The A_2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease

Abstract

Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A_2BR) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A_2BR or treatment with the A_2BR antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A_2BR attenuated vascular remodeling and hypertension in our model. Furthermore, direct A_2BR activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A_2BR antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.—Karmouty-Quintana, H., Zhong, H., Acero, L., Weng, T., Melicoff, E., West, J. D., Hemnes, A., Grenz, A., Eltzschig, H. K., Blackwell, T. S., Xia, Y., Johnston, R. A., Zeng, D., Belardinelli, L., Blackburn, M. R. The A_2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease.

Pulmonary hypertension (PH) is a disorder of the pulmonary vasculature characterized by increased vascular resistance leading to right ventricle (RV) hypertrophy and, ultimately, to right-sided heart failure and death (1). The pathology associated with this disorder is characterized by extensive remodeling of the vasculature that ultimately leads to increased proliferation of pulmonary artery endothelial and smooth muscle cells, muscularization of previously nonmuscular arteries, increased vascular tone, and formation of complex vascular lesions (2). Research and therapeutic efforts for this disorder have largely focused on pulmonary arterial hypertension (PAH), a form of PH with an incidence of 2–4 cases/million. However, development of PH is also a common complication of interstitial lung disease (ILD), with a prevalence of 30–40% among this patient group (3). The most common ILDs associated with PH are idiopathic pulmonary fibrosis (IPF), sarcoidosis, and systemic sclerosis (3). The prevalence of PH in patients with IPF has been reported to be up to 84% (4), and PH is a predictor of mortality in all patients with ILD (3). Despite its prevalence, little is known regarding the cellular and molecular mechanisms that lead to PH in patients with ILD (3) and as a result, effective treatment options are lacking.

In the pathogenesis of many ILDs, particularly IPF, repetitive injury processes lead to progressive fibrotic remodeling of the lung parenchyma (5). Due to the complex pathogenesis of PH secondary to ILD, where the development of PH is intimately linked to the progression of lung disease, it is essential to understand the interrelationship of fibroproliferation and PH. Several mediators have been identified that are involved in both fibroproliferative lung disease and vascular remodeling, including hypoxia (6, 7), reactive oxygen species (8, 9), endothelin-1 (ET-1; refs. 10–12), and interleukin-6 (IL-6; refs. 13–15). Interestingly, the production of these mediators is closely associated with the adenosine signaling system (16, 17), which has also been shown to regulate the progression of remodeling processes in ILDs (18). However, the interrelationship between adenosine signaling and these mediators has not been examined in the context of PH secondary to ILD.

Adenosine is an extracellular signaling molecule that is generated in response to cell injury, where it modulates tissue protection and repair (19). In chronic disease states, elevated adenosine levels have been implicated in disease progression and tissue remodeling (18). Adenosine exerts its actions through G-protein-coupled receptors on the cell surface that lead to stimulation of a wide array of signaling molecules (20). Four adenosine receptors have been identified, the adenosine 1, 2A, 2B, and 3 receptors (A₁R, A_2AR, A_2BR, and A₃R, respectively; ref. 20), and among these, the A_2BR has emerged as the receptor that appears to regulate many of the adenosine-driven remodeling responses seen in chronic lung disease (18). Levels of the A_2BR are elevated in patients with ILD (21, 22) and in various models of fibroproliferative lung disease (23–25). Moreover, genetic removal of the A_2BR or treatment with an A_2BR antagonist can attenuate remodeling responses in the lung in vivo (14, 25, 26). Pertinent to PH secondary to ILD, A_2BR signaling regulates the production of IL-6 (14, 17, 26) in chronic lung disease.

Taken together, these observations suggest that adenosine acts as a mediator of lung fibrosis and may play a role in the development of PH in ILD. The objective of this study was to investigate the role of the A_2BR in modulating vascular remodeling and PH in an experimental model of pulmonary fibrosis induced by repetitive bleomycin exposure. We hypothesized that adenosine acting via the A_2BR is a central player in the development of fibrotic lesions and vascular remodeling in the lung, leading to PH secondary to lung fibrosis. Our findings show that bleomycin-exposed mice present with features of vascular remodeling and develop elevated right ventricle systolic pressure (RVSP), a measure of PH, in association with adenosine elevations. These changes in the vasculature were accompanied by elevated levels of markers of PH, such as hypoxia-inducible factor 1α (HIF-1α), IL-6, and ET-1. Furthermore, we demonstrate, using a selective antagonist for the A_2BR, GS-6201, that blockade of this receptor inhibits vascular remodeling and increases in RVSP through the down-regulation of IL-6 and ET-1. These observations are confirmed in mice lacking the A_2BR that do not develop vascular changes or elevated RVSP following bleomycin exposure. In addition to restoring pulmonary vascular function, blockade or genetic removal of the A_2BR was also able to attenuate the development of pulmonary fibrosis. These results suggest that adenosine acting through the A_2BR is able to orchestrate changes in the lung that lead to fibrosis as well as vascular remodeling and PH, highlighting the therapeutic potential of blocking the A_2BR as a novel treatment for PH secondary to ILD.

