Wnt/β-catenin signaling pathways mediate monocrotaline-induced pulmonary arterial hypertension via glycolysis in rats

doi:10.21203/rs.3.rs-3237886/v1

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Wnt/β-catenin signaling pathways mediate monocrotaline-induced pulmonary arterial hypertension via glycolysis in rats

https://doi.org/10.21203/rs.3.rs-3237886/v1

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Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease. Metabolic abnormalities and immune inflammation are deeply involved in pulmonary vascular remodeling and in the formation of PAH. Cumulative evidence indicates that β-catenin and abnormal glucose metabolism play an important role in the development of PAH, but the underlying mechanism is still elusive. To elucidate this mechanism, we first established a rat model of PAH and then treated some rats with a specific β-catenin inhibitor (XAV939). After examining the protein expression of β-catenin, levels of indicators of glucose metabolism, right ventricular pressure (RVSP) and pulmonary artery histopathology, we found that monocrotaline (MCT)-exposed rats had an increase in levels of glycolytic metabolism markers and that this phenomenon could be blocked by β-catenin inhibitors. This study demonstrated that Wnt/β-catenin signaling promotes the inflammatory response involved in pulmonary vascular remodeling and participates in the formation of PAH by regulating glycolysis in macrophages. Inhibition of Wnt/β-catenin signaling pathways could improve the progression of PAH, which may provide novel therapeutic targets for the treatment of PAH.

WNT/β-catenin

glycolysis

inflammation

macrophages

NLRP3 inflammasome

pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) comprises a group of serious cardiopulmonary diseases caused by multiple factors (1). As pulmonary vascular resistance progressively increases, an irreversible pulmonary circulation disorder and a high load on the right ventricle of the heart are developed, ultimately leading to right heart ventricle failure and even premature mortality (2). The prevalence of PAH is approximately 1% of the global population but is up to 10% in people over 65 years of age (3). Currently, clinical treatment options for PAH are more limited and less effective than would be ideal. Targeted therapies such as prostaglandins, phosphodiesterase-5 inhibitors, endothelin receptor antagonists and soluble guanylate cyclase stimulators are mainly aimed at the endothelin, nitric oxide, and prostacyclin pathways (4, 5). Although these drugs can improve hemodynamics and relieve symptoms in patients, they cannot fundamentally curb the further exacerbation of PAH (5). A growing body of evidence indicates that the macrophage inflammatory response is the main driver of pathogenesis in PAH, and the number of macrophages present in PAH animal models and clinical patients is significantly higher than that in control groups (Florentin et al., 2018). Therefore, targeting the inflammatory response of macrophages may be a new option for the treatment of PAH.

Metabolic reprogramming involves and affects the development and outcome of a disease. Metabolic reprogramming maintains cellular redox homeostasis and ensures the synthesis of biomolecules by supplying energy, biosynthesis intermediates and reducing substances. It also promotes cell proliferation, migration and an antiapoptotic tumor-like malignant phenotype, leading to pulmonary vascular remodeling and right ventricular hypertrophy and accelerating the occurrence and development of PAH (6). The Warburg effect, which causes increased glucose uptake, is a phenomenon of metabolic reprogramming. The Warburg effect is common in tumor cells and leads to a reduction in the function of the mitochondrial oxidative phosphorylation (OXPHOS) system. Energy is thereby produced by glycolysis, even in the presence of abundant oxygen, to meet the high biosynthetic energy requirements (7). Glycolysis plays an important role in the proinflammatory response of M1 macrophages, as they preferentially metabolize glucose as an energy substrate. In addition, intermediates of glycolysis, such as phosphoenolpyruvate (PEP) and lactate, have been shown to affect the inflammatory status of cells, and glycolytic enzymes can act as posttranscriptional regulators of inflammatory genes (8).

As the sentinels of the frontline in tissue defense, macrophages are an important defense for the body to resist the invasion of pathogens, and their metabolic roles and functions are closely related to the development of many inflammatory diseases and tumors types (9). Metabolic changes affect the ability of macrophages to differentiate, polarize, mobilize, and exert antitumor effects (10). When stimulated by inflammation-inducing factors such as infection, endotoxin and hypoxia, resting macrophages undergo M1-type polarization, triggering glucose metabolism reprogramming (11), further activating the NLRP3 inflammasome, and producing large amounts of proinflammatory factors such as TNF-α and IL-1β to amplify the inflammatory response (12). These inflammatory cytokines induce pulmonary interstitial fibrosis and pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis and ultimately lead to the occurrence of PAH (13–16). We have reported that there is specific activation of NLRP3 in macrophages and that inhibition of NLRP3 can improve the progression of PAH (17–19). Therefore, blocking the reprogramming of glucose metabolism can effectively inhibit the inflammatory response.

