Canagliflozin ameliorates hypobaric hypoxia-induced pulmonary arterial hypertension by inhibiting pulmonary arterial smooth muscle cell proliferation

ABSTRACT

Pulmonary arterial hypertension (PAH) is a disease with a high mortality and few treatment options to prevent the development of pulmonary vessel remodeling, pulmonary vascular resistance, and right ventricular failure. Canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is originally used in diabetes patients which could assist the glucose excretion and decrease blood glucose. Recently, a few studies have reported the protective effect of SGLT2 inhibitor on monocrotaline-induced PAH. However, the effects of canagliflozin on hypobaric hypoxia-induced PAH as well as its mechanism still unclear. In this study, we used hypobaric hypoxia-induced PAH mice model to demonstrate if canagliflozin could alleviate PAH and prevent pulmonary vessel remodeling. We found that daily canagliflozin administration significantly improved survival in mice with hypobaric hypoxia-induced PAH compared to vehicle control. Canagliflozin treatment significantly reduced right ventricular systolic pressure and increased pulmonary acceleration time determined by hemodynamic assessments. Canagliflozin significantly reduced medial wall thickening and decreased muscularization of pulmonary arterioles compared to vehicle treated mice. In addition, canagliflozin inhibited the proliferation and migration of pulmonary arterial smooth muscle cells through suppressing glycolysis and reactivating AMP-activated protein kinase signaling pathway under hypoxia condition. In summary, our findings suggest that canagliflozin is sufficient to inhibit pulmonary arterial remodeling which is a potential therapeutic strategy for PAH treatment.

KEYWORDS:

• Canagliflozin
• pulmonary arterial hypertension
• sodium-glucose cotransporter 2 inhibitor
• pulmonary arterial smooth muscle cell proliferation
• AMP-activated protein kinase

Introduction

Pulmonary arterial hypertension (PAH) induced by hypobaric hypoxia is the main cause of death from altitude sickness in high-altitude regions (1). PAH is characterized by remodeling of the pulmonary vessels, resulting in an increase of pulmonary vascular resistance and right ventricular failure (2). The currently available therapies for PAH including calcium channel blockers, nitric oxide and prostacyclin mimetics and endothelin receptor antagonists all contribute to improve vasodilation of pulmonary arterioles which are not sufficient to improve the patient outcomes (3). It is due to that, currently, therapies are not able to stop or reverse the progression of vascular remodeling. Hypobaric and hypoxia is one of the important causes of PAH, and the incidence of PAH in high altitude regions is about 15% (4). The average altitude is greater than 4000 m in Qinghai-Tibetan Plateau which is the largest and highest plateau in the world. More than ten million people live at Qinghai-Tibetan Plateau currently (1). Thus, it is very urgent to explore other effective therapeutic approaches through revealing the mechanisms of hypobaric hypoxia-induced PAH development.

With a rising in-depth research, the proliferation of pulmonary arterial smooth muscle cells (PASMCs) and migration of PASMCs to the intima are considered as the central of the pathogenesis of pulmonary vascular remodeling (5,6). It was reported that secreted protein acidic and rich in cysteine could promote hypoxic pulmonary hypertension through activating PASMC proliferation (7). The RNA-binding protein, heterogeneous nuclear ribonucleoprotein A2B1, promotes the pro-proliferative/anti-apoptotic phenotype of PASMCs which is responsible for the development of PAH (8). Therefore, inhibition or reverse of PASMC proliferation might be the most effective strategy to alleviate pulmonary vascular remodeling and thus cure PAH.

Canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, was originally used in diabetes patients which could assist the glucose excretion and decrease blood glucose. However, numerous evidences were appeared that canagliflozin adverse cardiovascular events and HF hospitalization risk (9,10). Moreover, canagliflozin can affect pathophysiological processes of cardiovascular diseases, such as inhibition of vascular smooth muscle cells proliferation and migration (11). In addition, other members of SGLT2 inhibitor were also found to suppress the proliferation of vascular smooth muscle cells (11–14). For example, empagliflozin could prevent neointima formation by impairing smooth muscle cell proliferation (13), and it attenuates interleukin-17A-induced human aortic smooth muscle cell proliferation and migration (14). Above all, canagliflozin might play similar roles in PASMC proliferation.

In recent studies, the association of SGLT2 inhibitor and PAH was found (15–17). It was reported that SGLT2 inhibition with empagliflozin could lower mortality, reduce right ventricle systolic pressure, and attenuate maladaptive pulmonary remodeling in monocrotaline-induced PAH (15). Dapagliflozin, another SGLT2 inhibitor, could reduce right ventricular systolic pressure and pulmonary vascular remodeling in a rat model of PAH (16). In addition, dapagliflozin administration could reduce ventricular arrhythmia vulnerability in rats with PAH-induced right heart failure (17). These findings indicated the important relationship between SGLT2 and PAH. However, if canagliflozin would also reduce hypobaric hypoxia-induced pulmonary arterial remodeling and the mechanism of how dapagliflozin or empagliflozin reduced pulmonary arterial remodeling remained unknown.

Glycolysis was considered as an important factor for regulating PASMC proliferation. It was reported that suppression of glycolytic enzyme α-enolase prevents the hypoxia-induced metabolic shift from mitochondrial respiration to glycolysis in PASMC, and finally decreases PASMC proliferation (18). As a SGLT2 inhibitor, canagliflozin could suppress the glycolysis level of thyroid cancer cell (19). Thus, we hypothesized that canagliflozin could ameliorate hypobaric hypoxia-induced PAH by inhibiting glycolysis in PASMC which resulted in reducing PASMC proliferation and pulmonary arterial remodeling.

In this study, therefore, we present evidence that canagliflozin was involved in the proliferation of PASMCs. We present a new therapeutic approach focusing on canagliflozin, which we show to be highly effective in hypobaric hypoxia-induced PAH, as well as in vitro-cultured PASMCs isolated from pulmonary arteries of mice.

Methods

Animals and hypoxia-induced PAH model

Male C57BL/6J mice were allowed to acclimate 1 week before experiment with available food and drinking water ad libitum. To induce the PAH model, 8-week-old mice on a normal chow diet were housed in a hyperbaric oxygen chamber and exposed to continuous hypobaric hypoxia (simulated altitude 5000 m) for 4 weeks. The chambers were opened transitorily (less than 10–15 min) for food, water, and canagliflozin (Selleck, Shanghai, China) treatment by gavage. The animals were divided randomly into two groups: (a) Hypobaric hypoxia control group (treated with vehicle, n = 11); (b) Hypobaric hypoxia + canagliflozin group (treated with 10 mg/kg, n = 11). After 4 weeks, hemodynamic measurements and histopathological examination were performed. All animal studies were approved by the Institutional Animal Care and Use Committee of The General Hospital of Western Theater Command.

Hemodynamic measurements

Twenty-four hours after the last treatment, measurements of pulmonary artery acceleration time (PAAT) and tricuspid annular plane systolic excursion (TAPSE) were performed by vevo 2100 high-resolution ultrasound imaging system (VisualSonics Inc, Toronto, Canada) with an MS400 transducer (24–30 MHz) under isoflurane anesthesia (1.5%−2.5%). Right ventricular systolic pressure (RVSP) was recorded by jugular vein catheterization with 1.4 F microtip pressure-conductance micro-catheter (SPR-839, Millar Instruments Inc, Houston, Tex) under the above-mentioned circumstances using a closed-chest technique as described previously (5,18). The mice were euthanized after hemodynamic measurements, and the lung tissues were harvested for histological analysis.

