Call number: LD2668 .T4 1965 B71 / Master of Science

<p>Bronchiolitis Obliterans (BO) is a major cause of chronic airway dysfunction after toxic chemical inhalation. The pathophysiology of BO is not well understood, but epithelial cell injury has been closely associated with the development of fibrotic lesions in human studies and in animal models of both toxin- and transplant-induced BO. However, while almost all cases and models of BO include epithelial injury, not all instances of epithelial injury result in BO, suggesting that epithelial damage per se is not the critical event leading to the development of BO. In this dissertation, we describe a model of chlorine (Cl2)-induced BO in which mice develop tracheal and large airway obliterative lesions within 10 days of exposure to high (350 ppm), but not low (200 ppm), concentrations of Cl2 gas. Lesions develop in a series of well-demarcated pathological changes that include epithelial denudation, inflammatory cell infiltration by day 2 after exposure, fibroblast infiltration and collagen deposition by day 5, and in-growth of blood vessels by day 7, ultimately leading to lethal airway obstruction by days 9-12. Using this model, we were able to test our hypothesis that loss of epithelial progenitor cells is a critical factor leading to the development of obliterative airway lesions after chemical inhalation. Indeed, these lesions arise only under conditions and in areas in which basal cells, the resident progenitor cells for large airway epithelium, are eliminated by Cl2 exposure. </p><p>The molecular pathways contributing to BO development are not well understood. Mechanisms of epithelial injury differ across BO models, but we hypothesized that after the inciting injury, BO models share common pathways. We compared microarray analysis from day 5 non-BO- and BO-inducing chemical injuries and subsequently identified biological pathways that may contribute to BO pathogenesis. Our findings add support to pathways previously implicated in BO development and more importantly, suggest potential new pathways and molecular mediators of BO. Furthermore, we evaluated the efficacy of therapeutic inhibition of neovascularization or inflammation to prevent Cl2-induced BO. To date, our therapeutic interventions were ineffective. Nonetheless, our findings suggest that in the context of Cl2-induced BO, vascular endothelial growth factor receptor 2 (a mediator of neovascularization) and inducible nitric oxide synthase (a mediator of inflammation) are not critical in BO pathogenesis.</p><p>In sum, our work introduces and characterizes a novel Cl2-induced murine model of BO. Using this model we demonstrated that in the absence of basal cells, epithelial regeneration does not occur and regions of epithelial denudation persist from which an aberrant repair process is initiated, leading to obliterative airway lesions. Our findings suggest that, irrespective of the cause, loss of epithelial progenitor cells may be a critical factor leading to the development of BO. Furthermore, our gene expression analysis implicates novel mediators and signaling pathways in the development of BO. Our analysis lays the foundation for more rigorous exploration of these targets in the pathogenesis of BO.</p> / Dissertation

