DOCSLIB.ORG

Multiplexed Targeting of Mirna-210 in Stem Cell-Derived Extracellular

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Multiplexed Targeting of Mirna-210 in Stem Cell-Derived Extracellular Song et al. Experimental & Molecular Medicine (2021) 53:695–708 https://doi.org/10.1038/s12276-021-00584-0 Experimental & Molecular Medicine ARTICLE Open Access Multiplexed targeting of miRNA-210 in stem cell- derived extracellular vesicles promotes selective regeneration in ischemic hearts Byeong-Wook Song 1, Chang Youn Lee2,RanKim3,WonJungKim3, Hee Won Lee3,MinYoungLee4,JongminKim5, Jee-Yeong Jeong 6 and Woochul Chang3 Abstract Extracellular vesicles (EVs) are cell derivatives containing diverse cellular molecules, have various physiological properties and are also present in stem cells used for regenerative therapy. We selected a “multiplexed target” that demonstrates multiple effects on various cardiovascular cells, while functioning as a cargo of EVs. We screened various microRNAs (miRs) and identified miR-210 as a candidate target for survival and angiogenic function. We confirmed the cellular and biological functions of EV-210 (EVs derived from ASCmiR-210) secreted from adipose-derived stem cells (ASCs) transfected with miR-210 (ASCmiR-210). Under hypoxic conditions, we observed that ASCmiR-210 inhibits apoptosis by modulating protein tyrosine phosphatase 1B (PTP1B) and death-associated protein kinase 1 (DAPK1). In hypoxic endothelial cells, EV-210 exerted its angiogenic capacity by inhibiting Ephrin A (EFNA3). Furthermore, EV-210 enhanced cell survival under the control of PTP1B and induced antiapoptotic effects in hypoxic H9c2 cells. In cardiac fibroblasts, the fibrotic ratio was reduced after exposure to EV-210, but EVs derived from ASCmiR-210 did not communicate with fibroblasts. Finally, we observed the functional restoration of the ischemia/reperfusion-injured heart by maintaining miR-210 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; the intercommunication of EVs and cardiovascular cells derived from ASC . These results suggest that the multiplexed target with ASCmiR-210 is a useful tool for cardiovascular regeneration. Introduction cells. Furthermore, transient cellular constituents of the Cardiovascular diseases, including heart attacks and heart include lymphocytes, mast cells, and macrophages, strokes, result in impairment of circulating organs and a which can affect cardiac function4. For normal cardiac subsequently shortened life span. Coronary heart diseases function, cell populations of the heart are harmonized to (CHDs) are the most common type, inducing arrhythmias maintain intercellular homeostasis and function as cel- and myocardial infarction (MI)1. Unlike lower vertebrates, lular control networks, including autocrine, paracrine, adult mammals have difficulty in natural myocardial and cell–cell interactions. However, in the diseased state, regeneration, and many therapeutic approaches have been this innate condition cannot maintain cardiomyocyte researched for heart regeneration and repair2,3. The heart homeostasis and ultimately fails to restore the heart comprises cardiac myocytes and nonmyocytes, including performance5. fibroblasts, endothelial cells, and vascular smooth muscle Stem cells used to restore damaged hearts and wounded vessels are known to induce a multifaceted regenerative response through paracrine action by secreting various Correspondence: Woochul Chang ([email protected]) growth factors, that stimulate the survival and repair of 1Institute for Bio-Medical Convergence, Catholic Kwandong University host myocardium and recruit endogenous stem cells5,6. ’ International St. Mary s Hospital, Incheon, Republic of Korea Various stem cell types from the embryonic to the adult 2Department of Integrated Omics for Biomedical Sciences, Graduate School, Yonsei University, Seoul, Republic of Korea stage have been considered as treatments for the Full list of author information is available at the end of the article © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Song et al. Experimental & Molecular Medicine (2021) 53:695–708 696 regenerative process of cardiovascular diseases. Although Isolation of hASC-derived EVs stem cells are isolated from a single organ, they have hASCs were incubated for 1 day after seeding in a mixed cellular phenotypes that result in heterogeneous 100mmdish(1×106 cells/dish), washed twice with PBS, outcomes or cause undesirable results, such as carcino- and then cultured with 10 mL of DMEM without FBS for genic effects7,8. Therefore, a method has been proposed to 24 h. After 24 h of incubation, the medium was collected induce appropriate treatment of the damaged heart by and centrifuged at 3000 × g for15minat4°C,andthen, changing the phenotype of cells or differentiating them only the supernatant was transferred to a new tube. The into target cells