- Email: [email protected]
The Inhibitory Effects of Trastuzumab on Corneal Neovascularization
The Inhibitory Effects of Trastuzumab on Corneal Neovascularization METE GÜLER, TURGUT Y⌱LMAZ, ⌱˙BRAHIM ÖZERCAN, AND TAMER ELK⌱RAN ● PURPOSE:
To investigate the effect of systemic administration of trastuzumab in the prevention of experimentally induced corneal neovascularization in a rat model. ● DESIGN: An experimental animal study. ● METHODS: Sixteen male Wistar-Albino rats weighing 250 g to 300 g were used in the study. Silver nitrate sticks (75% silver nitrate, 25% potassium nitrate) were used to induce chemical cauterization on the corneas of 16 eyes. The rats were randomized to 1 of 2 groups: Group 1 (n ⴝ 8) received intraperitoneally 1 ml (4 mg/kg) trastuzumab and Group 2 (n ⴝ 8) received 1 ml saline. The corneal surface covered with neovascular vessels was measured on the photographs as the percentage of the total area of the cornea by using computer imaging analysis on the eighth day. The corneas obtained from rats were evaluated for vascular endothelial growth factor (VEGF) immunostaining semicantitatively. The number of the corneal neovascularizations were also determined on slides. The results were evaluated with the Mann–Whitney U test. ● RESULTS: The burn stimulus was similar between groups. The average neovascularization area in treatment group was statistically smaller than control (P ⴝ .008). The mean VEGF staining intensity of epithelial and endothelial layers of cornea in treatment group was less than control (P ⴝ .038 and P ⴝ .041, respectively). The stroma of the treatment group showed less staining, but the difference was not significant (P ⴝ .056). The number of corneal neovascularizations on slides in trastuzumab treated eyes were less than the control group (P ⴝ .02). ● CONCLUSION: Systemic administration of trastuzumab is effective in prevention of the corneal neovascularization. (Am J Ophthalmol 2009;147:703–708. © 2009 by Elsevier Inc. All rights reserved.)
N
EOVASCULARIZATION OR ANGIOGENESIS, THE
formation of new blood vessels, is a normal process that accompanies tissue growth, reproduction and repair of damaged tissue during the process of wound healing.1 But neovascularization in the eye may result in a sight-threatening condition and even blindness.
Accepted for publication Sep 27, 2008. From the Department of Ophthalmology (M.G., T.Y.), the Department of Pathology (I˙.Ö.), and the Department of Oncology (T.E.), School of Medicine, Fırat University, Elazıg, Turkey. Inquiries to Mete Güler, Fırat Universitesi Tıp Fak, Goz Hast A.D., 23119 Elazıg˘, Turkey; e-mail: [email protected] 0002-9394/09/$36.00 doi:10.1016/j.ajo.2008.09.022
©
2009 BY
A wide range of inflammatory, infectious, degenerative, or traumatic disorders may induce corneal neovascularization.2 A number of treatment modalities are currently used in corneal neovascularization. These include surgery, laser photocoagulation, and medication such as glucocorticosteroids, suleparoide, thalidomide, suramin, genistein, somatostatin, indomethacin, cyclosporin, methotrexate, rapamycin, and bevacizumab.3–15 There is still no clear consensus about the best treatment of corneal neovascularization. Trastuzumab (Herceptin; Genentech, South San Francisco, California, USA), a chimeric human monoclonal antibody against the human epidermal growth factor receptor 2 (HER2) oncoprotein, is a promising agent for molecular targeting therapy against breast cancer.16 Tumorigenesis is a multistep process and angiogenesis is critical for tumor growth and metastasis.17 Epidermal growth factor (EGF) is known to be an angiogenic factor, and it also up-regulates expression of potent angiogenic factors, vascular endothelial growth factor (VEGF), and interleukin-8. Epidermal growth factor receptor (EGFR) activation is often linked to angiogenesis as well as to invasion and metastasis, all processes thus able to be affected by EGFR antagonists.18 In this aspect, trastuzumab may be a multitargeted therapeutic approach in the prevention of corneal neovascularization. The aim of this study was to investigate the effect of trastuzumab in the prevention of experimentally induced corneal neovascularization in a rat model.
METHODS SIXTEEN MALE WISTAR-ALBINO RATS WEIGHTING 250 G TO
300 g were used in the study. Under general anesthesia (induced by intraperitoneally administered 94.7 mg/kg body weight ketamine hydrochloride and xylazine combination) supplemented by topical anesthesia (proparacaine hydrochloride 0.5%), the silver nitrate cauterization technique described by Mahoney and associates19 was used to induce corneal neovascularization. Briefly, one cornea of each animal was cauterized by pressing an applicator stick with a diameter of 2 mm coated with 75% silver nitrate/ 25% potassium nitrate to the central cornea for 10 seconds under the operating microscope. Excess silver nitrate was removed by rinsing the eyes with 5 ml of a balanced salt
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FIGURE 2. Peripheral corneal neovascularization is seen in the trastuzumab-treated group. FIGURE 1. Normal rat clear cornea is seen.
