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 , 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 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 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 -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 (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 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 , DNA Genes Sequence (Dnmts) and ten-eleven translocation Sirt1 59-GGGGTTTGCCCCATGGAAT-39 enzymes, maintain the DNA 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 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 water bath at 37°C for 60 min. The non- Tome fluorescence microscope (Carl Zeiss, Chicago, IL) vascular tissue was gently removed under a microscope (22). at 340 magnification.

Gene Expression Vascular Permeability cDNA was synthesized from the retinal microvessels, and Approximately 2 weeks before the experiment was was quantified with SYBR green–based terminated, vascular permeability was quantified by fluo- quantitative PCR using gene-specificprimers(Table1). rescein angiography using a Micron IV Retinal Imaging diabetes.diabetesjournals.org Mishra, Duraisamy, and Kowluru 747

Microscope (Phoenix Research Labs, Pleasanton, CA). Animals Acetylation of Dnmt1 promoter was determined by quan- were anesthetized with a mixture of ketamine (67 mg/kg) and tifying acetylated H3K9 using a immunoprecip- xylazine (10 mg/kg) (intraperitoneal injection), the pupil was itation technique, as reported previously (21). Dnmt1 protein dilated with 0.1% tropicamide ophthalmic solution, acetylation was analyzed by immunoprecipitating acetyl ly- and the cornea was lubricated with Goniovisc (hypromellose sine (Abcam), followed by Western blotting for Dnmt1 (22). 2.5%). The fundus was photographed using a fundus camera Electroretinography for small animals. AK-FLUOR (0.5% solution, 0.01 mL/g BW) Electroretinography (ERG) was performed in dark-adapted was then injected intraperitoneally, and photographs were mice anesthetized with ketamine and xylazine. After di- taken 10 min after fluorescein injection using a barrier filter for fluorescein angiography (25). lating the pupil with tropicamide ophthalmic solution and lubricating the cornea with Goniovisc, ERG responses were Vascular permeability was confirmed by quantifying al- measured using an OcuScience HMsERG Lab System by bumin leakage into the retina using the tech- placing a thread electrode embedded in silver over the nique (26). Evans blue (30 mg/kg) was injected into the tail cornea, above the lubricant solution (hypromellose 2.5%). A vein,andaftermicewerekeptonaheatingpadfor2h, contact lens was used to keep the electrode in place. ERG paraformaldehyde was perfused and the eye globes were responses were recorded using a series of Ganzfeld flashes enucleated immediately. The dye was extracted from the with intensities ranging from 100 to 25,000 mcds/m2.The retina using formamide, and absorbance was measured at 620 nm.

Vascular Density Vascular density was measured on fluorescein angiograms after they were converted to grayscale (to eliminate the fluorescein background) using AngioTool software from the National Cancer Institute (27) and by staining of the retinal flatmount with isolectin (28) using fluorescein isothiocyanate– conjugated Isolectin B4 (Alexa Fluor 488, 1:100 dilution; Technologies, Carlsbad, CA). The flatmounts were visu- alizing under a Leica SP5 confocal microscope at 310 mag- nification (Leica Microsystems, Wetzlar, Germany), and the images were converted to grayscale by AngioTool for analysis.

