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  • Plos One Transient Plasma Membrane Disruption Induced Calcium Waves in Mouse and Human Corneal Epithelial Cells - Figure Data Files

    Zhong, Chen; Lu, Xiaowen; Watsky, Mitchell; Department of Cellular Biology and Anatomy
    Data Files for figures 2-6 in manuscript titled "Transient Plasma Membrane Disruption Induced Calcium Waves in Mouse and Human Corneal Epithelial Cells" published in PLOS ONE.
  • OPTIMIZED ISOLATION AND QUANTIFICATION OF IN VIVO DISTRIBUTION OF EXOSOMES FOR POTENTIAL TARGETED THERANOSTIC APPLICATION

    Rashid, Mohammad Harun; Department of Biochemistry and Molecular Biology (Augusta University, 2019-07)
    Exosomes are critical mediators of intercellular crosstalk and regulators of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosome research has been thwarted by our limited knowledge of the most efficient isolation method and the in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 μm syringe filter and 100k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes compared to other available methods. We demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. Further, we also generated engineered exosomes expressing precision peptide for targeting CD206 positive M2-macrophages. M2-macrophages participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their pro-tumorigenic function and prevalence in most malignant tumors with lower survival, early in vivo detection and intervention of M2-macrophages may boost the clinical outcome. To determine in vivo distribution of M2-macrophages, we adopted 111In-oxine based radiolabeling of the targeted exosomes and SPECT. When injected these radiolabeled targeted exosomes into 4T1 breast tumor-bearing mice, exosomes accumulated at the periphery of the primary tumor, metastatic foci in the lungs, in the spleen, and liver. Ex vivo quantification of radioactivity also showed similar distribution. Injected DiI dye-labeled exosomes into the same mice showed the adherence of exosomes to the CD206 positive macrophages on ex vivo fluorescent microscopy imaging, confirming the targeting efficacy of the exosomes. In addition, we utilized these engineered exosomes to carry the Fc portion of mouse IgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. We have auspiciously demonstrated that M2-macrophage targeting therapeutic exosomes deplete M2-macrophages both in vitro and in vivo, and reduce tumor burden in a metastatic breast cancer model. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.
  • THE ROLE OF GPR109A IN REGULATION OF RETINAL ANGIOGENESIS AND BLOOD-RETINAL BARRIER AS A POTENTIAL THERAPEUTIC TARGET IN DIABETIC RETINOPATHY

    Abdelrahman, Ammar; Department of Biochemistry and Molecular Biology (Augusta University, 2020-12)
    Currently, treatments of diabetic retinopathy (DR) have limited therapeutic benefits and limited accessibility to the growing diabetic population at risk because of the high expenses and complicated procedures. Inflammation, endothelial dysfunction, and microvascular damage are common features of diabetic complications including DR. GPR109A is the metabolite sensing receptor of beta-hydroxybutyrate (BHB) the principal ketone body in humans. Our previous studies have shown the role of GPR109A expression in promoting anti-inflammatory response in retinal pigmented epithelial (RPE) cells and the relevance of the receptor in DR. Expression of the GPR109A in microvascular endothelial cells (ECs) has been reported recently. However, the relevance of GPR109A expression and activation to retinal EC functions are yet to be studied. Our goal in this study was to identify the role of GPR109A expression and activation in barrier and angiogenic functions of retinal ECs in context of diabetic retinopathy. We used electrical cell impedance sensing (ECIS) technology to evaluate barrier functions in primary human retinal endothelial cells (HRECs) which constitute the inner BRB. Knocking down GPR109A in HRECs with siRNA decreased the transendothelial electrical resistance (TEER) compared to scrambled siRNA. Treating HRECs with BHB increased their TEER and counteracted VEGF-induced barrier disruption through activation of GPR109A and increasing zonula occludens-1 (ZO-1) expression. Treatment of STZ-diabetic mice with exogenous BHB for one month protected against the pathologic albumin leakage induced by diabetes and improved the visual acuity of this animal model of diabetes. Using the mouse model of oxygen induced retinopathy (OIR), we showed that Gpr109a-/- mice had slower vascular recovery from pathologic angiogenesis compared to age matched wild type mice. Moreover, physiologic revascularization of vaso-oblitrated retinas was impaired by loss of GPR109a and associated with dysregulated inflammatory and angiogenic signaling. Collectively, these data point to a role for GPR109A in the regulation of barrier and angiogenic mechanisms in retinal ECs and, promote the receptor as a potential druggable target for impacting these mechanisms in microvascular retinal diseases such as DR.
  • A Molecular Basis of Chemoresistance in Bladder Cancer

