- Email: [email protected]
Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema
Vitreous Levels of Interleukin-6 and Vascular Endothelial Growth Factor Are Related to Diabetic Macular Edema Hideharu Funatsu, MD,1 Hidetoshi Yamashita, MD,2 Tomohiro Ikeda, MD,1,3 Tatsuya Mimura, MD,4 Shuichiro Eguchi, MD,5 Sadao Hori, MD6 Purpose: To investigate whether interleukin-6 (IL-6) or vascular endothelial growth factor (VEGF) is related to diabetic macular edema (DME) in subjects without posterior vitreous detachment (PVD). Design: Retrospective case-control study. Participants: Twenty-six subjects who had DME without PVD and 12 subjects who had nondiabetic ocular disease (the control group). Methods: Vitreous fluid samples were obtained at vitreoretinal surgery, and the IL-6 and VEGF levels in vitreous fluid and plasma were measured by enzyme-linked immunosorbent assay. Main Outcome Measures: Vitreous levels of IL-6 and VEGF in DME subjects without PVD. Results: The vitreous levels of both IL-6 and VEGF were significantly higher in the subjects with DME than in control subjects (P⬍0.0001 and P⬍0.0001, respectively). The vitreous level of IL-6 was significantly correlated with that of VEGF (P⬍0.0001). Vitreous levels of both IL-6 and VEGF were significantly higher in subjects with hyperfluorescent DME than in those with minimally fluorescent DME (P ⫽ 0.0008 and P ⫽ 0.0038, respectively). Conclusions: We found that the levels of both IL-6 and VEGF were elevated in the vitreous fluid of subjects with hyperfluorescent DME. Our results suggest that IL-6 and VEGF may promote an increase of vascular permeability in DME subjects without PVD. Interleukin-6 may possibly induce an increase of vascular permeability through a paracrine effect on VEGF in these subjects. Ophthalmology 2003;110:1690 –1696 © 2003 by the American Academy of Ophthalmology.
Diabetic macular edema (DME) is the most common cause of a decrease in visual acuity among diabetic subjects.1 It has been hypothesized that DME is related to (1) breakdown of the blood–retinal barrier (BRB) and subsequent leakage of intraretinal fluid from abnormal retinal capillaries and microaneurysms, (2) secretion of vasopermeability factors from the retina into the vitreous, and (3) vitreoretinal adhesions and traction on the macula.2,3 Recent studies have Originally received: July 1, 2002. Accepted: March 13, 2003. Manuscript no. 220444. 1 Department of Ophthalmology, Diabetes Center, Tokyo Women’s Medical University, Tokyo, Japan. 2 Department of Ophthalmology, Yamagata University School of Medicine, Yamagata, Japan. 3 Department of Ophthalmology, Hyogo Medical College, Nishinomiya, Japan. 4 Department of Ophthalmology, Tokyo University School of Medicine, Tokyo, Japan. 5 Eguchi Eye Hospital, Hakodate, Japan. 6 Department of Ophthalmology, Tokyo Women’s Medical University, Tokyo, Japan. Supported by a Health Science Research Grant (#10060101 to Drs Funatsu, Hori, and Yamashita) from the Japanese Ministry of Health, Labor and Welfare (Tokyo, Japan). Reprint requests to Hideharu Funatsu, MD, Department of Ophthalmology, Diabetes Center, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: [email protected].
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© 2003 by the American Academy of Ophthalmology Published by Elsevier Inc.
shown that vascular endothelial growth factor (VEGF) causes conformational changes in the tight junctions of retinal vascular endothelial cells4 and plays a major role in the increase of vascular permeability in diabetic eyes.2 Many of the effects of VEGF are mediated via other factors, and the events that induce VEGF production can initiate a cascade of factors, but it is still unclear how this occurs. Interleukin-6 (IL-6) is a multifunctional cytokine that may indirectly cause an increase of vascular permeability by inducing the expression of VEGF5 or else may directly increase endothelial permeability.6 We previously reported that the levels of both VEGF and IL-6 in the aqueous humor were correlated with the severity of diabetic retinopathy, the grade of fundus findings, and the severity of DME.7,8 Aiello and coauthors9 reported that the vitreous level of VEGF was elevated in subjects with proliferative diabetic retinopathy (PDR) when compared with nondiabetic subjects. Vascular endothelial growth factor levels in the vitreous fluid and aqueous humor may be related to each other, but they are not equivalent. Recently, the number of subjects having vitreous surgery for DME has increased, but the vitreous levels of VEGF or IL-6 in DME subjects without posterior vitreous detachment (PVD) have not been clarified. Accordingly, the present study was performed to investigate whether IL-6 was involved in the exacerbation of DME in subjects without PVD in cooperation with VEGF, which is considered to be the most potent factor promoting ISSN 0161-6420/03/$–see front matter doi:10.1016/S0161-6420(03)00568-2
Funatsu et al 䡠 Diabetic Macular Edema an increase of vascular permeability. We found that the vitreous levels of both IL-6 and VEGF were correlated with the severity of DME and also showed a strong correlation with each other. These results suggest that further investigation of the ocular cytokine network may help to improve our understanding of the pathogenesis of DME.
Research Design and Methods Subjects Undiluted vitreous fluid samples were harvested at the start of vitrectomy after informed consent was obtained from each subject following an explanation of the purpose and potential adverse effects of the procedure. This study was performed in accordance with the Helsinki Declaration of 1975 (the 1983 revision), and the Institutional Review Board of Tokyo Women’s Medical University also approved the protocol for collection of vitreous fluid and blood samples. Vitreous fluid samples were obtained from 26 subjects who had clinically significant DME without PVD and from 12 subjects with nondiabetic ocular disease. The DME subjects included 20 with diffuse macular edemas and 6 with cystoid macular edemas (CMEs). The subjects with nondiabetic ocular disease included 9 who had macular holes and 3 with epiretinal membranes, but none of these 12 subjects had associated proliferative vitreoretinopathy. The diabetic subjects (12 men and 14 women) were 64.2 ⫾ 16.8 years old (mean ⫾ standard deviation [SD]), with a duration of diabetes of 13.8 ⫾ 6.5 years and a hemoglobin A1c (HbA1c) of 7.6 ⫾ 2.0%. The 12 subjects with nondiabetic ocular disease (5 men and 7 women) were 62.8 ⫾ 13.6 years old. There was no significant age difference between the diabetic and nondiabetic subjects (P ⫽ 0.6012). Exclusion criteria included (1) previous ocular surgery, (2) a history of ocular inflammation, (3) PDR (such as fibrovascular proliferation), (4) ophthalmic disorders associated with macular edema (such as uveitis and branch or central retinal vein occlusion), (5) DME associated with posterior hyaloid traction, and (6) clinically detectable posterior vitreous separation (defined as vitreous separation at the disc or a visible Weiss ring). All operations were performed at Tokyo Women’s Medical University Hospital.
