All the "RAGE" in lung disease: The receptor for advanced glycation endproducts (RAGE) is a major mediator of pulmonary inflammatory responses
Impact Factor: 2.716
Level of Evidence: Level I
Study Type: Review article
Protocol: Systematic literature review on RAGEs in pulmonary diseases
Results: The results from several experimental studies were compiled to exhibit the role that RAGE’s (receptor for advanced glycation end products) play in different pulmonary pathologies due to its role in the amplification of cellular inflammatory responses. As a proinflammatory pattern recognition receptor, it has been found to be highly expressed in lung tissue on the basal membrane of type 1 alveolar epithelial cells. In clinical manifestations such as asthma, cystic fibrosis, acute lung injury, acute respiratory distress syndrome, and COPD, increased RAGE expression was correlated to worse clinical lung outcomes. On the flip side, in pulmonary fibrosis and lung cancers, upregulation of RAGE was correlated with better prognoses.
Conclusion: RAGE plays an important role in inflammation in pulmonary diseases and hence can be targeted in therapeutic strategies. sRAGE, a RAGE blocking antibody, can be used to interfere with the RAGE signaling cascade and treat patients with pulmonary diseases.
Oczypok EA, Perkins TN, Oury TD. All the "RAGE" in lung disease: The receptor for advanced glycation endproducts (RAGE) is a major mediator of pulmonary inflammatory responses. Paediatr Respir Rev. 2017 Jun;23:40-49. doi: 10.1016/j.prrv.2017.03.012. Epub 2017 Mar 18. PMID: 28416135; PMCID: PMC5509466.
The receptor for advanced glycation end products and its ligands: a new inflammatory pathway in lung disease?
Impact Factor: 5.915
Level of Evidence: Level III
Study Type: non-randomized controlled experimental design
Sample Size/Population: 42 lung samples
Protocol: Lung samples were taken through thoracoscopy and lobectomies. These samples included tissues from patients with pneumonias, sarcoidosis, tuberculosis, smoke induced lesions, and also nonsmoking patients. The samples were then reviewed to confirm the diagnoses and immunochemistry was performed on them with antibodies against RAGE ligands.
Results: In the normal lung, RAGE immunoreactivity was a low to intermediate level and S100A12 and S100B were expressed. Lung tissue samples with smoke related pulmonary disease, nonspecific inflammation, organizing pneumonia, granulomatous lung disorders, and usual interstitial pneumonia all showed high RAGE immunoreactivity and variable S100A12 and S100B expression.
Conclusion: The receptor for advanced glycation end products (RAGEs) inflammatory pathway may play a role in chronic lung diseases. The increased RAGE expression in tissue samples with lung disease was not only due to the increased number of reactive pneumocytes and macrophages but also in respects to normal conditions. RAGE has been shown, in this study and previous studies, to be more involved in the amplification of disease rather than the causative agent. This RAGE inflammatory pathway is not specific to one lung disease and can potentially be used to develop therapeutic agents based on RAGE blockage.
Morbini P, Villa C, Campo I, Zorzetto M, Inghilleri S, Luisetti M. The receptor for advanced glycation end products and its ligands: a new inflammatory pathway in lung disease? Mod Pathol. 2006 Nov;19(11):1437-45. doi: 10.1038/modpathol.3800661. Epub 2006 Aug 25. PMID: 16941014.
Receptor for advanced glycation end-products and environmental exposure related obstructive airways disease: a systematic review
Impact Factor: 12.339
Level of Evidence: Level I
Study Type: review article
Protocol: Systematic literature review examining the relationship between RAGE (receptor for advanced glycation end products) and obstructive airways disease (OAD) secondary to environmental exposure. A comprehensive search was done on PubMed and EMBASE.
Results: A compressive literature review was completed that examined the connection between RAGE and the development of OAD due to environmental exposures such as smoke and dust mites. RAGE was found to be a biomarker in pulmonary diseases such as emphysema, vascular injury, cardiovascular disease in COPD, and asthma. The environmental factor of smoking was studied in detail, and it was shown that cigarette smoke lowered sRAGE levels and elevated RAGE and NO levels. This was also connected to COPD, as studies showed that COPD patients and cigarette smokers developed pulmonary injuries through the AGE-RAGE pathway.
Conclusion: From the studies that were analyzed, RAGE was shown to be a contributor into OAD development. Further studies on the RAGE pathway and its role in inflammation associated with OAD can potentially be useful in identifying and developing therapeutic agents that can prevent the development and progression of lung diseases such as COPD.
Haider SH, Oskuei A, Crowley G, Kwon S, Lam R, Riggs J, Mikhail M, Talusan A, Veerappan A, Kim JS, Caraher EJ, Nolan A. Receptor for advanced glycation end-products and environmental exposure related obstructive airways disease: a systematic review. Eur Respir Rev. 2019 Mar 27;28(151):180096. doi: 10.1183/16000617.0096-2018. PMID: 30918021; PMCID: PMC7006869.
Advanced glycation end products in the skin are enhanced in COPD
Impact Factor: 6.159
Level of Evidence: V
Sample Size/Population: N= 395 (COPD: 202, Old Healthy: 83, Young Healthy: 110)
Protocol: Subject groups (COPD I-IV, Old Healthy, Young Healthy) performed a post-bronchodilator spirometry and body plethysmography in order to determine lung function and clinical characteristics. Skin autofluorescence (SAF) was also tested for using the AGE-Reader scanner. Three separate SAF measurements were recorded and expressed as the average of all three. Subject groups were compared between one another.
Statistical Analysis: Mann-Whitney U tests and Chi-square tests to compare baseline characteristics between groups. Linear regression analyses to observe associations between SAF and clinical characteristics as well as SAF and lung function.
Study Type: Cross-sectional study
Results: In terms of SAF values, there was a significant difference between COPD patients and healthy subjects, with COPD patients having higher SAF values. It was also noted that SAF values were significantly associated with lower lung function, regardless of age, gender, or pack-years in smokers. SAF levels were also observed to be similar between COPD groups I-IV. There was not a significant difference between healthy smokers and healthy non-smokers in each group, however.
Conclusion: AGE levels measured through SAF were significantly higher in individuals with COPD in comparison to healthy controls. However, given that SAF values were similar between COPD groups, it is indicated that the level of AGEs in the skin is not indicative of the level of disease in COPD. Researchers do suggest, however, that AGE formation may have a genetic component that contributes to onset of disease. This study was limited by the fact that it is a cross-sectional study and the possible broad overlap between groups. It is suggested that SAP values could be implemented as biomarkers for COPD in the clinic.
Hoonhorst SJ, Lo Tam Loi AT, Hartman JE, Telenga ED, van den Berge M, Koenderman L, Lammers JW, Boezen HM, Postma DS, Ten Hacken NH. Advanced glycation end products in the skin are enhanced in COPD. Metabolism. 2014 Sep;63(9):1149-56. doi: 10.1016/j.metabol.2014.06.006. Epub 2014 Jun 13. PMID: 25034386.
Soluble RAGE is deficient in neutrophilic asthma and COPD
Impact Factor: 12.339
Level of Evidence: Level III
Study Type: non-randomized controlled experimental design
Sample Size/Population: 16 patients with asthma, 37 with COPD, 18 healthy controls (Group1). 101 clinically stable patients with asthma, and 34 clinically stable patients with COPD (Group 2).
Protocol: Differential cell counts, sRAGE and RAGE ligands were measured from BL fluid samples from patients in group1. Differential cell counts and systemic sRAGE were analyzed using peripheral blood and sputum samples respectively from group 2. ELISA was used to detect total sRAGE and esRAGE and was also used to measure HMGB1 (IBL International, Hamburg, Germany; lower limit of detection 2.5 ng·mL−1), SAA (Anogen, Mississauga, ON, Canada; lower limit of detection 2.5 ng·mL−1), CXC chemokine ligand (CXCL)8 (R&D Systems; lower limit of detection 31.25 pg·mL−1) and myeloperoxidase (Zen™ Myeloperoxidase (MPO) ELISA, Invitrogen, Carlsbad, CA, USA; lower limit of detection 0.375 ng·mL−1).
Results: COPD patients with neutrophilic airway inflammation displayed a deficiency in lung sRAGE and systemic sRAGE. Lung sRAGE, older age, lower lung function and the presence of airway bacterial colonization were found to be independent predictors of neutrophilic airway inflammation. Systemic sRAGE was found to be an independent predictor of neutrophilic airway inflammation in COPD patients.
Conclusion: sRAGE is a potential biomarker for the prognosis and management of neutrophilic airway inflammation in airways disease. In asthma and COPD, deficiency in sRAGE was selectively associated with neutrophilic airway inflammation. Moreover, correcting deficiencies in sRAGE could represent a therapeutic strategy for neutrophilic asthma and COPD.
Sukkar MB, Wood LG, Tooze M, Simpson JL, McDonald VM, Gibson PG, Wark PA. Soluble RAGE is deficient in neutrophilic asthma and COPD. Eur Respir J. 2012 Mar;39(3):721-9. doi: 10.1183/09031936.00022011. Epub 2011 Sep 15. PMID: 21920897.
Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury
Impact Factor: 17.452
Level of Evidence: Level VII
Study Type: animal study
Sample Size/Population: Male C57BL/6J mice, 8–11 weeks of age and weighing 18–22 g
Protocol: BAL fluid was collected, and then Western blot analysis was done to determine the expression of sRAGE in the alveolar space. To determine the effect of sRAGE on LPS-induced lung injury, mice were divided into groups and injected with LPS or PBS then lung sampling was taken for NF-kB analysis. After injecting mice with HSA, the levels of HSA were measured in plasma and BAL fluid using ELISA. ELISA was also used to measure HMGB1 level in BAL fluid. Finally the lungs were sampled for pathology.
Statistical Analysis: One-way analysis of variance with the least squares differences test using SPSS Windows 14.0 statistical analysis software (SPSS, Inc., Chicago, IL). A value of P < 0.05 was considered to be statistically significant.
Results: sRAGE was found to be up-regulated in the alveolar space as a response to intratracheal LPS challenge. The results of the study showed that sRAGE mitigates LPS-induced inflammatory events in the lung, including neutrophil infiltration, increased lung permeability, edema formation, production of inflammatory cytokines, and NF-κB activation, as well as apoptosis of the lung cells.
Conclusion: In terms of the pathogenesis of LPS-induced lung injury and the blockade of RAGE signaling by sRAGE, RAGE plays a critical role and could be protective against the development of lung injury. It was found that sRAGE administered 1 hour after LPS challenge could sufficiently ameliorate lung injury. sRAGE may be secreted as a decoy receptor and contribute to the suppression of excessive inflammatory response during ALI/ARDS, yet further research has to be done on this.
Zhang H, Tasaka S, Shiraishi Y, Fukunaga K, Yamada W, Seki H, Ogawa Y, Miyamoto K, Nakano Y, Hasegawa N, Miyasho T, Maruyama I, Ishizaka A. Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury. Am J Respir Crit Care Med. 2008 Aug 15;178(4):356-62. doi: 10.1164/rccm.200707-1069OC. Epub 2008 Jun 5. PMID: 18535257.
The receptor for advanced glycation end products in ventilator induced lung injury
Impact Factor: 14.340
Study Type: Case-Control
Sample Size/Population: Eight- to ten-week-old male RAGE KO mice and C57BL/6 age matched wild type mice were split into 6 -9 per group in the study.
Protocol: Wild type and RAGE KO mice were randomized into a control group and a machine ventilator (MV) inflammation/injury group. The mice in the MV group were ventilated with an inspiratory pressure of 10 cm H2O (LVT) or inspiratory pressure of 18 cm H2O (HVT), with a respiratory rate of 110 breaths/min and 70 breaths/min respectively. Positive end-expiratory pressure was set at 2 cm H2O during both MV strategies.
A second set of wild type and RAGE KO mice were randomized into groups with pre-injured lungs. Injury was induced by inhalation of 5ug LPS. The groups then underwent the same ventilation setups previously stated or were kept off the ventilator.
In a third set of randomized wild type and RAGE KO groups, the presence of sRAGE (soluble RAGE) and HMGB1 (a RAGE ligand) were analyzed in the models. Additionally, RAGE KO mice with LPS included injury received 50 ug recombinant His-tagged murine sRAGE or saline intratracheally at the start of the HVT MV. Total protein was determined using a Bradford Protein Assay Kit. Interleukins, chemokines, TNF, and MIP were measured using ELISA. sRAGE was measured by Mouse RAGE Duo set ELISA. HMGB1 levels were determined via Western Blot.
Statistical Analysis: Data represent mean ± SEM. Human samples were analyzed by paired t test or Wilcoxon signed-rank test. One-way analysis of variance with Bonferroni or a Kruskall-Wallis test with Mann-Whitney U as post hoc analysis was used to analyze multiple groups. To compare two groups, a t-test or Mann-Whitney U test was used. p < 0.05 was considered statistically significant.
Results: MV enhanced RAGE expression in healthy murine lungs. Both HVT and LVT ventilation strategies significantly increased RAGE mRNA expression in lung tissue compared to the control group with no ventilation exposure.
RAGE KO mice lungs did not show any positive immunostaining for RAGE while the healthy non-ventilated control group expressed abundant levels of RAGE within their alveolar epithelium. There was no notable difference in abundance of RAGE between the HVT and LVT groups.
Wild type mice subjected to HVT MV produced marked increase in lung wet-to-dry ratio and total protein level compared to controls. Both ventilator strategies induced neutrophil influx into the alveolar compartment, with higher levels appearing in the HVT group.
HVT ventilated RAGE KO mice had the most enhanced LPS induced lung injury compared to controls. RAGE KO LPS induced mice also demonstrated elevated cytokine and chemokine levels. RAGE deficiency did not affect lung wet-to-dry ratio, total protein level, or cell influx in the LPS exposed and LPS exposed + MV groups.
Healthy HVT MV mice showed increased sRAGE concentration compared to the LVT MV and non-ventilated control groups. The highest levels of sRAGE were found in the HVT MV + LPS injured group. HMGB1 was only detectable in the HVT MV + LPS injured group.
Total protein count and neutrophil influx were not affected by sRAGE administration to the HVT MV +LPS injured group. However, cytokine and chemokine levels in sRAGE treated mice displayed significantly lower levels of IL6, KC, and MIP-2.
Conclusion: Rage expression is up regulated during 5h of MV in both human and murine lungs. RAGE contributes to inflammatory cell influx. RAGE deficiency is not protective, but rather resulted in enhanced inflammatory response when exposed to both MV and LPS. sRAGE administration to RAGE KO mice partly alleviated the tissue damage in the HVT MV+LPS group.
Kuipers MT, Aslami H, Tuinman PR, Tuip-de Boer AM, Jongsma G, van der Sluijs KF, Choi G, Wolthuis EK, Roelofs JJ, Bresser P, Schultz MJ, van der Poll T, Wieland CW. The receptor for advanced glycation end products in ventilator-induced lung injury. Intensive Care Med Exp. 2014 Dec;2(1):22. doi: 10.1186/s40635-014-0022-1. Epub 2014 Aug 2. PMID: 26215707; PMCID: PMC4678142.
Dietary phenotype and advanced glycation end-products predict WTC-obstructive airways disease: a longitudinal observational study.
Impact Factor: 4.043
Level of Evidence: IV
Sample Size/Population: N= 4,015 (Ever WTC-OAD: 921, Never WTC-OAD: 3,094).
Protocol: Subjects assessed for risk of developing World Trade Center obstructive airways disease (WTC-OAD) in association with dietary quality and AGE content. REAP-S nutritional scores with AGE quantification from annual FDNY-HP visits compiled and assessed.
Statistical Analysis: Paired sample t-tests comparing clinical parameters at two separate time periods, student t-tests comparing groups, one-way ANOVA to analyze lung function and dietary quality.
Study Type: observational prospective study.
Results: Subjects with WTC-OAD were found to consume significantly more processed meats and sugary drinks and consume significantly less grains and vegetables. Also, those who regularly consume AGE-rich foods from a single category were not at increased risk of developing WTC-OAD. In contrast, those who regularly consume AGE-rich foods from two categories had a significantly increased risk of developing WTC-OAD and those who regularly consume AGE-rich foods in at least three categories are at a highly significant increased risk.
Conclusion: Subjects with AGE-rich diets were significantly more likely to develop WTC-OAD. This is most likely due to the fact that AGEs induce oxidative stress and inflammation in the body, especially lung tissue. This study mimicked similar findings in other studies comparing high intakes of processed meats with risk of developing COPD. Limitations to the study included self-reporting of dietary habits which is subject to participant bias.
Lam R, Kwon S, Riggs J, Sunseri M, Crowley G, Schwartz T, Zeig-Owens R, Colbeth H, Halpren A, Liu M, Prezant DJ, Nolan A. Dietary phenotype and advanced glycation end-products predict WTC-obstructive airways disease: a longitudinal observational study. Respir Res. 2021 Jan 18;22(1):19. doi: 10.1186/s12931-020-01596-6. PMID: 33461547; PMCID: PMC7812653.
Intake of high fructose corn syrup sweetened soft drinks is associated with prevalent chronic bronchitis in U.S. Adults, ages 20-55 y
Impact Factor: 3.359
Level of Evidence: Level VI
Study Type: cross sectional study
Sample Size/Population: 2801 adults aged 20–55 y.
Protocol: National Health and Nutrition Examination Survey from 2003–2006 that collected data on self-reported existing chronic bronchitis or history of CB, non-diet soda consumption, age, sex, race-ethnicity, BMI, smoking, exposure to in-home smoking, pre-diabetes, diabetes, SES, total energy and total fruits and beverages consumption.
Statistical Analysis: Rao Scott Ҳ2 was used for prevalence differences and logistic regression for associations, adjusted for age, sex, race-ethnicity, BMI, smoking, exposure to in-home smoking, pre-diabetes, diabetes, SES, total energy and total fruits and beverages consumption.
Results: Increased consumption of non-diet soft drinks (more than 5 times a week) was correlated with an increased risk of developing chronic bronchitis.
Conclusion: The hypothesis that a higher intake of fructose was correlated with chronic bronchitis, a subset of COPD, was proven. The underlying mechanism behind this may be correlated with the previously proposed “intestinal enFruAGEs fructositis” hypothesis. This hypothesis states that when fructose is maldigested in the GI tract that it contributes to advanced glycation end product formation that then travels to the lungs.