mounted on poly-Lysine-coated slides, deparaffinized with xylene, and rehydrated through graded concentrations of ethanol. For antigen retrieval, sections were immersed in 10 mM sodium citrate buffer for 5 minutes (pH 6.0) and heated for 20 minutes in a microwave oven. Endogenous peroxidases were quenched in 0.3% (vol/vol) hydrogen peroxide for 5 minutes After washing in phosphatebuffered saline, slides were incubated with blocking reagent for 10 minutes. For the detection VEGF protein, sections were incubated for 30 minutes at 37 C with rabbit polyclonal antibody VEGF (Thermo Fisher Scientific, Cheshire, United Kingdom). The sections were further incubated with anti-polyvalent biotinylated antibody (ScyTek Laboratories, Logan, Utah, USA). The immune complexes were detected by using an AEC chromogen (ScyTek Laboratories) according to the manufacturer’s instructions. 21 Vascular endothelial growth factor immunostaining was evaluated in epithel, stroma, and endothel layers of corneas. Staining intensity was determined semicantitatively with previously described22 technique as no (0), weak (1), moderate (2), and intense (3). The numbers of the corneal neovascularizations were determined on slides immunostained with anti-CD31 antibodies, as described previously, by an examiner with no knowledge of the experimental procedures.21 Most sections were obtained from the central region of the cornea. The numbers of corneal neovascularization were evaluated on at least two sections from each eye from one limbus to another. The results between groups were compared with the Mann–Whitney U test. P values smaller than .05 were considered statistically significant.
solution and then gently blotting the eyes with tissue paper. To increase the reproducibility of the injuries, a single investigator (M.G.) cauterized all animals. For each eye, the extent of burn stimulus response was scored as 0 (no blister, not raised above corneal surface), ⫹1 (small blister, raised slightly above the surface), ⫹2 (medium blister, raised moderately above the surface), ⫹3 (large blister). Only the corneas with a burn stimulus score of ⫹2 or higher were included for the calculation of the mean burn stimulus and neovascularization scores in each group. Following cauterization, the experimental model was carried on according to a method described by Manzano and associates20 with minor modifications. The rats were randomized to 1 of 2 groups: Group 1 (n ⫽ 8) received intraperitoneally 1 ml (4 mg/kg) trastuzumab and Group 2 (n ⫽ 8) received 1 ml saline one time. Treatment started immediately after cauterization in the two groups. All animals were anesthetized as described above and their corneas were evaluated by slit-lamp biomicroscopy on the eighth day. Corneal photographs were taken with x40 magnification using a Sony digital camera (CCD-IRIS; model DXC 107 AP; Sony Corp, Tokyo, Japan) attached to the slit-lamp microscope. Neovascularization of each cornea was evaluated by an examiner (T.Y.) who was blinded as to the treatment groups to minimize the observer bias. The corneal surface covered with neovascular vessels was measured on the photographs as the percentage of the total area of the cornea by using computer imaging analysis (Topcon Image Net 2000; Itabashiku, Tokyo, Japan). A drawing of corneal blood vessels was made by one of the investigators to compare with digital photos. This is to be sure that no vascular area was missed during calculation. The animals were sacrificed on the eighth day.
RESULTS
● TISSUE PREPARATION AND VASCULAR ENDOTHELIAL GROWTH FACTOR IMMUNOSTAINING: The paraf-
THE BURN STIMULUS WAS SIMILAR BETWEEN THE GROUPS,
fin-embedded tissues were cut into 5-m-thick slices,
and stimulus scores were ⫹2 or higher. The number of
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FIGURE 3. Central corneal scarring and total corneal neovascularization are seen in the control group.
FIGURE 4. Minimal vascular endothelial growth factor (VEGF) immunostaining staining is seen in normal rat cornea, magnification ⴛ 100.
corneal neovascularizations in trastuzumab-treated eyes was less than in the control group at 8 days after cauterization (3.75 ⫾ 3.01 in treatment and 15.37 ⫾ 13.01 in controls, respectively; P ⫽ .02). The average neovascularization area to the total corneal surface in trastuzumabtreated eyes and in controls was 0.17 ⫾ 0.13 and 0.49 ⫾ 0.27, respectively. The average neovascularization area in treatment group was statistically smaller (P ⫽ .008) (Figures 1 to 3). The mean VEGF staining intensity of epithelial layer of cornea in treatment group was less than control (1.19 ⫾ 0.92 in treatment and 2.37 ⫾ 1.06 in control group; P ⫽ .038). The stroma of the treatment group showed less staning, but the difference is not significant (0.93 ⫾ 0.77 in treatment and 1.87 ⫾ 0.99 in control group; P ⫽ .056). VOL. 147, NO. 4
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FIGURE 5. Heavy VEGF immunostaining and neovascularization are seen in the control group, magnification ⴛ 100.
FIGURE 6. Moderate VEGF immunostaining with no neovascularization are seen in the trastuzumab-treated group, magnification ⴛ 100.
The mean endothelial VEGF staining intensity in trastuzumab group was less than control group (0.87 ⫾ 0.79 in treatment group and 2.00 ⫾ 1.06 in control; P ⫽ .041) (Figures 4 to 6).
