NPS2143 Modulates the Phenotypic Switching of PASMCs by Inhibiting Autophagy in Hypoxia-Induced Pulmonary Hypertension

Research Article

J Stem Cell Res Transplant. 2021; 8(2): 1038.

NPS2143 Modulates the Phenotypic Switching of PASMCs by Inhibiting Autophagy in Hypoxia-Induced Pulmonary Hypertension

Wang L¹, Shao H², Che B³, Wang N³, Peng X²* and Wei C³*

1Department of Respiratory Medicine, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China

2Department of Forensic Medicine, Harbin Medical University, Harbin, 150081, China

3Department of Pathophysiology, Harbin Medical University, Harbin, 150081, China

*Corresponding author: Xue Peng Department of Forensic Medicine, Harbin Medical University, Baojian Road, Harbin, 150081, China

Can Wei, Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin, 150081, China

Received: May 13, 2021; Accepted: June 09, 2021; Published: June 16, 2021


Background and Objectives: Pulmonary Artery Hypertension (PAH) is considered as a malignant tumor in cardiovascular disease. Our previous study found that Calcium-Sensing Receptor (CaSR) is involved in pulmonary vascular remodeling in hypoxic pulmonary hypertension (HPH). However, the relationship of Pulmonary Artery Smooth Muscle Cell (PASMC) phenotypic switching, proliferation, and autophagy in CaSR-related HPH remain unclear. The purpose of this study was to detect the role of a CaSR antagonist, NPS2143, on the vascular remodeling by autophagy modulation under hypoxia.

Methods: Hypoxic rat PAH model were simulated in vivo. Meanwhile, mean Pulmonary Artery Pressure (mPAP) was measured while RVI, WT%, and WA% indices were calculated. Immunohistochemistry and Western blot were used to detect phenotypic switching and cell proliferation in pulmonary arteriole. Cell viability was determined in vitro by CCK8 and cell cycle. Cell proliferation, phenotypic switching, autophagy level and PI3K/Akt/mTOR pathways were investigated in human PASMCs through mRNA or Western blot methods.

Results: Rats with hypoxic-induced PAH had an increased mPAP, RVI, WT% and WA%. Moreover, expression of CaSR was significantly increased, followed by activation of autophagy (increased LC3b and decreased p62), phenotypic switching of PASMCs (reduced calponin, SMA-a and increased OPN) and pulmonary vascular remodeling. However, NPS2143 weakened these hypoxic effects. The results using hypoxic-induced human PASMCs confirmed that NPS2143 suppressed autophagy and reversed phenotypic switching in vitro by inhibiting PI3K/Akt/mTOR pathways.

Conclusions: Our study demonstrates that NPS2143 was conducive to inhibit the proliferation and reverse phenotypic switching of PASMCs by regulating autophagy levels in HPH and vascular remodeling.

Keywords: NPS2143; Phenotypic switching; Autophagy; Hypoxia pulmonary vascular remodeling


Pulmonary Hypertension (PAH) is a life-threatening condition with no effective treatment at present. PAH is closely related to pulmonary vascular resistance and its irreversible remodeling. During the onset of PAH, excessive proliferation of pulmonary vascular smooth muscle cells (PASMCs) and decreased apoptosis accelerate the vascular remodeling [1]. In hypoxia-induced PAH, PASMCs undergo a contraction-synthesis phenotype transition, which is the main cause of pulmonary vascular remodeling [2]. However, the molecular mechanisms of pulmonary vascular remodeling in PAH are complex and still not fully understood.

Our previous study demonstrated that an increased CaSR expression involves phenotypic switching of PASMCs in small pulmonary artery in hypoxic rat [3]. NPS2143, a specific allosteric inhibitor of CaSR, is the first recognized CaSR antagonist [4,5]. NPS2143 has been reported to have a variety of biological properties, such as anti-cancer[6] and anti-inflammatory activities [7]. Recent studies also found that NPS2143 could inhibit the excessive proliferation of PASMCs in patients with idiopathic PAH [8]. Therefore, we hypothesize that interfering with NPS2143 can be a more effective method for treating hypoxic PAH.

Formation of hypoxic PAH involves many complex signaling and gene regulatory networks. Recent studies showed that autophagy acts as a self-protection mechanism for eukaryotic organisms to maintain intracellular environmental stability and participates in the occurrence and development of various types of PAHs [9]. Meanwhile, hypoxia can promote VSMC phenotype conversion and also be used as a stimulator of autophagy [10]. Therefore, we envisage that autophagy may be involved in hypoxia-promoting PASMCs phenotypic switching in the process of NPS2143 alleviating PAH. This study presents a more thorough understanding of the relationship between autophagy and phenotypic switching of PASMCs to provide a better therapeutic mechanism for the treatment of HPH with NPS2143.

