Thesis (Ph.D.)--Michigan State University. Dept. of Genetics, 2008. / Title from PDF t.p. (viewed on Sept. 11, 2009) Includes bibliographic references (p. 191-210). Also issued in print.

Abstract Idiopathic usual interstitial pneumonia (UIP) and bronchiolitis obliterans organizing pneumonia (BOOP) are fibrous pulmonary disorders in both of which there is newly formed connective tissue in distal air spaces. UIP is a progressive and usually fatal lung disease without any efficient treatment, while the prognosis of BOOP is good. In both diseases, an injury of the alveolar epithelium and its basement membrane (BM) leads to migration of fibroblasts and myofibroblasts into air spaces and production of extracellular matrix by these cells. In UIP, the newly formed intraluminal connective tissue lesions cause fusion of alveolar structures and interstitial remodeling, while in BOOP the newly formed connective tissue may resolve completely. One of the major aims of the research on pulmonary fibrosis is to define the mechanisms that lead to persistence of the newly formed connective tissue and thus to irreversible fibrosis in UIP. The aim of the present study was to compare the extent of capillarization, apoptotic activity and re-epithelialization of the newly formed connective tissue in BOOP and UIP. The number of capillaries per tissue surface area was measured. Furthermore, the expression of angiogenic growth factors vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) was evaluated in the same areas, in addition to the expression of Flt-1 and Flk-1, which serve as receptors for VEGF. Apoptotic activity was analyzed using TUNEL-method, and the immunohistochemical expression of apoptosis regulating proteins bcl-2, mcl-1, and bax was studied. Finally, the extent of re-epithelialization was studied with the immunohistochemical and ultrastructural localization of laminin-5 γ2 chain, and the sites of synthesis of laminin-5 γ2 chain mRNA. In BOOP, an efficient repair process with good capillarization along with high expression of VEGF and bFGF, and orderly re-epithelialization of the newly formed connective tissue takes place after lung injury. The apoptotic activity of the newly formed connective tissue is also high, presumably leading to resolution of the intraluminal connective tissue in BOOP. In UIP, the newly formed connective tissue showed poor capillarization, inadequate re-epithelialization and low apoptotic activity. The results suggest disturbed or delayed repair process in UIP, contributing to irreversible interstitial fibrosis and remodeling.

No description available.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease of unknown etiology, in which excessive accumulation of scar tissue in the interstitial spaces of the lung obstruct normal pulmonary function. Currently, the only curative treatment is lung transplantation. While pharmaceutical therapeutics have been recently approved for use in IPF in 2014, they are still unable to provide a truly curative treatment. While genetic risk factors have been identified, the most commonly occurring mutation is only detected in approximately 38% of IPF patients, leaving an uncertainty in the very existence of a common genetic factor in IPF. Cigarette smoke and other environmental particulates have been significantly linked to the diagnosis of IPF, implicating an initial immunological response to trigger the pathogenesis of IPF. Nintedanib, a potent tyrosine kinase receptor inhibitor was first developed in 1998 as a candidate for cancer treatment. Investigation of its effects in fibrosis in the past few decades has led to a significant discovery of its application in IPF. Nintedanib significantly inhibits the fibrotic activity of fibrotic myofibroblasts in the lungs by inhibiting signaling cascades necessary for cell proliferation and progression of the disease. However, nintedanib falls short in that it cannot fully inhibit the advancement of the disease and mortality rates of IPF still remain high. Pirfenidone, the other currently available pharmaceutical therapeutic, was discovered in 1976 as a potent inhibitor of inflammation. Subsequent experiments further reviled its potency as an anti-fibrotic drug. After decades of research, pirfenidone’s mechanism of antifibrotic characteristics were revealed as a potent inhibitor of fibrocyte recruitment and chemotaxis, and as an inhibitor of transcription growth factor beta (a growth factor heavily implicated in the activity of myofibroblasts) mediated pathways. However, like nintedanib, pirfenidone fails as a curative treatment, only delaying the progression of the disease. In the search for new molecular targets for pharmaceutical therapy, forkhead box M1 (FOXM1), programmed cell death protein-1 (PD-1), and prostaglandin E2, have been identified to play a mediatory role in many of the pathways involved in myofibroblast activity. Many of these targets have also been identified in other disease models such as cancer and immunological inflammatory disease. Avasimibe has been recently identified as a potent inhibitor of aldo-ketoreductase through a FOXM1 mediated pathway. Its molecular mechanism in osteosarcoma cancer disease model may prove to be a novel pharmaceutical therapeutic for IPF. BI 853250, a novel focal adhesion kinase (FAK) inhibitor also demonstrates potential to be a new pharmaceutical therapeutic for IPF patients. Exploring signaling pathways that involve these newly found targets and collaborative research with cancer and immunological diseases shows promise in providing steps to cure IPF in the future.

