UNIVERSITY of PENNSYLVANIA

19th AnnualRespiration

Research Retreat

Friday ~ June 14, 2013Villanova Conference Center ~ Villanova, PA

w8:00 am - 5:30 pm

Program and Abstracts2013

http://www.uphs.upenn.edu/ifem/rrr.html

���PROGRAM8:00 a.m. Registration/Continental Breakfast/Poster Set-up

8:55 a.m. Welcome, Vera P. Krymskaya, Ph.D.

9:00 a.m. The Robert E. Forster LectureGary Huffnagle, M.D.Division of Pulmonary and Critical Care MedicineUniversity of MichiganExploring the composition and function of the lung microbiome in health and diseaseSession Chair: Vera P. Krymskaya, Ph.D.

10:00 a.m. Break

10:15 a.m. Poster Presentations10:15 –11:05 a.m. even numbers, 11:05 –11:55 a.m. odd numbers

12:00 noon Lunch

1:00 p.m. Symposium: “Lung Immunity and Microbiome”Session Chair: G. Scott Worthen, M.D.Session Co-Chair: Horace Delisser, M.D.

Speakers:

1:00 p.m. Nilam Mangalmurti, M.D.Transfused red blood cells modulate the host inflammatory response to endotoxin

1:25 p.m. Kristin Hudock, M.D.Generation and function of induced pluripotent stem cell derived human neutrophils

1:50 p.m. Joshua Diamond, M.D.Evaluating the role of the lung microbiome after lung transplantation

2:15 p.m. Angela Haczku, M.D., Ph.D.Regulation of dendritic cell migration during pulmonary inflammation

2:40 p.m. Guang Yang, Ph.D.Oxidative stress and inflammation regulate the key circadian gene Rev-ERBa through conservedNKkB and NRF2 binding sites

2:55 p.m. John Reilly, M.D.Blood type A is associated with an increased risk of the acute respiratory distress syndrome amongCaucasian patients with severe sepsis

3:10 p.m. Kosuke Kato, Ph.D.MUC1 prevents virus-derived double-stranded RNA-induced epithelial cell inflammation andapoptosis by inhibiting recruitment of TRIF to TLR3

3:30 p.m. Break

3:45 p.m. The Alfred P. Fishman LectureDavid Artis, Ph.D.Department of MicrobiologyUniversity of PennsylvaniaSession Chair: Ronald Collman, M.D.

4:45 p.m. Wine and Cheese Reception/Poster Awards

���ORGANIZING COMMITTEE

u VERA P. KRYMSKAYA, PH.D., CHAIR

Associate Professor of MedicineUniversity of Pennsylvania

u STEVEN ALBELDA, M.D.

Associate Chief, Pulmonary MedicineHospital of the University of PennsylvaniaProfessor, Department Medicine, University of Pennsylvania

u JULIAN ALLEN, M.D.Professor, Department of Pediatrics, University of PennsylvaniaChief, Pulmonary Medicine SectionThe Children’s Hospital of Philadelphia

u MICHAEL F. BEERS, M.D.Professor, Department of Medicine, University of Pennsylvania

u PHYLLIS A. DENNERY, M.D.Professor, Department of Pediatrics, University of PennsylvaniaChief, Division of Neonatology,The Children’s Hospital of Philadelphia

u CLIFFORD DEUTSCHMAN, M.D.Professor, Department of Anesthesiology, University of Pennsylvania

u ARON B. FISHER, MDDirector, Institute for Environmental Medicine, Professor of Physiology and MedicineUniversity of Pennsylvania

u VLADIMIR MUZYKANTOV, M.D., PH.D.Professor, Department of Pharmacology, University of Pennsylvania

u REYNOLD PANETTIERI, JR., M.D.Professor, Department of Medicine, University of Pennsylvania

u TREVOR PENNING, PH.D.Professor, Department of Pharmacology, University of Pennsylvania

���SPONSORING PROGRAMS

v NRSA IN LUNG CELLULAR AND MOLECULAR BIOLOGY(T32-HL07748)

v NRSA IN PULMONARY IMMUNOLOGY AND CELLULAR BIOLOGY(T32-HL07586)

v NRSA IN ANESTHESIA RESEARCH(T32-GM07612)

v TRAINING PROGRAM IN PEDIATRIC PULMONOLOGY

v CENTER OF EXCELLENCE IN ENVIRONMENTAL TOXICOLOGY(ES013508)

v AIRWAYS BIOLOGY INITIATIVE

v INSTITUTE FOR ENVIRONMENTAL MEDICINE

PROGRAM IN ACUTE LUNG INJURY

v NEONATOLOGY & NEWBORN SERVICES

v PULMONARY, ALLERGY & CRITICAL CARE DIVISION

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ABSTRACTS

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Author CONTENTS Abstract # A. Lung Lipids and Surfactant Atochina-Vasserman, E.N., Abramova, H., Goncharov, D., Fehrenbach, M., Pastva, A.M., Beers M.F., Gow, A.J., Krymskaya, V.P. S-nitrosylation of SP-D in a mouse model of pulmonary Lymphangioleiomyomatosis (LAM). 1 Maguire, J.A., Malzer, E., Marciniak, S., Beers, M.F. Autophagy promotes cytoprotection by facilitating removal of aggregation-prone Surfactant Protein C L188Q BRICHOS mutant in vitro and in vivo. 2 Hawkins, A., Chatterjee, S., Deterding, R., Funkhouser, W.K., Goralski, J., Mulugeta, S., Beers, M.F. The Surfactant Protein C I73T missense mutation induces alterations in cellular macroautophagy and mitochondrial transmembrane potential. 3 Sharma, S.K., Koziol-White, C.J., Zhang, S., Yuan, C-X, Axelsen, P., Haczku, A. Structural changes in Surfactant Protein D (SP-D) in the mouse lung in response to ozone (O3) and allergen exposure. 4 Mulugeta, S., Tomer, Y., Russo, S.J., Zhao, M., Hawkins, A., Yu, K., Huang, S., Zhang, P., Guttentag, S.H., Beers, M.F. A mouse model for lung disease-associated ABCA3 mutations. 5 B. Oxidative Stress

Biswas, C., Yang, G., Shah, N., Muthu, M., Dennery, P.A. Regulation of Nrf2 by nuclear HO-1 provides cytoprotection through transcription of G6PDH. 6 Benipal, B., Feinstein, S.I., Fisher, A.B. Role of peroxiredoxin 6 phospholipase A2 activity in lung damage resulting from oxygen exposure. 7 Yang, G., Wright, C.J., Hinson, M., Fernando, A.P., Dennery, P.A. Oxidative stress and inflammation regulate the key circadian gene Rev-ERBα through conserved NFκB and NRF2 binding sites. 8 Abe, S., Wang, H., Hong, N.K., Yu, K., Debolt, K.M., Feinstein, S.I., Cantu, E., Christie, J.D., Fisher, A.B., Chatterjee, S. Mechanosensing with restart of flow drives KATP induced NOX2 activation in a model of lung ischemia reperfusion. 9 Zhou, S., Dodia, C., Shuvaeva, T., Sorokina, E., Harper, S., Speicher, D.W., Feinstein, S.I., Fisher, A.B. Effect of glutathione on the phospholipase A2 activity of peroxiredoxin 6. 10 Sorokina, E.M., Dodia, C., Yu, K., Hong, N.K., Feinstein, S.I., Fisher, A.B. Serine 32 targeted mutation abolishes Prdx6 trafficking to lamellar bodies in vivo. 11 Milovanova, T.N., Uzun, G., Sorokina, E.M., Moore, J.S., Thom, S.R. Hyperbaric oxygen stimulates adipose- derived mesenchymal stem cells growth and differentiation in streptozocin-induced diabetes mellitus type 1 mice in vivo. 12

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Author CONTENTS Abstract # Williams, M., Fernando, A.P., La, P., Dennery, P.A. HO-1 nuclear localization enhances DNA repair by increasing availability of the DNA repair enzyme OGG1. 13

C. Inflammation and Immune Response

Mangalmurti, N.S., Qing, D.Y., Faust, H., Lo, R., Lee, J.S., Worthen, G.S., Albelda, S.M. LPS- induced lung inflammation is attenuated in the absence of the Receptor for Advanced Glycation End products. 14 Scala, J., Koziol-White, C.J., Lee, W.M., Gern, J.E., Strong, P., Ito, K., Rapeport, G., Panettieri. R.A. Mediator release following rhinovirus exposure is selectively attenuated by a kinase inhibitor but not a steroid in human lung slices. 15 Ge, M.Q, Forbes, L., Hwang, J., Ducka, B., Redai, I.G., Stevenson, C., Haczku, A. O3 inhalation attenuates the budesonide effects on allergic airway inflammation and SP-D production in the lung of Balb/c mice. 16 Howard, M.D., Greineder, C.F., Hood, E., Muzykantov, V.R. Modulation of the endothelial inflammatory response by EUK-134 loaded immunoliposomes. 17 Kokalari, B., Ge, M., Forbes, L.R., Redai, I.G., Ogden, C.A., Raymond, H., Haczku, A. Surfactant Protein-D (SP-D) suppresses ozone (O3)-induced CCL17 release and epithelial accumulation of myeloid dendritic cells (DC) in mice. 18 Jiang, Z., Ducka, B., Ravaioli, G., Redai, I.G., Wang, S.S., Panettieri, R.A., Bhatnagar, S., Haczku, A. Social stress inhibited glucocorticoid receptor (GR) and CAAT enhancer binding protein (C/EBP)-² mediated surfactant protein D (SP-D) expression in the lung of mice in an asthma model. 19 Zern, B.J., Chacko, A.M., Greineder, C.F., Simone, E.A., Muzykantov, V.R. Detection of acute lung inflammation using molecular imaging. 20 Mondrinos, M.J., Kennedy, P., Sun, S., Kilpatrick, L. Pulmonary endothelial protein kinase C-delta(PKC-´ ) regulates neutrophil migration in acute lung inflammation. 21

Christofidou-Solomidou, M., Pietrofesa, R., Arguiri, E., Cengel, K.A., Witwer, K.W. Dietary flaxseed modulates the miRNA profile in irradiated and non-irradiated murine lungs-A novel mechanism of tissue radioprotection by flaxseed. 22

Kato, K., Kim, K.C. MUC1 prevents virus-derived double-stranded RNA-induced epithelial cell inflammation and apoptosis by inhibiting recruitment of TRIF to TLR3. 23

Kennedy, P.A., King, D.J., Zhang, T., Aksoy, M.O., Kelsen, S.G., Kilpatrick, L.E. Macrophage cytokine and UPR responses to ER stress are dependent on M1/M2 polarization. 24

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Author CONTENTS Abstract #

D. Lung Injury and Remodeling

Reilly, J.P., Shashaty, M.G.S., Herlim, M., Oz, N.L., Garcia, Z., Hiciano, I., Campbell, A., Christie, J.D., Meyer, N.J. Blood type A is associated with an increased risk of the Acute Respiratory Distress Syndrome among Caucasian patients with severe sepsis. 25 Lee, I., Dodia, C., Chatterjee, S., Feinstein, S., Fisher, A.B. Efficacy of MJ33, a novel nontoxic inhibitor of phospholipase A2 of peroxiredoxin 6, in lipopolysaccharide-induced acute lung injury. 26 Peng, T., Tian, Y., Cui, Z., Cantu, E., Christie, J., Morrisey, E. Stromal activation by Wnt signaling defines a cellular niche capable of clonal expansion after fibrotic lung injury. 27 Goncharova, E.A., Goncharov, D.A., Khavin, I.S., Krymskaya, V.P. TSC1/TSC2 modulate E-cadherin trafficking through Rac1 GTPase. 28 James, M.L., Goncharov, D.A., Li, H., Atochina-Vasserman, E.N., Guttentag, S., Gonzales, L.K., Schmidt, L.S., Linehan, W.M., Baba, M., Hong, S-B, Goncharova, E.A., Krymskaya, V.P. Folliculin and AMPK in epithelial cell survival: relevance to Birt-Hogg-Dubé Syndrome. 29 Suaud, L., Miller, K., Panichelli, A., Randell, R., Rubenstein, R.C. Modulation of Hsp70 expression by Elp2, a 4-phenylbutyrate inducible Elongator component, and STAT3 does not require STAT3 phosphorylation. 30 Grumbach, Y., Suaud, L., Rubenstein, R.C. ERP29 regulates ENAC functional expression by promoting channel cleavage. 31 Orndorff, R.L., Hong, N.K., Zern, B.J., Yu, K., Debolt, K., Huang, S., Fisher, A.B., Muzykantov, V.R., Chatterjee, S. Detecting cell adhesion molecules in intact lung using Quantum Dot conjugates targeted to endothelial cells. 32 Hubert, T.L., Mallilankaraman, K., Marcinkiewicz, M., Wu, J., Shaffer, T.H., Muniswamy, M., Wolfson, M.R. rhCC10 modulates lipopolysaccharide injury in alveolar like cell culture. 33

Pan, D., Zaitsev, S., Muzykantov, V.R. The effect of murine and primate erythrocytes bound fusion proteins when exposed to osmotic stress in pro-inflammatory and pro-thrombotic diseases. 34 Tian, Y., Kong, J., Lu, M., Morrisey, E.E. Regulation of miRNA-302-367 in lung epithelial progenitor cells during development and injury repair. 35 Diamond, J.M., Lee, J.C., Kawut, S.M., Shah, R.J., Localio, A.R., Bellamy, S., Lederer, D.J., Cantu, E., Kohl, B.A., Lama, V.N., Bhorade, S., Crespo, M., Demissie, E., Sonett, J., Wille, K., Orens, J., Shah, A.S., Weinacker, A., Arcasoy, S., Shah, P.D., Wilkes, D.S., Ware. L.B., Palmer, S.M., Christie, J.D. Clinical risk factors for primary graft dysfunction after lung transplantation. 36

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Author CONTENTS Abstract #

Shah, R.J., Diamond, J.M., Cantu, E., Flesch, J., Lee, J.C., Lederer, D.J., Lama, V.N., Orens, J., Weinacker, A., Wilkes, D.S., Bhorade, S., Wille, K.M., Ware, LB, Palmer, S.M., Crespo, M., Demissie, E.J., Kawut, S.M., Bellamy, S.L., Localio, A.R., Christie, J.D. Use of a simple prediction model improves pre-transplant risk stratification for primary graft dysfunction after lung transplantation. 37 E. Airway Smooth Muscle

Jude, J.A., Koziol-White, C.J., Scala, J., Jester, W.F., Maute, C., Dalton, P.H., Panettieri, R.A. Formaldehyde-induced airway hyperresponsiveness: Role of Ca2+ dynamics in human airway smooth muscle (HASM) cells. 38 Koziol-White, C.J., Li, S., Jiang, M., Scala, J., Morrisey, E.E., Panettieri, R.A. FOXP1/P4 knockout increases reactivity of murine airways, upregulates Neuropeptide Y (NPY), and stimulation with NPY increases reactivity of human and murine airways to methacholine. 39 Diener, B.L., Hu, A., Josephson, M.B., Grunstein, M.M. Differential effects of IL-13 on serine site-specific glucocorticoid receptor (GR) phosphorylation in human airway smooth muscle (HASM) cells. 40 Josephson, M.B., Hu, A., Diener, B.L., Grunstein, M.M. Phosphodiesterase 4 (PDE4) activity is intrinsically increased in cultured human asthmatic airway smooth muscle cells due to upregulated G protein βγ-subunit activation. 41 Hu, A., Diener, B.L., Josephson, M.B., Grunstein, M.M. Pro-asthmatic signaling via the G protein βγ-subunit is intrinsically activated in cultured human asthmatic airway smooth muscle cells. 42 F. Pulmonary Vasculature Kudryashova, T., Ziai, H., Goncharov, D., Thant Mon Soe, M., DeLisser, H., Kawut, S.M., Goncharova, E. Benefits of targeting mTORC2 signaling in human and experimental pulmonary hypertension. 43 Goncharov, D.A., Kudryashova, T., Ziai, H., Khavin, I., Ihida-Stansbury, K., Krymskaya, V.P., DeLisser, H., Tuder, R.M., Kawut, S.M., Goncharova, E.A. mTOR complex 2 stimulates vascular smooth muscle cell proliferation and survival In idiopathic pulmonary arterial hypertension by regulating cellular ATP levels and AMPK activity. 44 Winterbottom, C.J., Shah, R.J., Mangalmurti, N., Lederer, D.J., Bhorade, S., Palmer, S.M., Wille, K.M., Weinacker, A., Wilkes, D.S, Shah, A.S., Lama, V.N., Crespo, M., Orens, J.B., Ware, L.B., Kawut, S.M., Christie, J.D. The Receptor for Advanced Glycation Endproducts is increased in pulmonary hypertension due to advanced lung disease. 45 Ihida-Stansbury, K., Xia, Y., Lee, E., Nguyen, T., Chin, L., Delisser, H., Kawut, S., Yang, S. Control of pulmonary vascular smooth muscle cell growth by biochemical and biophysical ECM properties. 46

