Area Of Corneal Neovascularization Research Articles

DCZ3301, an aryl-guanidino agent, inhibits ocular neovascularization via PI3K/AKT and ERK1/2 signaling pathways

Neovascularization is a critical process in the pathophysiology of neovascular eye diseases. Although anti-VEGF therapy has achieved remarkable curative effects, complications, limited efficacy and drug resistance remain the prominent problems. DCZ3301, an aryl-guanidino compound, was reported to have anti-tumor activity in the previous studies. Here, we demonstrated the effects of DCZ3301 on human umbilical vein endothelial cell (HUVEC) in vitro, and performed choroid microvascular sprouting assay ex vivo and alkali-burn induced corneal neovascularization mouse model in vivo. We found that DCZ3301 inhibited the proliferation, migration, and tube formation of HUVECs, while inducing the spontaneous apoptosis of HUVECs by suppressing the activation of PI3K/AKT and ERK1/2 pathways. Furthermore, DCZ3301 inhibited the choroid microvascular sprouting, diminished the area of corneal neovascularization and attenuated the edema of corneal stroma after alkali burn. Together, these results suggested that DCZ3301 exerted anti-angiogenic properties, and might be regarded as a potential candidate for ocular neovascularization.

Novel Parameters to Assess the Severity of Corneal Neovascularization Using Anterior Segment Optical Coherence Tomography Angiography

Assessment of anterior segment-optical coherence tomography angiography (AS-OCTA) to determine severity of corneal neovascularization (CoNV). Retrospective, cross-sectional, single-center study. Patients of various CoNV etiologies were selected and classified into mild, moderate, and severe. Their AS-OCTA images were measured for CoNV anterior limit, CoNV posterior limit, CoNV thickness, CoNV depth%, CoNV vessel density, CoNV area, and CoNV volume. Further, AS-OCTA parameters were correlated to clinical parameters, such as classification, a numerical severity scale, vascular clock hours, and best-corrected visual acuity (BCVA). A total of 19 mild, 10 moderate, and 6 severe CoNV eyes were included with no significant age-gender differences. CoNV depth% and volume increased from mild to moderate (9.3 ± 1.1% to 17.7 ± 3.3%, P= .030, and 0.2 ± 0.1mm3 to 1.0 ± 0.3mm3, P= .025, respectively) and from moderate to severe CoNV (44.6 ± 5.3%, P < .001, and 2.0 ± 0.3mm3, P= .014, respectively). CoNV area and posterior limit increased from mild to moderate (1.7 ± 0.3mm2 to 4.6 ± 0.7mm2, P= .001, and 217.7 ± 16.8μm to 349.1 ± 54.9μm, P= .048, respectively), not from moderate to severe (P= .999 and P= .403, respectively). CoNV thickness increased from moderate to severe (218.2 ± 46.6μm to 340.2 ± 8.7μm, P= .020), but not from mild to moderate. CoNV area and volume showed good correlations to CoNV staging (r= 0.703 and r= 0.771, respectively; P < .001) and severity scale (r= 0.794 and r= 0.712, respectively; P < .001). CoNV area showed good correlation to clock hours (r= 0.749, P < .001). CoNV depth and volume showed good correlation to BCVA (r= 0.744 and r= 0.722, respectively; P < .001). CoNV anterior limit and vessel density showed no significant correlations (P ≥ .05). Severe CoNV shows greater CoNV posterior limit, thickness, depth%, area, and volume on AS-OCTA compared to mild. CoNV volume and depth strongly correlate to BCVA. AS-OCTA provides novel, quantitative, and noninvasive parameters for assessing CoNV severity.

Bilateral third eyelid ectopic cilia in an adult horse

A 15-year-old Cob mare presented with a 4-month history of chronic epiphora and intermittent blepharospasm in the right eye. On ophthalmic examination, two translucent aberrant hairs were identified at the third eyelid margin corresponding to an area of corneal fibrosis and neovascularization. Partial excision of the third eyelid was performed, and histopathology confirmed ectopic hair follicles. Two weeks later, clinical signs recurred in the same eye. Examination revealed another pair of aberrant hairs on the bulbar surface of the third eyelid near its leading edge. This portion of the third eyelid was also excised, and histopathology confirmed two additional ectopic hair follicles. Eight months later, the horse developed similar clinical signs in the left eye. Ophthalmic examination showed a single aberrant translucent hair at the third eyelid margin associated with focal fibrosis and neovascularization of the ventromedial cornea. Partial excision of the third eyelid was performed, and histopathology confirmed an ectopic hair follicle within the third eyelid conjunctiva. Excision was curative at 4years postoperatively with no further recurrence in either eye.

