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Abstract
Rationale: Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. No therapy stopping progress of the disease is available.
Objectives: To investigate the role of the soluble guanylate cyclase (sGC)–cGMP axis in development of lung emphysema and pulmonary hypertension (PH) and to test whether the sGC–cGMP axis is a treatment target for these conditions.
Methods: Investigations were performed in human lung tissue from patients with COPD, healthy donors, mice, and guinea pigs. Mice were exposed to cigarette smoke (CS) for 6 hours per day, 5 days per week for up to 6 months and treated with BAY 63-2521. Guinea pigs were exposed to CS from six cigarettes per day for 3 months, 5 days per week and treated with BAY 41-2272. Both BAY compounds are sGC stimulators. Gene and protein expression analysis were performed by quantitative real-time polymerase chain reaction and Western blotting. Lung compliance, hemodynamics, right ventricular heart mass alterations, and alveolar and vascular morphometry were performed, as well as inflammatory cell infiltrate assessment. In vitro assays of cell adhesion, proliferation, and apoptosis have been done.
Measurements and Main Results: The functionally essential sGC β1-subunit was down-regulated in patients with COPD and in CS-exposed mice. sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma, and inhibited adhesion of CS-stimulated neutrophils.
Conclusions: The sGC–cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.
The molecular mechanisms underlying chronic obstructive pulmonary disease are not fully understood. Currently no treatment that prevents the development of emphysema and pulmonary hypertension in chronic obstructive pulmonary disease is available.
We provide evidence that the soluble guanylate cyclase (sGC)-cGMP axis is affected by cigarette smoke exposure. Our data from investigations conducted in humans, mice, and guinea pigs using sGC stimulators demonstrate that maintenance of the sGC–cGMP pathway can prevent cigarette smoke–induced pulmonary hypertension and lung emphysema in animal models.
Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow obstruction associated with an enhanced inflammatory response in the airways and the lung to noxious particles or gases, most commonly cigarette smoke (CS) (1). COPD is caused by the obstruction of small airways and emphysema, which consists of the enlargement of air spaces, destruction of lung parenchyma, loss of elasticity, and closure of terminal airways (2, 3). The pathogenesis of COPD implicates the recruitment of macrophages, neutrophils, and lymphocytes in the small airways and the lung parenchyma, and the induction of oxidative stress, all of which result in airway remodeling and parenchymal destruction (4, 5). The mechanism of disease development is still not fully understood (1), despite COPD being the fourth leading cause of death worldwide, with a rising incidence. To date there is still no therapy available that can either prevent or even cure the progression of COPD. Currently, symptoms only can be alleviated to slightly improve the quality of life of patients with COPD.
Recent findings have suggested that endothelial dysfunction and pulmonary hypertension (PH) play a key role in the pathophysiology of COPD (6). This hypothesis is supported by the observation that a large proportion of patients with COPD also have PH, with percentages ranging from 30 to 70% (7), and that smokers not suffering from COPD can develop pulmonary vascular remodeling (8, 9). We recently showed that vascular remodeling and PH precede alveolar destruction in an emphysema model of mice chronically exposed to CS, suggesting that molecular alterations of the vasculature may trigger lung emphysema development (10, 11). The essential signaling molecule nitric oxide (NO), derived from inducible NO synthase (iNOS), was identified as a possible mediator of alveolar destruction and vascular remodeling through the subsequent formation of peroxynitrite (ONOO−) (10). However, the downstream targets of NO/ONOO− in the pathogenesis of CS-induced PH and lung emphysema have not yet been identified. Besides its pathophysiologic impact, NO is an effective and selective pulmonary vasodilator, which activates soluble guanylate cyclase (sGC). sGC normally exists as a heterodimer, consisting of a larger α-subunit and a smaller heme-binding β-subunit. Many studies showed that the β1 N-terminus constituted the heme-binding domain and it is known that NO binds to the heme of sGC (12) (supporting the importance of this subunit for sGC function) (12–14). Although an α2- and β2-subunit have been identified (12, 15, 16) it is well accepted that the α1- and β1-subunit form the physiologically relevant heterodimer. Several investigations suggested that dysfunction of the β1-subunit is key to development of diseases (14, 17, 18). The binding of NO to sGC results in the formation of the secondary messenger cGMP (14, 19). cGMP has not only been shown to mediate NO-driven pulmonary vasodilatation, but also to inhibit smooth muscle cell proliferation, platelet aggregation, and inflammatory cell recruitment (20–22). In addition, ONOO− has been suggested to oxidize and inactivate sGC (23–26). Stimulation of sGC has been shown to antagonize PH in experimental models (19, 27–30), and the novel sGC stimulator riociguat recently met the primary endpoint of improved exercise tolerance in a phase III study in patients with pulmonary arterial hypertension and is now approved for the treatment of pulmonary arterial hypertension and chronic thromboembolic PH (28, 31).
