A total of 12 Sprague-Dawley (SD) rats (8-10 weeks old, weighing 200-250 g) were used in the experiments. The rats were purchased from Beijing Vital River Experimental Animal Co., Ltd. (license no. SCXK Beijing 2016-0006). The animals were maintained in environmentally controlled conditions (adequate cage size; free access to food and water; temperature, 22±2°C; humidity, 50-55%) on a 12-h light/12-h dark cycle. All procedures involving animals were performed in accordance with ethical standards and approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Shihezi University School of Medicine (approval no. A 2020-165-01). Applicable guidelines were followed in accordance with the 'Guide for the Care and Use' published by the American Physiological Society (23).

A total of 12 rats were randomly and equally divided into two groups as follows: The control (CON) group and MCT group [60 mg/kg MCT (24-26) administered by intraperitoneal (i.p.) injection on the first day; Sigma-Aldrich; Merck KGaA].

The Doppler echo parameter, pulmonary artery acceleration time (PAAT) is considered an echocardiographic indicator of PH (27). PAAT is the time interval between the onset of systolic pulmonary arterial flow and peak flow velocity. Previous studies have demonstrated that PAAT is inversely proportional to pulmonary vascular resistance (28-30). If the pulmonary vascular resistance increases, the pulmonary artery pressure also increases. Doppler echocardiography was used to assess PH on the 28th day following model establishment in the SD rats. A transthoracic closed-chest echocardiography was performed using a Vivid E9 ultrasound system equipped with a 12-MHz transducer (GE Healthcare; Cytiva). The rats were anesthetized by administering an i.p. injection of 3% sodium pentobarbital (40 mg/kg). PAAT was measured near the pulmonary valve on the left side of the chest. EchoPAC™ BT11 software (v.6.5; GE Healthcare; Cytiva) was used to analyze the data.

For the measurement of right ventricular hypertrophy, the rats were euthanized by an i.p. injection of 3% sodium pentobarbital (100 mg/kg) combined with CO₂ anesthesia at a displacement of 70% vol/min. The heart tissue was collected and weighed to evaluate the right ventricular hypertrophy index (RVHI). The atrium and external blood vessels were separated from the isolated heart in 0.9% normal saline. The weights of both the right ventricle (RV) and left ventricle (LV) plus septum (S) were recorded. The RVHI was calculated using the formula: [RV/(LV + S)].

Lung tissues were harvested, fixed in 4% paraformaldehyde (Boster Co., Ltd.), dehydrated, cleared, waxed, embedded, sectioned, patched and cut into slices (4-µm-thick). Hematoxylin and eosin (H&E) staining or Masson's trichome staining (Beijing Solarbio Biotechnology Co., Ltd.) were used to evaluate the pulmonary artery morphology. Lung tissues were observed and photographed using a digital camera (BX51; Olympus Corporation). Image-Pro Plus v.6.0 software (Media Cybernetics, Inc.) was used for the quantitative analysis. Pulmonary vascular remodeling (PVR) was evaluated by calculating the percentage of the thickness of the vessel wall (WT%) and the percentage of the vessel wall area (WA%): WT%=[2× (blood vessel outer diameter-blood vessel inner diameter)]/(blood vessel outer diameter) ×100%; WA%=(total area of blood vessel-blood vessel internal area)/total area of blood vessel ×100% (31). Two professional pathologists randomly selected 20 different non-overlapping fields from each section and analyzed PVR and lung fibrosis with Image-Pro Plus v.6.0 software (Media Cybernetics, Inc.). The pulmonary fibrosis index was analyzed by calculating the ratio of the total area of collagen to the total area of connective tissue in each field of view, as previously described (32).

Human PASMCs (HPASMCs) (Shanghai iCell Bioscience Inc.) were cultured in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were incubated at 37°C for 24 h in a humidified 5% CO₂ atmosphere.

For the cell treatments, the cells were first pre-treated with the KIR2.1 pathway blocker, ML133 (20 µM, ApexBio) (14,17,33), or the TGF-β1/SMAD signaling pathway blocker, SB431542 (10 µM, ApexBio), for 24 h and were then treated with PDGF-BB (25 ng/ml, PeproTech, Inc.) (34-36) for 24 h. Cells in the control group were not treated. The experiment was repeated six times (n=6).

