Transgelin as a potential target in the reversibility of pulmonary arterial hypertension secondary to congenital heart disease

Abstract Background The reversibility of pulmonary arterial hypertension (PAH) in congenital heart disease (CHD) is of great importance for the operability of CHD. Proteomics analysis found that transgelin was significantly up‐regulated in the lung tissue of CHD‐PAH patients, especially in the irreversible group. However, how exactly it participated in CHD‐PAH development is unknown. Methods Immunohistochemical staining and Western blot were performed for further qualitative and quantitative analysis of transgelin in the lung tissues of CHD‐PAH patients. The mechanism of transgelin in CHD‐PAH development was explored in vitro. Primary human pulmonary arterial smooth muscle cells (hPASMCs) were cultured and infected with TAGLN siRNA or TAGLN lentiviral vector. Cell morphologic change (Coomassie Brilliant Blue staining), proliferation (cell count and EdU assay), apoptosis (terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling assay and Annexin‐V flow cytometry) and migration (transwell) were evaluated following the cell treatment. The mRNA and protein expression levels were detected in real‐time PCR and Western blot. Results In line with the proteomic findings, transgelin was obviously expressed in PASMC of the middle pulmonary arterioles, especially in the irreversible PAH group. Also, transgelin expression showed positive relation with pathological grading. Experiment in vitro demonstrated that transgelin overexpression promoted PASMC proliferation and migration, strengthened cytoskeleton and was accompanied by increased expression of synthetic phenotype markers (osteopontin, proliferating cell nuclear antigen) and anti‐apoptotic protein (bcl‐2). On the other hand, suppression of transgelin expression activated PASMC apoptosis, reducing cell proliferation and migration. Conclusions Transgelin may be a potential target in the development of irreversible CHD‐PAH through inducing PASMC phenotype change, proliferation, migration and reducing cell apoptosis.


| INTRODUCTION
Pulmonary arterial hypertension (PAH), characterized by increased pulmonary arterial pressure (PAP), pulmonary vascular resistance (PVR) and aggravated right heart function, is a common combination of congenital heart disease (CHD). Long-time exposure to high blood flow in CHD patients can lead to endothelial dysfunction and pulmonary vascular remodelling, which may finally result in irreversible pulmonary vasculopathy. Studies showed prevalence from 4% to 28%, 1,2 and 30% of unrepaired CHD patients have PAH. 3 Our previous data showed that 47.5% of Chinese CHD patients complicated by PAH. 4 Interventional and surgical repair as the main treatment for CHD has proved to be highly beneficial for the patients. But a study showed that 14% of patients were affected with PAH after the correction of complete atrioventricular septal defects, and the 1-year mortality of CHD-PAH patients who underwent cardiac operations reached 18.5%. 3 Studies even showed a worse 10-year postoperative survival for patients with persistent post-operative PAH than for those with Eisenmenger syndrome, 5,6 which was considered the severe and irreversible condition of CHD-PAH. It means that the pulmonary vascular lesion had become irreversible and the abnormality in haemodynamics cannot be reversed by surgical repair in these patients. Furthermore, the condition may be even worse because of the sudden haemodynamics change after surgery. Despite advances in our understanding of the pathophysiology and the management of PAH, significant translational and therapeutic gaps remained for the medication. 7 That raised the importance of accurate evaluation and gaining a better understanding about the reversibility of PAH in CHD patients before surgery. As of now little is known about that.
To explore the pathogenetic mechanism of irreversible CHD-PAH, proteomics comparison of the lung tissues in both reversible and irreversible CHD-PAH patients as well as in normal were performed. Proteomic analysis showed that transgelin was significantly up-regulated (2.5-fold) in irreversible CHD-PAH group compared to the reversible and control group. 8 Immunohistochemical staining and Western blot also confirmed the findings, and the expression of transgelin was significantly positively related with pathological grading of pulmonary arterioles. It indicated that transgelin may be an important potential target for the development of irreversible pulmonary vascular lesion in CHD patients.
Transgelin, a 22-kD protein of the calponin family which is also known as SM22α,WS3-10 or p27, is exclusively and abundantly expressed in the cytoskeleton of visceral and vascular smooth muscle cells of adult animals. It is also the earliest markers of smooth muscle differentiation. Studies have elucidated the regulating functions of transgelin, including actin cytoskeleton rearrangement, phenotypic modulation of vascular smooth muscle cells (SMC), SMC proliferation, cell migration and tumour suppression. [9][10][11][12] It is unknown how transgelin influence the PAH development in CHD. In immunohistochemical staining of the lung tissue, transgelin was obviously expressed in pulmonary arterial smooth muscle cells (PASMC) of the remodelled and thickened pulmonary arterioles, especially in irreversible CHD-PAH group. We speculated that transgelin may influence the PASMC function and then promote the pulmonary vascular remodelling and irreversible CHD-PAH occurrence.
Based on the research findings on lung tissue samples, further cellular experiment was carried out to illuminate the role of transgelin in regulating the function of PASMC. We demonstrated that knockdown and overexpression of transgelin can affect the PASMC phenotype, proliferation, apoptosis and migration.

