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Clinical and Experimental Ophthalmology 2015; 43: 415–421 doi: 10.1111/ceo.12477
Original Article Short-term effect of intravitreal ranibizumab on intraocular concentrations of vascular endothelial growth factor-A and pigment epithelium-derived factor in neovascular glaucoma Jia Wei Wang MD,1* Min Wen Zhou MD,1* Xiulan Zhang MD PhD,1 Wen Bin Huang MD,1 Xin Bo Gao MD,1 Wei Wang MD,1 Shida Chen MD,1 Xin Yu Zhang MD PhD,1 Xiao Yan Ding MD PhD1 and Jost B Jonas MD2 1
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen University, Guangzhou, China; and Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht-Karls-University, Heidelberg, Germany
2
ABSTRACT Background: This study aims to evaluate concentrations of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF)-A in aqueous of patients with neovascular glaucoma prior to, and shortly after, an intravitreal ranibizumab injection. Design: Interventional comparative study. Participants: The study included patients undergoing an intravitreal ranibizumab injection about one week before anti-glaucomatous surgery (study group) or who underwent routine cataract surgery (control group). Methods: Aqueous and blood samples were collected at the occasions of intravitreal injections, antiglaucomatous surgery or cataract surgery. They were analysed by enzyme-linked immunosorbent assay. Main Outcome Measures: Concentrations of PEDF and VEGF-A in aqueous.
Results: At baseline, concentrations VEGF-A (3698 ± 2105 pg/mL vs. 233 ± 98 pg/mL) and PEDF (18.9 ± 11.9 ug/mL vs. 2.2 ± 0.6 ug/mL) were higher (P < 0.001) in the study group (n = 20 patients) than control group (n = 20 patients). The VEGF-A/PEDF concentration ratio was higher in the study group (396 ± 554 vs. 110 ± 49; P = 0.02). One week after the ranibizumab injection, iris neovascularization had completely regressed in 17 (85%) eyes, and VEGF-A concentration decreased significantly (P < 0.001) to 184 ± 130 pg/mL. The PEDF concentration remained unchanged (19 ± 12 ug/mL). The VEGF-A/PEDF concentration ratio decreased to 13.2 ± 13.6. Plasma concentrations of VEGF-A and PEDF did not differ significantly between both groups (P = 0.65 and P = 0.15, respectively) nor were they significantly correlated with the aqueous concentrations (all P > 0.15). Conclusions: Aqueous concentrations of VEGF-A and PEDF were significantly elevated in eyes with neovascular glaucoma. Within one week after
■ Correspondence: Professor Xiulan Zhang, Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen University, 54 S. Xianlie Road, Guangzhou 510060, China. Email: [email protected] Received 9 August 2014; accepted 20 November 2014. *Jia Wei Wang and Min Wen Zhou contributed equally to this study and share the first authorship. Conflict of interest: Jost B. Jonas – Consultant for Allergan Inc.; Merck Sharp & Dohme Co., Inc.; Alimera Co.; Boehringer Ingelheim Co., Sanofi Co. Patent holder with CellMed AG, Alzenau, Germany. All other authors: No stated conflict of interest. Funding sources: This research was supported by the National Natural Science Foundation of China (81170849, 81371008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2014 Royal Australian and New Zealand College of Ophthalmologists
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intravitreal injection of ranibizumab, VEGF-A concentration decreased to subnormal levels, while the PEDF concentration remained unchanged and the VEGF-A/PEDF ratio decreased. Key words: intravitreal ranibizumab, neovascular glaucoma, pigment epithelium derived factor, vascular endothelial growth factor-A.
INTRODUCTION Neovascular glaucoma is characterized by the formation of new vessels on the iris surface and in the anterior chamber angle eventually blocking the access of aqueous to the trabecular meshwork. Neovascularization is caused by retinal ischaemia due to various diseases including diabetic retinopathy and ischaemic retinal vein occlusions. Landmark studies by Aiello et al. have shown that vascular endothelial growth factor (VEGF)-A is one of the key cytokines involved in the process of retinal neovascularization.1,2 Subsequent investigations revealed that specific inhibition of VEGF-A by molecules such as bevacizumab or ranibizumab resulted in a marked or complete regression of retinal or subretinal neovascularization.2,3 Shortly after the clinical introduction of intraocular bevacizumab as therapy of neovascular macular diseases, anti-VEGF drugs were also applied to treat neovascular glaucoma.4–9 In contrast to VEGF-A, pigment epitheliumderived factor (PEDF) is one of the most prominent endogenous inhibitors of ocular neovascularization.10 It is purified from conditioned media of human retinal pigment epithelium cells. Studies have shown that PEDF possesses dual activities to control angiogenesis. In the case of hypoxia, PEDF induces a permissive microenvironment for angiogenesis. In the case of normal oxygen tissue concentrations, however, PEDF forms an inhibitory environment against neovascularization, potentially by inhibiting the proliferation and migration of endothelial cells.10 PEDF plays thus a critical role in the homeostasis of angiogenesis in ocular tissues. In addition, PEDF exhibits a variety of other biological properties including anti-apoptotic, antiinflammatory, antioxidative and neuroprotective effects.11–15 As some of the most powerful stimulators and inhibitors of angiogenesis, VEGF-A and PEDF are interwoven with each other. PEDF acts as a major antagonistic component, while VEGF-A is the predominant cytokine in promoting angiogenesis.16 Furthermore, VEGF-A secreted by retinal pigment epithelium cells can stimulate the expression of PEDF.17 Under physiological conditions, the ratio of
the VEGF concentration to the PEDF concentration usually remains within a relatively narrow range. It has been assumed that a balance between the concentrations of VEGF-A and PEDF is important in preventing the development of angiogenesis, while any major change in the equilibrium between the concentrations of VEGF-A and PEDF may lead to neovascularization, as has been demonstrated in the case of proliferative diabetic retinopathy,18,19 retinal vein occlusion,20 exudative age-related degeneration,21,22 exudative myopic maculopathy23 and others.24 None of the studies however has been focused on the relationship between the concentrations of VEGF-A and PEDF in neovascular glaucoma or on the changes of the ratio after treatment. We therefore measured the intraocular concentrations of VEGF-A and PEDF in patients with neovascular glaucoma before and after anti-VEGF therapy and compared the measurements with those obtained in a control group of otherwise healthy patients undergoing routine cataract surgery.
