PHD, Kent State University, 2020, College of Arts and Sciences / School of Biomedical Sciences

Osteoarthritis (OA) is one of the leading causes of disability and is caused by a combination of mechanical and biochemical factors. Accumulating evidence suggests that inflammation has a key role in the pathogenesis of OA, and nitric oxide (NO) is considered as one of the major inflammatory mediators in OA that drives many pathological changes during the development and progression of OA. Excessive production of NO in chondrocyte promotes cartilage destruction and cellular injury, and its synthesis in chondrocytes is catalyzed by inducible nitric oxide synthase (iNOS), which is thereby an attractive therapeutic target for the treatment of OA. A number of direct and indirect iNOS inhibitors, bioactive compounds, and plant-derived small molecules have been shown to exhibit a chondroprotective effect by suppressing the expression of iNOS. Currently, there is no effective disease-modifying drug available for OA. Small molecules have proved to be powerful tools for modulating important molecular pathways in development and disease. Our preliminary screening of selected small molecules led us to select imperatorin (IMP) and peretinoin (PRT), which exhibit anti-inflammatory properties; however, their effect in chondrocytes is unknown. IMP is a plant-derived compound, while PRT is an acyclic retinoid and is currently in clinical trials for its efficacy to treat hepato-carcinoma. We found that IMP, as well as PRT, inhibited IL-1β induced expression of iNOS and production of NO in primary human OA chondrocytes by modulating the activation of mitogen-activated protein kinase (MAPK) pathway. Additionally, PRT inhibited matrix degradation by suppressing the expression of matrix metalloproteinase-13 (MMP-13). The work described in this dissertation demonstrates that PRT inhibits the expression of iNOS and production of NO in primary human OA chondrocytes and cartilage explants, identifies the mechanism, and shows OA suppressive effects in a mouse OA model.

Committee: Tariq Haqqi (Advisor); Fayez Safadi (Committee Member); Moses Oyewumi (Committee Member); Mohammad Ansari (Committee Member); Christine Crish (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2020, Pharmaceutical Sciences

Inflammation is the body's response to infection or injury. Endothelial cells are among the different players involved in an inflammatory cascade. In response to an inflammatory stimuli such as bacterial lipopolysaccharide (LPS), endothelial cells get activated which is characterized by the production of important mediators, such as inducible nitric oxide synthase (iNOS) which, catalyzes the production of nitric oxide (NO) and reactive nitrogen species and cyclooxygenase-2 (COX-2) that catalyzes the production of prostaglandins. Though the production of these mediators is required for an inflammatory response, it is important that their levels are regulated. Continued production of iNOS results in increased accumulation of reactive nitrogen species (RNS) that might lead to cytotoxicity, whereas lack of/suppression results in endothelial and vascular dysfunction. On the other hand, severe cardiovascular, intestinal and renal side effects are observed with significant suppression of COX-2. Thus, studying factors that could regulate the levels of iNOS and COX-2 could provide useful insights for developing novel therapeutic targets. Regulation of protein levels involves control of protein induction or turnover. Since protein induction requires transcription, in this dissertation we studied the role of a promoter of transcription “Cyclin-dependent kinase 7 (CDK7)” in iNOS and COX-2 protein induction. We first depleted CDK7 in mouse aortic endothelial cells (MAEC) using siRNA, followed by stimulus with lipopolysaccharide/interferon gamma (LPS/IFN) to induce iNOS and COX-2 protein. Interestingly, CDK7 depletion resulted in excessive induction of both iNOS and COX-2 protein levels compared to control. This observation was recapitulated using a covalent inhibitor of CDK7, THZ1. Further investigation showed the protein induction due to lack of CDK7 was not due to increased mRNA levels. CDK7 knockdown however decreased the turnover of iNOS, but not COX-2 protein. We id (open full item for complete abstract)

Committee: Dale Hoyt (Advisor); Liva Rakotondraibe (Committee Member); Moray Campbell (Committee Member); Keli Hu (Committee Member) Subjects: Biochemistry; Cellular Biology; Pharmacology

PhD, University of Cincinnati, 2005, Medicine : Pathobiology and Molecular Medicine