DISCUSSION THE EGFR FAMILY IS CHARACTERISTIC OF RECEPTOR TY-
rosine kinases. One of the subfamilies of EGFR, HER2 gene encodes an EGFR-related tyrosine kinase.23 The HER2 oncoproteins are composed of three membrane portions: 1) the internal tyrosine kinase is responsible for signal transduction, 2) a short transmembrane part, and 3) the extracellular domain; the latter being the site of binding for the ligand growth factors. These receptors are critical for mediating the proliferation and differentiation AND
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of normal cells, and their activation is associated with increased cell proliferation, tumor cell motility, and invasiveness, angiogenesis, and inhibition of apoptosis.24 Although the mechanisms by which Herceptin induces regression of HER2 overexpressing tumors are incompletely defined, several molecular and cellular effects have been observed in experimental in vitro and in vivo models. HER2 activates multiple cellular signaling pathways, including the phosphatidylinositol 3-kinase and mitogen activated protein kinase cascades. Trastuzumab reduces signaling from these pathways, and thus promotes cell cycle arrest and apoptosis. Diminished receptor signaling may result from trastuzumab mediated internalization and degradation of the HER2 receptor.25 Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle, with a concomitant reduction in proliferation.26 Clinical trials have demonstrated that trastuzumab, in contrast to many targeted agents, possesses cytotoxic properties. This ability to not only block proliferation but also to actually promote cell death may be related in part to induction of an immune response. Trastuzumab activated an antibody-dependent cellular cytotoxicity response in multiple breast cancer cell lines.27 Studies suggest that trastuzumab promotes deoxyribonucleic acid (DNA) damage and subsequently inhibits DNA repair, resulting in apoptosis.28,29 Human epidermal growth factor receptor 2 also acts to promote angiogenesis by pathways potentially distinct from those implicated in its cell-cycle regulatory functions. Overexpression of HER2 in human tumor cells is closely associated with increased angiogenesis and expression of VEGF. Treatment of HER2 overexpressing breast cancers with trastuzumab reduced tumor volume and decreased microvessel density in vivo and reduced endothelial cell migration in vitro. Furthermore, expression of multiple proangiogenic factors was reduced, while expression of antiangiogenic factors was increased in trastuzumabtreated tumors relative to control-treated tumors in vivo.30 VEGF and EGF exert their biological effects directly or indirectly on tumor growth and metastasis/invasion as well as on tumor angiogenesis. VEGF and HER2 signaling pathways are interlinked at the molecular level and both cooperate to promote cell proliferation, and HER2 signaling is known to control the expression of VEGF.24 VEGF binds its cognate receptor on endothelial cells and causes these cells to proliferate, migrate, and sprout new vessels. HER2 is implicated in up-regulating VEGF under hypoxic conditions, but has also been found recently to have hypoxic independent mechanisms.31 Guan and associates have shown that trastuzumab down-regulated both HER2 and VEGF expression in Ewing’s sarcoma cells in vitro and in vivo.32 Futhermore, it was shown that expression of the proangiogenic factors VEGF, transforming growth factor-␣, angiopoietin (Ang)-1, and plasminogen activator inhibitor-1 were all reduced, whereas expression of the antiangiogenic factor thrombospondin-1 was increased in 706
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trastuzumab-treated tumors relative to control-treated tumors in vivo, and VEGF expression was reduced by trastuzumab treatment in vitro.33 Ang-1 plays a role in maintaining and stabilizing mature vessels by promoting the interaction between endothelial cells and the surrounding support cells. Recent studies provide evidence that the Ang-2 gene is regulated by HER2 activity in breast cancer, and propose an additional mechanism for HER2 contributing to tumor angiogenesis and metastasis.34 Blocking Ang-2 activity by trastuzumab may offer an additional therapeutic target. These findings suggest that trastuzumab may inhibit neovascularization by multitargeted pathways. Recently, bevacizumab, a humanized monoclonal antibody to VEGF, was used for the treatment of corneal neovascularization. Some of the short-term results suggest that topical and subconjunctival bevacizumab is well tolerated but associated with a partial regression of corneal neovascularization.35,36 Bevacizumab inhibits VEGF signaling only by binding to free extracellular VEGF. The biological effects by VEGF and EGF are mediated through activation of their specific downstream signaling, but both factors also share common downstream signaling pathways. This is thus the potential for improved therapeutic efficacy by EGF/EGFR targeting or combination of both EGF/EGFRtargeting and VEGF/VEGF receptor-targeting drugs.37 So it is expected that the combination of various tyrosine kinase inhibitors or multitargeted inhibitors as trastuzumab might have better therapeutic benefits. Trastuzumab is remarkably well tolerated and generally not associated with the usual side effect profile of cytotoxic chemotherapy. Thus leukopenia, anemia, thrombocytopenia, and alopecia are exceedingly rare, and even though nausea has been reported as a side effect, it is seldom associated with vomiting, and antiemetics are rarely required. Fever and chills are clearly drug-related, with a trend toward increased incidence at higher doses. Trastuzumab can cause cardiotoxicity, including severe cardiotoxicity when given with or immediately after an anthracyclin. It is recommended that patients on longterm trastuzumab treatment should receive regular cardiac monitoring.38 We used systemic administration because we wanted to be sure that all animals received therapeutic dosage. It was shown that VEGF was up-regulated in inflamed and vascularized human corneas. Furthermore, in vascularized human corneal buttons, it was found that VEGF was expressed by corneal epithelial cells, endothelial cells, vascular endothelial cells of limbal blood vessels, and of newly formed blood vessels in the stroma, and weakly by keratocytes.22 VEGF was active in the wound-healing phase of cornea alcali burn healing, and was expressed in the epithelium and endothelium.39 The VEGF staining patterns in our corneal specimens were similar to previous studies. OF
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We found that both VEGF staining and corneal neovascularization was reduced in the trastuzumab-treated group. These findings indicate that systemic administration of trastuzumab is beneficial in prevention of the
corneal neovascularization. Further studies are needed to explain the optimal dosage and whether topical admininistration of trastuzumab is effective in the prevention of corneal neovascularization.
THE AUTHORS INDICATE NO FINANCIAL SUPPORT OR FINANCIAL CONFLICT OF INTEREST. INVOLVED IN DESIGN AND conduct of study (M.G., T.Y.); collection and management (M.G., T.Y.) and analysis and interpretation of the data (M.G., T.Y., I˙.Ö., T.E.); preparation of the manuscript (M.G.); and review and final approval of the manuscript (M.G., T.Y., I˙.Ö., T.E.). This study were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in Ophthalmic and Vision Research.