METHODS The prospective interventional comparative study included patients with neovascular glaucoma who attended the hospital in the study period from January 2013 to November 2013. The study was conducted in accordance with the provisions of the Declaration of Helsinki for research involving human subjects. The Ethical Review Committee of the Zhongshan Ophthalmic Center approved the study, and written informed consent was obtained from all study participants. All study participants were Han Chinese. Inclusion criteria for the study group were the presence of neovascularization of the iris and in the anterior chamber angle, uncontrolled intraocular pressure despite maximal tolerable anti-glaucomatous medication, and thus the need for glaucoma surgery which consisted of implantation of an anti-glaucomatous drainage device (FP-7 Ahmed glaucoma valve; New World Medical Inc., Rancho Cucamonga, CA, USA). All patients of the study group underwent an intravitreal ranibizumab injection about one week prior to antiglaucomatous surgery. The control group consisted of patients undergoing routine cataract surgery without any other ocular disease. Systemic diseases such as diabetes mellitus, rheumatoid arthritis arterial hypertension or malignant tumours were exclusion criteria. Medication against arterial hypertension was the only systemic medication allowed. All patients underwent a complete ophthalmic examination including measurement of bestcorrected visual acuity, tonometry, fundus examination and slit-lamp biomicroscopy of the anterior and
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Aqueous factors in neovascular glaucoma posterior segment of the eyes. Iris neovascularization and its change after the intravitreal ranibizumab injection was assessed by slit lamp assisted biomicroscopy of the anterior segment. The intravitreal injection of ranibizumab (Lucentis 0.5 mg/0.05 mL, Genentech Inc, San Francisco, CA, USA) was performed for all patients of the study group. It was conducted as described previously.25 Prior to the intravitreal injection, a paracentesis was performed, and an aqueous sample was obtained. Despite corneal oedema, present in some eyes due to the high intraocular pressure, the tip of the aspirating needle in the anterior chamber could be seen during the sampling of the aqueous. The volume of the aspirated aqueous samples was about 100 μL. Additionally, blood samples (5 mL) were collected from each patient. The blood samples were placed on ice for 30 min. They were then centrifuged to obtain plasma which was stored at −80°C.The aqueous samples were stored at −80°C immediately after collecting them. All patients of the study group underwent a second aqueous sampling when anti-glaucomatous surgery was performed approximately one week later. The participants of the control group underwent cataract surgery at the beginning of which an aqueous sample was obtained through a paracentesis. The concentrations of VEGF-A and of PEDF in plasma and aqueous were measured using an enzyme-linked immunosorbent assay (ELISA) kit(for VEGF-A: Quantitative detection of human VEGF-A ELISA kit, Cat. No.: RBMS277/2R, Biovendor, Modrice, Czech Republic; for PEDF: Quantitative detection of human PEDF ELISA kit, Cat. No.: RD191114200R, Biovendor, Modrice, Czech Republic). The test procedures were conducted in accordance with the manufacturer’s instructions. In brief, the samples were incubated in microtitration wells coated with polyclonal antihuman VEGF-A/PEDF antibody. Through several and repeated incubations and washing procedures, the reaction was terminated via the addition of acid to each well. The absorbance of the resulting yellow product was measured at 450 nm using a multi-well plate reader (Multiskan Ascent; Thermo Fisher Scientific GmbH, Schwerte, Germany). A standard curve was prepared by plotting absorbance values against concentrations of standards, and concentrations of any unknown samples were confirmed based on this curve. All samples were prepared and measured at the same day by the same technician using the same methods. Each measurement was performed thrice, and the mean values of the determinations were used for further statistical analysis. The statistical analysis was conducted using a commercially available statistical software program
417 (IBM-SPSS, version 21.0; Chicago, IL, USA). Normally distributed data were expressed as mean ±standard deviation. The independent sample t-test was used to compare two different groups, while the Mann–Whitney test was used when the data showed a non-parametric distribution. In order to compare the changes between pre- and post-injection within the groups and the correlation between correlative factors, the Wilcoxon-signed rank test and Spearman’s rank-order correlation analysis, respectively, were performed. Categorical covariates were assessed individually using the X2 test. For all calculations, P-values less than 0.05 were considered statistically significant.
RESULTS The study group included 20 patients (15 men and 5 women; 20 eyes)with a mean age of 52.6 ± 21.7 years, and the control group consisted of 20 patients (9 men and 11 women; 20 eyes)with a mean age of 60.9 ± 5.9 years. Both groups did not differ significantly in sex (P = 0.11) and age (P = 0.11). In the patients of the study group, mean intraocular pressure prior to the intravitreal ranibizumab injection was 45.7 ± 5.6 mmHg. Upon slit lamp examination of the anterior ocular segment and upon gonioscopy, all patients of the study group had different degrees of neovascularization of the iris and the anterior chamber angle. The retinal diseases underlying neovascular glaucoma in the study group were diabetic retinopathy (five patients), retinal vein occlusions (nine patients), proliferative vitreoretinopathy (two patients), retinal occlusive vasculitis (two patients) and unknown reasons (two patients). Eight (40%) patients in the study group had arterial hypertension and none in the control group. The time period between the intravitreal ranibizumab injection and the anti-glaucomatous surgery in the study group was 6.9 ± 1.4 days. Aqueous concentration of VEGF-A at baseline was significantly (P < 0.001) higher in the study group than in the control group (3698 ± 2105 pg/mL vs. 233 ± 98 pg/mL). In a parallel manner, the aqueous concentration of PEDF was significantly higher in the study group than in the control group (18.9 ± 11.9 μg/mL vs. 2.2 ± 0.6 μg/mL; P = 0.02). The aqueous concentrations of VEGF-A and of PEDF were not significantly correlated with each other, neither in the study group (P = 0.62) nor in the control group (P = 0.72). Neither the aqueous concentration of VEGF-Aor of PEDF was correlated with intraocular pressure (all P > 0.05). When anti-glaucomatous surgery was performed about one week after the intravitreal injection, iris neovascularization had completely regressed in 17 (85%) eyes and had partially regressed in three
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(15%) eyes. In a parallel manner, the neovascularization in the anterior chamber angle was reduced in all patients. Mean intraocular pressure (42.9 ± 3.0 mmHg) was still markedly elevated, despite application of 2.8 ± 1.1 anti-glaucomatous drugs. The high intraocular pressure mostly unchanged despite the preceding intravitreal injection of ranibizumab was the reason to proceed with the implantation of an Ahmed glaucoma drainage device. In the study group, the mean aqueous concentration of VEGF-A decreased significantly (P < 0.001) after the intravitreal ranibizumab injection from 3698 ± 2105 pg/mL to 184 ± 130 pg/mL (Fig. 1). The latter value did not differ significantly (P > 0.05) from the value obtained in the control group (233 ± 98 pg/mL). The aqueous concentration of PEDF did not differ significantly (P = 0.58) between the baseline examination and the examination performed after the ranibizumab injection (18.9 ± 11.9 μg/mL vs. 16.7 ± 11.3 μg/mL) (Fig. 2). The ratio of the concentrations of VEDF-A to PDEF was at baseline significantly higher in the study group than in the control group (396 ± 554 vs. 110 ± 49; P = 0.02), while after the ranibizumab injection, the ratio was significantly lower in the study group (13.2 ± 13.6; P < 0.001)(Fig. 3).
