Photoreceptor Cell Calcium Dysregulation and Calpain Activation Promote Pathogenic Photoreceptor Oxidative Stress and Inflammation in Prodromal Diabetic Retinopathy
2021; Elsevier BV; Volume: 191; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2021.06.006
ISSN1525-2191
AutoresAicha Saadane, Yunpeng Du, Wallace B. Thoreson, Masaru Miyagi, Emma M. Lessieur, Jianying Kiser, Xiangyi Wen, Bruce A. Berkowitz, Timothy S. Kern,
Tópico(s)Connexins and lens biology
ResumoThis study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1−/−) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2 months. Cytosolic Ca2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy. This study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1−/−) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2 months. Cytosolic Ca2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy. The pathogenesis of diabetic retinopathy (DR), a leading cause of vision loss and blindness, is not fully understood, and therefore effective treatment options remain limited. In experimental models, diabetes-induced degeneration of retinal capillaries and other lesions of the retina have been inhibited by antioxidant or anti-inflammatory strategies, suggesting that oxidative stress and inflammation play an important role in the early development of the retinopathy. Recent studies provide evidence that photoreceptor cells, the most numerous cell type in the retina, play a causal role in the microvascular disease of DR1Arden G.B. The absence of diabetic retinopathy in patients with retinitis pigmentosa: implications for pathophysiology and possible treatment.Br J Ophthalmol. 2001; 85: 366-370Crossref PubMed Scopus (111) Google Scholar, 2Berkowitz B.A. Preventing diabetic retinopathy by mitigating subretinal space oxidative stress in vivo.Vis Neurosci. 2020; 37: E002Crossref PubMed Scopus (4) Google Scholar, 3Kern T.S. Berkowitz B.A. Photoreceptors in diabetic retinopathy.J Diabetes Investig. 2015; 6: 371-380Crossref PubMed Scopus (79) Google Scholar and are a major source of reactive oxygen species (ROS) and inflammatory proteins and cytokines in diabetes.4Berkowitz B.A. Bissig D. Patel P. Bhatia A. Roberts R. Acute systemic 11-cis-retinal intervention improves abnormal outer retinal ion channel closure in diabetic mice.Mol Vis. 2012; 18: 372-376PubMed Google Scholar, 5Berkowitz B.A. Gradianu M. Schafer S. Jin Y. Porchia A. Iezzi R. Roberts R. Ionic dysregulatory phenotyping of pathologic retinal thinning with manganese-enhanced MRI.Invest Ophthalmol Vis Sci. 2008; 49: 3178-3184Crossref PubMed Scopus (24) Google Scholar, 6Berkowitz B.A. Grady E.M. Khetarpal N. Patel A. Roberts R. Oxidative stress and light-evoked responses of the posterior segment in a mouse model of diabetic retinopathy.Invest Ophthalmol Vis Sci. 2015; 56: 606-615Crossref PubMed Scopus (48) Google Scholar, 7Du Y. Veenstra A. Palczewski K. Kern T.S. Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina.Proc Natl Acad Sci U S A. 2013; 110: 16586-16591Crossref PubMed Scopus (210) Google Scholar Factors involved in promoting photoreceptor oxidative stress and inflammation during prodromal DR remain unclear. Maintaining Ca2+ homeostasis is vital for cell health and is tightly regulated.8Krizaj D. Copenhagen D.R. Calcium regulation in photoreceptors.Front Biosci. 2002; 7: d2023-d2044Crossref PubMed Google Scholar In vivo imaging studies using manganese-enhanced magnetic resonance imaging have shown that influx of Ca2+ and other ions in retinal cells (especially photoreceptors) is disrupted early in the course of diabetes. This defect can be corrected by a variety of antioxidant treatments, showing that oxidative stress and calcium dysregulation are interconnected.4Berkowitz B.A. Bissig D. Patel P. Bhatia A. Roberts R. Acute systemic 11-cis-retinal intervention improves abnormal outer retinal ion channel closure in diabetic mice.Mol Vis. 2012; 18: 372-376PubMed Google Scholar,9Berkowitz B.A. Gradianu M. Bissig D. Kern T.S. Roberts R. Retinal ion regulation in a mouse model of diabetic retinopathy: natural history and the effect of Cu/Zn superoxide dismutase overexpression.Invest Ophthalmol Vis Sci. 2009; 50: 2351-2358Crossref PubMed Scopus (64) Google Scholar, 10Berkowitz B.A. Roberts R. Stemmler A. Luan H. Gradianu M. Impaired apparent ion demand in experimental diabetic retinopathy: correction by lipoic acid.Invest Ophthalmol Vis Sci. 2007; 48: 4753-4758Crossref PubMed Scopus (47) Google Scholar, 11Gillardon F. Zimmermann M. Uhlmann E. Expression of c-Fos and c-Jun in the cornea, lens, and retina after ultraviolet irradiation of the rat eye and effects of topical antisense oligodeoxynucleotides.Br J Ophthalmol. 