DeChristopher LR, Uribarri J, Tucker KL. Intake of high fructose corn syrup sweetened soft drinks is associated with prevalent chronic bronchitis in U.S. Adults, ages 20-55 y. Nutr J. 2015 Oct 16;14:107. doi: 10.1186/s12937-015-0097-x. PMID: 26474970; PMCID: PMC4609055.
Increased advanced glycation end product and meat consumption is associated with childhood wheeze: analysis of the National Health and Nutrition Examination Survey
Impact Factor: 10.844
Level of Evidence: Level VI
Study Type: Cross sectional survey
Sample Size/Population: 4,388 children aged 2-17 years from the 2003 to 2006 National Health and Nutrition Examination Survey
Protocol: Between 2003 and 2006, 4,388 children with respiratory symptoms were assessed using the Food Frequency Questionnaire. Other factors were compiled via questionnaires such as age, sex, race/ethnicity, poverty level, BMI, asthma status, and caloric intake.
Statistical Analysis: Survey logistic regression models
Results: The results of the surveys showed a strong statistically significant correlation between consuming foods with high AGE (advanced glycation end products) scores and increased odds of wheezing. Furthermore there was a weaker, but still statistically significant correlation between eating cooked non-seafood meat and increased odds of wheezing.
Conclusion: With regards to the pediatric population, it can be concluded that there is a statistically significant correlation between consuming food with high AGE content and increased odds of wheezing. Further studies examining the impact of dietary AGEs on pediatric airways are justified.
Wang JG, Liu B, Kroll F, Hanson C, Vicencio A, Coca S, Uribarri J, Bose S. Increased advanced glycation end product and meat consumption is associated with childhood wheeze: analysis of the National Health and Nutrition Examination Survey. Thorax. 2021 Mar;76(3):292-294. doi: 10.1136/thoraxjnl-2020-216109. Epub 2020 Dec 21. PMID: 33443194; PMCID: PMC7892397.
Intakes of apple juice, fruit drinks and soda are associated with prevalent asthma in US children aged 2-9 years
Impact Factor: 3.182
Level of Evidence: Level VI
Study Type: cross sectional study
Sample Size/Population: 2–9-year-old children from the US that filled out the questionnaire from 2003 to 2006.
Protocol: Questionnaires were given out to children aged 2-9 years old in the US. These surveys attempted to correlate beverage consumption and a history or self-diagnosis of asthma. The beverages were categorized according to EFF beverages, including apple juice (AJ), non-diet soft drinks (ndSD) and fruit drinks (FD). Orange juice (OJ), not an EFF beverage, was included as a comparison. The questionnaire asked about asthma, beverage consumption, age, sex, race/ethnicity, BMI and total energy intake. These variables were then analyzed with Rao–Scott χ 2 analyses.
Statistical Analysis: Rao–Scott χ 2 analyses used for prevalence differences and logistic regression for associations, adjusted for age, sex, race/ethnicity, BMI and total energy intake.
Results: A correlation was found between EFF beverages and asthma in children aged 2-9 years old. Children who drank apple juice 5 times or more a week were extrapolated out and were found to be twice as likely to develop asthma when compared to the children that only had apple juice about once a month. Orange juice was found to be protective.
Conclusion: The results above support the hypothesis that an intake of sugary beverages sweetened with high fructose corn syrup lead to an increased risk of developing asthma in young children. This may also support the hypothesis that in situ intestinal formation of advanced glycation end products (enFruAGE) with EFF may be absorbed and play a role in RAGE-mediated asthma. More studies will have to be done to establish this.
DeChristopher LR, Uribarri J, Tucker KL. Intakes of apple juice, fruit drinks and soda are associated with prevalent asthma in US children aged 2-9 years. Public Health Nutr. 2016 Jan;19(1):123-30. doi: 10.1017/S1368980015000865. Epub 2015 Apr 10. PMID: 25857343.
RAGE: a new frontier in chronic airways disease
Impact Factor: 7.730
Level of Evidence: Level I
Study Type: systematic review
Protocol: Data was accumulated from animal and clinical investigations on ligand–RAGE axis in asthma and COPD.
Results: RAGE forms complexes with cell surface molecules and there is still limited knowledge regarding RAGE signaling and effects.
There may be collaboration between RAGE and TLR ligand recognition and signaling.
Soluble RAGE has agonist effects on monocyte and neutrophil responses.
Some studies showed that total soluble RAGE concentrations were positively associated with cardiovascular and all-cause mortality. Other studies showed soluble RAGE as a protective factor.
RAGE and its ligands are increased in patients with COPD and soluble RAGE is decreased.
RAGE expression may be increased with cigarette smoke.
Conclusion: RAGE and its ligands have been shown to be increased in chronic airways diseases while an inverse relationship has been shown with soluble RAGE. Environmental and cigarette smoke pollutants have shown to increase RAGE expression in the lungs which connects RAGE to the COPD pathogenesis. Further research needs to be done on the relationship between RAGE and TLR as well as between RAGE gene polymorphisms, environmental exposures, and susceptibility to asthma and COPD across different populations.
Sukkar MB, Ullah MA, Gan WJ, Wark PA, Chung KF, Hughes JM, Armour CL, Phipps S. RAGE: a new frontier in chronic airways disease. Br J Pharmacol. 2012 Nov;167(6):1161-76. doi: 10.1111/j.1476-5381.2012.01984.x. PMID: 22506507; PMCID: PMC3504985.
Tobacco smoke is a source of toxic reactive glycation products
Impact Factor: 9.412
Level of Evidence: Level III
Study Type: experimental
Sample Size/Population: cigarette smoke condensate and Salmonella strains TA98, TA100, TA1535, and TA1537
Protocol: Tobacco smoke condensate was produced from cigarettes. This was used on rat tail tendon collagen to do an in vitro AGE formation assay. The condensate was also used with a PBS/EDTA solution to measure glycotoxin cross linking activity and assess RNase A-AGE-specific fluorescence. Mutagenicity studies were done on Salmonella strains incubated with different amounts of the cigarette smoke condensate. Sandwich ELISA was used on human serum to measure AGE-ApoB. Competitive ELISA was used to measure AGEs in human sera.
Statistical Analysis: unpaired Student t test, standard deviation, mean
Results: The TA98 strain of Salmonella typhimurium saw a dose-dependent increase in mutations with the cigarette smoke condensate. In smokers, AGE-ApoB levels and serum AGE levels were significantly higher than in non-smokers. The aqueous tobacco and cigarette smoke contained highly reactive end products that enhanced the formation of AGEs on proteins in vivo and in vitro and caused DNA mutations in vitro.
Conclusion: In cigarette smoke, the glycotoxins induce AGE formation in hours due to their high reactivity. This AGE formation in vivo may be the reason why smokers have a high risk of developing atherosclerosis. The glycotoxins in cigarette smoke may also lead to increased incidences of cancer due to the reaction of glycation products with the amino groups of nucleic acids.
Cerami C, Founds H, Nicholl I, Mitsuhashi T, Giordano D, Vanpatten S, Lee A, Al-Abed Y, Vlassara H, Bucala R, Cerami A. Tobacco smoke is a source of toxic reactive glycation products. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13915-20. doi: 10.1073/pnas.94.25.13915. PMID: 9391127; PMCID: PMC28407.
Lung level of HMBG1 is elevated in response to advanced glycation end product-enriched food in vivo.
Impact Factor: 4.653
Level of Evidence: II
Sample Size/Population: N=18 (control: 6, bread crust: 6, coffee extract: 6, young adult: 9, senescent: 9)
Protocol: High mobiity group box protein 1 (HMBG1) are copounts that are used for normal regulation of transcription in somatic cells. In order to determine the impact of food-derived AGEs and age-related AGEs on lung tissue in regards to HMBG1, five separate rat animal model groups were formed. Food-derived AGEs were studied via a control mouse group, a group fed with bread crumbs, and a group fed with coffee extract. Level of AGEs were measured and observed after a notable feeding time period of 15 days. Age-derived AGEs were studied via an adult rat group and a senescent rat group. Level of AGEs were compared between the two groups in order to determine the effects of age.
Statistical Analysis: Groups compared using a student’s t-test and one-way ANOVA. A two-sided test was used to perform a linear regression in order to determine the significance of the data.
Study Type: Animal model control trial.
Results: Overall, feeding rats AGE-rich foods resulted in increased AGEs in lung tissue after the 15 day feeding period. This was true for both the bread crumb feeding group and the coffee extract feeding group in comparison to the control group. The same effects were not as pronounced in the age-dependent models, however, which suggests that dietary AGEs may play a larger role in determining AGE levels within lung tissue. In addition, when looking at the effects of AGE-rich foods it was shown that long term consumption resulted in decreased levels of HMGB1 in the lungs.
Conclusion: Given that AGE-rich foods presented with a large difference in AGE amounts between the groups, it is presumed that dietary AGEs play a big part in determining overall AGE levels in the lungs. This is especially true regarding regulation of HMGB1 in the lungs. This same phenomenon was not observed with aging ,however. This indicates that dietary AGE consumption is a better prediction of HMGB1 expression, an overall lung function, better than age-related AGE levels.
Bartling B, Fuchs C, Somoza V, Niemann B, Silber RE, Simm A. Lung level of HMBG1 is elevated in response to advanced glycation end product-enriched food in vivo. Mol Nutr Food Res. 2007 Apr;51(4):479-87. doi: 10.1002/mnfr.200600223. PMID: 17357979.
