Ling-Ing Lau 2010 Increase of 8-Hydroxy-2-Deoxyguanosine in Aqueous Humor of Patients With Exudative Age-Related Macular Degeneration

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  Increase of 8-Hydroxy-2  -Deoxyguanosine in AqueousHumor of Patients with Exudative Age-Related Macular Degeneration   Ling-Ing Lau, 1,2,3 Catherine Jui-ling Liu, 2,3 and Yau-Huei Wei  1,4,5 P   URPOSE .  Oxidative stress has been implicated as a major con-tributor to age-related macular degeneration (AMD). 8-Hy-droxy-2  -deoxyguanosine (8-OHdG) is one of the most abun-dant oxidative products of DNA damage and represents anoninvasive and sensitive biomarker of oxidative stress. Theauthors investigated the 8-OHdG levels in aqueous humor of patients with exudative AMD. M ETHODS .  Twenty-four eyes of 24 patients with active exuda-tive AMD and 31 eyes of 31 age-matched subjects who under- went cataract surgery were enrolled. Aqueous humor samples were collected from all subjects, and the 8-OHdG levels weredetermined by a commercially available enzyme-linked immu-nosorbent assay kit. The choroidal neovascularization (CNV)subtype was classified by fluorescein angiography. The macu-lar lesion, including CNV membrane, exudation, and retinalhemorrhage, was measured. The correlation between 8-OHdGlevel and the clinical features was analyzed. R  ESULTS .  The 8-OHdG level in the aqueous humor of AMDpatients was significantly higher than it was in controls(0.581  0.258 ng/mL vs. 0.251  0.116 ng/mL;  P   0.001),after adjusting for age and lens status. There was no differencein the 8-OHdG levels between AMD patients with classic/ predominantly classic and occult/minimally classic CNV (0.591   0.262 vs. 0.566  0.266 ng/mL;  P   0.639). The 8-OHdG level inaqueous humor was significantly correlated with the lesion size(     0.492;  P   0.017). C ONCLUSIONS .  The 8-OHdG level in aqueous humor washigher in patients with exudative AMD, and the level wascorrelated with the area of macular lesion. This suggests thatoxidative stress plays an important role in the disease courseof AMD. (   Invest Ophthalmol Vis Sci.  2010;51:5486–5490)DOI:10.1167/iovs.10-5663  A  ge-related macular degeneration (AMD) is one of the lead-ing causes of irreversible blindness among patients aged50 years or older, not only in Western countries 1–3 but also inTaiwan. 4 The central visual loss of AMD is attributable todegenerative or exudative changes at the macula. 5 Exudative AMD is characterized by the presence of choroidal neovascu-larization (CNV), which accounts for approximately 75% of cases with severe central vision loss. 6 Despite the high socio-economic burden it causes, the cause of AMD is not conclu-sively known.Oxidative damage has been implicated as a major contrib-utor to the pathogenesis of AMD. 7 Cigarette smoking andsunlight exposure, 8–10 the two most important environmentalrisk factors for AMD identified by large epidemiologic studies,are related to the level of oxidative stress caused by the gen-eration of reactive oxygen species. 7,11,12  Aging, the most im-portant inherent risk factor for AMD, is also associated with cumulative oxidative damage with decreased protection fromthe antioxidant enzymes. 6,13  AMD patients were found to havemore pronounced decreases in the protein and activity levelsof serum antioxidant enzymes than were age-matched sub- jects. 14,15 Despite all the reports, the in vivo intraocular oxi-dative stress status in AMD patients has remained poorly un-derstood.This study was undertaken to investigate the intraocular oxidative damage level in patients with exudative AMD. Among the various types of oxidative damage products, 8-hy-droxy-2  -deoxyguanosine (8-OHdG) is one of the most abun-dant products of oxidative DNA damage and represents anoninvasive, sensitive, and stable biomarker of oxidativestress. 