through proper modulation of stem supernatant was filtered through a 0.22 mm PVDF filter – cells9 11. However, cell contamination, cell death, and (Millipore, Burlington, MA, USA). An appropriate volume immune rejection significantly limit the clinical use of of ExoQuick-TC™ Exosome Precipitation Solution (System stem cells12. Alternative therapies such as cell-derived Biosciences, Palo Alto, CA, USA) was added to the filtered products have been proposed to address the disadvantages culture medium and mixed well by inverting. After refrig- of cardiovascular disease treatment using stem cells. eration for 12 h, the mixture was centrifuged at 1500×g for Extracellular vesicles (EVs) are derivatives of cells; 30 min, and all supernatant was removed by aspiration. EV they are small extracellular membrane fragments that pellets were resuspended in 100 µL of PBS (phosphate contain various types of cellular information and buffered saline; Gibco) and stored at −80 °C for further use. notable physiological properties. EVs predominantly consist of exosomes, small homogeneous vesicles, and In vitro ischemic condition microvesicles. EVs have a more heterogeneous dimen- For induction of hypoxia, 80% confluent hASCs were sional profile generated due to direct extrusion of the plated in cell culture dishes. The serum-free media were cytoplasmic membrane13, and contain numerous lipids, degassed and then exposed in a hypoxic chamber proteins, and RNAs, including noncoding RNAs. In the (Thermo Fisher Scientific, Waltham, MA, USA) main- process of cardiovascular disease and repair, the com- tained below 5% CO2,5%H2, and 1% O2 at 37 °C. H9c2 position and internal cargo of EVs play a role in cell-to- cells were harvested after an 18 h incubation period and cell communication. Notably, EVs may be a major incubated with TRIzol Reagent (Life Technologies, Fre- factor in paracrine effects, including humoral stimula- derick, MD, USA) for quantification of miR expression. tion of endogenous regeneration and preservation of pre-existing cells14.Fromtheviewpointofcelltherapy, Ischemia-reperfusion injury and cell transplantation stem cell EVs are exogenous factors that have the All experimental procedures for animal studies were capacity to stimulate myocardial regeneration and car- approved by the Committee for the Care and Use of dioprotective effects. Since cardiovascular function is Laboratory Animals of Catholic Kwandong University Col- dependent on cardiovascular cell interactions and lege of Medicine (CKU01-2015-003-1), and performed in communication, stem cell-derived EVs can increase accordance with the Committee’s Guidelines and Regula- several factors that are useful for survival and func- tions for Animal Care. Seven-week-old male Sprague- tional improvement. Despite this important mechan- Dawley rats (220 ± 30 g) were used for in vivo experi- ism, few studies have researched EVs derived from stem ments. After anesthetization via intraperitoneal injection of cells with specific traits regulating myocardial cells and zoletil (30 mg/kg) and xylazine (10 mg/kg), rats were venti- nonmyocardial cells within the diseased heart15.This lated via trachea using a ventilator (Harvard Apparatus, study demonstrates that miR-210 is a key regulator in Holliston, MA, USA) and then subjected to surgically ischemic myocardial cells and adipose-derived stem induced MI, followed by cell transplantation. MI was pro- cells (ASCs), and that ASC-derived exosomes (EV-210) duced by surgical occlusion of the left anterior descending overexpressing miR-210 selectively affect myocardial/ coronary artery by ligation using a 7-0 Prolene suture. The nonmyocardial cells. rats were randomized into five groups (normal, IR control, IR + ASC, IR + ASCmiR-210,andIR+ ASCmiR-210 GW4869). Materials and methods GW4869 is a potent inhibitor that blocks exosomes. For cell Culture of hASCs transplantation, ASCs (stained with PKH67) were suspended Human adipose-derived stem cells (hASCs) were pur-
Load more

Recommended publications
  • Epha Receptors and Ephrin-A Ligands Are Upregulated by Monocytic
    Mukai et al. BMC Cell Biology (2017) 18:28 DOI 10.1186/s12860-017-0144-x RESEARCHARTICLE Open Access EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/ maturation and promote cell adhesion and protrusion formation in HL60 monocytes Midori Mukai, Norihiko Suruga, Noritaka Saeki and Kazushige Ogawa* Abstract Background: Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/ spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/ macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation. Results: Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16− monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and β2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and β1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand- coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes.
