BASIC INVESTIGATION
Effect of Topical Epigallocatechin Gallate on Corneal Neovascularization in Rabbits Chang Hyun Koh, MD, Hyun Soo Lee, MD, PhD, and Sung Kun Chung, MD, PhD
Purpose: The aim of this study was to evaluate the efficacy of topical application of epigallocatechin gallate (EGCG) for the treatment of corneal neovascularization in a rabbit model.
Methods: Corneal neovascularization was induced in 12 rabbits by placing a black silk suture in the corneal stroma (24 eyes) for a week. After suturing, 1 randomly chosen eye of the 12 rabbits was treated with topical EGCG at 2 different concentrations: 0.01% (group 1) and 0.1% (group 2), whereas the contralateral eyes were treated with sterilized balanced salt solution as the control. All eye drops were applied for 2 weeks after suturing. The suture materials were removed from all eyes on day 7. The surface area of corneal neovascularization was measured and analyzed in all eyes on days 7 and 14. On day 14, all eyes were extracted to measure the concentrations of vascular endothelial growth factor (VEGF) messenger RNA and cyclooxygenase-2 (COX-2) protein.
Results: The surface area of induced corneal neovascularization was significantly smaller only in group 2 compared with that of the control group on days 7 and 14 (P , 0.001). The change in surface area of corneal neovascularization after removal of the suture material was not significantly different between all 3 groups. VEGF messenger RNA levels were significantly lower in group 2 than in the control group (P , 0.001). The concentration of COX-2 was significantly lower in group 2 than in the control group (P = 0.043), but no significant difference was observed between group 1 and the control group. Conclusions: Topical administration of EGCG effectively inhibits corneal neovascularization in rabbits. This inhibitory effect is probably related to the suppression of VEGF and COX-2 meditated angiogenesis. Key Words: corneal neovascularization, epigallocatechin gallate, VEGF, COX-2 (Cornea 2014;33:527–532)
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insults to the cornea.1–3 Angiogenic factors such as vascular endothelial growth factor (VEGF) play an important role in corneal neovascularization.4 Also, cyclooxygenase-2 (COX-2) has been shown to modulate the expression of the VEGF ligand and its receptors, and this enzyme isoform has been implicated as an important pro-angiogenic protein.5 Various drugs and chemical compounds have been suggested for the treatment of corneal neovascularization including steroids,6,7 bevacizumab,8 infliximab,9 methotrexate,10 sorafenib,11 and suramin.12 Still, there has been no definitive treatment of choice in clinical practice. Epigallocatechin gallate (EGCG), a secondary metabolite of Camellia sinensis, is a flavonoid present at high levels in green tea. This compound has been widely studied for its chemopreventive, antioxidative, and anti-inflammatory properties.13–16 Several studies have also reported that EGCG has antiangiogenic properties.17–19 However, most studies have used EGCG in oral form; no previous study has reported the antiangiogenic effect of topical EGCG eye drops. In this study, we examined whether topical administration of EGCG was effective in treating corneal neovascularization in a rabbit model.
MATERIALS AND METHODS This study was approved by the Institutional Review Board for animal experimentation at the Catholic University of Korea, St Mary’s Hospital. All in vivo experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Experimental Animals The study was conducted on 12 New Zealand white rabbits (24 eyes) weighing between 2.0 and 2.5 kg (Doo Yeol Biotech, Seoul, South Korea). All rabbits were confirmed to have normal corneas with no abnormalities before treatment.
orneal neovascularization is a condition of the cornea where blood vessels start growing into the otherwise clear and translucent cornea. This condition is usually affected by infectious, degenerative, traumatic, toxic, and inflammatory
Induction of Corneal Neovascularization
Received for publication November 25, 2013; revision received January 27, 2014; accepted January 30, 2014. Published online ahead of print March 7, 2014. From the Department of Ophthalmology, St Mary’s Hospital, College of Medicine, the Catholic University of Korea, Seoul, Korea. The authors have no funding or conflicts of interest to disclose. Reprints: Sung Kun Chung, Department of Ophthalmology, St Mary’s Hospital, College of Medicine, the Catholic University of Korea, #63-ro 10, Yeongdeungpo-gu, Seoul 130-709, Korea (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
All procedures were performed with animals under general anesthesia induced by intramuscular tiletamine (5 mg/kg body weight) and zolazepam (5 mg/kg body weight). Also, proparacaine (Alcaine; Alcon, Fort Worth, TX) was used for topical anesthesia to prevent and minimize possible movements. A 3-mm long corneal suture was made horizontally 1 mm away from the corneal limbus in the 12-o’clock direction with 7-0 black silk (Softsilk; Syneture, Quebec, Canada). All suture materials were placed bilaterally. After suturing, topical levofloxacin (Oculevo; Samil, Seoul, Korea) was applied
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4 times a day for 1 week to prevent infection. On day 7, after confirmation of corneal neovascularization in each rabbit eye, the suture material was removed.
Treatment With EGCG Eye Drops Twelve rabbits were divided into 2 groups of 6 rabbits each. One eye of each rabbit was randomly chosen to be the experimental eye, with the other eye serving as the control. Topical EGCG (Sigma–Aldrich, St Louis, MO) was diluted with 1% dimethyl sulfoxide in phosphate-buffered saline. The control eyes received 1% dimethyl sulfoxide in phosphatebuffered saline, whereas the experimental eyes were treated with topical EGCG at 2 different concentrations: 0.01% (0.01 mg/mL; group 1) and 0.1% (0.1 mg/mL; group 2). All eye drops were applied 4 times daily for 2 weeks after suturing.
Analysis of the Surface Area of Corneal Neovascularization Photographs of the area of corneal neovascularization were taken on days 7 and 14, using a camera (D-7; Contax, Stuttgart, Germany) attached to a light microscope (S21; Carl Zeiss, Jena, Germany) with ·25 magnification. We used ImageJ software (Wayne Rasband at the Research Services Branch, National Institute of Mental Health, Bethesda, MD) to analyze the vascularized corneal area in terms of pixels on the digital photographs. The change in neovascularized surface area after removal of the suture material was also measured between the day 7 and 14 photographs.