16  We measured the 8-OHdG concentration in the aque-ous humor of patients with active exudative AMD and corre-lated the 8-OHdG level with the CNV subtypes and the size of macular lesions. To the best of our knowledge, this is the firststudy to explore the 8-OHdG level in aqueous humor of pa-tients with exudative AMD and to correlate it with clinicalfeatures. S  UBJECTS AND  M ETHODS Subjects In this prospective comparative study, we investigated the level of 8-OHdG in the aqueous humor of AMD patients with active CNV.Patients were recruited from the Retinal Clinic of Taipei VeteransGeneral Hospital from July 2007 to July 2009. Patients who were older than 55 years and had active CNV confirmed by fluorescein angiogra-phy (FA) and who did not receive any treatment for CNV within 1 year  were enrolled as the study subjects. Patients with senile cataract without any other ocular diseases who underwent cataract surgery  were enrolled as the control subjects. Exclusion criteria included other ocular diseases apart from AMD and cataract, other ocular surgery apart from cataract surgery, diabetes mellitus complicated by diabeticretinopathy, malignancy, and current smoking habit (positive smokinghistory within 1 year). Patients with malignancy and current smokers were excluded because their serum 8-OHdG levels were higher thanthose of nonsmoking controls without malignancies. 17–20 The Institu-tional Review Board for Human Research of Taipei Veterans GeneralFrom the  1 Institute of Clinical Medicine, School of Medicine andthe  4 Department of Biochemistry and Molecular Biology, School of LifeSciences, National Yang-Ming University, Taipei, Taiwan; the  2 Depart-ment of Ophthalmology, Taipei Veterans General Hospital, Taipei,Taiwan;  3 Department of Ophthalmology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; and the  5 Department of Medi-cine, Mackay Medical College, Taipei, Taiwan.Supported by Taipei Veterans General Hospital Grant V95B1-018and National Science Council Grant NSC 96-2314-B-075-050-MY2.Submitted for publication April 8, 2010; revised May 12, 2010;accepted May 22, 2010.Disclosure:  L.-I. Lau , None;  C.J. Liu , None;  Y.-H. Wei  , NoneCorresponding author: Yau-Huei Wei, Department of Biochemis-try and Molecular Biology, National Yang-Ming University, 155 Li-NongStreet, Section 2, Taipei, Taiwan 112; Biochemistry and Molecular Biology Investigative Ophthalmology & Visual Science, November 2010, Vol. 51, No. 11 5486  Copyright © Association for Research in Vision and Ophthalmology   Hospital approved this study. Informed consent was obtained fromeach of the participants. All procedures adhered to the tenets of theDeclaration of Helsinki. All patients underwent complete ophthalmic examination, includ-ing best-corrected visual acuity, slit-lamp examination, intraocular pres-sure, and indirect ophthalmoscopy. Personal histories including sys-temic illness, smoking history, and vitamin supplementation wererecorded. Patients with dense cataract that precluded detail fundusexamination before cataract operation would be reexamined after cataract operation. Fluorescein angiography was performed in all AMDpatients to categorize the lesions into classic/predominantly classicCNV and occult/minimally classic CNV. Patients with polypoidal cho-roidal vasculopathy, diagnosed by indocyanine green angiography, were excluded from the study. Fundus photography and FA weretaken with a digital fundus camera (CF-60UD; Canon, Tokyo, Japan) at60-degree view. The area of the macular lesion, including the CNV membrane, exudation, and retinal hemorrhage, was measured with theuse of an industrial computer-aided design software package (GstarI-CAD 2008 Professional; The measured area was con- verted to the actual size on the retina in proportion to the magnifica-tion of the camera and the software.  