Mouse transcriptome array analysis
Two separate mouse atherosclerosis whole transcriptome array databases (MTA) were used [21, 22]. Firstly, a mouse model of atherosclerotic lesion formation induced by incomplete ligation of the common carotid artery and conical polyethylene cuff implantation was used on 12 weeks old male ApoE Knockout mice. 4 weeks after ligation with additional 4 days after cuff implantation, carotid arteries were harvested for RNA isolation and array analysis [21]. Secondly, a section of the aorta from the aortic root to the third rib was harvested every week from Ldlr −/− Apo100/100 Mttpflox/flox Mx1-Cre mice with a plasma lipoprotein profile similar to that of familial hypercholesterolemia (Ldlr2/2Apob100/100), which were fed on chow diet containing 4% fat for 60 weeks (GSE38574).
Human carotid artery plaque preparation for histology and RT-qPCR
Human carotid atherosclerotic plaques were from patients undergoing carotid endarterectomy and stored in Munich Vascular Biobank [23]. Control arteries were derived from deceased organ donors without any reported history of cardiovascular disease. The specimens were either fresh frozen at -80°C or fixed for 48 hours in 2% zinc-paraformaldehyde at room temperature, embedded in paraffin for histology study.
For gene expression analyses, specimens were treated with Trizol (Qiagen) and lysed with a tissue homogenizer (ProScientific, Oxford, MS, USA). RNA isolation was performed using the RNeasy MiniKit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Concentration of RNA was determined with a Nanodrop 2000 (Thermo Fisher Scientific, nc., Waltham, MA, USA). cDNA was generated using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). RT-qPCRs with TaqMan probes (Thermo Fisher) were applied to determine the changes in gene expression.
Human carotid atherosclerotic plaque single-cell Sequencing and data analysis
Human carotid arteries were obtained from patients undergoing carotid endarterectomy or open repair in our Department of Vascular and Endovascular Surgery (Munich Vascular Biobank) as previously published [24].
Single-cell RNA sequencing and analysis were performed as described [24]. The related Microarray data are available from Gene Expression Omnibus data sets (accession number GSE247238).
Isolation of primary human monocytes
Primary monocytes were isolated from whole blood of healthy individuals obtained from the blood bank of Karolinska University Hospital according to a standard protocol. Briefly, whole blood was diluted 1:1 with phosphate-buffered saline and layered on top of Ficoll-Premium (GE Healthcare) gradient to separate monocytes/lymphocytes from red blood cells and neutrophils by centrifugation (4000g, 30 min). The intermediate fraction, containing monocytes and lymphocytes, was collected and residual red blood cells were lysed using ACK (Ammonium-chloride-potassium) lysis buffer. To increase the purity of monocytes, a hyperosmotic Percoll (GE Healthcare) solution was used with subsequent centrifugation (5800 g, 15 min). Monocytes were cultured in Roswell Park Memorial Institute medium (RPMI 1640, Invitrogen), supplemented with 5% fetal bovine serum and 0.1% Penicillin-Streptomycin, differentiated with macrophage colony-stimulating factor (M-CSF, 100 ng/mL) treatment for 7 days, and transformed to macrophages on day 3 with M-CSF and fresh media.
THP1 cell culture
Human monocytic THP-1 cells were maintained in culture in RPMI culture medium containing 10% of heat inactivated fetal bovine serum and supplemented with 1% L-glutamine 1% Penicillin-Streptomycin. To transform THP1 monocytes to macrophages, 100 ng/ml PMA (phorbol 12-myristate-13-acetate) was added to the medium for 24 h and one day without PMA prior to the treatment.
Immunofluorescence staining
Monocytes plus M-CSF were seeded on coverslips (7 days), after treatment they have been fixed with 4% paraformaldehyde for 2 h. The cells were washed three times with PBS and permeabilized in 0.1% Saponin (20 min). After blocking with 2.5% BSA, the cells were incubated overnight with primary antibodies followed by staining with Alexa-dye labeled secondary antibodies. Images were obtained using LSM 700 Confocal Laser Scanning Microscope (Carl Zeiss). Images were processed either in Zen software (Carl Zeiss) or via Fiji (ImageJ).
Preparation of oxidized low-density lipoprotein
Lipoproteins used for foam cell formation were isolated through sequential ultracentrifugation from human plasma obtained from the blood bank. Briefly, plasma was ultracentrifuged for > 22 h at 285,000 g at 4°C. The intermediate fraction containing low-density lipoprotein (LDL) and high‐density lipoprotein (HDL) was collected. The density of the LDL/HDL fraction was adjusted to 1.063 g/ml with potassium bromide (Sigma‐Aldrich) and ultracentrifuged as described above. The upper fraction with LDL was collected and desalted using a PD‐10 column (GE Healthcare). LDL was oxidized overnight at 37°C using 20 µM copper sulphate [CuSO4] (Merck), and the reaction was stopped using 1 mM EDTA (Sigma‐Aldrich).
