1. Adenosine Protects Vascular Barrier Function in Hyperoxic Lung Injury Jonathan Davies 1, Harry Karmouty-Quintana 2, Thuy T. Le 2, Ning-Yuan Chen 2, Tingting Weng 2, Jose Molina 2 and Michael R. Blackburn 2 1 3rd year fellow, Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA. 2 Department of Biochemistry and Molecular Biology, The University of Texas – Houston Medical School, Houston, TX, USA. Results (cont) Abstract BPD remains a significant cause for morbidity and mortality in premature neonates. BPD is caused by multiple factors including supplemental oxygen that cause cellular injury, inflammation and disrupted alveolar development. Adenosine is a signaling molecule that signals through cell surface receptors and has been found to play a protective role in acute lung injury, however little is known about the role of adenosine signaling in hyperoxic lung injury and BPD. Our hypotheses are: 1. Hyperoxia-induced lung injury leads to increased extracellular adenosine. 2.CD73 and Adora2B receptor deficient mice will have more severe lung injury when exposed to hyperoxia. Results: Exposure of mice to hyperoxia produced characteristic pulmonary inflammation and edema associated with a 4-fold increase in adenosine concentrations. Loss of CD73-mediated extracellular adenosine production and Adora2B led to worsened pulmonary edema associated with loss of occluding signaling in pulmonary vasculature without affecting inflammatory cell infiltration. Additionally, hyperoxia did not produce increased expression of adenosine receptors as seen in other models of acute lung injury involving hypoxic environments. Conclusions: These results suggest that adenosine has a protective role in hyperoxic lung injury through Adora2B signaling and regulation of cellular adhesion proteins to enhance vascular barrier function. These results have important implications for the use of caffeine in premature neonates, and offers potential therapeutic targets aimed at enhancing adenosine signaling through up-regulation of adenosine receptors for the prevention of lung injury in patients exposed to hyperoxia. Texas Pediatric Society Electronic Poster Contest Introduction 25% of very low birth weight infants develop bronchopulmonary dysplasia (BPD) Hyperoxic lung injury contributes to the development of BPD Adenosine signaling has a protective role in various models of acute lung injury The A 2B R is up-regulated and plays an important role in adenosine’s signaling in acute injury Methods Results Hypothesis Exposure to high levels of oxygen leads to inflammation associated with elevated extracellular adenosine levels that signal through the A 2B R to protect tissue from hyperoxic lung injury. Conclusions Hyperoxic exposure leads to pulmonary inflammation and edema associated with increased extracellular adenosine. Adenosine promotes vascular barrier function in hyperoxic lung injury in part through the A 2B R. Adenosine signaling through A 2B R modulates occludin, and this could be the mechanism by which adenosine protects vascular barrier function. Protective effects of A 2B R up-regulation in other models of lung injury involving hypoxic conditions does not occur in hyperoxic lung injury. Implications Regulation of adenosine receptors offer potential targets for novel therapies to prevent hyperoxic lung injury Caffeine, an adenosine receptor antagonist used to treat apnea of prematurity in neonates may worsen hyperoxic lung injury acutely Implications Regulation of adenosine receptors offer potential targets for novel therapies to prevent hyperoxic lung injury Caffeine, an adenosine receptor antagonist used to treat apnea of prematurity in neonates may worsen hyperoxic lung injury acutely Adenosine levels are elevated in hyperoxic lung injury Loss of CD73 decreases extracellular adenosine and worsens pulmonary edema in hyperoxic lung injury Loss of the A 2B R worsens pulmonary edema in hyperoxic lung injury Occludin, a cellular adhesion protein, is decreased in CD73 -/- and A 2B R -/- mice in pulmonary endothelial cells in hyperoxia A 2B R is not up-regulated in hyperoxic lung injury Fig. 1. Wild-type mice were exposed to either room air or hyperoxia for 72 hrs. BALF analysis showed increase in cell count (A) and protein concentration (B) indicating increased inflammatory cell infiltration and pulmonary edema. Adenosine concentration was found to be elevated approximately 4-fold in mice exposed to hyperoxia (C). Fig. 2. CD73 -/- mice were exposed to room air or 95% oxygen environment for 72 hours. BALF analysis showed decreased adenosine concentrations in CD73 -/- mice in room air and hyperoxia (A). CD73 -/- mice had no change in BALF cell count in hyperoxia (B) but did have an increase in BALF protein concentration (C) indicating worsened pulmonary edema. Histology demonstrated increase in fluid accumulation perivascularly in hyperoxia compared to room air, and exagerated perivascular fluid in CD73 -/- with red blood cell extravasation (D). Fig. 3. A 2B R -/- mice were exposed to room air or 95% oxygen environment for 72 hours. A 2B R -/- mice had no change in BALF cell count in hyperoxia (A) but did have a significant elevation in BALF protein concentration (B) indicating worsened pulmonary edema. H&E staining of representative sections (C) shows an increase in perivascular fluid accumulation in hyperoxia compared to room air that is exaggerated and includes red blood cell extravasation in A 2B R -/- mice. Fig. 4. Representative immunohistochemistry staining for occludin, a cellular adhesion protein important in pulmonary vascular barrier function, in wild-type, CD73 -/- and A 2B R -/- mice in room air and hyperoxia (A). Occludin was decreased in wild-type animals exposed to hyperoxia, and decreased in CD73 -/- and A 2B R -/- mice in room air. However, occludin was nearly non-existent in CD73 -/- and A 2B R - /- mice exposed to hyperoxia. Occludin staining quantitation demonstrates these findings (B). Western analysis for occludin show similar decreases most noticeable in CD73 -/- and A 2B R -/- in hyperoxia. Fig. 5. Quantitative PCR analysis of whole lung lysate showed no increase in expression of the A 2B R in mice exposed to hyperoxic compared to room air.