The receptor for advanced glycation end-products enhances lung epithelial wound repair: An in vitro study
Impact Factor: 3.246
Study Type: Case-control
Sample Size/Population: In vitro study of Human adenocarcinoma-derived alveolar AT-II epithelial cells (A549)
Protocol: Treatment of AT-II cells with RAGE agonists high-mobility group 1 box protein (HMGB1) and AGES, alone or in addition to a RAGE antagonist and observing the effects of wound healing in the cells. Wound healing was determined via a scratch assay observed for 48 h. Duplicate measurements were taken from six independently set up experiments. After testing RAGE agonists HMGB1 and AGEs for their effect on wound area, their minimum effective concentration was used in subsequent experiments.
Statistical Analysis: All analyses were performed using Prism 6 and State version 15. The tests were two-sided with type 1 error set at 5%. Assumption of normality was assessed using Shapiro-wilk test. Continuous parameters were compared between experimental groups using analysis of variance or the Kruskal-Wallis test when t-test assumptions were not met.
Results: HMGB1 and AGEs both promote dose-dependent, and RAGE-dependent wound healing of lung alveolar epithelial cells. HMGB1 and AGEs have distinct effects of migration of AT-II cells during wound healing. HMGB1 treated cells showed higher migration at 12 h while AGE treated cells showed higher migration 48 h after injury. Additionally, both HMGB1 and AGEs increase AT-II proliferation in a RAGE-dependent manner during wound healing.
Conclusion: Alteration of alveolar epithelial barrier integrity plays a central role in the pathophysiology of acute respiratory distress syndrome (ARDS). HMGB1 and AGEs promote in vitro wound healing of AT-II cells in a RAGE-dependent manner. This promoted healing could be due to the increased cell migration and proliferation also experienced in HMGB1 and AGE treated cells.
Zhai R, Blondonnet R, Ebrahimi E, Belville C, Audard J, Gross C, Choltus H, Henrioux F, Constantin JM, Pereira B, Blanchon L, Sapin V, Jabaudon M. The receptor for advanced glycation end-products enhances lung epithelial wound repair: An in vitro study. Exp Cell Res. 2020 Jun 15;391(2):112030. doi: 10.1016/j.yexcr.2020.112030. Epub 2020 Apr 21. PMID: 32330509.
Advanced glycation end-products and receptor for advanced glycation end-products expression in patients with idiopathic pulmonary fibrosis and NSIP.
Impact Factor: 1.706
Level of Evidence: II
Sample Size/Population: N = 30 (Control: 10, IPF: 10, NSIP: 10)
Protocol: Lung samples from patients with non-specific interstitial pneumonia (NSIP), idiopathic pulmonary fibrosis (IPF), and a control group were analyzed using immunofluorescence assay, Western blot, and ELISA. Immunofluorescence assay was performed in order to determine the AGE and RAGE composition of the lung tissue. Results were compared between groups.
Statistical Analysis: This study used Tukey’s test to determine differences between groups as well as a one-way analysis of variance in order to determine specific differences. All of the data values were also expressed as means with standard deviations.
Study Type: Observational study.
Results: Despite the IPF and NSIP patients exhibiting decreased pulmonary function, there appeared to be no correlation between AGE levels and clinical parameters. With that being said, however, there was a significant difference between AGE and RAGE levels in patients with IPF compared to NSIP and control patients. No significant difference between NSIP and control or IPF subjects was observed. The Western blot demonstrated that patients with IPF not only have increased expression of AGEs, but also higher circulating levels of AGEs. The ELISA test revealed that these increased levels in IPF patients was significant.
Conclusion: The study confirmed previous evidence that AGE levels are significantly higher in patients with IPF in comparison to those with NSIP and controls. No significant difference in AGE expression was observed in patients with NSIP. In addition to the increased expression of AGE in IPF patients, circulating levels of AGE were also increased in these patients. Researchers indicated that this may mean that there is a connection between circulating AGE levels and the pathogenesis of IPF.
Kyung SY, Byun KH, Yoon JY, Kim YJ, Lee SP, Park JW, Lee BH, Park JS, Jang AS, Park CS, Jeong SH. Advanced glycation end-products and receptor for advanced glycation end-products expression in patients with idiopathic pulmonary fibrosis and NSIP. Int J Clin Exp Pathol. 2013 Dec 15;7(1):221-8. PMID: 24427342; PMCID: PMC3885476.
Immunohistochemical localisation of advanced glycation end products in pulmonary fibrosis.
Impact Factor: 2.460
Level of Evidence: II
Sample Size/Population: N = 17 (IPF: 7, Diffuse alveolar damage: 3, control: 7).
Protocol: A monoclonal mouse anti-AGE antibody (6D12) was produced in order to determine the level of AGEs in lung tissue. The 6D12 antibody was used since it reacts with the main AGE structure and would therefore cover a wide variety of AGEs. Immunochemistry was performed in order to evaluate the composition of AGEs found in the lung tissue of the three groups contained within the research study. Immunochemistry specifically for AGE on macrophages was performed.
Statistical Analysis: This study used a Mann-Whitney U test to compare the levels of immunoreactivity between groups of lung tissue. Probability levels were set at a significance level of 0.05. In order to compare level of immunoreactivity between groups, a semiquantitative scale was developed to use for evaluation.
Study Type: Observational case study.
Results: From immunochemistry analysis researchers found that five out of the seven control patients were negative for 6D12. However, all seven of the IDP patients exhibited 6D12 on immunohistochemistry. In addition, some of the cases with diffuse alveolar damage also exhibited 6D12 on immunohistochemistry. These results show that patients with idiopathic pulmonary fibrosis and diffuse alveolar damage exhibit advanced glycation end products in their lung tissue.
Conclusion: This study found that AGEs were detected in lung tissue samples from patients with idiopathic pulmonary fibrosis and diffuse alveolar damage. This was not found in the majority of the control group. Given that macrophages are thought to play a major role in the pathogenesis of pulmonary diseases such as IPF, researchers from this study suggest that AGEs, more specifically AGE modified proteins, may be a useful biomarker in determining oxidative stress of pulmonary tissue. The researchers also suggest that AGE may be a useful biomarker in the pathogenesis of pulmonary fibrosis.
da Silva LF, Skupien EC, Lazzari TK, Holler SR, de Almeida EGC, Zampieri LR, Coutinho SE, Andrades M, Silva DR. Advanced glycation end products (AGE) and receptor for AGE (RAGE) in patients with active tuberculosis, and their relationship between food intake and nutritional status. PLoS One. 2019 Mar 14;14(3):e0213991. doi: 10.1371/journal.pone.0213991. PMID: 30870511; PMCID: PMC6417785.
Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis
Impact Factor: 4.043
Level of Evidence: Level III
Study Type: non-randomized controlled experimental design
Sample Size/Population: 25 lung samples obtained through surgical lung biopsies Protocol: 16 lung samples with idiopathic pulmonary fibrosis (IPF) and 9 control lung samples were collected through surgical lung biopsy. RT-PCR, Western blot and immunohistochemistry were used to measure the differences in RAGE and AGE expression. The impact that AGEs have on cells in the lungs (fibroblasts and alveolar epithelial cells) was also examined.
Statistical Analysis: IBM SPSS Statistics 23 (IBM, USA) was used to analyze the data. A Student’s t-test, Mann–Whitney U-test, and one-way ANOVA were utilized. The p-value < 0.05 (*) or < 0.01 (**) were considered statistically significant.
Results: An increase of AGEs together with a decrease of RAGEs was observed in IPF lungs when compared with the control samples. Pentosidine and Nε-Carboxymethyl lysine were significantly increased in IPF samples. Immunohistochemistry showed higher staining of AGEs related to extracellular matrix (ECM) proteins and the apical surface of the alveolar epithelial cells (AECs). RAGE location was seen at the cell membrane of AECs in control lungs, while it was practically gone in pulmonary fibrotic tissue.
Conclusion: An increased ratio AGEs-RAGEs in IPF could be a relevant accelerating aging tissue reaction in the abnormal wound healing of the lung fibrotic process.
Machahua C, Montes-Worboys A, Llatjos R, Escobar I, Dorca J, Molina-Molina M, Vicens-Zygmunt V. Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis. Respir Res. 2016 Nov 5;17(1):144. doi: 10.1186/s12931-016-0460-2. PMID: 27816054; PMCID: PMC5097848.
Serum AGE/RAGEs as potential biomarker in idiopathic pulmonary fibrosis
Impact Factor: 4.043
Level of Evidence: Level III
Study Type: Observational
Sample Size/Population: Serum samples from 62 IPF, 22 chronic hypersensitivity pneumonitis (cHP), 20 fibrotic non-specific interstitial pneumonia (fNSIP); and 12 healthy controls
Protocol: Serum samples were collected from the patients above and the epidemiological/clinical factors were recorded such as race, sex, smoking habits, and lung function. ELISA was used to evaluate AGEs and sRAGE and pulmonary function tests were done. The ELISA results were then compared with the pulmonary function tests.
Statistical Analysis: One-way ANOVA or Student’s t test were used, followed by the appropriate post hoc analysis. Statistical software SPSS statistic 24 (IBM, USA) was used for statistical analyses. Significant differences were accepted when the p value was < 0.05 or < 0.01.