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Figure 1. Aqueous concentrations of vascular endothelial growth factor (VEGF)-A prior to, and about one week after an intravitreal injection of ranibizumab, and plasma concentrations of VEGF-A prior to the intravitreal ranibizumab injection in patients with neovascular glaucoma. The plasma concentration of VEGF-A (mean: 17.7 ± 7.2 pg/mL) was not significantly (P = 0.0.56) associated with the intraocular concentrations of VEGF-A. **P < 0.001; each point represents a measurement from a single patient.
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Figure 2. Aqueous concentrations of pigment epithelium derived factor (PEDF) prior to, and about one week after an intravitreal injection of ranibizumab, and plasma concentration of PEDF prior to the intravitreal ranibizumab injection in patients with neovascular glaucoma. The plasma concentration of PEDF prior to the ranibizumab injection (mean: 15.2 ± 2.7 μg/mL) was not significantly (P = 0.20) associated with the intraocular concentrations of PEDF. The aqueous concentration of PEDF did not differ significantly (P = 0.58) between the baseline examination and the examination performed after the ranibizumab injection (18.9 ± 11.9 μg/mL vs. 16.7 ± 11.3 μg/mL).
In the control group, the plasma concentration of VEGF-A and PEDF was 16.1 ± 4.2 pg/mL and 17.6 ± 5.9 μg/mL, respectively, and in the study group, the concentrations were 17.7 ± 7.2 pg/mL and 15.2 ± 2.7 μg/mL, with no significant differences between the two groups (P = 0.65, and P = 0.15, resp.). Neither in the study group nor in the control group, the aqueous concentrations of VEGF-A (P = 0.56 and P = 0.35, resp.) or of PEDF (P = 0.20 and P = 0.68, respectively) were significantly correlated with the plasma concentrations of VEGF-A or PEDF.
DISCUSSION Our study suggested that eyes with neovascular glaucoma have an abnormally high intraocular concentration of VEGF and of PEDF, that within one week after an intravitreal application of ranibizumab the VEGF concentration, but not the PEDF concentration, gets markedly reduced to concentrations significantly lower than normal levels, that prior to the injection, the ratio of VEGF concentration to PEDF concentration is markedly elevated, and that this
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Figure 3. Ratio of the vascular endothelial growth factor (VEGF)-A concentration divided by the pigment epithelium-derived factor (PEDF) Concentration (presented as mean ± standard deviation) in aqueous (a) and in plasma (b), in patients with neovascular glaucoma, prior to, and about one week after, an intravitreal injection of ranibizumab (a), and in individuals of a control group. *P < 0.05; **P < 0.001).The VEGF-A/PEDF ratio for the concentrations in plasma did not differ significantly (P = 0.20) between the control group and patients prior to the intravitreal injection of ranibizumab.
ratio profoundly decreases to subnormal levels shortly after the ranibizumab injection. The results of our study agree with findings obtained in previous investigations which revealed an abnormally high intraocular concentration of VEGF in eyes with ischaemic neovascular retinopathies and neovascular glaucoma and which also showed a marked reduction in the intraocular concentration of VEGF after an injection of anti-VEGF drugs.26–29 Abnormally high concentrations of PEDF as observed in our study were usually not detected in previous studies on patients with neovascular glaucoma.18–20,24,30 Interestingly, high PEDF concentrations were also observed in eyes with choroidal neovascularization secondary to age-related macular degeneration,22 and in highly myopic patients with exudative myopic maculopathy.23 The findings may suggest a complex relationship between VEGF-A and PEDF and the underlying neovascular diseases. One of the reasons responsible for the high aqueous concentration of PEDF in eyes with neovascular glaucoma may be that the high concentration of VEGF-A, induced by marked retinal ischaemia, secondarily leads to an elevation of the PEDF concentration. An elevated expression of PEDF has been considered to counteract an elevation of the VEGF-A concentration, and studies have shown that high VEGF concentrations upregulate the PEDF expression.17 Our finding that the aqueous concentration of PEDF did not show a significant change after the intravitreal injection of ranibizumab disagrees with a previous study in which the aqueous concentrations of PEDF were reduced at one month after the ranibizumab application.22 A reason for the discrepancy between the studies may be the difference in
the follow-up time of one week in our study and one month in the previous investigation. It could imply that the potential reduction in the PEDF concentration takes a longer time to occur compared with the decrease in the VEGF concentration. Interestingly, neither the intraocular concentrations of VEGF nor of PEDF were significantly correlated with intraocular pressure at baseline. It may suggest that the major determinant for the increase in intraocular pressure may be the extent of VEGF induced anterior synechias in the anterior chamber angle. A balance between the intraocular concentrations of VEGF-A and PEDF may be of importance for the prognosis of neovascular diseases, as studies have revealed that an abnormal ratio of VEGF to PEDF concentrations is related with major neovascular diseases.18–20,22–24,30 In our study, the VEGF-A/PEDF ratio was significantly higher (P < 0.001) in the eyes with neovascular glaucoma than in the eyes of the control group. After the intravitreal ranibizumab injection, the ratio decreased to subnormal levels. Although the marked decrease in the ratio shortly after the ranibizumab injection may be due to a time lag in a rapid decrease in the VEGF-A concentration and retarded decrease in the PEDF concentration, it has remained unclear which effects such profound changes in the concentration ratios of VEGF-A and PEDF may have. Potential imitations of our study should be mentioned. First, the sample size was relatively small. Despite of it, however, the statistical analysis showed significant results so that the small number of patients included into our study may only serve to strengthen the results and conclusions drawn. Second, we could not exclude the possibility that anti-glaucomatous drugs applied topically may have
