Inhibition Of Corneal Neovascularization Research Articles

(−)-Epigallocatechin 3-gallate (EGCG) at the ocular surface inhibits corneal neovascularization

Corneal neovascularization is often accompanied by inflammatory response and loss of their immune privilege which leads to significant visual impairment and worsens the prognosis of a subsequent penetrating keratoplasty. Several types of treatment are currently used. However, there are some associated limitations and complications. The consumption of (−)-epigallocatechin 3-gallate (EGCG) has been studied extensively as a potential treatment for a variety of carcinogenic and degenerative diseases due to its ability to suppress a variety of inflammatory and angiogenic factors such as NF-κB, IL-1β, COX2, VEGF, and matrix metalloproteinases. These factors are involved in the development of corneal neovascularization. The safety of long-term EGCG administration as well as the drug’s high solubility in water urge further investigation of the therapeutic potential of this drug. Therefore, we propose that the administration of EGCG to the ocular surface represents a new chemopreventive alternative to suppress the corneal neovascularization induced by inflammation.

The Induction of an Angiogenic Response in Corneal Myofibroblasts by Platelet-Activating Factor (PAF)

Purpose: Although the exact mechanisms underlying corneal neovascularization remain unclear, cytokines and growth factors play an important role in their development. We have shown previously that the inflammatory mediator platelet-activating factor (PAF) is a potent inducer of corneal neovascularization in vivo. In this study, we investigate the role of stromal myofibroblasts in neovascularization and the effect of PAF on this process.Methods: Myofibroblasts were obtained from rabbit corneal keratocytes and identified with anti-α-SMA antibody. Cells were treated with PAF (100 nM) for 24 hr. In some experiments, cells were pre-treated with the PAF antagonist LAU-0901 (150 nM). Expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) was examined by immunofluorescence and immunoblotting. To study the effect of myofibroblasts on vessel formation in vitro, Vybrant® CM-DiI labeled human umbilical vein endothelial cells (HUVECs) were cultured on myofibroblasts in a thin layer of collagen gel. CD31 was used as the cell marker of HUVEC.Results: VEGF and TSP-1 were not detectable in keratocytes, but they were positively stained in myofibroblasts. PAF induced a significant increase in VEGF expression and a decrease in TSP-1 expression. These changes were inhibited in the presence of LAU-0901. HUVECs co-cultured with corneal myofibroblasts formed a typical structure of vessel-like tubes within 1 week. The addition of PAF to the medium increased HUVEC-induced vessel-like tube formation, which was abolished by LAU-0901. Addition of anti-VEGF antibody to the medium completely prevented the formation of vessel-like tubes.Conclusion: We provide evidence for the role of stromal myofibroblasts in the corneal neovascularization process. By enhancing VEGF production and decreasing TSP-1 production in myofibroblasts, PAF augments the angiogenic response. The PAF antagonist LAU-0901 could represent a new therapeutic venue for inhibiting corneal neovascularization.

The effect of doxycycline temperature-sensitive hydrogel on inhibiting the corneal neovascularization induced by BFGF in rats

To study the effect of doxycycline temperature-sensitive hydrogel (DTSH) on inhibiting the corneal neovascularization (NV) induced by the basic fibroblast growth factor (bFGF). Corneal NV was induced by slow-release polymer pellets containing bFGF, using a rat corneal pocket model. After being implanted with bFGF pellets, the female Sprague-Dawley rats were randomly divided into seven groups (12 rats/group). The grouped rats were given topically normal saline solution and neutralized DTSH at a concentration of 0%, 0.01%, 0.05%, 0.1%, 0.5%, and 1% respectively, and treated for 6 consecutive days. After 6 days of treatment, the cornea was perfused with India ink. The length and area of the corneal vessel were measured and analyzed by Image Pro-Plus 5.1. Compared to the control group given saline solution, the study groups given DTSH at a concentration of 0.05%, 0.1%, 0.5%, and 1% showed significant reduction in the vessel length (respectively, 58%, 60%, 52%, and 37%) and the vessel area (respectively, 61%, 62%, 49%, and 39%) (p < 0.001). However, no such significant reduction was observed in the study group given 0.01% DTSH (p = 0.133 and 0.166 for vessel length and area respectively). Study groups given 0.05% and 0.1% DTSH showed better effects than groups given 0.01% and 1% DTSH with regard to reducing the vessel length and the vessel area (p < 0.05). The study results showed that topical DTSH effectively inhibited corneal NV at the ideal concentration of 0.05% and 0.1%. Therefore, topical DTSH could be considered as an alternative treatment for the clinical management of corneal NV.

