Hyperlipidemia Affects Tight Junctions and Pump Function in the Corneal Endothelium
2020; Elsevier BV; Volume: 190; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2019.11.008
ISSN1525-2191
AutoresJinghua Bu, Jingwen Yu, Yang Wu, Xiaoxin Cai, Kechun Li, Liying Tang, Nan Jiang, M. Vimalin Jeyalatha, Minjie Zhang, Huimin Sun, Hui He, Andrew J. Quantock, Yongxiong Chen, Zuguo Liu, Wei Li,
Tópico(s)Glaucoma and retinal disorders
ResumoHyperlipidemia impacts on various diseases, such as atherosclerosis, hypertension, and diabetes mellitus. However, its influence, if any, on ocular tissues is largely unknown. Herein, we developed hyperlipidemic murine models by feeding 4-week–old male wild-type mice with a high-fat diet and apolipoprotein E knockout (ApoE−/−) mice with a high-fat diet or standard diet to investigate the corneal endothelial change under hyperlipidemic conditions. Oil Red O staining showed an accumulation of lipid droplets in corneal endothelial cells (CECs) of hyperlipidemic mice. Other manifestations included a reduced cell density and distorted cell morphology, a disruption of the endothelial cell tight junctions and adhesion junctions, a reduced number of surface microvilli, down-regulation of Na+-K+-ATPase expression and function, activation of oxidative stress, changes in mitochondrial ultrastructure, and increased apoptosis. CEC recovery after injury, moreover, was diminished in hyperlipidemic mice; and high palmitate levels were found in the aqueous humor. In vitro hyperlipemia model, moreover, was found to be associated with dose-dependent CEC cytotoxicity, altered cell morphology, reduced pump function, and an induction of oxidative stress, leading to functional and pathologic changes in the corneal endothelium. Hyperlipidemia impacts on various diseases, such as atherosclerosis, hypertension, and diabetes mellitus. However, its influence, if any, on ocular tissues is largely unknown. Herein, we developed hyperlipidemic murine models by feeding 4-week–old male wild-type mice with a high-fat diet and apolipoprotein E knockout (ApoE−/−) mice with a high-fat diet or standard diet to investigate the corneal endothelial change under hyperlipidemic conditions. Oil Red O staining showed an accumulation of lipid droplets in corneal endothelial cells (CECs) of hyperlipidemic mice. Other manifestations included a reduced cell density and distorted cell morphology, a disruption of the endothelial cell tight junctions and adhesion junctions, a reduced number of surface microvilli, down-regulation of Na+-K+-ATPase expression and function, activation of oxidative stress, changes in mitochondrial ultrastructure, and increased apoptosis. CEC recovery after injury, moreover, was diminished in hyperlipidemic mice; and high palmitate levels were found in the aqueous humor. In vitro hyperlipemia model, moreover, was found to be associated with dose-dependent CEC cytotoxicity, altered cell morphology, reduced pump function, and an induction of oxidative stress, leading to functional and pathologic changes in the corneal endothelium. Hyperlipidemia is characterized by increased levels or deposition of circulating lipid,1Crispin S. Ocular lipid deposition and hyperlipoproteinaemia.Prog Retin Eye Res. 2002; 21: 169-224Crossref PubMed Scopus (69) Google Scholar and is considered to be a major risk factor in atherosclerosis, cardiovascular diseases, hypertension, and diabetes mellitus.2Chen H. Miao H. Feng Y.L. Zhao Y.Y. Lin R.C. Metabolomics in dyslipidemia.Adv Clin Chem. 2014; 66: 101-119Crossref PubMed Scopus (65) Google Scholar, 3Bahmani M. Mirhoseini M. Shirzad H. Sedighi M. Shahinfard N. Rafieian-Kopaei M. A review on promising natural agents effective on hyperlipidemia.J Evid Based Complement Altern Med. 2015; 20: 228-238Crossref PubMed Scopus (124) Google Scholar, 4Subramanian S. Chait A. Hypertriglyceridemia secondary to obesity and diabetes.Biochim Biophys Acta. 2012; 1821: 819-825Crossref PubMed Scopus (126) Google Scholar Among these systemic diseases, hyperlipidemia-induced vascular pathologies are common,5Li D. Zhang L. Dong F. Liu Y. Li N. Li H. Lei H. Hao F. Wang Y. Zhu Y. Tang H. Metabonomic changes associated with atherosclerosis progression for LDLR(-/-) mice.J