Simvastatin Inhibits Leukocyte Accumulation and Vascular Permeability in the Retinas of Rats with Streptozotocin-Induced Diabetes
2004; Elsevier BV; Volume: 164; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63728-5
ISSN1525-2191
AutoresShinsuke Miyahara, Junichi Kiryu, Kenji Yamashiro, Kazuaki Miyamoto, Fumitaka Hirose, Hiroshi Tamura, Hideto Katsuta, Kazuaki Nishijima, Akitaka Tsujikawa, Yoshihito Honda,
Tópico(s)Glaucoma and retinal disorders
ResumoLeukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common β-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy. Leukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common β-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy. Retinopathy is a major complication of diabetes and is one of the leading causes of adult blindness in many countries. In diabetic retinopathy, the expression of intercellular adhesion molecule-1 (ICAM-1) on the vascular endothelial cells is up-regulated,1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (650) Google Scholar, 2Joussen AM Poulaki V Mitsiades N Kirchhof B Koizumi K Dohmen S Adamis AP Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression.EMBO J. 2002; 16: 438-440Google Scholar, 3McLeod DS Lefer DJ Merges C Lutty GA Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid.Am J Pathol. 1995; 147: 642-653PubMed Google Scholar which leads to leukocyte adhesion to vascular endothelium and to accumulation of leukocytes within the retina.1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (650) Google Scholar, 4Miyamoto K Ogura Y Kenmochi S Honda Y Role of leukocytes in diabetic microcirculatory disturbances.Microvasc Res. 1997; 54: 43-48Crossref PubMed Scopus (41) Google Scholar, 5Miyamoto K Hiroshiba N Tsujikawa A Ogura Y In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats.Invest Ophthalmol Vis Sci. 1998; 39: 2190-2194PubMed Google Scholar Leukocytes that are adherent to vascular endothelium have been shown to cause capillary occlusion,1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (650) Google Scholar, 4Miyamoto K Ogura Y Kenmochi S Honda Y Role of leukocytes in diabetic microcirculatory disturbances.Microvasc Res. 1997; 54: 43-48Crossref PubMed Scopus (41) Google Scholar, 5Miyamoto K Hiroshiba N Tsujikawa A Ogura Y In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats.Invest Ophthalmol Vis Sci. 1998; 39: 2190-2194PubMed Google Scholar endothelial cell apoptosis,6Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 7Joussen AM Poulaki V Mitsiades N Cai WY Suzuma I Pak J Ju ST Rook SL Esser P Mitsiades C Kirchhof B Adamis AP Aiello LP Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes.EMBO J. 2003; 44: 117-123Google Scholar and, finally, blood-retinal barrier breakdown (BRB).1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (650) Google Scholar, 7Joussen AM Poulaki V Mitsiades N Cai WY Suzuma I Pak J Ju ST Rook SL Esser P Mitsiades C Kirchhof B Adamis AP Aiello LP Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes.EMBO J. 2003; 44: 117-123Google Scholar, 8Joussen AM Poulaki V Qin W Kirchhof B Mitsiades N Wiegand SJ Rudge J Yancopoulos GD Adamis AP Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo.Am J Pathol. 2002; 160: 501-509Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 9Joussen AM Poulaki V Tsujikawa A Qin W Qaum T Xu Q Moromizato Y Bursell SE Wiegand SJ Rudge J Ioffe E Yancopoulos GD Adamis AP Suppression of diabetic retinopathy with angiopoietin-1.Am J Pathol. 2002; 17: 76-78Google Scholar These damages result in clinical symptoms of diabetic retinopathy, such as areas of nonperfusion, retinal hemorrhage because of vascular vulnerability, and retinal edema each of which can cause serious loss of visual acuity. Simvastatin is one of the family of statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, that have been reported to exert many effects in addition to their cholesterol-lowering ability. 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In the study described herein, we evaluated quantitatively the inhibitory effects of simvastatin on leukocyte-endothelial cell interactions and BRB breakdown in experimental diabetic retinas. We also investigated the effects of simvastatin on the expression of several important mediators, such as ICAM-1, eNOS, and VEGF, in the diabetic retina. Consequently, the current study estimates the therapeutic efficacy of simvastatin on early diabetic retinopathy. Male pigmented Long-Evans rats (180 to 200 g, n = 72) were purchased from KIWA Laboratory Animals Co. Ltd. (Wakayama, Japan). Each rat received an intraperitoneal injection of streptozotocin (60 mg/kg; Sigma Chemical, St. Louis, MO) in 10 mmol/L citrate buffer (pH 4.5) after an overnight fast. Animals with blood glucose levels greater than 250 mg/dl 24 hours later were considered diabetic. We confirmed that the plasma glucose level in each rat was >250 mg/dl just before the experiment. All rats were kept in an air-conditioned room with a 12-hour light and 12-hour dark cycle and given free access to water and food until they were used for the experiments. Simvastatin was obtained from Merck/Banyu Pharmaceutical (Merck, Rahway, NJ; Banyu, Tokyo, Japan). Simvastatin (0.25, 2.5, and 25 mg/kg/day) was administered orally for 2 weeks from the day of diabetes induction. Animals injected with an equal volume of saline solution alone served as diabetic vehicle-treated controls. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Leukocyte accumulation in retinal microcirculation was evaluated with acridine orange (AO) digital fluorography, which has been described previously in detail elsewhere.1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (650) Google Scholar, 5Miyamoto K Hiroshiba N Tsujikawa A Ogura Y In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats.Invest Ophthalmol Vis Sci. 1998; 39: 2190-2194PubMed Google Scholar, 37Nishiwaki H Ogura Y Kimura H Kiryu J Honda Y Quantitative evaluation of leukocyte dynamics in retinal microcirculation.Invest Ophthalmol Vis Sci. 1995; 36: 123-130PubMed Google Scholar, 38Nonaka A Kiryu J Tsujikawa A Yamashiro K Miyamoto K Nishiwaki H Honda Y Ogura Y PKC-beta inhibitor (LY333531) attenuates leukocyte entrapment in retinal microcirculation of diabetic rats.Invest Ophthalmol Vis Sci. 2000; 41: 2702-2706PubMed Google Scholar Six different rats were used in each group. This technique uses a scanning laser ophthalmoscope (Rodenstock Instruments, Munich, Germany), coupled with a computer-assisted image analysis system, which makes continuous high-resolution images of fundus stained by AO (Wako Pure Chemicals, Osaka, Japan). AO, a metachromatic fluorochrome, is a widely used probe in biochemical and cytochemical studies. The dye emits a green fluorescence when it interacts with DNA. The argon blue laser was used for the illumination source, with a regular emission filter for fluorescein angiography, because the spectral properties of leukocytes stained with AO are similar to those of sodium fluorescein. Immediately before AO digital fluorography, rats were anesthetized with xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg) and their pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. A contact lens was placed on the cornea to maintain transparency throughout the experiments. Arterial blood pressure was monitored with a blood pressure analyzer (IITC, Woodland Hills, CA). Each rat had a catheter inserted into the tail vein and was placed on a movable platform. Body temperature was maintained at between 37°C and 39°C throughout the experiment. AO (0.1% solution in saline) was injected continuously through the catheter for 1 minute at a rate of 1 ml/min. Thirty minutes after the injection, the fundus was observed with the scanning laser ophthalmoscope for evaluation of leukocyte accumulation in the retinal microcirculation. The obtained images were recorded on a S-VHS videotape at the video rate of 30 frames/second for further analysis. Blood was collected to count the number of leukocytes in the peripheral blood by a hematology analyzer (ERMA, Tokyo, Japan) and to measure the serum cholesterol level. After the experiment, each rat was killed with an overdose of anesthesia. The video recordings were analyzed with a computer-assisted image analysis system consisting of a computer equipped with a video digitizer (Radius, San Jose, CA) that digitizes the video image in real time to 640 horizontal and 480 vertical pixels with an intensity resolution of 256 steps. The number of fluorescent dots in the retina within a circle of 1000 μm in diameter from the center of the optic disk was counted as the number of leukocytes accumulated in the retina for each rat. Leukocyte adhesion to rat retinal vessels was evaluated using the method described elsewhere, with slight modification.19Ishida S Usui T Yamashiro K Kaji Y Ahmed E Carrasquillo KG Amano S Hida T Oguchi Y Adamis AP VEGF164 is proinflammatory in the diabetic retina.Invest Ophthalmol Vis Sci. 2003; 44: 2155-2162Crossref PubMed Scopus (336) Google Scholar, 39Yamashiro K Tsujikawa A Ishida S Usui T Kaji Y Honda Y Ogura Y Adamis AP Platelets accumulate in the diabetic retinal vasculature following endothelial death and suppress blood-retinal barrier breakdown.Am J Pathol. 