Results: A significant increase of AGE/sRAGE serum concentration was seen in the IPF and cHP patients compared with fNSIP patients. An inverse correlation was found between AGEs and sRAGE levels in IPF. At diagnosis, serum sRAGE correlated with FVC and DLCO values. During the follow-up, changes in serum AGEs and sRAGE correlated with % change of FVC, DLCO and TLC. Poor survival rates were seen in patients with sRAGE levels below 428.25 pg/ml.
Conclusion: The increase of AGE/sRAGE ratio was higher in IPF than cHP patients. AGE/sRAGE increase correlates with respiratory functional progression and that ratio can potentially be useful as a diagnostic serum biomarker for making a differential diagnosis between the most common fibrosing interstitial lung diseases. The concentration of sRAGE in blood stream could be considered as a potential prognostic biomarker in IPF.
Machahua C, Montes-Worboys A, Planas-Cerezales L, Buendia-Flores R, Molina-Molina M, Vicens-Zygmunt V. Serum AGE/RAGEs as potential biomarker in idiopathic pulmonary fibrosis. Respir Res. 2018 Nov 8;19(1):215. doi: 10.1186/s12931-018-0924-7. PMID: 30409203; PMCID: PMC6225674.
Emphysema requires the receptor for advanced glycation end-products triggering on structural cells.
Impact Factor: 5.373
Level of Evidence: Level VII
Sample Size/Population: RAGE-/- and RAGE+/+ mice.
Protocol: RAGE-/- and RAGE+/+ mice were generated to study the role of RAGE in the development of emphysema.
Statistical Analysis: The groups were compared using a one-way ANOVA and the Tukey-Kramer test to compare multiple groups.
Study Type: Randomized controlled trial/animal research study.
Results: In terms of the RAGE-/- mice, they were found to be less susceptible to porcine pancreatic elastase (PPE) induced emphysema compared to RAGE+/+ mice. For RAGE+/+ mice, there was an increase in proinflammatory cytokine and RAGE ligand that led to an increase in recruitment of inflammatory cells such as neutrophils and macrophages to the lungs. In regards to both groups, the number of RAGE positive cells in the lungs was not significantly different between the groups. Finally, RAGE expression in the lungs was found to be spontaneous and not affected by PPE treatment.
Conclusion: Currently the role of RAGE in pulmonary emphysema is not fully understood. Based on the study, RAGE is involved in increased neutrophil-related cytokines and chemokines as a result of PPE-induced acute phase neutrophilic inflammation and emphysematous change in the lungs. As a result, it was concluded that RAGE could be targeted in emphysema cases for an improved clinical outcome.
Waseda K, Miyahara N, Taniguchi A, Kurimoto E, Ikeda G, Koga H, Fujii U, Yamamoto Y, Gelfand EW, Yamamoto H, Tanimoto M, Kanehiro A. Emphysema requires the receptor for advanced glycation end-products triggering on structural cells. Am J Respir Cell Mol Biol. 2015 Apr;52(4):482-91. doi: 10.1165/rcmb.2014-0027OC. PMID: 25188021.
The receptor for advanced glycation end products is a central mediator of asthma pathogenesis.
Impact Factor: 3.491 (The American Journal of Pathology)
Level of Evidence: Level III
Sample Size/Population: Wild-type and RAGE knockout mice.
Protocol: Wild type mice and RAGE knockout mice were exposed to house dust mites (HDM) via three different methods. After exposure they were sacrificed and their tissues were studied via qRT-PCR, immunoblotting, immunofluorescence microscopy, H&E and PAS staining, histologic scoring, and an ELISA for a smattering of different immune compounds including IgE and IL-4.
Statistical Analysis: Significance was determined by two-way variance analysis and in some cases an unpaired Student’s t-test with a P value <0.05.
Study Type: In-vivo mouse model.
Results: There was no statistically significant difference in overall RAGE when the whole wild type mouse lung was examined. Immunofluorescence microscopy also showed no change in bronchial or vascular RAGE in the treated wild type mice lung. The wild type mice did show eosinophilia, goblet cell hyperplasia, and elevated mucin expression during histologic evaluation. The RAGE knockout mice showed none of these histologic changes. The ELISA showed little differences between wild type, knockout, and controls. The only significant difference was RAGE knockout mice had higher baseline levels of IL-17 than the wild type. Interestingly, wild type mice showed induction of IL-17 after HDM exposure while the knockout mice did not. IL-17 was examined due to its connection to asthma in previous studies. Administration of soluble RAGE (sRAGE), which is potentially thought to decrease inflammatory responses, did show a decrease in bronchovascular inflammation. Overall, an asthmatic response is not initiated in RAGE knockout mice based on the pulmonary function parameters, histology, and cytokine profiling.
Conclusion: The seemingly protective effect that treatment with sRAGE had on lung tissue suggests that this kind of therapy could be beneficial in treatment of asthma. The absence of expression of IL-17 in the RAGE knockout mice treated with HDM extract implies that RAGE plays a role in IL-17 synthesis. Considering that IL-17 is known to contribute to the symptoms of asthma, this would mean that RAGE plays a role in the development of asthma, through its connection to IL-17. Also, the heightened baseline IL-17 levels in the RAGE knockout mice in the absence of the antigen suggests that some other process is upregulating to compensate for the lack of IL-17. More specifically, IL-17 is thought to contribute to neutrophilic asthma and also negatively regulating established allergic asthma. This means that the elevated baseline in the knockout mice may actually be inhibiting a primary asthmatic response. This also may be contributing to the subtle, unexpected differences in the other tests completed on these mice.
Overall, an asthmatic response is not initiated in RAGE knockout mice. This clearly is not straightforward, but there is some connection between RAGE and the development of asthma that should be further explored
Milutinovic PS, Alcorn JF, Englert JM, Crum LT, Oury TD. The receptor for advanced glycation end products is a central mediator of asthma pathogenesis. Am J Pathol. 2012 Oct;181(4):1215-25. doi: 10.1016/j.ajpath.2012.06.031. Epub 2012 Aug 11. PMID: 22889845; PMCID: PMC3463633.
The role of receptor for advanced glycation end products in airway inflammation in CF and CF related diabetes.
Impact Factor: 3.998 (Scientific Reports)
Level of Evidence: Level IV
Sample Size/Population: The study consisted of 10 healthy individuals, 5 cystic fibrosis patients (CF), 5 cystic fibrosis patients with associated diabetes (CFRD), and 7 diabetic patients.
Protocol: Five patients from each of the groups were selected sequentially to provide sputum samples. The following experiments considered both membrane bound RAGE (mRAGE) and free, soluble RAGE (sRAGE). Levels of mRAGE and sRAGE were stained for in sputum samples and quantified via independent observers. PCR was also used to quantify the amount of mRAGE and sRAGE mRNA in blood leukocytes and sputum. ELISA was also used to determine concentrations of RAGE in both blood and sputum samples.
Statistical Analysis: This group used ANOVA followed by Tukey’s test to examine differences between group means. The P value was <0.05.
Study Type: Case-control study.
Results: According to the PCR analysis of RAGE mRNA, CF individuals had higher RAGE mRNA than the healthy individuals and diabetics. Diabetic individuals had decreased amounts of RAGE mRNA. CFRD patients also had increased RAGE mRNA, but to a lesser extent than CF patients.
During immunocytochemistry, diabetic individuals had significantly higher mRAGE levels than healthy individuals. Also, CF patients had significantly more mRAGE than the healthy, CFRD, and diabetic groups.
Sputum samples showed a decreased amount of sRAGE in CF and CFRD patients than the healthy group. Sputum of CFRD patients also showed an increased ratio of mRAGE, which is proinflammatory, to sRAGE, which is said to be protective against inflammation. This was compared to other fluid samples from the body and suggests that there is localization of inflammation in the airways for this group of people. Overall, CF patients had low sRAGE and AGE levels. CFRD patients had increases in all variables measured.
Conclusion: CFRD patients have faster lung function decline than those with CF. The group hypothesized that this could be due to the increased RAGE and AGE levels in the body associated with diabetes leading to increased lung inflammation. CF patients showed downregulation of protective sRAGE and upregulation of mRAGE. CFRD patients had less mRAGE and even less sRAGE than the CF patients. Most importantly, the CFRD group had a much larger mRAGE/sRAGE ratio than any other group. This means that the tissue has more pro-inflammatory receptors and less protective receptors in the lung tissue. This is significant because lung function deteriorates when there is inflammation present. Serum RAGE levels were not increased in CF or CFRD patients which was unexpected considering results found in other studies on chronic lung diseases and suggests this point be examined more in further studies on chronic lung disease. Examining each piece of the RAGE system proved to be difficult and complex to monitor. All of the components were looked at simultaneously which also made the data much more difficult to present and decipher. Data may be better viewed as ratios with considerations of different parts of the system, such as the mRAGE/sRAGE ratio. It also would be beneficial to view these ratios over time to compare an individual’s healthy state to their unwell state.
Mulrennan S, Baltic S, Aggarwal S, Wood J, Miranda A, Frost F, Kaye J, Thompson PJ. The role of receptor for advanced glycation end products in airway inflammation in CF and CF related diabetes. Sci Rep. 2015 Mar 10;5:8931. doi: 10.1038/srep08931. PMID: 25754382; PMCID: PMC4354142.
Receptor for advanced glycation end products is protective during murine tuberculosis.