The Wnt signaling pathway, an open pathway with multiple chains and multiple action sites, plays a crucial role in processes such as cellular inflammation, immune regulation, energy metabolism, proliferation, differentiation and apoptosis (20). In the classical Wnt cascade, β-catenin is the key effector protein for the transduction of signals to the nucleus, and it triggers the transcription of Wnt-specific genes that control and determine the fate of many cells (21). β-Catenin promotes the abnormal proliferation, invasion and apoptosis of inflammatory cells and tumor cells by regulating their metabolism, leading to the development of many diseases (22, 23). There is some evidence that β-catenin is involved in the activation of the NLRP3 inflammasome (24–26), but the specific mechanism is still unclear.

There are many methods currently used to construct PAH models in vivo, and a classic and widely used drug that causes irreversible damage to pulmonary blood vessels through targeting is MCT (27). MCT can cause endothelial dysfunction, which can simulate clinical inflammation-related PAH (28). In this study, we illustrate that activation of the NLRP3 inflammasome in MCT-induced PAH is caused by enhanced glycolysis in macrophages and that β-catenin is a regulator of glycolysis. Increased glycolysis in macrophages, promoted by Wnt/β-catenin, is a contributor to the inflammatory response of pulmonary hypertension, and inhibition of Wnt/β-catenin signaling pathways may be a new direction for research into novel means for alleviating the progression of PAH.

Experimental animals

Pathogen-free inbred male Sprague–Dawley rats (220–250 g) were obtained from the Experimental Laboratory Animal Center of Guangxi Medical University (Nanning, China). The rats were placed in housing conditions with standard room temperature (21–22°C) and humidity (60–65%). All animals were provided adequate access to food and water.

Rat model of MCT-induced PAH and experimental groups

The PAH rat model was established by a single subcutaneous injection of MCT (60 mg/kg). To examine the effect of glycolysis inhibition on the progression of PAH,2-deoxy-d-glucose(2-DG), a competitive inhibitor of glucose metabolism (29), was administered to MCT-induced rats. A total of 24 male rats were randomly assigned to four experimental groups (n = 6/group): (1) saline + vehicle group (sham group), (2) saline + 2-DG group (sham + 2-DG group), (3) MCT + vehicle group (MCT group), and (4) MCT + 2-DG group (MCT + 2-DG group). The dose of 2-DG used in the study was based on a previous report (30). 2-DG (200 mg/kg/d) in phosphate buffered saline (PBS) was randomly injected intraperitoneally for 7 days after MCT administration for 3 weeks.

To examine the effect of β-catenin on glycolysis, XAV939, a widely used β-catenin inhibitor, was administered. A total of 24 male rats were randomly assigned to four experimental groups (n = 6/group): (1) saline + vehicle group (sham group), (2) saline + XAV939 group (sham + XAV939 group), (3) MCT + vehicle group (MCT group), and (4) MCT + XAV939 group (MCT + XAV939 group). The dose of XAV939 used in the study was based on a previous report (31). XAV939 (5 mg/kg/d) in phosphate buffered saline (PBS) was randomly injected intraperitoneally for 21 days after MCT administration. Body weight was measured once per week to adjust the dose accordingly. This investigation was approved by the Guangxi Medical University Animal Ethics Committee and carried out in compliance with the ARRIVE guidelines.

Echocardiography

Twenty-eight days after MCT administration, echocardiography and hemodynamic evaluations were performed on all rats. The rats were anesthetized by an intraperitoneal injection of chloral hydrate (300 mg/kg). Echocardiography was performed using a Resona 7 system with an L20-5U transducer (Mindray, China). Tricuspid annular plane systolic excursion (TAPSE), RV end diastolic dimension (RVEDD) and right ventricular ejection fraction (RVEF) were calculated by the following formula: RVEF (%) = (RVEDV - RVESV)/RVEDV × 100%. TAPSE was measured by M mode echocardiography. Measurements were recorded from 10 consecutive beats and were used to normalize beat-to-beat variations.

