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Elevated Vitreous Nitric Oxide Levels in Patients With Proliferative Diabetic Retinopathy

¨ RSEL YILMAZ, MD, PETER ESSER, MD, NORBERT KOCIEK, PHD, GU PINAR AYDIN, MD, PHD, AND KLAUS HEIMANN, MD

● PURPOSE:

The aim of this study was to determine nitric oxide levels in the vitreous of patients with proliferative diabetic retinopathy. ● METHODS: Using the spectrophotometric method based on Griess reaction, we measured levels of nitrite, the stable product of nitric oxide, in the vitreous of 21 eyes of 21 patients who underwent vitrectomy for the treatment of proliferative diabetic retinopathy with tractional retinal detachment, prospectively. Three samples were excluded from the study because of blood contamination. The control group was made up of vitreous samples from 15 eyes of 15 normal cadavers and five eyes of five patients who were undergoing vitrectomy for macular hole surgery. ● RESULTS: Nitrite levels were 0.524 ؎ 0.27 ␮M and 0.383 ؎ 0.17 ␮M in the vitreous of patients with proliferative diabetic retinopathy of diabetes type I and type II, respectively. In 15 cadaver eyes and five vitreous samples from patients who underwent macular hole surgery, nitrite levels were below the detection limit (less than 0.08 ␮M). There was no significant difference between nitrite levels in patients with type I and type II diabetes (P ‫ ؍‬.56), whereas there was a significant difference between diabetes groups and controls (P < .00001). ● CONCLUSION: Vitreous nitric oxide levels are elevated in patients with proliferative diabetic retinopathy with tractional retinal detachment. Nitric oxide may play a role in the pathogenesis of proliferative diabetic retinop-

athy. (Am J Ophthalmol 2000;130:87–90. © 2000 by Elsevier Science Inc. All rights reserved.)

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ITRIC OXIDE IS SYNTHESIZED IN CELLS THROUGH

Accepted for publication Jan 25, 2000. From the Department of Ophthalmology, Baskent University School of Medicine (Drs Yilmaz and Aydin), Ankara, Turkey; and Eye Clinic Vitreoretinal Department, Ko ¨ ln University Medical School (Drs Esser, Kociek, and Heimann), Ko ¨ ln, Germany. This work was supported by Ko ¨ ln Fortune, Ko ¨ ln, Germany; Deutsche Forschungsgemeinschaft, Bonn, Germany (Es 82/5-1, He 840/6-2) and the Turkish Scientific and Technical Research Council, Ankara, Turkey. Correspondence to Gu ¨ rsel Yilmaz, MD, Department of Ophthalmology, Baskent University School of Medicine, Fevzi C ¸ akmak Cad 10 Sok 06490 Bahc ¸ elievler, Ankara, Turkey; fax: 90-312-2237333; e-mail: gurselyilmaz@hotmail.com
0002-9394/00/$20.00 PII S0002-9394(00)00398-6

