Advanced glycation end products and its receptor (RAGE) are increased in patients with COPD

Respiratory Medicine (2011) 105, 329e336

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Advanced glycation end products and its receptor (RAGE) are increased in patients with COPD Lian Wu a,*, Li Ma b, Louise F.B. Nicholson b, Peter N. Black a a

Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand b Department of Anatomy with Radiology and the Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand Received 1 September 2010; accepted 4 November 2010 Available online 26 November 2010

KEYWORDS Advanced glycation end products; Receptor for advanced glycation end products; Chronic obstructive pulmonary disease; Immunohistochemistry

Summary Advanced Glycation End products (AGEs) are the products of nonenzymatic glycation and oxidation of proteins and lipids. Formation of AGEs is increased in response to hyperglycaemia, reactive oxygen species and ageing. AGEs are proinflammatory and can modify the extracellular matrix. RAGE (Receptor for Advanced Glycation End Products) mediates some of the effects of AGEs. Methods: Formalin-fixed lung tissue from patients who had lobectomy for bronchial carcinoma was used to investigate the presence of AGEs and RAGE. Subjects were divided into those with COPD and controls. Immunostaining for AGEs and RAGE was performed and the intensity of staining measured. Results: Subjects with COPD and controls were similar in age and smoking history but FEV1% predicted was lower for COPD than controls. Intensity of staining for AGEs was greater in the airways (p Z 0.025) and alveolar walls (p Z 0.004) in COPD. Intensity of staining for RAGE was also significantly increased in alveolar walls (p Z 0.03) but not the airways. FEV1% predicted was correlated with the intensity of staining for AGEs in the airways and alveoli. Conclusions: The increased staining for both AGEs and RAGE in COPD lung raises the possibility that the RAGEeAGEs interaction may have a role in the pathogenesis of COPD. ª 2010 Elsevier Ltd. All rights reserved.

Introduction The advanced glycosylation process is a spontaneous interaction between reducing sugars and a wide variety of proteins and lipids that induces a post-translational

modification of the protein or lipid and may increase the stiffness of matrix proteins. Advanced Glycation End products (AGEs) are the products of this nonenzymatic glycation and oxidation of proteins and lipids.1,2 Under normally physiological conditions AGEs are formed at a very slow but

* Corresponding author. Tel.: þ64 2102447838; fax: þ64 93737556. E-mail address: [email protected] (L. Wu). 0954-6111/$ - see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2010.11.001

330 steady rate and accumulated with ageing. Their production, however, is accelerated in inflammatory conditions such as hyperglycaemia and oxidative stress.3 Proteins with low turn-over rates within the extracellular matrix (e.g. collagen and elastin) are particularly prone to AGEs modification. In diabetic patients, evidence has been found indicating that neurofilaments (a very stable protein) cross-link with AGEs and accumulate in diabetic neuropathy.4 The acceleration of the formation of AGEs has been also shown to contribute to the development of diabetic complications such as retinopathy and vascular disease.5 AGEs have also been found to accumulate in the brain and are thought to play a role in the development and progression of Alzheimer disease through cellular responses to ROS (reactive oxygen species).6,7 Ageing and smoking are the two major risk factors of chronic obstructive pulmonary disease (COPD).8 However, how these two factors contribute to the development of COPD is still unclear. Ageing and smoking may also contribute to the acceleration of the formation of AGEs.9 AGEs are one of a number of structurally similar ligands for the receptor RAGE.10,11 Binding of AGEs to RAGE leads to activation of NFkB (nuclear factor-KappaB) and promotes inflammation.12 AGEs may therefore play a role in the development of COPD possibly through inflammation. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobin (Ig) superfamily.11 RAGE is a single membrane spanning receptor, consisting of a very short cytosolic domain and a large extracellular region containing three Ig-like domains.13 RAGE can bind with a myriad of ligands including AGEs, amphoterin, and S100/calgranulin.14 RAGE is expressed at low levels in normal tissues and in blood vessels but becomes up-regulated at sites where its ligands accumulate. Recently, Demling and colleagues confirmed that RAGE is abundantly expressed in normal lung tissue.15 By analyzing alveolar epithelial type II cells (AT II cells) transdifferentiating in vitro into AT I cells, they identified RAGE as a specific “differentiation marker” of human AT I cells. RAGE was also found to be expressed in endothelia and alveolar macrophages, other than type II alveolar pneumocytes. They also showed in vitro that RAGE promotes the adherence of epithelial cells to collagen and induces spreading.15 Based on previous published work from other groups, we were interested to know whether the RAGEeAGEs interaction might represent a novel pathway promoting inflammation in lung disease, and particularly in Chronic Obstructive Pulmonary Disease (COPD). COPD is characterised by poorly reversible airflow obstruction that is usually, but not invariably, due to cigarette smoking. The pathological changes in COPD are inflammation of the peripheral airways and destruction of the lung parenchyma (emphysema). The mechanism of development and progression of COPD, however, is still not clear. Chronic smoking and pollutant exposure, that cause a series of inflammatory stresses, are believed to play key roles in the pathogenesis of COPD.16 To date the role of AGEs and RAGE have not been investigated in COPD.17

