷ mol/L) with or without ASO-MALAT1 (100 µmol/L) for 24 hours and then washing by PBS for 2 times. Then, the cells were incubated in DCFH-DA at 37°C for 20 minutes and gently washed 3 times with PBS. Finally, the cells were photographed with an immunofluorescence microscope (Olympus IX71) and analyzed with Image J 1.44 software (NIH, MD).

Experimental Animals

SHRs were purchased from Vital River Laboratory Animal Technology Company (Beijing, China). All rats were fed in IVC environment, with constant temperature at 25°C and alternating light and dark for 12 hours, without restriction of diet and drinking water. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Guilin Medical University (GLMC202202230).

Protein Extraction and Western Blot Analysis

A10 cells for protein-level analysis were seeded in 6-well plates. After cells were treated, medium was removed and washed with PBS. Cells were then lysed in RIPA lysis buffer (Beyotime) containing protease inhibitor cocktail. Next, protein extraction, electrophoresis, transfer, antibody incubation, fluorescence imaging, and densitometric analysis were performed in sequence.²³ Antibodies were configured as follows: anti-GPX4 antibody (Proteintech, 67763-1-Ig; dilution 1: 500), anti-PCNA antibody (Cell Signaling Technology, 13110; dilution 1:500), anti-GAPDH antibody (Santa Cruz Biotechnology, sc-365062; dilution 1:1000), and anti-H3 antibody (CST, 4499; dilution 1:800), goat anti-Mouse lgG (H + L) antibody (Li-Cor Biosciences, WK355; dilution 1:5000, Lincoln, NE), and goat anti-Mouse lgG (H + L) antibody (Li-Cor Biosciences, BHR102; dilution 1:5000). The bounds were detected by the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Quantitative RT-PCR

Total RNA from A10 cells was extracted and reverse transcribed into cDNA following the manufacturer's instructions. Then, the mRNA expression level of GPX4 was detected with TB Green Premix ExTaqII (Takara, Shiga, Japan) taking β-actin as the internal reference. The primer sequences for GPX4 were 5′-GAG​GCA​AGA​CCG​AAG​TAA​ACT​AC-3' (forward) and 5′-CCG​AAC​TGG​TTA​CAC​GGG​AA-3' (reverse). The primer sequences for β-actin were 5′-GTG​GGT​ATG​GGT​CAG​AAG​GA-3' (forward) and 5′-AGC​GCG​TAA​CCC​TCA​TAG​AT-3' (reverse). The primer sequences for Nrf2 were 5′-CCA​TTT​ACG​GAG​ACC​CAC (forward) and 5′-TGA​GCG​GCA​ACT​TTA​TTC-3' (reverse).

Carotid Balloon Injury Models in SHRs

First, after male SHRs were anesthetized, a 3–3.5 cm midline incision was made in the neck to isolate the left common carotid artery and external carotid artery, and the distal end of the external carotid artery was ligated to temporarily block the blood flow of the left and common carotid arteries. After inserting a 2F Fogarty balloon catheter (Edwards Life Sciences, Irvine, CA) from the external carotid artery to the common carotid artery, the balloon was inflated. The balloon was slowly and repeatedly pumped back and forth for 3 times to ensure that the intima was damaged. The balloon was withdrawn, and the external carotid artery was ligated. After the skin suture, penicillin of 200,000 U was injected intramuscularly daily for 3 days to prevent infection. The normal control group underwent sham operation. SHRs in each group were injected with vehicle (1% DMSO; n = 6) or Fer-1 (3 mg/kg) intraperitoneally daily for 2 weeks.

Evaluation of Neointimal Formation

The injured carotid artery was cut and immersed in 4% paraformaldehyde for fixation, dehydrated, and embedded in paraffin and then sliced at 4 μm thick. Tissue sections were then stained with hematoxylin and eosin (H&E), observed under a light microscope (Olympus,Tokyo, Japan). The intimal (I) and medial (M) areas were measured using Image J 1.44 software (NIH, MD) in a blind manner, and I/M ratio was calculated.