Material and Methods

Animal models of HPH

A chronic hypoxia model was established as described in previous studies [11]. Male wistar rats (200-250 g) were randomly divided into 6 groups (n=5 per group), including control group, hypoxia 7d group, hypoxia 14d group, hypoxia 21d group, hypoxia 21d+NPS2143 group, control+NPS2143 group. Each hypoxia group rats were placed in a hypobaric chamber under normal pressure with a continuous flow of oxygen to stabilize its concentration in the chamber at (10±0.5)%, anoxic for 24 h per day. After hypoxia 7d, 14d, and 21d, the rats were removed from the chamber for subsequent experiments. The two NPS2143 groups were intraperitoneally injected with CaSR inhibitor, NPS2143 (5 mg/kg), every day.

All animal protocols used in this study were in accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all animal experiments were approved by the Scientific Investigation Board of Harbin Medical University, Harbin, China.

Evaluation of PAH

The rats were anesthetized with 1% pentobarbital sodium intraperitoneally, a polyethylene catheter was inserted into the right external jugular vein to the pulmonary trunk, and the mean Pulmonary Artery Pressure (mPAP) was measured using a PowerLab Physiological Signal Processing System (AD Instruments Inc., Australia). Hearts were dissected from the thoracic cavity and fixed in 4% formaldehyde for 2 d. The weight of Right Ventricle (RV) and the left ventricle plus ventricular septum (LV+S) were measured separately to calculate the Right Ventricular Hypertrophy Index (RVHI), which reflects the changes in right ventricular hypertrophy.

Hematoxylin and Eosin (H&E) staining

Lung tissue of rat was fixed with neutral formalin, embedded in paraffin, and stained with HE. The walls of pulmonary arterioles were observed under microscope.

Weigert’s staining

Conventional paraffin section was dewaxed, dehydrated. Counterstained with prepared VG staining solution, 95% ethanol rapidly differentiates, quickly dehydrated by anhydrous ethanol and transparent xylene, and sealed with neutral gum.

Pulmonary vascular morphometry

After HE and Weigert’s staining, the inner and outer diameters of the pulmonary vessels (80-120 μm in diameter) were measured using Imagepro-plus 6.0 software, and the wall thickness, wall area, total pulmonary vascular area, wall thickness/vascular outer diameter (WT%), and wall area/vessel area/ (WA%) were calculated.

Transmission electron microscopy

The pulmonary arterioles of each group were fixed with 2.5% glutaraldehyde, dehydrated with gradient ethanol and acetone, embedded, oven-cured, sliced with ultra-thin slicer, with a thickness of 70nm, and double stained with 3% uranyl acetate-lead lead. Finally, the transmission electron microscope was used to observe and film.


Conventional paraffin sections were dewaxed in water, each slice was subjected to antigen retrieval. 0.3% H2O2 blocked endogenous peroxidase. Each section was incubated with anti-CaSR, p62, LC3b, OPN, calponin, SMA-a and GAPDH (purchased from Cell Signaling Technology, Boston, USA). Secondary antibody was selected based on primary antibody, DAB color. Finally, neutral gum was used to slice. The brown stained area on the vessel wall was observed under a light microscope to be positive.

Cell culture

Human Pulmonary Arteries Smooth Muscle Cells (HPASMCs) and medium SMCM were purchased from American ScienCell Company. HPASMCs cells were cultured in SMCM in complete medium (89% DMEM + 10% fetal calf Serum + 1% growth factor) with 100 μg/mL penicillin and 100 μg/mL streptomycin. The cells were cultured at 37°C, 21% O2 and 5% CO2. The second to fourth generation cells were used for subsequent experiments. When the subcultured HPASMCs were grown to 60% of the bottom of the flask, the serum-free medium was replaced for 24 h and the following experiments were performed.

Hypoxia treatment of HPASMCs

A hypoxic environment was established by continuously mixing 95% N2, 1 % O2 and 5% CO2 in a hypoxic incubator. HPASMCs were first starved in serum-free DMEM for 24 h. Then they were exposed to one of the following 9 different experimental conditions for 24 h: (1) control: 21% O2 / 5% CO2 / balance N2; (2) hypoxia: 1 % O2 / 5% CO2/balance N2; (3) hypoxia with 1 μM NPS2143 (antagonist of CaSR); (4) normoxia with 1 μM NPS2143; (5) hypoxia with 5 mM 3-MA (inhibitor of autophagy); (6) normoxia with 5 mM 3-MA; (7) hypoxia with 5 μM R568 (an agonist of CaSR); (8) hypoxia with 10 μM NPS2143 and 5 μM R568; and (9) hypoxia with 50 nM NVPBEZ235 (a PI3K/mTOR dual inhibitors).

