NCAM1 Promotes the Proliferation and Migration of Pulmonary Arterial Smooth Muscle Cells via the ERK1/2 Pathway

doi:10.21203/rs.3.rs-2329028/v1

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NCAM1 Promotes the Proliferation and Migration of Pulmonary Arterial Smooth Muscle Cells via the ERK1/2 Pathway

https://doi.org/10.21203/rs.3.rs-2329028/v1

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Background: Pulmonary arterial hypertension (PAH) 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 relationship between NCAM1 and PAH is unknown.

Methods: Enzyme-linked immunosorbent assay, Bioinformatics methods, Echocardiography, Right heart catheterization, Hematoxylin-eosin staining, Immunofluorescent staining, Quantitative reverse transcription polymerase chain reaction, Western blotting, Cell Counting Kit-8, EdU staining, Scratch wound healing assay and Transwell migration assay were used to elucidate the role of NCAM1 in PAH.

Results: The protein expression levels of NCAM1 were increased in the plasma of PAH patients. Using bioinformatics methods, we found that the transcript levels of NCAM1 were also upregulated in the lung tissues of PAH patients. The expression levels of NCAM1 protein in the plasma of MCT-induced PAH rats were also increased. We found that both NCAM1 protein and mRNA expression levels were significantly enhanced in MCT rat lung tissues. Furthermore, we found that NCAM1 mRNA and protein levels were significantly upregulated in PDGF-BB-treated PASMCs. NCAM1 knockdown could partially reverse PDGF-BB-induced proliferation and migration of PASMCs. However, the effects were partially restored by tert-butylhydroquinone (TBHQ), an ERK1/2 activator. And recombinant rat NCAM1 protein promotes PASMC proliferation and migration and activates the ERK1/2 signaling pathway.

Conclusion: Our findings suggest that NCAM1 may be associated with pulmonary arterial hypertension and promotes the proliferation and migration of PASMCs via the ERK1/2 signaling pathway.

NCAM1

pulmonary arterial smooth muscle cells

PDGF-BB

ERK1/2

pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) 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 ≥ 3 Wood units ¹. PAH is characterized by a progressive increase in pulmonary arterial pressure that eventually leads to serious right heart failure and even death ¹. Vascular remodeling is considered the central pathophysiology in the multifactorial and complex pathophysiology of PAH 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 PAH is still not fully elucidated, and the malignant proliferation of PASMCs still cannot be completely blocked with the current target therapies for PAH. Therefore, the mechanism of PAH needs further exploration, and novel potential therapeutic targets found for the reversal of pulmonary vascular remodeling.

Neural cell adhesion molecule 1 (NCAM1, also referred to as NCAM, CD56) is a cell surface glycoprotein that belongs to the immunoglobulin (Ig) superfamily ^4,5. 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 ⁶. 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 ⁷. The RAS-MAPK/ERK pathway and CREB phosphorylation levels in neuronal cells can be increased by NCAM1 stimulation ⁸. However, whether NCAM1 is involved in the development of pulmonary hypertension remains unknown. This study aimed to investigate the role NCAM1 in PAH and explore the potential mechanism.

In the present study, we hypothesized that NCAM1 might promote the proliferation and migration of PASMCs through the ERK signaling pathway.

Reagents

Primary antibodies against β-actin, PCNA, and GAPDH and secondary antibodies (goat anti-rabbit) were purchased from Proteintech (Wuhan, China). Primary antibodies against ERK1/2, p-ERK1/2, and NCAM1 were obtained from Cell Signaling Technology (CST, Massachusetts, USA). Primary antibodies against α-SMA were purchased from BOSTER (Wuhan, China). NCAM1 ELISA kits for rats and humans were purchased from Jiangsu Meibiao Biotechnology Co., Ltd (Yancheng, China). Recombinant rat PDGF-BB was purchased from R&D Systems (State of California, USA). The Cell Counting Kit-8 (CCK-8) was purchased from APExBIO (Houston, USA). BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 was from Beyotime Institute of Biotechnology (Shanghai, China). TBHQ and recombinant rat NCAM1 protein were purchased from MedChem Express (New Jersey, USA).

Study population

The clinical study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. All the participants signed informed consent. A total of 39 subjects including 21 healthy controls and 18 patients with idiopathic pulmonary hypertension were included. PAH 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 September 2017 to April 2018. The collected blood samples were centrifuged (3000 rpm, 10 min) to obtain plasma. The collected plasma was then stored at − 80°C for subsequent experiments.

Enzyme-linked immunosorbent assay

The levels of NCAM1 protein in the plasma samples were detected by an ELISA kit (Ruixin Biotech, Quanzhou, China). First, the blank control wells, standard wells, and sample wells were set according to the instructions. Then, 100 µL of HRP-conjugated reagent was added to each well, except for the blank wells. The reaction plates were incubated for 60 min at 37°C and then washed five times. After washing, 100 µL of substrate mixture was added to each well and incubated at room temperature for 15 min in the dark. After adding 50 µL of stop solution to each well, the absorbance of each well was measured at a wavelength of 450 nm by zeroing the blank well.