Clinical research in cystic fibrosis (CF) requires study endpoints that are sensitive to airways disease, repeatable and non-invasive. Despite significant advances in the treatment of CF, lung function assessments continue to rely on the forced expiratory volume in 1 second (FEV1). Although simple to perform, it lacks sensitivity, is difficult for younger subjects, and changes over time. An alternative method of assessing lung physiology is to derive measures of ventilation heterogeneity from inert gas washout tests. In early lung disease, measures of gas mixing appear to be more sensitive than spirometry. In addition, since only tidal breathing is required, they are more physiological and are more straightforward for younger subjects. Widespread use has been impaired by the lack of a robust and cost effective gas analyser technology. The work presented in this thesis concerns the adaptation, validation and then use of a novel gas analyser (Innocor) in a clinical system for the performance of multiple breath washouts. Lung clearance index (LCI), a simple measure of ventilation heterogeneity, has been calculated from washouts in 52 adults with CF and 50 healthy controls. LCI was more sensitive to disease than FEV1 in CF, being elevated in 11 of the 12 CF patients with normal spirometry. In healthy subjects, LCI has been shown to be repeatable and reproducible, with a narrow range of normal that is stable over a wide age range. In a separate study of 19 patients, LCI has also been shown to improve with treatment of an exacerbation in CF. Correlation with changes in other biochemical (serum CRP, peripheral blood white cell count, sputum IL-8, sputum neutrophil) clinical (symptom score) or structural (computed tomography) markers was poor. Short term change in LCI has also been demonstrated in CF patients in response to chest physiotherapy, although there was considerable heterogeneity of response in terms of both LCI and volume of lung ventilated by tidal breathing (as measured by washout functional residual capacity). In addition to LCI, multiple breath phase III slope analysis has been performed on washouts of CF patients and healthy controls, and this has been compared to other measures of lung physiology. Proposed measures of convective and diffusive gas mixing have been shown to be unreliable in CF. These studies have also been the first to demonstrate multi-centre use of washout tests as endpoints. The technology described here offers the possibility of a simple and reliable system for performing multiple breath washouts, though at present it is not available commercially. The studies have added to the understanding of the utility and reliability of washout tests, as well as some of their limitations. It is hoped that in future LCI will be an important clinical endpoint in therapeutic intervention studies in CF, and that it will also offer new ways to follow changes in lung physiology in other diseases.

Cystic fibrosis (CF) lung disease is characterised by early airways infection and inflammation, chronic suppuration, frequent infective exacerbations and an increased influx of acute, and chronic inflammatory cells. The inflammatory process involves activation of many cell types including neutrophils, macrophages and epithelial cells, and leads ultimately to the development of progressive respiratory failure and death. Accurate assessment of the inflammatory process is a crucial part of disease monitoring and should allow appropriate evaluation of therapeutic interventions so as to maximize control of the respiratory sequelae of the disorder. Lung function markers such as FEV1 are insensitive and indirect. Direct but invasive methods such as fibreoptic bronchoscopy and biopsy are limited in application, repeatability and safety. Non-invasive methods of assessment are, therefore, attractive. Exhaled Breath Gases, Exhaled Breath Condensate and Induced Sputum provide potential for such measures. These techniques are safe, simple, repeatable and could assess all airways and can be used in children as young as 6 years. We hypothesised that biomarkers of inflammation in Cystic Fibrosis Lung Disease are measurable in samples collected noninvasively, and can be developed into clinically useful assays. These assays would have the ability to reflect the level of inflammation in the CF lungs as well as holding the potential to act as surrogate markers of CFTR function. Methods Non-invasive markers of inflammation in Cystic Fibrosis lung disease Methods. Exhaled breath gases, exhaled breath condensate, bronchoalveolar lavage fluid and induced sputum were investigated using a number of analysis techniques to identify the markers which best discriminated CF from non CF subjects. Analysis techniques used were electrochemical cells, chemiluminescene, ELISA, EIA, ion selective probes and mass spectrometry. Results Markers found to discriminate CF from non CF subjects were EBC pH and ammonium, and 38 proteomic markers were found in induced sputum. 21 proteomic markers were found in bronchoalveolar lavage fluid. One biomarker has been identified with confidence, Calgranulin A. Discussion A large component of the work of this thesis was focussed on exhaled breath condensate. Two markers, pH and Ammonium were different between the CF and control groups. The measurement of EBC pH and ammonium as markers of inflammation should be used in future gene therapy trials as they are cheap, quick and simple to perform Using clean techniques free from contamination, no proteins are repeatedly detectable in EBC using highly sensitive SELDI techniques. This technique reflects the highest sensitivity of any available proteomics instrument and therefore until new technologies become available, it would be incorrect to assay any proteins in EBC. The induced sputum proteomics study identified 38 independent markers of CF lung inflammation Therefore, sampling by collection of induced sputum should be used in gene therapy trials. The endpoints should be assessed by a combination of SELDI as an endpoint and by ELISA where this is available. The marker Calgranulin is likely to report on neutrophil recruitment to the lung. It is anticipated that this will be a sensitive marker of inflammation in the lung and it also has the potential to report on successful of gene transfer as it is raised in heterozygote carriers as well as homozygotes with CF. Therefore, the non-invasive technique induced sputum coupled to proteomic analysis would have the ability to reflect the level of inflammation in CF subjects and may also report on CFTR function.

