Inhibition Of Corneal Neovascularization Research Articles

The Effect of Subconjunctival Ranibizumab on Corneal and Anterior Segment Neovascularization: Study on an Animal Model

To evaluate the effect of subconjunctival anti-vascular endothelial growth factor (VEGF) ranibizumab on corneal and anterior segment neovascularization. In this experimental study and laboratory investigation, chemical cauterization was utilized to induce corneal neovascularization in 16 rabbits randomly divided in 2 equal groups. Cauterized eyes were either treated with 0.1 mL (1 mg) of subconjunctival ranibizumab or administered a sham injection. A third group of 4 rabbits served as control for side effects after ranibizumab administration. All animals were monitored daily for 14 days and the extent of corneal scarring and neovascularization was measured on days 1, 7, and 14. After enucleation, ocular tissues were separated under a surgical microscope and VEGF levels were measured with ELISA. Statistical analysis was performed to compare the extent of corneal neovascularization and VEGF levels between treated and untreated eyes. Subconjunctival ranibizumab inhibited corneal neovascularization significantly both in the first and the second week compared to untreated controls (p = 0.006 and p = 0.001, respectively). The VEGF levels were significantly lower in all anterior segment tissues like the cornea, iris, aqueous humor, and conjunctiva of the treated eyes (p<0.01). The reduction of VEGF levels ranged from 19% to 73% in different ocular tissues. Corneal scarring was not significantly affected by anti-VEGF treatment (p = 0.7). No side effects were noticed. Early subconjunctival administration of ranibizumab may successfully inhibit alkali-induced corneal neovascularization in an animal model. Subconjunctival ranibizumab reduces VEGF levels significantly not only in the cornea and the bulbar conjunctiva but also in the aqueous humor and the iris.

Recurrence of Corneal Neovascularization Associated With Lipid Deposition After Subconjunctival Injection of Bevacizumab

To report 7 cases of recurrent corneal neovascularization (NV) and lipid deposition after subconjunctival injection of bevacizumab for the treatment of corneal NV-induced lipid keratopathy. We conducted a prospective interventional case series that included 20 eyes of chronic lipid keratopathy that received bevacizumab injection for the treatment of corneal NV and lipid deposition, including 7 eyes with recurrent corneal NV and lipid deposition after ceasing treatment. Repeated subconjunctival injections of bevacizumab were performed in 5 patients with recurrence. Penetrating keratoplasty was performed in 1 patient who had severely recurrent lipid deposition. Clinical presentation of corneal NV and lipid deposition, best-corrected visual acuity, and complications after treatments were recorded. Bevacizumab inhibited corneal NV and lipid depositions in 19 patients. Seven of the 20 patients (35%) had different patterns of recurrence of corneal NV/lipid deposition 6 to 15 months after discontinuing treatment. Five of the 7 patients in whom corneal NV/lipid deposition was recurrent received another course of repeated bevacizumab treatments. Three eyes had partial response to the second course of treatment. Two eyes had too short re-treatment course to have conclusions. One patient who received penetrating keratoplasty had successful result after surgery. Corneal NV and lipid deposition may recur after ceasing the subconjunctival bevacizumab injections for lipid keratopathy. Some patients respond at least partially to repeated injections.

99Tc-MDP inhibits corneal neovascularization induced by alkali burn in rats

⁹⁹Tc-MDP inhibits corneal neovascularization induced by alkali burn in rats

The Artemisinin Derivative Artesunate Inhibits Corneal Neovascularization by Inducing ROS-Dependent Apoptosis in Vascular Endothelial Cells