Proteome Res. 2015; 14: 2237-2254Crossref PubMed Scopus (51) Google Scholar,6Davis N. Katz S. Wylie-Rosett J. The effect of diet on endothelial function.Cardiol Rev. 2007; 15: 62-66Crossref PubMed Scopus (56) Google Scholar in which vascular endothelial cells are at risk because hyperlipidemia can induce inflammasome activation7Koka S. Xia M. Chen Y. Bhat O.M. Yuan X. Boini K.M. Li P.L. Endothelial NLRP3 inflammasome activation and arterial neointima formation associated with acid sphingomyelinase during hypercholesterolemia.Redox Biol. 2017; 13: 336-344Crossref PubMed Scopus (64) Google Scholar and endoplasmic reticulum stress.8Cimellaro A. Perticone M. Fiorentino T.V. Sciacqua A. Hribal M.L. Role of endoplasmic reticulum stress in endothelial dysfunction.Nutr Metab Cardiovasc Dis. 2016; 26: 863-871Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar With regard to the eye, lipid deposition has been reported in the cornea (a condition known clinically as corneal arcus) during hyperlipidemia,9Zech Jr., L.A. Hoeg J.M. Correlating corneal arcus with atherosclerosis in familial hypercholesterolemia.Lipids Health Dis. 2008; 7: 7Crossref PubMed Scopus (51) Google Scholar and this has also been demonstrated in hyperlipidemic animal models in which the regression of corneal arcus is seen after the cessation of diet-induced hyperlipidemia.10Crispin S.M. Barnett K.C. Arcus lipoides corneae secondary to hypothyroidism in the Alsatian.J Small Anim Pract. 1978; 19: 127-142Crossref PubMed Scopus (23) Google Scholar Similarly, lipid keratopathy is characterized by a deposition of lipids in the stroma and is often accompanied with vascularization.11Reddy C. Stock E.L. Mendelsohn A.D. Nguyen H.S. Roth S.I. Ghosh S. Pathogenesis of experimental lipid keratopathy: corneal and plasma lipids.Invest Ophthalmol Vis Sci. 1987; 28: 1492-1496PubMed Google Scholar It has also been suggested that lipid keratopathy is more prevalent in patients with higher circulating cholesterol levels,12Cogan D.C. Kuwabara T. Lipid keratopathy and atheroma.Trans Am Ophthalmol Soc. 1958; 56 (discussion 109-119): 109-119PubMed Google Scholar and it is the case that women are more vulnerable to lipid keratopathy than men because of their higher high-density lipoprotein levels.13Crispin S.M. Lipid deposition at the limbus.Eye (Lond). 1989; 3: 240-250Crossref PubMed Scopus (16) Google Scholar To date, however, there is no evidence with regard to the possible effect of hyperlipidemia on the physiology of corneal endothelial cells (CECs). As previously reported, a high-fat diet (HFD) can increase circulating lipid levels and is widely used to induce hyperlipidemia in murine models.14Li S. Zeng X.Y. Zhou X. Wang H. Jo E. Robinson S.R. Xu A. Ye J.M. Dietary cholesterol induces hepatic inflammation and blunts mitochondrial function in the liver of high-fat-fed mice.J Nutr Biochem. 2016; 27: 96-103Crossref PubMed Scopus (23) Google Scholar,15Lee H.S. Nam Y. Chung Y.H. Kim H.R. Park E.S. Chung S.J. Kim J.H. Sohn U.D. Kim H.C. Oh K.W. Jeong J.H. Beneficial effects of phosphatidylcholine on high-fat diet-induced obesity, hyperlipidemia and fatty liver in mice.Life Sci. 2014; 118: 7-14Crossref PubMed Scopus (76) Google Scholar Apolipoprotein E (ApoE) is a cholesterol carrier that aids the transport of lipids and fat-soluble vitamins in the body,17Sato N. Nakamura M. Chikama T. Nishida T. Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats.Jpn J Ophthalmol. 1999; 43: 343-347Crossref PubMed Scopus (37) Google Scholar and in the cornea, its abnormal deposition has been associated with some corneal pathologic conditions.11Reddy C. Stock E.L. Mendelsohn A.D. Nguyen H.S. Roth S.I. Ghosh S. Pathogenesis of experimental lipid keratopathy: corneal and plasma lipids.Invest Ophthalmol Vis Sci. 1987; 28: 1492-1496PubMed Google Scholar, 18Hofker M.H. van Vlijmen B.J. Havekes L.M. Transgenic mouse models to study the role of APOE in hyperlipidemia and atherosclerosis.Atherosclerosis. 