Oxidative stress and tissue remodeling are involved in the development of fibrosis of the lung and heart. Extracellular superoxide dismutase (EC-SOD) is an important antioxidant enzyme that has been shown to limit inflammation and fibrosis. Idiopathic pulmonary fibrosis is a lung disease characterized by severe, progressive interstitial fibrosis. Cardiac fibrosis has various causes but is a fatal side effect of treatment with the chemotherapeutic doxorubicin. Both of these diseases involve oxidant/antioxidant imbalances and can be studied through animal models. It was hypothesized that one mechanism through which EC-SOD protects the lungs and heart from inflammation and fibrosis is by preventing oxidative shedding of extracellular matrix components, specifically syndecans. In the lung, wild type and EC-SOD KO mice were treated with titanium dioxide, asbestos, or Bleomycin. Over the course of injury, EC-SOD KO mice have significantly higher levels of shed syndecan-1 and -4 in their bronchoalveolar lavage fluid and tissue. By IHC staining, the lung distribution of EC-SOD decreases in areas of fibrosis while syndecan-1 increases. Furthermore, in vitro, EC-SOD prevents syndecan-1 shedding from epithelial cells through its antioxidant activity and by directly binding to syndecan-1. Shed syndecan-1 is chemotactic to neutrophils and inhibits wound healing. In the heart, the signifiance of EC-SOD on normal heart morphology, fibrosis, and cardiac function were evaluated in wild type and EC-SOD KO mice in a doxorubicin-induced LV fibrosis model. The lack of EC-SOD causes LV posterior wall thinning and ventricular dilation without an insult. After an oxidative insult induced by doxorubicin, EC-SOD KO mice lost significantly more cardiac function compared to wild-type mice, had enhanced inflammatory cell recruitment, increased shedding of syndecan-1 from the heart and increased caspase-3 activation or apoptosis. In summary, this study shows that EC-SOD is important in the maintenance of normal cardiac morphology, the development of cardiac fibrosis, inflammation, apoptosis, and the loss of function associated with oxidative cardiac injury. In the lung, this investigation shows that the loss of pulmonary EC-SOD leaves syndecan-1 vulnerable to oxidative stress and that oxidant-induced loss of cell surface syndecan-1 impairs re-epithelialization, induces inflammation, and promotes a fibrotic microenvironment in the lung.

Brain aging, the phenomenon by which the passage of chronological time is associated with reduced brain volume, is important because it is regarded as a key component of increased dementia risk. The main purpose of this thesis is to present a model and supporting data that enhances knowledge of the underlying processes that drive brain aging and dementia risk. This has been done using structural and functional MRI scans from the Cardiovascular Health Study-Cognition Study (CHS-CS), a longitudinal community cohort study of elderly individuals that possesses extensive clinical and neuropsychological data. The model defended in this dissertation states that the relationship between older age and lower gray matter volume is not strictly a function of the passage of chronological time. Rather, older age is correlated with vascular diseases that themselves are a driving force behind brain aging. Most importantly, the three entities of aging, vascular disease, and neurodegeneration overlap in key strategic areas of the brain. Thus, a large factor behind dementia risk is that there are common brain areas that serve as sites of synergy by which age, vascular disease, and neurodegeneration can summate and thus amplify the risk for cognitive impairment and dementia. Using a whole brain method for analyzing structural MRI scans, we have found that the age and vascular disease, as reflected by white matter hyperintense lesions (WMHL) jointly affect areas of the brain known to be targeted by age and neurodegeneration and that they interact in these key strategic brain regions. Finally, results from perfusion MRI imaging will be reported showing that hypertension as the main predictor of lower regional cerebral blood flow. Taken together, these data will be interpreted to support the following model: structural and functional changes in an aging brain are modulated by hypertensive vascular disease. Additionally, the damage exerted by neurodegenerative processes on the brain is also modified by vascular disease. Finally, there are common strategic anatomical sites in which this synergy occurs and they include areas with important cognitive functions such as the hippocampus, cingulate gyrus, and precuneus. This model has several broad implications. First, it suggests that age related brain atrophy and perhaps even neurodegenerative atrophy itself can be reduced in magnitude if underlying vascular diseases are either prevented or better managed. Second, such a reduction in brain aging may lower risk for dementia by providing additional brain reserve. Third, key strategic brain regions provide a basis for further study and therapeutic targets.