Apolipoprotein E (apoE) has been shown to exert anti-proliferative and anti-migratory effects on smooth muscle cells in vitro and protection against neointimal hyperplasia in vivo. In this study, apoE was detected in the medial layers of the carotid artery following vascular injury. The presence of a liver-specific human apoE transgene confirmed the recruitment from circulation, although detection of native apoE message by in situ hybridization indicated that local synthesis also occurs. Although the neointima-resistant C57BL/6 strain showed a transient apoE presence, the susceptible FVB/N strain showed a continuous presence. Interestingly, detection of iNOS expression was positive only in the C57BL/6 wildtype strain. Upon addition of the overexpressed apoE transgene, the FVB/N strain recovered expression of iNOS, while loss of the apoE gene in the C57BL/6 strain resulted in a concurrent loss of iNOS expression. ApoE and iNOS colocalization was demonstrated in the medial layer of mice that are resistant to injury-induced neointimal hyperplasia. NOS2-/- deficient C57BL/6 mice showed no difference in neointimal hyperplasia compared to wildtype, but a significant increase in medial thickness and area was observed. These data documented that injury-induced activation of iNOS requires apoE recruitment, both apoE and iNOS are necessary for suppression of cell proliferation, and apoE recruitment without iNOS expression resulted in medial smooth muscle hyperplasia without their migration to the intima. In addition, the apoE-dependent inhibition of smooth muscle cell migration is dependent on LRP-1 function in the smooth muscle cell. Interestingly, apoE accumulation in apoE transgenic mice followed a layer-specific pattern, and was inversely associated with smooth muscle alpha-actin expression. The vascular accumulation of apoE after endothelial denudation, and its association with alpha-actin-depleted smooth muscle cells, suggests that apoE inhibition of injury-induced neoi (open full item for complete abstract)

Committee: David Hui (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2012, Biochemistry Program, Ohio State

Nitric oxide (NO) generated by inducible NO synthase (iNOS) plays critical roles in inflammation and host defense. iNOS expression is induced by inflammatory stimuli and exhibits constant activity once expressed. Hence NO production from iNOS has been thought to be primarily controlled via enzyme expression. In this study, we identify an array of novel mechanisms showing that iNOS is regulated at multiple levels including gene expression, protein aggregation, and protein degradation. Calmodulin (CaM) has always been thought as an iNOS cofactor facilitating electron transfer amid NO syntheses. We now show that CaM is essential for iNOS induction. CaM inhibition or knockdown prevented iNOS expressions in macrophages stimulated by inflammatory mediators. Further studies revealed that CaM acted through Ca2+/CaM-dependent kinase II (CaMKII), which functioned as a cardinal initiator in iNOS gene transactivation via both LPS-NF-¿¿¿¿¿B and IFN-γ-STAT1 pathways. Our study also found that iNOS gene transactivation required cytosolic Ca2+ elevations. CaM or CaMKII inhibition was found to prevent iNOS induction in endotoxemic mice and improved survival rates. These studies extend the role of CaM from an enzyme cofactor to an essential modulator in iNOS gene transactivation. Once expressed, biological iNOS output is determined by the levels of functional enzyme, which is influenced by protein stability. Our studies found that while initially existing as a soluble protein, iNOS progressively formed inactive protein aggregates. Blocking NO production prevented iNOS aggregation inside cells. iNOS aggresome formation could be recaptured by exposing cells to exogenous NO. The finding that NO per se induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation. After elucidating NO-mediated iNOS aggregation in physiological conditions, we then investigated iNOS protein stability under pathological circumstances upon heat (open full item for complete abstract)

Committee: Yong Xia (Advisor); Terry Elton (Committee Member); Arthur Strauch (Committee Member); Mark Wewers (Committee Member); Qianben Wang (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2009, Integrated Biomedical Sciences

The NF-kappa B family of transcription factors has been implicated in regulating cellular processes such as immune response, cell survival, cellular proliferation, and differentiation. This dissertation will focus on the involvement of NF-kappa B in cellular proliferation and differentiation. Studies support that NF-kappa B functions in cellular proliferation through the transcriptional regulation of cyclin D1, but whether such regulation is attributed to a single NF-kappa B subunit remains unclear. In chapter two we examine endogenous cyclin D1 levels during cell cycle re-entry in mouse embryonic fibroblasts (MEFs) lacking specific NF-kappa B signaling subunits. We demonstrate that each of these subunits is dispensable for regulating cyclin D1 transcription. However, we found that resulting cyclin D1 protein was severely reduced in MEFs lacking only RelA/p65. Cycloheximide treatment revealed that this regulation was due to an increase in protein turnover. Similarly downregulation of cyclin D1 protein, but not RNA, was observed in vivo in multiple tissues lacking p65. Co-immunoprecipitation analysis also showed p65 and cyclin D1 were capable of interacting, thus providing a possible explanation for cyclin D1 protein stability. In addition, although the decrease in cyclin D1 in p65-/- MEFs was concomitant with lower CDK4 activity during cell cycle re-entry, this was not sufficient to affect S phase progression. Nevertheless, similar decreases in cyclin D1 protein in primary p65-/- myoblasts was adequate to accelerate cell cycle exit and myogenesis in these cells. A number of studies have identified classical NF-kappa B activity as an inhibitor of the myogenic program. Recent findings reveal that in newborn p65-/- mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-kappa B may be a contributing factor in early postnatal skeletal muscle development. In chapter 3 we show that in addition to p65 deficiency, repression of NF-kappa B w (open full item for complete abstract)