| Patient enrolment and lung biopsy
According to multidisciplinary and translational approaches guideline, 13 14 CHD-PAH patients diagnosed by right heart catheterization (RHC) who met the inclusion criteria and would undergo complete repair surgery were prospectively enrolled. Lung biopsy was performed during the repair surgery with patients' informed consent. The control normal lung tissues were from patients who would undergo surgery for bronchial carcinoma (n = 6). The tissues were sufficiently distal from the tumour to ensure the normal phenotype of tissue cells. After a year's follow-up, mean pulmonary arterial pressure (mPAP) were evaluated by RHC to determine the diagnosis of reversible (mPAP < 25 mm Hg, n = 10) and irreversible (mPAP ≥ 25 mm Hg, n = 4) PAH. Patients did not receive any drug treatment for PAH during the overall process. 8 This study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of Fuwai Hospital.

| Differential proteomic analysis of the lung tissue in reversible, irreversible CHD-PAH and control group
Protein supernatants of the lung tissue specimens (four from reversible group, four from irreversible group and six from control group) were extracted and reserved. iTRAQ reagent kit (Applied Biosystems,USA) were used for protein precipitation, digestion and iTRAQlabelling. The peptide mixture was separated with HPLC system (RIGOL 3220) and analysed by TripleTOFTM 5600 LC/MS/MS (Applied Biosystems Sciex). Protein identification and quantification for mass spectrometry was performed with the ProteinPilot software Beta (version 4.2, Applied Biosystems) in the Human UniProtKB/ Swiss-Prot database. The data was exported with PDST software system.

| Qualitative and quantitative analysis of the expression of transgelin in the lung tissue of each group
In MS analysis, transgelin expression was significantly increased in the lung tissue of CHD-PAH patients, and its expression level in irreversible group was 2.5-fold than that of the reversible group.
To confirm the proteomic findings of transgelin, immunohistochemical staining and Western blot were performed for qualitative and quantitative analysis of the expression of transgelin in the lung tissue of CHD-PAH patients and normal lung tissue.

| Immunohistochemical staining
Neutral formalin-fixed lung tissues were made into 4-5 μm sections after dehydration and paraffin embedding. After heating for antigen retrieval, 3% H 2 O 2 was used to block endogenous peroxidase.
The sections were incubated with transgelin (SM22α) antibody (Abcam corporation) at 4°C overnight. After incubation of second antibody for 30 minutes at room temperature and DAB colouration, the slides were covered with resin, and then observed under microscope.