Heart disease is the leading cause of death in the world and cellular cardiomyoplasty is an emerging therapeutic option to repair damaged myocardium. Muscle-derived stem cells (MDSCs) have been shown to have an improved regenerative capacity in bone, cartilage, and skeletal muscle when compared to myoblasts. After implantation into ischemic hearts, MDSCs display high levels of engraftment, induce neoangiogenesis, prevent cardiac remodeling, and elicit significant improvements in cardiac function. Notably, diversity in MDSC behavior has been associated with innate sex-related differences in skeletal muscle, bone, and cartilage. These results suggest that differences in inherent stem cell characteristics, including sex and age, could account for some of the outcome variability noted in clinical trials. Therefore we examined the effect of sex-related differences of MDSCs in cardiac repair. Transplantation of both cell sexes significantly improved cardiac function in comparison to saline. We also found that with increasing age, the proliferation and differentiation abilities of MDSCs decreased while their survival under stress conditions and vascular endothelial growth factor (VEGF) secretion remained unchanged. Our in vivo study demonstrates that the age of MDSCs does not impact their regenerative capacity but increasing the age of the host leads to decreased repair, which implies that the majority of age-related decreases in repair are due to changes in the microenvironment. We then examined methods to improve the efficacy of cell transplantation via preconditioning strategies. We first investigated the effects of mechanical stimulation on increasing the secretion of VEGF, which plays a major role in effecting cardiac repair after cell transplantation. Mechanical preconditioning significantly increased VEGF secretion, angiogenesis, and cardiac function after myocardial infarction, suggesting that this method of cell preconditioning could increase therapeutic efficacy. Another major issue with cell therapy is low cell survival after implantation. We hypothesized that increasing the level of antioxidants in MDSCs prior to transplantation could increase survival and therefore improve functional cardiac repair. We found that antioxidant pretreatment increased cell survival, cardiac function, and angiogenesis, and decreased scar formation. These pretreatment strategies have the potential to significantly improve the efficacy of cell transplantation and enhance the outcomes of heart disease patients.

The NF-B transcription factor family plays a central role in many aspects of the immune response, and activation of this family of transcription factors has been shown to trigger many disease processes. Thus, the ability to modulate NF-kB activity may be an attractive way to treat these diseases. We used an in vitro cell-based assay to test potential NF-kB inhibitors by measuring their effect on IL-1-induced expression of the NF-B dependent intracellular adhesion molecule-1 (ICAM-1, CD54). To develop the cell-based system we sorted IL-1b-responsive U373 human astrocytoma cells to obtain a population of cells with minimum background expression and maximum induced expression of CD54 following stimulation with IL-1. We tested ethyl pyruvate, a novel anti-inflammatory drug candidate, and the ability of related compounds to block activation of NF-kB activity by measuring the expression of CD54 on U373 cells exposed to IL-1. 4-hydroxyphenylpyruvic acid was the best inhibitor of CD54 upregulation. We further tested the compounds using the mouse macrophage-like RAW 264.7 cell line which produce a variety of cytokines and nitric oxide (NO) following exposure to lipopolysaccharide (LPS) in an NF-kB-dependent manner. The drugs downregulated LPS-induced IL-6 production, iNOS upregulation, and NO production following the same efficacy trend observed in the primary screening using CD54 expression in U373 cells. These studies show the ease of using an endogenous reporter gene (i.e., CD54) and FACS analysis to rapidly characterize the relative efficacy of pharmacologic inhibitors. A second completely unrelated topic of the dissertation dealt with the receptor for advanced glycation end-products (RAGE). RAGE is thought to be important in a variety of pathological conditions, including diabetes, sepsis, atherosclerosis, renal diseases, hypertension and Alzheimers disease. However, RAGE proximal signaling events are still unclear. We were able to establish that original RAGE, sequenced from bovine lung, is only present in the lung. This observation was based on antibody specificity, Northern blotting and N-glycosylation analysis. One of the antibodies that we used (H-300, Santa Cruz, CA) was very selective for lung RAGE and not cross-react with other RAGE isoforms. Only lung RAGE had a transcript size of 1.4 kb as determined by Northern blot and only lung RAGE was N-glycosylated. Non-lung tissues and cell lines appeared to express their own unique RAGE isoforms. Non-lung derived cell lines were permissive for lung RAGE isoform expression but lung derived cell lines were not. Interestingly, all transfected cell lines (of lung and non-lung origin) expressed RAGE mRNA transcripts. In addition, we established that previously described endogenous soluble RAGE (esRAGE) does not contain any of the canonical RAGE epitopes, but includes sequence encoded in intron 9. RAGE knockout mice lose esRAGE isoform along with the canonical one confirming that esRAGE originates from the RAGE gene. Signaling studies with pro-inflammatory stimuli in mouse lung slices of wild-type and knockout mice revealed the importance of RAGE in LPS and IL-1-induced inflammatory response, but not when reported RAGE ligands, including AGEs, HMGB1 and S100B, were applied.

