The receptor for advanced glycation end products (RAGE) contributes to the progression of emphysema in mice

Abstract

Several recent clinical studies have implied a role for the receptor for advanced glycation end products (RAGE) and its variants in chronic obstructive pulmonary disease (COPD). In this study we have defined a role for RAGE in the pathogenesis of emphysema in mice. RAGE deficient mice (RAGE-/-) exposed to chronic cigarette smoke were significantly protected from smoke induced emphysema as determined by airspace enlargement and had no significant reduction in lung tissue elastance when compared to their air exposed controls contrary to their wild type littermates. The progression of emphysema has been largely attributed to an increased inflammatory cell-mediated elastolysis. Acute cigarette smoke exposure in RAGE-/- mice revealed an impaired early recruitment of neutrophils, approximately a 6-fold decrease compared to wild type mice. Hence, impaired neutrophil recruitment with continued cigarette smoke exposure reduces elastolysis and consequent emphysema.

Fig 1. Lack of RAGE partially protects from progressive airspace enlargement.

RAGE-/- and wild type mice were exposed either to cigarette smoke or room-air and sacrificed after 6 months. Panel A-D show representative images of gills stained sections of the lungs from the different treatment groups. Panel E is a graphical representation of the mean chord lengths (μm) from the first experimental group. * Indicates a p-value < 0.05 by Kruskal-Wallis test. The bars indicate the standard error mean (SEM). Note, RAGE-/- mice have larger chord lengths at baseline but are protected from cigarette smoke-induced enlargement compared to their wild type counterparts.

Fig 2. Cigarette smoke exposed RAGE deficient mice show no reduction in lung tissue elastance on bronchocontriction when compared to their air-exposed controls.

Respiratory mechanics of RAGE-/- mice and wild type mice (n = 5–6 mice per strain per treatment group) previously exposed to cigarette smoke or room air was assessed using a mechanical ventilator (Flexivent) as described in the methods section. Panel A indicates a significant reduction in lung tissue elastance in wild type smokers (●) as compared to the air-exposed controls (◯) on bronchocontriction. However, RAGE-/- mice show no reduction in lung tissue elastance (H) on cigarette smoke exposure (■) (or methacholine challenge) compared to their controls (□). Panel B shows dynamic lung compliance (C) was not significantly altered by methacholine challenge in either mouse strain irrespective of cigarette smoke exposure. Standard 2-tailed student t-tests were performed and p-value < 0.05 indicate statistical significance (as denoted by *).

Fig 3. RAGE expression contributes to apoptosis with long-term smoke exposure.

Lung homogenates (50μg per well) harvested from RAGE-/- and wild type mice (n = 2/strain/treatmentgroup) exposed either to chronic cigarette smoke (Sm) or room air (NSm) was separated by SDS-PAGE and immunoblotted for caspase-3. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a molecular size of 37kDa was used as an internal control. Panel A- Western blot showing cleaved Caspase-3 and Pro-Caspase-3 bands in total lung homogenates. Cigarette smoke exposure led to significantly elevated caspase-3 cleavage in wild type but not RAGE-/- mice, when normalized either to Pro-Caspase-3 levels (Panel B) or GAPDH levels (Panel C). Band densities were analyzed using ImageJ and unpaired 2-tailed student T-test was used to analyze the normalized band intensities (*p<0.05).

Fig 4. RAGE mediates early recruitment of neutrophils in response to acute cigarette smoke exposure.

The bronchoalveolar lavage fluid (BALF) collected from the lungs of RAGE-/- (grey bars) and wild type (black bars) mice (n = 8–15 per strain per group) were used to stain and quantify cells that had migrated into the fluid lining the alveolus. Panel A indicates the total cell counts obtained from the BALF of the different treatment groups on acute cigarette smoke exposure. RAGE-/- mice displayed significantly reduced numbers of neutrophils at 4 hours post- smoke exposure as compared to their wild type counterparts, as shown in Panel B. There was no significant alteration observed in the number of macrophages or monocytes (Panel C) and lymphocytes (Panel D) recruited in either the RAGE-/- or wild type mice at 4 hours post-acute smoke exposure. The error bars indicate the standard error mean (SEM) within each group/treatment/time point (* p≤0.05 indicates statistical significance by unpaired 2-tailed student t-test).