MATERIALS AND METHODS

Animals

Male C57BL6 mice, 4–5 wk old, were purchased from Harlan Industries (Indianapolis, IN, USA). A_2BR-deficient (A_2BR^−/−) mice congenic on a C57BL/6 background were generated and genotyped as described previously (27). All mice were housed in ventilated cages equipped with microisolator lids and maintained under strict containment protocols. Mice were kept at an ambient temperature of 22°C and in a 12-h light-dark cycle. Animal care was in accordance with institutional and U.S. National Institute of Health guidelines. All studies were reviewed and approved by the University of Texas Health Science Center at Houston Animal Welfare Committee.

Experimental design

Mice were treated with 0.035 U/g of bleomycin (APP Pharmaceuticals, Schaumburg, IL, USA) or vehicle (PBS; Invitrogen, Carlsbad, CA, USA) 2×/wk for 4 wk intraperitoneally (i.p.). On d 15, mice were provided with chow containing GS-6201 (10 mg/kg/d) or normal chow (vehicle; Teklad Industries, Indianapolis, IN, USA) for the remainder of the experiment. GS-6201 (previously known as CVT-6883) has been characterized in vitro and in vivo as an A_2BR antagonist (26). Ad libitum GS-6201 in the diet resulted in GS-6201 plasma levels of 375 ng/ml by d 33 of the treatment protocol. The K_I for the human A_2BR is 22 nM compared to human A₁R (K_I 1940 nM), A_2AR (K_I 3280 nM), or A₃R (K_I 1070 nM). The K_i values for the mouse receptors have not been determined; however, the capacity of GS-6201 to inhibit cAMP accumulation demonstrates a K_B (binding potency) of 2.2 nM for both the mouse and human A_2BR (26), suggesting that exposure levels in our model were appropriate for A_2BR selective antagonism. On d 33, physiological readouts were performed, and the animals were then sacrificed for the collection of tissues and fluids for analysis. All analysis was conducted in a manner in which the investigators were blinded to the respective treatment groups or genotypes.

Arterial oxygen saturation

Physiological assessment measuring arterial oxygen saturation was conducted on conscious mice using MouseOx software analysis (Starr Life Sciences Corp., Oakmont, PA, USA). The hair around the neck was removed from mice in order to use a collar clip light sensor. The MouseOx provided real-time percentage oxygen saturation of functional arterial hemoglobin by utilizing pulse oximetry measurements of light absorption from the red and infrared light-emitting diodes.

Hemodynamic measurements: RVSP, systemic blood pressure, and RV hypertrophy

This procedure was performed based on studies described previously (28). Mice were given 0.75 mg/g of 2.5% Avertin (a mixture of tert-amyl alcohol and 2,2,2-tribromoethanol; Sigma-Aldrich, St. Louis, MO, USA) to induce a surgical plane of anesthesia. Mice were placed on a heated pad (Deltaphase Isothermal model 39; Braintree Scientific, Braintree, MA, USA) and secured with surgical tape. Systemic blood pressure and pulse were measured via a tail cuff and pulse transducer run through a PowerLab noninvasive blood pressure (NIBP) controller (AD Instruments, Colorado Springs, CO, USA). Mice were then tracheotomized with a 19-gauge blunt needle (Brico, Dayton, NJ, USA) and attached to a small animal ventilator (MiniVent; Hugo-Sachs Elektronik, March-Hugstetten, Germany) and ventilated at a stroke volume of 250 μl at 100 strokes/min. The surgical site was viewed using a surgical microscope (SMZ-2B; Nikon, Tokyo, Japan) and an incision of ∼1 cm in length was made just below the xiphoid process. An alm retractor (ALM-112; Braintree Scientific) was used to expose the abdominal cavity to visualize the diaphragm and the liver. An incision was then made on the diaphragm to expose the heart, and the pericardium was removed. The RV was then identified, and a puncture was made with a 27-gauge needle. A 1-french pressure catheter (SPR-1000; Millar Instruments, Houston, TX, USA) was inserted through the puncture, and the systolic blood pressure and heart rate were continuously recorded using a PowerLab 8-SP A/D (AD Instruments) converter, acquired at 1000 Hz. All RVSP results were recorded to a personal computer utilizing Chart5.3 software (AD Instruments). After completion of the measurements, blood was collected, and the lungs were excised and flash-frozen in liquid nitrogen for RNA extraction. The heart was excised, and the atria were removed. The RV was then surgically removed, and the dry weights of the RV and left ventricle (LV), including the septum (S), were obtained to determine the Fulton index (RV/LV+S).