Fundus Findings All subjects with DME and diabetic retinopathy presenting to the Diabetes Center at Tokyo Women’s Medical University Hospital were evaluated by careful biomicroscopic examination using a fundus contact lens, and subjects who had clinically significant DME without PVD were enrolled in this study. Fundus findings were confirmed preoperatively by standardized fundus color photography and fluorescein angiography (FA), which was performed with a Topcon TRC-501A fundus camera, an image-net system (Tokyo Optical Co Ltd., Tokyo, Japan), and a preset lens with a slit lamp.7,8 The severity of diabetic retinopathy was graded according to the modified Early Treatment Diabetic Retinopathy Study retinopathy severity scale.10,11
According to this scale, 3 eyes were level 35, 6 eyes were level 43, 9 eyes were level 47, and 8 eyes were level 53. All subjects with DME underwent retinal photocoagulation (mean ⫽ 592 shots; range ⫽ 186 –1112 shots) before vitreous surgery. Diffuse DME was diagnosed by the detection of leakage from diffusely dilated retinal capillaries throughout the posterior pole. A diagnosis of CME was indicated by a petalloid pattern of hyperfluorescence. Diabetic macular edema was classified as hyperfluorescent (18 eyes) if there was extensive fluorescein leakage in the macular region on FA, and as minimally fluorescent (8 eyes) if there was little fluorescein leakage.12 Posterior vitreous detachment was diagnosed intraoperatively: subjects without a Weiss ring or vitreoretinal separation had no PVD, whereas those with a Weiss ring had PVD, and those with posterior detachment of the vitreous from the retina (excluding the macular region) had partial PVD.12
Sample Collection Samples of vitreous fluid were collected into sterile tubes and were rapidly frozen at – 80°C. These samples were obtained at the time of vitreoretinal surgery. Plasma samples were also collected from the 26 subjects with DME. Blood was immediately placed on ice and centrifuged at 3000 g for 5 minutes at 4°C, after which the plasma was rapidly frozen and stored at – 80°C until assay.
Measurement of IL-6 and VEGF Levels Both IL-6 and VEGF levels were measured in vitreous samples from the same eye as well as in plasma samples by enzyme-linked immunosorbent assay (ELISA) using kits for human IL-6 and VEGF (R&D System, Minneapolis, MN).13,14 The VEGF kit was able to detect 2 of the 4 VEGF isoforms (VEGF121 and VEGF165), probably because these 2 shorter isoforms are secreted and the 2 longer isoforms are cell associated. Each assay was performed according to the manufacturer’s instructions. The standard solution (100 l for the VEGF ELISA and 200 l for the IL-6 ELISA) and the sample (10 or 100 l for the VEGF ELISA and 20 or 200 l for the IL-6 ELISA) were added to the wells of a 96-well plate coated with amonoclonal antibody. After incubation, the plate was washed, and an enzyme-labeled antibody was added. After further incubation, the plate was washed again, and the substrate was added. The reaction was stopped by adding the stop solution after color had developed, and the optical density was determined at 450 and 620 nm using an absorption spectrophotometer (Titertek Multiscan MCC/340; ICN, Tokyo, Japan). A standard curve was plotted from measurements made with the standard solution (from 15.6 to 1000 pg/ml for VEGF and from 0.156 to 100 pg/ml for IL-6) and was used to determine the concentration of VEGF or IL-6 in the sample. The levels of these factors in vitreous fluid and plasma were within the detection range of the relevant assay, with the minimum detectable concentration being 15.6 pg/ml for VEGF (the intra-assay coefficient of variation [CV] was 5.5% and the
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Ophthalmology Volume 110, Number 9, September 2003 interassay CV was 6.8%) and 0.156 pg/ml for IL-6 (intraassay CV of 5.6% and interassay CV of 6.9%).
Measurement of Protein The vitreous fluid level of protein (mg/ml) was measured by a previously validated microturbidimetric method using an autoanalyzer (Hitachi [Tokyo, Japan] 917).15 This method was based on the benzetonium chloride reaction and was highly specific for the detection of protein, showing a higher sensitivity and reproducibility than the classic Lowry method. The detection limit was 0.02 mg/ml, whereas the intra-assay and interassay CVs were 3.3 and 3.9%, respectively.
Statistical Analysis All analyses were performed with SAS System 6.12 software (SAS Institute, Inc., Cary, NC).16 Data are presented as frequencies or as the mean ⫾ SD. Data with a skewed distribution were transformed to a logarithmic scale, and the geometric mean was calculated together with the one SD range on either side of the mean. The Mann-Whitney U test was used to compare IL-6 and VEGF concentrations between the diabetic subjects and control subjects. To examine correlations, Spearman’s rank correlation coefficients were calculated by linear regression analysis. A two-tailed P value of ⬍0.05 was considered to indicate statistical significance.
Results Vitreous Levels of IL-6 and VEGF Vitreous fluid protein levels (median [range]) were significantly higher in the diabetic subjects with DME than in the nondiabetic control subjects (3.12 mg/ml [0.55– 8.42] vs. 0.55 mg/ml [0.21– 2.42], P ⫽ 0.0008). The vitreous fluid concentration of VEGF was also significantly elevated in the subjects with DME (818.0 pg/ml [36.8 –1902.4]) when compared with the control subjects (17.8 pg/ml [15.6 –26.2], P⬍0.0001, Fig 1A). Furthermore, the VEGF/ protein ratio showed a significant difference between the subjects with DME (263.6 pg/mg [24.2–748.3]) and the control subjects (20.2 pg/mg [5.6 –19.4], P⬍0.0001; Fig 1B). Vitreous fluid concentrations of IL-6 were also significantly elevated in the subjects with DME (188.1 pg/ml [18.0 –768.4]) when compared with control subjects (9.51 pg/ml [4.02–22.4], P⬍0.0001; Fig 2A). Moreover, the IL-6/protein ratio showed a significant difference between the subjects with DME (63.3 pg/mg [5.2–243.6]) and the control subjects (3.15 pg/mg [1.14 – 8.24], P⬍0.0001; Fig 2B). There was also a significant relationship between the vitreous concentration of VEGF and that of IL-6 in the diabetic subjects ( ⫽ 0.678, P⬍0.0001; Fig 3). Furthermore, the vitreous levels of VEGF and IL-6 detected in the subjects with hyperfluorescent DME (VEGF: 1008.6 pg/ml [52.8 –1902.4]; IL-6: 228.6 pg/ml [18.0 –768.4]) were significantly higher (P ⫽ 0.0008 and P ⫽ 0.0038, respectively) than those found in the subjects with minimally fluorescent DME (VEGF: 498.2 pg/ml [36.8 – 898.2]; IL-6: 98.6 pg/ml [18.0 –250.2]; Fig 4A, B). There was no significant difference of the vitreous VEGF concentration between DME subjects (792.6 pg/ml [36.8 –1591.2])
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Figure 1. A, Vascular endothelial growth factor (VEGF) concentration in the vitreous fluid of nondiabetic control subjects and subjects with diabetic macular edema (DME; *P ⬍ 0.0001). B, VEGF concentration per milligram of protein in the vitreous fluid of control subjects and DME subjects (*P⬍0.0001).
