For experiments involving human tissues, approval was obtained from the Ethics Committees of Nanjing Medical University (approval no. KY20190505-09; Nanjing, China), and all subjects provided written informed consent before the study. The inclusion criteria included patients with idiopathic PAH who underwent lung transplantation at Wuxi People's Hospital (Wuxi, China); patients with other types of PAH were excluded. Between June 2019 and April 2020, lung samples were collected from three patients (age range, 28-40 years; two female patients and one male patient) with idiopathic PAH [mean pulmonary arterial pressure (PAP), 68.7±13.5 mmHg; pulmonary vascular resistance, 19.9±0.9 Woods units]. Three normal unused donor lungs (age range, 42-50 years; two women and one man) were used as controls.

SMC-specific FLNA-knockout mice were generated according to a previous study (11). All of the animals were housed at constant temperature (22±2°C) and relative humidity (50%) under a 12-h light/dark cycle, and were allowed ad libitum access to food and water. As FLNA is located on the X chromosome, 16 male Tagln-Cre^ERT2 mice (Gempharmatech Co., Ltd.) were crossed with 32 female FLNA^flox/flox mice (Gempharmatech Co., Ltd.) to generate 24 FLNA^flox/Y Tagln-Cre^ERT2+ mice (FLNA-CKO). Male mice aged 6 weeks old and weighing 18-22 g were used. A total of 24 FLNA^flox/Y Tagln-Cre^ERT2− mice (FLNA-C; 6-week-old male mice; weight, 18-22 g) were used as controls. Tamoxifen (75 mg/kg; cat. no. T5648; MilliporeSigma) was intraperitoneally injected for 5 consecutive days to conditionally delete FLNA. On day 14 after the first tamoxifen injection, the mice were euthanized via overdose of pentobarbital sodium (200 mg/kg), and death was confirmed by cervical dislocation. FLNA expression was detected in the pulmonary artery and the aorta by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemical staining. In addition, DNA was extracted from tail clippings and the genotype was determined by PCR.

For tail genotyping, one 5-mm tail clipping was performed on each mouse <21 days of age without anesthesia. DNA was extracted from tail clippings using Animal Genomic DNA Quick Extraction Kit (cat. no. D0065S; Beyotime Institute of Biotechnology). The tail samples were lysed in lysis buffer containing DNA extraction solution and enzyme mix at 55°C for 15 min, followed by 95°C for 5 min. Subsequently, stop solution was added to stop the reaction. PCR was performed using the Quick Genotyping Assay Kit for Mouse Tail (cat. no. D7283S; Beyotime Institute of Biotechnology). The primer sequences were as follows: Wild-type FLNA, forward 5′-CCA AAC TGA ACC CGA AGA AA-3′ and reverse 5′-TTT TGC CTCTTC CTG GTG TC-3′; Cre, forward 5′-GCC TGC ATT ACC GGT CGA TGC-3′ and reverse 5′-CAG GGT GTT ATA AGCA ATC CC-3′. The samples were amplified using the following thermocycling conditions: Initial denaturation at 95°C for 3 min; followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 30 sec; and final extension at 72°C for 10 min. Subsequently, PCR products were separated by 1% agarose gel electrophoresis, and the bands were stained in 20 mg/ml ethidium bromide for 15 min at room temperature and verified under an UV transilluminator. All of the experimental protocols were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (approval no. 1905004), and conformed with the National Institutes of Health guide for the care and use of laboratory animals (13).

SU5416 is a selective vascular endothelial growth factor receptor inhibitor that may induce severe pulmonary hypertension if combined with chronic hypoxia. A commonly used protocol for the hypoxia/SU5416 model is 3 weeks hypoxic exposure, followed by 1 week normoxic exposure (14). However, several studies have argued that pulmonary hypertension may not be sustained after the hypoxic exposure has been discontinued (15,16). Therefore, in the present study, hypoxia exposure was prolonged to 4 weeks with weekly subcutaneous injections of 20 mg/kg SU5416 (cat. no. S8442; MilliporeSigma) (17). Briefly, 8-week-old male C57BL/6JGpt mice (weight, 22-24 g) were housed with ad libitum access to food and water at 22°C with 65% humidity and a 12-h light/dark cycle. The mice were subcutaneously injected with 20 mg/kg SU5416, and then exposed to chronic hypoxia (10% O₂) for the subsequent 4 weeks. Mice in the normoxic group were exposed to room air (10% O₂). A total of 36 mice were randomly divided into the following four groups (n=9/group): i) FLNA-C normoxic group; ii) FLNA-CKO normoxic group; iii) FLNA-C hypoxic group; and iv) FLNA-CKO hypoxic group.

