<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

Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.

Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.

ABSTRACT In humans, advanced malignancies are often targeted with broad-spectrum cytotoxic drugs that engender several detrimental side effects, in addition to their primary usage for eradicating cancerous cells. One of the lesser-researched of these effects, histological distortion of the olfactory system impedes a patient's ability to smell, perceive flavor, and ultimately may interfere with their nutritional intake and recovery from chemotherapy. Recent studies have indicated that cytotoxic drugs can damage gustatory epithelia immediately following administration (Mukherjee & Delay, 2011, 2013). We sought to observe the histological effects that cyclophosphamide (CYP), one of the oldest and most popular alkylating antineoplastic agents, may have on the murine main olfactory epithelium (MOE) and vomeronasal organ (VNO). We utilized two immunohistochemical antibodies to label cells in the olfactory epithelia: anti-Ki67, a marker strictly associated with cell proliferation; and, anti-Keratin 5, a marker for the cytoskeleton of horizontal basal cells. Twenty-eight C57BL/6 mice were administered a single intraperitoneal injection of CYP (75 mg/kg), while 20 control mice were administered saline, all at approximately seven weeks of age. Mice were euthanized at days one, two, six, 14, 30, and 45 post injection; subsequently, they were perfused with 4% paraformaldehyde, decalcified, cryoprotected, cryosectioned, and incubated with anti-Ki67 and anti-Keratin 5 antibodies, sequentially. Quantification results by fluorescent imaging of labeled sections revealed a significant decrease in the number of proliferative cells in the MOE and VNO of CYP-injected mice within the first 10 days post injection, followed by a compensatory period of increased cell proliferation through day 45 post injection, compared to saline-injected mice. Co-localization of horizontal basal cells and proliferative cells in the MOE and VNO of CYP-injected mice was significantly amplified at approximately 14 and 45 days post injection, respectively, compared to saline-injected mice. Our results suggest that administration of CYP can rapidly depress the populations of proliferative cells in the murine MOE and VNO; consequently, horizontal basal cells may afford restoration of the proliferative cell populations in the murine MOE and VNO, 14 to 45 days post injection, respectively.

O cão é a única espécie, além do homem, em que o câncer de próstata (CP), a neoplasia intraepitelial prostática (PIN) e a hiperplasia prostática benigna (HPB) ocorrem espontaneamente, permitindo dessa forma que se realize estudo comparativo de afecções benignas e malignas da próstata. Acredita-se que a existência de stem cells malignas, localizadas na camada de células basais da próstata, seja um dos fatores responsáveis pelo insucesso da terapia por ablação androgênica que ocorre na maioria dos carcinomas prostáticos avançados. O objetivo deste estudo foi caracterizar a próstata canina quanto a aspectos envolvidos na evolução para o carcinoma prostático, tentando identificar a origem celular e as alterações das lesões pré-neoplásicas. Foram obtidas 44 próstatas na necrópsia. Amostras prostáticas foram fixadas em metacarne, embebidas em parafina e seccionadas a 5µm para a coloração com hematoxilina eosina (HE) e avaliadas em relação à presença de hiperplasia, prostatite, PIN e neoplasia. Além disso, cortes corados em HE representando cada afecção foram utilizados na determinação da área nuclear média por morfometria computadorizada. Cortes histológicos obtidos em lâminas silanizadas foram utilizados na imunoistoquímica para células basais (p63 e 34E-12), conexinas 32 e 43, receptor de andrógeno (AR) e antígeno nuclear de proliferação celular (PCNA). Amostras foram coletadas também em nitrogênio líquido e mantidas a 80o C para a realização do PCR quantitativo em tempo real, para a determinação da expressão do RNAm do AR, e para a realização do Western blot, para a determinação da expressão da conexina 43. As afecções mais freqüentes foram a prostatite e a hiperplasia prostática benigna. Foi observada uma maior porcentagem de células basais e um alto índice proliferativo, como demonstrado pela imunoistoquímica para o PCNA, na neoplasia intraepitelial prostática. Além disso, observou-se nessas lesões marcação nuclear heterogênea para o AR, menor em relação à dos ácinos benignos. Ao contrário do observado na próstata humana, não foi observada expressão das conexinas 32 e 43 na próstata canina (normal ou com PIN). A área nuclear média, obtida pela morfometria computadorizada, foi maior em células epiteliais de ácinos apresentando PIN e/ou neoplasia em relação à de células epiteliais de ácinos benignos. Observou-se expressão variável do RNAm para o AR nas PINs e neoplasias, utilizando-se o PCR em tempo