Background
Pulmonary arterial hypertension (PAH) is a rare but life-threatening disease characterised by pulmonary vasoconstriction, endothelial cell proliferation, smooth muscle cell proliferation, and
in situ thrombosis, leading to progressive pulmonary hypertension and ultimately causing right ventricular (RV) failure and death [
1‐
3]. The current therapies licensed for PAH focus on vasodilation [
4]. Drugs targeting the prostacyclin, endothelin-1 receptor, and phosphodiesterase pathways improve symptoms and exercise tolerance, but persistent morbidity and mortality indicate that important pathogenic mechanisms are minimally affected [
5,
6]. There is accumulating evidence of a specific contribution of NLRP3 and related inflammasomes, and their regulated cytokines or receptors may represent novel diagnostic or therapeutic targets in pulmonary diseases, including PAH [
7‐
9].
The NLRP3 inflammasome comprising the apoptosis speck-like protein containing a caspase-recruitment domain (ASC), NLRP3, and procaspase-1, plays a key role in innate immunity and lung injury [
10]. The NLRP3 inflammasome is activated in response to cellular stresses through a two-component pathway involving a Toll-like receptor 4-ligand interaction (priming) followed by a second signal. In particular, extracellular ATP is the best-known danger signal in NLRP3 activation via stimulation of the P2X
7 purinergic receptor (P2X
7R) [
11]. Despite acting as a co‑stimulus or second signal for the formation of an NLRP3 inflammasome, the role of P2X
7R has not been previously characterised in models of PH. P2X
7R is a highly unusual ATP-gated non-selective cation channel expressed primarily on cells of haematopoietic origin, such as macrophages and microglia. P2X
7R signalling is involved in the regulation of many physiological and pathophysiological processes such as silica-induced lung-disease. High extracellular ATP levels are released into the extracellular medium due to cell damage, hypoxia or mechanical stress, alerting the immune system to sites of cell damage/injury [
12]. Recently, there has been growing evidence to implicate the ATP-P2X7-inflammasome-caspase 1-IL-1/18 axis in lung diseases such as murine models of hyperoxia-induced acute lung injury, smoke-induced airway inflammation and patients suffering from COPD [
13,
14]. P2X
7R, based on its role in the processing of the NLRP3 inflammasome and IL-1β, represents a reasonable target in the study of the pathogenesis of PAH.
Therefore, the purpose of this study was to determine the extent to which the inhibition of P2X
7R would suppress pulmonary vascular remodelling in an animal model with neointimal lesions resembling the neointimal lesions found in PAH. For pharmacological P2X
7R inhibition, we applied A-740003, which is a competitive antagonist of P2X
7R and is more potent and selective than any other antagonist with fewer species-dependent differences in various preclinical disease models [
12,
15,
16].
Methods
Animal models
Male Sprague-Dawley rats (weighing 300–330 g, obtained from the Laboratory Animal Center, Chinese Academy of Science, Beijing) were used in this experiment. The rats were housed at 20 ± 3 °C under a 12-h light/12-h dark cycle with free access to food and water. All procedures were conducted according to approved protocols and guidelines established by the Shandong University Institutional Animal Care and Use Committee.
Rats were randomly assigned to one of four possible groups: group A (
n = 15) was the sham group; group B (
n = 30) was the P/MCT group, in which PH rats received vehicle treatment; group C (
n = 20) was the P/MCT + CA group, in which PH rats received continuous and early administration of A-740003; and group D (
n = 20) was the P/MCT + DA group, in which PH rats received delayed administration of A-740003. PH was induced by left pneumonectomy plus MCT injection. For anaesthesia, the animals received 2% xylazine (4 mg/kg)/ketamine (100 mg/kg) and were intubated. After connection to a small-animal ventilator (HX-300S, TME, Chengdu, China), the animals received an adjusted rate of 60 breaths/min and a tidal volume set to 1.1–1.3 mL/100 g body weight, followed by a left unilateral pneumonectomy as described previously [
17]. MCT (Sigma, St. Louis, MO) was prepared as previously described. MCT (60 mg/kg) was injected subcutaneously 1 week later. All animals were monitored daily until they developed pulmonary hypertension symptoms such as weight loss and tachypnea.
