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• W2028528029 abstract "Purpose: Ranibizumab (Lucentis®) is a Fab-Fragment of a recombinant, humanized, monoclonal VEGF (anti-vascular endothelial growth factor) antibody. This study analyzed the ability of topical Ranibizumab to inhibit lymphangiogenesis in addition to hemangiogenesis after acute corneal inflammation in vivo. In addition, the effect of Ranibizumab on the proliferation of human lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) in vitro was studied. Methods: The inhibitory effect of Ranibizumab on LECs and BECs was studied in vitro using a proliferation enzyme-linked immunosorbent assay (ELISA) assay. To study the in vivo effects of Ranibizumab, the mouse model of suture induced inflammatory corneal neovascularization was used. Study mice received topical Ranibizumab as eye drops. After 1 week excised corneas were stained with LYVE-1 and CD31. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically by using a semiautomatic method based on the image analyzing program Cell^F. Results: An antiproliferative effect of Ranibizumab was seen in vitro on both human BECs and LECs with a significance of p < 0.0001 and p < 0.0004, respectively. In vivo experiments showed that topical application of Ranibizumab significantly inhibits both hemangiogenesis (p = 0.0026) and lymphangiogenesis (p = 0.0026) in the cornea. Conclusion: Ranibizumab is a potent inhibitor of inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo with a direct inhibitory effect on both endothelial cell types in vitro. This study for the first time demonstrates an inhibitory effect of Ranibizumab on lymphatic vessels which could have a wider range of clinical applications. The vascular endothelial growth factor A (VEGF-A) is required for vascular development and for pathological tumor, iris, retinal and choroidal neovascularization (Adamis et al. 1996; Amano et al. 1998; Ishida et al. 2003a,b). Pathological corneal neovascularization following an inflammatory insult is mainly mediated by VEGF-A and remains an unsolved therapeutic problem. Additionally the concurrent invasion of lymphatic vessels has implications for allograft transplant survival (Cursiefen et al. 2002, 2006a,b,c; Cursiefen 2007; Dietrich et al. 2010). The role of VEGF-A in inflammatory neovascularization has been well elucidated by the raised VEGF-A mRNA and protein levels in the cornea thus identifying VEGF-A as a functional endogenous corneal angiogenic factor (Amano et al. 1998; Joussen et al. 2003). Subsequent specific inhibition of VEGF-A bioactivity would suppress vessel growth (Krzystolik et al. 2002; Ishida et al. 2003a,b). Ranibizumab, a Fab-Fragment of a recombinant, humanized, monoclonal anti-VEGF antibody is licensed for the treatment of pathological choroidal neovascularization due to various etiologies and has demonstrated efficacy both in regressing new vessels and improving visual acuity (Boyer et al. 2007; Kaiser et al. 2007; Antoszyk et al. 2008; Regillo et al. 2008; Silva et al. 2008). Ranibizumab has been applied in animal models and off-label use in humans to inhibit corneal vascularisation including diseases with limbal stem cell deficiency like chemical injuries and vessel growth in neovascular glaucoma (Awadein 2007; Chilov et al. 2007; Dunavoelgyi et al. 2007; Erdurmus & Totan 2007; Bahar et al. 2008a,a,b; Bahar et al. 2008b; Carrasco 2008; Uy et al. 2008; Yoeruek et al. 2008). There are also studies showing that the anti-VEGF antibody Bevazicumab has an inhibitory effect on hem- and lymphangiogenesis in mouse and man (Bock et al. 2007; Koenig et al. 2009). In addition there are a number of studies showing an inhibitory effect of Ranibizumab on corneal neovascularisation. However, the patient series reported are small and there are still no long term data. Furthermore there is no study, neither experimental nor clinical, showing that VEGF-A-inhibitors can completely prevent or reverse the corneal neovascularisation. We have previously demonstrated that corneal hem- and lymphangiogenesis can be inhibited by competitively binding a specific neutralizing anti-VEGF antibody to VEGF-A with a specific neutralizing anti-VEGF antibody in an animal model and human subjects (Bock et al. 2007, 2008a,b). In addition