    Lahorewala, Sarrah; Biochemistry and Cancer Biology (Augusta University, 2020-12)
    Background: In advanced bladder cancer (BC), development of resistance to the frontline chemotherapeutic drugs Gemcitabine and Cisplatin contributes to the poor prognosis of patients. Newly discovered chondroitinase, HYAL-4 V1 (V1), drives malignant transformation in BC. We evaluated V1’s role and the downstream molecules involved in the mechanistic regulation of chemoresistance in BC. Experimental Design: HYAL-4 expression was evaluated by RT-qPCR and IHC in metastatic muscle-invasive BC patients who received Gemcitabine plus Cisplatin chemotherapy. HYAL-4 wild-type and V1 were stably expressed or silenced in three BC and one normal urothelial cell line. Transfectants were analyzed for Gemcitabine and Cisplatin sensitivity, and for Gemcitabine influx and efflux to determine the mechanism of Gemcitabine resistance. The effect of cytidine deaminase (CDA) inhibition on Gemcitabine sensitivity was evaluated in vitro and in xenograft models. Results: HYAL-4 expression was an independent predictor of disease-specific mortality and treatment failure in our clinical cohort, and stratified patients into higher risk for both those outcomes. V1-expressing BC and normal urothelial cells were resistant to Gemcitabine due to the upregulation of cytidine deaminase (CDA) expression and activity, resulting in increased Gemcitabine metabolism and efflux; treating cells with tetrahydrouridine (THU), a CDA inhibitor, abrogated the chemotherapeutic resistance. Gemcitabine-resistant V1 cells demonstrated increased expression of V1’s substrate CD44 and phosphorylated STAT3. Si-RNA mediated CD44 knockdown and STAT3 inhibition both sensitized cells to Gemcitabine in vitro. In xenograft models, treatment with a combination of Gemcitabine and THU completely inhibited tumor growth. Conclusions: This project discovered V1 as a novel determinant of Gemcitabine resistance and potential predictor of treatment response in BC. V1 drives resistance to Gemcitabine through CD44-STAT3 mediated upregulation of CDA, and inhibiting this pathway sensitizes tumor cells to the therapy in preclinical models of BC.
  • OSTEOPONTIN AS A NOVEL IMMUNE CHECKPOINT

    Klement, John; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
    The host adaptive immune system functions to discriminate self from non-self, eliminating threats from viral infection to tumors. Cytotoxic lymphocytes (CTLs) are the primary effector arm of adaptive immunity. To prevent aberrant activation and autoimmunity, immune checkpoints function physiologically to restrain the CTL response. Tumors pathologically express these checkpoints, preventing immune-driven tumor clearance. Accordingly, immune checkpoint inhibitors (ICIs) have shown remarkable clinical success. However, many types of malignancies, as well as many individual patients with responsive tumor types, fail to benefit from current ICI immunotherapies. This conundrum suggests that as-yet undiscovered immune checkpoints exist. We observed that mice deficient in the transcription factor interferon regulatory factor eight (IRF8) tolerated allogenic tumor grafts and demonstrated impaired CTL activation with an accumulation of CD44hi memory-like CTLs. We sought to investigate the mechanism of this immunosuppression. Conditional deletion of IRF8 in T cells, as well as a mixed chimera model, demonstrated that IRF8 did not directly control CTL activation or differentiation into a CD44hi population. Instead, global loss of IRF8 lead to an expansion of an immature myeloid CD11b+Ly6G+Ly6Clo population which highly expressed osteopontin (OPN), a physiological ligand for CD44. Elevated levels of OPN were shown to suppress murine CTL activation and proliferation. A similar IRF8-OPN-CD44 axis was observed in murine and human colorectal cancer, which is refractory to current ICI therapies. Malignant cells and human patients displayed enhanced OPN levels relative to healthy donor controls. This was shown to be mediated by loss of IRF8 expression, which directly bound to the OPN promoter to repress its transcription. Elevated levels of OPN similarly prevented human CTL activation, and higher levels of OPN were correlated with decreased survival in human patients. We have shown that the IRF8-OPN-CD44 axis functions as a novel immune checkpoint in both myeloid and tumor cells. Blockade of OPN may have potent anti-tumor activity, expanding the pool of patients responsive to ICI therapy.
  • Gene regulation by the putative Campylobacter jejuni diguanylate cyclase CbrR