Cardiac fibroblast to myofibroblast phenoconversion is a critical step during the development of cardiac fibrosis. Myofibroblasts chronically remodel extracellular matrix that results in myocardial stiffening, cardiac dysfunction and eventually heart failure. Previously we showed that Meox2, a homeobox transcription factor, can inhibit myofibroblast phenoconversion. Here we show that Zeb2, a repressor of Meox2, plays a crucial role during this phenoconversion process. Zeb2 overexpression significantly upregulates the expression of three key myofibroblast markers: α-SMA, SMemb and ED-A fibronectin in primary rat cardiac myofibroblast. We show that Zeb2 is highly expressed in myofibroblast nuclei whereas it is minimally expressed in fibroblast nuclei. Zeb2 overexpression in myofibroblasts results in a less migratory and more contractile mature myofibroblast phenotype. Moreover, Zeb2 overexpression represses Meox2 expression in endothelial cells. Thus, the current study enhances our understanding of the mechanism behind myofibroblast phenoconversion and provides a basis for developing Zeb2-based novel anti-fibrotic drug in the future. / February 2016

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.

Age-related degeneration of the equine endometrium is an established and important cause of fertility problems in thoroughbred mares, causing great loss to the industry. As a part of the age-related endometrial degeneration complex, an excessive deposition of collagen leading to endometrial fibrosis is particularly important due to the limitations it causes to uterine function. The consequences include reduced efficacy of uterine defence mechanisms and a decrease in the uterine capacity for foetal nutrition. Extensive research into the process of fibrosis in other organs has shown that this condition results from the malfunction of physiological tissue repair mechanisms. These mechanisms revolve around tissue fibroblasts that due to continuous stimulation secrete excessive amounts of collagen and inhibit the activation of factors essential to the normal collagen degradation occurring in scar resolution. Among these factors are the MMPs, an enzyme family with the ability to degrade extracellular matrix components such as collagen during the normal repair mechanisms following tissue injury. The malfunction in the regulation of these enzymes is important in the development of fibrosis in the liver and other organs. In this study it was demonstrated that MMPs are involved in the acute uterine inflammatory response and that they were secreted by infiltrating inflammatory cells. The cellular mechanisms observed during endometritis in normal mares were comparable to the normal repair mechanisms known to be altered in the fibrosis of other organs. These enzymes were present in equine foetal fluids, and their regulation may be important in the process of abortion and stillbirth. It was demonstrated that inbreeding may be correlated with increased deposition of endometrial collagen in a study population of the Icelandic horse breed even though this breed appears to exhibit less severe endometrial degeneration than what is known in lighter breeds. It is likely that genetic predisposition leads to the disruption of normally self-limiting inflammatory and repair mechanisms in the endometrium, resulting in constant activation of collagen synthesis by local and infiltrating cells. This thesis has shown that tissue repair mechanisms involving MMPs are likely to be involved in endometrial fibrosis in the mare. An inherent alteration in these mechanisms may play a role in the pathogenesis of this condition, and might arise due to genetic predisposition. Further understanding of the pathways leading to excess collagen amounts in the endometrium may produce preventative measures, and even therapeutic targets.