Lung function

Lung function experiments were performed based on previously published experiments (29, 30). On d 33, mice were anesthetized with xylazine hydrochloride (7 mg/kg, i.p.), followed by the administration of sodium pentobarbital (50 mg/kg, i.p.). Once surgical anesthesia was obtained, the mouse was tracheotomized using a 19-gauge metal cannula (Brico) and connected via an endotracheal cannula to a flexiVent system (Scireq Inc., Montreal, QC, Canada) and ventilated at a respiratory rate of 150 breaths/min and tidal volume of 10 ml/kg against a positive end expiratory pressure (PEEP) of 3 cmH₂O. The linear single-compartment model was used to assess total respiratory system resistance and elastance, whereas the constant-phase model was used to further partition responses within the lungs. In addition, quasi-static pressure-volume relationships were established. Blood was then obtained, and the lungs were inflated with 10% phosphate buffered formalin (Fisher Scientific, Fair Lawn, NJ, USA) for immunohistochemistry staining.

Bronchoalveolar lavage (BAL) and histology

Avertin was used to anesthetize mice, and airways were lavaged twice with 1 ml PBS, and 2 ml BAL fluid was recovered. Total cell counts were determined using a hemocytometer, and cellular differentials were determined by cytospinning BAL aliquots onto microscope slides and staining with Diff-Quick (Dade Behring, Deerfield, IL, USA). After lavage, the lungs were inflated with 10% buffered formalin at 25 cm of pressure and fixed at 4°C overnight. Lungs were dehydrated in ethanol gradients and embedded in paraffin, and 5-μm tissue sections were collected on microscope slides and stained with hematoxylin and eosin (H&E; Shandon-Lipshaw, Pittsburgh, PA, USA) or Masson's trichrome (EM Science, Gibbstown, NJ, USA) according to manufacturer's instructions.

Adenosine, collagen, and IL-6 content in BAL fluid

Adenosine levels in BAL fluid were quantified using high-pressure liquid chromatography (HPLC), as described previously (31). IL-6 levels were quantified in 50 μl clarified BAL samples using an ELISA kit from BD Biosystems (San Diego, CA, USA) following the manufacturer's instructions. The Sircol assay (Biocolor, Carrick, UK) was used to quantify soluble collagen levels in BAL fluid.

Immunohistochemistry

Immunohistochemistry was performed on 5-μm sections cut from formalin-fixed, paraffin-embedded lungs. Sections were rehydrated through graded ethanol to water, antigen retrieval was performed using a solution of 10 mM citric acid and heated for 20 min at 90°C, and endogenous avidin and biotin were blocked with the Biotin-Blocking System (Dako, Glostrup, Denmark). Slides were incubated with primary antibodies for mouse α-smooth muscle actin (α-SMA; 1:1000 dilution, mouse monoclonal 1 h at 37°C; Sigma-Aldrich), ET-1 (1:800 dilution, rabbit polyclonal 4°C overnight; Abcam). All sections were incubated with ABC-AP reagents and appropriate secondary antibodies. Sections were developed with Vector Red Alkaline Phosphatase Substrate Kit (Vector Laboratories, Burlingame, CA, USA) and counterstained with Gill's hematoxylin.

Morphometry

Muscularized arterioles of the lung parenchyma were observed at ×20. Muscularized arterioles were then photographed at ×40, and micropictographs were analyzed using Image Pro-Plus software (MediaCybernetics Inc, Bethesda, MD, USA). The overall area of the muscularized portion was measured for each arteriole. To account for size, the largest diameter for each arteriole was also measured. The area of the arteriole was then divided by the largest diameter to give a relative measurement of muscularization. In additional observations, 10 micropictographs of the parenchymal area were taken at ×20, the number of muscularized vessels (those positive for α-SMA) were counted for each micropictograph, and the total number per animal was obtained and averaged within the group. All experiments were conducted in a manner in which examiners were blinded to group status.

Quantitative RT-PCR

Total RNA was isolated from frozen whole-lung tissue using TRIzol reagent (Invitrogen). RNA samples were then DNase treated and subjected to quantitative real-time RT-PCR. Specific transcript levels for the mouse A_2BR, IL-6, ET-1, and α1-procollagen were determined by normalization to 18S rRNA and presented as mean normalized transcript levels using the comparative C_t method (2^ΔΔCt).

Cell culture and measurement of IL-6 and ET-1

Primary normal human pulmonary arterial smooth muscle cells and endothelial cells were obtained from Lonza (Basel, Switzerland) and cultured according to instructions provided by Lonza. Cells were washed twice in HBSS, and incubated in serum-free basal medium (Lonza) with or without agonist or antagonist of adenosine receptors for 18 h before measurement of IL-6 and ET-1. The concentrations of IL-6 and ET-1 in cell medium and plasma were determined using ELISA kits obtained from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's instructions.

