Thesis (Ph. D.)--Ohio State University, 20043. / Title from first page of PDF file. Document formatted into pages; contains xiv, 115 p.; also includes graphics. Includes abstract and vita. Advisor: Clay B. Marsh, Dept.of Pathology. Includes bibliographical references (p. 102-115).

This thesis reviews the current literature on idiopathic pulmonary fibrosis (IPF), a progressive, scarring lung condition largely affecting older adults that is experiencing an increasing incidence in the U.S. and abroad. Two troubling clinical aspects of IPF are the difficulty of timely diagnosis and uncertain progression once diagnosed. The need for early detection is driven by the condition’s median survival rate post-diagnosis of about 3 years. Environmental and familial risk factors are important predictors of IPF, but cannot alone determine who is at risk for the condition. High-resolution computed tomography is currently the best non-invasive diagnostic tool, but many efforts are now underway to identify biological markers, which may aid not only in diagnosis, but illuminate both susceptibility and progression of the disease. Although the pathogenesis of IPF remains unclear, a compelling correlation has surfaced between the mechanics of IPF and herpes virus infection, which also may lead to a biological marker for the condition. Likewise, some genetic factors have shown promise in revealing pathogenesis and possible diagnosis. The only treatment currently available to ameliorate IPF is lung transplantation, but it is a last resort effort. In terms of pharmaceutical treatment, the most significant development has been the recent approval and use of two anti-fibrotic drugs, pirfenidone and nintedanib, that appear to slow the progression of the disease, but do not eliminate the fibrotic condition that impairs patients’ breathing. As efforts progress in addressing affirmative treatments for IPF, there is consensus that not enough is being done to address palliative and psychological needs of IPF. In sum, a review of the current literature suggests tremendous accomplishments have made in treating what remains a fatal condition, but much work remains to truly understand how and why IPF occurs, and whether, short of lung transplantation, there are treatments that can improve, not just maintain, patients’ health.

Abstract Fibrotic pulmonary and pleural disorders represent a group of intrathoracic disorders with different etiologies and prognoses. A prominent part of both pulmonary and pleural fibrotic disorders remains etiologically unknown. An essential feature for all these disorders is an increase and disarray of many extracellular matrix proteins which take part in the remodeling of the fibrotic tissue. Further, the injury in pulmonary as well as in pleural fibrosis occurs often at the border between the epithelial or mesothelial and the mesenchymal cells breaking the epithelial basement membrane. Tenascin is an oligomeric matrix glycoprotein of the extracellular matrix. The best known isoforms are tenascin -C, -X, -R, -Y and -W. Tenascin-C is synthesized during embryonic development, expressed in a variety of tumors, being absent or scantily expressed in most adult tissues. The function of tenascin-C is still unclear. In lung, tenascin-C has been shown to be expressed in fetal lung during branching morphogenesis, benign and malignant lung tumors, idiopathic pulmonary fibrosis, sarcoidosis and asthma. The aim of the present study was to study tenascin-C (later called tenascin) expression in various types of pulmonary fibrosis such as usual interstitial pneumonia (UIP), desquamative interstitial pneumonia (DIP), nonspecific interstitial pneumonia (NSIP), bronchiolitis obliterans organizing pneumonia (BOOP), sarcoidosis and extrinsic allergic alveolitis as well as in fibrotic and inflammatory disorders of the pleura of different etiologies. Further, the aims were to compare the accumulation of tenascin with the prognosis in UIP, to confirm the immunohistochemical findings in UIP by Western blotting and immunoelectron miscroscopic (immuno-EM) studies, to investigate which cells synthesize tenascin in UIP and in pleural fibrosis by mRNA in situ hybridization, and to determine whether epithelial lining fluid (ELF) and serum tenascin concentration are increased in patients with UIP, sarcoidosis and extrinsic allergic alveolitis. Tenascin was shown to be increased by immunohistochemical studies in all types of pulmonary and pleural fibrotic disorders included in the study. In UIP, increased tenascin expression was associated with a shortened survival time of the patients. In immuno-EM, labeling for tenascin was seen within type II pneumocytes. UIP cases showed reactivity for a polypeptide of Mr = 200 000 by Western blotting. Myofibroblasts and type II pneumocytes were mainly shown to synthesize tenascin in UIP. Also in pleural fibrosis myofibroblasts, and in addition possibly mesothelial cells, were observed to be responsible for its synthesis. ELF and serum tenascin concentrations were increased in UIP, sarcoidosis and extrinsic allergic alveolitis. In conclusion, tenascin expression is increased in pulmonary and pleural fibrotic disorders, especially in newly formed fibrosis. In UIP, tenascin is actively synthesized at the sites of recent epithelial injury, suggesting that it plays an important role in the fibrogenesis in the lung.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xi, 145 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 134-142).

