TEPP-46

Pyruvate kinase M2 activation protects against the proliferation and migration of pulmonary artery smooth muscle cells

Aikai Zhang • Fenfang Yu • Wande Yu • Peng Ye • Pengfei Liu • Yue Gu • Shaoliang Chen • Hang Zhang
1 Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210029, China
2 Department of Respiratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210029, China

Abstract
Pyruvate kinase M2 (PKM2), which is encoded by PKM, is a ubiquitously expressed intracellular protein and is associated with proliferation cell phenotype. In PAH patients and PAH models, we found higher levels of PKM2 tyrosine 105 phosphorylation (phospho-PKM2 (Y105)) than in controls, both in vivo and in vitro. Here, we demonstrate that PKM2 stimulates inflammatory and apoptosis signalling pathways in pulmonary artery smooth muscle cells (PASMCs) and promotes PASMC migration and proliferation. PKM2 phosphorylation promoted the dimerization activation and nuclear translocation of STAT3, a transcription factor regulating proliferation, growth, and apoptosis. TLR2, a transmembrane protein receptor involved in both innate and adaptive immune responses, promoted PKM2 phosphorylation in hypoxia-induced PASMCs. Therefore, we hypothesized that PKM2 also affects the proliferation and migration of PASMCs. The proliferation of hypoxia-induced normal human pulmonary artery smooth muscle cells (normal-HPASMCs) was found to be inhibited by TEPP-46 (PKM2 agonist) and PKM2 siRNA using wound healing, 5-ethynyl-2′-deoxyuridine (EdU), and immunofluorescence (Ki67) assays. PASMCs isolated from PAH patients (PAH-HPASMCs) and hypoxia-treated rats (PAH-RPASMCs) also confirmed the above results. TEPP-46 treatment was found to improve hypoxia-induced pulmonary artery remodelling and right heart function in mice, and the link between PKM2 and STAT3 was also confirmed in vivo. In conclusion, PKM2 plays crucial roles in the proliferation and migration of PASMCs.

Introduction
Pulmonary artery hypertension refers to a type of disease in which the mean pulmonary artery pressure (mPAP) is ≥ 25 mmHg measured by the right heart catheter (RHC) (Hurdman et al. 2012). Pulmonary hypertension is character- ized by pulmonary vascular remodelling and vessel muscularization and has a high incidence worldwide. In many studies, hypoxia has been shown to confer proliferation ad- vantages and promote PASMC growth. Pulmonaryhypertension due to hypoxia (WHO group 3 PAH) affects millions of patients worldwide and is the second most com- mon disease in all PAH classifications (Hu et al. 2019). Although there are many studies on PAH, there is still no effective treatment method so far, and its pathogenesis has not been clearly explained.
Pyruvate kinase (PK) is a key enzyme in the glycolytic pathway that catalyses the production of pyruvate by phos- phoenolpyruvate (PEP), which determines glycolytic activity (Luo et al. 2011). Pyruvate kinase M2 isoform (PKM2) arises from alternative splicing of PKM pre-mRNA by including exon 10 (PKM2) and excluding exon 9 (PKM1), which ismediated by polypyrimidine tract-binding protein (PTBP).
