(7) Caffeine Modulates Expression of Inflammatory and Epithelial to Mesenchymal Transition (EMT) Pathway Genes in Lung alveolar Epithelial Cells

Hamood Malik, Children's Hospital of the University of Illinois, Chicago, IL, United States; De-Ann Pillers, University of Illinois at Chicago, United States; Luo Wenxiang, University of Illinois, United States

Background: Caffeine is a routine medication used to treat apnea of prematurity, and may decrease the rate of bronchopulmonary dysplasia (BPD). BPD is associated with lung injury induced by hyperoxia and infection, leading to abnormal alveolar and vascular development and fibrosis. Epithelial mesenchymal transition (EMT) has been linked to BPD in mice, and previous studies have suggested that caffeine may modulate EMT genes. This study investigated the caffeine effects on expression of EMT and inflammation related genes (α-SMA,SERPINE1,occludin (OCLN,an epithelial marker), and interleukin-6 (IL6)) in two different mouse pulmonary alveolar epithelial cell lines treated under hyperoxic conditions.

Objectives: The objective of this study is to determine if 100 µM of caffeine (tissue concentration) in vitro modulates expression of EMT related genes in mouse MLE 12 and C10 alveolar epithelial cells (AEC) that were treated under hyperoxic condition.

Design/Methods: Quantitative real-time PCR was used to measure mRNA expression in response to hyperoxia, with and without 100 µM caffeine treatment, in MLE12 and C10 AEC. Hyperoxia-treated cells were incubated in 95%O2/5% CO2, and control cells incubated in room air with 5% CO2.Data were analyzed by ANOVA and t-test, with a P value < 0.05 as a significant difference. Isolation of mRNA with Magnetight oligo (dT) magnetic beads.Quantstudio 3 PCR machine for Quantitative RT-PCR reactions. Housekeeping genes to adjust quantitative results.

Results: Four EMT associated genes analyzed: α-SMA, SERPINE1 and OCLN, as well as inflammatory marker, IL-6. Hyperoxia induced SERPINE1 and IL6 and suppressed OCLN-in both MLE12 and C10 AEC.Although α-SMA was induced with hyperoxia in MLE 12 AEC, it was not induced in C10 AEC. With 100 µM caffeine citrate treatment, there was decreased induction with hyperoxia of α-SMA and SERPINE1 in MLE12 AEC. In C10 AEC,100µM caffeine treatment decreased induction in SERPINE1 (P=0.04); α-SMA could not be evaluated as it was not induced by hyperoxia. No statistical change with caffeine treatment in the response of IL6 and OCLN to hyperoxia.

Conclusion: Hyperoxia induced expression of fibrotic markers and suppressed the epithelial marker in both MLE12 and C10 AEC.Our data showed that caffeine modulated fibrotic markers that were altered by hyperoxia in both cell lines. This requires further investigation on the effects of different oxygen concentrations and caffeine dosages to ameliorate progression of EMT in BPD.