Aim of this study To investigate the presence of RAGE and its ligand AGEs in lung tissue from control subjects and patients with COPD.

L. Wu et al.

Methods Subjects Blocks of archival, formalin-fixed lung tissue were used for this study. Blocks were from patients who had had one or more lobes of the lung resected for bronchial carcinoma. The specimens were identified through the computerized records of the Department of Pathology, GreenLane Hospital. The operations were performed between January 1992 and September 1996. All of the blocks used were from sites remote from the bronchial carcinoma. Further information, including smoking history, past medical history, medication, and preoperative lung function, were obtained from the patient’s hospital notes. Patients were excluded if they had a history of asthma. Patients were classified as having COPD if their FEV1 was <80% predicted and the ratio of FEV1 to FVC was <70%. This is equivalent to GOLD Stage 2e4. Patients with FEV1 >80% predicted and FEV1/FVC >70% were served as controls. Both groups were non-diabetic. (see Table 1 for summary of patient groups).

Immunostaining methods Sections were cut at 5 mm on a microtome and mounted on poly-L-lysineetreated microscope slides. Immediately before staining, the slides were warmed to 37  C in an incubator for at least 1 h. Sections were then deparaffinized in xylene and rehydrated in a grading series of alcohol dilutions and 0.01 M phosphate-buffered saline (PBS) for 5 min. Sections were then treated with DAKO (R) Target Retrieval Solution (Dako, Glostrup Denmark) for 30 min at 98  C to enhance antibody labeling. After 3 washes of 5 min in PBS, sections were incubated overnight with mouse anti-human AGEs (clone 6D12, 1:200, Cosmo Bio, Japan) or goat anti-human RAGE (Biodesign, 1:200, Meridian Life Science, ME USA) antibody at 4  C. After a further three washes in PBS for 5 min each, the secondary antibody (Jackson immunoresearch, USA), goat anti-mouse biotin conjugated, was applied to the sections overnight at 4  C for AGE staining. Sections were washed in PBS three times for 5 min then treated with ABC (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA USA) solution for 2 h at room temperature and the reaction visualized with diaminobenzidine (Sigma, USA) and H2O2. For RAGE staining, rabbit anti-goat IgG peroxidase conjugated (1:1500, Biodesign), was applied as secondary antibody to the sections for 4 h at room temperature. The reaction was visualized with 0.05% diaminobenzidine (Sigma, USA) 0.01% H2O2. Negative control sections were incubated with PBS in the absence of the primary antibody. Sections of mouse brain were used as a negative control.