Hemodynamic measurements and tissue processing

RVSP was measured by thoracotomy compression measurement. Briefly, rats were anesthetized and placed on a thermostatic heating pad at 37°C, intubated and connected to the ventilator (tidal volume 5.0 ml, respiratory rate 80 times/min). The surgeon performed the right heart puncture immediately after opening the chest and then connected it to a pressure sensor. The data were collected by the cardiac pressure volume system ADV500 (Transonic, Beijing, China) through a computer. The animals were euthanized after completion of RVSP data collection. Lungs and hearts from rats were rapidly excised and weighed. The dry weights of the right ventricular free wall (RV) and the left ventricle (LV) plus septum (LV + S) were measured, and then the RV/LV + S was calculated. Parts of the right ventricle and left lung tissue from all experimental groups were fixed in 4% paraformaldehyde for 24 h before being paraffin-embedded for histopathological examination. The right lung was excised for ELISA, glycolysis-related index measurements and Western blotting (stored at -80°C).

Histological analysis

Paraffin sections were first heated at 60°C for 2 h, followed by dewaxing using xylene and gradient ethanol. After antigen repair, endogenous peroxidase action was extinguished with 0.3% hydrogen peroxide and sections were blocked with 10% normal goat serum for 1 h. Then, the segments were incubated with α-smooth muscle actin (α-SMA, 1:150, BIOSS, Beijing, China) antibodies overnight at 4°C, washed in PBS and incubated with HRP-labeled goat anti-mouse/rabbit IgG polymer for 30 min. After incubation with horseradish for 10 min and DAB staining for 3–5 min, the samples were completely rinsed with tap water, and hematoxylin and anti-blue were retained, dehydrated, clarified and sealed. Images were examined using an Olympus BX51 microscope. The thickness of the PA wall or smooth muscle layer was measured in the cross-sectional images of the lung, and the remodeling of the pulmonary artery (PA) was quantified by morphometric analysis of distal small PAs (50–100 mm).

Primary rat PASMC and alveolar macrophage (AM) isolation

Rat PASMCs obtained from the medial smooth muscle layer of pulmonary arteries were isolated as described previously (32). Briefly, rat pulmonary artery samples were cut longitudinally with ophthalmic scissors, and the inner and outer membranes were removed by gentle scraping back and forth 2 to 3 times with a blade and rinsed with PBS. The PA was cut into 1 mm³ pieces and placed in the bottom of a T-25 culture flask. The flask was incubated at 37°C with 5% CO₂ for 3–4 h, and then 3 mL of high-glucose DMEM containing 20% FBS was added to the flask. After 5–7 days of culture, a small number of cells grew from the edge of the tissue block. Once grown to approximately 70% confluence, PASMCs with good growth were taken from the second through sixth passages and used for subsequent experiments. Anti-α-actin immunohistochemical staining was used to identify PASMCs.

Primary rat AMs were collected by bronchoalveolar lavage as described previously (33). Briefly, the lungs of rats were harvested for multiple lavages with PBS, and the rat alveolar lavage fluid was recovered. After centrifugation (1000 rpm for 5 min), red blood cells were taken, and the cells were lysed for 5 min. Subsequently, AMs were isolated and cultured with IMDM containing 20% FBS. Anti-CD86 immunohistochemical staining was used to identify AMs 3 days later.

Rat alveolar macrophage cell line (NR8383)

The rat alveolar macrophage cell line (NR8383) was purchased from the Chinese Academy of Sciences Cell Bank. Cells were cultured in HamF-12K culture medium (Pricella, China) containing 20% FBS (Sigma, Australia) at 37°C in a 5% CO₂ incubator. The culture medium was changed every 2 to 3 days, and cell passaging was performed once every 3 to 4 days.

Coculture of PASMCs with NR8383

First, lithium chloride (LiCl) was selected as a β-catenin agonist for in vitro β-catenin intervention experiments. NR8383 cells were divided into 5 groups: control group (diluent), LPS group, LPS + XAV939 (10 mM, Selleck, USA) group, and LPS + LiCl (10 mM, Selleck, USA) group. Before administering the cells XAV939 and LiCl for 48 h, the cells were stimulated by LPS (100 ng/ml) for 6 h. The interaction between PASMCs and macrophages was investigated, as well as the mechanisms of numerous PAH-related events, using an in vitro coculture model. Macrophages from each group were seeded in the upper chamber of the Transwell chamber, while PASMCs were seeded in the lower chamber of the Transwell. The cells and supernatant were collected after the experiment for ELISA, Western blot, and a CCK-8 assay was used to examine the proliferation of PASMCs.

