A Potential Role for the Receptor for Advanced Glycation End-Products (RAGE) in the Development of Secondhand Smoke-Induced Chronic Sinusitis.

Chronic sinusitis (CS) is characterized by sinonasal inflammation, mucus overproduction, and edematous mucosal tissue. CS impacts one in seven adults and estimates suggest up to 15% of the general U.S. population may be affected. This research sought to assess a potential role for receptors for advanced glycation end-products (RAGE), an inflammatory receptor expressed in tissues exposed to secondhand smoke (SHS). Human sinus tissue sections were stained for RAGE and S100s, common RAGE ligands. Wild-type mice and mice that over-express RAGE in sinonasal epithelium (RAGE TG) were maintained in room air (RA) or exposed to secondhand smoke (SHS) via a nose-only delivery system five days a week for 6 weeks. Mouse sections were stained for RAGE and tissue lysates were assayed for cleaved caspase 3, cytokines, or matrix metalloproteases. We discovered increased RAGE expression in sinus tissue following SHS exposure and in sinuses from RAGE TG mice in the absence of SHS. Cleaved caspase-3, cytokines (IL-1ß, IL-3, and TNF-α), and MMPs (-9 and -13) were induced by SHS and in tissues from RAGE TG mice. These results expand the inflammatory role of RAGE signaling, a key axis in disease progression observed in smokers. In this relatively unexplored area, enhanced understanding of RAGE signaling during voluntary and involuntary smoking may help to elucidate potential therapeutic targets that may attenuate the progression of smoke-related CS.

Examining the effects of early patient care and biomedical science integration on predoctoral dental student competence and confidence.

INTRODUCTION: Our study investigates early experiential learning as a method of curricular integration by allowing students to begin their clinical experience in the first year of the programme, as well as distributing biomedical classes throughout the predoctoral dental school curriculum. MATERIALS AND METHODS: This study utilises a quasi-experimental design with two different groups, Standard Curriculum Group and Integrated Curriculum Group, n = 87. Data were collected from 2017 to 2021. RESULTS: We found that, on average, it took 608 h less for the participants in an integrated curriculum group to reach clinical competence in comparison to peers who did not experience the same methods of integration in their programme. These data were collected through daily faculty evaluations of students' progression as well as participants' own self-assessment. Our results indicate that participants in the Integrated Curriculum Group also experienced a positive effect on their confidence in their ability to apply the biomedical sciences to patient care. DISCUSSION/CONCLUSION: Our findings demonstrate that predoctoral dental programmes may be able to bring about positive outcomes for students' clinical confidence and competence by providing patient care opportunities early in the programme and sequencing the biomedical sciences throughout the curriculum. As such, it appears that early experiential learning may be a viable option for curricular integration that can have a positive effect on both students' confidence in their clinical abilities and their progression to clinical competence.

Inflammation and Invasion in Oral Squamous Cell Carcinoma Cells Exposed to Electronic Cigarette Vapor Extract.

Electronic cigarettes (eCig) represent a new avenue of tobacco exposure that involves heating oil-based liquids and the delivery of aerosolized flavors with or without nicotine, yet little is known about their overall health impact. The oral cavity is an anatomic gateway for exposure that can be compromised by activating myriad of signaling networks. Oral squamous cell carcinoma (OSSC) is a common malignancy affecting 30,000 people in the United States each year. Our objective was to determine the impact of eCig and nicotine on gingival OSSC invasion and their secretion of pro-inflammatory molecules. Gingiva-derived Ca9-22 cells and tongue-derived Cal27 cells were exposed to eCig vapor extract (EVE) generated from Red Hot or Green Apple (Apple) flavored eCig solution +/- nicotine for 6 hours. Isolation of protein lysates and collection conditioned media was done after treatment. Real-time cellular invasion was assessed using a RTCA DP instrument. Protein expression was determined using western blot. Compared to controls, we observed: elevated NF-kB, TNF-α, ERK, JNK, MMP-13 and cell invasion by Ca9-22 treated with Apple EVE; increased TNF-α and JNK by Ca9-22 treated with Red Hot EVE; and increased TNF-α and JNK by Cal27 cells treated with both Apple and Red Hot EVE. We conclude that eCig flavoring and nicotine orchestrated differential cell invasion and inflammatory effects. This study provides an important initial step in dissecting mechanisms of cancerous invasion and molecular avenues employed by OSCC.