recent works have now shown that VEGF-A is also implicated in lymphangiogenesis (Hirakawa et al. 2005; Halin et al. 2007). Anti-VEGF agents have been previously administered topically and injected but given the fact that there are no published prospective controlled clinical trials on anti-VEGF treatment for ocular surface disease, its exact administration has yet to be determined (Awadein 2007; Bock et al. 2008a,b; Uy et al. 2008). Most recently Sener et al. (2011) described in an animal model a profound effect of locally applied Ranibizumab on corneal neovascularisation. In addition, Dana et al. successfully completed a phase I clinical study analysing the antiangiogenic effect of topical Ranibizumab (http://clinicaltrials.gov/ct2/show/NCT00681889?term=Dana+Ranibizumab&rank=1). Further studies using Ranibizumab at the ocular surface are currently in progress (http://clinicaltrials.gov/ct2/results?term=ranibizumab+cornea). Since there are so far no data available on the effect of Ranibizumab on lymphatic vessels, this study was initiated to analyze the inhibitory effect of topical Ranibizumab on corneal lymphangiogenesis. Human lymphatic microvascular endothelial cells (HLMVEC; Cambrex Bio Science, Walkersville, MD, USA) and blood endothelial cells (BECs) were cultured in EGM2-MV medium (Cambrex Bio Science) following manufacturer`s instructions (Bock et al. 2007). For the enzyme-linked immunosorbent assay (ELISA), cells were seeded in a 96-well plate in EGM2-MV medium at a density of 2 × 103 cells/well and left over night to attach. For each group, 30 wells were supplied with either verum or control substance. Medium was replaced with serum-, basic fibroblat growth factor (bFGF)- and VEGF-A-free EGM2-MV medium. Ranibizumab (10 nm, 48 ng/ml; Novartis International AG, Basel, Switzerland) and Bromodeoxyuridine (BrdU) (0.1 μl/ml; Cell Proliferation ELISA, BrdU; Roche, Penzberg, Germany) were added. After 3 hr the medium was supplemented with 0.0025 ± 0.002 μg/ml (costumer data) human VEGF-A (EGM2-MV supplement; Cambrex Bio Science.). Cells were fixed and stained after 3 days according to manufacturer`s instructions (Cell Proliferation ELISA, BrdU, Roche). Colorimetric analyses were performed with the ELISA reader SLT Spectra (SLT Labinstruments Deutschland GmbH, Crailsheim, Germany). Absorbance at 450 nm with a reference wavelength of 690 nm is defined as the BrdU labeling index. For this assay female BALB/c mice (aged 6–8 weeks) were used. The protocols were in accordance with the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmology and Vision Research and institutional guidelines regarding animal experimentation were followed. Prior to surgery, each animal was deeply anesthetized with an intramuscular injection of Ketanest®S (8 mg/kg) and Rompun (0.1 ml/kg). Three 11-0 nylon sutures (Serag Wiessner, Naila, Germany) were placed intrastromally extending over 120° of corneal circumference each. The outer point of entry was near the limbus, and the inner exit point was the corneal centre equidistant from limbus to obtain standardized angiogenic responses in three parts of the cornea. Sutures were left in place for duration of experiment. The central 2 mm of the epithelium was debried prior to suturing. Treatment group (n = 25) received Ranibizumab (Roche, Madison, WI, USA) as topical drops (5 μl/drop; conc.: 5 mg/ml) four times daily for 5 days (equals 0.5 mg, dosage advise by the manufacturer for intravitreal injections) while the control group (n = 25) received saline solution. The mice were euthanized after 1 week. Same experiments were performed without central debridement of the epithelium prior to suturing (n = 5 received Ranibizumab, n = 5 saline solution). Eye drops were given as in the group of mice with debridement prior to suturing (four times a day for 5 days, 0.5 mg Ranibizumab). Mice were euthanized after 7 days as it is the best point of time with the maximum of hem- and lymphatic vessels (Cursiefen et al. 2006a,b,c). The blood and lymphatic vessels in the corneal wholemounts were stained with LYVE-1 and CD31 antibodies as described previously (Cursiefen et al. 2002; Chen et al. 2004; Bock et al. 2007). In short, lymphatic vessels were stained with rabbit anti-mouse LYVE-1 (1:500; AngioBio, Del Mar, CA, USA) and blood