Aqueous Humor Sampling  Patients with exudative AMD received an intravitreal injection of either bevacizumab or triamcinolone acetonide, or both, for their active CNV.Undiluted aqueous humor (0.1–0.15 mL) was collected before intrav-itreal injection to prevent a surge in intraocular pressure after theinjection. All injections and sample collections were performed under a standard sterilization procedure that included disinfection of theocular surface and periocular skin with 5% povidone iodine solution. Inthe control subjects who underwent cataract surgery, aqueous humor  was collected at the beginning of the surgery. All aqueous humor samples were collected using a 30-gauge needle connected to a tuber-culin syringe. The samples were frozen immediately in liquid nitrogenand stored at  80°C until analysis. Quantitative Analysis of 8-OHdG  The concentration of 8-OHdG in aqueous humor was determined by the highly sensitive 8-OHdG enzyme-linked immunosorbent assay (ELISA) kit (Japan Institute for the Control of Aging, Fukuroi, Japan)according to the manufacturer’s instructions. The detection range of 8-OHdG concentration was 0.125 to 10 ng/mL. ELISA is simpler andmore efficient than the conventional high-performance liquid chroma-tography with electrochemical detection (HPLC-ECD) approach with respect to the equipment, analysis time, and sample volume requiredfor quantitative analysis of 8-OHdG. Results of ELISA have been shownto correlate well with the data obtained from HPLC-ECD. 21 Statistical Analysis Statistical analysis was performed (SPSS for Windows, version 15.0;SPSS Inc., Chicago, IL). Data of the two groups were analyzed by theStudent’s  t  -test for continuous variables and    2 test for categorical variables. The concentration of 8-OHdG in the aqueous humor be-tween the two groups was compared by the Student’s  t  -test. Thecorrelation between the macular lesion size and the 8-OHdG level wasexamined by the Spearman rank correlation test. The 8-OHdG level in AMD patients with different CNV subtypes was analyzed by the Mann- Whitney   U   test. Multiple linear regression was performed to evaluatethe factors that affected the 8-OHdG level in aqueous humor.  P   0.05 was considered statistically significant. R  ESULTS There were 24 AMD patients and 33 control patients enrolledconsecutively throughout the study period. Two patients fromthe control group were excluded for further analysis becauseof the presence of diabetic retinopathy found after cataractextraction. Univariate analysis revealed no significant differ-ences in age and sex ratio between the two groups (Table 1);41.7% patients in the AMD group and 45.2% patients in thecontrol group took multivitamin supplements (Table 1) whoseantioxidant concentrations were lower than those suggestedby the Age-related Macular Degeneration Study. 22 The mean8-OHdG concentration in aqueous humor was 0.581    0.258ng/mL in the AMD group, which was significantly higher thanthat in the control group (0.251  0.116 ng/mL;  P   0.0001;Fig. 1). The difference remained highly significant under mul-tiple linear regression analysis after adjusting the factors of ageand lens status (Table 2). Age was also found as a significantfactor influencing the 8-OHdG level in aqueous humor. Lensstatus between the two groups was significantly different be-cause all subjects in the control group were phakic at themoment of aqueous humor collection. However, it was not asignificant factor that affected the 8-OHdG level in aqueoushumor after examination by multiple linear regression (Table2). Further analysis of 8-OHdG levels in the subgroup of phakic AMD patients also revealed significantly higher concentrationsthan found in the controls (0.533    0.285 vs. 0.251    0.116ng/mL;  P   0.006).  T  ABLE  1.  