HPASMCs in the logarithmic growth phase were plated as monolayers on six-well plates and cultured at 37°C with 5% CO₂ until the cell density reached 80%. A cell-free band was uniformly generated at the center of each well using a 1-ml pipet tip. The cells were washed twice with PBS and then incubated with 2 ml 2% DMEM/F12 low-serum medium and pretreated with 0 µM ML133 for 24 h, then treated with 25 ng/ml PDGF-BB for 24 h. Images were recorded and assessed at 0 and 24 h using an Olympus inverted microscope (Olympus Corporation). The migration distance was estimated using ImageJ v1.8.0 software (National Institutes of Health). The cell migration rate (%)=[(0 h average scratch area-24 h average scratch area)/0 h average scratch area] ×100% (37-39).

The Transwell™ chamber (Corning, Inc.) was placed in a 24-well culture plate. Subsequently, 200 µl of the cell suspension (cell density of 10⁴ cells/ml) were inoculated into the upper chamber. Complete DMEM/F12 containing 10% FBS (600 µl) was then added to the lower chamber. 20 µM ML133 was added for 24 h after the cells adhered to the well, then treated with 25 ng/ml PDGF-BB for 24 h. Following the intervention, the cells were cultured with fresh medium at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. Subsequently, the cells below the membrane were fixed with 4% paraformaldehyde for 10 min at room temperature and stained with 0.1% crystal violet for 30 min at room temperature. A cotton swab was used to gently remove the cells from the upper surface of the chamber. Following observation with a light microscope and imaging with a digital camera (BX51, Olympus Corporation), five different fields of view were randomly selected; the cells that invaded the submembrane surface were counted, and the number of invaded cells reflected the strength of the invasive ability of the HPASMCs (37-39).

The paraffin-embedded tissue sections were dewaxed, and antigen retrieval was performed. The cells were seeded on six-well glass slides for fixation. Triton X (0.3%) was used to permeabilize the membrane. The cells were then incubated overnight at 4°C with the following antibodies: Anti-KIR2.1 (1:100 dilution, cat. no. ab109750), anti-osteopontin (OPN; 1:500 dilution, cat. no. ab8448), anti-proliferating cell nuclear antigen (PCNA; 1:200 dilution, cat. no. ab29) and anti-α-smooth muscle actin (α-SMA; 1: 500; cat. no. ab124964) (all from Abcam). The following day, the cells were incubated with FITC-labeled goat anti-rabbit IgG (1:100; cat. no. ZF-0311) or TRITC-labeled anti-mouse IgG (1:100; ZF-0313) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) antibodies in a dark box at 37°C for 2 h. The nuclei were stained with DAPI (1:1,000, D9542; Sigma-Aldrich; Merck KGaA) at 37°C in a dark box for 20 min. The cells were observed and photographed under a fluorescence inverted microscope (LSM710; Carl Zeiss AG). Proteins were semi-quantitatively analyzed using Image-Pro Plus 6.0 software (National Institutes of Health).

Total protein was extracted from HPASMC suspensions or pulmonary vascular tissue homogenates from SD rats with RIPA Lysis Buffer (Thermo Fisher Scientific, Inc.) in western blot analysis. The BCA protein determination method was used to determine the protein concentration. Proteins aliquots (40 µg/lane) were separated using standard sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, (8-10% gel)) and transferred to 0.45 µm nitrocellulose membranes (Invitrogen; Thermo Fisher Scientific, Inc.). The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-GAPDH (1:10,000 dilution, cat. no. ab8245), anti-KIR2.1 (1:1,000 dilution, cat. no. ab109750), anti-TGF-β1 (1:1,000 dilution, cat. no. ab92486), anti-SMAD2 (1:1,000 dilution, cat. no. ab40855), anti-SMAD3 (1:1,000 dilution, cat. no. ab40854), anti-phosphorylated (p-)SMAD2 (1:1,000 dilution, cat. no. ab188334), anti-p-SMAD3 (1:1,000 dilution, cat. no. ab52903), anti-OPN (1:1,000 dilution, cat. no. ab8448) and anti-PCNA (1:1,000 dilution, cat. no. ab29) (all from Abcam). Subsequently, the membranes were incubated with HRP-labeled anti-mouse IgG (1:20,000 dilution; cat. no. ZB-2305) or anti-rabbit IgG (1:10,000 dilution; cat. no. ZB-5301; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 2 h. Luminescence reagents were obtained from the SuperSignal™ West Pico luminescence substrate kit (Thermo Fisher Scientific, Inc.) and incubated with the membranes. Protein bands were quantified using ImageJ v1.8.0 software (National Institutes of Health).