vessels were stained with an anti-CD-31-FITC (Acris Antibodies GmbH, Hiddenhausen, Germany) antibody. LYVE-1 was detected with a Cy3-conjugated secondary antibody. Isotype control was assured with a FITC-conjugated normal rat2A IgG for CD31-FITC and with a normal rabbit IgG (both Santa Cruz Biotechnology, Santa Cruz, CA, USA) for LYVE-1. Double stained wholemounts were analyzed with a fluorescence microscope (BX51; Olympus Optical Co., Hamburg, Germany) and digital pictures were taken with a 12-bit monochrome CCD camera (F-View II; Olympus). Each wholemount picture was assembled out of nine pictures taken at 100× magnification. The areas covered with blood or lymphatic vessels were detected with an algorithm established in the image analyzing program Cell^F (Olympus) as described previously (Bock et al. 2008a,b). The quantification of the vascularized area was done on multi image alignments covering the whole cornea (Bock et al. 2008a,b). Statistical analysis was done by Microsoft® Excel 2000 and InStat 3 Version 3.06 (GraphPad Software Inc, San Diego, CA, USA). Graphs were drawn using prism4, Version 4.03 (GraphPad Software Inc). Student’s t-test was used to detect the difference between the groups. Ranibizumab inhibits BECs proliferation in vitro along with a previously not shown antilymphangiogenic effect on lymphatic endothelial cells (LECs). The student’s t-test showed that the proliferation of both cell cultures was significantly inhibited by Ranibizumab treatment (BECs: p < 0.0001; LECs: p = 0.0003) (Fig. 1). Ranibizumab inhibits proliferation of blood endothelial cells (BECs), (left panel: Ranibizumab 10 nm; inhibition by 20%, p < 0.0001) and lymphatic endothelial cells (LECs) in vitro (right panel: Ranibizumab 10 nm: Inhibition by 10%, p = 0.0003). Proliferation was measured by a cell proliferation enzyme-linked immunosorbent assay (ELISA) with BrdU. We provided quantitative analysis of the neovascularised corneal area identified by immunohistochemical staining as shown previously (Bock et al. 2008a,b) which revealed that topical application of Ranibizumab in mice with epithelium debridement significantly reduces the density of blood and lymphatic vessels in the corneas of treated mice by binding to murine VEGF-A relative to animals that received normal saline after 7 days of topical application [control: 100% (SE mean: 4.87%) (Fig. 2), Ranibizumab: 79.52% (SE mean: 4.19%), p = 0.0026 (Fig. 2A)]. For lymphatic vessels we were able to demonstrate an equally significant inhibition [control: 100% (SE mean: 8.44%), Ranibizumab: 69.52% (SE mean: 4.54%), p = 0.0026 (Fig. 2B)]. Inhibition of hem- (control: 100% (SE mean: 11.4%), Ranibizumab: 76.7% (SE mean: 10.2%), p = 0.0157] (Fig. 2G) and lymphangiogenesis [control: 100% (SE mean: 16.8%), Ranibizumab: 53.1% (SE mean: 15.7%), p = 0.0035] (Fig. 2H) in corneas without debrided epithelium was comparable to those that underwent debridement. Ranibizumab inhibits proliferation of blood and lymphatic vessels in vivo (A–H). Fluorescent pictures of corneal whole mounts showing Ranibizumab treated corneas (C, D) with reduced growth of blood vessels and lymphatic vessels as compared to controls (A, B); Graphs showing (in debrided corneas) that hemangiogenesis was inhibited by approximately 20% as compared to controls, p = 0.0026 and lymphangiogenesis by approximately 30%, p = 0.0026 (E, F). Also vascularised corneas with epithelium showed inhibition of hem- (approximately 25%, p = 0.0157) and lymphangiogenesis (approximately 45%, p = 0.0035) when treated with Ranibizumab compared to controls (G, H). Our experiments demonstrate for the first time an antiproliferative effect of Ranibizumab on LECs and BECs in vitro and a significant effect of Ranibizumab on corneal lymphangiogenesis in the cornea in vivo. The majority of the laboratory experiments with Ranibizumab has focused on the intravitreal use of the drug (Husain et al. 2005; Kim et al. 2006, 2011; Lu & Adelman 2009; Zayit-Soudry et al. 2010; Lai et al. 2011). Our corneal and in vitro experiments comprehensively show that Ranibizumab has an antiproliferative effect not only on BECs but also on LECs in vitro and additionally shows a significant effect on inflammatory lymphangiogenesis in the cornea even after short time application. These