• 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.
    • MECHANISM OF 12/15 LIPOXYGENASE-INDUCED RETINAL MICROVASCULAR DYSFUNCTION IN DIABETIC RETINOPATHY

      Elmasry, Khaled; Department of Biochemistry and Molecular Biology / Cancer Center (5/22/2018)
      Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leukocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.
    • 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.
    • 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.