Preferential PKM2 isoform expression results from the tran- scription of hnRNPI, hnRNPA1, and hnRNPA2 (heteroge- neous nuclear ribonucleoproteins) activated by the oncoprotein c-Myc. PKM1 is often referred to as the adult- specific isoform and is the major kinase that catalyses the above reactions, promoting oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, but its activity cannot be regulated. In contrast, the kinase activity of PKM2 is relatively low, contributing to the Warburg effect by promoting aerobic glycolysis, and the enzyme activity can be regulated by vari- ous factors, such as fructose 1,6-biophophate (FBP), Y105 phosphorylation, K433 acetylation, and C358 oxidation. PKM2 is present in either a low-activity dimeric or high- activity tetrameric form, whereas PKM1 only exists as a high-activity tetrameric form. The low-activity dimeric form of PKM2 is preferentially expressed in highly proliferating cells, including cancer cells and embryonic stem cells, where- as the high-activity tetrameric form is present in normal pro- liferating cells (Dayton et al. 2016; Singh et al. 2017; Chen et al. 2019). The active form of PKM2 is a homotetramer, which can be dissociated by phosphorylation to form a dimer with a greatly reduced activity. The previous studies in rat PASMCs have shown that elevated levels of ROS induced by hypoxia mediated increased phosphorylation of PKM2, which caused the shift of glucose metabolism from the tricar- boxylic acid cycle to the pentose phosphate pathway (PPP). This would confer PASMCs proliferative activity and pro- mote the progression of pulmonary artery hypertension (Guo et al. 2016).The increase in the PKM2/PKM1 ratio caused by increased PKM2 expression in pulmonary artery fibroblasts and endothelial cells was considered to be the cause of meta- bolic reprogramming with a shift from mitochondrial oxida- tive phosphorylation towards glycolysis (Warburg effect) (Caruso et al. 2017; Zhang et al. 2017). The dimer can be activated by the highly specific small-molecule activator TEPP-46 (Zhang et al. 2017). Recent cancer studies have shown that in addition to the activity of pyruvate kinase, PKM2 also has non-glycolytic functions and protein kinase activity, which may play an important role in the occurrence and development of tumours (Cheng et al. 2018). The charac- teristics of smooth muscle cells from pulmonary hypertension patients are similar to tumour cells in some ways; however, it is still unclear whether and how PKM2 specifically affects cell proliferation and migration.
Signal transducer and activator of transcription 3 (STAT3) belongs to a family of transcription factors involved in immu- nity, inflammation, cell proliferation, and antiapoptosis. STAT3 can be phosphorylated on Y705, which can lead to its homodimerization, nuclear translocation, binding to DNA, and promotion of the expression of target genes (Soutto et al. 2019). Toll-like receptor 2 (TLR2) belongs to the Toll-like receptor (TLR) family (type I transmembrane protein recep- tors), which is closely related to the innate and adaptive im- mune response and inflammation (Cen et al. 2019).
In our experiments, we investigated the relationship be- tween phosphorylation (Y105) of PKM2 and hypoxia- induced PASMC proliferation and explored possible underly- ing mechanisms. This may help to understand the pathogene- sis of PAH and perhaps discover a novel target for the treat- ment of PAH. PASMC proliferation could be decreased by reducing PKM2 tyrosine 105 phosphorylation with TEPP-46 (PKM2 agonist) or knocking down PKM2 with siRNA in vitro. Our experiments demonstrate that PKM2 affects the proliferation of PASMCs and plays an important role in the development of PAH.

Materials and methods
Reagents
Antibodies against PKM2, phospho-PKM2 (Y105), Toll-like receptor 2 (TLR2), and β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against Ki67 and smooth muscle α-actin were purchased from Abcam (Cambridge, UK). Antibodies against STAT3 and phospho-STAT3 (Y705) were purchased from BioWorld Technology, Inc. (Tulare County, CA). Antibodies against histone H3 were obtained from Servicebio (Wuhan, China). Secondary antibodies were purchased from BioWorld Technology, Inc. (Tulare County, CA) and applied at a 1:10,000 dilution. SU5416, the vascular endothelial growth factor receptor (VEGFR) inhibitor, was purchased from Sigma (St. Louis, MO).
SU5416 were purchased from Sigma (St. Louis, MO). The PKM2 inhibitor TEPP-46 and the TLR2 inhibitor 5H- benzocycloheptene-8-carboxylic acid, 3,4,6-trihydroxy-2- methoxy-5-oxo-, and hexyl ester (Cu-CPT22) were purchased from MedChemExpress.