1998; 137: 1-11Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Accordingly, elevated serum cholesterol levels have been noted in apolipoprotein E knockout (ApoE−/−) mice, which during HFD feeding exhibit an extreme hypercholesterolemic phenotype compared with mice fed with a standard diet (SD).19van Ree J.H. van den Broek W.J. Dahlmans V.E. Groot P.H. Vidgeon-Hart M. Frants R.R. Wieringa B. Havekes L.M. Hofker M.H. Diet-induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E-deficient mice.Atherosclerosis. 1994; 111: 25-37Abstract Full Text PDF PubMed Scopus (133) Google Scholar ApoE−/− mice, therefore, represent a valuable resource to support research into hyperlipidemia.20Li C. Dong F. Jia Y. Du H. Dong N. Xu Y. Wang S. Wu H. Liu Z. Li W. Notch signal regulates corneal endothelial-to-mesenchymal transition.Am J Pathol. 2013; 183: 786-795Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar To investigate the potential effect of hyperlipidemia on the corneal endothelium, CECs were studied in vivo and in an ex vivo cell culture model with a focus on lipid accumulation, cell morphology, tight junction formation, pump function, oxidative damage, and mitochondrial health. Pathologic changes of CECs were discovered under various degrees of hyperlipidemia via feeding ApoE−/− and wild-type (WT) mice with either an SD or HFD, indicating that hyperlipidemia compromises CEC function via an oxidative stress pathway. The following antibodies were used in this study: anti–zonula occludens protein 1 (ZO-1; QA21310; Life Technologies, Carlsbad, CA), anti–N-cadherin (13116S; Cell Signaling Technology, Danvers, MA), anti–Na+-K+-ATPase (ab76020; Abcam, Cambridge, UK), anti–4-hydroxynonenal (4-HNE; ab46545; Abcam), anti-NADPH oxidase 4 (NOX4; ab13303; Abcam), anti–8-dihydroxy-2'-deoxyguanosine (8-OHdG; sc-393871; Santa Cruz Biotechnology, Dallas, TX), anti-Tom20 (11802-1-AP; Proteintech, Rosemont, IL), and anti-Tim23 (11123-1-AP; Proteintech). The secondary antibodies were Alexa Fluor 594–conjugated IgG (A11508; Invitrogen, Carlsbad, CA) and Alexa Fluor 488–conjugated IgG (A11055; Invitrogen). ApoE−/− mice on a C57BL/6 background and wild-type C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co, Ltd (Beijing, China). New Zealand white rabbits were purchased from Shanghai SLAC Laboratory Animal Center (Shanghai, China). All animals were kept in standard pathogen-free environment with alternate 12-hour light-dark cycles (8:00 am to 8:00 pm). In vitro cell culture experiments used corneal endothelia from rabbits rather than mice to avoid the need to sacrifice large numbers of animals to ascertain optimal cultivation protocols, which tend to be species specific. All animal studies were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Ethical Committee of Xiamen University. At 4 weeks of age, WT and ApoE−/− mice were fed with either an SD (1022; Beijing HFK Bioscience, Beijing, China; 10 kcal% fat) or an HFD (D12492; Research Diets, New Brunswick, NJ; 60 kcal% fat). After 16 weeks of receiving the respective diet, mice were sacrificed by cervical dislocation. Before SD or HFD feeding commenced and after 16 weeks of diet feeding, blood was collected from 16 hours fasted mice (n = 7) by cardiac puncture at the time of sacrifice, after which the fasting glucose concentration was measured using a glucose analyzer (ACCU-CHEK; Roche, Basel, Switzerland). Blood was similarly collected from 6 to 8 hours fasted mice (n = 7), after which serum total cholesterol was measured enzymatically using a commercially available kit (ab65390; Abcam). Oil Red O staining was performed to evaluate lipid accumulation in CECs. Corneal whole-mount tissues were fixed in 4% paraformaldehyde for 10 minutes, washed in phosphate-buffered saline (PBS) for 5 minutes, and then stained for 10 minutes in freshly prepared Oil Red O solution. After rinsing with PBS for 5 minutes, the tissues were mounted in 90% glycerol and observed under light microscope (Eclipse 50i; Nikon, Tokyo, Japan). Three samples from each group were used to conduct Oil Red O corneal endothelial whole-mount staining. Five areas with the same surface area in each sample were chosen to quantify and analyze staining intensity, avoiding the folded area. The mean staining intensity was measured by image analysis software (NIS Elements version 4.1; Nikon, Melville, NY). After general anesthesia, mice corneas (n = 7) were examined with a laser-scanning in vivo confocal microscope with a corneal