Germinal peptide eye drops promote corneal wound healing and decrease inflammation after alkali injury

Germinal peptide is being developed to treat corneal injuries. The purpose of this study was to investigate its effect on corneal epithelial cells in vitro and its ability to promote healing in an alkali injury model in vivo. Cultured rabbit corneal epithelial cells were treated with germinal peptide at three concentrations. Cell proliferation and migration were assessed and compared with the effect of recombinant human epidermal growth factor (rh-EGF). In vivo, the corneas of New Zealand albino rabbits were chemically burned with 1 mol/l NaOH for 30 s. The injured eyes were topically treated with germinal peptide (10, 20, and 40 μg/ml), rh-EGF, or phosphate-buffered saline thrice daily. At fixed time points post injury, the healing of the cornea and its histopathology were evaluated. There was no difference in the effect of germinal peptide on cultured cell proliferation. However, cell migration was significantly higher than that in the control groups, with germinal peptide at concentrations of 20 and 40 μg/ml being the most efficacious. In vivo, 20 and 40 μg/ml germinal peptide significantly alleviated corneal opacity and edema. By day 21, the areas of corneal neovascularization in the germinal peptide-treated groups were smaller than those in the rh-EGF and control groups. The repaired corneas in the germinal peptide- and rh-EGF-treated groups also had more corneal epithelial layers and fewer inflammatory cells than the controls. Germinal peptide may be developed as a novel topical treatment agent for corneal wound healing in clinical settings.

Protective Effect of Vitamin C against Infancy Rat Corneal Injury Caused by Acute UVB Irradiation.

Purpose Studies have shown that corneas of young children were more susceptible to Ultraviolet B (UVB) radiation damage. However, there exist limited information about the harm of UVB to eyes and preventive measures on infancy. Vitamin C as an antioxidant is widely used to prevent many diseases. Therefore, the aim of this study was to explore the protective effect of vitamin C on the cornea of infant rats with acute UVB injury. Method Thirty-six infant rats were randomly divided into three groups: control (CON) group, UVB (UVB) group, and UVB+vitamin C (UVB+VitC) group. The UVB group was exposed to UVB irradiation (8 J/cm2, 15 min/d, 7 d) and the UVB+vitamin C group suffered the same UVB irradiation treated with vitamin C at the dose of 40 mg/kg via intraperitoneal injection. Then, corneal morphology was detected in vivo and in vitro at 7 d post-UVB exposure. Furthermore, serum inflammatory factors (IL-1, IL-6, and TNF-α) and oxidative status (4-HNE and MDA) were detected by ELISA, and the expression of vascular endothelial growth factor-α (VEGF-α) and superoxide dismutase (SOD) in the cornea was detected by western blot or immunofluorescent staining. Results Slit lamp detection revealed that the area of corneal desquamation and corneal neovascularization in the UVB+VitC group was significantly less than those in the UVB group at 7 d post-UVB exposure (all p < 0.05). OCT results showed that the thickness of the central cornea in the UVB+VitC group was decreased than that in the UVB group (p < 0.05). The serum inflammatory factors (IL-1, IL-6, and TNF-α) and oxidative status (4-HNE and MDA) in the UVB group were significantly increased compared with the CON group (all p < 0.05), while those factors in the UVB+VitC group were decreased compared with those in the UVB group. Furthermore, the expression of VEGF-α in the UVB+VitC group was dramatically decreased compared with that in the UVB group (p < 0.05), and the expression of SOD2 in the UVB+VitC group was dramatically increased compared with that in the UVB group at 7 d post-UVB exposure (p < 0.05). Conclusion Vitamin C could protect infant rats from corneal injury induced by UVB via alleviating corneal edema, improving corneal inflammatory reaction, and decreasing VEGF-α expression.