Animals in Group C received continuous and early A-740003 (Sigma-Aldrich), a selective P2X
7R inhibitor, intraperitoneally at a dose of 50 mg/kg from the time of pneumonectomy to day 28 [
18‐
20]. Animals in Group D received delayed administration of A-740003 drug 2 weeks before harvest (2 weeks after MCT). Once initiated, A-740003 in Group D was continued until harvest. Sham control animals received water alone. Haemodynamic, morphologic, and biochemical assessments were performed on day 28 after MCT injection.
Echocardiography and haemodynamic measurements
The rats in the experimental groups were anaesthetised by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The room temperature was maintained at approximately 25 °C. A Visual Sonics Vevo 770 echocardiographic machine (Visual Sonics, Toronto, Canada) equipped with a 14-MHz linear transducer was used to assess cardiac function. The measurements were performed in a blinded manner by an echocardiographic expert. Short- and long-axis B-dimensional parasternal views of both ventricles at the level of the papillary muscles were acquired to visualise the areas of the left ventricle (LV) and the right ventricle (RV). Cardiac output and stroke volume were obtained from the B-mode long axis according to Simpson’s method, while the pulmonary artery diameter and RV wall thickness were obtained in M-mode. Doppler was applied to the pulmonary artery to obtain the pulmonary artery acceleration time [
21].
Blood pressure was evaluated with the tail-cuff method using a non-invasive automatic blood pressure recorder (BP-98A; Softron, Tokyo, Japan). Each value was the average of at least three consecutive measurements [
22]. Prior to sacrifice of the animals, RV systolic pressure (RVSP) was transduced from the right jugular vein into the vena cava and then into the right atrium followed by the right ventricle using a 1.4 F Millar Mikro-Tip catheter transducer (Millar Instruments Inc., Houston, TX). The position of the catheter into the right ventricle was validated by an acutely increased pressure wave accompanied by the loss of resistance, and RVSP was then measured with Power Lab monitoring equipment (Millar Instruments). Haemodynamic values were automatically calculated using a LabChart 7.0 physiological data acquisition system (AD Instruments, Sydney, Australia). The animals were then euthanised prior to sacrifice.
Tissue processing and histology
After acquiring the above measurements, cardiac arrest was induced by injection of 2 mmol KCl through the catheter. The left lungs were then weighed. The lung was then separated longitudinally into two parts: one was removed and frozen in liquid nitrogen for western blot analysis, and the other was inflated with 0.5% low-melting agarose at a constant pressure of 25 cm H
2O, fixed in 10% formalin for 24 h and used for small pulmonary artery and IHC analyses. Next, the heart was excised, and the weight ratio of the right ventricle to the left ventricle plus the septum (RV/LV + S) was determined using Fulton’s index [
23].
Western blot
For immunoblot analyses, modified RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China) was used to extract total protein from frozen lung tissue [
24]. Extraction proteins from tissues and cells were measured using the BCA protein assay reagent kit (Pierce). An equal amount of total protein (80 μg of protein/lane) was resolved on a 5–12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat dry milk in PBST (containing 0.05% Tween 20). Incubation with the antibodies was performed using the following dilutions: 1:750 for P2X
7R (Abcam, USA) and 1:1000 for caspase-1, procaspase-1, caspase-1, IL-1β (all of them from Cell Signaling Technology, USA) and total NLRP3 (Biosource, Belgium). Primary antibodies were detected with horseradish peroxidase-conjugated antibodies, 1:5000 for anti-mouse (ZSJQ-BIO, Beijing, China) and 1:5000 for anti-rabbit (ZSJQ-BIO, Beijing, China), at room temperature for 1.5 h. Blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore) and visualised using a FluroChem E Imager (Protein-Simple, Santa Clara, CA, USA). Blot bands were qualified using NIH ImageJ software.