To produce FITC-labelled oxLDL, Fluorescein Isothiocyanate (FITC, Sigma-Aldrich) was dissolved in DMSO and added to LDL (10 µM). Then, incubated in dark under rotation for 4 h at 4°C. The remaining FITC was removed by PD10 column (GE Healthcare).
Foam cell formation
Human monocyte differentiated macrophages were exposed to 25 µM FITC-labelled oxLDL for 24,48 and 72 h, foam cell formation was confirmed by fluorescence microscopy and observation of lipid droplets of the macrophages.
Live cell imaging for apoptosis
Human monocyte differentiated macrophages were seeded in a 12- or 24-well plate. To assess the effect of PIEZO1 on apoptosis, CellEvent Caspase-3/7 Green Detection Reagent (ThermoFisher) was used according to the manufacturer’s instructions and the images were obtained with the use of live cell imaging IncuCyte HD system (Essen BioScience). Cultures were maintained at 37°C in an XL-3 incubation chamber (Carl Zeiss) and run-in quadruplicates.
Phagocytosis assay
Human monocyte differentiated macrophages were seeded on glass coverslips in 24 well plate and stimulated with 50 µM Yoda1 (Tocris Bioscience, UK) for 1h. Without changing the medium, pHrodo™ Green Zymosan Bioparticles™ were added for an additional 90 min followed by addition of LysoTracker Red (1 µM) before terminating the experiment. Cells were fixed with 4% paraformaldehyde and mounted on the slide for imaging.
PIEZO1 silencing
To knockdown PIEZO1 gene expression in monocyte differentiated macrophages, siRNA targeting human PIEZO1 or scrambled (negative control) was purchased from Dharmacon (L-020870-03-0005, D-001810-10-05)[25]. 20nM siRNA were transfected using Lipofectamine RNAiMax (Invitrogen) for 3 days.
Calcium influx measurement
Human monocyte differentiated macrophages were seeded on glass-bottom dishes (ibidi) and treated with Yoda1 (50 µM) or DMSO as solvent control for 2 h. Thereafter, they were stained with Fluo4-AM (1 µM, Life Technologies) for 30 min before starting imaging on 63× oil immersion objective of LSM 700 Confocal Laser Scanning Microscope (Carl Zeiss).
Live cell mitochondrial fragmentation, mtROS, and cytosolic ROS assays
Mitochondria fragmentation, mitochondrial reactive oxygen species (mtROS), and cytosolic ROS were assessed on cultured human monocyte differentiated macrophages seeded on glass-bottom dishes (ibidi GMbH, Gewerbehof Gräfelfing, Germany). Yoda1 (50 µM) or DMSO solvent control was added to medium for 2 h, then 30 min before imaging either MitoTracker Deep Red (1 µM, ThermoFisher) or MitoSOX (1 µM, ThermoFisher) or H2DCFDA (10 µM, ThermoFisher) was added to the medium. Images were obtained using 63× oil immersion objective of LSM 700 Confocal Laser Scanning Microscope (Carl Zeiss).
Extracellular ROS assay
Monocyte differentiated macrophages were seeded in 96 well plate and treated immediately with Lucigenin-enhanced chemiluminescence (500 µM) and Yoda1 (50 µM) or DMSO solvent control. Luminescence values were detected by Glomax multi detection system (Promega).
RNA isolation and processing of macrophages
RNA was extracted from cells using RNeasy Mini Kit according to a modified manufacturer’s protocol (Qiagen). Reverse transcription was carried out using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. RT-qPCR was carried out using TaqMan gene expression assays (Thermo Fisher Scientific) and StepOne plus Real-Time PCR System (Thermo Fisher Scientific). Relative gene expression was determined using the ΔΔCT method (online methods)
Western blot
Monocyte-derived macrophages were lysed in a buffer containing 50 mmol/L HEPES (pH 7.4), 1% Triton X-100 (v/v), complete protease inhibitor cocktail, and PhosSTOP phosphatase inhibitor cocktail (both from Roche Diagnostics). Equal amounts of protein were loaded to SDS‐PAGE using 4–20% Criterion XT Bis‐Tris precast gels (Bio‐Rad, Hercules, CA) followed by transfer onto polyvinylidene fluoride membrane (Immobilon‐P; Millipore). Incubation with primary antibodies was performed in the same buffer overnight at 4°C. Peroxidase‐conjugated IgG was used as secondary antibody. The SuperSignal West Dura Extended Duration Substrate from Thermo Fisher Scientific was used for enhanced chemiluminescence detection. The signals were visualized and evaluated on a ChemiDoc Touch Imaging System (Bio‐Rad).