Post-operative adhesion is a common complication after abdominal surgery, with high impact on patient wellbeing and healthcare costs. The repair of peritoneum is a complex process involving orderly phases which share some common features to normal wound healing. These include coagulation, infiltration of inflammatory cells, cell proliferation, extracellular matrix (ECM) deposition and remodelling, often with overlap between phases. The unique feature of peritoneal repair is that both small and large peritoneal wounds heal in a similar time. The peritoneum is a monolayer of elongated, flattened, squamous-like peritoneal mesothelial cells (PMC). Local mesothelial cell proliferation, centripetal cell migration from the wound edge, as well as incorporation of free-floating mesothelial cells may all contribute to repair of injured peritoneum. To date, the only well-characterised pathologic mechanism underlying post-operative adhesion formation at the molecular level is the formation of the fibrin layer and regulation of peritoneal fibrinolytic capacity. However, the contributions of collagen deposition and ECM remodelling to the peritoneal repair mechanism are not well understood. This thesis focuses on the role of PMC in the regulation of ECM deposition and remodelling in response to inflammatory stimuli in both in vivo and in vitro models, aiming to identify other key pro-fibrotic factors involved in the development of post-operative adhesion. We first identified that lysyl oxidase (LOX) played a key role in the progression of peritoneal fibrosis by regulating collagen cross-linking and deposition in vivo. The inhibition of LOX enzyme activity prevented the formation of fibrotic tissue by reducing collagen deposition. Meanwhile, dexamethasone (DEX) treatment also minimized the fibrotic response. Furthermore, in vitro studies showed that the induction of collagen deposition factors in PMC, including LOX and pro-collagen I, required both IL-1 and TGF-β signalling pathways. Thus, the combination of IL-1 + TGF-β was adopted in an in vitro model to mimic the inflammatory environment during peritoneal repair. Treatment of PMC with IL-1+TGF-β caused an epithelial-to-mesenchymal transition (EMT). These transformed PMC had enhanced cell motility and were more adherent to fibronectin. Finally, a real-time quantitative PCR-based microarray was used for genomic analysis of ECM-adhesion-related PMC genes in response to IL-1 and TGF-β treatment. The results showed that IL-1 was more involved in regulating ECM degradation by inducing expression of matrix metalloproteinase (MMP) genes, whereas TGF-β mainly affected genes involved in ECM deposition, including collagens and other ECM components. However, both cytokines were shown to regulate some key genes involved in the development of adhesion, including COL16A1, COL7A1, FN1, ITGA5, and TGFB1. Moreover, IL-1 was shown to reduce ITGA4 and ITGB6 expression affecting adherence of PMC to basement membrane, while TGF-β increased MMP14 and MMP16 expression, which could facilitate invasion of EMT-transformed PMC to the site of tissue repair. In summary, this thesis indicates that LOX plays an important role in peritoneal fibrosis. Secondly, a combination of IL-1 and TGF-β1 treatment demonstrates how these factors can act in concert to orchestrate tissue remodelling during peritoneal repair. Finally, genomic analysis of ECM-adhesion genes increases our understanding of aspects of the pathology of post-operative adhesion and identifies novel potential therapeutic targets to prevent adhesion formation.