imaging module (HRT3-RCM; Heidelberg Engineering, Heidelberg, Germany). Briefly, a drop of transparent carbomer gel (Alcon Laboratories, Fort Worth, TX) was placed on the microscope objective lens with a sterile disposable plastic cap (Tomocap; Heidelberg Engineering) affixed over the gel-coated lens. The center of the cap was applanated onto the central cornea by adjusting the controller, and the central corneal endothelium was examined. More than 30 in vivo digital images were captured once the CEC was located. The cell counting function of the HRT3-RCM machine software (Heidelberg Eye Explorer version 1.9.10.0; Heidelberg Engineering, Heidelberg, Germany) was used to perform corneal endothelial cell counting (Supplemental Video S1). To determine hexagonality, the number of cell sides directly apposed to adjacent cells within the endothelial mosaic of each sample were counted; hexagonal cells were marked and counted to calculate the percentage of hexagonal cells. For these measurements, three to four selected images in each eye were analyzed. All operations were performed by a single investigator (M.Z.) masked to the experimental conditions. CEC damage was performed as previously described with modifications.21Chen L. Xie B. Li L. Jiang W. Zhang Y. Fu J. Guan G. Qiu Y. Rapid and sensitive LC–MS/MS analysis of fatty acids in clinical samples.Chromatographia. 2014; 77: 1241-1247Crossref Scopus (14) Google Scholar In brief, mice were anesthetized with i.p. injection of 0.2% chloral hydrate, after which the ocular surface was further anesthetized with a drop of 0.4% oxybuprocaine hydrochloride (Santen Pharmaceutical, Osaka, Japan) before surgery. A stainless steel probe cooled with liquid nitrogen was applied to central cornea of right eye of the mice (n = 7) for 30 seconds. Ofloxacin eye ointment (Santen Pharmaceutical) was instilled in each eye twice a day after the procedure. During the follow-up, corneas were observed under a slit-lamp microscope (Kanghua Science & Technology Co, Ltd, Chongqing, China), and corneal thickness at different time points was measured by in vivo confocal microscopy. Animals were sacrificed 7 days after the freeze injury to assess the status of the corneal endothelium. Corneas from experimental groups (n = 3 for each group) were fixed in a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in PBS (pH 7.4) at 4°C for 2 hours. They were cut into 4 × 2-mm sized pieces, without touching the endothelium, and further embedded, sliced, and stained. The ultrastructure of corneal endothelium was examined and imaged using scanning electron microscope (JSM6390LV; JEOL, Tokyo, Japan) or transmission electron microscopy (JEM2100HC; JEOL). Mitochondria with cristae abnormalities and vacuoles were considered to be abnormal. Mitochondria in six to eight transmission electron microscopy images taken from each cornea at ×10,000 magnification were counted, and the ratio of the number of abnormal mitochondria/the total number of mitochondria was calculated. After general anesthesia, a drop of 0.4% oxybuprocaine hydrochloride was used to anesthetize the ocular surface before aqueous humor procurement. Aqueous humor was obtained from the anterior chamber of mice via a paracentesis using a sharp capillary straw (Supplemental Video S2). The samples were immediately frozen at −80°C after extraction, and palmitate levels were measured by the use of a high-performance liquid chromatography–tandem mass spectrometry system, as described previously.22Salvioli S. Ardizzoni A. Franceschi C. Cossarizza A. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis.FEBS Lett. 1997; 411: 77-82Crossref PubMed Scopus (895) Google Scholar A total of 4 to 5 μL of aqueous humor was collected per mouse (n = 18), and three samples were used in each group (one sample consisted of pooled aqueous humor from six mice). Because the amount of aqueous humor from one mouse is small, samples from six mice were pooled within each group to reach the minimum volume required for HPLC analysis. The total amount of pooled aqueous humor was thus approximately 25 μL in each group. Rabbit, rather than mouse, CECs (rCECs) were used for the ex vivo cell culture experiments as culture protocols are well established in our laboratory and in the published literature. This is not the case for mouse CECs. Rabbit corneas (n = 18) were harvested after sacrificing the animals, and the endothelial cells with Descemet membrane attached were mechanically separated from the corneal stroma under a dissecting microscope. These specimens were digested in 1 mg/mL collagenase I in supplemented hormonal epithelial medium for 16 hours to digest the Descemet membrane, as previously reported.21Chen L. Xie B. Li L. Jiang W. Zhang Y. Fu J. Guan G. Qiu Y. Rapid and sensitive LC–MS/MS analysis of fatty acids in clinical samples.Chromatographia. 