Interstitial Fluid Shear Stress Induces the Synthetic Phenotype Switching of VSMCs to Release Pro-calcified Extracellular Vesicles via EGFR-MAPK-KLF5 Pathway

Phenotypic switching (from contractile to synthetic) of vascular smooth muscle cells (VSMCs) is essential in the progression of atherosclerosis. The damaged endothelium in the atherosclerotic artery exposes VSMCs to increased interstitial fluid shear stress (IFSS). However, the precise mechanisms by which increased IFSS influences VSMCs phenotypic switching are unrevealed. Here, we employed advanced numerical simulations to calculate IFSS values accurately based on parameters acquired from patient samples. We then carefully investigated the phenotypic switching and extracellular vesicles (EVs) secretion of VSMCs under various IFSS conditions. By employing a comprehensive set of approaches, we found that VSMCs exhibited synthetic phenotype upon atherosclerotic IFSS. This synthetic phenotype is the upstream regulator for the enhanced secretion of pro-calcified EVs. Mechanistically, as a mechanotransducer, the epidermal growth factor receptor (EGFR) initiates the flow-based mechanical cues to MAPK signaling pathway, facilitating the nuclear accumulation of the transcription factor krüppel-like factor 5 (KLF5). Furthermore, pharmacological inhibiting either EGFR or MAPK signaling pathway blocks the nuclear accumulation of KLF5 and finally results in the maintenance of contractile VSMCs even under increased IFSS stimulation. Collectively, targeting this signaling pathway holds potential as a novel therapeutic strategy to inhibit VSMCs phenotypic switching and mitigate the progression of atherosclerosis.

temperature.After staining, the sections were washed three or five times with water.In the case of HASMCs, the culture medium was removed, and the cells were washed three times with sterile PBS.The cells are fixed in 95% ethanol for 30 min, followed by three washes with ultrapure water at room temperature.Subsequently, the cells were stained with Alizarin Red S staining solution (pH = 4.2) in the dark for 30 min at room temperature.After removing the staining solution, the cells were washed three times with ultrapure water and then observed and photographed under a microscope.

Von Kossa Staining
Von Kossa silver staining solution was applied to the vessel sections and then irradiated with UV for 60 min.
Afterward, the sections were washed with distilled water for 1 min, treated with 5% sodium thiosulfate for 2 min, and washed three times with PBS.Following the staining process, the vessel sections were dehydrated using gradient ethanol and xylene, dried, and sealed.Imaging was carried out using a film sweeper (SLIDEVIEW VS200, Olympus).

Transmission Electron Microscopy (TEM)
To validate the characteristics of EVs isolated from mice tissue or HASMCs, transmission electron microscopy (TEM) was employed.The TEM observation was performed following the previously described [1].Images were captured by transmission electron microscopy (HT7800, Hitachi tech).

Oil Red O Stain
The aortas from C56BL/6 and ApoE -/-atherosclerotic mice were dissected, with the removal of any excess connective and adipose tissue.The aortas were then fixed in a 4% paraformaldehyde solution for 48 h.Subsequently, the aortas were sectioned from the aortic arch to the iliac artery and rinsed in water for 10 min before being soaked in 60% formaldehyde for an additional 10 min.The vessels were then stained with an Oil Red O staining solution while being agitated at 90 rpm for 10 min.After staining, the vessels were washed sequentially in formaldehyde and water for 10 min respectively and then photographed.

Wound-Healing Assay
The wound-healing assay was performed according to a previously described method [2].Briefly, HAMSCs were cultured on sterile slides until reaching confluence.The cells were then treated with the serum-free DMEM medium or appropriate inhibitors 24 h before the experiment.Subsequently, the cells were exposed to IFSS of 0.5 dyn/cm 2 or 3 dyn/cm 2 for 8 h, respectively.A straight and uniform wound area was created with a tip of a sterile micropipette.The slides were incubated at 37°C in a 5% CO2 environment, and photographs of the same wound area were taken at the indicated time points (0 and 24 h) using an inverted microscope (Olympus).The images were analyzed using ImageJ software, and the experiment was repeated three times in parallel.

