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Clinical and Experimental Ophthalmology 2015; 43: 458–465 doi: 10.1111/ceo.12473
Original Article Rofecoxib inhibits retinal neovascularization via down regulation of cyclooxygenase-2 and vascular endothelial growth factor expression Ning-ning Liu PhD, Yi-zhou Sun MD, Ning Zhao MD and Lei Chen MD Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China
ABSTRACT Background: To explore the anti-angiogenesis mechanism of Rofecoxib and determine whether Rofecoxib can be a therapeutic agent for the prevention of retinal neovascularization using a model of retinopathy of prematurity (ROP). Methods: ROP was induced by exposing mice to 75% oxygen from postnatal day 7 (P7) to P12, then to room air from P12 to P17. Sixteen mice were in each of the three groups: untreated ROP group as positive control, Rofecoxib-treated ROP group and the normal group (age-matched mice maintained in room air from birth to P17 as negative control). The localized expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) protein and mRNA in retinal blood vessels was assessed using immunohistochemistry, Western blot analysis and reverse transcription polymerase chain reaction. Results: Mice in the Rofecoxib-treated group had a significantly reduced retinal neovascular tufts compared with those in the untreated ROP group. COX-2 and VEGF protein and mRNA expression levels were increased in the untreated ROP group, compared with the normal group. Rofecoxib decreased retinal angiogenesis by inhibiting COX-2 and VEGF expression. The expression levels of VEGF and COX-2 were positively correlated at mRNA and protein levels.
Conclusions: COX-2 and VEGF expressions were both involved in the regulation of angiogenesis and had the same cellular localization. Expression of COX-2 correlated positively with VEGF in retinal neovascularization. Rofecoxib attenuated retinal angiogenesis by inhibiting the expression of COX-2 and VEGF mRNA and protein. Key words: retinal neovascularization, Rofecoxib, vascular endothelial growth factor.
INTRODUCTION Pathological angiogenesis, or neovascularization of the eye, is a complex process and major cause of blindness in developed countries.1,2 It is clear that ischaemia-induced hypoxia is a primary aetiological factor causing ocular neovascularization.3 Vascular endothelial growth factor (VEGF) is the most prominently potent angiogenic factor in response to hypoxia.4,5 Highly expressed VEGF were found in the diabetic retinopathy, neovascular age-related macular degeneration, and inflammation-associated corneal neovascularization.6–8 The binding of VEGF with its receptors initiates signal transduction cascades, causing angiogenic endothelia cell behaviours. The inhibition of the VEGF/VEGF-receptor pathway may potentially be an efficacious therapeutic strategy to suppress experimental ocular neovascularization.
■ Correspondence: Professor Lei Chen, Department of Ophthalmology, The First Affiliated Hospital of China Medical University, No. 155, Nanjing North Street, Heping Area, Shenyang, Liaoning, China 110001. Email: [email protected] Received 17 June 2014; accepted 12 November 2014. Conflict of interest: No stated conflict of interest. Funding sources: This research was supported by Doctoral Scientific Research Foundation of China (No. 20091115). © 2014 Royal Australian and New Zealand College of Ophthalmologists
Rofecoxib and retinal neovascularization Cyclooxygenase (COX)-prostaglandin pathway has recently been found to work in conjunction with VEGF and plays a vital role in the formation of new blood vessels. COX enzymes catalyse the production of prostanoids (prostaglandins and thromboxanes) and have at least two isoforms: COX-1 and COX-2. COX-1 is a housekeeping enzyme and presented in most tissues, whereas COX-2 is a rate-limiting, inducible enzyme that converts arachidonic acid into various prostaglandins.9,10 High expression of COX-2 is linked to VEGF overexpression in tumours suggesting that COX-2 plays a part in the regulation of VEGF expression in the angiogenesis of tumour blood vessels.11,12 Studies also found that, in response of hypoxia, high expression of COX-2 is associated with upregulation of VEGF production in retina.13,14 Compounds that induce COX-2 expression can induce VEGF, and COX-2 inhibitors also inhibit VEGF.15 These findings suggest a key regulatory and potential role for COX-2 in the pathways of VEGF expression in neovascularization. Retinal angiogenesis is the common pathophysiological basis of all retinal vascular diseases. Laser photocoagulation is usually applied clinically to inhibit the development of retinal neovascularization and has shown some therapeutic effects.16 Laser treatment, however, only delays the progression of lesions and may potentially further damage retinal tissue.17 Drug therapy inhibiting vascular proliferation in intraocular neovascularization has become an important area in ophthalmological research, but the clinical therapeutic effects are yet to be satisfactory.18 Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are inhibitors of both COX-1 and COX-2, and are generally used for the treatment of pain and arthritis. Increasing evidence showed that high dose of aspirin administration decreased retinal vascular leakage and abnormalities in diabetic rates and reduced the incidence of diabetic retinopathy in humans.19,20 The gastrointestinal side effects of NSAIDs resulting from the blockage of COX-1 prompted the development of selective COX-2 inhibitors, which in recent years have proven to exhibit potent anti-tumour and anti-angiogenic effects; thus, they are considered to be novel candidate agents for the treatment of vascular proliferative diseases.21 In a prostate cancer model, inhibition of COX-2 suppresses angiogenesis and tumour growth through suppressing VEGF expression.22 In cancer cell lines, treatment with selective COX-2 inhibitor actually induced the expression of VEGF.23 In the eyes, the antiangiogenesis properties of COX-2 inhibitor have been demonstrated in experimental models.24,25 However, the exact molecular mechanisms through which the COX-2 inhibition downregulate patho-
459 logical neovascularization remain unclear. Using a laboratory model of oxygen-induced retinopathy to induce mouse retinopathy of prematurity (ROP), we investigated the retinal localization and expression of COX-2 and VEGF at both mRNA and protein levels with and without treatment of Rofecoxib, a highly selective COX-2 inhibitor. Our hypothesis was that Rofecoxib can be a preventive agent against pathological neovascularization and exhibits anti-angiogenesis effects through suppressing expression of COX-2 and VEGF in retinas.
