Karisa Wasley and Dr. Paul Reynolds, Physiology and Developmental Biology
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The causes of high morbidity and mortality associated with inflammatory respiratory diseases are not well understood. My research project helped to shed light on cellular signaling pathways associated with inflammatory disease, particularly those caused by or worsened by air pollutants. Asthmatics, in particular, have a difficult time dealing with common pollutants found in cities, including diesel particulate matter (PM). I focused specifically on the inflammation induced by air pollutants such as diesel PM and the activation of signaling pathways initiated by their binding with the receptors for advanced glycation end-products (RAGE). RAGE is involved developmentally in the lungs and specifically involved in alveolar type II cells as they transition to alveolar type I cells necessary for gas exchange. RAGE is also activated in several lung diseases, particularly in the pathobiochemistry observed in epithelial cells in pulmonary fibrosis (Mei et al, 2007), COPD (Morbini et al. 2006), and asthma (Fu et al. 2008). Patients with COPD and asthma endure particularly severe exacerbation in their diseased state when they encounter excessive particulate air pollution exposure.
Since beginning my work on this project last year, my preliminary data demonstrate that RAGE was significantly up-regulated in alveolar and bronchial epithelial cells exposed to diesel PM, capable of penetrating deep into the alveoli of the lungs. My preliminary work also implicated RAGE signaling in NF-κβ-mediated inflammation. In this study, I evaluated PM effects on R3/1 cells, an alveolar epithelial cell line. These cells have been shown to duplicate native effects in primary cells in human lungs. In concert with preliminary data that demonstrate selective up-regulation of RAGE by PM, I hypothesized that PM induces RAGE directly and activates endogenous RAGE ligands to perpetuate an inflammatory response. Briefly, I exposed cells to pre-determined concentrations of PM and evaluated RAGE mRNA and protein levels, NF-κβ activation and nuclear translocation, and cytokine elaboration including IL-8 and MCP-1. I targeted RAGE using sRAGE (soluble RAGE) as a RAGE blocker and determined direct effects of RAGE silencing. The methods required for this work were optimized and I was privileged to add them to my growing list of proficient lab techniques. For example, in addition to my training in cell culture and transfection of cultured cells, I extended my in vitro work and assessed the role of RAGE in PM-induced inflammation in mouse lungs. I nasally instilled RAGE knockout mice and wild type mice to PM, PM and sRAGE, or control conditions and assessed the efficacy of RAGE attenuation in the inflammatory response. This ORCA grant helped to provide time to continue my mastery of these animal techniques and obtain meaningful data that is clinically relevant.
In concert with my hypothesis, our study reports additional biological information regarding the details of RAGE-mediated signaling pathways activated by diesel particulate matter. Namely, R3/1 cells exposed to DPM showed significant increases in RAGE mRNA and protein synthesis levels as well as NF-κβ activation and nuclear translocation. Finally, DPM exposure induced the secretion of two pro-inflammatory cytokines, MCP-1 and IL-8. siRAGE incorporation significantly decreased DPM-induced NF-κβ activity and cytokine secretion in R3/1 cells.
Now completed, this research contributes to the overall understanding of inflammation and may provide insight into potential therapeutic interventions aimed at dampening pulmonary inflammation. Specifically, data may also be used to identify native inhibitors or perhaps allow for synthetically engineered inhibitors that possess the correct geometry necessary to bind and potentially inactivate RAGE in order to treat pulmonary inflammatory diseases. In addition, we suggest that RAGE expression influences sustained inflammation by potentially functioning as a sensor of adverse stimuli in respiratory epithelium. This insight makes RAGE a potential target to protect against insults that enhance pulmonary inflammation such as smoke, hyperoxia, and PM.
Ongoing research seeks to translate the presented in vitro data into an in vivo environment. As mentioned, the role and efficacy of RAGE in potentially attenuating the inflammatory response elicited by DPM exposure is currently being assessed in a mouse model. Further research may demonstrate that RAGE is an important target in the successful pharmacological treatment of DPM-exacerbated chronic lung diseases such as COPD and asthma.
As a result of this research, I was privileged to present our findings at the Experimental Biology 2010 research convention in Anaheim, California. I submitted a qualifying abstract and presented my data to people from all over the world in a poster format. In connection with this meeting, I applied for and was selected as a David Bruce Award Finalist for excellence in undergraduate research. Finally, in the near future we hope to compile and submit a manuscript of our data and findings to Cell and Tissue Research journal. As a result, we anticipate these new data will not only assist in future research within our own laboratories, but will also add to the growing amount of information available to fellow researchers examining related mechanisms.
References
- He M., Kubo H, Ishizawa K., Hegab AE., Yamamoto H., Yamaya M. The role of the receptor for advanced glycation end-products in lung fibrosis. AJP- Lung Cellular and Molecular Physiology. 2007;293(6):L1427-36.
- 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;19(11):1437-45.
- Fu L, Cai SX, Zhao HJ, Li WJ, & Tong WC (2008). Effect of N-acetylcysteine on HMGB1 and RAGE expression in the lungs of asthmatic mice. Nan Fang Yi Ke Da Xue Xue Bao. 28(5):692-5. Chinese.