Without therapeutic intervention, corneal neovascularization rapidly compromises visual acuity, and is a leading cause of blindness. Artesunate was reported to inhibit angiogenesis in tumors, although, the effects of artesunate on nontumor angiogenesis have not been investigated. This study was designed to investigate the effect of artesunate on corneal neovascularization and delineate its underlying mechanism of action. Rats with alkali-burned corneas were treated with artesunate for 11 days. Corneal neovascularization was evaluated by measuring the length and area of corneal vasculature in the rats. Apoptotic cells were stained with AnnexinV and propidine iodide (PI), and measured with flow cytometry analysis. Apoptosis-related and p38 mitogen-activated protein kinases (p38MAPK) signaling were evaluated by Western blot analysis. Artesunate significantly inhibited corneal neovascularization and inflammation via specifically inducing apoptosis of vascular endothelial cells. In vascular endothelial cells, artesunate increased the Bax/Bcl-2 ratio, reduced mitochondrial membrane potential, stimulated release of cytochrome C, and cleavage of caspase 9 and 3, suggesting that the mitochondrial apoptotic pathway was involved. Artesunate activated p38MAPK, and specific p38MAPK inhibitors suppressed artesunate-induced apoptosis in endothelial cells. Reactive oxygen species (ROS) levels were increased by artesunate. N-acetyl-L-cysteine blocked p38MAPK activation and protected endothelial cells from artesunate-induced apoptosis. Ferrous salt increased ROS levels and elevated the cytotoxic effect of artesunate on endothelial cells, while the iron chelating agent deferoxamine decreased ROS levels and artesunate-induced apoptosis. Artesunate had no effect on expression of Fas, Fas Ligand, or caspase 8 cleavage. These results suggest that artesunate induces apoptosis of endothelial cells via an iron/ROS-dependent p38MAPK-mitochondrial pathway.

Inhibition of Corneal Neovascularization by Subconjunctival and Topical Bevacizumab and Sunitinib in a Rabbit Model

To compare the effects of subconjunctival injection and topical application of bevacizumab and sunitinib on experimentally induced corneal neovascularization (CNV). CNV was induced by sutures in the right eyes of 36 rabbits. After suture removal, the rabbits were divided into 6 groups with 6 rabbits in each group. In groups 1, 2, and 3, the eyes received a subconjunctival injection of 0.1 mL of normal saline, 2.5 mg/0.1 mL of bevacizumab, and 0.25 mg/0.1 mL of sunitinib, respectively, immediately after suture removal. A booster injection of the same agent was repeated 1 week later in each group. In groups 4, 5, and 6, the eyes received topical applications of saline, bevacizumab (5 mg/mL), and sunitinib (0.5 mg/mL), respectively. These solutions were applied twice a day for 2 weeks, starting immediately after suture removal. CNV was analyzed through biomicroscopy and through histological examination using hematoxylin and eosin and CD31 immunohistochemical staining. On day 14, the mean percentages of areas of CNV in sunitinib-treated eyes were smaller compared with saline-treated or bevacizumab-treated eyes in both the subconjunctival (P = 0.003 and 0.032, respectively) and topical groups (P < 0.001 in both). The topical administration of sunitinib was significantly more effective than the subconjunctival injection of the same drug at 1 week (P = 0.011). Upon histological examination of samples from the topical group, sunitinib-treated eyes showed lower vascularity than saline-treated and bevacizumab-treated eyes (P = 0.036 and 0.046, respectively). These results suggest that sunitinib is more effective than bevacizumab for the inhibition of CNV. Furthermore, topical administration of sunitinib yields better results than a subconjunctival injection of the same medication.

Comparison of subconjunctivally injected bevacizumab, ranibizumab, and pegaptanib for inhibition of corneal neovascularization in a rat model.

To compare the efficacies of subconjunctival bevacizumab, ranibizumab, and pegaptanib sodium injections for the inhibition of corneal neovascularization in an experimental rat model. Sixteen corneas of 16 rats were chemically cauterized and randomized into four groups: bevacizumab group that treated with 0.05mL/1.25mg bevacizumab, ranibizumab group that treated with 0.05mL/0.5mg ranibizumab, pegaptanib group that treated with 0.05mL/0.15mg pegaptanib sodium, and control group that treated with 0.05mL saline solution. Digital photographs of the corneas were taken and analyzed using an image analysis software program. All corneas were excised and examined histologically on the 15(th) day. Each treatment group had significantly less neovascularized corneal areas and fewer blood vessels than the control group (all P<0.05). In addition, bevacizumab group had significantly less neovascularized corneal areas and fewer blood vessels than ranibizumab and pegaptanib groups (both P<0.05). However, there was no significant difference between the ranibizumab and pegaptanib groups regarding percentage of neovascularized corneal areas and number of blood vessels (both P>0.05). Subconjunctival bevacizumab, ranibizumab, and pegaptanib sodium were effective with no corneal epitheliopathy for inhibiting corneal neovascularization after corneal burn in rats. Bevacizumab was more effective than ranibizumab and pegaptanib sodium.

