Study population

The clinical study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (2023–154). All the participants signed informed consent. A total of 39 subjects including 21 healthy controls and 18 patients with idiopathic pulmonary hypertension were included. PH was diagnosed according to the following criteria: mPAP ≥ 25 mmHg, PAWP ≤ 15 mmHg, and PVR ≥ 3 Wood units measured at rest and at sea level by right heart catheterization (RHC). Clinical samples were collected at the First Affiliated Hospital of Chongqing Medical University from April 2015 to March 2021. The collected blood samples were centrifuged to obtain plasma at 3000 rpm for 10 min. The collected plasma was then stored at −80 °C for subsequent experiments.

Animals

All animal experiments were conducted following the ethical guidelines of the 1964 Declaration of Helsinki and received approval from the Institutional Animal Care and Use Committee at Chongqing Medical University (IACUC-CQMU-2023-0007).

Male Sprague–Dawley rats (180–200 g) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). All rats were placed under standard pathogen-free conditions with controlled temperature (22 ± 2 °C) and 12/12 h light/dark cycle. Rats in the monocrotaline (MCT) group were intraperitoneally injected with MCT (50 mg/kg, Mengbio, Chongqing, China), while rats in the control group were injected with the same volume of saline. Then, all rats were feed in the same conditions for 4 weeks.

Adult male mice (aged approximately 8 weeks), weighing between 22 and 26 g were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. The corresponding target RNA cloning constructs and serotype 9 adenovirus-associated virus (AAV9) were packaged by Genechem, Shanghai, China. Mice were randomly allocated into various groups and, following isoflurane anesthesia, were administered 2 * 1011 genome equivalents of the AAV9 vector in 50 μL of sterile PBS solution via nasal drops. 7 days later, the mice were allocated to environments of either normoxia or hypoxia, with the hypoxic conditions set at 10% oxygen concentration, mice feed and bedding were replenished and replaced weekly.

Cell culture

Primary PASMCs were isolated from Sprague–Dawley rats (6–8 weeks, 180–200 g) as previously described [22]. Briefly, rat lung tissues were rapidly isolated after the rats were sacrificed. The pulmonary artery tissues were quickly isolated, and the surrounding connective tissue was carefully removed. Pulmonary artery tissues were then minced into pieces, evenly dispersed in Petri dishes, transferred to a culture dish with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12) containing 20% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and incubated in a humidified incubator at 37 °C with 5% CO2 and 21% O2. Cells were cultured for 3–8 passages for subsequent experiments.

Enzyme-linked immunosorbent assay

The levels of NCAM1 protein in plasma samples were quantified using ELISA kits (Ruixin Biotech, Quanzhou, China or Jiangsu Meibiao Biotechnology Co., Ltd). Briefly, blank control wells, standard wells, and sample wells were prepared according to the manufacturer’s instructions. Subsequently, 100 µL of HRP-conjugated reagent was added to each well, excluding the blank wells. The plates were incubated at 37 °C for 60 min and then subjected to five washing cycles. Following the washes, 100 µL of substrate solution was added to each well and incubated at room temperature for 15 min in the dark. The reaction was terminated by adding 50 µL of stop solution to each well, and the absorbance was measured at 450 nm using a microplate reader, with the blank well serving as the zero reference. For the ROC curve analysis, plasma levels of NCAM1 protein were measured in both the control group and the PH patient group using ELISA. The resulting data were imported into GraphPad Prism 9.0 software, where the Wilson/Brown method was employed to calculate the 95% confidence intervals for the ROC curve construction.

Echocardiography

After anesthesia was administered, the rats or mice were positioned on their back, and the fur on the anterior chest area was shaved. The structure and function of the right heart were assessed using an ultrasound system to perform echocardiography. Measurements were taken of the right atrium transverse diameter (RATD) and right ventricle transverse diameter (RVTD) from the heart’s four-chamber view in two-dimensional ultrasound mode. Additionally, tricuspid annular plane systolic excursion (TAPSE) was evaluated in the same four-chamber view using M-mode ultrasound. Pulsed-wave Doppler was utilized to assess pulmonary acceleration time (PAT) and ejection time (PET).