Measurement of the Concentrations of Corneal VEGF Messenger RNA and COX-2 Protein On day 14, all eyes were extracted and the neovascularized corneal tissues were sectioned to a 3 · 3-mm size and stored in a 270°C freezer. Half of the samples were homogenized in TRIzol reagent (Gibco BRL, Grand Island, NY) to extract total messenger RNA (mRNA). Single-stranded complementary DNA was synthesized by the reverse transcriptase– polymerase chain reaction (PCR) using a First-Strand Synthesis System (Superscript III; Invitrogen, Carlsbad, CA) and random primers, and the resulting complementary DNA was used as a template for PCR. Expression of the target gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR amplification was performed with a primer (sense, 59-AGCGGAGAAAGCATTTGTTT-39; antisense, 59-TGCAACGCGACTCTGTGTTT-39) for VEGF mRNA. The conditions were a 5-minute hot start at 94°C, followed by 30 cycles of denaturation for 1 minute at 94°C, annealing for 1 minute at 58°C, and extension for 1 minute at 72°C. Amplified products were separated by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining. To investigate the relative expression of VEGF, band densities were assessed by densitometric analysis (ImageMaster VDS 2.0; Pharmacia Biotech Inc, San Francisco, CA). For Western blot analysis, the other half of the neovascularized corneal samples was placed in 100 mL lysis
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buffer (Proprep Protein Extraction Solution; iNtRON Biotechnology, Sungnam, Korea) and homogenized using a Precellys 24-bead–based homogenizer (Bertin Technologies, Villeurbanne, France). Fifteen-microgram aliquots of protein per sample were electrophoresed through 10% sodium dodecyl sulfate–polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) and blocked with a 3% bovine serum albumin solution. Immunoblotting was performed using mouse monoclonal antibodies, anti–COX-2 and anti-b-actin (Santa Cruz Biotechnology, Santa Cruz, CA). After washing with Tris-buffered saline–Tween 0.05%, blots were incubated with the respective secondary peroxidase-labeled antibodies for 1 hour at room temperature. The blots were then washed 4 times with Tris-buffered saline– Tween 0.05% and processed for chemiluminescence detection of immunoreactive proteins by Western blot analysis using a peroxidase substrate (Lumigen PS-3; Lumigen, Southfield, MI). The densities of immunoreactive bands were measured by densitometric analysis (ImageMaster VDS 2.0; Pharmacia Biotech Inc, San Francisco, CA).
Statistical Analysis The Mann–Whitney U test was used to evaluate the significance of differences among groups regarding the surface area of corneal neovascularization and the concentrations of VEGF mRNA and COX-2 protein. SPSS 15.0 software was used for statistical analyses (SPSS Inc, Chicago, IL). P , 0.05 was considered statistically significant.
RESULTS Surface Area of Corneal Neovascularization After suture placement, corneal neovascularization was induced in all rabbits. No infections or other complications were detected during the study period. The mean surface areas of corneal neovascularization were measured at the time when the corneal suture material was removed on day 7 and at the end of the follow-up period on day 14 (Fig. 1). On day 7, the mean surface area of corneal neovascularization in the control group was 141.34 6 18.02 pixels; in group 1, 130.58 6 21.61 pixels; and in group 2, 118.13 6 14.27 pixels. The mean surface area of corneal neovascularization in group 2 was significantly smaller than that in the control group (P , 0.001). On day 14, the mean surface area of corneal neovascularization in the control group was 78.71 6 15.71 pixels; in group 1, 67.34 6 18.08 pixels; and in group 2, 42.97 6 11.28 pixels. Group 2 showed a significantly smaller surface area of corneal neovascularization than in the control group (P , 0.001). However, the mean surface area of corneal neovascularization in group 1 was not significantly different than that in the control group on either day 7 or day 14 (day 7, P = 0.425; day 14, P = 0.796) (Table 1). No statistically significant differences were found between group 1 and group 2 on day 7 and on day 14 (day 7, P = 0.127; day 14, P = 0.071). The change in surface area of corneal neovascularization after removal of the suture material was also measured. 2014 Lippincott Williams & Wilkins
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FIGURE 1. Microscopic examination of the neovascularized area of the cornea. The control group (A, D), the group treated with topical 0.01% EGCG (B, E) and the group treated with topical 0.1% EGCG (C, F). One week after treatment, the suture material was removed. Corneal neovascularization was induced in all groups (A–C), but the neovascularized area of the group treated with topical 0.1% EGCG (C) was significantly smaller than that of the control group (A). Two weeks after treatment, the neovascularized area had decreased in size in all groups (D–F). However, only the neovascularized areas of the group treated with topical 0.1% EGCG (F) was significantly smaller than that of the control group (D). The boundaries of the area of corneal neovascularization were highlighted with yellow lines.
The change in surface area of corneal neovascularization in the control group was 63.36 6 16.74 pixels; in group 1, 64.23 6 20.24 pixels; and in group 2, 74.91 6 18.28 pixels. There was no significant difference between group 1, group 2, and the control group (group 1, P = 0.816; group 2, P = 0.107) (Table 2). No statistically significant difference was found between group 1 and group 2 (P = 0.226).
Concentrations of VEGF mRNA and COX-2 Protein
Concentrations of corneal COX-2 protein were measured in each group by Western blot analysis. The mean optical density rate of COX-2 protein was 0.63 6 0.17 in the control group; 0.58 6 0.28 in group 1; and 0.43 6 0.15 in group 2. The concentration of corneal COX-2 protein in group 2 was significantly lower than that in the control group (P = 0.043). However, there was no significant difference between group 1 and the control group (P = 0.562) (Fig. 3). Also, no statistically significant difference was found between group 1 and group 2 (P = 0.191).
Concentrations of corneal VEGF mRNA were compared among groups. The mean optical density rate of VEGF mRNA was 0.57 6 0.13 in the control group; 0.49 6 0.21 in group 1; and 0.24 6 0.09 in group 2. The group 2 showed significantly decreased expression of VEGF mRNA compared with that of the control group (P , 0.001). However, there was no significant difference between group 1 and the control group (P = 0.417) (Fig. 2). Also, no statistically significant difference was found between group 1 and group 2 (P = 0.094).