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affected the concentrations of VEGF-A and PEDF in the aqueous. Third, the follow-up of one week after the intravitreal ranibizumab was relatively short so that no information was gained on the intraocular concentrations of the cytokines after a longer followup. Fourth, we did not collect blood samples after the intravitreal ranibizumab injection so that only the plasma concentrations of the cytokines measured prior to the injection could be compared with the intraocular cytokine concentrations. One might discuss that the elevated concentrations of VEGF-A and PEDF at baseline of the study were due to a leakage of the cytokines from the blood into the aqueous so that the intraocular cytokine production might not have been elevated. The serum concentrations of VEGF-A and PEDF, however, were not related with the intraocular concentrations of VEGF-A and PEDF. It made it likely that the increased intraocular concentrations of VEGF-A and PEDF were due to an increased intraocular production. In summary, eyes with neovascular glaucoma have an abnormally high intraocular concentration of VEGF-A and of PEDF. Within one week after an intravitreal application of ranibizumab, the VEGF-A concentration, but not the PEDF concentration, is markedly reduced to subnormal concentrations. Consequently, the concentration ratio of VEGF-A to PEDF concentration is markedly elevated in neovascular glaucoma (NVG) and gets profoundly reduced shortly after an intravitreal ranibizumab injection. Future studies may address whether the change in the VEGFA/PEDF ratio, in addition to the single concentrations of VEGF-A and PEDF, play a role in the therapy and prognosis of neovascular glaucoma in particular and ischaemic retinopathies in general.
REFERENCES 1. Aiello LP, Avery RL, Arrigg PG et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994; 331: 1480–7. 2. Aiello LP, Pierce EA, Foley ED et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci U S A 1995; 92: 10457–61. 3. Rosenfeld PJ, Brown DM, Heier JS et al. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 2006; 355: 1419–31. 4. Iliev ME, Domig D, Wolf-Schnurrbursch U, Wolf S, Sarra GM. Intravitreal bevacizumab (Avastin) in the treatment of neovascular glaucoma. Am J Ophthalmol 2006; 142: 1054–6. 5. Davidorf FH, Mouser JG, Derick RJ. Rapid improvement of rubeosis iridis from a single bevacizumab (Avastin) injection. Retina 2006; 26: 354–6.
6. Grisanti S, Biester S, Peters S, Tatar O, Ziemssen F, Bartz-Schmidt KU. Intracameral bevacizumab for iris rubeosis. Am J Ophthalmol 2006; 142: 158–60. 7. Yazdani S, Hendi K, Pakravan M. Intravitreal bevacizumab (Avastin) injection for neovascular glaucoma. J Glaucoma 2007; 16: 437–9. 8. Wakabayashi T, Oshima Y, Sakaguchi H et al. Intravitreal bevacizumab to treat iris neovascularization and neovascular glaucoma secondary to ischemic retinal diseases in 41 consecutive cases. Ophthalmology 2008; 115: 1571–80, 1580.e1–3. 9. Yazdani S, Hendi K, Pakravan M, Mahdavi M, Yaseri M. Intravitreal bevacizumab for neovascular glaucoma: a randomized controlled trial. J Glaucoma 2009; 18: 632–7. 10. Dawson DW, Volpert OV, Gillis P et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 1999; 285: 245–8. 11. Zhang SX, Wang JJ, Gao G, Shao C, Mott R, Ma JX. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor. FASEB J 2006; 20: 323–5. 12. Vigneswara V, Berry M, Logan A, Ahmed Z. Pigment epithelium-derived factor is retinal ganglion cell neuroprotective and axogenic after optic nerve crush injury. Invest Ophthalmol Vis Sci 2013; 54: 2624–33. 13. Sanchez A, Tripathy D, Yin X, Luo J, Martinez J, Grammas P. Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2. Neurosci Res 2012; 72: 1–8. 14. Zhou X, Li F, Kong L, Chodosh J, Cao W. Antiinflammatory effect of pigment epithelium-derived factor in DBA/2J mice. Mol Vis 2009; 15: 438–50. 15. Bilak MM, Corse AM, Bilak SR, Lehar M, Tombran-Tink J, Kuncl RW. Pigment epitheliumderived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration. J Neuropathol Exp Neurol 1999; 58: 719–28. 16. Zhang SX, Wang JJ, Gao G, Parke K, Ma JX. Pigment epithelium-derived factor downregulates vascular endothelial growth factor (VEGF) expression and inhibits VEGF-VEGF receptor 2 binding in diabetic retinopathy. J Mol Endocrinol 2006; 37: 1–12. 17. Ohno-Matsui K, Yoshida T, Uetama T, Mochizuki M, Morita I. Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells. Biochem Biophys Res Commun 2003; 303: 962–7. 18. Li S, Fu XA, Zhou XF, Chen YY, Chen WQ. Angiogenesis-related cytokines in serum of proliferative diabetic retinopathy patients before and after vitrectomy. Int J Ophthalmol 2012; 5: 726–30. 19. Ogata N, Nishikawa M, Nishimura T, Mitsuma Y, Matsumura M. Unbalanced vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic retinopathy. Am J Ophthalmol 2002; 134: 348–53. 20. Noma H, Mimura T, Eguchi S. Association of inflammatory factors with macular edema in branch
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retinal vein occlusion. JAMA Ophthalmol 2013; 131: 160–5. Machalinska A, Safranow K, Mozolewska-Piotrowska K, Dziedziejko V, Karczewicz D. PEDF and VEGF plasma level alterations in patients with dry form of age-related degeneration – a possible link to the development of the disease. Klin Oczna 2012; 114: 115–20. Ahn JK, Moon HJ. Changes in aqueous vascular endothelial growth factor and pigment epitheliumderived factor after ranibizumab alone or combined with verteporfin for exudative age-related macular degeneration. Am J Ophthalmol 2009; 148: 718–24, e1. Shin YJ, Nam WH, Park SE, Kim JH, Kim HK. Aqueous humor concentrations of vascular endothelial growth factor and pigment epithelium-derived factor in high myopic patients. Mol Vis 2012; 18: 2265–70. Gao G, Li Y, Zhang D, Gee S, Crosson C, Ma J. Unbalanced expression of VEGF and PEDF in ischemiainduced retinal neovascularization. FEBS Lett 2001; 489: 270–6. Zhou M, Chen S, Wang W et al. Levels of erythropoietin and vascular endothelial growth factor in surgeryrequired advanced neovascular glaucoma eyes before and after intravitreal injection of bevacizumab. Invest Ophthalmol Vis Sci 2013; 54: 3874–9.