Antibodies against phosphorylated (p)-Akt (Ser473) (cat. no. 9271), Akt (cat. no. 9272), p-ERK (Thr202/Tyr204; cat. no. 9101), ERK (cat. no. 9102), p-PAK1 (Ser144; cat. no. 2606), PAK1 (cat. no. 2602) and proliferating cell nuclear antigen (PCNA; cat. no. 2586) were purchased from Cell Signaling Technology, Inc. Antibodies against FLNA (cat. no. ab76289), Ras-related C3 botulinum toxin substrate 1 (Rac1; cat. no. ab33186) and α-smooth muscle actin (α-SMA; cat. no. ab124964) were purchased from Abcam. Secondary antibodies used for western blotting were obtained from OriGene Technologies, Inc. (HRP-conjugated goat anti-rabbit IgG, cat. no. ZB-2301; HRP-conjugated goat anti-mouse IgG, cat. no. ZB-2305). Secondary antibodies used for immunofluorescence were obtained from OriGene Technologies, Inc. (TRITC-conjugated goat anti-rabbit IgG, cat. no. ZF-0316; FITC-conjugated goat anti-rabbit IgG, cat. no. ZF-0311). FLNA small interfering RNA (siRNA) (cat. no. sc-35374) and the control siRNA (cat. no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. pcDNA-myc-FLNA WT was obtained from John Blenis (Addgene #8982) (18). The control plasmid pcDNA™ 3.1⁽⁺⁾ was purchased from Thermo Fisher Scientific, Inc. (cat. no. V79020).

Right ventricular systolic pressure (RVSP) was measured as previously described (17). Before measurement, mice were intraperitoneally anesthetized with 45 mg/kg pentobarbital sodium. After RVSP was recorded, all mice were sacrificed by cardiac puncture under anesthesia and death was confirmed by cervical dislocation. Subsequently, the heart and lung tissues were removed. The right ventricle (RV) was separated from the left ventricle plus ventricular septum (LV + S). The weight ratio of RV/(LV + S) was calculated to assess the right ventricular hypertrophy index (RVHI).

After anesthesia with 2% isoflurane, echocardiographic images were obtained with the Vevo 2100 imaging system (VisualSonics, Inc.). The following parameters were assessed: RV free wall thickness (RVWT), RV internal diameter (RVID), pulmonary artery acceleration time (PAAT) and pulmonary artery ejection time (PAET). The PAAT/PAET ratio was then calculated as an indirect measure of systolic PAP.

The lung tissues were fixed in 10% neutralbuffered formalin for 24 h at room temperature followed by embedding in paraffin wax, and then cut into 5-µm slices. Paraffin-embedded slices were deparaffinized in xylene, rehydrated in a graded ethanol series to phosphate-buffered saline, and the slices were then stained with hematoxylin and eosin for 1 min, and elastic van Gieson for 3 min at room temperature, respectively.

For immunohistochemical staining, after deparaffinization and rehydration, antigen retrieval was performed by heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) at 95°C for 3 min. Subsequently, the sections were stained with anti-FLNA (1:200) or anti-PCNA (1:2,000) primary antibodies at 4°C overnight. Subsequently, the sections were incubated with biotinylated secondary antibody for 30 min at room temperature, followed by reaction with diaminobenzidine. The images were observed under an optical microscope (Olympus BX61; Olympus Corporation).

For immunofluorescence analysis, the slices were permeabilized with 0.3% Triton X-100 for 10 min and blocked by 1% bovine serum albumin (cat. no. ST023; Beyotime Institute of Biotechnology) for 1 hat room temperature. Then, primary antibodies against FLNA (1:200) and α-SMA (1:200) were applied at 4°C overnight. The sections were then incubated with the respective secondary antibody at room temperature for 1 h, followed by DAPI counterstaining. Images were collected using laser scanning confocal microscopy (LSM 710; Carl Zeiss AG).