    Fulmer, Claudia; Department of Biochemistry and Cancer Biology (Augusta University, 2020-05)
    As a leading cause of bacterial gastroenteritis, Campylobacter jejuni incurs health care costs estimated at $290 million a year in the US and up to 40,000 deaths in children aged 5 and younger worldwide. As such, determining those proteins that regulate C. jejuni virulence factors are prime targets to possibly develop a prophylactic therapy, that as of yet does not exist. CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), the class of enzyme that catalyzes the synthesis of cyclic di-GMP, a universal bacterial second message molecule, from GTP. In C. jejuni strain DRH212, an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+, were constructed. Soft agar motility tests, biofilm formation assays, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were performed. Site-directed mutagenesis was performed on cbrR to make alanine substitutions in both the autoinhibitory site (I-site) and active site and differential radial capillary of action ligand (DRaCALA) assays were performed to determine nucleotide binding by wild-type CbrR and the CbrR point mutants. Soft agar motility assays indicated a hyper-motile phenotype associated with the C. jejuni cbrR- mutant, whereas motility was all but negated in the cbrR+ complement. Biofilm assays and SEM demonstrated similar formation and robustness of biofilms between wild type and cbrR- mutant, however cbrR+ was unable to form significant biofilms in 72 hours. TEM images showed similar cellular morphology between cbrR-, wild type, and cbrR+, however cbrR+ cells had fewer flagella. DRaCALA assays showed wild-type CbrR and the active site mutant were both able to bind GTP and cyclic di-GMP, whereas the I-site mutant lost the ability to bind cyclic di-GMP, indicating the product binding site on CbrR. The highly conserved diguanylate cyclase CbrR is the only annotated DGC in the C. jejuni genome. Though the active site sequence is highly variant when compared to the consensus sequence, this protein is able to bind both substrate and product of the chemical synthesis of cyclic di-GMP and has now been shown to be a negative regulator of motility, a critical virulence factor in C. jejuni pathogenesis.
  • GLYCOSAMINOGLYCANS, CHONDROITINASE, AND MOLECULAR SUBTYPES IN BLADDER CANCER

    Morera, Daley S; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
    There is a need for novel prognostic biomarkers and targeted treatments in bladder cancer (BC), even more so for muscle-invasive disease (MIBC). Discovery of molecular markers to predict outcome in BC patients may lead to identification of impactful therapeutic targets. The hyaluronic acid (HA) family of molecules and distinct molecular subtypes have both been investigated as potential prognostic markers. HA family and chondroitin sulfate proteoglycans, such as CD44, have been implicated in driving aggressiveness of disease; however, a chondroitinase enzyme that cleaves chondroitin sulfate proteoglycans has not been identified in any eukaryotic system. We evaluated molecular markers of BC and the first known eukaryotic/human chondroitinase, that we identified, for their ability to predict clinical outcome in patients, and for their roles as drivers of disease. We also investigated the anti-tumor effects of HA synthesis inhibitor 4-methylumbelliferone (4MU), a non-toxic orally bioavailable supplement. This study demonstrates that transcript levels of HA family members can predict metastasis and poor survival in BC patients. HA- family expression also correlated with epithelial mesenchymal transition (EMT) markers β -Catenin, Twist, Snail, and E-Cadherin. HA signaled through its receptors CD44/RHAMM and the PI3-K/AKT axis. 4MU targeted HA signaling, inhibiting proliferation and motility/invasion, inducing apoptosis in vitro, and preventing tumor growth in vivo. We discovered that a previously unidentified splice variant of HYAL-4 was elevated in bladder tumors. We named this variant "V1". Our studies showed that V1 had chondroitinase activity, cleaved chondroitin-6-sulfate from CD44, and consequently increased CD44 secretion. In vivo, V1-expressing urothelial cells formed muscle-invasive tumors and V1-expressing cancer cells developed metastatic tumors. Evaluation of the prognostic significance of the molecular subtypes of MIBC that were recently identified by other groups, showed the subtypes to have little to no predictive ability for clinical outcome in multivariate analyses that included standard clinical parameters. Consistently, clinical parameters such as histopathologic tumor grade, T-stage, and lymph node status, outperformed the molecular subtypes. Contrarily, V1 levels could independently predict metastasis and survival with high efficacy, suggesting that focusing on V1 as a functional biomarker may be a better strategy to improve clinical outcome of BC patients.
  • HLA-G DIMER PROLONGS KIDNEY ALLOGRAFT SURVIVAL BY INHIBITING CD8+T CELL ACTIVATION AND GRANZYME B EXPRESSION

    Ajith, Ashwin; Biomedical Sciences (Augusta University, 2019-12)
    Solid organ transplantation is the preferred therapy for many patients diagnosed with end stage organ failure, however allograft rejection is a significant barrier for graft survival. Patient care involves heavy immunosuppressive drug treatment leading to elevated risk for cancer and other opportunistic infections. Hence there is a need to develop effective alternative approaches to minimize graft rejection. We focused on Human leukocyte antigen G (HLA-G), a nonclassical HLA class Ib molecule critically involved in the maintenance of maternal tolerance to semi-allogeneic fetal tissues during pregnancy and has emerged as a potential therapeutic target to control allograft rejection. We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants. Using genomics, molecular and cellular analyses of cells from T-cell–mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the downregulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.
  • Investigating the Role of the Hdac3 Co-Repressor Complex in Glucocorticoid Signaling-Mediated Bone Marrow Lipid Storage with Age