Corneal neovascularization can be caused by infectious, degenerative, traumatic, toxic, or inflammatory insults and can cause significant visual impairment by reducing the transparency of the cornea.1–3 Several methods to prevent or treat corneal neovascularization have been proposed. Surgical methods, including amniotic membrane transplantation,20 limbal stem-cell transplantation,21 laser photocoagulation,22 and photodynamic therapy23 have been attempted. Furthermore,
TABLE 1. Mean Surface Area of Corneal Neovascularization Before and After the Application of EGCG Eye Drop
TABLE 2. Change in the Surface Area of Corneal Neovascularization After Removal of the Suture Material
DISCUSSION
EGCG-Treated Groups Day 7, pixels P Day 14, pixels P
Control
0.01%
0.1%
141.34 6 18.02
130.58 6 21.61 0.425 67.34 6 18.08 0.796
118.13 6 14.27 ,0.001 42.97 6 11.28 ,0.001
78.71 6 15.71
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EGCG-Treated Groups Control Change in neovascularized surface area, pixels P
0.01%
0.1%
63.36 6 16.74 64.23 6 20.24 74.91 6 18.28
0.816
0.107
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FIGURE 2. Semiquantitative reverse transcriptase– PCR analysis of VEGF mRNA extracted from corneas 2 weeks after treatment. VEGF mRNA expression in the group treated with topical 0.1% EGCG was significantly reduced compared with that of the control group (*P , 0.05).
drug regimens including nonsteroidal anti-inflammatory drugs, thalidomide, prolactin,24 steroids,25 cyclosporine A,26 and angiostatin27 have been used to treat corneal neovascularization. Recently, we investigated the effect of methotrexate,10 ascorbic acid,28 curcumin,29 suramin,12 and a combination treatment of triamcinolone and bevacizumab8 to prevent and treat corneal neovascularization. However, to date, there is no definitive treatment of choice for corneal neovascularization, which highlights the need for further clinical investigations. Angiogenic factors such as VEGF play an important role in corneal neovascularization. This factor regulates proliferation, permeability, and the survival of vascular endothelial cells.30 Under normal conditions, VEGF promotes endothelial migration and development and maintains normal vessels by preventing apoptosis of endothelial cells. However, overexpression of VEGF is associated with several vascular diseases such as diabetic retinopathy,31 corneal neovascularization,2 retinopathy of prematurity,32 and choroidal neovascularization.5 Treatment of these diseases therefore involves reduction of VEGF production or blockage of preexisting VEGF.2
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COX-2 is a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Numerous studies have shown that these eicosanoids, in particular thromboxane A2 and prostaglandin E2, are inducers of VEGF.33–36 Thus, mediators of inflammation such as COX-2 also play a key role in corneal neovascularization. EGCG is a major active component of the polyphenolic fraction of green tea. It has been studied widely for its excellent chemopreventive properties in the context of most types of cancer,15,17 as well as chronic diseases such as pulmonary fibrosis.14 EGCG reduces cardiovascular risk factors by scavenging reactive oxygen species, limiting the proliferation of endothelial cells, inhibiting angiogenesis, and reducing inflammation.13–16 Numerous studies have examined the mechanism by which EGCG reduces inflammation and angiogenesis. Shimizu et al17 and Sen et al18 demonstrated that oral consumption of green tea is associated with a decrease in the activation of various types of tyrosine kinase receptors, such as the VEGF/VEGFR system. Kim et al37 and Singh et al38 reported that EGCG directly inhibits nuclear factor kappa B transcriptional activity, resulting in significant inhibition of 2014 Lippincott Williams & Wilkins
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Effect of Topical Epigallocatechin Gallate
FIGURE 3. Western blot analysis of COX-2 protein extracted from corneas 2 weeks after the treatment. The concentration of COX-2 protein in the group treated with topical 0.1% EGCG was significantly lower than that in the control group (*P , 0.05).
proinflammatory and proangiogenic mediators, including interleukin 1b, matrix metalloproteinases, VEGF, and COX-2. In this study, we examined the effect of topical EGCG on corneal neovascularization in a rabbit model. Topical application of 0.1% EGCG eye drops reduced the development of corneal neovascularization after suture placement. However, it did not significantly stimulate the regression of newly formed vessels after removal of the suture material. These findings suggest that the antiangiogenic mechanism of EGCG is mostly related to the blockage of earlier phases of corneal neovascularization. The concentrations of corneal VEGF mRNA and COX2 protein were significantly lower in the group treated with 0.1% EGCG than in the control group. However, the group treated with 0.01% EGCG eye drop did not show a significant difference from the control group in the surface area of corneal neovascularization, the VEGF mRNA expression, and the amount of COX-2 protein. Therefore, when using topical EGCG eye drop for patients with corneal neovascularization, the concentration needs to be above 0.1%. Although we demonstrated that EGCG treatment inhibited VEGF mRNA expression and COX-2 activation and reduced the induction of corneal neovascularization in topical 2014 Lippincott Williams & Wilkins
EGCG-treated eyes, the precise mechanism by which EGCG exerts these antiangiogenic effects was not examined. We were only able to determine that the antiangiogenic effect of EGCG was related to VEGF expression and COX-2 activation. In addition, the study was limited by the small number of rabbits. Furthermore, because the treatment period was only 2 weeks, we could not identify potential complications from long-term application of topical EGCG eye drop. However, this is the first report of the effectiveness of topical EGCG eye drops at preventing corneal neovascularization in a rabbit model. In conclusion, based on our results, topical EGCG eye drops seem to be effective at preventing corneal neovascularization by inhibiting VEGF- and COX-2-mediated angiogenic and inflammatory processes. Further long-term clinical studies are required to determine the precise antiangiogenic mechanisms, target molecules, correct doses, and possible complications of EGCG use. REFERENCES 1. Dana MR, Streilein JW. Loss and restoration of immune privilege in eyes with corneal neovascularization. Invest Ophthalmol Vis Sci. 1996;37: 2485–2494.