RESULTS

Vascular remodeling following bleomycin exposure

Chronic intraperitoneal administration of bleomycin leads to progressive and irreversible pulmonary fibrosis (14); however, little is known regarding the involvement of hypoxia and adenosine production and signaling in this model. To address this problem, pulse oximetry was used to examine the degree of oxygen saturation on d 33 of the protocol. Results revealed a significant reduction in arterial oxygen saturation (Fig. 1A). Consistent with this finding, transcript and protein levels of HIF-1α were significantly elevated in the lungs of mice exposed to bleomycin (Fig. 1B). In addition, there were increases in the levels of adenosine in BAL fluid of mice exposed to bleomycin, and transcript levels of the A_2BR were elevated in whole-lung RNA extracts (Fig. 1C, D). These results are in line with published observations suggesting that hypoxia can promote increases in the levels adenosine and expression of the A_2BR (32) and suggest that these pathways are active in our model of pulmonary fibrosis resulting from chronic bleomycin exposure.

Figure 1.

Hypoxic markers following bleomycin (BLM) exposure. All analyses were performed on d 33 of PBS or BLM exposure. A) Changes in arterial oxygen saturation. B) HIF-1α transcript levels and Western blot images on whole-lung lysates. C) Adenosine levels, measured by HPLC, from BAL fluid of mice. D) A_2BR transcript levels from fresh frozen lungs. Results are presented as means ± se, n = 4–6/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS group; ANOVA.

PH is associated with vascular remodeling in the lung; these changes include increased vascular smooth muscle mass and neomuscularization of previously nonmuscular vessels (33). To study whether these changes occurred following chronic bleomycin exposure, we stained lung sections for α-SMA, a marker of myofibroblasts and vascular smooth muscle cells. The results show that following bleomycin exposure, increased α-SMA staining is present throughout the lung parenchyma (Fig. 2A); a phenomenon consistent with increased myofibroblasts in lung fibrosis (14). In addition, increased vascular smooth muscle mass was apparent following bleomycin exposure (Fig. 2A). Morphometric analysis aimed at identifying the extent of muscularization of the vessels showed increased thickness of the muscular wall of vessels from bleomycin-exposed mice (Fig. 2B). In addition, exposure to bleomycin led to an increase in the number of muscularized vessels compared to PBS-exposed mice (Fig. 2C). These findings demonstrate that chronic bleomycin exposure results in substantial vascular remodeling consistent with the development of PH secondary to pulmonary fibrosis.

Figure 2.

Vascular remodeling following bleomycin (BLM) exposure and the effects of GS-6201. All analyses were performed on d 33 of PBS or BLM exposure. A) Immunostaining for α-SMA to identify myofibroblasts (pink signal) in the parenchyma (top panels) and the muscular wall of vessels (arrows and bottom panels). B, C) Morphometric analysis was conducted to determine the extent of muscularization present in 5–7 vessels for each mouse in all treatment groups (B) and the number of muscularized vessels observed in 10 random micropictographs of the lung parenchyma of each mouse in all groups (C). Results are presented as means ± se, n = 5–8/group. ***P < 0.001 vs. PBS group; ^#P < 0.05, ^##P < 0.01 vs. BLM group; ANOVA.

Treatment with an A_2BR antagonist attenuates vascular remodeling

We next sought to examine the importance of A_2BR signaling in the vascular remodeling seen following chronic bleomycin exposure. This was accomplished by providing mice with a selective A_2BR antagonist, GS-6201, in the diet. Our goal was to initiate treatment with an A_2BR antagonist at a stage after pulmonary fibrosis was established to examine the therapeutic benefit to established disease. Our analysis revealed increased lavage cellularity (Fig. 3A), collagen content (Fig. 3B) and reduced arterial oxygen saturation (Fig. 3C) by d 10 of bleomycin exposure. Thus, we initiated GS-6201 treatment on d 15 and examined endpoints on d 33 (Fig. 3D). Mice exposed to bleomycin and treated with GS-6201 exhibited reduced α-SMA staining in the parenchyma and around muscular vessels of the lung compared to mice exposed to bleomycin and treated with a normal diet (Fig. 2A). These observations correlated with morphometric evaluation of the vasculature of the lung, showing that GS-6201 was able to inhibit the increase in vascular smooth muscle mass and neomuscularization of vessels induced by chronic bleomycin exposure (Fig. 2B, C). In A_2BR^−/− mice, exposure to bleomycin did not lead to an increase in vascular smooth muscle area and the number of muscularized vessels (Fig. 2B, C). In addition, no significant differences in vascular remodeling were seen between control groups (Fig. 2B, C). These results suggest that activation of the A_2BR plays an important role in the processes of vascular remodeling following chronic bleomycin exposure.