Based on these observations, we hypothesized that sGC is involved in the development of CS-induced emphysema and PH, and that the sGC–cGMP axis may therefore be a potential target for the treatment of COPD and COPD-associated PH. To address this hypothesis, we investigated possible alterations of sGC expression in patients with COPD. In addition, we targeted the sGC–cGMP axis in two experimental models of COPD: mice and guinea pigs chronically exposed to CS (32–34). We assessed the effects of the sGC stimulator compounds BAY 63-2521 (riociguat) and BAY 41-2272, which have a dual mode of action, sensitizing sGC to endogenous NO and directly stimulating sGC independently of NO, thereby restoring the NO–sGC–cGMP pathway (14, 19, 35, 36). No relevant differences with respect to the biochemical and pharmacologic profile of both compounds are known. We aimed to compare the effect of two different but tightly related compounds in two different species on smoke-induced emphysema and PH as a strategy that, if the outcome was positive, would substantiate the relevance of the effects of such drugs.
Some of the results of these studies have been previously reported in form of abstracts (37–41).
Detailed descriptions of methods are provided in the online supplement.
Human lung tissues were obtained from transplanted patients with COPD with and without PH (42) and donor control subjects. Patient characteristics are given in Table E3 in the online supplement. The studies were approved by the Ethics Committee of the Justus-Liebig-University School of Medicine, Giessen, Germany and by the Ethics Committee of the Hospital Clinic, Barcelona, Spain.
Mouse experiments were approved by the governmental ethics committee for animal welfare (Regierungspräsidium Giessen, Germany). Male C57BL/6J mice (body weight, 19–20 g) were divided into following groups (16 mice per group): (1) healthy control (no CS exposure), (2) CS exposure only, (3) placebo treatment (CS exposure), (4) sGC stimulator (BAY 63-2521) treatment (3 mg/kg body weight; CS exposure), and (5) BAY 63-2521 treatment (10 mg/kg body weight; CS exposure).
Smoke-challenged animals were exposed to the smoke of 3R4F cigarettes 6 hours per day, 5 days per week for 6 months. Nonexposed mice were treated similarly, but without CS exposure. The 200-μl solutions of BAY 63-2521 were freshly prepared each day and suspended in 4.0% methocel and 1.3% polyethylene glycol. On CS exposure days, the suspension was applied by gavage 1 hour before exposure. This time point of the last application was chosen to allow determination of peak plasma levels of BAY 63-2521 on termination of the experiments. The solvent was used as placebo. After 6 months, half of each group was randomly divided into two subgroups. In Subgroup 1 lung function tests were performed and in Subgroup 2 hemodynamics were quantified. From these subgroups, half of the lungs were fixed for alveolar morphometry and half were fixed for vascular morphometry. Portions of the expression studies were done in a separate group of mice exposed for 8 months to tobacco smoke, because it was previously shown that no major differences exist between 6 and 8 months of smoke exposure except the slightly increasing severity of lung emphysema and PH with time (10).
Procedures involving guinea pigs were approved by the Ethics Committee for Animal Experimentation of the University of Barcelona, Spain. Thirty-nine male Dunkin Hartley guinea pigs (starting 300 g) were randomly distributed in four groups: (1) a nonexposed control group (Control, n = 7), (2) a nonexposed group receiving BAY 41-2272 (Control + sGCS, n = 8), (3) a group exposed to CS receiving placebo (CS, n = 12), and (4) a group exposed to CS receiving BAY 41-2272 (CS + sGCS, n = 12). Animals from CS groups were exposed to the smoke generated from six nonfiltered 3R4F cigarettes per day for 3 months, 5 days per week using a nose-only system (32). BAY 41-2272 was administered orally by gavage at a dose of 3 mg/kg/day.
In vivo assessments in mice included right ventricular systolic pressure, tidal volume, and dynamic compliance. In the guinea pig pulmonary artery pressure, systemic arterial pressure, aortic flow, and unrestrained whole-body plethysmography were assessed. Right ventricular hypertrophy was assessed at the end of the experiments.
Assessments in lung tissue included the following:
| 1. | Alveolar morphometry: mean linear intercept, air space, and septal wall thickness | ||||
| 2. | Muscularization and medial wall thickness of lung vessels | ||||
| 3. | Inflammatory cell (neutrophils, alveolar macrophages) counts | ||||
| 4. | Soluble GCβ1, sGCβ2, sGCα1, and sGCα2 expression by real-time polymerase chain reaction | ||||
| 5. | Soluble GCβ1, sGCβ2, sGCα1, and sGCα2 expression by Western blot | ||||
| 6. | Localization of sGCβ1 and 3-nitrotyrosine | ||||
These studies included proliferation and apoptosis assays, as well as mRNA-expression analyses in lung endothelial and alveolar type II epithelial cells (AECII); and adhesion assays to fibrinogen of human polymorphonuclear leukocytes (hPMNL), treated and untreated with CS extract (CSE).