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2. Chang JH, Gabison EE, Kato T, et al. Corneal neovascularization. Curr Opin Ophthalmol. 2001;12:242–249. 3. Lee P, Wang CC, Adamis AP. Ocular neovascularization: an epidemiologic review. Surv Ophthalmol. 1998;43:245–269. 4. Azar DT. Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc. 2006;104:264–302. 5. Castro MR, Lutz D, Edelman JL. Effect of COX inhibitors on VEGFinduced retinal vascular leakage and experimental corneal and choroidal neovascularization. Exp Eye Res. 2004;79:275–285. 6. Epstein RJ, Stulting RD, Hendricks RL, et al. Corneal neovascularization. Pathogenesis and inhibition. Cornea. 1987;6:250–257. 7. Crum R, Szabo S, Folkman J. A new class of steroids inhibits angiogenesis in the presence of heparin or a heparin fragment. Science. 1985;230: 1375–1378. 8. Kang S, Chung SK. The effect of subconjuctival combined treatment of bevacizumab and triamcinolone acetonide on corneal neovascularization in rabbits. Cornea. 2010;29:192–196. 9. Kim JW, Chung SK. The effect of topical infliximab on corneal neovascularization in rabbits. Cornea. 2013;32:185–190. 10. Byun YS, Chung SK. The effect of methotrexate on corneal neovascularization in rabbits. Cornea. 2011;30:442–446. 11. Seo JW, Chung SH, Choi JS, et al. Inhibition of corneal neovascularization in rats by systemic administration of sorafenib. Cornea. 2012;31: 907–912. 12. Lee HS, Chung SK. The effect of subconjunctival suramin on corneal neovascularization in rabbits. Cornea. 2010;29:86–92. 13. Valcic S, Muders A, Jacobsen NE, et al. Antioxidant chemistry of green tea catechins. Identification of products of the reaction of (-)-epigallocatechin gallate with peroxyl radicals. Chem Res Toxicol. 1999;12: 382–386. 14. Donà M, Dell’Aica I, Calabrese F, et al. Neutrophil restraint by green tea: inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis. J Immunol. 2003;170:4335–4341. 15. Fujiki H. Green tea: health benefits as cancer preventive for humans. Chem Rec. 2005;5:119–132. 16. Wolfram S. Effects of green tea and EGCG on cardiovascular and metabolic health. J Am Coll Nutr. 2007;26:373S–388S. 17. Shimizu M, Shirakami Y, Moriwaki H. Targeting receptor tyrosine kinases for chemoprevention by green tea catechin, EGCG. Int J Mol Sci. 2008;9:1034–1049. 18. Sen T, Moulik S, Dutta A, et al. Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7. Life Sci. 2009;84:194–204. 19. Rodriguez SK, Guo W, Liu L, et al. Green tea catechin, epigallocatechin3-gallate, inhibits vascular endothelial growth factor angiogenic signaling by disrupting the formation of a receptor complex. Int J Cancer. 2006; 118:1635–1644.
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20. Phillips K, Arffa R, Cintron C, et al. Effects of prednisolone and medroxyprogesterone on corneal wound healing, ulceration, and neovascularization. Arch Ophthalmol. 1983;101:640–643. 21. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2000;343:86–93. 22. Mendelsohn AD, Stock EL, Lo GG, et al. Laser photocoagulation of feeder vessels in lipid keratopathy. Ophthalmic Surg. 1986;17:502–508. 23. Primbs GB, Casey R, Wamser K, et al. Photodynamic therapy for corneal neovascularization. Ophthalmic Surg Lasers. 1998;29:832–838. 24. Riazi-Esfahani M, Peyman GA, Aydin E, et al. Prevention of corneal neovascularization: evaluation of various commercially available compounds in an experimental rat model. Cornea. 2006;25:801–805. 25. Murata M, Shimizu S, Horiuchi S, et al. Inhibitory effect of triamcinolone acetonide on corneal neovascularization. Graefes Arch Clin Exp Ophthalmol. 2006;244:205–209. 26. Benelli U, Ross JR, Nardi M, et al. Corneal neovascularization induced by xenografts or chemical cautery. Inhibition by cyclosporin A. Invest Ophthalmol Vis Sci. 1997;38:274–282. 27. Ambati BK, Joussen AM, Ambati J, et al. Angiostatin inhibits and regresses corneal neovascularization. Arch Ophthalmol. 2002;120:1063–1068. 28. Lee MY, Chung SK. Treatment of corneal neovascularization by topical application of ascorbic acid in the rabbit model. Cornea. 2012;31:1165–1169. 29. Kim JS, Choi JS, Chung SK. The effect of curcumin on corneal neovascularization in rabbit eyes. Curr Eye Res. 2010;35:274–280. 30. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol. 2001;61:253–270. 31. Salam A, Mathew R, Sivaprasad S. Treatment of proliferative diabetic retinopathy with anti-VEGF agents. Acta Ophthalmol. 2011;89:405–411. 32. Leske DA, Wu J, Mookadam M, et al. The relationship of retinal VEGF and retinal IGF-1 mRNA with neovascularization in an acidosis-induced model of retinopathy of prematurity. Curr Eye Res. 2006;31:163–169. 33. Toomey DP, Murphy JF, Conlon KC. COX-2, VEGF and tumour angiogenesis. Surgeon. 2009;7:174–180. 34. Cianchi F, Cortesini C, Bechi P, et al. Up-regulation of cyclooxygenase 2 gene expression correlates with tumor angiogenesis in human colorectal cancer. Gastroenterology. 2001;121:1339–1347. 35. Huang SP, Wu MS, Shun CT, et al. Cyclooxygenase-2 increases hypoxia-inducible factor-1 and vascular endothelial growth factor to promote angiogenesis in gastric carcinoma. J Biomed Sci. 2005;12:229–241. 36. Williams CS, Tsujii M, Reese J, et al. Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest. 2000;105:1589–1594. 37. Kim SJ, Jeong HJ, Lee KM, et al. Epigallocatechin-3-gallate suppresses NF-kappaB activation and phosphorylation of p38 MAPK and JNK in human astrocytoma U373MG cells. J Nutr Biochem. 2007;18:587–596. 38. Singh R, Ahmed S, Islam N, et al. Epigallocatechin-3-gallate inhibits interleukin-1beta-induced expression of nitric oxide synthase and production of nitric oxide in human chondrocytes: suppression of nuclear factor kappaB activation by degradation of the inhibitor of nuclear factor kappaB. Arthritis Rheum. 2002;46:2079–2086.