    [Show full text]
  • Supplemental Material 1
    Supplemental Fig. 1 12 1mg/ml A1AT p<0.001 10 8 p=0.004 6 p=0.029 (fold change) 4 mRNA ofmRNA angptl4 at h 1 2 0 Control Prolastin Aralast A1AT Sigma Supplemental Figure 1. Adherent PBMCs were incubated with 1 mg/ml A1AT from different sources (Prolastin, Grifols; Aralast, Baxter; or Sigma Aldrich) for 1h and mRNA levels of angplt4 were determined by RT-PCR. Bars represent the mean ± SD of four experiments. Supplemental Fig. 2 12 8 h p=0.005 10 8 6 mRNA angplt4 mRNA change Fold 4 2 0 Control A1AT 0.5 mg/ml Supplemental Figure 2. HMVEC-L were incubated with 0.5 mg/ml A1AT (Calbiochem) for 8 h and mRNA levels of angptl4 were determined by RT-PCR. Bars represent the mean ± SEM of three experiments. Supplemental Table I. Influence of A1AT (1 mg/ml, 8 h) on expression of angiogenesis genesa in HMVEC-L Unigene Symbol Description Gname fold changeb Hs.525622 AKT1 V-akt murine thymoma viral AKT/MGC99656/PKB/PKB- 0,94 0,14 oncogene homolog 1 ALPHA/PRKBA/RAC/RAC- ALPHA Hs.369675 ANGPT1 Angiopoietin 1 AGP1/AGPT/ANG1 3,61 5,11 Hs.583870 ANGPT2 Angiopoietin 2 AGPT2/ANG2 0,98 0,01 Hs.209153 ANGPTL3 Angiopoietin-like 3 ANGPT5/FHBL2 1,10 0,59 Hs.9613 ANGPTL4 Angiopoietin-like 4 ANGPTL2/ARP4/FIAF/HFAR 5,79 1,66 P/NL2/PGAR/pp1158 Hs.1239 ANPEP Alanyl (membrane) APN/CD13/GP150/LAP1/P150 1,03 0,01 aminopeptidase /PEPN Hs.194654 BAI1 Brain-specific angiogenesis FLJ41988/GDAIF 0,89 0,26 inhibitor 1 Hs.54460 CCL11 Chemokine (C-C motif) ligand 11 MGC22554/SCYA11 1,04 0,15 Hs.303649 CCL2 Chemokine (C-C motif) ligand 2 GDCF- 1,09 0,14 2/HC11/HSMCR30/MCAF/M CP-
    [Show full text]
  • Ephrin-A Binding and Epha Receptor Expression Delineate the Matrix Compartment of the Striatum
    The Journal of Neuroscience, June 15, 1999, 19(12):4962–4971 Ephrin-A Binding and EphA Receptor Expression Delineate the Matrix Compartment of the Striatum L. Scott Janis, Robert M. Cassidy, and Lawrence F. Kromer Department of Cell Biology and Interdisciplinary Program in Neuroscience, Georgetown University Medical Center, Washington, DC 20007 The striatum integrates limbic and neocortical inputs to regulate matrix neurons. In situ hybridization for EphA RTKs reveals that sensorimotor and psychomotor behaviors. This function is de- the two different ligand binding patterns strictly match pendent on the segregation of striatal projection neurons into the mRNA expression patterns of EphA4 and EphA7. anatomical and functional components, such as the striosome Ligand–receptor binding assays indicate that ephrin-A1 and and matrix compartments. In the present study the association ephrin-A4 selectively bind EphA4 but not EphA7 in the lysates of ephrin-A cell surface ligands and EphA receptor tyrosine of striatal tissue. Conversely, ephrin-A2, ephrin-A3, and kinases (RTKs) with the organization of these compartments ephrin-A5 bind EphA7 but not EphA4. These observations im- was determined in postnatal rats. Ephrin-A1 and ephrin-A4 plicate selective interactions between ephrin-A molecules and selectively bind to EphA receptors on neurons restricted to the EphA RTKs as potential mechanisms for regulating the com- matrix compartment. Binding is absent from the striosomes, partmental organization of the striatum. which were identified by m-opioid
    [Show full text]
  • Molecular Regulation of Visual System Development: More Than Meets the Eye
    Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Molecular regulation of visual system development: more than meets the eye Takayuki Harada,1,2 Chikako Harada,1,2 and Luis F. Parada1,3 1Department of Developmental Biology and Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA; 2Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan Vertebrate eye development has been an excellent model toderm, intercalating mesoderm, surface ectoderm, and system to investigate basic concepts of developmental neural crest (Fig. 1). The neuroectoderm differentiates biology ranging from mechanisms of tissue induction to into the retina, iris, and optic nerve; the surface ecto- the complex patterning and bidimensional orientation of derm gives rise to lens and corneal epithelium; the me- the highly specialized retina. Recent advances have shed soderm differentiates into the extraocular muscles and light on the interplay between numerous transcriptional the fibrous and vascular coats of the eye; and neural crest networks and growth factors that are involved in the cells become the corneal stroma sclera and corneal en- specific stages of retinogenesis, optic nerve formation, dothelium. The vertebrate eye originates from bilateral and topographic mapping. In this review, we summarize telencephalic optic grooves. In humans, optic vesicles this recent progress on the molecular mechanisms un- emerge at the end of the fourth week of development and derlying the development of the eye, visual system, and soon thereafter contact the surface ectoderm to induce embryonic tumors that arise in the optic system.
    [Show full text]
  • ALL1 Fusion Proteins Induce Deregulation of Epha7 and ERK Phosphorylation in Human Acute Leukemias
    ALL1 fusion proteins induce deregulation of EphA7 and ERK phosphorylation in human acute leukemias Hiroshi Nakanishi*, Tatsuya Nakamura*, Eli Canaani†, and Carlo M. Croce*‡ *Department of Molecular Virology, Immunology, and Medical Genetics and Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210; and †Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Edited by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL, and approved July 25, 2007 (received for review April 6, 2007) Erythropoietin-producing hepatoma-amplified sequence (Eph) re- studies revealed deregulation of some of the Eph/ephrin genes in ceptor tyrosine kinases and their cell-surface-bound ligands, the human malignancies, including up-regulation of ephrin-A1 or -B2 ephrins, function as a unique signaling system triggered by cell- in melanoma (2, 3), up-regulation of EphB2 in stomach cancer to-cell interaction and have been shown to mediate neurodevel- (4) and in breast cancer (5), and up-regulation of EphA2 in opmental processes. In addition, recent studies showed deregula- prostate (6), breast (7), and esophageal cancers (8), some of tion of some of Eph/ephrin genes in human malignancies, which were shown to be associated with tumor invasion or tumor suggesting the involvement of this signaling pathway in tumori- metastasis and therefore associated with poor prognosis. Con- genesis. The ALL1 (also termed MLL) gene on human chromosome versely, mutational inactivation of EphB2 was detected in pros- 11q23 was isolated by virtue of its involvement in recurrent tate (9) and colon cancers (10), suggesting tumor suppressor chromosome translocations associated with acute leukemias with function of this Eph receptor in the relevant tumors.