The Effect of Topical Infliximab on Corneal Neovascularization in Rabbits

To determine the efficacy of topical application of infliximab [tumor necrosis factor-α (TNF-α) monoclonal antibody] for the treatment of corneal neovascularization in the rabbit model. In 12 rabbits (24 eyes), the corneal stroma was sutured to induce corneal neovascularization. One week after suture, corneal neovascularization was confirmed and the subjects were divided into 4 groups of 3 rabbits. Sterilized balanced salt solution was applied in the control group, and the experimental groups were treated with infliximab eye drops of varying concentrations (1, 2, and 4 mg/mL) twice daily for a week. The area of corneal neovascularization was measured and analyzed in all groups on day 3 and day 7. On day 7, all eyes were extracted to compare the TNF-α messenger RNA concentration by reverse transcriptase-polymerase chain reaction, and the vascular endothelial growth factor activity of corneal neovascular tissue was observed by fluorescence immunostaining. On day 7, all 3 experimental groups (1, 2, and 4 mg/mL) had a significantly reduced area of corneal neovascularization compared with the control group (P = 0.043, 0.027, and 0.01, respectively) and significantly reduced TNF-α messenger RNA (P = 0.038, 0.031, and 0.022, respectively). Fluorescence immunostaining confirmed the reduced expression of vascular endothelial growth factor in all 3 experimental groups compared with the control group. The application of infliximab is expected to effectively inhibit corneal neovascularization. Further clinical studies are necessary.

Korneal Neovaskularizasyonun Subkonjonktival Ranibizumab ve Bevacizumab Enjeksiyonu ile Tavşan Korneasında İnhibisyonu

Korneal Neovaskularizasyonun Subkonjonktival Ranibizumab ve Bevacizumab Enjeksiyonu ile Tavşan Korneasında İnhibisyonu

Inhibition of Multiple Pathogenic Pathways by Histone Deacetylase Inhibitor SAHA in a Corneal Alkali-Burn Injury Model

Neovascularization (NV) in the cornea is a major cause of vision impairment and corneal blindness. Hemangiogenesis and lymphangiogenesis induced by inflammation underlie the pathogenesis of corneal NV. The current mainstay treatment, corticosteroid, treats the inflammation associated with corneal NV, but is not satisfactory due to such side effects as cataract and the increase in intraocular pressure. It is imperative to develop a novel therapy that specifically targets the hemangiogenesis, lymphangiogenesis, and inflammation pathways underlying corneal NV. Histone deacetylase inhibitors (HDACi) have been in clinical trials for cancer and other diseases. In particular, HDACi suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The functional mechanism of SAHA in cancer and especially in corneal NV remains unclear. Here, we show that topical application of SAHA inhibits neovascularization in an alkali-burn corneal injury model. Mechanistically, SAHA inhibits corneal NV by repressing hemangiogenesis, inflammation pathways, and previously overlooked lymphangiogenesis. Topical SAHA is well tolerated on the ocular surface. In addition, the potency of SAHA in corneal NV appears to be comparable to the current steroid therapy. SAHA may possess promising therapeutic potential in alkali-burn corneal injury and other inflammatory neovascularization disorders.

Evaluation of the effects of resveratrol and bevacizumab on experimental corneal alkali burn

PurposeTo evaluate the effects of resveratrol and bevacizumab on experimental corneal neovascularization. MethodA corneal alkali burn was performed in 62 eyes of 31 male white Vienna rabbits. Resveratrol (group 1), dimethyl sulfoxide (group 2), bevacizumab (group 3) and 0.9% NaCl (group 4) were administered to both eyes of the rabbits by subconjunctival injection for 7 days. Corneal photos were taken at 15 days after alkali injury. Inflammatory index scores and neovascularization areas were calculated. ResultsIn bevacizumab group both inflammatory index scores and the calculation of the corneal neovascularization area was significantly less than the groups. ConclusionThe subconjunctival administration of bevacizumab inhibits corneal neovascularization effectively in the rabbit corneal alkali burn model. No effect of resveratrol to the corneal neovascularization on experimental model of the corneal alkali burn was seen at the doses of usage.

Comparison of the effects of bevacizumab and ranibizumab injection on corneal angiogenesis in an alkali burn induced model.