REFERENCES 1. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86: 353–364. 2. Chang JH, Gabison EE, Kato T, Azar DT. Corneal neovascularization. Curr Opin Ophthalmol 2001;12:242–249. 3. Mendelsohn AD, Lo GG, Stock EL. Laser photocoagulation of feeder vessels in lipid keratopathy. Ophthalmic Surg 1986;17:502–508. 4. Goto S. Q-switched Nd:YAG laser treatment for corneal neovascularization. Jpn J Ophthalmol 1992;36:291–300. 5. BenEzra D, Griffin BW, Maftzir G, Sharif NA, Clark AF. Topical formulations of novel angiostatic steroids inhibit rabbit corneal neovascularization. Invest Ophthalmol Vis Sci 1997;38:1954 –1962. 6. Benelli U, Bocci G, Danesi R, et al. The heparan sulfate suleparoide inhibits rat corneal angiogenesis and in vitro neovascularization. Exp Eye Res 1998;67:133–142. 7. D’Amato RJ, Loughnan MS, Flynn E, Folkman J. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci USA 1994;91:4082– 4085. 8. Bocci G, Danesi R, Benelli U, et al. Inhibitory effect of suramin in rat models of angiogenesis in vitro and in vivo. Cancer Chemother Pharmacol 1999;43:205–212. 9. Fotsis T, Pepper M, Adlercreutz H, et al. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc Natl Acad Sci USA 1993;90:2690 –2694. 10. Wu PC, Liu CC, Chen C, et al. Inhibition of experimental angiogenesis of cornea by somatostatin. Graefes Arch Clin Exp Ophthalmol 2003;241:63– 69. 11. Deutsch TA, Hughes WF. Suppressive effects of indomethacin on thermally induced neovascularization of rabbit corneas. Am J Ophthalmol 1979;87:536 –540. 12. Lipman RM, Epstein RJ, Hendricks RL. Suppression of corneal neovascularization with cyclosporine. Arch Ophthalmol 1992;110:405– 407. 13. Joussen AM, Kruse FE, Volcker HE, Kirchhof B. Topical application of methotrexate for inhibition of corneal angiogenesis. Graefes Arch Clin Exp Ophthalmol 1999; 237:920 –927. 14. Shi W, Gao H, Xie L, Wang S. Sustained intraocular rapamycin delivery effectively prevents high-risk corneal allograft rejection and neovascularization in rabbits. Invest Ophthalmol Vis Sci 2006;47:3339 –3344. 15. Bock F, König Y, Kruse F, Baier M, Cursiefen C. Bevacizumab (Avastin) eye drops inhibit corneal neovascularization Graefes Arch Clin Exp Ophthalmol 2008;246:281– 284.
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16. Kim R, Toge T. Changes in therapy for solid tumors: potential for overcoming drug resistance in vivo with molecular targeting agents. Surg Today 2004;34:293– 303. 17. Gasparini G, Longo R, Torino F, Morabito A. Therapy of breast cancer with molecular targeting agents. Ann Oncol 2005;16:S28 –S36. 18. Baker CH, Kedar D, Mc Carty MF, et al. Blockade of epidermal growth factor receptor signaling on tumor cells and tumor-associated endothelial cells for therapy of human carcinomas. Am J Pathol 2002;161:929 –938. 19. Mahoney JM, Waterbury LD. Drug effects on the neovascularization response to silver nitrate cauterization of the rat cornea. Curr Eye Res 1985;4:531–535. 20. Manzano RP, Peyman GA, Khan P, et al. Inhibition of experimental corneal neovascularization by bevacizumab (Avastin). Br J Ophthalmol 2007;91:804 – 807. 21. Lu P, Li L, Mukaida N, Zhang X. Alkali-induced corneal neovascularization is independent of CXCR2-mediated neutrophil infiltration. Cornea 2007;26:199 –206. 22. Philipp W, Speicher L, Humpel C. Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas. Invest Ophthalmol Vis Sci 2000;41:2514 –2522. 23. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–182. 24. Rowinsky EK. Signal events: Cell signal transduction and inhibition in cancer. The Oncologist 2003;8:5–17. 25. Baselga J, Albanell J, Molina MA, Arribas J. Mechanism of action of trastuzumab and scientific update. Semin Oncol 2001;28;4 –11. 26. Lane HA, Motoyama AB, Beuvink I, Hynes NE. Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling. Ann Oncol 2001;12: S21–S22. 27. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 2000;6:443– 446. 28. Mohsin SK, Weiss HL, Gutierrez MC, et al. Neoadjuvant trastuzumab induces apoptosis in primary breast cancers. J Clin Oncol 2005;23:2460 –2468. 29. Mayfield S, Vaughn JP, Kute TE. DNA strand breaks and cell cycle perturbation in Herceptin treated breast cancer cell lines. Breast Cancer Res Treat 2001;70:123– 129. 30. Nahta R, Esteva Francisco J. Herceptin: mechanisms of action and resistance. Cancer Lett 2006;232:123–138. AND
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35. Bahar I, Kaiserman I, Mc Allum P, Rootman D, Slomovic A. Subconjunctival bevacizumab injection for corneal neovascularization. Cornea 2008;27:142–147. 36. Bahar I, Igor Kaiserman I, McAllum P, Rootman D, Slomovic A. Subconjunctival bevacizumab injection for corneal neovascularization in recurrent pterygium. Curr Eye Res 2008;33:23–28. 37. Ono M, Kuwano M. Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs. Clin Cancer Res 2006;12:7242–7251. 38. Moore S. Managing treatment side effects in advanced breast cancer. Semin Oncol Nurs 2007;23:S23–S30. 39. Gan L, Fagerholm P, Palmblad J. Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in the regulation of corneal neovascularization and wound healing. Acta Ophthalmol Scand 2004:82:557–563.