Staining quantification Immuno stained sections were observed on a Nikon microscope (Nikon, Tokyo, Japan). Digital images were taken using a Nikon digital microscope camera system (Nikon, Tokyo, Japan). For all quantification processes of staining the observer was blind to COPD status of each subject. The intensity of staining for AGEs and RAGE was measured using Image J software (National Institutes of

AGEs and RAGE in COPD Table 1

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Summary of the two patient groups.

Age Smoking pack yrs FEV1% of predicted FVC % of predicted FEV1/FVC% Number of subjects

COPD mean (SD)

Non COPD mean (SD)

P-value

64.8 (1.7) 43.4 (4.8) 62.7 (1.8) 80.3 (2.4) 58.0 (1.6) 20 (13M/7F)

62.7 (2.0) 44.1 (4.9) 94.1 (2.8) 92.6 (3.7) 77.4 (1.4) 18 (11M/7F)

Not significant Not significant P < 0.01 P < 0.05 P < 0.01 Not significant

The study was approved by the North Health Ethics Committee, Auckland, New Zealand.

Health, Bethesda, Maryland, USA). All small airways (bronchioles without cartilage, normally 3e6 for each section) were photographed digitally. Intensities were determined for successive fields across the section. Background staining intensities were determined for adjacent or close by control sections (with the primary antibody omitted.) and subtracted from intensities determined for the antibody-stained sections. For each positive stained region, 10 measuring spots were sampled randomly and an average intensity  SD was calculated. The number of cells positive for AGEs and RAGE was counted manually. Briefly, the middle field (20) of each section was chosen for analysis. The total number of cells and the number of positive cells were counted in successive fields at a magnification of 400, starting at the centre of the top edge of the section and proceeding straight down to the bottom of the section until a vertical column had been counted. 10e15 successive fields for each section were counted.

Statistics analysis All the data were analysed using a SAS system software (version: enterprise 9.1) package. All P values of <0.05 are taken to be significant. Student’s t-test was used to compare the difference between two groups and Spearman Correlation Coefficients test was used for the correlation between the staining intensity and FEV1% of predicted value.

Results The characteristics of the subjects are shown in Table 1. Subjects with COPD were similar to the controls with respect to age, gender and smoking history but, had lower lung function. FEV1 was 62  1.8% of predicted in the subjects with COPD compared with 94  2.8% for the controls.

AGEs staining Positive staining of AGEs was mainly confined to the cell membrane of the single layer of epithelium of alveolar walls. In the small airway areas, positive staining of AGEs was found both on the surface and in the cytoplasm of goblet cells and epithelial cells. The cilia of small airways also stained positively in most sections. Positive staining was also occasionally found in the stroma of small airways and in the pulmonary macrophages in the lumen of airways (Fig. 1). While the number of cells positive for AGEs was not significantly different between the COPD group and non-

COPD controls, the intensity of the positive staining was significantly stronger in the COPD group than in the nonCOPD control group (p Z 0.004) (Fig. 2). Furthermore the mean intensity of AGEs staining around the small airway areas (bronchiolesenoncartilaginous conducting airways) was stronger in the COPD group than in the non-COPD control group (p Z 0.025) (Fig. 3).

RAGE staining The positive staining of RAGE was mainly located on the cell membrane of alveolar walls. Positive staining of RAGE was also observed in the cytoplasm. (Fig. 4). The intensity of staining for RAGE was stronger in the COPD group than in non-COPD control group. (p Z 0.027) (Fig. 5). The mean staining intensity for RAGE in the small airways of the COPD group was, however, not significantly different to that of the non-COPD control group. (p Z 0.26) (Fig. 6). However, patients with low FEV1% of predicted values had a stronger intensity of RAGE staining than subjects with higher FEV1% of predicted values (p Z 0.0069) and the intensity of staining was strongly associated with FEV1% of predicted alone (Fig. 7).