Mitochondrial DNA Mitochondrial DNA (mtDNA) damage was determined by analyzing the sequence variants in the regulatory region of the mtDNA (displacement loop [D-Loop])—the region that experiences more damage in diabetes than other regions of the mtDNA (29)—using a Surveyor Detection Kit (IDT Inc., Coralville, IA). Sequence variants were deter- mined by digesting the amplicons with a surveyor , a mismatch-specific endonuclease with high specificity for the sites of base substitution sequence variants. The digested products were electrophoresed on a 2% agarose gel and analyzed upon visualization under an ultraviolet transillu- minator, as reported previously (29). Figure 1—Effect of Sirt1 overexpression on retinal capillary cell dam- mtDNA copy numbers were quantified in retinal micro- age in diabetes. Trypsin-digested retinal microvessels from C57BL/6J CytB WT and Sirt1-overexpressing mice, which had diabetes for ;8months, vessels using primers for cytochrome B ( )asamarker were stained with TUNEL. TUNEL-positive cells were counted through- for mtDNA and b-Actin for nuclear DNA (Table 1). SYBR out the entire retinal vasculature. The microvasculature was then green–based quantitative PCR was carried out by amplifica- stained with periodic acid Schiff–hematoxylin, providing representa- tion at 95°C for 10 min, followed by 40 cycles at 95°C for tive microvasculature (A). The arrowhead indicates an acellular capil- lary, and the arrow points to a pericyte ghost. B: Sirt1 expression was 15 s and 60°C for 60 s. The ratio of mtDNA to nuclear DNA quantified in the retinal cryosections by immunofluorescence (fluoresc) (CytB:b-Actin) within each sample was used to calculate staining using DyLight 488–labeled (green) secondary antibodies. Retinal mtDNA copy numbers (29). microvessels prepared with the hypotonic shock method were analyzed for Sirt1 gene expression by SYBR green–based quantitative PCR (C) Epigenetic Modifications and protein expression by Western blotting (D). Values are presented 6 n – P , DNA methylation of the Sirt1 promoter was determined by as the mean SD ( =57 mice/group). * 0.05 compared with age-matched WT-N; #P , 0.05 compared with WT-D. Sirt-D, diabetic quantifying 5-methylcytosine (5mC) using a methylated DNA Sirt1-overexpressing mice; Sirt-N, normal Sirt1-overexpressing mice; immunoprecipitation kit (Epigentek, Farmingdale, NY) (19). WT-D, diabetic WT C57BL/6J mice; WT-N, normal WT C57BL/6J mice. 748 Sirt1 and Diabetic Retinopathy Diabetes Volume 67, April 2018 amplitudes and the implicit times of both a-waves and were analyzed at three random places using ImageJ soft- b-waves were measured using ERGview software (23). ware.

Retinal Thickness Statistical Analysis Retinal thickness was quantified by optical coherence Statistical analysis was performed using SigmaStat software tomography (OCT) using an OCT module customized to (Systat Software Inc., San Jose, CA). Comparison among image the retinas of small animals on the Micron IV micro- groups was analyzed using one-way ANOVA followed by the scope. The anesthetized animals were positioned in front of Student-Newman-Keuls test for data with a normal distri- the camera, and a high-resolution B-scan of the retinal cross bution. For data for which a normality test failed, one-way sections was obtained from both eyes by averaging and ANOVA was performed, followed by the Dunn test. A spatially aligning 50 individual B-scans along the same P value ,0.05 was considered statistically significant. horizontal axis through the optic disc (30). Thickness of the ganglion cell layer (GCL) 1 inner plexiform layer (IPL), and RESULTS of the inner nuclear layer (INL), was measured at a 200- to Retinal vasculature from diabetic WT mice had a signifi- 400-mm distance on either side of the optic disc using the cantly higher number of acellular capillaries compared with caliper tool available in InSight software. age-matched normal WT mice, as expected (13): ;8acellu- To confirm changes in the retinal layers, retinal cryosections lar capillaries in normal mice compared with ;20 in di- were stained with hematoxylin-eosin (31), and the images abetic mice (Fig. 1A). Similarly, the number of pericytes