Right heart catheterization (RHC)

After administering anesthesia, the rats or mice were secured to the operating table, and the fur on the right side of the neck was shaved. The right external jugular vein was surgically exposed in preparation for catheter insertion. For rats, the ventricular pressure catheter was advanced through the external jugular vein into the right atrium, passed through the right ventricle, and positioned into the pulmonary artery, guided by waveform analysis. In mice, the catheter was positioned in the right ventricle. Pressure readings were subsequently recorded from these locations.

Hematoxylin–eosin (H&E) staining

The rat and mouse lung tissues were immersed in 4% paraformaldehyde and fixed overnight. It was then dehydrated, cleared, embedded in paraffin, and cut into 5 µm thick paraffin sections. H&E staining was performed on lung tissue paraffin sections. Images of distal pulmonary arterioles were acquired with a microscope (Leica Microsystems DFC550, Germany). The obtained results were analyzed using Image-Pro Plus 6.0 software. Then we used Image Pro Plus 6.0 image analysis software to measure the wall thickness (WT) and external diameter (ED) of the pulmonary arterioles in rats and mice from each group. The percentage of pulmonary arterial wall thickness (WT%) was defined as: WT% = 2 * WT/ED × 100%.

Immunofluorescent staining

Paraffin sections of rat lung tissues were dewaxed with gradient alcohol and then the antigen was retrieved with EDTA antigen repair fluid. After blocking with 3% BSA for 30 min, sections were incubated overnight with rabbit anti-NCAM1(Cat # 99746S, Cell Signaling Technology, USA) or PBS and mouse anti-α-SMA(Cat # BM0002, BOSTER Biological Technology, China). The sections were incubated with the corresponding secondary fluorescently-labeled antibody for 50 min before the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, the images were acquired using a fluorescence microscope (Leica Microsystems DFC550, Germany).

Immunohistochemistry staining

The lung tissue sections were deparaffinized and rehydrated in graduated alcohol. The endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide at room temperature for 25 min. Then the sections were blocked with 3% bovine serum albumin (BSA) (Servicebio, Wuhan, China) for 30 min and then incubated with a polyclonal rabbit anti-rat α-SMA (Cat # BM0002, BOSTER Biological Technology, China) antibodies overnight, followed by incubation with the secondary antibody [Goat Anti-Rabbit IgG (H + L) -HRP, AIFang biological, Changsha, China] for 50 min. After the colour development through incubation with diaminobenzidine, the sections were counterstained with haematoxylin. The developed tissue sections were imaged under a microscope (Leica Microsystems DFC550, Germany). The obtained results were analyzed using Image-Pro Plus 6.0 software. Then we used Image Pro Plus 6.0 software to measure the area of α-SMA immunohistochemical positive regions in the pulmonary artery sections. We then calculated the percentage of this area relative to the total area of the image and normalized the results to the control group.

Quantitative reverse transcription PCR (QRT-PCR)

Trizol reagent (TaKaRa, Japan) was used to extract total RNA from rat lung tissues and PASMCs. Then, cDNA was obtained from the total RNA by reverse transcription using the M5 super plus qPCR RT kit with gDNA remover (Mei5 Biotech, Beijing, China). Real-time quantitative PCR was conducted using TSINGKE TSE202-2 × T5 Fast qPCR Mix (Tsingke, Chongqing, China) on an ABI7500 quantitative PCR instrument. The sequences of primers were as follows: NCAM1, forward 5′-AGC CAA GGA GAA ATC AGC GT-3′, reverse 5′-GCG TTG TAG ATG GTG AGG GT-3′; β-actin, forward 5′-AGA TCA AGA TCA TTG CTC CT-3′, reverse 5′-ACG CAG CT CAG TAA CAG TCC-3′. The relative mRNA expression was calculated by the 2−ΔΔCt method, and β-actin was used as the internal control.

