The protocols and procedures in this present study were approved by the Experimental Ethics Committee of Zunyi Medical University. All protocols were consistent with the Guide for the Care and Use of Experimental Animals at the Animal Center of Zunyi Medical University.

A total of 30 SPF (specific pathogen-free) male Sprague–Dawley (SD) rats (body weight:300–400 g) were used in this study. The rats were randomly divided into three groups and subjected to the following treatments: the sham group (n = 10): received a sham operation with the same volume of saline by gavage daily; the control group (n = 10): model induction with the same volume of saline by gavage daily; the ICS-II group (n = 10): model induction with ICS-II 20 mg/kg by gavage daily. The protocols for the rat carotid artery balloon injury model were described previously (Lv et al., 2018). Briefly, the rats were anesthetized with 5% isoflurane in oxygen. A midline cervical incision was made to expose the bilateral carotid arteries. A 2F Fogarty balloon catheter (Edwards Lifesciences Corporation; United States) was inserted into the left common carotid artery (CCA) three times to disrupt the endothelium through the external carotid artery (ECA). After that, the ECA was sutured and the left CCA was re-perfused. All processes during the operation were performed by a single researcher. All rats were euthanatized on day 14 for the following experiments.

For the morphometric analysis, artery samples were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned into 4 mm thickness. After deparaffinization and rehydration, sections were stained with hematoxylin and eosin (H&E) to observe the morphological changes. For each section, five randomly selected non-continuous microscopic fields were pictured to determine the mean thickness value of the intima and media, respectively. Finally, the thickness ratio of intima to media of each section was determined.

IHC experiments were implemented for assessing the protein expression level of the VSMC synthetic marker OPN in vitro. Protocols for the IHC experiments were described previously (Wen et al., 2020). In brief, after de-paraffinization and rehydration, sections were incubated with 3% H₂O₂ to deactivate endogenous peroxidase followed by a heat-induced antigen retrieval process. Then, sections were incubated with primary and secondary antibodies before image acquisition.

The arterial tissue samples were lysed with an SDT (4% SDS in 0.1 M Tris-HCl, pH = 7.6) solution, transferred into a Lysing Matrix A (MP, cat: 6910-100-99219) tube, and homogenized and broken with an MP homogenizer (MP Fastprep-24 5G, 24 × 2, 6.0 M/S, 60 s, twice). The supernatant was filtered through a 0.22 µm centrifuge tube and the filtrate was collected. Protein quantification was performed using the BCA (Beyotime Biotechnology, P0012) method. About 80 μg of the protein solution was taken for each sample, and DTT (dithiothreitol, Sigma, 43819-5G) was added to a final concentration of 100 mM, boiled for 5 min, and cooled to room temperature. Then, 200 μL of the UA (8 M urea in 0.15 M Tris-HCl, pH = 8.5) buffer was added, mixed, and transferred to a 30 kDa ultracentrifuge tube (Sartorius, VN01H22); the mixture was centrifuged at 12,500 g for 15 min, and the filtrate (repeat this step once) was discarded. Further, 100 μL of IAA (iodoacetamide, Sigma, I1149-5G) buffer (100 mM IAA in UA) was added and the mixture was shaken at 600 rpm for 1 min and centrifuged at room temperature for 30 min at 12,500 g for 15 min. Then, 100 μL of the UA buffer was added and centrifuged at 12,500 g for 15 min. Subsequently, 100 μL of 40 mM NH₄HCO₃ solution was added, centrifuged at 12,500 g for 15 min, and the procedure was repeated twice. After that, 40 μL of trypsin buffer (4 μg of trypsin in 40 μL of 40 mM NH₄HCO₃ solution) was added and the mixture was shaken at 600 rpm for 1 min, and allowed to stand at 37°C for 16–18 h. The collection tube was replaced with a new one and centrifuged at 12,500 g for 15 min; then 20 μL of 40 mM NH₄HCO₃ solution was added and centrifuged at 12,500 g for 15 min, and the filtrate was collected. The peptide was desalted by C18 Cartridge (Waters, WAT023590), and 40 μL of 0.1% formic acid (Thermo Fisher Scientific, A117) solution was added after the peptide was lyophilized; the peptide was quantified (OD 280).

For LC–MS/MS analysis, each sample was analyzed using the Easy nLC system (Thermo Fisher Scientific). Buffer solution A was 0.1% formic acid in water and solution B was 0.1% formic acid with 80% acetonitrile in water. The chromatographic column was equilibrated with 100% solution A. Samples were separated by autosampler loading onto an analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50 μm × 15 cm, nano Viper, P/N164943) at a flow rate of 300 nL/min. The samples were separated by chromatography and analyzed by mass spectrometry using a Q Exactive (Thermo Fisher Scientific) mass spectrometer. Precursor mass spectra were recorded in a 350–1,800m/z (mass/charge ratio) mass range at 70,000 resolution and 17,500 resolution for fragment ions. Data obtained were analyzed using MaxQuant (version 1.5.5.1) against the UniProt database (Uniprot_RattusNorvegicus_36080_20180123) based on the LFQ (label-free quantitation) method (Cox et al., 2014) with the following parameters: enzyme, trypsin; max missed cleavages, two; main search, 4.5 ppm; first search, 20 pp; MS/MS tolerance, 20 ppm; fixed modification, carbamidomethyl (C); dynamic modification, oxidation (M), acetyl (protein N-term); peptide and protein FDR (false discovery rate), <0.01.