All data are given as mean ± SEM. For comparison of more than two groups analysis of variance followed by Student-Newman-Keuls post hoc test was performed. For comparison of two groups, a Student t test was applied. Correlations between variables were done by Pearson analysis. P values less than 0.05 were considered significant.
Analysis of sGC subunit regulation in lungs from patients with COPD compared with healthy donors revealed a down-regulation of sGCβ1 and sGCβ2 expression at the mRNA level (Figure 1A) and a down-regulation of the sGCβ1 subunit at the protein level (Figure 1B). Comparing lungs from patients with COPD with and without PH we did not find significant differences in the protein levels of the functionally essential sGC subunit β1 (Figure 1C). Similar to human COPD the sGCβ1 mRNA subunit was down-regulated in 3 months CS-exposed guinea pigs (Figure 1D). Focusing on mice again a similar down-regulation of the sGCβ1 protein was found after 3 and 8 months of CS exposure (Figures 1E and 1F). In addition, as assessed on the mRNA level a down-regulation of sGCα1 and sGCβ1 was observed after 3 months, and a down-regulation of sGCα2 and sGCβ1 was observed after 8 months of CS-exposure in this species (Figures 1G and 1H). Immunofluorescence staining suggested that the sGCβ1 protein was located throughout the lung in bronchi, vessels, and septa. Focusing on the pulmonary vasculature it was evident that the sGCβ1 subunit was colocalized with α-smooth muscle actin–positive cells. Because vascular localization was not restricted to this cell type endothelial and adventitial cells may also express sGCβ1 (Figure 1I).
Figure 1. Soluble guanylate cyclase (sGC) subunit regulation in human lungs from patients with chronic obstructive pulmonary disease (COPD) with and without pulmonary hypertension (PH), in 3 months cigarette smoke (CS)–exposed guinea pigs and in 3 and 8 months CS-exposed mouse lungs and localization in mouse lungs. mRNA-expression was analyzed by quantitative real-time polymerase chain reaction for the expression of the sGCα1, sGCα2, sGCβ1, and sGCβ2 subunits isolated from (A) lung homogenate of healthy donors (n = 7) or patients with COPD (n = 9) after explantation. (B) Western blot analysis for the sGCβ1 subunit of lung homogenates from healthy donors and patients with COPD after explantation. (C) Western blot analysis for the sGCβ1 subunit of lung homogenates from patients with COPD with (n = 7) or without PH (n = 9). (D) mRNA expression of sGCβ1-subunit of 3 months CS-exposed guinea pig lungs in comparison with time-matched control animals. (E and F) Western blot analysis for the sGCβ1-subunit of mouse lungs after (E) 3 months and (F) 8 months of CS exposure compared with non–CS-exposed, time-matched control animals. Data are from six lungs per group and given as mean ± SEM. Values were normalized to β-actin. For B, C, E, and F a representative blot (right) and densitometry (left) are shown. (G and H) mRNA expression of sGC subunits in (G) mouse lungs after 3 months (n = 4–5) and (H) mouse lungs after 8 months (n = 4–10) of CS exposure compared with non–CS-exposed time-matched control animals. All data are duplicate measurements and are given as mean ± SEM. Ct values of individual genes were normalized to the housekeeping gene B2M (human), porphobilinogendeaminase (mouse), or β-actin (guinea pigs). Bars indicate significant differences between healthy donors and COPD lungs or CS-exposed and non–CS-exposed mice, respectively (*P < 0.05; **P < 0.01; ***P < 0.001). (I) Localization of sGCβ1 in cryosections of mouse lungs. Data are shown for non–CS-exposed control mice (0 mo), and mice exposed to CS for 3 and 8 months. Left-hand panels show anti-sGC (green), middle panels show anti–α-smooth muscle actin (α-SMA; red), and right-hand panels show merged images (V = vessel; B = bronchi; S = septa).
[More] [Minimize]Mice exposed to CS for 6 months developed PH and pulmonary vascular remodeling, as shown by quantification of right ventricular systolic pressure (Figure 2A), right heart hypertrophy (Figure 2B), degree of vascular muscularization (Figures 2C–2E), and the medial wall thickness (Figures 2F–2H). Treatment with BAY 63-2521, either at doses of 3 or 10 mg/kg body weight/day, resulted in complete protection against the development of PH, based on all four parameters of PH characterization. Systemic arterial pressure was reduced on smoke exposure and was normalized by treatment with BAY 63-2521 (Figure 2I).