METHODS Establishment of mouse ROP model and experimental groups All use of animals was performed following the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were certified by the Institutional Animal Care and Use Committee of Medical University Ophthalmic Center. ROP was induced in C57BL/6J mice (Laboratory Animal Center of China Medical University, China), using the method described by Smith et al.26 Postnatal day 7 (P7) mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of 75 ± 3% oxygen for 5 days and were then removed and placed in room air for an additional 5 days. The incubator temperature was maintained at 23 ± 2°C, and oxygen levels were continuously monitored using a portable oxygen analyser (Model CY-12C, Electrochemical Analytical Instruments, Ltd, Meicheng, Zhejiang, China). The mice were randomly allocated into three experimental groups with 16 mice in each group: normal group – age-matched mice were maintained in room air from birth to P17 and were served as negative control; untreated ROP group – ROP was induced as described above without treatment and served as positive control; Rofecoxib-treated ROP group – ROP mice were treated daily with Rofecoxib (15 mg/kg body weight, intraperitoneally) (Sigma, St. Louis, MO, USA) from P12 to P17.24 Rofecoxib was dissolved in a 0.5% aqueous methylcellulose solution before administration. At the end of the 17-day experimental period, eight randomly chosen mice from each group were sacrificed. The right eye from each mouse was immediately enucleated and fixed for histology and immunohistochemistry. Meanwhile, the remaining eight mice from each group were sacrificed, the retinas of left eyes were collected for Western blot array and the retinas from the right eyes were collected and frozen for subsequent analysis of mRNA expression.
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Histological analysis of blood vessel profiles (BVPs) in the inner retina The eight right eyes from chosen mice were immersed in 4% paraformaldehyde in phosphate buffered saline (PBS) for at least 24 h, and embedded in serial paraffin sections (6 μm) of whole eyes which were cut sagittally through the cornea and parallel to the optic nerve. Randomly chosen eight sections of one eye from each animal were deparaffinized, and then stained with haematoxylin and eosin. Between two and four sections on each side of the optic nerve and 30 to 90 μm apart, BVPs were counted using an established technique24,26,27 by two independent observers in a double-masked fashion. Vascular cell nuclei were considered to be associated with new vessels, and were present on the vitreal side of the internal limiting membrane (ILM). BVPs were counted in the inner retina and included vessels adherent to the ILM. The inner retina comprised the ILM, ganglion cell layer (GCL) and inner plexiform layer. A BVP was defined as an endothelial cell or a blood vessel with a lumen.
Immunohistochemistry for COX-2 and VEGF As described in the histological analysis, randomly selected sections were carried out for immunohistochemical staining for VEGF and COX-2 expression using the strep avidin-biotin complex method. Antibodies were polyclonal COX-2 antibody (1:300 in PBS), polyclonal VEGF antibody (1:400 in PBS) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Sections incubated in PBS rather than primary antiserum were used as negative controls. Cells positive for VEGF and COX-2 showed light yellow or dark brown colouration in the cytoplasm. The integrated areas of VEGF and COX-2 staining were determined using automatic microscope and image analysis system (LUZEX-F, Nireco Company, Tokyo, Japan) and the integral optical density (IOD) values were detected as an indicator of VEGF and COX-2 expression.
Western blot array for VEGF and COX-2 Retinas were separated on an iced plate and immediately frozen in liquid nitrogen for further use. Retinas were lysed in 200 μL of lysis buffer using protease inhibitors (Sigma Chemical Co., St. Louis, MO, USA). Protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The protein were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane that was incubated with rabbit polyclonal anti-COX-2 anti-
body (1:600, Santa Cruz Biotechnology, Inc.) or polyclonal anti-VEGF antibody (1:400, Santa Cruz Biotechnology, Inc.) and β-actin monoclonal antibody (1:500, Santa Cruz Biotechnology, Inc.) overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit antibody, 1:1500, Santa Cruz Biotechnology, Inc.). The proteins were detected with enhanced chemiluminescence detection (Santa Cruz Biotechnology, Inc.). β-actin was used as an internal standard. The experiments were repeated three times.
Measurement of VEGF and COX-2 mRNA by reverse transcription polymerase chain reaction (RT-PCR) Retinas were separated on an iced plate and immediately frozen in liquid nitrogen for further use. Total retinal RNA was extracted using Tri-Zol reagent (Life Technologies, Glasgow, UK) according to the manufacturer’s instructions. Semi-quantitative RT-PCR assays were described previously.28 Reverse transcription was performed with approximately 0.5 μg of total RNA, reverse transcriptase (SuperScript II; Life Technologies, Gaithersburg, MD, USA) and 5.0 μM oligo-d(T) primer. Aliquots of cDNA were used for polymerase chain reaction (PCR) amplification with primers specific for VEGF, COX-2 and β-actin. The primer sequences were VEGF (460 bp): 5'-CAG AAA CAC GAC AAA CCC ATCC-3', 5'-TAA GCC ACT CAC ACA CAC AGCC-3'; COX-2 (450 bp): 5'-CAG CAC TTC ACG CAT CAG TT-3', 5'-TCT GGT CAA TGG AAG CCT GT-3'; and β-actin (450 bp): 5'-TCT ACA ATG AGC TGC GTG TGG-3', 5'-GGA ACC GCT CAT TGC CAA TG-3' (TaKaRa Company, Dalian, China). Standard PCR amplification was performed at 95°C for 1 min, 58°C for 1 min and 72°C for 1 min for 30 cycles, 7 min at 72°C .The amplified products were visualized on 2% agarose gels. The electrophoretic bands were determined with 100 bp gradient makers (TaKaRa Company). The levels of target gene mRNA expression were calculated by the ratio of absorbance of target gene/absorbance of β-actin.
Statistical analysis The results were expressed as mean ± standard deviation. One way analysis of variance was used to analyse multiple variables followed by Student– Newman–Keuls. Analysis of correlation between VEGF and COX-2 expression was performed by linear correlation. All analyses were performed using SPSS 10.0 software (SPSS, Inc, Chicago, IL, USA) and a P value < 0.05 was considered to be statistically significant.