Comparison of Topical Pigment Epithelium-Derived Factor (PEDF) with Topical Bevacizumab for Accelerating the Regression of Corneal Neovascularization in an Experimental Model of Rabbit Corneal Angiogenesis

ABSTRACT Purpose: To evaluate the anti-angiogenic effect of topical administration of Pigment epithelium-derived factor (PEDF) on the reduction of corneal neovascularization (NV) in comparison to topical Bevacizumab. Methods: 18 eyes of 18 New Zealand rabbits were enrolled. Corneal NV was induced by a 7–0 silk suture. After suture removal, rabbits were randomly divided into three groups. In every group, one eye randomly treated with topical bevacizumab or topical PEDF or saline for 14 days. The area and length of neovascularization were measured by Image J. Histological studies were done in three groups. Results: After 14 days, the mean decrease of corneal NV length was 1.84 ± 0.17 mm (P < .001) in PEDF group and 1.6 ± 0.07 mm (P < .001) in bevacizumab group which was significantly more than the saline group (P = .001 and P < .001, respectively). There was no significant difference between PEDF and bevacizumab group in the reduction of corneal NV length (P = .85). The mean decrease of corneal NV area was 4.94 ± 0.55 mm2 (P < .001) in PEDF group and 4.23 ± 0.29 mm2 in the bevacizumab group (P < .001). PEDF and bevacizumab significantly decreased corneal NV area in comparison to the saline group (p = .017, p = .001, respectively). The mean decrease of corneal NV area did not show a significant difference between PEDF and bevacizumab groups (P = .72). Conclusion: Topical PEDF might be an effective and safe treatment option as bevacizumab in a short-term use, indicating that it is as good as the standard. However, long-term effect is required to be investigated.

Comparison of the effect of topical bevacizumab and sorafenib in experimental corneal neovascularization

Purpose The purpose of this study was to compare the neovascularization inhibiting the effect of topical bevacizumab and sorafenib and to determine the effective dose of sorafenib. Material and Methods Forty-two healthy Wistar albino rats were randomly divided into six groups. The right corneas of all rats except group 1 were cauterised with silver nitrate. Group 2 received DMSO, group 3 received topical bevacizumab (5 mg/dL, 3 times a day) and group 4, 5 and 6 received topical sorafenib (2.5 mg/dl, 5 mg/dL, 7.5 mg/dL, 2 times a day respectively), between days 1 and 7. Corneal photographs were taken on day 8 and the corneal neovascular area percentage was calculated. Following decapitation, the corneas were removed to determine the levels of VEGF ELİSA and corneal immune staining. The Mann–Whitney U-test was used for statistical analysis Results The neovascular corneal area percentage was statistically significantly lower in the treatment groups than group 2 (p < 0.05). The intensity of VEGF immune staining was also lower in groups 3, 5 and 6 from the group 2. Group 3, 5 and 6 were no significant differences compared to group 1. The VEGF ELİSA levels were statistically significantly lower in group 3, 5 and 6 compared to group 2 (p < 0.05). There was no statistically difference between VEGF ELİSA levels of group 2 and 4 (p > 0.05) Conclusions Sorafenib was as effective as bevacizumab in the regression of corneal neovascularization. The effect of sorafenib seems to be dose-dependent. The low doses and twice a day administration are important advantages of sorafenib.

Keratocytes promote corneal neovascularization through VEGFr3 induced by PPARα-inhibition

As the peroxisome proliferator – activated receptor alpha (PPARα) agonist, fenofibrate has been widely used to be a good lipid-regulating drug in the clinical application. In this study, we investigated the mechanism by which keratocytes inhibit the corneal neovascularization (CNV) through PPARα - activation. To do this, the CNV model was established by alkali burn, followed by being divided into three groups including control, fenofibrate and vehicle group. The expression of VEGFr3, MMP13 and PPARα in corneas of normal mouse and alkali-burned mouse was determined via quantitative RT- PCR (qRT-PCR) and Western blot analysis (WB). The CNV area was observed under a slit lamp microscope. The location of PPARα expression in the corneas was determined via immunohistochemistry. In cultured primary keratocytes, the effect of fenofibrate on PPARα, VEGFr3 and MMP13 expression was determined by qRT-PCR and WB. Besides, PPARα knockout (PPARα−/−) mouse CNV and keratocytes model were established to further confirm the effect of PPARα on VEGFr3 and MMP13 expression. We found that PPARα was expressed in epithelium, stroma and endothelium of the normal cornea, however, with relatively low level in the corneal stroma. Meanwhile, its expression was decreased markedly in the cornea during the stage of CNV formation. After treatment of fenofibrate, PPARα expression was promoted and the expression of VEGFr3 and MMP13 was inhibited in both CNV mice model and primary keratocytes, and CNV areas were decreased in CNV mice model. However, the results in PPARα−/− CNV and keratocytes model were opposite. Our results suggest that keratocytes could promote the expression of VEGFr3 and MMP13, and CNV formation through PPARα downregulation.

