Experimental models and research in diffuse parenchymal lung diseases

printing supported by . Visit Chiesi at Stand D.30 SUNDAY, SEPTEMBER 25TH 2011 P670 The stimulator of soluble guanylate cyclase riociguat protects against bleomycin-induced pulmonary fibrosis in mice Oleg V. Evgenov1, Johannes-Peter Stasch2, Mari Mino-Kenudson3, Eugene J. Mark3, Emmanuel S. Buys1, Lin Zou1, Ming Zhang1, Michael J. Raher1, Yan Li1, Yan Feng1, Rosemary C. Jones1, Wei Chao1. 1Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; 2Institute of Pharmacy, Martin Luther University, Halle, Germany; 3Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States Background: Effective therapies for pulmonary fibrosis (PF) are currently lacking. Patients with PF develop pulmonary hypertension (PH), in part due to impaired production of endogenous nitric oxide (NO) that activates soluble guanylate cyclase (sGC). We hypothesized that the NO-independent stimulation of sGC might attenuate PF. Methods: Male C57/BL6 mice (10-12 wks) were subjected to intratracheal bleomycin (0.5 U/kg) and gavage-feeding with the sGC stimulator riociguat (1, 3 or 10 mg/kg/day), the phosphodiesterase 5 (PDE5) inhibitor sildenafil (100 mg/kg/day), a combination of riociguat (1 mg/kg/day) and sildenafil (100 mg/kg/day), or vehicle alone for two weeks. Results: Bleomycin-induced PH (an increase in the right ventricle systolic pressure and a decrease in the pulmonary acceleration time/ejection time ratio) and the right ventricular hypertrophy were attenuated by riociguat and the combination of riociguat and sildenafil to a greater extent than by sildenafil alone. In the vehicle-treated mice, fibrosis and inflammation diffusely involved lung parenchyma. Riociguat and its combination with sildenafil but not sildenafil alone markedly ameliorated PF and inflammation that was mainly confined to subpleural areas and/or peripheral lung in a patchy distribution. Riociguat increased plasma cGMP levels and also reduced mortality. Conclusions: Pharmacological stimulation of sGC with riociguat attenuates PF, PH, right ventricular hypertrophy and mortality in the bleomycin-exposed mice. This therapeutic approach appears to be superior to treatment with sildenafil. Stimulation of sGC might represent a new modality for treating PF and related conditions. P671 Experimental model resembling the histological pattern of usual interstitial pneumonia and reinforcing the epithelial injury as pathway Marcella Soares Pincelli, João Barbas-Filho, Walcy Teodoro, Vera Capelozzi, Edwin Roger Parra. Pathology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil The present experimental model in mice was developed to confront the histopathological features with that of the idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP), as we suppose both are caused by the injury of type II pneumocyte (TIIp) and by the increase of collagen system. Material and methods: Twenty male Balb/c mice are injected ip with 400mg/kg of butylated hydroxitolueno (BHT) and kept breathing for six days at a 70% oxygen atmosphere. The mice were killed after one month. The lungs were fixed and stained by Hematoxylin & Eosin and imunofluorescence for collagen I and III. Also, TUNEL and electron microscopy were used to evaluate the epithelial apoptosis index. We used 5 balb/c mice as controls. Results: Pulmonary specimens of this model confirmed UIP pattern, such as progressive increase of interstitial deposition. They showed significant increase of collagen I and III deposition and significant increase of epithelial apoptosis when compared to control group (p<0.05).The apoptosis was more prominent in the TIIp observed by electron microscopy. Conclusion: This experimental model showed the same histopathological patterns of UIP, and also reinforced the increase of apoptosis TIIp after injury or apoptosis of these cells and collagen deposition as an early feature in the pathogenesis of IPF. Financial support: FAPESP CNPq. P672 Can flaxseed oil reduce experimental lung fibrosis in rats? Anouar Abidi1, Raja Srairi2 , Nadia Kourda3, Fatma Tritar4, Sonia Maalej4, Leila Douik5, Moncef Fekih1, Saloua Ben Khamsa1. 1Physiology, University of Medicine, Tunis, Tunisia; 2Physiology, Superior Institute of Science and Technic Health, Tunis, Tunisia; 3Anatomopathology, Charles Nicole Hospital, Tunis, Tunisia; 4Pneumology, Abderahmen Mami Hospitl, Ariana, Tunis, Tunisia; 5Pneumolgy, Charles Nicole Hospital, Tunis, Tunisia Background: The anti-inflammatory effects of poylunsaturated fatty acids (PUFA) is actually demonstrated. In this study we evaluated protective and therapeutic effects of Flaxeeed Oil (FO) on Bleomycin (BLM) lung fibrosis in rats. Methods: 30 males wistar rats were randomly divided into 3 equal groups: untreated group (G1) and 2 treated groups (FO, 1g/kg bw/day). G2 received FO during two months before inducing lung fibrosis than G3 received FO 2 days after inducing