Second, D-fructose 1,6-bisphosphate trisodium salt octahydrate (FBP) was chosen as an agonist of glycolysis for the glycolysis intervention experiments. NR8383 cells were divided into the control group (diluent), LPS group, LPS + 2-DG (4 mM, MCE) group, LPS + FBP (4 mM, Alladin, Shanghai, China) group, and LPS + XAV939 + FBP group. Cells were stimulated with LPS (100 ng/ml) for 6 h before administration of 2-DG and FBP. Relevant markers were tested after coculture with PSAMCs.

Cell counting kit-8 assay

A CCK-8 assay kit (Dojindo Laboratory, Kumamoto, Japan) was used to determine PASMC viability according to the manufacturer’s instructions. Briefly, cells were plated at a density of 2.0×10⁵ cells/well in 96-well plates. After the corresponding treatment, cell proliferation was assessed with a CCK-8 kit according to the manufacturer’s instructions. The amounts of cells were determined relative to the control cells in triplicate experiments.

Phosphofructokinase (PFK) and lactate dehydrogenase (LDH) activities and content of glucose and lactate (LA) determination

PFK and LDH activities and levels of glucose and LA were determined by a commercially available colorimetric glucose assay kit and LA assay kit (Solarbio, Beijing) according to the manufacturer's instructions.

ELISA

Cell culture supernatants and rat lung tissue homogenates were analyzed after centrifugation. The levels of IL-6, TNF-α, IL-1β and IL-18 were measured using commercially available ELISA kits (Jiang Lai creatures, Shanghai, China) according to the manufacturer’s instructions.

Western blot analysis

Tissues and cellular proteins were extracted using RIPA lysis buffer (Solarbio, Beijing, China) mixed with protease and phosphatase inhibitors (Beyotime, Shanghai, China). The concentrations of the extracted proteins were measured using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were probed with antibodies against β-catenin (1:1000, Servicebio, Wuhan, China), hexokinase 2 (HK2),M2-pyruvate kinase (PKM2) (1:4000, Proteintech, Wuhan, China), ASC, pro-caspase-1, caspase-1, NLRP3 (1:1000; all Cell Signaling Technology, USA), β-actin and GAPDH (1:2500, Servicebio, Wuhan, China). After blocking with 5% (w/v) dry milk for 1 h, antibodies were added and incubated overnight at 4°C. After washing the membranes, they were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; ZSGB-Bio, Beijing, China) for 2 h. PVDF was immersed in sensitized ECL and then exposed to X-rays via a gel imaging system (UVP, Upland, CA) to develop the protein bands. ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify the density of the bands and normalize them to GAPDH or β-actin levels.

5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay

PASMCs were stimulated with NR8383 cell supernatant from each group for 24 h. The proliferation of PASMCs was detected by EdU assay. PASMCs were stained with the BeyoClick™ EdU Apollo488 in vitro Imaging Kit (Beyotime, Shanghai, China) according to the instructions. Nuclei were stained with Hoechst.

Cell migration

Cell migration was determined by a scratch wound assay. Briefly, PASMCs were cultured in six-well plates in starvation medium and wounded with a sterile pipette tip to generate a cell-free gap of 1 mm width, and the wound location in the culture dish was marked as previously described. Cells were photographed to record the wound width at 0 h. Afterward, cells were treated as previously described.24 h later, photographs were taken again at the marked wound location for migration measurement.

Statistical analysis

GraphPad 9.0 software was used for all data statistical analyses and bar chart production. The data are expressed as the mean ± standard deviation (SD). Comparisons between two groups (which followed a normal distribution) were determined by unpaired Student’s t tests. One-way analysis of variance (ANOVA), followed by the Newman–Keuls multiple comparison test for normally distributed data, was used for comparisons among multiple groups. Statistical significance was characterized as ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed).