Detection of Malondialdehyde (MDA) and Total Cellular Antioxidant Capacity (T-AOC)

Using the T-AOC and MDA Assay kits purchased from Beyotime, the content of total antioxidant capacity (T-AOC) and MDA in the injured carotid artery of SHRs was calculated using chemical colorimetry and spectrophotometer colorimetry.

Statistical Analyses

All experiments were repeated at least for 3 times. All analyses were performed using SPSS 19.0. Data are expressed as mean ± SEM. The comparison within groups was made by repeated-measures ANOVA (or paired t test when only 2 groups were compared), and the comparison among groups (or t test when only 2 groups were compared) was made by factorial ANOVA and Duncan's test. Statistical significance was assumed at P < 0.05.

RESULTS

ASO-MALAT1 Inhibited AngII-Induced Rat VSMCs Proliferation and Migration

In hypertension and vascular remodeling, VSMCs proliferation and migration play a major role.²⁴ To explore the inhibitory effect of MALAT1 on VSMCs proliferation, we selected an appropriate concentration of AngII to continuously induce the VSMCs proliferation model, which was determined by the uptake of MTT. The results show that AngII stimulates VSMCs proliferation in a concentration-dependent manner (10-9∼10-6M) (Fig. 1A). QT-PCR showed that AngII increased the expression of MALAT1 (Fig. 1B). We used ASO-MALAT1 to knock down MALAT1 (Fig. 1C). The decrease in the level of MALAT1 in VSMCs confirmed the effectiveness of ASO-MALAT1. MTT assay and EdU incorporation analysis detect DNA synthesis in proliferating cells. ASO-MALAT1 inhibited the cell proliferation caused by AngII in VSMCs and the increase in the number of EdU-positive cells (Figs. 1D, E). PCNA is an important part of DNA replication and a key marker involved in DNA replication and cell proliferation. AngII increased the expression of PCNA in VSMCs, while ASO-MALAT1 reversed this effect (Fig. 1F).

FIGURE 1.

ASO-MALAT1 inhibited AngII-induced proliferation in VSMCs. A, Effects of AngII on proliferation in VSMCs. VSMCs were treated with different concentrations (10⁻⁹–10⁻⁶ M) of AngII for 24 hours. Cell viability was determined by MTT assay. AngII with 10⁻⁷ M was chosen to induce a VSMCs proliferation model in the upcoming experiments (*P < 0.05 vs. control, n = 8/group). B, Real-time PCR analysis of MALAT1 expression in A10 cells treated with AngII (10⁻⁷ M) or vehicle (*P < 0.05 vs. control, n = 8/group). C, Knockdown efficiencies of MALAT1 by ASO and real-time PCR analysis of MALAT1 expression in A10 cells (*P < 0.05 vs. ScASO, n = 8/group). D, Effects of ASO-MALAT1 on AngII-induced proliferation in VSMCs. A10 cells were treated with ASO-MALAT1 without or with AngII stimulation. Cell viability was determined by MTT assay (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only group, n = 8/group). E, EdU staining was used to detect the proliferation of VSMCs (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only group, n = 8/group). F, PCNA immunofluorescence staining (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only group, n = 8/group).

The effect of ASO-MALAT1 on VSMCs migration was also studied by transwell and scratch wound migration tests. Although AngII significantly increased VSMCs migration, ASO-MALAT1 effectively inhibited the AngII-mediated VSMCs migration, which was quantitatively evaluated by migration cell counting and migration area calculation (Figs. 2A–D).

FIGURE 2.

ASO-MALAT1 inhibited AngII-induced migration in VSMCs. A10 cells were coincubated with ASO-MALAT1 and/or AngII (10⁻⁷ M) in transwell assay and scratch wound assay to reflect the abilities of VSMCs migration. Representative images are shown in A and B, respectively (scale bar = 100 µm). Cell migration number in transwell assay (C) and migration area in scratch wound assay (D) were analyzed (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only group, n = 9/group).