Figure 2. Effect of soluble guanylate cyclase stimulation with BAY 63-2521 on cigarette smoke (CS)–induced pulmonary hypertension in mice assessed by structural and functional parameters. Mice were exposed to CS for 6 months in combination with BAY 63-2521 (3 or 10 mg/kg body weight/day). Control animals were either untreated or received the solvent (placebo). (A) Right ventricular systolic pressure quantified by right heart catheterization in anesthetized animals. (B) Right heart hypertrophy, given as the ratio of right ventricular (RV) mass to left ventricular plus septum (LV+S) mass, from dried heart tissue; left ventricular weights did not differ between the different groups. (C−E) Degree of muscularization of (C) small pulmonary arteries (outer diameter 20–70 μm), (D) medium vessels (outer diameter >70–150 μm), and (E) large vessel (outer diameter >150 μm), as a percentage of total vessel count for fully muscularized (full), partially muscularized (partial), and nonmuscularized (non) vessels. (F–H) Wall thickness of (F) small pulmonary vessels (% of vessel diameter, outer diameter 20–70 μm), (G) medium vessels (% of vessel diameter, outer diameter >70–150 μm), and (H) large vessel (% of vessel diameter, outer diameter >150 μm). (I) Systemic arterial pressure. (J) Representative histology from lung sections stained with antibodies against α-smooth muscle actin and von Willebrand factor. The respective right image is an enlarged view of the left image (frame). Data are given as mean ± SEM. Bars indicate significant differences (A–E and I, n = 8; F–H, n = 4–6; *P < 0.05; **P < 0.01; ***P < 0.001).
[More] [Minimize]Guinea pigs exposed to CS for 3 months also showed a higher pulmonary arterial pressure (Figure 3A) and total pulmonary resistance (Figure 3B). Cardiac output (Figure 3C) and systemic arterial pressure (Figure 3D) did not differ significantly from nonexposed animals. Treatment with BAY 41-2272 (3 mg/kg body weight/day) during CS exposure resulted only in a tendency toward a decrease of pulmonary resistance, which did not differ from nonexposed guinea pigs, and clearly reduced pulmonary vascular remodeling induced by CS exposure (Figure 3E). The number of muscularized intrapulmonary vessels with a diameter less than 50 μm and positive immune reactivity to α-smooth muscle actin was greater in CS-exposed animals than in control animals (71 ± 10% and 28 ± 12%, respectively; P < 0.05). By contrast, in guinea pigs exposed to CS and treated with BAY 41-2272, the percentage of muscularized vessels was significantly lower than in CS-exposed only (P < 0.05) (Figure 3E). In addition, total pulmonary resistance and intrapulmonary vessel muscularization were significantly related (P < 0.001; r = 0.59).
Figure 3. Effect of soluble guanylate cyclase (sGC) stimulation with BAY 41-2272 on cigarette smoke (CS)–induced pulmonary hypertension in guinea pigs. Guinea pigs were exposed to cigarette smoke for 3 months in combination with BAY 41-2272. Control animals received the solvent (placebo). (A) Pulmonary arterial pressure, (B) total pulmonary resistance (pulmonary artery pressure/cardiac output), (C) cardiac output, and (D) systemic arterial pressure. (E) Percentage of muscularized intrapulmonary vessels less than 50 μm diameter. Control+sGCS = control and BAY 41-2272 3 mg/kg/day; CS = cigarette smoke; CS+sGCS = cigarette smoke and BAY 41-2272 3 mg/kg/day. Data are given from n = 7–12. Bars indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001).
[More] [Minimize]Figure 4 shows that mice exposed to CS also developed lung emphysema, quantified by determination of the airspace, the septal wall thickness, and the mean distance between alveolar septa (mean linear intercept) (Figures 4A−4C). This was reflected by an increase in compliance and tidal volume on the functional side (Figures 4D and 4E). Treatment with the sGC stimulator BAY 63-2521 in parallel with CS exposure prevented the development of lung emphysema, as shown by both structural and functional parameters (Figures 4A–4E and 4I). No difference in efficacy could be detected between the two doses of BAY 63-2521 used. Quantification of PCNA staining as an estimate for proliferation revealed that BAY 63-2521 treatment increased proliferation in alveolar septa compared with lungs from untreated smoke-exposed mice (see Figures E1A–E1D). Addressing apoptosis in the in vivo situation, we performed caspase-3 staining (marker for apoptosis) on murine lung sections. Referring to Figure E1E-I, the amount of caspase-3–positive cells is increased by CS compared with nonexposed mice, which was prevented by both doses of BAY 63-2521. Moreover, the additional stainings of the same lungs with the epithelial marker SP-C (see Figure E1E-II) and the endothelial marker von Willebrand factor (see Figure E1E-III) suggest these cells as caspase-3–positive cells. Those results from the lung sections confirmed our in vitro experiments using isolated vascular endothelial and AECII, which showed increased apoptosis on ONOO− treatment, which was attenuated by BAY 63-2521 treatment (Figure 6B). Adverse effects of BAY 63-2521 treatment (e.g., in terms of fibrosis development or inflammation) were not observed (see Figures E2 and E3).