Figure 3.

Pulmonary inflammation and fibrosis on d 10 of bleomycin (BLM) exposure. A, B) Total leukocyte cell counts (A) and soluble collagen levels (B) in BAL fluid of mice treated with BLM at baseline or d 10 of BLM exposure. C) Arterial oxygen saturation at baseline and d 10 of BLM exposure. D) Diagrammatic representation of the disease model used. Results are presented as means ± se, n = 4/group. *P < 0.05; ANOVA.

Effects of chronic bleomycin exposure on cardiovascular function and hypertrophy

To determine the effect of chronic bleomycin exposure and A_2BR blockade on cardiovascular function, hemodynamic measurement, heart rate, and extent of myocardial hypertrophy were determined. These included RVSP, a common readout that is used to identify PH (28); the Fulton index, which assesses RV hypertrophy; systemic systolic pressure; and heart rate. Our results show that mice exposed to bleomycin develop increased RVSP and that treatment with GS-6201 led to a reduction in this increased RVSP (Fig. 4A). This effect was comparable to A_2BR^−/− mice that did not develop elevated RVSP when exposed to bleomycin (Fig. 4A). Similarly, bleomycin exposure resulted in an increased Fulton index compared to control groups, demonstrating that bleomycin exposure can lead to RV hypertrophy (Fig. 4B). However, pharmacological blockade of the A_2BR or use of A_2BR^−/− mice did not significantly reduce the extent of RV hypertrophy seen (Fig. 4B). Measurements of systemic systolic blood pressure (data not shown) and heart rate (Fig. 4C) did not show significant differences between treatment groups. These results demonstrate that blockade of the A_2BR is able to restore normal pulmonary vascular function following chronic exposure to bleomycin.

Figure 4.

Cardiovascular physiology after bleomycin (BLM) treatment and the effects of GS-6201. All analyses were performed on d 33 of PBS or BLM exposure. A) RVSP measured in fully anesthetized mice. B) RV hypertrophy determined by measuring the dry weight of RV and LV with the septum. C) Systemic systolic blood pressure measured with a mouse tail cuff in lightly anesthetized mice. D) Mouse heart rates, determined during RVSP measurements. Results are presented as means ± se, n = 6–8/group. **P < 0.01, ***P < 0.001 vs. PBS group; ^###P < 0.001 vs. BLM group; ANOVA.

Perivascular fibrosis following A_2BR antagonist treatment

Increased fibrotic deposition around vessels is commonly observed in PH (33). To determine whether increased perivascular fibrotic deposition was present in bleomycin-exposed mice, we performed staining with Masson's trichrome to reveal collagen bundles. Our results show increased collagen fibers next to vessels compared to control groups (Fig. 5A). This increase in collagen was reduced following GS-6201 treatment and in A_2BR^−/− mice (Fig. 5A). These observations are in line with collagen 1A transcript levels from fresh frozen lung tissue (Fig. 5B) and soluble collagen protein levels from BAL fluid (Fig. 5C), where increases in transcript and protein levels of collagen were observed in bleomycin-exposed mice but not in control groups (Fig. 5B, C). Similarly, blockade of the A_2BR was capable of reducing both transcript and protein collagen levels, and elevations were absent in A_2BR^−/− mice (Fig. 5B, C).

Figure 5.

Perivascular fibrosis and total collagen levels in the lung. All analyses were performed on d 33 of PBS or bleomycin (BLM) exposure. A) Representative histological sections stained with Masson's trichrome to reveal collagen fibers (blue signal). Asterisk denotes the region where the fibrotic fibers are present. B) Collagen 1A transcript levels from fresh frozen lung samples were normalized to expression of 18sRNA using the ΔΔC_t method. C) Soluble collagen protein levels in BAL fluid were quantified using the Sircol assay. Results are presented as means ± se, n = 5–8/group. ***P < 0.001 vs. PBS group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. BLM group; ANOVA.

We next examined the effect of chronic bleomycin exposure and the role of the A_2BR in modulating changes in lung function that have been previously reported in fibrotic airways (34). In line with such studies, we report an increase in total lung resistance (Fig. 6A), tissue damping (Fig. 6B) and static elastance (Fig. 6C) in bleomycin-exposed mice. These changes were not observed in mice treated with GS-6201 or in A_2BR^−/− mice (Fig. 6A–C). In addition, measurement of arterial oxygen saturation levels revealed that GS-6201 treatment was able to improve oxygen saturation in the lungs of bleomycin-exposed mice, an effect that was also seen in A_2BR^−/− mice (Fig. 6D). Together, these results show that blockade or genetic removal of the A_2BR is able to attenuate collagen deposition in the lungs and in areas surrounding vessels that contributes to increased stiffness of the lung.