Background Prostate cancer is the second leading cause of cancer related death in men. Current therapies for metastatic prostate cancer can only prolong progression, as most men eventually succumb to metastasis and then death. Therefore, there is continued urgency to identify novel therapeutic targets for advanced disease. Previous reports have identified an increase in Translocator Protein (TSPO) expression in numerous cancer models, including prostate. Functionally, TSPO has been implicated in the regulation of apoptosis and cell proliferation. Here, the role of TSPO in advanced prostate cancer is evaluated in an effort to establish the potential value of TSPO as a therapeutic target in advanced disease. Methodology and Principle Findings Immunohistochemical analysis using tissue microarrays was used to determine the expression profile of TSPO in human prostate cancer tissues. We observed that TSPO expression increases with disease progression, as prostate cancer metastases had the highest expression. To demonstrate the effect of TSPO ligands PK11195 and lorazepam in prostate cancer, we utilized cell proliferation assays, cell death ELISAs, and a prostate cancer mouse xenograft study. Our findings provide the first evidence of the anti-tumor effects of lorazepam acting on TSPO. To determine the effect of modulating TSPO expression, we performed overexpression and knockdown studies. These studies provided further evidence that lorazepam is acting through TSPO, as overexpression of TSPO conferred increased susceptibility to lorazepam while TSPO knockdown decreased susceptibility. Lastly, we investigated the role of TSPO multimers in prostate cancer. We found that TSPO multimers can be induced by reactive oxygen species and may be formed through a di-tyrosine covalent bond. Conclusions and Significance TSPO expression increases with prostate cancer progression. The benzodiazepine lorazepam exerts its anti-cancer effects through its binding to TSPO. Collectively, these data suggest that TSPO is an excellent therapeutic target for advanced disease and that our preclinical results demonstrating that the already existing FDA-approved drug lorazepam has anti-tumor effects could be easily translated to the prostate cancer patient population. These studies could lead to a significant change in the management of prostate cancer by providing a treatment option with minimal toxicity for use in advanced disease and could ultimately prevent prostate cancer deaths.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super-family of cell surface receptors whose activation has been suggested to contribute to various pathologies. RAGE has been primarily studied in diabetes where its upregulation has been linked to disease in the kidney, vasculature, and nervous system. This protein is highly expressed in the lung under normal conditions, but its function is unknown. We therefore investigated the normal function of RAGE in the lung and its pulmonary expression in two disease states. Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with both high morbidity and mortality. Unfortunately, there are currently no effective therapies for IPF necessitating mechanistic insight into the disease pathogenesis. We found that pulmonary fibrosis led to a depletion of RAGE in both animal models and tissue from patients with idiopathic pulmonary fibrosis. In contrast to other diseases in which RAGE signaling promotes pathology, we found that aged RAGE null mice spontaneously develop pulmonary fibrosis-like alterations and more severe fibrosis in response to asbestos injury. In addition, we found that RAGE null mice were fully protected from the fibrotic effects of bleomycin. In addition, we investigated the expression of RAGE in the lungs of diabetic rodents. Diabetes has been shown to alter RAGE expression in a number of tissues that do not normally express RAGE. We hypothesized that diabetes would alter pulmonary RAGE expression and contribute to the susceptibility to pulmonary injury. We found that pulmonary RAGE expression was unaltered in five rodent models suggesting that diabetes does not effect RAGE expression in the lung. Lastly, we identified that RAGE has a very high affinity for components in the basement membrane of the lung. A few RAGE studies suggested that it might serve a role as an adhesion molecule. We found that RAGE extensively colocalized with the alveolar basement membrane and had very high affinity for collagen I, collagen IV, and laminin, but not fibronectin. These findings along with the fact that RAGE null mice spontaneously develop fibrosis suggest a potential homeostatic function of RAGE in the lung. This is in stark contrast to the vast majority of studies, which suggest that its expression is solely pathologic.

