Diabetes Volume 67, April 2018 745 Sirt1: A Guardian of the Development of Diabetic Retinopathy Manish Mishra, Arul J. Duraisamy, and Renu A. Kowluru Diabetes 2018;67:745–754 | https://doi.org/10.2337/db17-0996 Diabetic retinopathy is a multifactorial disease, and the molecular mechanism of the development of diabetic reti- exact mechanism of its pathogenesis remains obscure. nopathy remains to be established. Sirtuin 1 (Sirt1), a multifunctional deacetylase, is impli- Sirtuin 1 (Sirt1), a member of the silent information cated in the regulation of many cellular functions and in regulator 2 family, is a class III histone deacetylase that gene transcription, and retinal Sirt1 is inhibited in di- interacts with target proteins and regulates many cellular abetes. Our aim was to determine the role of Sirt1 in the functions including cell proliferation, apoptosis, and inflam- development of diabetic retinopathy and to elucidate the matory responses (6–8). Sirt1 is mainly a nuclear protein, Sirt1 molecular mechanism of its downregulation. Using - and its activity depends on cellular NAD availability (9). It is overexpressing mice that were diabetic for 8 months, Sirt1 expressed throughout the retina, and upregulation of COMPLICATIONS structural, functional, and metabolic abnormalities were protects against various ocular diseases including retinal investigated in vascular and neuronal retina. The role of degeneration, cataract, and optic neuritis (10). Our previous epigenetics in Sirt1 transcriptional suppression was inves- work has shown that Sirt1 expression and activity are de- tigated in retinal microvessels. Compared with diabetic wild-type mice, retinal vasculature from diabetic Sirt1 mice creased in the retina and its capillary cells in diabetes (11). did not present any increase in the number of apoptotic However, the direct role of Sirt1 in the development of cells or degenerative capillaries or decrease in vascular diabetic retinopathy remains elusive. density. Diabetic Sirt1 mice were also protected from mi- Sirt1 also regulates gene transcription, and this is medi- tochondrial damage and had normal electroretinogra- ated either by altering the acetylation status of the tran- phy responses and ganglion cell layer thickness. Diabetic scription factor or by regulating epigenetic modifications at wild-type mice had hypermethylated Sirt1 promoter DNA, the transcriptional factor binding site of a gene (12). In the which was alleviated in diabetic Sirt1 mice, suggesting pathogenesis of diabetic retinopathy, inhibition of Sirt1 is a role for epigenetics in its transcriptional suppression. implicated in the hyperacetylation and activation of nuclear Thus strategies targeted to ameliorate Sirt1 inhibition have transcription factor-kB(NF-kB), and NF-kB plays a major role the potential to maintain retinal vascular and neuronal ho- in the transcriptional activation of mitochondria-damaging meostasis, providing opportunities to retard the develop- matrix metalloproteinase (MMP)-9 (11,13,14). Sirt1 is also ment of diabetic retinopathy in its early stages. a redox-sensitive enzyme (15), and oxidative stress, in addition to regulating NAD levels, affects Sirt1 activity by reg- ulating posttranslational modifications and protein-protein Diabetic retinopathy remains the major cause of acquired interactions (16); in diabetes, regulation of oxidative stress blindness in working-age adults, and high circulating glucose prevents decreases in Sirt1 activity in the retinal vasculature is considered to be the major instigator of deleterious (11). How diabetes regulates Sirt1 is, however, not clear. functional, structural, and metabolic changes (1–3). Chronic The expression of a gene, along with its DNA sequence, hyperglycemia increases oxidative stress, activates protein is also regulated by epigenetic modifications (17,18). Diabe- kinases and polyol pathways, and results in neuronal and tes alters the epigenetic machinery (activates/inhibits) in vascular damage including loss of ganglion cells and for- the retina, and many genes considered to play important mation of degenerative capillaries (1,2,4,5), but the exact roles in mitochondrial homeostasis are epigenetically modified Kresge Eye Institute, Wayne State University, Detroit, MI M.M. and A.J.D. contributed equally to this study. Corresponding author: Renu A. Kowluru, [email protected]. © 2018 by the American Diabetes Association. Readers may use this article as fi Received 21 August 2017 and accepted 29 December 2017. long as the work is properly cited, the use is educational and not for pro t, and the work is not altered. More information is available at http://www.diabetesjournals This article contains Supplementary Data online at http://diabetes .org/content/license. .