Cell & Molecular Biology

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    Chemical Reprogramming of Mouse and Human Müller glia into Retinal Ganglion-Like Cells (RGCs)
    (2019-03-05) Zhang, Wei; Chavala, Sai; Fan, Yan
    Chemical Reprogramming of Mouse and Human Müller glia into Retinal Ganglion-Like Cells (RGCs) Yan Fan, Wei Zhang, Sai Chavala Department of Pharmacology and Neuroscience, Graduate School of Biomedical Sciences, UNT Health Science Center, Fort Worth, TX 76107 Purpose: Glaucoma is a leading cause of irreversible blindness with increasing prevalence as the population ages. Glaucoma is characterized by loss of retinal ganglion cells (RGCs), and current therapies, whether surgical, pharmacological, or neuroprotective, do not reverse the degeneration. Stem cell approaches to replace lost RGCs are a viable option. However, the use of stem cells for RGC replacement currently faces several barriers: 1) absence of a safe, non-immunogenic, and ethical stem cell source of RGCs, 2) inefficient differentiation protocols that can take more than 40 days, and 3) RGC donor integration into the degenerate host retina are major issues that need to be overcome to be considered for clinical use. A radically new approach to restoring vision for glaucoma patients that can overcome these limitations would be to reengineer a resident cell in the retina, such as Müller glia, that could serve as a reservoir for new RGCs, and avoid the need for cell transplantation. In this study, we take the first step in realizing our long-term goal of using a chemically reprogramming strategy to replace lost RGCs and restore vision. Materials and Methods: Primary Müller glia were isolated from mouse or human retina. Small molecules were purchased from Sigma or Cayman. For the in vitro studies, Müller glia were fixed after small molecule conversion with 4% PFA. Immunofluorescence staining was performed to detect RGC specific markers: Brn3a, Brn3b, ISL-1, RBPMS, and Tuj1. Total RNA was isolated and subjected for real-time PCR for detection of neuronal marker NeuN and RGC markers: Brn3a, Brn3b, ISL-1, NefH, and Nefl. For the in vivo studies, intravitreal injection was performed to deliver small molecules and other growth factors. BrdU was injected intravitreally or intraperitoneally to label proliferating and regenerating cells. 24 hours after the final injection, eyes were enucleated, fixed, and embedded for frozen sections. Immunofluorescence staining was performed to detect proliferation with BrdU and Ki67. After 7, 21, and 35 days of final injection, BrdU and RGC markers were co-stained to detect newly generated RGC-like cells. Results: Both mouse and human Müller glia can be converted to RGC-like cells within 24 hours in vitro. In vivo injection of small molecule and growth factor cocktail into the vitreous stimulated cell proliferation, which was located between the outer nuclear layer and the inner nuclear layer where the cells stained positive with GFAP. After 7 days, BrdU positive cells migrated to the inner edge of INL. After 21 days, BrdU positive cells reached the ganglion cell layer (GCL) but no co-staining with a RGC marker was observed at this time point. Strikingly after 35 days, we detected co-stained BrdU and ISL-1 or RBPMS in the GCL. There was no retinal toxicity observed from the small molecule injection. Conclusion: Our small molecule cocktail is highly efficient in converting mouse and human Müller glia to RGC-like cells in vitro, enabling us to generate RGC-like cells in approximately one day with more than a 90% conversion efficiency. The small molecule cocktail can be injected intravitreally to stimulate cell proliferation and RGC regeneration in vivo. Additional retinal toxicity testing will be needed to evaluate the safety profile of the intravitreal small molecule cocktail.
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    Inhibition of Mitochondrial Respiratory Chain Complex I Induces Vascular Endothelial Cell Apoptosis and Release of Mitochondrial DNA
    (2019-03-05) Cushen, Spencer; Phillips, Nicole; Goulopoulou, Styliani; Riaz, Maryam
    Purpose: Vascular endothelial oxidative stress is a common feature of preeclampsia, a pregnancy specific hypertensive syndrome with high incidence of maternal and fetal mortality and morbidity. Cellular oxidative stress can lead to cell death, which promotes the release of cellular constituents (e.g. mitochondrial fragments) into the extracellular space. Circulating cell-free mitochondrial DNA (mtDNA) concentrations are increased in pregnant women with preeclampsia. The main objective of this study was to determine the mechanisms by which vascular endothelial cells may contribute to this increase in cell-free mtDNA. The hypothesis was that mitochondrial complex I inhibition results in extrusion of mtDNA from vascular endothelial cells via cell death-dependent mechanisms. Methods: Human umbilical vein endothelial cells (HUVEC, Lonza) were grown to 80-90% confluency before treatment with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 μM - 4 h). Immunocytochemistry was used to confirm that HUVEC maintained endothelial cell characteristics. Cell death (apoptotic and non-apoptotic) was quantified using flow cytometry (staining for Annexin V and propidium iodide). mtDNA was measured on total nucleic acid extracts from cell culture supernatants using absolute real-time PCR techniques. Results: Treatment of HUVECs with rotenone increased early apoptosis and late apoptosis/necrosis [5μM (n=7), Veh: 11.16 ± 1.96% vs Rotenone: 14.74 ± 1.96% p=0.0159; 10μM (n=7), Veh: 10.54 ± 1.93% vs Rotenone: 14.83 ± 2.60% p=0.0033; 25μM (n=7), Veh: 10.34 ± 1.85% vs Rotenone: 15.87 ± 3.023% p=0.0002; 1-way ANOVA followed by Sidak’s post-hoc test]. Concentrations of mtDNA in HUVEC supernatant were increased in HUVECs treated with 5 μM of Rotenone [Veh (n=5): 2.45 ± 0.05 pg/mL vs. Rotenone (n=6): 3.65 ± 0.39 pg/mL, p=0.0700; Sidak’s post-hoc test]. Higher concentrations of Rotenone had no effect on concentrations of extracellular mtDNA (p [greater than] 0.84). Conclusions: Mitochondrial oxidative stress due to inhibition of mitochondrial respiratory chain complex I induces vascular endothelial cell death. Extrusion of mtDNA from apoptotic and necrotic endothelial cells may contribute to increased circulating mtDNA concentrations in preeclamptic pregnancies. Future studies will test this hypothesis using integrative pharmacological and physiological approaches.