In vivo investigation of the effectiveness of reactive oxygen species-responsive nanomedicine in suppressing corneal neovascularization

Objective To evaluate the effectiveness of reactive oxygen species (ROS)-responsive nanomedicine in suppressing corneal neovascularization (CNV) in vivo. Methods ROS-responsive nanomedicine (ROS-TK-5/siVEGF), which consists of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-luc-KI transgenic mice were used in this study, of which 30 mice were randomly divided into a normal control group, a PBS control group, an ROS-TK-5/NC group, an ROS-TK-5/siVEGF group, and a ranibizumab group, with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium (DHE) staining in the other 9 mice.In each group, alkali-burned mice were subconjunctivally injected with 10 μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system (IVIS) at 7, 14, and 21 days after alkali burning. The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO) and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital (No.2018-X14-82). Results After treathrent with an aqueous solution without ROS, only 5%-10% of the siVEGF was released from the nanoparticles within 10 hours.In contrast, about 70% of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255, respectively, which was significantly greater than those in the normal control group (1.003±0.015) (both at P<0.05). The CNV areas were statistically different among the four groups at various time points (Fgroup=49.855, P<0.01; Ftime=65.556, P<0.01). The CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared with the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling, and the CNV area was more effectively reduced in the ROS-TK-5/siVEGF group than the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At day 21 after modeling, the CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05). IVIS showed that the corneal fluorescent intensity was statistically different among the four groups at various times (Fgroup=27.193, P=0.003; Ftime=51.062, P<0.01). The corneal fluorescent intensities were significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling; in addition, the corneal fluorescent intensity was more effectively reduced in the ROS-TK-5/siVEGF group in comparison with the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At 21 days after modeling, the corneal fluorescent intensity was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05). Conclusions ROS-TK-5/siVEGF nanomedicine effectively attenuates alkali burn-induced CNV formation and appears to have a better effect in comparison with ranibizumab at an early stage. Key words: In vivo imaging system; Alkali burn; Corneal neovascularization; Reactive oxygen species; Nanomedicine

Sex differences in corneal neovascularization in response to superficial corneal cautery in the rat.

Sex-based differences in susceptibility have been reported for a number of neovascular ocular diseases. We quantified corneal neovascularization, induced by superficial silver nitrate cautery, in male and female inbred albino Sprague-Dawley, inbred albino Fischer 344, outbred pigmented Hooded Wistar and inbred pigmented Dark Agouti rats of a range of ages. Corneal neovascular area was quantified on haematoxylin-stained corneal flatmounts by image analysis. Pro-and anti-angiogenic gene expression was measured early in the neovascular response by quantitative real-time polymerase chain reaction. Androgen and estrogen receptor expression was assessed by immunohistochemistry. Male rats from all strains, with or without ocular pigmentation, exhibited significantly greater corneal neovascular area than females: Sprague-Dawley males 43±12% (n = 8), females 25±5% (n = 12), p = 0.001; Fischer 344 males 38±10% (n = 12) females 27±8% (n = 8) p = 0.043; Hooded Wistar males 32±6% (n = 8) females 22±5% (n = 12) p = 0.002; Dark Agouti males 37±11% (n = 9) females 26±7% (n = 9) p = 0.015. Corneal vascular endothelial cells expressed neither androgen nor estrogen receptor. The expression in cornea post-cautery of Cox-2, Vegf-a and Vegf-r2 was significantly higher in males compared with females and Vegf-r1 was significantly lower in the cornea of males compared to females, p<0.001 for each comparison. These data suggest that male corneas are primed for angiogenesis through a signalling nexus involving Cox-2, Vegf-a, and Vegf receptors 1 and 2. Our findings re-enforce that pre-clinical animal models of human diseases should account for sex-based differences in their design and highlight the need for well characterized and reproducible pre-clinical studies that include both male and female animals.