Histological analysis

The lungs were fixed with 4% paraformaldehyde (PFA) after harvest immediately. After paraffin embedding and sectioning, the lung slides (4 μm thickness) were next subjected to hematoxylin and eosin (HE) staining for quantitative assessment of pulmonary vascular remodeling. To evaluate the degree of medial wall thickness, the arteries with diameters 20–70 μm from each lung section were randomly outlined in different groups. The medial wall thickness was calculated as follows: 100 × (external diameter-internal diameter)/external diameter. An immunofluorescence assay was performed to determine the proportion of muscularization of pulmonary arterioles using an antibody to α-smooth muscle actin (α-SMA) (Proteintech, Wuhan, China, Mouse, 1:400). More than 20 pulmonary arterioles were enumerated from each lung sections and classified as fully muscularized (M, >75% α-SMA staining around the vessel), partially muscularized (P, 25–75% α-SMA staining around the vessel) or non-muscularized (N, <25% α-SMA staining around the vessel) depending on the percentage of α-SMA staining around the artery.

Isolation and culture of PASMCs

According to the previously described protocol, primary PASMCs were isolated and obtained from pulmonary arteries of mice euthanized by cervical dislocation (20). Cell purity was evaluated by immunofluorescence staining for α-SMA, a specific smooth muscle cell marker. The cells were maintained in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gibco, Australia) supplemented with 1% penicillin-streptomycin (Beyotime, Shanghai, China) and 10% fetal bovine serum (FBS, Gibco) in a standard incubator flushed with 5% CO₂. Only passages 3–6 were used for the present experiments, during which PASMCs routinely exhibited typical spindle-shaped morphology.

Immunofluorescence analysis

The lung sections were blocked with 5% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 1 h at room temperature after antigen retrieval with 1 mM EDTA in boiling water for 20 min. PASMCs were washed twice with PBS and then fixed in 4% PFA/PBS solution for 15 min. The fixed cells were washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and then blocked with PBS containing 0.1% Triton and 3% BSA for 30 min at room temperature. Then, the PASMCs or lung sections were incubated with primary antibodies overnight at 4°C. The cardiomyocytes or heart sections were subsequently washed 3 times with PBS and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555 (Thermo Fisher Scientific) for 2 h at 37°C. The slides were mounted in antifade mounting medium. Primary antibodies used are following: anti-Ki67 antibody (Proteintech, Rabbit, 1:100) and anti-α-SMA antibody (Proteintech, Mouse, 1:400). DAPI was used for nuclear staining. To quantify the Ki67 positive rate of PASMCs, five different fields and positions per sample were captured at 20× magnification. ImageJ software was used to quantify the number of Ki67 positive cells and total cells.

EdU incorporation assay

Click-iT® EdU Imaging Kits (Thermo Fisher Scientific, Waltham, MA) was used to detect proliferation of PASMCs according to the manufacturer’s instructions. Briefly, after reaching available confluence, the cells were exposed to prolonged hypoxia (3% O₂) or normoxia in the presence or absence of canagliflozin (10 μM). The cells were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 2 h. Then, the cells were fixed in 4% PFA for 30 min at room temperature followed by a 30 min incubation at room temperature in phosphate buffer saline (PBS) containing 0.1% Triton X-100. The cells were then incubated with a premixed click additive solution for 30 min at room temperature and finally incubated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime) for 5 min at room temperature. To quantify the EdU positive rate of PASMCs, five different fields and positions per sample were captured at 20× magnification. ImageJ software was used to quantify the number of EdU positive cells and total cells.

Wound healing assay

For the wound healing assay, PASMCs (2 × 10⁵ cells/mL) were seeded into a 12-well plate. When the cells reached a confluent state, a straight scratch was drawn using 10 µL pipette tip across the center of the well. The cells were subjected to hypoxia or normoxia after being incubated in FBS-free DMEM/F12 alone or containing canagliflozin (10 μM). The images of the scratches were taken at 0 and 24 h to record wound width for comparison. ImageJ software was used to quantify the width of the scratches.

