Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Physiology. Title from resource description page. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xi, 148 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 124 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Pulmonary fibrosis is a potentially deadly pathology characterized by excessive deposition of extracellular matrix (ECM), increased tissue stiffness, and loss of tissue structure and function. Recent evidence has suggested epithelial to mesenchymal transition (EMT), the transdifferentiation of an epithelial cell into a mesenchymal fibroblast, is one mechanism that results in the accumulation of myofibroblasts and excessive deposition of ECM. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin mediated interactions with ECM proteins and soluble growth factors including TGFβ. TGFβ, a potent inducer of EMT, can be activated by cell contraction mediated mechanical release of the growth factor from a macromolecular latent complex. Therefore, TGFβ activity and subsequent EMT may be influenced by both the biochemical composition and biophysical state of the surrounding ECM. Based on these knowns it was first investigated how changes in the biochemical composition of the matrix and changes in tissue rigidity together modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin specific interactions with fibronectin (Fn) variants displaying both the RGD and PHSRN binding sites facilitate cell binding through α3β1 and α5β1 integrins, and that these interactions maintain an epithelial phenotype despite engagement of increased tissue rigidities. Conversely, Fn fragments that facilitate cell binding through αv integrins drive TGFβ activation and subsequent EMT even while engaging soft underlying substrates. Adding to the complexity of studying mechanisms that contribute to pulmonary fibrosis, is exposure of the lung to injuries from environmental particulates. Therefore, we investigated how EMT is altered in response to particulate matter (PM). Here we show that PM exposure further drives TGFβ activation, EMT, and increases intracellular levels of reactive oxygen species (ROS). Additionally, cells binding the ECM through α5β1 and α3β1 integrins only partially recover an epithelial phenotype, suggesting ROS may be a secondary driver of TGFβ and EMT. Taken together these results suggest dynamic changes to the ECM microenvironment are major contributors to the control of EMT responses and provide insights into the design of biomaterial-based microenvironments for control of epithelial cell phenotype.

The purpose of this study was to investigate whether or not gender differences may be present in the expression of a number of urinary proteins which may serve as markers of inflammation and oxidative stress. Males and females have different patterns of illness and different life spans, suggesting basic biological traits exert significant control on the incidence of rhabdomyolysis, renal failure, atherosclerosis, myocardial ischemia, myocardial contraction band formation, autoimmune disorders and general inflammatory diseases. Men are at greater risk for cardiovascular disease; however women, particularly elderly women, have higher fatality rates due to heart failure. Renal diseases progress far more quickly in men, possibly due to testosterone. Men also have higher kidney bulk related to androgen expression. Gender disparity may be most obvious in autoimmune disorders; of the estimated 8.5 million people diagnosed with autoimmune disorders, approximately 80% are women. Hashimoto’s thyroiditis, the most common form of hypothyroidism, is up to 10 times more common in women. Systemic Lupus Erythematosus (SLE), an autoimmune disease characterized by acute and chronic inflammation, is 9 times more common in women. Rheumatoid arthritis (RA), an autoimmune disease affecting approximately 1.3 million people in the United States, is four times more common in women. Diabetes mellitus (DM), affecting more than 17 million people – the majority of which are women, is linked to microvascular and macrovascular diseases such as kidney failure, strokes and atherosclerosis. These conditions are linked to physiological changes that may alter the expression of certain biomarkers of inflammation and oxidative stress. Over the past several decades, it has become increasingly clear that the role of diet, smoking, and other lifestyle choices clearly influence the etiology and pathophysiology of these diseases. The use of drugs, both licit and illicit, has been clearly linked to many of these diseases. Illicit substances, particularly cocaine, have been demonstrated to produce pathophysiological changes to many systems in the body which can greatly influence the progression of existing and drug-induced disease states leading to systemic damage. A relationship between the expression of markers of inflammation, oxidative stress, cardiac damage, or other systemic injury, gender and cocaine use has not been clearly established. Urine is an important medium for assessment of general health status. It has classically been used to monitor disease states; glucosuria as an indicator of diabetes and renal dysfunction, microorganisms signifying urinary tract or bladder infection, and biomarkers such as human chorionic gonadotropin to confirm pregnancy. Recently urine has been used to assess biomarker expression and disease states. Urine is an ideal clinical tool for toxicological screens; it is readily accessible, non invasive and typically supplied in sufficient quantity to accommodate multiple tests. In this study, urine specimens were collected and analyzed for creatinine, cocaine, total protein, aldosterone, c-reactive protein (hsCRP), myeloperoxidase (MPO), microalbumin (MAB), neutrophil gelatinase-associated lipocalin (NGAL), heat shock protein 90α (hsp90α), vascular endothelial growth factor (VEGF), myoglobin, pro atrial natriuretic peptide (proANP) and interleukins 1α, 1 β , and 6 using ELISA and colorimetric assays. Urine specimens that tested negative for all illicit substances in the standard National Institute on Drug Abuse (NIDA) 10 panel showed differences in a number of these biomarkers which strongly suggested significant differences between males and females for aldosterone, IL1α and IL1β. In addition, significance is suggested for MPO and CRP. Although sex specific differences in serum expression have been noted for some of the markers in both animal and human models, this has not been previously demonstrated in human urine. This may have implications for what is typically referred to as ‘normal’ values. Gender specific differences were not apparent in urine specimens that tested positive for cocaine. Also, in males only, the levels of myoglobin and aldosterone significantly increased.

