Dr. Paul Reynolds, Department of Physiology & Developmental Biology
Academic Objectives Met and Results Observed
Funding available through this MEG award has provided an opportunity for me to meet many academic objectives I’ve determined to be important in the early stages of my faculty appointment at BYU. First of all, I have been able to move my on site research program forward and involve many undergraduates in the process. Specific to the current MEG award, involved students and I have been able to accomplish the proposed research. We have been successful in conducting pilot studies that reveal possible roles for RAGE in mechanisms of lung inflammation induced by both diesel particulate matter (PM) and cigarette smoke (CS). Despite improvement in air quality during the past few decades, the rationale for studying mechanisms leading to adverse health effects from air pollution remains important. This MEG award specifically provided opportunities for students to conduct meaningful research that led to the observation that alveolar epithelial cells and bronchiolar epithelial cells increased RAGE transcription after 24 hours of PM exposure. Because previous data reveals that molecules with binding affinity for RAGE are also induced, it was therefore an intriguing paradigm to consider the fact that the required molecules for RAGE signaling, while basally expressed under normal conditions, can be intensely activated in both the proximal and distal lung with the appropriate PM trigger.
With the assistance of undergraduates funded by this MEG award, we have made significant progress in characterizing conditional transgenic mice (SPC-rtTA/TetO-RAGE) that over-express RAGE in the respiratory epithelium. The summary of the research is as follows:
Receptors for advanced glycation end-products (RAGE) are multiligand cell-surface receptors highly expressed in pulmonary tissue that likely contribute to the transitioning of alveolar type (AT) II to ATI cells required for normal alveolar formation. However, the precise contribution of RAGE in the interplay between pulmonary epithelium and splanchnic mesenchyme during lung organogenesis remains uncertain. In order to test the hypothesis that RAGE misexpression adversely affects lung morphogenesis, conditional transgenic mice were generated that over-express RAGE in ATII cells. Mice that over-express RAGE in ATII cells throughout embryogenesis experienced 100% mortality and were significantly smaller and cyanotic when compared to single or non-transgenic control littermates. Histological evaluation of lungs from RAGE transgenic mice revealed significant lung hypoplasia coincident with large, vacuous areas in the periphery when compared to normal airway and alveolar architecture observed in non-transgenic control mouse lungs. Immunohistochemistry employing cell-specific markers for distal (FoxA2) and respiratory epithelium (TTF-1), ATII cells (proSP-C), and ATI cells (T1-α) demonstrated anomalies in key epithelial cell populations resulting from RAGE up-regulation. Flow cytometry specifically confirmed a significant decrease in the percentage of ATI cells in lungs from transgenic mice when compared to controls. These results reveal that precise regulation of RAGE expression is required during lung formation. Furthermore, over-expression of RAGE results in profound alterations in epithelial cell differentiation that culminate in severe respiratory distress and perinatal lethality.
I are happy to report that there are clear research directions now possible due to the initial work made possible by this MEG award. I am making progress on a series of award applications, involving both internal and external funding mechanisms, which will move the work forward.
Funding available through the MEG program, and specifically the award I have received in 2008, has allowed for the generation of a successful mentoring environment. I currently provide mentoring experiences for 17 undergraduates. The students in my lab will continue to be assembled in strata of varying degrees of experience. For example, the students currently working in the lab are committed for an average of about two years. They will be invaluable in the instruction of students that enter the lab during the current year. My goal is to layer the students in such a way that experienced graduate and undergraduate students lead teams of newer individuals so that both mutually benefit. As the students progress, they learn not only the methodology of the required techniques, but valuable problem solving skills, the ability to technically address scientific questions, and insight regarding how their specific research integrates into pulmonary biology as a whole. In addition to one-on-one and small group interactions with me as the faculty mentor regarding specific research issues, regularly scheduled laboratory meetings and broader discussion groups will continue to aid significantly in the development of these students. Research conducted in this type of mentoring environment will not only buttress their broad BYU undergraduate education, but it will also enhance the students’ motivation and opportunity for continued professional and graduate training at prominent research institutions throughout the United States.
Students Involved and Academic Deliverables
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Since receiving my MEG funding in 2008, I have mentored 29 undergraduate students. Eleven of these undergraduate students have been co-authors on abstracts submitted for presentation at national and international scientific conferences. These abstracts have or will be presented at the International Society for Developmental Biologists in Edinburgh Scotland (Sept 2009), Experimental Biology Meetings in Anaheim, CA (April 2010), the American Thoracic Society International Meeting in New Orleans, LA (May 2010), and at the Experimental Biology Meetings in Washington DC (April 2011). Additionally, eleven student co-authors and I have submitted manuscripts to peer reviewed journals including Environmental Health Perspectives (IF 7.1, accepted), Respiratory Research (IF 3.9, accepted), American Journal of Respiratory Cell and Molecular Biology (IF 4.3, accepted), Journal of Biological Chemistry (IF 7.4, submitted), and Journal of Histochemistry and Cytochemsitry (IF 2.6, submitted). Three additional manuscripts are in various stages of preparation. Lastly, there has been a high rate of success relating to professional schools admission by students that have worked or are currently working in the lab. I anticipate similar success from lab alumni in the future.
For a complete listing of individual students and their accomplishments, please see the following page:
Description of the Project-Specific Results
The main results have been published in the journal Environmental Health Perspectives, the flagship journal for the National Institute of Environmental Health Sciences:
Background: Receptors for advanced glycation end-products (RAGE) are cell-surface receptors expressed in alveolar type I (ATI) epithelial cells and are implicated in mechanisms of alveolar development and sustained pulmonary inflammation.
Objectives: In the present study, we test the hypothesis that diesel particulate matter (DPM) up-regulates RAGE in rat ATI-like R3/1 cells and human primary airway epithelial cells (SAECs) leading to an inflammatory response.
Methods and Results: Using real-time RT-PCR and immunoblotting, we demonstrate that RAGE mRNA and protein are up-regulated in cells exposed to DPM for two hours. Utilization of a luciferase reporter containing NF-κB response elements revealed decreased NF- κB activation in cells transfected with small interfering RNA for RAGE (siRAGE) prior to DPM exposure when compared to cells transfected with scrambled control siRNA (siControl). In addition, immunostaining revealed diminished nuclear translocation of NF-κB in DPM-exposed cells transfected with siRAGE compared to cells transfected with siControl prior to DPM stimulation. ELISA demonstrated DPM-induced secretion of MCP-1 and IL-8 by R3/1 cells, two cytokines induced by NF-κB and associated with leukocyte chemotaxis during an inflammatory response. Incorporation of siRAGE was sufficient to significantly decrease DPM-induced MCP-1 and IL-8 secretion when compared to cells transfected with siControl.
Conclusions: These data offer novel insights into potential mechanisms whereby RAGE influences pulmonary inflammation exacerbated by DPM exposure. Further research may demonstrate that molecules involved in RAGE signaling are potential targets in lessening the degree of particulate matter-induced exacerbations of inflammatory lung disease.
With this initial publication in the field of environmental health and possible contributions of RAGE biology in the progression of inflammation, we are positioned well to move the research forward in a collaborative fashion. I have made several initial plans with a team of researchers to procure external funding in necessary to expand the research.
The expenses for the project are categorized as undergraduate travel/salaries and lab supplies. Specifically, about $1,800 has been spent to offset undergraduate travel expenses incurred in relation to the meetings cited above. Approximately $8,000 has been spent on salaries and the remainder, $12,200, on lab supplies necessary for the completion of the work.