Cell cultures
Human umbilical vein endothelial cells (HUVECs), a human endothelial cell lineage, were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. LysoPC(Sigma Aldrich; Merck KGaA) was dissolved in PBS and preserved at –20 °C. When the cells reached about 80% confluence, they were treated with lysoPC for 24 h. Cells in the GDF11 and inhibitor groups were pretreated with GDF11 and 4-PBA for 1 h prior to exposure to 50 ug/mL lysoPC for another 24 h.
Cell viability assay
Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Briefly, cells were seeded in 96-well plates at a density of 1×104 cells/well overnight. After treatment for 24 h as described in Cell Cultures, 10 μL of CCK-8 solution was added to the culture medium in each well, and the cells were then incubated in humidified 95% air and 5% CO2 for 1 h at 37 °C. The absorbance at a wavelength of 450 nm was measured using a microplate reader (Thermo Fisher Scientific, NYC, USA).
Determination of the LDH activity
Cell incubation was carried out as previously described in Cell Cultures, after which cytotoxicity was determined by monitoring LDH release with an LDH cytotoxicity assay kit (Beyotime, Shanghai, China). Briefly, after the predetermined period of time, the cell culture plate was centrifuged at 400 g for 5 min. We took 120 μL of supernatant from each well and added it to the corresponding well of a new 96-well plate, and then added 60 μL of LDH detection solution to each. This plate was incubated at room temperature for 30 min, after which the absorbance at 490 nm was measured using a microplate reader (Thermo Fisher Scientific, NYC, USA).
Hoechst 33342 and propidium iodide (PI) staining
HUVECs were seeded into 6-well plates at a density of 2×105 cells/well. After treatment, the cells in each group were washed with PBS 3 times and stained with 5 μL of Hoechst 33342 and 5 μL of PI (Beyotime, Shanghai, China) for 20 min at 4 ℃. The stained cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) and evaluated using the Image J software.
Western blot analysis
As usual, HUVECs were seeded into 6-well plates at a density of 2×105 cells/well. After treatment, the HUVECs were analyzed using Western blotting [24]. Briefly, the cells were lysed with RIPA buffer containing 1% proteinase inhibitor, PMSF, at 4 ℃. Then, they underwent centrifugation for 10 min at 4 ℃, the supernatant was collected, and the protein concentration was measured with a BCA kit (Beyotime, Shanghai, China). The protein samples were separated by SDS–polyacrylamide gel electrophoresis and transferred onto a poly (vinylidene fluoride) (PVDF) membrane. After blocking with 5% non-fat milk for 1 h at room temperature, the membrane was incubated with a primary antibody against CHOP (Beyotime, Shanghai, China), Casp1 p20 (Cell Signaling Technology, MA, USA), GDF11, LOX-1, NLRP3, IL-1β, GSDMD-N, or GRP78 (Abcam, Cambridge, UK) at 4 °C overnight. This was followed by incubation with a secondary antibody for 1 h at 37 °C. The proteins were scanned using the Odyssey Imaging System, and the band intensities were quantified using Image J. GAPDH (Abcam, Cambridge, UK) was used as an internal control. The protein expression levels of NLRP3, Casp1 p20, and IL-1β in the harvested artery specimens were also determined using Western blotting.
RNA extraction and qPCR
The total RNA of HUVECs was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer’s instructions. The concentration of RNA was measured usinga NanoDrop (Thermo Fisher Scientific, NYC, USA). Reverse transcription and qPCR were performed as described previously [25]. To carry out mRNA quantification, we used a reverse transcription kit (Vazyme, China) for the reverse transcription of total RNA according to the manufacturer’s instructions. Following this, the SYBR Green Master Mix (Nanjing, China) was utilized to perform qPCR, and a Roche Light Cycler system (Roche, Switzerland) was used for analysis. The sequences of the primers used were as follows: LOX-1, F: 5′-GAGTGAACATATCCATCATC-3’, R: 5′-TGGAGACATATGAATCTCAA-3’; GAPDH, F: 5′-AAGAAGGTGGTGAAGCAGGC-3’, R: 5′-TCCACCACCCAGTTGCTGTA-3’.
Small-interfering RNA transfection
The small-interfering RNA against LOX-1 (si-LOX-1) was synthesized by RiboBio Biotechnology Company (Guangzhou, China). For transfection, 50 nM si-LOX-1 and scrambled siRNA were delivered into cells using the Lipofectamine™ 2000 transfection reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. HUVECs were harvested for other experiments 48 h after transfection.
Animal model and adenovirus transduction
Animals were treated in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and this experiment was approved by the Animal Ethics Committee of the Tongji University School of Medicine. Male Sprague-Dawley rats (weight: 300–350 g) were anesthetized by the intraperitoneal injection of pentobarbital solution (30 mg/kg, Sigma) and then injected with heparin (100 U/kg) in the tail vein. The procedure for the rat-carotid-artery balloon-injury model has been described previously [26]. In brief, a 2F balloon catheter (Fogarty, E-060-2F, Baxter) was inserted via the right external carotid artery into the common carotid artery, and the balloon was inflated with saline and drawn towards the arteriotomy site three times to ensure complete intimal injury. The injured artery was washed with PBS. Then, an adenovirus encoding GDF11 or control GFP (1×109 pfu, Genechem Co., Ltd, Shanghai, China) was injected into the balloon-injured rat carotid arteries via the incision of the right external carotid artery and kept in situ for 30 min. The solution was subsequently withdrawn from the infected area, and blood flow was restored. To demonstrate the efficiency of adenovirus delivery into the carotid artery, carotid arteries from Ad-GFP infected rats were observed under fluorescence microscopy (Olympus, Tokyo, Japan).
Evans blue staining
To measure the re-endothelialization area 7 days after vascular injury, 5% Evans blue dye (Sigma) was injected via the tail vein. After 30 min, arteries were dissected from the carotid bifurcation and opened longitudinally to analyze the denuded and recovered areas. Images were obtained using a digital camera (Cannon, Japan). Re-endothelialization was measured using image analyzer software (Image-Pro Plus 6.0).
Immunohistochemistry
After the rats were sacrificed, the injured aortic tissues were fixed with 4% paraformaldehyde solution, embedded in paraffin, and cut into 6 μm sections, which were prepared and incubated with rabbit anti-CD31 (Servicebio, China) overnight at 4 °C. After routine immunohistochemistry, the sections were observed under a microscope (Olympus, Tokyo, Japan). The Image Pro Plus 6.0 software was used to calculate the ratio of CD31-expressing cells to the total number of cells in the arterial lumen wall to measure the re-endothelialization rate for each cross-section.
Hematoxylin and eosin (HE) staining
Paraffin sections of 6 μm were deparaffinized with xylene and hydrated with ethanol. The sections were dyed with hematoxylin and then stained in eosin dye solution. Images were obtained with an inverted phase contrast microscope (Olympus, Tokyo, Japan). Neointima formation (intimal, medial, and intimal/medial area) was measured using the Image-Pro Plus 6.0 image analysis software.
Statistical analysis
All the data are expressed as means ± SEMs, and all the experiments were performed in triplicate. Comparisons between groups were evaluated using unpaired Student’s t tests. Analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was utilized to test for differences among groups. The results were analyzed using Graphpad Prism 9 (Graphpad Software Inc., USA). P<0.05 was regarded as indicating a statistically significant difference.