Therapeutic effects of three human-derived materials in a mouse corneal alkali burn model*

Purpose: To compare the therapeutic effects of human derivatives in a mouse alkali burn model.Methods: The right eyes of mice were injured using NaOH. After alkali injury, one of the following agents was topically administered for 7 d: human amniotic membrane (hAM) suspension, human umbilical cord serum (hUCS), and human peripheral blood serum (hPBS), or saline. The epithelial defect areas on days 1, 2, and 3 degrees of opacity on days 2, 3, and 7, and corneal neovascularization (NV) areas on day 7 were evaluated. Histologic examination and mRNA expression levels of tumour necrosis factor (TNF)-α, interleukin (IL)-6, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-8, and MMP-9 were also evaluated on day 7.Results: The epithelial defect areas in the hUCS group were smaller than those in the control and hPBS groups on day 3 (p < .05, respectively). The epithelial defect areas in the hAM suspension group showed smaller than those in the control and hPBS groups on days 1 and 2 (p < .05, respectively). The degrees of opacity were lower in all treatment groups than that of the saline control group on day 7 (p < .05, respectively). Corneal NV areas were not different among groups on day 7 (p = 0.20). The expression levels of TNF-α, IL-6, MMP-8, and MMP-9 mRNA and the infiltration of the inflammatory cells in all treatment groups were lesser than those in the control group on day 7 (p< .05, respectively).Conclusions: All treatments reduced inflammatory reactions and corneal opacity development. Corneal reepithelialization was faster in the hUCS group.

Comparison of the Effects of Dovitinib and Bevacizumab on Reducing Neovascularization in an Experimental Rat Corneal Neovascularization Model

To compare the inhibitory effects of dovitinib and bevacizumab for treatment of corneal neovascularization (CNV). Thirty-nine adult female Sprague Dawley rats weighing 180 to 250 g were used. CNV was induced by silver nitrate in the right eye of each rat. After the chemical burn, the animals were randomized into 5 groups. Group 1 did not receive any chemical substance. Group 2 received dimethyl sulfoxide, group 3 received bevacizumab 5 mg/mL, group 4 received dovitinib 5 mg/mL, and group 5 received bevacizumab 5 mg/mL + dovitinib 5 mg/mL topically administered twice daily for 14 days. On the 14th day, slit-lamp examination was performed, and anterior segment photographs were taken. The corneal neovascular area was measured on photographs as the percentage of the cornea's total area using computer imaging analysis. The corneal sections were stained with hematoxylin and eosin for histopathological examination. A statistically significant decrease in the percentage of CNV was found in all treatment groups (group 3, group 4, and group 5) compared with the control group (group 1) (P < 0.01). A statistically significant difference in the percentage of CNV was found among group 3, group 4, and group 5 (P = 0.003). The percentage of CNV in group 4 was significantly higher than that in group 3 and group 5 (P1 = 0.004; P2 = 0.006). There was no statistically significant difference in the percentage of CNV between group 3 and group 5 (P = 0.228). Dovitinib is a newly developed multitargeted tyrosine kinase inhibitor. Topical administration of dovitinib effectively inhibited CNV, but this effect of dovitinib was found less than topical bevacizumab.

Cucurbita argyrosperma Seed Extracts Attenuate Angiogenesis in a Corneal Chemical Burn Model.

Severe corneal inflammation produces opacity or even perforation, scarring, and angiogenesis, resulting in blindness. In this study, we used the cornea to examine the effect of new anti-angiogenic chemopreventive agents. We researched the anti-angiogenic effect of two extracts, methanol (Met) and hexane (Hex), from the seed of Cucurbita argyrosperma, on inflamed corneas. The corneas of Wistar rats were alkali-injured and treated intragastrically for seven successive days. We evaluated: opacity score, corneal neovascularization (CNV) area, re-epithelialization percentage, and histological changes. Also, we assessed the inflammatory (cyclooxigenase-2, nuclear factor-kappaB, and interleukin-1β) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2, Il-1β, and Vegf-a mRNA were also determined. After treatment, we observed a reduction in corneal edema, with lower opacity scores and cell infiltration compared to untreated rats. Treatment also accelerated wound healing and decreased the CNV area. The staining of inflammatory and angiogenic factors was significantly decreased and related to a down-expression of Cox-2, Il-1β, and Vegf. These results suggest that intake of C. argyrosperma seed has the potential to attenuate the angiogenesis secondary to inflammation in corneal chemical damage.