Integrin-mediated cell-extracellular matrix (ECM) adhesion is essential for the survival of normal epithelial cells, and loss of this cell-ECM adhesion leads to anoikis. In this dissertation study, we first identify migfilin, a novel focal adhesion protein, as a key sensor of cell-ECM adhesion in epithelial cells. Loss of cell-ECM adhesion significantly reduces migfilin protein levels in untransformed epithelial cells and concomitantly induces anoikis. Migfilin RNAi is sufficient to induce apoptosis in MCF-10A cells while overexpression of FLAG-migfilin partially protects these cells from anoikis, strongly suggesting that migfilin plays a critical role in cell adhesion-mediated cell survival signaling. Cell detachment-induced migfilin reduction is, at least partially, responsible for the induction of anoikis. Further signaling studies reveal that migfilin regulates cell survival and anoikis by influencing Src activation. Immunoflorescence staining shows that migfilin co-localizes with active Src in focal adhesions, and immunoprecipitation and GST pull-down assays demonstrate that migfilin directly interacts with Src. Moreover, the detailed structural studies show that migfilin strongly binds to the Src SH3 domain via the second PXXP cluster (140-173aa) in its proline-rich region, and weakly binds to the Src SH2 domain via an atypical binding sequence (E6KRVASS12) in its N-terminal. A working model is proposed in which migfilin promotes Src activation via direct interaction, and loss of cell-ECM adhesion triggers the degradation of migfilin protein, thereby causing Src inactivation which contributes to the initiation of anoikis. Interestingly, this migfilin-Src signaling pathway is dysfunctional in some anoikis-resistant cancer cells. During cell detachment, migfilin proteins are stabilized in these cancer cells, and phosph-Y419 Src levels are not reduced concomitantly, representing a novel mechanism for anoikis resistance during tumorigenesis. In addition, migfilin is found to negatively regulate p27 protein level. Depletion of migfilin significantly increases p27 protein levels in different cell lines. In HCT116 cells, migfilin RNAi increases both cytoplasmic and nuclear p27, and inhibits cell cycle progression. These findings indicate that migfilin provides a linkage between p27 and integrin-mediated cell-ECM adhesion.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide due to poor prognosis and limited therapeutic options. Point mutations affecting phosphorylation sites such as serine-45 in Beta-catenin gene are evident in around 30% of HCC. We developed a transgenic mouse that expresses Ser45 mutated Beta-catenin (TG) in hepatocytes. While this mutation did not induce spontaneous tumorigenesis, it promoted diethylnitrosamine (DEN)-induced HCC through cyclin-D1 overexpression and other factors. The Wnt/Beta-catenin signaling is important in stem cell self-renewal. The adult progenitor cell of the liver, or oval cells that emanate from atypical ductular proliferation (ADP), maybe involved in liver regeneration and/or hepatocarcinogenesis and can be observed after exposure to DDC diet, which induces hepatic and biliary injury. When challenged with chronic-DDC diet, Beta-catenin transgene led to a cellular disparity in the form of increased appearance of atypical hepatocytes (positive for ductular marker A6), which was associated with better resolution of intrahepatic cholestasis. We also utilized DDC diet in conditional Beta-catenin knockout mice (KO) that lacked Beta-catenin in hepatocytes and cholangiocytes. ADP was blunted after short-term DDC feeding in KO mice; however, long-term feeding resulted in gradual increase in ADP, hepatic fibrosis and HCC. Interestingly, the KO livers begin to exhibit periportal Beta-catenin-positive hepatocytes, which eventually populate the entire livers over the course of this process. Finally, we explored targeting of the Wnt pathway with pegylated interferon-alpha2A (Peg-IFN). We found that Peg-IFN decreased Beta-catenin activity in mouse liver and several human hepatoma cell lines. The mechanism seemed to be at least partly due to upregulation of a nuclear export factor, RanBP3. Thus, this study characterizes an animal model utilizing Beta-catenin mutation, which is evident in HCC patients. DEN-exposure in these animals led to HCC development, thus providing a valuable tool to study mechanisms of hepatocarcinogenesis and providing a model to test therapeutic inhibition of Beta-catenin by agents such as peg-IFN and others. Our studies also provide evidence that Wnt activation may resolve intrahepatic cholestasis. Finally, we show that chronic damage to the liver in KO led to appearance of Beta-catenin-positive hepatocytes, which continued to proliferate and in the face of continued injury and fibrosis, led to development of HCC, which is also relevant clinically.

Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol-anchored, cell-surface heparan sulfate proteoglycans. The GPC3 gene is located on the X chromosome, and is highly expressed during embryogenesis and organogenesis. Loss-of-function mutations of GPC3 in humans result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and post-natal liver and other organ overgrowth. GPC3 is one of the most over-expressed proteins in human hepatocellular carcinoma and is used as a novel diagnostic marker. However, its role in normal liver regeneration is still not well characterized. In this study, we investigated the role and effects of GPC3 in hepatocyte proliferation and liver regeneration, using 2/3 partial hepatectomy (PHx) model in rats and hepatocyte-targeted GPC3 transgenic mice. We found in rats that GPC3 mRNA and protein increase in a time frame which coincides with the termination of proliferative activities of either hepatocytes (day 2 after PHx and day 8-12 in culture) or non-parenchymal cells (day 5-6 after PHx). Blocking GPC3 expression using morpholino oligonucleotides promoted rat hepatocyte growth in vitro. We further generated GPC3 transgenic mice with hepatocyte-targeted over-expression of GPC3. These transgenic mice develop normally compared with their non-transgenic littermates, but have a suppressed rate of hepatocyte proliferation and liver regeneration after 2/3 PHx. Therefore we hypothesize that GPC3 is a negative regulator of hepatocyte proliferation and liver regeneration. The yeast two-hybrid assay revealed that GPC3 interacts with several interesting proteins including CD81, a cell membrane tetraspanin. CD81 levels changed in the same manner as GPC3 after rat PHx, and their interaction was confirmed by co-immunoprecipitation and co-immunofluorescence. The co-localization of GPC3 and CD81 after PHx indicates an important regulator interaction between the two proteins. Moreover, gene array analysis revealed a series of changes in the expression profiles in GPC3 transgenic mice. After PHx, a panel of cell cycle related genes and some oncogenes are either up- or down-regulated, which was confirmed by western blotting. Our results indicate that GPC3 plays a negative regulatory role in hepatocyte proliferation and liver regeneration in rats and hepatocyte-targeted transgenic mice, in which several potential proteins and multiple pathways are involved and affected.

Amyotrophic lateral sclerosis (ALS) is the most common form of adult onset motor neuron disease and is characterized by the progressive degeneration and death of motor neurons. The pathologic mechanisms underlying ALS are poorly understood although our laboratory identified decreased levels of transthyretin (TTR), a protein that impacts the retinoid signaling pathway, in the cerebrospinal fluid of ALS patients. Differential expression of retinoid signaling components has been reported in ALS patients and transgenic animal models of familial ALS. We sought to further characterize TTR and retinoid signaling proteins in ALS and to evaluate the role of retinoid signaling in motor neuron cell death. Mass spectrometry and immunoblotting were used to investigate TTR. Immunohistochemistry using lumbar spinal cord tissue from ALS patients and non-neurologic disease controls was used to characterize retinoid signaling pathway proteins. Spinal cord tissue homogenates were used for co-immunoprecipitation studies and electrophoretic mobility shift assays. Motor neuron-enriched cultures established from embryonic day 14 rats were utilized for in vitro studies. RAR-mediated signaling was modulated with pan-agonists and isotype-specific agents and hydrogen peroxide used to model oxidative stress/injury. Altered post-translational modifications and high molecular weight species of the TTR protein were observed in ALS. Cellular retinoic acid binding protein-II (CRABP-II) and retinoic acid receptor beta (RARβ) exhibited increased nuclear localization in motor neurons of sporadic ALS patients. Protein-protein interactions (between CRABP-II and RARα or RARβ) did not differ although retinoic acid response element binding was increased in ALS as compared to controls. Treatment with a pan-RAR or RARβ-specific agonist significantly decreased oxidative stress-induced motor neuron cell death in vitro and genes downstream of RARβ were increased with treatment. Our results indicate that TTR genetic polymorphisms do not represent a novel susceptibility factor for ALS, although protein modification and aggregation appear to be altered in ALS. Localization of proteins of the retinoid signaling pathway is altered in ALS patients and these changes translate to the transcriptional level. Our in vitro work indicates that stimulating the RARs (particularly RARβ) is neuroprotective and that pharmacologic agents that target this nuclear receptor may be of value in slowing the progression of ALS.