| Western blot
The tissue of each group was digested with RIPA lysis buffer. Prepared protein specimens of each group were loaded to 4%-12% PAGE electrophoresis precast gels (Invitrogen). IBlot ® transfer stacks that contained the required buffers and transfer membrane (nitrocellulose) were used for transfer with iBlot ® 2 dry blotting device (Invitrogen). After blocking by TBST with 5% non-fat dry milk at room temperature for 2 hours, nitrocellulose membrane was incubated with antibody of transgelin at 4°C overnight. The NC membrane was washed with TBST three times followed by incubation with horseradish peroxidase-conjugated anti-rabit IgG for 2 hours. After a second wash with TBST, immunoblots were detected using a chemiluminescence kit (Thermo Scientific Piece, Waltham, MA, USA), and the radiographs were quantitated via densitometry (Science Imaging System, BioRad, Hercules, CA, USA).

| Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA from the cultured cells of each group was extracted using TRIZOL (Invitrogen, USA). Quantification was performed with a twostep reaction process: reverse transcription (RT) and PCR. The RNA was reversely transcribed to cDNA using the PrimeScript ™ RT Master Mix kit (TaKaRa, Japan). PCR was performed on the 7500 real-

| Cell proliferation assay
Cell count and EdU staining assay were used to evaluate the cell proliferation after transfection. The cells were washed with PBS gentlely for two times to clear the residual medium. After detaching by 0.25% trypsin-EDTA, the cells were resuspended in 1 ml DMEM.
Number of the cells in each group was manually counted using a haemocytometer. Cell proliferation after treatment was also observed under microscope. Proliferation was presented as % proliferative cells. The assay was repeated for at least three times to achieve statistical analysis.

| Cell apoptosis assay
Cell apoptosis was determined by terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and Annexin-V flow cytometry.

| TUNEL assay
Cells were cultured on the coverslips. Apoptosis of the treated cells were detected using one-step TUNEL cell apoptosis detection kit (red fluorescent protein labelling with TRITC) (KeyGEN BioTECH, China). The cells were fixed with 4% paraformaldehyde for 30 minutes, rinsed with PBS and permeabilized with 1% Triton X-100 for 20 minutes. The cells then were incubated with TdT reaction mixture for 1 hour at 37°C, protected from the light. After washing with PBS, the cells were treated with streptavidin-TRITC diluted with labelling buffer for 30 minutes at 37°C, protected from the light. Cell nuclei were stained with DAPI solution for 10 minutes at room temperature. The coverslips were then washed and sealed by neutral balsam. Images were acquired using a fluorescence microscope (Leica Microsystem, Germany). The TUNEL-positive (apoptotic) cells were characterized with pink nuclei. Apoptosis was presented as normalize ratio of the percentage of the positive cells in experimental group and control group. The assay was repeated for at least three times to achieve statistical analysis.

| Annexin-V flow cytometry
The cell apoptosis was detected using Annexin V/APC-7ADD apoptosis detection kit (KeyGEN BioTECH, China) according to the manufacturer's instructions in the meanwhile. Treated cells were harvested and washed twice with ice-cold phosphate buffer solution.
Then the cells were resuspended in 500 ul binding buffer, incubated with 5 ul Annexin V-APC and 5 ul 7-amino-actinomucin D (7-ADD) for 15 minutes. All the samples were immediately analysed using a flow cytometer (Accuri C6, BD Biosciences, USA). The assay was repeated for at least three times to achieve statistical analysis.

| Transgelin influences proliferation of hPASMC
Cell count analysis presented that the number of living cells was much less in LV-siTAGLN group while significantly more in

| Transgelin influence apoptosis of hPASMC
The results of TUNEL assay ( Figure 7) and Annexin-V flow cytometry

| Transgelin influenced migration of hPASMC
Transwell migration assays showed a distinct effect of transgelin on the migration of hPASMC, as it shown in Figure 9.