Animals and treatment
All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (Nanjing, China). Male wild-type C57/BL6 mice at 8 weeks of age (average weight 25 g) and male Sprague Dawley (SD) rats weighing 200–250 g at 6 weeks of age were purchased from the Laboratory Animal Center of Nanjing Medical University. SU-PAH mice subjected to SU5416/hypoxia were injected subcutaneous- ly with 20 mg/kg SU5416 (vascular endothelial growth factor receptor 2 inhibitor) once a week and followed by exposure to chronic hypoxia (10% O2) in a ventilated chamber. TEPP-46 (30 mg/kg/days, 3 weeks) or vehicle was administered to mice as gavage (Li et al. 2020). After 3 weeks, the mice were sacrificed, and lung tissues were removed and embedded in paraffin for H&E staining to evaluate vascular remodelling. The rats (n = 8) were injected subcutaneously with SU5416 (20 mg/kg) follow- ed by 3-week chronic normobaric hypoxia (10% O2) as the hypoxia group, and the remaining 6 rats were housed in an atmospheric environment as the control group (Yung et al. 2016).

PASMC isolation and culture
Rat pulmonary artery smooth muscle cells (RPASMCs) were obtained from peripheral small pulmonary arteries of Sprague Dawley (SD) rats (200–250 g) as previously described (Li et al. 2009). We isolated and cultured PASMCs from periph- eral small pulmonary arteries (< 1000-μm diameter) of healthy donors and PAH patient lung tissue samples as previ- ously described. Normal-HPASMCs and PAH-HPASMCs were cultured at 37 °C in 5% CO2 and 95% air in smooth muscle cell medium (SMCM, ScienCell) containing 2.5% FBS, 1% PS, and 1% SMCGS. RPASMCs were cultured in Dulbecco’s modified essential medium (DMEM; Gibco BRL, Rockville, MD) containing 4.5 mmol/L of D-glucose supple- mented with 20% foetal bovine serum (FBS, Gibco BRL) (Li et al. 2009). We identified the purity of the extracted PASMCs by smooth muscle α-actin immunofluorescence. In all exper- iments, PASMCs were starved in serum-free DMEM for 24 h before treatment to rule out the effects of FBS (Pozeg et al. 2003). siRNA transfection The PKM2, TLR2, and negative control small interfering RNAs (siRNAs) were obtained from GenePharma (Shanghai, China) and transfected into PASMCs using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA) ac- cording to the manufacturer’s protocol. The primer sequences were as follows: H u m a n P K M 2 s i R N A s , 5 ′ - CCAUAAUCGUCCUCACCAATT-3′ (sense) 5 ′ - UUGGUGAGGACGAUUAUGGTT-3′ (sense)Human TLR2 siRNAs, 5′-GCCCUCUCUACAAACUUUATT-3′ (sense)5′-UAAAGUUUGUAGAGAGGGCT experimental method was as described in the kit protocol. The appropriate number of cells was cultured in a 6-well plate (if necessary, a coverslip was added). After the cells were cul- tured overnight and returned to a normal state, the desired drug treatment or other stimulation treatment was performed, and a 2X EdU working solution (20 μM) preheated to 37 °C was added to the 6-well plate in an equal volume to make the final concentration of EdU 1X. The cells were incubated for 2 h. After the cells were labelled with EdU, the culture medi- um was removed, and 1 ml of 4% paraformaldehyde was added for 15 min at room temperature. Cells were perme- abilized with 0.3% Triton X-100 for 10–15 min at room tem- perature. Click reaction was added and incubated for 30 min at room temperature in the dark. Hoechst 33342 was used for nuclear staining, and PASMCs were incubated at room tem- perature for 10 min in the dark (Salic and Mitchison 2008). Immunofluorescence PASMCs were washed three times with PBS and then fixed in 4% paraformaldehyde for 20 min at room temperature after treatment with TEPP-46 or siRNA-PKM2. After being perme- abilized with 0.2% Triton X-100 for 20 min and washed three times with PBS, PASMCs were exposed to 3% BSA blocking buffer for 1 h. Then, the cells were incubated with the rabbit monoclonal anti-Stat3 antibody (dilution 1:100, BioWorld BS9907M) overnight at 4 °C. After washing with PBS, the cells were incubated with goat anti-rabbit IgG Alexa Fluor® 488 (dilution 