1995; 79: 277-281Crossref PubMed Scopus (12) Google Scholar It remains unclear whether abnormal calcium handling in diabetes can also promote oxidative stress, the focus of this study. One important consequence of Ca2+ dysregulation is activation of Ca2+-dependent proteases in the calpain family. Calpains are responsible for limited proteolysis of target proteins, potentially leading to the activation or inhibition of enzymes and kinases. Excessive calpain activation has been implicated in the development of complications in a variety of tissues, including complications of diabetes, at least in the heart and brain.12Azuma M. Shearer T.R. The role of calcium-activated protease calpain in experimental retinal pathology.Surv Ophthalmol. 2008; 53: 150-163Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Frustaci A. Kajstura J. Chimenti C. Jakoniuk I. Leri A. Maseri A. Nadal-Ginard B. Anversa P. Myocardial cell death in human diabetes.Circ Res. 2000; 87: 1123-1132Crossref PubMed Scopus (681) Google Scholar, 14Hirata M. Shearer T. Azuma M. Hypoxia activates calpains in the nerve fiber layer of monkey retinal explants.Invest Ophthalmol Vis Sci. 2015; 56: 6049-6057Crossref PubMed Scopus (6) Google Scholar, 15Joyal J.-S. Sun Y. Gantner M.L. Shao Z. Evans L.P. Saba N. Fredrick T. Burnim S. Kim J.S. Patel G. Juan A.M. Hurst C.G. Hatton C.J. Cui Z. Pierce K.A. Bherer P. Aguilar E. Powner M.B. Vevis K. Boisvert M. Fu Z. Levy E. Fruttiger M. Packard A. Rezende F.A. Maranda B. Sapieha P. Chen J. Friedlander M. Clish C.B. Smith L.E.H. Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1.Nat Med. 2016; 22: 439-445Crossref PubMed Scopus (127) Google Scholar, 16Paquet-Durand F. Azadi S. Hauck S.M. Ueffing M. van Veen T. Ekström P. Calpain is activated in degenerating photoreceptors in the rd1 mouse.J Neurochem. 2006; 96: 802-814Crossref PubMed Scopus (117) Google Scholar, 17Vanderklish P.W. Bahr B.A. The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states.Int J Exp Pathol. 2000; 81: 323-339Crossref PubMed Scopus (174) Google Scholar Calpains regulate the pathogenic mitochondrial oxidative stress in diabetic cardiomyopathy, which can be inhibited by overexpression of mitochondrial calpastatin, an endogenous calpain inhibitor (CI).18Ni R. Zheng D. Xiong S. Hill D.J. Sun T. Gardiner R.B. Fan G.C. Lu Y. Abel E.D. Greer P.A. Peng T. Mitochondrial calpain-1 disrupts ATP synthase and induces superoxide generation in type 1 diabetic hearts: a novel mechanism contributing to diabetic cardiomyopathy.Diabetes. 2016; 65: 255-268Crossref PubMed Scopus (82) Google Scholar Because photoreceptor cells in the retina are the major site of retinal superoxide generation in diabetes,7Du Y. Veenstra A. Palczewski K. Kern T.S. Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina.Proc Natl Acad Sci U S A. 2013; 110: 16586-16591Crossref PubMed Scopus (210) Google Scholar and contain approximately 75% of the retina's mitochondria, we postulated that calpains might play a role in diabetes-induced photoreceptors oxidative stress and induction of proinflammatory proteins in the retina in diabetes. This study addresses the above knowledge gaps by measuring i) Ca2+ levels from rod photoreceptor cells in retinal slices of diabetic mice, ii) calpain expression and activity in retinas of diabetic mice, iii) the impact of in vivo administration of a CI or genetic deletion of Capn1 on diabetes-induced oxidative stress and expression of inflammatory proteins in the retina, and iv) diabetes-induced abnormalities of the photoreceptor proteome that are influenced by calpain activity. Finally, this study identified Wwox as a novel substrate of calpain that contributes to the hyperglycemia-induced generation of superoxide by the retina. This study was performed in strict accordance with the NIH's Guide for the Care and Use of Laboratory Animals,19Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research,20Association for Research in Vision and OphthalmologyStatement for the Use of Animals in Ophthalmic and Vision Research. ARVO, Rockville, MD2016Google