In this thesis I examined the role of circulating monocytes and lung macrophages in the pathogenesis of the early fibrotic, progressive fibrotic and resolution phases of pulmonary fibrosis. Pulmonary fibrosis with destruction of lung architecture and consequent respiratory failure and death represents a massive worldwide health burden. Although idiopathic pulmonary fibrosis (IPF) is the archetypal and most common cause of lung fibrosis, numerous respiratory diseases can progress to pulmonary fibrosis, and this usually signifies a worse prognosis. Importantly, the incidence and prevalence of IPF continue to rise and it remains one of the few respiratory conditions for which there are no effective therapies. The mechanisms resulting in pulmonary fibrosis are controversial. Early work in the 1980s and 1990s suggested that lung macrophages were important. However, at the turn of the 21st century there was a shift to a belief that pulmonary fibrosis resulted from aberrant wound healing as a consequence of repetitive epithelial injury from an as yet unknown cause. However, with the ever expanding knowledge of the importance of macrophages in other fibrotic conditions such as the kidney and liver, the potential importance of macrophages in pulmonary fibrosis has become more pertinent. Using an in vivo depletional strategy in several murine models of lung fibrosis, in conjunction with human studies, I sought to characterise the role of circulating monocytes and lung macrophages in the pathogenesis of pulmonary fibrosis. I have established that circulating monocytes and lung macrophages are not critical for the development of early lung fibrosis. In contrast, circulating monocytes and lung macrophages are important during the progressive fibrotic phase of lung fibrosis. Furthermore, my data suggest that the pro-fibrotic alternatively activated macrophages may be the sub-class of macrophages that mediate this fibrogenic effect. In addition and in contrast, I have established that lung macrophages are required for the resolution of fibrosis. This finding is in keeping with important work performed in the field of liver fibrosis. There is an ever increasing literature examining the role of matrix metalloproteinases (MMPs) during tissue fibrosis and repair. My work has suggested that during lung fibrosis there may be compartmental specific functions of MMPs that regulate lung fibrogenesis, although more work is required before this exciting finding can be properly defined.

IPF is a progressive disease, characterized by dysregulated fibrosis of the extracellular matrix (ECM). The pathobiology of the disease is still unknown, and the median survival post-diagnosis is about 3-5 years. The two current US FDA approved drugs for IPF (nintedanib and pirfenidone) slow, but fail to reverse, disease progression. There is cumulating research that suggests the ECM is an active player in fibrosis. In this thesis, we summarized the current knowledge of ECM-cell interactions in the context of pulmonary fibrosis. To gain more mechanistic insight into the ECM characteristics that dictate cell behavior, we established a 3D ECM ex vivo system to assess the nonfibrotic and fibrotic ECM’s effect on fibroblasts. The ECM appears to promote both pathological and physiological cellular changes, depending on its structural and compositional properties. We also used this 3D ex vivo system as a preclinical tool to test the effect of directly inhibiting mechanotransduction in the fibrotic ECM – fibroblast profibrotic relationship. Lastly, since the fibrotic ECM seems to play a key role in progressive fibrosis, we evaluate if researchers are appropriately using the bleomycin model by starting interventions after ECM fibrosis is established. Over the past decade in the field, there has been an overall improvement in the appropriate therapeutic timing. In the preventative studies, however, there is still an inadequate characterization of inflammation. There is also poor transparency of preclinical-bleomycin data for clinically tested interventions for IPF. Addressing these shortcomings may improve the utility of the model at predicting an intervention’s success in clinical trials. These findings illustrate the ECM’s role in driving pulmonary fibrosis. Therefore, the ECM should be further investigated to understand disease progression, and reproduced in preclinical models to test interventions. This will improve the transition of pathobiological findings into efficient drug development for this devastating disease. / Thesis / Candidate in Philosophy / Idiopathic pulmonary fibrosis (IPF) (idiopathic - unknown cause; pulmonary - lungs; fibrosis - scarring) is characterized by progressive scarring of the lung extracellular matrix (ECM). The ECM is an organ’s backbone that provides structural and biochemical support to surrounding cells. Continued ECM scarring can lead to difficulty breathing, cough, and ultimately death. The cause of IPF is unknown, however, studies suggest that the scarred ECM can promote further scarring, and cause disease progression. In this thesis, we summarized the current knowledge of how the ECM interacts with cells. Using a 3D model we see that depending on the ECM’s structure and composition, it can promote both disease and healthy cellular changes. Lastly, we evaluate if researchers are appropriately using the bleomycin model (most common preclinical model for pulmonary fibrosis) by testing interventions after ECM fibrosis is established. We propose changes to improve its usefulness as a preclinical tool for IPF.