Figure 4. Effect of soluble guanylate cyclase stimulation on cigarette smoke (CS)–induced lung emphysema development assessed by structural and functional parameters. Mice were exposed to CS for 6 months in combination with BAY 63-2521 (3 or 10 mg/kg body weight/day). Control animals were untreated or received the solvent (placebo). (A) Air space, (B) septal wall thickness, (C) mean linear intercept, (D) in vivo dynamic lung compliance, and (E) tidal volume (data are given from n = 8, each). Guinea pigs were exposed to CS for 3 months in combination with BAY 41-2272 (3 mg/kg body weight/day). Control animals received the solvent (placebo). (F) Mean linear intercept of alveolar septa (n = 7–12), (G) relative bronchial muscularization (n = 3–6), and (H) Penh (n = 7–9). (I) Representative histology from mice lung sections stained with hematoxylin and eosin. The respective right image is an enlarged view of the left image (frame). Data are given as mean ± SEM. Bars indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001).
[More] [Minimize]Similar findings were observed in guinea pigs: animals exposed to CS showed increased mean linear intercept, whereas in those exposed to CS and treated with BAY 41-2272 emphysema development was prevented (Figure 4F). Guinea pigs exposed to CS showed muscularization of noncartilaginous bronchi and increased respiratory resistance (enhanced pause) (Figures 4G and 4H). Concomitant treatment with the sGC stimulator BAY 41-2272 did not modify either the bronchial or functional changes induced by CS.
We previously suggested that ONOO− mediated the development of PH and lung emphysema on CS-exposure through the subsequent formation of 3-nitrotyrosine. We therefore investigated the effect of sGC stimulation on lung 3-nitrotyrosine formation in CS-exposed mice, to determine whether sGC stimulation prevents the development of PH and lung emphysema by affecting signaling downstream of ONOO−. CS exposure resulted in increased 3-nitrotyrosine formation, which was not altered by treatment with BAY 63-2521(Figures 5A and 5B). However, possible downstream effects of ONOO− signaling were affected by BAY 63-2521 treatment. On the cellular level ONOO− exposure of isolated endothelial cells and AECII resulted in increased apoptosis and reduced proliferation (Figures 6A and 5B). Although 8-bromo-cGMP and the sGC stimulator BAY 63-2521 had no effect on the reduced proliferation (Figure 6A), both compounds antagonized the increase in apoptosis induced by ONOO− (Figure 6B). A respective up-regulation of Fgf10 mRNA could be confirmed for the in vivo situation in the BAY 63-2521–treated mice (Figure 6C). In addition, BAY 63-2521 treatment reversed the ONOO−-induced down-regulation of Fgf10 mRNA in AECII (Figure 6D). Similarly, Sod1 was down-regulated in isolated epithelial cells by ONOO− exposure, again counterregulated by BAY 63-2521 treatment (Figure 6E). Sod1 and Vegfa were also up-regulated in endothelial cells by BAY 63-2521 treatment after ONOO− exposure (Figures 6F and 6G) and the inflammatory markers Ccl25 and Csf2 were down-regulated by sGC stimulator treatment on the mRNA level (Figures 6H and 6I). Along these lines, again focusing on the in vivo situation in a different species, vascular endothelial growth factor (VEGF) was up-regulated by BAY 41-2272 treatment when assessed from homogenized guinea pig lungs (see Figure E4).
Figure 5. 3-Nitrotyrosine formation in lungs from BAY 63-2521–treated cigarette smoke (CS)–exposed mice. (A) 3-Nitrotyrosine staining (violet) of the lungs of mice exposed to tobacco smoke for 6 months and non–smoke-exposed (control) mice, compared with BAY 63-2521–treated mice. Placebo-treated animals received the vehicle only. (B) Western blot analysis of the respective groups. A representative blot (upper) and data from densitometry (lower) are shown. Data were normalized to β-actin and are given as mean ± SEM (n = 6). Bars indicate significant differences (*P < 0.05).