Scholar and with authorization of the institutional animal and care use committees at Case Western Reserve University, University of California, Irvine, Wayne State University, and the University of Nebraska Medical Center. All efforts were made to minimize suffering within the context of the diabetic protocol including administration of insulin to prevent weight loss. Wild-type (WT) C57Bl/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Calpain1 knockout (Capn1−/−) mice (C57Bl/6j background) were obtained from the laboratory of Dr. Chishti (Tufts University School of Medicine, Boston, MA).21Azam M. Andrabi S.S. Sahr K.E. Kamath L. Kuliopulos A. Chishti A.H. Disruption of the mouse mu-calpain gene reveals an essential role in platelet function.Mol Cell Biol. 2001; 21: 2213-2220Crossref PubMed Scopus (213) Google Scholar In all studies, male mice (2-month–old) were randomly assigned to become diabetic or remain as nondiabetic controls. Diabetes was induced by five sequential daily intraperitoneal injections of a freshly prepared solution of streptozotocin in citrate buffer (pH 4.5) at 55 to 60 mg/kg of body weight. Hyperglycemia was verified at least three times during the second week after streptozotocin administration, and mice having three consecutive measurements of fasting blood glucose >275 mg/dL were classified as being diabetic. Insulin was given as needed to prevent weight loss without preventing hyperglycemia and glucosuria (0 to 0.2 U of NPH insulin subcutaneously 0 to 3 times per week). All animals were maintained on a standard 12-hour light (~10 lux)-dark cycle and were provided standard rodent chow (Purina TestDiet 5001; Purina, Richmond, IN) and water ad libitum. Blood glucose and hemoglobin A1c were measured as reported previously.7Du Y. Veenstra A. Palczewski K. Kern T.S. Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina.Proc Natl Acad Sci U S A. 2013; 110: 16586-16591Crossref PubMed Scopus (210) Google Scholar,22Liu H. Tang J. Du Y. Saadane A. Samuels I. Veenstra A. Kiser J.Z. Palczewski K. Kern T.S. Transducin1, phototransduction and the development of early diabetic retinopathy.Invest Ophthalmol Vis Sci. 2019; 60: 1538-1546Crossref PubMed Scopus (26) Google Scholar Body weight was measured weekly. Once declared diabetic, some mice received a daily intraperitoneal injection (10 mg/kg body weight) of CI (carbobenzoxy-valinyl-phenylalaninal, also known as MDL 28170; Calbiochem, Burlington, MA) dissolved in dimethyl sulfoxide. At 2 months of diabetes (4 months of age), retinal structure and function were characterized, and then animals were euthanized and eyes collected. Regarding the measurement of calcium in retinal photoreceptor cells, the experiments were done between 10 AM and 2 PM during circadian day, and the tissue was light adapted (because of the bright 340/380-nm fluorescence used to measure calcium changes, the eyes were not dissected in the dark). For calpain activity and calpain1 immunohistochemistry studies, mice were in the normal light cycle, and the eyes were collected before 1:30 PM. The authors conducted an anatomic characterization of Capn1−/− and WT mice using spectral domain-optical coherence tomography (OCT; the 840 HHP spectral domain-OCT system; Bioptigen, Durham, NC), and electroretinographic (ERG; Diagnosys Celeris rodent ERG device; Diagnosys, Lowell, MA) recordings as described,23Orban T. Leinonen H. Getter T. Dong Z. Sun W. Gao S. Veenstra A. Heidari-Torkabadi H. Kern T.S. Kiser P.D. Palczewski K. A combination of G protein-coupled receptor modulators protects photoreceptors from degeneration.J Pharmacol Exp Ther. 2018; 364: 207-220Crossref PubMed Scopus (12) Google Scholar,24Saadane A. Mast N. Charvet C.D. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol.Am J Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar except that for ERG analysis, mice were anesthetized using isoflurane. Retinal morphology was further confirmed by histology. After enucleation, eyes were fixed in formalin (10% neutral buffered) for 48 hours. Paraffin-embedding, sectioning, hematoxylin and eosin staining, and image scanning were performed by the Histowiz company (Brooklyn, NY). For Ca2+ imaging in mouse vertical retinal slices, mice were euthanized by carbon dioxide asphyxiation under dim red light. Subsequent dissections were performed under infrared illumination using GenIII night vision goggles (Nitemate NavIII; Litton Industries, Watertown, CT) mounted on a dissecting microscope. After enucleation, the eye was hemisected and the retina mounted vitreal side down on a nitrocellulose membrane (5 × 10 mm; type AAWP, 0.8-μm pores; Millipore, Billerica, MA). The retina was cut into 125-μm slices using a razor blade tissue slicer (Stoelting, Wood Dale, IL). Slices were rotated 90 degrees to view the retinal layers and anchored in the recording chamber by embedding the ends of the nitrocellulose membrane in vacuum grease so that measurement could be make from cells in the photoreceptor layer. The recording chamber was mounted on an upright fixed-stage microscope (Nikon E600FN; Nikon Instruments, Melville, NY), and slices were superfused at approximately 1 mL per minute with Ames' medium (US Biological, Salem, MA) bubbled with 95% O2 and 5% CO2 (35°C). For measurements of intracellular Ca2+ [Ca2+]i, the ratiometric dye Fura-2 (Invitrogen, Carlsbad, CA) was used. Retinal slices were loaded with Fura-2 by incubating them at room temperature for 2 hours with 0.5 mL of 10 μmol/L Fura-2/AM in Hibernate-A medium (Brain Bits LLC, Springfield, IL) in darkness. An epifluorescence lamp (150 W, Xe) was attached to a Lambda 10 to 2 filter wheel (Sutter Instruments, Novato, CA), equipped with 340- and 380-nm interference filters, and coupled to the microscope (E600FN; Nikon Instruments) through a liquid light guide (Sutter Instruments). Images were acquired through a 60× (1.0 numerical aperture ; Nikon Instruments) water immersion objective using a cooled electron multiplying charge-coupled device camera (Rolera MGi Plus; QImaging, Surrey, BC, Canada) and Nikon NIS-Elements AR 4.50.0 software. [Ca2+]i was determined using the formula: [Ca2+]i = Kd (Fmin/Fmax) [(R-Rmin)/(Rmax-R)] where Kd = 224 nmol/L Ca2+, r = ratio of fluorescence emitted at 510 nm by excitation with 340- and 380-nm light, Fmin = highest fluorescence emission measured with 380-nm excitation, and Fmax = lowest 380-nm emission. The minimum 340/380 ratio (Rmin) was determined by bath application of ionomycin (10 μmol/L) in a Ca2+-free solution containing 5 mmol/L EGTA. The maximum 340/380 ratio (Rmax) was then measured by bath application of ionomycin in control Ames' solution. Resting Ca2+ values in rods were measured at 5-second intervals in regions of interest placed on rod somas and averaged over the first 1 to 2 minutes of recording. Measurements were averaged from 2 to 10 rods in each slice preparation to yield a single value for each eye. Retinal calpain activity was determined using three methods. In the first, the right eye of diabetic and nondiabetic mice was injected intravitreally with 1 μL of fluorogenic calpain substrate IV (Catalog # 208,773; Calbiochem, Burlington, MA) at 10 μmol/L under visualization from a dissecting microscope. Two hours later, mice were sacrificed, and eyes were fresh frozen in OCT medium (Tissue-Tek; Sakura, Torrance, CA) and stored at −80°C until cryosectioned. In the second method, whole eyes from nondiabetic and diabetic mice were embedded in OCT and stored at −80°C until cryosectioned. Twelve-micron thick sections were air dried for 30 minutes at room temperature, hydrated 3 × 5 minutes in phosphate-buffered saline (PBS), and incubated with fluorogenic calpain substrate at room temperature for 1 hour (Millipore Sigma, St. Louis, MO) with or without CI (10 μmol/L), then rinsed in distilled water and mounted with Prolong Gold containing DAPI (Invitrogen) for nuclear staining and viewed using an inverted fluorescence microscope. In the third method, calpain activation was monitored by the proteolysis of a calpain substrate, spectrin, using an antibody against the cleaved spectrin fragment. Twelve-micron retinal sections were warmed to room temperature for 30 minutes and washed three times for 5 minutes in PBS. Sections were then blocked for 1 hour with 5% normal goat serum (Invitrogen) in PBS containing 0.05% Tween 20 (blocking buffer). Sections were incubated overnight at 4°C with rabbit anti-calpain1 (A1172; ABclonal, Woburn, MA). The next day, slides were washed three times for 5 minutes in PBS containing 0.05% Tween 20 and incubated for 1 hour in the dark with Alexa Fluor 647 secondary antibody (111 to 605-144; Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were washed three