ASO-MALAT1 Inhibited AngII-Induced ROS Production, Cell Proliferation, and Migration by Upregulating GPX4 Expression in VSMCs

A large number of studies have shown that ROS promotes the activation of VSMCs migration and proliferation, which is a key feature of hypertensive vascular restenosis.²⁵ We also confirmed that ASO-MALAT1 could reduce the production of total ROS in VSMCs stimulated by AngII (Fig. 3A). The role of GPX4, as a ROS scavenger molecule, is unclear in AngII-stimulated VSMCs. Therefore, we first checked the changes in GPX4 expression. As shown in Figures 3B-C, within 24 hours, AngII time dependently reduced the mRNA expression of GPX4 in VSMCs, but the downregulation of GPX4 gene transcription was reversed in the presence of ASO-MALAT1. Western blot also confirmed that ASO-MALAT1 reversed the protein expression level of GPX4 (Figs. 3D, 8A).

FIGURE 3.

ASO-MALAT1 inhibited AngII-induced ROS production in VSMCs through GPX4. A, A10 cells were coincubated with ASO-MALAT1 and/or AngII (10⁻⁷ M). Total ROS production was compared with vehicle-treated cells. Representative images were shown. (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, n = 7/group). B, A10 cells were incubated with AngII (10⁻⁷ M) for incremental hours (3–24 hours). GPX4 expression in cells was determined by q-PCR. (*P < 0.05 vs. control, n = 5/group). C, A10 cells were treated with ASO-MALAT1 and/or AngII (10⁻⁷ M) for 24 hours before GPX4 mRNA detection by q-PCR (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, n = 6/group). D, A10 cells were treated with ASO-MALAT1 and/or AngII (10⁻⁷ M). Protein expression of GPX4 was determined by immunoblotting (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, n = 6/group). E, A10 cells were coincubated with ASO-MALAT1, AngII (10⁻⁷ M), and/or sir-GPX4. Total ROS production was compared with vehicle-treated cells. Representative images were shown (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, &P < 0.05 vs. AngII and ASO-MALAT1, n = 7/group). F, A10 cells were coincubated with ASO-MALAT1, AngII (10⁻⁷ M), and/or sir-GPX4. EdU staining was used to detect the proliferation of VSMCs (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, &P < 0.05 vs. AngII and ASO-MALAT1, n = 8/group).

FIGURE 8.

Original Western blot images. A: Refer to Figure 3D. B, Refer to Figure 4B. C, Refer to Figure 5B. D, Refer to Figure 5D. E, Refer to Figure 5F. F, Refer to Figure 7C. G, Refer to Figure 7F. H, Refer to Supplemental Digital Content 1 (see Figure, http://links.lww.com/JCVP/B20).

To further clarify the role of GPX4 in the downregulation of ROS generation in VSMCs by ASO-MALAT1, GPX4 siRNA was used to interfere with the expression of GPX4. The interference effect of candidate siRNA is shown in Supplemental Digital Content 1 (see Figure, http://links.lww.com/JCVP/B20; Fig. 8H). Select the siRNA with the best interference effect for the next GPX4 interference test. Our results indicate that the silencing of GPX4 expression eliminates the inhibitory effect of ASO-MALAT1 on AngII-induced ROS generation and cell proliferation (Figs. 3E, F). These results indicate that GPX4 is a downstream signal of MALAT1 and plays a major role in the inhibitory effect of ASO-MALAT1 on AngII-induced ROS generation in VSMCs.

