Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation

It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.


Introduction
Pulmonary arterial hypertension (PAH) is a cardiopulmonary dysfunctional disease, which is associated with a marked increase in pulmonary vascular resistance and pulmonary artery pressure [1,2]. All forms of PAH share a common pathogenesis mechanisms which include persistent pulmonary vasoconstriction, vascular remodeling, and thrombosis in situ [3]. Pulmonary vascular remodeling is the most prominent pathological feature of PAH, and the proliferation of pulmonary arterial smooth muscle cells (PASMCs) plays a major role in pulmonary vascular remodeling [4]. Therefore, elucidating the molecular mechanisms underlying the PASMCs proliferation and pursuing appropriate targets to inhibit vascular remodeling are critical in the treatment of PAH. Autophagy, a highly conserved self-degradative process, is preserved in eukaryotic cells and has essential roles in cellular homeostasis and energy balance [5][6][7]. Autophagy has been reported to regulate diverse biological activities, such as cell proliferation, differentiation, survival, and tissue remodeling [8]. Accumulating evidence suggests that dysregulation of autophagy is involved in PAH. However, the role of autophagy in the pathogenesis of PAH remains controversial. Some studies have suggested that autophagy contributes to the development of PAH, while others reported that autophagy protected against PAH [9,10]. In the current study, we explored the role and underlying mechanism of autophagy in PAH.
Forkhead box M1 (FOXM1), a member of forkhead box family of transcription factors, regulates a variety of cellular processes including cell proliferation, migration, apoptosis, and angiogenesis by directly binding to enhancers of target genes [11,12]. It has been demonstrated that FOXM1 promotes vascular remodeling in hypoxia-induced PAH mice models, and inhibition of FOXM1 suppresses the proliferation of PASMCs [13]. Meanwhile, a study has suggested that FOXM1 induces the phosphorylation of focal adhesion kinase (FAK) by binding to the promoter of integrin β1, which promotes the proliferation of triple-negative breast cancer cells [14]. Furthermore, FOXM1 has been reported to promote triple-negative breast cancer cells proliferation through induction of autophagy [15]. However, it is still unclear whether FOXM1 induces the activation of autophagy in PAH. Therefore, we examined whether FOXM1 promoted the development of MCT-induced PAH by increasing the phosphorylation of FAK and consequent activation of autophagy.

Animals
Ninety male Sprague-Dawley (SD) rats (200 to 250 g) were used in the current study. All animal care and experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Xi'an Jiaotong University Animal Experiment Center. All protocols used in this study were approved by the Laboratory Animal Care Committee of Xi'an Jiaotong University. The rats with similar baseline characteristics were randomly divided into six groups: control group (Con group, n = 15), monocrotaline (MCT)treated group (MCT group, n = 15), MCT and vehicle (dimethyl sulfoxide, DMSO)-treated group (MCT + vehicle group, n = 15), MCT and thiostrepton-treated group (MCT + thiostrepton group, n = 15), MCT and 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)-treated group (MCT + Y15 group, n = 15), and MCT and chloroquine phosphate (CQ)-treated group (MCT + CQ group, n = 15). All rats were housed in a 12 h light-dark cycle at 22 ± 2 °C and free accessed to food pellets and tap water.

Generation of PAH Models and Drug Treatment
MCT (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.1 mol/L HCl, the solution was then titrated to pH 7.4 with 0.1 mol/L NaOH with the final concentration of 30 mg/mL. Thiostrepton (Selleckchem, USA) was dissolved in DMSO, and then diluted with 0.9% NaCl to a final concentration of 10 mg/mL. DMSO was diluted with 0.9% NaCl to a final concentration of 0.5% as a vehicle for thiostrepton. Y15 (Selleckchem, USA) and CQ (Aladdin Bio-Chem Technology Co., Shanghai, China) were dissolved in 0.9% NaCl with the final concentration of 7.5 mg/mL and 20 mg/ mL. The PAH model was established by intraperitoneally (IP) injection of MCT (60 mg/kg) on day 1. Once PAH was established (2 weeks after MCT injection), FOXM1 inhibitor thiostrepton (20 mg/kg) or its vehicle was administrated to the rats by IP injection every day for 2 weeks. After the establishment of MCT-induced PAH rat model, FAK inhibitor Y15 (15 mg/kg) or CQ (40 mg/kg) was daily administered to the rats by IP. The healthy control rats were IP injected with the same volume of vehicle solution (DMSO or 0.9% NaCl).