The statistical software packages SPSS 21.0 and GraphPad Prism 8.0 were used to analyze the experimental results. All data are presented as the mean ± standard deviations (means ± SD). The Kolmogorov-Smirnov test was used for each set of data and the data were found to be normally distributed. Differences between two groups were analyzed using unpaired t-tests. Differences in data from more than two groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test. A value of P<0.05 was considered to indicate a statistically significant difference.

The echocardiogram of the blood flow in the pulmonary artery was detected using the Doppler ultrasonic diagnostic instrument. Compared with the CON group (Fig. 1A), the MCT group exhibited a midsystolic notch, with the peak shifting forward, and the PAAT value was decreased (P<0.01, n=6; Fig. 1B). The RVHI% of the rats in the MCT group was significantly higher than that in the CON group (P<0.01, n=6; Fig. 1C).

H&E staining was performed to observe the structural differences between the small pulmonary arteries (diameter, 15-50 µm) (Fig. 1D) and the middle pulmonary arteries (diameter, 50-150 µm) (Fig. 1E). The pulmonary artery cells in the CON group were evenly distributed, continuous and structurally intact, while the pulmonary artery cells in the MCT group exhibited a disordered arrangement, the area of the lumen was reduced, and the thickness of the tube wall was significantly increased. The pulmonary artery WT% and WA% of the MCT group were significantly higher than that of the CON group (P<0.01, n=6; Fig. 1F).

Masson's trichrome staining was used to observe collagen deposition and lung fibrosis (Fig. 1G). The analysis revealed evident collagen deposition in the pulmonary blood vessels and lung tissues of the MCT group of rats (P<0.01, Fig. 1H). The lung tissue was evidently fibrotic.

Immunohistochemistry and immunofluorescence staining were performed to detect changes in the expression of KIR2.1 (Fig. 2A and B), the migration-related protein, OPN (Fig. 2A and C), and the proliferation-related protein, PCNA (Fig. 2A and D), in rat lung tissue. The KIR2.1, OPN and PCNA proteins were widely expressed in lung tissues and pulmonary blood vessels. The semi-quantitative analysis of the fluorescence intensity revealed that KIR2.1, OPN, and PCNA expression was significantly increased in the MCT group (P<0.01, n=6) compared with that in the CON group (Fig. 2E).

Western blot analysis was conducted to further detect KIR2.1, OPN and PCNA protein levels in the tissue homogenate of rat pulmonary blood vessels (Fig. 2F). The results were consistent with those of the semi-quantitative analysis of immunofluorescence staining. Significantly higher levels of the KIR2.1, OPN and PCNA proteins were detected in the MCT group compared with the CON group (P<0.01 or P<0.05, n=6; Fig. 2G). Furthermore, western blot analysis was used to assess the expression of proteins in the TGF-β1/SMAD2/3 signaling pathway in tissue homogenates of pulmonary blood vessels (Fig. 2F). Following treatment with MCT, the levels of the TGF-β1 and p-SMAD2/3 proteins were significantly increased compared with those in the CON group (P<0.01 or P<0.05, n=6; Fig. 2G).

HPASMCs were treated with PDGF-BB and cell proliferation, migration and changes in KIR2.1 protein expression were observed to further investigate the role of KIR2.1 in PVR. Cell proliferation and migration were analyzed using scratch and Transwell assays (Fig. 3A and C). Following stimulation with PDGF-BB, the scratch healing ability of the HPASMCs was enhanced, and the number of cells that migrated to the lower surface of the Transwell™ chamber increased (P<0.01, n=6). Moreover, cell scratch healing and the number of cells migrating through the Transwell™ were reduced following pre-treatment with the KIR2.1 protein inhibitor, ML133 (P<0.01, n=6; Fig. 3B and D). The expression of the migration-related protein, OPN, and the proliferation-related protein, PCNA, in HPASMCs was detected using western blot analysis (Fig. 4A). The results were consistent with the phenotype. Following PDGF-BB intervention, the protein levels of OPN and PCNA in the HPASMCs were significantly increased (P<0.01, n=6), and OPN and the OPN and PCNA protein levels were significantly decreased in cells pre-treated with ML133 (P<0.01, n=6; Fig. 4C and D).