No description available.

Thesis (M.S.P.H.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 3, 2009). Includes bibliographical references (p. 43-48).

Final publication is avilable at http://www.sciencedirect.com/science/article/pii/S0954611115300172 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19577号 / 医博第4084号 / 新制||医||1013(附属図書館) / 32613 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 平家 俊男, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Orientador: Vera Luiza Capelozzi / Coorientador: Claudia Aparecida Rainho / Banca: João Lauro Viana de Camargo / Banca: Thais Helena A. Thomaz Queluz / Banca: Alexandra Muxfeldt Ab'Saber / Banca: Rimarcs Gomes Ferreira / Resumo: A fibrose pulmonar é a base patológica de uma variedade de doenças crônicas incuráveis. A IL-17A, uma glicoproteína secretada de células Th17, é uma citocina pró-fibrótica relacionada recentemente à síntese de colágeno V e fibrose pulmonar. O remodelamento parenquimatoso pulmonar da matriz extracelular pelo colágeno I e V, apoptose e resposta Th17 foi estudado em camundongos Balb/c, C57/B6J selvagens e com knockout para o receptor A da IL-17; para determinar as vias fisiopatológicas que prolongam a fase tardia do processo fibrótico induzido por bleomicina e paraquat. Microscopia eletrônica, imunofluorescência, imunohistoquímica, detecção in situ da apoptose, morfometria, reconstrução tridimensional e reação em cadeia da polimerase em tempo real foram usados para avaliar a quantidade, estrutura e cadeias moleculares dos tipos de colágenos, apoptose e células imunes. Verificamos um aumento da síntese e secreção do colágeno V que promove a perpetuação da fibrose pulmonar de maneira IL-17A dependente. Além disso, observou-se que marcadores críticos da resposta Th17 como IL17, STAT3, TGF-β, IL-6, IL- 21, IL-23 e células T CD4+ foram significantemente aumentados em cepas susceptíveis a fibrose pulmonar e intensificadas na ausência do receptor A da IL-17. O aumento de marcadores Th17 resulta em um aumento de células T CD4+ através de uma resposta imune que efetivamente bloqueia a degradação do colágeno V, o qual contribui para o bloqueio da apoptose. Nosso estudo indica que o colágeno V participa da perpetuação da fibrose pulmonar por mecanismos IL-17 dependente e independentes, indicando o potencial alvo terapêutico do colágeno V e das vias de sinalização da resposta IL-17 no tratamento das doenças fibroproliferativas pulmonares / Abstract: Pulmonary fibrosis is the pathologic basis for a variety of incurable human chronic lung diseases. IL-17A, a glycoprotein secreted from IL-17- producing cells, has recently been shown to be a profibrotic cytokine involved in type V collagen synthesis and pulmonary fibrosis. Remodeling of the extracellular matrix by collagen I and V, cell death and Th-17 immune response were evaluated in Balb/c, wild and IL-17 receptor A knockout C57/B6J mice, to determine the pathways that prolong the late phase of the fibrotic process induced by bleomycin and paraquat. Electron microscopy, immunofluorescence, immunohistochemistry, in situ detection of apoptosis, morphometry, tridimensional reconstruction and a real-time PCR were used to evaluate the amount, structure and molecular chains of collagen types, apoptosis and immune cells. We verified increased synthesis and secretion of type V collagen that promoted the maintenance of pulmonary fibrosis in a IL-17A dependent manner. However, we observed that the critical Th17 markers, IL-17, STAT3, TGF-β, IL-6, IL-21, IL-23 and CD4+ T cells, were significantly increased in the fibrosis-susceptible strain and intensified in the absence of IL-17 receptor A. Increased Th17 markers resulted in an increase in CD4+ T cells in fibrotic lung tissue toward an immune response that effectively blocked degradation of collagen V, which contributes to block apoptosis. Our studies indicate that collagen V participates in the maintenance of pulmonary fibrosis in both Th-17 - dependent and -independent manners and that collagen V and the components of the Th-17 signaling pathway are potential therapeutic targets for the treatment of fibroproliferative lung diseases / Doutor