Measurement of RVSP and RVHI
At the endpoint of the study, all survived rats were anesthetized by a spontaneous inhalation of isoflurane. After stable anesthesia, we isolated the right internal jugular vein of rats, and then inserted a polyethylene catheter into the right ventricle (RV). A Grass polygraph was used to detect the right ventricle systolic pressure (RVSP). Then hearts and lungs of rats were excised. The RV was dissected from the left ventricle (LV) and interventricular septum (S), and then each part was weighed. The ratio of the weight of RV to the LV plus S [RV/ (LV + S)] was calculated to assess the index of right ventricular hypertrophy (RVHI).

Histologic Analysis
Harvested pulmonary tissues from marginal right lower lobes were immersed in 4% paraformaldehyde, and then embedded in paraffin. Tissue blocks were cut in a thickness of 5 μm, and then stained with hematoxylin and eosin (HE). The medial wall thickness (%MT) of vessels (20-70 μm diameters, n = 15 per rat) was observed using a light microscope to assess pulmonary arterioles vascular remodeling. The %MT was calculated as follows: %MT = (2 × medial wall thickness) × 100/external diameter.

Western Blot Analysis
Harvested lung tissues were immersed in RIPA lysis buffer (HEART, Xi'an, China), and the protein concentrations were measured by BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Equal amounts of proteins were resolved and separated on 8%-15% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with nonfat dry milk (5%, w/v) for 1 h and then incubated overnight at 4 °C with the specific primary antibodies against β-actin (YM3028, Immunoway, TX, USA,  b Change of RVHI in different groups of rats (n = 10-15). c Hematoxylin and eosin staining of small pulmonary arteries in different groups (n = 10-15) (magnification × 400). d Quantitative analysis of the medial wall thickness of pulmonary arteries (n = 10-15). e The degree of muscularization of pulmonary arteries was evaluated by immunostaining of α-SMA. f Percentage of muscular, partially muscular and non-muscular arteries in different groups (25 arteries each slide, diameter 20-200 μm). g PASMCs were stained for Ki67 by immunohistochemistry to assess cell proliferation; cells with brownstained nuclei in the medial layer of pulmonary artery are Ki67-positive PASMCs. h The percentage of Ki67-positive PASMCs in different groups (25 arteries each slide, diameter 20-100 μm). *P < 0.05 versus control group; # P < 0.05 versus MCT group were soaked in horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (AP156P or AP127P, Sigma-Aldrich, St. Louis, MO, USA, 1:5000) for 1 h. Blots were visualized by the enhanced chemiluminescence detection system (Amersham Bioscience), and the band densities were measured using Quality One software (Bio-Rad).

Statistical Analysis
Experimental data are presented as means ± standard deviation. Group comparisons were analyzed using one-way analysis of variance followed by a Tukey post hoc test. P < 0.05 was considered statistically significant.

Autophagy is Activated in MCT-Induced PAH Model
To examine whether autophagy is increased in MCT-induced PAH rats, the protein level of LC3B was measured in lung lysates. In MCT-treated rats, the level of LC3B and LC3B-II/LC3B-I ratio was increased to 1.75-fold and 2.01-fold over control, respectively (P < 0.05 versus control; Fig. 1a, b). CQ, an autophagy inhibitor, disrupts autophagy by inhibiting the acidification of the lysosomes that fuse with the autophagosomes, thereby preventing the degradation of metabolic stress products and enhancing the accumulations of autophagy markers (LC3A, LC3B and Beclin 1) [17]. As shown in Fig. 1a and b, administration of CQ further increased LC3B and LC3B-II/LC3B-I ratio to 3.00-fold and 2.59-fold over control (P < 0.05 versus MCT-treated rats). Meanwhile, immunohistochemistry was used to detect the expression of LC3B. Figure 1c and d indicate that the percentage of LC3B-positive PASMCs was increased from (6.2 ± 1.4) % in control rats to (23.5 ± 3.1) % in MCTtreated PAH rats (P < 0.05 versus control). The presence of CQ further increased the ratio of LC3B-positive PASMCs to (31.5 ± 2.4) % in MCT and CQ co-treated rats (P < 0.05 versus MCT-treated rats, Fig. 1c, d). These results imply that autophagy is activated in MCT-induced PAH rats.