times for 5 minutes in PBS, then one time in distilled water, blotted dry, and then mounted with ProLong Gold anti-fade reagent with DAPI (P36935; Invitrogen, Carlsbad, CA). The calpain1 staining was imaged on a Keyence microscope (Keyence Corporation of America, Itasca, IL). Superoxide levels were measured chemically with lucigenin (bis-N-methylacridinium nitrate), as reported previously.25Du Y. Miller C.M. Kern T.S. Hyperglycemia increases mitochondrial superoxide in retina and retinal cells.Free Radic Biol Med. 2003; 35: 1491-1499Crossref PubMed Scopus (301) Google Scholar Briefly, freshly isolated retinas were preincubated in 200 μL of Krebs-HEPES buffer (pH 7.2) with 5 or 30 mmol/L glucose for 10 minutes at 37°C in 5% CO2. Luminescence indicating the presence of superoxide was measured 5 minutes after addition of lucigenin (5 mmol/L). Luminescence intensity is reported in arbitrary units per milligram of protein. Superoxide levels in 661W cells were measured similar to retinal samples, except about 200,000 cells were used, and luminescence intensity is reported in arbitrary units/106 cells. A vibratome was used to bisect fresh unfixed retina into outer and inner retinas as previously described.26Tonade D. Liu H. Kern T.S. Photoreceptor cells produce inflammatory mediators that contribute to endothelial cell death in diabetes.Invest Ophthalmol Vis Sci. 2016; 57: 4264-4271Crossref PubMed Scopus (38) Google Scholar The retinas were freshly isolated, and a flat mount of the retina with appropriate radial cuts was laid flat (photoreceptor side up) on a 4% gelatin block, and 20% warm gelatin was used to cover and seal the entire retina. Cold Dulbecco's modified Eagle's medium was placed in the vibratome chamber containing the retinas. Based on the measured thickness of the photoreceptor layer of mouse retina,26Tonade D. Liu H. Kern T.S. Photoreceptor cells produce inflammatory mediators that contribute to endothelial cell death in diabetes.Invest Ophthalmol Vis Sci. 2016; 57: 4264-4271Crossref PubMed Scopus (38) Google Scholar a conservative estimate of a 40-μm thick slice was used to enrich the photoreceptor layer from the remaining retina using a Leica VT1000 S vibratome. The isolated section of photoreceptor cells was immediately placed in liquid nitrogen and stored at −80°C. Elapsed time from eye enucleation to freezing the photoreceptor layer in an Eppendorf tube was about 15 to 25 minutes. One retina from each mouse was sonicated in 70 μL of lysis buffer (50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Nonidet P-40, 0.1% SDS, and complete EDTA-free protease inhibitor mixture from Roche (Lakwood, CA). Retinal homogenates were incubated on ice for 30 minutes followed by centrifugation at 12,000 × g for 15 minutes at 4°C. Supernatants were used for SDS-PAGE (50 μg of protein/lane) followed by Western blot analysis, which was performed as described.27Tang J. Du Y. Lee C.A. Talahalli R. Eells J.T. Kern T.S. Low-intensity far-red light inhibits early lesions that contribute to diabetic retinopathy: in vivo and in vitro.Invest Ophthalmol Vis Sci. 2013; 54: 3681-3690Crossref PubMed Scopus (65) Google Scholar Proteins were visualized with the following primary antibodies: 1:200 for intercellular adhesion molecule 1 (ICAM-1; sc-71292; Santa Cruz Biotechnology, Santa Cruz, CA), 1:200 for inducible nitric oxide synthase (iNOS; 610,328; BD Biosciences, San Jose, CA), 1:100 for pIκBα (2859; Cell Signaling Technology, Danvers, MA), 1:1000 inhibitor of κBα (IκBα; 9242; Cell Signaling), and 1:1000 for cleaved spectrin (ABN2264; EMD Millipore, Temecula, CA). The secondary antibody was goat anti-rabbit IRDye 800CW (925 to 32,211; Li-Cor, Lincoln, NE; dilution 1:5000). Membranes were also incubated with primary antibody against β-actin (1:5000), which was used as a loading control (ab8226; Abcam, Cambridge, MA), and secondary goat anti-mouse IRDye 680RD (925 to 68,070; Li-Cor; dilution 1:5000). Membranes were imaged using the Odyssey infrared imaging system (Li-Cor). The densitometry results of Western blots were expressed as means ± SD. Both retinas from each mouse were combined (total of three to four mice per group), and total RNA was isolated with TRIzol Reagent (Life Technologies, Grand Island, NY). Total RNA (1 μg) was converted to cDNA by SuperScript III Reverse Transcriptase (Invitrogen) and used for quantitative