Inhibition of corneal neovascularization by vascular endothelia growth inhibitor gene.

To evaluate the effect of Effectene™ lipofectine mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA(4)-VEGI) gene on corneal neovascularization (CNV). Forty New Zealand albino rabbits were sutured by 5-0 silk on the superior cornea to establish the animal model and divided into 4 random group, ten per each group: group A: transfected by pcDNA(4)-VEGI gene mediated by Effectene™ lipofectine transfection, group B: by Plasmid pcDNA(4), group C: by Effectene™, and group D: by normal saline. Length and area of CNV were measured under slit lamp every day after transfection, immunohistochemistry was used to detected the expression of VEGI protein in cornea at 3, 7, 14 and 21 days. Average occurrence of CNV in the pcDNA(4)-VEGI gene transfected group (group A) was 6.3 days, in plasmid pcDNA(4) control group (group B) was 3.1 days, in Effectene™ lipofectine control group (group C) was 3.2 days, in normal saline control group (group D) was 3.2 days. Differences between groups A and B, C, D were statistically significant (P<0.01), while differences in groups B, C and D were meaningless (P>0.05). Lenth and average area of CNV in each period in group A was meaningful different from that in groups B, C, and D (P<0.01), while differences in group B, C and D were meaningless (P>0.05). Immunohistochemistry result: VEGI positive cells could be seen in epithelium, stroma, endothelium and the cliff of CNV in group A at 3 days after transfection. VEGI cells changed with the decrease of CNV. None positive cells were in the control groups (groups B, C and D) all the time. Effectene™ lipofectine transfection technique can be effectively used in transfecting pcDNA(4)-VEGI gene into rabbit cornea and the lenth and areas of CNV can be inhibited by VEGI gene.

Therapeutic effect of topical bevacizumab on corneal neovascularization following keratitis

Abstract Purpose To evaluate clinical results of the treatment with topically applied bevacizumab in patients with corneal neovascularization secondary to keratitis. Methods A retrospective noncomparative case series report. 7 eyes of 7 patients with corneal neovascularization following ulcerative keratitis were treated with topical bevacizumab eye drops (5mg/ml) for 4 to 22 weeks. Underlying etiology of keratitis included 4 cases of herpetic infection, 2 cases of Pseudomonas aeruginosa infection and 1 case of keratitis e lagophthalmo. The follow‐up time was 4 to 25 weeks. Results After the course of topical bevacizumab the decrease of corneal neovascularization was observed in all of the cases due to reduction of the diameter of the blood vessels and an involution of the small vessels’ network. At the baseline examination the mean extent of the neovascularized area of the cornea was 4.43 (SD 2.82, range 1‐9) clock hours in comparison with 2,57 (SD 2.15, range 1‐6) clock hours after the treatment, however, this difference was not statistically significant. The neovascularized area diminished in 5 eyes, the mean reduction being 1.86 (SD 1.57, range 1‐4) clock hours. In 2 eyes former epithelial defects of the cornea persisted without significant changes in the size or depth during the treatment. The rest of the eyes showed an epitheliazed, stable and noninflamed ocular surface. No local or systemic adverse effects of topical bevacizumab were noticed during the follow‐up time. Conclusion Topical bevacizumab is an effective and well tolerated treatment for inhibiting corneal neovascularization secondary to keratitis of different etiologies.