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LUNG LIPIDS AND

SURFACTANT

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1 S-nitrosylation of SP-D in a mouse model of pulmonary Lymphangioleiomyomatosis (LAM). E. N. Atochina-Vasserman1, H. Abramova2, D. Goncharov1, M. Fehrenbach1, A.M. Pastva3, M.F. Beers1, A.J. Gow2, V.P. Krymskaya. 1Philadelphia, PA/US, 3Piscataway/US, 2Durham, NC/US Pulmonary Iymphangioleiomyomatosis (LAM), a rare lung disease, is associated with mutations of the Tuberous Sclerosis Complex (TSC1 or TSC2) tumor suppressor genes. LAM is manifested by neoplastic growth of atypical smooth muscle-like LAM cells, cystic lung destruction, obstruction of lymphatics, and spontaneous pneumothoraces. However, it is not well understood how LAM cell growth induces airspace enlargement in LAM. Surfactant Protein D (SP-D), a pulmonary collectin, is a typical representative of the innate host defense proteins and has well-established immuno-modulatory properties. We have recently shown that the S-nitrosylation of SP-D (SNO-SP-D) leads to a shift in the balance of pro- versus anti-inflammatory signaling in alveolar macrophages and alters their function, makes SP-D an intriguing protein in lung injury. In many inflammatory diseases, upregulated of iNOS expression within the cells of the alveolar space induced S-nitrosylation of SP-D and disruption of its multimeric structure which is related to lung emphysema. Thus we hypothesized that S-nitrosylation of SP-D might be involved in airspace enlargement manifested in LAM. Using our experimental TSC2-null murine LAM model (Goncharova et al. Science Transl. Med. 2012; 4:154ra134) we demonstrated time-dependent growth of multiple TSC2-null lesions resulting in progressive increases of airspace enlargement. In addition, these mice demonstrated elevated levels of BAL inflammatory cells, MMPs, proinflammatory cytokines upregulation and dramatic decline in mouse survival. TSC2-null lesions promote lung inflammation that leads to an increase in nitrate level at day 10 after TSC2-null cell injection. BAL from mice with TSC2-null lesion growth contains SNO-modified SP-D as demonstrated by a biotin-switch assay. Marked increase of SNO-SP-D was time-dependent and consistent with progression of TSC2-null lesion growth. Thus, these data suggest that increased SNO-SP-D in the lungs occurs specifically due to growth of TSC2-null lesions and may contribute to airspace enlargement. Funding: 1RO1HL114085-01 (VPK), LAM foundation grant LAM089E0112 (VPK), HL086621 (AJG)

2 Autophagy promotes cytoprotection by facilitating removal of aggregation-prone surfactant protein C L188Q BRICHOS mutant in vitro and in vivo. Jean Ann Maguire1, Elke Malzer2 Stefan Marciniak2, and Michael F. Beers1. 1Department of Medicine, Univ. of Pennsylvania SOM, Phila. PA, 2Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Macroautophagy is a critical cellular quality control pathway for clearance of oligomeric mutant protein aggregates. Mutations in Surfactant Protein C (SP-C), an alveolar type II cell specific protein, have been associated with chronic interstitial lung disease in children and adults. We have previously shown that transient expression of COOH-terminal SP-C mutants (BRICHOS) in mammalian epithelial cells leads to activation of the unfolded protein response, proteasomal inhibition, cytosolic aggregation of SP-C propeptide, and apoptosis. We hypothesized that SP-C BRICHOS mutants could utilize macroautophagy to facilitate elimination of intracellular aggregates. Expression of the aggregate-prone human SP-CL188Q mutant in HEK293 and HeLa cells induced LC3 (atg8) isoform conversion. Compared with transfected wild-type SP-C (SP-CWT), inhibition of autophagy by 3-methyladenine resulted in a significant increase in levels of mutant SP-C while Torin-1 decreased levels suggesting this pathway plays a prominent role in mutant clearance. Inhibition of autophagy in cells expressing SP-C BRICHOS mutants led to pronounced caspase 3 activation. To assess the role of macroautophagy in vivo we expressed native and EGFP tagged SP-C isoforms in the eye, salivary gland, and fat body of Drosophila melanogaster using the UAS-GAL4 system. By Western Blotting, F1 progeny of UAS-EGFP-SP-CL188Q Drosophila crossed with GMR-GAL4 driver flies exhibited eyes which had time-dependent increases in EGFP-SP-CL188Q levels that markedly exceeded those of UAS-EGFP-SP-CWT. Confocal microscopy of salivary glands of 3rd instar larvae showed expressed EGFP-SP-CWT localized in plasma membrane and subplasma membrane vesicles, while EGFP-SP-CL188Q was observed to be in a diffuse cytosolic punctate pattern. Untagged SP-CL188Q induced activation of autophagy in fat bodies co-expressing a GFP-atg8 reporter, while RNAi knockdown of atg8 caused significant toxicity in the eyes of GMR-GAL4 X EGFP-SP-CL188Q Drosophila. These results indicate that macroautophagy is activated in response to expression of SP-C BRICHOS mutants and plays an important role in the degradation of mutant SP-C propeptide in vitro and in vivo. We speculate that the autophagic response is important for lung epithelial cell proteostasis, and that Drosophila offer an important new paradigm for high throughput, in vivo evaluation of quality control pathways and therapies for cellular dysfunction induced by mutant SP-C expression. Supported by NIH HL19737 (MFB), VA Merit Award BX001176 (MFB)

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3 The Surfactant Protein C I73T missense mutation induces alterations in cellular macroautophagy and mitochondrial transmembrane potential. Arie Hawkins1, Shampa Chatterjee2, Robin Deterding3, William K. Funkhouser4, Jennifer Goralski5, Surafel Mulugeta1, and Michael F. Beers1. 1 Pulm. & Crit. Care Div.; Univ. of Penn. School of Med., Phila., PA 2 Inst. for Environmental Med.; Univ. of Penn. SOM., Phila., PA 3 Dept. of Pediatrics; Univ of Colorado SOM., Denver, CO 4 Dept. of Path. and Lab Medicine; Univ. of North Carolina, Chapel Hill NC 5 Dept. of Pediatrics; Univ. of North Carolina, Chapel Hill NC A missense substitution of isoleucine for threonine at codon 73 (I73T) of the lung specific SFTPC gene accounts for a significant portion of all Surfactant Protein C (SP-C) mutations associated with interstitial lung disease in children and adults. We previously showed that SP-CI73T is the prototype of a distinct subset of SP-C mutations which are mistargeted to the plasma membrane ultimately accumulating in endosomal/lysosomal compartments and causing impaired surfactant lipid endocytosis and degradation. Given the emerging role of autophagy in cytoprotection and its previously described disruption in lysosomal storage diseases, we hypothesized that SP-CI73T expression leads to epithelial cell dysfunction via impairment of autophagolysosome function. To test this we generated HEK cells stably expressing EGFP- or DsRed-tagged proSP-C isoforms. By electron microscopy SP-CI73T expressing cells were marked by abnormally large electron-dense, double membrane limited organelles containing amorphous and organellar debris. Transmission EM of a lung biopsy from a patient with SP-CI73T associated interstitial lung disease revealed similar ultrastructural changes in alveolar type 2 cells. Biochemically, when compared to wild type SP-C, HEK cells expressing SP-CI73T showed an increased expression of endogenous LC3 as well as p62 which was exaggerated by treatment with the vacuolar H+ ATPase inhibitor, bafilomycin A1. Additionally, in dose-response studies, SP-CI73T expressing cells exhibited an enhanced sensitivity of LC3 expression in response to bafilomycin A1 versus SP-CWT cells consistent with an impairment of distal autophagic flux. Using 2 different cell permeable fluorescent dyes, mitochondria in SP-CI73T cells had marked increases in mitochondrial transmembrane potential (depolarization). These results demonstrate that SP-CI73T induces a cellular phenotype consistent with an acquired block in macroautophagy and defective mitochondrial function. Supported By NIH 2T32HL007586-26 (AH), HL 090732 (SM), HL19737 (MFB), and VA Merit Award BX001176 (MFB)

4 Structural changes in Surfactant Protein D (SP-D) in the mouse lung in response to ozone (O3) and allergen exposure. S.K. Sharma, C.J. Koziol-White, S. Zhang, C.-X. Yuan, P. Axelsen, A. Haczku University of Pennsylvania - Philadelphia, PA/US BACKGROUND: O3 promotes exacerbation of the asthmatic airway inflammation, likely due to failure of protective immune mechanisms. SP-D is a critical pulmonary innate immune regulator with both suppressive and proinflammatory effects. We hypothesized that exposure to allergen or O3 induce oxidative modifications in key areas of the SP-D molecule that can induce a functional anti-inflammatory to proinflammatory switch. METHODS: Balb/c mice were sensitized and challenged with Aspergillus fumigatus (Af) and four days later exposed to O3 (3.0 ppm for 2 h). The bronchoalveolar lavage (BAL) supernatant was assessed for cellular and molecular inflammatory changes 12h later. SP-D was studied by native gel electrophoresis and biotin switch assay using an in-house biotinylated monoclonal anti-SP-D. Recombinant SP-D was treated with air or O3 (3.0 ppm for 30 min). Bands were extracted from BAL run on SDS-PAGE gels, digested with trypsin and analyzed using Nano LC/MS/MS with Sequest and Scaffold software. In additional experiments recombinant SP-D was denatured, reduced and alkylated before in-solution or in-gel trypsin digestion with or without PNGase F de-glycosylation and analyzed using TSQ Vantage and LTQ Orbitrap. RESULTS: In the BAL of O3 and Af exposed mice airway inflammation was associated with de-oligomerisation of the SP-D dodecamer. Because presence of nitrosothiols directly reflects cysteine modifications, we used the biotin switch method to investigate nitrosylation of SP-D cysteines and found that SP-D was indeed nitrosylated in response to Af or Af+O3 inhalation. To verify location of the modified cysteines we used LTQ Mass spectrometric analysis of trypsinized BAL samples. With this technique however we were able to reveal peptides only in the C-terminal region with coverage of up to 49%. The modifications observed (oxidation and deamidation) were present in controls as well as in O3 exposed SP-D samples with no differences between the BAL from naïve and treated mice. Additional cyanogen bromide treatment of SP-D after trypsin also failed to reveal N-terminal peptides that were only uncovered after de-glycosylation of the samples. Targeted analysis verified peptide recognition covering both N-terminal cysteines (SVPNTCTLVMCSPTENGLPG R, 726.01 da, Z=3). CONCLUSIONS: Cysteine residues in strategic positions for example at 15 and 20 in the N-terminal region of the SP-D molecule are protected by post translational glycosylation. In spite of this, O3-induced exacerbation of allergic airway inflammation in mice was associated with cysteine nitrosylation of SP-D in the BAL. This modification may be responsible for the de-oligomerisation of the native SP-D structure.

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5 A mouse model for lung disease-associated ABCA3 mutations. Surafel Mulugeta1, Yaniv Tomer1, Scott J. Russo1, Ming Zhao1, Arie Hawkins1, Kevin Yu2, Shaohui Huang2, Ping Zhang3, Susan H. Guttentag3, and Michael F. Beers1. 1Department of Medicine, Division of Pulmonary, Allergy, and Critical Care; 2Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA; 3Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, PA Idiopathic pulmonary fibrosis (IPF) is a devastating chronic and progressive form of interstitial lung disease (ILD) of unknown etiology characterized by fibrosis of the alveolar interstitium. Intrinsic epithelial cell injury and/or malfunction contribute to the development of ILD/IPF. The ATP-binding cassette class A3, ABCA3, is a member of the ABC superfamily of transporters that function in the translocation of a wide variety of substrates across cell membranes. ABCA3 functions as a lipid and phospholipid transporter and, in the lung, is critical for the biogenesis of alveolar type II (ATII) cell lamellar bodies. A relatively large number of mutations in the ABCA3 gene have been identified in association with adult forms of ILD and IPF. The most common mutation, a valine for glutamate missense mutation at residue 292 (E292V) causes functional impairment of the transporter. Using homologous recombination (recombineering) strategies, we have generated knock-in (KI) mice carrying the E292V mutation within the mouse ABCA3 locus, directly mimicking the allelic gene mutation observed in humans. Evaluation of 14-16 week old mice homozygous for the E292V mutation (KI/KI) revealed: 1) Decreased bronchoalveolar lavage (BAL) phospholipid (PL) content and increased protein concentration; 2) Reduced lamellar body size; 3) Interstitial, peribronchial infiltration of the distal lung by inflammatory leukocytes and macrophages; and 5) Disrupted alveolar architecture including alveolar wall thickening and fibrosis. Mice heterozygous for the E292V mutation exhibited an intermediate phenotype between wild type and KI/KI mice, with preservation of LB size and BAL PL content but elevated BAL protein content, suggestive of a dose responsive phenotype. These results suggest that ABCA3 mutations expressed in ATII cells of mice can phenocopy the human disease. This model offers a platform for future studies to understand the pathogenesis of human ILD and to develop pre clinical testing for novel, targeted therapeutic strategies. Supported by NIH HL090732 (SM) , NIH HL19737 (MFB), and VA Merit Award BX001176(MFB)

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OXIDATIVE

STRESS

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6 Regulation of Nrf2 by nuclear HO-1 provides cytoprotection through transcription of G6PDH. Chhanda Biswas‡§, Guang Yang‡, Nidhi Shah‡, Manasa Muthu‡, Phyllis A. Dennery‡§, ‡Division of Neonatology, Children's Hospital of Philadelphia, §Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 RATIONALE: In response to oxidative stress Nrf2 bypasses proteasomal degradation and translocates to nucleus. There it binds to the antioxidant response elements (ARE) and promotes transcription of various cytoprotective genes. Nrf2 mediated induction of G6PDH regulates metabolic pathways and cell proliferation via pentose phosphate pathway. Induction of heme oxygenase 1 (HO-1) is another defense mechanism. This enzyme although is anchored to the smooth endoplasmic reticulum, in oxidative stress, undergoes C terminus truncation and nuclear translocation; this form of HO-1 is devoid of enzymatic activity. Studies have shown increased nuclear localization of HO-1 in proliferating and some cancer cells. However the function of nuclear HO-1 is not fully understood. OBJECTIVES: Our objective is to demonstrate in cell-based assays that oxidative stress-mediated induction, truncation and nuclear migration of HO-1 influences specific Nrf2 activation and transcription of G6pdx. METHODS: The HO-1 knock out (KO) mouse embryonic fibroblasts (MEF) were generated at embryonic day 13.5 and were rescued with FLAG-fused full length HO-1 (FL), truncated HO-1 (TR) devoid of C-terminal 51 amino acid and Vector (V, control). These rescued cells and the WT and control cells were exposed to 95%O2/5%CO2 (hyperoxia, O2) or 95% air/5% CO2 (normoxia, Air) for 0, 4 and18 hours with or without MG132 (proteasome inhibitor). These cells were further transiently trasnfected with V5 peptide fused Nrf2 full length and N-terminal deletion mutant. The cytoplasmic and nuclear fractions of these cells were immunoblotted for HO-1 and Nrf2. Immunoprecipitation of FL and TR, endogenous Nrf2 and V5-fused Nrf2 from these cells lysates were performed using anti-FLAG, anti-Nrf2 and anti-V5 antibodies, respectively. The pulled down immunoprecipitates were immunoblotted for Nrf2, HO-1 and ubiquitin. Furthermore HO-1 in WT and TR cells was stably knocked down and these cells were compared with control cells for steady state mRNA levels of HO-1, Nrf2 several genes downstream of Nrf2 including G6pdx by qRT-PCR. MAIN RESULTS: Hyperoxia induced and truncated HO-1 that allowed its nuclear migration. The cells enriched with nuclear HO-1 were also enriched with a 38kDa Nrf2 arising from an N-terminal deletion of native Nrf2. The 38kDa Nrf2 localized in the nucleus in a complex with the truncated nuclear HO-1. Disruption of HO-1 or in the presence of cytoplasm restricted FL, although the 38kDa Nrf2 was rescued from proteasomal degradation by MG132, its enrichment in the nucleus was limited. The cells abundant with nuclear HO-1 exhibited increased transcription and activation of G6PDH and cell proliferation. CONCLUSIONS: We conclude that in oxidative stress nuclear HO-1 facilitates stabilization and nuclear translocation of a 38kDaNrf2 as HO-1/Nrf2 complex, which is crucial for the transcription of G6PDH leading to cell survival and proliferation.