    [Show full text]
  • Epha3 Function Is Regulated by Multiple
    EPHA3 FUNCTION IS REGULA TED BY MULTIPLE PHOSPHOTYROSINE RESIDUES 2 1 Guanfang Shi\ Gang Yue , Mike Sheng\ Suzie Chen\ and Renping Zhou IDepartment of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854; 2Department of Oral Biology, New Jersey Dental School, University of Medicine & Dentistry of New Jersey, Newark, NJ 07101. Address correspondence to: Renping Zhou, Ph. D. Phone: (732) 445-3400 ext. 264; Fax: (732) 445-0687; E-mail: rzhou @rci.rutgers.edu EphA3, a member of the Eph family of tyrosine carcinoma was significantly correlated with kinase receptors, has been implicated in a regional lymph node metastasis (5). variety of tumors including melanoma, Overexpression of ephrin-B2, a ligand for EphB glioblastoma, pancreatic carcinoma, lung and receptors, in B 16 melanoma cells enhanced colon cancers. The loss of EphA3 function has integrin-mediated ECM-attachment and migration been associated with colorectal cancer. To (6). EphB2 positively regulates glioma cell identify potential roles in tumor formation, we adhesion, growth, and invasion via R-Ras signaling examined effects of EphA3 expression and (7). In contrast to promote tumor metastasis and activation on melanoma cell migration, and cancer cell line migration, Eph receptors may also identified key signal transducer docking sites of regulate the cell motility negatively. Hafner and the receptor. This analysis showed that the coworkers compared EphB6 mRNA level in human melanoma cell line WMl15 lacks benign nevi, primary melanomas, and metastatic EphA3 expression, while two other melanoma melanomas, and found a progressive and cell lines, WM239A and C8161, both retain significant reduction of EphB6 mRNA expression expression of the receptor.
    [Show full text]
  • Ephrin A4-Ephrin Receptor A10 Signaling Promotes Cell Migration
    www.nature.com/scientificreports OPEN Ephrin A4‑ephrin receptor A10 signaling promotes cell migration and spheroid formation by upregulating NANOG expression in oral squamous cell carcinoma cells Yu‑Lin Chen1, Yi‑Chen Yen1, Chuan‑Wei Jang1, Ssu‑Han Wang1, Hsin‑Ting Huang1, Chung‑Hsing Chen2,3, Jenn‑Ren Hsiao4, Jang‑Yang Chang1 & Ya‑Wen Chen1,5* Ephrin type‑A receptor 10 (EPHA10) has been implicated as a potential target for breast and prostate cancer therapy. However, its involvement in oral squamous cell carcinoma (OSCC) remains unclear. We demonstrated that EPHA10 supports in vivo tumor growth and lymphatic metastasis of OSCC cells. OSCC cell migration, epithelial mesenchymal transition (EMT), and sphere formation were found to be regulated by EPHA10, and EPHA10 was found to drive expression of some EMT‑ and stemness‑ associated transcription factors. Among EPHA10 ligands, exogenous ephrin A4 (EFNA4) induced the most OSCC cell migration and sphere formation, as well as up‑regulation of SNAIL, NANOG, and OCT4. These efects were abolished by extracellular signal‑regulated kinase (ERK) inhibition and NANOG knockdown. Also, EPHA10 was required for EFNA4‑induced cell migration, sphere formation, and expression of NANOG and OCT4 mRNA. Our microarray dataset revealed that EFNA4 mRNA expression was associated with expression of NANOG and OCT4 mRNA, and OSCC patients showing high co‑expression of EFNA4 with NANOG or OCT4 mRNA demonstrated poor recurrence‑free survival rates. Targeting forward signaling of the EFNA4‑EPHA10 axis may be a promising therapeutic approach for oral malignancies, and the combination of EFNA4 mRNA and downstream gene expression may be a useful prognostic biomarker for OSCC.