31. Klos KS, Zhou X, Lee S, et al. Combined trastuzumab and paclitaxel treatment better inhibits ErbB-2-mediated angiogenesis in breast carcinoma through a more effective inhibition of Akt than either treatment alone. Cancer 2003; 98:1377–1385. 32. Guan H, Jia SF, Zhou Z, Stewart J, Kleinerman ES. Herceptin down-regulates HER-2/neu and vascular endothelial growth factor expression and enhances taxol-induced cytotoxicity of human Ewing’s sarcoma cells in vitro and in vivo. Clin Cancer Res 2005;11:2008 –2017. 33. Izumi Y, Xu L, Tomaso E, Fukumura D, Jain RK. Tumour biology: herceptin acts as an anti-angiogenic cocktail. Nature 2002;21:279 –280. 34. Niu G, Carter WB. Human epidermal growth factor receptor 2 regulates angiopoietin-2 expression in breast cancer via AKT and mitogen-activated protein kinase pathways. Cancer Res 2007;67:1487–1493.
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Biosketch Mete Güler graduated from Fırat University School of Medicine, Elazıg˘, Turkey in 2003. He is currently continuing ophthalmology residency and fellowship at Fırat University School of Medicine.
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Biosketch Turgut Yılmaz graduated from Fırat University School of Medicine, Elazıg˘, Turkey in 1992. He completed his ophthalmology residency and fellowship at Fırat University School of Medicine. Dr Yılmaz is currently an Assistant Professor at Medical Park Hospital in Elazıg˘. He specializes in vitreoretinal surgery.
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To investigate the effect of systemic administration of trastuzumab in the prevention of experimentally induced corneal neovascularization in a rat model. ● DESIGN: An experimental animal study. ● METHODS: Sixteen male Wistar-Albino rats weighing 250 g to 300 g were used in the study. Silver nitrate sticks (75% silver nitrate, 25% potassium nitrate) were used to induce chemical cauterization on the corneas of 16 eyes. The rats were randomized to 1 of 2 groups: Group 1 (n ⴝ 8) received intraperitoneally 1 ml (4 mg/kg) trastuzumab and Group 2 (n ⴝ 8) received 1 ml saline. The corneal surface covered with neovascular vessels was measured on the photographs as the percentage of the total area of the cornea by using computer imaging analysis on the eighth day. The corneas obtained from rats were evaluated for vascular endothelial growth factor (VEGF) immunostaining semicantitatively. The number of the corneal neovascularizations were also determined on slides. The results were evaluated with the Mann–Whitney U test. ● RESULTS: The burn stimulus was similar between groups. The average neovascularization area in treatment group was statistically smaller than control (P ⴝ .008). The mean VEGF staining intensity of epithelial and endothelial layers of cornea in treatment group was less than control (P ⴝ .038 and P ⴝ .041, respectively). The stroma of the treatment group showed less staining, but the difference was not significant (P ⴝ .056). The number of corneal neovascularizations on slides in trastuzumab treated eyes were less than the control group (P ⴝ .02). ● CONCLUSION: Systemic administration of trastuzumab is effective in prevention of the corneal neovascularization. (Am J Ophthalmol 2009;147:703–708. © 2009 by Elsevier Inc. All rights reserved.)
N
EOVASCULARIZATION OR ANGIOGENESIS, THE
formation of new blood vessels, is a normal process that accompanies tissue growth, reproduction and repair of damaged tissue during the process of wound healing.1 But neovascularization in the eye may result in a sight-threatening condition and even blindness.
Accepted for publication Sep 27, 2008. From the Department of Ophthalmology (M.G., T.Y.), the Department of Pathology (I˙.Ö.), and the Department of Oncology (T.E.), School of Medicine, Fırat University, Elazıg, Turkey. Inquiries to Mete Güler, Fırat Universitesi Tıp Fak, Goz Hast A.D., 23119 Elazıg˘, Turkey; e-mail: [email protected] 0002-9394/09/$36.00 doi:10.1016/j.ajo.2008.09.022
©
2009 BY
A wide range of inflammatory, infectious, degenerative, or traumatic disorders may induce corneal neovascularization.2 A number of treatment modalities are currently used in corneal neovascularization. These include surgery, laser photocoagulation, and medication such as glucocorticosteroids, suleparoide, thalidomide, suramin, genistein, somatostatin, indomethacin, cyclosporin, methotrexate, rapamycin, and bevacizumab.3–15 There is still no clear consensus about the best treatment of corneal neovascularization. Trastuzumab (Herceptin; Genentech, South San Francisco, California, USA), a chimeric human monoclonal antibody against the human epidermal growth factor receptor 2 (HER2) oncoprotein, is a promising agent for molecular targeting therapy against breast cancer.16 Tumorigenesis is a multistep process and angiogenesis is critical for tumor growth and metastasis.17 Epidermal growth factor (EGF) is known to be an angiogenic factor, and it also up-regulates expression of potent angiogenic factors, vascular endothelial growth factor (VEGF), and interleukin-8. Epidermal growth factor receptor (EGFR) activation is often linked to angiogenesis as well as to invasion and metastasis, all processes thus able to be affected by EGFR antagonists.18 In this aspect, trastuzumab may be a multitargeted therapeutic approach in the prevention of corneal neovascularization. The aim of this study was to investigate the effect of trastuzumab in the prevention of experimentally induced corneal neovascularization in a rat model.