2014 Lippincott Williams & Wilkins
Effect of Topical Epigallocatechin Gallate on Corneal Neovascularization in Rabbits Chang Hyun Koh, MD, Hyun Soo Lee, MD, PhD, and Sung Kun Chung, MD, PhD
Purpose: The aim of this study was to evaluate the efficacy of topical application of epigallocatechin gallate (EGCG) for the treatment of corneal neovascularization in a rabbit model.
Methods: Corneal neovascularization was induced in 12 rabbits by placing a black silk suture in the corneal stroma (24 eyes) for a week. After suturing, 1 randomly chosen eye of the 12 rabbits was treated with topical EGCG at 2 different concentrations: 0.01% (group 1) and 0.1% (group 2), whereas the contralateral eyes were treated with sterilized balanced salt solution as the control. All eye drops were applied for 2 weeks after suturing. The suture materials were removed from all eyes on day 7. The surface area of corneal neovascularization was measured and analyzed in all eyes on days 7 and 14. On day 14, all eyes were extracted to measure the concentrations of vascular endothelial growth factor (VEGF) messenger RNA and cyclooxygenase-2 (COX-2) protein.
Results: The surface area of induced corneal neovascularization was significantly smaller only in group 2 compared with that of the control group on days 7 and 14 (P , 0.001). The change in surface area of corneal neovascularization after removal of the suture material was not significantly different between all 3 groups. VEGF messenger RNA levels were significantly lower in group 2 than in the control group (P , 0.001). The concentration of COX-2 was significantly lower in group 2 than in the control group (P = 0.043), but no significant difference was observed between group 1 and the control group. Conclusions: Topical administration of EGCG effectively inhibits corneal neovascularization in rabbits. This inhibitory effect is probably related to the suppression of VEGF and COX-2 meditated angiogenesis. Key Words: corneal neovascularization, epigallocatechin gallate, VEGF, COX-2 (Cornea 2014;33:527–532)
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insults to the cornea.1–3 Angiogenic factors such as vascular endothelial growth factor (VEGF) play an important role in corneal neovascularization.4 Also, cyclooxygenase-2 (COX-2) has been shown to modulate the expression of the VEGF ligand and its receptors, and this enzyme isoform has been implicated as an important pro-angiogenic protein.5 Various drugs and chemical compounds have been suggested for the treatment of corneal neovascularization including steroids,6,7 bevacizumab,8 infliximab,9 methotrexate,10 sorafenib,11 and suramin.12 Still, there has been no definitive treatment of choice in clinical practice. Epigallocatechin gallate (EGCG), a secondary metabolite of Camellia sinensis, is a flavonoid present at high levels in green tea. This compound has been widely studied for its chemopreventive, antioxidative, and anti-inflammatory properties.13–16 Several studies have also reported that EGCG has antiangiogenic properties.17–19 However, most studies have used EGCG in oral form; no previous study has reported the antiangiogenic effect of topical EGCG eye drops. In this study, we examined whether topical administration of EGCG was effective in treating corneal neovascularization in a rabbit model.
MATERIALS AND METHODS This study was approved by the Institutional Review Board for animal experimentation at the Catholic University of Korea, St Mary’s Hospital. All in vivo experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Experimental Animals The study was conducted on 12 New Zealand white rabbits (24 eyes) weighing between 2.0 and 2.5 kg (Doo Yeol Biotech, Seoul, South Korea). All rabbits were confirmed to have normal corneas with no abnormalities before treatment.
orneal neovascularization is a condition of the cornea where blood vessels start growing into the otherwise clear and translucent cornea. This condition is usually affected by infectious, degenerative, traumatic, toxic, and inflammatory
Induction of Corneal Neovascularization
Received for publication November 25, 2013; revision received January 27, 2014; accepted January 30, 2014. Published online ahead of print March 7, 2014. From the Department of Ophthalmology, St Mary’s Hospital, College of Medicine, the Catholic University of Korea, Seoul, Korea. The authors have no funding or conflicts of interest to disclose. Reprints: Sung Kun Chung, Department of Ophthalmology, St Mary’s Hospital, College of Medicine, the Catholic University of Korea, #63-ro 10, Yeongdeungpo-gu, Seoul 130-709, Korea (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
All procedures were performed with animals under general anesthesia induced by intramuscular tiletamine (5 mg/kg body weight) and zolazepam (5 mg/kg body weight). Also, proparacaine (Alcaine; Alcon, Fort Worth, TX) was used for topical anesthesia to prevent and minimize possible movements. A 3-mm long corneal suture was made horizontally 1 mm away from the corneal limbus in the 12-o’clock direction with 7-0 black silk (Softsilk; Syneture, Quebec, Canada). All suture materials were placed bilaterally. After suturing, topical levofloxacin (Oculevo; Samil, Seoul, Korea) was applied
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4 times a day for 1 week to prevent infection. On day 7, after confirmation of corneal neovascularization in each rabbit eye, the suture material was removed.
Treatment With EGCG Eye Drops Twelve rabbits were divided into 2 groups of 6 rabbits each. One eye of each rabbit was randomly chosen to be the experimental eye, with the other eye serving as the control. Topical EGCG (Sigma–Aldrich, St Louis, MO) was diluted with 1% dimethyl sulfoxide in phosphate-buffered saline. The control eyes received 1% dimethyl sulfoxide in phosphatebuffered saline, whereas the experimental eyes were treated with topical EGCG at 2 different concentrations: 0.01% (0.01 mg/mL; group 1) and 0.1% (0.1 mg/mL; group 2). All eye drops were applied 4 times daily for 2 weeks after suturing.
Analysis of the Surface Area of Corneal Neovascularization Photographs of the area of corneal neovascularization were taken on days 7 and 14, using a camera (D-7; Contax, Stuttgart, Germany) attached to a light microscope (S21; Carl Zeiss, Jena, Germany) with ·25 magnification. We used ImageJ software (Wayne Rasband at the Research Services Branch, National Institute of Mental Health, Bethesda, MD) to analyze the vascularized corneal area in terms of pixels on the digital photographs. The change in neovascularized surface area after removal of the suture material was also measured between the day 7 and 14 photographs.