Cell invasion, RAGE expression, and inflammation in oral squamous cell carcinoma (OSCC) cells exposed to e-cigarette flavoring.

OBJECTIVE: Electronic cigarettes have given rise to a new, largely unregulated market within the smoking industry. While generally supposed to be less harmful than traditional tobacco smoke, awareness of the biological effects of electronic cigarette liquid is still scarce. Our objective was to determine the impact of electronic cigarette flavoring and nicotine on gingival squamous cell carcinoma invasion, RAGE expression, and the elaboration of pro-inflammatory molecules. METHODS AND MATERIALS: Gingival and tongue squamous cell carcinoma cells were exposed to Red Hot or Green Apple flavored electronic cigarette flavoring with or without nicotine. Immunofluorescence determined RAGE expression. Real-time cellular invasion was assessed using a RTCA DP instrument. Culture medium was assayed for cytokine secretion. RESULTS: Compared to controls we observed: increased cell invasion in gingival cells with Red Hot electronic cigarette flavoring and decreased cell invasion with Green Apple; decreased cell invasion in tongue cells treated with Red Hot electronic cigarette flavoring and no differences in invasion with Green Apple; flavor and nicotine dependent increases in RAGE expression; and differential expression of IL-1α, IL-8, and MMP-13. CONCLUSION: We conclude that electronic cigarette flavoring and nicotine orchestrate differential regulation of oral squamous cell carcinoma (OSCC) cell invasion and inflammatory effects. This study provides an important initial step in dissecting RAGE-mediated mechanisms of cancerous invasion and molecular avenues employed by OSCC.

Gas6 protein induces invasion and reduces inflammatory cytokines in oral squamous cell carcinoma.

BACKGROUND: Gas6 protein is involved in the progression of cancers and has been demonstrated to have a role in inflammation. Oral squamous cell carcinoma is a common form of oral cancer, and it commonly expresses Gas6. Our objective was to determine the effects of Gas6 on oral squamous cell carcinoma invasion and identify signaling molecules and cytokines associated with Gas6-mediated invasion. METHODS: Ca9-22 cells were cultured in the presence or absence of Gas6. Real-time cell invasion was evaluated, and cultured cells were lysed for Western blot analysis. Cell medium was collected and assayed for cytokine elaboration. RESULTS: Treatment of cells with Gas6 resulted in: (i) increased invasion, (ii) increased expression of Gas6 and AXL receptor, (iii) reduced invasion when AXL was inhibited, (iv) decreased ERK activation, (v) increased AKT activation, and (vi) decreased secretion of G-CSF, IL-2, IL-6, and IL-8. CONCLUSIONS: Gas6 increases invasion of oral squamous cell carcinoma, and the invasion correlates with the increased AKT and the downregulation of pro-inflammatory cytokines. These results may prove useful in providing avenues that explain the role of Gas6 in the development and progression of oral squamous cell carcinoma.

Cigarette smoke extract induces oral squamous cell carcinoma cell invasion in a receptor for advanced glycation end-products-dependent manner.