lung fibrosis during 10 days. Pulmonary fibrosis was induced by BLM (4 mg/kg, intratracheally single dose). Inflammatory index, fibrosis score (Ashcroft) and TGF-β density was evaluated in different areas of damaged lung by anatomo-histological and immuno-histological analysis. Results: Independent-samplesTtest revealed that comparatively to control group (G1). FO reduced significantly fibrosis score in G2 and TGF-β density in fibrocytes in G3 (p<0,05). Figure 1. Distribution of TGF-β in fibrocytes of treated groups (G2, G3) and control group (G1). Figure 2. Score of fibrosis of treated groups (G2, G3) and control group (G1). Conclusion: These data demonstrated a protective activity of FO against bleomycin-induced lung fibrosis model in rats. More investigations will be realized in human to prove relationship between diet and interstitial lung diseases. P673 Inhibition of PAI-1 in the cigarette smoke induced epithelial-mesenchymal transition Jeong-Sup Song1, Chun-Mi Kang1, Ki-Hoon Park1, Hyung-Kyu Yoon1, Hwa-Sik Moon1, Sung-Hak Park2. 1Internal Medicine, St Mary’s Hospital, Catholic University Medical College, Seoul, Korea; 2Internal Medicine, Seoul St Mary’s Hospital, Catholic University Medical College, Seoul, Korea Introduction: Plasminogen activator inhibitor-1 (PAI-1) has been known to play an essential role in pulmonary fibrosis by inhibiting plasminogen activator (PA). Recently, it is assumed that epithelial-mesenchymal transition (EMT) play a role in the pathogenesis of IPF. We tried to find out whether PAI-1 is involved in the bleomycin-induced pulmonary fibrosis in the rat and in smoking-induced rat alveolar EMT in vitro. Methods: First, rats were received intratracheal bleomycin (4U/kg) and then administered tiplaxtinin (1mg/kg) at day 1, 3, 5, 7, 10 orally. Rats were sacrificed at day 14. Next, the type II alveolar epithelial cells were isolated from normal rats by percoll gradient methods. Type II epithelial cells were transfected with PAI-1-siRNA and were stimulated with cigarette smoking extract (CSE, 5%). Results: In bleomycin-induced pulmonary fibrosis model, tiplaxitinin decreased the bleomycin-induced pulmonary fibrosis by Ashcroft score and also decreased the PAI-1 and TGF-β concentrations in the BAL fluids. In epithelial cell experiment, exposure to CSE increased the α-SMA and PAI-1 mRNA expression in real-time PCR. However, they were attenuated either after transfection with siRNA-targeted PAI-1 or treatment with tiplaxitinin (50 μM). TGF-β concentrations in the CSE exposed cell culture supernatants were also decreased either by PAI-1-siRNA transfection or treatment with tiplaxitinin. The up-regulation of Snail and pSMAD by smoking exposure in epithelial cells were decreased either by transfection with PAI-1-siRNA or treatment with tiplaxitinin. 106s Thematic Poster Session Hall 2-9 12:50-14:40 Abstract printing supported by . Visit Chiesi at Stand D.30printing supported by . Visit Chiesi at Stand D.30 SUNDAY, SEPTEMBER 25TH 2011 Conclusions: PAI-1 is involved in the EMT by directly activating the Snail and SMAD2 in the CSE exposed rat type II alveolar epithelial cells. P674 Bleomycin-induced remodeling – Inflammation and fibrosis are developing in parallel during initiation Kristina Rydell-Törmänen1, Gunilla Westergren-Thorsson2. 1Rheumatology, Dept. Clinical Sciences, Lund University, Lund, Sweden; 2LungBiology, Dept. Experimental Medical Science, Lund University, Lund, Sweden Background: Pulmonary fibrosis is a facet of Diffuse Parenchymal Lung Disease and has been considered a sequel to chronic inflammation. Yet the relation between connective tissue and immune system during the initiation phase has not been thoroughly investigated. Our aim was to investigate the initiation phase and the interplay between immune system and connective tissue. Methods: C57/Bl6 mice were given subcutaneous bleomycin injections (controls received saline) 3 times/week for 1, 2, 3 and 4 weeks. Following sacrifice, lungs were embedded in paraffin and 4 μm thick sections were used for analysis of lung parenchyma; Masson Trichrome (total collagen) and immunohistochemistry to detect neutrophils, macrophages and B-cells. Results: Total collagen was significantly increased at 1, 2 and 4 weeks. A transient neutrophilia was found at 1w (2779±820 cells/mm2), but no difference compared to controls was found at 2, 3 and 4w. Macrophages were significantly increased at 2w (135±29 cells/mm2), whereas B-cells were significantly decreased at 3 and 4w, compared to controls. Conclusions: Subcutaneous administration of bleomycin induced rapid pulmonary remodeling, exemplified by increased total collagen. The immune response was markedly different from the one following intratracheal administration, illustrated by the moderate and transient neutrophilia. Increase of macrophages at 2w in combination with decreased neutrophils suggests ongoing clearance. Decrease of B-cells may sugg