Discussion In this study, subjects from two groups (COPD and nonCOPD) were well controlled with no significant difference in age, gender, smoking history and medical histories; both groups were non-diabetic patients with bronchial carcinoma. The only major uncontrolled factor between these two groups was their lung function. Subjects from the COPD group had significantly lower ratios in FEV1% of predicted value and FEV1/FVC which together represent a poorer airflow in the lungs. We found that in the COPD group, subjects had a significantly higher staining intensity for AGEs in lung parenchyma and in the small airways than in the non-COPD control group. COPD subjects also had a significantly higher staining intensity for RAGE in lung parenchyma than in the non-COPD control group, and that the intensity of RAGE staining was strongly correlated with the patients’ lung function measured by FEV1% of predicted. Together these results suggest that the AGEseRAGE interaction may be more common and more intense in the lungs of COPD patients and around small airways than in the non-COPD controls. Our findings are consistent with the results from a recent study by Ferhani and colleagues.18 In that study, they found

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Figure 1 A: AGEs staining in COPD bronchioles. In the small airways, the positive staining of AGEs was found on the surface (Arrow a) and in the cytoplasm (Arrow b) of epithelium cells. The cilia (Arrow c) of small airways were also stained positively in most sections. Positive staining was also occasionally found in the stroma (Arrow d) of bronchioles and in the pulmonary macrophages in lumen of airways. B: AGEs staining in non-COPD bronchioles. While the positive staining of AGEs had a similar pattern to COPD bronchioles the intensity of staining was significantly weaker (Arrow a: positive staining on the surface of epithelium cells. Arrow b: positive staining in the cytoplasm. Arrow c: positive staining of the cilia of small airways.). C: AGEs staining in alveolar walls of COPD. The positive staining of AGEs was seen in the pulmonary macrophages in lumen of the alveoli (Arrows a and c). The positive staining of AGEs was mainly confined to the cell membrane of the single layer of squamous epithelium of alveolar walls (Arrow b). D: AGEs staining in alveolar walls of non-COPD. Positive staining was seen in the epithelium cells of alveolar walls (Arrows a and b). Positive staining was also seen in the pulmonary macrophages in lumen of the alveoli (Arrow c). The scale bar Z 5um (A, B, C and D).

AGEs and RAGE in COPD

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AGE mean intensity of positive cells

200 175 150 125 100 75 50 25 0 COPD

NON-COPD

* p=0.004 Figure 2 The intensity of the positive staining was significantly stronger in the COPD group than in non-COPD control group (*p Z 0.004). Bars above and below the means show SD.

that RAGE was overexpressed in the airway epithelium and smooth muscle of patients with COPD and RAGE colocalized with HMGB1 (High-mobility group box 1). They also found that smokers with COPD had higher levels of HMGB1 in their BAL fluid than did smokers without COPD, or never-smoker healthy individuals. The authors suggest that the existence of an autocrine/paracrine loop in which binding of HMGB1 to IL-1b, and possibly to RAGE and potentially other HMGB1 receptors, amplifies inflammatory and remodelling signals that are involved in COPD pathogenesis. In an animal study,19 the authors reported that feeding rats with AGEs-enriched foods resulted in an increased level of AGEs in lung tissue after 2 wk of feeding. Furthermore, the mRNA expression of HMGB1 was also increased in response to the AGEs-rich foods but not in response to the ageing associated AGEs

AGE airway mean intensity

200

150

100

50

0 COPD

NON-COPD

* p=0.025 Figure 3 In bronchioles, the intensity of positive staining of AGEs was stronger in the COPD group than in non-COPD control group (*p Z 0.025). Bars above and below the means show SD.

