PROINFLAMMATORY CYTOKINES AND ANGIOGENIC AND ANTI-ANGIOGENIC FACTORS IN VITREOUS OF PATIENTS WITH PROLIFERATIVE DIABETIC RETINOPATHY AND EALES

DISEASE
PONNALAGU MURUGESWARI, MSC,* DHANANJAY SHUKLA, MS, ANAND RAJENDRAN, FRCS, RAMASAMY KIM, NB, PERUMALSAMY NAMPERUMALSAMY, MS, FAMS, VEERAPPAN MUTHUKKARUPPAN, PHD*
Purpose: To investigate the mechanism of angiogenesis in proliferative diabetic retinopathy (PDR) and Eales disease (ED) on the basis of the levels of proinammatory cytokines, angiogenic growth factor, and antiangiogenic factor in the vitreous humor. Methods: Twenty-ve patients with PDR, 10 patients with ED, and 25 with macular hole (MH) as control subjects were studied. The concentration of the proinammatory cytokines interleukin-6 (IL-6), IL-8, IL-1; chemokine-monocyte chemoattractant protein-1 (MCP-1); angiogenic factor-vascular endothelial growth factor (VEGF); and antiangiogenic factorpigment epithelium derived factor (PEDF) in the vitreous uid obtained from the eyes during vitrectomy were measured by sandwich enzyme linked immunosorbent assay (ELISA). Results: IL-6, IL-8, MCP-1, and VEGF levels in the vitreous were signicantly higher in PDR (P 0.0001) and ED (P 0.0001) than in MH patients. Conversely, the vitreous level of PEDF was signicantly reduced in PDR (P 0.0001) but not in ED. A signicant correlation was observed between VEGF and IL-6 in ED patients. Conclusion: The authors demonstrate the importance of VEGF in retinal neovascularization of ED which is an idiopathic inammatory venous occlusion. Further study is required to understand the interrelationship between VEGF and inammatory cytokines in PDR and ED. RETINA 28:817 824, 2008

etinopathy is one of the most frequent chronic microvascular complications associated with diabetes mellitus. A population-based Chennai Urban Rural Epidemiology Study (CURES) revealed that for

From the *Department of Immunology, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation; and Retina Clinic, Aravind Eye Hospital, Madurai, Tamilnadu, India. Supported by grants from Department of Science and Technology and TIFAC-CORE in Diabetic Retinopathy, India. Reprint requests: Dr. V. R. Muthukkaruppan, Director-Research, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, 1, Anna Nagar, Madurai-20, Tamilnadu, India; e-mail: drvrm2002@yahoo.com; muthu@aravind.org

every 5-year increase in the duration of diabetes, the risk for diabetic retinopathy (DR) increased 1.89 fold.1 Proliferative diabetic retinopathy (PDR) is a leading cause of blindness among people of working age.2 It is characterized by hyperglycemia and pericyte loss, manifested as microaneurysms, macular edema and subsequently retinal neovascularization, vitreous hemorrhage, and tractional complications.3 Eales disease (ED) is an idiopathic inammatory venous occlusion that primarily affects the peripheral retina of healthy young adult men (20 30 years). In India, it occurs in 1 of 200 to 250 ophthalmic pa817