Figure 2—Effect of Sirt1 overexpression on vascular leakage and capillary density. A: Fluorescein angiography was performed using a Micron IV Retinal Imaging Microscope containing a barrier filter. The images show representative angiograms from mice in each group; the arrow in the inset indicates vascular leakage. B:Tailvein–injected Evans blue dye in the retinal extract was quantified spectrophotometrically at 620 nm. C and D: Vascular density was determined in fluorescein angiograms (C) and by isolectin staining of retinal flatmounts using fluorescein iso- thiocyanate–conjugated Isolectin B4 under a confocal microscope (D). The grayscale images, converted using AngioTool software, were analyzed, and the accompanying graph represents vessel area. The values obtained from normal WT mice are considered 100%. Each group had five or six mice. *P , 0.05 compared with normal WT C57BL/6J mice (WT-N); #P , 0.05 compared with diabetic WT C57BL/6J mice (WT-D). Sirt-D, diabetic Sirt1-overexpressing mice; Sirt-N, normal Sirt1-overexpressing mice. diabetes.diabetesjournals.org Mishra, Duraisamy, and Kowluru 749

Because vascular leakage is one of the hallmarks of diabetic retinopathy (1), the effect of Sirt1 overexpression on vascular health was determined by fluorescein angiog- raphy. Although some vascular leakage was observed in di- abetic WT mice, consistent with normal WT and Sirt1 mice, no leakage was observed in diabetic Sirt1 mice (Fig. 2A). Similarly, protection by Sirt1 against diabetes-induced reti- nal vascular leakage was also confirmed using the Evans blue method (Fig. 2B). The effect of Sirt1 on retinal vascular health was evaluated further by quantifying capillary den- sity. Both fluorescein angiography and isolectin staining showed a significant decrease in the retinal vascular density in diabetic WT mice compared with normal WT mice (Fig. 2C and D); however, diabetes had no effect on retinal vas- cular density in Sirt1 mice, and the density was similar to that seen in normal WT mice. Figure 3—Effect of Sirt1 upregulation on mtDNA damage and bio- To investigate the effect of Sirt1 on mitochondrial dam- A genesis. : Retinal microvessels were analyzed for DNA damage by age, mtDNA damage was analyzed in the retinal microvas- measuring sequence variants in the amplified D-Loop region using a mismatch-specific surveyor endonuclease, followed by analysis on a 2% culature. Consistent with our previous results (29), diabetic agarose gel. B: The parent band amplicon intensity was quantified; the WT mice had a higher number of sequence variants in the intensity of the amplicons from normal WT C57BL/6J mice (WT-N) D-Loop and a lower parent amplicon band intensity (Fig. 3A was considered 100%. C: Mitochondrial copy numbers were quanti- B Sirt1 fi and ), which was prevented in diabetic mice. Sirt1 ed in the total DNA isolated from retinal microvessels by quantitative fi PCR, using CytB as an mtDNA marker and b-Actin as a nuclear DNA also plays a signi cant role in mtDNA biogenesis (32), and marker. The results are representative of five or six microvessel prepara- in diabetes, mtDNA copy numbers are decreased (33). Com- tions per group. *P , 0.05 compared with WT-N; #P , 0.05 compared pared to normal WT mice, mtDNA copy numbers were with diabetic WT C57BL/6J mice (WT-D). M, marker; Sirt-D, diabetic fi Sirt1-overexpressing mice; Sirt-N, normal Sirt1-overexpressing mice. signi cantly lower in the retinal microvessels of diabetic WT mice (Fig. 3C), but diabetic Sirt1 mice had an amount of copy numbers similar to those obtained from normal mice (WT or Sirt1). and TUNEL-positive cells was increased from 5–8 in normal Because upregulation of MMP-9 in diabetes is implicated to 16–25 in diabetic mice. However, both diabetic and nor- in the mitochondrial damage seen in the retina and its mal Sirt1 mice had approximately nine acellular capillaries vasculature (11,13), we evaluated the effect of Sirt1 on and six to eight TUNEL-positive capillary cells. Figure 1B–D MMP-9 expression. The retinal microvasculature of diabetic shows a significant decrease in Sirt1 expression (protein Sirt1 mice was also protected from an increase in MMP-9 and mRNA) in retinal vasculature in diabetic WT mice, expression, and the values obtained from normal WT and and its prevention in diabetic Sirt1 mice. Sirt1 mice and diabetic Sirt1 mice were not different from