Inhibitory effect of bromfenac sodium hydrate ophthalmic solution on alkali burn induced-corneal neovascularization in rats

Objective To investigate the inhibitory effect of bromfenac sodium hydrate ophthalmic solution on corneal neovascularization (CNV) induced by alkali burn. Methods A total of 192 specific pathogen free (SPF) degree adult male Sprague-Dawley (SD) rats were used in this study.One hundred and seventy-two rats were chosen to establish CNV model with alkali burn in the right eyes.Following alkali burn, rats were randomly divided into CNV group, model control group, bromfenac sodium group and fluorometholone group, with 43 rats (43 eyes) in each group.Another 20 rats (40 eyes) served as normal control group.One day after modeling, the model control group, bromfenac sodium group and fluorometholone group received phosphate buffer saline (PBS), bromfenac sodium hydrate ophthalmic solution and 0.1% fluorometholone eye drops, respectively.The state of cornea and anterior chamber and the growth of CNV of rats in each group were observed by slit-lamp microscope every day after modeling.At 1, 3, 7, 14, 21 and 28 days after modeling, the anterior segment photos of the experimental eyes were captured, and the percent of cornea areas covered by CNV was calculated.At 7, 14 and 28 days after modeling, the eye tissue sections were stained with hematoxylin and eosin staining and immunohistochemistry staining to evaluate the expressions of CD45 and VEGF-A.Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of COX-2 and VEGF mRNA and protein level.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO). Results Each model group showed corneal edema and opacification 1 day after modeling.The corneal edema was aggravated 7 days after modeling.On the 14th day after modeling, the degree of corneal opacity and edema decreased gradually.On the 28th day after modeling, leucoma was observed in CNV group and model control group, and nebula was observed in bromfenac sodium group and fluorometholone group.At 7, 14, 21 and 28 days after modeling, the percentages of CNV areas in bromfenac sodium group and fluorometholone group were significantly lower than those in CNV group and model control group (all at P 0.05). On the 7th day after modeling, the thinning of corneal epithelial layer, edema and arrangement disorder of stroma layer were observed, and the expression of VEGF-A was positive in all model groups; a small amount of CD45 positive inflammatory cell infiltrations were observed in CNV group and model control group.On the 14th and 28th day after modeling, CNV was seen in the center of cornea in CNV group and model control group; the epithelial keratosis and reduction of corneal edema were seen in each group, and no inflammatory cell infiltration was observed in each group.On the 7th day after modeling, the expressions of COX-2 and VEGF mRNA in CNV group and model control group were significantly higher than those in normal control group, bromfenac sodium group and fluorometholone group (all at P<0.05), the expressions of COX-2 and VEGF protein in bromfenac sodium group were significantly lower than those in CNV group (all at P<0.05). The corneal perforation rate in model control group and bromfenac sodium group was 10% (1 case in 10 rats). The corneal perforation rate in fluorometholone group was 30% (3 cases in 10 rats). In each model group, 10% to 30% rats had hyphema. Conclusions Bromfenac sodium hydrate ophthalmic solution can inhibit the formation and growth of CNV after alkali burn in rats.This effect may be mediated by regulating COX-2 expression, reducing inflammation and inhibiting VEGF production. Key words: Corneal neovascularization; Alkali burn; Bromfenac sodium; Cyclooxygenase-2

Efficacy of Sunitinib, Sunitinib-Hesperetin, and Sunitinib-Doxycycline Combinations on Experimentally-Induced Corneal Neovascularization