Inhibition of Corneal Neovascularization by Topical Bevacizumab (Anti-VEGF) and Sunitinib (Anti-VEGF and Anti-PDGF) in an Animal Model

To evaluate the effects of topically applied bevacizumab and sunitinib on experimentally induced corneal neovascularization. Experimental animal study. Thirty-six New Zealand rabbits were involved. One eye per rabbit was used. Corneal neovascularization was induced by placing 5 silk sutures in the upper cornea. Rabbits were randomized to 1 of 3 groups (12 rabbits each): Group 1 received saline 0.9%, Group 2 bevacizumab 5 mg/mL, and Group 3 sunitinib 0.5 mg/mL. All treatments were administered 3 times daily for 14 days. Photographs were taken on a slit lamp on days 7 and 14, and angiographic photographs were taken on day 14. The area of neovascularization was measured in mm(2), percentage of the total corneal area, and percentage of the corneal surface covered by sutures. On day 14, corneal neovascularization area in Group 1 (25.92 ± 5.08 mm(2), 18.78% ± 3.5% of corneal surface, 105.59% ± 18.9% of corneal surface with sutures) was larger than in Groups 2 (18.52 ± 7.94 mm(2), 13.67% ± 5.8%, 76.35% ± 33.2%) (1-way analysis of variance, P = .041) and 3 (4.57 ± 2.32 mm(2), 3.40% ± 1.7%, 18.94% ± 9.2%)(P < .001). Neovascularization in Group 2 was larger than in Group 3 (P < .001). Compared to saline, corneal neovascularization was inhibited 28.5% by bevacizumab and 82.3% by sunitinib. Sunitinib settled on the iris. Topical administration of both bevacizumab and sunitinib inhibits corneal neovascularization in rabbits. But vascular endothelial growth factor (VEGF) pathway blockade by bevacizumab was not sufficient for a profound inhibition. Blocking both VEGF and platelet-derived growth factor pathways using sunitinib was 3-fold more effective.

Parstatin suppresses ocular neovascularization and inflammation.

Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the PAR1 receptor. The authors recently showed that parstatin is a potent inhibitor of angiogenesis. The purpose of the present study was to evaluate the therapeutic effect of parstatin on ocular neovascularization. Choroidal neovascularization was generated in mice using laser-induced rupture of Bruch's membrane and was assessed after 14 days after perfusion of FITC-dextran. Oxygen-induced retinal neovascularization was established in neonatal mice by exposing them to 75% O(2) at postnatal day (P)7 for 5 days and then placing them in room air for 5 days. Evaluation was performed on P17 after staining with anti-mouse PECAM-1. The effect of parstatin was tested after intravitreal administration. The effects of subconjunctival-injected parstatin on corneal neovascularization and inflammation in rats were assessed 7 days after chemical burn-induced corneal neovascularization. Retinal leukostasis in mice was assessed after perfusion with FITC-conjugated concanavalin A. Parstatin potently inhibited choroidal neovascularization with an IC(50) of approximately 3 μg and a maximum inhibition of 59% at 10 μg. Parstatin suppressed retinal neovascularization with maximum inhibition of 60% at 3 μg. Ten-microgram and 30-μg doses appeared to be toxic to the neonatal retina. Subconjunctival parstatin inhibited corneal neovascularization, with 200 μg the most effective dose (59% inhibition). In addition, parstatin significantly inhibited corneal inflammation and VEGF-induced retinal leukostasis. In all models tested, scrambled parstatin was without any significant effect. Parstatin is a potent antiangiogenic agent of ocular neovascularization and may have clinical potential in the treatment of angiogenesis-related ocular disorders.