The goal of this study is to utilize polymers as macromolecular ligands for the construction of catalysts by formation of coordination complexes with transition metals with the main focus on complexes of Cu(II) and Fe(III) and further determine (a) their catalytic efficiency (b) mechanism of action (c) similarities to enzymatic systems and synthetic metal complexes. The reactions of interest are (1) hydrolytic cleavage of a series of phosphoesters(2) oxidation of catechol type of substrates (3) hydroxylation of phenolic substrates and chlorinated phenols (4) activation of molecular oxygen and/ or hydrogen peroxide (5)oxidative cleavage of DNA plasmid. The major premise of the study is that by mimicking the macromolecular nature and some structural features of enzymes, polymers can in principle, catalyze chemical transformations with similar efficiencies and specificities and can offer alternatives to peptide based catalysts or simple metal complexes with the advantage of a wider range of building blocks, increased stability and the potential of reusability. The crosslinked resins used contained the functional groups iminodiacetate (chelex resin), diethylenetriamine and tris(2-aminomethylamine) and were based on styrene-divinylbenzene backbone. The catalytic proficiencies of the Fe(III) and the Cu(II) complexes of chelex resin and diethylenetriamine approached 100 and 1000 respectively towards the model phosphodiester BNPP at pH 8.0 and 25°C. Moreover, the Fe(III) complexes of linear copolymers with repeating unit of three vinylpyridines to one acrylamide (P1) showed selectivity towards phosphodiester hydrolysis over monoesters and phosphonate esters and exhibited catalytic proficiencies approaching 50,000 towards BNPP hydrolysis. Further exploration of the catalytic capabilities of copolymer P1 revealed that Cu(II) complexes of this macromolecular ligand are potentially capable of assembling to active dicopper intermediates found in the catalytic pathways of copper oxygenases like tyrosinase and catechol oxidase and thus were able to accelerate catechol oxidation to ortho-quinones with rate accelerations approaching 10,000 and hydroxylate phenols with rate accelerations close to one million. The results suggest that these Cu(II)-polymer systems can potentially be used as model systems to further understand metal centered reactive oxygen species (ROS) generated in vivo and can be very promising remediation agents for the dechlorination of persistant chlorine containing pollutants.

Studies on a series of polymethine cyanine dyes and analogs (1-24) show that certain near-infrared cyanines are capable of damaging DNA in the absence of light and external reducing agents. Experimental results imply that in this DNA thermal cleavage, the cyanine reduces Cu(II) to Cu(I) which reacts with O2 to generate the reactive oxygen species (ROS) O2∙- and ∙OH. The formation of these ROS is also thought to be responsible for the irreversible bleaching of the dyes in aqueous solutions. A correlation between structural features and DNA thermal cleavage activity as well as dye bleaching is suggested. Long polymethine chains appear to confer instability to cyanines in aqueous solutions and further contribute to undesired thermal DNA cleavage. These drawbacks can be overcome by introducing an electron-withdrawing group to the polymethine bridge of the cyanine dye.