Increased expression of β-catenin in AMs from rats with MCT-induced PAH

To examine the distribution and expression of β-catenin in PAH, an MCT-induced PAH model in rats was constructed. First, we used double immunofluorescence staining to determine the localization of β-catenin in MCT-induced PAH lungs, and the results showed that β-catenin was mainly distributed in AMs (Fig. 1A). To confirm the expression of β-catenin in M1 AMs, we isolated and cultured AMs and repeated double fluorescence staining and obtained the same results (Fig. 1B). Then, we examined the protein levels of β-catenin in the lung tissue of sham and MCT-treated rats. The results showed that β-catenin protein levels were significantly increased in lung tissue in the MCT-induced model compared to those in the lung tissue of the sham group (Fig. 1C). Moreover, we obtained identical results in the isolated and cultured AM homogenates (Fig. 1D). Additionally, to determine the involvement of β-catenin in PAH formation, the β-catenin inhibitor XAV939 was administered for 3 weeks beginning on the day of MCT injection. Immunofluorescence results showed that XAV939 significantly downregulated the expression of β-catenin in the lungs of MCT-induced PAH rats (Fig. 1E-F). These results demonstrate that the Wnt/β-catenin pathway is activated in the MCT-induced PAH model and that β-catenin might play a role in the progression of PAH in AMs.

An inhibitor of β-catenin (XAV939) attenuated the progression of PAH induced by MCT in rats

Hemodynamic measurements were taken for all the rats, and we found that RVSP levels were significantly increased in MCT-exposed rats compared to those of Sham groups, and XAV939 effectively reduced this phenomenon (Fig. 2A-B). In addition, RV structure and function were examined. The β-catenin inhibitor XAV939 attenuated the decrease in RV/(LV + S) in MCT-administered rats (Fig. 2C). Furthermore, HE staining showed that XAV939 reduced the rate of pulmonary vascular wall thickening as well as the right ventricle enlargement caused by MCT. α-SMA (a smooth muscle cell marker) immunohistochemistry demonstrated a relief of the increased muscularization rate of pulmonary blood vessels induced by MCT after the application of XAV939 (Fig. 2D-F). Moreover, rats exposed to MCT exhibited a significant increase in RVEDD and decreases in TAPSE and RVEF compared to controls, and these effects were reversed by XAV9393 administration (Fig. 2G-J). These data suggest that XAV939 attenuates changes in RV structure and improves right ventricular function in PAH rats.

β-catenin regulates glycolysis in macrophages

It has been reported that glycolysis is a critical factor in the pathogenesis of PAH (34). We hypothesized that β-catenin has a stimulatory role in the regulation of glycolysis, which would imply that the reduction in β-catenin expression suppresses the levels of glycolysis. Therefore, we used XAV939 to inhibit β-catenin, and Western blotting showed successful inhibition (Fig. 3A). In the lung tissue homogenate of MCT-administered rats, we detected that the lactate levels were significantly higher than those in the sham group, while the glucose content showed the opposite trend (Fig. 3B-C). Concurrently, the levels of HK2, PFK,

PKM2 and LDH were increased in the MCT-administered group. These results demonstrate that reprogramming of glucose metabolism occurred in MCT-administered rats. As expected, in our inhibition-of-function study, XAV939 alleviated the above indices when compared to those of the MCT-model rats, (Fig. 3D-F).

In addition, we performed in vitro cell assays. Administration of LPS in NR8383 cell lines promoted M1 polarization to simulate inflammatory changes in PAH, and the cells were then treated with an inhibitor (XAV939) and an agonist (LiCl) of β-catenin. First, we determined the protein level of β-catenin to ensure successful intervention (Fig. 3G). Then, the supernatants of NR8383 cells from all groups were collected for analysis. The results showed that lactate levels in the supernatant increased after LPS intervention, and this trend was decreased after XAV939 administration. As expected, lactic acid levels were highest in the LiCl group supernatant. (Fig. 3H). In addition, glucose levels in all groups were as expected (Fig. 3I). At the same time, HK2, PFK, PKM 2, and LDH-A levels were significantly decreased in macrophages in the XAV939 group compared with the LPS group, while the opposite trend occurred in the LiCl group (Fig. 3J-L). Taken together, these results indicate that β-catenin positively regulates glycolysis in macrophages. Conversely, we also tested the relevant indicators of oxidative phosphorylation (citrate, acetyl-coenzyme A, A-ketoglutarate), but the results were not as expected.

Enhanced glycolysis promotes the activation of the NLRP3 inflammasome in macrophages.