Oral squamous cell carcinoma (OSCC) affects approximately 30,000 people and is associated with tobacco use. Little is known about the mechanistic effects of second-hand smoke in the development of OSSC. The receptor for advanced glycation end-products (RAGE) is a surface receptor that is upregulated by second-hand smoke and inhibited by semi-synthetic glycosaminoglycan ethers (SAGEs). Our objective was to determine the role of RAGE during cigarette smoke extract-induced cellular responses and to use SAGEs as a modulating factor of Ca9-22 OSCC cell invasion. Ca9-22 cells were cultured in the presence or absence of cigarette smoke extract and SAGEs. Cell invasion was determined and cells were lysed for western blot analysis. Ras and nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) activation were determined. Treatment of cells with cigarette smoke extract resulted in: (i) increased invasion of OSCC; (ii) increased RAGE expression; (iii) inhibition of cigarette smoke extract-induced OSCC cell invasion by SAGEs; (iv) increased Ras, increased AKT and NF-κB activation, and downregulation by SAGEs; and (v) increased expression of matrix metalloproteinases (MMPs) 2, 9, and 14, and downregulation by SAGEs. We conclude that cigarette smoke extract increases invasion of OSCC cells in a RAGE-dependent manner. Inhibition of RAGE decreases the levels of its signaling molecules, which results in blocking the cigarette smoke extract-induced invasion.

LPS from P. gingivalis Negatively Alters Gingival Cell Mitochondrial Bioenergetics.

OBJECTIVE: Oral inflammatory pathologies are linked to increased oxidative stress, thereby partly explaining their relevance in the etiology of systemic disorders. The purpose of this work was to determine the degree to which LPS from Porphyromonas gingivalis, the primary pathogen related to oral inflammation, altered gingival mitochondrial function and reactive oxygen species generation. METHODS: Human gingival fibroblast (HGF-1) cells were treated with lipopolysaccharide of P. gingivalis. Mitochondrial function was determined via high-resolution respirometry. P GINGIVALIS: Mitochondrial function was determined via high-resolution respirometry. RESULTS: LPS-treated HGF-1 cells had significantly higher mitochondrial complex IV and higher rates of mitochondrial respiration. However, this failed to translate into greater ATP production, as ATP production was paradoxically diminished with LPS treatment. Nevertheless, production of the reactive H2O2 was elevated with LPS treatment. CONCLUSIONS: LPS elicits an increase in gingival cell mitochondria content, with a subsequent increase in reactive oxygen species production (i.e., H2O2), despite a paradoxical reduction in ATP generation. These findings provide an insight into the nature of oxidative stress in oral inflammatory pathologies.

High-Mobility Group Box 1 Disrupts Metabolic Function with Cigarette Smoke Exposure in a Ceramide-Dependent Manner.

We have previously found that cigarette smoke disrupts metabolic function, in part, by increasing muscle ceramide accrual. To further our understanding of this, we sought to determine the role of the cytokine high-mobility group box 1 (HMGB1), which is increased with smoke exposure, in smoke-induced muscle metabolic perturbations. To test this theory, we determined HMGB1 from lungs of human smokers, as well as from lung cells from mice exposed to cigarette smoke. We also treated cells and mice directly with HMGB1, in the presence or absence of myriocin, an inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in ceramide biosynthesis. Outcomes included assessments of insulin resistance and muscle mitochondrial function. HMGB1 was significantly increased in both human lungs and rodent alveolar macrophages. Further testing revealed that HMGB1 treatment elicited a widespread increase in ceramide species and reduction in myotube mitochondrial respiration, an increase in reactive oxygen species, and reduced insulin-stimulated Akt phosphorylation. Inhibition of ceramide biosynthesis with myriocin was protective. In mice, by comparing treatments of HMGB1 injections with or without myriocin, we found that HMGB1 injections resulted in increased muscle ceramides, especially C16 and C24, which were necessary for reduced muscle mitochondrial respiration and compromised insulin and glucose tolerance. In conclusion, HMGB1 may be a necessary intermediate in the ceramide-dependent metabolic consequences of cigarette smoke exposure.

Cigarette smoke extract (CSE) induces RAGE-mediated inflammation in the Ca9-22 gingival carcinoma epithelial cell line.