The polymorphonuclear neutrophil granulocytes (neutrophils) are gaining renewed interest regarding their involvement in chronic inflammatory disorders, including rheumatoid arthritis (RA). Besides phagocytic and destructive capabilities, neutrophils have regulatory roles, e.g. by influencing responses from dendritic cells and lymphocytes. Several animal models have revealed that neutrophils are crucial for the initiation and maintenance of chronic inflammatory diseases. Neutrophil function is highly dependent on their ability to produce superoxide, an oxygen radical which can be further metabolized to other free radicals. Whether or not neutrophils are capable of producing the oxygen radical nitric oxide (NO˙) has been a matter of debate. In this thesis it was shown that freshly isolated neutrophils from the joint cavity of patients with RA, but not from other arthritis patients, had ongoing intracellular production of superoxide, indicating the processing of ingested material. The finding that joint neutrophils, but seemingly not circulating cells, expressed the NO-inducing enzyme iNOS, led to a series of experiments aimed to elucidate where in the exudative process this enzyme could first be detected. We could finally, for the first time, present evidence that human neutrophils actually express iNOS constitutively. Our data also suggest that neutrophil iNOS may be membrane associated, thus differing from the cytosolic location in other cell types. Since NOS activity was not demonstrated in isolated cells, the notion that neutrophil iNOS is regulated primarily at the transcriptional level must be questioned. NO production from iNOS requires the presence of its substrate, L-arginine. To test the hypothesis that neutrophil arginase prevents neutrophil NO-production, we investigated whether arginase inhibition affects neutrophil NO-dependent oxidative function. Initial data revealed a difference in the effect of arginase inhibition comparing neutrophil stimulus with a soluble formylated tri-peptide (fMLF) and integrin-mediated stimulation with particle-bound collagen type-1. This led to the hypothesis that integrin-ligation on neutrophils induces extracellular liberation of arginase, which was confirmed both by measuring arginase and its enzyme activity. The findings in this thesis may be important not only regarding the role of neutrophils in chronic joint inflammation, but also as a link in the accelerated atherosclerosis observed in chronic inflammatory disorders, e.g. RA. / Vid reumatiska ledinflammationer ansamlas mycket stora mängder polymorfkärniga neutrofila granulocyter (neutrofiler) inne i den vätskefyllda ledhålan. Neutrofiler har kraftfull destruktiv potential och anses kunna bidra till uppkomst av skada i leden. Då flera djurmodeller av ledinflammation har visat sig omöjliga att initiera i frånvaro av neutrofiler, har intresset för denna celltyp åter ökat efter att de under lång tid har stått i skuggan av andra typer av vita blodkroppar. En viktig del i avdödning av mikroorganismer och cellsignalering är förmågan att bilda fria syreradikaler, t.ex. superoxid (˙O2-) och kväveoxid (NO˙). Denna avhandling belyser aspekter kring produktionen av dessa reaktiva syreprodukter och mekanismer av potentiell betydelse vid ledinflammation. I det första arbetet visas att neutrofiler isolerade ur ledvätska från patienter med ledgångsreumatism (RA) har ett unikt beteende avseende superoxidproduktion jämfört med motsvarande celler från patienter med andra reumatiska sjukdomar. RA-neutrofiler från ledvätska (men inte från blod) producerar superoxid intracellulärt redan i vila och stimulering via vidhäftningsmolekyler ger en snabb ytterligare ökning av denna aktivitet. Fyndet antyder att cellerna är engagerade med hantering av endocyterade partiklar och/eller immunkomplex/immunaggregat. I de båda nästkommande arbetena undersöktes förekomst av det NO˙-producerande enzymet iNOS i neutrofiler. En rad tidigare publikationer har rapporterat motsägelsefulla resultat i denna fråga. Efter en serie experiment kunde vi konstatera att humana neutrofiler uttrycker iNOS konstitutivt, men att både dess cellulära lokalisation och reglering skiljer sig från andra celler. Neutrofiler har nyligen även visats innehålla arginas, ett enzym som konkurrerar med iNOS om bindningen till L-arginin och som därmed kan hämma NO˙-produktion. I det fjärde arbetet undersökte vi därför om hämning av arginas påverkade neutrofilernas funktion och produktion av superoxid. Vi fann att effekterna av arginashämning var större hos celler som stimulerats genom vidhäftning av kollagenklädda partiklar jämfört med en löslig formylerad tri-peptid (fMLF). Vidare, kunde vi visa att vidhäftning av kollagenklädda partiklar medför större extracellulär frisättning av arginas. Med stöd av dessa fynd kunde vi i påföljande försök bekräfta hypotesen att extracellulär frisättning av arginas är större efter vidhäftning av kollagen-partiklar än med fMLF-stimulering. Fysiologiskt är fyndet logiskt då det skulle medföra ökade vidhäftningsmöjligheter för neutrofilen inne i blodbanan genom att begränsa blodkärlets egen NO˙ produktion. Fyndet är också förenligt med den ökade frekvensen hjärt- och kärlsjukdomar vid RA, då en intensiv kontinuerlig utvandring av neutrofiler skulle medföra ökad arginas frisättning, sänkta argininnivåer och endotelial dysfunktion.