Inhibition of corneal neovascularization after alkali burn: comparison of different doses of bevacizumab in monotherapy or associated with dexamethasone

To compare the effects of different doses of bevacizumab with both saline and dexamethasone on inflammatory angiogenesis in the rat cornea induced by small chemical lesions. Corneal chemical cauterization was performed on 24 rats. Animals were divided randomly into six groups and received a daily subconjunctival injection for 7 days of: balanced salt solution 0.1 mL or dexamethasone phosphate 4 mg/day or bevacizumab 2.5 mg/day, 3.75 mg/day, 5.0 mg/day or bevacizumab 5.0 mg/day + dexamethasone phosphate 4 mg/day. Clinical examination under slitlamp was performed daily for 7 days to evaluate corneal opacity and vessel size evolution. Computer assisted quantitative image analysis was used to measure the total corneal area covered by neovascularization. At final examination, the dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups showed a significant lowering in corneal opacity score as compared with control (P = 0.024, P = 0.006 and P = 0.013, respectively). Also, a significant reduction on new vessels size score was observed. Surface of corneal neovascularization was significantly reduced in dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups compared with control (P =0.045, P = 0.047 and P = 0.044, respectively). Our study demonstrates the ability of a 5.0 mg/day bevacizumab subconjunctival injection, in monotherapy or associated with dexamethasone, to cause a short-term involution of corneal neovascularization after corneal alkali burn. Combination of both of these treatments may have advantages to monotherapy approaches.

The Different Effects of Early and Late Bevacizumab (Avastin) Injection on Inhibiting Corneal Neovascularization and Conjunctivalization in Rabbit Limbal Insufficiency

To compare the effects of early, mid, and late subconjunctival injection of bevacizumab on corneal neovascularization (NV) and conjunctivalization in a rabbit limbal insufficiency model. Limbal insufficiency was created surgically, and subconjunctival injections of 2.5 mg bevacizumab twice weekly for 1 month were started immediately (early group), 1 week (mid group), and 1 month after injury (late group). The corneal epithelial alterations, stromal opacity, centricity, extent, and PICS (percentage of involved corneal surface) of the corneal NV were evaluated. The expression of cytokeratins K3, K4, K12, K13, and MUC5 by the corneal surface cells was examined by immunohistochemistry. Bevacizumab significantly inhibited corneal NV in the early and mid, but not the late, treatment groups at 4 weeks after treatment (PICS: P < 0.01 in the early group, P < 0.01 in the mid group, and P > 0.05 in the late group). Early and mid treatment produced significant inhibition of corneal alteration (P < 0.01 in the early group and P = 0.03 in the mid group) and stromal opacity (P < 0.01 in the early group and P = 0.02 in the mid group) at 4 months after treatment but not in the late group. The immunohistochemistry of cytokeratin on the corneal surface cells at 1 month after treatment was K3(+), K4(-), K12(+), K13(-), and MUC5(-) in the early group; K3(+), K4(+), K12(+), K13(+), and MUC5(-) in the mid group; and K3(+), K4(+), K12(-), K13(+), and MUC5(+) in the late treatment group. Early and mid bevacizumab injection inhibited corneal NV, epithelial alteration, and stromal opacity in limbal insufficiency, but late treatment did not. Early treatment with bevacizumab seems to be clinically beneficial in the management of limbal injury such as chemical burn.

Inhibition of corneal neovascularization by vascular endothelia growth inhibitor gene.

To evaluate the effect of Effectene™ lipofectine mediated plasmids encoding human pcDNA(4)-vascular endothelia growth inhibitor (pcDNA(4)-VEGI) gene on corneal neovascularization (CNV). Forty New Zealand albino rabbits were sutured by 5-0 silk on the superior cornea to induce CNV and divided into 4 random teams, ten per each team: team A: transfected by pcDNA(4)-VEGI gene mediated by Effectene(™) lipofectine transfection; team B: by plasmid pcDNA(4); team C: by Effectene(™), and team D: by normal saline. Length and area of CNV were observed under slit lamp every day after transfection. Immunohistochemistry was performed to detect the expression of VEGI protein in corneas at day 3, 7, 14 and 21. 1) Average occurrence of CNV was 6.3 days in team A, 3.1 days in team B, 3.2 days in team C, and 3.2 days in team D. Difference was significant between A and other teams (P<0.01); 2) Length and average area of CNV in each period in team A was significantly different from those in team B, C and D (P<0.01); 3) VEGI expressions were observed in epithelium, stroma, endothelium and the cliff of CNV in team A at 3 days after transfection by immunohistochemical staining. None VEGI positive cells were found in the control teams (team B, C and D) all the time. Effectene(™) lipofectine transfection technique can effectively transfect pcDNA(4)-VEGI gene into rabbit cornea and the length and CNV areas can be inhibited by VEGI gene.