To investigate the effects of bevacizumab and ranibizumab on corneal neovascularization in an alkali burn-induced model of corneal angiogenesis. Fifteen Wistar albino rats were divided randomly into 3 groups after chemical cauterization of the cornea. The first group received a single dose of 0.1mL saline solution as a control group whereas second and third groups received a single dose of 2.5mg bevacizumab or 1mg ranibizumab by subconjunctival injection, respectively. After three weeks, the rat corneas were evaluated by biomicroscopy and corneal photographs were taken. The percentage of neovascularization area, length of the longest new vessel, corneal edema and corneal opacity scores were assessed. The analysis of digital photographs showed that the percentage of neovascularization area to the total corneal area, the length of the longest new vessel, corneal edema and opacity scores were significantly lower in both study groups compared to the control group (P<0.05). Additionally, the percentage of corneal neovascularization area, the length of the longest new vessel and corneal opacity score were less with bevacizumab than ranibizumab. Subconjunctival bevacizumab and ranibizumab treatments may be effective methods in reducing corneal neovascularization. Furthermore, bevacizumab is more effective than ranibizumab in the inhibition of corneal neovascularization.

Inhibition of Corneal Neovascularization in Rats by Systemic Administration of Sorafenib

To evaluate the effect of orally administered sorafenib on corneal neovascularization in rat models. In male Sprague-Dawley rats, a silver nitrate applicator was placed on the central cornea in both eyes to elicit angiogenesis. Rats were divided into 3 groups, the control group and the 2 sorafenib-treated groups (low dose, 30 mg · kg(-1) · day(-1); high dose, 60 mg · kg(-1) · day(-1)). The area of corneal neovascularization was measured by image analysis. Vascular endothelial growth factor receptor 2 (VEGFR2) messenger RNA expression was measured in rat corneas by reverse transcription-polymerase chain reaction, and the expression of phosphorylated extracellular signal-regulated kinase (ERK) was measured by Western blot analysis 1 week after cauterization. The area of corneal neovascularization was significantly reduced by 44% in the 30 mg · kg(-1) · day(-1) group and by 66% in the 60 mg · kg(-1) · day(-1) group, compared with the control group (P = 0.014 and P < 0.0001). Corneal VEGFR2 messenger RNA expression was higher in the control group than in the sorafenib-treated groups. The expression of phosphorylated ERK in rat corneas was suppressed in the sorafenib-treated groups but not in the control group. Oral administration of a multikinase inhibitor (sorafenib) significantly reduced the development of experimental corneal neovascularization in a dose-dependent manner. This inhibitory effect is probably related to the suppression of ERK phosphorylation by sorafenib.

Trehalose treatment accelerates the healing of UVB-irradiated corneas. Comparative immunohistochemical studies on corneal cryostat sections and corneal impression cytology.

The UVB-irradiated cornea is damaged by oxidative stress. Toxic oxygen products induced by UVB radiation in the cornea are insufficiently removed by antioxidants, whose numbers decrease with increasing UVB irradiation. In addition, the UVB-irradiated cornea suffers from hypoxic conditions because damaged corneal cells cannot utilize oxygen normally, although the supply of oxygen to the cornea is unchanged (normal). This contributes to attenuated re-epithelialization, corneal neovascularization and apoptotic cell death. Our previous publications reported that trehalose applied on the corneal surface during irradiation significantly suppressed UVB-induced corneal oxidative damage. The results of this study provide for the first time important evidence that trehalose applied on the surface of corneas for two weeks following repeated UVB irradiation (312 nm, daily dose 0.5 J/cm2) accelerated corneal healing, restored corneal transparency and suppressed corneal neovascularization. Compared to buffered saline treatment, following which caspase-3, nitrotyrosine, malondialdehyde and urokinase-type plasminogen activator were still strongly expressed in the corneal epithelium two weeks after irradiation and corneal neovascularization was evident, apoptotic cell death was already significantly reduced after one week of trehalose application. The expression of other markers of injury returned to normal levels during two weeks of trehalose treatment. In conclusion, our results show that trehalose accelerated healing of the UVB irradiated cornea, very probably via suppression of hypoxia-response injury. In addition, immunohistochemical results on corneal cryostat sections corresponded with those obtained using corneal impression cytologies, thus confirming that corneal impression cytologies are useful for diagnostic purposes.