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Rofecoxib and retinal neovascularization
461 of VEGF was 4.32 ± 0.17 (Fig. 2a,b). The IODs of COX-2 and VEGF in the untreated mice were 5.96 ± 1.53 and 9.21 ± 1.25, respectively. There was more COX-2 and VEGF protein expression in the inner nuclear layer and the GCL, and more neovascularization breaking through the inner retina than those in the normal group (P < 0.01, P < 0.01, respectively) (Fig. 2c,d). In Rofecoxib-treated ROP mice, there were very few COX-2 protein expressions in the ganglion cell and in the retinal blood vessels. Little VEGF protein was expressed, except for that in the cytoplasm of some ganglion cells (Fig. 2e,f). COX-2 and VEGF protein expressions were significantly decreased in the retinas of Rofecoxib-treated ROP mice compared with those in the ROP group (P < 0.01, P < 0.01, respectively). The IODs of COX-2 and VEGF were 1.16 ± 0.07 and 2.34 ± 0.36, respectively.
RESULTS Histopathology and quantification of BVPs As shown in Figure 1a–d, blood vessels were confined to the inner retina in the normal group, and there were no nuclei of new vascular endothelial cells breaking through the inner retina compared with those in the untreated ROP group which had large clusters of blood vessels adherent to the ILM (0.34 ± 0.15 vs. 38.14 ± 2.01). In the Rofecoxib-treated ROP group, blood vessels attached to the ILM were reduced by 68% compared with those in the untreated ROP mice (12.09 ± 0.49), but greater than those in the normal group.
Immunohistochemistry for COX-2 and VEGF expression
Western blot array for COX-2 and VEGF protein expression
In the normal group, the IOD of COX-2 was 0.53 ± 1.32, and there was scanty COX-2 protein expression in any of the retinal tissue layers. VEGF protein was mainly expressed in the GCL. The IOD
As shown in Figure 3a,b, there was little expression of COX-2 and VEGF protein in the normal group.
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Figure 1. Histopathological analysis and blood vessel profiles of all the groups: In the normal group, there were no nuclei of new vascular endothelial cells breaking through the inner retinal layer (a) and blood vessels confined to the inner retina (arrow). Large clusters of blood vessels (BVPs) were adherent to the internal limiting membrane in untreated retinopathy of prematurity (ROP) group (b) (arrow). In ROP mice treated with Rofecoxib, the number of nuclei of endothelial cells was significantly reduced, but more than the normal group (c). A small amount of BVPs was still presented in the Rofecoxib-treated ROP group (arrow) (magnification × 200). (d) The average numbers of BVPs in the inner retina (means ± SD, n = 8 retinas per group) of the Rofecoxib-treated ROP group mice were compared with the untreated ROP group and normal group mice using one-way analysis of variance (ANOVA). The untreated ROP group versus normal group (P < 0.001); Rofecoxib-treated ROP group versus the untreated ROP group (P < 0.01); Rofecoxib-treated ROP group versus normal group (*P < 0.05). © 2014 Royal Australian and New Zealand College of Ophthalmologists
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Figure 2. Immunolabelling for cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) protein expression of all the groups. In the normal group, there was scanty COX-2 protein expression in any tissue layer of the retina (a). VEGF was mainly expressed in the ganglion cell layer (GCL) (b) (arrow). In untreated retinopathy of prematurity (ROP) group, there was greater COX-2 and VEGF protein expression in the inner nuclear layer, the ganglion cell layer, as well as neovascularization breaking through the inner retinal layer, compared with the normal group (c, d arrows). In ROP mice treated with Rofecoxib, there were few COX-2 protein expressions in the ganglion cell and in retinal blood vessels and little VEGF protein expression, except for that in the cytoplasm of some ganglion cells (e, f arrows). Magnification × 200.
Normal ROP group ROf-treated ROP group P > 0.05
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Figure 3. The protein expression levels of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) in all of the groups. The protein levels of COX-2 and VEGF were elevated significantly in the retinas of untreated retinopathy of prematurity (ROP) group compared with those of the normal group (**P < 0.01). After the treatment of Rofecoxib, the expression of these proteins was decreased markedly compared with those of untreated ROP group. (P < 0.01), but the protein levels of VEGF was still stronger than those in the normal group (n = 8 retinas per group) (*P < 0.05).
A significantly increased protein expression in COX-2 and VEGF was detected in untreated ROP group when compared with the normal group. Compared with the untreated ROP group, the protein expression levels for COX-2 and VEGF in the Rofecoxib-treated group were markedly decreased, but the expression of VEGF remained stronger than those in the normal group. The protein level of COX-2 in the Rofecoxib-treated group had no statistical difference compared with the normal group (P > 0.05).
COX-2 and VEGF mRNA expression The expression of COX-2 and VEGF mRNA had a similar pattern with their corresponding protein expression. COX-2 mRNA expression was 4.57 times greater, and VEGF mRNA expression was 4.33 times greater in the untreated ROP group compared with the normal group. In the Rofecoxib-treated ROP group, COX-2 mRNA decreased by approximately 76% and VEGF mRNA decreased by approximately 60% when compared with the untreated ROP
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Rofecoxib and retinal neovascularization Normal ROP group ROf-treated ROP group 12 P < 0.01 P < 0.01
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Figure 4. The expression levels of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) mRNA in all of the groups. There was marked upregulation of COX-2 mRNA and VEGF mRNA in the retinas of untreated retinopathy of prematurity (ROP) mice compared with those of the normal group (**P < 0.01). There was a significant decrease of COX-2 mRNA and VEGF mRNA in the retinas of ROP mice treated with rofecoxib, compared with those of untreated ROP mice (P < 0.01), but the mRNA expression of VEGF was more than those in the normal group (n = 8 retinas per group) (*P < 0.05).
group (Fig. 4a,b), but the mRNA levels of VEGF were more than those in the normal group.
Correlation analysis There was a strong positive correlation between the protein levels of VEGF and COX-2 (r = 0.834, P < 0.001), and between the mRNA expression of VEGF and COX-2 (r = 0.785, P < 0.001).
