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NCAM1 modulates the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary hypertension

Respiratory Research volume 25, Article number: 435 (2024) Cite this article

Abstract

Background

Pulmonary hypertension (PH) is a malignant vascular disease characterized by pulmonary arterial remodeling. Neural cell adhesion molecule 1 (NCAM1) is a cell surface glycoprotein that is involved in a variety of diseases, including cardiovascular disease. However, the role of NCAM1 in PH remains underexplored.

Methods

Pulmonary hypertension models were established using monocrotaline in rats and hypoxia in mice. NCAM1 protein levels in plasma from patients and rats were measured by ELISA. Expression of NCAM1 in rat lung tissues were evaluated using qRT-PCR, Western blotting, and immunofluorescence. The effects of NCAM1 on rat pulmonary artery smooth muscle cells were studied by stimulating these cells with PDGF-BB.

Results

Elevated levels of NCAM1 protein and mRNA were observed in both PH patients and monocrotaline-induced PH rats. NCAM1 knockdown ameliorated hypoxia-induced PH, highlighting its role in pulmonary artery remodeling. In PASMCs, NCAM1 expression was upregulated by PDGF-BB stimulation, enhancing cell proliferation and migration. This effect was attenuated by NCAM1 knockdown but partially restored by an ERK1/2 pathway activator (tert-butylhydroquinone, TBHQ), suggesting NCAM1’s involvement in PASMC dynamics through the ERK1/2 signaling pathway.

Conclusion

Our findings confirm the role of NCAM1 in pulmonary arterial hypertension and demonstrate its promotion of PASMC proliferation and migration through the ERK1/2 signaling pathway.

Introduction

Pulmonary hypertension (PH) is defined as a mean pulmonary arterial pressure (mPAP) of >20 mmHg, mean pulmonary artery wedge pressure (PAWP) of ≤15 mmHg, and pulmonary vascular resistance (PVR) of ≥2 Wood units. PH is characterized by a progressive increase in pulmonary arterial pressure that eventually leads to serious right heart hypertrophy and even death by right heart failure [1]. Vascular remodeling is considered the central pathophysiology in the multifactorial and complex pathophysiology of PH [2]. The proliferation, migration, and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) play a key role in pulmonary vascular remodeling [3]. At present, the pathophysiological mechanism of PH is still not fully elucidated, and the malignant proliferation of PASMCs still cannot be completely blocked with the current target therapies for PH. Therefore, the mechanism of PH needs further exploration, and novel potential therapeutic targets found for the reversal of pulmonary vascular remodeling.

It is reported that platelet-derived growth factor (PDGF) is involved in the pathological process of pulmonary hypertension. Increased PDGF activity may lead to unrestricted PASMC proliferation and migration [4, 5]. It has been reported that PDGF-BB is able to activate multifarious signal transduction pathways that can regulate PASMCs proliferation and migration [6,7,8]. Pulmonary vascular remodeling was mitigated by inhibiting the PDGF-BB/PDGFR signaling pathway [9]. PDGF-BB/PDGFR signaling pathway leads to receptor dimerization, autophosphorylation, and then signal transduction mainly through mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) cascade, leading to transcription of targeted genes that promote cell proliferation and migration [7, 10]. Therefore, the prevention and treatment of PH can be achieved by inhibiting the abnormal proliferation and migration of PASMCs that induced by PDGF-BB.

Neural cell adhesion molecule 1 (NCAM1, also referred to as NCAM, CD56) is a cell surface glycoprotein that belongs to the immunoglobulin (Ig) superfamily [11, 12]. NCAM1 is reported to be a signal transduction receptor molecule capable of regulating a variety of biological phenomena, including cell adhesion, proliferation, migration, differentiation, survival, and synaptic plasticity [13]. Studies have reported that NCAM1 regulates the proliferation and migration of human melanoma cells, which is achieved through the regulation of the Src/Akt/mTOR/Cofilin signaling pathway [14]. NCAM1 is involved in the occurrence and development of a variety of tumor and non tumor diseases, including salivary adenoid cystic carcinoma [15], ameloblastoma [16], thyroid cancer [17] and coronary atherosclerotic diseases [18]. Currently, more and more studies have shown that there are many similarities between the pathogenesis of pulmonary hypertension and tumors, such as abnormal cell proliferation [19] and the Warburg effect [20]. The RAS-MAPK/ERK pathway and CREB phosphorylation levels in neuronal cells can be increased by NCAM1 stimulation [21]. However, whether NCAM1 is involved in the development of pulmonary hypertension remains unknown. This study aimed to investigate the role NCAM1 in PH and explore the potential mechanism.