1:200; CST 4408). Then, the cells were incubat- ed with DAPI according to the manufacturer’s instructions (dilution 1:50; Beyotime). Images were acquired and proc- essed by laser scanning confocal microscopy (LSM 710; Carl Zeiss, Germany). Ki67 and smooth muscle α-actin fluo- rescence staining were performed in the same way. Cardiac function evaluation by echocardiography T-3′ (antisense) R a t P K M 2 s i R N A s , 5 ′ - CAUCUACCACUUGCAAUUATT-3′ (sense) 5 ′ - UAAUUGCAAGUGGUAGAUGTT- 3′ (antisense) Wound-healing assay The cells were scratched in a Petri dish in the presence of serum-free DMEM to exclude the effects of serum, and cell migration was observed 24 h later. This experimental method has been described in detail previously (Chignalia et al. 2012). EdU staining We used EdU staining to detect cell proliferation, and the BeyoClick EdU-488 kit was purchased from Beyotime. The cardiac function of mice was assessed using the Vevo2100 system (Fujifilm VisualSonics, Inc., Toronto, ON, Canada) with a (30 MHz) MS-400 transducer. The pul- monary artery acceleration time (PAAT), pulmonary ejection time (PET), and peak E/A ratio (E/A) were measured by the value of left ventricle ejection fraction (LVEF) and fraction shortening (FS). The LVEF percentage was calculated as ([LV vol, d-LV vol, s]/LV vol, d) × 100, and the FS percentage was calculated as ([LVID, d-LVID, s]/LVID, d) × 100. Human tissue sample collection The present study was first approved by the Ethics Committees of the Nanjing Medical University for experi-ments involving human tissues, and informed consent was obtained from each individual before collecting tissues. Human lung tissue samples were obtained from 8 PAH pa- tients (mean pulmonary artery pressure 78 ± 12 mmHg) re- ceiving lung transplantation at the Affiliated Wuxi People’s Hospital of Nanjing Medical University (Wuxi, China). Eight- matched control samples were collected from 4 donor subjects (Wuxi, China) and 4 individuals without PAH undergoing lung resection for tumour nodules at the Thoracic Surgery Department, Nanjing First Hospital (Nanjing, China). PAH was previously diagnosed by right heart catheterization ac- cording to current guidelines. Immunoblotting Samples from human (donor and PAH patients) and mouse lungs (control, SU-PAH) were cut into pieces and then ho- mogenized in cold phosphate-buffered saline (PBS). After centrifugation and removal of PBS, tissue homogenates and cells were lysed in RIPA buffer incubated on ice for 30 min, with proteinase inhibitor cocktail, and phosphatase inhibitors were added before use and centrifuged at 12,000 rpm at 4 °C for 20 min. The protein concentration in the supernatant was detected by spectrophotometry (BCA assay; Pierce, Rockford, IL) with bovine serum albumin as the standard. Total protein was electrophoresed in 10% sodium dodecyl sulphate- polyacrylamide(SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes. The target protein was incubated with primary antibody overnight, and the sec- ondary antibody was used for 2 h at room temperature. Fractionation of cytoplasmic and nuclear proteins Nuclear protein extraction was performed using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s protocol. Statistical analysis All experimental data are shown as the mean ± standard error of the mean (SEM). The statistical significance of these data was evaluated by using Student’s t test to compare two groups or one-way analysis of variance for multiple group analysis. All statistical analyses were calculated using GraphPad Prism 5.0 software (La Jolla, CA). A P value less than 0.05 was considered statistically significant. Results PKM2 phosphorylation (Y105) level is upregulated in PAH To understand the relationship between PKM2 and PAH, we compared the PKM2 phosphorylation (Y105) level in lung tissues between patients with PAH and healthy donors with- out PAH. In the lung tissue of patients with PAH, the phos- phorylation (Y105) of PKM2 was elevated, while the total amount of PKM2 was unchanged (Fig. 1a, b). We