METHODS SIXTEEN MALE WISTAR-ALBINO RATS WEIGHTING 250 G TO
300 g were used in the study. Under general anesthesia (induced by intraperitoneally administered 94.7 mg/kg body weight ketamine hydrochloride and xylazine combination) supplemented by topical anesthesia (proparacaine hydrochloride 0.5%), the silver nitrate cauterization technique described by Mahoney and associates19 was used to induce corneal neovascularization. Briefly, one cornea of each animal was cauterized by pressing an applicator stick with a diameter of 2 mm coated with 75% silver nitrate/ 25% potassium nitrate to the central cornea for 10 seconds under the operating microscope. Excess silver nitrate was removed by rinsing the eyes with 5 ml of a balanced salt
ELSEVIER INC. ALL
RIGHTS RESERVED.
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FIGURE 2. Peripheral corneal neovascularization is seen in the trastuzumab-treated group. FIGURE 1. Normal rat clear cornea is seen.
mounted on poly-Lysine-coated slides, deparaffinized with xylene, and rehydrated through graded concentrations of ethanol. For antigen retrieval, sections were immersed in 10 mM sodium citrate buffer for 5 minutes (pH 6.0) and heated for 20 minutes in a microwave oven. Endogenous peroxidases were quenched in 0.3% (vol/vol) hydrogen peroxide for 5 minutes After washing in phosphatebuffered saline, slides were incubated with blocking reagent for 10 minutes. For the detection VEGF protein, sections were incubated for 30 minutes at 37 C with rabbit polyclonal antibody VEGF (Thermo Fisher Scientific, Cheshire, United Kingdom). The sections were further incubated with anti-polyvalent biotinylated antibody (ScyTek Laboratories, Logan, Utah, USA). The immune complexes were detected by using an AEC chromogen (ScyTek Laboratories) according to the manufacturer’s instructions. 21 Vascular endothelial growth factor immunostaining was evaluated in epithel, stroma, and endothel layers of corneas. Staining intensity was determined semicantitatively with previously described22 technique as no (0), weak (1), moderate (2), and intense (3). The numbers of the corneal neovascularizations were determined on slides immunostained with anti-CD31 antibodies, as described previously, by an examiner with no knowledge of the experimental procedures.21 Most sections were obtained from the central region of the cornea. The numbers of corneal neovascularization were evaluated on at least two sections from each eye from one limbus to another. The results between groups were compared with the Mann–Whitney U test. P values smaller than .05 were considered statistically significant.
solution and then gently blotting the eyes with tissue paper. To increase the reproducibility of the injuries, a single investigator (M.G.) cauterized all animals. For each eye, the extent of burn stimulus response was scored as 0 (no blister, not raised above corneal surface), ⫹1 (small blister, raised slightly above the surface), ⫹2 (medium blister, raised moderately above the surface), ⫹3 (large blister). Only the corneas with a burn stimulus score of ⫹2 or higher were included for the calculation of the mean burn stimulus and neovascularization scores in each group. Following cauterization, the experimental model was carried on according to a method described by Manzano and associates20 with minor modifications. The rats were randomized to 1 of 2 groups: Group 1 (n ⫽ 8) received intraperitoneally 1 ml (4 mg/kg) trastuzumab and Group 2 (n ⫽ 8) received 1 ml saline one time. Treatment started immediately after cauterization in the two groups. All animals were anesthetized as described above and their corneas were evaluated by slit-lamp biomicroscopy on the eighth day. Corneal photographs were taken with x40 magnification using a Sony digital camera (CCD-IRIS; model DXC 107 AP; Sony Corp, Tokyo, Japan) attached to the slit-lamp microscope. Neovascularization of each cornea was evaluated by an examiner (T.Y.) who was blinded as to the treatment groups to minimize the observer bias. The corneal surface covered with neovascular vessels was measured on the photographs as the percentage of the total area of the cornea by using computer imaging analysis (Topcon Image Net 2000; Itabashiku, Tokyo, Japan). A drawing of corneal blood vessels was made by one of the investigators to compare with digital photos. This is to be sure that no vascular area was missed during calculation. The animals were sacrificed on the eighth day.
RESULTS
● TISSUE PREPARATION AND VASCULAR ENDOTHELIAL GROWTH FACTOR IMMUNOSTAINING: The paraf-
THE BURN STIMULUS WAS SIMILAR BETWEEN THE GROUPS,
fin-embedded tissues were cut into 5-m-thick slices,
and stimulus scores were ⫹2 or higher. The number of
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FIGURE 3. Central corneal scarring and total corneal neovascularization are seen in the control group.
FIGURE 4. Minimal vascular endothelial growth factor (VEGF) immunostaining staining is seen in normal rat cornea, magnification ⴛ 100.
corneal neovascularizations in trastuzumab-treated eyes was less than in the control group at 8 days after cauterization (3.75 ⫾ 3.01 in treatment and 15.37 ⫾ 13.01 in controls, respectively; P ⫽ .02). The average neovascularization area to the total corneal surface in trastuzumabtreated eyes and in controls was 0.17 ⫾ 0.13 and 0.49 ⫾ 0.27, respectively. The average neovascularization area in treatment group was statistically smaller (P ⫽ .008) (Figures 1 to 3). The mean VEGF staining intensity of epithelial layer of cornea in treatment group was less than control (1.19 ⫾ 0.92 in treatment and 2.37 ⫾ 1.06 in control group; P ⫽ .038). The stroma of the treatment group showed less staning, but the difference is not significant (0.93 ⫾ 0.77 in treatment and 1.87 ⫾ 0.99 in control group; P ⫽ .056). VOL. 147, NO. 4
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FIGURE 5. Heavy VEGF immunostaining and neovascularization are seen in the control group, magnification ⴛ 100.
FIGURE 6. Moderate VEGF immunostaining with no neovascularization are seen in the trastuzumab-treated group, magnification ⴛ 100.
The mean endothelial VEGF staining intensity in trastuzumab group was less than control group (0.87 ⫾ 0.79 in treatment group and 2.00 ⫾ 1.06 in control; P ⫽ .041) (Figures 4 to 6).