GPX4 overexpression in mice can improve oxidative stress, regulate lipid oxidation levels, inhibit the expression of adhesion molecules and the adhesion of monocytes to endothelial cells, repair vascular damage, and improve atherosclerosis of apolipoprotein E deficiency (ApoE(−/−)) mice.²⁶ Whether GPX4 regulates VSMCs dysfunction remains unclear. Therefore, after transfection of GPX4 plasmid in A10 cells, it was observed by immunofluorescence staining and confocal microscopy that GPX4 was highly expressed and appeared red (Fig. 4A). The successful overexpression of GPX4 was further verified by Western blot (Figs. 4B, 8B). By MTT assay, transwell, and scratch migration assays, it was found that GPX4 overexpression inhibited the proliferation and migration of VSMCs (Figs. 4C–G).

FIGURE 4.

Overexpression of GPX4 inhibited AngII-induced proliferation and migration in VSMCs. A–B, A10 cells were transfected with plasmids carrying GPX4 overexpression genes. A, Representative immunofluorescence images of GPX4 (red), α-SMA (green), and DAPI (blue) (scale bar = 20 µm). B, Protein expression of GPX4 was determined by immunoblotting (*P < 0.05 vs. control, n = 7/group). C, GPX4 overexpression A10 cells were treated with AngII (10⁻⁷ M) for 24 hours, and cell proliferation was determined by MTT assay (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, n = 6/group). D–G, Cell migration was determined by transwell assay (D) and scratch wound test (F). Representative images were shown. Cell migration number (E) and migration area (G) were also analyzed (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, n = 6/group).

ASO-MALAT1 Regulated GPX4 Expression through Nrf2 Signaling Pathway

MALAT1 regulates the expression of Nrf2 through epigenetic suppression mediated by EZH2, and Nrf2 can regulate the expression of GPX4.²⁷ Therefore, we wondered whether ASO-MALAT1 regulated GPX4 expression through Nrf2. We found that ASO-MALAT1 upregulated the protein expression of GPX4 in A10 cells (Figs. 5A, B, 8C). Then, we found that ASO-MALAT1 upregulated the mRNA expression of Nrf2 (Fig. 5C). The protein expression of GPX4 was decreased when A10 cells were transfected with siRNA of Nrf2 gene (Figs. 5D, 8D). To determine that ASO-MALAT1 might regulate the expression of GPX4 through Nrf2 signaling pathway, we used ML385, which is an Nrf2 inhibitor. The results showed that ML385 effectively blocked the upregulation of GPX4 mRNA and protein expression by ASO-MALAT1 (Figs. 5E, F, 8E).

FIGURE 5.

ASO-MALAT1 regulates GPX4 expression through Nrf2 pathway in VSMCs. A, A10 cells were treated with ASO-MALAT1 or control. Representative immunofluorescence images of GPX4 (red), α-SMA (green), and DAPI (blue) (scale bar = 20 µm). B, Protein expression of GPX4 was determined by immunoblotting (*P < 0.05 vs. ScASO, n = 6/group). C, Nrf2 expression in cells was determined by q-PCR (*P < 0.05 vs. ScASO, n = 6/group). D, A10 cells were transfected with siRNA of Nrf2 genes. Protein expression of GPX4 was determined by immunoblotting (*P < 0.05 vs. scrambled, n = 6/group). E–F, ML385-treated A10 cells were treated with ASO-MALAT1 for 24 hours. GPX4 expression in cells was determined by q-PCR (E) and immunoblotting (F) (*P < 0.05 vs. ScASO-only, #P < 0.05 vs. ASO-MALAT1-only, n = 6/group).

To further confirm that Nrf2 signaling is a key signal regulating ASO-MALAT1 to improve AngII-induced VSMCs proliferation and migration, we used ML385 (Nrf2 inhibitor) to coincubate with ASO-MALAT1. The results showed that the ML385 reversed the inhibitory effect of ASO-MALAT1 on AngII-induced VSMCs proliferation (Figs. 6A, B) and the migration of VSMCs, confirmed by transwell and scratch migration test (Figs. 6C–F). The above evidence indicates that ASO-MALAT1 upregulates the expression of GPX4 through the Nrf2 signaling pathway, thereby inhibiting the proliferation and migration of VSMCs.