OBJECTIVE: The oral environment is anatomically positioned as a significant gateway for exposure to environmental toxicants. Cigarette smoke exposure compromises oral health by orchestrating inflammation. The receptor for advanced glycation end-products (RAGE) has been implicated in smoke-induced inflammatory effects; however, its role in the oral cavity is unknown. The purpose of this study was to determine RAGE expression by immortalized gingival carcinoma cells and the degree to which RAGE-mediated signaling influences inflammation. DESIGN: Gingival epithelia cells (Ca9-22) were exposed to 10% cigarette smoke extract (CSE) for six hours and screened for RAGE expression and inflammatory mediators. RESULTS: Quantitative PCR and immunoblotting revealed increased RAGE expression following exposure. Furthermore, exposure activated RAGE signaling intermediates including Ras and NF-κB. IL-6 and IL-1ß were also elevated in cell culture medium from CSE-exposed cells when compared to controls. A family of anionic, partially lipophilic sulfated polysaccharide derivatives known as semi-synthetic glycosaminoglycan ethers (SAGEs) were used in an effort to block RAGE signaling. Co-treatment of CSE and SAGEs ameliorated inflammatory responses. CONCLUSIONS: These results provide a new perspective on a mechanism of cigarette smoke induced oral inflammation. Further work may show RAGE signaling as a potential target in the treatment of diseases of the oral cavity exacerbated by tobacco smoke exposure.

Decreased activation of placental mTOR family members is associated with the induction of intrauterine growth restriction by secondhand smoke in the mouse.

Cigarette smoke is known to be a risk for the development of intrauterine growth restriction (IUGR). Our objective was to assess the effects of secondhand smoke (SHS) during pregnancy and to what extent it regulates the activation of mTOR family members and murine trophoblast invasion. Mice were treated to SHS for 4 days. Placental and fetal weights were recorded at the time of necropsy. Immunohistochemistry was used to determine the level of placental trophoblast invasion. Western blots were utilized to assess the activation of caspase 3, XIAP, mTOR, p70 and 4EBP1 in treated and control placental lysates. As compared to controls, treated animals showed: (1) decreased placental (1.4-fold) and fetal (2.3-fold) weights (p < 0.05); (2) decreased trophoblast invasion; (3) significantly decreased active caspase 3 (1.3-fold; p < 0.02) and increased active XIAP (3.6-fold; p < 0.05) in the placenta; and (4) a significant decrease in the activation of placental mTOR (2.1-fold; p < 0.05), p70 (1.9-fold; p < 0.05) and 4EBP1 (1.3-fold; p < 0.05). Confirmatory in vitro experiments revealed decreased trophoblast invasion when SW71 cells were treated with 0.5 or 1.0 % cigarette smoke extract (CSE). Similar to primary smoking, SHS may induce IUGR via decreased activation of the mTOR family of proteins in the placenta. Increased activation of the placental XIAP protein could be a survival mechanism for abnormal trophoblast cells during SHS exposure. Further, CSE reduced trophoblast invasion, suggesting a direct causative effect of smoke on susceptible trophoblast cells involved in IUGR progression. These results provide important insight into the physiological consequences of SHS exposure and smoke-mediated placental disease.

Cigarette smoke and decreased oxygen tension inhibit pulmonary claudin-6 expression.