modifications in vivo. Based on these findings, it is very possible that the elevated AGEs levels in lung tissues from COPD patients may cause the increase of HMGB1 expression. Advanced Glycation End products (AGEs) are the result of a chain of chemical reactions following an initial glycation reaction. AGEs are a heterogenous class of compounds formed by diverse stimuli, including hyperglycaemia, oxidative stress, inflammation, renal failure, and innate ageing.5 There is considerable evidence of increased oxidative stress in the lungs of patients with COPD with a number of studies documenting increased expression of markers of oxidative stress in the lungs of patients with COPD, compared to healthy subjects or smokers with a similar smoking history who have not developed COPD.18 Since a possible consequence of the cumulated chronic oxidative stress in COPD patients is increased AGEs, the higher staining intensity for AGEs we observed is consistent with findings in other studies. In addition, the possibility of poor clearance of AGEs in COPD patients may also contribute to the observed increase in AGEs. In diabetics, it has been found that suffering from increase AGE production, subsequent kidney damage (by AGE production in the glomerulus) reduces the subsequent urinary clearance of AGEs, forming a positive feedback loop and further increasing the rate of damage.20 RAGE (receptor for advanced glycation end products) was initially isolated from endothelial cells of bovine lung.11 RAGE transgenic and knockout mice have provided functional genomic models to help identify the roles of RAGE. It was found that transgenic mice with high levels of expression of full length RAGE developed the diabetic pathogenic changes and were susceptible to the development of diabetic nephropathy.21 The RAGE knockout mice model also demonstrates that RAGE is associated with atherosclerosis.22 In homozygous RAGE null mice, a significant decrease in neointimal expansion after arterial injury and decreased smooth muscle cell proliferation, migration, and expression of extracellular matrix proteins was observed. In a sepsis knockout mouse model,23 the deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. For hemorrhagic shock, the RAGE knockout mice model24 showed that activation of RAGE-dependent signaling is a key factor leading to gut mucosal barrier dysfunction after hemorrhagic shock and resuscitation. In most AGEs-RAGE related diseases, RAGE expression is up-regulated in response to the disease. For example, in the Alzheimer’s disease brains, RAGE is up-regulated and serves to activate microglia as well as mediate amyloid-b transport across the bloodebrain barrier.25 In diabetic vasculature that is prone to atherosclerosis, RAGE expression is upregulated and together with its ligands AGEs and S100/ calgranulins, initiates proinflammatory signaling that exacerbates plaque formation.26 In contrast, in both mouse models and human idiopathic pulmonary fibrosis (IPF) lungs, RAGE and sRAGE expression is significantly down regulated after profibrotic pulmonary injury. COPD does however have an opposing mechanism of action to that of IPF. IPF is associated with overexpression of collagen and elastin in the lung tissue while COPD is believed to be associated with the decrease of collagen and elastin in lung. While AGEs and RAGE were found to be decreased in the lung tissues with IPF27,28 AGEs and RAGE staining intensity were both found to

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Figure 4 A: RAGE staining in COPD alveolar walls. The positive staining of RAGE was mainly located to the cell membrane of the epithelial cells of alveolar walls (Arrows a and b). Positive staining of RAGE was also observed in the cytoplasm of epithelial cells (Arrow c). B: RAGE staining in non-COPD alveolar walls. The positive staining of RAGE had a similar pattern to that seen in COPD sections, however, the staining intensity was much lower than in the COPD alveolar walls (Arrow a and c). Positive staining was also found in the cytoplasm (Arrow b). The scale bar Z 5um (A and B).

be significantly higher in the non-diabetic COPD group compared to the non-COPD control group in our study. The role of RAGE and AGEs in maintaining and amplifying inflammation has been documented only recently. RAGEmediated tissue degradation has been implicated in the chronic inflammatory pathogenesis in many organs. In lung, the RAGEeAGEs interaction has been associated with a number of lung diseases including acute lung injury,29 cystic fibrosis,30 common interstitial diseases and post-

obstructive pneumonia.31 In a recent review by Schmidt and colleagues, authors suggest that the RAGEeAGEs interaction may not play a causative role or initiating factor in these lung diseases, but rather acts as a mediator of amplification of chronic cellular activation such as cellular dysfunction and tissue destruction.3 It is possible, the AGEs eRAGE interaction also accelerates the processes of COPD by promoting inflammatory and perhaps other pathways.