Bevacizumab (Avastin) inhibits corneal neovascularization in rabbits

Objective:To evaluate the effect of subconjunctival injection of Bevacizumab(Avastin) on corneal neovascularization(CNV) in rabbits.Methods:Forty-eight New Zealand rabbits were randomly divided into three groups:normal control group(group A,3 rabbits),neovascularization group(group B,9 rabbits) and Bevacizumab treatment group(group C,36 rabbits).Group C was further divided into group C1(1 day small dose group),C2(1 day high dose group),C3(14 days small dose group) and C4(14 days high dose group),with 9 rabbits each group.CNV model was made by suture in group B and C.Animals in group C were injected subconjunctivally with 0.1 ml or 0.2 ml Bevacizumab(25 mg/ml) in the left eyes.The growth of CNV was observed every day after operation and the neovascularization areas calculated.Expression of VEGF in the cornea was detected by immunohistochemistry on day 7,14 and 28 after suture.VEGF content in the aqueous humor was determined by ELISA assay.Results:CNV growth in group C1 and C2 was inhibited significantly compared with that in group B on day 7,14 and 28(P0.01).On day 28 the growth in group C3 and C4 was significantly inhibited compared with that in group B(P0.01),that in group C1 was significantly inhibited than that in group C3,and that in group C2 was significantly inhibited compared with that in group C4(P0.01).VEGF levels in the cornea and aqueous humor in group B increased as time passed by,and they were positively associated with CNV area(P0.01).On day 7,14,and 28,the VEGF levels in the cornea and aqueous humor in C1 and C2 groups were significantly lower than that in group B(P0.01).On day 28,those in group C3 and C4 were significantly lower than those in group B(P0.01);those in group C1 were significantly lower than those in group C3(P0.01);and those in group C2 were significantly lower than those in group C4(P0.01).The CNV area and VEGF levels in the cornea and aqueou humor were similar between C1 and C2 groups and between C3 and C4 groups.Conclusion:Subconjunctival administration of Bevacizumab can effectively inhibit corneal neovascularization in experimental CNV model,and early administration has a better outcome than late administration.Bevacizumab may exert its effect by down-regulating VEGF in cornea and aqueous humor.

Small interference RNA targeting vascular endothelial growth factor inhibits rat corneal neovascularization

To test the effects of VEGF-siRNA-transfected corneal epithelium on corneal neovascularization (CNV). It was an experimental study. Cultured rat corneal epithelial cells and keratocytes were transfected with synthesize VEGF siRNA by lipofectamine 2000. The level of VEGF mRNA was analyzed by real time PCR, and the protein levels were determined by enzyme-linked immunosorbent assay (ELISA). CNV was induced by cauterization with 1 mol/L sodium hydroxide in rat corneas. The VEGF-siRNA-transfected-corneal epithelium cells were transplanted to the CNV lesions. Immediately after transplantation, the VEGF-siRNA combined with lipofectamine 2000 were directly transfected rat cornea through injecting into the anterior chamber. After surgery, the surface areas occupied by new vessels were measured, and VEGF protein was localized by immunohistochemistry. The levels of VEGF expression at both mRNA and protein in the VEGF-siRNA transfected corneal epithelial cells and keratocytes were significantly lower than those of control cells. VEGF siRNA could inhibit the expression of VEGF mRNA in corneal epithelial cells and keratocytes to 57% - 85% and 59% - 78%, respectively. The VEGF-siRNA-transfected-corneal epithelium transplantation significantly decreased the surface areas occupied by new vessels. VEGF expression level in interference groups was lower than that in the control group. The development of CNV is markedly suppressed by VEGF-siRNA transfection in vivo.