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    Store-Operated Calcium Entry in Mesangial Cells And Glomerular Inflammatory Responses: Role of Interleukin-6
    (2019-03-05) Yazdizadehshotorbani, Parisa; Wang, Yanxia; Ma, Rong; Chaudhari, Sarika
    Purpose: Emerging evidence indicates that immunological and inflammatory mechanisms play a significant role in the development of diabetic nephropathy (DN). The early features of DN include accumulation of extracellular matrix (ECM) in the glomerular mesangium. Inflammatory cell infiltration mediated by locally produced cytokines/chemokines contributes to the histological impairment in DN. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines/chemokines in response to diabetes and a major contributor to mesangial expansion in DN. Interleukin-6 (IL-6) has dual roles acting as an inflammatory or anti-inflammatory cytokine in a cell context manner. We have previously demonstrated that the Orai-1 mediated store-operated calcium entry (SOCE) suppressed ECM protein production by MCs. The aim of this study was to determine if and how SOCE in MCs regulated IL-6 by MCs and macrophage infiltration into glomerulus. Methods: In cultured human MCs, levels of IL-6 were examined using ELISA in the presence of normal glucose (5 mM D-glucose) with/without an activator (thapsigargin at 1 µM) of SOCE. Immunoblot analysis was used to study the expression of various proteins in the whole cell lysates of MCs. In the human MCs, IL-6 was overexpressed using IL-6 plasmid while Orai 1 was knockdown using siRNA against human Orai1 using transfection reagents. In wild type C57BL6 mice, Orai-1 channel protein in MCs was knocked down using the targeted nanoparticle-siRNA delivery system at the age of 16 weeks. Immunohistochemistry was performed on the paraffin embedded kidney sections to examine macrophages infiltration in glomeruli using F4/80 as a marker. Results: In cultured human MCs, activation of SOCE by thapsigargin significantly increased IL-6 expression level which was attenuated by inhibitor of SOCE, GSK7975A and knockdown of Orai 1. IL-6 overexpression reduced the expression of ECM proteins in MCs. In vivo knockdown of Orai1 in MCs induced infiltration of F4/80 stained macrophages into the glomeruli in the mice treated with nanoparticle/Orai1 siRNA for 5 days compared to the control mice. Conclusion: SOCE positively regulates IL-6 expression in MCs which in turn suppresses the ECM proteins. Orai-1 mediated SOCE inhibits the glomerular macrophage infiltration in mice. Thus SOCE in MCs has protective responses against glomerular inflammation and fibrosis.
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    Poly lactic-co-glycolic acid (PLGA) mediated gene delivery to astrocytes requires arginine-modified polyethylenimine (PEI) polymer to facilitate gene expression
    (2019-03-05) Joshi, Chaitanya; Labhasetwar, Vinod; Borgmann, Kathleen; Ghorpade, Anuja; Proulx, Jessica
    Purpose: The global burden of neurodegenerative diseases and disorders devastate not only their victims but also their loved ones. Astrocyte dysfunction is a hallmark of central nervous system injury or infection. As a primary contributor to neurodegeneration, astrocytes are an ideal therapeutic target to combat neurodegenerative conditions. Gene therapy has arisen as an innovative technique that provides excellent prospect for disease intervention. Poly lactic-co-glycolic-acid (PLGA) and polyethylenimine (PEI) are polymeric nanoparticles commonly used in gene delivery, each manifesting their own set of advantages and disadvantages. While PLGA nanoparticles are FDA approved and well established for their biocompatibility, they fail to facilitate exogenous gene expression in primary human astrocytes. Furthermore, PEI polymers illustrate high delivery efficiency but induce cytotoxicity. The purpose of this study is to develop viable and biocompatible nanoparticles for astrocyte-targeted gene therapy. Methods: Successful gene expression by PLGA nanoparticles alone or in combination with arginine-modified PEI polymers (A5P10) was accessed by a luciferase reporter gene encapsulated in PLGA nanoparticles. Cytoplasmic release and nuclear localization were investigated using fluorescent confocal imaging with YOYO-labeled DNA. Nanoparticle-mediated cytotoxicity was assessed via lactate dehydrogenase (LDH) in primary human astrocytes and neurons. Results: Confocal imaging of YOYO-labeled DNA confirmed PLGA nanoparticles delivered DNA to the cytoplasm in a dose and time dependent manner. However, co-staining revealed DNA delivered by PLGA did not localize to the nucleus. The addition of A5P10 improved nuclear localization and successfully achieved gene expression in primary human astrocytes. Moreover, these formulations were biocompatible with both astrocytes and neurons. Conclusion: By integrating two polymeric nanoparticles, we developed an improved system for gene delivery and expression in primary human astrocytes. These findings provide a biocompatible and clinically translatable method to regulate astrocyte function during neurodegenerative diseases and disorders.