results underline the recent finding that VEGF-A seem to play a crucial role in the development of inflammatory induced lymphangiogenesis (Chen et al. 2004; Cursiefen 2007). We observed a stronger impact on lymphangiogenesis in the in vivo model than in the in vitro system. Although it was shown, that VEGF- A has a direct proliferative effect on LECs (Bock et al. 2007), Maruyama et al. (2005) showed, that the prolymphangiogenic effect of VEGF-A is mainly indirect via the recruitment of macrophages, which in turn release prolymphangiogenic growth factors like VEGF-C and D. Blocking VEGF and with it lymphangiogenesis has a profound effect on transplant survival (Dietrich et al. 2010). With Ranibizumab only blocking VEGF-A and not all of the various angiogenic factors in vivo, as, for example, bFGF, angiostatin, endostatin, or pigment epithelium–derived factor (PEDF), it will be not possible to completely block hem- and lymphangiogenesis. With Ranibizumab given as eye drops there is always a uncertainty of the exact used dosage. We followed the advised concentration for intravitreal Ranibizumab (0.5 mg) as explained in the methods. Like the progenitor Bevacizumab (Avastin®) the IgG1 kappa isotype monoclonal antibody fragment Ranibizumab competitively binds with all isoforms of VEGF (Chen et al. 1999; Liang et al. 2006). In addition, Ranibizumab lacks the Fc region of the antibody and is less likely to cause complement-mediated inflammation after application which might be an additional benefit in inflamed eyes (Ferrara et al. 2006). These aforementioned effects and advantages of Ranibizumab have implications for the ongoing clinical trials for the use of topical Ranibizumab in patients with progressive neovascularisation at the ocular surface (http://clinicaltrials.gov/ct2/show/NCT00681889?term=Dana+Ranibizumab&rank=1). The important role of an anti-VEGF therapy to prolong graft survival in high risk cornea with regressed or new blood and lymphatic vessels has recently been demonstrated (Bachmann et al. 2008; Hos et al. 2008; Dietrich et al. 2010). Having shown here that Ranibizumab also inhibits lymphangiogenesis in the cornea further research will show whether Ranibizumab also promotes graft survival by interfering with the afferent and efferent arm of the immune reflex arc as shown earlier (Chen et al. 2004; Dana 2006). Experiments without debridement of the epithelium showed an even stronger effect on hem- and lymphangiogenesis. There might be two facts which contribute to this effect. Dastjerdi et al. (2011) has recently shown, that vascularized corneas are sufficiently penetrated by the anti-VEGF-A antibody bevacizumab. In addition it is known, that the corneal epithelium is not only a physical barrier but also contains decoy-mechanisms, for example soluble VEGFR1, R2 or R3 (Cursiefen et al. 2006a,b,c; Ambati et al. 2007; Albuquerque et al. 2009), which maintain the avascularity of the cornea. So the debridement on the one hand guarantees a good penetration of the drug and on the other hand lowers the antiangiogenic properties of the cornea. Therefore the angiogenic stimulus after abrasion might be higher than with the intact epithelium, so that a higher antiangiogenic effect at the same concentration of ranibizumab can be seen with epithelium. Further studies to determine optimal dose for topical use and penetration through intact epithelium along with the elucidation of the mechanism of action of Ranibizumab on lymphangiogenesis provide additional avenues for research. In conclusion Ranibizumab inhibits not only hem-, but also inflammatory corneal lymphangiogenesis in vivo. Topical Ranibizumab as a pharmacological agent has not only the ability to reduce inflammation associated neovascularization but can have additional clinical indications which have to be further explored. Support: International Council of Ophthalmology (A.P); Interdisciplinary Center for Clinical Research (IZKF) Erlangen (A9); DFG (SFB 643/B10)." @default.
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• W2028528029 date "2012-09-20" @default.
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• W2028528029 title "Topical Ranibizumab inhibits inflammatory corneal hem- and lymphangiogenesis" @default.
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