Inhibition of corneal neovascularization with plasmid pigment epithelium-derived factor (p-PEDF) delivered by synthetic amphiphile INTeraction-18 (SAINT-18) vector in an experimental model of rat corneal angiogenesis

The use of Synthetic Amphiphile INTeraction-18 (SAINT-18) carrying plasmid pigment epithelium-derived factor (p-PEDF) as an anti-angiogenesis strategy to treat corneal neovascularization in a rat model was evaluated. Four partially dried forms (Group A: 0 μg, B: 0.1 μg, C: 1 μg, D: 10 μg) of a p-PEDF–SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus at the temporal side. The 1 μg of plasmid-basic fibroblast growth factor–-SAINT-18 (p-bFGF–SAINT-18) (1 μg) was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. Inhibition of neovascularization was observed and quantified from day 1 to day 60. PEDF (50-kDa) and bFGF (18-kDa) protein expression were analyzed by biomicroscopic examination, Western blot analysis, and immunohistochemistry. Gene expression in corneal and conjunctival tissue was observed as early as 3 days after gene transfer and stably lasted for over 3 months with minimal immune reaction. Subconjunctival injection of a highly efficient p-PEDF–SAINT-18 successfully inhibited corneal neovascularization. Successful gene expression of bFGF, PEDF and a mild immune response of HLA-DR were shown by immunohistochemistry staining. We concluded that SAINT-18 was capable of directly delivering genes to the ocular surface by way of subconjunctival injection, and delivered sustained, high levels of gene expression in vivo to inhibit angiogenesis.

Suppression of corneal neovascularization by human amniotic homogenate in rabbits

目的 观察人羊膜匀浆上清液对家兔角膜新生血管(CNV)的抑制作用.方法 63只新西兰白兔随机选取3只作为正常对照组,余60只制作双眼碱烧伤模型,随机分为4个组:A组为碱烧伤对照组滴用PBS液,B、C、D组为实验组分别滴用50、200、400μg/mL的羊膜匀浆上清液.术后1、5、7、14、28d每组分别随机处死3只兔子,行组织病理学检查,测量微血管数量、多形核白细胞(PMNL)含量、血管内皮生长因子(VEGF)的表达量.结果 碱烧伤1、5、7、14、28d各时间点CNV的生长面积、微血管计数、PMNL浸润、VEGF的表达4个组差异均有统计学意义(P＜0.01),其中D组均数最小.VEGF、PMNL、微血管计数三者呈正相关(P＜0.01).结论 羊膜匀浆上清液有减轻角膜血管化的作用,呈质量浓度依赖性,质量浓度达到400μg/mL时抑制作用显著增强。

Inhibition of Corneal Neovascularization by Ranibizumab (Lucentis): An Experimental Study in Rabbit Cornea

Summary The purpose of this study was to evaluate the effects of the use of the subconjunctival injection of ranibizumab (Lucentis) on angiogenesis in the rabbit cornea. Corneas of 12 New Zealand Rabbits were cauterized with silver nitrate crystal. Animals were divided in two groups: control group (GC) that received 0.02 ml of 0.9% saline solution; group ranibizumab (GR) that received 0.5 mg of ranibizumab subconjunctivally at the 24th h after the lesion was formed. Animals corneas were extracted on the 14th day under general anesthesia. The newly formed vessels digital photographs were obtained and analyzed in a computerized system (google sketch-up program). In the control group, neovascularization covered 64.66%±20.81 (mean±standard deviation [SD]) of the corneal surface, compared with 34.17%±4.53 (mean±SD) in the GR group. When vascular density is compared between treated groups, statistical differences were observed (P<0.002). The results showed an inhibition of angiogenesis when the control group was compared with ranibizumab treated groups. These results suggest that subconjunctival injection of ranibizumab is able to inhibit corneal angiogenesis.