adminis- tered 10% hypoxia and SU5416 treatment to rats and then extracted PASMCsfromtherats. Wefoundthatthephosphor- ylation level was also increased under hypoxia, and the total amount of PKM2 was unchanged, which was consistent with the results on patient lung tissues (Fig. 1c, d). This suggests that hypoxia may be the cause of PKM2 phosphorylation and that PKM2 phosphorylation was also accompanied by PAH. Therefore, we exposed normal-HPASMCs to 3% hypoxic treatment in a time gradient and found that PKM2 phosphor- ylation levels showed a time-dependent increase, and the highest level was observed at 48 h; however, total PKM2 was unchanged (Fig. 1e, f). Thus, the following cell experi- ments were performed with 3% hypoxia treatment for 48 h. PKM2 phosphorylation (Y105) is elevated in PAH mice To further explore the relationship between PKM2 phos- phorylation and PAH, we constructed an SU/hypoxia mouse model. Echocardiography showed that the pulmonary artery acceleration time (PAAT)/pulmonary artery ejection time (PET) was significantly reduced in the SU/hypoxia group, suggesting that the model successfully caused an increase in pulmonary vascular resistance (PVR)(Fig. 2a). H&E stain- ing of mouse lungs showed a significant increase in the vas- cu la r m uscu la riza tion perce n tage (Fig . 2b ). Immunofluorescence of αSMA in the lung tissue of the mice demonstrated the thickening of pulmonary artery smooth muscle (Fig. 2c, d). The right ventricular hypertrophy index (RVHI) was obtained by dividing RV weight by LV plus septum weight (RV/[LV + S]). The RV/[LV + S] of the SU/ hypoxia group was significantly higher than that of the con- trol group, indicating that the pulmonary vascular remodel- ling of the SU/hypoxia group had successfully caused right heart hypertrophy and remodelling, which further verified the success of the PAH model (Fig. 2e). Then, the mice were anaesthetized, and their lungs were taken and used for west- ern blotting. We found that the phosphorylation level of PKM2 in the lung tissue of the SU/hypoxia group was sig- nificantly higher than that of the control group, and total PKM2 did not change, which is consistent with the above experimental results (Fig. 2f, g). These results indicate that hypoxia is an important inducer of PKM2 phosphorylation (Y105) in PASMCs in vitro and in vivo. PKM2 phosphorylation promoted the activation of STAT3 To further elucidate the role of PKM2, we transfected normal- HPASMCs with PKM2 siRNAs. The results showed that the hypoxia-induced p-STAT3 upregulation was dramatically at- tenuated by PKM2 siRNA transfection (Fig. 3a–c). To verify the above results, we then treated the PASMCs from hypoxia intervention with TEPP-46 and found that the same results were obtained (Fig. 3d–f). Next, we treated PAH-HPASMCs with si-PKM2 and TEPP-46 to reduce PKM2 phosphorylation and found that they could reverse the increase in STAT3 phosphorylation, which further confirmed the link between PKM2 phosphorylation and STAT3 phosphorylation (Fig. 3g, h). Hypoxia-treated normal-HPASMCs had more STAT3 in the nucleus than the control group, whereas TEPP-46-treatednormal-HPASMCs combined with hypoxia treatment resulted in a decrease in STAT3 in the nucleus com- pared with hypoxia treatment alone ( Fig. 3i, j). Immunofluorescence of STAT3 showed that hypoxia caused nuclear translocation and activation of STAT3, and TEPP-46 treatment reversed the hypoxia-induced STAT3 activation (Fig. 4a–c). This suggests that PKM2 may affect cell function by causing STAT3 phosphorylation/activation, dimerization, and nuclear translocation. TLR2 could promote phospho-PKM2 (Y105) To determine whether human TLR2 promoted the levels of phospho-PKM2 (Y105) in PASMCs, we knocked down the intracellular TLR2 gene with TLR2-specific siRNAs. TLR2- specific siRNAs reduced the expression of TLR2 after 48 h, as was determined with immunoblotting. We found that the phosphorylation levels of PKM2 decreased when PASMCs were treated with si-TLR2. This indicates that the changes of the