In the present study, we demonstrated that NCAM1 promotes the development of pulmonary hypertension. In vitro experiments indicates NCAM1 may increase proliferation and migration of pulmonary arterial smooth muscle cells via the ERK1/2 signaling pathway, thereby participating in pulmonary vascular remodeling in pulmonary hypertension.

Materials and methods

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.

Results

NCAM1 is upregulated in plasma in PH patients

To investigate the role of NCAM1 in PH, we used ELISA to detect the level of NCAM1 in plasma samples of PH patients (n = 18) and healthy controls (n = 21). As shown in Fig. 1A, the expression level of NCAM1 protein in plasma from patients with PH was significantly higher than in healthy controls. We generated a receiver operating characteristic (ROC) curve based on the results of the ELISAs to assess the diagnostic value of plasma NCAM1. As shown in Fig. 1B, the area under the ROC curve (AUC) reached 0.9365 (p < 0.01).

Fig. 1
figure 1

Neural cell adhesion molecule 1 (NCAM1) expression is upregulated in the plasma and lung tissues of pulmonary hypertension (PH) patients. A The protein level of NCAM1 in the plasma of PH patients (n = 18) and healthy controls (n = 21) was detected by ELISA; B a ROC curve based on the protein level of NCAM1 in PH patients and healthy controls; C volcano plot based on DEGs of RNA expression profiling of lung tissue in PH patients (n = 18) and healthy controls (n = 13) in the GSE15197 dataset; D statistical results of NCAM1 transcript levels in PH patients and healthy controls. The results are expressed as the mean ± SD, * p < 0.05 vs. Control group, ** p < 0.01 vs. Control group

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Furthermore, in the GEO dataset GSE15197, which included RNA expression profiling of lung tissue from 13 controls and 18 PH patients, we screened out 8074 differentially expressed genes (DEGs). Among the 8074 DEGs, 4224 were upregulated genes (including NCAM1) and 3850 were downregulated genes compared with the controls (Fig. 1C, D). This is consistent with the enhanced protein expression levels in the patients in our hospital.

NCAM1 is upregulated in plasma and lung tissues in MCT-induced PH rats

To investigate the expression level of NCAM1 in an animal model of PH, we used the monocrotaline (MCT)-induced rat model of PH. To verify the PH rat model, echocardiography and right heart catheterization (RHC) was performed after 28 days. The RATD and RVTD of the MCT group were significantly increased compared with the control group (Fig. 2A, B). The TAPSE of the MCT group was significantly decreased compared with the control group (Fig. 2C). Compared with the control rats, the right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were significantly increased in the MCT group rats (Fig. 2D, E). Compared with the control group, the percentage of myometrium thickness (WT%) in the pulmonary artery wall of the rats in the MCT group was significantly higher (Fig. 2F), indicating that the MCT-induced pulmonary hypertension model was established.

Fig. 2
figure 2

NCAM1 expression increased in plasma and lung tissues in monocrotaline (MCT)-induced PH rats. A–C Right atrium transverse diameter (RATD), right ventricle transverse diameter (RVTD), and tricuspid annular plane systolic excursion (TAPSE) of rats were measured by ultrasound; D–E RVSP and mPAP of rats were measured by a right heart catheter; F H&E staining of rat lung tissues. Scale bar, 50 µm; G immunofluorescent staining of rat lung tissues stained with alpha smooth muscle actin (α-SMA) (green), NCAM1 (red) or PBS, and DAPI (blue). Scale bar, 20 µm; H the protein levels of NCAM1 in the plasma of rats were detected by ELISA. I The mRNA expression of NCAM1 in the lung tissues of rats was detected by qRT-PCR. J The protein expression of NCAM1 in rat lung tissues was detected by WB. The results are expressed as the mean ± SD, ** p < 0.01 vs. Control group

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To address the protein level of NCAM1, immunofluorescence double-labeling was used to evaluate the localization and expression of NCAM1 in pulmonary arteries. We detected the expression of NCAM1 protein mainly in the smooth muscle layer of pulmonary arteries, and its expression was increased in the MCT group compared with the control group (Fig. 2G). Using an ELISA, we found that the NCAM1 plasma level in the MCT group was significantly enhanced compared with the control group (Fig. 2H). We detected NCAM1 mRNA levels and protein expression in rat lung tissues and found that they were significantly increased in the MCT group compared with the control group (Fig. 2I, J).