the conversion of L-arginine to L-citrulline, with the action of the enzyme nitric oxide synthase.1,2 Nitric oxide synthase exists in three major isoforms: neuronal, endothelial, and inducible. Neuronal and endothelial isoforms are constitutively expressed and generate small amounts of nitric oxide when activated by the calcium/calmodulin complex. In contrast, the inducible isoform is not expressed constitutively. It is expressed in many cell types after challenge by immunologic or inflammatory stimuli and acts independently of calcium, generating large amounts of nitric oxide over longer periods of time.3–5 In the eye, neuronal nitric oxide synthase is thought to be responsible for producing nitric oxide in photoreceptors and bipolar cells,6,7 whereas endothelial nitric oxide synthase is present in vascular endothelial cells.7,8 However, inducible nitric oxide synthase, which is found in Mu ¨ ller cells and in the retinal pigment epithelium, may be involved in phagocytosis of the photoreceptor outer segment9; infectious,10 inflammatory,11–14 and ischemic processes; and in the pathogenesis of diabetic retinopathy.15,16 Diabetic retinopathy is the leading cause of blindness in young adults in the western countries and in the United States,17 yet the mechanism behind the development of retinopathy is not fully understood. Elevated vitreous levels of cytokines and inflammatory mediators, such as interleukin-1, tumor necrosis factor, and interferon have been reported in cases of diabetic retinopathy.18,19 Also, it has been proved that these cytokines induce expression of the inducible nitric oxide synthase isoform in retinal pigment epithelium and Mu ¨ ller cells.20 –22 A recent report has documented increased nitric oxide synthase activity in the retinas of diabetic rats compared with normal rats.23 Considering all the experimental evidence to date, it is plausible that nitric oxide is a contributing factor in the development of diabetic retinopathy. We are unaware of
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where Hammes and associates25 reported that aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. degrees of freedom [df]. After obtaining patient consent. P Ͻ . Specimens were stored at Ϫ70 C until they were analyzed. Data are given as mean values ϩ SEM. Demographics of the patients are shown in Table 1.08 to 1. Vitreous samples were centrifuged for 5 minutes to separate the cell contents. These results indicate that there is a significant increase in vitreous nitric oxide level in patients with proliferative diabetic retinopathy. undiluted vitreous samples were collected from 21 eyes of 21 individuals undergoing pars plana vitrectomy as treatment for proliferative diabetic retinopathy with tractional retinal detachment.383 Ϯ 0. Thus. which appears to be responsible for the excess production of nitric oxide linked to disease status. indicates that these diabetic complications may result. The nitrite concentration was determined by measuring absorbance at 550 nm in comparison with standard solutions of sodium nitrite.524 Ϯ 0. P Ͻ . There was a significant difference in nitric oxide levels between patients with proliferative diabetic retinopathy and controls (diabetes type I: chisquare. and nitrite levels were measured in the supernatant using the spectrophotometric method based on the Griess reaction. 27.383 Ϯ 0. diabetes type II Control 7 11 20 0.8 Ϯ 3.26 –28 Furthermore. but later it was clearly demonstrated that aminoguanidine selectively inhibits the cytokineinducible isoform of nitric oxide synthase.00001). 0.05 were considered significant. whereas those with diabetes type II had a mean of 0. 31. Seven samples were taken from patients with proliferative diabetic retinopathy associated with diabetes type I and 11 from patients with proliferative diabetic retinopathy of diabetes type II.08 as the cutoff value and compared the diabetic groups with controls using the chi-square test. which was allowed to react at room temperature for 10 minutes. Background absorbance was subtracted from all values. diabetes type II: chi-square.5 Ϯ 2.56).4 59. 1. PDR ϭ proliferative diabetic retinopathy.9). All eyes with proliferative diabetic retinopathy had panretinal photocoagulation before vitrectomy. Also. The beneficial effect of aminoguanidine was initially attributed to its inhibition of advanced glycosylation product formation.27 ␮M (range. The control group was made up of vitreous samples from five eyes of five patients who were undergoing vitrectomy for idiopathic macular hole and from 15 eyes of 15 normal cadaver subjects. diabetes type I PDR. 1. RESULTS THE NITRITE LEVELS IN THE VITREOUS OF PATIENTS WITH METHODS THIS STUDY WAS CONDUCTED ACCORDING TO THE TENETS of the Declaration of Helsinki.17 ␮M (range. The vitreous nitrite levels in patients with macular hole and in normal cadaver eyes were below the detection limit (less than 0. Tilton and associates27 proved that aminoguanidine prevents or markedly ameliorates diabetes-induced vascular dysfunction in retinal and uveal vessels. diabetes type II Control 7 11 20 33.08 ␮M. The fact that aminoguanidine prevents diabetes-induced vascular and neural dysfunction. There was no significant difference between levels in patients with proliferative diabetic retinopathy of diabetes type I and type II (P ϭ . Patient Demographics Age (Mean ϩ SEM) Gender (Female/Male) TABLE 2.18).1% naphthyl-ethylenediamine. samples were mixed with 1% sulfanilamide and 0. DISCUSSION PREVIOUS STUDIES HAVE DESCRIBED THE ROLE OF NITRIC oxide in diabetic retinopathy in rats.7 52.17 Ͻ0. Nitrite Levels Measured in the Vitreous Samples N Nitrite (␮M) Group n PDR. at least in part. 88 AMERICAN JOURNAL OF proliferative diabetic retinopathy were higher than those in the control group. prospectively. we sought to explore this and make comparisons with appropriate controls. Statistical analysis showed that patient characteristics of gender were not associated with elevation in nitrite level (P ϭ .524 Ϯ 0.00001. Three samples with blood contamination were excluded from the study.TABLE 1.24 Briefly. df. Patients with proliferative diabetic retinopathy of diabetes type I had a mean vitreous concentration of 0. 0.72). diabetes type I PDR. and there was no active neovascularization. previous reports of clinical human study on nitric oxide levels in the vitreous of patients with proliferative diabetic retinopathy and could find no reference to it in a computerized search using MEDLINE. as well as vascular structural changes. from OPHTHALMOLOGY JULY 2000 . P values less than .8 Ϯ 1.08 to 1. Table 2).27 0.08 PDR ϭ proliferative diabetic retinopathy. we considered 0.1 3/4 5/6 8/12 PDR. Results were analyzed statistically using the Student t test and were expressed as mean Ϯ SEM when comparing type I and II diabetes groups.