expression of TLR2 affect the levels of phospho- PKM2 (Y105) (Fig. 4d–f). We also used the TLR2-specific agonist CU-CPT22 to block TLR2 activity. For this, PASMCs were treated with 8 μmol/l CU-CPT22. CU-CPT22 treatment is a potent protein complex of Toll-like receptor 1 and 2 (TLR1/2) inhibitor and competes with the synthetic triacylated lipoprotein binding to TLR1/2. Similar results were observed after CU-CPT22 treatment, indicating that TLR2 promoted phospho-PKM2 (Y105) without the effects on the total ex- pression of PKM2 in HPASMCs (Fig. 4g, h). PKM2 plays an important role in the proliferation and migration of PASMCs Smooth muscle cell proliferation is critical in PAH vascular remodelling. Therefore, we suspected that PKM2 affects PAH by altering PASMC proliferation. We treated normal- HPASMCs with PKM2-specific small interfering RNA (si- PKM2) or a PKM2-specific agonist (TEPP-46, 100 μmol/l), which reduced PKM2 phosphorylation, and found that they inhibited hypoxia-induced PASMC proliferation and migra- tion. It was also shown that hypoxia could increase the pro- portion of Ki67-positive HPASMCs, while si-PKM2 and TEPP-46 could decrease the proportion of Ki67-positive HPASMCs, indicating that hypoxia-induced normal- HPASMC proliferative activity can be reduced by decreasing PKM2 phosphorylation levels (Fig. 5a–d). The PASMCs from PAH patients treated with si-PKM2 or TEPP-46 showed reduced proliferation and migration capacity, as demonstrated by the wound-healing assay and Ki67 immunofluorescence (Fig. 5e–i). We isolated hypoxia-treated rat pulmonary artery smooth muscle cells (RPASMCs), and infected the cells with si-PKM2 or treated them with TEPP-46. We found that si- PKM2 or TEPP-46 treatment reduced the proliferation and migration of RPASMCs, according to the wound-healing as- say (Fig. 5j,k). The EdU incorporation assay showed that hyp- oxia induced an increase in proliferating RPASMCs, whereas administration of si-PKM2 or TEPP-46 on a hypoxic back- ground reduced the number of proliferating cells (Fig. 5l, m, n). TEPP-46 attenuates PAH in vivo Since we have verified the roles of phospho-PKM2 (Y105) on hypoxia-induced proliferation of PASMCs in vitro, we then further investigated its effects in vivo. We randomly divided the mice into three groups and TEPP-46 (30 mg/kg/day, 3 weeks, administered as gavage), or vehicle was administered to mice exposed to hypoxia. H&E staining of mouse lungs showed a significant difference of medial wall thickness per- centage between the SU-PAH group and TEPP-46 group (Fig. 6a, b). In this study, the right ventricular hypertrophy index (RVHI), calculated by RV/(left ventricle + septal),decreased significantly when the SU-PAH group were treated with TEPP-46(Fig. 6c). TEPP-46 treatment could in- crease the PAAT/PET ratio of PAH mice, with a P value of 0.0713, indicating that TEPP-46 could improve the pulmo- nary arterial vascular remodelling (Fig. 6d). The right ventric- ular systolic pressure (RVSP) of the SU-PAH group was higher than that of the control group, while TEPP-46 signifi- cantly reduced the RVSP of the PAH model (Fig. 6e, f). According to western blot, we found that phospho-PKM2 (Y105) in lung tissue of the SU-PAH group was increased, and TEPP-46 could significantly reduce the phosphorylation rate of PKM2, thereby exerting the function of a PKM2- specific agonist (Fig. 6g–i). This treatment further reduced phospho-STAT3 (Y705), which further validated the link be- tween PKM2 and STAT3. Discussion Previous studies have shown that small pulmonary vessel re- modelling is an important pathogenesis of PAH, and the pro- liferation and migration of pulmonary artery smooth muscle cells are important factors in vascular remodelling. In PAH, small pulmonary vessel remodelling causes progressively in- creasing pulmonary vascular resistance (PVR) and leads to right heart failure and sudden death (Deng et al. 2015). Our study suggests that PKM2 phosphorylation is an important cause of PASMC proliferation and migration and may be a key target for