The mouse lung tissues were fixed in 10% neutral-buffered formalin for 24 h at room temperature. The TUNEL Apoptosis Assay kit (cat. no. C1098; Beyotime Institute of Biotechnology) was used to detect apoptotic cells in paraffin-embedded lung tissues, which were cut into 5-µm slices. The sections were deparaffinized and rehydrated, followed by incubation with 50 µl biotin labeling solution at 37°C for 1 h in the dark. Subsequently, samples were incubated with 50 µl streptavidin-HRP solution for 30 min at room temperature and then with diaminobenzidine solution for 15 min at room temperature. The nuclei were counterstained with hematoxylin. The nuclei of positive cells were stained brown. The positively stained cells in five different fields of view were observed under an optical microscope (Olympus BX61; Olympus Corporation). The cell apoptotic rate was calculated as the percentage of TUNEL-positive cells.

Human pulmonary arterial smooth muscle cells (HPASMCs; cat. no. 3110) were obtained from ScienCell Research Laboratories, Inc. and cells at passages 4-9 were used for the present study. The cells were cultured with complete smooth muscle cell medium (cat. no. 1101; ScienCell Research Laboratories, Inc.) at 37°C in a 5% CO₂-humidified incubator. Ethics approval for the use of HPASMCs was obtained from the Ethics Committees of the Nanjing Medical University. FLNA was knocked down and overexpressed in HPASMCs to investigate the effects of FLNA on cell proliferation and migration. FLNA was knocked down using siRNA and was overexpressed by transfection with overexpression plasmids. PASMCs were seeded into 6-well culture plates and when the cells had reached 70% confluence, they were transfected with FLNA siRNA (50 nmol/l), control siRNA (50 nmol/l), pcDNA-myc-FLNA WT (2 µg) or control plasmid (2 µg) per well under serum-free conditions using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 6 h at 37°C, the infection solution was replaced by complete medium. After transfection for 48 h, cells were harvested for further analysis.

PASMC viability and proliferation was assessed using Cell Counting Kit 8 (CCK-8) and EdU staining assays. For the CCK-8 assay, a cell suspension was prepared and 2×10³ cells/well in 100 µl medium was added into 96-well plates. CCK-8 (cat. no. C0038; Beyotime Institute of Biotechnology) solution was added to the 96-well plate and incubated at 37° for 3 h. Absorbance was determined at a wavelength of 450 nm.

For the EdU staining assay, PASMCs were seeded at a density of 1×10⁵ cells/well in a 6-well plate. After transfection for 48 h, the cells were incubated with 10 µM EdU (Beyotime Institute of Biotechnology, cat. no. C0071S) for 2 h at 37°C. Then, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized by 0.3% Triton X-100 in PBS at room temperature for 15 min. Finally, click reaction cocktail was added to inactivate endogenous peroxidase for 30 min at room temperature in the dark. Cell nuclei were stained with DAPI and images were captured via fluorescence microscopy. Proliferation was expressed as the percentage of EdU-positive cells.

Transwell migration and wound-healing assays were used to assess PASMC migration (19). For the woundhealing assay, PASMCs were seeded into 6-well plates until the cell density approached 90%. A sterile 10-µl pipette tip was used to produce a straight scratch to the cell monolayer and the cells were then incubated with serum-free medium. Migration was determined as the percentage of the wound closure; images were captured at 0 and 24 h under an optical microscope.

For the Transwell assay, the cells were seeded in the Transwell chamber (cat. no. CLS3428; Corning, Inc.) located in a 24-well plate. Subsequently, 1×10⁵ cells in 200 µl serum-free medium were seeded in the upper chamber and 600 µl complete medium containing 20% FBS was added to the lower chamber. The cells were cultured for 24 h at 37°C. Then, the cells on the upper membrane surface were removed, whereas the cells that had migrated to the lower membrane surface were fixed with 4% paraformaldehyde for 30 min, and then stained with crystal violet for 30 min at room temperature. Finally, the number of cells in five random fields on each membrane was counted under a light microscope.