RT-PCR
Total RNA was extracted from lung tissues with TRIzol reagent (Invitrogen). cDNA was synthesised from 2 μg RNA using a Prime Script RT Reagent Kit (TaKaRa, Dalian, China) as described previously. mRNA expression was determined using gene-specific primers and SYBR Green 1 with a Bio-Rad iQ5 Multicolor Real-Time PCR machine (Bio-Rad Laboratories). For each sample, both GAPDH and the target gene were amplified in triplicate in separate tubes. The relative gene expression was calculated using the 2
-ΔΔCT method [
25] and normalised to GAPDH expression. The primers used in this study were as follows:
NLRP3: forward, 5′-CTGCATGCCGTATCTGGTTG-3′, reverse, 5′-GCTGAGCAAGCTAAAGGCTTC-3′;
Caspase-1: forward, 5’-ACTCG TACAC GTCTTGCCCTCA-3’, reverse, 5’-CTGGGCAGGCAGCAAATTC-3’;
P2X7R: forward, 5′-CTACTCTTCGGTGGGGGCTT 3′, reverse, 5′- AACCCTGGTCAGAATGGCAC 3′;
IL-1β: forward, 5′-GCACAGTTCCCCAACTGGTA-3′, reverse, 5′-TGTCCCGACCATTGCTGTTT-3′;
GAPDH: forward, 5′-AGATCCACAACGGATACATT-3′, reverse, 5′-TCCCTCAAGATTGTCAGCAA-3′;
Immunohistochemistry
The left lung lobes were longitudinally cut and processed as described previously [
21] by preparing standard formalin-fixed, paraffin-embedded tissues for HE or regular immunohistochemistry staining. Tissue samples were sectioned at a thickness of 5 μm [
26]. In each lung section, 30 small PAs (50–100 μm in diameter) were analysed at × 40 in a blinded manner. The medial wall thickness was expressed as the summation of two points of medial thickness/external diameter × 100 (%). Intra-acinar (precapillary) PAs (20–30 μm in diameter, 25 vessels each) were assessed for occlusive lesions as Grade 0 for no evidence of neointimal lesion, Grade 1 for less than 50% luminal occlusion, and Grade 2 for more than 50% luminal occlusion [
27]. There was no evidence of neointimal lesion formation in any PAs from normal rats (all PAs were graded as 0). Fibrosis was evaluated on heart tissue sections stained with Masson’s trichrome (Jiancheng, China) according to standard protocols [
28]. Anti-P2X
7R (1:100, Abcam), α-SMA (1:500; Abcam) and anti-CD68 (1:150; Abcam) antibodies were used as primary antibodies. Subsequently, slides were incubated using an ABC Elite Kit (Vector Laboratories) and DAB substrate (Vector Laboratories) and counterstained with haematoxylin.
For immunofluorescence, samples were incubated with anti-P2X7R antibody (1:50; Abcam) and α-SMA (1:200; Abcam) or FITC-CD68 (1:100; Abcam) overnight at 4 °C, followed by a 2-h incubation with Alexa 545-conjugated goat anti-rabbit (1:100; Peprotech) or FITC-conjugated goat anti-rabbit (1:200; Abcam) and FITC-conjugated rabbit anti-mouse (1:200; BioLegend) secondary antibodies. The α-SMA -positive cells in each group were double-immunostained with anti-proliferating cell nuclear antigen (PCNA) (1:300; Abcam). The sections were counterstained with DAPI (Life Technologies) to identify nuclei. The contribution of α-SMA to PCNA expression was measured semi-quantitatively by the proportion of colocalization cells (i.e. yellow staining in merged images) divided by the number of corresponding staining SMA cells.
A pathologist blinded to the study reviewed 10 sections per lung. All images were obtained using an Olympus LCX100 Imaging System and analysed with ImageJ software (version 1.38x; National Institutes of Health). The Institutional Review Board of Shandong University approved the studies.
Bronchoalveolar lavage fluid (BALF)
Bronchoalveolar lavage was collected and analysed for macrophage influx and cytokines such as TNF-α and IL1-β as described previously [
29]. Briefly, the trachea was cannulated, and BALF was obtained by administering three consecutive injections of phosphate-buffered saline (PBS) to a final volume of 1.0 mL. The BALF was then centrifuged at 400
g for 10 min (Mikro 22 R, Hettich), and the supernatant was stored at −20 °C. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit (Peprotech, NJ) was used to detect serum TNF-α and IL-1β concentrations according to the manufacturer’s instructions. The intra- and inter-sample variability for each kit was less than 8%.