2014; 77: 1241-1247Crossref Scopus (14) Google Scholar Supplemented hormonal epithelial medium was prepared with equal volume of HEPES-buffered Dulbecco's modified Eagle's medium and Ham's F12 supplemented with 5% fetal bovine serum, 0.5% dimethyl sulfoxide, 2 ng/mL mouse epidermal growth factor (Life Technologies), 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium, 0.5 μg/mL hydrocortisone, 50 μg/mL gentamicin, and 1.25 μg/mL amphotericin B. After digestion, rCECs were dispersed into a cell suspension and cultured in supplemented hormonal epithelial medium at 37°C with 5% CO2. The medium was changed every 3 days. Primary rCECs at passage 0 were used after reaching confluence. Palmitate (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.1 mol/L NaOH (shaken in water bath at 75°C for 30 minutes) and diluted 1:1 in preheated 1 × PBS containing 40% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) to give a concentration of 20 mmol/L (stock palmitate solution). The BSA should be free of fatty acids and endotoxins. Therefore, the stock palmitate solution was passed through a 0.2-μm filter and diluted in supplemented hormonal epithelial medium to obtain concentration of 0 to 1000 μmol/L for use. Cell viability to evaluate cytotoxicity of palmitate on rCECs was assessed by the use of a Cell Counting Assay Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan), according to the manufacturer's instructions. CECs were seeded at density of 1 × 104 cells per well in 96-well plates overnight and were treated with palmitate at varying concentrations (0 to 1000 μmol/L). BSA was used as vehicle control. After 24 hours in culture, the medium was replaced by Cell Counting Assay Kit-8 constituted media and incubated for 4 hours at 37°C in the dark. Absorbance at 450 nm was measured using a microplate reader (Bio-Tek, Winooski, VT). Cryosections (5 μm thick) of mouse eyes, corneal whole mounts, or cultured cells were fixed in acetone at −20°C for 10 minutes. The samples were washed three times with PBS for 10 minutes per wash and then incubated in 0.2% Triton X-100 for 10 minutes. After rinsing the sections three times in PBS for 5 minutes, they were blocked with 2% BSA for 1 hour at room temperature and incubated with anti–ZO-1 (1:100), anti–N-cadherin (1:100), anti–Na+-K+-ATPase (1:100), anti–4-HNE (1:200), anti–8-OHdG (1:50), anti-Tom20 (1:100), or anti-Tim23 (1:100) primary antibodies overnight at 4°C. The next day, samples were washed three times in PBS for 10 minutes and incubated with secondary antibodies Alexa Fluor 594–conjugated IgG (1:300) or Alexa Fluor 488–conjugated IgG (1:300) for 1 hour at room temperature in the dark. After three washes in PBS and counterstaining with DAPI (H-1200; Vector, Burlingame, CA), laser confocal scanning microscope (Fluoview 1000; Olympus, Tokyo, Japan) was used to study the immunofluorescence. JC-1 (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazole-carbocyanide iodide) is a cationic fluorescent dye that, when added to live cells, is only located in mitochondria, and is particularly attracted to those in good physiological condition characterized by a sufficient mitochondrial membrane potential.16Ong J.M. Zorapapel N.C. Rich K.A. Wagstaff R.E. Lambert R.W. Rosenberg S.E. Moghaddas F. Pirouzmanesh A. Aoki A.M. Kenney M.C. Effects of cholesterol and apolipoprotein E on retinal abnormalities in ApoE-deficient mice.Invest Ophthalmol Vis Sci. 2001; 42: 1891-1900PubMed Google Scholar Mitochondrial function was assessed by tracking the mitochondrial transmembrane potential through detection of the fluorescent