Pulmonary fibrosis is a chronic and debilitating condition that proposes several challenges to both veterinary and medical clinicians. Despite considerable research, many fibrotic lung diseases remain elusive in terms of aetiology, pathogenesis and treatment. Furthermore, progress is hindered by the lack of a translatable animal model with durable and persistent fibrosis. Asinine Pulmonary Fibrosis (APF) is a spontaneous syndrome of aged donkeys with high prevalence (35%). No previous detailed characterisation of APF has been performed and disease diagnosis remains a challenge. APF was studied with regard to clinical, pathological and molecular features and the suitability of this condition as a model for a rare fibrotic lung disease in humans known as pleuroparenchymal fibroelastosis (PPFE) was assessed. In addition, target activatable optical imaging reagents for the real time detection of two key molecular markers of fibrosis: matrix metalloproteinases (MMPs) and lysyl oxidases (LOXF) were evaluated in spontaneous ex vivo models of fibrosis. Such reagents may be used alongside fibred confocal fluorescence microscopy (FCFM), a relatively noninvasive and cutting edge diagnostic tool, to detect and monitor fibroproliferation in animals and man. Whole lungs were collected from 32 aged donkeys at routine necropsy. Gross examination revealed pulmonary fibrosis in 19 donkeys (APF cases), while 13 (controls) had grossly normal lungs. HRCT images and histology sections were reviewed independently and blindly for each of the lungs. Ten of 19 APF lungs were categorised as being ‘consistent with’ PPFE according to previously defined histological and imaging criteria. All 10 PPFE-like lungs had marked pleural and subpleural fibrosis, predominantly within the upper lung zone, with accompanying intra-alveolar fibrosis and elastosis. An activatable Smartprobe for the detection of LOXF, TWB-219, was synthesised by The Bradley Group, Department of Chemistry (UoEDC). The probe was based on a tandem amine oxidation and β-elimination mechanism, resulting in signal amplification detected at the 488nm wavelength. The probe showed increased fluorescence in the presence of diamine oxidase as well as on incubation with aged human lung tissue cell-free homogenate as determined by a fluorescent plate reader. This signal amplification could be inhibited by β-aminopropionitrile, a recognised LOX inhibitor as well as by an in-house inhibitor specific to LOX. An evolutionary family of MMP probes with varying cleavage sequences and structures, synthesised by the UoEDC, was evaluated at each stage of progression with regard to signal to noise ratio, sensitivity and specificity. Probes were tested against recombinant enzymes from the MMP family as well as neutrophil elastase and plasmin. Signal amplification was also assessed on incubation with human and ovine ex vivo lung tissue. The final ‘lead’ MMP probe, SVC-186, was cleaved by MMP-2, -9 and -13. Signal amplification was also seen following incubation with both human and ovine tissue with significant inhibition in the presence of the pan- MMP inhibitor, marimastat. In conclusion, APF is an emerging condition of aged donkeys that shares key pathological and imaging features with human PPFE. Diagnosis of APF and other fibrotic lung conditions across species remains a challenge to veterinary and medical professionals. As such, optical imaging tools may provide dynamic, real time information on the presence and progression of fibroproliferation in the lung. TWB- 219 and SVC-186 produce a detectable increase in fluorescent signal at the 488nm wavelength when activated by LOXF and MMPs respectively. These probes have been shown to function in human ex vivo tissue as assessed by FCFM.

ROGDI, a novel gene, locates on human¡¦s chromosome 16p13.3. According to Gene Ontology Annotation database, ROGDI is related to hemopoiesis and positive regulation of cell proliferation. In order to investigate the function of this novel gene in pulmonary fibrosis, fibrotic models in vivo and in vitro were created. Mice which received single intra-tracheal bleomycin injection were sacrificed on various intervals. Rogdi and other pro-fibrotic mediators, including CCL2 and TGF-£]1, were up-regulated in the early phase(< 10 days). On contrary, the anti-fibrotic mediators IL-10, IFN-£^ and heme oxygenase(HO)-1 were up-regulated in the late phase(> 10 days). The precursor microRNA 21 (miR-21) was up-regulated as the fibrotic severity increased. The human embryonic fibroblasts(WI-38 cells) showed fibrogenic phenotype and up-regulation of precursor miR-21 and ROGDI after bleomycin treatment. Human embryonic fibroblasts transfected by coding sequence of ROGDI showed up-regulated precursor miR-21 and £\-SMA compared to those transfected by empty vectors after bleomycin treatment. Two signaling molecules related to positive regulation of cell proliferation, Akt and Erk, showed over-expressed after ROGDI transfection and bleomycin treatment compared to those with empty vector transfection. Our results imply that ROGDI is up-regulated in pulmonary fibrosis and turns fibroblasts into fibrogenic phenotype through positive regulation of miR-21. The increase of precursor, but not primary miR-21, after ROGDI transfection and bleomycin treatment indicates that ROGDI may regulate the TGF-£] signaling pathway in human embryonic fibroblasts. Our results support that ROGDI is a novel gene for pulmonary fibrosis and warrants for further investigation. £[