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tients.4 Retinal changes include periphlebitis, peripheral nonperfusion, and neovascularization. In addition, visual loss is characteristically caused by bilateral recurrent vitreous hemorrhage.5,6 The symptoms and signs of the two diseases run parallel to each other with some differences. Like DR, patients with ED also manifest clinical signs of microaneurysms, capillary nonperfusion, retinal neovascularization, and subsequent vitreous hemorrhage.7 While retinal neovascularization and vitreous hemorrhage are common to both PDR and ED, the diabetic patients, who commonly have visual deterioration due to macular edema, typically manifest the disease bilaterally.2 In contrast, the patients with ED are typically healthy, young, and nondiabetic, and generally show uniocular involvement of peripheral retina, at least initially. Recently, similarities have been revealed in the pathogenic mechanisms of the two diseases, specically, the presence of inammatory cytokines and chemo-attractant molecules in DR (infra vide), leading to the landmark hypothesis that DR may be a low-grade, subclinical inammatory disease.8 Early studies have attempted to correlate systemic abnormalities with ED and tuberculosis has been implicated.5 However, none of the associations has been proven. Immunologic studies on Eales patients indicate that there was no difference between ED patients and controls in their immune response to tubercular antigens and no correlation was observed between the ocular lesion and Mantoux positivity.9 Biswas et al reported higher frequencies of HLA B5, DR1, and DR4 in ED.10 However, the mechanism of neovascularization in ED remains unclear. Many inammatory cytokines (IL-6, IL-8, and IL1), angiogenic growth factor (VEGF), and antiangiogenic factor (PEDF) have been studied to understand the pathogenesis of DR.1116 IL-6 is a multifunctional cytokine which is said to indirectly cause an increase in vascular permeability and neovascularization by inducing the expression of vascular endothelial growth factor (VEGF).17 IL-8 is the prototype of C-X-C chemokine, which has been recognized as potent chemoattractant, activator of neutrophils and T-lymphocytes but not monocytes.18 Monocyte chemoattractant protein-1 (MCP-1) exhibits chemoattractant potential for monocytes and lymphocytes but not for neutrophils.19 Therefore, it is possible that IL-8 and MCP-1 are involved in the recruitment of inammatory cells. To date, VEGF is the most attractive candidate for stimulating new vessel formation in DR among the members of angiogenic factors. VEGF is a mitogen for endothelial cells and its expression both in vivo and in vitro can be induced by hypoxia.20 Pigment epithelium derived factor (PEDF), a 50 kD protein, is

a potent inhibitor of angiogenesis in the vitreous compared to angiostatin and other naturally occurring ocular angiostatic agents.21 Recent studies have shown that an imbalance between PEDF (angiogenic inhibitor) and VEGF (angiogenic growth factor) may contribute to retinal neovascularization.21,22 Though the cause of DR is hyperglycemia, the precise pathogenic mechanism is unclear. Neither the etiology nor the pathogenic mechanism of ED is known. Therefore, to delineate the importance of cytokines, angiogenic and antiangiogenic factors in relation to retinal neovascularization, we measured the concentrations of IL-6, IL-8, IL1-, MCP-1, VEGF, and PEDF in vitreous of PDR, ED, and idiopathic macular hole (MH) patients. Subjects and Methods Study Subjects The clinical diagnosis of ED was made on the basis of peripheral venous sheathing in multiple quadrants in the fellow eye, or the operated eye, as observed intraoperatively. The patients with conditions which could secondarily cause venous sheathing, such as noninammatory retinal vein occlusions, diabetic or hypertensive retinopathy, sickle cell retinopathy, ocular inammatory conditions like choroiditis, or pars planitis, or associated systemic infective/autoimmune/ other inammatory disease (as revealed by history, examination, or investigations), were excluded from the study. The indications for vitrectomy included best-corrected visual acuity less than 20/40 due to vitreous hemorrhage of at least 2 months duration with/without epiretinal membranes; rhegmatogenous/ combined-mechanism retinal detachment; or a tractional retinal detachment involving/threatening the macula. All the diabetic patients included in this study had Type 2 diabetes mellitus. Like ED patients, they were also surgical inpatients, who presented with advanced PDR, as dened by the ETDRS study.23 The indications for vitrectomy were the same as mentioned above for ED patients. Patients with idiopathic MH were interviewed and evaluated to rule out a history or signs of ocular trauma. The main preoperative investigation in MH was optical coherence tomography, to assess the morphology of macular hole for surgical feasibility and prognosis. Undiluted vitreous samples were obtained from 25 PDR, 10 ED, and 25 MH patients. Patients were recruited in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board of Aravind Eye Hospital. The PDR patients had vitreous hemorrhage (VH) (n 25) of whom eight had macular tractional retinal detachment. The ED