FIGURE 6.

ASO-MALAT1 inhibits AngII-induced proliferation and migration of VSMCs through Nrf2 pathway. A–B, Effects of ASO-MALAT1 on AngII-induced proliferation of VSMCs. A10 cells were treated with ASO-MALAT1 and/or ML385 before coincubated with AngII (10⁻⁷ M) for 24 hours. Cell viability was determined by MTT assay (A) and cell counting (B) (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, &P < 0.05 vs. AngII and ASO-MALAT1, n = 6/group). C–F, A10 cells were treated with ASO-MALAT1 and/or ML385 before coincubated with AngII (10⁻⁷ M) in transwell assay and scratch wound assay of VSMCs migration. Representative images are shown C and E, respectively (scale bar = 100 µm). Cell migration number in transwell assay (D) and migration area in scratch wound assay (F) were analyzed. (*P < 0.05 vs. control, #P < 0.05 vs. AngII-only, &P < 0.05 vs. AngII and ASO-MALAT1, n = 6/group).

Ferrostatin-1 Inhibited Intimal Hyperplasia In Vivo

Hypertensive patients undergoing angioplasty of PCI are highly predisposed to vascular restenosis. The proliferation and migration of VSMC induced by oxidative stress is the main pathophysiological process. We constructed the SHR common carotid artery injury model. We used Fer-1 (5 mg/kg), the GPX4 (an antioxidant enzyme) agonist, to confirm the effect of GPX4 signal on intimal hyperplasia in SHRs. Vascular injury was detected by arterial H&E staining (Figs. 7A, B), the expression of PCNA was detected using Western blot (Figs. 7C, 8F), and the area of intima/media was calculated using ImageJ. The results showed that the injured carotid artery intima of SHRs was significantly thickened 2 weeks after the vascular injury. Although Fer-1 administration effectively improved intimal neogenesis, increased the total antioxidant capacity (T-AOC) and decreased the MDA level (Figs. 7D–E), and increased the expression of GPX4 in the injured common carotid arteries (Figs. 7F, 8G), Fer-1 can effectively antagonize hypertensive vascular stenosis.

FIGURE 7.

Ferrostatin-1 inhibited intimal hyperplasia after balloon injury of carotid artery in SHRs. Intimal hyperplasia of carotid artery was induced after balloon injury in SHRs. Fer-1 (5 mg/kg) or vehicle was intraperitoneally injected daily for 2 weeks. Then, carotid artery was harvested for neointima examination. A, Representative images of hematoxylin-eosin (H&E) cross-sections in different groups were shown at 10× and 20× magnifications. B, The ratio of intima (I) to media (M) was compared between vehicle and Fer-1 groups (*P < 0.05 vs. vehicle, n = 5/group). C, Protein expression of PCNA was determined by immunoblotting (*P < 0.05 vs. sham, #P < 0.05 vs. vehicle, n = 6/group). The level of T-AOC (total cellular antioxidant capacity) activity (D) and MDA (E) in carotid artery were measured (*P < 0.05 vs. sham, #P < 0.05 vs. vehicle, n = 6/group). F, The effect of Fer-1 on GPX4 protein expression in carotid artery determined by Western blot (*P < 0.05 vs. sham, #P < 0.05 vs. vehicle, n = 6/group).

DISCUSSION

VSMCs are important constituent cells of the vascular wall, whose proliferation and migration play an important role in the formation and development of vascular remodeling, atherosclerosis, vascular stenosis, and restenosis in hypertension.²⁸ High oxidative stress is a key factor in VSMCs homeostasis imbalance.²⁹ The main new finding of this study is that ASO-MALAT1 inhibits the oxidative stress, proliferation, and migration of VSMCs induced by AngII through Nrf2/GPX4. In addition, GPX4 agonist ferrostatin-1 attenuates oxidative stress and intimal hyperplasia of SHRs.