Downregulation of NCAM1 ameliorated the development of PH

To explore the role of NCAM1 in pulmonary hypertension, we administered adeno-associated virus (AAV9) vectors encoding NCAM1 (AAV9-NCAM1) or a control vector (AAV9-NC) nasally to mice, which were subsequently exposed to a 10% oxygen environment for 4 weeks. NCAM1 protein levels in the lung tissue of mice significantly increased following hypoxia treatment, whereas treatment with AAV9-NCAM1 markedly inhibited this change (Fig. 3A). Additionally, we observed a significant reduction in right ventricular systolic pressure in AAV9-NCAM1-treated mice compared to those treated with the control vector (Fig. 3B). Treatment with AAV9-NCAM1 also significantly improved the pulmonary acceleration time to ejection time ratio (PAT/PET) and tricuspid annular planc systolic excursion in hypoxic mice compared to those treated with AAV9-NC (Fig. 3C, D). Furthermore, immunofluorescence analysis was employed to examine NCAM1 expression and localization in mouse lung tissue, revealing that NCAM1 is predominantly expressed in the smooth muscle layer of the pulmonary arteries (Fig. 4A). To assess pulmonary vascular remodeling, we performed hematoxylin and eosin (H&E) staining along with α-smooth muscle actin (α-SMA) immunohistochemistry. The results demonstrated that NCAM1 knockdown significantly attenuated pulmonary vascular remodeling (Fig. 4B, C).

Fig. 3
figure 3

NCAM1 is involved in the development of pulmonary hypertension. A Western bloting of NCAM1 in mouse lung tissues. B Right ventricular pressure of mice were measured by a right heart catheter; C,D the pulmonary artery acceleration time to ejection time ratio (PAT/PET) and tricuspid annular plane systolic excursion (TAPSE) were measured using cardiac ultrasound. The results are expressed as the mean ± SD, * p < 0.05 vs. Control group, ** p < 0.01 vs. Control group, # p < 0.05 vs. NC group, ## p < 0.01 vs. NC group

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Fig. 4
figure 4

NCAM1 is involved in pulmonary vascular remodeling. A Immunofluorescent staining of mouse lung tissues stained with alpha smooth muscle actin (α-SMA) (green), NCAM1 (red) or PBS, and DAPI (blue). B,C Hematoxylin and eosin (H&E) staining and α-smooth muscle actin (α-SMA) immunohistochemistry of mouse lung tissues. The results are expressed as the mean ± SD, ** p < 0.05 vs. Control group, ## p < 0.01 vs. NC group. Scale bar, 50 µm

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NCAM1 expression is dramatically upregulated in PASMCs stimulated by PDGF-BB

PDGF-BB induces vascular remodeling and participates in the development of PH [4]. The proliferation and migration of PASMCs mediated by PDGF-BB play a crucial role in pulmonary vascular remodeling [23]. In this study, we investigated the role of NCAM1 in the proliferation and migration of PASMCs induced by PDGF-BB stimulation. We found that the expression of NCAM1 at both mRNA (Fig. 5A) and protein (Fig. 5B) levels was dramatically enhanced in PASMCs exposed to PDGF-BB.

Fig. 5
figure 5

NCAM1 expression is upregulated in PASMCs treated with PDGF-BB. A The mRNA expression of NCAM1 in platelet derived growth factor-BB (PDGF-BB) (20 ng/mL, 24 h)-induced PASMCs was detected by qRT-PCR; B the protein expression of NCAM1 and proliferating cell nuclear antigen (PCNA) in PDGF-BB (20 ng/mL, 24 h)-induced PASMCs were detected by WB. The results are expressed as the mean ± SD, * p < 0.05 vs. control group

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NCAM1 contributes to the proliferation and migration of PDGF-BB-induced PASMCs

To investigate the potential role of NCAM1, we knocked down NCAM1 mRNA with siRNA (Fig. 6A). Knockdown of NCAM1 partly blocked the PDGF-BB-induced PCNA increase in PASMCs (Fig. 6B). Similarly, the CCK-8 assay proved that PDGF-BB increased the cell number of PASMCs, which were partially suppressed by NCAM1 knockdown (Fig. 6C). As shown in Fig. 6D, PDGF-BB increased proliferation as measured by EdU. In addition, we also found that treatment with PDGF-BB induced the migration of PASMCs, while the increased cell migration was inhibited by NCAM1 knockdown (Fig. 6E, F).