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patients (nondiabetic) had VH (n 10). None of the MH patients had associated proliferative vitreoretinopathy and diabetes. All the cases had no history of ocular surgery. The PDR patients (21 male; 4 female) were aged 54.9 8.9 years (mean SD) had diabetes duration of 13.6 8.3 years (mean SD) which included both active (n 16) and inactive PDR (n 9). Sixteen of these PDR patients had undergone panretinal photocoagulation (PRP). The ED patients (10 male; 0 female) were aged 29.3 6.1 years. The control group MH patients (6 male; 19 female) were aged 61.8 8.1 years. Sample Collection At the beginning of vitrectomy, undiluted vitreous (200 700 L) was aspirated via pars plana with a vitreous cutter, before opening the infusion port. These undiluted vitreous samples were immediately frozen in aliquots in polypropylene tube at 80C until assay. Cytokine Assay The concentrations of IL-6, IL-8, IL1-, MCP-1, VEGF, and PEDF were determined in the vitreous samples using sandwich enzyme linked immunosorbent assay (ELISA) (BD, R&D systems, Chemicon international) according to the manufacturers instructions. The standard curve was prepared using recombinant human cytokines. A calibration run on a few vitreous samples showed that a dilution of 1:5 was found to be appropriate for IL-6, IL-8, MCP-1, VEGF;

1:4 for PEDF; and 1:2 for IL1-. The minimum detectable concentrations were found to be 7.8 (IL-1 and MCP-1), 15.0 (IL-6), 3.1 (IL-8), and 31.3 (VEGF) pg/mL and 0.98 ng/mL for PEDF, respectively. Statistical Analysis Mann-Whitney U-test was used to analyze the difference between PDR, ED, and control groups. Values are reported as medians with ranges. To examine the correlations between VEGF and other cytokines, Spearmans rank correlation test was used and graphically represented by means of Pearsons correlations. The results were considered signicant at P 0.05. Results Vitreous Levels of IL-6, IL-8, IL-1, and MCP-1 The median levels of IL-6 and IL-8 were signicantly higher in PDR (80.7 pg/mL, P 0.001; 59.6 pg/mL, P 0.001) and ED patients (201.9 pg/mL, P 0.001; 148.2 pg/mL, P 0.001) when compared with the MH patients (0, 0 pg/mL; 0, 137.1 pg/mL). However, no signicant difference was observed in IL-1 (Figure 1). In PDR (1812.9 pg/mL, P 0.0001) and ED (1754 pg/mL, P 0.001) the median vitreous MCP-1 concentration was signicantly higher when compared to MH patients (0, 1168.3 pg/mL) (Figure 2). Vitreous Levels of VEGF and PEDF The median VEGF concentration in vitreous was signicantly higher in PDR (1123.3 pg/mL; P

Fig. 1. IL-6, IL-8, and IL-1 levels in the vitreous of patients with proliferative diabetic retinopathy (PDR), Eales disease (ED), and macular hole (MH).

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Fig. 2. MCP-1 levels in the vitreous of patients with proliferative diabetic retinopathy (PDR), Eales disease (ED), and macular hole (MH).

0.0001) and ED patients (1219.9 pg/mL; P 0.0001) than in the MH patients (0 pg/mL) (Figure 3). On the other hand, PEDF levels were signicantly decreased in PDR (0 ng/mL, P 0.0001) than in MH (17.3 ng/mL), while the PEDF levels in ED (13.6 ng/mL) were not signicantly reduced (Figure 4). Furthermore, a signicant correlation between VEGF and IL-6 was detected in ED patients (Figure 5) but not in PDR. In comparison of PDR and ED, the vitreous

level of PEDF was signicantly lower in PDR than in the ED patients (P 0.026). No signicant difference was observed between PDR and ED in terms of proinammatory cytokines. There were no correlations between VEGF and inammatory cytokines (IL-8, MCP-1) and angiogenic inhibitor (PEDF) in PDR and ED. No signicant difference of VEGF, PEDF, IL-6, IL-8, and MCP-1 was observed between active and inactive PDR pa-

Fig. 3. VEGF levels in the vitreous of patients with proliferative diabetic retinopathy (PDR), Eales disease (ED), and macular hole (MH).