PURPOSE: Chronic obstructive pulmonary disease is a condition involving perturbed barrier integrity coincident with both emphysema and inflammation of the airways, and smoking is considered a major risk factor. Claudins (Cldns) stabilize barriers and contribute to tight junctions by preventing paracellular transport of extracellular fluid constituents. METHODS: To determine Cldn6 was differentially influenced by tobacco smoke, Cldn6 was evaluated in cells and tissues by q-PCR, immunoblotting, and immunohistochemistry following exposure. Cldn6 transcriptional regulation was also assessed using luciferase reporter constructs. RESULTS: Q-PCR and immunoblotting revealed that Cldn6 was decreased in alveolar type II-like epithelial cells (A549) and primary small airway epithelial cells when exposed to cigarette smoke extract (CSE). Cldn6 was also markedly decreased in the lungs of mice exposed to acute tobacco smoke delivered by a nose-only automated smoke machine compared to controls. Luciferase reporter assays incorporating 0.5-kb, 1.0-kb, or 2.0-kb of the Cldn6 promoter revealed decreased transcription of Cldn6 following exposure to CSE. Cldn6 transcriptional regulation was also assessed in hypoxic conditions due to low oxygen tension observed during smoking. Hypoxia and hypoxia inducible factor-1 alpha caused decreased transcription of the Cldn6 gene via interactions with putative response elements in the proximal promoter sequence. CONCLUSIONS: These data reveal that tight junctional proteins such as Cldn6 are differentially regulated by tobacco-smoke exposure and that Cldns are potentially targeted when epithelial cells respond to tobacco smoke. Further research may show that Cldns expressed in tight junctions between parenchymal cells contribute to impaired structural integrity of the lung coincident with smoking.

Oral Gingival Cell Cigarette Smoke Exposure Induces Muscle Cell Metabolic Disruption.

Cigarette smoke exposure compromises health through damaging multiple physiological systems, including disrupting metabolic function. The purpose of this study was to determine the role of oral gingiva in mediating the deleterious metabolic effects of cigarette smoke exposure on skeletal muscle metabolic function. Using an in vitro conditioned medium cell model, skeletal muscle cells were incubated with medium from gingival cells treated with normal medium or medium containing suspended cigarette smoke extract (CSE). Following incubation of muscle cells with gingival cell conditioned medium, muscle cell mitochondrial respiration and insulin signaling and action were determined as an indication of overall muscle metabolic health. Skeletal muscle cells incubated with conditioned medium of CSE-treated gingival cells had a profound reduction in mitochondrial respiration and respiratory control. Furthermore, skeletal muscle cells had a greatly reduced response in insulin-stimulated Akt phosphorylation and glycogen synthesis. Altogether, these results provide a novel perspective on the mechanism whereby cigarette smoke affects systemic metabolic function. In conclusion, we found that oral gingival cells treated with CSE create an altered milieu that is sufficient to both disrupted skeletal muscle cell mitochondrial function and insulin sensitivity.

Cardiomyocyte mitochondrial respiration is reduced by receptor for advanced glycation end-product signaling in a ceramide-dependent manner.

Cigarette smoke exposure is associated with an increased risk of cardiovascular complications. The role of advanced glycation end products (AGEs) is already well established in numerous comorbidities, including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with the AGE carboxy-methyllysine before mitochondrial respiration assessment. We discovered that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when cotreated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed wild-type and RAGE knockout mice to secondhand cigarette smoke and found reduced mitochondrial respiration in the left ventricular myocardium from wild-type mice, but RAGE knockout mice were protected from this effect. Finally, conditional overexpression of RAGE in the lungs of transgenic mice elicited a robust increase in left ventricular ceramides in the absence of smoke exposure. Taken together, these findings suggest a RAGE-ceramide axis as an important contributor to AGE-mediated disrupted cardiomyocyte mitochondrial function.

Cigarette smoke increases cardiomyocyte ceramide accumulation and inhibits mitochondrial respiration.