Figure 4—Effect of Sirt1 overexpression on diabetes-induced retinal MMP-9. A:TheMMP-9 gene transcript was quantified in retinal micro- vasculature by SYBR green–based quantitative PCR using 18S as the housekeeping gene. Values are the mean 6 SD of four to six samples per group. B: Expression of MMP-9 in retinal cryosections was evaluated with immunofluorescence (fluoresc) using DyLight 488–conjugated (green) and Texas Red–conjugated (red) secondary antibodies for Sirt1 and MMP-9, respectively; cells were mounted in DAPI mounting medium (blue). The insets show magnified areas. Values are mean 6 SD (4–6 samples/group). *P , 0.05 compared with normal WT C57BL/6J mice (WT-N); #P , 0.05 compared with diabetic WT C57BL/6J mice (WT-D). Sirt-D, diabetic Sirt1-overexpressing mice; Sirt-N, normal Sirt1-overexpressing mice. 750 Sirt1 and Diabetic Retinopathy Diabetes Volume 67, April 2018

retinal microvasculature from diabetic WT mice had over 3-fold more 5mC at the Sirt1 promoter, and ;2.5-fold higher Dnmt1 expression (Fig. 5A). However, in diabetic Sirt1 mice, although 5mC levels were higher than those in normal mice, these values were significantly lower than those obtained from diabetic WT mice, suggesting a role for DNA methylation in diabetes-induced Sirt1 suppression. In a similar way, a diabetes-induced increase in Dnmt1 ex- pression was ameliorated in diabetic Sirt1 mice (Fig. 5B). Immunohistochemical data confirmed the protective effect of Sirt1 against an increase in Dnmt1 staining (Fig. 5C and D). Sirt1 can deacetylate both histones and proteins, and deacetylation of Dnmt1 by Sirt1 deactivates it (35). To in- vestigate the role of Sirt1 in a diabetes-induced increase in Dnmt1, we investigated the acetylation of histone at the Dnmt1 promoter. As shown in Fig. 5E, although diabetes increased acetylated H3K9 levels, which were ameliorated in diabetic Sirt1 mice, it had no effect on Dnmt1 protein acet- ylation (Supplementary Fig. 2). Diabetes also affects nonvascular cells of the retina, re- sulting in abnormal electrical responses (5). ERG was per- formed to investigate the effect of Sirt1 on functional changes in nonvascular cells. Compared with normal WT mice, diabetic WT mice showed an ;20%decreaseina-waveandb-wave amplitudes; these changes were prevented in diabetic Sirt1 mice, and the values were similar to those obtained from age- matched normal WT mice (Fig. 6A–C). Similarly, the implicit times of a- and b-waves were also increased by 15–20%. Consistent changes in a- and b-waves were also observed at 3,000 and 10,000 mcds/m2. Because of the accelerated loss of retinal cells in diabetes, the retinal layers commonly become thinner (36); we de- termined the effect of Sirt1 overexpression on diabetes- — Figure 5 DNA methylation of retinal Sirt1. Retinal microvessels were induced changes in retinal layer thickness. Diabetic WT mice used to quantify 5mC levels using the methylated DNA immunopre- fi 1 cipitation technique (A)andDnmt1 gene transcripts through quan- showed a signi cant decrease in the thickness of the GCL titative PCR (B), using 18S as the housekeeping gene. C:Dnmt1 IPL 200 mm from the optic disc (40.43 6 1.10 mm) com- expression in the cryosections was determined by immunofluores- pared with normal WT mice (44.00 6 1.26 mm; P , cence using secondary antibodies conjugated with DyLight 488 (green) 6 for Sirt1 and with Texas Red for Dnmt1. DAPI mounting medium (blue) 0.0001). Similar decreases were seen in the INL (22.83 was used to mount the sections. D: The graph shows the mean fluo- 1.72 vs. 20.29 6 1.3 mm; P , 0.013). However, in diabetic rescence intensity (fluoresc) of Dnmt1. E: Acetylated H3K9 levels at Sirt1 mice, thicknesses of both the GCL 1 IPL and the INL Dnmt1 fi the promoter in retinal microvessels were quanti ed by immu- were significantly different from those observed in di- noprecipitating genomic DNA with H3K9Ac antibody, followed by 6 6 m quantitative