Statistics
The data are presented as the mean ± standard deviation (SD). The unpaired t-test was used to compare values between two groups. Analysis of variance (ANOVA) was used to compare differences among more than two groups, followed by a Newman-Keuls test. Analyses were performed using SPSS 17.0 software (SPSS Inc. Chicago, IL, USA). A p-value < 0.05 was considered statistically significant.
Discussion
To our knowledge, this is the first study to demonstrate the role of P2X7R in the pathogenesis of PAH. Pretreatment with A-740003, a P2X7R inhibitor, attenuated the development and progression of MCT-induced pulmonary hypertension, RV hypertrophy, and pulmonary arterial neointimal formation in pneumonectomised rats. Moreover, administration of A-740003 even at 2 weeks after MCT could retard the progression of pulmonary hypertension.
Considerable circumstantial evidence suggests that inflammation plays an important role in the pathobiology of PAH. In clinical work, subjects with idiopathic or heritable PAH with higher levels of inflammatory cytokines are associated with higher mortality in PAH [
35]. In response to injury and stress, lung vascular cells produce inflammatory mediators, thereby recruiting monocytes/macrophages. Inflammatory cells might continue the release of cytokines and growth factors, forming positive feedback loops. These are characteristic features of the pulmonary inflammatory process and could lead to vascular remodelling by matrix remodelling, collagen deposition, and vascular cell proliferation and migration in PAH. Therefore, the control of inflammation is important for the prevention or treatment of PAH.
In this study, MCT-challenged left pneumonectomised rats showed a marked increase in the recruitment of macrophages into perivascular and peri-alveolar areas of pulmonary tissues and bronchoalveolar lavage samples, consistent with previous reports [
36]. Studies have shown that the extracellular P2X
7R axis contributes to the development of lung inflammation [
37], as well as the proliferation and migration of vascular smooth muscle cells [
38]. The above data provide evidence for a potential role of P2X
7R signalling in the development of PAH. We observed increased P2X
7R activation in the lung tissues of PH rats, most likely due to the early signal of ATP release secondary to stretch-activated channels caused by the elevated pulmonary pressure and MCT-induced lung EC injury [
39,
40]. Activated P2X
7R co-localised with both myeloid lineage cells and constitutive vascular cells. Therefore, we further explored the extent to which P2X
7R participated in the process of pulmonary vessel remodelling by P2X
7R inhibitor—A-740003, which is broadly used in preclinical disease models [
12]. Our data showed that either pretreatment or treatment with A-740003 could decrease macrophage recruitment. Furthermore, the downregulated expression of pro-inflammatory cytokines identified by ELISA validated the suppressive effect of P2X
7R-targeted therapy on inflammation in PAH, demonstrating a direct link between P2X
7R and a pro-inflammatory phenotype during the inflammatory process of PAH. Eventually, A-740003 ameliorated pulmonary vascular remodelling and pulmonary hypertension in both preventive and therapeutic manners. However, the exact mechanism by which A-740003 prevented PH and inflammation remained elusive until the discovery of the NLRP3 inflammasome/IL-1β pathway.
P2X
7R activation acts as the second signal in NLRP3 inflammasome formation, which results in the cleavage of procaspase-1 into active caspase-1 [
41]. Caspase-1 could further cleave the IL-1β precursor to form the mature and secreted forms [
42]. P2X
7R is highly expressed in mononuclear macrophage cell lines. Moreover, most of the pharmacological evidence regarding the therapeutic potential of targeting P2X
7R was focused on the processing and release of IL-1β from activated monocytes/macrophages and microglia [
43‐
45]. Hence, P2X
7 may orchestrate macrophage-dominated inflammation during the PH process. In our study, activated P2X
7R highly co-localised with CD68 positive macrophages in the lung tissue of PH rats. The blockade of P2X
7 was associated with the inhibition of NLRP3 activation and the decrease of mature IL-1β release, compatible with a scenario in which sustained P2X
7R activation of alveolar macrophages somehow provided the second signal necessary for NLRP3/ASC/caspase-1 inflammasome aggregation, proteolytic maturation/activation of caspase-1, pro-IL-1β cleavage, and subsequent IL-1β release. The mature IL-1β is a prototypic multifunctional cytokine that is involved in pulmonary inflammation and could stimulate chemokines and adhesion molecules, such as MCP-1 and MIP-1α for macrophage recruitment [
46,
47]. Therefore, the mechanisms responsible for the decrease in macrophage infiltration by A-740003 may be caused by a specific decrease in IL-1β. Both caspase-1 and IL-1β have been shown to participate in the pathogenesis of experimental PH in gene knockout and specific antagonist studies [
8,
48,
49]. Therefore, P2X
7R potentially has roles in PH by mediating NLRP3 inflammasome activation and IL-1β secretion. In contrast, A-740003 likely decreased IL-1β production due to the loss of macrophage infiltration and caspase-1 activation, participating in the reversal of PH. We speculate that ATP release acts as an early signal via P2X
7R activation to trigger pulmonary cell responses including inflammasome activation, leading to mature IL-1β and factor expression remodelling.