    Pierce, Jessica Liane; Biomedical Sciences (Augusta University, 2019-12)
    Aging bone is characterized by loss of tissue density, marrow fat accumulation, and dysregulated bone marrow stromal cell (BMSC) differentiation. The contribution of the epigenetic regulator histone deactylase 3 (Hdac3) is of increasing interest in bone biology. Hdac3 expression decreases with aging, and the current model for conditional deletion of Hdac3 in Osterix-expressing osteoprogenitor cells (Hdac3-CKOOsx) exhibited an aged bone phenotype in young mice along with the novel finding of osteoblastic (Runx2+ osteogenic cells) lipid droplet storage. In addition, bone-specific loss of Hdac3 activity increases expression of the glucocorticoid (GC)-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (Hsd11b1), suggesting a mechanism for the increased lipid accumulation in aged and Hdac3-deficient BMSC-derived osteoblasts. The cofactor nuclear receptor corepressor 1 (NCoR1), which mediates Hdac3 enzymatic activity in a co-repressor complex (CRC), was proposed as a regulator of Hdac3 activity in bone. Both Hdac3 and NCoR1 expression decreased in aged osteoblasts, and the two factors exhibited synergy in downregulating the promoter activity of glucocorticoid-responsive elements. Because of the relationship between increased GC signaling and osteoporosis, the glucocorticoid receptor (GR) was investigated as a mediator of the marrow fat phenotype, with the hypothesis that loss of GR function in bone would be protective against common forms of osteoporosis. Chronic caloric restriction in WT and GR-deficient (GR-CKOOsx) mice was used as a short-term stressor to induce an osteoporotic phenotype, while aging of GR-deficient mice (where Hdac3 CRC expression naturally decreases) was used as a biologically-relevant model for dual loss of Hdac3 and the GR. Surprisingly, the loss of GR function in osteoprogenitors exacerbated bone loss and marrow fat accumulation in both models—and induced a chronic stress phenotype by increasing cellular bioenergetics and whole-body metabolic rate—providing evidence of a role for the GR in facilitating healthy bone maintenance as well as evidence for compensatory mechanisms that regulate bone biology through GC signaling. GR-deficient bone also induced changes to whole-body physiology (e.g., sarcopenia, decreased physical activity, metabolic dysfunction) that further demonstrate the intricacies of bone as an endocrine organ. The current study provides new avenues to investigate cell signaling, bioenergetics, and tissue crosstalk in osteoporotic bone.
  • CHOLINESTERASE INHIBITOR TOXICITY: MECHANISTIC STUDIES AND THERAPEUTIC STRATEGIES FOCUSED ON AXONAL TRANSPORT

    Naughton, Sean X; Biomedical Sciences (Augusta University, 2019-12)
    Organophosphates (OPs) are a broad class of chemicals with a variety of uses that include pesticides, chemical warfare agents, fuel additives, and plasticizers. Due to their sheer number of applications and known toxicological profile, OPs represent a persistent concern to human health worldwide. Furthermore, the effects of OPs that occur independently of their well-known mechanism of acute toxicity (AChE inhibition) have not been well studied. The presented research seeks to expand upon our understanding of AChE-independent mechanisms of OP toxicity as well as to identify potential therapies for treating these negative effects. In Manuscript 1 we demonstrate that the OP diisopropylfluorophosphate (DFP) induced axonal transport deficits occur in vivo at exposure levels that were not associated with cholinergic toxicity. Additionally, we observed deficits in white matter integrity following sub-acute DFP exposure. In Manuscript 2 we present a series of experiments, which were conducted to identify potential therapeutic compounds for the treatment of OP induced deficits in axonal transport. Here, we utilized a phenotypic drug-screening assay in order to identify compounds that could be protective against DFP. In Manuscript 3 we present data which demonstrates that the carbamate physostigmine does not impair axonal transport, as has been previously demonstrated with OPs. These experiments were critical to demonstrating the AChE independence of OP-induced axonal transport deficits and further elucidate the unique nature of OP toxicity in comparison to other AChE inhibitors. Collectively, these studies contribute to a better understanding of the full spectrum of toxicological effects of OPs and provide insightful findings into potential therapeutics for the treatment of OP related toxicity.
  • Primary Tumor-Induced Immunity Is Suppressed By Surgery-Induced Inflammation In The Presence Of Residual Tumor Cells