Fig. 6
figure 6

NCAM1 contributes to the proliferation and migration of PDGF-BB-induced PASMCs. PASMCs were treated with or without NCAM1 siRNA for 24 h before PDGF-BB (20 ng/mL, 24 h) treatment. A,B The protein expression of NCAM1 and PCNA in PASMCs was detected by WB. C Cell number of PASMCs was detected by CCK8 assay. D Proliferation of PASMCs was detected by EdU. Scale bar, 200 µm. E,F the migration of PASMCs was detected by the scratch wound healing assay (scale bar, 500 µm) and transwell migration assay (scale bar, 200 µm). The results are expressed as the mean ± SD, * p < 0.05 vs. Control group, ** p < 0.01 vs. Control group, # p < 0.05 vs. NC group, ## p < 0.01 vs. NC group

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NCAM1 regulates the proliferation and migration of PASMCs through ERK1/2 activation

The ERK1/2 signaling pathway has been demonstrated to be involved in PASMCs in response to PDGF-BB [7]. Thus, we examined the effects of NCAM1 on the ERK1/2 signaling pathway by detecting levels of ERK1/2 and p-ERK1/2 in PASMCs. As indicated in Fig. 7A, treatment with TBHQ, an ERK1/2 agonist, failed to reverse the inhibitory effects of NCAM1 siRNA. However, treatment with TBHQ significantly reversed the inhibitory effect of NCAM1 knockdown on the expression level of p-ERK1/2 (Fig. 7B). Similarly, the decreased protein level of PCNA and cell number caused by NCAM1 knockdown in PDGF-BB-induced PASMCs was significantly increased after treatment with TBHQ (Fig. 7C, D). Furthermore, NCAM1 knockdown suppressed the effects on proliferation as measured by EdU in the PDGF-BB-induced PASMCs and was also markedly abrogated by TBHQ in PASMCs (Fig. 7E). In addition, we observed that the inhibitory effects of NCAM1 knockdown on cell migration in PDGF-BB-induced PASMCs were reversed by TBHQ (Fig. 7F, G).

Fig. 7
figure 7

NCAM1 promotes the proliferation and migration of PDGF-BB-induced PASMCs by the ERK1/2 signaling pathway. PASMCs were treated with NCAM1 siRNA for 24 h, then treated with TBHQ (50 µM) for 30 min, followed by PDGF-BB for another 24 h (A, C, D–G) or 30 min (B), and then the protein expression in PASMCs was detected by WB. A–C The protein level of NCAM1, p-ERK1/2, and PCNA in PASMCs was detected by WB. D Cell number was detected by CCK8 assay. E Proliferation of PASMCs was detected by EdU. Scale bar, 200 µm. F,G Migration of PASMCs was detected by the scratch wound healing assay (scale bar, 500 µm) and transwell migration assay (scale bar, 200 µm). The results are expressed as the mean ± SD, ** p < 0.01 vs. Control group, ## p < 0.01 vs. NC group, & p < 0.05 vs. NCAM1-SiRNA group, && p < 0.01 vs. NCAM1-SiRNA group

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Recombinant rat NCAM1 protein promotes PASMC proliferation and migration and activates the ERK1/2 signaling pathway

To clarify the role of NCAM1 in PASMCs, we found that recombinant rat NCAM1 protein could promote increased cell number, and the effects were strongest at 100 ng/mL (Fig. 8A). Therefore, this concentration was selected for subsequent experiments. As shown in Fig. 8B and C, recombinant rat NCAM1 promoted the proliferation of PASMCs. Recombinant rat NCAM1 also promoted the migration of PASMCs (Fig. 8D, E).

Fig. 8
figure 8

Exogenous NCAM1 promotes the proliferation and migration of PASMCs and activates the ERK1/2 signaling pathway. PASMCs were treated with recombinant rat NCAM1 protein for 24 h (A, C, D–F) or 30 min (B). A PASMCs were treated with different concentrations of recombinant rat NCAM1 protein for 24 h, cell number of PASMCs was detected by CCK8 assay. B,C The levels of p-ERK1/2 and PCNA in PASMCs were detected by WB. D Proliferation of PASMCs was detected by EdU. Scale bar, 200 µm. E,F The migration of PASMCs was detected by the scratch wound healing assay (scale bar, 500 µm) and transwell migration assay (scale bar, 200 µm). The results are pressed as the mean ± SD, * p < 0.05 vs. Control group, ** p < 0.01 vs. Control group

