This clinical study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. Written informed consent was obtained from all the participants. In this study, control subjects (n = 21) were recruited from healthy volunteers, and IPAH patients (n = 14) were enrolled at the First Affiliated Hospital of Chongqing Medical University from april 2016 to December 2020. IPAH was diagnosed according to the following criteria: mean pulmonary arterial pressure (mPAP) > 20 mmHg, pulmonary artery wedge pressure (PAWP) ≤ 15 mmHg and PVR ≥3 Wood units measured at rest and at sea level by right heart catheterization (RHC) (Rosenkranz et al., 2019). No etiology or familial history of PAH was identified in IPAH patients. All clinical data and samples were collected during the patient admission at the first visit. Plasma samples were collected as follows: whole blood was collected and centrifuged (3,000 rpm, 10 min), and plasma was collected and stored at −80°C.

Plasma levels of chemerin were measured using ELISA kit (Ruixin Biotech, Quanzhou, China). Blank control, standard and sample wells were set following the manufacturer’s instructions. Then, each well was added with HRP-labeled antibody (100 μL/well) followed by shaking gently. After being incubated at 37°C for 30 min, the reaction plates were rinsed repeatedly with a washing liquor. Next, 100 μL of substrate mixture was added to each well and incubated at 37°C for 15 min. Finally, 50 μL of stop solution was added per well and the absorbance was read at 450 nm wavelength. The plasma concentrations of chemerin were calculated using the standard curve.

Specific pathogen-free (SPF) male Sprague-Dawley (SD) rats (n = 20,180–200 g) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China) and raised in individual ventilated cages (IVCs) in the animal experiment room. All experiments were conducted in accordance with the guidelines of the Ethics Committee of Chongqing Medical University and in compliance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, United States). Twenty SD rats were randomly divided into the control and monocrotaline (MCT, Mengbio, Chongqing, China) groups (n = 10 per group). MCT (500 mg) was dissolved in anhydrous alcohol (10 ml) and saline (40 ml). Rats in the MCT group were intraperitoneally (i.p.) injected with MCT (55 mg/kg), and the remaining rats were injected with equal volumes of a mixture of alcohol and saline (1:4) as controls. And other twelve rats were randomly divided into the nomoxia and hypoxia groups (n = 6 per group). Rats in the hypoxia group were housed in a closed chamber with 12% oxygen concentration for 4 weeks to induce PH and there was no intervention in the control rats.

On the 21st day after MCT injection, the rats were anesthetized with 2% pentobarbital sodium (Merck KGaA; 60 mg/kg, i. p.), and the fur on the left chest was removed with depilatory cream. Then, echocardiography was performed with an ultrasound system (IE33, Philip, Holland) to examine right ventricular structure and function.

Hemodynamic data of rats were measured by RHC. A polyethylene catheter (inner diameter, 0.5 mm; outside diameter, 0.9 mm) filled with heparinized saline was connected to the MP150 multi-channel physiological recorder (BIOPAC Systems, CA, United States). Subsequently, the anesthetized rats were fixed on the operating table and the right external jugular vein was exposed through surgery. Finally, the catheter was inserted into the right ventricle (RV) by cutting a V-shaped incision on the external jugular vein and introduced into pulmonary artery (PA) under the guidance of the pressure waveforms. The right ventricular systolic pressure (RVSP) and mPAP were measured by recorded waveforms of RV and PA, respectively.

Rat pulmonary tissues were fixed with 4% paraformaldehyde at 4°C, embedded in paraffin, and sectioned at a thickness of 5 µm for hematoxylin-eosin (HE) staining (Servicebio, Wuhan, China). Images of pulmonary arterioles were captured with a microscope (Leica Microsystems DFC550, Germany).