421 26. Grover S, Gupta S, Sharma R, Brar VS, Chalam KV. Intracameral bevacizumab effectively reduces aqueous vascular endothelial growth factor concentrations in neovascular glaucoma. Br J Ophthalmol 2009; 93: 273–4. 27. Matsuyama K, Ogata N, Jo N, Shima C, Matsuoka M, Matsumura M. Levels of vascular endothelial growth factor and pigment epithelium-derived factor in eyes before and after intravitreal injection of bevacizumab. Jpn J Ophthalmol 2009; 53: 243–8. 28. Lim TH, Bae SH, Cho YJ, Lee JH, Kim HK, Sohn YH. Concentration of vascular endothelial growth factor after intracameral bevacizumab injection in eyes with neovascular glaucoma. Korean J Ophthalmol 2009; 23: 188–92. 29. Sasamoto Y, Oshima Y, Miki A et al. Clinical outcomes and changes in aqueous vascular endothelial growth factor levels after intravitreal bevacizumab for iris neovascularization and neovascular glaucoma: a retrospective two-dose comparative study. J Ocul Pharmacol Ther 2012; 28: 41–8. 30. Pons M, Marin-Castano ME. Nicotine increases the VEGF/PEDF ratio in retinal pigment epithelium: a possible mechanism for CNV in passive smokers with AMD. Invest Ophthalmol Vis Sci 2011; 52: 3842–53.
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Clinical and Experimental Ophthalmology 2015; 43: 415–421 doi: 10.1111/ceo.12477
Original Article Short-term effect of intravitreal ranibizumab on intraocular concentrations of vascular endothelial growth factor-A and pigment epithelium-derived factor in neovascular glaucoma Jia Wei Wang MD,1* Min Wen Zhou MD,1* Xiulan Zhang MD PhD,1 Wen Bin Huang MD,1 Xin Bo Gao MD,1 Wei Wang MD,1 Shida Chen MD,1 Xin Yu Zhang MD PhD,1 Xiao Yan Ding MD PhD1 and Jost B Jonas MD2 1
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen University, Guangzhou, China; and Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht-Karls-University, Heidelberg, Germany
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ABSTRACT Background: This study aims to evaluate concentrations of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF)-A in aqueous of patients with neovascular glaucoma prior to, and shortly after, an intravitreal ranibizumab injection. Design: Interventional comparative study. Participants: The study included patients undergoing an intravitreal ranibizumab injection about one week before anti-glaucomatous surgery (study group) or who underwent routine cataract surgery (control group). Methods: Aqueous and blood samples were collected at the occasions of intravitreal injections, antiglaucomatous surgery or cataract surgery. They were analysed by enzyme-linked immunosorbent assay. Main Outcome Measures: Concentrations of PEDF and VEGF-A in aqueous.
Results: At baseline, concentrations VEGF-A (3698 ± 2105 pg/mL vs. 233 ± 98 pg/mL) and PEDF (18.9 ± 11.9 ug/mL vs. 2.2 ± 0.6 ug/mL) were higher (P < 0.001) in the study group (n = 20 patients) than control group (n = 20 patients). The VEGF-A/PEDF concentration ratio was higher in the study group (396 ± 554 vs. 110 ± 49; P = 0.02). One week after the ranibizumab injection, iris neovascularization had completely regressed in 17 (85%) eyes, and VEGF-A concentration decreased significantly (P < 0.001) to 184 ± 130 pg/mL. The PEDF concentration remained unchanged (19 ± 12 ug/mL). The VEGF-A/PEDF concentration ratio decreased to 13.2 ± 13.6. Plasma concentrations of VEGF-A and PEDF did not differ significantly between both groups (P = 0.65 and P = 0.15, respectively) nor were they significantly correlated with the aqueous concentrations (all P > 0.15). Conclusions: Aqueous concentrations of VEGF-A and PEDF were significantly elevated in eyes with neovascular glaucoma. Within one week after
■ Correspondence: Professor Xiulan Zhang, Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen University, 54 S. Xianlie Road, Guangzhou 510060, China. Email: [email protected] Received 9 August 2014; accepted 20 November 2014. *Jia Wei Wang and Min Wen Zhou contributed equally to this study and share the first authorship. Conflict of interest: Jost B. Jonas – Consultant for Allergan Inc.; Merck Sharp & Dohme Co., Inc.; Alimera Co.; Boehringer Ingelheim Co., Sanofi Co. Patent holder with CellMed AG, Alzenau, Germany. All other authors: No stated conflict of interest. Funding sources: This research was supported by the National Natural Science Foundation of China (81170849, 81371008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2014 Royal Australian and New Zealand College of Ophthalmologists
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intravitreal injection of ranibizumab, VEGF-A concentration decreased to subnormal levels, while the PEDF concentration remained unchanged and the VEGF-A/PEDF ratio decreased. Key words: intravitreal ranibizumab, neovascular glaucoma, pigment epithelium derived factor, vascular endothelial growth factor-A.