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Fig. 4. Levels of PEDF in the vitreous of proliferative diabetic retinopathy (PDR), Eales disease (ED), and macular hole (MH) patients..

tients. The vitreous levels of cytokines (IL-6, IL-8), VEGF, and PEDF showed no signicant difference in PDR patients, who had undergone PRP treatment before vitrectomy, when compared with patients who had no PRP treatment. Concentration of all the cytokines, angiogenic and antiangiogenic factors in the vitreous of PDR, ED, and control subjects are mentioned in Table 1 as median ranges. We have analyzed the proinammatory cytokines and growth factors in serum of PDR, ED, and MH patients. The cytokine IL-6 and antiangiogenic factor PEDF were below the detectable levels in the serum of PDR, ED, and MH

patients. The serum levels of IL-8 (25.1 142.2; median SD), VEGF (165.9 145.7), and MCP-1 (319.9 92.6) were below the vitreous levels of PDR patients. Discussion Earlier studies on PDR involved the detection of the levels of angiogenic stimulator and inhibitors alone22,24 or any one cytokine and angiogenic factor in the vitreous.2527 However, the present report is a comprehensive study to estimate the inammatory cytokines (IL-6, IL-8, IL1-), chemokines (MCP-1), angiogenic factor (VEGF), and antiangiogenic factor (PEDF) all together in each of the vitreous samples of PDR, ED, and MH patients. This was carried out to elucidate whether neovascularization in PDR and ED are mediated by inammatory cytokines and growth factors and further to understand the relationship between the cytokines and growth factors. To our knowledge, this is the rst report to demonstrate that the vitreous levels of VEGF, IL-6, IL-8, and MCP-1 were signicantly and simultaneously increased in both PDR and ED when compared with MH patients. ED is associated with localized inammation of retinal blood vessel walls and is suggested to be an immune-mediated disease, as shown by the increase in the levels of -1 acid glycoprotein.28 DR may not display most of the macroscopic attributes of inammation, but all the microscopic signs of inammation,

Fig. 5. Correlation between VEGF and IL-6 in vitreous of Eales disease patients; r 0.698, P 0.025.

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i.e., vasodilatation, altered ow, uid exudation, and leukocyte migration/accumulation, are observed in DR.8 This path-breaking information has overseen a surge of myriad studies evaluating corticosteroids in the treatment of DR, particularly macular edema, culminating into the National Eye Institutesponsored trials on the role of steroids in the treatment of DR (http://www.nei.nih.gov/neitrials). Gao et al have demonstrated the elevated levels of carbonic anhydrase-1 (CA-1) in vitreous of DR patients. They proved that injection of CA-1 into rats induced alkalinization of vitreous which increased kallikrein activity and its generation of factor XIIa, revealing that inhibition of extracellular CA-1 and kallikrein-mediated innate inammation could provide new therapeutic opportunities for the treatment of DR.29 Though PDR and ED differ in their etiology and early pathogenic mechanisms the result in uncontrolled disease is neovascularization. The role of cytokines in the pathogenesis of PDR and ED is not completely understood. However, there are reports suggesting that cytokine IL-6 can increase endothelial cell permeability in vitro by rearranging actin laments and by changing the shape of endothelial cells.30 Further, the increased vitreous levels of VEGF and IL-6 correlated with the progression of PDR in the outcome of vitreous surgery.31 Even though there are reports on high levels of IL-6, IL-8, and MCP-1 in the vitreous of PDR,25,3234 we demonstrate for the rst time the increased levels of IL-6, IL-8, MCP-1, and VEGF in the vitreous of ED patients. The source of the high levels of cytokines and chemokine detected within the vitreous of PDR and ED patients remains unclear. A possibility is that cells in the vitreous could be the main cause accounting for the high levels of these cytokines and chemokine. The vitreous of MH patients are largely devoid of inammatory cells.35 Further, El-Ghrably and associates reported that macrophages, monocytes, retinal pigment epithelial cells, and glial cells are found in the vitreous of patients with PDR and the majority of these cells are capable of producing cytokines in vitro.35 We suggest that increased levels of IL-6, IL-8, and MCP-1 are involved in the pathogenesis of inducing neovascularization in PDR and ED. Further studies on this issue are needed to understand the mechanism of PDR and ED. There are varying reports on the status of PEDF and VEGF in diabetic retinopathy. The PEDF level in MH is equal to the normal bovine eyes.36 We conrm the earlier report that higher concentration of VEGF and lower concentration of PEDF in the vitreous of PDR may be related to angiogenesis.21,22 On the other hand,