    [Show full text]
  • Pro‑Angiogenic Microrna‑296 Upregulates Vascular Endothelial Growth Factor and Downregulates Notch1 Following Cerebral Ischemic Injury
    MOLECULAR MEDICINE REPORTS 12: 8141-8147, 2015 Pro‑angiogenic microRNA‑296 upregulates vascular endothelial growth factor and downregulates Notch1 following cerebral ischemic injury JIE FENG1, TIANXIANG HUANG2, QING HUANG1, HUA CHEN1, YU LI1, WEI HE1, GUI-BIN WANG1, LE ZHANG1, JIAN XIA1, NING ZHANG1 and YUNHAI LIU1 1Institute of Neurology; 2Institute of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China Received June 8, 2014; Accepted June 3, 2015 DOI: 10.3892/mmr.2015.4436 Abstract. The present study examined the association between regional cerebral blood supply is critical for improving stroke microRNA (miR)-296 and angiogenesis following cerebral outcomes and post-stroke functional recovery (2). Therefore, ischemic injury, and the underlying mechanisms. A cerebral angiogenesis has become a focus in the field of ischemic stroke ischemic model was established in rats via right middle cere- investigation. bral artery occlusion. The animals were randomly divided into MicroRNAs (miRNAs), which are 21~23 nucleotide four groups (baseline, 1 day, 3 day and 7 day). Quantitative non‑protein‑coding RNA molecules, have been identified as polymerase chain reaction and western blot analyses were negative regulators of gene expression in a post-transcriptional performed to examine the expression levels of miR-296 and manner (3). Several previous studies have demonstrated that hepatocyte growth factor-regulated tyrosine kinase substrate miRNAs are essential determinants of vascular endothelial (HGS), respectively. Angiogenesis was assessed by examining cell biology and angiogenesis, and contribute to stroke patho- microvessel density. The results demonstrated that miR-296 genesis (4-7). A previous study demonstrated that glioma cells and angiogenesis were significantly upregulated, while HGS and angiogenic growth factors elevate the level of miRNA was significantly downregulated following ischemic injury.
    [Show full text]
  • BEACH-DISSERTATION-2016.Pdf
    Towards mammalian retinal regeneration: cell signaling in re-differentiating mouse Müller glia and transcriptional regulation of the axonal guidance receptor EphA5 By Krista Marie Beach B.S, O.D. DISSERTATION In partial satisfaction of the requirements for the degree of PhD in PHYSIOLOGICAL OPTICS Presented to the Graduate Faculty of the College of Optometry University of Houston May, 2016 Approved: Dr. Deborah C. Otteson, PhD (Chair) Dr. Laura J. Frishman, PhD Dr. Donald A. Fox, PhD Dr. Steven W. Wang, PhD Committee in Charge DEDICATION I dedicate this dissertation to my parents, Col. Thomas N. Beach, M.D., and Jean M. Beach, B.A., and to my two sisters, Paula J. Beach, M.S., B.S. and Laura E. Beach, B.A., B.A. for their unconditional love and support during these years of incessant study and work. ii ACKNOWLEDGMENTS I wish to acknowledge my committee members for their guidance and support: Deborah C. Otteson, PhD – chair Laura J. Frishman, PhD Donald A. Fox, PhD Steven W. Wang, PhD I wish to acknowledge the past and present members of the Otteson lab, for their personal support, friendship, and occasional help with experiments: Amanda Gall, M.S Tia Petkova, O.D., PhD Tanya Geranpayeh, B.S Laila Pillai, B.S Mary Guirguis, B.S M. Joseph Phillips, PhD Steven Huynh, B.S Jian Bo Wang, PhD Micah Mesko, B.S Kuichen Zhu, PhD I wish to acknowledge also the following people: Librarians Suzanne Ferimer and Pamela Forbes of the Weston Petty UH Library for acquiring a great many hard-to-find articles and resources.