    Piranlioglu, Raziye; Department of Biochemistry and Molecular Biology (Augusta University, 2019-12)
    It is widely thought that tumor cells disseminate from a primary site into the circulation during the early stages of tumor development. However, the fate of these early disseminated tumor cells (DTCs) has been elusive. By utilizing the murine mammary tumors, 4T1 and EMT6, in a syngeneic mouse model, we show that both tumors disseminate into secondary organs but only 4T1 tumors are able to generate metastasis. In contrast, EMT6 primary tumors induce an anti-tumor response that leads to elimination of DTCs. This anti-tumor immunity is CD8+ T cell-dependent and provides long-term immunity. Furthermore, the mice are free of DTCs within a couple of days when primary tumors are completely resected and they reject subsequently injected tumors, whereas mice with residual tumors following surgery show enhanced local recurrence and outgrowth of DTCs at metastatic sites; this effect may be explained by elevated levels of granulocyte colony-stimulating factor (G-CSF). This increase is accompanied by an accumulation of immature myeloid-derived suppressor cells (MDSCs) in the spleen and lungs, the main target organ for metastasis. Moreover, the infiltration of a granulocytic subset of MDSCs (gMDSCs) leads to a decrease in a subset of T cells that have a role in long-term immunity. Our goal for this study is to elucidate how immune components of distant organs affect the fate of DTCs and the role of surgery induced-inflammation in generating a pre-metastatic niche. Our studies may also provide a molecular explanation of improved overall survival in breast cancer patients following complete resection of primary tumors with negative margins.
  • DISCOVERY AND VALIDATION OF A NOVEL NEUTROPHIL ACTIVATION MARKER ASSOCIATED WITH OBESITY

    Pan, Yue; Biomedical Sciences
    Obesity and its related comorbidities such as cardiovascular disease (CVD) have imposed a huge burden on public health worldwide. Identification of the mechanistic pathways by which obesity impacts cardiovascular health is urgently needed to provide new targets for prevention of obesity and its associated CVDs. Low-grade systemic inflammation accompanies obesity and etiologically contributes to obesity-induced CVD. Neutrophils represent the most abundant type of leukocytes in humans and neutrophil activation is a fundamental process in the inflammatory response. Growing evidence supports that neutrophils are most likely to be the target peripheral leukocyte subtype initiating the adipose tissue inflammatory cascade in response to obesity. As depleting neutrophils is not a choice in humans, identification of obesity induced neutrophil activation markers becomes a prerequisite to develop targeted treatment. Therefore, our central hypothesis is that there are neutrophil activation markers that can specifically respond to obesity status and mediate obesity’s effect on CVD risks. The goal of this study is to identify these markers and their roles on CVD risk using a step-wise approach including an unbiased omic step (i.e. the discovery phase) and a target step (i.e. the validation phase). To achieve this goal, we used the biological samples of 688 subjects from multiple cohorts that generally have neutrophils, white blood cells, and plasma stored. CVD risk factors including blood pressure, insulin resistance, lipid profile, and pulse wave velocity have been measured. In the discovery phase, genome wide DNA methylation, RNA-sequencing and quantitative proteomics were obtained from the purified neutrophils. A significant difference was found for one gene, ALPL, across 3 omics platforms. In the validation phase, ALPL expression and the cellular protein levels were found to be higher in obese compared with lean subjects. Within the obese population, we observed ALPL expression level positively associated with CVD risk factors including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), carotid intima–media thickness (IMT), triglycerides (TG), and fasting insulin. This study identified one novel marker of neutrophil activation in response to obesity and provided evidence that obesity induced changes in ALPL expression were associated with CVD risk factors.
  • ROLE OF ARGINASE IN OBESITY-INDUCED VISCERAL ADIPOSE TISSUE DYSREGULATION AND ENDOTHELIAL DYSFUNCTION