DISCUSSION With long durations of reduced oxygen tension, cascades of compensatory responses are triggered leading to improved tissue oxygenation through processes such as angiogenesis. This process is regulated by multiple molecular effectors which activate angiogenesis, mediate endothelial cell proliferation, migration and tube formation. VEGF and its cognate receptors are the most important factors for both retinal and choroidal neovascularization (NV).28 Although recent studies found that inhibiting the COX enzymes is an effective means to downregulate VEGF expression and inhibit NV in models of ocular disease,13,29 the studies investigating the mechanisms of VEGF expression and the role of COX-2 inhibition on VEGF regulation in retina are sparse. The present study showed that the expression of both COX-2 and VEGF mRNA and protein was highly variable in untreated ROP mice, and both of protein expressions were observed in the inner nuclear layer, the GCL and neovascularization. Furthermore, a strong positive correlation between COX and VEGF at both mRNA and protein levels was noted. It prompted that they have interdependence relationship in the formation of neovascularization. In addition, we showed that treating ROP mice with a COX inhibitor resulted in significantly
decreased expression of COX-2 and VEGF. These findings indicate that both COX-2 and VEGF may be involved in the processes of retinal angiogenesis and they may be coordinated activation in retinal neovascularization. This appears to be the mechanism by which Rofecoxib exerts its effect, as exhibited by the reduction in retinal VEGF mRNA and protein in Rofecoxib-treated mice with ischaemic retinopathy. This finding is consistent with studies demonstrating that increased COX activity enhances, whereas treatment with NSAIDs decreases VEGF expression.30 It is the first time that we confirmed the anti-angiogenesis effect of selective COX-2 inhibitor in the retinal neovascularization by downregulating the expression of VEGF. Apart from its inhibitory effect on COX-2 which results in decreasing prostaglandin synthesis, Rofecoxib also reduces vascular prostacyclin production.31 The latter phenomenon may be associated with increased risk of cardiovascular events, which leads to its removal from the United States in 2004.31 However, as Rofecoxib is more potent and highly selective to COX-2 than Celecoxib32 with more than 800 times of the selectivity relative to COX-1,33 we used Rofecoxib as a COX-2 inhibitor in the present study. Our data demonstrated an obvious inhibitory effect of Rofecoxib on the angiogenesis in the expression of COX-2 protein and mRNA. These findings are consistent with the idea that if the tumour cells expressed COX-2, tumour growth and angiogenesis could be suppressed by selective COX-2 inhibitors only.34 Our study also suggests that retinopathyassociated COX-2 is required for angiogenesis and the expression of COX-2 is suppressed directly by Rofecoxib. Besides the role of the COX enzymes in angiogenesis, the potential involvement of COX-2 in
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lymphangiogenesis has been recently suggested based on the significant association between the expression of COX-2 and the expression of lymphangiogenic molecule, VEGF-C, in gastric adenocarcinomas.35 These findings are backed by our results, which showed a positive correlation between COX-2 and VEGF expression. Although Wilkinson-Berka et al.24 have proved that COX-2 is localized to sites in retinal blood vessels, they did not compare with the cellular location of VEGF, specifically, the location changes of VEGF and COX-2 after Rofecoxib treatment. Our findings provide evidence that the inter-relationship of COX-2 and VEGF is involved in the regulation of angiogenesis in retinal neovascularization, and COX-2 inhibition by Rofecoxib is achieved via downregulation of VEGF expression. It should be pointed out that the effect of Rofecoxib on VEGF expression seems to be partially dependent on COX-2 inhibition. We found in our study that suppressing COX-2-VEGF signalling did not return VEGF to normoxic levels, and therefore decrease, but did not eliminate retinal neovascularization, suggesting Rofecoxib might indirectly block VEGF production. The incomplete inhibition of VEGF upregulation documents that the angiogenic activity mediated by hypoxia is a complex process and other mediators, such as hypoxia, Sp1 promoter and COX-2/PGE2 signalling, also contribute to increasing VEGF expression in the retina. Another possible mechanism is that Rofecoxib in the local concentration does not reach high enough to completely prevent retinal neovascularization. To achieve a higher local concentration, either the concentration or dose of Rofecoxib can be increased or other modes of administration such as subretinal injection and intravitreal injections can be considered. The presence of alternative molecular pathways that bypass the effect of COX inhibition on expression of VEGF can also be an option. To more clearly define the role of COX-2 in this process, it is important to assess VEGF production in COX-2 knockout animals and the cells derived from these animals. Further studies are needed. In summary, our results demonstrated that COX-2 and VEGF expressions were activated and had the same cellular localization in the hypoxia-induced retinal neovascularization. The expression of COX-2 played an important regulatory role in the expression of VEGF. Rofecoxib exhibited potent antiangiogenic activities by impairing hypoxia-induced expression of COX-2 and VEGF. Although the inhibition of VEGF by Rofecoxib appeared to be incomplete, it provided a novel insight of a selective COX-2 inhibitor for the treatment of ischemic ocular diseases.
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Rofecoxib and retinal neovascularization 16. Petrovic V, Bhisitkul RB. Lasers and diabetic retinopathy: the art of gentle destruction. Diabetes Technol Ther 1999; 1: 177–87. 17. Sramek CK, Leung LS, Paulus YM, Palanker DV. Therapeutic window of retinal photocoagulation with green (532-nm) and yellow (577-nm) lasers. Ophthalmic Surg Lasers Imaging 2012; 43: 341–7. 18. Cheung N, Wong IY, Wong TY. Ocular anti-VEGF therapy for diabetic retinopathy: overview of clinical efficacy and evolving applications. Diabetes Care 2014; 37: 900–5. 19. Qaum T, Xu Q, Joussen AM et al. VEGF-initiated blood–retinal barrier breakdown in early diabetes. Invest Ophthalmol Vis Sci 2001; 42: 2408–13. 20. Powell ED, Field RA. Studies on salicylates and complement in diabetes. Diabetes 1966; 15: 730–3. 21. Li WW, Long GX, Liu DB et al. Cyclooxygenase-2 inhibitor celecoxib suppresses invasion and migration of nasopharyngeal carcinoma cell lines through a decrease in matrix metalloproteinase-2 and -9 activity. Pharmazie 2014; 69: 132–7. 22. Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, Levine AC. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol 2000; 164: 820–5. 23.Xu K, Gao H, Shu HK. Celecoxib can induce vascular endothelial growth factor expression and tumor angiogenesis. Mol Cancer Ther 2011; 10: 138–47. 24. Wilkinson-Berka JL, Alousis NS, Kelly DJ, Gilbert RE. COX-2 inhibition and retinal angiogenesis in a mouse model of retinopathy of prematurity. Invest Ophthalmol Vis Sci 2003; 44: 974–9. 25. Masferrer JL, Leahy KM, Koki AT et al. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res 2000; 60: 1306–11. 26. Smith LE, Wesolowski E, McLellan A et al. Oxygeninduced retinopathy in the mouse. Invest Ophthalmol Vis Sci 1994; 35: 101–11.