Consistent with previous studies, glycolysis contributes to inflammasome activation (35). Enhanced glycolysis was present in MCT-induced PAH rats, accompanied by activation of the NLRP3 inflammasome. Levels of key enzymes of glycolysis and of lactate were significantly increased in the lung tissue of PAH rats compared with the control group. After 2-DG intervention in MCT rats, the above trend was reversed (Fig. 4A-G), along with the downregulation of NRLP3 inflammasome-associated protein expression levels, including those of NLRP3, ASC, pro-caspase-1, and caspase-1 (Fig. 4H-I).Moreover, 2-DG markedly decreased the release of IL-18, IL-1β, IL-6 and TNF-α(Fig. 4J-M).

Similarly, to verify that glycolysis in macrophages promotes NLRP3 inflammasome activation, we also performed in vitro cellular assays. We first grouped the cells by corresponding stimulation, detected the lactate levels and key glycolytic enzyme levels to confirm successful intervention in all the groups (Fig. 5A-G), and then performed Western blotting and ELISA analyses. Compared with the control group, levels of NLRP3 inflammasome-related proteins and key enzymes of glycolysis were increased in the LPS group. Protein expression of the NLRP3 inflammasome was inhibited after administration of the glycolytic inhibitor 2-DG, while the levels were upregulated in the group administered FBP compared to the LPS group(Fig. 5H-I). Furthermore, the same trend was seen for levels of inflammatory factors (including IL-18, IL-1β, IL-6 and TNF-α) detected in cell culture supernatants by ELISA experiments (Fig. 5J-M). These results indicate that macrophage glycolysis participates in the activation of the NLRP3 inflammasome.

Inhibition of glycolysis can inhibit smooth muscle proliferation in the pulmonary arteries.

Abnormal proliferation of PASMCs is one of the markers of the pathogenesis of PAH. We hypothesized that glycolysis affects the proliferation of PASMCs. After treating normal rat PASMCs with macrophage supernatants from each group for 24 h, cells were collected to perform the scratch assay, Transwell migration assay, CCK-8 assay, and EdU fluorescence assay to detect their proliferation. The 24 h scratch results showed that PASMCs of the LPS + FBP group had the fastest wound healing rate, followed by the LPS and LPS + 2-DG groups; the slowest wound healing rate was observed in the control group. The results of the Transwell migration assay were similar to those of the scratch assay. The CCK-8 assay results showed that compared with the LPS group, supernatants of macrophages treated with LPS + FBP significantly promoted PASMC proliferation, while LPS + 2-DG inhibited PASMC proliferation. The EdU assay results were consistent with the CCK-8 assay results (Fig. 6A-F). This indicates that inhibition of glycolysis attenuates the proliferation of PASMCs.

β-catenin activation triggers NLRP3 inflammasome activation by glycolysis in macrophages.

Huang (24) noted that β-catenin expression contributes to the activation of the NLRP3 inflammasome. Consistent with their results, we found that levels of proteins related to the NLRP3 inflammasome in lung tissue from the MCT + XAV939 group were decreased compared to those in lung tissue from the MCT rats. Concurrently, the ELISA results showed that cytokine levels in the lung tissue supernatants of rats from the MCT + XAV939 group were also decreased compared to those of the MCT group (Fig. 7A-F). These results indicate that β-catenin expression may regulate the activation of the NLRP3 inflammasome.

To further elucidate the effect of β-catenin on NLRP3 activation in macrophages, we extracted cell proteins and examined the expression levels of proteins related to the NLRP3 inflammasome. Compared with those in the control group, the levels of NLRP3, ASC, pro-caspase-1, and caspase-1 were all increased in the LPS group. Using the LPS group as a reference, the above indicators showed decreased levels in the LPS + XAV939 group; the opposite results were seen in the LPS + LiCl group. In addition, levels of the key enzymes of glycolysis and of NLRP3 inflammasome-related proteins were decreased in the LPS + XAV939 + FBP group compared with the LPS + XAV939 group. Moreover, the detection of cytokine levels in the supernatants of macrophages of each group showed the same trend as those in the lung tissue (Fig. 8A-F). Thus, we conclude that β-catenin participates in the activation of the NLRP3 inflammasome by regulating glycolysis in macrophages.

Inhibition of β-catenin attenuates PASMC proliferation.

