Induction of ferroptosis promotes vascular smooth muscle cell phenotypic switching and aggravates neointimal hyperplasia in mice

Chemicals and reagents

RAS-selective lethal (RSL3, S8155) and ferrostatin-1 (Fer-1, S7243) were purchased from Selleck Chemicals (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO, V900090, Sigma-Aldrich). RSL3 and Fer-1 were prepared for intraperitoneal (i.p.) injection as follows: 1% RSL3 or Fer-1 + 30% PEG300 (S6704, Selleck) + 5% Tween 80 (S6702, Selleck) + 64% H2O. N-Acetyl-l-cysteine (NAC, A9165, Sigma-Aldrich) was used. An optimal cutting temperature compound (OCT, 14020108926) was obtained from Leica (Wetzlar, Germany).

Animals

Animal experiments were approved by the Animal Research Ethic Committee of Guangzhou Medical University (Protocol number: 2019-061) and performed following the NIH Guide for the Care and Use of Laboratory Animals. Male 8-week-old C57BL/6J mice were purchased from Shanghai Model Organisms (Shanghai, China). All mice were fed a chow diet, given autonomous access to water and food, maintained in the specific pathogen-free (SPF) facility, and kept on a 12 h light–dark cycle. For ligation of the common right carotid artery, mice were anesthetized with isoflurane on a heated stage. All hair on the neck between the mandible and sternum was gently removed using a suitable amount of depilatory agent, and an incision was made in the middle of the neck to find the common right carotid artery, which was ligated with 6-0 silk suture. Mice were randomly assigned to the vehicle (i.p. injection of DMSO, daily), RSL3 (i.p. injection of 10 mg/kg, daily), or Fer-1 (i.p. injection of 1 mg/kg, daily) groups. Cross-sections of carotid arteries were stained with hematoxylin and eosin (H&E). Intimal and medial areas were measured using Image J software (National Institutes of Health, Bethesda, MD, USA).

Cell culture

Primary cultures of VSMCs were obtained from the media of aortas of SPF Sprague-Dawley (SD) rats (body weight 150–180 g) by tissue explant method, as described previously (Chi et al. 2017). The rat VSMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, C11995500BT, Gibco) containing 10% fetal bovine serum (FBS, 10270-106, Gibco), and 100 U/ml penicillin/streptomycin (15140-122, Gibco). Primary rat VSMCs from 4 to 7 passages were used for the experiments. Cells with ~ 70% confluency were treated.

Western blotting

Cells were washed twice with PBS after treatment. 50 μl pre-cooled RIPA lysis buffer (FD008, FUDE) was added to cells and placed on ice for 10 min. The protein extraction was collected using a cell scraper into an Eppendorf (EP) tube, followed by centrifugation at 13,000g and 4 °C. The supernatant was taken and transferred into a new EP tube. After the protein concentration was determined using the bicinchoninic acid (BCA, P0009, Beyotime) method, 4× loading buffer was added to the samples, mixed evenly, and heated at 100 °C for 10 min. 30 μg protein was separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes (66485, PALL). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight. Membranes were then washed and incubated with HRP-conjugated secondary antibodies, washed again, and visualized with HRP chemiluminescent substrates (WBKLS0500, Millipore). Two pre-stained protein markers (abs923, absin; MP102-02, Vazyme) were used. Primary antibodies were as follows: smooth muscle myosin heavy chain (SM-MHC, ab53219, Abcam), calponin 1 (ABT129, Millipore), osteopontin (OPN, ab11503, Abcam), and β-actin (sc-47778, Santa Cruz). β-actin was used as the internal control. Quantification of band densitometry was done using AI 600 (GE, MA, USA).

Quantitative RT-PCR (qRT-PCR)

Total RNA from rat VSMCs was extracted using TRIzol reagent (21101, AG). The purity and concentration of the RNA were determined by Thermo Scientific NanoDrop One. A reverse transcription kit (11706, AG) was applied to synthesize cDNA, and SYBR Green real-time PCR premix kit (11701, AG) was used to measure the mRNA levels. qRT-PCR was performed on a LightCycler® 480 Instrument II (Roche Applied Science, CA, USA). The following primers were used (5′-3′): Myh11 (SM-MHC) forward: ATCACGGGGGAGCTGGAAAA; Myh11 (SM-MHC) reverse: AATGAACTTGCCAAAGCGGG; Acta2 (α-SMA) forward: CATCCGACCTTGCTAACGGA; Acta2 (α-SMA) reverse: AGAGTCCAGCACAATACCAGT; Cnn1 (Calponin 1) forward: GCCCAGAAATACGACCACCA; Cnn1 (Calponin 1) reverse: TGGAGCTTGTTGATAAATTCGCA; Spp1 (OPN) forward: CAGTCGATGTCCCTGACGG; Spp1 (OPN) reverse: GTTGCTGTCCTGATCAGAGG; Ptgs2 (COX-2) forward: TCCTCCTGTGGCTGATGACT; Ptgs2 (COX-2) reverse: CGGGATGAACTCTCTCCTCA; Gpx4 forward: CCATTCCCGAGCCTTTCAAC; Gpx4 reverse: CGGTTTTGCCTCATTGCGAG; and Gapdh forward: ATTGTCAGCAATGCATCCTG; Gapdh reverse: ATGGACTGTGGTCATGAGCC.

Immunofluorescence

Immunofluorescence staining was performed after fixation in 4% (v/v) formaldehyde (G1101, Servicebio) and permeabilization with 0.5% Triton X-100 in PBS, supplemented with 10% goat serum for 1 h. Samples were incubated with primary antibodies for cyclooxygenase-2 (COX-2, ab15191, Abcam) and α-smooth muscle actin (α-SMA, ab5694, Abcam) at 4 °C overnight and then incubated with Goat anti-Rabbit Alexa Fluor Plus 555 (1:1000) secondary antibody (A32732, Invitrogen) for 1 h at room temperature. After washing three times in PBS, samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, D1306, Invitrogen) for 15 min, and fluorescence was preserved using Fluoromount-G (0100-01, SouthernBiotech). Images were captured by a Nikon A1R confocal microscope (Tokyo, Japan). Mean fluorescence intensity (MFI, AU) and relative fluorescence intensity was calculated using Image J software.

Perls’ Prussian blue staining

Perls’ Prussian blue staining was used to analyze iron deposition in neointima. The cross-sections of ligated carotid arteries were fixed with 4% (v/v) formaldehyde (G1101, Servicebio) and washed with PBS 3 times. Sections were incubated in Perl’s solution (G1422, Solarbio) for 30 min, and deionized water was used to rinse three times. After incubation with Nuclear Fast Red (G1422, Solarbio) for 7.5 min, the sections were set in 75%, 85%, 95%, and 100% ethanol for rapid gradient dehydration. Iron+ cells were counted at ×200 magnification.

Statistical analysis

Statistical analyses were conducted using Graph Pad Prism 8 (Graph Pad Software). Data were presented as the mean ± SEM. Two groups were compared using a two-tailed unpaired Student’s t-test. Multiple groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. P < 0.05 was considered statistically significant.

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