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Discussion

The present study demonstrated that the protein levels of NCAM1 were significantly increased in the plasma of PH patients. The mRNA expression level of NCAM1 was also enhanced in the lung tissues of PH patients. In addition, NCAM1 expression was up-regulated in rats with MCT-induced PH and in PDGF-BB-induced PAMSCs. Furthermore, we found that knockdown of NCAM1 expression in vivo resulted in amelioration of hypoxia-induced pulmonary hypertension. We also found that NCAM1 regulated PDGF-BB-induced proliferation and migration of PASMCs, which might be mediated by the ERK1/2 signaling pathway. Therefore, NCAM1 may play an important role in the development of PH.

It has been reported that the expression of NCAM1 is upregulated in viable cardiomyocytes within ischemic scars adjacent to the acute infarct in ischemic cardiomyopathy [24]. The observation that single-nucleotide polymorphisms in NCAM1 contribute to left ventricular wall thickness in hypertensive families suggests that NCAM1 may play an important role in the development of human heart diseases [25]. Plasma levels of NCAM1 were also increased in multiple sclerosis patients compared to healthy individuals [26]. Through bioinformatics, we found that the mRNA level of NCAM1 in the lung tissue of PH patients was significantly increased. We also found that NCAM1 protein expression was significantly upregulated in the plasma of PH patients. Moreover, ROC curve analysis showed that the AUC was 0.9365, indicating that NCAM1 might be a potential biomarker for PH.

Multifarious pathogenesis involving multiple genes is involved in the development of PH [27]. As a cell surface glycoprotein, NCAM1 is expressed early in the development of various cell types [28]. NCAM1 dysregulation is associated with diverse diseases because it is an important protein involved in a variety of cellular processes and cell–cell interactions [29]. NCAM1 molecules on adjacent cells adhere mainly to one another but can also activate or be activated by other signaling molecules (e.g., ECM components and cell-surface receptors) [30]. A previous study reported that NCAM1 was significantly upregulated in ameloblastoma tissues at the mRNA and protein levels [16]. Here, our findings demonstrate that NCAM1 expression levels were elevated in both PH patients and animal models of pulmonary hypertension. Moreover, our animal studies revealed that in vivo knockdown of NCAM1 expression attenuated the progression of pulmonary hypertension. These observations underscore the critical role of NCAM1 in the pathophysiology of pulmonary hypertension and highlight its potential as a therapeutic target.

Excessive proliferation and migration of PASMCs promote the development of PH because they have a crucial role in pulmonary vascular remodeling [31]. The abnormal surplus of PDGF-BB is involved in the basic pathological process of PH and facilitates PH development. PDGF levels have been reported to be significantly increased in plasma from patients with systemic SSc [32]. As we know, PH is a serious complication of SSc and is accompanied by increased expression of IL-32 and other proinflammatory cytokines [33]. Experimental PH was attenuated because reversed vascular remodeling was achieved by blocking PDGF-BB/PDGFR-β signaling [34]. Previous reports have indicated that NCAM1 was able to stimulate proliferation, epithelial–mesenchymal transition, and migration of human melanoma cells [14]. NCAM1 deficiency significantly impaired the migration of mesenchymal stem cells (MSCs) [35]. In the present study, we explored the role of NCAM1 in the PDGF-BB-induced proliferation and migration of PASMCs in vitro. The results showed that the knockdown of NCAM1 attenuated PDGF-BB-induced proliferation and migration. In addition, compared with controls, PASMCs stimulated by recombinant rat NCAM1 protein showed higher p-ERK1/2 levels and enhanced proliferation and migration. In conclusion, these data indicate that NCAM1 is involved in the regulation of PASMC proliferation and migration.