and CME subjects (837.2 pg/ml [36.8 –1902.4], P⫽0.1645; Table 1). There was also no significant difference of the vitreous IL-6 concentration between DME subjects (171.7 pg/ml [18.0 – 602.3]) and CME subjects (188.1 pg/ml [18.0 –768.4], P ⫽ 0.2638; Table 1). The vitreous level of VEGF was significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.648, P⬍0.0001; Fig 5A), and the vitreous level of IL-6 was also significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.596, P⬍0.0001; Fig 5B).
Funatsu et al 䡠 Diabetic Macular Edema
Figure 3. Relationship between the vitreous levels of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in subjects with diabetic macular edema ( ⫽ 0.678, P⬍0.0001).
Discussion
Figure 2. A, Interleukin-6 (IL-6) concentration in the vitreous fluid of nondiabetic control subjects and subjects with diabetic macular edema (DME; *P⬍0.0001). B, IL-6 concentration per milligram of protein in the vitreous fluid of control subjects and DME subjects (*P⬍0.0001).
Vitreous and Plasma Levels of IL-6 and VEGF The vitreous fluid concentration of VEGF was significantly higher than the plasma VEGF level (48.2 pg/ml [15.6 –344.0]) in the subjects with DME (P⬍0.0001), and the vitreous fluid concentration of IL-6 was also significantly higher than the plasma IL-6 level (17.5 pg/ml [4.0 – 46.0]) in these subjects (P ⫽ 0.0046). There was no significant relationship between HbA1c (7.6% [4.8 – 12.3]) and the vitreous levels of VEGF or IL-6 ( ⫽ 0.220, P ⫽ 0.1712 and ⫽ 0.253, P ⫽ 0.0626, respectively).
In the present and previous studies,7,8 we have shown that aqueous levels of both IL-6 and VEGF are correlated not only with the severity of diabetic retinopathy and the grade of fundus findings, such as retinal hemorrhages and hard exudates, but also with the severity of DME. We have also shown that the aqueous level of IL-6 is significantly correlated with that of VEGF.7,8 Furthermore, both VEGF and IL-6 concentrations are higher in the aqueous humor than in the plasma.7,8 Although it has been reported that posterior hyaloid traction on the macula may play a role in the development and progression of DME and that vitrectomy is beneficial for DME associated with posterior hyaloid traction,3,17 vitrectomy was recently shown to be an effective procedure for reducing DME in eyes with complete PVD or eyes without visible evidence of posterior hyaloid thickening and traction.3,18,19 Diabetic macular edema is also influenced by the severity of diabetic retinopathy, including the extent of proliferative changes. Therefore, we excluded subject who had DME with posterior hyaloid thickening and traction, complete PVD, or PDR, and we investigated the relationship between IL-6 and VEGF levels in the vitreous fluid of DME subjects without PVD who had nonproliferative diabetic retinopathy. We also investigated whether there was a significant difference in the vitreous levels of IL-6 and VEGF between hyperfluorescent and minimally fluorescent DME, because the fluorescence on FA reflects changes of vascular permeability. This study showed that the vitreous fluid levels of both VEGF and IL-6 were significantly elevated in DME subjects when compared with nondiabetic subjects, not only in
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Ophthalmology Volume 110, Number 9, September 2003 Table 1. Comparison of Vitreous Levels of VEGF and IL-6 between Diffuse Macular Edema and Cystoid Macular Edema
VEGF (pg/ml) IL-6 (pg/ml)
DME (n ⴝ 20)
CME (n ⴝ 6)
P Value
792.6 ⫾ 203.4 171.7 ⫾ 98.4
837.2 ⫾ 278.2 188.1 ⫾ 121.6
0.1645 0.2368
DME ⫽ diffuse macular edema; CME ⫽ cystoid macular edema; VEGF ⫽ vascular endothelial growth factor; IL-6 ⫽ interleukin-6.
decrease of occludin (a membrane-spanning tight junction protein), which could account for the increase of retinal vascular permeability in subjects with DME.4 However, the
Figure 4. A, Vascular endothelial growth factor (VEGF) levels in the vitreous fluid of subjects with minimally fluorescent and hyperfluorescent diabetic macular edema (DME; *P ⫽ 0.0008). B, Interleukin-6 (IL-6) levels in the vitreous fluid of subjects with minimally fluorescent and hyperfluorescent DME (*P ⫽ 0.0038).
absolute terms but also after adjusting for the total vitreous protein level. In addition, there was a significant correlation between the vitreous concentration of IL-6 and that of VEGF. It has been reported that VEGF is expressed by a number of retinal cells20 and that intraocular synthesis is the main factor contributing to the high vitreous VEGF concentration observed in PDR.21 It is also well known that VEGF is a powerful inducer of an increase in vascular permeability22 and that an increase of VEGF in the vitreous causes a
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Figure 5. A, Correlation between the severity of diabetic retinopathy and the vitreous level of vascular endothelial growth factor (VEGF). The severity of retinopathy was evaluated using the Early Treatment Diabetic Retinopathy Study (ETDRS) retinopathy severity scale. VEGF levels in vitreous fluid were significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.648, P⬍0.0001). B, Correlation between the severity of diabetic retinopathy and the vitreous level of interleukin-6 (IL-6). Vitreous fluid levels of IL-6 were significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.596, P⬍0.0001). F ⫽ hyperfluorescent diabetic macular edema; E ⫽ minimally fluorescent diabetic macular edema.