The DEP Package (Zhang et al., 2018) in R software (version: 4.0.2) was applied for differentially expressed protein analysis. Proteins were filtered to remove contaminants and missing values in more than one sample per group. Then, background correction and normalization by the variance-stabilizing transformations method (Huber et al., 2002) were performed. The remaining missing values were estimated and imputed by impute.MinProb function for left-censored missing data (Lazar, 2015). A differential expression analysis based on protein-wise linear models with empirical Bayes statistics was then performed on the imputed dataset. Proteins with the absolute value of fold change >1.5 and p-value < 0.05 were identified as significant DEPs.

Construction of the PPI network was implemented with the Search Tool for Retrieval of Interacting Genes/Proteins (STRING: https://string-db.org/) database (Szklarczyk et al., 2019) with criteria of medium confidence (interaction score >0.4). Then, the derived PPI network was visualized and analyzed by the analysis network function via Cytoscape (version 3.8.0) (Shannon et al., 2003) software. Proteins in the aforementioned PPI network with a degree >5 were selected for visualization and subsequent enrichment analysis.

Enrichment analysis regarding Gene Ontology (GO) (Ashburner et al., 2000) terms describing the biological process (BP), molecular function (MF), and cellular component (CC), as well as the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Ogata et al., 1999) pathway, were performed by using the clusterProfiler (Yu et al., 2012) package in R. Enriched terms with FDR <0.05 were selected and visualized.

Rat VSMCs were primarily isolated and cultured by the explant method as described previously (Li et al., 2020). In brief, thoracic aortas were gently dissected from 8-week-old male SD rats and the surrounding connective tissue was removed. Aortas were longitudinally dissected, and the epithelial monolayer was detached by scraping. Tissue fragments were delivered to culture plates with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. After 2 days, tissue fragments were removed and the sprouting VSMCs were maintained until 80% confluence. Cells were trypsinized and seeded on a culture flask for in vitro experiments. All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C, and passages between three and six were used in the present study.

The primarily cultured VSMCs were randomly divided into three experimental groups: the control group, 0.5% DMSO; the PDGF group, 25 ng/ml PDGF; and the ICS-II group, 20 μM ICS-II + 25 ng/ml PDGF. All experimental cell groups were incubated with corresponding drugs for 24 h, respectively.

Briefly, cells were seeded onto coverslips and incubated for 24 h in 6-well plates. After that, the cells were fixed with formaldehyde and permeabilized with 0.25% Triton X-100 in PBS and 1% bovine serum albumin (BSA) for 5 min. Cells were then blocked with 5% goat serum in PBS for 1 h. Then, the cells were incubated with primary antibodies overnight at 4°C and treated with secondary antibodies in 3% BSA in PBS for 2.5 h at room temperature. Then, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescence microscope. Negative isotypes were incubated with PBS under the same conditions.

Briefly, the total proteins from different groups were extracted by lysing VSMCs with RIPA buffer (Beyotime, Shanghai, China), and the protein concentration was determined by using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Then, the protein samples were separated by SDS–PAGE and subsequently transferred to PVDF membranes (Millipore, United States). After being blocked in 5% BSA at room temperature for 2 h, membranes were incubated for 12 h at 4°C with primary antibodies. After being washed three times with PBS, HRP-conjugated secondary antibodies were used to incubate the membranes for 1 h at room temperature. Finally, protein bands were visualized using chemiluminescence equipment.

The primarily cultured VSMCs were seeded into 6-well plates at a cell density of 5×10⁵ until 80% confluence. The cells were scratched with a 10 μL pipette tip, the residue was rinsed, and the corresponding drugs with the medium containing 1% serum were added. The cell scratch assay was observed under an inverted microscope (Olympus, Japan), and photographed at 0 and 24 h, respectively.

The VSMC transwell migration assay was implemented using 24-well transwell chambers (8 μm pore size; BD Biosciences, United States). Briefly, a total of 10,000 cells per well were re-suspended in a serum-free medium with corresponding interventions, respectively. Cells were then added into the top chamber with the same medium containing 10% FBS at the bottom of the chambers. After 24 h, the migrated cells through the biofilms were fixed and stained with a crystal violet staining solution (C0121, Beyotime, China) for morphological observation with the microscope (Olympus, Japan).