7 Role of Peroxiredoxin 6 phospholipase A2 activity in lung damage resulting from oxygen exposure. Bavneet Benipal, Sheldon I. Feinstein and Aron B. Fisher. Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA 19104 Hyperoxia-induced lung injury results in increased pulmonary permeability, inflammation and respiratory failure. The lung damage is caused by an imbalance between ROS generation and ROS scavenging by antioxidant defense. Peroxiredoxin 6 is the only antioxidant enzyme that has both peroxidase and PLA2 activities and plays a major role in protecting lung cells against oxidative stress. Previous studies in cells have shown that cell protection by Prdx6 involves both peroxidase and PLA2 activities. However, Prdx6 PLA2 activity activates Nox2, leading to ROS generation. MJ33 is considered a potent inhibitor of Prdx6 PLA2 activity and blocks agonist-induced activation of Nox2. Studies also suggest that unlike the cell model, in which Prdx6 PLA2 was protective, in the mouse model, Prdx6 PLA2 may promote lung damage due to activation of Nox2 in neutrophils that enter the lung. We hypothesize that blocking Prdx6 PLA2 activity using MJ33 or by mutation will be protective in a mouse model of exposure to hyperoxia. To test this hypothesis, we evaluated the effect of blocking Nox2 in protection against hyperoxia and the effect of MJ33 on Nox2-mediated ROS production in the intact lung following hyperoxia. For survival or lung injury studies, mice were kept in an O2 chamber and exposed to 100% O2. Three groups of mice were used. The first group was kept at room air. The second and third groups were kept in an O2 chamber (exposed to 100% O2). The second group was injected with PBS and the third group was injected with MJ33 (50nmoles) at 0 and 48 hours. For the survival study, mice were monitored till signs of stress were observed, then sacrificed and survival time recorded. For the lung-injury study, mice were sacrificed at 80 hours, BALF and lung tissue were used to perform oxidative stress studies. Survival study data showed that Nox2 KO mice survived longer than WT mice in hyperoxia suggesting that blocking Nox2 activity is protective. Studies are in process to determine the effect of hyperoxia on lung damage by blocking Prdx6 PLA2 activity using MJ33 or Prdx6 mutant mice. Supported by NIH/RO1-HL105509 and NIH-T32-HL07748-17.

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8 Oxidative stress and inflammation regulate the key circadian gene Rev-ERBα through conserved NFκB and NRF2 binding sites. 1Guang Yang, 3Clyde J. Wright, 1Maurice Hinson, 1Amal P. Fernando, 1,2 Phyllis A. Dennery. 1Children’s Hospital of Philadelphia, 2 University of Pennsylvania, Philadelphia, PA, USA. 3Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado Denver School of Medicine, Aurora, CO. USA.

Rational: In mammals, the circadian rhythm is key in controlling cellular homeostasis because it alters cell metabolism and cell growth. Rev-ERBalpha, a nuclear receptor, is found in the lung and is a key gene for sustaining the circadian rhythm. We have previously shown that the activity of a 0.9 kb promoter luciferase construct of Rev-ERBalpha was increased in hyperoxia. Linear or targeted deletion mutants, aimed for the cis elements of NRF2 and NF-kappaB transcription factor binding sites, showed decreased promoter activity at baseline and in hyperoxia. Objective: In this work we further verified occupancy and DNA binding of NRF2 and NF-kappaB on the Rev-ERBalpha promoter. Using serum shock as a model for circadian rhythmicity, we tested whether oxidative stress or inflammatory stimuli would alter circadian function of Rev-ERBalpha. Methods: Mouse lung fibroblasts (Mlg) were exposed to hyperoxia (95% O2 and 5% CO2), normoxia (95% air and 5% CO2) or incubated with TNFalpha for 24 hours. Chromatin immuno precipitation assay (ChIP) was performed for occupancy of NrF2, p65 and p50. In other experiments nuclear fractions were evaluated for DNA binding to NrF2 and NF-kappaB using EMSA. For testing Rev-ERBalpha circadian function, Mlg cells were either exposed to hyperoxia for 4 h or stably disrupted with p50 shRNA prior to serum shock experiments as described as following: cells were starved with 0.5% FBS overnight and shocked with 50% horse serum for 2 h. Total RNA was collected 1 h after the shock and thereafter every 3 h for a total of 24 hours. Levels of Rev-ERBalpha mRNA and protein were measured using RT-PCR. Results: Occupancy of NrF2 and p50 was increased on a proximal region of the Rev-ERBalpha promoter in hyperoxia. DNA binding to NrF2 sequence was also enhanced. These data are in agreement with our previous work that the NF-kappaB and NrF2 binding sites mediate hyperoxia induced Rev-ERBalpha promoter activity. However with TNFalpha incubation, p65 occupancy was diminished. Additionally both p50 and p52 subunits of the NF-kappaB were recruited to the promoter. When p50 was disrupted the TNFalpha mediated down regulation of Rev-ERBalpha was abolished suggesting the NF-kappaB binding site differentially regulates Rev-ERBalpha promoter activity under inflammatory condition via repressors of the NF-kappaB subunits. Hyperoxia and p50 disruption altered the amplitude of the rhythmicity of Rev-ERBalpha. Conclusion: We conclude that both NrF2 and NF-kappaB binding sites mediate Rev-ERBalpha transcription. Hyperoxia activates Rev-ERBalpha via the NrF2 site and inflammation deactivates Rev-ERBalpha via the NF-kappaB site. This work was sponsored by R01 HL058752 (P.A.D).

9 Mechanosensing with restart of flow drives KATP induced NOX2 activation in a model of lung ischemia reperfusion. Shigeru Abe, Hui Wang, Nankang Hong, Kevin Yu, Kris M. Debolt, Sheldon Feinstein, Edward Cantu, Jason D. Christie , Aron B Fisher and Shampa Chatterjee From our earlier studies in lung ischemia, we concluded that endothelial mechanotransduction with stop of flow in the lung activates the generation of reactive oxygen species (ROS) as a result of KATP closure that leads to NOX2 activation. As the next phase, we evaluated the role of mechanosignaling with reperfusion. We now propose that the increase in shear with reperfusion triggers a mechanosignaling cascade similar to that associated with ischemia. Isolated perfused mouse lungs and pulmonary endothelial cells showed a 2 fold increase in ROS production (as monitored by ROS sensitive dyes H2DCFDA and DHE) with ischemia that is further enhanced by 3-3.5 fold upon reperfusion. This was not observed in lungs and cells from NOX2 null and KATP channel null mice. Lungs subjected to ischemia or I/R show ~1.8 fold increase in lung damage (as assessed by 8-isoprostanes, TBARS, permeability) with ischemia and a ~2.5 fold increase during the reperfusion period. Understanding the initiation of reperfusion signaling has potential application in lung transplantation which requires reperfusion of a previously ischemic lung; thus this model is relevant to design strategies that can reduce NOX2 activation and resultant injury.

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10 Effect of glutathione on the phospholipase A2 activity of peroxiredoxin 6. Suiping Zhou1, Chandra Dodia1, Tea Shuvaeva1, Elena Sorokina1, Sandra Harper2, David W. Speicher2, Sheldon I. Feinstein1 and Aron B Fisher1. 1Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA, 2Center for Systems and Computational Biology, The Wistar Institute, Philadelphia, PA, 19104, USA,

Peroxiredoxin 6 (Prdx6) is a unique non-seleno 1-Cys peroxidase with both peroxidase and phospholipase A2 (PLA2) activities.We have shown that phosphorylation of Prdx6 at Thr177 increases its PLA2 activity and broadens its pH-activity spectrum (Biochem. J. 419:669, 2009). Our recent study indicates that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhanced enzymatic activity at pH 7.4 (Biochemistry. 51:5521-30. 2012). Here, we studied the effect of glutathione (GSH) on the PLA2 activity of Prdx6 using biochemical and biophysical approaches. With 3H-DPPC as substrate, basal Prdx6 PLA2 activity was 0.17 ± 0.1 nmole/min/mg and increased to 49 ± 1 nmole/min/mg upon addition of 5 mM GSH. Addition of other thiols (GSSG, DTT) had no effect. The enhancing effect of GSH on the PLA2 activity of Prdx6 depended on the redox status of substrates and phosphorylation status of the enzyme. GSH has no effect on PLA2 activity if the substrate is oxidized by Cu(2+)-ascorbate or if the enzyme is phosphorylated. By isothermal titration calorimetry (ITC), GSH interacts with Prdx6. Circular dichroism (CD) indicates that GSH induces a change of Prdx6 conformation. Thus, a change in the conformation of Prdx6 in the presence of GSH is the basis for enhancing effect of GSH on PLA2 activity of non-phosphorylated Prdx6 in the presence of reduced substrate. This work was supported by NIH grants HL102016 and HL19737

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Serine 32 targeted mutation abolishes Prdx6 trafficking to lamellar bodies in vivo. Elena M. Sorokina, Chandra Dodia, Kevin Yu, NanKang Hong, Sheldon I. Feinstein and Aron B. Fisher. Institute for Environmental Medicine, University of Pennsylvania, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104 Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid (PL) turnover and protects lung epithelium from oxidative stress. Prdx6 null mice accumulate PL in lung tissue, compatible with the role of PLA2 activity in lung phospholipid turnover. Prdx6 has been localized to cytosolic and acidic compartments (lamellar bodies (LB) and lysosomes) in lung epithelium. Our previous studies indicated that serine at position 32 (S32) is necessary for proper Prdx6 targeting to LB. We have generated a mouse model with an S32T mutation in the Prdx6 LB targeting sequence. Homozygous mice carrying the S32T mutation exhibited phospholipid (PL) accumulation in LB (PL/protein ratio 14.36) vs. WT (PL/protein ratio 9.51). Western blot analysis demonstrated absence of Prdx6 in LBs isolated from S32T mice despite equal amounts of Prdx6 in lung homogenates. Immunofluorescence analysis of mouse lung tissue sections showed co-localization of Prdx6 with LAMP1, a marker of lysosome-related organelles, in wild type but not in S32T mouse alveolar type II cells. These findings indicate that S32 is required for Prdx6 targeting to LB in mouse lung epithelium. [HL102016 and HL19737]

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12 Hyperbaric oxygen stimulates adipose- derived mesenchymal stem cells growth and differentiation in streptozocin-induced diabetes mellitus type 1 mice in vivo. Tatyana N. Milovanova1, Gunalp Uzun1, Elena M Sorokina, Jonni S. Moore3 and Stephen R. Thom 1,2. 1Institute for Environmental Medicine, Departments of 2Pathology and Laboratory Medicine and 2Emergency Medicine, University of Pennsylvania Medical Center, Phila., , Pennsylvania; 3Department of Flow Cytometry, University of Pennsylvania Medical Center, Phila. Hyperbaric oxygen modified function of stem/progenitor cell (SPCs) in new blood vessels formation which is a required step in wound healing in diabetic patients. In wounds healing VEGF secreted from adipose-derived mesenchymal stem cells (ASCs) induces migration and proliferation of endothelial cells, increasing the vascularity of wound bed. We hypothesized that oxidative stress from hyperbaric oxygen (HBO2, 2.8 ATA for 90 min daily) exerts a trophic effect on adipose- derived mesenchymal stem cells in streptozocin-induced diabetes mellitus type 1 mouse model via reactive oxygen /reactive nitrogen species (ROS/RNS)-dependent mechanism. Adipose-derived stem/progenitor cells (ASCs:Sca-1+/CD31-/DAPI-), were sorted from omentum of additional WT mouse, labeled with the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), added to one of the Matrigel plugs subcutaneously injected one on either side of the thoracic vertebrae and stimulated by HBO2. In combination, HBO2 and ASCs in vivo demonstrated cumulative effects. Vascular channels lined by CD34+ SPCs were identified and they were significantly reduced in diabetic animals. In the Matrigel with seeded ASCs after HBO2

simulation accelerated channel development, cell differentiation based on surface marker expression and cell cycle entry was identified. CD34+ SPCs of diabetic animals in blood and bone marrow defined down regulated thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3, phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and stromal cell-derived factor-1 in. Cell recruitment to Matrigel and protein synthesis responses was abrogated in STZ-mice. Thioredoxin system activation leads to elevations in HIF-1 and -2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on SPCs. By

causing an oxidative stress, HBO2 activates a physiological redox-active autocrine loop in recruited SPCs and increased paracrine secretion in seeded ASCs, resulted in the stimulated neovasculogenesis.

13 HO-1 nuclear localization enhances DNA repair by increasing availability of the DNA repair enzyme OGG1. Monica Williams, MS1, Amal P Fernando, MD1, Ping La, PhD1, Phyllis A Dennery, MD1, 2 Children’s Hospital of Philadelphia1, University of Pennsylvania School of Medicine2 Rational: Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme and an integral protein of the smooth endoplasmic reticulum. Its inducible isoform, HO-1, is highly expressed in the neonatal lung. We have shown that HO-1 protein, truncated at the C-terminus, becomes catalytically inactive and can migrate to the nucleus under oxidative stress. The role of nuclear HO-1 remains poorly defined. Neonatal rodents are more tolerant to hyperoxia than adults and have increased nuclear HO-1. Preliminary data show that several DNA repair proteins associate with nuclear HO-1 after oxidative stress. Thus we hypothesized that nuclear HO-1 provides cytoprotection by enhancing the function of DNA repair proteins thereby mitigating the effects of hyperoxic stress. Objective: We sought to determine the role of nuclear HO-1 after hyperoxic injury. Methods: HO-1 null mutant (KO) mouse embryonic fibroblast (MEF) cells were stably infected with retroviruses expressing full-length (FL) or C-terminal truncated (TR) HO-1 cDNA, or empty vector (VEC) as a control. To evaluate the response to oxidative stress, cells were exposed to either 95% O2/5%CO2 (hyperoxia) or room air/ 5%CO2 (normoxia) for 28 hours. Live and dead cells were counted at 0, 4, 8, and 24 hrs to assess cell viability using the Moxi Z automated cell counter (Orflo). DNA fragmentation, a marker of apoptosis, was evaluated by DNA laddering and oxidative DNA damage was assessed by immunofluorescence staining for 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), an oxidative DNA adduct. Steady state mRNA levels and protein levels of 8-oxoguanine DNA glycosylase (OGG-1), a DNA repair enzyme, were evaluated using quantitative polymerase chain reaction (qPCR), western analysis and immunofluorescence staining for OGG-1 signal. Results: After 24 hrs of hyperoxia, TR infected cells showed improved survival compared to both VEC and FL infected cells. In addition, DNA laddering and 8-oxo-dG immunofluorescence staining were decreased, demonstrating less apoptosis and less oxidatively damaged DNA. Although qPCR showed a three fold increase in OGG-1 mRNA in TR infected cells at 28 hours of hyperoxia compared to VEC, cellular OGG-1 protein was not increased at this time point. Interestingly, immunofluorescence staining for OGG-1 protein showed enhanced nuclear localization in TR infected cells at 28 hours of hyperoxia. Conclusion: Enhanced nuclear HO-1 is associated with decreased apoptosis, lesser oxidative DNA damage and improved survival in MEF cells. The likely mechanism involves enhanced binding and nuclear translocation of OGG-1 rather than up-regulation of OGG-1 mRNA and protein, since this occurred later. We speculate that nuclear translocation of HO-1 mitigates hyperoxic injury in neonates by enhancing repair or obviating oxidative damage of DNA. This work was funded by grant NIH RO1 HL058752 (P.A.D)

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INFLAMMATION

AND IMMUNE RESPONSE

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14 LPS-induced lung inflammation is attenuated in the absence of the Receptor for Advanced Glycation End products. NS Mangalmurti, DY Qing, H Faust, R Lo, JS Lee, GS Worthen and SM Albelda. 1Perelman School of Medicine, University of Pennsylvania, Phila., PA, 2 University of Pittsburgh School of Medicine, Pittsburgh, PA, 3Children’s Hospital of Phila., University of Penn., Phila., PA. Rationale: The Receptor for Advanced Glycation End products (RAGE) is a multi-ligand pattern recognition receptor highly expressed in the lung. The role of RAGE in LPS-induced inflammation remains unclear as some studies implicate RAGE as a direct receptor for LPS and others have demonstrated no differences in the lung inflammatory response to LPS between WT and RAGE KO mice. Furthermore, although RAGE signaling has been implicated in the late response to LPS induced inflammation, its role in early LPS induced inflammation has not been studied. We therefore examined whether early LPS induced lung inflammation was mediated by RAGE. Methods: To investigate the role of RAGE in LPS-induced lung inflammation, RAGE KO or WT (C57Bl/6J) mice were challenged with low dose LPS (1.5 ng/g, intranasally). BAL chemokines, neutrophil counts and total protein was examined following intranasal LPS challenge. In vitro inflammatory responses to LPS challenge were examined utilizing peritoneal macrophages that were obtained from RAGE KO or WT (C57Bl/6J) mice following thioglycollate injection. Macrophages were stimulated with LPS (10 ng/mL) and supernatants and cell lysates were collected at the designated time points. Supernatants were assayed for TNF-±, KC and MIP-2 utilizing ELISA. Cell lysate IKB± was measured by immunoblotting. Results: RAGE KO mice demonstrated attenuated neutrophil recruitment at 4 and 6 hours and attenuated BAL KC (1514 ± 486 pg/mL v 3072 ± 32 pg/mL, p=0.019) but not MIP-2 at 2 hours when compared with WT mice in response to intranasal LPS challenge. In vitro, SN from RAGE KO macrophages demonstrated less KC and TNF± in response to LPS at all time-points assayed (p <0.008 for KC, p <0.02 for TNF± ,Table 1). MIP-2 secretion was decreased in RAGE KO macrophages at 6, 8 and 24 hours (p <0.01, Table 1). In response to LPS challenge, Iº b± degradation was attenuated in the RAGE KO macrophages. Conclusions: RAGE KO mice demonstrated diminished lung inflammation in response to low dose LPS. In vitro, cytokine and chemokine release by peritoneal macrophages in response to LPS was attenuated in the absence of RAGE. These effects appear to be associated with altered NfºB activation as IºB± degradation following LPS challenge was abrogated in RAGE KO macrophages when compared with WT macrophages. Thus, RAGE mediates LPS induced lung inflammation and activation of the RAGE axis may be one mechanism by which LPS incites pro-inflammatory responses in macrophages.