| Transgelin and CHD-PAH development
Studies indicated that the severity of pulmonary vasculature remodelling adversely influenced the natural history and operative mortality in CHD-PAH patients. [14][15][16] But the mechanism of pulmonary remodelling is poorly understood in irreversible CHD-PAH.
Transgelin as an important cytoskeletal protein has been demon-

| Transgelin and PASMC proliferation, migration, apoptosis and phenotype
In studies, transgelin was demonstrated with a divergent function on cell proliferation, migration and apoptosis under different conditions.
Daniel's study demonstrated that transgelin was up-regulated in repopulating mesangial cells and promoted their migratory and proliferative nature after injury. 18 It was reported in another study that increased alveolar epithelial type II cell expression of transgelin contributed to cell migration in lung fibrosis through TGF-β/Smad3 pathway. 19 In contrast, study also found that overexpression of transgelin inhibited VSMC proliferation and neointima formation via blockade of the Ras-extracellular signal-regulated kinase (ERK) 1/2 pathway. 20 Transgelin even presented different regulating effects of migration on VSMC under different cell phenotype. 21 Transgelin which was considered as a tumour suppressor was shown to suppress proliferation of HepG2 cells, while the transgelin -overexpressing cells became resistant to apoptotic cell death caused by cytotoxic agents. 22 In the present study, transgelin overexpression activated PASMC proliferation and migration distinctly, and meanwhile the PASMC was likely anti-apoptotic as the anti-apoptotic protein (bcl-2) was sig- in HPH. [26][27][28] Meanwhile, transgelin was suggested to maintain the contractile phenotype of SMCs and inhibits the phenotypic modulation of SMCs from contractile to synthetic/proliferative cells during atherosclerosis. 29 However, the molecular mechanism of phenotypic transition presented unknown, the reorganization of the actin cytoskeleton seemed to be involved in that process. [30][31][32] In this in vitro experiment, transgelin seemed to regulate the Previous studies have demonstrated various regulating functions of STAT3 signalling in PAH development. 33,34 STAT3 activation was found in both human and experimental models of PAH. 35 Haemoodynamic forces which were induced by pressure overload, shear stress and cyclic strain were also upstream activators of STAT3. 34  and interfered with the contractile phenotype of VSMC, 40 and RAGE activation-induced apoptosis in VSMC. 41 We may also consider RAGE as an important upstream regulating factor of transgelin in CHD-PAH although the increased RAGE repression did not reach a significant level in our previous proteomics analysis.
However, the expression of transgelin in our study of CHD-PAH was significantly up-regulated, and the increased transgelin promoted PASMC proliferation and resistance to apoptosis. The regulatory mechanism of RAGE and transgelin seemed to be complicated and we suspect that some other signals may also be involved in.
Proteomics analysis of our previous work also showed obvious activation of adhesion-integrin-mediated cell adhesion and migration. FAK, which is activated by growth factor receptors and integrin clustering is required for cell adhesion, migration and other aspects of the remodelling process. FAK activation was closely related with VSMC proliferation, migration and phenotypes. 42,43 FRNK, a specific FAK inhibitor, was selectively expressed in large arterioles when SMC are transitioning from a synthetic to contractile phenotype, and inhibited FAK-dependent SMC proliferation and migration. FAK activation significantly reduced SM22 (transgelin) and α-SMA expression, 42 leading to cell proliferation and modulation from a contractile phenotype to a synthetic phenotype. 44 Studies also showed that FAK activation requires signalling by both the growth factor receptors and the fibronectin-binding integrin. 44 TGF-β could induce FRNK expression and then restrained FAK activation, which finally stimulated SM marker gene expression. 42 These results may remind us that FAK is an upstream regulating factor of transgelin, and the abnormal expres-

| LIMITATION
We also have some limitations in this study. First, we lack the animal experimental research to test and verify the role of transgelin in the development of CHD-PAH and irreversible pulmonary vasculopathy.
Second, we failed to explore the upstream mechanism of the increased expression of transgelin in the pulmonary arterioles of CHD-PAH patients under the present research conditions on lung tissue and in vitro cell lines. Our future research work will explore these discrepancies.

| CONCLUSION S
We conclude that transgelin is a potential important target in the

CONFLI CT OF INTEREST
The authors declare no relevant financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.