DISCUSSION THE EGFR FAMILY IS CHARACTERISTIC OF RECEPTOR TY-
rosine kinases. One of the subfamilies of EGFR, HER2 gene encodes an EGFR-related tyrosine kinase.23 The HER2 oncoproteins are composed of three membrane portions: 1) the internal tyrosine kinase is responsible for signal transduction, 2) a short transmembrane part, and 3) the extracellular domain; the latter being the site of binding for the ligand growth factors. These receptors are critical for mediating the proliferation and differentiation AND
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of normal cells, and their activation is associated with increased cell proliferation, tumor cell motility, and invasiveness, angiogenesis, and inhibition of apoptosis.24 Although the mechanisms by which Herceptin induces regression of HER2 overexpressing tumors are incompletely defined, several molecular and cellular effects have been observed in experimental in vitro and in vivo models. HER2 activates multiple cellular signaling pathways, including the phosphatidylinositol 3-kinase and mitogen activated protein kinase cascades. Trastuzumab reduces signaling from these pathways, and thus promotes cell cycle arrest and apoptosis. Diminished receptor signaling may result from trastuzumab mediated internalization and degradation of the HER2 receptor.25 Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle, with a concomitant reduction in proliferation.26 Clinical trials have demonstrated that trastuzumab, in contrast to many targeted agents, possesses cytotoxic properties. This ability to not only block proliferation but also to actually promote cell death may be related in part to induction of an immune response. Trastuzumab activated an antibody-dependent cellular cytotoxicity response in multiple breast cancer cell lines.27 Studies suggest that trastuzumab promotes deoxyribonucleic acid (DNA) damage and subsequently inhibits DNA repair, resulting in apoptosis.28,29 Human epidermal growth factor receptor 2 also acts to promote angiogenesis by pathways potentially distinct from those implicated in its cell-cycle regulatory functions. Overexpression of HER2 in human tumor cells is closely associated with increased angiogenesis and expression of VEGF. Treatment of HER2 overexpressing breast cancers with trastuzumab reduced tumor volume and decreased microvessel density in vivo and reduced endothelial cell migration in vitro. Furthermore, expression of multiple proangiogenic factors was reduced, while expression of antiangiogenic factors was increased in trastuzumabtreated tumors relative to control-treated tumors in vivo.30 VEGF and EGF exert their biological effects directly or indirectly on tumor growth and metastasis/invasion as well as on tumor angiogenesis. VEGF and HER2 signaling pathways are interlinked at the molecular level and both cooperate to promote cell proliferation, and HER2 signaling is known to control the expression of VEGF.24 VEGF binds its cognate receptor on endothelial cells and causes these cells to proliferate, migrate, and sprout new vessels. HER2 is implicated in up-regulating VEGF under hypoxic conditions, but has also been found recently to have hypoxic independent mechanisms.31 Guan and associates have shown that trastuzumab down-regulated both HER2 and VEGF expression in Ewing’s sarcoma cells in vitro and in vivo.32 Futhermore, it was shown that expression of the proangiogenic factors VEGF, transforming growth factor-␣, angiopoietin (Ang)-1, and plasminogen activator inhibitor-1 were all reduced, whereas expression of the antiangiogenic factor thrombospondin-1 was increased in 706
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trastuzumab-treated tumors relative to control-treated tumors in vivo, and VEGF expression was reduced by trastuzumab treatment in vitro.33 Ang-1 plays a role in maintaining and stabilizing mature vessels by promoting the interaction between endothelial cells and the surrounding support cells. Recent studies provide evidence that the Ang-2 gene is regulated by HER2 activity in breast cancer, and propose an additional mechanism for HER2 contributing to tumor angiogenesis and metastasis.34 Blocking Ang-2 activity by trastuzumab may offer an additional therapeutic target. These findings suggest that trastuzumab may inhibit neovascularization by multitargeted pathways. Recently, bevacizumab, a humanized monoclonal antibody to VEGF, was used for the treatment of corneal neovascularization. Some of the short-term results suggest that topical and subconjunctival bevacizumab is well tolerated but associated with a partial regression of corneal neovascularization.35,36 Bevacizumab inhibits VEGF signaling only by binding to free extracellular VEGF. The biological effects by VEGF and EGF are mediated through activation of their specific downstream signaling, but both factors also share common downstream signaling pathways. This is thus the potential for improved therapeutic efficacy by EGF/EGFR targeting or combination of both EGF/EGFRtargeting and VEGF/VEGF receptor-targeting drugs.37 So it is expected that the combination of various tyrosine kinase inhibitors or multitargeted inhibitors as trastuzumab might have better therapeutic benefits. Trastuzumab is remarkably well tolerated and generally not associated with the usual side effect profile of cytotoxic chemotherapy. Thus leukopenia, anemia, thrombocytopenia, and alopecia are exceedingly rare, and even though nausea has been reported as a side effect, it is seldom associated with vomiting, and antiemetics are rarely required. Fever and chills are clearly drug-related, with a trend toward increased incidence at higher doses. Trastuzumab can cause cardiotoxicity, including severe cardiotoxicity when given with or immediately after an anthracyclin. It is recommended that patients on longterm trastuzumab treatment should receive regular cardiac monitoring.38 We used systemic administration because we wanted to be sure that all animals received therapeutic dosage. It was shown that VEGF was up-regulated in inflamed and vascularized human corneas. Furthermore, in vascularized human corneal buttons, it was found that VEGF was expressed by corneal epithelial cells, endothelial cells, vascular endothelial cells of limbal blood vessels, and of newly formed blood vessels in the stroma, and weakly by keratocytes.22 VEGF was active in the wound-healing phase of cornea alcali burn healing, and was expressed in the epithelium and endothelium.39 The VEGF staining patterns in our corneal specimens were similar to previous studies. OF
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We found that both VEGF staining and corneal neovascularization was reduced in the trastuzumab-treated group. These findings indicate that systemic administration of trastuzumab is beneficial in prevention of the
corneal neovascularization. Further studies are needed to explain the optimal dosage and whether topical admininistration of trastuzumab is effective in the prevention of corneal neovascularization.