PCR using primers for the Dnmt1 promoter. Values are abetic WT mice (43.83 0.82 and 23.00 0.60 m, presented as the mean 6 SD of four to six retinal microvessel prep- respectively; P , 0.0001). Similar differences in the thick- arations per group. *P , 0.05 vs. normal WT C57BL/6J mice (WT-N); ness in diabetic WT and Sirt1 animals were also observed #P , 0.05 vs. diabetic WT C57BL/6J mice (WT-D). Sirt-D, diabetic m A Sirt1-overexpressing mice; Sirt-N, normal Sirt1-overexpressing mice. 300 mfromtheopticdisc(Fig.7 ). Retinal cryosections stained with hematoxylin-eosin also presented similar pro- tection against retinal layer thinning in diabetic Sirt1 mice (Fig. 7B). each other (Fig. 4A). Figure 4B further confirms the de- crease in immunostaining of MMP-9 in the retinal cryosec- tions from diabetic Sirt1 mice compared with that in DISCUSSION diabetic WT mice. Diabetic retinopathy is a multifactorial disease, and many Cytosine methylation results in transcriptional repres- structural, biochemical, molecular, and functional abnor- sion (17,18,34), and DNA methylation machinery is acti- malities are implicated in its development (1,2). Sirt1 is vated in diabetes (19,20). To understand the mechanism a cellular energy sensor that plays a critical role in linking responsible for Sirt1 suppression, its promoter DNA meth- metabolic stress with cellular response (6,7); in diabetes, ylation was investigated. Compared with normal WT mice, Sirt1 is downregulated in the retina and its vasculature (11). diabetes.diabetesjournals.org Mishra, Duraisamy, and Kowluru 751

The Sirtuin family of proteins comprises highly con- served NAD-dependent deacetylases; humans encode seven different sirtuins (Sirt1–Sirt7); among them Sirt1 is the most extensively studied member (6,8). Sirt1 is linked to cellular energy metabolism and the redox state through multiple signaling and survival pathways, and it is also im- plicated in the pathophysiology of many chronic diseases, including diabetes, neurodegenerative disorders, and cardio- vascular disease (37). Sirt1 controls the redox environment and counteracts oxidative damage by converting NAD to its reduced form, NADH (8); in coronary heart and cerebrovas- cular diseases, activation of Sirt1 protects against oxidative stress at the cellular level and increases survival at the systemic level to further limit these diseases (38). Sirt1- transgenic mice are protected from diabetes induced by obesity (through both diet and ) (39). However, although Sirt1 is a core systemic regulator of cellular me- tabolism, mice overexpressing Sirt1 have a normal life span, and the protection is restricted to specific tissues (40). Here Figure 6—Effect of Sirt1 on retinal neuronal function. ERG was per- we provide convincing data showing a direct role for Sirt1 formed in dark-adapted mice using the OcuScience HMsERG Lab Sys- in diabetic retinopathy: the retinal vasculature of diabetic tem. The electrical response was recorded using a series of Ganzfeld mice overexpressing Sirt1 is protected from the develop- 2 flashes with intensities ranging from 100 to 25,000 mcds/m . A:A ment of histopathology and from accelerated apoptosis, representative ERG response at 1,000 mcds/m2 from one mouse in each group. B and C:a-Waveamplitude(B) and b-wave amplitude (C) a phenomenon that precedes the development of histopa- are presented as the percentage of normal; the values obtained from thology (41), implying that Sirt1 activators could potentially normal WT C57BL/6J mice (WT-N) were considered to be 100%. *P , impede the development of retinopathy in patients with 0.05 compared with normal WT C57BL/6J mice (WT-N); #P , 0.05 diabetes. Consistent with this, decreased Sirt1 mRNA and compared with diabetic WT C57BL/6J mice (WT-D). Sirt-D, diabetic Sirt1-overexpressing mice; Sirt-N, normal Sirt1-overexpressing mice. activity are also seen in retinal microvasculature and pe- ripheral blood mononuclear cells from human donors with diabetic retinopathy (11,42). A decrease in Sirt1, via regu- lating acetylation of the liver X , has been shown Downregulation of Sirt1 hyperacetylates many regulatory to increase both hyperglycemia and