    Atawia, Reem T.; Biomedical Sciences (Augusta University, 2019-11-05)
    An obesity epidemic continues to rise worldwide. Visceral (central) obesity is an important concern as it correlates with metabolic and cardiovascular pathologies. Arginase is a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial). We found that visceral adipose tissue (VAT) from obese WT mice fed a high fat/high sucrose diet (HFHS) showed a significantly higher expression of A2 compared to mice fed normal chow diet (ND). We also observed that A2 expression is upregulated 3-fold in differentiated 3T3- L1 adipocytes exposed to high levels of palmitate and glucose, a mimic of the obese state, compared to control media. Our study focused on the involvement of A2 in obesity associated metabolic and vascular disorders. WT mice and those globally lacking A2 (A2-/-) were fed HFHS or ND for 16 weeks. The HFHS diet-induced increases in body and VAT weights and total adiposity were prevented or reduced in A2-/- mice. In concert, metabolic chamber studies revealed that energy expenditure and fatty acid oxidation rates were significantly higher in A2-/- compared to WT HFHS mice. VAT from A2-/- mice fed HFHS had higher levels of active AMPK-α, the master regulator of fatty acid metabolism, as well as higher adipocyte expression of genes involved in fatty acid β-oxidation and oxidative phosphorylation, along with preserved mitochondrial density compared to WT HFHS. A2 deletion also prevented HFHS-induced fibrous tissue deposition and inflammation in VAT, which contributed to adipocyte metabolic dysfunction. These results indicate that A2 is involved in metabolic dysfunctions. To gain insights into the role of A2 in adipocytes, primary preadipocytes isolated from VAT of A2-/- mice and differentiated in vitro showed increased expression of adiponectin and better mitochondrial function. Adenoviral overexpression of A2 in differentiated 3T3-L1 cells showed impaired mitochondrial function and increased mitochondrial ROS. Obesity-related metabolic disorders increase the risk of cardiovascular diseases, the leading global cause of death. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2-/- mice, but more prominently prevented in A1+/- mice. In conclusion, A2 is critically involved in HFHS-induced obesity, VAT inflammation and metabolic dysregulation. Both A1 and A2 are involved in HFHS-induced vascular endothelial dysfunction.
  • DNA METHYLATION AS A KEY PLAYER IN INFLAMMATION-MEDIATED COLON TUMORIGENESIS

    Ibrahim, Mohammed Mahmoud Labib; Biomedical Sciences (Augusta University, 2019-05)
    A causal link between chronic inflammation and tumorigenesis is now well established in the literature with a great deal of supporting evidences from genetic, epigenetic, pharmacological and epidemiological perspectives. In particular, inflammatory bowel diseases represent an important risk factor for colon cancer development. Moreover, it seems that even sporadic colon cancers that do not develop as a complication of chronic colitis are also driven by inflammation. However, the molecular mechanisms behind inflammation-mediated colon tumorigenesis have remained largely unknown. Colitis associated cancer development is thought to be multifaceted due to a combination of genetic and epigenetic aberrations. Recently, epigenetic alterations -particularly aberrant DNA methylation- have gained great attention in cancer biology and have been observed to play a key role in the pathogenesis of inflammation-associated tumors; especially in colitis-associated cancer. IRF8, a key transcription factor originally identified in myeloid cells, has been reported to play a crucial role in myeloid cells differentiation and immune response regulation. IRF8 deficiency is associated with deregulation of myeloid cell differentiation and accumulation of immature myeloid subsets phenotypically and functionally resemble MDSCs (Myeloid Derived Suppressor Cells). IRF8 is thought to function as a tumor suppressor and was found to be silenced in different types of cancers including colon cancer. Myeloid derived-IRF8 has been extensively studied. However, the role of epithelial-derived IRF8 in colon inflammation and colon cancer initiation remains a point to be addressed. In this study, we generated conditional Irf8cKO mice in which IRF8 is specifically deleted in colon epithelium. Irf8cKO mice exhibit a more aggressive pattern of colitis associated cancer with higher tumor incidence and severe loss of body weight. Additionally, we provide evidence that chronic inflammation promotes the accumulation and infiltration of CD11b+Gr1+ MDSCs, which plentifully secrete IL10 in colon tissue. IL10 then induces STAT3 phosphorylation and nuclear translocation to bind to Dnmt1 and Dnmt3b promoters to upregulate their expression, leading to DNA hyper-methylation at the Irf8 promoter to silence IRF8 expression in colonic epithelial cells and promote colon tumorigenesis. Collectively, our data pinpoint the MDSC-IL10-STAT3-DNMT3b-IRF8 axis as a novel bridge between chronic inflammation and colon cancer formation.
  • The c-MYC oncogene deregulates global DNA methylation and hydroxymethylation to control genome-wide gene expression for tumor maintenance in leukemia/lymphoma