ERK1/2, an important member of the regulatory protein kinase family, is widely involved in many physiological and pathological processes [36]. ERK1 and 2 share about 85% similarity in amino acids and are widely expressed in various tissues and cells. They regulate the proliferation, differentiation, and apoptosis of various cells (such as smooth muscle cells and nerve cells) [37] during body growth and development and damage repair [38]. Phospho-ERK1/2 can promote mitosis, and the sustained activity of ERK1/2 in the S phase is key to promoting cell progression from the G1 phase to the S phase [39]. The ERK1/2 signaling pathway has been shown to be significantly activated in a variety of animal models of PH [40,41,42]. Erk1/2 knockdown can effectively inhibit pulmonary vascular remodeling and thus prevent the development of PH [43]. According to previous reports, NCAM1 could activate ERK1/2 signaling pathway in neuronal cells [44] and mesenchymal stromal cells [35]. Thus, in the present study, we examined the role of ERK1/2 in the effects of NCAM1. The results showed that the knockdown of NCAM1 inhibited PDGF-BB-induced activation of ERK1/2. Moreover, activation of ERK1/2 by TBHQ treatment reversed the effects of NCAM1 on PDGF-BB-regulated proliferation and migration of PASMCs. We also found that recombinant rat NCAM1 protein could promote an increase in p-ERK1/2 protein expression levels and promote the proliferation and migration of PASMCs. Collectively, these data suggested that NCAM1 regulates proliferation and migration of PASMCs partly via the ERK1/2 signaling pathway.

This study has several limitations. This study did not conduct in vivo rescue experiments to validate the mechanistic role of NCAM1 in pulmonary hypertension. The precise function of NCAM1 in treated pulmonary hypertension models and cellular systems remains to be elucidated. Additionally, we did not confirm the role and mechanism of NCAM1 using human pulmonary artery smooth muscle cells (PASMCs). Our investigation was limited to the ERK1/2 signaling pathway, leaving the potential involvement of other pathways unexplored. Further research is necessary to fully delineate the specific roles and mechanisms of NCAM1 in pulmonary hypertension.

Conclusion

In summary, our study reveals that NCAM1, implicated in pulmonary vascular remodeling, is upregulated in PH patients and animal models. We clarified that NCAM1 regulates the proliferation and migration of PASMCs through the ERK1/2 signaling pathway. These findings provide new insights into the molecular mechanism of PH, which may have important implications for treating PH.

Availability of data and materials

No datasets were generated during the current study. Specifically, we analyzed the GSE15197 dataset from the GEO database. The supporting data of the present study are available in the article and its supplementary information. For any further information needed to reanalyze the data presented in this paper, please feel free to contact the corresponding author.

Abbreviations

PAH:

Pulmonary arterial hypertension

NCAM1:

Neural cell adhesion molecule 1

MCT:

Monocrotaline

AAV:

Adenovirus-associated virus

PASMCs:

Pulmonary arterial smooth muscle cells

PDGF-BB:

Platelet derived growth factor-BB

TBHQ:

Tert-butylhydroquinone

mPAP:

Mean pulmonary arterial pressure

PVR:

Pulmonary vascular resistance

RHC:

Right heart catheterization

DEGs:

Differentially expressed genes

RATD:

Right atrium transverse diameter

RVTD:

Right ventricle transverse diameter

TAPSE:

Tricuspid annular plane systolic excursion

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Funding

This research was funded by the National Natural Science Foundation of China (82270061), Natural Science Foundation Project of Chongqing (cstc2021jcyj-msxmX0474), Natural Science Foundation Project of Chongqing (cstc2020jcyj-msxmX0542), Chongqing Municipal Health and Health Commission and Chongqing Science and Technology Bureau (2020FYYX225).

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Author notes

  1. Yunwei Chen and Ningxin Liu have contributed equally to this work.

Authors and Affiliations

  1. Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China

    Yunwei Chen, Ningxin Liu, Yunjing Yang, Lingzhi Yang, Yan Li, Zhuo Qiao, Yumin Zhang, Ailing Li, Rui Xiang, Li Wen & Wei Huang

  2. Institute of Life Science, Chongqing Medical University, Chongqing, China

    Yunwei Chen, Yan Li, Zhuo Qiao & Yumin Zhang

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  1. Yunwei Chen
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Contributions

Participated in research design: Yunwei Chen, Ningxin Liu, Rui Xiang and Wei Huang; Conducted experiments: Yunwei Chen, Ningxin Liu, Yunjing Yang, Lingzhi Yang, Yan Li, Zhuo Qiao, Yumin Zhang; Ailing Li; Analyzed and interpreted the data: Yunwei Chen and Li Wen; Contributed to the writing of the manuscript: Yunwei Chen, Li Wen and Wei Huang. All authors reviewed the manuscript.

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Correspondence to Li Wen or Wei Huang.

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Chen, Y., Liu, N., Yang, Y. et al. NCAM1 modulates the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary hypertension. Respir Res 25, 435 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03068-7

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Respiratory Research

ISSN: 1465-993X

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