the treatment of PAH. This argument is based on the following points. (a) In PAH patients and hypoxia-induced animal model lung tissue, PKM2 phosphorylation increased, while the total amount of PKM2 remained unchanged, and the same results were observed in hypoxia-induced cell models. (b) Hypoxia caused an increase in PASMC proliferation and migration, which could be reversed by PKM2-specific small interfering RNA (si-PKM2) or a PKM2-specific agonist (TEPP-46, 100 μmol/l). (c) PKM2 could act through the PKM2/STAT3 signalling pathway, and TLR2 may be a stable upstream molecule of PKM2. (d) PKM2 activation inhibited PAH progression and reduced STAT3 phosphorylation (Y105) in vivo. There is solid evidence showing that hypoxia could pro- mote remodelling of the pulmonary vasculature, which results in pulmonary vascular resistance and PAH. Hypoxia led to dysfunction of pulmonary artery endothelial cells and smooth muscle cells, decreased the secretion of vasodilatory, anti- proliferative mediators (nitric oxide (NO) and prostaglandin- I2), increased expression of vasoconstrictive pro-proliferative factors (endothelin (ET)-1, angiotensin II, thromboxane A2), and caused an imbalance in vasoactive mediators, pulmonary vasoconstriction, and pulmonary artery smooth muscle cell proliferation, resulting in pulmonary vascular remodelling. In SMCs, hypoxia could increase Ca2+ concentrations in the cytoplasm and lead to the activation of mitogen-activated pro- tein kinases (MAPKs), thus modulating SMC proliferation and growth. The link between p38 MAPK and hypoxia induc- ible factor HIF-1α, a key transcription factor in the biochem- ical response to hypoxia, has been confirmed in previous stud- ies. The link between PKM2 and HIF-1α has also been prov- en. Thus, the relationship between PKM2 and p38 MAPK and their effects on the progression of PAH are also of great im- portance for research (Pak et al. 2007; Gao and Raj 2011; Azoitei et al. 2016; Wang et al. 2018). In a previous study of fibroblasts and endothelial cells in PAH, the increase in the PKM2/PKM1 ratio caused by increased PKM2 expression was considered to be the cause of the Warburg effect, showing that under aerobic conditions, cells still used glycolysis as the main mode of energy rather than the tricarboxylic acid cycle, but the authors did not explore changes in PKM2 (Caruso et al. 2017; Zhang et al. 2017). In a previous PASMC study of PAH, the decrease in activity caused by phosphorylation of PKM2 was thought to induce the shift of glucose me- tabolism from the tricarboxylic acid cycle to the pentose phosphate pathway (PPP)(Guo et al. 2016). In a study of human glioblastoma multiforme (GBM) cells, the nonmetabolic functions of PKM2, whereby PKM2 transactivates β-catenin by increasing the levels of β- catenin phosphorylation, were considered to be an impor- tant cause of tumour proliferation and invasion. ERK1/2 is thought to phosphorylate PKM2 and convert it from a tetramer to a monomer which could induce it to translo- cate into the nucleus upon receptor tyrosine kinase acti- vation where it acts as a histone kinase, and PKM2-mediated upregulation of c-Myc and cyclin D1 expression promotes the Warburg effect and cell cycle progression, respectively (Yang et al. 2011; Yang et al. 2012; Yang and Lu 2013). The proliferation of PASMCs in PAH is considered to be similar to the proliferation of tumour cells, so we have reason to believe that PKM2 is associ- ated with the proliferation and migration of PASMCs in PAH. At present, there are few studies on the role of PKM2 in PAH, and the role of the non-metabolic func- tions of PKM2 in PAH is minimal. Therefore, we wanted to understand the effect of PKM2 protein kinase activity on PASMC proliferation and migration. Phospho-PKM2 (Y105) was elevated in human breast can- cer cells, while reduce of PKM2 expression or mutation of Y105 site could inhibit tumour progression (Zhou et al. 2018). In breast cancer cells, PKM2 could be methylated by co-activator-associated arginine methyltransferase 