Results are expressed as mean ± SEM (standard error of the mean) and the statistical significance between the means of the two groups was determined by the two-tailed t-test or Mann–Whitney test. The p-value < 0.05 was considered statistically significant. GraphPad Prism (version 8.2.1, United States) was used for all statistical analyses.

To investigate the role of ICS-II in vascular remodeling, a rat carotid artery balloon injury model was implemented, which is capable of mimicking vascular remodeling as well as VSMC phenotypic transition validly in vivo (Tulis, 2007; Zhang and Trebak, 2014). Results from the morphometric analysis showed that ICS-II significantly suppressed intima thickness and intima/media ratio (Figure 1A–C) at day 14 compared to the control group (p < 0.05). Immunohistochemistry experiments showed that SMA-α, a contractile phenotype marker of VSMC, was significantly enhanced in the arterial tissue compared with the control group (p < 0.05), as shown in Figures 1D,E. In contrast, the expression level of synthetic marker OPN was attenuated after ICS-II administration (Figure 1F,G). The data aforementioned indicated that ICS-II effectively attenuated the vascular remodeling process and restored VSMC contractile phenotype in vivo.

To further elucidate the proteomic changes after ICS-II intervention in vascular remodeling, the label-free quantitative proteomic analysis was implemented. With the steps of filtering, normalization, and imputing missing values embedded in the DEP algorithm (Figure 2A–D), a total of 145 differential proteins (including 70 up-regulated proteins and 75 down-regulated proteins) in the ICS-II group compared to the control group, were identified. The volcano plot of the differential analysis is presented in Figure 2E. In addition, the expression levels of PTK2, PXN, ZYX, and ELN are plotted in Figure 2F, respectively. Results indicated significantly higher protein expression levels of PTK2, PXN, ZYX, and ELN in the ICS-II group compared to the control group (p < 0.05).

To further elucidate the underlying protein–protein interactions and intricated networks, the PPI network, and enrichment analyses were conducted. Seventy up-regulated and 75 down-regulated proteins in the ICS-II group were imported into the STRING database to derive the PPI network, as shown in Figure 3A,F, respectively. Proteins with degrees >5 were further selected for the enrichment analysis. The biological process enrichment analysis showed that up-regulated DEPs were mainly enriched in the artery development, actomyosin structure organization, response to transforming growth factor β, actin filament bundle assembly, and organization. (Figure 3B). The cellular components enrichment analysis showed that up-regulated DEPs were mainly enriched in the stress fiber, contractile actin filament bundle, actin filament, focal adhesion, actin cytoskeleton, myofibril, etc. (Figure 3C). For molecular function, enriched terms mainly included actin binding, actinin binding, muscle alpha-actinin binding, etc. (Figure 3D). The KEGG pathway enrichment analysis revealed that up-regulated DEPs were mainly correlated with focal adhesion, regulation of actin cytoskeleton, VEGF pathway, etc. (Figure 3E). For down-regulated DEPs, enriched terms mainly included fatty acid metabolism, respiratory chain complex, regulation of reactive oxygen species biosynthetic and metabolic processes, amino acid metabolism, etc. (Figure 3G–J). The aforementioned bioinformatic results indicated that ICS-II attenuated vascular remodeling through the focal adhesion pathway by enhancing the VSMC contractile phenotype and suppressing signaling pathways related to cellular metabolism.

To further verify that ICS-II restored the VSMC contractile phenotype during vascular remodeling, we used primarily cultured rat aortic VSMC to conduct in vitro experiments (Figure 4A,B). Excessive proliferation is the major feature during VSMC phenotypic modulation. Western blot results showed that ICS-II significantly attenuated the protein expression levels of PCNA and CCND1 (Figure 4C–E), which indicated that ICS-II inhibited VSMC proliferation. Cell migration represents another essential hallmark for the VSMC phenotypic transition. Therefore, in vitro scratch assay and transwell assay were implemented to assess the ability of VSMC migration after ICS-II intervention. Results of the scratch assay revealed the suppressed migration ability of VSMC after ICS-II intervention compared to the PDGF-BB group (p < 0.05, Figure 4F,G). The transwell assay showed similar results, as shown in Figure 4H,I.

We next examined the protein expression level of SMA-α and OPN by cellular IF experiments. Results revealed that the ICS-II intervention significantly enhanced the SMA-α expression level while it inhibited the OPN protein expression compared to the PDGF-BB group (p < 0.05, Figures 5A–D). The aforementioned results showed that ICS-II restored the VSMC contractile phenotype in vitro.

To elucidate the role of the focal adhesion pathway in the ICS-II-induced VSMC contractile phenotype, we next examined the protein expression levels of PTK2, PXN, and ZYX after ICS-II intervention. Western blot showed significantly enhanced PTK2, PXN, and ZYX protein levels in the ICS-II group compared to PDGF-BB stimulation (p < 0.05, Figures 5E–H). Taken together, ICS-II restored VSMC contractile phenotype by enhancing focal adhesion signaling activity.