Figure 6.

Lung function measurements after bleomycin (BLM) treatment and the effects of GS-6201. All analyses were performed on d 33 of PBS or BLM exposure. A) Dynamic resistance of the lungs. B) Tissue damping (resistance) parameters. C) Quasi-static elastance, reflecting the elastic recoil pressure on the lungs at a given volume. These measurements were performed using a Flexivent system in tracheotomized and anesthetized mice. D) Arterial oxygenation levels were determined in awake mice by pulse oximetry using the MouseOx system. Results are presented as means ± se, n = 8–9/group. ***P < 0.001 vs. PBS group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. BLM group; ANOVA.

Mediators of vascular remodeling

IL-6 has been implicated in the pathogenesis of PH (15) and is up-regulated in patients with pulmonary fibrosis (21). In our model of PH secondary to bleomycin-induced lung fibrosis, elevated IL-6 transcripts were observed following chronic bleomycin exposure (Fig. 7A). These increases correlated with increased IL-6 protein levels in the BAL fluid and plasma of bleomycin-exposed mice (Fig. 7B, C). Treatment with GS-6201 resulted in modest reductions in whole-lung IL-6 transcript levels (Fig. 7A), whereas protein levels in BAL fluid and plasma were markedly reduced (Fig. 7B, C). Similar effects were seen in A_2BR^−/− mice (Fig. 7A, B).

Figure 7.

IL-6 levels after bleomycin (BLM) and the effects of GS-6201. All analyses were performed on d 33 of PBS or BLM exposure. A) IL-6 transcript levels from fresh frozen lung samples, normalized to the expression of 18sRNA. B, C) IL-6 protein levels in BALF (B) and plasma (C), determined by using ELISA. Results are presented as means ± se, n = 4–6/group. *P < 0.05, ***P < 0.001 vs. PBS group; ^#P < 0.05, ^###P < 0.001 vs. BLM group; ANOVA.

ET-1 is recognized as a key mediator in PH (10). Our examination of ET-1 revealed elevations in ET-1 protein levels in whole-lung lysates following bleomycin exposure (Fig. 8A). In addition, ET-1 protein levels were elevated in the plasma following bleomycin exposure (Fig. 8B). Interestingly, these increases in plasma ET-1 levels were attenuated following GS-6201 treatment (Fig. 8B). In line with these observations, immunohistochemistry for ET-1 revealed positive staining for ET-1 in pulmonary vascular tissue following bleomycin exposure that was not observed in mice treated with GS-6201 or in A_2BR^−/− mice (Fig. 8C). These observations suggest that activation of the A_2BR modulates the release of important mediators that are capable of influencing vascular remodeling following chronic bleomycin exposure.

Figure 8.

Endothelin-1 expression following treatment with bleomycin (BLM). All analyses were performed on d 33 of PBS or BLM exposure. A) Transcript levels of ET-1 from fresh frozen lungs of mice normalized to β-actin expression using the ΔΔC_t method. B) Protein levels of ET-1 in plasma determined by ELISA. Results are presented as means ± se, n = 4/group. *P < 0.05, vs. PBS group, ^#P < 0.05 vs. BLM group; ANOVA.C) Immunofluorescent staining for the ET-1 (red signal) in lung sections from mice treated with PBS, BLM, and BLM+GS-6201, and from A_2BR^−/− mice treated with BLM. Arrows denote the location of the vessel wall.

To directly determine the effect of A_2BR signaling on IL-6 and ET-1 production, cultured human pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs) were exposed to increasing concentrations of 5′-N-ethyl-carboxamido-adenosine (NECA), an adenosine receptor agonist. Results revealed a concentration-dependent increase in IL-6 and ET-1 release in PASMCs and PAECs, respectively (Fig. 9A, B). NECA stimulated increases in IL-6 and ET-1 were inhibited by treatment with GS-6201 (Fig. 9A, B) suggesting that A_2BR signaling is able to directly modulate the release of these mediators implicated in the development of vascular remodeling. To gain additional insight into the significance of the A_2BR mediated release of ET-1 or other vascular remodeling factors from PAECs, medium from NECA-stimulated PAECs was added to PASMC cultures, and increases in cell numbers as an index of proliferation were measured (Fig. 9C). Results demonstrated that medium from NECA-stimulated PAECs could promote proliferation of PASMCs. Together, these results suggest that A_2BR-driven production of IL-6 and ET-1 in vascular cells may account for the vascular remodeling seen secondary to pulmonary fibrosis.

Figure 9.