    [Show full text]
  • Electrical Stimulation Induces Retinal Müller Cell Proliferation and Their Progenitor Cell Potential
    cells Article Electrical Stimulation Induces Retinal Müller Cell Proliferation and Their Progenitor Cell Potential Sam Enayati 1,2,3,4, Karen Chang 1, Hamida Achour 1,4, Kin-Sang Cho 1 , Fuyi Xu 5, Shuai Guo 1, Katarina Z. Enayati 1, Jia Xie 1, Eric Zhao 1, Tytteli Turunen 1, Amer Sehic 6, Lu Lu 5, Tor Paaske Utheim 1,2,3,6,7 and Dong Feng Chen 1,* 1 Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA; [email protected] (S.E.); [email protected] (K.C.); [email protected] (H.A.); [email protected] (K.-S.C.); [email protected] (S.G.); [email protected] (K.Z.E.); [email protected] (J.X.); [email protected] (E.Z.); [email protected] (T.T.); [email protected] (T.P.U.) 2 Department of Medical Biochemistry, Oslo University Hospital, 0372 Oslo, Norway 3 Department of Ophthalmology, Drammen Hospital, Vestre Viken Hospital Trust, 3004 Drammen, Norway 4 Institute of clinical medicine, University of Oslo, 0318 Oslo, Norway 5 Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, TN 38163, USA; [email protected] (F.X.); [email protected] (L.L.) 6 Department of Oral Biology; Faculty of Dentistry, University of Oslo, 0372 Oslo, Norway; [email protected] 7 Department of Plastic and Reconstructive Surgery, Oslo University Hospital, 0027 Oslo, Norway * Correspondence: [email protected]; Tel.: +617-912-7490 Received: 1 February 2020; Accepted: 17 March 2020; Published: 23 March 2020 Abstract: Non-invasive electrical stimulation (ES) is increasingly applied to improve vision in untreatable eye conditions, such as retinitis pigmentosa and age-related macular degeneration.
    [Show full text]
  • Epha5-Ephrina5 Interactions Within the Ventromedial
    ORIGINAL ARTICLE EphA5-EphrinA5 Interactions Within the Ventromedial Hypothalamus Influence Counterregulatory Hormone Release and Local Glutamine/Glutamate Balance During Hypoglycemia Barbara Szepietowska,1 Wanling Zhu,1 Jan Czyzyk,2 Tore Eid,3 and Robert S. Sherwin1 Activation of b-cell EphA5 receptors by its ligand ephrinA5 from typical warning symptoms. These protective responses are adjacent b-cells has been reported to decrease insulin secretion often disrupted in type 1 diabetic patients receiving intensive during hypoglycemia. Given the similarities between islet and insulin therapy who have a history of hypoglycemia (7). As ventromedial hypothalamus (VMH) glucose sensing, we tested a result, the fear of hypoglycemia is the major factor limiting the hypothesis that the EphA5/ephrinA5 system might function the benefits of intensive insulin treatment (8). within the VMH during hypoglycemia to stimulate counterregula- Activation of counterregulation requires effective de- tory hormone release as well. Counterregulatory responses and tection of falling glucose levels. Although a complex net- glutamine/glutamate concentrations in the VMH were assessed work of glucose sensors has been described in the central during a hyperinsulinemic-hypoglycemic glucose clamp study in – chronically catheterized awake male Sprague-Dawley rats that nervous system (9 11) and peripherally (12), the brain received an acute VMH microinjection of ephrinA5-Fc, chronic appears to have the dominant role during hypoglycemia VMH knockdown, or overexpression of ephrinA5 using an and, specifically, the ventromedial region of the hypothala- adenoassociated viral construct. Local stimulation of VMH EphA5 mus or VMH (13,14). Interestingly, VMH neurons contain receptors by ephrinA5-Fc or ephrinA5 overexpression increased, much of the same glucose-sensing machinery (e.g., gluco- whereas knockdown of VMH ephrinA5 reduced counterregula- kinase [15], ATP-sensitive K+ channels [16–18]) as pancre- tory responses during hypoglycemia.
    [Show full text]
  • Isolation and Functional Analysis of Xenopus Ephrin-A3
    Isolation and Functional Analysis of Xenopus Ephrin-A3 Taslima Khan A Thesis for the Degree of Doctor of Philosophy 1999 Division of Developmental Biology and Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 lAA. University College, London, Gower Street, London, WCIE 6BT. ProQuest Number: 10014866 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10014866 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ''We shall not cease from exploration And the end of all our exploring Will be to arrive where we started And know the place for the first time. Through the unknown, remembered gate When the last o f the earth left to discover Is that which was the beginning; ” Little Gidding, T. S. Eliot. To My Family For Encouraging Me, Entertaining Me and For Trying, So Often, To Understand This PhD ABSTRACT ABSTRACT Segmentation is a primary requirement for the establishment of the vertebrate body plan and it is therefore of great interest to identify proteins involved in these patterning events.
    [Show full text]