The physiological level of ROS is critical to cell function.³⁰ However, exposure to ROS that exceeds the capacity of the antioxidant system can cause oxidative stress and cause VSMCs proliferation and migration.³¹ AngII greatly promotes the pathogenesis of hypertension and vascular remodeling. lncRNA MALAT1 is highly expressed in the thoracic aorta of hypertensive rats.¹⁶ In addition, another study showed that the level of lncRNA MALAT1 in the serum of patients with white coat hypertension and coronary atherosclerotic heart disease was significantly increased.³² We found that MALAT1 in SHRs and AngII-induced VSMCs was highly expressed and ROS levels were significantly increased, indicating that MALAT1 has a higher ROS capacity and a lower antioxidant capacity environment. MALAT1 is a kind of lncRNA. The gene regulation functions reported so far mainly focus on its role in regulating transcription and RNA processing. MALAT1 knockdown reduces ROS levels induced by AngII, indicating that MALAT1 knockdown may reduce the proliferation and migration response of AngII, and is protected from oxidative stress. To further study the role of MALAT1 in regulating AngII-induced ROS, we conducted pathway analysis in MALAT1 VSMCs and found that compared with the wild type, the antioxidant genes regulated by GPX4 were activated. We confirmed that ASO-MAMLAT1 inhibited AngII-induced glutathione peroxidase GPX4 decline and overexpression of GPX4 also inhibited AngII-induced VSMCs proliferation and migration.

GPX4, also known as phospholipid hydrogen peroxide glutathione peroxidase, is the fourth GPX member containing selenium. There are 7 exons in the full-length GPX4 gene, and the theoretical molecular weight is approximately 19 kDa. Several members of the GPX family have been found in mammals, including GPX1 to GPX8. Among them, GPX4 can effectively scavenge peroxides depending on its specific amino acid sequence and spatial structure, thereby preventing oxidative stress.³³ GPX4 is widely expressed in normal tissues throughout the body. In addition to detoxification of nonenzymatic oxidized lipids, GPX4 can also reduce lipid peroxides produced by lipoxygenase, cyclooxygenase and acetyl-CoA, thereby reducing the synthesis of leukotrienes,³⁴ prostaglandins E,³⁵ and platelet activating factor.³⁶ As we all know, AngII is highly hypertensive. According to reports, GPX4 plays a role in inflammation and atherosclerosis. Overexpression of GPX4 has been shown to protect cells from oxidants and cytokines. Overexpression of GPX4 has been shown to protect cells from lipid oxidation, inhibit oxidative stress, regulate endoplasmic reticulum stress, repair endothelial dysfunction, and inhibit the expression of vascular adhesion molecules, improving monocyte and endothelial cell adhesion.³⁷ Overexpression of GPX4 in mice can effectively improve atherosclerosis.³⁸

PCI is an important means of treating coronary blood flow obstruction caused by hypertension.²⁶ Postoperative intimal neogenesis and restenosis are the main postoperative complications, and there is no effective intervention.³⁹ To better elucidate the mechanism and intervention targets of restenosis, our findings confirm that the GPX4 agonist Fer-1 can effectively prevent restenosis caused by balloon injury in SHRs and improve the oxidative stress state after vascular injury. Fer-1 treatment increased the total antioxidant capacity (T-AOC) and the expression level of GPX4, inhibit the production of MDA, improve the level of oxidative stress, and promote the PCNA expression of injured common carotid artery. This study showed that Fer-1 improved the oxidative stress and vessel restenosis of SHRs after balloon injury.

CONCLUSION

This study revealed a crucial role of MALAT1 in balloon injury-induced intimal hyperplasia in SHRs. We are the first to show that Fer-1 can attenuate neointimal formation through its antiproliferation and antimigration effects on VSMCs. ASO-MALAT1/Nrf2/GPX4 could be a potential therapeutic target to prevent vascular restenosis after angioplasty in hypertensive patients.

Supplementary Material

jcvp-84-515-s001.pptx ^{(1.1MB, pptx)}