Release of IL-6 and ET-1 from cultured vascular smooth muscle and endothelial cells. A, B) Cultured pulmonary vascular smooth muscle and endothelial cells were treated with increasing concentrations of the adenosine analog, NECA. After 18 h, the supernatant was removed, and IL-6 levels were determined in vascular smooth muscle cells (A) and ET-1 levels were determined in pulmonary endothelial cells (B) using ELISA. C) Control or NECA-conditioned medium from PAECs was added to PASMCs for 18 or 42 h, and cell numbers were determined using a hemocytometer. Data are presented as means ± se, n = 4/group. *P < 0.05 vs. control, ^#P < 0.05 vs. NECA (10 μM); 1-way ANOVA followed by Newman-Keuls test.

DISCUSSION

Adenosine is a signaling molecule that is generated following cellular injury or stress. Elevated adenosine signaling has been observed in patients with chronic lung diseases, including chronic obstructive pulmonary disease and IPF (21). These are conditions where a high prevalence of PH exists, yet the role of adenosine in orchestrating vascular changes leading to PH in ILD has not been investigated. In this study, we demonstrate that chronic exposure to bleomycin is able to induce fibrotic lesions in the lungs that are accompanied by vascular remodeling and the development of PH in association with increases in tissue levels of adenosine. Moreover, our results show that pharmacological blockade or genetic removal of the A_2BR was able to inhibit the progression of fibrotic and vascular damage leading to PH, implicating a central role for adenosine and the A_2BR in the modulation of ILD and PH secondary to lung fibrosis. Our data suggest that the mechanisms leading to the development of PH associated with pulmonary fibrosis involve A_2BR-modulated release of ET-1 and IL-6 from vascular cells. These data have important clinical significance as they suggest that molecules targeting the A_2BR may be of potential benefit to patients with PH secondary to ILD, a condition lacking effective therapies.

The exact prevalence of PH in ILD is not fully known, with figures ranging greatly from 8% (35) up to 84% (4). A reason for the differences in the reported prevalence of PH in ILD is the fact that many of the symptoms of PH are masked by those of the underlying pulmonary disease, which include dyspnea, fatigue, and exercise limitation (3, 7). As a result, diagnosis of ILD may be missed until signs of heart failure develop (36–38). Consequently, it is not surprising to observe that the highest prevalence of PH in ILD is present in patients with advanced disease who are listed for transplantation (4, 39, 40). In addition to the problems in diagnosing PH, there is a need for better experimental models of PH secondary to ILD (3). In the present study, we investigated the process of vascular remodeling leading to PH in a model of chronic bleomycin exposure where fibrotic injury is apparent (14). These studies are in line with previous experiments demonstrating that a single intratracheal dose of bleomycin can induce PH associated with pulmonary fibrosis (41–45). A major difference between these experiments and our findings is the exposure regimen of bleomycin. In our studies, mice were exposed chronically to bleomycin through biweekly intraperitoneal injections. This experimental regimen results in progressive and nonreversible fibrosis (46) that leads to subpleural scarring that is similar to the human condition (46). This differs from single-dose intratracheal bleomycin administration, where there is a prominent inflammatory response that resembles acute lung injury and a bronchocentric distribution of fibrosis (34, 46) that is reversible (34). For these reasons, we suggest that the intraperitoneal bleomycin model is a novel and effective representation of PH secondary to pulmonary fibrosis.

A major finding in our study was the development of vascular remodeling and PH in mice chronically exposed to bleomycin. These changes consisted of elevated RVSP (a readout of PH), increased vascular smooth muscle mass, neomuscularization of previously nonvascular vessels, and increased collagen deposition adjacent to pulmonary vessels. These physiological and histological observations were accompanied by increased levels of HIF-1α (a marker of hypoxia), reduced arterial oxygen saturation levels, defects in lung function, and increases in IL-6 and ET-1. Our findings mimic observations seen in patients with PH, where hypoxemia, deteriorated lung function, and vascular lesions characterized by increased vascular smooth muscle deposition and neovascularization are prevalent (3, 7). In addition, IL-6 and ET-1 have been implicated in modulating both the fibroproliferative and vascular remodeling processes in PH secondary to ILD (10, 15); however, the mechanisms that control the production and release of these mediators are not fully understood.