Despite being the most important pathophysiological response triggered by the activation of the P2X
7R/NLRP3 inflammasome axis and release of IL-1β, we showed that P2X
7R was also strongly expressed by PA-SMCs. Therefore, we could not rule out the possibility that P2X
7 may function on PA-SMCs directly instead of through the NLRP3/IL-1β pathway. The complex vascular lesions associated with PAH appear to be governed by the same traits that control cancer growth, the absence of apoptotic cells and the presence of anti-apoptotic proteins in the lesion cells. Recently, it has been clearly shown that P2X
7R can also support cell growth, mainly by increasing the endoplasmic reticulum Ca
2+ content and the mitochondrial potential, thereby activating NFATc1, preventing apoptosis and promoting cell proliferation [
50,
51]. In addition, P2X
7R has a clear role in the activation of immune cells, and P2X
7R also mediates the release of factors that can modulate the inflammatory state of vessel wall P2 receptors, which are potential targets for the treatment of hypertension [
52]. Histological and molecular analysis revealed that A-740003 treatment abrogated significant ECM deposition (collagens) and PA remodelling in the lungs, supporting the dual effect of P2X
7R on macrophage migration and activation as well as on PA-SMC proliferation. Does P2X
7R activation exert direct proliferative and pro-survival roles for the pulmonary artery? To answer this question, future studies focusing on the effects of vascular P2X
7R signalling on ECs and SMA proliferation should be conducted.
Clinical implications
Our study proposes that P2X
7R-specific inhibitors may be a promising approach to improve the life span and quality of life for patients with idiopathic PAH (iPAH). In addition to iPAH, CTD-associated PAH accounts for nearly half of patient disease aetiologies in the oldest age group, which benefits less from iPAH-targeted therapy [
53]. Therapy employing the blockade of P2X
7R pathways has been proven to be effective in various connective tissue diseases (CTDs) [
54]. Therefore, A-740003-directed therapeutic strategies also seem to be justified in other forms of severe PAH. The poor prognosis of patients afflicted by this disease despite treatment with currently available vasodilator drugs makes the development of new treatment strategies imperative. If vascular remodelling can be developed from redundant mediators, a combination “cocktail” consisting of anti-remodelling agents such as endothelin inhibitors, prostacyclin, and anti-inflammatory agents such as A-740003 may be more beneficial for patients with PAH than any single agent alone [
55].
Limitations
First, our study lacked specific NLRP3 antagonist interference or NLRP3 gene deficiency analysis in animals to confirm the role of NLRP3 in PAH. Second, other PH models like hypoxia or pulmonary artery banding in rodents must be further investigated in the context of anti-P2X7R treatment. Third, A-740003 may function as an off-target effect other than acting on P2X7R.
Acknowledgements
The work was supported by the National Natural Science Foundation of China (NSFC, 81570305;81370157), the Doctoral Fund of the Ministry of Education of China (20130131110069), Science and Technology Development Planning of Shandong Province (2013GGB14056), the Independent Innovation Foundation for Jinan Science and Technology Developmental Planning (201311020), Projects of medical and health technology development program in Shandong province (2016WS0457) and the Shandong Taishan Scholarship (Suhua Yan).