real. Estes achados sugerem que células basais malignas desempenham papel na origem da neoplasia intraepitelial prostática e possuem capacidade de proliferar a despeito da expressão heterogênea do receptor de andrógeno. / Dogs are the only animal other than man to develop prostate cancer, prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (HPB) spontaneously, allowing the comparison between benign and malignat affections of prostate. Malignant stem cells among the basal cell layer of the prostate are believed to play an important role in the failure of androgen-ablation therapy that occurs in most advanced prostate cancer. The goal of this study was to characterize the canine prostate in relation to evolution to prostatic carcinoma, trying to identify the cellular origin and the alterations of pre-neoplastic lesions. Forty-four canine prostates were obtained at necropsy. Prostatic samples were fixed in methacarn, embedded in paraffin wax and sectioned into 5µm-thick slices for hematoxylin eosin (HE) staining and evaluated for the presence of hyperplasia, prostatitis, PIN and neoplasia. Moreover, HE stained sections representing each affection were used to determine the mean nuclear area by computerized morphometry. Tissue sections obtained in silanized slides were used in immunohistochemical staining for basal cells (p63 and 34E-12), connexins 32 and 43, androgen receptor (AR) and proliferating-cell nuclear antigen (PCNA). Quantitative real-time PCR to determine the expression level of AR at the mRNA level and Western blot to protein levels of connexin 43 were examined in samples collected using liquid nitrogen and kept at 80o C. The most common lesions were prostatitis and benign prostatic hyperplasia. The prostatic intraepithelial neoplasia exhibited a higher percent of basal cells and was highly proliferative, as demonstrated by PCNA immunohistochemistry. Moreover, these lesions exhibited heterogeneous nuclear AR staining, lower in comparision with benign acini. In contrast to human prostate, the canine prostate (normal or harboring PIN) did not express the connexins 32 and 43. The mean nuclear area measured by computerized morphometry was greater in epithelial cells of PIN and neoplastic acini than that of benign acini. We found variable RNAm AR expression in prostatic intraepithelial neoplasia and neoplasia by real-time PCR. These findings suggest that malignant basal cells may play a role in the origin of PIN and can proliferate despite the heterogeneous AR expression.

O cão é a única espécie, além do homem, em que o câncer de próstata (CP), a neoplasia intraepitelial prostática (PIN) e a hiperplasia prostática benigna (HPB) ocorrem espontaneamente, permitindo dessa forma que se realize estudo comparativo de afecções benignas e malignas da próstata. Acredita-se que a existência de stem cells malignas, localizadas na camada de células basais da próstata, seja um dos fatores responsáveis pelo insucesso da terapia por ablação androgênica que ocorre na maioria dos carcinomas prostáticos avançados. O objetivo deste estudo foi caracterizar a próstata canina quanto a aspectos envolvidos na evolução para o carcinoma prostático, tentando identificar a origem celular e as alterações das lesões pré-neoplásicas. Foram obtidas 44 próstatas na necrópsia. Amostras prostáticas foram fixadas em metacarne, embebidas em parafina e seccionadas a 5µm para a coloração com hematoxilina eosina (HE) e avaliadas em relação à presença de hiperplasia, prostatite, PIN e neoplasia. Além disso, cortes corados em HE representando cada afecção foram utilizados na determinação da área nuclear média por morfometria computadorizada. Cortes histológicos obtidos em lâminas silanizadas foram utilizados na imunoistoquímica para células basais (p63 e 34E-12), conexinas 32 e 43, receptor de andrógeno (AR) e antígeno nuclear de proliferação celular (PCNA). Amostras foram coletadas também em nitrogênio líquido e mantidas a 80o C para a realização do PCR quantitativo em tempo real, para a determinação da expressão do RNAm do AR, e para a realização do Western blot, para a determinação da expressão da conexina 43. As afecções mais freqüentes foram a prostatite e a hiperplasia prostática benigna. Foi observada uma maior porcentagem de células basais e um alto índice proliferativo, como demonstrado pela imunoistoquímica para o PCNA, na neoplasia intraepitelial prostática. Além disso, observou-se nessas lesões marcação nuclear heterogênea para o AR, menor em relação à dos ácinos benignos. Ao contrário do observado na próstata humana, não foi observada expressão das conexinas 32 e 43 na próstata canina (normal ou com PIN). A área nuclear média, obtida pela morfometria computadorizada, foi maior em células epiteliais de ácinos apresentando PIN e/ou neoplasia em relação à de células epiteliais de ácinos benignos. Observou-se expressão variável do RNAm para o AR nas PINs e neoplasias, utilizando-se o PCR em tempo real. Estes achados sugerem que