To determine the effect of β-catenin expression on the proliferation of PASMCs, we used macrophage supernatants from the control, LPS, LPS + XAV939, LPS + LiCl and LPS + XAV939 + FBP groups to treat normal rat PASMCs for 24 h. Subsequently, we performed scratch experiments, Transwell migration assays, CCK-8 assays, and EdU fluorescence detection. The 24 h scratch results showed that the PASMCs had the fastest wound healing rate in the LPS + XAV939 group, followed by the LPS and LPS + LiCl groups; the slowest wound healing rate was observed in the control group. The same trend was foreseen in the Transwell migration assay. The CCK-8 assay results showed that the supernatants of macrophages treated with LPS + LiCl significantly promoted PASMC proliferation compared with the LPS group, while XAV939 inhibited PASMC proliferation. The EdU assay results were consistent with the CCK-8 assay results (Fig. 9A- F). These results indicate that β-catenin inhibition attenuated the proliferation of PASMCs.

β-catenin is involved in the pathogenesis of many diseases, but its role in PAH remains unclear. In this study, we have demonstrated that (1) upregulation of β-catenin is observed in a PAH rat model induced by MCT and (2) the Wnt/β-catenin inhibitor XAV939 has a protective effect against PAH. Further, (3) Wnt/β-catenin signaling pathways mediate glycolysis in macrophages, (4) enhanced glycolysis promotes the activation of the NLRP3 inflammasome in macrophages, and (5) Wnt/β-catenin signaling increased the secretion of IL-18, IL-1β and TNF-α inflammatory cytokines that can promote PASMC proliferation. These results show that Wnt/β-catenin signaling participates in PAH by promoting the inflammatory response through the upregulation of glycolysis in macrophages, suggesting that inhibition of Wnt/β-catenin signaling is a potential therapeutic option for PAH.

Pulmonary hypertension is a group of chronic progressive diseases in which inflammation and immune mechanisms play an important role in the development and pathogenesis. Higher levels of inflammation and increased infiltration of immune cells in lung tissue of individuals with PAH are strong evidence for the involvement of inflammation in PAH. Moreover, abnormal metabolism, especially aerobic glycolysis or the Warburg effect, also plays an important role in the development of PAH. Existing studies suggest that changes in levels of key enzymes of glycolysis and their metabolites contribute to the regulation of innate immune function involved in the inflammatory response (36–38). Some scholars even call it a “close criminal partner” in glycolysis and inflammation. Key enzymes of glycolysis directly play critical roles in the initiation, assembly, maturation, and secretion of the NRLP3 inflammasome through various pathways (39). In our study, glycolysis had a positive effect on the activation of the NLRP3 inflammasome. The glycolytic agonist FBP promoted the activation of the NLRP3 inflammasome and increased the secretion of inflammatory factors, while the glycolytic inhibitor 2-DG caused decreased secretion of inflammatory factors.

High expression of β-catenin was associated with pulmonary hypertension, and pharmacological inhibition of β-catenin by XAV939 attenuated PASMC cell proliferation (40). The Wnt/β-catenin pathway plays a key role in maintaining tissue homeostasis. Dysregulation of Wnt/β-catenin signaling often leads to a variety of severe lung diseases, including chronic obstructive pulmonary disease (COPD), lung inflammation, idiopathic pulmonary fibrosis (IPF), hyperoxic injury, bronchopulmonary dysplasia (BPD) silicosis, and lung cancer (41). Sustained activation of β-catenin may drive inflammatory and fibrotic changes in the lungs (42), but the specific mechanism of action is not clear. We hence sought to delineate the characteristics of β-catenin in the development of PAH in our study. It has been reported that activation of the Wnt/β-catenin pathway leads to increased glycolysis (43–45), and in agreement with these findings, we identified β-catenin as a stimulus of glycolysis in this study. Our results show that β-catenin activation can indeed cause an increase in HK2, PKM 2, and LDHA, key glycolytic enzymes, which is consistent with the study of Lidi Deng et al (46). With the increase in glucose consumption and lactate secretion and the upregulation of glycolytic metabolic enzyme levels in macrophages, blocking β-catenin alleviated inflammation and limited glycolysis in the lung.