15 Mediator release following rhinovirus exposure is selectively attenuated by a kinase inhibitor but not a steroid in human lung slices. J. Scala, C.J. Koziol-White, W.M. Lee, J.E. Gern, P. Strong, K. Ito, G. Rapeport, and R.A. Panettieri. University of Pennsylvania, Philadelphia, PA; RespiVert Ltd., Imperial College, London; University of Wisconsin-Madison, Madison, WI RATIONALE: Viral-induced respiratory infections, a leading cause of asthma exacerbations, are primarily evoked by rhinovirus. Direct effects of virus on small airways of humans and approaches to attenuate viral-mediated exacerbations of airways disease remain therapeutic challenges. We hypothesized that RV16 induces mediator release from human small airways, which may be modulated by steroid treatment or kinase inhibition. METHODS: Human precision cut lung slices (PCLS) from normal healthy donors, each containing a small airway (d 2 mm), were stimulated ex vivo with increasing concentrations of RV16 (105-107 Pfu) for 48 hr, with culture supernatants assessed for mediator release. Slices were pre-incubated with the narrow spectrum kinase inhibitor RV1088 or dexamethasone for 1 hr prior to rhinovirus stimulation. RESULTS: Exposure of human small airways to RV16 induced IL-8, IL-6, and IP-10 secretion, with levels of all mediators markedly increasing. RV1088 decreased RV16-stimulated IL-8, IL-6, and IP-10 levels, whereas dexamethasone exhibited little effect on either mediator release. Qualitative cilia beat frequency was unchanged by treatment with any of the treatments. CONCLUSIONS: These data suggest that virus selectively increases chemokine and cytokine secretion shortly following viral exposure. Narrow spectrum kinase inhibition attenuates virus-induced IL-8, IL-6, and IP-10 release but steroid treatment has little to no effect. Given these data, kinase inhibitors may serve to modulate the inflammatory mediator profile upregulated following viral exposure. This research was sponsored, in part, by RespiVert Ltd.

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16 O3 inhalation attenuates the budesonide effects on allergic airway inflammation and SP-D production in the lung of Balb/c mice. Moyar Q. Ge1, Lisa Forbes1, Jin Hwang1, Blerina Ducka1, Imre G. Redai1, Christopher Stevenson2, Angela Haczku1. 1University of Pennsylvania, Phila., PA. 2Inflammation Discovery,pRED Pharma Research & Early Development, Hoffmann-La Roche, Nutley NJ BACKGROUND: Our previous studies on airway responses to allergen or O3 exposure showed that C57BL/6 mice were significantly more protected against developing inflammation and airway hyperresponsiveness than Balb/c mice that were relatively deficient in their ability to produce the immunoprotective surfactant protein D (SP-D) in their airways. Presence of SP-D in the lung is important to prevent constitutive activation of the pulmonary innate immune system. Glucocorticoid treatment can upregulate SP-D production. The aim of this study was to investigate whether the anti-inflammatory and SP-D inducing effects of glucocorticoid treatment are altered in allergen or O3 exposed mice. METHODS: We studied BALB/c and C57BL/6 mice sensitized and challenged with Aspergillus fumigatus (Af), exposed to air or O3 and treated with different doses budesonide (0. 0.1 and 1 uM). Lung function and airway inflammation were assessed. BAL SP-D content was analyzed by Western blot and ELISA. Lung cellular composition was analyzed by FACS. RESULTS: Treatment with budesonide significantly reduced inflammatory changes and airway hyperresponsiveness in mice that received allergen sensitization and challenge alone. Budesonide inhibited allergic airway hyperresponsiveness to MCh in a dose-dependent manner. Budesonide also induced a protective increase in SP-D in Af sensitized and challenged BALB/c mice together with inhibition of the inflammatory cell numbers, macrophages and CD4 T cells in the lung. O3 induced exacerbation of allergic inflammation 4 days after allergen challenge in Balb/c (but not C57BL/6) mice. This was characterized by enhanced airway eosinophilia, neutrophil influx and increased release of IL-6, TNF-alpha, eotaxin and CCL17 indicating activation of the innate immune system in the lung. O3-induced exacerbation of the inflammatory changes was associated with an impaired expression of SP-D in the BAL. The effects of budesonide on inflammatory cells were attenuated in the O3-exposed Balb/c mice. Importantly, the budesonide induced increase in SP-D production was abolished by O3 exposure (p<0.05, n=6). Our results indicate that Balb/c mice are more susceptible for O3-induced exacerbation of allergic airway inflammation than C57BL/6 mice. This susceptibility is associated with an impaired ability to express SP-D. Balb/c mice sensitized and challenged with Af are highly responsive to glucocorticoid treatment. However this responsiveness together with the protective induction of SP-D is abolished when the mice are also exposed to O3.CONCLUSIONS: We speculate that the beneficial action of glucocorticoids in allergic airway inflammation maybe mediated at least in part by an induction of SP-D production in the lung and that O3 inhalation attenuates the glucocorticoid effects. Funding: Roche, R01AI072197; RC1ES018505; P30ES013508; CIS Baxter Senior Fellowship(LRF), PIDTC Senior Fellowship(LRF)

17 Modulation of the endothelial inflammatory response by EUK-134 loaded immunoliposomes. M.D. Howard, C.F. Greineder, E. Hood, V. R. Muzykantov. Department of Pharmacology and Institute for Translational Medicine and Therapeutics, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104, USA

Oxidative stress has been implicated in a number of acute vascular pathologies, yet poor delivery of antioxidants limits their therapeutic benefit. To overcome this challenge, endothelial-targeted immunoliposomes loaded with EUK-134 (EUK-ILs), an SOD/catalase mimetic, were prepared and evaluated for their ability to inhibit the endothelial inflammatory response. Comparisons were made between ILs coated with anti-platelet endothelial cell adhesion molecule (PECAM) or control IgG. EUK-134 could successfully be loaded into ILs characterized by a small size (<200 nm) and low polydispersity (<0.2). Encapsulated drug concentration was shown to increase linearly with added drug, up to a concentration of 0.36 mg/ml. Partial drug activity was retained in the intact ILs; full activity could be recovered upon bilayer disruption. Similar antibody surface coverage was attained with two separate conjugation chemistries (biotin-streptavidin and maleimide-SATA). Targeted EUK-ILs showed enhanced binding to HUVECs (~6-fold) and lung accumulation (~16-fold) as compared to control IgG EUK-ILs. Treatment of HUVECs with anti-PECAM EUK-ILs prior to insult with the cytokine TNF eliminated VCAM upregulation while use of IgG EUK-ILs resulted in only a minor suppression. This trend was also observed in vivo. Pretreatment with targeted EUK-ILs successfully reduced BSA leakage from the lungs in LPS-challenged mice, resulting in >60% protection as compared to only 6% protection with IgG EUK-ILs. These preliminary studies provide evidence that targeted EUK-ILs may be useful for modulating the endothelial inflammatory response.

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18 Surfactant Protein-D (SP-D) suppresses ozone (O3)-induced CCL17 release and epithelial accumulation of myeloid dendritic cells (DC) in mice. Blerina Kokalari1, Moyar Ge1, Lisa R. Forbes1, Imre G. Redai1, Carol Anne Ogden2, Holly Raymond2, Angela Haczku1. 1University of Pennsylvania, Philadelphia, PA, 2Johnson and Johnson, King of Prussia, PA BACKGROUND: The asthmatic airways are highly susceptible to O3 inhalation induced worsening of inflammation but the underlying mechanisms are unclear. Myeloid pro-inflammatory DC mediate allergen and infection-associated airway pathologies but the role of these cells in O3- provoked lung injury is unclear. SP-D plays a protective role in airway inflammation by regulating innate immune cell function. We hypothesized that SP-D counteracts epithelial homing of myeloid DC by inhibiting release of the autocrine chemokine CCR4 ligand, CCL17. METHODS: Wild type (WT) C57BL/6 and SP-D-/- mice and the relatively low SP-D expressor Balb/c mice were compared in a model of O3-induced (3 ppm for 2 hours) exacerbation of allergic airway inflammation generated by Aspergillus fumigatus (Af) exposure. The direct effects of SP-D were investigated on bone-marrow derived DC cultured from c-kit+ stem cells. CCL17 protein and mRNA levels were studied in lung tissue extracts. Cells from the bone marrow, dissociated lung tissue and bronchoalveolar lavage (BAL) were assessed for CCL17 and CCR4 expression by FACS analysis. Cell migration was studied by PKH21 injection (i.v.). RESULTS: In response to O3 alone, in the BAL, Balb/c mice had a heightened cell influx together with a greater induction of CCL17 but a reduced SP-D expression in comparison with C57BL/6 mice (p<0.05). O3-induced exacerbation of allergic airway inflammation in Balb/c (but not C57BL/6) mice was associated with increased eosinophilia and CCL17 and decreased SP-D protein expression 12h after O3 inhalation. In comparison with wild type, SP-D-/- CD11c+/MHC-II+ DC had heightened cytoplasmic CCL17 expression both at baseline and after O3 exposure. 96h after Af challenge SP-D-/- mice had significantly increased expression of the CCL17 gene in the lung associated with greater PKH21+ DC accumulation. O3 exposure further increased the number of PKH21+ cells in the BAL and the expression of CCL17 mRNA in the lung of SP-D-/- mice. Thus, SP-D-/- DC expressed high levels of CCL17 and showed a significantly increased epithelial homing of PKH21-ingested cells. In bone marrow stem cell-derived DC culture recombinant SP-D (1-5µg/ml) directly inhibited cytoplasmic CCL17 expression in a time and dose dependent manner in vitro. CONCLUSION: Ozone induced CCL17 release in the lung tissue may be important in attracting proinflammatory DC into the airways. Our results also suggest that SP-D plays a key role in regulating DC migration by suppressing production of this CCR4 ligand by DC. We speculate that these mechanisms are important in O3-induced exacerbation of allergic airway inflammation. Funding: Johnson & Johnson, R01AI072197; RC1ES018505; P30ES013508; CIS Baxter Senior Fellowship(LRF), PIDTC Senior Fellowship(LRF)

19 Social stress inhibited glucocorticoid receptor (GR) and CAAT enhancer binding protein (C/EBP)-² mediated surfactant protein D (SP-D) expression in the lung of mice in an asthma model. Zhilong Jiang, Blerina Ducka, Giulia Ravaioli, Imre G. Redai, Sheri S. Wang, Reynold A Panettieri, Seema Bhatnagar, Angela Haczku. Airway Biology Initiative, Pulm., Allergy & Critical Care Div., Dept. of Med., Univ.of Penn., Phila. PA. BACKGROUND: Psychosocial stress alters corticosteroid responsiveness in the lung. Production SP-D, an epithelial immune regulator, requires constitutive glucocorticoid action on the airway epithelial cells. Corticosteroid-induced SP-D release plays an important immunoprotective role in allergic airway inflammation. We have previously shown that social stress in mice induced glucocorticoid resistance with impaired GR function. Here we hypothesized that GR impairment due to social stress results in abnormal transcriptional regulation of SP-D mRNA. METHODS: Combined models of social stress and allergic sensitization with Aspergillus fumigatus were studied on inflammatory cell influx, cytokine release and SP-D production in Balb/c and CD1 mice. Stress induced behavioral changes were assessed by the open field test. mRNA expression for GR and C/EBP-² (a steroid-responsive transcription factor responsible for induction of SP-D transcription) were studied in lung tissue and in isolated lung epithelial cells in vitro. SP-D mRNA regulation by GR and C/EBP-² was investigated in primary human lung epithelial cells and A549 cell line by CHIP assay. RESULTS: Social stress significantly enhanced airway responses to allergen challenge and attenuated levels of SP-D in the lung of sensitized CD1 mice but not Balb/c mice. Stress exposed CD1 mice also exhibited a reduced exploratory behavior in the open field test. While the SP-D promoter does not contain any proximal GR responsive elements, there are several C/EBP binding elements located in the promoter region. To test the hypothesis that GR may utilize C/EBP regions to regulate SP-D transcription we performed CHIP assay. Dexamethasone treatment activated binding to the C/EBP DNA region by both the GR and C/EBP-² in both A549 cells and primary, human lung epithelial cells. Further, this binding induced SP-D mRNA and protein production by epithelial cells in a time and dose-dependent manner. Attenuation of the SP-D response during stress was associated with decreased mRNA expression for GR as well as C/EBP-² . CONCLUSIONS: Our data suggest that impaired production of the immunoprotective SP-D is associated with social stress-induced enhancement of allergic inflammation and a reduced corticosteroid sensitivity. This effect maybe due to a sustained increase in circulating glucocorticoids together with high levels of inflammatory mediators. Importantly, we demonstrate here that glucocorticoids activate the C/EBP binding DNA region in the promoter of SP-D suggesting tethering of the GR with C/EBP-² . Thus, stress may affect glucocorticoid function by modifying not only the GR but also additional transcription factors responsible for mediating the glucocorticoid effects. Funding: R01AI072197; P30ES013508

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20 Detection of acute lung inflammation using molecular imaging. Blaine J. Zern, Ann-Marie Chacko, Colin F. Greineder, Eric A. Simone, Vladimir R. Muzykantov Introduction: The targeting of therapeutic or imaging agents to the endothelium may improve specificity and effectiveness of treatment for many diseases. One strategy to achieve this goal is the use of nanoparticles (NPs) targeted to the endothelium by ligands of protein determinants present on the endothelium, including cell adhesion molecules, peptidases, and cell receptors. Further, the endothelium plays a critical role in regulating the inflammatory response and cell adhesion molecules present on the luminal surface are intimately involved in this response, representing attractive molecular targets. The current clinical standard for PET imaging of inflammation is 18F-FDG, as well as detecting other maladies such as edema and tumor growth. Yet, it does not distinguish between these pathologies and its resolution is limited by false-positive signals from normal highly metabolically active cells (e.g. heart, muscle). The work presented here introduces a NP platform based on the polymer polyvinylphenol (PVPh) that permits the PET-imaging of ICAM-1 for the detection of acute inflammation in the lung. Methods: PVPh NPs were prepared by a solvent diffusion formulation, resulting in nano-scale spherical particles suspended in water. NPs were directly labeled with either [125I]NaI or [124I]NaI and then coated with the appropriate antibody for endothelial targeting. Following isolation from free antibody, in vivo targeting was studied in naïve or LPS-challenged C57BL/6 mice. Results: Prepared PVPh NPs exhibited a tight size range and optimal radiostability with both 125I and 124I isotopes. Endothelial targeting of 125I-NPs was demonstrated using three distinct molecular determinants (PECAM, ICAM, and Thrombomodulin), all exhibiting lung accumulation of at least 2-fold greater lung accumulation than paternal antibody. From these molecular targets, ICAM was investigated for its ability to be a predictive signature of inflammation. Mice were administered LPS and targeted for ICAM 24 hours after challenge. Lung accumulation of ICAM-targeted NPs demonstrated a ~ 2-fold increase in LPS-challenged mice relative to naive. These findings were then translated to a PET (124I-NPs) imaging platform and investigated for its capacity to detect acute lung inflammation. ICAM-targeted NPs were able to detect lung inflammation comparable to the clinical standard, 18F-FDG, and correlated with the radiotracing done with 125I-NPs. Conclusion: PVPh NPs represent an attractive tool for the diagnosis of acute lung inflammation using ICAM-1 as a molecular target. PET imaging of ICAM-targeted NPs demonstrated comparable detection of lung inflammation relative to the clinical standard, 18F-FDG. Further, NPs can be tracked longitudinally due to the half-life of 124I and this could potentially be used for the optimization of drug targeting in acute lung inflammation.