    Poole, Candace Jean; Biomedical Sciences (Augusta University, 2019-05)
    Aberrant DNA methylation is a characteristic feature of tumor cells. However, our knowledge of how DNA methylation patterns are established and maintained to contribute to tumorigenesis is limited. Inactivation of the c-MYC oncogene triggers tumor regression in T-cell acute lymphoblastic leukemia (T-ALL) resulting in dramatic changes to the chromatin landscape including DNA methylation. In this study, I investigated how MYC regulates DNA methylation and hydroxymethylation patterns to contribute to gene expression programs important for tumor maintenance in T-ALL and Burkitt lymphoma. I report that MYC maintains 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) patterns by regulating the DNA methylation machinery, which is important for gene expression in T-ALL. DNA methyltransferases (DNMTs) initiate 5mC marks, while Ten-eleven translocation methylcytosine dioxygenases (TETs) oxidize 5mC to produce 5hmC as an intermediate modification, ultimately leading to active DNA de-methylation. I demonstrated that DNMT1 and DNMT3B are MYC target genes and that their expression is dependent on high MYC levels. Knockdown of DNMT3B in T-ALL reduced cell proliferation through cell cycle arrest and caused the reactivation of gene transcription through reversing promoter/CpG island methylation. Furthermore, I demonstrated that TET1 and TET2 expression is MYC-dependent, as high TET1 and low TET2 levels depend on oncogenic MYC. Knockdown of TET1 in T-ALL reduced cell proliferation through cell cycle arrest and caused genome-wide changes in 5mC and 5hmC corresponding to changes in gene programs important for ribosomal biosynthesis and protein synthesis. In contrast, ectopic expression of TET2 reduced tumor cell proliferation through apoptosis/necrosis and caused genome-wide changes in 5mC and 5hmC corresponding to changes in transcriptional regulatory gene programs. My finding that a coordinated interplay between components of the DNA methylation machinery is necessary for MYC-driven tumor maintenance highlights the potential of targeting specific DNMT or TET proteins for therapeutic strategies.
  • The Role of GPR109A in NAD+ Metabolism in Aging RPE

    Fuller, Jasmine; Department of Biochemistry and Cancer Biology (Augusta University, 2018-12-28)
    Age-related macular degeneration (AMD) is the leading cause of blindness in people over the age of 50 worldwide. The retinal pigment epithelium (RPE), located in the back of the eye, is most affected in AMD. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme common to most metabolic pathways. Reductions in NAD+ and NAD+- dependent enzymes (e.g., SIRT1) have been linked causally to the development/progression of many age-related pathologies. None, however, have evaluated NAD+ directly or the mechanisms governing its biosynthesis and related availability in RPE. In our previous study, we have shown that NAD+ levels decline with age in the RPE. This correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression. SIRT1 expression and activity was also significantly reduced. Using the human RPE cell line, ARPE-19, primary mouse RPE cells and FK866, a highly specific, noncompetitive NAMPT inhibitor, we simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence. Using this model, we demonstrated the positive impact that therapies that provide supplemental or alternate energy sources such as nicotinamide mononucleotide (NMN) and β-hydroxybutyrate (B-HB) have on RPE viability and the possible role of G-protein coupled receptor, GPR109A in this process.
  • Immune regulation of tumor cell plasticity: A promising molecular target in breast cancer metastasis

    LEE, EUNMI; Department of Biochemistry and Molecular Biology / Cancer Center (2018-11-29)
    It is widely accepted that phenotypic plasticity of malignant cells is required during metastatic cascade. However, the specific mechanism of how the tumor microenvironment regulates tumor cell plasticity in metastasis is under intense investigation. We demonstrate here that monocytic and granulocytic subsets of myeloid-derived suppressor cells (MDSC), hereafter called mMDSCs and gMDSCs, infiltrate in the primary tumor and distant organs with different time kinetics and regulate spatiotemporal tumor plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumor-infiltrating mMDSCs facilitate dissemination from the primary site by inducing the EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support metastatic growth by reverting the EMT/CSC phenotype and promoting tumor cell proliferation. We also observe that lung-derived gMDSCs isolated from tumor-bearing mice enhance metastatic growth of already disseminated tumor cells. Our ongoing studies reveal that calprotectin (S100A8 and S100A9 heterotetramer) is an important regulator of gMDSCs, which play a critical role in promoting breast cancer metastasis by inducing MET-like CSCs as well as suppressing anti-tumor immunity within the pre-metastatic niche. Furthermore, we develop a novel gMDSC-targeting compound that potentially binds to calprotectin and validate its therapeutic utility in a preclinical breast cancer model. Our goal for this study is to elucidate the molecular co-evolution of tumor and immune cells in cancer development and to identify molecular targets to provide alternative therapeutic options for women with metastatic disease.
  • THE TUMOR SECRETORY FACTOR ZAG PROMOTES WHITE ADIPOSE TISSUE BROWNING AND ENERGY WASTING IN CACHEXIA