Immunofluorescence Staining
Artery tissue sections from different groups of mice were treated with xylene and varying concentrations of ethanol, followed by heating in a microwave for 15 min and soaking in a sodium citrate solution to repair the antigens.The sections were then maintained at room temperature.For HASMCs after IFSS loading, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature, followed by PBS washing.Different primary antibodies (refer to Table S1) were dissolved in PBS containing 1% BSA, mixed in appropriate proportions, and added uniformly to the tissue or cells overnight at 4°C.Subsequently, the secondary antibodies (including TRITC-conjugated goat anti-rabbit antibody, FITC-conjugated goat antimouse antibody, FITC-conjugated goat anti-rabbit antibody, ZSGB-BIO, goat anti-rabbit IgG H&L; Alexa Fluor® 594, and Alexa Fluor® 647, Abcam) were incubated for 1 h at room temperature.Nuclei staining was performed using 4'6'-diamidino-2-phenylindole (DAPI).The Tyramide SuperBoost TM kit with Alexa Flour (Thermo Fisher) was employed for multi-color staining.The samples were stored in 1×PBS in the dark and observed by confocal laser scanning microscopy (CLSM, Zeiss, LSM710).The images were then postprocessed using FIJI Image J software.

Quantitative Real-time Polymerase Chain Reaction Detecting System (qRT-PCR)
Total RNA was extracted from cells upon IFSS stimulation for 8 h using the Cellular Total RNA Isolation Kit (FOREGENE) following the manufacturer's instruction and was quantified through NanoDrop spectrophotometer (ThrmoFisher Scientific, Waltham, MA, USA).cDNA was synthesized from 1 μg of total RNA using the Evo M-MLV RT Mix Kit and gDNAClean for qPCR (ACCURATE BIOLOGY).qRT-PCR was performed using 2×Taq SYBRGreen® qPCR PreMix (Innovagene) and the CFX Connect Real-Time PCR detection system (Bio-Rad Laboratories Inc) to assess the expression levels of target genes.The reference gene GAPDH was used to normalize relative expression values, which were obtained using the delta-Ct method.The primer sequences are available in Table S2 (Ykang Biotech Company).

Nuclear and Cytoplasmic Protein Extraction
The nuclear and cytoplasmic protein extraction was performed as described previously [1].The nuclear and cytoplasmic extracts were prepared using the nuclear protein extraction kit (Solarbio), following the manufacturer's instructions.Lamin B was used as the internal control for nuclear protein in subsequent western blot analysis.

Western Blot
Western blot was performed according to previously described protocols [3].Briefly, cells were lysed in cold RIPA buffer containing 1% protease inhibitor, 1% phosphatase inhibitor (SAB) and 1% phenylmethanesulfonyl fluoride (PMSF) on slides.Proteins in the supernatant were collected after centrifugation at 12,000 g for 10 min at 4°C and quantified using the BCA kit (Beyotime Biotechnology).
Each sample was then electrophoresed on 8% or 12% sodium dodecyl sulfate-polyacrylamide gel for 2-3 h and transferred to 0.22 μm Immobilon-FL polyvinylidene difluoride membranes (PVDF; Merck Millipore Ltd, Tullagreen, Carrigtwohill, Co., Cork, IRL) with TBST buffer containing 5% skimmed milk (20 mM Tris-HCl [pH 7.4], 500 mM NaCl, 0.1% Tween 20) for 2 h at room temperature.The primary antibodies listed in Table S1 were incubated overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies (1:3000, Solarbio) for 2 h at room temperature.Enhanced chemiluminescence (ECL, 4ABIO.) was used to visualize the bands, and the Molecular Image®ChemiDocTM XRS+ system (Bio-Rad Laboratories Inc) with Image LabTM software was utilized.Each experiment was repeated three times, and quantitative analysis was performed using FIJI Image J software.