dyslipidemia in type 2 proteins, including transcription factors associated with ox- diabetes, contributing to the progression of diabetic retinop- idative stress and apoptosis (6,8,11). Here we show that athy (43). Furthermore, administration of the Sirt1 activa- Sirt1 is also intimately associated with the development of tor resveratrol ameliorates age-related increases in retinal diabetic retinopathy and related retinal vascular and neuro- degeneration and cell apoptosis in rats (44). nal damage. Our novel data clearly demonstrate that diabetic Clinical and experimental studies have documented mice overexpressing Sirt1 are protected from the develop- many alterations in the retinal vasculature in diabetic reti- ment of degenerative capillaries, a histopathological char- nopathy, including increased vascular permeability, leuko- acteristic of retinopathy, and their retinas have normal stasis, and capillary degeneration (1,2), and the vascular vascular density and do not leak. Sirt1 also protects mito- density is decreased (45). Damage to the blood-retina bar- chondria from diabetes-induced increases in mtDNA dam- rier is considered to be an early clinical sign of the devel- age and decreases in copy numbers, and it obliterates the opment of diabetic retinopathy (1). We show that, whereas activation of mitochondria-damaging MMP-9. Diabetic Sirt1 the retinas of diabetic WT mice have less vascular density mice are spared from retinal neuronal damage; ERG responses and more vascular leakage than normal WT mice, diabetic are normal and GCL-IPL and INL thicknesses are not dif- Sirt1 mice have normal retinal vascular density with no ferent than those seen in normal WT or Sirt1 mice, suggest- vascular leakage, suggesting that, in addition to preventing ing a major role of Sirt1 in preventing diabetes-induced increased capillary cell apoptosis and the formation of de- damage to the overall retinal health. Furthermore, we elu- generative capillaries, Sirt1 overexpression also helps to cidated the role of DNA methylation in diabetes-induced maintain the overall health of the retinal vasculature. Sirt1 transcriptional suppression and showed that, to the Mitochondrial damage plays a critical role in the path- contrary, Sirt1 regulates its transcription by modulating his- ogenesis of diabetic retinopathy; in diabetic rodents, in- tone acetylation of the Dnmt1 promoter. These results creased functional and structural damage to mitochondria together imply a significant role for Sirt1 in the develop- is observed before capillary cell apoptosis and the number ment of diabetic retinopathy, and the mechanism of Sirt1 of degenerative capillaries increase within the retinal suppression seems to be the epigenetic modification of its vasculature (13,29). Compromised mtDNA biogenesis and promoter. fewer mtDNA copy numbers are observed in the retinal 752 Sirt1 and Diabetic Retinopathy Diabetes Volume 67, April 2018

Figure 7—Effect of Sirt1 overexpression on retinal layer thickness. A: OCT was performed using an OCT module, customized for retinal imaging of small animals, in the Micron IV Retinal Imaging System. An average high-resolution B-scan of retinal cross sections was obtained by spatially aligning 50 individual B-scans along the same horizontal axis through the optic disc, marked with a green arrow on the fundus images (left). The right panels show representative B-scans from each group; the layer thickness measurement points are marked. The thickness of the GCL 1 IPL and INL, at 200, 300, and 400 mm on either side of the optic disc, was measured using InSight software. In the graph, negative integers represent measurements on the left side of the optic disc, and positive integers represent those on the right side of the optic disc. B: Retinal cryosections were stained with hematoxylin-eosin, and the thickness of the layers was analyzed at three random places using ImageJ software. *P , 0.05 vs. normal WT C57BL/6J mice; #P , 0.05 vs. diabetic WT C57BL/6J mice. ONL, outer nuclear layer; Sirt-D, diabetic Sirt1-overexpressing mice; Sirt-N, normal Sirt1-overexpressing mice; WT-D, diabetic WT C57BL/6J mice; WT-N, normal WT C57BL/6J mice.