Committee: Denis Guttridge Ph.D. (Advisor); Kay Huebner Ph.D. (Committee Member); Lawrence Kirschner MD./Ph.D. (Committee Member); Matthew Ringel MD. (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology

Master of Science, The Ohio State University, 2009, Pathology

The human immune system is a network of cells, physical barriers, and antimicrobial proteins that keep foreign microorganisms from causing serious disease. Despite many layers of protection, certain microbes have developed methods of evading the immune system and in some cases, using the host immunity to their advantage. The potential biowarfare agent Francisella tularensis is a highly infectious bacterium that has evolved mechanisms to use immune cells for its benefit. In this thesis we examined two aspects of the innate immune response to F. tularensis. Initially we investigated the role of the cytokine IFN-γ and the signaling molecule Akt in the production of nitric oxide in macrophages. This investigation included a titration of amount of IFN-γ required to ‘prime' the macrophages, a temporal profile of the production of NO as well as inhibitor/transgenic studies to gauge Akt's impact on the iNOS cascade. Secondly, we tested the immune response to different subspecies of Francisella. Our results showed a striking difference in the level of the immune response to some virulent strains, but not others. Curiously, the moderately pathogenic strain of Francisella seems to evade the host immune response more effectively than strains of greater virulence. Our examination into this immunity gap involved measuring cytokine responses, bacterial growth, intracellular signaling and microarray analysis of infected cells. The studies included in this thesis offer two different approaches to investigating the unknowns during Francisella infection – research into the molecular mechanisms responsible and descriptive study of a novel global phenomenon during infection.

Committee: Susheela Tridandapani PhD (Advisor); Jonathan Godbout PhD (Committee Member); James Waldman PhD (Committee Member) Subjects: Biomedical Research; Immunology; Microbiology

Doctor of Philosophy, The Ohio State University, 2009, Pharmacy

Endothelial cells are a primary target for injury and cardiovascular risk factors. Endothelial inflammatory activation and dysfunction has been critically implicated in diverse cardiovascular diseases. Inflammatory cytokines and bacterial products such as lipopolysaccharide (LPS) together with interferon-gamma (IFN-γ), can activate vascular endothelial cells and induce expression of pro-inflammatory proteins including such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which may promote endothelial cell injury and play an important role in the pathogenesis of many cardiovascular diseases. The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), is a cis-trans peptidyl-prolyl isomerase that catalyzes isomerization of phospho-proteins, affects their conformation and regulates most aspects of protein function. PIN1 is known to increase levels or activity of several transcription factors that are activated by phosphorylation at serine/threonine-proline and that can induce iNOS and COX-2. PIN1 can regulate these various proteins by increasing their mRNA, by reducing their protein turnover, and by regulating their subcellular distribution as well. However, the role of PIN1 in expression of iNOS and COX-2 in endothelial cells is unexplored. Thus, the original hypothesis of my dissertation was that PIN1 is necessary for induction of inflammatory proteins in endothelial cells. Here, the effect of PIN1 depletion and gene knockout on induction of iNOS and COX-2 by LPS and IFN-γ in murine aortic endothelial cells (MAEC) was determined. In contrast to the original hypothesis, knockdown of PIN1 with an shRNA-lentiviral system, transient transfection with small interfering RNA, and gene knockout each enhanced the induction of iNOS and COX-2 proteins by LPS/IFN. There was no effect on induction of their mRNA, suggesting a post-transcriptional effect. Knockdown of PIN1 increased the stability of iNOS and COX-2 proteins in cyclo (open full item for complete abstract)

Committee: Dale Hoyt (Advisor); Lakhu Keshvara (Advisor); Terry Elton (Committee Member); Anthony Young (Committee Member) Subjects: Pharmacology

Doctor of Philosophy, The Ohio State University, 2007, Animal Science

Failure to terminate the inflammatory response results in chronic inflammation that may lead to disease or cancer, especially in epithelial cells. We explored mammary epithelial cells as a model to identify mechanisms of anti-inflammatory activity in epithelia alone in the absence of immune cells. Bacterial endotoxin (ET) added to SCp2 mammary secretory epithelial cells: (1) induced both interleukin-6 (IL-6) secretion and nitric oxide (NO) production, but with unexpected delay in expression of mRNA for iNOS compared to IL-6; and (2) NFκB activation by 1 h after ET application (post-ET) that was transient for NFκB/p65 but persisted for NFκB/p50. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in ET-induced IL-6 and iNOS regulation via NFκB activation. Serum supplementation but not soluble extracellular matrix (EHS) enhanced ET-induced IL-6 mRNA expression and protein secretion without affecting iNOS mRNA expression or NO production, confirming the different modes of regulation of IL-6 and iNOS expressions. Culturing SCp2 cells on a confluent monolayer of SCg6 mouse mammary myoepithelial cells increased IL-6 secretion dramatically even in the absence of ET, with ET treatment further increasing IL-6 secretion but having little effect on induction of NO production over that for SCp2 cells alone; showing importance of microenvironment and cell-cell interaction in regulation of inflammation and likely its link to cancer in epithelia. ET-induced inflammation in SCp2 cells was used to screen and identify anti-inflammatory fractions of methanol extracts of wild Lebanese Centaurea ainetensis, used in Lebanese folk medicine to treat inflammatory diseases. A partially purified solid phase (SPE columns) methanolic elution fraction of C. ainetensis followed by methanol gradient elution on reverse phase HPLC chromatography (RP-HPLC) strongly inhibited ET-induc (open full item for complete abstract)