Microarray data acquisition

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.

Data processing and DEG determining

The easylabel package in R (version 4.1.0) software was used to screen differentially expressed genes (DEGs) between controls and patients with PAH. A p < 0.05 was considered statistically significant. The DEGs obtained were used to draw the volcano map using the easylabel package.

Animal experiments

Male Sprague-Dawley rats (180–200 g) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). All rat experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Chongqing Medical University. 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.

Echocardiography

After anesthesia, the rats were fixed in the supine position, and the fur on the anterior chest was removed. The right heart structure and function of the rats were examined by echocardiography using an ultrasound system. The right atrium transverse diameter (RATD) and right ventricle transverse diameter (RVTD) were measured in the four-chamber section of the heart under two-dimensional ultrasound mode, and then the tricuspid annular plane systolic excursion (TAPSE) was measured in the four-chamber section of the heart under M-mode ultrasound mode.

Right heart catheterization (RHC)

After anesthesia, the rats were fixed on the operating table, and the right neck fur was removed before surgically exposing the right external jugular vein. Finally, the catheter was filled with heparin saline, and the connected multi-lead physiological recorder (MP150, BIOPAC System, USA) was inserted into the right atrium through the external jugular vein. It was passed through the right ventricle to the pulmonary artery based on the waveform. The pressure was recorded.

Hematoxylin-eosin (H&E) staining

The rat lung tissue was 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).

Immunofluorescent staining

Paraffin sections of rat lung tissue 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 and mouse anti-α-SMA. 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).

Preparation of PASMCs

Primary PASMCs were isolated from Sprague-Dawley rats (6–8 weeks, 180–200 g) as previously described ⁹. 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.

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^−△△Ct method, and β-actin was used as the internal control.

Western blotting

The protein samples were separated on 10% gels and then transferred to PVDF membranes (Bio-Rad Laboratories, California, USA). The membranes were blocked in 5% non-fat milk for 1.5 h at room temperature and incubated with the primary antibodies [β-actin (1:5000), GAPDH (1:10000), ERK-1/2 (1:1000), p-ERK-1/2 (1:1000), PCNA (1:2000), NCAM1 (1:1000)] overnight at 4°C. The membranes were incubated with secondary antibodies (1:5000) for another 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 the following sequence of siRNA for NCAM1: sense 5′-GGU UCA UAG UCC UAU CCA ATT-3′ and antisense 5′-UUG GAU AGG ACU AUG AAC CTT-3′. The siRNA was transfected into PASMCs using Lipofectamine™3000 (Invitrogen) according to the manufacturer’s protocol once the cells had reached 30–40% confluence. The transfection efficiency was detected by western blot.

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 minutes 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 minutes in the dark. PASMC nuclei were stained with Hoechst-33342. Finally, cells were photographed under a fluorescence microscope.

Cell viability 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.

Statistical analysis

Statistical results of all experiments are presented as the mean ± standard error (SD). GraphPad 9.0 software was used for statistical analysis. The t-test was used to compare the mean values between two groups, and one-way ANOVA was used to analyze the mean values of three or more groups. A p-value < 0.05 was considered statistically significant.

NCAM1 is upregulated in plasma in PAH patients

To investigate the role of NCAM1 in PAH, we used ELISA to detect the level of NCAM1 in plasma samples of PAH patients (n = 18) and healthy controls (n = 21). As shown in Fig. 1A, the expression level of NCAM1 protein in plasma from patients with PAH 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).

Furthermore, in the dataset GSE15197, which included RNA expression profiling of lung tissue from 13 controls and 18 PAH 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, 1D). 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 PAH rats

To investigate the expression level of NCAM1 in an animal model of PAH, we used the monocrotaline (MCT)-induced rat model of PAH. To verify the PAH 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, 2B). 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, 2E). 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.

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, 2J).

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

PDGF-BB induces vascular remodeling and participates in the development of PAH ¹⁰. The proliferation and migration of PASMCs mediated by PDGF-BB play a crucial role in pulmonary vascular remodeling ¹¹. 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. 3A) and protein (Fig. 3B) levels was dramatically enhanced in PASMCs exposed to PDGF-BB.

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. 4A). Knockdown of NCAM1 partly blocked the PDGF-BB-induced PCNA increase in PASMCs (Fig. 4B). Similarly, the CCK-8 assay proved that PDGF-BB increased the cellular activities in PASMCs, which were partially suppressed by NCAM1 knockdown (Fig. 4C). As shown in Fig. 4D, 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. 4E, 4F).

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 ¹². 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. 5A, 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. 5B). Similarly, the decreased protein level of PCNA and cell activity caused by NCAM1 knockdown in PDGF-BB-induced PASMCs was significantly increased after treatment with TBHQ (Fig. 5C, 5D). 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. 5E). In addition, we observed that the inhibitory effects of NCAM1 knockdown on cell migration in PDGF-BB-induced PASMCs were reversed by TBHQ (Fig. 5F, 5G).