Table 1. Vitreous Levels of Cytokines, Angiogenic and Anti-Angiogenic Factors in Proliferative Diabetic Retinopathy, Eales Disease, and Macular Hole Patients

ED and MH P Value PDR and MH P Value Macular Hole (MH) Sample No. Sample No. Median (Minimum, Maximum) Median (Minimum, Maximum) Eales Disease (ED)

10 10 9 10 10 10 IL-6 (pg/mL) IL-8 (pg/mL) IL1- (pg/mL) MCP-1 (pg/mL) VEGF (pg/mL) PEDF (ng/mL) 25 25 20 25 25 25 80.7 (0, 1645.9) 59.6 (0, 777.2) 0.0 (0, 57.9) 1812.9 (0, 5830.9) 1123.3 (0, 8092.8) 0.0 (0, 21.8)

201.9 (0, 1200) 148.2 (13, 748.7) 0.0 (0, 23.87) 1754.0 (0, 4104.1) 1219.9 (121.8, 9900.2) 13.6 (0, 93.8)
Values are presented as median ranges (minimum, maximum).

Proliferative Diabetic Retinopathy (PDR)

Cytokines

Sample No.

Median (Minimum, Maximum)

25 25 25 25 25 25

0.0 (0) 0.0 (0, 137.08) 0.0 (0, 103.76) 277.7 (0, 1168.3) 0.0 (0, 139.75) 17.3 (0, 298.7)

0.0001 0.0001 0.127 0.0001 0.0001 0.0001

0.0001 0.0001 0.127 0.001 0.0001 0.432

0.546 0.322 0.753 0.674 0.475 0.026

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similar decrease in PEDF was not observed in ED. Duh et al demonstrated a signicant increase rather than a decrease in the vitreous of PEDF levels in PDR patients.24 Furthermore, Ogata and coworkers suggested that signicantly elevated levels of PEDF in the plasma of PDR patients may be related to the progression of diabetic retinopathy.37 Moreover, a concentration of 50 ng/mL of PEDF completely inhibits VEGFinduced migration of cultured microvascular endothelial cells.36 The lowest baseline vitreous concentration of PEDF reported among all the studies is 20-fold higher than the concentration that was maximally effective in vitro and a 1:20 dilution of human vitreous completely inhibited VEGF-induced migration of vascular endothelial cells. This inhibitory activity was also neutralized by anti-PEDF antibody.36 The mechanism of increase and decrease in PEDF levels is not clear in the modulation of ocular neovascularization. The decreased level of PEDF in PDR when compared with ED and MH suggests that the regulation of the inhibitory effect of PEDF may differ in ED. Further, the signicant correlation between VEGF and IL-6 in ED suggests that the roles and induction mechanism of VEGF also differ between PDR and ED. In conclusion, we suggest that increased levels of IL-6, IL-8, MCP-1, and VEGF may act as a key regulator of neovascularization in PDR and ED. The regulatory role of VEGF and the inhibitory effect of PEDF may differ in the modulation of neovascularization in PDR and ED. Even though the etiology is different in both diseases, we demonstrated the levels of cytokines and growth factors in PDR and ED. Moreover, the regulation of VEGF and the inhibitory role of PEDF in inducing the neovascularization cannot be clearly explained. Further, investigation is required to understand the regulatory role of neovascularization in PDR and ED. Key words: cytokines, Eales disease, neovascularization, proliferative diabetic retinopathy, pigment epithelium derived factor, vascular endothelial growth factor.

5. 6. 7.

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