465 27. Moravski CJ, Kelly DJ, Cooper ME et al. Retinal neovascularization is prevented by blockade of the renin-angiotensin system. Hypertension 2000; 36: 1099– 104. 28. Kwak N, Okamoto N, Wood JM, Campochiaro PA. VEGF is an important stimulator in a model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2000; 41: 3158–64. 29. Ayalasomayajula SP, Kompella UB. Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozotocin-induced diabetic rat model. Eur J Pharmacol 2003; 458: 283–9. 30. Ma JX, Sun YL, Wang YQ, Wu HY, Jin J, Yu XF. Triptolide induces apoptosis and inhibits the growth and angiogenesis of human pancreatic cancer cells by downregulating COX-2 and VEGF. Oncol Res 2013; 20: 359–68. 31. Fitz Gerald GA, Patrono C. The coxibs, selective inhibitors of cyclooxygenase-2. N Engl J Med 2002; 345: 433–42. 32. Ouellet M, Riendeau D, Percival MD. A high level of cyclooxygenase-2 inhibitor selectivity is associated with a reduced interference of platelet cyxlooxygenase-1 inactivation by aspirin. Proc Natl Acad Sci USA 2001; 98: 14583–8. 33. Dogan E, Saygili U, Posaci C et al. Regression of endometrial explants in rats treated with the cyclooxygenase-2 inhibitor rofecoxib. Fertil Steril 2004; 82: 1115–20. 34. Sawaoka H, Tsuji S, Tsujii M et al. Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab Invest 1999; 79: 1469–77. 35. Gou HF, Chen XC, Zhu J et al. Expressions of COX-2 and VEGF-C in gastric cancer: correlations with lymphangiogenesis and prognostic implications. J Exp Clin Cancer Res 2011; 30: 14–22.
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Clinical and Experimental Ophthalmology 2015; 43: 458–465 doi: 10.1111/ceo.12473
Original Article Rofecoxib inhibits retinal neovascularization via down regulation of cyclooxygenase-2 and vascular endothelial growth factor expression Ning-ning Liu PhD, Yi-zhou Sun MD, Ning Zhao MD and Lei Chen MD Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China
ABSTRACT Background: To explore the anti-angiogenesis mechanism of Rofecoxib and determine whether Rofecoxib can be a therapeutic agent for the prevention of retinal neovascularization using a model of retinopathy of prematurity (ROP). Methods: ROP was induced by exposing mice to 75% oxygen from postnatal day 7 (P7) to P12, then to room air from P12 to P17. Sixteen mice were in each of the three groups: untreated ROP group as positive control, Rofecoxib-treated ROP group and the normal group (age-matched mice maintained in room air from birth to P17 as negative control). The localized expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) protein and mRNA in retinal blood vessels was assessed using immunohistochemistry, Western blot analysis and reverse transcription polymerase chain reaction. Results: Mice in the Rofecoxib-treated group had a significantly reduced retinal neovascular tufts compared with those in the untreated ROP group. COX-2 and VEGF protein and mRNA expression levels were increased in the untreated ROP group, compared with the normal group. Rofecoxib decreased retinal angiogenesis by inhibiting COX-2 and VEGF expression. The expression levels of VEGF and COX-2 were positively correlated at mRNA and protein levels.
Conclusions: COX-2 and VEGF expressions were both involved in the regulation of angiogenesis and had the same cellular localization. Expression of COX-2 correlated positively with VEGF in retinal neovascularization. Rofecoxib attenuated retinal angiogenesis by inhibiting the expression of COX-2 and VEGF mRNA and protein. Key words: retinal neovascularization, Rofecoxib, vascular endothelial growth factor.
INTRODUCTION Pathological angiogenesis, or neovascularization of the eye, is a complex process and major cause of blindness in developed countries.1,2 It is clear that ischaemia-induced hypoxia is a primary aetiological factor causing ocular neovascularization.3 Vascular endothelial growth factor (VEGF) is the most prominently potent angiogenic factor in response to hypoxia.4,5 Highly expressed VEGF were found in the diabetic retinopathy, neovascular age-related macular degeneration, and inflammation-associated corneal neovascularization.6–8 The binding of VEGF with its receptors initiates signal transduction cascades, causing angiogenic endothelia cell behaviours. The inhibition of the VEGF/VEGF-receptor pathway may potentially be an efficacious therapeutic strategy to suppress experimental ocular neovascularization.