Paraffin section of lung tissue were dewaxed and progressively rehydrated in alcoholic baths. Nonspecific binding sites were blocked with 3% bovine serum albumin (BSA) (Servicebio, Wuhan, China); After overnight incubation with a polyclonal rabbit anti-rat CMKLR1 (Affinity Antibodies, United States) or a polyclonal rabbit anti-rat chemerin (Invitrogen Antibodies, United States) and a polyclonal rabbit anti-rat α-SMA (Boster, Wuhan, China), sections were incubated for 50 min with corresponding secondary fluorescent-labeled antibodies [Goat Anti-Rabbit IgG(H + L)-Cy3, AIFang biological, Changsha, China]. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole[(DAPI) (Servicebio, Wuhan, China)]. Images were taken using a fluorescence microscope (Leica Microsystems DFC550, Germany).

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 (Boster Antibodies, Wuhan, 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).

Primary PASMCs were obtained from SD rats (6–8 weeks, 180–200 g) as previously described (Tang et al., 2020). Briefly, the lungs were rapidly excised after the rats were anesthetized with 2% pentobarbital sodium (Merck KGaA; 120 mg/kg, i. p.). The pulmonary arteries were isolated from the lungs in phosphate-buffered saline (PBS, HyClone, Utah, United States) and cut along the long axis of the vessel with ophthalmic scissors. Then, the endothelial cells and adventitial fibroblasts were gently removed by rubbing the internal and external surfaces of the pulmonary artery with tweezers. Subsequently, the remaining smooth muscle layer was cut into pieces, transferred to a culture dish with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12, HyClone, Utah, United States) containing 20% fetal bovine serum (FBS, PAN-Biotech, Adenbach, Germany), and cultured in a humidified incubator at 37°C with 5% CO₂. The purity of PASMCs was identified by immunofluorescence staining with an α-SMA antibody. Cells at passages 3-6 were used for subsequent experiments.

Total RNA was extracted from rat lung tissue with TRIzol reagent (Invitrogen, CA, United States). Then, cDNA was prepared by reverse transcription using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer’s instructions. Subsequently, real-time quantitative PCR was conducted using SYBR^® Premix Ex Taq (TaKaRa, Japan) on an ABI7500 quantitative PCR instrument. PCR primers were designed using NCBI Primer-BLAST and synthesized by Tsingke (Chongqing, China). The sequences of primers used in the present study were as follows: Chemerin, 5′-AGG​GCC​TCT​CTA​AAG​CAA​CGA-3’ (Forward), 5′-CAA​GCT​CTG​TCC​CGT​GTA​TGT-3’ (Reverse); CMKLR1, 5′-TGT​GCT​TCC​TCG​GGA​TCC​TA-3’ (Forward), 5′-GGT​GAT​GTG​GAT​GGG​CAA​GA-3’ (Reverse); and β-actin, 5′-TCA​GGT​CAT​CAC​TAT​CGG​CAA​T-3’ (Forward), 5′-ACT​GTG​TTG​GCA​TAG​AGG​TCT​T-3’ (Reverse). The relative mRNA expression was calculated by the 2^-△△Ct method. β-actin was used as an internal control for normalizing the expression of the target genes.

Proteins were extracted from rat pulmonary tissues and PASMCs in RIPA lysis buffer with PMSF on ice. The protein concentration was determined by a BCA protein assay kit (MultiSciences, Hangzhou, China). Protein samples (15–25 μg) were separated by 12% SDS-PAGE and then transferred onto PVDF membranes (Bio-Rad, California, United States). After being blocked with 5% nonfat milk at room temperature for 1.5 h, the membranes were incubated with primary antibodies overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (MultiSciences, Hangzhou, China) at room temperature for 1 h. Immunoreactive bands were visualized using an enhanced ECL kit (Beyotime, Shanghai, China). GAPDH,α-Tubulin or β-actin served as an endogenous control for equal sample loading. The primary antibodies used in this study were as follows: Chemerin (Invitrogen, CA, United States), CMKLR1 [(Bauer et al., 2011) (Abcam, Cambridge, UK)], proliferating cell nuclear antigen (PCNA) [(Hu et al., 2019) (Proteintech, Wuhan, China)], p-ERK1/2 (Thr202/Tyr204)[ (Karadeniz et al., 2021) (Cell Signaling Technology, Massachusetts, United States)], ERK1/2 [(Karadeniz et al., 2021) (Cell Signaling Technology, Massachusetts, United States)], β-actin [(Zhao et al., 2018) (Proteintech, Wuhan, China)], α-Tubulin [(Zhang et al., 2019) (Proteintech, Wuhan, China) ]and GAPDH [(Zhang et al., 2018) (Proteintech, Wuhan, China)].

