Isolation and culture of VSMCs

Cells were extracted from healthy bovine aorta mesentery after obtaining appropriate animal ethical approvals. The bovine aorta mesentery is a part of the bovine aorta that is left behind after the removal of the inner and outer membranes and is mainly composed of VSMCs and an elastin layer. The connective tissue around the aorta was removed and the inner and outer membranes were peeled off. The aortic mesentery was cut into small pieces. Subsequently, the mid-aortic membrane was affixed to T75 plastic culture flasks and incubated at 37 °C with 5% CO2. The T75 plastic culture flasks were inverted so that the side with the mesentery tissue block faced upwards and remained attached for 4 h. Then, the flasks were turned over and 15 mL of culture medium containing DMEM F-12 (Gibco, USA), 10% FBS (Corning, USA), and 1% penicillin–streptomycin (Gibco, USA) was added. After 7 days, the first VSMCs grew around the aortic mesentery. Once the cells reached 80–90%, they were treated with 0.25% trypsin (Gibco, USA) and passaged in cell culture plates. The experiment was approved by the Medical Ethics Committee of Guangdong Provincial People's Hospital (Guangdong Provincial Academy of Medical Sciences; reference number: KY2023-192-01).

MMC construction

VSMCs were inoculated into cell culture plates at 2.0 × 104 cells/cm2. After 24 h, the original medium was replaced with MMC agent. The MMC agent was 225 μg/mL CR (Sigma Aldrich, USA). The medium was changed every 2–3 d. All cells were used at passages 3–5, and samples were analyzed on day 5.

Immunocytochemistry

The cell layer was washed with phosphate buffer solution (PBS, Sigma-Aldrich, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for 30 min at room temperature. After three washes with PBS, the cells were permeabilized with 0.25% Triton X-100 for 20 min. Non-specific sites were blocked with 3% bovine serum albumin (Sigma Aldrich, USA) for 30 min. The cells were incubated with a primary antibody overnight at 4 °C. Antibodies used were α-SMA (1:200, NBP1-30,894, Novus Biologicals, USA), calponin (1:200, 13,938–1-AP, Proteintech, USA), and smoothelin (1:200, orb158429, Biorbyt, UK). After three washes in PBS, α-SMA, calponin, and smoothelin were incubated with goat anti-rabbit antibodies; Dylight488 and Dylight596, respectively. The nuclei were stained with DAPI. Images were captured using an Olympus IX-81 inverted fluorescence microscope (Olympus IX-81, Olympus Corporation, Japan).

Dynamic light scattering (DLS) measurements

According to previous studies [23, 24], DLS characterizes the particle size in solution and can be used to detect the charge, particle size, and polydispersity coefficients of MMC reagents. The study examined the hydrodynamic radius (RH) and volume occupancy fraction (FVO) of 25 and 225 μg/mL CR using Malvern particle sizer (Zetasizer Ultra, Malvern Instruments, UK). Crowding solutions were prepared in PBS to simulate physiological conditions. FVO was calculated based on the RH values as described in the literature [25].

Calculations for estimating the fractional volume occupancy are as follows:

$$\bullet \qquad } = /\pi }^} = /\pi \, \left( }_}} } \right)^}$$

(1)

The MW of carrageenan is ~ 550,000 Da.

The number of carrageenan molecules present in X mg was calculated as follows:

$$\left( } \times 0^}} } \right) \, \times \, \left( .0 \times 0^}} } \right) \, \div 0,000$$

(2)

The volume occupied by these number of CR molecules in 1 mL was calculated by multiplying 1 and 2 and then expressed in percentage to estimate the fraction volume occupancy:

$$}\% \, =\,/\pi \, \left( }_}} } \right)^} \times \, \left( } \times 0^}} } \right) \, \times \, \left( .0 \times 0^}} } \right) \, \div 0,000.$$

Calcein/PI cell activity and cytotoxicity assay

Cell survival was assessed on days three and five using a calcein/PI cell activity and cytotoxicity assay kit (Beyotime Biotechnology). After removing the medium from the 96-well plates, cells were washed with PBS. Subsequently, they were stained with 100 μL of calcein AM/PI assay workup and incubated at 37 ℃ away from light for 30 min. The staining was observed under an inverted fluorescence microscope (Olympus IX-81, Olympus Corporation, Japan), with green fluorescence indicating calcein AM and red fluorescence indicating PI.