21 Pulmonary endothelial protein kinase C-delta (PKC-´ ) regulates neutrophil migration in acute lung inflammation. Mark J. Mondrinos1,2, Paul Kennedy1,2, Shuang Sun1,2 and Laurie Kilpatrick1,2 1 - Center for Inflammation, Translational and Clinical Lung Research. 2 - Dept. of Physiology Temple University School of Medicine, Philadelphia, Pennsylvania, USA Rationale: Excessive neutrophil migration into the lung and release of oxidants and proteases are key elements in the pathogenesis of acute lung injury. Previously, we identified Protein Kinase C-delta (PKC´ ) as an important regulator of proinflammatory signaling in human neutrophils and demonstrated that PKC´ inhibition was lung protective in a rat model of sepsis. Key to neutrophil-mediated lung damage is neutrophil migration across the pulmonary endothelium. We hypothesized that attenuation of pulmonary endothelial adhesion molecule expression by PKC´ inhibition could be a mechanism of lung protection through the reduction of neutrophil influx. Methods: Polymicrobial sepsis and indirect lung injury were induced in adult rats by cecal ligation and puncture (CLP). Following CLP, a TAT-conjugated PKC´ inhibitory peptide was delivered via intratracheal instillation. Controls included sham operations and instillation of vehicle (PBS) only following CLP. The numbers of myeloperoxidase (MPO)-expressing cells in lung tissue sections and endothelial expression of ICAM-1/VCAM-1 were assessed by IHC. To further delineate the role of endothelial PKC´ in regulating neutrophil migration in a human cell-based culture model, monolayers of IL-1² -treated human pulmonary microvascular endothelial cells (PMVEC) seeded on transwells were subjected to genetic and pharmacological PKC´ inhibition in order to assess the effect on endothelial expression of proinflammatory mediators and neutrophil transmigration. Results: In support of our hypothesis, intra-tracheal administration of a PKC´ inhibitor in our septic rat model significantly decreased neutrophil influx into the lung with concomitant attenuation of sepsis-induced ICAM-1 and VCAM-1 expression. In line with these in vivo findings, inhibition of PMVEC PKC´ decreased IL-1² -mediated neutrophil transmigration in vitro. PKC´ was essential for IL-1² -mediated neutrophil adherence and NFº B-dependent expression of ICAM-1 and VCAM-1. IL-1² -mediated ROS production and activation of the redox-sensitive NFº B was PKC´ -dependent suggesting an upstream signaling role. Conclusions: PKC´ has an important role in regulating neutrophil-endothelial cell interactions and recruitment to the lung. Inhibition of PKC´ in the setting of acute inflammation attenuates endothelial adhesion molecule expression and neutrophil transmigration both in vivo in rodents and in vitro in a human cell-based system. Taken together these preclinical findings suggest PKC´ has a conserved role in lung inflammation and is therefore a promising therapeutic target.

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22 Dietary flaxseed modulates the miRNA profile in irradiated and non-irradiated murine lungs-A novel mechanism of tissue radioprotection by flaxseed. *Melpo Christofidou-Solomidou,*Ralph Pietrofesa, *Evguenia Arguiri, †Keith A. Cengel and +Kenneth W. Witwer. *Depts. of Med., Pulm. Allergy & Critical Care Div., † Radiation Oncology, Univ. of Pennsylvania Med. Ctr., Phila., PA and + Dept. of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD. Introduction: As a dietary supplement, wholegrain flaxseed (FS), displays antioxidant and anti-inflammatory properties. We have previously shown that dietary FS protects against radiation pneumonopathy in a rodent model of thoracic X-ray radiation therapy (XRT). Specifically, FS enhanced survival and prevented adverse radiation effects including pulmonary fibrosis, inflammation and oxidative lung damage when administered prior to radiation exposure and for the duration of the study (4 months). Rationale: The mechanisms whereby dietary FS exerts radioprotective effects in lung are incompletely understood. MicroRNAs (miRNAs) are short oligonucleotides that act as important post-transcriptional regulators of inflammatory response networks and are also thought to participate in cell-to-cell communication. Responses of miRNA profiles to diet and to radiation exposure have been reported, but the potential contribution of miRNAs to diet-related radioprotection has never been tested. Methods: Mice were fed 10% FS or a 0% FS isocaloric control diet for 3 weeks, a time needed for bioactive FS lignan metabolites to reach a steady level in the systemic circulation and exposed or not to a single-dose 13.5 Gy thoracic XRT. Mice were sacrificed 48 hours post XRT, RNA was extracted from lung tissue of three animals for each of the four experimental groups (+/- FS, +/- XRT), and small RNAs were profiled by OpenArray. Differential regulation of small RNAs was assessed by two-way ANOVA. Several individual two-group comparisons were also made. Results: FS resulted in statistically significant expression differences for multiple miRNAs, including 7 with p<0.001. miR-150 was downregulated approximately 2.9-fold in the FS groups and is disproportionately integrated into immune response-related networks. Although few miRNAs were significantly changed by radiation exposure, interaction between diet and radiation was observed, e.g. for miR-29c, which was greatly downregulated in the FS control group (10 to 50-fold) but slightly upregulated in the FS radiation group. Compared with FS control, the FS/XRT group experienced a 50% decrease of the p53-responsive miR-34a, which regulates senescence- and apoptosis-related factors. Conclusions: The induction by dietary FS of significant changes in lung miRNA profile suggests that i) modulation of small RNA by dietary supplements may represent a novel strategy to prevent the adverse effects of thoracic radiotherapy and ii) that this may be one of the mechanisms by which FS exerts its known radioprotective effects in lung. Our model provides a useful system to further explore and optimize such small RNA-based therapies. Funded in part by: NIH-R01 CA133470, NIH-RC1AI081251, NIH-P30 CA016520 (MCS).

23 MUC1 prevents virus-derived double-stranded RNA-induced epithelial cell inflammation and apoptosis by inhibiting recruitment of TRIF to TLR3. Kosuke Kato and K. Chul Kim. Department of Physiology and Center for Inflammation, Translational and Clinical Lung Research, Temple University School of Medicine, Philadelphia, PA

Background: Respiratory influenza virus (RIV) infection is a major global pandemic health problem. It is not clear how this virus is able to survive after an anti-virus response has been mounted. However, one explanation is that RIV is able to proliferate following infection of respiratory epithelial cells (REC) by suppressing the host inflammatory response, and allowing the infected epithelial cells to survive (i.e., to escape apoptosis). Since it has been shown that viral-derived double-stranded (ds)RNA-induced apoptosis is mediated through its interaction with TLR3 and that MUC1 mucin (a cell surface membrane-tethered glycoprotein) suppresses TLR signaling (Ueno et al. AJRCMB 38:263, 2008), we hypothesized that MUC1 expressed on the surface of REC may play an important role during the RIV infection. Objective: To determine whether manipulation of MUC1 levels can modify dsRNA-induced inflammatory responses, and apoptosis, in REC. Methods: REC with varying degrees of MUC1 expression were treated with poly(I:C), an experimental agonist of TLR3, and TLR3 signaling (both inflammatory response and apoptosis) were monitored by immunological, biochemical and molecular techniques. Results: (1) Poly(I:C)-induced apoptosis (measured by both MTT assay and activation of pro-apoptotic caspase-8) was suppressed by expression of MUC1, whereas it was enhanced by silencing MUC1 expression; (2) The poly(I:C)-induced inflammatory response (measured by IFN-² expression) was also associated with MUC1 levels in the same way; (3) Overexpression of MUC1 inhibited the recruitment of TRIF to TLR3, the first essential step for TLR3 signaling. Conclusion: MUC1 mucin expressing in REC attenuates poly(I:C)-induced inflammatory responses and preventing apoptosis through suppression of TLR3. This is the first report suggesting that MUC1 mucin may be involved in REC inflammatory responses and viability during RIV infection, and might provide an important strategy for controlling respiratory viral infection. (Supported by NIH RO1 HL-47125 and HL-81825)

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24 Macrophage cytokine and UPR responses to ER stress are dependent on M1/M2 polarization P.A. Kennedy, D.J. King, T. Zhang, M. O. Aksoy, S.G. Kelsen and L.E. Kilpatrick. Center for Inflammation, Translational and Clinical Lung Research, Temple University, Philadelphia, PA Rationale: Macrophages are central mediators of the inflammatory response and play a pivotal role in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Macrophages are heterogeneous cells expressing distinct phenotypes which can be classified as M1 (pro-inflammatory) or M2 (immunomodulatory). Macrophages exhibit plasticity and the pulmonary composition of M1 and M2 phenotypes can shift in COPD. Oxidant stress is increased in COPD, resulting in protein misfolding and induction of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) restores ER homeostasis by activating pathways regulating protein folding, degradation and translation. However, when ER stress is severe or prolonged, UPR signaling pathways induce apoptosis. While there is considerable crosstalk between the UPR and inflammatory signaling pathways, it is not known whether cell phenotype alters macrophage responsiveness to ER stress-mediated UPR and inflammatory signaling. Methods: Human blood monocytes were cultured and differentiated to monocyte-derived macrophages (MDMs). MDMs were biased toward M1 or M2 phenotypes by addition of IFN³ /LPS or IL-4/IL-13, respectively. ER stress was induced by thapsigargin (TG) and tunicamycin (TN). Inflammatory gene expression was characterized using PCR Arrays and at the protein level by ELISA. UPR effectors, phos-eIF2±/eIF2±, ATF4, GADD34, CHOP, sXBP1, and ATF6 were assessed by qPCR-array, western blotting, or ICC. Apoptosis was determined by YO-PRO1/PI staining. Results: Treatment with IFN³ /LPS or IL-4/IL-13 promoted expression of specific cell surface phenotype markers of M1 (CCR7) and M2 (CD206) polarization. ER stress induced a robust up-regulation of 33 pro-inflammatory genes (>4-fold) in M1 macrophages compared to M2 macrophages including IL-8, TNF, IL-6, CXCL9 and CCL8. This response was confirmed by RT-PCR and ELISA. In M1 but not M2 macrophages, ER stress activated the inflammasome and increased release of IL-1² . In contrast, ER stress produced a more robust UPR response in M2 macrophages as evidenced by a larger and more sustained activation of all three branches of the UPR. Moreover, the blunted UPR responses in M1 macrophages were associated with increased cell death. Conclusions: In M1 macrophages, ER stress augments cytokine/chemokine expression and apoptotic cell death. In contrast, the UPR is differentially regulated in M2 macrophages and is not associated with cell death. Thus, the phenotypic modulation of inflammatory and UPR networks may contribute to the survival of M2 macrophages and may account for the increased presence of M2 macrophages in chronic lung inflammation such as COPD.

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LUNG INJURY AND

REMODELING

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25 Blood type A is associated with an increased risk of the Acute Respiratory Distress Syndrome among Caucasian patients with severe sepsis. John P. Reilly MD, Michael G.S. Shashaty MD, MS, Maximilian Herlim, Nathaniel L. Oz BS, Zachery Garcia BA, Isabel Hiciano BA, Ana Campbell MD, Jason D. Christie MD, MS, Nuala J. Meyer MD, MS Rationale: The ABO gene encodes a glycosyltransferase that catalyzes antigen modifications, characterizing the ABO blood group. ABO variants have been associated with risk of infections, vascular events, and circulating inflammatory and endothelial injury markers, including von Willebrand factor and soluble intracellular adhesion molecule 1. In a trauma cohort, we previously identified an association between blood type A and 1) increased ARDS risk among Caucasians and 2) increased mortality among African Americans with ARDS. We hypothesize that ABO blood types are similarly associated with ARDS risk and mortality in a population of patients with severe sepsis. Methods: We conducted a prospective cohort study of critically ill patients admitted to a single academic medical intensive care unit with severe sepsis using ACCP consensus criteria. Patients were phenotyped for ARDS daily for 5 days following presentation according to the Berlin Definition. ABO blood type was determined by RBC typing. Associations between ABO blood types and ARDS risk and mortality were tested using multivariable logistic regression. Caucasians and African Americans were analyzed separately given population differences in the ABO structure and divergent genetic backgrounds across racial groups. Results: Blood type was available in 973 of 1,062 (92%) septic patients. Of the 973 patients, 222 (23%) developed ARDS. Blood type A was associated with increased ARDS risk among Caucasians with 31% developing ARDS versus 21% in other blood groups (adjusted OR 1.82, 95% CI 1.20,2.76, p=0.005, Table 1). We were unable to demonstrate an increased ARDS risk of blood type A among African Americans (adjusted OR 1.30, 95% CI 0.71,2.40, p=0.400). Associations were robust to adjustment for age, severity of illness, sex, and pulmonary or extrapulmonary infection. Of 222 patients who developed ARDS, 142 (62%) died prior to hospital discharge. There was an increased risk of death among African Americans with ARDS and blood type A (adjusted OR 2.06, 95% CI 0.67, 6.33, p=0.207), however this result failed to achieve statistical significance due to low power. Conclusions: Blood type A is associated with an increased risk of ARDS among Caucasians and may be associated with an increased risk of death among African Americans who develop ARDS associated with sepsis. These results are consistent with our previous findings in trauma patients and further support a role for ABO glycans and glycosyltransferases in ARDS. Mechanistic investigations of ABO glycobiology and its role in modifying ARDS risk and outcomes are warranted, and may identify novel therapeutic targets.

26 Efficacy of MJ33, a novel nontoxic inhibitor of phospholipase A2 of peroxiredoxin 6, in lipopolysaccharide-induced acute lung injury. Intae Lee, Chandra Dodia, Shampa Chatterjee, Sheldon Feinstein, and Aron B. Fisher Institute for Environmental Medicine, Univ. of Pennsylvania, Philadelphia, PA We found in our previous studies (Lee et al., JPET 345:284-296, 2013) that MJ33 showed very high lung uptake (67-86%), high inhibition of peroxiredoxin 6 (Prdx6)-phospholipase A2 (PLA2) activity (>85%), and a high margin of safety in the intact mouse (toxic dose= >25µmol/kg; PLA2

inhibitory dose= ~0.02µmol/kg). It is well known that Prdx6 on PLA2 activity is required for activation of NOX2 and subsequent generation of ROS which results in lipopolysaccharide (LPS)-induced acute lung injury (ALI). MJ33 can negate LPS-induced oxidative stress by inhibiting Prdx6-PLA2 activity. LPS (0111:B4 from E. coli, 1-5mg/kg) was administered intratracheally (IT) followed by an IT injection of 4nmol MJ33 in unilamellar liposomes either concurrently or 2 h post LPS. LPS at all doses significantly increased lung infiltration, increased the number of inflammatory cells, lung permeability (protein levels, lung wet to dry ratio, leakage of FITC-dextran into the lung lavage fluid), lipid peroxidation (TBARS and 8-isoprostanes), protein oxidation (protein carbonylation), pro-inflammatory cytokins IL-6 and TNF-α levels, MIP-2 levels, and NF-kB levels. MJ33 dramatically reduced all of these measured parameters. These findings show that MJ33 can suppress the manifestation of oxidant stress associated with the activation of NOX2 as well as lung inflammation. Inhibiting Prdx6-PLA2 activity by MJ33 also limited excessive ROS production and cell injury that accompanies lung inflammation. We identified MJ33 as a potent and specific inhibitor of Prdx6-PLA2 activity on NOX2-dependent ROS generation and a potential protector against LPS-induced lung injury. (Supported by NHLBI-R01-HL105509, P.I.; Aron B. Fisher, MD)

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27 Stromal activation by Wnt signaling defines a cellular niche capable of clonal expansion after fibrotic lung injury. Tien Peng1, Ying Tian2, Zheng Cui2, Edward Cantu3, Jason Christie1, and Edward Morrisey2,4,5. 1Division of Pulmonary, Allergy, and Critical Care, 2Cardiovascular Institute, 3Division of Cardiovascular Surgery, 4Cell and Developmental Biology, 5Institute of Regenerative Medicine Idiopathic pulmonary fibrosis (IPF) is characterized pathologically by a heterogeneous proliferation of stromal fibroblasts that leads to progressive effacement of the normal alveolar architecture, resulting in respiratory failure and death. Although the abnormal proliferative features of IPF are well known, little is understood about the cellular origin of the fibrotic foci or the signals that promote fibroproliferative behavior. Here we show that stromal activation by canonical Wnt signaling is present in both human IPF lung samples and an animal model of pulmonary fibrosis, and that Wnt-activated stromal fibroblasts are capable of clonal proliferation into distinct foci within the injured lung parenchyma. Immunohistochemistry of explanted IPF lung samples obtained at lung transplantation demonstrates aberrant Axin2 (a reporter of Wnt activation) expression in the stroma. To identify and prospectively study the cellular compartments of the lung activated by Wnt signaling, we generated an Axin2CreERT2 mouse lines to lineage trace Wnt-activated (Axin2+) cells in the lung during homeostasis and fibrotic injury. Lineage tracing in the uninjured lung demonstrates Wnt-activation of PDGFRα+/β+ stromal fibroblasts within the alveolar interstitium with minimal proliferation and cellular turnover under homeostatic condition. After bleomycin administration to induce lung fibrosis, Wnt activation is upregulated as indicated by an increase in Axin2 expression within the stroma. Furthermore, lineage-traced Axin2+ (Wnt-activated) stromal fibroblasts proliferate during fibrotic injury within the damaged alveoli, with a subset capable of myofibroblastic differentiation indicated by the acquisition of αSMA expression. Multicolored clonal analysis with the R26RConfetti reporter demonstrates that Axin2+ fibroblasts clonally proliferate after injury and migrate to form spatially-proximate fibroblast clusters within the injured lung. Taken together, these studies identify a Wnt-activated cellular niche within the lung stroma that is capable of clonal expansion after fibrotic injury. Studies are currently underway to address the cell-autonomous role of Wnt signaling in regulating the proliferation, differentiation, and migration of lung stromal fibroblasts in response to fibrotic stimuli.