Total RNA was isolated using RNA isolater Total RNA Extraction Reagent (cat. no. R401-01; Vazyme, Inc.) from the PASMCs and mouse tissues, and RT was performed usig the HiScript II Q Select RT SuperMix (cat. no. R232-01; Vazyme, Inc.) at 50°C for 15 min and terminated at 85°C for 5 sec. qPCR was carried out using Taq Pro Universal SYBR qPCR Master Mix (cat. no. Q712-02; Vazyme, Inc.). The reference genes were β-actin for human PASMCs and GAPDH for mouse lung tissues. PCR amplification was performed as follows: Initial denaturation at 95°C for 3 min; followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 30 sec. The primers used were as follows: FLNA (human), forward 5′-CAG TGC TAT GGG CCT GGT AT-3′, reverse 5′-CCA CTT TGT ACA TGC CAT CG-3′; β-actin (human), forward 5′-AAA CGT GCT GCT GAC CGA GR-3′, reverse 5′-TAG CAC AGC CTG GAT AGC AAC-3′; FLNA (mouse), forward 5′-CCA AAC TGA ACC CGA AGA AA-3′, reverse 5′-TTT TGC CTC TTC CTG GTG TC-3′, GAPDH (mouse), forward 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′. Results were qualified using the 2^−ΔΔCq method (20).

Total protein was extracted from the tissues and PASMCs with RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing protease and phosphatase inhibitor cocktail (cat. no. P1046; Beyotime Institute of Biotechnology). Protein concentration was measured using the bicinchoninic acid method (cat. no. P0010S; Beyotime Institute of Biotechnology) and 50 µg protein/lane was separated by SDS-PAGE on 8-12% gels. The proteins were subsequently transferred to PVDF membranes, which were blocked with 10% milk for 1 h at room temperature and incubated overnight at 4°C with the primary antibodies (1:2,000 for FLNA; 1:1,000 for other proteins). The membranes were subsequently incubated with the corresponding secondary antibody (1:1,000) for 2 h at 37°C and were detected using the enhanced chemiluminescence system (cat. no. P0018S; Beyotime Institute of Biotechnology). The bands were semi-quantified using Quantity One version 4.6.6 software (Bio-Rad Laboratories, Inc.).

The degree of activation of Rac1 was measured using a G-LISA Ras Activation Assay Biochem kit (cat. no. BK131; Cytoskeleton, Inc.) according to the manufacturer's instructions (21).

All continuous variables are presented as the mean ± SD. At least three repeats were performed for each experiment. Unpaired Student's t-test was used to determine the statistical difference between two groups. Differences between multiple groups were compared with one-way ANOVA followed by Bonferroni's post hoc test. All analyses were performed using SPSS version 22.0 (IBM Corporation). P<0.05 was considered to indicate a statistically significant difference.

As determined by western blotting and immunofluorescence staining, the expression levels of FLNA were markedly increased in lung tissues from patients with PAH compared with in control individuals (Fig. 1). FLNA was mainly expressed in the PASMCs of pulmonary arterioles, as evidenced by α-SMA co-immunostaining (Fig. 1C). These results suggested that FLNA may be an important factor in pulmonary vascular remodeling.

As FLNA-knockout mice exhibit embryonic lethality (22), the female FLNA^flox/flox mice were crossed with male Tagln-Cre^ERT2+ mice to produce male hemizygote FLNA^flox/Y Tagln-Cre^ERT2+ mice (Fig. 2A). Using PCR, the presence of floxed alleles and Cre recombinase was detected in male FLNA^flox/Y hemizygotes. The FLNA^flox/Y mice without the Cre recombinase were used as controls (Fig. 2B). SMC-specific FLNA deficiency (FLNA-CKO) was induced in 8-week-old FLNA^flox/Y Tagln-Cre^ERT2+ mice by intraperitoneal injection of tamoxifen for 5 consecutive days, and FLNA depletion was confirmed by RT-qPCR and immunohistochemical staining at day 14 after the initial tamoxifen injection. RT-qPCR analysis of the aorta and pulmonary artery demonstrated a significant decrease in FLNA expression in FLNA-CKO mice (Fig. 2D). Immunohistochemical staining further confirmed the downregulation of FLNA in the aorta and pulmonary artery (Fig. 2C). These results indicated that tamoxifen efficiently induced FLNA deficiency in vascular smooth muscle cells. Furthermore, TUNEL staining suggested that the apoptotic rate was significantly increased in FLNA-CKO mice (Fig. S1).