■ Correspondence: Professor Lei Chen, Department of Ophthalmology, The First Affiliated Hospital of China Medical University, No. 155, Nanjing North Street, Heping Area, Shenyang, Liaoning, China 110001. Email: [email protected] Received 17 June 2014; accepted 12 November 2014. Conflict of interest: No stated conflict of interest. Funding sources: This research was supported by Doctoral Scientific Research Foundation of China (No. 20091115). © 2014 Royal Australian and New Zealand College of Ophthalmologists
Rofecoxib and retinal neovascularization Cyclooxygenase (COX)-prostaglandin pathway has recently been found to work in conjunction with VEGF and plays a vital role in the formation of new blood vessels. COX enzymes catalyse the production of prostanoids (prostaglandins and thromboxanes) and have at least two isoforms: COX-1 and COX-2. COX-1 is a housekeeping enzyme and presented in most tissues, whereas COX-2 is a rate-limiting, inducible enzyme that converts arachidonic acid into various prostaglandins.9,10 High expression of COX-2 is linked to VEGF overexpression in tumours suggesting that COX-2 plays a part in the regulation of VEGF expression in the angiogenesis of tumour blood vessels.11,12 Studies also found that, in response of hypoxia, high expression of COX-2 is associated with upregulation of VEGF production in retina.13,14 Compounds that induce COX-2 expression can induce VEGF, and COX-2 inhibitors also inhibit VEGF.15 These findings suggest a key regulatory and potential role for COX-2 in the pathways of VEGF expression in neovascularization. Retinal angiogenesis is the common pathophysiological basis of all retinal vascular diseases. Laser photocoagulation is usually applied clinically to inhibit the development of retinal neovascularization and has shown some therapeutic effects.16 Laser treatment, however, only delays the progression of lesions and may potentially further damage retinal tissue.17 Drug therapy inhibiting vascular proliferation in intraocular neovascularization has become an important area in ophthalmological research, but the clinical therapeutic effects are yet to be satisfactory.18 Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are inhibitors of both COX-1 and COX-2, and are generally used for the treatment of pain and arthritis. Increasing evidence showed that high dose of aspirin administration decreased retinal vascular leakage and abnormalities in diabetic rates and reduced the incidence of diabetic retinopathy in humans.19,20 The gastrointestinal side effects of NSAIDs resulting from the blockage of COX-1 prompted the development of selective COX-2 inhibitors, which in recent years have proven to exhibit potent anti-tumour and anti-angiogenic effects; thus, they are considered to be novel candidate agents for the treatment of vascular proliferative diseases.21 In a prostate cancer model, inhibition of COX-2 suppresses angiogenesis and tumour growth through suppressing VEGF expression.22 In cancer cell lines, treatment with selective COX-2 inhibitor actually induced the expression of VEGF.23 In the eyes, the antiangiogenesis properties of COX-2 inhibitor have been demonstrated in experimental models.24,25 However, the exact molecular mechanisms through which the COX-2 inhibition downregulate patho-
459 logical neovascularization remain unclear. Using a laboratory model of oxygen-induced retinopathy to induce mouse retinopathy of prematurity (ROP), we investigated the retinal localization and expression of COX-2 and VEGF at both mRNA and protein levels with and without treatment of Rofecoxib, a highly selective COX-2 inhibitor. Our hypothesis was that Rofecoxib can be a preventive agent against pathological neovascularization and exhibits anti-angiogenesis effects through suppressing expression of COX-2 and VEGF in retinas.
METHODS Establishment of mouse ROP model and experimental groups All use of animals was performed following the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were certified by the Institutional Animal Care and Use Committee of Medical University Ophthalmic Center. ROP was induced in C57BL/6J mice (Laboratory Animal Center of China Medical University, China), using the method described by Smith et al.26 Postnatal day 7 (P7) mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of 75 ± 3% oxygen for 5 days and were then removed and placed in room air for an additional 5 days. The incubator temperature was maintained at 23 ± 2°C, and oxygen levels were continuously monitored using a portable oxygen analyser (Model CY-12C, Electrochemical Analytical Instruments, Ltd, Meicheng, Zhejiang, China). The mice were randomly allocated into three experimental groups with 16 mice in each group: normal group – age-matched mice were maintained in room air from birth to P17 and were served as negative control; untreated ROP group – ROP was induced as described above without treatment and served as positive control; Rofecoxib-treated ROP group – ROP mice were treated daily with Rofecoxib (15 mg/kg body weight, intraperitoneally) (Sigma, St. Louis, MO, USA) from P12 to P17.24 Rofecoxib was dissolved in a 0.5% aqueous methylcellulose solution before administration. At the end of the 17-day experimental period, eight randomly chosen mice from each group were sacrificed. The right eye from each mouse was immediately enucleated and fixed for histology and immunohistochemistry. Meanwhile, the remaining eight mice from each group were sacrificed, the retinas of left eyes were collected for Western blot array and the retinas from the right eyes were collected and frozen for subsequent analysis of mRNA expression.
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Histological analysis of blood vessel profiles (BVPs) in the inner retina The eight right eyes from chosen mice were immersed in 4% paraformaldehyde in phosphate buffered saline (PBS) for at least 24 h, and embedded in serial paraffin sections (6 μm) of whole eyes which were cut sagittally through the cornea and parallel to the optic nerve. Randomly chosen eight sections of one eye from each animal were deparaffinized, and then stained with haematoxylin and eosin. Between two and four sections on each side of the optic nerve and 30 to 90 μm apart, BVPs were counted using an established technique24,26,27 by two independent observers in a double-masked fashion. Vascular cell nuclei were considered to be associated with new vessels, and were present on the vitreal side of the internal limiting membrane (ILM). BVPs were counted in the inner retina and included vessels adherent to the ILM. The inner retina comprised the ILM, ganglion cell layer (GCL) and inner plexiform layer. A BVP was defined as an endothelial cell or a blood vessel with a lumen.
Immunohistochemistry for COX-2 and VEGF As described in the histological analysis, randomly selected sections were carried out for immunohistochemical staining for VEGF and COX-2 expression using the strep avidin-biotin complex method. Antibodies were polyclonal COX-2 antibody (1:300 in PBS), polyclonal VEGF antibody (1:400 in PBS) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Sections incubated in PBS rather than primary antiserum were used as negative controls. Cells positive for VEGF and COX-2 showed light yellow or dark brown colouration in the cytoplasm. The integrated areas of VEGF and COX-2 staining were determined using automatic microscope and image analysis system (LUZEX-F, Nireco Company, Tokyo, Japan) and the integral optical density (IOD) values were detected as an indicator of VEGF and COX-2 expression.
Western blot array for VEGF and COX-2 Retinas were separated on an iced plate and immediately frozen in liquid nitrogen for further use. Retinas were lysed in 200 μL of lysis buffer using protease inhibitors (Sigma Chemical Co., St. Louis, MO, USA). Protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The protein were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane that was incubated with rabbit polyclonal anti-COX-2 anti-
body (1:600, Santa Cruz Biotechnology, Inc.) or polyclonal anti-VEGF antibody (1:400, Santa Cruz Biotechnology, Inc.) and β-actin monoclonal antibody (1:500, Santa Cruz Biotechnology, Inc.) overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit antibody, 1:1500, Santa Cruz Biotechnology, Inc.). The proteins were detected with enhanced chemiluminescence detection (Santa Cruz Biotechnology, Inc.). β-actin was used as an internal standard. The experiments were repeated three times.