Suppression of corneal neovascularization by culture supernatant of human amniotic epithelial cells in vitro

Background Corneal neovascularization (CNV) is a common eye disease.The researches on the pathogenesis and treatment of CNV are focus in Ophthalmology. Objective This study was to investigate the effects of culture supernatant from human amniotic epithelial cells (AECs) on CNV in vitro and its mechanism.Methods Human AECs were obtained from a placenta and cultured in serum-free medium for 48 hours,and the supernatant was collected.The levels of interleukin-1 receptor antagonist (IL-1 Ra) and pigment epithelium-derived factor (PEDF) in the human AECs culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).Rabbit corneal epithelial cells (CECs) were obtained and cultured in different concentrations of human AECs culture supernatant for 48 hours,serum-free medium was used as the control group.The expression of vascular endothelial growth factor (VEGF) mRNA and basic fibroblast growth factor (bFGF) mRNA in rabbit CECs were measured by quantitative real-time PCR (QRT-PCR).Human umbilical cord vein endothelial cells (UVECs) were cultured in the three mediums above,and the proliferation of human UVECs (absorbance value,A value) was tested by the cell counting kit 8 ( CCK8 ).Migration assay was performed by the wound healing method for the human UVECs.The membrane ultra-structure of human UVECs was examined under the atomic force microscope (AFM).Results Cultured and passaged human AECs showed a positive response for keratin.The expression of VEGF mRNA (1.00±0.22) and bFGF mRNA (1.00±0.36) in rabbit CECs was suppressed by the human AECs culture supernatant,with a significant reduction in comparison with the serum-free DMEM group (2.98±0.46,2.55±0.48 )(P=0.001,0.002).The A value was significantly lowered in the human AECs culture group for 72 hours compared with the serum-free DMEM group ( 1.941 ± 0.036 versus 2.144 ± 0.059 ) ( P =0.000 ),and the bFGF-induced migration rate of human UVECs was strongly inhibited by the culture supernatant of human AECs in comparison with serum-free DMEM.The plasma membrane of human UVECs cultured with the human AECs culture supernatant was full of bumps,and decreased intercellular connection and cellular pseudopodia were found on the AFM image.The concentration of IL-1Ra was (153.56±0.36)ng/L and that of PEDF was (70.41 ±0.68 )μ,g/L in the human AECs culture supernatant.Nothing was deteched in serum-free DMEM group. Conclusions Human AECs culture supernatant suppressed the expression of VEGF and bFGF in CECs as well as the migration and proliferation of vascular endothelial cells.The effect may be associated with IL-1Ra and PEDF secreted by human AECs.These results suggest that human AECs may be a potential therapy for the inhibition of CNV. Key words: Human amniotic epithelial cell; Corneal neovascularization; Vascular endothelial growth factor; Human umbilical cord vein endothelial cell; Basic fibroblast growth factor; Ultrastructure

Netrin-1 Simultaneously Suppresses Corneal Inflammation and Neovascularization

To investigate the effect of netrin-1 on alkali burn-induced corneal inflammation and neovascularization. The expression of netrin-1 and its receptors UNC5A, UNC5B, UNC5C, UNC5D, adenosine 2b receptor (A2BAR), deleted in colorectal cancer (DCC), and neogenin in normal and alkali-burned rat cornea were determined by RT-PCR and/or Western blot analysis, or immunostaining. Topical netrin-1 protein was applied to treat rat corneal alkali-burn injury for 14 consecutive days, started right after the injury or 10 days postinjury. Corneal inflammation and neovascularization were observed under slit lamp microscope. The apoptosis of corneal cells was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Corneal inflammatory cell infiltration was evaluated by immunostaining of anti-PMN and anti-ED1 antibodies. The expression of epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), and pigment epithelium-derived factor (PEDF) in rat cornea was determined by Western blot analysis. Netrin-1 and its receptor UNC5B were expressed in normal rat corneal epithelium and stromal cells, and their expression decreased after corneal alkali burn. Exogenous netrin-1 administered on rat ocular surfaces resolved alkali burn-induced corneal inflammation, and also suppressed corneal neovascularization. Furthermore, netrin-1 could reverse neovascularization in alkali-burned cornea. The authors found that netrin-1 executed the functions through various mechanisms, including upregulating EGF expression, accelerating epithelial wound healing, inhibiting neutrophil and macrophage infiltration, reducing corneal cell apoptosis, and restoring the equilibrium of VEGF and PEDF in the wounded cornea. Netrin-1 could dampen inflammation, inhibit, and reverse neovascularization in alkali-burned cornea.