Changes in intracellular redox homeostasis are implicated in both normal cell signaling and as pathophysiological mechanisms contributing to a variety of age-related diseases, including diabetes, atherosclerosis, neurodegenerative conditions, and cancer. Though a variety of well described mechanisms exist to counterbalance the overproduction of cellular oxidants and maintain optimal intracellular redox poise, the understanding of the mechanism(s) through which cellular redox homeostasis regulates cell signaling functions is less well understood. Here, we demonstrate that signaling by the immediate early gene / orphan nuclear hormone receptor NGFI-B (Nur77, TR3), which functions pleiotropically in the regulation of cell growth, metabolism, differentiation and death in diverse tissues, is redox-regulated at both the level of induction and NGFI-B-dependent gene transcription. Using co-immunoprecipitation experiments in cells, we also identified a novel interaction between NGFI-B and the cytoplasmic thiol-reducing catalyst thioredoxin1 (Trx1), that, similar to DTT, blocks NGFI-B-dependent gene expression in a manner that depends on the Trx1 active site cysteines. Together these observations add NGFI-B-dependent gene expression to a growing portfolio of transcription factor pathways that are redox-regulated. NGFI-B, in addition, appears to regulate the mitochondrial membrane potential in L6 skeletal myoblasts. NGFI-B is indispensible for T-cell receptor-mediated apoptosis and induces cell death in a variety of cell types in response to diverse pro-apoptotic stimuli. Like p53, translocation of NGFI-B from the nucleus to the mitochondria may be a critical aspect of its pro-apoptotic function. Interestingly, we found that enforced NGFI-B expression in L6 skeletal muscle myoblasts led to a significant decrease of MMP that peaked 48hr after transfection and did not require a cell death-inducing stimulus. Moreover, NGFI-B transfected cells had no increase in mitochondrial cytochrome C release despite loss of MMP at 48 hr. Combined, these data suggest that loss of MMP in muscle cells may be an early event in the apoptotic process regulated by NGFI-B. This, along with the redox regulation of NGFI-B, provides unique evidence of a relationship between the mitochondria, mitochondrial by-products, ROS, and the regulation of and by the transcription factor NGFI-B. / text

Τις τελευταίες δεκαετίες υπάρχει ένα αυξανόμενο ενδιαφέρον για το ρόλο των ROS στην πειραματική και κλινική ιατρική (Alexandre et al., 2007; Zheleva et al., 2007; Videla et al., 2007). Οι ROS, όπως το ανιόν του υπεροξειδίου, το υπεροξείδιο του υδρογόνου (ΗΡ) και η υδροξυλική ρίζα, είναι γνωστό ότι διαδραματίζουν ένα διττό ρόλο στα βιολογικά συστήματα, όπου είναι επιβλαβείς στις υψηλές και ευεργετικές στις χαμηλές συγκεντρώσεις (Valko et al., 2004). Στις υψηλές συγκεντρώσεις οι ROS φαίνεται να διαμεσολαβούν την απόπτωση. Ενδογενή ή εξωγενή ερεθίσματα οδηγούν στη συσσώρευση των ROS, το λεγόμενο «οξειδωτικό στρες», που χαρακτηρίζεται από βλάβη των κυτταρικών δομών, των λιπιδίων, των μεμβρανών, των πρωτεϊνών και των νουκλεϊκών οξέων (Poli et al., 2004). Αντίθετα, οι χαμηλές συγκεντρώσεις των ROS μέσα στα κύτταρα δρούν ως δεύτεροι αγγελιοφόροι σε αρκετά ενδοκυτταρικά σηματοδοτικά μονοπάτια που οδηγούν στην ενεργοποίηση κινασών τυροσίνης, των ΜΑΡ κινασών, της πρωτεϊνικής κινάσης C, του υποδοχέα του επιδερμικού αυξητικού παράγοντα, πρωτεϊνικών