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0996/-/DC1. 746 Sirt1 and Diabetic Retinopathy Diabetes Volume 67, April 2018 – (2,3,11,19 21). We have shown that dynamic activation Table 1—Primer sequences of DNA methylating–hydroxymethylating enzymes, DNA Genes Sequence methyltransferases (Dnmts) and ten-eleven translocation Sirt1 59-GGGGTTTGCCCCATGGAAT-39 enzymes, maintain the DNA methylation status of the ret- 59-GAGCCCATCCCCACACTGTA-39 inal MMP-9 promoter to keep it transcriptionally active Sirt1 MMP-9 59-GGGGTTTGCCCCATGGAAT-39 (19). The role of epigenetics in the regulation of in 59-GAGCCCATCCCCACACTGTA-39 the pathogenesis of diabetic retinopathy, however, remains Dnmt1 59-CCTAGTTCCGTGGCTACGAGGAGAA-39 to be explored. 59-TCTCTCTCCTCTGCAGCCGACTCA-39 Sirt1 is a multifunctional protein implicated in a wide 18S 9 9 – 5 -GCCCTGTAATTGGAATGAGTCCACTT-3 range of molecular and epigenetic pathways (6 8). The goal 59-CTCCCCAAGATCCAACTACGAGCTTT-39 of this study was to determine whether regulation of Sirt1 Dnmt1 promoter 59-TCCTCTGCAAGAGCAGCACTA-39 ameliorates the development of diabetic retinopathy and to 59-ATGTACCACACAGGGCAAGA-39 elucidate the mechanism responsible for Sirt1 regulation. Sirt1 9 9 Sirt1 promoter 5 -GGCAGCACACACCTTTTACA-3 Using mice overexpressing , we investigated its role in 59-AGATTTGCCTGCATCTGCCT-39 structural, functional, and metabolic abnormalities critically D-Loop 59-AGCACCCAAAGCTGGTATTCT-39 associated with the development of diabetic retinopathy. 59-CCAGGACCAAACCTTTGTGTTT-39 Sirt1 The possible mechanism of transcriptional suppres- CytB 59-AGACAAAGCCACCTTGACCCGAT-39 sion is elucidated through investigation of the DNA meth- 59-ACGATTGCTAGGGCCGCGAT-39 ylation status of its promoter. b-Actin 59-AAAGGAAGCGCAGACCGGCC-39 59-GCGCAGTGTAGGCGGAGCTT-39 RESEARCH DESIGN AND METHODS Mice 18S Diabetes was induced in wild-type C57BL/6J (WT) and Ribosomal RNA was used as a housekeeping gene, DD Sirt1-overexpressing (St, C57BL/6-Actbtm3.1 [Sirt1] Npa/J; and the fold change was calculated using the Ct method Sirt1) mice (The Jackson Laboratory, Bar Harbor, ME), (19,22). ; with a body weight (BW) of 20 g (either sex), by strepto- Histopathology and Apoptosis in Retinal Microvessels zotocin injection (55 mg/kg BW for four consecutive days). The whole retina was isolated from the formalin-fixed eyes . Mice presenting blood glucose 250 mg/dL 2 days after the and rinsed overnight in running water and then incubated last injection were considered to have diabetes (19,22). Age- in 3% crude trypsin (Thermo Fisher Scientific, Waltham, Sirt1 matched normal WT and mice were used as their MA) containing 200 mol/L sodium fluoride at 37°C for respective controls. Compared with normal WT mice, al- 45–70 min. After gently brushing away the neuroretinal fi though Sirt1 expression was signi cantly increased in the tissue under a microscope, the vasculature was stained Sirt1 retina of mice, we found no increase in kidneys from with terminal deoxyribonucleotide TUNEL (In Situ Cell the same animals (Supplementary Fig. 1). Mice were sacri- Death Kit; Roche Molecular Biochemicals, Indianapolis, IN). fi ; ced 8 months after diabetes was induced; one eye was As a control, retinal vasculature treated with DNAse fi xed in 10% buffered formalin, and the retina from the was also stained with TUNEL. The TUNEL-positive capil- other eye was removed immediately to obtain biochemical lary cells were counted under a microscope, and the slides measurements. Glycated hemoglobin was measured after then were stained with periodic acid Schiff–hematoxylin ; the mice had diabetes for 6monthsusingakitfrom to count acellular capillaries and pericyte ghosts by light Helena Laboratories (Beaumont, TX), and serum HDL was microscopy (23). quantified as described previously (23). Normal Sirt1 and WT mice had similar glucose and HDL levels, and the se- Immunofluorescence Staining verity of hyperglycemia (blood glucose and glycated hemo- Using specific primary antibodies, 8-mm-thick retinal cryo- globin) was also similar in WT and Sirt1 mice with diabetes sections were stained for MMP-9, Sirt1 (Abcam, Cambridge, (Supplementary Table 1). The treatment of animals con- MA), and Dnmt1 (Sigma-Aldrich, St. Louis, MO) following formed to the Association for Research in Vision and Oph- the method reported previously (24). The slides then were thalmology Resolution on the Use of Animals in Research incubated with fluorescence-labeled secondary antibodies: and to Wayne State University’s guidelines. DyLight 488 (green) for Sirt1 and Texas Red for Dnmt1 Retinal microvessels were prepared with a hypotonic and MMP-9. The sections were mounted with medium con- shock method. The retina was incubated in 5–6mLdeionized taining DAPI (blue) and photographed under a ZEISS Apo- water in a shaking