28 TSC1/TSC2 modulate E-cadherin trafficking through Rac1 GTPase. Elena A. Goncharova, Dmitry A. Goncharov, Irene S. Khavin, and Vera P. Krymskaya. Pulmonary, Allergy & Critical Care Division, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Loss of function mutations of the tuberous sclerosis complex 1 (TSC1) and TSC2 is associated with pulmonary lymphangioleiomyomatosis (LAM), a rare disease characterized by diffuse neoplastic proliferation of smooth muscle-like cells in the lung and formation of renal tumors angiomyolipomas. Accumulating evidence support metastatic nature of cell dissemination in LAM, but the mechanism is not completely understood. Recent study suggests the link between TSC2 and E-cadherin in regulating renal cell metastatic potential. We previously reported that LAM cell survival, migration and invasiveness depend on small GTPase Rac1, known regulator of E-cadherin-dependent cell-cell adhesion. The goal of this study was to determine molecular mechanism by which TSC1/TSC2 regulate E-cadherin localization. Here, we report that TSC1-null and TSC2-null cells from Eker rat renal carcinoma and from kidney lesions of TSC2+/- and TSC1+/- mice have reduced levels of membrane-bound E-cadherin, accumulation of E-cadherin in cytoplasmic vesicles, and increased activity of Rac1 GTPase. Re-expression of TSC1 restores membrane localization of E-cadherin in TSC1-null cells. siRNA-induced knock-down of TSC1 or TSC2 in normal epithelial cells modestly increases percentage of cells with aberrant cytoplasmic E-cadherin localization while combined TSC1/TSC2 depletion induces re-localization of E-cadherin into cytoplasmic vesicles in ~80% of cells. Using a panel of truncated TSC1 constructs, we found that TSC2-binding site of TSC1 is required to prevent E-cadherin cytoplasmic re-localization and activation of Rac1 in TSC1-null epithelial cells. siRNA-induced Rac1 knock-down restored membrane E-cadherin localization in TSC1- and TSC2-null cells while transfection with constitutively active Rac1 prevented E-cadherin membrane re-localization caused by TSC1 expression showing that TSC1/TSC2 loss promotes cytoplasmic E-cadherin re-localization through Rac1. Inhibitor-based analysis revealed that selective proteasome inhibitor MG-132 restores membrane E-cadherin localization in TSC2-null cells, increases fraction of Rab5-bound and reduces fraction of Rab-11-bound E-cadherin, and decreases Rab11 levels in TSC1- and TSC2-null cells. Further, MG-132 inhibited anchorage-independent growth of TSC1-null and TSC2-null cells at the levels comparable to mTORC1 inhibitor rapamycin. Taken together, our data suggest that TSC1/TSC2 loss in epithelial cells promotes Rac1-dependent deregulation of E-cadherin trafficking potentially via restricting its Rab11-dependent transport from early to recycling endosomes. That leads to cytoplasmic E-cadherin accumulation, reduction of membrane-bound E-cadherin levels, and increased metastatic potential of TSC1/TSC2-null epithelial cells. Funded by NIH/NHLBI RO1HL090829, RO1HL114085 and The LAM Foundation (LAM089E01-12) (V.P.K.).

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29 Folliculin and AMPK in epithelial cell survival: relevance to Birt-Hogg-Dubé Syndrome. Melane L. James1, Dmitry A. Goncharov1, Hua Li1, Elena N. Atochina-Vasserman1, Susan Guttentag2, Linda K. Gonzales2, Laura S. Schmidt3,4, W. Marston Linehan3, Masaya Baba3, Seung-Beom Hong1, Elena A. Goncharova1, Vera P. Krymskaya1. 1Department of Medicine, University of Pennsylvania, Philadelphia, PA; 2Division of Neonatology, Children’s Hospital of Philadelphia, Philadelphia, PA; 3Urologic Oncology Branch, National Cancer Institute, Bethesda, MD; 4BSP, SAIC-Frederick, Inc., Frederick National Lab for Cancer Research, Frederick, MD Mutational inactivation of the tumor suppressor gene folliculin (FLCN) is associated with Birt-Hogg-Dubé (BHD) syndrome. BHD manifests by cystic airspace enlargement, spontaneous pneumothorax, renal tumors, and cutaneous fibrofolliculomas. How the loss of FLCN induces lung cyst formation has not been established. Conditional inducible deletion of FLCN in SP-C-expressing cells of FLCNf/f:SP-C-Cre mice causes emphysematous-like changes in the lungs due to alveolar cell apoptosis. Epithelial cell survival depends on cell-cell contact integrity and nutrient availability. These processes are regulated by the energy sensor 5’-AMP-activated protein kinase (AMPK) by unknown mechanisms. FLCN acts within the nutrient and energy sensing signaling networks regulated by AMPK, but the role of FLCN in AMPK-dependent epithelial cell-cell contact integrity and survival has not been studied. Here, we show that targeted FLCN knock-down in distal lung epithelial cells induces epithelial cell apoptosis, promotes alveolar airspace enlargement, and impairs lung function in mice. In cell culture of primary ATII mouse lung epithelial cells from FLCNf/f mice Cre-recombinase-induced FLCN knock-down promoted disruption of cell-cell contacts and formation of gaps between cells as compared to control cells. Further, siRNA-induced depletion of FLCN in wild type epithelial cells induced changes in cell morphology due to disrupted cell-cell contacts, loss of the typical epithelial cell monolayer, altered membrane localization of E-cadherin, decreased viability, and increased apoptosis (11.22 ± 1.87% vs 3.78 ± 1.49% for cells transfected with control siRNA, p < .003). The siRNA-dependent FLCN depletion significantly reduced the protein levels of LKB1 (29 ± 9% vs 100 ± 16% for siRNA control, p < .001), a major regulator of AMPK. Expression of the constitutively active AMPK-CA or pharmacological activation (by AICAR) of AMPK rescued FLCN siRNA-induced disruption of the epithelial cell morphology and epithelial cell apoptosis. Furthermore, re-expression of FLCN in FLCN-null human epithelial cells increased expression of LKB1 in cytoplasm (1.00 to 1.40 fold change; p < .05) and membrane (1.00 to 1.86 fold change; p < .001) fractions as compared to controls. These data demonstrate that FLCN and AMPK cooperate in maintaining the epithelial cell-cell contact integrity, morphology and survival, and suggest that AMPK activation is a potential therapeutic target in preventing the emphysematous changes in the lung. Funded by: NIH/NHLBI RO1 HL110551

30 Modulation of Hsp70 expression by Elp2, a 4-phenylbutyrate inducible Elongator component, and STAT3 does not require STAT3 phosphorylation. Laurence Suaud1 , Katelyn Miller1, Ashley Panichelli1 Rachel Randell1 and Ronald C. Rubenstein1,2 . 1Division of Pulmonary Medicine, Children’s Hospital of Philadelphia, 2Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. In our efforts to understand how Sodium 4-Phenylbutyrate (4PBA) corrects trafficking of ∆F508-CFTR in Cystic Fibrosis (CF) epithelia, we recently demonstrated that a transient increase in Hsp70 expression in CF epithelial cells treated with 4PBA occurs through a process mediated by STAT3 (signal transducer and activator of transcription-3) and Elp2 (Elongator Protein 2, Suaud, et al, JBC, 2011). This transient increase in Hsp70 expression may promote correction of ∆F508-CFTR trafficking. Elp2 (also known as STAT3-interacting protein 1 or StIP-1) is a scaffold protein that is required for activation of STAT3, and is also a component of the Elongator complex that appears to regulate a number of cellular processes including RNA polymerase II activity. Previously, we demonstrated that 4PBA caused transient phosphorylation and translocation of STAT3 into the nucleus. Since Elp2 does not itself contain a nuclear localization signal (NLS), we hypothesized that STAT3 activation/phophosrylation and STAT3’s nuclear localization signal (NLS) is required to “bring” Elp2/Elongator into the nucleus. We tested this hypothesis using luciferase reporter assays where luciferase expression was controlled by the human Hsp70 promoter, and with overexpression STAT3 and mutant STAT3 constructs, including a constituvely active STAT3 (STAT-3C), STAT3-Y705F that inhibits STAT3 tyrosine phosphorylation and STAT3-S727A that blocks STAT3 serine phosphorylation. In control experiments in transfected IB3-1 CF bronchiolar epithelial cells, 4PBA increased Hsp70 promoter driven luciferase expression 1.5-2 fold, while siRNA mediated depletion of Elp2 decreased this luciferase expression by 50%. Overexpression of WT STAT3 or STAT3C also increased luciferase expression, but did not overcome the negative effect of Elp2 depletion on Hsp70 promoter function. Interestingly, transfection of STAT3-S727A also increased luciferase expression to a similar extent as WT STAT3, and transfection of STAT3-Y705F increased luciferase reporter expression to a 2-fold greater extent than WT or STAT3-S727A. Again, these effects were diminished by siRNA-mediated depletion of Elp2. In control experiments, all of these mutants STAT3’s interacted with Elp2 in co-immunoprecipitation experiments. These data suggest that the phosphorylation state of STAT3 is not relevant to the STAT3/Elp2 regulation of Hsp70 expression in response to 4PBA. Supported by grant R01 DK-58046.

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31 ERP29 regulates ENAC functional expression by promoting channel cleavage. Yael Grumbach1, Laurence Suaud1 and Ronald C. Rubenstein1,2. 1Division of Pulmonary Medicine and Cystic Fibrosis Center, Children’s Hospital of Philadelphia, 2Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. The Epithelial Na+ Channel (ENaC) is a key regulator of airway surface liquid volume. In the Cystic Fibrosis (CF) airway, relative hyperactivity of ENaC is hypothesized to cause depletion of the periciliary liquid, which impairs mucociliary clearance and portends bacterial colonization of the airway. In our group’s studies of the correction of ∆F508-CFTR trafficking by 4-phenylbutyrate (4PBA), we demonstrated that ERp29, a novel 29 kDa molecular chaperone of the lumen of the endoplasmic reticulum (ER), had increased abundance in CF epithelial cells after treatment with 4PBA, and that ERp29 promoted trafficking of both WT and ∆F508 CFTR. ERp29 is homologous to the thioredoxins, which are enzymes that catalyze the formation and rearrangement of disulfide bridges. However, ERp29 has only a single cysteine residue (C157) and lacks the C-X-X-C motif that is characteristic of the thioredoxins. We tested the hypotheses that ERp29 regulates ENaC trafficking and functional expression, and that its single cysteine, C157, would have a critical role in this regulation. To test this hypothesis in epithelial cells, we selected ENaC-overexpressing MDCK cell lines with doxycycline (Dox)-inducible expression of either WT ERp29 or a mutant ERp29 where C157 was changed to serine (C157S ERp29). In Ussing chamber, Dox-induced overexpression of WT ERp29 increased amiloride-sensitive Isc, and the abundance of the cleaved form of ENaC’s γ-subunit. In contrast, overexpression of C157S ERp29 had the opposite effects. We also tested the influence of ERp29 on the fraction of cleaved, higher Po versus uncleaved, lower Po ENaC at the apical surface by examining the influence of trypsin on amiloride-sensitive Isc in Ussing chambers. Cells overexpressing WT ERp29 had a smaller fractional increase in amiloride-sensitive Isc in response to apical application of trypsin compared to the control cells, suggesting that ERp29 promoted ENaC cleavage. In contrast, cells overexpressing C157S ERp29 had a significantly greater fractional increase in amiloride-sensitive Isc in response to trypsin, suggesting that C157S ERp29 inhibited ENaC cleavage. The effect of siRNA-mediated depletion of ERp29 expression was similar to that of C157S ERp29; cells treated with ERp29-directed siRNA had decreased amiloride-sensitive Isc and a greater fractional increase in amiloride-sensitive Isc in response to trypsin compared to controls. These data support the hypothesis that ERp29 regulates ENaC functional expression by facilitating ENaC subunit cleavage, and that ERp29’s single cysteine residue is critical for this effect.

32 Detecting cell adhesion molecules in intact lung using Quantum Dot conjugates targeted to endothelial cells. Rebecca L. Orndorff1, Nankang Hong1, Blaine J. Zern2, Kevin Yu1, Kristine Debolt1, Shaohui Huang1, Aron B. Fisher1, Vladimir R. Muzykantov1,2, 3, Shampa Chatterjee1. 1Institute for Environmental Medicine, 2Center for Targeted Therapeutics & Translational Nanomedicine and 3Department of Pharmacology, University of Pennsylvania Perelman School of Medicine, Phila., PA Quantum dots (QD) coupled with fluorescence imaging techniques are a powerful tool for detecting proteins in vivo. Here we used dual QD labeling to detect cellular adhesion molecules, CAM (endothelial marker, PECAM and vascular cell adhesion molecule, VCAM) expression in a LPS model of lung inflammation. QDs were functionalized via an adaptor protein and linked to anti-PECAM and anti-VCAM Abs to create QD655-PECAM (QDP) and QD525-VCAM (QDV), respectively. We established probe specificity in vitro in a PECAM expressing cell line and in LPS treated endothelial cells (for VCAM). For in situ experiments, mice lungs were removed at 24h after LPS instillation, and perfused with medium containing QDV and QDP. For in vivo lung studies, QDs were injected into the jugular vein at 24h after LPS treatment and lungs were removed and processed after overnight fixation. Imaged sections from both studies showed that LPS treated mice had 8-10 fold higher QDV signal, compared to naïve lungs, which significantly colocalized with QDP, indicating increased VCAM expression in pulmonary endothelium. Thus, QD labeling for intravital microscopy will be useful for detection and localization of proteins in pulmonary tissue.

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rhCC10 modulates lipopolysaccharide injury in alveolar like cell culture. T.L. Hubert, K. Mallilankaraman, M. Marcinkiewicz, J. Wu, T.H. Shaffer, M. Muniswamy, M.R. Wolfson Temple University School of Medicine - Philadelphia, PA/US

Rationale: CC10, is a pleiotrophic protein produced by airway clara cells, secreted into the extracellular fluids, and known to have anti-inflammatory affects by inhibiting sPLA2 and fibronectin. In preclinical and clinical studies, CC10 has also been used effectively to decrease NF-kB mediated inflammation in the immature injured lung. Toll-Like Receptors (TLRs), as danger associated molecular pattern receptors, have been determined to be integral to lung inflammation. However, the effect of rhCC10 in response to TLR specific mediated injury is not known. Objective: Assess TLR activity and expression in response to rhCC10 treatment of a TLR agonist induced injury. Design: Alveolar-like A549 cells were maintained in multiple six well culture dishes with 5% CO2 balance air at 37 oC and assigned to one of four groups (n=6/group): normal growth media; growth media + rhCC10 (1 mg); growth media + LPS (10 ug/mL); or growth media + LPS and rhCC10. After 24 hours, cells were trypsinized, counted, and homogenized. RNA was extracted from the cell lysate, cDNA synthesis was performed, and qPCR was used to measure TLR 3, TLR 4, and IFN-³ mRNA expression as normalized by 18S mRNA expression. Conclusions: These data demonstrate that rhCC10 treatment decreased both TLR expression and activity in response to LPS treatment to be no different from control A549 cells in normal growth media alone. Additionally, rhCC10 itself did not increase TLR 3 and TLR 4 mRNA expression as we have shown in the preterm lung. Further analysis of TLR protein and activity by western blot is ongoing.

34 The effect of murine and primate erythrocytes bound fusion proteins when exposed to osmotic stress in pro-inflammatory and pro-thrombotic diseases. Daniel Pan, Sergei Zaitsev, Vladimir Muzykantov. The pulmonary vasculature is a major target of inflammation and thrombosis in sepsis and acute lung injury (ALI). At the site of inflammation, a complex series of interactions triggered by damage to endothelium induces intravascular blood coagulation. Studies have revealed that thrombomodulin (TM) is expressed on the surface of endothelial cells. This is attractive in the context of the pulmonary disorders such as sepsis and ALI where the expression of endothelium TM is severely down-regulated, causing an impaired activation of protein C, which further aggravates inflammation and thrombosis. Attaching protein drugs onto the surface of erythrocytes for target delivery to the pulmonary endothelium for inflammation and thrombosis treatment by genetically engineered antibody fragments such as a single chain Fv (scFv) has shown to be effective. It has been demonstrated that attaching protein drugs, TM, to murine erythrocytes via scFv circulates much longer, inhibits activation of platelets and coagulation, and alleviates acute inflammation by lowering TNF-±, MIP-2, ALT, and BALF more effectively than soluble thrombomodulin (sTM) alone, since sTM and activated protein C (APC) are rapidly eliminated from blood circulation. However, its functionality, biocompability, stability, activity on when exposed to osmotic stress as well as on primate erythrocytes have not been studied. Here we report the murine erythrocyte exhibited greater osmotic sensitivity compared to their human counterparts. Murine erythrocytes start to lyse at approximately 73 mM NaCl and exhibited 100% hemolysis at 55mM NaCl; human erythrocytes displayed slight hemolysis at 64 mM NaCl and exhibited complete hemolysis at 45 mM NaCl. There was also a significant difference in amount of hemolysis between 6-8 week old mice (40-50%) vs. 16 week old mice (80%) at 64 mM NaCl, suggesting the old erythrocytes are more sensitive to osmotic stress than younger erythrocytes. In addition, SAGM additive solution provided optimum human erythrocyte viability for more than 14 days of storage; however, it had very little effect on murine erythrocyte viability after 4 days of storage.