Western blot analysis

The total protein was extracted from primary PAMCs which were exposed to prolonged hypoxia (3% O₂) or normoxia in the presence or absence of canagliflozin (10 μM). The proteins containing 50 μg of protein were separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, California). After treatment with blocking buffer (Beyotime), the blots were probed with the primary antibodies at 4°C overnight. The membranes were washed in TBST and incubated with the appropriate secondary antibodies (Li-Cor, IRDye 800CW, Goat anti-Rabbit or Goat anti-Mouse, 1:10000) for 2 h at room temperature. Membranes were washed and visualized with an Odyssey Imaging System. Primary antibodies used are following: anti-AMP-activated protein kinase (AMPK) alpha antibody (Cell Signaling Technology, Danvers, MA, Rabbit, 1:1000) or anti-phospho-AMPKα (Thr172) (Cell Signaling Technology, Rabbit, 1:1000). Anti-β-Actin (Proteintech, Mouse, 1:2000) was used for the normalization of protein expressions.

Seahorse glycolysis measurement

Extracellular acidification rate (ECAR) was measured using Seahorse XF24 Cellular Flux Analyzer (Agilent Technologies, Santa Clara, CA). Thirty thousand PASMCs per well were seeded in Seahorse XF24 cell culture microplates and cultured in a 37°C incubator containing 5% CO₂ for 12 h. Then, PASMC was treated with normoxia or hypoxia for 24 h. After washing with assay medium (bicarbonate and glucose-free DMEM, containing 2 mM glutamine) PASMC were kept in a CO₂-free incubator at 37°C for 1 h before the assay. After the addition of canagliflozin or vehicle, three injections were performed as follows: glucose (20 mM), oligomycin (2 μM), and 2-Deoxy-D-glucose (100 mM). The glycolysis parameters were calculated as follows: glycolysis = (maximal ECAR before oligomycin injection) – (non-glycolytic acidification), glycolytic capacity = (maximal ECAR) – (non-glycolytic acidification).

Cell viability assay

Cell viability was detected by the cell counting kit-8 (CCK-8, Beyotime) assay. PASMCs were seeded in 96-well microplates at 1 × 10⁴ cells/well and serum starved for 24 h. Then, cells were treated with canagliflozin (10 μM) or control reagent under normoxia or hypoxia (3% O₂) condition. After cultured for 24 h, CCK8 solution (10 µL) was added to each well and subsequently incubated for 2 h, and the absorbance was evaluated at 450 nm by using a microplate reader (Bio-Rad).

Statistics

The data are expressed as mean ± SEM. All data collected and analyzed were assumed to be distributed normally. Statistical assessments were performed using Prism 8 software. Comparison between the two groups was made by student’s unpaired t test. For multiple group comparisons, one-way ANOVA was performed followed by Holm-Sidak test. Log-rank test was performed in survival analysis. A value of P < .05 was considered statistically significant.

Results

Canagliflozin prolonged the survival of hypobaric hypoxia-induced PAH mice

To determine the effect of canagliflozin on hypobaric hypoxia-induced PAH, adult mice were exposed to hypobaric hypoxia (simulated altitude 5000 m) in a workstation with or without canagliflozin treatment. We first determined if canagliflozin treatment would prolong survival in hypobaric hypoxia-treated mice. We found that the survival rate of hypobaric hypoxia-induced PAH mice was significantly increased after daily canagliflozin treatment (Figure 1). This result indicated that canagliflozin treatment could significantly prolong survival in hypobaric hypoxia-induced PAH mice.

Figure 1. Canagliflozin prolonged the survival of hypobaric hypoxia-induced PAH mice. Mice were under hypobaric hypoxia condition on day 0 and administered canagliflozin or vehicle for 4 weeks. Survival curves demonstrated a significant survival benefit in canagliflozin-treated mice compared to control group. n = 11; *P < .05 compared to control.