BACKGROUND: Cigarette smoking is a common and lethal worldwide habit, with considerable mortality stemming from its deleterious effects on heart function. While current theories posit altered blood lipids and fibrinogen metabolism as likely mediators, none have explored the role of the sphingolipid ceramide in exacerbating heart function with smoke exposure. Ceramide production is a consequence of cigarette smoke in the lung, and considering ceramide's harmful effects on mitochondrial function, we sought to elucidate the role of ceramide in mediating smoke-induced altered heart mitochondrial respiration. METHODS: Lung cells (A549) were exposed to cigarette smoke extract (CSE) and heart cells (H9C2) were exposed to the lung-cell conditioned medium. Adult male mice were exposed sidestream cigarette smoke for 8 wk with dietary intervention and ceramide inhibition. Ceramides and heart cell or myocardial mitochondrial respiration were determined. RESULTS: Lung cell cultures revealed a robust response to cigarette smoke extract in both production and secretion of ceramides. Heart cells incubated with lung-cell conditioned medium revealed a pronounced inhibition of myocardial mitochondrial respiration, though this effect was mitigated with ceramide inhibition via myriocin. In vivo, heart ceramides increased roughly 600% in adult mice with long-term sidestream cigarette smoke exposure. This resulted in a significant ceramide-dependent reduction in left myocardial mitochondrial respiration, as heart mitochondria from the mice exposed to both smoke and myriocin injections respired normally. CONCLUSIONS: These results suggest ceramide to be an important mediator of altered myocardial mitochondrial function with cigarette smoke exposure. Thus, anti-ceramide therapies might be considered in the future to protect heart mitochondrial function with smoke exposure.

Antenatal exposure of maternal secondhand smoke (SHS) increases fetal lung expression of RAGE and induces RAGE-mediated pulmonary inflammation.

BACKGROUND: Receptors for advanced glycation end-products (RAGE) are immunoglobulin-like pattern recognition receptors abundantly localized to lung epithelium. Our research demonstrated that primary tobacco smoke exposure increases RAGE expression and that RAGE partly mediates pro-inflammatory signaling during exposure. However, the degree to which RAGE influences developing lungs when gestating mice are exposed to secondhand smoke (SHS) has not been determined to date. METHODS: Timed pregnant RAGE null and wild type control mice were exposed to 4 consecutive days of SHS from embryonic day (E) 14.5 through E18.5 using a state of the art nose-only smoke exposure system (Scireq, Montreal, Canada). RAGE expression was assessed using immunofluorescence, immunoblotting, and quantitative RT-PCR. TUNEL immunostaining and blotting for caspase-3 were performed to evaluate effects on cell turnover. Matrix abnormalities were discerned by quantifying collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. Lastly, TNF-α and IL-1ß levels were assessed in order to determine inflammatory status in the developing lung. RESULTS: Pulmonary RAGE expression was elevated in both dams exposed to SHS and in fetuses gestating within mothers exposed to SHS. Fetal weight, a measure of organismal health, was decreased in SHS-exposed pups, but unchanged in SHS-exposed RAGE null mice. TUNEL assessments suggested a shift toward pulmonary cell apoptosis and matrix in SHS-exposed pups was diminished as revealed by decreased collagen IV and increased MMP-9 expression. Furthermore, SHS-exposed RAGE null mice expressed less TNF-α and IL-1ß when compared to SHS-exposed controls. CONCLUSIONS: RAGE augmentation in developing pups exposed to maternal SHS weakens matrix deposition and influences lung inflammation.

Ceramides mediate cigarette smoke-induced metabolic disruption in mice.