Funatsu et al 䡠 Diabetic Macular Edema mechanism that underlies the increase of VEGF production is uncertain. Because VEGF production is known to be upregulated by ischemia, advanced glycation end-products, protein kinase C and insulin-like growth factor-1,23–25 it seems likely that changes of other cytokines might also promote the expression of VEGF. Hyperglycemia stimulates the synthesis and secretion of IL-6 by human peripheral monocytes in vitro.26 In addition, IL-6 has been shown to induce an increase of endothelial permeability in vitro secondary to rearrangement of actin filaments and morphological changes of endothelial cells.6 Furthermore, the ability of VEGF to increase vascular permeability suggests that an IL-6-induced increase of VEGF production could contribute to DME. The following possibilities can be suggested: (1) both VEGF and IL-6 may directly cause an increase of vascular permeability, (2) IL-6 may indirectly cause an increase of vascular permeability via upregulation of VEGF, and (3) VEGF alone may cause vascular permeability to increase, with the elevated vitreous level of IL-6 being related to hyperglycemia and not having an influence on vascular permeability. Therefore, further investigations are needed to clarify the ocular interaction between IL-6 and VEGF as well as the role of IL-6 in the pathogenesis of DME. We have also suggested that the levels of VEGF and IL-6 in the vitreous fluid might vary with respect to the pattern of fluorescein leakage in subjects with DME. In the present study, we found that the vitreous levels of VEGF and IL-6 were significantly higher in hyperfluorescent DME than in minimally fluorescent DME. In subjects with DME, breakdown of the BRB is followed by leakage of intraretinal fluid from abnormal retinal capillaries. Detection of hyperfluorescence on FA reflects the leakage of fluorescein associated with BRB breakdown, and minimally fluorescent DME represents a different phase or stage of the disease. It is possible that minimally fluorescent DME is an earlier phase of this disease, or it could be resolving after treatment. By classifying DME according to the pattern of fluorescein leakage on FA, we showed that the levels of both VEGF and IL-6 in the vitreous fluid were higher in subjects with higher vascular permeability. In our previous study, VEGF and IL-6 levels in the aqueous humor were significantly correlated with the severity of DME (graded as no DME, focal DME, and diffuse DME or CME).8 In subject with hyperfluorescent DME, VEGF and IL-6 may induce an increase of vascular permeability and stimulate cellular migration into the attached premacular posterior vitreous hyaloid. These cells in the posterior hyaloid could subsequently produce growth factors that induce an increase of vascular permeability.27 Further investigations are needed to compare subjects with and without PVD, as well as a prospective study on the outcome of vitreous surgery for DME. The underlying biochemical cause of DME in diabetic subjects would seem to be chronic hyperglycemia. Therefore, we suspected that the HbA1c level might show a correlation with the IL-6 concentration in vitreous fluid, but no significant relationship was found. Likewise, the VEGF concentration in the vitreous fluid showed no significant relationship with HbA1c. Because some subjects underwent tightening of glycemic control before vitreous surgery, the
HbA1c level at the time of operation may not necessarily have indicated their long-term glycemic control. Therefore, we could not assess the relationship between hyperglycemia and IL-6 or VEGF from these results. In the present study, we found that the concentrations of both VEGF and IL-6 were significantly higher in the vitreous fluid of subjects with DME than in nondiabetic subjects and that elevation of both VEGF and IL-6 was related to hyperfluorescent DME. In addition, the vitreous level of IL-6 was significantly correlated with that of VEGF. These findings suggest that IL-6 might promote an increase of vascular permeability in DME subjects together with and/or via VEGF. Acknowledgments. We thank Drs Shigehiko Kitano, Erika Shimizu, Hidetaka Noma, Yuichiro Nakanishi, Koji Makita, Kensuke Haruyama, and Shinko Nakamura for their assistance in collecting the vitreous and plasma samples and in performing the ophthalmological examinations. We also thank Drs Yasuhiko Iwamoto and Naoko Iwasaki for their assistance in performing the internal medical examinations. Finally, we would like to thank Katsunori Shimada (Department of Biostatistics, STATZ Institute Co., Ltd.) for his assistance in conducting the statistical analysis.
References 1. Moss SE, Klein R, Klein BE. The 14-year incidence of visual loss in a diabetic population. Ophthalmology 1998;105:998 – 1003. 2. Antcliff RJ, Marshall J. The pathogenesis of edema in diabetic maculopathy. Semin Ophthalmol 1999;14:223–32. 3. Lewis H. The role of vitrectomy in the treatment of diabetic macular edema. Am J Ophthalmol 2001;131:123–5. 4. Penn State Retina Research Group, Antonetti DA, Barber AJ, et al. Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Diabetes 1998;47:1953–9. 5. Cohen T, Nahari D, Cerem LW, et al. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 1996;271:736 – 41. 6. Maruo N, Morita I, Shirao M, Murota S. IL-6 increases endothelial permeability in vitro. Endocrinology 1992;131: 710 – 4. 7. Funatsu H, Yamashita H, Shimizu E, et al. Relationship between vascular endothelial growth factor and interleukin-6 in diabetic retinopathy. Retina 2001;21:469 –77. 8. Funatsu H, Yamashita H, Noma H, et al. Increased levels of vascular endothelial growth factor and interleukin-6 in the aqueous humor of diabetics with macular edema. Am J Ophthalmol 2002;133:70 –7. 9. Aiello LP, Avery RL, Arrigg PG, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994;331: 1480 –7. 10. Early Treatment Diabetic Retinopathy Study Research Group. Grading diabetic retinopathy from stereoscopic color fundus photographs—an extension of the modified Airlie House classification. ETDRS Report Number 10. Ophthalmology 1991; 98(5 suppl):786 – 806. 11. Early Treatment Diabetic Retinopathy Study Research Group. Fundus photographic risk factors for progression of diabetic retinopathy. ETDRS Report Number 12. Ophthalmology 1991;98(5 Suppl):823–33.