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 activity, and the effects were strongest at 100 ng/mL (Fig. 6A). Therefore, this concentration was selected for subsequent experiments. As shown in Fig. 6B and 6C, recombinant rat NCAM1 promoted the proliferation of PASMCs. Recombinant rat NCAM1 also promoted the migration of PASMCs (Fig. 6D, 6E).

The present study demonstrated that the protein levels of NCAM1 were significantly increased in the plasma of PAH patients. The mRNA expression level of NCAM1 was also enhanced in the lung tissues of PAH patients. In addition, NCAM1 expression was up-regulated in rats with MCT-induced PAH and in PDGF-BB-induced PAMSCs. 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 PAH.

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 ¹³. 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 disease ¹⁴. Plasma levels of NCAM1 were also increased in multiple sclerosis patients compared to healthy individuals ¹⁵. Through bioinformatics, we found that the mRNA level of NCAM1 in the lung tissue of PAH patients was significantly increased. We also found that NCAM1 protein expression was significantly upregulated in the plasma of PAH patients. Moreover, ROC curve analysis showed that the AUC was 0.9365, indicating that NCAM1 might be a potential biomarker for PAH.

Multifarious pathogenesis involving multiple genes is involved in the development of PAH ¹⁶. As a cell surface glycoprotein, NCAM1 is expressed early in the development of various cell types ¹⁷. NCAM1 dysregulation is associated with diverse diseases because it is an important protein involved in a variety of cellular processes and cell-cell interactions ¹⁸. 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) ¹⁹. A previous study reported that NCAM1 was significantly upregulated in ameloblastoma tissues at the mRNA and protein levels ²⁰. Here, for the first time, our data revealed that both NCAM1 mRNA levels and protein expression were upregulated in PAH patients and PAH animal models.

Excessive proliferation and migration of PASMCs promote the development of PAH because they have a crucial role in pulmonary vascular remodeling ²¹. The abnormal surplus of PDGF-BB is involved in the basic pathological process of PAH and facilitates PAH development. PDGF levels have been reported to be significantly increased in plasma from patients with systemic SSc ²². As we know, PAH is a serious complication of SSc and is accompanied by increased expression of IL-32 and other proinflammatory cytokines ²³. Experimental PAH was attenuated because reversed vascular remodeling was achieved by blocking PDGF-BB/PDGFR-β signaling ²⁴. Previous reports have indicated that NCAM1 was able to stimulate proliferation, epithelial–mesenchymal transition, and migration of human melanoma cells ⁷. NCAM1 deficiency significantly impaired the migration of bone mesenchymal stem cells (BMSCs) ²⁵. 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 ²⁶. 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) ²⁷ during body growth and development and damage repair ²⁸. 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 ²⁹. The ERK1/2 signaling pathway has been shown to be significantly activated in a variety of animal models of PAH ^30–32. Erk1/2 knockdown can effectively inhibit pulmonary vascular remodeling and thus prevent the development of PAH ³³. According to previous reports, NCAM1 could activate ERK1/2 signaling pathway in neuronal cells ³⁴ and mesenchymal stromal cells ²⁵. 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.

In conclusion, our results show that the expression levels of NCAM1 are elevated in PAH patients and a PAH animal model. 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 PAH, which may have important implications for treating PAH.

PAH: pulmonary arterial hypertension; NCAM1: neural cell adhesion molecule 1; MCT: monocrotaline; 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.

Ethical Approval:

The clinical study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. All rat experiments were approved by the Ethics Committee of Chongqing Medical University.

Consent for publication

Not applicable.

Competing interests

The authors have no competing interest to declare.

Author Contributions:

Participated in research design: Yunwei Chen and Wei Huang; Conducted experiments: Yunwei Chen, Yunjing Yang, Lingzhi Yang, Yan Li, Ailing Li; Analyzed and interpreted the data: Yunwei Chen; Contributed to the writing of the manuscript:Yunwei Chen and Wei Huang.

Sources of Funding:

This work was supported by the National Natural Science Foundation of China (82270061) and Chongqing Natural Science Foundation (cstc2021jcyj-msxmX0474).

Availability of data and materials

All the relevant raw data and materials are freely available from the corresponding author on reasonable request.

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No competing interests reported.

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NCAM1 Promotes the Proliferation and Migration of Pulmonary Arterial Smooth Muscle Cells via the ERK1/2 Pathway

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Abstract

Figures

Introduction

Materials And Methods

Reagents

Study population

Enzyme-linked immunosorbent assay

Microarray data acquisition

Data processing and DEG determining

Animal experiments

Echocardiography

Right heart catheterization (RHC)

Hematoxylin-eosin (H&E) staining

Immunofluorescent staining

Preparation of PASMCs

Quantitative reverse transcription PCR (qRT-PCR)

Western blotting

Cell transfection

EdU staining

Cell viability assay

Scratch wound healing assay

Transwell migration assay

Statistical analysis

Results

NCAM1 is upregulated in plasma in PAH patients

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

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

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

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

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

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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