2003; 163: 253-259Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Six different rats were used in each group. After the induction of deep anesthesia, the chest cavity was carefully opened and a 20-gauge perfusion cannula was introduced into the aorta. Drainage was achieved by opening the right atrium. The animals were then perfused with 100 ml of phosphate-buffered saline (PBS) to wash out blood cells in the vessels, such as erythrocytes and nonadherent leukocytes. After PBS perfusion, the animals were perfused with 25 ml of fluorescein isothiocyanate (FITC)-labeled concanavalin A lectin (ConA; 40 μg/ml in PBS, pH 7.4) (Vector Laboratories, Burlingame, CA). ConA was used to label leukocytes adherent to the vessel walls and vascular endothelial cells. Residual unbound ConA was removed with PBS perfusion. The retina was carefully removed and fixed with 1% paraformaldehyde, and flat mounts were prepared using a fluorescence anti-fading medium (Vector Laboratories). The retinas were then observed using fluorescence microscopy (FITC filter; Olympus Optical, Tokyo, Japan), and the total number of adherent leukocytes per retina was determined. Two weeks after the onset of diabetes, one eye from each of six rats in the simvastatin (25 mg/kg/day)-treated, vehicle-treated, and nondiabetic control groups was enucleated. Total RNA was isolated from the retina using TRIzol reagent (Life Technologies, Grand Island, NY). The extracted RNA was quantified, and then 2 μg of the RNA was used to make cDNA with a kit (Omniscript Reverse Transcriptase; Qiagen, Valencia, CA). For semiquantitative PCR, 2 μl of each first-strand reaction was then amplified using eNOS-, ICAM-1-, and β-actin-specific oligonucleotide primers.40Miyahara S Kiryu J Tsujikawa A Katsuta H Nishijima K Miyamoto K Yamashiro K Nonaka A Honda Y Argatroban attenuates leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia.Stroke. 2003; 34: 2043-2049Crossref PubMed Scopus (22) Google Scholar PCR amplification was performed with the following conditions: denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and polymerization at 72°C for 1 minute. The reaction was performed for 32 cycles for eNOS, 34 cycles for ICAM-1, and 29 cycles for β-actin. The primers were GCATGGGCAACTTGAAGAGT (sense) and CTGGGAACCATCCTTTTGA (anti-sense) for eNOS, AGCCTCAGGCCTAAGAGGAC (sense) and AGGGGTCCCAGAGAG-GTCTA (anti-sense) for ICAM-1, and GGCATCCTGACCC-TGAAGTA (sense) and GCCATCTCTTGCTCGAAGTC (anti-sense) for β-actin. After completion, 10 μl of the reactions were analyzed by agarose gel electrophoresis and ethidium bromide staining to determine the levels of transcript relative to the control transcript β-actin RNA. The animals were killed with an overdose of anesthesia, and the eyes were immediately enucleated. The retina was carefully isolated, placed in 150 μl of lysis buffer (20% glycerol, 10 mmol/L KCl, 1 mmol/L MgCl2, 0.1% Triton, 300 mmol/L NaCl, 0.5 mmol/L dithiothreitol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 20 mmol/L HEPES, pH 7.9) and sonicated. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and both the VEGF levels and the ICAM-1 levels in the supernatant were determined with the VEGF kit and the ICAM-1 kit (Quantikine; R&D Systems, Minneapolis, MN), respectively, according to the manufacturer's protocol. Total protein was determined using the bicinchoninic acid kit (Bio-Rad, Hercules, CA) and was used to normalize the VEGF protein levels. The surface expression of CD18 on rat leukocytes was determined using flow cytometry using the method described elsewhere with slight modification.41Barouch FC Miyamoto K Allport JR Fujita K Bursell SE Aiello LP Luscinskas FW Adamis AP Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes.Invest Ophthalmol Vis Sci. 2000; 41: 1153-1158PubMed Google Scholar, 42Lefer AM Campbell B Shin YK Scalia R Hayward R Lefer D Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts.Circulation. 1999; 100: 178-184Crossref PubMed Scopus (322) Google Scholar CD18 was evaluated by direct immunofluorescence staining of whole blood using an established lyse/wash procedure (BD PharMingen, Franklin Lakes, NJ). After the deep anesthesia, the whole blood anti-coagulated with ethylenediaminetetraacetic acid was obtained from each of six rats in the simvastatin (25 mg/kg/day)-treated, vehicle-treated, and nondiabetic control groups. FITC-conjugated mouse (BALB/c) anti-rat CD18 IgG and FITC-conjugated mouse (BALB/c) IgG (as an isotype-matched control) were purchased from BD PharMingen. Immediately after blood collection, 10 μg of FITC-labeled antibody in 100 μl of staining buffer (PBS with 0.1% sodium azide and 1% fetal bovine serum) were incubated with 100 μl of rat whole blood for 30 minutes at room temperature. Erythrocytes were lysed for 15 minutes by addition of 2 ml of 1× fluorescence-activated cell sorting lysing solution (BD PharMingen). The tubes were centrifuged at 200 × g for 5 minutes. The cell pellets were resuspended in 2 ml of staining buffer. The tubes were centrifuged at 200 × g for 5 minutes and the leukocytes were resuspended in a fixation solution (2% formaldehyde prepared in PBS). Leukocytes were gated by the typical forward and side light scatter profiles. The fluorescence of 104 cells was measured on a FACScan (Becton Dickinson, Franklin Lakes, NJ). Leukocytes were gated on the basis of their characteristic forward and s
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