Measurement of the Concentrations of Corneal VEGF Messenger RNA and COX-2 Protein On day 14, all eyes were extracted and the neovascularized corneal tissues were sectioned to a 3 · 3-mm size and stored in a 270°C freezer. Half of the samples were homogenized in TRIzol reagent (Gibco BRL, Grand Island, NY) to extract total messenger RNA (mRNA). Single-stranded complementary DNA was synthesized by the reverse transcriptase– polymerase chain reaction (PCR) using a First-Strand Synthesis System (Superscript III; Invitrogen, Carlsbad, CA) and random primers, and the resulting complementary DNA was used as a template for PCR. Expression of the target gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR amplification was performed with a primer (sense, 59-AGCGGAGAAAGCATTTGTTT-39; antisense, 59-TGCAACGCGACTCTGTGTTT-39) for VEGF mRNA. The conditions were a 5-minute hot start at 94°C, followed by 30 cycles of denaturation for 1 minute at 94°C, annealing for 1 minute at 58°C, and extension for 1 minute at 72°C. Amplified products were separated by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining. To investigate the relative expression of VEGF, band densities were assessed by densitometric analysis (ImageMaster VDS 2.0; Pharmacia Biotech Inc, San Francisco, CA). For Western blot analysis, the other half of the neovascularized corneal samples was placed in 100 mL lysis
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buffer (Proprep Protein Extraction Solution; iNtRON Biotechnology, Sungnam, Korea) and homogenized using a Precellys 24-bead–based homogenizer (Bertin Technologies, Villeurbanne, France). Fifteen-microgram aliquots of protein per sample were electrophoresed through 10% sodium dodecyl sulfate–polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) and blocked with a 3% bovine serum albumin solution. Immunoblotting was performed using mouse monoclonal antibodies, anti–COX-2 and anti-b-actin (Santa Cruz Biotechnology, Santa Cruz, CA). After washing with Tris-buffered saline–Tween 0.05%, blots were incubated with the respective secondary peroxidase-labeled antibodies for 1 hour at room temperature. The blots were then washed 4 times with Tris-buffered saline– Tween 0.05% and processed for chemiluminescence detection of immunoreactive proteins by Western blot analysis using a peroxidase substrate (Lumigen PS-3; Lumigen, Southfield, MI). The densities of immunoreactive bands were measured by densitometric analysis (ImageMaster VDS 2.0; Pharmacia Biotech Inc, San Francisco, CA).
Statistical Analysis The Mann–Whitney U test was used to evaluate the significance of differences among groups regarding the surface area of corneal neovascularization and the concentrations of VEGF mRNA and COX-2 protein. SPSS 15.0 software was used for statistical analyses (SPSS Inc, Chicago, IL). P , 0.05 was considered statistically significant.
RESULTS Surface Area of Corneal Neovascularization After suture placement, corneal neovascularization was induced in all rabbits. No infections or other complications were detected during the study period. The mean surface areas of corneal neovascularization were measured at the time when the corneal suture material was removed on day 7 and at the end of the follow-up period on day 14 (Fig. 1). On day 7, the mean surface area of corneal neovascularization in the control group was 141.34 6 18.02 pixels; in group 1, 130.58 6 21.61 pixels; and in group 2, 118.13 6 14.27 pixels. The mean surface area of corneal neovascularization in group 2 was significantly smaller than that in the control group (P , 0.001). On day 14, the mean surface area of corneal neovascularization in the control group was 78.71 6 15.71 pixels; in group 1, 67.34 6 18.08 pixels; and in group 2, 42.97 6 11.28 pixels. Group 2 showed a significantly smaller surface area of corneal neovascularization than in the control group (P , 0.001). However, the mean surface area of corneal neovascularization in group 1 was not significantly different than that in the control group on either day 7 or day 14 (day 7, P = 0.425; day 14, P = 0.796) (Table 1). No statistically significant differences were found between group 1 and group 2 on day 7 and on day 14 (day 7, P = 0.127; day 14, P = 0.071). The change in surface area of corneal neovascularization after removal of the suture material was also measured. 2014 Lippincott Williams & Wilkins
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FIGURE 1. Microscopic examination of the neovascularized area of the cornea. The control group (A, D), the group treated with topical 0.01% EGCG (B, E) and the group treated with topical 0.1% EGCG (C, F). One week after treatment, the suture material was removed. Corneal neovascularization was induced in all groups (A–C), but the neovascularized area of the group treated with topical 0.1% EGCG (C) was significantly smaller than that of the control group (A). Two weeks after treatment, the neovascularized area had decreased in size in all groups (D–F). However, only the neovascularized areas of the group treated with topical 0.1% EGCG (F) was significantly smaller than that of the control group (D). The boundaries of the area of corneal neovascularization were highlighted with yellow lines.
The change in surface area of corneal neovascularization in the control group was 63.36 6 16.74 pixels; in group 1, 64.23 6 20.24 pixels; and in group 2, 74.91 6 18.28 pixels. There was no significant difference between group 1, group 2, and the control group (group 1, P = 0.816; group 2, P = 0.107) (Table 2). No statistically significant difference was found between group 1 and group 2 (P = 0.226).
Concentrations of VEGF mRNA and COX-2 Protein
Concentrations of corneal COX-2 protein were measured in each group by Western blot analysis. The mean optical density rate of COX-2 protein was 0.63 6 0.17 in the control group; 0.58 6 0.28 in group 1; and 0.43 6 0.15 in group 2. The concentration of corneal COX-2 protein in group 2 was significantly lower than that in the control group (P = 0.043). However, there was no significant difference between group 1 and the control group (P = 0.562) (Fig. 3). Also, no statistically significant difference was found between group 1 and group 2 (P = 0.191).
Concentrations of corneal VEGF mRNA were compared among groups. The mean optical density rate of VEGF mRNA was 0.57 6 0.13 in the control group; 0.49 6 0.21 in group 1; and 0.24 6 0.09 in group 2. The group 2 showed significantly decreased expression of VEGF mRNA compared with that of the control group (P , 0.001). However, there was no significant difference between group 1 and the control group (P = 0.417) (Fig. 2). Also, no statistically significant difference was found between group 1 and group 2 (P = 0.094).