Measurement of VEGF and COX-2 mRNA by reverse transcription polymerase chain reaction (RT-PCR) Retinas were separated on an iced plate and immediately frozen in liquid nitrogen for further use. Total retinal RNA was extracted using Tri-Zol reagent (Life Technologies, Glasgow, UK) according to the manufacturer’s instructions. Semi-quantitative RT-PCR assays were described previously.28 Reverse transcription was performed with approximately 0.5 μg of total RNA, reverse transcriptase (SuperScript II; Life Technologies, Gaithersburg, MD, USA) and 5.0 μM oligo-d(T) primer. Aliquots of cDNA were used for polymerase chain reaction (PCR) amplification with primers specific for VEGF, COX-2 and β-actin. The primer sequences were VEGF (460 bp): 5'-CAG AAA CAC GAC AAA CCC ATCC-3', 5'-TAA GCC ACT CAC ACA CAC AGCC-3'; COX-2 (450 bp): 5'-CAG CAC TTC ACG CAT CAG TT-3', 5'-TCT GGT CAA TGG AAG CCT GT-3'; and β-actin (450 bp): 5'-TCT ACA ATG AGC TGC GTG TGG-3', 5'-GGA ACC GCT CAT TGC CAA TG-3' (TaKaRa Company, Dalian, China). Standard PCR amplification was performed at 95°C for 1 min, 58°C for 1 min and 72°C for 1 min for 30 cycles, 7 min at 72°C .The amplified products were visualized on 2% agarose gels. The electrophoretic bands were determined with 100 bp gradient makers (TaKaRa Company). The levels of target gene mRNA expression were calculated by the ratio of absorbance of target gene/absorbance of β-actin.
Statistical analysis The results were expressed as mean ± standard deviation. One way analysis of variance was used to analyse multiple variables followed by Student– Newman–Keuls. Analysis of correlation between VEGF and COX-2 expression was performed by linear correlation. All analyses were performed using SPSS 10.0 software (SPSS, Inc, Chicago, IL, USA) and a P value < 0.05 was considered to be statistically significant.
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Rofecoxib and retinal neovascularization
461 of VEGF was 4.32 ± 0.17 (Fig. 2a,b). The IODs of COX-2 and VEGF in the untreated mice were 5.96 ± 1.53 and 9.21 ± 1.25, respectively. There was more COX-2 and VEGF protein expression in the inner nuclear layer and the GCL, and more neovascularization breaking through the inner retina than those in the normal group (P < 0.01, P < 0.01, respectively) (Fig. 2c,d). In Rofecoxib-treated ROP mice, there were very few COX-2 protein expressions in the ganglion cell and in the retinal blood vessels. Little VEGF protein was expressed, except for that in the cytoplasm of some ganglion cells (Fig. 2e,f). COX-2 and VEGF protein expressions were significantly decreased in the retinas of Rofecoxib-treated ROP mice compared with those in the ROP group (P < 0.01, P < 0.01, respectively). The IODs of COX-2 and VEGF were 1.16 ± 0.07 and 2.34 ± 0.36, respectively.
RESULTS Histopathology and quantification of BVPs As shown in Figure 1a–d, blood vessels were confined to the inner retina in the normal group, and there were no nuclei of new vascular endothelial cells breaking through the inner retina compared with those in the untreated ROP group which had large clusters of blood vessels adherent to the ILM (0.34 ± 0.15 vs. 38.14 ± 2.01). In the Rofecoxib-treated ROP group, blood vessels attached to the ILM were reduced by 68% compared with those in the untreated ROP mice (12.09 ± 0.49), but greater than those in the normal group.
Immunohistochemistry for COX-2 and VEGF expression
Western blot array for COX-2 and VEGF protein expression
In the normal group, the IOD of COX-2 was 0.53 ± 1.32, and there was scanty COX-2 protein expression in any of the retinal tissue layers. VEGF protein was mainly expressed in the GCL. The IOD
As shown in Figure 3a,b, there was little expression of COX-2 and VEGF protein in the normal group.
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Figure 1. Histopathological analysis and blood vessel profiles of all the groups: In the normal group, there were no nuclei of new vascular endothelial cells breaking through the inner retinal layer (a) and blood vessels confined to the inner retina (arrow). Large clusters of blood vessels (BVPs) were adherent to the internal limiting membrane in untreated retinopathy of prematurity (ROP) group (b) (arrow). In ROP mice treated with Rofecoxib, the number of nuclei of endothelial cells was significantly reduced, but more than the normal group (c). A small amount of BVPs was still presented in the Rofecoxib-treated ROP group (arrow) (magnification × 200). (d) The average numbers of BVPs in the inner retina (means ± SD, n = 8 retinas per group) of the Rofecoxib-treated ROP group mice were compared with the untreated ROP group and normal group mice using one-way analysis of variance (ANOVA). The untreated ROP group versus normal group (P < 0.001); Rofecoxib-treated ROP group versus the untreated ROP group (P < 0.01); Rofecoxib-treated ROP group versus normal group (*P < 0.05). © 2014 Royal Australian and New Zealand College of Ophthalmologists
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Figure 2. Immunolabelling for cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) protein expression of all the groups. In the normal group, there was scanty COX-2 protein expression in any tissue layer of the retina (a). VEGF was mainly expressed in the ganglion cell layer (GCL) (b) (arrow). In untreated retinopathy of prematurity (ROP) group, there was greater COX-2 and VEGF protein expression in the inner nuclear layer, the ganglion cell layer, as well as neovascularization breaking through the inner retinal layer, compared with the normal group (c, d arrows). In ROP mice treated with Rofecoxib, there were few COX-2 protein expressions in the ganglion cell and in retinal blood vessels and little VEGF protein expression, except for that in the cytoplasm of some ganglion cells (e, f arrows). Magnification × 200.
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Figure 3. The protein expression levels of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) in all of the groups. The protein levels of COX-2 and VEGF were elevated significantly in the retinas of untreated retinopathy of prematurity (ROP) group compared with those of the normal group (**P < 0.01). After the treatment of Rofecoxib, the expression of these proteins was decreased markedly compared with those of untreated ROP group. (P < 0.01), but the protein levels of VEGF was still stronger than those in the normal group (n = 8 retinas per group) (*P < 0.05).
A significantly increased protein expression in COX-2 and VEGF was detected in untreated ROP group when compared with the normal group. Compared with the untreated ROP group, the protein expression levels for COX-2 and VEGF in the Rofecoxib-treated group were markedly decreased, but the expression of VEGF remained stronger than those in the normal group. The protein level of COX-2 in the Rofecoxib-treated group had no statistical difference compared with the normal group (P > 0.05).