células basais malignas desempenham papel na origem da neoplasia intraepitelial prostática e possuem capacidade de proliferar a despeito da expressão heterogênea do receptor de andrógeno. / Dogs are the only animal other than man to develop prostate cancer, prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (HPB) spontaneously, allowing the comparison between benign and malignat affections of prostate. Malignant stem cells among the basal cell layer of the prostate are believed to play an important role in the failure of androgen-ablation therapy that occurs in most advanced prostate cancer. The goal of this study was to characterize the canine prostate in relation to evolution to prostatic carcinoma, trying to identify the cellular origin and the alterations of pre-neoplastic lesions. Forty-four canine prostates were obtained at necropsy. Prostatic samples were fixed in methacarn, embedded in paraffin wax and sectioned into 5µm-thick slices for hematoxylin eosin (HE) staining and evaluated for the presence of hyperplasia, prostatitis, PIN and neoplasia. Moreover, HE stained sections representing each affection were used to determine the mean nuclear area by computerized morphometry. Tissue sections obtained in silanized slides were used in immunohistochemical staining for basal cells (p63 and 34E-12), connexins 32 and 43, androgen receptor (AR) and proliferating-cell nuclear antigen (PCNA). Quantitative real-time PCR to determine the expression level of AR at the mRNA level and Western blot to protein levels of connexin 43 were examined in samples collected using liquid nitrogen and kept at 80o C. The most common lesions were prostatitis and benign prostatic hyperplasia. The prostatic intraepithelial neoplasia exhibited a higher percent of basal cells and was highly proliferative, as demonstrated by PCNA immunohistochemistry. Moreover, these lesions exhibited heterogeneous nuclear AR staining, lower in comparision with benign acini. In contrast to human prostate, the canine prostate (normal or harboring PIN) did not express the connexins 32 and 43. The mean nuclear area measured by computerized morphometry was greater in epithelial cells of PIN and neoplastic acini than that of benign acini. We found variable RNAm AR expression in prostatic intraepithelial neoplasia and neoplasia by real-time PCR. These findings suggest that malignant basal cells may play a role in the origin of PIN and can proliferate despite the heterogeneous AR expression.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

At present, prostate cancer is a significant public health issue throughout the world and is the second leading cause of cancer deaths in older men. The prostate specific antigen or PSA (which is encoded by the kallikrein 3/KLK3 gene) test is the current most valuable tool for the diagnosis and management of prostate cancer. However, it is insufficiently sensitive and specific for early diagnosis, for staging of prostate cancer or for discriminating between benign prostatic hyperplasia (BPH) and prostate cancer. Recent research has revealed another potential tumour marker, glandular kallikrein 2 (KLK2 gene/hK2 protein), which may be used alone or in conjunction with PSA to overcome some of the limitations of the PSA test. Twelve new kallikrein gene family members have been recently identified and, like hK2 and PSA, many of these genes have been suggested to be involved in carcinogenesis. In this study, the cellular localisation and level of expression of several of these newer kallikreins (KLK4, KLK5, KLK7, KLK8 and KLK11) was examined in prostate tissue, to provide an understanding of the association of their expression with prostatic diseases and their potential as additional biomarkers. Like PSA and hK2, the present observation indicated that each of these proteins, hK4, hK5, hK7, hK8 and hK11, was detected within the cytoplasm of the secretory cells of the prostate glands. For the first time, all of these newly-identified proteins were shown to be expressed in prostatic intraepithelial neoplasia (PIN) lesions, in comparison to normal glands and cancer lesions. In addition to cytoplasmic secretory cell expression, the localisation of hK4 to the basal cells and nuclei in prostatic lesions was intriguing. The intensity of hK4 staining in prostate tissue was strongest in comparison to the other newly-identified kallikrein proteins (hK5, hK7, hK8 and hK11). Therefore, KLK4/hK4 expression was characterised further to define this cellular localisation and examined in non-prostatic tissue and also in a larger number of prostate tissues in an attempt to determine its potential value as a biomarker for prostate disease. Three hK4 antipeptide polyclonal antibodies, derived against N-terminal, mid-region and C-terminal hK4 amino acid sequences, were used. The hK4 N-terminal antipeptide antibody was used to demonstrate the cellular localisation of hK4 in kidney, salivary glands, liver, testis, colon carcinoma, heart, endometrium