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35 Regulation of miRNA-302-367 in lung epithelial progenitor cells during development and injury repair. Ying Tian1, Jun Kong1, Minmin Lu1, Edward E. Morrisey1, 2, 3. 1Department of Medicine, 2Department of Cell and Developmental Biology, 3Cardiovascular Institute and Institute of Regenerative Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 While the importance of lung epithelial progenitor cells has been studied during lung development and injury repair, little is known about the mechanisms controlling their behaviors including proliferation and differentiation during development and the progression of airway injury repair. MicroRNAs (miRNAs) regulate messenger RNA (mRNA) translation and stability at the post-transcriptional level. They are known to be key modulators of both organ development and remodeling in response to injury. Our previous work has demonstrated that miR-302-367 is a direct target of Gata6 (a transcription factor essential for proper lung development and airway regeneration) and regulates the balance of lung epithelial progenitor cell proliferation and differentiation as well as cell apical-basal polarity. To further characterize the role of miR-302-367 in both embryonic and postnatal lung, we have generated ROSA-miR-302-367 and floxed miR-302-367 mouse lines to conditionally regulate miR-302-367 expression. To specifically and efficiently overexpress miR-302-367 in early lung epithelium, SHH-Cre line has been crossed into ROSA-miR-302-367 line. Our preliminary data show that ROSA-miR-302-367: SHH-Cre mutant recapitulated the phenotypes observed in our previous Sftpc:miR-302-367 transgenics. These include ROSA-miR-302-367: SHH-Cre mutant exhibit severe sacculation defect with thickened intersaculative interstitium, increased expression of Sox2 and Sox9 epithelial progenitors as well as neuro-endocrine cells (Pgp9.5) in the developing epithelium. In contrast, the expression of CC10 and SP-C, which mark proximal and distal epithelial cells, were greatly reduced. These preliminary studies suggest that the overexpression of miR-302-367 in early lung endoderm disrupted lung epithelium development. Moreover, our studies reveal that expression of miR-302-367 is markedly increased in naphthalene-injured lungs, suggesting a potential role for miR-302-367 in mediating lung epithelial gene transcription and the injury response during airway repair and regeneration. We have begun to assess the endogenous targets of miR-302-367 in mouse embryonic stem cells using Argonaute high-throughput sequencing of RNAs isolated by crossing immunoprecipitation analysis. Several members of the Hippo pathway were identified as the targets of miR-302-367. We are currently validating these targets and determining how they interact with miR-302-367 during lung development and injury repair.

36 Clinical risk factors for primary graft dysfunction after lung transplantation. Joshua M. Diamond, James C. Lee, Steven M. Kawut, Rupal J. Shah, A. Russell Localio, Scarlett Bellamy, David J. Lederer, Edward Cantu, Benjamin A. Kohl, Vibha N. Lama, Sangeeta Bhorade, Maria Crespo, Ejigayehu Demissie, Joshua Sonett, Keith Wille, Jonathan Orens, Ashish S. Shah; Ann Weinacker, Selim Arcasoy, Pali D. Shah, David S. Wilkes, Lorraine B. Ware, Scott M. Palmer and Jason D. Christie Rationale: Primary graft dysfunction (PGD) is the main cause of early morbidity and mortality after lung transplantation. Previous studies have yielded conflicting results for PGD risk factors. Objective: We sought to identify donor, recipient, and peri-operative risk factors for PGD. Methods: We performed a 10 center prospective cohort study enrolled between March 2002 and December 2010 (the Lung Transplant Outcomes Group). The primary outcome was ISHLT grade 3 PGD at 48 or 72 hours post transplant. The association of potential risk factors with PGD was analyzed using multivariable conditional logistic regression. Measurements and Main Results: 1255 patients from 10 centers were enrolled, 211 subjects (16.8%) developed grade 3 PGD. In multivariable models, independent risk factors for PGD were any history of donor smoking (OR=1.8, 95%CI 1.2, 2.6, p=0.002), FiO2 during allograft reperfusion (OR=1.1 per 10% increase in FiO2, 95%CI 1.0, 1.2; p=0.01), single lung transplant (OR=2.0, 95%CI 1.2, 3.3; p=0.008), use of cardiopulmonary bypass (OR=3.4, 95%CI 2.2, 5.3; p<0.001), overweight (OR=1.8, 95%CI 1.2, 2.7; p=0.01) and obese (OR=2.3, 95%CI 1.3, 3.9; p=0.004) recipient BMI, pre-operative sarcoidosis (OR=2.5, 95%CI 1.1, 5.6; p=0.03) or pulmonary arterial hypertension (OR=3.5, 95%CI 1.6, 7.7; p=0.002), and mean pulmonary artery pressure (OR=1.3 per 10mmHg increase, 95%CI 1.1, 1.5; p<0.001). PGD was significantly associated with 90 day (relative risk (RR)=4.8, absolute risk increase (ARI)=18%, p<0.001) and 1 year (RR=3.0, ARI=23%, p<0.001) mortality. Interpretation: We identified grade 3 PGD risk factors, several of which are potentially modifiable and should be prioritized for future research aimed at preventative strategies.

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37 Use of a simple prediction model improves pre-transplant risk stratification for primary graft dysfunction after lung transplantation. RJ Shah MD, MS*1,2, JM Diamond MD, MS*1,2, E Cantu MD*3, J Flesch1,2, JC Lee MD1, DJ Lederer MD, MS3, VN Lama MD4, J Orens MD5, A Weinacker MD6, DS Wilkes MD7, S Bhorade MD8, KM Wille MD9, LB Ware MD10, SM Palmer MD11, M Crespo MD12, EJ Demissie MSN2, SM Kawut MD, MS,1,2, SL Bellamy ScD2, AR Localio PhD2, JD Christie MD, MS1,2 , 1Pulmonary, Allergy, and Critical Care Division, 2Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, PA,3 Division of Cardiovascular Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA, 3Division of Pulmonary, Allergy, and Critical Care Medicine, Columbia University College of Physicians and Surgeons, New York, New York, 4Division of Pulmonary, Allergy, and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan, 5Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Johns Hopkins University Hospital, Baltimore, Maryland, 6Department of Pulmonary and Critical Care, Stanford University, Palo Alto, CA, 7Division of Pulmonary, Allergy, and Critical Care Medicine, Indiana University School of Medicine, Indianapolis, IN, 8Division of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, 9Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, Alabama, 10Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, 11Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University, Raleigh-Durham, North Carolina, 12Division of Pulmonary, Allergy, and Critical Care, University of Pittsburgh, Pittsburgh, Pennsylvania Background: Primary graft dysfunction (PGD), a form of acute lung injury occurring in 10-30% of lung transplant recipients, is a major cause of early mortality after transplant. We aimed to develop a prediction model for PGD to improve pre-transplant risk stratification and enhance donor-recipient matching. Methods: We performed a prospective cohort study of lung transplant recipients from 10 centers in the Lung Transplant Outcomes Group enrolled between March 2002 and December 2010. Our primary outcome was grade 3 PGD on day 2 or 3 post-transplantation. We evaluated 15 pre-transplant recipient and donor variables for inclusion in the prediction model. We used logistic regression to identify factors that were significant in a multivariable model conditioned on center. We determined predicted risk of PGD based on significant factors using logistic regression. PGD grade was 100% complete and multiple imputation was used to account for missing data in other covariates. Results: Of the 1255 subjects enrolled, 211 (17%) subjects had grade 3 PGD on day 2 or 3 post-transplantation. Significant risk factors for PGD included recipient BMI, pre-transplant diagnosis, presence of pulmonary hypertension, and any history of donor smoking. The combination of a normal BMI (18.5-25 kg/m2), chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF) as a pre-transplant diagnosis, and lack of significant pulmonary hypertension (mPAP< 40mmHg) defined a low-risk recipient. The low-risk group (24% of the cohort) had a predicted PGD risk of 6% (95% CI: 2%, 10%). The high-risk group, defined as having an abnormal BMI, any diagnosis other than CF or COPD, and pulmonary hypertension, had a predicted PGD risk of 16% (95% CI: 13%, 19%). The use of a lung from a donor with a smoking history in a high-risk recipient resulted in an absolute risk increase of approximately 10%, generating a PGD risk 26% (95% CI: 21%, 31%), while there was not a significant increase in PGD risk in the low-risk group (9%, 95% CI: 4%,15%). Conclusions: We have identified a combination of recipient pre-transplant factors that increase risk for PGD. In high-risk but not low-risk recipients, recipient of a lung from a donor with a smoking history substantially increases PGD risk. If validated, these findings may be useful in pre-transplant risk stratification to improve donor recipient matching.

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AIRWAY SMOOTH MUSCLE

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38 Formaldehyde-induced airway hyperresponsiveness: Role of Ca2+ dynamics in human airway smooth muscle (HASM) cells. Jude JA, Koziol-White CJ, Scala J, Jester WF, 1Maute C, 1Dalton PH & Panettieri RA. Airway Biology Initiative, University of Pennsylvania & 1Monell Institute of Chemical Senses, Philadelphia, PA. Background: Asthma is a chronic airway disorder clinically characterized by airway spasms, resulting from hyper-responsive airway smooth muscle (ASM). Altered Ca2+ handling mechanisms, including Ca2+-sensitization, can contribute to hyper-responsive ASM cell. Formaldehyde (FA) is an important indoor air pollutant with known roles in asthma exacerbation and airway hyperresponsiveness (AHR). The mechanisms involved in FA-induced AHR are not clearly understood. Hypothesis: We hypothesized that FA induces AHR through enhancing the contractile response of ASM. Methods: Precision-cut human lung slices (PCLS) or human ASM (HASM) cells obtained from healthy donors were exposed to vehicle (saline) or FA (0.2, 0.8 or 2 ppm) for 1 h and incubated for 24 h in fresh medium. In PCLS, the contractile response to carbamylcholine (Cch), ciliary beat frequency (CBF) and secretion of IL-6 and IL-8 were determined. In HASM cells, agonist-induced cellular Ca2+ response ([Ca2+]i), IL-6 secretion and Rho-associated Kinase (ROCK) activities were determined. Results: Exposure to 2.0 ppm FA enhanced Cch-induced contractile response in PCLS without significant effects on IL-6/IL-8 secretion. The ciliary beat frequency was reduced in FA-treated PCLS. In HASM cells, agonist-induced [Ca2+]i and IL-6 secretion were not significantly altered by exposure to FA. In HASM cells exposed to 0.2 ppm FA, ROCK activity was elevated compared to that of vehicle-treated cells. Conclusions: FA enhances Cch-induced airway contractility in PCLS. Ca2+ sensitization through Rho-associated kinase and elevated ASM cell shortening may be the potential mechanisms of FA-enhanced airway contractility. Acknowledgment: This work is funded through NIH T32 training grant: T32-ES019851-01A1.

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FOXP1/P4 knockout increases reactivity of murine airways, upregulates Neuropeptide Y (NPY), and stimulation with NPY increases reactivity of human and murine airways to methacholine. C.J. Koziol-White, S. Li, M. Jiang, J. Scala, E.E. Morrisey, and R.A. Panettieri. University of Pennsylvania, Philadelphia, PA RATIONALE: FOXP1 and FOXP4, transcription factors that modulate inflammatory responses in diseases such as asthma, may play a role in regulating epithelial cell lineage determination and in epithelial regeneration in the lungs. We hypothesized that deficiency of FOXP1/P4 may also modulate airway responsiveness to contractile stimuli. METHODS: Lung resistance was measured in FOXP1/P4 knockout (P1/P4 KO) mice and wild type mice. mRNA was then isolated from both the large and small airways of the P1/P4 KO and transcriptomics were examined. Interestingly, Neuropeptide Y (NPY) mRNA levels markedly increased in the KO mice. Accordingly, naïve Balb/c mice lung slices were treated overnight with NPY and examined for carbachol-induced bronchoconstriction. Additionally, human precison cut lung slices (PCLS) from normal healthy donors also were stimulated with NPY and agonist-induced bronchoconstriction assessed. RESULTS: Compared to wild type mice, FOXP1/P4 mice exhibit increased airway hyperreactivity to methacholine. Microarray analysis of the P1/P4 KO lungs showed a 40 fold increase in transcripts for NPY. In both naïve Balb/c mouse and human PCLS, NPY increased the sensitivity of the airways to carbachol-induced bronchoconstriction. CONCLUSIONS: These data suggest that knockout of FOXP1/P4 modulates not only cell lineage fate, but may also promote airway hyper-responsiveness. Given these data, further study defining the molecular mechanisms by which knockout of these transcription factors alters responsiveness of human airways may offer novel therapeutic targets for airways diseases.

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40 Differential effects of IL-13 on serine site-specific glucocorticoid receptor (GR) phosphorylation in human airway smooth muscle (HASM) cells. B. L. Diener, A. Hu, M. B. Josephson, and M. M. Grunstein. The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. RATIONALE: We previously reported that HASM cells exposed to the Th2 cytokine, IL-13, exhibit upregulated GR signaling due to ligand-independent phosphorylation of the GR at its functionally relevant serine (Ser) residues, GR-Ser203, -Ser211, and -Ser226, a phenomenon that confers heightened glucocorticoid (GC) ligand-stimulated GR transcriptional activity. This study aimed to identify the regulatory role of each of these serine sites by examining the effects of disruption of their phosphorylation on IL-13-induced release of the pro-asthmatic chemokines, eotaxin-3, from HASM cells, both in the absence and presence of GC ligand. METHODS: Confluent cultures of HASM cells were treated for varying durations with IL-13 (50 ng/ml) alone and in combination dexamethasone (DEX; 1µM). Both IL-13-induced ligand-independent and DEX-stimulated GR activation and signaling were then examined by: 1) immunoblot analysis of the temporal patterns of phosphorylation of the GR at its Ser211, Ser203, and Ser226 sites; and 2) assessing whether cells transfected with either human wild-type (WT) GR or site-specific serine-substituted with alanine (A) phosphorylation-deficient mutant GR plasmids exhibit altered eotaxin-3 release at 24 hr following IL-13 administration. RESULTS: The data demonstrated that: 1) relative to DEX-induced early (0.5 hr) peak selective phosphorylation of GR-Ser211, IL-13 alone elicited delayed sequential phosphorylation of GR-Ser203, -Ser226, and -Ser211, peaking at 3, 12, and 24 hr, respectively; 2) IL-13-treated HASM cells transfected with WT GR exhibited a 5.08 ± 0.61-fold increase above baseline in eotaxin-3 release; 3) similar induction of eotaxin-3 release by IL-13 was detected in HASM cells transfected with the S203A or S211A mutant GR, amounting to 1.57 ± 0.02- and 0.918 ± 0.19-fold above baseline, respectively; and 4) by comparison, in cells transfected with the S226A mutant GR, IL-13-induced eotaxin-3 release was strikingly enhanced to 19.17 ± 0.29-fold above baseline. Finally, while having little suppressive effect on eotaxin-3 release from IL-13-exposed S203A- or S211A-transfected HASM cells, pretreatment with DEX markedly suppressed IL-13-induced eotaxin-3 release from both WT- and S226A-transfected cells. CONCLUSION: Collectively, these novel data demonstrate that IL-13 elicits ligand-independent GR activation in HASM cells wherein GR-Ser226 phosphorylation serves to homeostatically suppress the magnitude of IL-13-induced eotaxin-3 release and, thereby, facilitate the GR-Ser203 and –Ser211-mediated inhibitory effect of GC ligand treatment on IL-13-induced eotaxin-3 release. Thus, these findings support the consideration that disruption of this GC ligand-independent cytokine-stimulated homeostatic GR signaling mechanism may account, at least in part, for the reduced efficacy of glucocorticoid therapy exhibited in subgroups of asthmatic individuals. Funded By: NIH HLBI Grant: HL-097739

41 Phosphodiesterase 4 (PDE4) activity is intrinsically increased in cultured human asthmatic airway smooth muscle cells due to upregulated G protein βγ-subunit activation. M. B. Josephson, A. Hu, B. L. Diener, and M. M. Grunstein. The Children’s Hospital of Phila., Univ. of Pennsylvania Perelman SOM, Philadelphia, Pennsylvania. RATIONALE: We recently reported that signaling by the βγ-subunits of pertussis toxin (PTX)-sensitive Gi protein (Giβγ), leading to c-Src-induced ERK1/2-dependent upregulation of phosphodiesterase-4, mediates the airway hyperresponsiveness and inflammation exhibited in an in vivo rabbit model of allergic asthma, as well as the altered contractility detected in atopic asthmatic sensitized airway smooth muscle (PLoS One. 7(2):e32078, 2012). Given this evidence, together with that in an accompanying study by Hu et al. demonstrating that primary cultures of human asthmatic airway smooth muscle (HASM) cells exhibit preserved intrinsically upregulated Gβγ activity, this study examined: 1) whether human asthmatic HASM (A-HASM) cells, as compared to normal (N-HASM) cells, exhibit intrinsically increased Gβγ-regulated PDE4 activity; and 2) whether comparable Gβγ-dependent upregulation of PDE4 activity is elicited in N-HASM exposed to the key pro-asthmatic Th2 cytokine, IL-13. METHODS: Primary cultures of N-HASM and A-HASM cells isolated from normal and asthmatic individuals, respectively, were compared with respect to differences in constitutive and IL-13-stimulated PDE activity, detected using a enzymatic assay kit, as well as temporal changes in PDE activity elicited by treatment with 1 µM of either an anti-Gβγ blocking peptide or gallein, a small molecule inhibitor of Gβγ activation. RESULTS: The data demonstrated that: 1) A-HASM cells exhibit constitutively increased PDE activity, averaging ~ 4-fold above that detected in N-HASM cells; 2) this upregulated PDE activity is acutely suppressed in A-HASM cells to near basal N-HASM cell levels at 15 min following Gβγ inhibition with either the anti-Gβγ blocking peptide or gallein; 3) the suppressive effect of Gβγ inhibition is more effectively sustained (i.e., for up to 24 hr) in A-HASM cells treated with the anti-Gβγ blocking peptide vs. gallein; and 4) unlike A-HASM cells, basal PDE activity in N-HASM cells is relatively little affected by treatment with either Gβγ inhibitor. Finally, in contrast to A-HASM cells that showed relatively little effect, exposure of N-HASM cells to IL-13 elicited increased Gβγ-regulated PDE activity. CONCLUSION: Collectively, these data are the first to demonstrate that the Gβγ signaling mechanism recently identified in mediating PDE-dependent pro-asthmatic changes in airway responsiveness is intrinsically upregulated in cultured asthmatic HASM cells. This novel finding implies that asthmatic HASM cells preserve their altered phenotype in primary cell cultures, a phenomenon that may be attributed to genetic/epigenetic changes that are inherently present in the asthmatic airway smooth muscle. This relevant consideration warrants future systematic investigation that, ultimately, may lead to new targeted therapies for asthma. Funded By: NIH HLBI Grant: HL-097739.