Canagliflozin improved cardiac function

Next, we investigated the effect of canagliflozin on heart function using a hemodynamic assay. We found that hypobaric hypoxia (HH) treatment significantly increased in RVSP, which indicated the successful establishment of PAH with HH treatment in our study. Canagliflozin (10 mg/kg/day) treatment could significantly decreased RVSP compared to vehicle-treated mice (Figure 2a). PAAT, an important parameter for the diagnosis of PAH, was also assessed in this study by echocardiography. It was shown that HH treatment decreased PAAT compared with the normoxia group. Canagliflozin appeared to block the inhibitory effect of HH treatment on PAAT (Figure 2b,c). In addition, the HH treatment for 4 weeks lowered TAPSE which could also be blocked by canagliflozin treatment (Figure 2d). These findings indicated that canagliflozin improved cardiac dysfunction in PAH after HH treatment.

Figure 2. Canagliflozin improved cardiac function. Echocardiography followed by hemodynamic measurements were carried out on mice exposed to HH for 4 weeks or maintained under normoxic conditions to determine (a) RVSP, (b and c) PAAT and (d) TAPSE of the mice. n = 5 normoxia; n = 6 HH; n = 10 HH + canagliflozin. *P < .05 compared to normoxia; ^#P < .05 compared to HH.

Canagliflozin attenuated pulmonary artery remodeling

To verify the effect of canagliflozin on pulmonary artery remodeling, the examination of lung sections was performed. The result showed that more pulmonary artery muscularization was found in HH group compared with normoxia group while canagliflozin treatment could reverse the effect of HH on pulmonary artery muscularization (Figure 3a,b). Quantitative assessments were performed to confirm that HH increased medial wall thickening compared to normoxia group, while there was less medial wall thickening in HH + canagliflozin treatment group (Fig 3c,d). These results indicated that canagliflozin could attenuate pulmonary artery remodeling in HH-treated mice.

Figure 3. Canagliflozin attenuated pulmonary artery remodeling. (a) HE staining of lung sections. (b) immunofluorescence staining of α-SMA in small pulmonary vessels followed by morphometric analysis. (c) medial wall thickness of pulmonary arteries. (d) quantitative assessments of muscularized (M), partially muscularized (P) and nonmuscularized (N) pulmonary arteries shown as percentages. n = 5 normoxia; n = 6 HH; n = 10 HH + canagliflozin. *P < .05 compared to normoxia; ^#P < .05 compared to HH. Scale bars = 20 μm.

Canagliflozin suppressed pulmonary artery smooth muscle cell proliferation and migration

To explore the effect of canagliflozin on pulmonary artery proliferation, the primary PASMCs were also used to detect the inhibitory effect of canagliflozin on PASMC proliferation and migration. The result showed that hypoxia (3% O₂) treatment could robustly increase EdU and Ki67 positive rate of PASMCs (Figure 4a–d). However, the EdU and Ki67 positive rate of PASMCs decreased after canagliflozin treatment (Figure 4a–d). In addition, the effect of canagliflozin on PASMC proliferation was further detected by CCK-8 assay. The result showed that canagliflozin could inhibit PASMC proliferation significantly under hypoxia condition (Figure 4e). Otherwise, canagliflozin could also reverse the effect of hypoxia on migration of PASMCs (Figure 4f,g). These results indicated that canagliflozin could suppress pulmonary artery smooth muscle cell proliferation and migration under hypoxia condition.

Figure 4. Canagliflozin suppressed pulmonary artery smooth muscle cell proliferation and migration. (a and b) EdU positive rate of PASMC in the presence or absence of canagliflozin. Values are expressed as a percentage relative to the normoxia group. (c and d) Ki67 positive rate of PASMC in the presence or absence of canagliflozin. Values are expressed as a percentage relative to the normoxia group. (e) CCK-8 determination of the viability of PASMCs treated with or without canagliflozin for 24 hours and cultured under normoxia and hypoxia condition. Values are expressed as a percentage relative to the normoxia group. (f and g) wound scratch assay was used to assay the migration of PASMCs in the presence or absence of canagliflozin. n = 3 for each group. *P < .05 compared to normoxia; ^#P < .05 compared to hypoxia. Scale bars = 100 μm.