Cell proliferation assays

HPASMCs were seeded in a 48-well plate at 1×104 cells/well, and cultured at 37°C under 5% CO2 until the cells were 90% confluent, and then incubated with serum-free DMEM/F12 medium for 2 h to synchronize the cells. 10 μL of CCK-8 (purchased from Invitrogen, CA, USA) solution was added to each well. After the incubation, A450 was measured using a microplate reader.

Cell cycle assays

For cell cycle analysis, the HPASMCs to be tested were washed and centrifuged at 1000 r/min for 5 min. Supernatant was discarded and cells were resuspended in 80% ethanol or added propidium iodide. Cell cycle analysis was performed with a CytoFLEX S Flow Cytometer (manufactured by Beckman Coulter).

Western blotting

Pulmonary arterioles or HPASMCs were used to detect the target proteins. The following primary antibodies for rabbit anti-mouse CaSR, calponin, SMA-a, OPN, PCNA, Ki67, LC3b, p62, p-AKT, AKT, p-PI3K, PI3K, p-mTOR, mTOR, caspase-3, HIF-1a and GAPDH (purchased from Cell Signaling Technology, Boston, USA) were used. The resulting protein strip image was detected using Quality One software to analyze the image to GAPDH light density. The degree value is used as an internal parameter to correct the optical density value of the target protein.

Real-time fluorescence quantitative PCR

Total RNA was extracted by Trizol (DRR037A, Takara, Japan) method and its concentration and quality were measured. Reverse transcription of synthetic DNA was done according to the reverse transcription kit instructions (Takara, Japan). The expression levels of CaSR, PCNA, Ki67, LC3b, p62, calponin, SMA-a OPN, and β-actin were detected on a Takara fluorescence quantitative PCR instrument according to the SYBR Premix Ex TaqTMII (Tli RNase H Plus) protocol. Three repetitions were made for each concentration. Difference analysis of expression was performed on the qRT-PCR results using the 2-ΔΔCT method. The following primers were used:

CaSR: C T T T G T G C T G G G T G T C T T C A (forward), A A C A A G G A G C T G G A G A A G C A (reverse); PCNA: A C G C C G C C C G A A C T G A T (forward), C G T G C G T G A C A T T A A A G A G (reverse); Ki67: G G T G T C A A G C A C G A A T G (forward), G G G A C C T G G C A C G A A T A G A T (reverse); P62: T G A A G G C T A T T A C A G C C A G A G T C A A (forward), C C T T C A G T G A T G G C C T G G T C (reverse); LC3b: T T G A C C T C C C A A A G T G C (forward), T C C A A G C C T G T A A A C C C (reverse); calponin: G A A C T T G T C T G G G T C A T C T C G (forward), A C G C T G G T G G T C G T A T T T C T (reverse); SMA-a: A C C A T C G G G A A T G A A C G C T T (forward), T T G C G T T C T G G A G G A G C A A T (reverse); OPN: G C C G A G G T G A T A G C T T G G C T T A (forward), T T G A T A T C C T C A T C G G A C T C C T G (reverse); β-actin: C A A C C T T C T T G C A G C T C C T C (forward), C G G T G T C C C T T C T G A G T G T T (reverse).


Data analysis was done using SPSS21.0 statistical software, all results were calculated according to the mean ±SEM, the comparison between the different groups was tested in One-way ANOVA, p < 0.05 was considered statistically significant.


Hypoxia induces pulmonary vascular remodeling, the expressions of CaSR, autophay protein, HIF-1a, PASMCs proliferation and phenotypic switching in hypoxic PAH rats

We first established a hypoxic PAH model. It was found that the level of mPAP and RVI were increased at hypoxic 14d and 21d (Figure 1A and 1B). HE and Weigert’s elastic staining (Figure 1C) showed thickening of the pulmonary vascular wall and increased WT% and WA% (Figure 1D) of the pulmonary arterioles. Next, the expression of CaSR and HIF-1a in the pulmonary arterioles was significantly increased with the increased expression of LC3b and decreased expression of p62 in hypoxic 14d and 21d, compared to control rats (Figure 1E-1F). In addition, we used TEM to observe the formation of autophagosomes in pulmonary arterioles in hypoxic 21d (Figure 1G). These results suggested that there may be some association between CaSR and autophagy in HPH rats.