and ovarian cancer, for the first time. The presence of hK4 in these non-prostate tissues was consistent with the previous reports using RT-PCR. The dual cytoplasmic and nuclear localisation of hK4 observed in the prostate above was also seen in these tissues. Although hK4 was found widely expressed in many human tissue types, indicating that it is not prostate specific in its expression, the highest expression level of hK4 was seen in the prostate. Therefore, detailed expression patterns and levels of KLK4 mRNA and hK4 protein in the normal prostate and prostatic diseases and histopathological lesions were investigated and reported for the first time in this study. Twelve benign prostatic hyperplasia (BPH), 19 adenocarcinoma (Gleason grade 2-5) and 34 bone metastases from prostate cancer were analysed. Using in situ hybridisation, the expression of KLK4 mRNA was detected in the cytoplasm of the secretory cells of both normal and diseased prostate tissue. KLK4 mRNA was also noted in both secretory and basal cells of PIN lesions, but the basal cells of normal glands were negative. Using the hK4 N-terminal and mid-region antipeptide antibodies, hK4 was predominantly localised in the cytoplasm of the secretory cells. The intensity of hK4 staining appeared lowest in normal and BPH, and increased in PIN lesions, high Gleason grade prostate cancer and bone metastases indicating the potential of hK4 as a histopathological marker for prostatic neoplasias. Further studies are required with a larger cohort to determine its utility as a clinical biomarker. Small foci of atypical cells, which were found within normal glands, were also intensely stained. Surprisingly, hK4 protein was found in the nucleus of the secretory cells (but not the basal cells) of high grade PIN and Gleason grade 3 prostate cancer. The detection of KLK4 mRNA and hK4 protein in PIN lesions and small foci of atypical cells suggests that up-regulation of KLK4 expression occurs early in the pathology of prostate carcinogenesis. The finding of basal cell expression is not typical for the kallikreins and it is not clear what role hK4 would play in this cell type. With the use of the hK4 C-terminal antipeptide antibody, the staining was mainly localised in the nuclei of the secretory cells of the prostate glands. Although the nuclear localisation was readily noted in more than 90% of epithelial cells of the prostate gland with the C-terminal antibody, no difference in staining intensity was observed among the histopathological lesions of the prostate. The prominent nuclear localisation with the C-terminal antipeptide antibody was also shown to be distributed throughout the nucleus by using confocal microscopy. Further, by using gold-labelled particles for electron microscopy, the intracellular localisation of these hK4 antipeptide antibodies was reported here for the first time. Similar to the immunohistochemical results, the cytoplasm was the major site of localisation with the N-terminal and mid-region antipeptide antibodies. To further characterise the involvement of KLK4/hK4 in human prostate cancer progression, the transgenic adenocarcinoma mouse prostate (TRAMP) model was used in this study. In this study, mouse KLK4 (also known as enamel matrix serine protease -1, EMSP-1) was shown to be expressed in the TRAMP prostate for the first time. Previous studies had only shown the developing tooth as a site of expression for EMSP-1. The level of EMSP-1 mRNA expression was increased in PIN and prostate cancer lesions of the TRAMP model, while negative or low levels of EMSP-1 mRNA were seen in normal glands or in control mouse prostate tissue. The normal mouse prostate did not stain with any the three hK4 antipeptide antibodies. hK4 N-terminal and mid-region antipeptide antibodies showed positive staining in the cytoplasm of the epithelial cells of PIN and cancer lesions of the mouse prostate. The C-terminal antipeptide antibody showed distinctively nuclear staining and was predominantly localised in the nuclei of the glandular cells of PIN and cancer lesions of the mouse prostate. The expression patterns of both the mRNA and protein level for mouse KLK4 strongly supported the observations of KLK4/hK4 expression in the human prostate and further support the utility of the TRAMP model. Overall, the findings in this thesis indicate a clear association of KLK4/hK4 expression with prostate cancer progression. In addition, several intriguing findings were made in terms of cellular localisation (basal as well as secretory cells; nuclear and cytoplasmic) and high expression in atypical glandular cells and PIN, perhaps indicating an early involvement in prostate disease progression and, additionally, utility as basal cell and PIN histological markers. These findings provide the basis for future studies to confirm the utility of hK4 as a biomarker for prostate cancer progression and identify functional roles in the different cellular compartments.