Inhibition of Autophagy Prevents the Increase of RVSP, RVHI, and Pulmonary Arterial Remodeling in MCT-Induced PAH Model
In MCT-treated PAH rats, RVSP was significantly increased to 43.63 ± 2.33 mmHg versus 22.36 ± 1.69 mmHg in control group (P < 0.05 versus control; Fig. 2a). This suggests that PAH was successfully induced in MCT-treated rats. While treatment of PAH rats with CQ reduced RVSP to 28.66 ± 1.71 mmHg (P < 0.05 versus MCT-treated rats; Fig. 2a). Similar changes were observed in RVHI. As shown in Fig. 2b, RVHI was markedly raised from 0.34 ± 0.02 in control rats to 0.59 ± 0.04 in MCT-treated PAH rats (P < 0.05 versus control). After administration of CQ in MCT-treated rats, the RVHI declined to 0.36 ± 0.02 (P < 0.05 versus MCT-treated rats; Fig. 2b). These results indicate that inhibition of autophagy effectively prevents the development of PAH.
As shown in Fig. 2c and d, medial wall thickness in small pulmonary arteries in MCT-treated rats [(32.46 ± 2.15) %] was effectively elevated compared with that in control rats [(21.67 ± 1.48) %] (P < 0.05 versus control). However, treatment of PAH rats with CQ decreased medial wall thickness to (23.31 ± 1.96) % (P < 0.05 versus MCT-treated rats; Fig. 2c, d). We further assessed the pulmonary arterial muscularization by α-SMA immunostaining. Figure 2e and f shows that the percentage of pulmonary arterial muscularization was significantly increased in MCT-treated PAH rats compared with control rats (P < 0.05 versus control). In addition, the percentage of Ki67-positive PASMCs was increased from (6.20 ± 1.0) % in control rats to (19.12 ± 2.2)  Fig. 2g, h). However, CQ prevented MCT-induced increase of the pulmonary arteries muscularization and proliferation of PASMCs. In MCT and CQ-treated rats, the ratio of Ki67-positive PASMCs was dropped to (12.23 ± 1.8) % (P < 0.05 versus MCT-treated rats; Fig. 2e-h). These results indicate that inhibition of autophagy suppresses the pulmonary arterial remodeling in MCT-induced PAH.
In this study, we examined the protein levels of FAK and p-FAK in lysates of rat lung tissue samples. Figure 3 shows that p-FAK protein level was increased to 3.24-fold over control in MCT-treated rats (P < 0.05 versus control). After administration of FAK inhibitor, Y15 decreased the level of p-FAK to 1.33-fold over control (P < 0.05 versus MCTtreated rats; Fig. 3). These results indicate that FAK activity is increased in MCT-induced PAH.

Inhibition of FAK Prevents MCT-Induced PAH by Suppression of Autophagy
As depicted in Fig. 4a and b, the increased RVSP and RVHI in MCT-treated rats were significantly decreased from 43.63 ± 2.33 mmHg and 0.59 ± 0.04 to 26.14 ± 1.56 mmHg and 0.35 ± 0.02 in MCT and Y15 co-treated rats, respectively (both P < 0.05 versus MCT-treated rats). Similarly, . c Immunohistochemistry was used to detect the protein level of LC3B in the lung tissues from different groups; cells with brown-stained cytoplasm in the medial layer of pulmonary artery are LC3B-positive PASMCs. d The percentage of LC3B-positive PASMCs in different groups (25 arteries each slide, diameter 20-100 μm). *P < 0.05 versus control group; # P < 0.05 versus MCT group elevated medial wall thickness in small pulmonary arteries was dramatically ameliorated in MCT and Y15-treated rats (Fig. 4c, d). Quantitative morphometric analysis of the medial wall thickness showed that the increased %MT in MCT-induced PAH rats reduced from (32.46 ± 2.15) % to (22.54 ± 1.78) % in MCT and Y15-treated rats (P < 0.05 versus MCT-treated rats; Fig. 4c, d). Moreover, treatment of PAH rats with Y15 reduced the pulmonary arteries muscularization and PASMCs proliferation, the percentage of Ki67-positive PASMCs dropped from (19.12 ± 2.2) % in MCT-treated rats to (14.53 ± 2.0) % in MCT and Y15 co-treated PAH rats (P < 0.05 versus MCT-treated rats, Fig. 4e-h). The results suggest that inhibition of FAK attenuates pulmonary vascular remodeling and the development of PAH.
In order to determine whether FAK mediates autophagy activation in rats with MCT-induced PAH, the protein level of LC3B was examined. In MCT-induced PAH rats, the level of LC3B and LC3B-II/LC3B-I ratio was increased to 4.35fold and 1.67-fold over control (P < 0.05 versus control), while which was declined to 2.26-fold and 1.01-fold over control in MCT and Y15-treated rats, respectively (P < 0.05 versus MCT-treated rats; Fig. 5a, b). At the same time, the percentage of LC3B-positive PASMCs was decreased from (23.5 ± 3.1) % in MCT-treated rats to (12.3 ± 2.0) % in MCT and Y15 co-treated rats (P < 0.05 versus MCT-treated rats; Fig. 5c, d). These results indicate that inhibition of FAK suppresses the activation of autophagy in MCT-induced PAH.