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Pro-asthmatic signaling via the G protein βγ-subunit is intrinsically activated in cultured human asthmatic airway smooth muscle cells. A. Hu, B. L. Diener, M. B. Josephson, and M. M. Grunstein The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. RATIONALE: We recently reported that signaling by the βγ-subunits of pertussis toxin (PTX)-sensitive Gi protein (Giβγ), leading to c-Src-induced ERK1/2-dependent upregulation of phosphodiesterase-4, mediates the airway hyperresponsiveness and inflammation exhibited in an in vivo rabbit model of allergic asthma, as well as the altered contractility detected in atopic asthmatic sensitized airway smooth muscle (PLoS One. 7(2):e32078, 2012). Given this evidence, this study examined whether human asthmatic ASM (HASM) cells, as compared to normal HASM cells, exhibit intrinsically altered constitutive and regulated expression of this Gβγ-coupled signaling mechanism. METHODS: Primary cultures of HASM cells isolated from normal (N-HASM) and asthmatic (A-HASM) individuals were compared with respect to differences in free and Gα-bound Gβγ expression, the latter assessed by co-immunoprecipitation and immunoblotting, and phosphorylation of c-Src at Tyr416 (p-Src), which denotes its activated autophosphorylated state, and phosphorylated ERK1/2 (p-ERK1/2), both detected by immunoblotting using phospho-specific targeted antibodies. RESULTS: The data demonstrated that, in contrast to N-HASM cells, A-HASM cells exhibited constitutively increased intracellular free Gβ protein levels and heightened levels of p-SRC and p-ERK1/2. Moreover, unlike N-HASM cells that showed little change, inhibition of Gβγ signaling in A-HASM cell by treatment with 1 µM of either an anti-Gβγ blocking peptide or gallein, a small molecule inhibitor of Gβγ activation, acutely reduced free Gβ levels, in association with increased Gβ co-localization with membrane-bound Gα protein, and also suppressed both p-Src and p-ERK1/2 levels. Of note, these effects of inhibition of Gβγ signaling were detected in the absence of any appreciable change in β-actin protein expression. Finally, contrasting the effects of inhibition of Gβγ activation, treatment of A-HASM cells with the MEK-ERK1/2 inhibitor, U0126 (10 µM), had no effect on either free Gβ or p-Src levels. CONCLUSION: Collectively, these data are the first to demonstrate that the Gβγ signaling mechanism that leads to c-Src-mediated ERK1/2-dependent induction of pro-asthmatic changes in airway contractility is intrinsically activated in cultured isolated asthmatic HASM cells. This novel finding suggests that asthmatic HASM cells preserve their altered phenotype in cell culture, a phenomenon that may be attributed to genetic/epigenetic changes that are inherently present in the asthmatic airway smooth muscle. This relevant consideration warrants future systematic investigation that, ultimately, may lead to new targeted therapies for asthma. Funded By: NIH HLBI Grant: HL-097739

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43 Benefits of targeting mTORC2 signaling in human and experimental pulmonary hypertension. Tatiana Kudryashova1, Houman Ziai1, Dmitry Goncharov1, Mi Thant Mon Soe1, Horace DeLisser1, Steven M. Kawut2,3, and Elena Goncharova1,2. 1Pulmonary, Allergy & Critical Care Division, 2Pulmonary Vascular Disease Program, 3Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA Increased proliferation and survival of pulmonary arterial vascular smooth muscle cells (PAVSMC) are critical components of pulmonary vascular remodeling, a major and currently irreversible feature of pulmonary arterial hypertension (PAH). We recently reported that activation of mammalian target of rapamycin (mTOR) in two distinct complexes, mTORC1 and mTORC2, is required for elevated PAVSMC proliferation in the rat hypoxia model of pulmonary hypertension (PH). Here, we show that mTORC1 and mTORC2 signaling pathways are up-regulated in distal (50-150 µm) pulmonary arteries (PAs) and PAVSMC of subjects with idiopathic PAH (IPAH). Suppression of mTORC1 by siRNA raptor or rapamycin affected IPAH PAVSMC proliferation, but not survival. siRNA rictor-induced inhibition of mTORC2 signaling or dual mTORC1/mTORC2 inhibitor PP242, reduced proliferation and promoted apoptosis in IPAH PAVSMC. Importantly, PP242 reversed pulmonary vascular remodeling and right ventricular (RV) hypertrophy in the rat chronic hypoxia model of PH, as assessed by a significant increase in internal radius, a decrease in vessel wall area of distal PAs and marked reduction of RV/(LV+S) and RV/BW ratios. Collectively, these data show that mTORC2 is required for enhanced IPAH PAVSMC proliferation and survival and suggest that targeting mTORC2 may represent an attractive therapeutic approach to inhibit PAVSMC proliferation, induce apoptosis and attenuate pulmonary vascular remodeling in PH. Funded by: NIH/NHLBI 1R01HL113178 (EG), American Lung Association RG196551 (EG), Cardiovascular Medical Research and Educational Fund (HD, SK), and NIH/NHLBI K24 HL103844 (SK).

44 mTOR complex 2 stimulates vascular smooth muscle cell proliferation and survival In idiopathic pulmonary arterial hypertension by regulating cellular ATP levels and AMPK activity. Dmitry A. Goncharov1, Tatiana Kudryashova1, Houman Ziai1, Irene Khavin1, Kaori Ihida-Stansbury2,3, Vera P. Krymskaya1,3,5, Horace DeLisser1,3, Rubin M. Tuder6, Steven M. Kawut1,3,4, and Elena A. Goncharova1,3 1Pulmonary, Allergy & Critical Care Division, 2Department of Pathology and Laboratory Medicine, 3Pulmonary Vascular Disease Program, 4Center for Clinical Epidemiology and Biostatistics, 5Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 6Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, CO, USA Enhanced proliferation and resistance to apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMC) coupled with metabolic shift to glycolysis are key pathophysiological components of pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH), the mechanisms of which are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of cell metabolism and proliferation that acts through two distinct complexes, rapamycin-sensitive mTORC1 (mTOR-raptor) and rapamycin-resistant mTORC2 (mTOR-rictor). We previously demonstrated that chronic hypoxia, a recognized trigger of pulmonary vascular remodeling, activates mTORC1 and mTORC2 that both are required for increased PAVSMC proliferation. Here we report that distal PAVSMC from patients with idiopathic PAH (IPAH) have activated mTORC1-S6 and mTORC2-Akt signaling pathways, down-regulated AMP-activated protein kinase (AMPK) signaling, and deficiency of pro-apoptotic protein Bim that associated with elevated glycolytic ATP generation, increased DNA synthesis, growth and viability compared to controls. siRNA-based and pharmacological analysis showed that while both mTORC1 and mTORC2 contribute to increased proliferation, only mTORC2 is required for elevated ATP generation and survival of IPAH PAVSMC. Further, siRNA rictor-induced inhibition of mTORC2 activated AMPK signaling, inhibited mTORC1-S6 and increased Bim protein levels in IPAH PAVSMC. AMPK activator AICAR suppressed mTORC1-S6 signaling and increased Bim protein levels without significant effect on mTORC2-Akt pathway. In contrast, siRNA AMPK rescued siRNA rictor-dependent inhibition of mTORC1 and proliferation, and prevented siRNA rictor-induced Bim expression and apoptosis of IPAH PAVSMC. Taken together, these data provide a novel mechanistic link from mTORC2 via energy sensor AMPK to increased proliferation and survival of PAVSMC in IPAH and suggest that mTORC2-AMPK signaling may be considered as a potential therapeutic target pathway for human PAH. Funded by: NIH/NHLBI 1R01HL113178 (EAG), American Lung Association RG196551 (EAG), Cardiovascular Medical Research and Educational Fund (HD, SK,KIS), and NIH/NHLBI K24 HL103844 (SK).

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45 The Receptor for Advanced Glycation Endproducts is increased in pulmonary hypertension due to advanced lung disease. Christopher J. Winterbottom, MD, Rupal J. Shah, MD, Nilam Mangalmurti, MD, David J. Lederer, MD, Sangeeta Bhorade, MD, Scott M. Palmer, MD, MHS, Keith M. Wille, MD, Ann Weinacker, MD, David S. Wilkes, MD, Ashish S. Shah, MD, Vibha N. Lama, MD, Maria Crespo, MD, Jonathan B. Orens, MD, Lorraine B. Ware, MD, Steven M. Kawut, MD, Jason D. Christie, MD. Background: The Receptor for Advanced Glycation Endproducts (RAGE) is a widely expressed multi-ligand pattern recognition receptor that is implicated in the development of systemic vascular disease. Engagement of RAGE leads to sustained cellular dysfunction marked by increased Nfº B activation, and reactive oxygen species generation. Although RAGE ligands have been implicated in the development of pulmonary arterial hypertension (PAH), the role of RAGE in the development of secondary pulmonary hypertension (PH) remains unknown. We therefore hypothesized that sRAGE would be elevated in patients undergoing lung transplantation with secondary pulmonary hypertension when compared with those without pulmonary hypertension, and that higher plasma levels would be associated with more severe PH. Methods: We conducted a cross-sectional analysis of a multi-center prospective cohort of patients enrolled in the Lung Transplant Outcomes Group study who underwent lung transplantation at 10 centers between 2002 and 2010. Plasma sRAGE was measured preoperatively utilizing ELISA. PH was defined by WHO criteria as mean pulmonary artery pressure > 25mmHg at time of transplant. Wilcoxon rank sum test and logistic regression were used to compare sRAGE levels between groups. Spearman’s rank correlation was used to examine associations of RAGE levels with disease severity. Results: The study sample included 714 patients (289 with COPD and 236 with IPF). The mean age was 52 and 47% were female. Four hundred and twenty (59%) had PH at the time of transplantation. sRAGE levels were significantly higher in those with secondary PH than in those without (median 772 pg/ml, IQR 444-1563 vs. 667, IQR 362-1265, p <0.01 and correlated with increasing mean PA pressure (Spearman’s Á=0.09, p=0.02). There was a significant interaction with diagnosis in analysis of the two largest diagnosis groups (COPD and IPF, p = 0.06). In a stratified analysis of these two groups, increased sRAGE was associated with increased odds of PH in patients with COPD but not IPF (OR per SD of RAGE 2.00 in COPD, 95%CI 1.06-3.80, p=0.03; OR of 0.98 in IPF, 95%CI 0.69-1.40, p=0.92). Conclusions: Plasma sRAGE levels are higher in patients with PH at the time of lung transplantation than those of patients without PH, and correlate with severity of PH. This association appears to be driven primarily by COPD. Future studies to elucidate the mechanisms by which RAGE mediates the development of pulmonary hypertension in patients with COPD are warranted.

46 Control of pulmonary vascular smooth muscle cell growth by biochemical and biophysical ECM properties. K. Ihida-Stansbury1,2, Y. Xia3, E. Lee3, T. Nguyen2, L. Chin2, H. Delisser4, S. Kawut4, S. Yang3. 1Dept. Path & Lab Medicine, 2Institute for Medicine & Engineering, 3Dept. Material Sci, 4Dept Perelman School of Medicine, University of Pennsylvania. Pulmonary arterial hypertension (PAH) is characterized by increasing pulmonary arterial (PA) resistance accompanied by thickening of the artery wall through SMC hyperplasia and extracellular matrix (ECM) remodeling. Previously we have shown that expression of ECM protein tenascin-C (TN-C) is upregulated in and around pulmonary vessel wall and altered SMC behavior such as proliferation and migration in PAH patients. Although recent studies have demonstrated that microenvironmental cues such as architecture, composition, biophysical properties (i.e. stiffness) and biochemical properties (i.e growth factors) alter the cell growth, how ECM properties contribute to the vascular SMC growth in PAH is not well understood. In this study, first, we characterized the ECM properties of PA from control and PAH patients using decellularized PA scaffolds. We have found that the mechanical property, stiffness, was higher and their ultrastructure of fiber formation was dramatically altered in PAH arteries with disorganized fine fiber formation comparing to the control PAs thicker organized bundled ECM fiber structure. Based on these results, to elucidate the ECM structure-dependent SMC growth, we utilized synthetic polymer substrates to mimic the PAH and control PA ECM (i.e.archtecture, stiffness, composition) for cell culture system. Uniquely designed 1D channel and/or wrinkled membranes with or without pillars were coated with collagen I and/or TN-C, seeded the SMC then cell migration, proliferation and differentiation were examined. Control PASMCs cultured on collagen coated wrinkled/1D channel substrates showed elongated functional SMC-like morphology along the channel/wrinkle with width and depth dependent manner, while SMC cultured on substrates of disturbed wrinkled channel with pillars which is mimicking the PAH ECM possessed less aspect ratio and showed mesenchymal-like morphology. Furthermore, SMC seeded on TN-C coated substrate also showed mesenchymal-like morphology regardless the architecture of ECM. α-SMA immunostaining support these observation that altered ECM ultrastructure and composition induce de-differentiation of the SMC. These results suggest that altered PA ECM architecture and composition in PAH play a key role in altering SMC growth and induce remodeling of arterial walls.

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Antioxidant enzyme delivery by nanocarriers protects endothelial cells from oxidative stress. Elizabeth Hood*,#, Michael Chorny +,#, Colin Greineder*,#, Ivan Alferiev+,#, Robert J. Levy +,#, Vladimir R. Muzykantov *,#, 1. * Institute for Translational Medicine and Therapeutics, Department of Pharmacology, University of Pennsylvania School of Medicine, +Department of Pediatrics, The Children’s Hospital of Philadelphia and #Institute for Translational Medicine and Therapeutics, University of Pennsylvania. Targeted magnetic nanoparticle (MNP) delivery of antioxidant enzymes (AOEs) protects against inflammatory injury to the pulmonary endothelium in models of oxidative stress in vitro and in vivo. Delivery of superoxide dismutase or catalase targeted to adhesion molecules expressed by endothelial cells that line the pulmonary vasculature enable protection by suppressing damage along different oxidative pathways. Unlike other carriers these nanoparticles, formed by controlled precipitation, not only enable access to enzyme substrate but also provide protection of enzyme cargo from in vivo proteolysis. By including either biotin or maleimide functionalized Pluronic F127 in the particle composition modified antibodies were linked to formed MNPs in a dose dependent manner. The antibody decorated AOE MNP localize to the endothelium in cultured cells and in mouse lungs. Anti-PECAM modified MNPs bind specifically to cultured endothelial cells whereas untargeted IgG modified MNPs show minimal binding. Additionally, intravenously-administered, PECAM-targeted catalase MNPs localized in mouse lungs after 60 mins circulation time at over 200% injected dose per gram of lung tissue (mouse lung mass = ~ 0.15 g). Additionally, catalase delivery to PECAM protects against oxidative stress to endothelial cells in vitro, and from increased permeability in vivo. MNPs loaded with catalase protect endothelial cells from hydrogen peroxide mediated apoptosis at 75% of untreated reference. In mice challenged with endotoxin lipopolysaccharide (LPS) PECAM targeted catalase MNPs reduced brochoalveolar lavage protein compared to that of untreated or untargeted IgG control catalase as a marker of increased endothelial permeability and also decreased infiltration of white blood cells. In SOD MNP treated endothelial cells treated with tissue necrosis factor (TNF) there was a reduction of expressed VCAM, a marker of endothelial inflammation. Cell adhesion molecule targeted AOE MNPs enable delivery of both catalase and SOD as protective agents to the endothelium and are potentially clinically viable drug carriers in a myriad of acute inflammatory conditions.