    Elattar, Sawsan; Department of Biochemistry and Molecular Biology / Cancer Center (8/7/2018)
    SAWSAN ELATTAR The Tumor Secretory Factor ZAG Promotes White Adipose Tissue Browning and Energy Wasting in Cachexia (Under the direction of SATYANARAYANA ANDE) Cachexia is a complex tissue-wasting syndrome characterized by inflammation, hyper-metabolism, increased energy expenditure and anorexia. Browning of white adipose tissue (WAT) is one of the significant factors that contribute to energy wasting in cachexia. Tumors secrete an array of secretory factors, such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon γ (IFNγ) and zinc-α2-glycoprotein (ZAG), that have been implicated in altering metabolism and promoting cachexia. Previous studies have demonstrated that ZAG can induce lipolysis; however, whether ZAG plays a role beyond lipolysis remains unclear. Here, by utilizing a cell implantation model, we demonstrate that the lipid-mobilizing factor, ZAG, induces WAT browning in mice. Increased circulating levels of ZAG not only induced lipolysis in the adipose tissues, but also caused robust browning in the WAT. Stimulating white adipose progenitors with ZAG recombinant protein or expression of ZAG in mouse embryonic fibroblasts (MEFs) strongly enhanced brown-like differentiation. At the molecular level, ZAG stimulated peroxisome proliferator-activated receptor gamma (PPARƔ) and early B cell factor 2 (Ebf2) expression and promoted their recruitment to the PR/SET domain 16 (Prdm16) promoter, leading to enhanced expression of Prdm16, which determines brown cell fate. In the brown adipose tissue (BAT), ZAG stimulated the expression of PPARƔand peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and promoted recruitment of PPARƔ to the uncoupling protein 1 (UCP1) promoter, leading to increased expression of UCP1. Collectively, by promoting WAT browning and by activating thermogenesis in the BAT, ZAG increased body energy expenditure. Overall, our results revealed a novel function of ZAG in WAT browning and highlight that targeting ZAG may have therapeutic applications in humans with cachexia. KEYWORDS: (Cachexia, beige adipocyte, brown fat, adipose atrophy, Zinc-α2-glycoprotein, Ebf2, Prdm16, PPARƔ, UCP1)
  • Canonical Wnt Signaling in Antigen Presenting Cells Regulates Microbiota-Induced Inflammation and Immune Cell Homeostasis in the Colon

    Swafford, Daniel Joseph; Department of Biochemistry and Molecular Biology / Cancer Center (8/3/2018)
    Aberrant Wnt/β-catenin-signaling occurs in several inflammatory diseases including inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. Here, we investigated the importance of canonical Wnt signaling in CD11c+ antigen presenting cells (APCs) in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6) in CD11c+ APCs in mice (LRP5/6ΔCD11c mice) resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors such as IL-10, retinoic acid (RA), and IDO. In addition, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis, which led to a loss of systemic tolerance to oral antigen. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced acute intestinal inflammation with decreased histopathological severity of colonic tissue. Loss of canonical Wnt signaling in CD11c+ APCs also results in an increase in colonic polyp formation and exacerbation of chronic inflammation/injury. This was also heavily dependent on the presence and composition of the gut microbiota, as fecal transfers from LRP5/6ΔCD11c mice to floxed control (LRP5/6FL/FL) mice that were administered an antibiotic cocktail produces a polyp load and weight loss similar to that of LRP5/6ΔCD11c mice without treatment. Additionally, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice reduces severity of inflammation-associated colon carcinogenesis in these mice. Furthermore, we show that treatment of LRP5/6ΔCD11c mice with either RA or IL-10 reduces severity of inflammation-associated colon carcinogenesis. Mechanistically, RA and IL-10 may independently reduce key inflammatory factors at the acute phase of colitis. These results ultimately reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt signaling pathway, which may serve as a promising target for chronic inflammatory disorders.
  • CXCR2 EXPRESSING TUMOR CELLS DRIVE VASCULAR MIMICRY IN ANTI-ANGIOGENIC THERAPY RESISTANT GLIOBLASTOMA

    Angara, Kartik Prasad; Department of Biochemistry and Molecular Biology (Augusta University, 7/20/2018)
    Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels, control abnormal vasculatures and prevent recurrence. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties, lay down vascular patterned networks reminiscent of host endothelial blood vessels and served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands in the event of the ensuing hypoxia resulting from AAT. In our studies, we have demonstrated that AAT accelerates VM. We observed that Vatalanib (a VEGFR2 tyrosine kinase inhibitor) induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer side. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at the periphery of the tumors. During our extensive studies to understand the tumor-inherent mechanisms of AAT-induced resistance, we identified a crucial chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. IL-8 has been well established as a highly prevalent cytokine in GBM with potent pro-migratory and pro-angiogenic functions. AAT-treated groups had significantly higher populations of CXCR2+ glioma stem cells and endothelial-like subpopulations and these populations were decreased following treatment with HET0016 and SB225002 (a CXCR2 antagonist). CXCR2+ GBM tumor cells were shown to form VM-like vascular channels carrying functional RBCs. Knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that HET0016 and SB225002 have potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.

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