We demonstrated that blockade of the A_2BR by GS-6201 was able to attenuate the development of vascular remodeling and PH as well as inhibit the fibroproliferative process. These observations were accompanied by a reduced presence of IL-6 and ET-1 in mice treated with GS-6201. It is plausible that adenosine (that is elevated following bleomycin exposure), acting via the A_2BR, modulates the release of IL-6 and ET-1, which, in turn, regulate fibrotic processes and vascular remodeling, leading to PH. In favor of this hypothesis, we demonstrate that PAECs and PASMCs are able to release ET-1 and IL-6, respectively, following stimulation with NECA, an analog of adenosine, suggesting that these cells express the A_2BR and respond by enhancing the production of critical remodeling factors. In support of our findings, it has been demonstrated that human lung fibroblasts are capable of releasing IL-6 after stimulation with NECA (17). In addition, elevated IL-6 levels have been detected in patients with IPF and in experimental models of fibrosis (13, 14, 21, 47–49) that have implicated adenosine and A_2BR signaling (14, 21). These data, together with our findings, demonstrate an important function for A_2BR-mediated IL-6 release in the fibroproliferative process. Moreover, transgenic overexpression of IL-6 leads to the development of increased RVSP and vascular remodeling that are exacerbated in hypoxic conditions (50). We show elevated IL-6 levels at stages where vascular remodeling is evident following bleomycin exposure and demonstrate that treatment with GS-6201 leads to an attenuation of the remodeling process and a reduction of IL-6 levels. Together, these findings suggest that repeated injury to the lungs results in A_2BR-mediated release of IL-6 that, in turn, modulates fibrotic and vascular remodeling processes that contribute to PH. Interestingly, these findings support observations in other models of lung disease, including airway remodeling and asthma (25) and adenosine-driven lung disease in adenosine deaminase-deficient mice (26). Current investigations are focused on determining whether A_2BR-liberated IL-6 contributes to PH in these diverse models.

In addition to IL-6, there is ample evidence for the role of ET-1 in PH, particularly within the context of PAH. ET-1 is a potent vasoconstrictor and a growth factor for endothelial cells and myofibroblasts (51–53). ET-1 exerts its actions through activation of the endothelin A and B (ET_A and ET_B) receptors (53). Activation of ET_A leads to vasoconstriction, and as a result, ET_A antagonists are used in the treatment of PAH due to their vasodilatory properties (54, 55). The role of ET_B is less clear; it is believed to regulate the actions following ET_A activation, through the release of NO and prostacyclin, and is also thought to restrict the release of ET-1 from endothelial cells (54, 56). Elevated plasma levels of ET-1 have been detected in patients with PH (57), correlating with increased vascular resistance and pulmonary arterial pressure (58–60). In PH secondary to ILD, increased levels of ET-1 have also been reported (10, 12, 54, 61). Moreover, recent preclinical studies have shown that therapies directed against the ET-1 system are able to attenuate the development of experimental fibrosis (43); suggesting that ET-1 is a dual player, modulating both fibrotic and vascular pathophysiological changes. Consistent with this suggestion, we show that chronic bleomycin exposure leads to an increase in plasma levels of ET-1. Moreover, treatment with GS-6201 was able to reduce circulating levels of ET-1. This is the first study to demonstrate that activation of the A_2BR can regulate plasma ET-1 levels, and suggests that this may represent an important mechanism for the progression of PH secondary to ILD.

Specific therapy for PH includes the use of agents that promote vasodilation, such as prostanoids, endothelin antagonists, and phosphodiesterase-5 inhibitors (7); however, the use of these drugs provides symptomatic relief but does not halt or reverse the progression of the disease. Moreover, the effect of these drugs in patients with ILD-associated PH has only been evaluated in a few studies with a small population size, and although a reduction in pulmonary vascular resistance was observed, worsening of hypoxemia was also reported (7). Taken together, these observations warrant the development of new therapeutics for patients with PH associated with ILD. Targeting the remodeling processes in the lung that lead to PH could provide a novel therapeutic alternative to patients with PH that may translate to better clinical outcomes, particularly for patients with PH secondary to ILD. The A_2BR antagonist GS-6201 has been shown to be therapeutically beneficial in the treatment of experimental pulmonary fibrosis (26). Moreover, several A_2BR antagonists, including GS-6201, have been selected for preclinical development (62), and GS-6201 has been shown to be safe and well tolerated in phase I clinical trials (62). These observations, together with our results, support the use of GS-6201 as a therapeutic agent that is able to target remodeling processes in the lung. Although these studies provide insight into the use of the A_2BR antagonist in the treatment of chronic remodeling events, there is evidence to suggest that activation of this receptor may play important roles in the regulation of lipogenesis (63) or protection from acute injury processes (64, 65). Thus, it will be important to carefully define patient populations and disease states when considering the clinical use of the A_2BR antagonist.

In summary, our results highlight the role of adenosine acting via the A_2BR as an upstream mediator involved in the fibroproliferative and vascular remodeling processes that contribute to PH in the fibrotic lung. Using A_2BR-deficient mice and GS-6201, a selective A_2BR antagonist, we demonstrate that activation of this receptor appears to regulate the development of fibrosis and vascular lesions leading to PH. We suggest that activation of the A_2BR on vascular cells may be the mechanism by which adenosine is able to modulate IL-6 and ET-1 release that contributes to vascular remodeling and PH. These studies support the development of A_2BR antagonists for the treatment of PH secondary to ILD.