1 (CARM1), resulting in substantial metabolic reprogramming and promoting tumorigenesis (Liu et al. 2017). Small- molecule activators (TEPP-46 or DASA-58) could bind PKM2 at the subunit interaction interface to improve its olig- omerization and suppress tumorigenesis (Anastasiou et al. 2012). These results indicate that increased expression or de- creased activity of PKM2 promotes tumour progression. PKM2 can be phosphorylated by some non-receptor tyrosine kinases and receptor tyrosine kinases, such as Src, FGFR1, BCR-ABL, and JAK2. Phospho-PKM2 could cause PKM2 oligomerization, paving the way to metabolic reprogramming (Prakasam et al. 2018) . In cancer cells, dimeric PKM2 can phosphorylate tyro- sine residues in STAT3 using phosphoenolpyruvate (PEP) as a phosphate donor, suggesting that STAT3 is an im- portant regulator and transcription factor for PKM2 to exert its biological activity (Shirai et al. 2016). Reactive oxygen species (ROS) generated by NADPH oxidases (Nox) activate the JAK2/STAT3 pathway, after which STAT3 directly binds to the IL6 promoter and increases IL6 mRNA levels and the subsequent secretion of growth factors to promote the proliferation and survival of normal and starved HeLa cells in starvation-induced autophagy of cancer cells (Yoon et al. 2014). A549 human lung cancer cells exposed to hypoxia (1% O2) showed increased in- tracellular ROS concentrations, causing inhibition of PKM2 through the oxidation of Cys358, which promoted the metabolic changes required for proliferation and con- ferred an additional advantage to cancer cells to withstand oxidative stress (Anastasiou et al. 2011). Toll-like recep- tor (TLR1, TLR2, and TLR4) activation results in mito- chondria being recruited to macrophage phagosomes and augments mitochondrial ROS (mROS) production, which involves translocation of tumour necrosis factor receptor- associated factor 6 (TRAF6) to mitochondria, where it can collaborate with the protein ECSIT (evolutionarily con- served signalling intermediate in Toll pathways), which is involved in mitochondrial respiratory chain assembly. Increased mROS induced by TLRs is an important com- ponent of the antibacterial responses of macrophages, en- hancing their ability to kill intracellular bacteria and es- tablishing mitochondria as hubs for innate immune signal- ling (West et al. 2011). Therefore, we hypothesized that ROS may be an important mediator of hypoxia-induced proliferation of PASMCs, transmitting the signal from TLR2 to PKM2 and causing its nuclear translocation to synergize with transcription factors such as STAT3, en- hancing the expression of proliferation-related genes, and ultimately accelerating the progression of PAH, which is a point that could be studied in depth in the future. Our study showed that TEPP-46 treatment could reduce PVR and right ventricular remodelling, but it is a deficiency that we cannot determine whether the improvement of right ventricle remodelling was due to the decrease in PVR or the therapeutic effect of TEPP-46 on the right heart. Other studies have also shown that cardiac hypoxia itself is an independent factor that stimulates right heart remodelling (Bogaard et al. 2009). In the previous study, expression of PKM2 was elevat- ed in the cardiac hypertrophy hearts of rats undergoing TAC at the 6-week time point (Liu et al. 2016). The effects of PKM2 on cardiac remodelling is worthy of further study, including hypertrophy induced by stress, sympathetic nerve stimulation, and hypoxia. In conclusion, PKM2 phosphorylation is significantly ele- vated in PAH both in vitro and vivo. PKM2 affects the pro- liferation and migration of PASMCs, including normal- PASMCs and PAH-PASMCs. PKM2 could influence the di- merization activation and nuclear translocation of STAT3, and TLR2 may be responsible for the increase in PKM2 protein kinase activity. To the best of our knowledge, this is the first study to explore PKM2 protein kinase activity in pulmonary artery hypertension. 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