A BeyoClick™ EdU-555 Cell Proliferation Kit (Beyotime Bio, Shanghai, China) was used for the cell proliferation assay. The PASMCs were incubated with10 μmol/L EdU for 2 h at 37 °C after treatment. The cells were permeabilized with Enhanced Immunostaining Permeabilization Buffer for 15 min after fixation with 4% formaldehyde for 15 min at room temperature. After three washes with PBS, the cells were incubated with click additive solution for 30 min. PASMC nuclei stained with Hoechst-33,342 were used for the cell counts and examined using fluorescence microscope.

PASMCs at 90% confluence were wounded with a 200 µL pipette tip in 6-well plates. Then, the cells were washed three times to remove cell debris, pretreated with or without PD98059 (MCE, New Jersey, United States) for 30 min and cultured with recombinant human chemerin (R&D, Minneapolis, United States) as indicated. Wound closure was observed and photographed at 0 and 12 h with a microscope (Leica Microsystems DFC550, Germany).

The Transwell migration assay was performed in 24-well plates with Transwell chambers (8-μm pores; Biofil, Guangzhou, China). Briefly, PASMCs were trypsinized and resuspended in serum-free medium. Subsequently, 1 × 10⁴ cells were added to the upper chamber, while 500 μL of complete medium containing chemerin with or without PD98059 was placed in the lower chamber. After 12 h, the cells that failed to migrate from the upper chamber were removed with cotton swabs. Then, the migrated cells on the lower side of the membrane were photographed after being stained with 1% crystal violet (Mengbio, Chongqing, China).

The results are presented as the mean ± standard error of the mean (SEM), median or percentages. Unpaired t-tests were used for comparisons between two groups. One-way ANOVA followed by the LSD test was used to assess statistical significance among groups. The diagnostic value of plasma chemerin for PAH was evaluated by receiver operating characteristic (ROC) curve analysis. A value of p < 0.05 was considered statistically significant.

To evaluate the PH rat model, HE staining showed that the walls of pulmonary arterioles were significantly thickened and that the lumens were obviously narrowed in PH rats (Figure 1A). Moreover, Immunohistochemistry and Immunofluorescent staining of pulmonary arteries stained with α-SMA were used to further evaluate the PH model, and indicated that the walls of PH groups’ pulmonary arterioles were significantly thickened (Figures 1B,C). These data suggested that the PH rat models were successfully established by MCT administration and hypoxia.

Next, to localize and identify chemerin and its receptor CMKLR1, we co-immunostained lung tissues, with either antibody against chemerin and its receptor CMKLR1 together with a smooth muscle-specific marker, the α-smooth muscle actin. In control rat lungs, microscopic analysis revealed a strong staining of chemerin and its receptor CMKLR1 within the intimal layer (Figures 1D,E). However, as illustrated in Figures 1D,E, chemerin/CMKLR1 was upregulated within the media of PH rat lungs compared to the controls.

Then, the mRNA and protein expression levels of chemerin and CMKLR1 were measured in the rat lungs. As shown in Figure 2A, the mRNA levels of chemerin and CMKLR1 in the MCT group were significantly higher than those in the control group (both p < 0.01, Figure 2A), and these elevations were also verified by Western blot analysis (chemerin, p < 0.01, Figures 2C,E; CMKLR1, p < 0.05, Figures 2C,D). We also observed this phenomenon in the hypoxia group (both p < 0.01, Figure 2B; chemerin, p < 0.05, Figures 2F,H; CMKLR1, p < 0.05, Figures 2F,G).