Western blotting

The protein samples were separated on 10% SDS-PAGE gels and then transferred to PVDF membranes (Bio-Rad Laboratories, California, USA). The membranes were blocked with 5% non-fat milk for 1.5 h at room temperature and then incubated overnight at 4 °C with primary antibodies against β-actin (1:5000, Cat # 20536-1-AP, Proteintech Group, China), GAPDH (1:10,000, Cat # 10494-1-AP, Proteintech Group, China), ERK-1/2 (1:1000, Cat # 9102S, Cell Signaling Technology, USA), p-ERK-1/2 (1:1000, Cat # 9101S, Cell Signaling Technology, USA), PCNA (1:2000, Cat # 10205-2-AP, Proteintech Group, China), and NCAM1 (1:1000, Cat # 99746S, Cell Signaling Technology, USA). The membranes were then incubated with secondary antibodies (1:5000) for 1.5 h at room temperature. Immunoreactive bands were visualized using an enhanced ECL kit.

Cell transfection

For the knockdown of NCAM1, we transfected cells with NCAM1-siRNA, with the following sequence: sense 5′-GGU UCA UAG UCC UAU CCA ATT-3′ and antisense 5′-UUG GAU AGG ACU AUG AAC CTT-3′. The sequence for the negative control siRNA (NC-siRNA) is: sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′. The siRNA concentration used in the experiments was 50 nM. According to the manufacturer’s protocol, siRNA was transfected into PASMCs using Lipofectamine™ 3000 (Invitrogen). However, in our actual experimental process, we chose to perform the transfection at 30–40% confluence. Transfection efficiency was assessed by western blot analysis.

EdU staining

The proliferation of PASMCs was detected using a BeyoClick™ EdU-555 Cell Proliferation Kit. The treated PASMCs were cultured with 10 μmol/L EdU for 2 h. After EdU labeling, the culture medium was removed and the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After washing the cells with PBS three times, cells were permeabilized with enhanced immunostaining permeabilization buffer for 15 min at room temperature. After washing the cells three times, the cells were incubated with click additive solution at room temperature for 30 min in the dark. PASMC nuclei were stained with Hoechst-33342. Finally, cells were photographed under a fluorescence microscope.

CCK-8 assay

PASMCs (5 × 103 cells/well) were seeded in 96-well plates, cultured for 24 h, and then treated differently according to the grouping arrangement. Ten microliters Cell Counting Kit-8 was added to the treated cells. Then, the absorbance was measured at a wavelength of 450 nm with a microplate reader.

Scratch wound healing assay

PASMCs were seeded into a 6-well plate and cultured until 80% confluence before processing for the first step. Cells were wounded using a 1000 µL pipette tip and then treated differently according to the grouping arrangement. Wound closures were observed and photographed at 0 h and 24 h with a microscope (Leica Microsystems DFC550, Germany).

Transwell migration assay

Using a Transwell chamber with 8-µm pore size to test PASMCs migration. The PASMCs (1 × 104) were seeded in the upper chamber and cultured in a serum-free medium, while complete medium containing 10% serum was added to the lower chamber. PASMCs were fixed with 4% paraformaldehyde before staining with 1% crystal violet. Finally, the migrating cells on the lower side of the membrane were photographed.

Bioinformatics analysis

The GSE15197 microarray dataset was downloaded from a public database called the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). This dataset included lung tissue samples from 18 subjects with pulmonary hypertension and 13 normal controls. The easylabel package in R (version 4.2.1) software was used to screen differentially expressed genes (DEGs) between controls and patients with PH. * p < 0.05 was considered statistically significant. The DEGs obtained were used to draw the volcano map using the easylabel package.

Statistical analysis

Experimental results are presented as average values plus or minus the standard deviation (SD). The Shapiro–Wilk test was used to verify if the data distributions were normal. For assessing differences between two groups, data following normal and non-normal distributions were analyzed with Unpaired 2-tailed Student t tests and Mann–Whitney U tests, respectively. For comparisons involving three or more groups, data were analyzed with one-way analysis of variance (ANOVA) with a Bonferroni correction for multiple comparisons, or with the Kruskal–Wallis test followed by Dunn’s multiple comparison test, as appropriate. All statistical analyses were performed using GraphPad version 9.0, and a p value less than 0.05 was considered statistically significant.