Impact Factor: 3.641
Sample Size/Population: 12-16 wild type (Wt) mice C57Bl/6 and 12-16 RAGE−/− mice.
Protocol: Both Wt and RAGE−/− mice were infected intranasally with M. tuberculosis. 7-8 mice from each group were then sacrificed at 3 weeks and again at 6 weeks after infection. The remaining mice were sacrificed at 28 weeks after infection. Lungs and livers were removed from the mice used to obtain the several parameters of infection compared in this study. The lungs were immunostained for RAGE and analysed by a pathologist unaware of the sample's genotype. To score inflammation and damage, each sample was screened for interstitial inflammation, alveolar inflammation, vasculitis, bronchitis, edema, and pleuritis. Each parameter was graded on a scale of 0-4 (0 being absent, 4 being severe) and the “total lung inflammation score” was then expressed as a sum of the parameter scores for each sample (max = 24). Lung sample infiltrates were characterized by immunostaining for cell surface molecules using directly labeled antibodies against CD3, CD4, CD8, or GR-1. Percentage of polymorphonuclear cells (PMNs), macrophages, and lymphocytes were determined by detection of these antibody types. Cytokine measurements were taken from lung homogenates as well. Interferon (IFN)-, interleukin (IL)-4, TNF-, IL-10, IL-1, IL-6, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2) were measured via an ELISA using matched antibody pairs.
Statistical Analysis: Values were expressed as mean ± SE. Mann-Whitney U- test analyzed differences between the two groups, log rank test compared survival curves, two way ANOVA was used to compare the two groups at multiple time points, bacterial counts were transformed to log values before further statistical comparison, and statistical significance was considered P<0.05.
Study Type: Case-Control study.
Results: M. Tuberculosis pneumonia results in enhanced RAGE expression in the lungs, as indicated by predominant RAGE immunostaining in the interalveolar septae of infected mice. The RAGE-/- mice demonstrated enhanced pulmonary inflammation as indicated by higher mean total histology score, heavier weighing lungs (indicative of inflammation and edema), and higher amount of leukocytes (profoundly neutrophils and lymphocytes). RAGE -/- mice also demonstrated elevated levels of proinflammatory cytokines and chemokines compared to the Wt mice. Additionally, the RAGE -/- mice displayed a “transiently enhanced” outgrowth of M. tuberculosis. At 3 weeks it was observed that the M. tuberculosis counts were 2-4 fold higher in the RAGE-/- mice, but at 6 weeks the bacterial counts between the two groups were similar. So to observe the semination of M. tuberculosis to distant organs, bacterial load was determined from the sample’s livers as well. No statistical difference was observed in the bacterial counts obtained from the liver at either 3 or 6 weeks. Finally, RAGE -/- mice demonstrated enhanced lethality after M. tuberculosis infection. RAGE -/- mortality was 58% while the Wt mortality was 12%.
Conclusion: RAGE deficiency resulted in enhanced inflammatory response in the lungs of mice infected with M. tuberculosis. This is reflected by the accelerated weight loss and increased mortality observed in the RAGE-/- group. The authors showed that healthy Wt mice abundantly express RAGE and that RAGE expression is also upgradulared in healthy Wt lung tissue inoculated with M. tuberculosis. Uniquely, the data collected in this study demonstrates enhanced, rather than diminished, leukocyte recruitment in RAGE -/- mice. The high accounts of neutrophils and lymphocytes can most likely be attributed to a combination of the RAGE receptors cell migration capabilities and the unbalanced immune response observed in the RAGE -/- mice. The higher bacterial counts observed in the RAGE -/- mice could also possibly be attributed to the less controlled inflammatory reaction. The findings in the study suggest that RAGE is important for a balanced inflammatory reaction in lungs infected with M. tuberculosis.
van Zoelen MA, Wieland CW, van der Windt GJ, Florquin S, Nawroth PP, Bierhaus A, van der Poll T. Receptor for advanced glycation end products is protective during murine tuberculosis. Mol Immunol. 2012 Oct;52(3-4):183-9. doi: 10.1016/j.molimm.2012.05.014. Epub 2012 Jun 13. PMID: 22698798.
Relationship between advanced glycation end-product accumulation in the skin and pulmonary function.
Impact Factor: 3.691
Level of Evidence: Level III
Sample Size/Population: 272 patient that went through a medical checkup at Ginza Hospital.
Protocol: Subjects went through the Anti-Aging Dock and then were divided into categories of >65 and <65 years old. The subjects measurements for AGEs (Advanced Glycation End products), pulmonary function, and blood parameters were recorded. Skin autofluorescence (SAF) was assessed using an AGE Reader to determine AGE accumulation in the skin. A spirometer was used to assess forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and percent predicted FEV1 (%FEV1). A medical history was also taken for each subject and all the data was then statistically analyzed.
Statistical Analysis: Unpaired Student’s t-test, Fisher’s exact test, Spearman’s rank correlation coefficient, and multiple linear regression analysis using the backward-selection method. P<0.05 was considered statistically significant. All analyses were performed using SPSS 19.0 for Windows.
Study Type: Cross sectional study.
Results: SAF was found to be an independent factor that is negatively associated with FEV1/FVC in elderly people with normal spirometry results, but not in younger people. Pack years of smoking was significantly associated with decreased FEV1/FVC in the elderly group which was accelerated by AGE accumulation. Between the elderly and younger groups, there were no significant differences in gender ratio, BMI, HDL-C, LDL-C, TG, HbA1c, WBC, DL, lipid-lowering and other drugs, drinking status, smoking status, %FVC, and %FEV1.
Conclusion: Pulmonary function was shown to potentially be maintained by preventing AGE accumulation in the body. In elderly people with normal spirometry results, AGE accumulation that was assessed through SAF was found to be an independent factor negatively associated with FEV1/FVC.
Kubo A, Kato M, Sugioka Y, Mitsui R, Fukuhara N, Nihei F, Takeda Y. Relationship between advanced glycation end-product accumulation in the skin and pulmonary function. J Phys Ther Sci. 2018 Mar;30(3):413-418. doi: 10.1589/jpts.30.413. Epub 2018 Mar 2. PMID: 29581662; PMCID: PMC5857449.
Plasma advanced glycation end-products and skin autofluorescence are increased in COPD.
Impact Factor: 12.339 (European Respiratory Journal)
Level of Evidence: Level IV
Sample Size/Population: This study involved 114 stable COPD patients, and 61 healthy individuals who are not current smokers. In the end, 6 controls and 26 COPD patients were excluded due to poor data collection.
Protocol: The main focus of this study was examining the amount of AGEs in the body via a skin autofluorescence reader (AFR) which can identify AGEs due to their yellow-brown fluorescence coloring. This fluorescence was examined on the palmar side of the dominant forearm of each participant. This data was collected via spectrometry as a ratio of the light administered to the skin compared to the light emitted back from the skin. Spirometry was also used to assess lung function. Plasma was collected to assess levels of CML, pentosidine, and CEL, some of the better studied AGEs. Oxidative damage of plasma protein was determined via examination of carbonyls in the plasma collected. Other factors such as high-density lipoproteins (HDL), triglycerides, glucose, c-reactive protein (CRP), and creatine were also measured in the blood.
Statistical Analysis: Comparison of characteristics between groups was done by t-test for continuous data and Chi-squared test for categorical data. Multiple linear regression analysis was used in examining if COPD and lung function were correlated to AGE levels. A linear regression model was used to investigate the correlation of AFR reported AGE levels and plasma AGE levels. Both of these last two methods were adjusted for the secondary data collected such as age, BMI, and CRP levels.
Study Type: Case-control trial.
Results: The following variables were shown to have no difference between groups: BMI, HDL cholesterol, triglycerides, glucose, plasma creatine, and oxidative stress markers. CRP was increased in COPD patients compared to controls. COPD patients had smoked more pack-years than controls. Plasma levels of CML were lower in COPD patients than controls, while plasma CEL levels were increased in COPD patients. Plasma pentosidine showed no difference between the two groups. AFR was higher in the COPD patients than the controls. Both plasma and AFR levels did not differ between the never- and ex- smokers in the control group. Interestingly, plasma and AFR levels of AGEs also did not differ between ex-smoker controls and currently smoking COPD patients. Increased levels of CEL and CML were associated with higher AFR readings, while increased pentosidine was not.
Conclusion: When all the data is said and done, CML was shown to be a negative determinant of disease state and CEL and AFR were positive determinants of disease state. However, it is unclear how these compounds and findings are implicated in the disease state. More work should be done to investigate the mechanisms of AGEs, specifically CEL, in the lungs and how it may contribute to COPD.
Gopal P, Reynaert NL, Scheijen JL, Engelen L, Schalkwijk CG, Franssen FM, Wouters EF, Rutten EP. Plasma advanced glycation end-products and skin autofluorescence are increased in COPD. Eur Respir J. 2014 Feb;43(2):430-8. doi: 10.1183/09031936.00135312. Epub 2013 May 3. PMID: 23645408.
Effect of high advanced glycation end-product diet on pulmonary inflammatory response and pulmonary function following gastric aspiration.
Impact Factor: 3.203
Level of Evidence: Level VII
Sample Size/Population: Male, specific-pathogen-free CD-1 mice.