To mimic PH conditions in vitro, PASMCs were exposed to hypoxia (2%) for 12 and 24 h, respectively. As shown in Figure 3, the protein level of CMKLR1 was markedly increased in the hypoxia groups compared to the control group (both p<0.01).

PASMCs were treated with various concentrations of exogenous recombinant chemerin protein. As shown in Figures 4A,C, after stimulation for 24 h, the expression of CMKLR1 was significantly increased in the chemerin groups (10–300 ng/ml) compared to the control group (10 ng/ml, 100 ng/ml, and 200 ng/ml, all p<0.05; 300 ng/ml, p < 0.01). Furthermore, as the concentration of chemerin increased, the protein expression of PCNA was gradually upregulated compared to that in the control group (100 ng/ml, 200 ng/ml, 300 ng/ml, and 400 ng/ml, all p < 0.05, Figures 4B,D). To further elucidate whether the chemerin promotes the proliferation of PASMCs, we conducted the EdU assay. As shown in Figures 4E,F, chemerin can significantly promote the proliferation of PASMCs measured by EdU compared to control group (10 ng/ml, 100 ng/ml, 400 ng/ml, all p < 0.05, 200 ng/ml, 300 ng/ml, p < 0.01).

As shown in Figures 5A,B, as the concentration of chemerin increased, the migration distance of PASMCs gradually increased (100 ng/ml, 200 ng/ml, 300 ng/ml, and 400 ng/ml, all p < 0.01, Figures 5A,B). Consistent with the scratch assay results, the number of migrated cells was similarly increased by chemerin stimulation, as shown in the Transwell migration assay (100 ng/ml, p < 0.05; 200 ng/ml, 300 ng/ml, and 400 ng/ml, all p < 0.01, Figures 5C,D).

To investigate the potential mechanism of chemerin-induced phenotypic changes in PASMCs, the key regulatory proteins in the ERK1/2 signaling pathway were analyzed by Western blotting. As shown in Figures 6A,B, under exogenous chemerin stimulation, the ERK1/2 signaling pathway was significantly activated, as indicated by the increased p-ERK/ERK ratios in the chemerin group compared with the control group, and the increase was reduced by PD98059 (an inhibitor of ERK1/2 signaling) (*p < 0.05 vs. control group, &&p < 0.01 vs. chemerin group). Furthermore, PD98059 pretreatment eliminated the increase in PCNA expression in chemerin-treated PASMCs (*p < 0.05 vs. control group, &p < 0.05 vs. chemerin group, Figures 6C,D). Similarly, The proliferation promoting effect of chemerin on PASMCs can be decreased by PD98059 (*p < 0.05 vs. control group, &p < 0.05 vs. chemerin group, Figures 6E,F). In addition, the migration assays showed that chemerin induced sharp increases in the migration distance and number of migrated PASMCs, which were reversed by PD98059 (**p < 0.01 vs. control group, &&P < 0.01 vs. chemerin group, Figures 6G,H and Figures 6I,J).

The clinical characteristics of all participants were summarized in Table 1. The median age of control individuals was 25 years and that of IPAH patients was 31.5 years. The proportion of females was 85.7% in the IPAH group, and 61.9% in the control group. The median mPAP was 53 mmHg, the median PAWP was 7.5 mmHg, the median PVR was 13.06 Wood units, and median cardiac output (CO) was 3.85 L/min in the IPAH group.

ELISA assay result showed that compared to the control group, the plasma level of chemerin was markedly increased in the IPAH group (581.7 ± 24.9 pg/ml vs. 407.5 ± 10.23 pg/ml, p < 0.01, Figure 7).

To assess the diagnostic value of plasma chemerin, we generated a ROC curve using the expression data measured by ELISA. As shown in Figure 8, the area under the ROC curve (AUC) reached 0.949 [95% confidence interval (CI): 87.3–100%], and plasma chemerin had a satisfactory sensitivity (85.7%) and specificity (100%) at a concentration of 471.76 pg/ml (p<0.001), which indicated considerable diagnostic value for IPAH.