13. Ann Rev Biochem 1994. J Neurochem 1994.42:71– 82.27. Mu ¨ ller cells. 20. A synthesis of perspectives. Merimee TJ. Courtois Y. 6. 8. Ostwald P. Maimone D. Invest Ophthalmol Vis Sci 1996. 4. Differential regulation of inducible nitric oxide synthase by fibroblast growth factors and transforming growth factor beta. NO. Invest Ophthalmol Vis Sci 1994. The localization of nitric oxide synthase in the eye and related cranial ganglia. Bredt DS. Thillage B. J Immunol 1995. 18. 21. Nathan C. et al. Abu el Asrar AM. and interleukin-1. Kleinert H.35:3278 –3288. Reder AT. In the light of previous findings concerning the origin of nitric oxide in this type of retinopathy. tolls. Courtois Y. J Cell Physiol 1994. it is conceivable that retinal pigment epithelium. It seems reasonable to suggest that nitric oxide is at least partly involved in the pathogenesis of proliferative diabetic retinopathy and that its role may be of particular importance in the pathology of this condition.90:4276 – 4280. Franks WA. 22. Goureau O. Corbett JA. McNally J. Biochem Biophys Res Comm 1994. and pharmacology. Courtois Y. Chakravarthy U.154:6518 – 6523. Curr Eye Res 1992. Diabetic retinopathy.28 These findings support the evidence that diabetes-induced vascular dysfunction is mediated and/or modulated by nitric oxide. 5. Goureau O. Nitric oxide: physiology. Becquet F.63:310 –317. 11(suppl):187–191. We hypothesize that the expression of inducible nitric oxide synthase by the cytokines that are elevated in the vitreous of patients with proliferative diabetic retinopathy may occur in the eye and lead to nitric oxide synthesis during proliferative diabetic retinopathy. so further studies are necessary to identify clearly the role of nitric oxide in proliferative diabetic retinopathy. Neuroscience 1993. et al. Neurosci Lett 1994. other research has confirmed that the vitreous of patients with proliferative diabetic retinopathy contains elevated levels of cytokine tumor necrosis factor. Courtois Y.198:120 –126. Stitt AW. Goureau O. Morse PH. Expression of inducible nitric oxide synthase in cytomegalovirus-infected glial cells of retinas from AIDS patients. Nitric oxide: a physiologic messanger molecule. Proc Natl Acad Sci USA 1993.20 –22 More recently. 130. and even pericytes and capillary endothelial cells may be involved. Cell 1994. Palmer RMJ.54:189 –200. 11. The two fundamental abnormalities in diabetic retinopathy are increased retinal vascular permeability and progressive retinal vessel closure. Sitaramayya A. Yoshida A. Tilton RG. Nitric oxide synthases: roles. Nitric oxide as a secretory product of mammalian cells. Cytokines in the vitreous of patients with proliferative diabetic retinopathy. Fo ¨ rstermann U. Bellot J.159: 259 –262. Induction and regulation of nitric oxide synthase in retinal Mu ¨ ller glial cells.15. Reddy VN. Lipopolysaccharide and PROLIFERATIVE DIABETIC RETINOPATHY 89 . Moncada S.63:175–195. Limb GA. Surv Ophthalmol 1997. 16. Chang K. Endotoxin-induced uveitis in the rat is attenuated by inhibition of nitric oxide production. Nitric oxide: a review of its role in retinal function and disease. the inducible nitric oxide synthase isoform has been detected in vitro in retinal pericytes and capillary endothelial cells after cytokine treatment.6:3051–3064. Stone RA. Becquet F. Human retina pigment epithelial cells produce nitric oxide in response to cytokines. Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes.352:351–364. Vis Neurosci 1995. Treatments based on the inhibition of inducible nitric oxide synthase hopefully may lead to novel therapeutic approaches for patients with proliferative diabetic retinopathy.29 It is well known that nitric oxide is a potent vasodilator and has a role in ischemic processes. Hicks D. Kozak Y. particularly diabetic retinopathy.37:1187–1196. Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitic rats. 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