Immunohistochemical Staining
Immunohistochemistry was conducted to detect target proteins following previously described methods [2].
Briefly, antigen repair was performed by boiling in citrate buffer (pH 6.0), and endogenous peroxidase activity was blocked with hydrogen peroxide.Aortic vascular tissue sections were stained using the appropriate primary antibody (refer to Table S1).Staining was visualized using the DAB method (TA-125QHPX, Thermo Scientific) according to the manufacturer's instructions.

RNA-Sequencing
Total RNA was extracted from the sample by using the TRIzol reagent (Invitrogen Company) following the manufacturer's instructions.Next-generation sequencing was performed by Aksomics, Inc [3,4].Quality control of total RNA was assessed using agarose gel electrophoresis, and quantification was checked using NANODROP.Agilent 2100 was used for library quality control, and library quantification was performed using qPCR.Sequencing was conducted using Illumina Hiseq 4000.After using FastQC software to detect sequence quality, the abundance of transcripts in each sample was estimated by StringTie.The FPKM value (≥0.5) of genes and transcripts were assessed using the R package Ballgown.Differentially expressed genes and transcripts were filtered based on a fold change of 1.5 or greater.Principal component analysis (PCA) was performed based on gene expression levels, and correlation analysis was conducted.Hierarchical clustering, Gene Ontology (GO), and pathway analysis were performed using genes with a fold change of 1.5 or greater.

Plasmid construction, lentiviral production, and transfection
To generate KLF5 knockdown cells, oligonucleotides were cloned into psi-LVRU6GP with the BamHI/EcoRI restriction enzyme cutting sites.The sequences of the oligonucleotides are as follows: KLF5-sense, 5'-GGTTACCTTACAGTATCAACA-3'; KLF5-antisense, 5'-TGTTGATACTGTAAGGTAACC-3'.An empty vector was used as a control, referred to as shNT.Plasmids were transformed, propagated, and purified from DH5a E. coli host cells.To establish a HASMCs cell line with KLF5 stably silenced, HEK293-T cells were co-transfected with the viral plasmid and lentiviral packaging plasmids psPAX2 and pMD2.G.The medium after 48 h and 72 h transfection was collected, filtered through a 0.45 µm filter, and centrifuged at 4000 rpm for 30 min.The PEG precipitation was then resuspended with serum-free DMEM medium (Gibco) and used to infect HASMCs cells.Cells were screened by high glucose DMEM culture medium containing 0.5 μg/mL puromycin.After 1 week of puromycin screening, the expression levels of the KLF5 were detected using qRT-PCR and western blot.GAPDH is used as a loading control, n=3

Figure S3 .
Figure S3.The screening of IFSS magnitude and in vitro verification.(A) Immunostaining demonstrated alterations in the expression of the contractile marker α-SMA (red) and the synthetic marker MMP9 (green) within HASMCs under physiological IFSS stimulation in vitro.The contractile HASMCs maintained by heparin treatment were set up as the negative control.Scale bar, 20 μm, n=3.(B) Immunostaining reveals

Figure S6 .
Figure S6.Deficiency of KLF5 suppresses IFSS-induced phenotypic switching of VSMCs.(A) Dot plots illustrated gene ontology (GO) enrichment terms of differentially expressed genes (DEGs) in HASMCs after

Figure S7 .
Figure S7.MAPK signal pathway contributes to IFSS-induced VSMCs phenotypic switching.(A-D) Western blot analysis revealed the activation of MAPK pathway in HASMCs with or without PD98059 treatment and quantitative analysis of key factors containing MAPK pathway upon the IFSS stimulation.GAPDH is used as a loading control, n=3.(E, F) Immunofluorescence staining for ERK and p-ERK in HASMCs pre-treated with PD98058 upon IFSS stimulation.ERK (green), p-ERK (red), and nuclei was counterstained with DAPI (blue).Scale bar, 20 μm.(G, H) Immunofluorescence staining for contractile
. Unpaired t-tests were used to compare variables with normal distribution and homogeneity of variance.All data are presented as mean ± SEM.