Pulmonary fibrosis (PF) is a serious lung disease, as its life expectancy is only 3-5 years upon occurrence and more than 50 % of the cases are idiopathic, i.e., unknown cause. Two drugs, pirfenidone (PIR) and nintedanib, have recently been approved; however, their efficacies are moderate without evidence of prolonged survival. While this is primarily due to our insufficient knowledge about key PF pathogenesis, inductions of oxidative stress and transforming growth factor-b1 (TGF-b1) have been suggested in PF lungs. Hence, anti-oxidative melatonin (MEL) and curcumin (CUR) have been studied yet their efficacies remain moderate without clear understanding about the mechanisms of action. Accordingly, this project hypothesized that a novel hybrid molecule of MEL and CUR, AM24, was a more potent inhibitor against oxidative stress and TGF-b1 induced PF pathobiologic events than MEL or CUR, so that its pulmonary delivery enabled therapeutic intervention in an animal model of PF. Free radical scavenging activity and various in vitro lung cell-based anti-fibrotic activities of AM24 were determined and compared with those of MEL and CUR as well as their admixture (MEL+CUR) and PIR. Pulmonary administration of AM24 was then examined for therapeutic intervention in a rat model of bleomycin (BLM)-induced experimental PF. AM24 was equipotent to MEL, but less potent than CUR in the hydrogen peroxide-induced free radical (ABTS) scavenging assay, ranked with the half-maximal inhibitory concentration (IC50) of 25.7, 32.0 and 11.4 uM, respectively. However, in the in vitro human lung fibroblast systems, AM24 was shown to be more potent than MEL or CUR and notably than MEL+CUR or PIR in the TGF-b1 induced 1) collagen synthesis by the picrosirius red assay, 2) proliferation by the MTT assay; and 3) differentiation to myofibroblast by western blot analysis of a myofibroblast marker, a-smooth muscle actin (a-SMA). In detail, at 10 uM, AM24 inhibited TGF-b1 induced 1) collagen synthesis by 90 %; 2) proliferation by ~72 %; and 3) differentiation to myofibroblast completely, while MEL, CUR, MEL+CUR and PIR resulted in 30-55 % or insignificant inhibition. In addition, in the in vitro human lung alveolar epithelial cell system, AM24 at 10 uM almost completely inhibited TGF-b1 induced epithelial-mesenchymal transition (EMT), as measured with western blot expressions of an epithelial marker, E-cadherin, and a mesenchymal marker, vimentin. Again, MEL, CUR, MEL+CUR and PIR exerted much less inhibitory activities. Hence, all these results consistently suggested that AM24 was a unique hybrid molecule of MEL and CUR and possessed highly potent anti-fibrotic activities in addition to the free radical scavenging activity. AM24 was then examined for therapeutic intervention in an in vivo rat model of BLM-induced PF. BLM was orotracheally spray-dosed to the lungs at 0.6 mg/kg on day 1 to develop experimental PF in 14 days. Lung administrations of AM24 at 0.1 mg/kg commenced at 6 hours of BLM induction on day 1 and continued thrice weekly over two weeks. Functional treadmill exercise endurance was measured on day 12 and 15; and lungs were harvested upon sacrifice on day 16. Overall, AM24 showed significant intervention activities as follows: 1) exercise endurance was reduced only ~20%, much lower than 78% of the untreated PF rats; 2) reduced fibrotic tissue area and alveolar structural destruction were seen by histological examinations; and 3) lung’s induced collagen deposition was inhibited by ~78 %. However, unlike the literature, the lung’s TGF-b1, PCNA (a cell proliferation marker), and a-SMA (a differentiation marker), were not largely induced in the BLM-induced PF model, so that the intervention activities of AM24 to these markers were not clearly shown. In contrast, induced EMT was seen in the BLM-induced model, represented by increased mesenchymal marker, vimentin, and by decreased epithelial marker, E-cadherin; and AM24 appeared to counter this induced EMT. Accordingly, while the BLM-induced PF model may need further optimizations for clearer pathogenic changes, AM24 exerted certain degree of in vivo efficacies with a lung dose of 0.1 mg/kg, which was much lower than the effective doses of MEL, CUR, PIR and nintedanib seen in the literature with BLM induced PF model. In conclusion, this thesis study has provided an early proof-of-concept for AM24, a novel MEL-CUR hybrid molecule, being potently anti-oxidative and anti-fibrotic in the in vitro lung cell-based assessments. As a result, AM24 enabled therapeutic intervention just with a lung dose of 0.1 mg/kg in the BLM-induced rat model of experimental PF.