Protocol: Mice were separated into two groups: one group was fed a high AGE (HAGE) diet and the other a low AGE (LAGE) diet for four weeks. After the four weeks, the mice were further separated into 1.- LAGE uninjured controls; 2. HAGE uninjured controls; 3. LAGE + gastric aspiration injury; and 4. HAGE + gastric aspiration injury. The mice lung tissue was then assessed for resistance, plasma AGE concentrations, whole lung myeloperoxidase activity, albumin concentrations, cytokines and chemokines, and histopathology.
Statistical Analysis: GraphPad Prism 5® was used for all of the analysis. A two-way ANOVA was done then Prism performed the post test Bonferroni’s correction. Comparisons were considered significantly different for p <0.05.
Study Type: Randomized controlled study.
Results: Elevated number of PMNs in BAL and whole lung homogenate MPO enzymatic activity and increased expression of TNFsRII showed that increased blood AGE levels were associated with more intense pulmonary inflammatory responses.
Conclusion: High circulatory AGEs levels secondary to diet exacerbate acute lung injury following gastric aspiration. This implies an important pathogenic interaction between AGEs and the acute inflammatory response in the lung following gastric aspiration. Thus, this study supports further research in the role of RAGE in the lung and the benefits of restricting AGEs in the diets of patients with lung injury after gastric aspiration.
Guo WA, Davidson BA, Ottosen J, Ohtake PJ, Raghavendran K, Mullan BA, Dayton MT, Knight PR 3rd. Effect of high advanced glycation end-product diet on pulmonary inflammatory response and pulmonary function following gastric aspiration. Shock. 2012 Dec;38(6):677-84. doi: 10.1097/SHK.0b013e318273982e. PMID: 23143059; PMCID: PMC3532929.
AGE-RAGE Stress in the Pathophysiology of Pulmonary Hypertension and its Treatment.
Level of Evidence: I
Study: Review Article
Mechanism: AGE-RAGE stress is the term used to describe the interaction between AGE and its receptor RAGE in terms of stressors to anti-stressors. Several mechanisms of AGE-RAGE stress and its negative impacts on pulmonary hypertension have been suggested in the literature. All of such mechanisms deal with the vasoconstrictive nature of the AGE-RAGE axis. One such mechanism is that increased AGEs contribute to pulmonary stiffness due to increased cross-linking of collagen with AGEs. This leads to increased hypertrophy and resistance in pulmonary vessels. Another such mechanism is that interaction between AGE with RAGE triggers increased fibrosis, thickness, and ultimately stiffness of pulmonary arteries. Finally, production of reactive oxygen species (ROS) can be triggered by AGE-RAGE interaction which encourages multiplication of vascular smooth muscle cells, ultimately contributing to pulmonary hypertension. Of note, sRAGE is a competitive binder for AGE against RAGE. Interaction between sRAGE and AGE does not activate intracellular signaling and therefore may be helpful in reducing AGE-RAGE stress in the body.
Therapeutic Approaches: Reducing dietary intake of AGEs has been shown to acutely decrease serum levels which would lead to a decreased available pool to interact with RAGE in the lungs. It has also been suggested that certain vitamins such as vitamin C, E, and D can help to prevent the formation of AGEs and may be used as therapeutic treatment in patients with pulmonary hypertension. Also, many drugs such as statins, angiotensin converting enzyme inhibitors, and antidiabetic drugs like rosiglitazone have been shown to increase levels of sRAGE which can help to lower AGE levels and ultimately decrease AGE-RAGE interaction.
Conclusion: Current data indicates that AGE-RAGE stress is a possible etiology for pulmonary hypertension via induction of hypertrophy and smooth muscle cell proliferation in pulmonary tissue.
Prasad K. AGE-RAGE Stress in the Pathophysiology of Pulmonary Hypertension and its Treatment. Int J Angiol. 2019 Jun;28(2):71-79. doi: 10.1055/s-0039-1687818. Epub 2019 Apr 19. PMID: 31384104; PMCID: PMC6679961.
Age-related expression, enzymatic solubility and modification with advanced glycation end-products of fibrillar collagens in mouse lung.
Impact Factor: 3.376
Level of Evidence: II
Study Type: randomized control study
Sample Size/Population: N= 49 (young: 21, adult: 11, old: 17)
Protocol: Three groups of mice (young, adult, and old), assessed for integrity of various aspects of lung tissue, including mRNA expression, collagen, amount of AGEs, and types of cells. AGE load was compared between different types of collagen fibers found in lung tissue and analyzed for impact.
Statistical Analysis: Data presented as median with interquartile range for ease of reference. Student’s t test was implemented in order to compare between two separate groups and then an ANOVA test was performed in order to compare multiple groups. A regression analysis was also performed. Correlation coefficient of the linear regression analysis was tested using a two-sided test.
Results: Researchers found that mRNA expression for collagen types I, II, II, and IV were reduced in old mice. It was also found that collagen was highest in adult mice and lowest in young mice samples. Looking at the AGE load in each of the fibrillar collagens, there was an age-dependent increase in AGEs within collagen fibers, with young mice having the fewest and old mice having the highest.
Conclusion: When looking at mouse lung tissue, there appears to be an increase in AGE load in fibrillar lung collagens as age increases. With inspection of transcription analyses, this could in part be due to the fact that collagen turnover rate decreases with age. As collagen turnover rate decreases, this would disproportionately increase the AGE load seen in the tissue. In addition, AGEs have the ability to modify collagens and reduce their turnover rate. Overall, this leads to an increase in AGEs within the fibrillin collagen tissue of the mouse lung. Modifications to collagen fibers by AGEs also have the possibility of impairing lung mechanics as well which can impair lung function.
Rolewska P, Al-Robaiy S, Navarrete Santos A, Simm A, Silber RE, Bartling B. Age-related expression, enzymatic solubility and modification with advanced glycation end-products of fibrillar collagens in mouse lung. Exp Gerontol. 2013 Jan;48(1):29-37. doi: 10.1016/j.exger.2012.04.012. Epub 2012 May 15. PMID: 22595698.
Advanced glycation end products and its receptor (RAGE) are increased in patients with COPD
Impact Factor: 3.095
Level of Evidence: level 3
Summary: This study was performed in order to determine if the amount of both AGEs and RAGE in lung tissue differs between those with COPD and those without. The lung tissue was taken from individuals who had a history of bronchial carcinomas. The groups did not differ significantly in gender, age, or smoking history. There was a total of 38 samples, 20 with COPD and 18 without. The samples were then stained for both AGEs and RAGE via immunostaining. Digital photos were taken, and the intensity of staining was quantified by computer software. The number of cells positive for AGEs and RAGE were also counted manually. Student t-test, with a P value <0.05, was used to compare the control to the COPD group. The Spearman Correlation Coefficients test was used for the correlation of staining intensity. The number of cells positive for AGEs did not differ between the COPD and control group. However, the intensity of AGE staining was significantly stronger in the COPD group. Specifically, the intensity of AGE staining in the COPD group was stronger around the small airway areas. The intensity of RAGE staining was also more intense in the COPD tissues than the control tissues, but there was no significant difference around the small airways. Overall, the COPD group stained more intensely for both AGEs and RAGE than the control. This could suggest that AGEs-RAGE interactions are more common or more intense in the lungs of those with COPD. AGEs-RAGE interactions could participate in the development of COPD via airway and alveolar destruction, cytokine production, and inflammation. These are all processes that AGEs, and RAGE have been previously shown to participate in. More work should be done to precisely identify the function of both AGEs and RAGE in the lungs, and to determine if a blockade of either component could slow the progression of COPD development.
Wu L, Ma L, Nicholson LF, Black PN. Advanced glycation end products and its receptor (RAGE) are increased in patients with COPD. Respir Med. 2011 Mar;105(3):329-36. doi: 10.1016/j.rmed.2010.11.001. Epub 2010 Nov 26. PMID: 21112201.
Advanced glycation end products are elevated in cystic fibrosis-related diabetes and correlate with worse lung function
Impact Factor: 4.759 (Journal of Cystic Fibrosis)
Level of Evidence: IV
Study Type: Case-control study
Sample Size/Population: This study contained 5 control individuals, 15 cystic fibrosis (CF) individuals, and 12 cystic fibrosis-related diabetes (CFRD) patients.
Protocol: Plasma samples were collected from each study participant. Soluble RAGE (sRAGE), RAGE ligands, specifically AGEs, S100A12, and HMGB-1, were measured using ELISAs. Lung capacities were also examined using forced vital capacity (FVC) and forced expiratory volume in one second (FEV1).
Statistical Analysis: Kruskal-Wallis one-way analysis was used for comparisons between groups with the Dunn method used for pairwise multiple comparison procedures. Statistical significance was determined using a P-value <0.05. Correlations were determined using Spearman’s ranks.
Results: The CFRD group had significantly poorer lung function than the control and CF patients, as expected. Plasma AGE levels were significantly higher in CFRD patients compared to CF patients. S100A12 was not significantly elevated in CFRD patients when compared to both CF and control individuals, but it was elevated when CFRD patients were compared to just controls. HMGB-1 levels were significantly lower in CFRD patients than CF patients. There was no statistically significant difference in sRAGE levels in any of the three groups.
Conclusion: RAGE and AGEs are correlated to increased inflammation, which is a major player in CF and CFRD patient’s decreased lung functions. The data from this study further support this concept in that the AGE levels were significantly higher in CFRD patients, and these patients also had significantly poorer ling function. The details of each different AGE and their impacts on inflammation and lung function need further investigation.