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Figure 6. Cellular effects of soluble guanylate cyclase (sGC) stimulation on proliferation, apoptosis, adhesion, and signaling. (A and B) Mouse alveolar type II epithelial cells (AECII) and vascular endothelial cells (EC) were exposed to ONOO− in the presence or absence of 8-bromo-cGMP or BAY 63-2521. Control animals were either not treated with ONOO− or received staurosporine as a positive control. (A) Assessment of proliferation in EC (1 × 105 cells [24-well plate]; n = 5–6) and AECII (3.5 × 105 cells [24-well plate]; n = 5–6); the negative untreated control was set at 100%. All other values are calculated in relation to the negative control. (B) Determination of apoptosis in EC (2 × 104 cells [96-well plate]; n = 8–9) and AECII (4 × 104 cells [96-well plate]; n = 4). Values were normalized to untreated cells (negative control, set at 100%) and are given as mean ± SEM. Bars indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001). Assessment of mRNA expression levels of (C) Fgf10 in lung homogenate, (D) Fgf10 in murine AECII, (E) Sod1 in murine AECII, (F) Sod1 in EC, (G) Vegfa in EC, (H) Ccl25 in EC, and (I) Csf2 in murine EC. In D–I #P < 0.05 and ##P < 0.01 = significant to respective control (n = 5). For guinea pig experiments animals were exposed to cigarette smoke for 3 months in combination with BAY 41-2272 treatment. Control animals received the solvent (placebo). (J) Presence of intraseptal neutrophils (cells x10−7/μm2), (K) alveolar macrophages (cells per field), correlations between (L) neutrophils (r = 0.784, P < 0.001) and (M) macrophages (r = 0.535, P < 0.001) cell counts and the mean interseptal distance. Control+sGCS = control and BAY 41-2272 3 mg/kg/day; CS = cigarette smoke; CS+sGCS = cigarette smoke and BAY 41-2272 3 mg/kg/day. (N) In vitro human polymorphonuclear cells adhesion capacity to fibrinogen when incubated with basal medium (RPMI), cigarette smoke extract (1/5), or BAY 41-2272 (1 μM) (sGCS) (n = 7–12).
[More] [Minimize]It is well known that inflammatory cells, such as neutrophils and macrophages, actively participate in the pathogenesis and progression of COPD. Thus, we analyzed the influence of sGC stimulation on the number of inflammatory cells and inflammatory markers in CS-exposed guinea pigs. Compared with nonexposed animals, the number of neutrophils was three times greater (P < 0.05) in alveolar septa of CS-exposed guinea pigs. Animals treated with sGC stimulator (sGCS) and exposed to CS showed similar values in the neutrophil count as control animals (1.73 ± 0.06 and 1.88 ± 0.05 10−7 cells/μm2 for CS+sGCS and Control+sGCS groups, respectively; P < 0.05) (Figure 6J). Similarly, alveolar macrophages increased in CS-exposed guinea pigs, an effect that was partially abrogated in animals treated concomitantly with BAY 41-2272 (Figure 6K). Counts of both neutrophils and macrophages correlated with the alveolar space size (Figures 6L and 6M). In addition, there was a trend for a higher proportion of guinea pigs with detectable plasma levels of IL-8 among those exposed to CS (40%) than in those exposed to CS and treated with BAY 41-2272 (14%) or in control animals (P = 0.06).
To better understand the effects of sGC stimulation on the inflammatory response and assess its effects on human cells, the adhesion capacity of hPMNL was assessed in vitro in presence of both BAY 41-2272 and CSE. Assays revealed a twofold increased adhesion to a fibrinogen matrix capacity of hPMNL when incubated with CSE with respect to baseline conditions. Addition of the sGC stimulator BAY 41-2272 (1 μM) inhibited the adhesion capacity of neutrophils in presence of CSE, keeping it similar to baseline conditions (1.19 ± 0.45 and 1.23 ± 0.46 folds, respectively) (Figure 6N). A screening of mouse pulmonary vascular endothelial cells exposed to CSE showed a down-regulation of β2-integrin on sGC stimulator treatment and a trend to lower expression of intercellular adhesion molecule-1 and P-selectin (see Figure E5).
Our data suggest that the sGC–cGMP axis is critical for the development of lung emphysema and PH, and that the sGC–cGMP axis is a potential target for treatment of CS-induced PH and alveolar destruction. We provide evidence that sGC is partially inactivated by CS, thereby suppressing the protective effect of cGMP on the structural and functional deterioration of the lung in CS-induced emphysema and PH. This conclusion is based on the following findings: (1) the sGCβ1 subunit protein, which is the functionally essential sGC subunit (12), was significantly down-regulated in lungs from patients with severe COPD (with and without PH), and in the mouse and in the guinea pig model of emphysema induced by long-term exposure to CS; (2) treatment with the sGC stimulators BAY 63-2521 and BAY 41-2272 prevented the development of lung emphysema, vascular remodeling, and/or PH in two different rodent models of COPD, without adverse effects of treatment; (3) BAY 41-2272 abrogated inflammatory cell recruitment in lung parenchyma; (4) induction of apoptosis by ONOO− in both endothelial and AECII could be prevented by the application of either a cGMP analog or a sGC stimulator; and (5) BAY 63-2521 or BAY 41-2272 induced an up-regulation of Fgf10, the superoxide scavenger Sod1, and Vegfa as well as a down-regulation of markers for inflammation or adhesion as assessed in vivo, in ONOO−-, or CSE-challenged pulmonary vascular endothelial.