No description available.

Bleomycin-induced pulmonary fibrosis (BIPF) is a disease caused by the chemotherapeutic bleomycin in which normal lung tissue is replaced with fibrous tissue that is unable to fulfill the normal functions of oxygen exchange in the lung. The development of this disease is dictated, in part, by a set of genes governing susceptibility. Knowledge of such genetic modifiers offers novel therapeutic targets and improved understanding of the pathways involved in the disease process. Our method integrates different data types to identify genes that have a single nucleotide polymorphism (SNP) disrupting a transcription factor binding site that modifies the outcome of BIPF. Our current approach examines over 7 million SNPs, phenotypes from 11 inbred mice strains, mRNA expression data, linkage data, and over 600 transcription factor binding sites from the TRANSFAC database. Each gene is scored with respect to each data type and then integrated using a weighted multiplicative model. Our integrative method produces a list of potential genetic modifiers that will be validated using allelic imbalance tests, existing knockout mice if available, siRNA or antibodies. By investigating these genes, we have identified several that are related to known genetic modifiers or others that make biological sense such as H2-Q2, an antigen presentation gene, and Runx1, a transcription factor known to interact with the known BIPF genetic modifiers Smad3 and Cdkn1a.

Background and objective: Asbestos can cause various pulmonary diseases including asbestosis, pleural plaques and pleural thickening. Animal and in vitro studies suggest that the toxicity of asbestos is due to the iron content of the fibres which not only generate oxidants directly, but also activate the inflammatory cells in the lung that contribute to oxidative stress. This study therefore sought to establish data in man to corroborate the animal and in vitro evidence. Exhaled breath condensate (EBC) collection is a novel, non-invasive technique to collect samples from the lung for investigating inflammatory biomarkers of lung diseases. This technique is harmless, rapid and easily repeatable which leads itself to the investigation of lung diseases such as asbestos-related diseases and pulmonary fibrosis (PF) that are otherwise difficult to study. The hypothesis tested was that oxidative and nitrosylative stress markers will be elevated in the EBC of patients with asbestos-related diseases and PF compared to normal control subjects. Methods: The study design was a cross-sectional and observational in vivo study whereby EBC was collected and fractional exhaled nitric oxide (FeNO) and carbon monoxide (eCO) were measured. EBC markers including pH, hydrogen peroxide (H2O2), total nitrogen oxides (NOx), 3-nitrotyrosine (3-NT), 8-isoprostane (8-iso), total protein and transforming growth factor-β1 (TGFβ1) were measured by microelectrode analysis, colorimetric and enzyme immunoassays. 3-NT and 8-iso were further examined by immunohistochemical techniques in samples of lung tissue. Results: Subjects with asbestosis had significantly raised levels of EBC H2O2, 8-iso, total protein and FeNO compared with healthy individuals. The same markers except H2O2, but with 3-NT and eCO were again significantly increased in those with other causes of PF, compared with control subjects. Heavy nitrotyrosine staining was found on the lung sections from patients with asbestosis and PF. Conclusions: This study confirmed that increased production of reactive oxygen and nitrogen species is associated with asbestos exposure and pulmonary fibrosis in vivo confirming animal and in vitro studies. Analysis of EBC may prove a useful non-invasive tool in exploring the basic pathophysiology of lung diseases in clinical research and may in the future be used to monitor progress in asbestosis and pulmonary fibrosis.