probe, JC-1. Following the manufacturer's instructions (Beyotime, Shanghai, China), CECs cultured in confocal dishes were treated with JC-1 staining solution for 20 minutes at 37°C in the dark. Cells were subsequently washed with JC-1 staining buffer twice and observed under a laser scanning confocal microscope (Fluoview 1000). The activity of Na+-K+-ATPase, which is central to the workings of the corneal endothelial pump, was determined by measuring the hydrolysis of ATP. Released inorganic phosphate from ATP was detected using a commercial assay kit (Solarbio, Shanghai, China) in accordance with the manufacturer's instructions. Briefly, CECs cultured in a 6-well plate were collected and lysed in lysis buffer for 2 minutes. Cellular lysates were centrifuged at 8000 × g for 10 minutes to obtain a supernatant, after which 100 μL of supernatant from each sample was suspended in a reaction mixture containing 40 mmol/L NaCl, 8 mmol/L KCl, 2 mmol/L MgCl2, 5 mmol/L EGTA, and 20 mmol/L Tris-HCl, pH 7.4. Addition of 2 mmol/L ATP started the reactions, which was terminated after 10 minutes of incubation at 37°C. The reaction mixtures were then centrifuged at 4000 × g for 10 minutes to get a supernatant, after which 200 μL of coloring reagent was mixed with 20 μL of the supernatant. After incubation at 40°C for 10 minutes, the absorbance was read at 660 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and results were expressed as percentage change compared with control values. Cell apoptosis detection was performed using the Fluorometric TUNEL System (G3250; Promega, Madison, WI). The whole corneas were rehydrated and incubated with Proteinase K Tris/HCL, pH 7.4 (10 mmol/L) for 30 minutes at 37°C. The whole corneas were washed three times with PBS for 5 minutes each. Then, 50 μL of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling reaction mixture was added and the corneas were placed in the dark for 1 hour at 37°C. The whole corneas were rinsed three times with PBS for 5 minutes each, counterstained with DAPI, mounted, and imaged with a microscope (DM2500; Leica Microsystems, Wetzlar, Germany). Formalin-fixed eyeballs were processed in paraffin and divided into sections (5 μm thick). Sections were rehydrated and endogenous peroxidase activity was blocked (0.3% hydrogen peroxide in PBS), after which sections were permeabilized with 1% Triton X-100 for 10 minutes. After rinsing three times with PBS for 5 minutes, the sections were blocked using 2% BSA for 1 hour at room temperature and incubated with anti-NOX4 (1:200) at 4°C overnight. After three rinses with PBS for 10 minutes, the sections were further incubated with biotinylated anti-rabbit IgG (1:50) for 1 hour, followed by Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) for 30 minutes. The reaction product was then developed with diaminobenzidine for 1 minute, mounted with mounting medium (H-5000; Vector), and examined under a light microscope (Eclipse 50i; Nikon, Tokyo, Japan). Murine corneal endothelia were stripped using toothed forceps, and total RNA was isolated using PicoPure RNA isolation kit (Arcturus, Mountain View, CA). Total RNA from the cultured rCECs was extracted using Trizol Reagent (Invitrogen), according to manufacturer's protocol. Seven samples per group for the in vivo studies and five samples per group for the in vitro analyses were used. A sample consisted of the following: pooled corneal endothelia from both eyes of the same animal or one well of cultured CECs seeded in a 24-well plate. RNA was transcribed to cDNA using a reverse transcription kit (PrimeScript RT reagent kit; TaKaRa, Shiga, Japan). Quantitative real-time PCR was performed with a StepOne Real-Time detection system (Applied Biosystems, Foster City, CA) using an SYBR Premix Ex Taq Kit (TaKaRa), according to the manufacturer's instructions. The primer sequences used to amplify specific gene products are listed in Table 1 and Table 2. The amplification program included an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Subsequently, a melt curve analysis was performed to access amplification specificity. Differential gene expression was calculated according to the comparative threshold cycle (CT) method and normalized to β-actin