The NO–sGC–cGMP signaling pathway plays a critical role in the physiology and pathophysiology of the pulmonary vasculature (30). A dysregulation of this pathway caused by reduced bioavailability of, or responsiveness to, endogenous NO may contribute to pulmonary diseases and PH (17, 27, 43). NO generated by NOS binds to sGC, which produces cGMP from GTP. The effects of cGMP can be mediated by cGMP-dependent protein kinases (26) that modulate apoptosis, proliferation, migration, and extracellular matrix protein expression (21, 44, 45).
Soluble GC consists of a large (α) and small heme-binding (β) subunit. For both subunits the isoforms α1 (synonym α3), α2, β1, and β2 have been described (12, 13). No sGC heterodimer including the β2-subunit has yet been found (13); the β1-subunit therefore seems essential for sGC function (12). Besides the expression of the sGC subunits the activity of the enzyme may also contribute to the observed smoke effect. Although not determined in our study it was suggested that sGC α1β1 are the essential subunits determining sGC activity (12). Moreover, the sGC–cGMP system is essential for vascular homeostasis and regulation of vascular tone, and can interfere with the pulmonary vascular structure and remodeling (14, 19). In our study we show that this essential β1-subunit is down-regulated in patients with COPD, in mice, and in guinea pigs chronically exposed to CS. In addition to such a down-regulation, sGC-dependent cGMP formation can also be suppressed by oxidation (25). The redox state of sGC can be altered by reactive oxygen and nitrogen species, such as superoxide (O2.−) and ONOO− (17, 26, 47) through the oxidation of the ferrous heme group of the β1-subunit, leading finally to a heme-free sGC. In this regard, it was previously shown that CS-induced emphysema and PH in mice is affected by iNOS up-regulation, most likely caused by subsequent ONOO− formation (10).
Furthermore, it was previously shown that ONOO− induced apoptosis in AECII and vascular endothelial cells, and reduced proliferation of AECII (10). Our data now reveal that (1) the induction of apoptosis by ONOO− could be inhibited by both sGC stimulators and cGMP analogs; (2) sGC stimulators can increase mediators of vascular integrity and lung maintenance, such as Vegfa (3) and Fgf10 (47, 48); (3) sGC stimulator treatment can increase antioxidant enzymes, such as Sod1 (which can reduce sGC oxidation and thus inactivation); and (4) sGC stimulator treatment can reduce inflammation under ONOO− exposure.
We speculate that a deactivation of sGC by oxidation, besides its down-regulation, can be antagonized by a cGMP-increasing treatment. Therefore, either the down-regulation of cGMP can lead to emphysema and PH development on CS exposure, and/or cGMP can antagonize the non–sGC-targeting effects of ONOO−.
It is also well accepted that inflammatory cells, such as neutrophils and macrophages, actively participate in the pathogenesis and progression of COPD and emphysema by releasing interleukins, elastase, and metalloproteinases (3, 10). In our CS-exposed guinea pig model, air space size was related to the number of neutrophils and macrophages in lung tissue, suggesting that the abrogation of the development of emphysema could also be related to the attenuation of the inflammatory response induced by CS exposure. A prevention of emphysema was proved by normal lung distensibility. However, there was no improvement in the respiratory resistance, likely because of the concomitant effects of CS on the bronchial structure, which were not prevented by sGC stimulation. Accordingly, prevention of emphysema development in CS animals can be explained, at least in part, by the antiinflammatory effects of sGC stimulation.