Comparisons between Patients with Exudative AMD and Control Subjects  AMD (  n  24) Control (  n  31)  P  Age, y, mean  SD 81.7  5.9 78.7  6.7 0.090Sex, M:F 19:5 22:9 0.704Diabetes mellitus Yes,  n  (%) 2 (8.3) 5 (16.1)No,  n  (%) 22 (91.7) 26 (83.9) 0.451Hypertension Yes,  n  (%) 13 (54.2) 14 (45.2)No,  n  (%) 11 (45.8) 17 (54.8) 0.591Multivitamin supplements 10 (41.7) 14 (45.2) 0.796LensPhakic,  n  (%) 12 (50) 31 (100)Pseudophakic,  n  (%) 12 (50) 0 (0)   0.0001IOP, mm Hg, mean  SD 13.1  2.8 13.5  2.2 0.552Types of CNV Classic/predominantly classic,  n  (%) 14 (58.3) NA Occult/minimally classic,  n  (%) 10 (41.7) NA  Aqueous 8-OHdG, ng/mL, mean  SD 0.581  0.258 0.251  0.116   0.0001NA, not applicable.  IOVS,  November 2010, Vol. 51, No. 11  Increased 8-OHdG in Aqueous of Exudative AMD 5487  Fourteen AMD patients had classic/predominantly classicCNV and 10 had occult/minimally classic CNV. There was nodifference in the 8-OHdG level between these two groups(0.591  0.262 vs. 0.566  0.266 ng/mL;  P   0.639). Further analysis found positive correlations between the level of 8-OHdG in aqueous humor and macular lesion size (     0.492;  P   0.017; Fig. 2). D ISCUSSION Oxidative stress has been associated with various ocular dis-eases. Increased 8-OHdG level has been observed in the tra-becular meshwork of glaucoma patients, 23 in the pterygium, 24 in leukocytes of patients with Eales’ disease 25 and Leber’shereditary optic neuropathy, 26 and in the urine of patients with Grave’s ophthalmopathy. 27 Our study has demonstratedthat the 8-OHdG level in aqueous humor was significantly higher in AMD patients and was correlated with macular lesionsize.8-OHdG is one of the most abundant forms of oxidativedamage products of DNA. 28 The modified DNA is excised andexported into serum, urine, or other extracellular fluids with-out further metabolism. 29–31 Excretion of 8-OHdG into urinerepresents the average rate of oxidative damage in the wholebody. 32 The 8-OHdG level in the extracellular fluid that isisolated from the systemic circulation, such as cerebrospinalfluid (CSF) and aqueous humor, may reflect oxidative damageof the organ. 33–35 The 8-OHdG level in the CSF of patients with neurodegenerative diseases was significantly higher than in theCSF of control patients, 34,36 and the level was positively cor-related with the duration of illness. 35,37  Aqueous humor is anintraocular fluid isolated from the systemic circulation (blood-aqueous barrier); its 8-OHdG content may reflect intraocular oxidative damage status. Moreover, the 8-OHdG concentrationin the aqueous humor was positively correlated with the areaof macular lesion that represented the area of retinal damage. We are unable to postulate at this stage whether the increased8-OHdG concentration in aqueous humor is a primary event or is secondary to CNV growth. In vitro study found that oxidativestress on RPE can upregulate the secretion of vascular endo-thelial growth factor (VEGF), 38  which is a potent stimulator of neovascularization. Several transcription factors that enhanceexpression of VEGF are also regulated by reactive oxygenspecies. 39,40  Animal studies found more severe neovasculariza-tion growth in the mice deficient in superoxide dismutase 1(  Sod1   /    ), which had increased constitutive and stimulatedoxidative damage in the retina, 41 compared with that in the Sod1   /   mice under the stimulation of VEGF. 42 On the other hand, retinal neovascularization may also cause retinal oxida-tive damage. Animal studies of the  Vldlr    /   mice, which had adefective gene for VLDL receptor and developed aberrant sub-retinal neovascularization, showed increased oxidative stressand neuronal degeneration limited to the region with neovas-cularization even in the absence of clinically significant leakageor hemorrhage, and the degeneration was attenuated signifi-cantly by antioxidants. 43 Further studies to analyze the oxida-tive stress level in patients with extensive early stage AMD without CNV growth may help to delineate this issue.It is intriguing that our study found an age-dependent in-crease of 8-OHdG level in aqueous humor. Age-related in-creases in oxidative DNA damage have been found in varioushuman organs, 44,45 and age-related increases in 8-OHdG levelhave been found in both plasma and CSF. 33 Our study resultsalso showed an age-related increase in oxidative DNA damageproduct in the intraocular fluid, providing further support thatoxidative DNA damage is associated with aging. 