Inhibition of Corneal Neovascularization with Propolis Extract

Neovascularization of the normally avascular cornea is seen in many pathological conditions including trauma, corneal transplantation, inflammation and eye diseases. Various growth factors and proteinases are involved in corneal neovascularization. Data supporting a causal role for vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are extensive. Inhibition of angiogenesis is a main strategy for treating corneal neovascularization. Several findings have shown that corneal neovascularization can be reduced by using anti-VEGF and anti-MMPs agents. Efficacy of a propolis extract has been demonstrated for reducing angiogenesis in vitro and in vivo. Propolis extracts containing artepillin C and caffeic acid phenyl ester significantly reduced the number of newly formed vessels and expression of MMPs and VEGF production from various cells. So far, propolis extract is a potential candidate as an anti-angiogenic agent and can inhibit cell proliferation, migration and capillary tube formation. We hypothesize that topical application of propolis is potentially useful for inhibiting corneal neovascularization and restoration of corneal clarity.

Subconjunctival Injection of Bevacizumab (Avastin) on Corneal Neovascularization in Different Rabbit Models of Corneal Angiogenesis

Bevacizumab is a potent recombinant humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). The purpose of this study was to evaluate the therapeutic effect of subconjunctival injection of bevacizumab on corneal neovascularization (NV) in different rabbit models. Several rabbit models of corneal NV were used, including (1) a corneal micropocket assay with VEGF pellet, (2) a corneal micropocket assay with basic fibroblast growth factor (b-FGF) pellets, (3) mechanical limbal injury-induced corneal NV, and (4) an alkali-induced model of corneal NV. Subconjunctival injections of bevacizumab (0.25-2.5 mg) were applied twice per week for 2 to 8 weeks. Digital photographs of the cornea were analyzed to determine the length of corneal NV and the area of cornea covered by NV as a percentage of the total corneal area. Immunohistochemical staining with anti-human IgG antibody labeled with Cy3 was used to determine the detection of intracorneal distribution of bevacizumab after injection. Subconjunctival injection of bevacizumab caused significant inhibition of corneal NV formation as measured by length or surface area in all animal models (P<0.05). No significant ocular complications were found. Staining of bevacizumab was found in the corneal stroma for 3 to at least 14 days in the different rabbit models. Subconjunctival injection of bevacizumab is effective in inhibiting corneal NV in several rabbit models. Bevacizumab may diffuse into the corneal stroma and persist for a few days after injection. It may be useful in preventing corneal NV in the acute phase of various kinds of corneal inflammation.

Inhibition of corneal neovascularization by vascular endothelial growth inhibitor gene

To evaluate the effect of Effectene lipofectene mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene on corneal neovascularization (CNV). It was an experimental study. Forty healthy New Zealand albino rabbits (40 eyes) were divided into 4 teams according to the random digits table: team A (10 rabbits) was the team which transfected by lipofectine mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene; team B (10 rabbits) was the team transfected by empty vector; team C (10 rabbits) was the team transfected by lipofectine; team D (10 rabbits) was the empty control group which was transfected by saline. (1) Human VEGI gene fragment was connected with expressional vector plasmid pcDNA4 to construct the pcDNA4-VEGI gene expression vector. Computer automatic sequence analysis was used to identify the gene. (2) New Zealand albino rabbits were sutured by 5 - 0 silk in the superior cornea to establish the animal model and were transfected by pcDNA4-VEGI gene mediated by Effectene lipofectene transfection. Length and area of CNV were measured by slit lamp every day after transfection, immunohistochemistry was used to detected the expression of VEGI protein in cornea at the time 3, 7, 14 and 21 d. (1) Computer automatic sequence analysis confirmed the correct recombination of pcDNA4-VEGI gene. (2) Average occurrence of CNV in the pcDNA4-VEGI gene transfected team was 6.3 days while that in the control teams were 3.1, 3.2, 3.2 days (F = 39.838, P < 0.01). Average length and area of CNV were also different in the VEGI team and the control teams (q between team A and B on average length of CNV was 17.386, 20.944 and 8.892 on 7, 14 and 21 d; q between team A and C on average length of CNV was 19.488, 19.795 and 7.483 on 7, 14 and 21 d; q between team A and C on average length of CNV was 19.583, 20.413 and 8.941 on 7, 14 and 21 d, and P were all < 0.01. And q between team A and B on average area of CNV was 30.238, 57.820 and 35.543 on 7, 14 and 21 d; q between team A and C on average area of CNV was 32.607, 57.843 and 36.653 on 7, 14 and 21 d; q between team A and C on average area of CNV was 33.873, 57.590 and 34.724 on 7, 14 and 21 d, and P were all < 0.01). Integrated epithelium, light stroma edema, less CNV and less inflammation could be seen in the gene-therapy team. And immunohistochemistry results showed VEGI positive cells in epithelium, stroma, endothelium and the cliff of CNV in the gene therapy team. PCR, enzyme cutting, recombination and other genetic techniques could be used to construct expressional pcDNA4-VEGI gene. Effectene lipofectene transfection technique could be effectively used in transfecting pcDNA4-VEGI gene into rabbit cornea to inhibit CNV.