INTRODUCTION Neovascular glaucoma is characterized by the formation of new vessels on the iris surface and in the anterior chamber angle eventually blocking the access of aqueous to the trabecular meshwork. Neovascularization is caused by retinal ischaemia due to various diseases including diabetic retinopathy and ischaemic retinal vein occlusions. Landmark studies by Aiello et al. have shown that vascular endothelial growth factor (VEGF)-A is one of the key cytokines involved in the process of retinal neovascularization.1,2 Subsequent investigations revealed that specific inhibition of VEGF-A by molecules such as bevacizumab or ranibizumab resulted in a marked or complete regression of retinal or subretinal neovascularization.2,3 Shortly after the clinical introduction of intraocular bevacizumab as therapy of neovascular macular diseases, anti-VEGF drugs were also applied to treat neovascular glaucoma.4–9 In contrast to VEGF-A, pigment epitheliumderived factor (PEDF) is one of the most prominent endogenous inhibitors of ocular neovascularization.10 It is purified from conditioned media of human retinal pigment epithelium cells. Studies have shown that PEDF possesses dual activities to control angiogenesis. In the case of hypoxia, PEDF induces a permissive microenvironment for angiogenesis. In the case of normal oxygen tissue concentrations, however, PEDF forms an inhibitory environment against neovascularization, potentially by inhibiting the proliferation and migration of endothelial cells.10 PEDF plays thus a critical role in the homeostasis of angiogenesis in ocular tissues. In addition, PEDF exhibits a variety of other biological properties including anti-apoptotic, antiinflammatory, antioxidative and neuroprotective effects.11–15 As some of the most powerful stimulators and inhibitors of angiogenesis, VEGF-A and PEDF are interwoven with each other. PEDF acts as a major antagonistic component, while VEGF-A is the predominant cytokine in promoting angiogenesis.16 Furthermore, VEGF-A secreted by retinal pigment epithelium cells can stimulate the expression of PEDF.17 Under physiological conditions, the ratio of
the VEGF concentration to the PEDF concentration usually remains within a relatively narrow range. It has been assumed that a balance between the concentrations of VEGF-A and PEDF is important in preventing the development of angiogenesis, while any major change in the equilibrium between the concentrations of VEGF-A and PEDF may lead to neovascularization, as has been demonstrated in the case of proliferative diabetic retinopathy,18,19 retinal vein occlusion,20 exudative age-related degeneration,21,22 exudative myopic maculopathy23 and others.24 None of the studies however has been focused on the relationship between the concentrations of VEGF-A and PEDF in neovascular glaucoma or on the changes of the ratio after treatment. We therefore measured the intraocular concentrations of VEGF-A and PEDF in patients with neovascular glaucoma before and after anti-VEGF therapy and compared the measurements with those obtained in a control group of otherwise healthy patients undergoing routine cataract surgery.
METHODS The prospective interventional comparative study included patients with neovascular glaucoma who attended the hospital in the study period from January 2013 to November 2013. The study was conducted in accordance with the provisions of the Declaration of Helsinki for research involving human subjects. The Ethical Review Committee of the Zhongshan Ophthalmic Center approved the study, and written informed consent was obtained from all study participants. All study participants were Han Chinese. Inclusion criteria for the study group were the presence of neovascularization of the iris and in the anterior chamber angle, uncontrolled intraocular pressure despite maximal tolerable anti-glaucomatous medication, and thus the need for glaucoma surgery which consisted of implantation of an anti-glaucomatous drainage device (FP-7 Ahmed glaucoma valve; New World Medical Inc., Rancho Cucamonga, CA, USA). All patients of the study group underwent an intravitreal ranibizumab injection about one week prior to antiglaucomatous surgery. The control group consisted of patients undergoing routine cataract surgery without any other ocular disease. Systemic diseases such as diabetes mellitus, rheumatoid arthritis arterial hypertension or malignant tumours were exclusion criteria. Medication against arterial hypertension was the only systemic medication allowed. All patients underwent a complete ophthalmic examination including measurement of bestcorrected visual acuity, tonometry, fundus examination and slit-lamp biomicroscopy of the anterior and
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Aqueous factors in neovascular glaucoma posterior segment of the eyes. Iris neovascularization and its change after the intravitreal ranibizumab injection was assessed by slit lamp assisted biomicroscopy of the anterior segment. The intravitreal injection of ranibizumab (Lucentis 0.5 mg/0.05 mL, Genentech Inc, San Francisco, CA, USA) was performed for all patients of the study group. It was conducted as described previously.25 Prior to the intravitreal injection, a paracentesis was performed, and an aqueous sample was obtained. Despite corneal oedema, present in some eyes due to the high intraocular pressure, the tip of the aspirating needle in the anterior chamber could be seen during the sampling of the aqueous. The volume of the aspirated aqueous samples was about 100 μL. Additionally, blood samples (5 mL) were collected from each patient. The blood samples were placed on ice for 30 min. They were then centrifuged to obtain plasma which was stored at −80°C.The aqueous samples were stored at −80°C immediately after collecting them. All patients of the study group underwent a second aqueous sampling when anti-glaucomatous surgery was performed approximately one week later. The participants of the control group underwent cataract surgery at the beginning of which an aqueous sample was obtained through a paracentesis. The concentrations of VEGF-A and of PEDF in plasma and aqueous were measured using an enzyme-linked immunosorbent assay (ELISA) kit(for VEGF-A: Quantitative detection of human VEGF-A ELISA kit, Cat. No.: RBMS277/2R, Biovendor, Modrice, Czech Republic; for PEDF: Quantitative detection of human PEDF ELISA kit, Cat. No.: RD191114200R, Biovendor, Modrice, Czech Republic). The test procedures were conducted in accordance with the manufacturer’s instructions. In brief, the samples were incubated in microtitration wells coated with polyclonal antihuman VEGF-A/PEDF antibody. Through several and repeated incubations and washing procedures, the reaction was terminated via the addition of acid to each well. The absorbance of the resulting yellow product was measured at 450 nm using a multi-well plate reader (Multiskan Ascent; Thermo Fisher Scientific GmbH, Schwerte, Germany). A standard curve was prepared by plotting absorbance values against concentrations of standards, and concentrations of any unknown samples were confirmed based on this curve. All samples were prepared and measured at the same day by the same technician using the same methods. Each measurement was performed thrice, and the mean values of the determinations were used for further statistical analysis. The statistical analysis was conducted using a commercially available statistical software program
417 (IBM-SPSS, version 21.0; Chicago, IL, USA). Normally distributed data were expressed as mean ±standard deviation. The independent sample t-test was used to compare two different groups, while the Mann–Whitney test was used when the data showed a non-parametric distribution. In order to compare the changes between pre- and post-injection within the groups and the correlation between correlative factors, the Wilcoxon-signed rank test and Spearman’s rank-order correlation analysis, respectively, were performed. Categorical covariates were assessed individually using the X2 test. For all calculations, P-values less than 0.05 were considered statistically significant.