RAGE mean staining intensity in bronchioles 200 RAGE airway mean intensity

RAGE mean intensity of positive cells

200

150

100

50

150

100

50

0 COPD

0 COPD

NON-COPD

* p=0.027 Figure 5 The intensity of staining for RAGE in alveolar walls was stronger in the COPD group than in non-COPD control group (*p Z 0.027). Bars above and below the means show SD.

NON-COPD

p=0.26 Figure 6 The mean staining intensity for RAGE in the bronchioles of the COPD group was not significantly different to that of the non-COPD control group (p Z 0.26). Bars above and below the means show SD.

AGEs and RAGE in COPD

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Relationship between RAGE staining intensity and % predicted FEV1

RAGE Staining Mean Intensity

150

COPD Non COPD

125

100

75

to the development of COPD in a number of ways including airway and alveolar destruction, cytokine production and inflammation. Further studies are now required to determine the precise role of the AGEseRAGE interaction and to investigate whether the blockade of this receptoreligand interaction slows the progression of COPD development.

Dedication

50

25

0 0

10 20 30 40 50 60 70 80 90 100 110 120 130 % Predicted FEV1

r Spearman= -0.45, * P=0.0069

Figure 7 Patients with the lower FEV1% of predicted value had a stronger intensity of RAGE staining in alveolar walls than subjects with higher FEV1% of predicted values (*p Z 0.0069).

The AGEs and RAGE interaction can increase intracellular oxidative stress by activating NADPH-oxidase (Nicotinamide adenine dinucleotide phosphate), a central player in the production of superoxide radicals.32 Recently it has been reported that NADPH-oxidase enhances susceptibility to the proinflammatory effects leading to airspace enlargement and alveolar damage.33 Other components of the signal transduction pathways of AGEs downstream of RAGE are p21ras and ERK1/2.34 The MAPKs (including Erk1/2) have been reported to mediate transcription of proteases and cytokines in response to a variety of stimuli. Smoking or other components of cigarettes indeed activate the MAPK-Erk1/2 pathway in fibroblasts, smooth muscle cells, pulmonary epithelial cells, and bronchoalveolar cells which may contribute the development of COPD.35 The AGEseRAGE interaction can also activate signal transduction pathways that include transcription factors such as NF-kB36,37 that bind to the promoter sites of many proinflammatory cytokines such as IL-1, IL-6, TNF-a and the inducible nitric oxide synthase.38 Cytokines or other proinflammatory stimuli can themselves activate NF-kB, and this turn induces RAGE expression and subsequent RAGE-mediated perpetuated NF-kB activation creating a positive autoregulatory loop.39,40 It is therefore quite possible that the increase in AGEseRAGE interactions in COPD lung amplify the inflammatory response through the activation of NF-kB. Currently, increasing evidence suggests that COPD is also involved with systemic inflammation. Serum TNF-a, IL-6 and TIMP-1 concentrations are significantly higher in patients with stable COPD than in control patients.41,42 The AGEseRAGE interaction may also contribute to this increase in these cytokines.

Conclusions The staining intensities of AGEs and RAGE were significantly higher in our non-diabetic COPD group than our non-COPD control group. The AGEseRAGE interaction may contribute

This Paper is dedicated to our close friend and mentor Professor Peter Black who died suddenly in January 2010. Peter was instrumental in the conception and design of this study. His death is a huge loss to us personally and to the COPD research community.

Acknowledgements The authors thank Mr. Gregory D Gamble for his excellent assistance with statistical analysis. This study was funded by Auckland Medical Research Fund.

Conflict of interest statement All authors confirm that none has any conflicts of interest, financial or personal, relating to the findings in this publication.

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