Inhibition of Corneal Neovascularization by Blocking the Angiotensin II Type 1 Receptor

To determine the role of angiotensin II type 1 receptor (AT1R) signaling in corneal neovascularization. Corneal neovascularization was induced by suturing 10-0 nylon 1 mm away from limbal vessels in C57 BJ6 mice. Angiotensinogen and its receptor (AT1R) gene expression levels were evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The expression of angiotensin II (Ang2) and AT1R was confirmed by Western blotting and immunohistochemistry. To investigate the function of Ang2 in corneal neovascularization, infiltrating macrophages in vascularized corneas and the neovascularized area were investigated after intraperitoneal injection of an AT1R antagonist (telmisartan, 10 mg/kg). Further, corneal mRNA of VEGF, MCP-1, IL-6, ICAM-1, and TNF-alpha was examined in control and telmisartan-treated mice. Ang2 and AT1R markedly increased in the neovascularized corneas compared with normal corneas. Ang2 and AT1R were expressed in epithelium and stromal cells (vascular endothelium, infiltrating leukocytes, and keratocytes) in neovascularized cornea at protein levels and were weakly detected in normal corneal epithelium. Infiltrating macrophages were reduced in telmisartan-treated mice on day 7 after suturing. Neovascularized area in the cornea of telmisartan-treated mice was 70% smaller than that of control mice on day 7 after suturing. A PPAR-gamma antagonist partially, but significantly, reversed the suppressive effect of telmisartan on induction of corneal neovascularization. The expression of VEGF, MCP-1, IL-6, and ICAM-1 was significantly inhibited in telmisartan-treated mice. These findings indicate that Ang2, abundantly expressed in neovascularized corneas, has a significant role in inflammation-related driven corneal neovascularization. AT1R may be a therapeutic target for the suppression of corneal neovascularization.

Suppression of Alkali Burn-Induced Corneal Neovascularization by Dendritic Cell Vaccination Targeting VEGF Receptor 2

To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization caused by alkali injury. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGF receptor 2 (VEGFR2(400-408)) was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of VEGFR2(400-408)- or gp70-pulsed mature DCs three times at 6-day intervals. After the third immunization, corneal neovascularization was induced by alkali injury. Two weeks after the injury, the corneal vascularized area was evaluated by lectin angiography. To confirm the peptide-specific CTL activities in C57BL/6 mice, CD8(+) T cells from immunized mice were subjected to ELISA for interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and (51)Cr-release cytotoxicity assay. To determine the in vivo effector T cells, the immunized mice were intraperitoneally injected with an anti-CD4 or -CD8 depletion antibody. Corneal neovascularization was significantly attenuated in mice immunized with VEGFR2(400-408) compared with those not immunized or immunized with gp70. VEGFR2(400-408) or gp70, but not beta-gal(96-103), application led to dose-dependent induction of IFN-gamma and TNF-alpha in the CD8(+) T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of major histocompatibility complex-matched cells expressing VEGFR2, but not beta-gal(96-103). In vivo depletion of CD8(+), but not CD4(+), T cells significantly reversed the suppressive effect of VEGFR2(400-408) immunization on corneal neovascularization to the level observed in nonimmunized or gp70-immunized animals. These results indicate the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for corneal chemical injury.