RESULTS The study group included 20 patients (15 men and 5 women; 20 eyes)with a mean age of 52.6 ± 21.7 years, and the control group consisted of 20 patients (9 men and 11 women; 20 eyes)with a mean age of 60.9 ± 5.9 years. Both groups did not differ significantly in sex (P = 0.11) and age (P = 0.11). In the patients of the study group, mean intraocular pressure prior to the intravitreal ranibizumab injection was 45.7 ± 5.6 mmHg. Upon slit lamp examination of the anterior ocular segment and upon gonioscopy, all patients of the study group had different degrees of neovascularization of the iris and the anterior chamber angle. The retinal diseases underlying neovascular glaucoma in the study group were diabetic retinopathy (five patients), retinal vein occlusions (nine patients), proliferative vitreoretinopathy (two patients), retinal occlusive vasculitis (two patients) and unknown reasons (two patients). Eight (40%) patients in the study group had arterial hypertension and none in the control group. The time period between the intravitreal ranibizumab injection and the anti-glaucomatous surgery in the study group was 6.9 ± 1.4 days. Aqueous concentration of VEGF-A at baseline was significantly (P < 0.001) higher in the study group than in the control group (3698 ± 2105 pg/mL vs. 233 ± 98 pg/mL). In a parallel manner, the aqueous concentration of PEDF was significantly higher in the study group than in the control group (18.9 ± 11.9 μg/mL vs. 2.2 ± 0.6 μg/mL; P = 0.02). The aqueous concentrations of VEGF-A and of PEDF were not significantly correlated with each other, neither in the study group (P = 0.62) nor in the control group (P = 0.72). Neither the aqueous concentration of VEGF-Aor of PEDF was correlated with intraocular pressure (all P > 0.05). When anti-glaucomatous surgery was performed about one week after the intravitreal injection, iris neovascularization had completely regressed in 17 (85%) eyes and had partially regressed in three
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(15%) eyes. In a parallel manner, the neovascularization in the anterior chamber angle was reduced in all patients. Mean intraocular pressure (42.9 ± 3.0 mmHg) was still markedly elevated, despite application of 2.8 ± 1.1 anti-glaucomatous drugs. The high intraocular pressure mostly unchanged despite the preceding intravitreal injection of ranibizumab was the reason to proceed with the implantation of an Ahmed glaucoma drainage device. In the study group, the mean aqueous concentration of VEGF-A decreased significantly (P < 0.001) after the intravitreal ranibizumab injection from 3698 ± 2105 pg/mL to 184 ± 130 pg/mL (Fig. 1). The latter value did not differ significantly (P > 0.05) from the value obtained in the control group (233 ± 98 pg/mL). The aqueous concentration of PEDF did not differ significantly (P = 0.58) between the baseline examination and the examination performed after the ranibizumab injection (18.9 ± 11.9 μg/mL vs. 16.7 ± 11.3 μg/mL) (Fig. 2). The ratio of the concentrations of VEDF-A to PDEF was at baseline significantly higher in the study group than in the control group (396 ± 554 vs. 110 ± 49; P = 0.02), while after the ranibizumab injection, the ratio was significantly lower in the study group (13.2 ± 13.6; P < 0.001)(Fig. 3).
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Figure 1. Aqueous concentrations of vascular endothelial growth factor (VEGF)-A prior to, and about one week after an intravitreal injection of ranibizumab, and plasma concentrations of VEGF-A prior to the intravitreal ranibizumab injection in patients with neovascular glaucoma. The plasma concentration of VEGF-A (mean: 17.7 ± 7.2 pg/mL) was not significantly (P = 0.0.56) associated with the intraocular concentrations of VEGF-A. **P < 0.001; each point represents a measurement from a single patient.
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Figure 2. Aqueous concentrations of pigment epithelium derived factor (PEDF) prior to, and about one week after an intravitreal injection of ranibizumab, and plasma concentration of PEDF prior to the intravitreal ranibizumab injection in patients with neovascular glaucoma. The plasma concentration of PEDF prior to the ranibizumab injection (mean: 15.2 ± 2.7 μg/mL) was not significantly (P = 0.20) associated with the intraocular concentrations of PEDF. The aqueous concentration of PEDF did not differ significantly (P = 0.58) between the baseline examination and the examination performed after the ranibizumab injection (18.9 ± 11.9 μg/mL vs. 16.7 ± 11.3 μg/mL).
In the control group, the plasma concentration of VEGF-A and PEDF was 16.1 ± 4.2 pg/mL and 17.6 ± 5.9 μg/mL, respectively, and in the study group, the concentrations were 17.7 ± 7.2 pg/mL and 15.2 ± 2.7 μg/mL, with no significant differences between the two groups (P = 0.65, and P = 0.15, resp.). Neither in the study group nor in the control group, the aqueous concentrations of VEGF-A (P = 0.56 and P = 0.35, resp.) or of PEDF (P = 0.20 and P = 0.68, respectively) were significantly correlated with the plasma concentrations of VEGF-A or PEDF.