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Ophthalmology Volume 110, Number 9, September 2003 12. Funatsu H, Yamashita H, Ikeda T, et al. Relationship of diabetic macular edema to cytokines and posterior vitreous detachment. Am J Ophthalmol 2003;135:321–7. 13. Hyodo I, Doi T, Endo H, et al. Clinical significance plasma vascular endothelial growth factor in gastrointestinal cancer. Eur J Cancer 1998;34:2041–5. 14. Grey A, Mitnick MA, Shapses S, et al. Circulating levels of interleukin-6 and tumor necrosis factor-alpha are elevated in primary hyperperparathyroidism and correlate with markers of bone resorption—a clinical research center study. J Clin Endocrinol Metab 1996;81:3450 – 4. 15. Hernandez C, Burgos R, Canton A, et al. Vitreous levels of vascular cell adhesion molecule and vascular endothelial growth factor in patients with proliferative diabetic retinopathy: a case control study. Diabetes Care 2001;24:516 –21. 16. SAS/STAT Software: changes and enhancements through release 6.12. Cary, NC: SAS Publishing; 1997. 17. Lewis H, Abrams GW, Blumenkranz MS, Campo RV. Vitrectomy for diabetic macular traction and edema associated with posterior hyaloidal traction. Ophthalmology 1992;99: 753–9. 18. Yamamoto T, Akabane N, Takeuchi S. Vitrectomy for diabetic macular edema: the role of posterior vitreous detachment and epimacular membrane. Am J Ophthalmol 2001;132:369 – 77. 19. Tachi N, Ogino N. Vitrectomy for diffuse macular edema in cases of diabetic retinopathy. Am J Ophthalmol 1996;122: 258 – 60.
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20. Lutty GA, McLeod S, Merges C, et al. Localization of vascular endothelial growth factor in human retina and choroid. Arch Ophthalmol 1996;114:971–7. 21. Burgos R, Simo R, Audi L, et al. Vitreous levels of vascular endothelial growth factor are not influenced by its serum concentrations in diabetic retinopathy. Diabetologia 1997;40: 1107–9. 22. Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983–5. 23. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 1992;359:843–5. 24. Aiello LP, Bursell SE, Clermont A, et al. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 1997;46:1473– 80. 25. Punglia RS, Lu M, Hsu J, et al. Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes 1997;46:1619 –26. 26. Morohoshi M, Fujisawa K, Uchimura I, Numano F. Glucosedependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro. Diabetes 1996; 45:954 –9. 27. Jumper MJ, Embabi SN, Toth CA, et al. Electron immunocytochemical analysis of posterior hyaloid associated with diabetic macular edema. Retina 2000;20:63– 8.
Diabetic macular edema (DME) is the most common cause of a decrease in visual acuity among diabetic subjects.1 It has been hypothesized that DME is related to (1) breakdown of the blood–retinal barrier (BRB) and subsequent leakage of intraretinal fluid from abnormal retinal capillaries and microaneurysms, (2) secretion of vasopermeability factors from the retina into the vitreous, and (3) vitreoretinal adhesions and traction on the macula.2,3 Recent studies have Originally received: July 1, 2002. Accepted: March 13, 2003. Manuscript no. 220444. 1 Department of Ophthalmology, Diabetes Center, Tokyo Women’s Medical University, Tokyo, Japan. 2 Department of Ophthalmology, Yamagata University School of Medicine, Yamagata, Japan. 3 Department of Ophthalmology, Hyogo Medical College, Nishinomiya, Japan. 4 Department of Ophthalmology, Tokyo University School of Medicine, Tokyo, Japan. 5 Eguchi Eye Hospital, Hakodate, Japan. 6 Department of Ophthalmology, Tokyo Women’s Medical University, Tokyo, Japan. Supported by a Health Science Research Grant (#10060101 to Drs Funatsu, Hori, and Yamashita) from the Japanese Ministry of Health, Labor and Welfare (Tokyo, Japan). Reprint requests to Hideharu Funatsu, MD, Department of Ophthalmology, Diabetes Center, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: [email protected].
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shown that vascular endothelial growth factor (VEGF) causes conformational changes in the tight junctions of retinal vascular endothelial cells4 and plays a major role in the increase of vascular permeability in diabetic eyes.2 Many of the effects of VEGF are mediated via other factors, and the events that induce VEGF production can initiate a cascade of factors, but it is still unclear how this occurs. Interleukin-6 (IL-6) is a multifunctional cytokine that may indirectly cause an increase of vascular permeability by inducing the expression of VEGF5 or else may directly increase endothelial permeability.6 We previously reported that the levels of both VEGF and IL-6 in the aqueous humor were correlated with the severity of diabetic retinopathy, the grade of fundus findings, and the severity of DME.7,8 Aiello and coauthors9 reported that the vitreous level of VEGF was elevated in subjects with proliferative diabetic retinopathy (PDR) when compared with nondiabetic subjects. Vascular endothelial growth factor levels in the vitreous fluid and aqueous humor may be related to each other, but they are not equivalent. Recently, the number of subjects having vitreous surgery for DME has increased, but the vitreous levels of VEGF or IL-6 in DME subjects without posterior vitreous detachment (PVD) have not been clarified. Accordingly, the present study was performed to investigate whether IL-6 was involved in the exacerbation of DME in subjects without PVD in cooperation with VEGF, which is considered to be the most potent factor promoting ISSN 0161-6420/03/$–see front matter doi:10.1016/S0161-6420(03)00568-2
Funatsu et al 䡠 Diabetic Macular Edema an increase of vascular permeability. We found that the vitreous levels of both IL-6 and VEGF were correlated with the severity of DME and also showed a strong correlation with each other. These results suggest that further investigation of the ocular cytokine network may help to improve our understanding of the pathogenesis of DME.
Research Design and Methods Subjects Undiluted vitreous fluid samples were harvested at the start of vitrectomy after informed consent was obtained from each subject following an explanation of the purpose and potential adverse effects of the procedure. This study was performed in accordance with the Helsinki Declaration of 1975 (the 1983 revision), and the Institutional Review Board of Tokyo Women’s Medical University also approved the protocol for collection of vitreous fluid and blood samples. Vitreous fluid samples were obtained from 26 subjects who had clinically significant DME without PVD and from 12 subjects with nondiabetic ocular disease. The DME subjects included 20 with diffuse macular edemas and 6 with cystoid macular edemas (CMEs). The subjects with nondiabetic ocular disease included 9 who had macular holes and 3 with epiretinal membranes, but none of these 12 subjects had associated proliferative vitreoretinopathy. The diabetic subjects (12 men and 14 women) were 64.2 ⫾ 16.8 years old (mean ⫾ standard deviation [SD]), with a duration of diabetes of 13.8 ⫾ 6.5 years and a hemoglobin A1c (HbA1c) of 7.6 ⫾ 2.0%. The 12 subjects with nondiabetic ocular disease (5 men and 7 women) were 62.8 ⫾ 13.6 years old. There was no significant age difference between the diabetic and nondiabetic subjects (P ⫽ 0.6012). Exclusion criteria included (1) previous ocular surgery, (2) a history of ocular inflammation, (3) PDR (such as fibrovascular proliferation), (4) ophthalmic disorders associated with macular edema (such as uveitis and branch or central retinal vein occlusion), (5) DME associated with posterior hyaloid traction, and (6) clinically detectable posterior vitreous separation (defined as vitreous separation at the disc or a visible Weiss ring). All operations were performed at Tokyo Women’s Medical University Hospital.