However, the involvement of β-catenin in lung inflammation may be related to the status of AMs. Wnt/β-catenin signaling can inhibit AM self-renewal and promote inflammation (47). Several studies have shown that the interaction of β-catenin and the hypoxia-inducing factor HIF-1 α (HIF1-α), a key protein that regulates glycolysis metabolism, promotes glycolysis-dependent AM inflammation while inhibiting mitochondrial metabolism and inhibiting the proliferation of AMs (47, 48). However, this is only one of the regulatory mechanisms involved, and the role of β-catenin in the inflammatory response is complex. It may participate in inflammation in other ways, such as in the processes of apoptosis and autophagy. Zhang et al. verified that H19 facilitated proliferation and inhibited apoptosis by modulating the Wnt/β-catenin signaling pathway (49). Fan et al. verified that Wnt/β-catenin signaling pathway activation mediated the effect of autophagy on HCC cell glycolysis (50). Although sustained activation of Wnt/β-catenin signaling is associated with PAH, the mechanism remains largely unexplored.

Furthermore, there is endogenous crosstalk between Wnt/β-catenin signaling and the NLRP3 inflammasome, and β-catenin inhibitor XAV939 inhibition can impair NLRP3 inflammasome activation (24), which is consistent with our results. We first demonstrated that XAV939 can reduce the expression of NLRP3 inflammasome-associated proteins and inhibit the secretion of inflammatory factors, indicating that the activation of the NLRP3 inflammasome was inhibited. Second, when LiCl was used to agonize β-catenin, glycolysis was upregulated, and the NLRP3 inflammasome was activated, promoting PASMC proliferation.

There are some limitations in our study. First, only the MCT-induced PAH model was used in our experiments, and the results should be validated in other PAH models, such as hypoxic rats, hypoxic mice, and hypoxia + SU5416-treated rats. Second, given the pathophysiological distinctions between mice and humans, our findings should be further validated in clinical studies. Third, we overexpressed β-catenin in the lungs, and whether this phenomenon occurs in other organs, such as the kidney and liver, should be further investigated. Fourth, although our data suggest a therapeutic role for β-catenin, the inhibition of the PAH glycolysis pathway through activation of NLRP3 inflammasome signaling still requires further investigation of pulmonary artery remodeling.

In conclusion, our study reveals a critical role for the Wnt/β-catenin signaling pathway in controlling the inflammatory response via the regulation of glycolysis. This reprogramming of glucose metabolism by Wnt/β-catenin is essential for NLRP3 inflammasome activation. Therefore, the Wnt/β-catenin/glycolytic pathway in macrophages may represent a target for novel treatments of PAH.

PAH Pulmonary arterial hypertension

NLRP3 NOD-like receptor family pyrin domain containing 3

PASMCs Pulmonary artery smooth muscle cells

AM Alveolar macrophages

FBS Foetal bovine serum

ELISA Enzyme-linked immunosorbent assay

TNF-α Tumor necrosis factor alpha

IL-1β Interleukin-1β

HK2 Hexokinase 2

PKM2 M2-pyruvate kinase

LA Lactate

PFK Phosphofructokinase

LDH Lactate dehydrogenase

Data availability

The datasets supporting the findings of this article are included within the paper .

Acknowledgments

Not applicable.

Funding

This study was supported by grants from the National Natural Science Foundation of China (Grant no. 82060051); the projects of Guangxi Medical and Health Appropriate Technology Development and Promotion Application (Grant no. S2020030); the Precision Medicine Foundation of Guangxi Key Laboratory of Cardio-cerebrovascular Disease (Grant no. GXXNXG202001); Guangxi medical “139” Program for Training High-level Backbone Talents (Grant no. G201903053) and Project of training one thousand young and middle aged backbone teachers of Guangxi.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

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No competing interests reported.

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Wnt/β-catenin signaling pathways mediate monocrotaline-induced pulmonary arterial hypertension via glycolysis in rats

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Abstract

Figures

Introduction

Materials and methods

Experimental animals

Rat model of MCT-induced PAH and experimental groups

Echocardiography

Hemodynamic measurements and tissue processing

Histological analysis

Primary rat PASMC and alveolar macrophage (AM) isolation

Rat alveolar macrophage cell line (NR8383)

Coculture of PASMCs with NR8383

Cell counting kit-8 assay

Phosphofructokinase (PFK) and lactate dehydrogenase (LDH) activities and content of glucose and lactate (LA) determination

ELISA

Western blot analysis

Cell migration

Statistical analysis

Results

Increased expression of β-catenin in AMs from rats with MCT-induced PAH

An inhibitor of β-catenin (XAV939) attenuated the progression of PAH induced by MCT in rats

β-catenin regulates glycolysis in macrophages

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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