Corneal neovascularization can be caused by infectious, degenerative, traumatic, toxic, or inflammatory insults and can cause significant visual impairment by reducing the transparency of the cornea.1–3 Several methods to prevent or treat corneal neovascularization have been proposed. Surgical methods, including amniotic membrane transplantation,20 limbal stem-cell transplantation,21 laser photocoagulation,22 and photodynamic therapy23 have been attempted. Furthermore,
TABLE 1. Mean Surface Area of Corneal Neovascularization Before and After the Application of EGCG Eye Drop
TABLE 2. Change in the Surface Area of Corneal Neovascularization After Removal of the Suture Material
DISCUSSION
EGCG-Treated Groups Day 7, pixels P Day 14, pixels P
Control
0.01%
0.1%
141.34 6 18.02
130.58 6 21.61 0.425 67.34 6 18.08 0.796
118.13 6 14.27 ,0.001 42.97 6 11.28 ,0.001
78.71 6 15.71
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EGCG-Treated Groups Control Change in neovascularized surface area, pixels P
0.01%
0.1%
63.36 6 16.74 64.23 6 20.24 74.91 6 18.28
0.816
0.107
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FIGURE 2. Semiquantitative reverse transcriptase– PCR analysis of VEGF mRNA extracted from corneas 2 weeks after treatment. VEGF mRNA expression in the group treated with topical 0.1% EGCG was significantly reduced compared with that of the control group (*P , 0.05).
drug regimens including nonsteroidal anti-inflammatory drugs, thalidomide, prolactin,24 steroids,25 cyclosporine A,26 and angiostatin27 have been used to treat corneal neovascularization. Recently, we investigated the effect of methotrexate,10 ascorbic acid,28 curcumin,29 suramin,12 and a combination treatment of triamcinolone and bevacizumab8 to prevent and treat corneal neovascularization. However, to date, there is no definitive treatment of choice for corneal neovascularization, which highlights the need for further clinical investigations. Angiogenic factors such as VEGF play an important role in corneal neovascularization. This factor regulates proliferation, permeability, and the survival of vascular endothelial cells.30 Under normal conditions, VEGF promotes endothelial migration and development and maintains normal vessels by preventing apoptosis of endothelial cells. However, overexpression of VEGF is associated with several vascular diseases such as diabetic retinopathy,31 corneal neovascularization,2 retinopathy of prematurity,32 and choroidal neovascularization.5 Treatment of these diseases therefore involves reduction of VEGF production or blockage of preexisting VEGF.2
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COX-2 is a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Numerous studies have shown that these eicosanoids, in particular thromboxane A2 and prostaglandin E2, are inducers of VEGF.33–36 Thus, mediators of inflammation such as COX-2 also play a key role in corneal neovascularization. EGCG is a major active component of the polyphenolic fraction of green tea. It has been studied widely for its excellent chemopreventive properties in the context of most types of cancer,15,17 as well as chronic diseases such as pulmonary fibrosis.14 EGCG reduces cardiovascular risk factors by scavenging reactive oxygen species, limiting the proliferation of endothelial cells, inhibiting angiogenesis, and reducing inflammation.13–16 Numerous studies have examined the mechanism by which EGCG reduces inflammation and angiogenesis. Shimizu et al17 and Sen et al18 demonstrated that oral consumption of green tea is associated with a decrease in the activation of various types of tyrosine kinase receptors, such as the VEGF/VEGFR system. Kim et al37 and Singh et al38 reported that EGCG directly inhibits nuclear factor kappa B transcriptional activity, resulting in significant inhibition of 2014 Lippincott Williams & Wilkins
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FIGURE 3. Western blot analysis of COX-2 protein extracted from corneas 2 weeks after the treatment. The concentration of COX-2 protein in the group treated with topical 0.1% EGCG was significantly lower than that in the control group (*P , 0.05).
proinflammatory and proangiogenic mediators, including interleukin 1b, matrix metalloproteinases, VEGF, and COX-2. In this study, we examined the effect of topical EGCG on corneal neovascularization in a rabbit model. Topical application of 0.1% EGCG eye drops reduced the development of corneal neovascularization after suture placement. However, it did not significantly stimulate the regression of newly formed vessels after removal of the suture material. These findings suggest that the antiangiogenic mechanism of EGCG is mostly related to the blockage of earlier phases of corneal neovascularization. The concentrations of corneal VEGF mRNA and COX2 protein were significantly lower in the group treated with 0.1% EGCG than in the control group. However, the group treated with 0.01% EGCG eye drop did not show a significant difference from the control group in the surface area of corneal neovascularization, the VEGF mRNA expression, and the amount of COX-2 protein. Therefore, when using topical EGCG eye drop for patients with corneal neovascularization, the concentration needs to be above 0.1%. Although we demonstrated that EGCG treatment inhibited VEGF mRNA expression and COX-2 activation and reduced the induction of corneal neovascularization in topical 2014 Lippincott Williams & Wilkins
EGCG-treated eyes, the precise mechanism by which EGCG exerts these antiangiogenic effects was not examined. We were only able to determine that the antiangiogenic effect of EGCG was related to VEGF expression and COX-2 activation. In addition, the study was limited by the small number of rabbits. Furthermore, because the treatment period was only 2 weeks, we could not identify potential complications from long-term application of topical EGCG eye drop. However, this is the first report of the effectiveness of topical EGCG eye drops at preventing corneal neovascularization in a rabbit model. In conclusion, based on our results, topical EGCG eye drops seem to be effective at preventing corneal neovascularization by inhibiting VEGF- and COX-2-mediated angiogenic and inflammatory processes. Further long-term clinical studies are required to determine the precise antiangiogenic mechanisms, target molecules, correct doses, and possible complications of EGCG use. REFERENCES 1. Dana MR, Streilein JW. Loss and restoration of immune privilege in eyes with corneal neovascularization. Invest Ophthalmol Vis Sci. 1996;37: 2485–2494.