Animal gain and loss function experiments
Male ApoE−/− mice (6–8 weeks old) and high fat diet were purchased from Nanjing Qingzilan Technology Co., Ltd (Nanjing, China). All mice were fed on chow diet until they were 16 weeks old and then changed to a high fat diet (1.5% cholesterol, 10% lard, 4% milk powder, 0.5% sodium cholate) for 4 weeks before and during the 4-week treatment period (online Fig. 4A). The mice were randomly divided into three treatment groups (n = 6 per group): vehicle (saline) control; PIEZO1 antagonist (GsMTx4); and PIEZO1 agonist (Yoda1). GsMTx4 (270 µg/kg, ab141871, Abcam)[26] and Yoda1 (70 µg/kg, SML1558-25MG, Sigma-Aldrich)[27] diluted in saline and 200 µl of each treatment were injected intraperitoneally twice a week for 4 weeks. Tissues were collected 3 days after the last intraperitoneal injection. All mice were housed in a specific pathogen-free environment on a 12 h light/dark cycle at 25°C with free access to high fat diet and water. The animal protocol was approved by the Harbin Medical University Ethics Committee (13766830089). The use and care of the animals conformed to the US National Institutes of Health Guide for Care and Use of Laboratory Animals.
Mouse blood pressure measurement
The CODA™ mouse/rat tail-cuff system (USA) was used to noninvasively monitor the mice’s blood pressure before and after injection every week. The blood pressure was measured in a quiet standardized animal room on a heating pad at 30 to 35°C.
Mouse serum lipid profiling
Mouse peripheral blood was collected. After standing at room temperature for 2 h, the blood was centrifuged under the condition of 4°C, 3000 r/min for 15 min. Serum was separated for the determination of total cholesterol, low-density lipoprotein cholesterol and triglycerides with the kits (A111-2; A110-1-1, A113-1-1, Nanjing Jiancheng Bioengineering Institute, China).
Evaluation of mouse peritoneal macrophage oxLDL uptake
High fat-diet fed ApoE−/− mice were euthanized with CO2 and immersed in 75% alcohol for 5 min. Peritoneal macrophages were harvested via peritoneal lavage with 5 ml cold phosphate-buffered saline (PBS). The cells were cultured in 35-mm petri dishes in RPMI-1640 medium containing 15% FBS. Adherent peritoneal macrophages were used for subsequent experiments. The cells were transformed into foam cells by incubating with 25 µg/ml oxidized low-density lipoprotein (oxLDL) in serum-free RPMI-1640 medium for 24 h. Cells were fixed with 4% paraformaldehyde. The staining was measured according to the manufacturer’s instructions of the Oil Red O staining kit (Solarbio) and were observed under light microscope (Olympus) with quantification using Image-Pro Plus 6.0 software (Media Cybernetics).
Histology and immunohistochemistry
For immunohistochemistry, consecutive tissue sections of 3µm thickness were incubated for 1h at room temperature with primary antibodies detailed in Table S1. Secondary antibody and DAB-mediated detection reagents were provided with the DAKO REAL Detection Kit Rabbit/Mouse. Nuclear counterstaining was performed with hematoxylin staining.
Mouse atherosclerotic lesions were evaluated using H&E and Oil Red O (Solarbio kit) staining with cryosections of the aortic roots. Masson’s trichrome staining was performed to assess the collagen contents in atherosclerotic plaques.
Images were obtained using an Olympus microscope. The positive area relative to the plaque area was analyzed using a computer-assisted color image analysis system (Image-Pro Plus, version 6.0, Media Cybernetics, Inc.)
Immunofluorescence staining was performed on cryosections of murine aortic roots. The sections were incubated with anti-PIEZO1 antibody, anti-CD206 antibody, and anti-iNOS antibody at 4°C overnight. Rinsed sections were then incubated with FITC conjugated anti-F4/80 PE antibody for 1 h at room temperature in the dark and 4’,6-diamidino- 2-phenylindole (DAPI) for additional 2 min. The fluorescent images were obtained using an Olympus BX53F microscope (Olympus). All parameters were measured by computer-assisted color image analysis (Image-Pro Plus, version 6.0, Media Cybernetics, Inc.).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Cell apoptosis was determined by In Situ Cell Death Detection Kit(Roche, 11684817910). Aortic root sections were incubated with TUNEL reaction mixture and anti-F4/80-PE. Followed by DAPI incubation. Sections were analyzed with a fluorescence microscope (Olympus BX53F) and the percentage of TUNEL positive cells was analyzing using an ImageJ software.
Statistics
All data is presented as the mean ± standard deviation. The difference among groups was analyzed via Students t-test or one-way analysis of variance (ANOVA) followed by the Tukey's multiple comparisons test. Differences with P < 0.05 were statistically significant (* P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). The statistical analyses were performed using GraphPad Prism 8.0.