DISCUSSION Our study suggested that eyes with neovascular glaucoma have an abnormally high intraocular concentration of VEGF and of PEDF, that within one week after an intravitreal application of ranibizumab the VEGF concentration, but not the PEDF concentration, gets markedly reduced to concentrations significantly lower than normal levels, that prior to the injection, the ratio of VEGF concentration to PEDF concentration is markedly elevated, and that this
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Figure 3. Ratio of the vascular endothelial growth factor (VEGF)-A concentration divided by the pigment epithelium-derived factor (PEDF) Concentration (presented as mean ± standard deviation) in aqueous (a) and in plasma (b), in patients with neovascular glaucoma, prior to, and about one week after, an intravitreal injection of ranibizumab (a), and in individuals of a control group. *P < 0.05; **P < 0.001).The VEGF-A/PEDF ratio for the concentrations in plasma did not differ significantly (P = 0.20) between the control group and patients prior to the intravitreal injection of ranibizumab.
ratio profoundly decreases to subnormal levels shortly after the ranibizumab injection. The results of our study agree with findings obtained in previous investigations which revealed an abnormally high intraocular concentration of VEGF in eyes with ischaemic neovascular retinopathies and neovascular glaucoma and which also showed a marked reduction in the intraocular concentration of VEGF after an injection of anti-VEGF drugs.26–29 Abnormally high concentrations of PEDF as observed in our study were usually not detected in previous studies on patients with neovascular glaucoma.18–20,24,30 Interestingly, high PEDF concentrations were also observed in eyes with choroidal neovascularization secondary to age-related macular degeneration,22 and in highly myopic patients with exudative myopic maculopathy.23 The findings may suggest a complex relationship between VEGF-A and PEDF and the underlying neovascular diseases. One of the reasons responsible for the high aqueous concentration of PEDF in eyes with neovascular glaucoma may be that the high concentration of VEGF-A, induced by marked retinal ischaemia, secondarily leads to an elevation of the PEDF concentration. An elevated expression of PEDF has been considered to counteract an elevation of the VEGF-A concentration, and studies have shown that high VEGF concentrations upregulate the PEDF expression.17 Our finding that the aqueous concentration of PEDF did not show a significant change after the intravitreal injection of ranibizumab disagrees with a previous study in which the aqueous concentrations of PEDF were reduced at one month after the ranibizumab application.22 A reason for the discrepancy between the studies may be the difference in
the follow-up time of one week in our study and one month in the previous investigation. It could imply that the potential reduction in the PEDF concentration takes a longer time to occur compared with the decrease in the VEGF concentration. Interestingly, neither the intraocular concentrations of VEGF nor of PEDF were significantly correlated with intraocular pressure at baseline. It may suggest that the major determinant for the increase in intraocular pressure may be the extent of VEGF induced anterior synechias in the anterior chamber angle. A balance between the intraocular concentrations of VEGF-A and PEDF may be of importance for the prognosis of neovascular diseases, as studies have revealed that an abnormal ratio of VEGF to PEDF concentrations is related with major neovascular diseases.18–20,22–24,30 In our study, the VEGF-A/PEDF ratio was significantly higher (P < 0.001) in the eyes with neovascular glaucoma than in the eyes of the control group. After the intravitreal ranibizumab injection, the ratio decreased to subnormal levels. Although the marked decrease in the ratio shortly after the ranibizumab injection may be due to a time lag in a rapid decrease in the VEGF-A concentration and retarded decrease in the PEDF concentration, it has remained unclear which effects such profound changes in the concentration ratios of VEGF-A and PEDF may have. Potential imitations of our study should be mentioned. First, the sample size was relatively small. Despite of it, however, the statistical analysis showed significant results so that the small number of patients included into our study may only serve to strengthen the results and conclusions drawn. Second, we could not exclude the possibility that anti-glaucomatous drugs applied topically may have
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affected the concentrations of VEGF-A and PEDF in the aqueous. Third, the follow-up of one week after the intravitreal ranibizumab was relatively short so that no information was gained on the intraocular concentrations of the cytokines after a longer followup. Fourth, we did not collect blood samples after the intravitreal ranibizumab injection so that only the plasma concentrations of the cytokines measured prior to the injection could be compared with the intraocular cytokine concentrations. One might discuss that the elevated concentrations of VEGF-A and PEDF at baseline of the study were due to a leakage of the cytokines from the blood into the aqueous so that the intraocular cytokine production might not have been elevated. The serum concentrations of VEGF-A and PEDF, however, were not related with the intraocular concentrations of VEGF-A and PEDF. It made it likely that the increased intraocular concentrations of VEGF-A and PEDF were due to an increased intraocular production. In summary, eyes with neovascular glaucoma have an abnormally high intraocular concentration of VEGF-A and of PEDF. Within one week after an intravitreal application of ranibizumab, the VEGF-A concentration, but not the PEDF concentration, is markedly reduced to subnormal concentrations. Consequently, the concentration ratio of VEGF-A to PEDF concentration is markedly elevated in neovascular glaucoma (NVG) and gets profoundly reduced shortly after an intravitreal ranibizumab injection. Future studies may address whether the change in the VEGFA/PEDF ratio, in addition to the single concentrations of VEGF-A and PEDF, play a role in the therapy and prognosis of neovascular glaucoma in particular and ischaemic retinopathies in general.
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421 26. Grover S, Gupta S, Sharma R, Brar VS, Chalam KV. Intracameral bevacizumab effectively reduces aqueous vascular endothelial growth factor concentrations in neovascular glaucoma. Br J Ophthalmol 2009; 93: 273–4. 27. Matsuyama K, Ogata N, Jo N, Shima C, Matsuoka M, Matsumura M. Levels of vascular endothelial growth factor and pigment epithelium-derived factor in eyes before and after intravitreal injection of bevacizumab. Jpn J Ophthalmol 2009; 53: 243–8. 28. Lim TH, Bae SH, Cho YJ, Lee JH, Kim HK, Sohn YH. Concentration of vascular endothelial growth factor after intracameral bevacizumab injection in eyes with neovascular glaucoma. Korean J Ophthalmol 2009; 23: 188–92. 29. Sasamoto Y, Oshima Y, Miki A et al. Clinical outcomes and changes in aqueous vascular endothelial growth factor levels after intravitreal bevacizumab for iris neovascularization and neovascular glaucoma: a retrospective two-dose comparative study. J Ocul Pharmacol Ther 2012; 28: 41–8. 30. Pons M, Marin-Castano ME. Nicotine increases the VEGF/PEDF ratio in retinal pigment epithelium: a possible mechanism for CNV in passive smokers with AMD. Invest Ophthalmol Vis Sci 2011; 52: 3842–53.
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