The Expression of FOXM1 is Up-Regulated in MCT-Induced PAH Model
To explore whether the expression of FOXM1 is increased in rats with MCT-induced PAH, the protein level of FOXM-1was examined using western blotting. In MCT and vehicletreated PAH rats, the protein level of FOXM1 was increased to 2.47-fold over control (P < 0.05 versus control; Fig. 6). Administration of thiostrepton, an inhibitor of FOXM1, reduced FOXM1 level to 0.52-fold over control (P < 0.05 versus MCT and vehicle-treated rats; Fig. 6). These results suggest that FOXM1 protein level is up-regulated in MCTinduced PAH.

FOXM1 Mediates FAK and Autophagy Activation in MCT-Induced PAH Model
Suppression of FOXM1 has been shown to inhibit vascular remodeling in MCT-induced PAH rats, here we further examined whether this effect is associated with the inhibition of autophagy. Figure 8 shows that the protein level of p-FAK was increased to 3.12-fold over control in MCT and vehicle co-treated rats (P < 0.05 versus control), while treatment of PAH rats with thiostrepton decreased the level of p-FAK to 1.45-fold over control (P < 0.05 versus MCT and vehicle-treated rats). Similarly, thiostrepton decreased the level of LC3B and LC3B-II/ LC3B-I ratio from 3.12-fold and 2.62-fold in MCT and Fig. 6 The expression of FOXM1 is up-regulated in MCT-induced PAH model. The level of FOXM1 in lung tissues from different groups was determined using immunoblotting. Representative Western blot and quantification of bands are shown. (n = 4 each group). *P < 0.05 versus control group; # P < 0.05 versus MCT and vehicletreated rats Fig. 7 Inhibition of FOXM1 prevents the increase of RVSP, RVHI, and pulmonary arterial remodeling in MCT-induced PAH model. a Change of RVSP in different groups of rats (n = 10-15). b Change of RVHI in different groups of rats (n = 10-15). c Hematoxylin and eosin staining of small pulmonary arteries in different groups (n = 10-15) (magnification × 400). d Quantitative analysis of the medial wall thickness of pulmonary arteries (n = 10-15). e The degree of muscularization of pulmonary arteries was evaluated by immunostaining of α-SMA. f Percentage of muscular, partially muscular and non-muscular arteries in different groups (25 arteries each slide, diameter 20-200 μm). g PASMCs were stained for Ki67 by immunohistochemistry to assess cell proliferation; cells with brownstained nuclei in the medial layer of pulmonary artery are Ki67-positive PASMCs. h The percentage of Ki67-positive PASMCs in different groups (25 arteries each slide, diameter 20-100 μm). * P < 0.05 versus control group; # P < 0.05 versus MCT and vehicle-treated rats vehicle-treated rats to 1.49-fold and 1.25-fold in MCT and thiostrepton co-treated rats, respectively (P < 0.05 versus MCT and vehicle-treated rats; Fig. 9a, b). Meanwhile, the percentage of LC3B-positive PASMCs was decreased from (22.1 ± 2.6) % in MCT and vehicle-treated rats to (15.2 ± 2.2) % in MCT and thiostrepton-treated rats (P < 0.05 versus MCT and vehicle-treated rats; Fig. 9c, d). These results suggest that FOXM1 induces the activation of FAK and autophagy in MCT-induced PAH rats.