Hunt WR, Helfman BR, McCarty NA, Hansen JM. Advanced glycation end products are elevated in cystic fibrosis-related diabetes and correlate with worse lung function. J Cyst Fibros. 2016 Sep;15(5):681-8. doi: 10.1016/j.jcf.2015.12.011. Epub 2016 Jan 23. PMID: 26817932.
Critical Role of Methylglyoxal and AGE in Mycobacteria Induced Macrophage Apoptosis and Activation
Impact Factor: 2.740
Level of Evidence:
Study Type: Case-control
Sample Size/Population: Compared Murine Alveolar Macrophage cell line (MH-S) cells with and without M. tuberculosis infection. Observed AGE within surgical samples of pulmonary lesions of TB patients, but number of patients is not mentioned.
Protocol: Measured methylglyoxal (MG) and AGE levels in MH-S cells with and without M. tuberculosis along with apoptosis patterns to determine the effect of MG/AGE on inducing apoptosis within infected cells. MG levels were determined by HPLC, apoptosis was observed via PI chromosomal staining, and AGE levels were determined by FACS analysis. MG induced apoptosis was also observed in the presence/ absence of c-Jun N-terminal kinase (JNK). Additionally, reduced glutathione (GSH) was added to the cell culture before infection in an attempt to observe if the antioxidant properties of GSH could counteract the reactive oxygen radical species induced by MG. Furthermore, the mechanism of apoptosis induced by MG/AGE was investigated through global gene expression profiling.
Statistical Analysis: MG induced apoptosis statistical significance determined by Student’s T-test. Significance of reversing effects of GSH on apoptotic effects of MG/AGE were determined by Student’s T-test. TNF-a, cytokine, and chemokine induction statistical significance were also all determined by Student’s T-tests.
Results: MG and AGE levels were both elevated in M. tuberculosis infected macrophages. MG levels were 2.0-7.0 times higher in infected macrophages compared to noninfected macrophages. MG induced elevated levels of apoptosis in infected cells, however the percentage of apoptotic cells peaked at a concentration of 0/8mM. Apoptosis induced by MG has been correlated with activation of JNK, and this experiment suggests that activation of JNK actually preceded the onset of apoptosis by initiating the intracellular pathway that leads to cells death and therefore JNK is required for MG induced apoptosis to take place. AGE levels were elevated within the infected macrophages and was even observed within TB lesions, suggesting that accumulation of infected macrophages is one of the main sources of AGE production during TB infection. Furthermore, this study showed that both MG and AGEs are required to induce apoptosis in infected cells. This was shown via reduced apoptosis and AGE formation observed in preincubated GSH cells. Global gene expression profiling of MG treated macrophages showed that TNF-a was induced at all time points, confirming that MG and AGE can induce TNF-a production. Additionally, MG induced a number of apoptotic, macrophage activation, and immunity induction genes via chemokine and cytokine families.
Conclusion: It is possible that mycobacterial infection of macrophages triggers shits in metabolic pathways leading to increased MG/AGE production. Both MG and AGE are required to carry out MG induced apoptosis. MG and AGE play pivotal roles in granulomatous lesion formation during TB infection.
Rachman H, Kim N, Ulrichs T, Baumann S, Pradl L, Nasser Eddine A, Bild M, Rother M, Kuban RJ, Lee JS, Hurwitz R, Brinkmann V, Kosmiadi GA, Kaufmann SH. Critical role of methylglyoxal and AGE in mycobacteria-induced macrophage apoptosis and activation. PLoS One. 2006 Dec 20;1(1):e29. doi: 10.1371/journal.pone.0000029. PMID: 17183656; PMCID: PMC1762319.
Pulmonary receptor for advanced glycation end-products promotes asthma pathogenesis through IL-33 and accumulation of group 2 innate lymphoid cells
Impact Factor: 14.110
Study Type: Case-Control
Sample Size/Population: 8-week-old wild type C57BL/6 mice and RAGE KO mice, split into groups treated with IL-33, house dust mite extract, or Alternaria alternata extract to induce asthmas/allergic airway inflammation (AAI). 3-6 mice per strain/treatment group.
Protocol: The chronic model of AAI was induced by treating wild type or RAGE KO mice intranasally four times per week for seven weeks with 40 μg of HDM extract in 25 μL of saline. Mice were sacrificed 48 hours after the last treatment. In an acute model of AAI, mice were treated intranasally with 25 μg of Alternaria alternata extract in 25 μL of saline on days 0, 3, 6, and 9 and sacrificed on day 10. IL-33 AAI was induced by intranasal protocol and intraperitoneal protocol. Via the intranasal protocol mice were treated with 1 μg IL-33 in 25 μL saline daily for 4 days, then sacrificed after 24 hours. For the IL-33 intraperitoneal protocol, mice were treated with 0.5 μg IL-33 in 200 μL saline daily for 3 days, then sacrificed after 24 hours. In all models, control mice were treated with saline alone.
Data was collected through various methods using samples collected from the sacrificed mice. Hyperresponsiveness was measured on a flexiVent machine with methacholine challenge. Bronchoalveolar and peritoneal lavage samples recovered from the mice underwent cell counts and cell type differentials using flow cytometry. H&E and PAS staining was used to visualize gross samples. ILC2 identification was completed using flow cytometry. Immunoblotting, ELISA, and qRT-PCR were all utilized in identification of different interleukins and antibodies present in the samples.
Statistical Analysis: GraphPad Prism 5 was used to perform all statistical analysis. Results are expressed as mean ± standard error. Statistical significance was determined using 2-way ANOVA and unpaired Student’s t-test. A value of P<0.05 was considered significant.
Results: The chronic model of AAI showed increased expression of IL-33 and mRNA in the wild type mice, but not in the RAGE KO mice. This suggests that RAGE mediates type 2 immune response by stimulating pulmonary IL-33 expression in response to allergen signals. The acute allergen model, induced by Alternaria alternata, triggers a type 2 inflammatory response dependent on IL-33. The wildtype mice were found to develop eosinophilic inflammation and mucus hypersecretion while the RAGE KO mice did not. In fact, the RAGE KO Alternaria treated mice had little to no airway hyperresponsiveness, while the wild type had significant airway hyperreactivity. Together the chronic and acute models show that RAGE is necessary for the development of type 2 inflammatory responses and appears to do so by upregulating IL-33 expression in the lung.
Hypothesizing that RAGE acted as a sensor upstream of IL-33, the researchers substituted IL-33 for other cytokines (IL-5 and IL-13) to see if it would produce an allergic reaction in the RAGE KO mice. Surprisingly, the RAGE KO mice still failed to display any AAI in response to intranasal exposure to IL-33. This finding suggests that RAGE both enables IL-5 and IL-13 producing cells to respond to IL-33 and promotes IL-33 up-regulation in response to allergens.
In order to determine RAGE involvement in ILC2 recruitment, proliferation, and retention within the lung, IL-33 and Alternaria model mice samples were evaluated via flow cytometry for ILC2 cell counts. In both models the wild type mice accumulated ILC2s in the lungs while the RAGE KO mice did not, suggesting that RAGE contributes to IL-33 induced AAI by promoting ILC2 accumulation in the lungs.
Bone marrow chimeras were utilized to determine the contribution of stromal versus hematopoietic RAGE in AAI. The results found that inflammation was greater in the wild type recipient mice compared to the RAGE KO mice regardless of the strain of bone marrow donated. This finding suggests that stromal RAGE drives AAI within the models rather than hematopoietic RAGE.
Intraperitoneal administration of IL-33 was used to determine gastrointestinal type 2 inflammatory responses in light of global RAGE deletion. Both the wild type and RAGE KO mice developed goblet cell hyperplasia in the gut ileum and produced increased levels of IL-5 and IL-13 within their peritoneal lavage fluid. The lungs of both models failed to develop any inflammation. Furthermore, splenic ILC2 accumulation was analyzed to determine the induction of ILC2s in circulation. The wild type and RAGE KO mice had comparable splenic ILC2 concentrations. Together this data suggests that hematopoietic RAGE is not critical for the development of type 2 immune responses.
Conclusion: RAGE is an essential factor for the upregulation of IL-33 and accumulation of ILC2s in the lung. RAGE also mediates the downstream effects of IL-33 induces AAI by promoting the accumulation of ILC2s in the lungs. RAGE itself may not directly recruit ILC2s into the lung, but it may promote the expression of a downstream mediator. Lack of RAGE inhibits allergic responses from IL-33, IL-5, and IL-13 pathways, therefore is necessary for both chronic and acute allergic responses to occur. Due to its unique position both upstream and downstream of IL-33 in an allergic asthmatic response, RAGE manipulation may be able to alleviate symptoms associated with AAI like mucus hypersecretion, airway hyperresponsiveness, and eosinophilic inflammation.
Oczypok EA, Milutinovic PS, Alcorn JF, Khare A, Crum LT, Manni ML, Epperly MW, Pawluk AM, Ray A, Oury TD. Pulmonary receptor for advanced glycation end-products promotes asthma pathogenesis through IL-33 and accumulation of group 2 innate lymphoid cells. J Allergy Clin Immunol. 2015 Sep;136(3):747-756.e4. doi: 10.1016/j.jaci.2015.03.011. Epub 2015 Apr 28. PMID: 25930197; PMCID: PMC4562894.