Fundus Findings All subjects with DME and diabetic retinopathy presenting to the Diabetes Center at Tokyo Women’s Medical University Hospital were evaluated by careful biomicroscopic examination using a fundus contact lens, and subjects who had clinically significant DME without PVD were enrolled in this study. Fundus findings were confirmed preoperatively by standardized fundus color photography and fluorescein angiography (FA), which was performed with a Topcon TRC-501A fundus camera, an image-net system (Tokyo Optical Co Ltd., Tokyo, Japan), and a preset lens with a slit lamp.7,8 The severity of diabetic retinopathy was graded according to the modified Early Treatment Diabetic Retinopathy Study retinopathy severity scale.10,11
According to this scale, 3 eyes were level 35, 6 eyes were level 43, 9 eyes were level 47, and 8 eyes were level 53. All subjects with DME underwent retinal photocoagulation (mean ⫽ 592 shots; range ⫽ 186 –1112 shots) before vitreous surgery. Diffuse DME was diagnosed by the detection of leakage from diffusely dilated retinal capillaries throughout the posterior pole. A diagnosis of CME was indicated by a petalloid pattern of hyperfluorescence. Diabetic macular edema was classified as hyperfluorescent (18 eyes) if there was extensive fluorescein leakage in the macular region on FA, and as minimally fluorescent (8 eyes) if there was little fluorescein leakage.12 Posterior vitreous detachment was diagnosed intraoperatively: subjects without a Weiss ring or vitreoretinal separation had no PVD, whereas those with a Weiss ring had PVD, and those with posterior detachment of the vitreous from the retina (excluding the macular region) had partial PVD.12
Sample Collection Samples of vitreous fluid were collected into sterile tubes and were rapidly frozen at – 80°C. These samples were obtained at the time of vitreoretinal surgery. Plasma samples were also collected from the 26 subjects with DME. Blood was immediately placed on ice and centrifuged at 3000 g for 5 minutes at 4°C, after which the plasma was rapidly frozen and stored at – 80°C until assay.
Measurement of IL-6 and VEGF Levels Both IL-6 and VEGF levels were measured in vitreous samples from the same eye as well as in plasma samples by enzyme-linked immunosorbent assay (ELISA) using kits for human IL-6 and VEGF (R&D System, Minneapolis, MN).13,14 The VEGF kit was able to detect 2 of the 4 VEGF isoforms (VEGF121 and VEGF165), probably because these 2 shorter isoforms are secreted and the 2 longer isoforms are cell associated. Each assay was performed according to the manufacturer’s instructions. The standard solution (100 l for the VEGF ELISA and 200 l for the IL-6 ELISA) and the sample (10 or 100 l for the VEGF ELISA and 20 or 200 l for the IL-6 ELISA) were added to the wells of a 96-well plate coated with amonoclonal antibody. After incubation, the plate was washed, and an enzyme-labeled antibody was added. After further incubation, the plate was washed again, and the substrate was added. The reaction was stopped by adding the stop solution after color had developed, and the optical density was determined at 450 and 620 nm using an absorption spectrophotometer (Titertek Multiscan MCC/340; ICN, Tokyo, Japan). A standard curve was plotted from measurements made with the standard solution (from 15.6 to 1000 pg/ml for VEGF and from 0.156 to 100 pg/ml for IL-6) and was used to determine the concentration of VEGF or IL-6 in the sample. The levels of these factors in vitreous fluid and plasma were within the detection range of the relevant assay, with the minimum detectable concentration being 15.6 pg/ml for VEGF (the intra-assay coefficient of variation [CV] was 5.5% and the
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Ophthalmology Volume 110, Number 9, September 2003 interassay CV was 6.8%) and 0.156 pg/ml for IL-6 (intraassay CV of 5.6% and interassay CV of 6.9%).
Measurement of Protein The vitreous fluid level of protein (mg/ml) was measured by a previously validated microturbidimetric method using an autoanalyzer (Hitachi [Tokyo, Japan] 917).15 This method was based on the benzetonium chloride reaction and was highly specific for the detection of protein, showing a higher sensitivity and reproducibility than the classic Lowry method. The detection limit was 0.02 mg/ml, whereas the intra-assay and interassay CVs were 3.3 and 3.9%, respectively.
Statistical Analysis All analyses were performed with SAS System 6.12 software (SAS Institute, Inc., Cary, NC).16 Data are presented as frequencies or as the mean ⫾ SD. Data with a skewed distribution were transformed to a logarithmic scale, and the geometric mean was calculated together with the one SD range on either side of the mean. The Mann-Whitney U test was used to compare IL-6 and VEGF concentrations between the diabetic subjects and control subjects. To examine correlations, Spearman’s rank correlation coefficients were calculated by linear regression analysis. A two-tailed P value of ⬍0.05 was considered to indicate statistical significance.
Results Vitreous Levels of IL-6 and VEGF Vitreous fluid protein levels (median [range]) were significantly higher in the diabetic subjects with DME than in the nondiabetic control subjects (3.12 mg/ml [0.55– 8.42] vs. 0.55 mg/ml [0.21– 2.42], P ⫽ 0.0008). The vitreous fluid concentration of VEGF was also significantly elevated in the subjects with DME (818.0 pg/ml [36.8 –1902.4]) when compared with the control subjects (17.8 pg/ml [15.6 –26.2], P⬍0.0001, Fig 1A). Furthermore, the VEGF/ protein ratio showed a significant difference between the subjects with DME (263.6 pg/mg [24.2–748.3]) and the control subjects (20.2 pg/mg [5.6 –19.4], P⬍0.0001; Fig 1B). Vitreous fluid concentrations of IL-6 were also significantly elevated in the subjects with DME (188.1 pg/ml [18.0 –768.4]) when compared with control subjects (9.51 pg/ml [4.02–22.4], P⬍0.0001; Fig 2A). Moreover, the IL-6/protein ratio showed a significant difference between the subjects with DME (63.3 pg/mg [5.2–243.6]) and the control subjects (3.15 pg/mg [1.14 – 8.24], P⬍0.0001; Fig 2B). There was also a significant relationship between the vitreous concentration of VEGF and that of IL-6 in the diabetic subjects ( ⫽ 0.678, P⬍0.0001; Fig 3). Furthermore, the vitreous levels of VEGF and IL-6 detected in the subjects with hyperfluorescent DME (VEGF: 1008.6 pg/ml [52.8 –1902.4]; IL-6: 228.6 pg/ml [18.0 –768.4]) were significantly higher (P ⫽ 0.0008 and P ⫽ 0.0038, respectively) than those found in the subjects with minimally fluorescent DME (VEGF: 498.2 pg/ml [36.8 – 898.2]; IL-6: 98.6 pg/ml [18.0 –250.2]; Fig 4A, B). There was no significant difference of the vitreous VEGF concentration between DME subjects (792.6 pg/ml [36.8 –1591.2])
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Figure 1. A, Vascular endothelial growth factor (VEGF) concentration in the vitreous fluid of nondiabetic control subjects and subjects with diabetic macular edema (DME; *P ⬍ 0.0001). B, VEGF concentration per milligram of protein in the vitreous fluid of control subjects and DME subjects (*P⬍0.0001).