Canagliflozin inhibited pulmonary artery smooth muscle cells glycolysis

As a sodium-glucose co-transporter inhibitor, we investigated the function of canagliflozin on glucose utilization of PASMCs. Seahorse analysis was used to assay the glycolysis in PASMCs. We found that hypoxia increased glycolysis and glycolytic capacity (Figure 5a–c). Canagliflozin that could inhibit the transport of sodium and glucose into cytoplasm reduced glycolysis and glycolytic capacity in PASMCs (Figure 5a–c). These data indicated that canagliflozin could inhibit PASMC glycolysis under hypoxia condition.

Figure 5. Canagliflozin inhibited pulmonary artery smooth muscle cells glycolysis. (a) measurement of ECAR in PASMCs. Dotted line indicated the time points of adding glucose, oligomycin and 2-DG. (b and c) glycolysis and glycolytic capacity were calculated by ECAR in PASMCs after adding glucose, oligomycin and 2-DG. n = 6 normoxia; n = 7 hypoxia and hypoxia + canagliflozin. * P < .05 compared to normoxia; ^#P < .05 compared to hypoxia.

Canagliflozin suppressed pulmonary artery smooth muscle cell proliferation through activation of AMPK signaling pathway

Next, we further investigated the mechanism of how canagliflozin inhibited PASMC proliferation. As a key regulator of cellular energy homeostasis via the phosphorylation of multiple proteins involved in metabolic pathways, AMPKα phosphorylation was detected after hypoxia and canagliflozin treatment. We found that the phosphorylation of AMPKα was decreased after hypoxia treatment, while canagliflozin could reactivate AMPK signal through increasing the phosphorylation of AMPKα (Figure 6a–c). This indicated that AMPK signaling pathway was involved in canagliflozin reduced PASMC proliferation. Then, the AMPK inhibitor, Compound C, was used to investigate the effect of AMPKα phosphorylation on canagliflozin reduced PASMC proliferation. We found that the increase of AMPKα phosphorylation after canagliflozin treatment could be blocked by Compound C (Figure 6d–f). Compound C also blocked the inhibitory effect of canagliflozin on PASMC proliferation under hypoxia condition (Figure 6g–i). These results indicated that canagliflozin suppressed PASMC proliferation through activation of AMPK signaling pathway under hypoxia condition.

Figure 6. Canagliflozin suppressed pulmonary artery smooth muscle cell proliferation through activation of AMPK signaling pathway. (a-c) Western blot analysis of AMPK phosphorylation levels in PASMCs. (d-f) Western blot analysis of the effect canagliflozin on AMPK phosphorylation in the presence or absence of compound C under hypoxia condition. (g and h) immunofluorescence staining analysis of the effect canagliflozin on PASMC proliferation in the presence or absence of compound C under hypoxia condition. Values are expressed as a percentage relative to the hypoxia group. (i) CCK-8 determination of the viability of PASMCs treated with canagliflozin or compound C for 24 hours and cultured under hypoxia condition. Values are expressed as a percentage relative to the hypoxia group. n = 3 for each group. *P < .05 compared to hypoxia; NS = not significant. Scale bars = 100 μm.

Discussion

In this study, we investigated the effect of canagliflozin on hypobaric hypoxia-induced PAH in mice model. We first demonstrated that canagliflozin could protect the development of hypobaric hypoxia-induced PAH through inhibition of pulmonary artery remodeling. Canagliflozin suppressed glycolysis, activated AMPK signaling pathway, and thus inhibited the proliferation and migration of PASMCs. Therefore, our results provide evidence that canagliflozin is a potential strategy against hypobaric hypoxia-induced PAH.