THE AUTHORS INDICATE NO FINANCIAL SUPPORT OR FINANCIAL CONFLICT OF INTEREST. INVOLVED IN DESIGN AND conduct of study (M.G., T.Y.); collection and management (M.G., T.Y.) and analysis and interpretation of the data (M.G., T.Y., I˙.Ö., T.E.); preparation of the manuscript (M.G.); and review and final approval of the manuscript (M.G., T.Y., I˙.Ö., T.E.). This study were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in Ophthalmic and Vision Research.
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16. Kim R, Toge T. Changes in therapy for solid tumors: potential for overcoming drug resistance in vivo with molecular targeting agents. Surg Today 2004;34:293– 303. 17. Gasparini G, Longo R, Torino F, Morabito A. Therapy of breast cancer with molecular targeting agents. Ann Oncol 2005;16:S28 –S36. 18. Baker CH, Kedar D, Mc Carty MF, et al. Blockade of epidermal growth factor receptor signaling on tumor cells and tumor-associated endothelial cells for therapy of human carcinomas. Am J Pathol 2002;161:929 –938. 19. Mahoney JM, Waterbury LD. Drug effects on the neovascularization response to silver nitrate cauterization of the rat cornea. Curr Eye Res 1985;4:531–535. 20. Manzano RP, Peyman GA, Khan P, et al. Inhibition of experimental corneal neovascularization by bevacizumab (Avastin). Br J Ophthalmol 2007;91:804 – 807. 21. Lu P, Li L, Mukaida N, Zhang X. Alkali-induced corneal neovascularization is independent of CXCR2-mediated neutrophil infiltration. Cornea 2007;26:199 –206. 22. Philipp W, Speicher L, Humpel C. Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas. Invest Ophthalmol Vis Sci 2000;41:2514 –2522. 23. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–182. 24. Rowinsky EK. Signal events: Cell signal transduction and inhibition in cancer. The Oncologist 2003;8:5–17. 25. Baselga J, Albanell J, Molina MA, Arribas J. Mechanism of action of trastuzumab and scientific update. Semin Oncol 2001;28;4 –11. 26. Lane HA, Motoyama AB, Beuvink I, Hynes NE. Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling. Ann Oncol 2001;12: S21–S22. 27. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 2000;6:443– 446. 28. Mohsin SK, Weiss HL, Gutierrez MC, et al. Neoadjuvant trastuzumab induces apoptosis in primary breast cancers. J Clin Oncol 2005;23:2460 –2468. 29. Mayfield S, Vaughn JP, Kute TE. DNA strand breaks and cell cycle perturbation in Herceptin treated breast cancer cell lines. Breast Cancer Res Treat 2001;70:123– 129. 30. Nahta R, Esteva Francisco J. Herceptin: mechanisms of action and resistance. Cancer Lett 2006;232:123–138. AND
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35. Bahar I, Kaiserman I, Mc Allum P, Rootman D, Slomovic A. Subconjunctival bevacizumab injection for corneal neovascularization. Cornea 2008;27:142–147. 36. Bahar I, Igor Kaiserman I, McAllum P, Rootman D, Slomovic A. Subconjunctival bevacizumab injection for corneal neovascularization in recurrent pterygium. Curr Eye Res 2008;33:23–28. 37. Ono M, Kuwano M. Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs. Clin Cancer Res 2006;12:7242–7251. 38. Moore S. Managing treatment side effects in advanced breast cancer. Semin Oncol Nurs 2007;23:S23–S30. 39. Gan L, Fagerholm P, Palmblad J. Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in the regulation of corneal neovascularization and wound healing. Acta Ophthalmol Scand 2004:82:557–563.
31. Klos KS, Zhou X, Lee S, et al. Combined trastuzumab and paclitaxel treatment better inhibits ErbB-2-mediated angiogenesis in breast carcinoma through a more effective inhibition of Akt than either treatment alone. Cancer 2003; 98:1377–1385. 32. Guan H, Jia SF, Zhou Z, Stewart J, Kleinerman ES. Herceptin down-regulates HER-2/neu and vascular endothelial growth factor expression and enhances taxol-induced cytotoxicity of human Ewing’s sarcoma cells in vitro and in vivo. Clin Cancer Res 2005;11:2008 –2017. 33. Izumi Y, Xu L, Tomaso E, Fukumura D, Jain RK. Tumour biology: herceptin acts as an anti-angiogenic cocktail. Nature 2002;21:279 –280. 34. Niu G, Carter WB. Human epidermal growth factor receptor 2 regulates angiopoietin-2 expression in breast cancer via AKT and mitogen-activated protein kinase pathways. Cancer Res 2007;67:1487–1493.
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Biosketch Mete Güler graduated from Fırat University School of Medicine, Elazıg˘, Turkey in 2003. He is currently continuing ophthalmology residency and fellowship at Fırat University School of Medicine.
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Biosketch Turgut Yılmaz graduated from Fırat University School of Medicine, Elazıg˘, Turkey in 1992. He completed his ophthalmology residency and fellowship at Fırat University School of Medicine. Dr Yılmaz is currently an Assistant Professor at Medical Park Hospital in Elazıg˘. He specializes in vitreoretinal surgery.
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