and CME subjects (837.2 pg/ml [36.8 –1902.4], P⫽0.1645; Table 1). There was also no significant difference of the vitreous IL-6 concentration between DME subjects (171.7 pg/ml [18.0 – 602.3]) and CME subjects (188.1 pg/ml [18.0 –768.4], P ⫽ 0.2638; Table 1). The vitreous level of VEGF was significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.648, P⬍0.0001; Fig 5A), and the vitreous level of IL-6 was also significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.596, P⬍0.0001; Fig 5B).
Funatsu et al 䡠 Diabetic Macular Edema
Figure 3. Relationship between the vitreous levels of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in subjects with diabetic macular edema ( ⫽ 0.678, P⬍0.0001).
Discussion
Figure 2. A, Interleukin-6 (IL-6) concentration in the vitreous fluid of nondiabetic control subjects and subjects with diabetic macular edema (DME; *P⬍0.0001). B, IL-6 concentration per milligram of protein in the vitreous fluid of control subjects and DME subjects (*P⬍0.0001).
Vitreous and Plasma Levels of IL-6 and VEGF The vitreous fluid concentration of VEGF was significantly higher than the plasma VEGF level (48.2 pg/ml [15.6 –344.0]) in the subjects with DME (P⬍0.0001), and the vitreous fluid concentration of IL-6 was also significantly higher than the plasma IL-6 level (17.5 pg/ml [4.0 – 46.0]) in these subjects (P ⫽ 0.0046). There was no significant relationship between HbA1c (7.6% [4.8 – 12.3]) and the vitreous levels of VEGF or IL-6 ( ⫽ 0.220, P ⫽ 0.1712 and ⫽ 0.253, P ⫽ 0.0626, respectively).
In the present and previous studies,7,8 we have shown that aqueous levels of both IL-6 and VEGF are correlated not only with the severity of diabetic retinopathy and the grade of fundus findings, such as retinal hemorrhages and hard exudates, but also with the severity of DME. We have also shown that the aqueous level of IL-6 is significantly correlated with that of VEGF.7,8 Furthermore, both VEGF and IL-6 concentrations are higher in the aqueous humor than in the plasma.7,8 Although it has been reported that posterior hyaloid traction on the macula may play a role in the development and progression of DME and that vitrectomy is beneficial for DME associated with posterior hyaloid traction,3,17 vitrectomy was recently shown to be an effective procedure for reducing DME in eyes with complete PVD or eyes without visible evidence of posterior hyaloid thickening and traction.3,18,19 Diabetic macular edema is also influenced by the severity of diabetic retinopathy, including the extent of proliferative changes. Therefore, we excluded subject who had DME with posterior hyaloid thickening and traction, complete PVD, or PDR, and we investigated the relationship between IL-6 and VEGF levels in the vitreous fluid of DME subjects without PVD who had nonproliferative diabetic retinopathy. We also investigated whether there was a significant difference in the vitreous levels of IL-6 and VEGF between hyperfluorescent and minimally fluorescent DME, because the fluorescence on FA reflects changes of vascular permeability. This study showed that the vitreous fluid levels of both VEGF and IL-6 were significantly elevated in DME subjects when compared with nondiabetic subjects, not only in
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Ophthalmology Volume 110, Number 9, September 2003 Table 1. Comparison of Vitreous Levels of VEGF and IL-6 between Diffuse Macular Edema and Cystoid Macular Edema
VEGF (pg/ml) IL-6 (pg/ml)
DME (n ⴝ 20)
CME (n ⴝ 6)
P Value
792.6 ⫾ 203.4 171.7 ⫾ 98.4
837.2 ⫾ 278.2 188.1 ⫾ 121.6
0.1645 0.2368
DME ⫽ diffuse macular edema; CME ⫽ cystoid macular edema; VEGF ⫽ vascular endothelial growth factor; IL-6 ⫽ interleukin-6.
decrease of occludin (a membrane-spanning tight junction protein), which could account for the increase of retinal vascular permeability in subjects with DME.4 However, the
Figure 4. A, Vascular endothelial growth factor (VEGF) levels in the vitreous fluid of subjects with minimally fluorescent and hyperfluorescent diabetic macular edema (DME; *P ⫽ 0.0008). B, Interleukin-6 (IL-6) levels in the vitreous fluid of subjects with minimally fluorescent and hyperfluorescent DME (*P ⫽ 0.0038).
absolute terms but also after adjusting for the total vitreous protein level. In addition, there was a significant correlation between the vitreous concentration of IL-6 and that of VEGF. It has been reported that VEGF is expressed by a number of retinal cells20 and that intraocular synthesis is the main factor contributing to the high vitreous VEGF concentration observed in PDR.21 It is also well known that VEGF is a powerful inducer of an increase in vascular permeability22 and that an increase of VEGF in the vitreous causes a
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Figure 5. A, Correlation between the severity of diabetic retinopathy and the vitreous level of vascular endothelial growth factor (VEGF). The severity of retinopathy was evaluated using the Early Treatment Diabetic Retinopathy Study (ETDRS) retinopathy severity scale. VEGF levels in vitreous fluid were significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.648, P⬍0.0001). B, Correlation between the severity of diabetic retinopathy and the vitreous level of interleukin-6 (IL-6). Vitreous fluid levels of IL-6 were significantly correlated with the severity of diabetic retinopathy ( ⫽ 0.596, P⬍0.0001). F ⫽ hyperfluorescent diabetic macular edema; E ⫽ minimally fluorescent diabetic macular edema.