The results showed an elevation in the RVSP and reduction in the PAAT and TAPSE concomitant with a significant increase in pulmonary artery medial wall thickening and pulmonary artery muscularization in mice exposed to hypobaric hypoxia for 4 weeks, suggesting that the hypobaric hypoxia-induced PAH mice model was successfully established. Canagliflozin reversed the increased RVSP and decreased PAAT and TAPSE in the hypobaric hypoxia-induced PAH. Additionally, the abnormally increased of pulmonary artery medial wall thickening and pulmonary artery muscularization were significantly blocked by canagliflozin treatment. Due to the important role of pulmonary vascular remodeling and right ventricular pressure in PAH therapy (21), our findings indicated that canagliflozin administration could successfully repress the development of hypobaric hypoxia-induced PAH. These findings were in accord with the effect of empagliflozin and dapagliflozin on experimental pulmonary hypertension which was induced by monocrotaline in rats (15,16).

The proliferation and migration of PASMCs play an important role in PAH formation and development (5,6), while treatments targeting these processes are deficient. In our study, we first demonstrated that canagliflozin could inhibit PASMC proliferation and migration under hypoxia condition. The mechanism was partially due to the function of canagliflozin on glucose transport. It was reported that canagliflozin could suppress the glycolysis level of thyroid cancer cell by using Seahorse XF Extracellular Flux assay (19). In this study, glycolysis level of PASMC was also assayed after canagliflozin treatment. Results showed that canagliflozin could inhibit PASMC glycolysis level under hypoxia condition. Previous studies have shown that suppression of glycolytic enzyme α-enolase prevents the hypoxia-induced metabolic shift from mitochondrial respiration to glycolysis in PASMC, and finally decreases PASMC proliferation (18). These indicated that canagliflozin might decrease PASMC proliferation and migration via suppression of PASMC glycolysis.

AMPK, a key regulator of cellular energy homeostasis, was involved both in the cell glycolysis and proliferation (18,22,23). AMPK is activated by glucose deprivation, which is due to the impaired production of ATP from reduced glucose metabolism and triggers the increasing levels of AMP/ADP (22). Decreased endothelial cell glucose uptake and glycolysis could lead to energy depletion and the activation of the cellular energy sensor AMPK (23). Suppression of glycolytic enzyme α-enolase prevents glycolysis in PASMC and decreases PASMC proliferation through activating AMPK-AKT pathway (18). Additionally, disintegrin and metalloproteinase with thrombospondin motifs 8 overexpression could increase PASMC proliferation with downregulation of AMPK (24). Prior incubation of PASMC with metformin induced a dramatic AMPK activation and significantly blocked platelet-derived growth factor-induced cell proliferation (25). Therefore, the function of AMPK in PASMC after canagliflozin treatment was investigated in our study. It was found that canagliflozin could reactivate AMPK signal through increasing the phosphorylation of AMPKα which could be blocked by Compound C. Besides, the anti-proliferation effect of canagliflozin on PASMC could also be blocked by Compound C. These indicated that canagliflozin suppressed PASMC proliferation through activation of AMPK signaling pathway.

In conclusion, canagliflozin was confirmed to reduce RVSP, improve PAAT and TAPSE, and inhibit pulmonary arteriolar remodeling in hypobaric hypoxia-induced PAH mice model in this study. We also demonstrated that canagliflozin inhibited the proliferation and migration of PASMCs through suppression of glycolysis and reactivation of AMPK signaling pathway in hypobaric hypoxia-induced PAH mice.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the [Natural Science Foundation of Sichuan Province] under Grant [number 2023NSFSC1651]; [Project of The General Hospital of Western Theater Command] under Grant [number 2021-XZYG-A01]; and [Popularization and Application Project of Sichuan Health Commission] under Grant [number 21PJ069].