NO-cGMP signaling exerts antiinflammatory and antithrombotic effects by preventing the activation and adherence of circulating inflammatory cells and platelets. Mechanisms underlying these effects have been suggested to involve changes in the expression pattern of adhesion molecules. P-selectin, together with L-selectin, β2-integrin, and intercellular adhesion molecules 1 and 2 are necessary for the inflammatory cells to roll and adhere to vascular endothelium before their migration into lung structures. Reduced expression of P-selectin on the endothelial surface results in an impaired capacity of inflammatory cells to infiltrate tissues (22, 49, 50). Along these lines, sGC stimulator treatment down-regulated β2-integrin in pulmonary vascular endothelial cells exposed to CSE and a trend in the same direction for intercellular adhesion molecule-1 and P-selectin. In the present study, sGC stimulation inhibited the adhesion of CS-stimulated neutrophils to a fibrinogen matrix indicating at least a direct pharmacologic action on inflammatory cells (51, 52) additional to the effects described on endothelial cells. Furthermore, it has been recently shown that sGC mediates the angiogenic and permeability-promoting activities of VEGF (53), pointing to the key role of sGC as a downstream effector of VEGF-triggered responses. Consistent with this concept, we could show that BAY 41-2272 increased Vegf expression in comparison with smoke in guinea pigs. Along these lines sGC stimulators also increase Vegfa in isolated mouse endothelial cells challenged with ONOO−, indicating that sGC stimulators could exert partially their protective role preventing emphysema through this growth factor (3). Similar results have been shown with statin, which stimulates NO production, thereby reinforcing the critical role of the NO–cGMP pathway in lung alterations of COPD (54).
We also investigated the development of CS-induced PH in detail. There is currently discussion on whether in COPD changes in pulmonary circulation emerge secondary to changes in the airways and lung parenchyma, or if they can antecede or even trigger emphysema development. The latter concept has recently been supported by investigations showing that smokers who have not developed COPD can display conspicuous abnormalities in pulmonary vessels (8, 9). Moreover, recent work has shown that more than 50% of patients with COPD may suffer from PH (7–9, 29, 55). We have recently determined in mice exposed to CS that (1) PH clearly precedes emphysema development, as shown for other species (32); (2) PH development is not driven by hypoxia; and (3) emphysema and PH are induced by different pathways, with emphysema development being dependent on non-bone-marrow–derived iNOS-containing cells, and PH development being dependent on bone-marrow–derived iNOS-containing cells (10).
Based on these findings, it was proposed that the up-regulation of iNOS and the subsequent production of ONOO− in the vascular compartment can trigger emphysema development independently from vascular remodeling (10). We now show that maintenance of cGMP levels can also prevent pulmonary vascular remodeling and the development of PH induced by CS. These effects are in line with the ability of sGC stimulators to antagonize PH induced by mechanisms other than CS, including PH in hypoxic mice, PH induced by monocrotaline in rats, PH in neonatal rats (14, 19, 27, 30), in lambs (56), and more recently in men (28, 31, 57, 58). Of note, in the guinea pig model, sGC stimulation prevented pulmonary vascular remodeling but did not modify significantly the increase in pulmonary artery pressure induced by CS exposure, presumably because of the lower dose used in that experiment (3 mg/kg). Similar observations were made in a hypertensive rat model in which a low dose of BAY 41-2272 attenuated the structural changes without modifying hemodynamic parameters (59). In support of our data and the important role of cGMP, it was shown that sildenafil, a PDE5 inhibitor that prevents the degradation of cGMP (60), preserved alveolar growth and lung angiogenesis, and decreased pulmonary vascular resistance, right ventricular hypertrophy, and medial wall thickness in oxygen-induced lung injury in newborn rats (61).
In conclusion, we provide evidence that the sGC–cGMP axis is perturbed by CS exposure. Our data from investigations conducted in humans, mice, and guinea pigs using sGC stimulators demonstrate that maintenance of the sGC–cGMP pathway can prevent CS-induced lung emphysema and PH. If these data are transferable to the human situation (with a compound that is already approved for treatment of other diseases in humans), stopping the progress of the disease would be a major advance. Further investigations should, however, focus on the potential of sGC stimulators to reverse established emphysema.
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* These authors contributed equally.
Supported by Bayer HealthCare; German Research Foundation, Excellence Cluster Cardiopulmonary System; the Hessian Government (LOEWE); grant PS09-0536 from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness; and Fundació La Maratò TV3 (#040430).
Author Contributions: N.W., J.A.B., conception, study design, analysis and interpretation of data, drafting and revising the manuscript. B.L., A.P., N.P., and M.S., design of the study, acquisition, analysis, and interpretation of data, drafting the manuscript. R.P.-P., E.F., V.I.P., D.D.-F., I.B., O.T.-C., and J.G., acquisition, analysis, and interpretation of data, drafting the manuscript. A.F., J.-P.S., H.A.G., N.C.-B., R.F., R.T.S., M.B., E.T., R.P.B., W.K., P.J., W.S., and F.G., study design, interpretation of data, drafting the manuscript. J.G.-L., study design, acquisition and interpretation of data, drafting the manuscript. R.R.-R., acquisition, analysis, and interpretation of data.
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Originally Published in Press as DOI: 10.1164/rccm.201311-2037OC on April 16, 2014
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