Funatsu et al 䡠 Diabetic Macular Edema mechanism that underlies the increase of VEGF production is uncertain. Because VEGF production is known to be upregulated by ischemia, advanced glycation end-products, protein kinase C and insulin-like growth factor-1,23–25 it seems likely that changes of other cytokines might also promote the expression of VEGF. Hyperglycemia stimulates the synthesis and secretion of IL-6 by human peripheral monocytes in vitro.26 In addition, IL-6 has been shown to induce an increase of endothelial permeability in vitro secondary to rearrangement of actin filaments and morphological changes of endothelial cells.6 Furthermore, the ability of VEGF to increase vascular permeability suggests that an IL-6-induced increase of VEGF production could contribute to DME. The following possibilities can be suggested: (1) both VEGF and IL-6 may directly cause an increase of vascular permeability, (2) IL-6 may indirectly cause an increase of vascular permeability via upregulation of VEGF, and (3) VEGF alone may cause vascular permeability to increase, with the elevated vitreous level of IL-6 being related to hyperglycemia and not having an influence on vascular permeability. Therefore, further investigations are needed to clarify the ocular interaction between IL-6 and VEGF as well as the role of IL-6 in the pathogenesis of DME. We have also suggested that the levels of VEGF and IL-6 in the vitreous fluid might vary with respect to the pattern of fluorescein leakage in subjects with DME. In the present study, we found that the vitreous levels of VEGF and IL-6 were significantly higher in hyperfluorescent DME than in minimally fluorescent DME. In subjects with DME, breakdown of the BRB is followed by leakage of intraretinal fluid from abnormal retinal capillaries. Detection of hyperfluorescence on FA reflects the leakage of fluorescein associated with BRB breakdown, and minimally fluorescent DME represents a different phase or stage of the disease. It is possible that minimally fluorescent DME is an earlier phase of this disease, or it could be resolving after treatment. By classifying DME according to the pattern of fluorescein leakage on FA, we showed that the levels of both VEGF and IL-6 in the vitreous fluid were higher in subjects with higher vascular permeability. In our previous study, VEGF and IL-6 levels in the aqueous humor were significantly correlated with the severity of DME (graded as no DME, focal DME, and diffuse DME or CME).8 In subject with hyperfluorescent DME, VEGF and IL-6 may induce an increase of vascular permeability and stimulate cellular migration into the attached premacular posterior vitreous hyaloid. These cells in the posterior hyaloid could subsequently produce growth factors that induce an increase of vascular permeability.27 Further investigations are needed to compare subjects with and without PVD, as well as a prospective study on the outcome of vitreous surgery for DME. The underlying biochemical cause of DME in diabetic subjects would seem to be chronic hyperglycemia. Therefore, we suspected that the HbA1c level might show a correlation with the IL-6 concentration in vitreous fluid, but no significant relationship was found. Likewise, the VEGF concentration in the vitreous fluid showed no significant relationship with HbA1c. Because some subjects underwent tightening of glycemic control before vitreous surgery, the
HbA1c level at the time of operation may not necessarily have indicated their long-term glycemic control. Therefore, we could not assess the relationship between hyperglycemia and IL-6 or VEGF from these results. In the present study, we found that the concentrations of both VEGF and IL-6 were significantly higher in the vitreous fluid of subjects with DME than in nondiabetic subjects and that elevation of both VEGF and IL-6 was related to hyperfluorescent DME. In addition, the vitreous level of IL-6 was significantly correlated with that of VEGF. These findings suggest that IL-6 might promote an increase of vascular permeability in DME subjects together with and/or via VEGF. Acknowledgments. We thank Drs Shigehiko Kitano, Erika Shimizu, Hidetaka Noma, Yuichiro Nakanishi, Koji Makita, Kensuke Haruyama, and Shinko Nakamura for their assistance in collecting the vitreous and plasma samples and in performing the ophthalmological examinations. We also thank Drs Yasuhiko Iwamoto and Naoko Iwasaki for their assistance in performing the internal medical examinations. Finally, we would like to thank Katsunori Shimada (Department of Biostatistics, STATZ Institute Co., Ltd.) for his assistance in conducting the statistical analysis.
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Ophthalmology Volume 110, Number 9, September 2003 12. Funatsu H, Yamashita H, Ikeda T, et al. Relationship of diabetic macular edema to cytokines and posterior vitreous detachment. Am J Ophthalmol 2003;135:321–7. 13. Hyodo I, Doi T, Endo H, et al. Clinical significance plasma vascular endothelial growth factor in gastrointestinal cancer. Eur J Cancer 1998;34:2041–5. 14. Grey A, Mitnick MA, Shapses S, et al. Circulating levels of interleukin-6 and tumor necrosis factor-alpha are elevated in primary hyperperparathyroidism and correlate with markers of bone resorption—a clinical research center study. J Clin Endocrinol Metab 1996;81:3450 – 4. 15. Hernandez C, Burgos R, Canton A, et al. Vitreous levels of vascular cell adhesion molecule and vascular endothelial growth factor in patients with proliferative diabetic retinopathy: a case control study. Diabetes Care 2001;24:516 –21. 16. SAS/STAT Software: changes and enhancements through release 6.12. Cary, NC: SAS Publishing; 1997. 17. Lewis H, Abrams GW, Blumenkranz MS, Campo RV. Vitrectomy for diabetic macular traction and edema associated with posterior hyaloidal traction. Ophthalmology 1992;99: 753–9. 18. Yamamoto T, Akabane N, Takeuchi S. Vitrectomy for diabetic macular edema: the role of posterior vitreous detachment and epimacular membrane. Am J Ophthalmol 2001;132:369 – 77. 19. Tachi N, Ogino N. Vitrectomy for diffuse macular edema in cases of diabetic retinopathy. Am J Ophthalmol 1996;122: 258 – 60.
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20. Lutty GA, McLeod S, Merges C, et al. Localization of vascular endothelial growth factor in human retina and choroid. Arch Ophthalmol 1996;114:971–7. 21. Burgos R, Simo R, Audi L, et al. Vitreous levels of vascular endothelial growth factor are not influenced by its serum concentrations in diabetic retinopathy. Diabetologia 1997;40: 1107–9. 22. Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983–5. 23. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 1992;359:843–5. 24. Aiello LP, Bursell SE, Clermont A, et al. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 1997;46:1473– 80. 25. Punglia RS, Lu M, Hsu J, et al. Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes 1997;46:1619 –26. 26. Morohoshi M, Fujisawa K, Uchimura I, Numano F. Glucosedependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro. Diabetes 1996; 45:954 –9. 27. Jumper MJ, Embabi SN, Toth CA, et al. Electron immunocytochemical analysis of posterior hyaloid associated with diabetic macular edema. Retina 2000;20:63– 8.