microvasculature, and damaged mtDNA continues to fuel factors NF-kB and activator protein-1, their binding at the vicious cycle of free radicals by compromising the elec- the MMP-9 promoter is increased, resulting in MMP-9 tran- tron transport chain system (2,3). Here we show that the scriptional activation (11,14). Furthermore, Sirt1 also regu- retinal microvasculature of these diabetic Sirt1 mice are also lates the binding of PARP-1 at the MMP-9 promoter, which protected from mtDNA damage, and their mtDNA copy regulates MMP-9 expression by manipulating the binding of numbers are normal. In support of this, Sirt1 is critically NF-kB/activator protein-1 (22). The results presented here involved in maintaining mitochondrial quality; it regulates clearly show that the retinal vasculature of the same di- mtDNA biogenesis and turnover of damaged mitochondria abetic Sirt1 mice protected from the development of dia- (32), and manipulation of Sirt1 has been shown to regulate betic retinopathy also have normal MMP-9 expression, hyperglycemia-induced mitochondrial dysfunction and apo- further confirming the role of Sirt1 in the mitochondrial ptosis in human umbilical cord vascular cells (46). Further- damage seen in diabetes. more, retinal poly(ADP-) polymerase (PARP) is also Diabetes downregulates Sirt1 in many tissues, including activated in diabetes (22), and chronic activation of PARP-1 retina and kidney (11,49); one of the mechanisms implicated inactivates Sirt1 by depleting NAD and results in mitochondrial in its inactivation is increased oxidative stress (8). In a dis- dysfunction (47), further fueling mitochondrial damage. ease state, epigenetic modifications also regulate gene tran- Activation of proteinases, especially MMP-2 and MMP-9, scription, and diabetes favors many of these epigenetic is implicated in the retinal mitochondrial damage seen modifications, including DNA methylation and histone in diabetes (13,24). The MMP-9 promoter has multiple modifications (2,19). DNA methylation is considered to be transcriptional factor binding sites, and many transcrip- a gene suppressor (34), and the machinery responsible for tional factors are involved in its transcription (48). We have maintaining DNA methylation is activated in the retina in shown that, because of hyperacetylation of transcriptional diabetes (19). Our results demonstrating hypermethylation diabetes.diabetesjournals.org Mishra, Duraisamy, and Kowluru 753 of the Sirt1 promoter in retinal microvessels from diabetic to Prevent Blindness to the Department of Ophthalmology, Wayne State University, mice are supported by other reports showing increased Sirt1 Detroit, MI. promoter methylation in peripheral blood from patients Duality of Interest. No potential conflicts of interest relevant to this article were with Alzheimer disease (50). Consistent with the regulation reported. Author Contributions. M.M. researched data, performed the literature of DNA methylation of the Sirt1 promoter, in the same Sirt1 Dnmt1 search, and edited the manuscript. A.J.D. researched data and performed the literature diabetic mice, the promoter (the only member search. R.A.K. created the experimental plan, performed the literature search, and of the Dnmt family that is upregulated in the retina in wrote and edited the manuscript. R.A.K. is the guarantor of this work and, as such, diabetes [19]) is protected from increased acetylation of had full access to all the data in the study and takes responsibility for the integrity of H3K9, which results in the regulation of Dnmt1 transcrip- thedataandtheaccuracyofthedataanalysis. tion. These results suggest that Sirt1 ameliorates Dnmt1 acti- vation by inhibiting H3K9 acetylation at the promoter of References Dnmt1.InhibitionofDnmt1,inturn,impedesSirt1 pro- 1. Frank RN. Diabetic retinopathy. N Engl J Med 2004;350:48–58 moter DNA methylation, and regulates transcription of 2. Kowluru RA, Kowluru A, Mishra M, Kumar B. Oxidative stress and epigenetic fi Sirt1. modi cations in the pathogenesis of diabetic retinopathy. Prog Retin Eye Res 2015; – Although the histopathology of diabetic retinopathy is 48:40 61 3. Kowluru RA. Diabetic retinopathy, metabolic memory and epigenetic mod- observed in the retinal vasculature, neuronal cells also ifications. 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