Glucocorticoids are steroid hormones that have potent anti-inflammatory actions. Endogenous glucocorticoid action is modulated by 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyses the interconversion of active glucocorticoids (cortisol, corticosterone) and intrinsically inert forms (cortisone, 11-dehydrocorticosterone). There are 2 isozymes; 11β-HSD type 1 regenerates active glucocorticoids in vivo whereas 11β-HSD type 2 inactivates glucocorticoids. Although 11β-HSD1 is highly expressed in the lung, its role there has been little explored. In this study, the expression and localization of 11β-HSD1 mRNA in lung was confirmed by in situ hybridization. Immunohistochemical staining of mouse lung localized 11β-HSD1 to the cytoplasm of fusiform cells in alveolar walls, in a multivesicular pattern characteristic of interstitial fibroblasts. A lung fibrosis model of inflammation was used to test the role and regulation of 11β-HSD1. The results suggest that levels of 11β-HSD1 mRNA and enzyme were not changed during bleomycin-induced lung inflammation. However, 11β-HSD1-deficient mice showed a more severe inflammatory response than congenic wild-type controls, with greater inflammatory cell infiltration into the lung, and increased levels of HO-1 and iNOS mRNA 14 days following bleomycin installation into lung. Picrosirius red staining of lung sections suggested more collagen deposition in 11β-HSD1-deficient mice than in wild-type controls during the course of the lung inflammatory response. Moreover, whereas naïve 11β-HSD1-deficient mice had significantly lower collagen content in lung (84% of WT levels, p<0.05). 28d after bleomycin there was no significant difference between genotypes (KO having 94% of WT levels, p=0.42) confirming more collagen production in 11β-HSD1-deficient mice following bleomycin. Fibroblasts are critical in the regulation of inflammatory responses and are essential in the model of bleomycin-induced lung injury. Lung fibroblasts may have a different transcriptional regulation of 11β-HSD1 compared to other tissues. In the majority of tissues, 11β-HSD1 can be transcribed from 2 promoters; the P1 promoter is the main promoter used in lung, with other tissues mainly using the P2 promoter. To address the relevance of the P1 promoter in lung and to identify the cell type using the P1 promoter, mouse lungs were collagenase-digested to isolate primary fibroblast and epithelial cells. Isolated lung fibroblasts highly expressed 11β-HSD1, predominantly from the P1 promoter. During passage, primary lung fibroblasts switched promoter usage from P1 to P2. In fibroblast primary culture, treatment with TGF-β for 72h markedly decreased 11β-HSD1 expression to 38% of untreated levels, an effect which was reversed by SB431542, a TGF-β receptor antagonist. Whilst TGF-β reduced levels of mRNA initiating at the P2 promoter, initiation from the P1 promoter was completely repressed. Treatment with TGF-β receptor antagonist increased levels of P1-initiated 11β-HSD1 mRNA by 6.6-fold compared to untreated cells. These data suggest that the switch in 11β-HSD1 promoter usage may be regulated by TGF-β during an inflammatory response. Furthermore, as the P1 and P2 promoters are differentially regulated (e.g. by C/EBPβ, a cytokine-responsive transcription factor), the promoter switch may place 11β-HSD1 under a different transcriptional regulation during inflammation. Taken together, these results suggest that 11β-HSD1 deficiency worsens lung inflammation and results in greater lung fibrosis. Therefore, amplification of intracellular glucocorticoids levels, by 11β-HSD1, may represent an important mechanism to limit the inflammatory response and shape fibroblast function, limiting subsequent collagen production and fibrosis.

Class of 2012 Abstract / Specific Aims: We sought to determine the influence of genetic variation of ADRB2 on the airway response to albuterol in patients with CF when compared to matched healthy controls at baseline and at 60 minutes following the administration of albuterol (2.5mg diluted in 3ml normal saline). Methods: Baseline pulmonary function (forced vital capacity, FVC, forced expiratory flow in 1-second, FEV1, mid-maximal expiratory flow, MMF, and forced expiratory flow at 50% of the FVC) was assessed in 17 patients with CF and 31 healthy subjects. Main Results: As expected, the healthy group had higher baseline pulmonary function when compared to the CF group (FVC=97±3 vs. 83±5; FEV1=95±3 vs. 72±6; MMF=90±4 vs. 54±8, % predicted for healthy and CF, respectively, mean±SE, p<0.05 for all. We compared Arg16Arg to Arg16Gly/Gly16Gly subjects. There was no effect of genotype on the response to albuterol in healthy subjects. However, in the CF group, we found that the Arg16Arg group (n=6) had an attenuated response to β-agonist when compared to the Gly-containing group (n=11) (FVC=0±0.9 vs. 6±3: FEV1=3±1 vs. 7±4: MMF=12±3 vs. 12±5 % change, for Arg16Arg and Gly-containing groups, respectively, p<0.05 for FVC, p=0.06 for FEV1). Conclusions: These results demonstrate a differential response to β-agonists according to genetic variation of the ADRB2 at amino acid 16. Due to the differences in FVC and FEV1 but not in MMF, these data suggest that the genetic difference in airway function is primarily in bronchodilation of the larger airways.