Discussion
We have indicated in this study that FOXM1 significantly increases the phosphorylation of FAK, which subsequently induces the activation of autophagy, and thereby promotes the development of MCT-induced PAH. Autophagy, a catabolic pathway, plays a crucial role in removing misfolded or aggregated proteins, clearing damaged organelles, and eliminating intracellular pathogens [22]. The role of autophagy in the development of PAH is conflicting [10]. Multiple reports have discovered that autophagy promotes pulmonary artery remodeling in MCT or hypoxic-induced PAH rats [23,24]. However, other studies have shown that autophagy provides a protective role in the pathogenesis of PAH [10]. Our previous studies have suggested that autophagy is activated in sphingosine-1-phosphate (S1P)stimulated PASMCs and MCT-induced model of PAH in rats [25][26][27]. In this study, we found that the level of LC3B and LC3B-II/LC3B-I ratio was increased in MCT-induced PAH rats, and inhibition of autophagy by CQ suppressed pulmonary artery remodeling and the development of PAH.
FAK is a cytoplasmic tyrosine kinase and scaffold protein, which is localized to focal adhesions [18]. It has been shown that FAK is implicated in integrating signals such as growth factors receptors, G-protein coupled receptors, and cytokine receptors [28,29]. A study has demonstrated that FAK is activated in PASMCs from PAH patients and animal models of PAH [30]. In this study, we confirmed that the phosphorylation of FAK was significantly increased in MCT-induced PAH rats. A previous study has reported that inhibition of FAK reduces the activation of signal transducer and activator of transcription 3 (STAT3) in PAH-PASMCs [30]. In addition, FAK has been reported to be related to autophagy in the development of numerous types of human diseases. Sun et al. have shown that osteopontin-enhanced autophagy inhibits early brain injury by activation of FAK [31]. However, Zheng et al. have shown that osteoprotegerin increases FAK phosphorylation, which participates in the suppression of autophagy in cultured H9C2 cells [32]. In our study, we found that inhibition of FAK reduced the activation of autophagy in MCT-treated rats and accompanied with the suppression of pulmonary artery remodeling. FOXM1, a forkhead box transcription factor, is widely expressed in proliferating cells [33,34]. Studies in FOXM1 were found to be increased in distal pulmonary arteries and isolated PASMCs from PAH patients and multiple animal models of PAH [35][36][37]. Inhibition of FOXM1 has been shown to decrease the expression of cell division cycle protein 2 in PASMCs from PAH patients [38]. Importantly, FOXM1 has been reported to promote the activation of FAK by binding to integrin β1 in triple-negative breast cancer [14]. In addition, studies have suggested that inhibition of FOXM1 blocks starvation and rapamycin-induced autophagy in breast cancer [15]. In this study, we found that FOXM1 induced autophagy activation by phosphorylating FAK in MCT-induced PAH. In addition, the reduction in autophagy seen with inhibition of FOXM1 or FAK may also be the result of reduced PAH.
In conclusion, we identify that FOXM1 promotes the development of PAH by increasing the phosphorylation of FAK and subsequent activation of autophagy. Our study may contribute to the development of more effective PAH therapeutic strategies. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by CZ, NZ, JW, MC, and JL. The first draft of the manuscript was written by CZ, SL, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding This work was supported by the National Natural Science Foundation of China (Grant Numbers 82103179 to Dr. Huan Liu), the Natural Science Foundation of Shaanxi Province (2020JQ-508 to Dr. Shaojun Li), and the Research Foundation of Education Department of Shaanxi Provincial (21JS040 to Dr. Meng Cao).

Data Availability
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Declarations
Competing interests The authors have no relevant financial or nonfinancial interests to disclose.
Ethical Approval All animal care and experiments were performed in accordance with Xi'an Jiaotong University Animal Care Policy following the Guide for the Care and Use of Laboratory Animals. Ethics approval for the experimental protocols was received from the Laboratory Animal Care and Use Committee of Xi'an Jiaotong University. (n = 4 each group). c Immunohistochemistry was used to detect the protein level of LC3B in the lung tissues from different groups; cells with brown-stained cytoplasm in the medial layer of pulmonary artery are LC3B-positive PASMCs. d The percentage of LC3B-positive PASMCs in different groups (25 arteries each slide, diameter 20-100 μm). *P < 0.05 versus control group; # P < 0.05 versus MCT and vehicle-treated rats