Committee: Floyd Schanbacher (Advisor); Charles Brooks (Other); James DeWille (Other); Joy Pate (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Pharmacy

Atherogenesis shares many features with inflammatory and immunal reactions. Bacterial endotoxin (LPS), together with T-lymphocytes cytokine interferon-gamma (IFNγ), causes inflammation and immune response. Because of its strategic anatomic position, endothelial cells are a primary target for injury and cardiovascular risk factors. A simple method for isolating and culturing mouse aortic endothelial cells (MAEC) was developed for studying the endothelial injury and activation by these stimuli in vitro (Section I). The effects of LPS and IFN, in the absence and presence of hypercholesterolemia, a traditional risk factor for atherosclerosis, on endothelial function and pro-inflammatory gene expression in vivo were also studied (Section II). We found that LPS and IFN synergistically activated MAEC by upregulating pro-inflammatory genes, such as inducible nitric oxide synthase and vascular cell adhesion molecule-1. Several signal transduction pathways, such as the Janus kinase/signal transducer and activator of transducer-1 (STAT1), the p38 mitogen-activated protein kinase, and the protein kinase A pathways, were involved in this regulation. Other effects of LPS included inducing the production of superoxide anion and hydrogen peroxide by MAEC. These reactive oxygen species, especially oxidized low-density lipoprotein cholesterol, caused MAEC injury, DNA breakage and gene expression. The in vivo effects of inflammation in endothelial dysfunction and atherogenesis were studied using a mouse model (C57BL/6) on an atherogenic diet. A single low dose of LPS (1.0 mg/kg, intraperitoneal injection) caused an impairment of endothelial-dependent vasorelaxation in response to acetylcholine in these mice but not their normal diet fed littermates. Endothelial-independent vasorelaxation remained unaffected. Chronic treatment with LPS (once a week for 12 weeks) or 16-week of the atherogenic diet each caused endothelial dysfunction. A more significant effect was observed with the combi (open full item for complete abstract)

Committee: Dale Hoyt (Advisor) Subjects: Health Sciences, Pharmacy

Master of Science in Chemistry, Cleveland State University, 2008, College of Science

Macrophages and other leukocytes play a vital role during inflammation. IFN-γ which is secreted by Th1 cells activates monocyte-macrophages and this activation leads to induction as well as suppression of certain genes expressed during inflammation. This suppression of genes may be potentially vital for resolution of inflammation. Upon IFN-γ stimulation, a Gamma-activated inhibitor of translation complex (GAIT) is formed, which binds to 3'-UTR of target mRNAs thereby silencing their translation. VEGF-A and ceruloplasmin are two proteins whose translation is suppressed by this pathway. The GAIT complex proteins have been identified as ribosomal protein L13a, glutamyl-prolyl tRNA synthatase (EPRS), NS-1 associated protein 1 (NSAP-1), and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (Mazumder et al., 2003; Sampath et al., 2004). The cellular abundance of the proteins involved in GAIT complex formation is in excess of Cp or VEGF mRNA, so we hypothesize that there exists additional mRNAs that are subject to GAIT-mediated translational control. Hence further studies were done to investigate other possible targets of this pathway. A probable list of targets containing putative GAIT-like elements in 3'-UTR were obtained using pattern search, a bioinformatic approach (Ray and Fox, 2007). Among these targets, inducible nitric oxide synthase (iNOS) was found to be of particular interest as nitric oxide which is produced by enzymatic activity of nitric oxide synthases, is central for the function of macrophages and also for early immune responses to invading microorganisms. Apart from iNOS, translational silencing of putative GAIT element bearing mRNAs of ADAM10, GLUT10, LITAF, GABA B receptor and mouse iNOS in their 3'-UTR was also investigated in IFN-γ treated monocytic cells which suggests that there might be additional targets of this pathway.

Committee: Paul L. Fox PhD (Committee Chair); Aimin Zhou PhD (Advisor); Valentin Gogonea PhD (Committee Member) Subjects: Cellular Biology