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2. Chang JH, Gabison EE, Kato T, et al. Corneal neovascularization. Curr Opin Ophthalmol. 2001;12:242–249. 3. Lee P, Wang CC, Adamis AP. Ocular neovascularization: an epidemiologic review. Surv Ophthalmol. 1998;43:245–269. 4. Azar DT. Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc. 2006;104:264–302. 5. Castro MR, Lutz D, Edelman JL. Effect of COX inhibitors on VEGFinduced retinal vascular leakage and experimental corneal and choroidal neovascularization. Exp Eye Res. 2004;79:275–285. 6. Epstein RJ, Stulting RD, Hendricks RL, et al. Corneal neovascularization. Pathogenesis and inhibition. Cornea. 1987;6:250–257. 7. Crum R, Szabo S, Folkman J. A new class of steroids inhibits angiogenesis in the presence of heparin or a heparin fragment. Science. 1985;230: 1375–1378. 8. Kang S, Chung SK. The effect of subconjuctival combined treatment of bevacizumab and triamcinolone acetonide on corneal neovascularization in rabbits. Cornea. 2010;29:192–196. 9. Kim JW, Chung SK. The effect of topical infliximab on corneal neovascularization in rabbits. Cornea. 2013;32:185–190. 10. Byun YS, Chung SK. The effect of methotrexate on corneal neovascularization in rabbits. Cornea. 2011;30:442–446. 11. Seo JW, Chung SH, Choi JS, et al. Inhibition of corneal neovascularization in rats by systemic administration of sorafenib. Cornea. 2012;31: 907–912. 12. Lee HS, Chung SK. The effect of subconjunctival suramin on corneal neovascularization in rabbits. Cornea. 2010;29:86–92. 13. Valcic S, Muders A, Jacobsen NE, et al. Antioxidant chemistry of green tea catechins. Identification of products of the reaction of (-)-epigallocatechin gallate with peroxyl radicals. Chem Res Toxicol. 1999;12: 382–386. 14. Donà M, Dell’Aica I, Calabrese F, et al. Neutrophil restraint by green tea: inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis. J Immunol. 2003;170:4335–4341. 15. Fujiki H. Green tea: health benefits as cancer preventive for humans. Chem Rec. 2005;5:119–132. 16. Wolfram S. Effects of green tea and EGCG on cardiovascular and metabolic health. J Am Coll Nutr. 2007;26:373S–388S. 17. Shimizu M, Shirakami Y, Moriwaki H. Targeting receptor tyrosine kinases for chemoprevention by green tea catechin, EGCG. Int J Mol Sci. 2008;9:1034–1049. 18. Sen T, Moulik S, Dutta A, et al. Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7. Life Sci. 2009;84:194–204. 19. Rodriguez SK, Guo W, Liu L, et al. Green tea catechin, epigallocatechin3-gallate, inhibits vascular endothelial growth factor angiogenic signaling by disrupting the formation of a receptor complex. Int J Cancer. 2006; 118:1635–1644.
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20. Phillips K, Arffa R, Cintron C, et al. Effects of prednisolone and medroxyprogesterone on corneal wound healing, ulceration, and neovascularization. Arch Ophthalmol. 1983;101:640–643. 21. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2000;343:86–93. 22. Mendelsohn AD, Stock EL, Lo GG, et al. Laser photocoagulation of feeder vessels in lipid keratopathy. Ophthalmic Surg. 1986;17:502–508. 23. Primbs GB, Casey R, Wamser K, et al. Photodynamic therapy for corneal neovascularization. Ophthalmic Surg Lasers. 1998;29:832–838. 24. Riazi-Esfahani M, Peyman GA, Aydin E, et al. Prevention of corneal neovascularization: evaluation of various commercially available compounds in an experimental rat model. Cornea. 2006;25:801–805. 25. Murata M, Shimizu S, Horiuchi S, et al. Inhibitory effect of triamcinolone acetonide on corneal neovascularization. Graefes Arch Clin Exp Ophthalmol. 2006;244:205–209. 26. Benelli U, Ross JR, Nardi M, et al. Corneal neovascularization induced by xenografts or chemical cautery. Inhibition by cyclosporin A. Invest Ophthalmol Vis Sci. 1997;38:274–282. 27. Ambati BK, Joussen AM, Ambati J, et al. Angiostatin inhibits and regresses corneal neovascularization. Arch Ophthalmol. 2002;120:1063–1068. 28. Lee MY, Chung SK. Treatment of corneal neovascularization by topical application of ascorbic acid in the rabbit model. Cornea. 2012;31:1165–1169. 29. Kim JS, Choi JS, Chung SK. The effect of curcumin on corneal neovascularization in rabbit eyes. Curr Eye Res. 2010;35:274–280. 30. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol. 2001;61:253–270. 31. Salam A, Mathew R, Sivaprasad S. Treatment of proliferative diabetic retinopathy with anti-VEGF agents. Acta Ophthalmol. 2011;89:405–411. 32. Leske DA, Wu J, Mookadam M, et al. The relationship of retinal VEGF and retinal IGF-1 mRNA with neovascularization in an acidosis-induced model of retinopathy of prematurity. Curr Eye Res. 2006;31:163–169. 33. Toomey DP, Murphy JF, Conlon KC. COX-2, VEGF and tumour angiogenesis. Surgeon. 2009;7:174–180. 34. Cianchi F, Cortesini C, Bechi P, et al. Up-regulation of cyclooxygenase 2 gene expression correlates with tumor angiogenesis in human colorectal cancer. Gastroenterology. 2001;121:1339–1347. 35. Huang SP, Wu MS, Shun CT, et al. Cyclooxygenase-2 increases hypoxia-inducible factor-1 and vascular endothelial growth factor to promote angiogenesis in gastric carcinoma. J Biomed Sci. 2005;12:229–241. 36. Williams CS, Tsujii M, Reese J, et al. Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest. 2000;105:1589–1594. 37. Kim SJ, Jeong HJ, Lee KM, et al. Epigallocatechin-3-gallate suppresses NF-kappaB activation and phosphorylation of p38 MAPK and JNK in human astrocytoma U373MG cells. J Nutr Biochem. 2007;18:587–596. 38. Singh R, Ahmed S, Islam N, et al. Epigallocatechin-3-gallate inhibits interleukin-1beta-induced expression of nitric oxide synthase and production of nitric oxide in human chondrocytes: suppression of nuclear factor kappaB activation by degradation of the inhibitor of nuclear factor kappaB. Arthritis Rheum. 2002;46:2079–2086.
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