After exposure to hypoxia, the RVSP was significantly increased in the hypoxic groups of both FLNA-C and FLNA-CKO mice. However, compared with in FLNA-C mice, FLNA-CKO mice exhibited a significantly lower RVSP (Fig. 3A and B). In addition, RVHI was much lower in FLNA-CKO mice than that in FLNA-C mice after hypoxic exposure (Fig. 3C). Echocardiography revealed that RV dilatation and RV thickness were attenuated in FLNA-CKO mice after hypoxic exposure (Fig. 3D and E). Moreover, right heart function after exposure to chronic hypoxia was improved in FLNA-CKO mice, as revealed by PAAT/PAET ratio (Fig. 3F).

Histological studies revealed that FLNA-CKO could ameliorate pulmonary vascular remodeling. The medial wall thickness and medial wall area were increased after hypoxia exposure, and were significantly decreased in FLNA-CKO mice compared with those in FLNA-C mice (Fig. 3G and H). Immunofluorescence staining confirmed that FLNA was mainly expressed in α-SMA-positive SMCs in pulmonary arterioles; FLNA expression was increased in α-SMA-positive smooth muscle cells after hypoxic exposure, but was markedly decreased in FLNA-CKO compared with in FLNA-C mice (Fig. 4A). Immunohistochemical analyses also revealed that FLNA knockout significantly inhibited hypoxia-induced PASMC proliferation, as evidenced by a reduction in the number of PCNA-positive cells (Fig. 4B and C). Thus, loss of FLNA in SMCs may confer protective effects in hypoxia-induced pulmonary vascular remodeling and PASMC hyperplasia.

In in vitro studies, FLNA siRNA was used to knockdown FLNA expression. The efficient knockdown of FLNA in PASMCs was identified by western blotting (Fig. 5A and B) and RT-qPCR (Fig. 5C). Both CCK-8 and EdU staining assays indicated that FLNA knockdown significantly inhibited PASMC viability and proliferation, respectively (Fig. 5D-F). Similarly, Transwell migration and wound-healing assays demonstrated a marked effect of FLNA knockdown on the migration of PASMCs; PASMC migration was significantly decreased in the FLNA siRNA-treated group compared with that in the control group (Fig. 5G-J).

To determine the molecular mechanisms behind the FLNA knockdown-induced decreases in cell proliferation and migration, the expression levels of downstream signaling molecules, including p-PAK1, p-AKT, p-ERK and Rac1, were determined. FLNA siRNA-transfected PASMCs exhibited significantly reduced p-PAK1 levels (Fig. 6A and B); however, there was no change in p-AKT, p-ERK and Rac1 expression (Fig. 6A and C-E). BY contrast, G-LISA assay revealed that Rac1 activity was decreased by 56% in PASMCs transfected with FLNA siRNA (Fig. 6F).

FLNA overexpression was achieved by transfection with FLNA plasmids, and confirmed by western blotting (Fig. 7A and B) and RT-qPCR (Fig. 7C). The CCK-8 and EdU proliferation assays showed that PASMC viability and proliferation was significantly higher in FLNA plasmid-transfected cells compared with that in the control group (Fig. 7D-F). Similarly, the results of the migration assays confirmed a significant increase in PASMC migration after FLNA overexpression (Fig. 7G-J). Moreover, western blotting revealed that p-PAK1 levels were significantly increased in cells transfected with the FLNA plasmid (Fig. 8A and B); however, there was no change in p-AKT, p-ERK and Rac1 expression (Fig. 8A and C-E). Furthermore, G-LISA assay revealed that Rac1 activity was increased in PASMCs transfected with the FLNA plasmid (Fig. 8F). These findings indicated that FLNA overexpression induced PASMC proliferation and migration, whereas FLNA knockdown suppressed the proliferation and migration of these cells.