PEDF-derived peptide inhibits corneal angiogenesis by suppressing VEGF expression

Pigment epithelium-derived factor (PEDF) a glycoprotein that belongs to the superfamily of serine protease inhibitors, has been recently shown to be the most potent inhibitor of angiogenesis in the mammalian eye. However, which active domain of PEDF protein could be involved in its anti-angiogenic properties remains unknown. Therefore, in this study, we examined which PEDF-derived synthetic peptides could inhibit corneal neovascularization induced by chemical cauterization in vivo. Rats treated with topical application of PEDF protein had 31% less corneal neovascularization at day 7 after the injury than phosphate-buffered saline (PBS)-treated rats. P5-2 and P5-3 peptides (residues 388–393 and 394–400 of PEDF protein, respectively) significantly suppressed the corneal neovascularization after chemical cauterization at day 7, and its anti-angiogenic potential was almost equal to that of full-length PEDF protein. Further, full-length PEDF protein and P5-3 peptide significantly decreased 8-hydroxy-2′-deoxyguanosine and vascular endothelial growth factor (VEGF) levels in the corneal. Our present study suggests that PEDF-derived synthetic peptide, P5-3 could inhibit the corneal neovascularization induced by chemical cauterization in rats by suppressing VEGF expression via its anti-oxidative properties.

Inhibitory effect of RGDRGD-endostatin on the expression of vascular endothelial growth factor and its receptor in corneal neovascularization

Background Endostatin (ES) is currently the strongest endogenous angiognesis inhibitor,and it can inhibit the occurrence of neovascularization.Various studies demonstrated that the poly RGD sequence can enhance the function of the ES gene. Objective This study was to evaluate the use of gene therapy of modified ES for alkaline burn-induced corneal neovascularization (CNV). Methods One hundred and two clean SD rats were randomly divided into the normal control group,the pCI empty vector group,the pCI-ES group,and the pCI-RGDRGDES group.Corneal neovascularization models were established by placing a piece of 3 mm filter paper with 1 mol/L NaOH at the central cornea for 40 seconds.3 μg of the pCI blank vector,ES-tranfected pCI blank vector,or RGDRGD-ES-transfected pCI vector was injected into the superior bulbar conjunctiva after the alkali burn twice at 1-week intervals.Area of CNV and edema of the cornea in the various groups of rats were examined daily under the slit lamp biomicroscope.1,4,7 and 14 days after operation,the rats were sacrificed by the excessive anesthesia method and corneal tissues were obtained to evaluate pathological changes.The expression of CD34 in vascular endothelial cells was detected by immunochemistry to calculate the corneal neovascular density.The expressions of VEGF mRNA and Flk-1 protein in the corneas were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis.The use and maintenance of animals followed the Statement of ARVO. Results Seven to fourteen days after corneal alkali-burning,the corneal neovascular area was smaller in the pCI-ES group and pCI-RGDRGD-ES group compared with the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and nevascular area in the pCI-RGDRGD-ES group was smaller than that in the pCI-ES group (P＜0.05).The expression level of CD34 was significantly lower in the pCI-ES group and pCI-RGDRGD-ES group than that in the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and the expression level of CD34 was further declined in the pCI-RGDRGD-ES group compared with the pCI-ES group (P＜0.05 ).Compared with the normal control group and pCI vector group,the expressions of the Flk-1 protein and VEGF mRNA were decreased in the pCI-ES group and pCI-RGDRGD-ES group on the fourth day after corneal alkali-burning (P＜0.01,P＜0.05 ),and those in the pCI-RGDRGD-ES group were less than the pCI-ES group (P＜ 0.05,P＜ 0.05 ). Conclusions Subconjunctival injection of both ES and modified RGDRGD-ES genes result in significant suppression of CNV in vivo,and modified RGDRGD-ES appears to be more effective than native ES.The main mechanism of ES in inhibiting neovascularization is to downregulate the expression of VEGF and Flk-1. Key words: Endostatin; Cornea/neovascularization; Gene therapy; Vascular endothelial growth factor receptor 2