RT-PCR (qRT-PCR) conducted on a LightCycler 480 instrument (Roche). The sequences of the primers for gene quantifications were taken from qPrimerDepot, a primer database for qRT-PCR.28Cui W. Taub D.D. Gardner K. qPrimerDepot: a primer database for quantitative real time PCR.Nucleic Acids Res. 2007; 35: D805-D809Crossref PubMed Scopus (135) Google Scholar GAPDH was used as a housekeeping gene (Ct number approximately 14) over β-actin (Ct number ∼ 28). PCR reactions were performed in triplicate and normalized to GAPDH. The authors used a cone photoreceptor cell line, 661W, which they had previously confirmed by positive identification of cone opsin mRNA.29Du Y. Cramer M. Lee C.A. Tang J. Muthusamy A. Antonetti D.A. Jin H. Palczewski K. Kern T.S. Adrenergic and serotonin receptors affect retinal superoxide generation in diabetic mice: relationship to capillary degeneration and permeability.FASEB J. 2015; 29: 2194-2204Crossref PubMed Scopus (36) Google Scholar 661W cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5 mmol/L (normal glucose) or 30 mmol/L glucose (high glucose) in 6-well plates for 5 days (the medium was changed every other day). In a different set of experiments, 661W cells were incubated with 5 mmol/L, 25 mmol/L, or 30 mmol/L glucose or equivalent concentration of D-mannitol (osmotic control), to determine the conditions that lead to the highest superoxide levels and to ascertain that the measured superoxide level is due to elevated glucose as opposed to hyperosmolar conditions. Retinas were isolated from nondiabetic, diabetic, diabetic treated daily with CI (n = 3 to 4) for 2 months or their nondiabetic age-matched controls (total age 4 months) under carbon dioxide anesthesia, and stored briefly in PBS. The photoreceptor-enriched block (outer retina) was isolated from the rest of the retina using a vibratome as described above. Proteins from each photoreceptor-enriched block were extracted with SDS-sample buffer, 50 μg of which was separated by SDS-PAGE. The gel was stained by Coomassie R-250, and each sample (lane) was cut into 12 segments. The proteins in each segment were in-gel digested using trypsin,30Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3342) Google Scholar and the digests were analyzed by liquid chromatography with tandem mass spectrometry (MS/MS) as described below. Liquid chromatography with MS/MS analysis was performed using a Finnigan LTQ-Orbitrap Elite hybrid mass spectrometer system (Thermo Scientific, Bremen, Germany). The tryptic digests were chromatographed on a reversed-phase 0.075 × 150-mm C18 Acclaim PepMap 100 column (Dionex Inc., Sunnyvale, CA) using a linear gradient of acetonitrile from 2% to 40% over 5 to 110 minutes in aqueous 0.1% formic acid at a flow rate of 300 nL per minute. The eluent was directly introduced into the mass spectrometer operated in a data-dependent MS to MS/MS switching mode, with the 15 most intense ions in each MS scan subjected to MS/MS analysis. The full MS scan was performed at a resolution of 60,000 in the Orbitrap detector, and the MS/MS scans were performed in the ion trap detector in collision-induced dissociation mode. The fragmentation was performed using the collision-induced dissociation mode with a normalized collision energy of 35 eV. RAW MS files were imported to MaxQuant software version 1.5.2.8 (Max-Planck-Institute of Biochemistry, Planegg, Germany)31Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (8402) Google Scholar with Andromeda as the search engine for protein identification and label-free quantitation (LFQ).32Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3183) Google Scholar The protein samples from the three experimental groups were analyzed as one set. Proteins were identified by comparing all the experimental peptide MS/MS spectra against the UniProt mouse canonical database (UniProt, https://www.uniprot.org, last accessed July 2017). Carbamidomethylation of cysteine was set as a fixed modification, whereas oxidation of methionine to methionine sulfoxide and acetylation of N-terminal amino group were set to be variable modifications. LFQ was enabled, and the LFQ minimum ratio count was set to 1. Remaining options were kept as default. The output file, ProteinGroups.txt, from the MaxQuant database search was imported to Perseus software ve
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