φωσφατασών, των καναλιών καλίου και των μεταγραφικών παραγόντων AP-1 και NF-κB (Droge, 2002). Πρόσφατα έχουμε δείξει ότι υπό φυσιολογικές, μη-στρεσογόνες συνθήκες, το ΗΡ επάγει τον πολλαπλασιασμό και τη μετανάστευση στα ανθρώπινα κύτταρα καρκίνου του προστάτη LNCaP, μέσω ενεργοποίησης της έκφρασης του αυξητικού παράγοντα HARP (Polytarchou et al., 2005). Η HARP ή πλειοτροπίνη είναι ένας εκκρινόμενος αυξητικός παράγοντας 18 kDa που παρουσιάζει υψηλή συγγένεια για την ηπαρίνη. Η HARP είναι υψηλά συντηρημένη μεταξύ των διαφόρων ειδών, όπως ο άνθρωπος, το ποντίκι, ο αρουραίος, τα βοειδή, τα ψάρια, ο βάτραχος και τα έντομα, και το γονίδιό της εκφράζεται σε ένα ιδιαίτερα περιορισμένο χρονικό και χωρικό μοτίβο κατά τη διάρκεια της ανάπτυξης. Φαίνεται ότι η HARP είναι μια σημαντική πρωτεΐνη που συμβάλλει ενδεχομένως στη λειτουργία διαφόρων ρυθμιστικών συστημάτων. Αρκετά δεδομένα υποδεικνύουν ότι η HARP εμπλέκεται στον πολλαπλασιασμό, τη μετανάστευση και τη διαφοροποίηση των κυττάρων και διαδραματίζει σημαντικό ρόλο στις διάφορες κυτταρικές διαδικασίες (Kadomatsu and Muramatsu, 2004; Papadimitriou et al., 2004). Επιπλέον, η HARP ανιχνεύεται σε μια πληθώρα καρκινωμάτων λειτουργώντας ως πρωτο-ογκογονίδιο και φαίνεται να παίζει κύριο ρόλο στη φυσιολογική και την επαγόμενη από καρκινικούς όγκους αγγειογένεση (Kadomatsu and Muramatsu, 2004; Papadimitriou et al., 2004; Hatziapostolou et al., 2005;2006; Polykratis et al., 2005; Christman et al., 2005; Zhang et al., 2006). Είναι πλέον γνωστό ότι οι χαμηλές συγκεντρώσεις ROS ενεργοποιούν ενδοκυτταρικά σηματοδοτικά μονοπάτια που οδηγούν σε ενισχυμένη αγγειογένεση in vivo (Maulik and Das, 2002; Polytarchou and Papadimitriou, 2004; Ushio-Fukai, 2006; Kim et al., 2006; Chen et al., 2007) και ενεργοποίηση των ενδοθηλιακών κυττάρων in vitro (Lelkes et al., 1998; Maulik and Das, 2002; Stone and Collins, 2002; Yasuda et al., 1999; Polytarchou and Papadimitriou, 2004; Ushio-Fukai, 2006; Kim et al., 2006; Chen et al., 2007; Guo et al., 2007). Επίσης, οι ROS, όπως το ανιόν του υπεροξειδίου και το ΗΡ, φαίνεται να διαμεσολαβούν τα αγγειογενετικά σήματα που ξεκινούν από αυξητικούς παράγοντες, όπως ο VEGF (Lin et al., 2003 Ushio-Fukai et al., 2002). Προηγούμενη μελέτη της ερευνητικής μας ομάδας έδειξε ότι το ανιόν του υπεροξειδίου και το ΗΡ είναι πιθανοί επαγωγείς των λειτουργιών των ενδοθηλιακών κυττάρων in vitro, ενδεχομένως μέσω επαγωγής της ενεργοποίησης της ενδοθηλιακής συνθάσης του μονοξειδίου του αζώτου (eNOS), που οδηγεί σε αυξημένη παραγωγή του ενδογενούς μονοξειδίου του αζώτου (NO) και επακόλουθη ενεργοποίηση της διαλυτής γουανυλικής κυκλάσης (sGC) (Polytarchou and Papadimitriou, 2005). Ο κύριος στόχος της παρούσας μελέτης ήταν να διερευνηθεί η πιθανή εμπλοκή της eNOS στην επαγωγή της παραγωγής της HARP από χαμηλές συγκεντρώσεις ΗΡ. Τα αποτελέσματά μας δείχνουν ότι η ενεργοποίηση του ανθρώπινου γονιδίου της HARP επάγεται από το ΗΡ μέσω ενεργοποίησης της eNOS στα κύτταρα HUVEC και LNCaP. Όπως αναφέρθηκε και παραπάνω, περίσσεια ROS σε συνδυασμό με ανεπάρκεια των κυτταρικών αντιοξειδωτικών συστημάτων οδηγεί σε οξειδωτικό στρες (Halliwell and Whiteman, 2004) και ενεργοποίηση μονοπατιών απόπτωσης και νέκρωσης (Paraskevas et al., 2000; Fleury et al., 2002; Nakano et al., 2004). Ένα δεύτερο μέρος της παρούσας εργασίας ασχολείται με τον τρόπο που οι υψηλές συγκεντρώσεις ΗΡ επηρεάζουν την ανάπτυξη των ενδοθηλιακών κυττάρων από στεφανιαία φλέβα βοός (CVEC). / -