Inhibition of corneal neovascularization by Avastin in rabbits

Objective To evaluate the effect of Aavastin to inhibit experimental corneal neovascularization. Methods Twenty rabbits were sutured on the superior cornea. Animals were randomly divided into 2 groups. After 7 days, 10 eyes in the treatment group were given Aavastin drops(4 rog/mi) twice daily, whereas 10 eyes in the control group received placebo (normal saline drops twice daily). The occurrence and developement of CNV was observed on the 3, 7, 14, 21,28 days after suturing by slit-lamp microscope, and the area of CNV was calculated. Fluorescein staining was detected for the integrity of corneal epithelium. Results The occurrence of CNV in group 1 was (3.43±0.53)d and group 2 (3.14±0.69)d. No significant difference in the occurrence time of CNV was found between group 1 and group 2. The CNV areas of group 1 were significantly smaller than that of group 2 at every time poin(P<0.05). The diameter of CNV was significantly smaller after Avastin treatment. Conclusion Avastin can inhibit and restrain corneal neovascularization at some degree without significant side effects. Key words: Avastin; corneal neovascularization; rabbit

Topical dihydroartemisinin inhibits suture-induced neovascularization in rat corneas through ERK1/2 and p38 pathways.

To determine if topical instillation of dihydroartemisinin (DHA) inhibits corneal neovascularization (NV) in rats and to investigate the role of the extracellular regulated kinases (ERK) 1/2 and p38 pathways in this process. Suture-induced corneal NV was produced in rats and the eyes were topically treated with different concentrations of DHA (20mg/L, 10mg/L or 5mg/L) or normal saline 4 times a day for 7 days. The corneal NV was quantified as the proportion of NV area to the whole cornea. Western blot was used to determine the expressions of vascular endothelial growth factor (VEGF) and the phosphorylation status of VEGF receptor-2, ERK1/2 and p38 in the corneas. Immunofluorescent staining was used to determine the expressions of phospho-ERK1/2 and phospho-p38 in the corneal tissues from the eyes treated with 20 mg/L DHA (DHA group) or normal saline (control group). The proportion of corneal NV area in the eyes treated with normal saline or DHA at dosages of 20mg/L, 10mg/L or 5mg/L was (23.74±3.00)%, (15.73±2.88)%, (19.53±2.42)%, and (23.38±2.79)%, respectively. In the eyes treated with 20mg/L or 10mg/L DHA, the corneal NV area was significantly reduced when compared to that in eyes with normal saline (P<0.05). Western blot analyses revealed that 20mg/L DHA significantly inhibited the expressions of VEGF and phospho-VEGFR-2. Both 20mg/L and 10mg/L DHA inhibited the expressions of phospho-ERK1/2 and phospho-p38. Immunofluorescent staining further demonstrated that 20mg/L DHA lowered the expression levels of phospho-ERK1/2 and phospho-p38 in the corneas with suture-induced NV. Suture-induced NV in rat corneas was significantly inhibited by topical treatment with 20mg/L and 10mg/L DHA. The results suggest that the effects could be partially dependent on the DHA-mediated inhibitions of the ERK1/2 and p38 pathways.

Effects of amniotic membrane transplantation on cytokines expression in chemically burned rat corneas.

To investigate the effect of amniotic membrane transplantation (AMT) on the expressions of inflammatory-related, angiogenic-related and growth-related cytokines in rat corneas after chemical injury. Alkali wounds were inflicted on the central corneas of rats by applying a round filter paper soaked in 1mol/L NaOH for 40 seconds. One week after alkali burn, 12 rats were randomly divided into 2 groups: the AMT group and the control group, and AMT was performed on the rats in the AMT group. Corneal opacity and neovascularization were observed by slit-lamp microscopy. The protein levels of interleukin (IL)-2, interferon (IFN)-γ, IL-10 and transforming growth factor (TGF)-β were determined by enzyme-linked immunosorbent assay 2 weeks after AMT. The mRNA levels of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were evaluated by real-time quantitative PCR. In the AMT group, the corneal opacity was improved (P=0.011) and the area of corneal neovascularization was significantly decreased (P=0.005) compared with the control group. The amount of IL-2 and IFN-γ secreted by Th1 cells were decreased after AMT, whereas the amount of IL-10 and TGF-β secreted by Th2 cells were increased (P<0.05). The level of MMP-2 was significantly down-regulated (P=0.013) at the mRNA level in the AMT group, while the expression of EGF was significantly higher (P=0.022) compared with controls. [corrected] AMT may suppress corneal neovascularization after chemical injury by modulating the expressions of soluble factors.