Immunofluorescence staining and western blot analysis were also performed to determine KIR2.1 protein expression in the HPASMCs (Figs. 3E and 4A). The results of immunofluorescence staining revealed that KIR2.1 was mainly located in the cell membrane and cytoplasm. The semi-quantitative analysis of the fluorescence intensity revealed a significantly higher protein expression of KIR2.1 in the HPASMCs stimulated with PDGF-BB than that in the CON group (P<0.01, n=6; Fig. 3E). However, KIR2.1 protein expression was decreased in the PDGF-BB + ML133 group (P<0.01, n=6; Fig. 3F). The results of western blot analysis were consistent with those from the semi-quantitative analysis of immunofluorescence staining. Following stimulation with PDGF-BB, KIR2.1 protein expression in the HPASMCs was significantly increased (P<0.01, n=6), whereas it was significantly decreased following pre-treatment with ML133 (P<0.01, n=6; Fig. 4B).

The levels of TGF-β1/SMAD2/3 signaling pathway proteins in HPASMCs treated with PDGF-BB were examined using western blot analysis to investigate the underlying molecular mechanisms (Fig. 4A). PDGF-BB increased the levels of TGF-β1, p-SMAD2 and p-SMAD3 in HPASMCs (P<0.01 or P<0.05, n=6) and activated the TGF-β1/SMAD2/3 signaling pathway. Following pre-treatment with ML133, the levels of TGF-β1 and p-SMAD2/3 were significantly decreased (P<0.01 or P<0.05, n=6), and the TGF-β1/SMAD2/3 signaling pathway was inhibited (Fig. 4E-G).

The HPASMCs were pre-treated with the TGF-β1/SMAD2/3 inhibitor, SB431542, and the changes in cell proliferation were observed following the PDGF-BB intervention to further elucidate the role of the TGF-β1/SMAD2/3 signaling pathway in HPASMC proliferation and migration. Compared with the PDGF-BB group, the cell scratch healing level was reduced in the PDGF-BB + SB431542 group (Fig. 5A and C), and the number of cells migrating through the Transwell™ was decreased (P<0.01 or P<0.05, n=6; Fig. 5B and D), indicating that cell proliferation and migration were decreased. The expression of the OPN and PCNA proteins was examined using western blot analysis (Fig. 6A), and the results were consistent with the phenotype. Following pre-treatment with SB431542, OPN and PCNA protein expression was significantly decreased (P<0.01 or P<0.05, n=6; Fig. 6C and D).

Based on the aforementioned results, it was found that PDGF-BB upregulates KIR2.1 protein expression in HPASMCs and activates the TGF-β1/SMAD2/3 signaling pathway. The present study then further investigated the upstream and downstream association between KIR2.1 and the TGF-β1/SMAD2/3 signaling pathway by detecting changes in KIR2.1 levels following pre-treatment with SB431542. The results of immunofluorescence staining and western blot analysis (Figs. 5E and 6A) revealed that the SMAD2/3 signaling pathway was inhibited following pre-treatment with SB431542 (P<0.01 or P<0.05, n=6; Fig. 6E and F); however, no significant differences in KIR2.1 protein expression were observed between the PDGF-BB + SB431542 group and the PDGF-BB group (P>0.05, n=6; Figs. 5F and 6B).

On the whole, PDGF-BB activated the TGF-β1/SMAD2/3 signaling pathway and upregulated the expression of OPN and PCNA to promote cell proliferation and migration. ML133 inhibited KIR2.1 channel activation and modulated the downstream TGF-β1/SMAD2/3 signaling pathway. The activation of the TGF-β1/SMAD2/3 signaling pathway was blocked by SB431542; however, the expression of KIR2.1 was not affected, indicating that KIR2.1 is located upstream of the TGF-β1/SMAD2/3 signaling pathway (Fig. 7).