Cigarette smoke exposure increases lung ceramide biosynthesis and alters metabolic function. We hypothesized that ceramides are released from the lung during cigarette smoke exposure and result in elevated skeletal muscle ceramide levels, resulting in insulin resistance and altered mitochondrial respiration. Employing cell and animal models, we explored the effect of cigarette smoke on muscle cell insulin signaling and mitochondrial respiration. Muscle cells were treated with conditioned medium from cigarette smoke extract (CSE)-exposed lung cells, followed by analysis of ceramides and assessment of insulin signaling and mitochondrial function. Mice were exposed to daily cigarette smoke and a high-fat, high-sugar (HFHS) diet with myriocin injections to inhibit ceramide synthesis. Comparisons were conducted between these mice and control animals on standard diets in the absence of smoke exposure and myriocin injections. Muscle cells treated with CSE-exposed conditioned medium were completely unresponsive to insulin stimulation, and mitochondrial respiration was severely blunted. These effects were mitigated when lung cells were treated with the ceramide inhibitor myriocin prior to and during CSE exposure. In mice, daily cigarette smoke exposure and HFHS diet resulted in insulin resistance, which correlated with elevated ceramides. Although myriocin injection was protective against insulin resistance with either smoke or HFHS, it was insufficient to prevent insulin resistance with combined CS and HFHS. However, myriocin injection restored muscle mitochondrial respiration in all treatments. Ceramide inhibition prevents metabolic disruption in muscle cells with smoke exposure and may explain whole body insulin resistance and mitochondrial dysfunction in vivo.

Acute secondhand smoke-induced pulmonary inflammation is diminished in RAGE knockout mice.

The receptor for advanced glycation end-products (RAGE) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in proinflammatory signaling, and its role in irreversible pulmonary remodeling. The current research evaluated the in vivo effects of short-term secondhand smoke (SHS) exposure in RAGE knockout and control mice compared with identical animals exposed to room air only. Quantitative PCR, immunoblotting, and immunohistochemistry revealed elevated RAGE expression in controls after 4 wk of SHS exposure and an anticipated absence of RAGE expression in RAGE knockout mice regardless of smoke exposure. Ras activation, NF-κB activity, and cytokine elaboration were assessed to characterize the molecular basis of SHS-induced inflammation in the mouse lung. Furthermore, bronchoalveolar lavage fluid was procured from RAGE knockout and control animals for the assessment of inflammatory cells and molecules. As a general theme, inflammation coincident with leukocyte recruitment was induced by SHS exposure and significantly influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to SHS. However, ongoing research is still warranted to fully explain roles for RAGE and other receptors in cells coping with involuntary smoke exposure for prolonged periods of time.

Conditional over-expression of RAGE by embryonic alveolar epithelium compromises the respiratory membrane and impairs endothelial cell differentiation.

BACKGROUND: Receptors for advanced glycation end-products (RAGE) are cell surface receptors prominently expressed by lung epithelium. Previous research demonstrated that over-expression of RAGE by murine alveolar epithelial cells during embryogenesis caused severe lung hypoplasia and neonatal lethality. However, the effects of RAGE over-expression on adjacent matrix and endothelial cells remained unknown. METHODS: RAGE transgenic (TG) mice were generated that conditionally over-expressed RAGE in alveolar type II cells when fed doxycycline (dox) from conception to E18.5. To evaluate effects on the basement membrane, immunostaining and immunoblotting were performed for collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. To assess changes in vasculature, immunostaining, immunoblotting and qRT-PCR were performed for Pecam-1, a platelet endothelial cell adhesion marker also known as CD31. Lastly, to characterize potential regulatory mechanisms of endothelial cell differentiation, immunoblotting and qRT-PCR for FoxM1, a key endothelium-specific transcription factor of the Forkhead Box (Fox) family, were completed. RESULTS: Qualitative immunostaining for collagen IV was less in RAGE TG mice compared to controls and immunoblotting revealed decreased collagen IV in the RAGE TG mouse lung. Additionally, elevated MMP-9 detected via immunostaining and immunoblotting implicated MMP-9 as a possible down stream effector in matrix destabilization mediated by RAGE signaling. Lastly, Pecam-1 assessment revealed a decrease in the prevalence of microvascular endothelial cells coincident with FoxM1 abrogation in RAGE TG mice compared to controls. CONCLUSIONS: RAGE over-expression by alveolar epithelium weakened the basement membrane and associated matrix via increased MMP-9 activity. Furthermore, over-expression of RAGE inhibited FoxM1, suggesting that anomalous transcriptional control contributes to decreased endothelial cell prevalence in the TG mouse lung.