COX-2 and VEGF mRNA expression The expression of COX-2 and VEGF mRNA had a similar pattern with their corresponding protein expression. COX-2 mRNA expression was 4.57 times greater, and VEGF mRNA expression was 4.33 times greater in the untreated ROP group compared with the normal group. In the Rofecoxib-treated ROP group, COX-2 mRNA decreased by approximately 76% and VEGF mRNA decreased by approximately 60% when compared with the untreated ROP
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Rofecoxib and retinal neovascularization Normal ROP group ROf-treated ROP group 12 P < 0.01 P < 0.01
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Figure 4. The expression levels of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) mRNA in all of the groups. There was marked upregulation of COX-2 mRNA and VEGF mRNA in the retinas of untreated retinopathy of prematurity (ROP) mice compared with those of the normal group (**P < 0.01). There was a significant decrease of COX-2 mRNA and VEGF mRNA in the retinas of ROP mice treated with rofecoxib, compared with those of untreated ROP mice (P < 0.01), but the mRNA expression of VEGF was more than those in the normal group (n = 8 retinas per group) (*P < 0.05).
group (Fig. 4a,b), but the mRNA levels of VEGF were more than those in the normal group.
Correlation analysis There was a strong positive correlation between the protein levels of VEGF and COX-2 (r = 0.834, P < 0.001), and between the mRNA expression of VEGF and COX-2 (r = 0.785, P < 0.001).
DISCUSSION With long durations of reduced oxygen tension, cascades of compensatory responses are triggered leading to improved tissue oxygenation through processes such as angiogenesis. This process is regulated by multiple molecular effectors which activate angiogenesis, mediate endothelial cell proliferation, migration and tube formation. VEGF and its cognate receptors are the most important factors for both retinal and choroidal neovascularization (NV).28 Although recent studies found that inhibiting the COX enzymes is an effective means to downregulate VEGF expression and inhibit NV in models of ocular disease,13,29 the studies investigating the mechanisms of VEGF expression and the role of COX-2 inhibition on VEGF regulation in retina are sparse. The present study showed that the expression of both COX-2 and VEGF mRNA and protein was highly variable in untreated ROP mice, and both of protein expressions were observed in the inner nuclear layer, the GCL and neovascularization. Furthermore, a strong positive correlation between COX and VEGF at both mRNA and protein levels was noted. It prompted that they have interdependence relationship in the formation of neovascularization. In addition, we showed that treating ROP mice with a COX inhibitor resulted in significantly
decreased expression of COX-2 and VEGF. These findings indicate that both COX-2 and VEGF may be involved in the processes of retinal angiogenesis and they may be coordinated activation in retinal neovascularization. This appears to be the mechanism by which Rofecoxib exerts its effect, as exhibited by the reduction in retinal VEGF mRNA and protein in Rofecoxib-treated mice with ischaemic retinopathy. This finding is consistent with studies demonstrating that increased COX activity enhances, whereas treatment with NSAIDs decreases VEGF expression.30 It is the first time that we confirmed the anti-angiogenesis effect of selective COX-2 inhibitor in the retinal neovascularization by downregulating the expression of VEGF. Apart from its inhibitory effect on COX-2 which results in decreasing prostaglandin synthesis, Rofecoxib also reduces vascular prostacyclin production.31 The latter phenomenon may be associated with increased risk of cardiovascular events, which leads to its removal from the United States in 2004.31 However, as Rofecoxib is more potent and highly selective to COX-2 than Celecoxib32 with more than 800 times of the selectivity relative to COX-1,33 we used Rofecoxib as a COX-2 inhibitor in the present study. Our data demonstrated an obvious inhibitory effect of Rofecoxib on the angiogenesis in the expression of COX-2 protein and mRNA. These findings are consistent with the idea that if the tumour cells expressed COX-2, tumour growth and angiogenesis could be suppressed by selective COX-2 inhibitors only.34 Our study also suggests that retinopathyassociated COX-2 is required for angiogenesis and the expression of COX-2 is suppressed directly by Rofecoxib. Besides the role of the COX enzymes in angiogenesis, the potential involvement of COX-2 in
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lymphangiogenesis has been recently suggested based on the significant association between the expression of COX-2 and the expression of lymphangiogenic molecule, VEGF-C, in gastric adenocarcinomas.35 These findings are backed by our results, which showed a positive correlation between COX-2 and VEGF expression. Although Wilkinson-Berka et al.24 have proved that COX-2 is localized to sites in retinal blood vessels, they did not compare with the cellular location of VEGF, specifically, the location changes of VEGF and COX-2 after Rofecoxib treatment. Our findings provide evidence that the inter-relationship of COX-2 and VEGF is involved in the regulation of angiogenesis in retinal neovascularization, and COX-2 inhibition by Rofecoxib is achieved via downregulation of VEGF expression. It should be pointed out that the effect of Rofecoxib on VEGF expression seems to be partially dependent on COX-2 inhibition. We found in our study that suppressing COX-2-VEGF signalling did not return VEGF to normoxic levels, and therefore decrease, but did not eliminate retinal neovascularization, suggesting Rofecoxib might indirectly block VEGF production. The incomplete inhibition of VEGF upregulation documents that the angiogenic activity mediated by hypoxia is a complex process and other mediators, such as hypoxia, Sp1 promoter and COX-2/PGE2 signalling, also contribute to increasing VEGF expression in the retina. Another possible mechanism is that Rofecoxib in the local concentration does not reach high enough to completely prevent retinal neovascularization. To achieve a higher local concentration, either the concentration or dose of Rofecoxib can be increased or other modes of administration such as subretinal injection and intravitreal injections can be considered. The presence of alternative molecular pathways that bypass the effect of COX inhibition on expression of VEGF can also be an option. To more clearly define the role of COX-2 in this process, it is important to assess VEGF production in COX-2 knockout animals and the cells derived from these animals. Further studies are needed. In summary, our results demonstrated that COX-2 and VEGF expressions were activated and had the same cellular localization in the hypoxia-induced retinal neovascularization. The expression of COX-2 played an important regulatory role in the expression of VEGF. Rofecoxib exhibited potent antiangiogenic activities by impairing hypoxia-induced expression of COX-2 and VEGF. Although the inhibition of VEGF by Rofecoxib appeared to be incomplete, it provided a novel insight of a selective COX-2 inhibitor for the treatment of ischemic ocular diseases.
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