expression as an internal control.Table 1Mouse Sequences Used for Quantitative Real-Time PCRGeneSense primerAntisense primerActb5′-GTGGGAATGGGTCAGAAGGA-3′5′-CTTCTCCATGTCGTCCCAGT-3Tjp15′-ACGATCTCCTGACCAACGTT-3′5′-GCTTTGGGTGGATGATCGTC-3′Ncad5′-AGAACAGGGTGGACGTCATT-3′5′-CTGTTGGGGTCTGTCAGGAT-3′Atp1b15′-GCGACATCAATCACGAACGA-3′5′-CCCCTCTCTGTAGCCGTAAG-3′ZO-1, zonula occludens protein 1. Open table in a new tab Table 2Rabbit Sequences Used for Quantitative Real-Time PCRGeneSense primerAntisense primerActb5′-CGGCTACAAAGACGGCAAAC-3′5′-GAACAGGCAGCACATTTGGG-3′Tjp15′-AGTTTGGCAGCAAGAGATGG-3′5′-GCTGTCAGAAAGGTCAGGGA-3′Ncad5′-CAGAAAACTCCCGAGGACCT-3′5′-ACGATCCAGAGGCTTTGTCA-3′Atp1b15′-CGGCTACAAAGACGGCAAAC-3′5′-GAACAGGCAGCACATTTGGG-3′Nox45′-TGCTGTATAACCAAGGGCCA-3′5′-GGATGAGGCTGCAATTGAGG-3′Nrf25′-AAGTGGCTGCTCAGAATTGC-3′5′-GCTCATCCCGTAACATGCTG-3′Cat5′-CTGAACATCATCACGGCAGG-3′5′-CACCTTCGCCTTGCTGTATC-3′Gpx15′-GCCCAACTTCATGCTCTTCC-3′5′-ATGAACTTGGGGTCGGTCAT-3′Sod15′-ACCTGGGTAATGTGACTGCA-3′5′-AATGACACCACAGGCCAAAC-3′ Open table in a new tab ZO-1, zonula occludens protein 1. Statistical analysis was performed with SPSS 16.0.0 (SPSS Inc., Chicago, IL). All summary data are reported as means ± SD. Statistical analysis was performed with unpaired t-test for two-group comparisons or one-way analysis of variance for more than two group comparisons using GraphPad Prism 6.0 software (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant. Body weight and blood lipid levels of 4-week–old ApoE−/− mice were significantly higher compared with those of age-matched WT mice, whereas the plasma glucose concentrations were similar (Supplemental Figure S1). Body weight increased in WT mice after HFD feeding (WT+HFD; WH) for 16 weeks, compared with the body weight of WT mice with an SD (WT+SD; WS). ApoE−/− mice fed with SD (ApoE−/−+SD; AS) also showed increased body weight compared with WS mice and had similar body weight as WH mice. ApoE−/− mice fed with an HFD (ApoE−/−+HFD; AH) showed a significant body weight increase compared with AS mice (Figure 1A). Previously, it was reported that an ApoE mutation and an HFD both influenced total cholesterol level in serum,17Sato N. Nakamura M. Chikama T. Nishida T. Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats.Jpn J Ophthalmol. 1999; 43: 343-347Crossref PubMed Scopus (37) Google Scholar and it was found that HFD feeding induced a significant increase of serum total cholesterol in both WT and ApoE−/− mice. Compared with WS mice, ApoE−/− mice exhibited a significant increase in total cholesterol when fed with an SD and a much higher level when fed with HFD (Figure 1B). There was no significant differences of fasting blood sugar concentrations among the groups after 16 weeks feeding with an SD or HFD (Figure 1C). Oil Red O staining of CECs in whole-mount corneal tissues clearly delineated the cell borders of CECs and revealed that there was no lipid deposition in WS mice. However, an accumulation of small lipid droplets was observed in the cytoplasm of CECs in WH mice, with the accumulation being more prominent around the CEC border in AS mice (Figure 1D). Oil Red O staining intensity analysis showed that there was a higher lipid accumulation in the corneal endothelia of WH and AS mice compared with the endothelia of WS mice. The highest lipid accumulation was observed in AH mice (Figure 1E), indicating that hyperlipidemia induces lipid deposition in CECs. In vivo confocal microscope showed that CECs in WS mice were well organized and exhibited hexagonal shapes in a regular array. In WH and AS mice, numerous CECs lost their typical hexagonal morphology and the cell boundaries became distorted. In addition, after HFD in ApoE−/− mice, severe cell boundary damage and a heterogeneous cell morphology were noted (Figure 1F). Central CEC density in WS mice averaged 2443 ± 158 cells/mm2. In WH mice, average endothelial cell density decreased by 17% to 2021 ± 143 cells/mm2 compared with WS mice. A small (6%) decline in CEC density was observed in AS mice (1895 ± 101 cells/mm2) compared with WH mice, although this difference was insignificant. Overall, a 39% decrease in mean cell density was observed in AH mice (1500 ± 103
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