46 Previous studies on human eye specimens show decreasedantioxidant enzyme activity with aging 47–49 ; this decrease ismore pronounced in eyes with AMD, implicating the role of oxidative stress in the pathogenesis of AMD. There is an age-related decrease in metallothionein, a scavenger of hydroxylradicals, in submacular RPE. 47 The catalase activity of theexcised RPE from human donor eye also shows an age-relateddecline, and the decline is more pronounced in subjects with  AMD. 48 The heme oxygenase-1 and heme oxygenase-2 immu-noreactivity in the submacular RPE tends to decrease with increased age, especially in the RPE lysosomes of exudative AMD. 49 The present study showed that the concentration of 8-OHdG in the aqueous humor was higher in patients with  F IGURE  2.  8-OHdG level in aqueous humor is positively correlated with macular lesion size of patients with exudative AMD, examined by Spearman rank correlation test (     0.492;  P   0.017). F IGURE  1.  8-OHdG levels in the aqueous humor of patients with exudative AMD and control subjects.  Horizontal lines : mean 8-OHdGconcentrations, which were significantly different between the twogroups (   P   0.0001, Student’s  t  -test).  T  ABLE  2.  Multiple Linear Regression of the Factors Influencing8-OHdG Concentration in Aqueous Humor   Variables Coefficient   SE  P Groups (AMD vs. controls) 0.255  0.063   0.001 Age 0.009  0.004 0.027Lens 0.096  0.075 0.205  R 2  0.498. 5488 Lau et al.  IOVS,  November 2010, Vol. 51, No. 11  exudative AMD, providing further support that oxidative dam-age plays an important role in AMD pathogenesis. Aqueous humor was chosen in this study to explore intraoc-ular oxidative damage status in AMD patients because collect-ing aqueous humor is an easier and less risky procedure thancollecting vitreous humor. Moreover, aqueous humor can becollected readily during intravitreal injection or cataractsurgery. Previous studies have demonstrated that the cyto-kine levels in aqueous humor are correlated with those inthe vitreous humor, reflecting the activities of the retinaldiseases. 50–52 There are limitations to this study. The sample size wassmall, and other oxidative stress markers in aqueous humor  were not evaluated. The amount of aqueous humor drawn wasinadequate for the assay of other oxidative stress markersbecause 8-OHdG analysis was performed in duplicate with undiluted aqueous humor. Nevertheless, we have shown for the first time that 8-OHdG can be detected in aqueous humor and that the level is significantly higher in patients with exu-dative AMD than in healthy controls. Further studies with larger sample size and longitudinal follow-up are warranted toenhance our understanding of the intraocular oxidative stressstatus in AMD patients.  Acknowledgments The authors thank Hui-Chen Lee (Biostatistics Task Force and Divisionof Experimental Surgery, Department of Surgery, Taipei-Veterans Gen-eral Hospital) for help with statistical analysis.  References 1. Kocur I, Resnikoff S. Visual impairment and blindness in Europeand their prevention.  Br J Ophthalmol.  2002;86:716–722.2. Klein R, Klein BE, Linton KL. Prevalence of age-related macu-lopathy: the Beaver Dam Eye Study.  Ophthalmology.  1992;99:933–943.3. Mitchell P, Smith W, Attebo K, Wang JJ. Prevalence of age-relatedmaculopathy in Australia: the Blue Mountains Eye Study.  Ophthal- mology.  1995;102:1450–1460.4. Hsu WM, Cheng CY, Liu JH, Tsai SY, Chou P. Prevalence andcauses of visual impairment in an elderly Chinese population inTaiwan: the Shihpai Eye Study.  Ophthalmology.  2004;111:62–69.5. 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