Western blotting

After 5 days of culture, the cells were collected and washed three times with pre-cooled PBS. The cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, China) and protease inhibitors (Thermo Fisher, USA) on ice for 30 min. The lysate was centrifuged at 12,000 rpm and 4 ℃ for 15 min, and the supernatant was collected. The protein concentration in each sample was determined using a BCA assay. Protein samples were separated using 10% SDS-PAGE gels with 15 μg per well, electro-transferred to nitrocellulose membranes, and blocked with milk. The membranes were then incubated with α-SMA (1:2000, Rabbit polyclonal, NBP1-30,894, Novusbio, USA), CNN 1 (1:2000, Rabbit polyclonal, 13,938–1-AP, Proteintech, USA), and anti-histone 3 (1:2000, Rabbit polyclonal, 4499 s, Cell signaling, USA). The protein samples were incubated with goat anti-rabbit conjugated horseradish peroxidase (1:20,000; Abbkine, China). Blots were developed using an enhanced chemiluminescence method according to the manufacturer’s protocol (Biosharp, China).

VSMCs contraction assay

VSMCs in the logarithmic growth stage were inoculated in 12-well plates at a density of 10,000 cells per well. Next, 1 mL of the medium was added to each well. After 24 h, the medium was replaced with CR or no-CR medium. After 5 days, the medium was discarded, and 4% PFA was added for termination. The cells were then subjected to staining with CoraLite®594-Phalloidin (PF00003, Proteintech, USA) and imaged using an inverted fluorescence microscope (Olympus IX-81 Olympus Corporation, Japan). The length of 80 cells in each group was measured using ImageJ software.

Cell proliferation assessment

VSMCs proliferation was assessed using Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). VSMCs were seeded in 96-well plates, and the cells were divided into blank, control, and experimental groups. The CCK-8 assay was performed on days 0, 1, 3, and 5. The medium and CCK8 solution were mixed in a 10:1 ratio. Next, 100 μL of the mixture was added to each well and incubated for 4 h at 37 ℃ with 5% CO2. Finally, the absorbance was measured at 450 nm using a full-wavelength enzyme labeling instrument.

Cell migration assessment

VSMCs migration was assessed using the wound healing assay [26]. In a 12-well plate, VMSCs were inoculated and scratches were made with a 200-μL plastic pipette gun tip once they reached 90% confluence. After washing with PBS, the cells were cultured in serum-free medium containing CR and a common serum-free medium. The wound area was imaged using a microscope at 0, 12, 30, and 48 h.

Transcript sequencing and data analysis

After 5 days of culture, VSMCs were analyzed using reference transcriptome sequencing. RNA was isolated and purified from total samples using TRIzol (Thermo Fisher, USA). Total RNA amount and purity were assessed using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was evaluated using Bioanalyzer 2100 (Agilent, CA, USA). The concentration was > 50 ng/μL, and the RIN value was > 7.0. A total RNA amount of > 1 μg was deemed sufficient for downstream experiments. The library was fragmented with divalent cations at high temperatures, followed by polymerase chain reaction to construct a library with a fragment size of 300–50 bP. Bipartite sequencing was performed using an Illumina Novaseq™ PE6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) with PE150 as the sequencing mode. After transcriptome sequencing using the Illumina paired-end RNA-seq method, raw data were subjected to quality control using FastQC (0.10.1, https://github.com/OpenGene/fastp) software to remove junctions, repetitive sequences, and low-quality sequences. Default parameters were used to compare the sequencing data to the genome using HISAT2 (2.2.1, https://ccb.jhu.edu/software/hisat2) and the species was Bos taurus. Genes were then assembled and quantified using FPKM (FPKM = [total_exon_/mapped_reads (million) × exon_length (kB)]. The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and with a parametric F-test comparing nested linear models (p value < 0.05) using the R package edge (https://bioconductor.org/packages/release/bioc/html/edgeR.html). Finally, interacting genes clusters and GO and KEGG enrichment analyses were performed using STRING and DAVID software.

RT-qPCR

RNA was extracted from the VSMCs after 5 days of culture using an RNA extraction kit (Beyotime Biotechnology, China). The concentration and purity of the RNA were then measured. Next, 400 ng of RNA was taken from each sample and used to generate 20 μL of cDNA through reverse transcription using the Reverse Transcription Kit (Yeason, China). Genes were amplified separately using cDNA as a template, with GAPDH serving as an internal reference gene. The two-step PCR amplification standard procedure involved pre-denaturation at 95 ℃ for 5 min in one cycle, followed by PCR translation at 95 ℃ for 10 s and 60 ℃ for 30 s in 40 cycles. The solubilization curve was achieved by heating at 95 ℃ for 15 s, cooling at 60 ℃ for 30 s, and then heating again at 95 ℃ for 15 s. Please refer to Table 1 for the primer sequences.

Table 1 Primer sequences for RT-qPCRStatistical analyses

Statistical analysis data are presented as mean ± standard deviation. GraphPad Prism 9.5 software was used for the statistical analysis. All experiments were conducted at least thrice. A non-matching ratio t-test was used for the two groups that followed a normal distribution, and a Chi-square test was used for non-parametric data. A two-way ANOVA test was used to compare multiple groups of data, and a P-value of less than 0.05 was considered statistically significant.