Steven Knapp and Paul Reynolds, PDBIO

Introduction

The goal of this project was to characterize the predisposing susceptibility of mice that overexpress receptors for advanced gylcation end products (RAGE) to apoptosis (cell death) and autophagy. Work has already been done to characterize this mouse model as pro-apoptotic; this new project will allow us to identify powerful markers that track DNA damage prior to cell death. RAGE is found in the lungs and aids in the transitioning of cell types (Reynolds P et Al., 2011). The expression of RAGE increases during instances of disease including COPD, diabetes, and lung cancer (Celli BR et Al., 2004, Wu L et Al., 2011). Conditional RAGE overexpression in lungs of adult mice elicits apoptosis and causes airspace enlargement resulting in increased inflammation (Stogsdill et Al., 2013). Overexpression of RAGE through its signaling pathway is known to lead to apoptosis, however the precise timing and threshold before a cell reaches cell death is not known.

Methodology

Two transgenic lines of mice were bred to give us 5 transgenic pups and 5 control (wild type) pups. These 10 pups were weaned at day 20 and then placed on DOX, a special food that overexpresses RAGE in our mouse model. The animals were sacrificed at day 110 after being on DOX for 90 days. The lungs were extracted and samples were collected. This process took a lot of time since ensuring that we have the correct cross requires many attempts.

RNA and protein were isolated and quantified. Immunohistochemical staining was also performed. Tests were run to determine if there was a significant difference between the control group and the transgenic group.

This project required a significant amount of time to perform all of the assays, such that it is still ongoing. Westerns blots, which test for protein presence, must be performed and adjusted to ensure proper results are gathered. Immunohistochemical staining can be a hit or a miss and must be adjusted accordingly as well.

Results and Discussion

In accordance with previous research, qPCR found a significant increase in RAGE expression among transgenic mice as compared with wild type. As shown previously, an increase in RAGE leads to increased inflammation, apoptosis, and airspace enlargement.

Westerns for EGR-1, PCNA, and RAGE were ran and showed results dissimilar with previous data. Westerns will be ran again to ensure results. Further assays, including staining and blots, will be ran for Caspase 3, S-100, TTF, EGR-1, and PCNA. An ELISA was ordered and will be ran on our samples to test for numerous indicators. A protocol with lab specific concerns is also being assembled for the comet assay.

Conclusion

This field of research is very exciting to be involved in. RAGE has numerous implications in various diseases and the development in our understanding of RAGE can lead to potential therapeutic targets. Thank you for funding this research project as it has not only helped me to develop an appreciation for the scientific method, but it has also trained and prepared me and various other students in numerous lab techniques. RAGE is an exploding topic and I look forward to the continued research.

References

Reynolds P, Stogsdill J, Stogsdill M, Heimann N. Up-regulation of receptors for advanced glycation end-products by alveolar epithelium influences cytodifferentiation and causes severe lung hypoplasia. American journal of respiratory cell and molecular biology. 2011;45(6):1195-202.

Celli BR and MacNee W Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J. 2004; 23(6):932-946.

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; 105(3), 329-36.

Stogsdill M, Stogsdill J, Bodine BG, Fredrickson A, Sefcik T, Wood T, et al. Conditional RAGE overexpression in the adult murine lung causes airspace enlargement and induces inflammation. American journal of respiratory cell and molecular biology. 2013.