ABSTRACTPurpose: To investigate the preventive effects of topical sunitinib, sunitinib-hesperetin and sunitinib-doxycycline combinations on corneal neovascularization (CNV), apoptosis and fibrosis in a corneal alkali burn model.Materials and Methods: The corneas of 32 Wistar albino rats were cauterized with silver nitrate to induce CNV. Four groups were created receiving artificial tears (sham), sunitinib (0.5 mg/ml), sunitinib-hesperetin (0.5 mg/ml-0.2 mg/ml), and sunitinib-doxycycline (0.5 mg/ml-20 mg/ml) treatments. Corneal photographs were taken on days 0, 7 and 15‎. Photographs of the cornea were digitally analyzed to measure the size of the neovascularization area in comparison to the total corneal surface area. On the 15th day, the animals were euthanized, and the eyes were enucleated for immunohistochemical staining to investigate neovascularization, apoptosis, and fibrosis.Results: CNV areas on the 7th day in the sunitinib (4.8% ± 0.07%) and sunitinib-hesperetin (1.1% ± 0.03%) groups were smaller than those in the sham group (33.9% ± 0.12%) (p = 0.001 and, p < 0.001 respectively). On the 15th day, the CNV area in the sunitinib-hesperetin (20.8% ± 0.37%) group was significantly smaller than that of the sham group (74.6% ± 0.32%) (p = 0.039). The combination groups had lower levels of VEGF, TUNEL and α-SMA positivity than the sunitinib monotherapy group. TUNEL positivity was lowest in the sunitinib-hesperetin and sunitinib-doxycycline groups, and α-SMA positivity was lowest in the sunitinib-hesperetin group.Conclusion: Topical sunitinib-hesperetin was more effective than sunitinib alone and the sunitinib-doxycycline combination in the treatment of CNV. The combination of sunitinib and hesperetin seems to be a promising treatment for preventing corneal fibrosis and apoptosis.

Inhibited corneal neovascularization in rabbits following corneal alkali burn by double-target interference for VEGF and HIF-1α.

Expression of hypoxia-inducible factor (HIF) 1α has been observed in corneal neovascularization (CNV). Vascular endothelial growth factor (VEGF), one of the most well-known angiogenic factors in CNV, is under the regulation of HIF-1. The present study aims to investigate the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV in rabbits. The models of rabbits in corneal alkali burn were established. SiRNA recombinant adenovirus was used to explore the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV. CNV area and ultrastructure of cornea were observed. The expression of VEGF and HIF-1α was detected. CNV was observed in rabbits following alkali burn. In addition, overexpressed VEGF and HIF-1α was also observed in rabbits following alkali burn. Then, silencing HIF-1α or silencing VEGF decreased area of CNV, inhibited neovascularization and improved pathological changes, while double-target interference for VEGF and HIF-1α decreased area of CNV inhibited neovascularization, and improved pathological changes to a greater extent. Our study provides evidences emphasizing the distinct notion that VEGF and HIF-1α play the contributory role in alkali burn-induced CNV as a result of double-target interference for VEGF and HIF-1α inhibiting CNV in rabbits following corneal alkali burn.

Effect of ERK inhibitor on corneal neovascularization induced by alkali burn in mice and its mechanism

The objective of this study is to explore the effect of extracellular signal–regulated kinase (ERK) inhibitors on corneal neovascularization induced by alkali burn in mice and its mechanism. A total of 30 standard diet (SD) healthy mice were divided into normal group, alkali burn group, and inhibitor group. Normal group was not treated. Alkali burn group and inhibitor group were used to establish corneal neovascularization model induced by alkali burn. After successful modeling, ERK inhibitor was used to intervene in inhibitor group, and saline of equal volume was used in normal group and alkali burn group. The area of corneal neovascularization was calculated and the expression of vascular endothelial growth factor (VEGF), c-Fos, c-Jun, ERK1/2, and p-ERK1/2 protein in cornea tissue of three groups of mice was detected. The relative expression of vascular area, length, VEGF, c-Fos, c-Jun, ERK1/2, and p-ERK1/2 protein in cornea tissue of mice in alkali burn group was significantly higher than that in normal group and inhibitor group. The relative expression of vascular area, length, VEGF, c-Fos, c-Jun, ERK1/2, and p-ERK1/2 protein in cornea tissue of mice in inhibitor group was higher than that in normal group, and the expression level of PEDF was lower than that in normal group ( P &lt; 0.05). ERK inhibitors inhibit the formation of corneal neovascularization by inhibiting the expression of VEGF, c-Fos, and c-Jun proteins through the action of ERK signaling pathway.

Effect of Collagen Cross-Linking on Alkali Burn-Induced Corneal Neovascularization in Rabbits.

Objective This study aims at investigating the effects and molecular mechanism of riboflavin-ultraviolet-A-induced cross-linking (corneal collagen cross-linking, CXL) on corneal neovascularization (CNV) in a rabbit alkali burn model. Methods A total of 60 rabbits were injured with alkali burns to induce CNV in the right eye and were randomly divided into six groups: Group A—injury and no treatment; Groups B, C, and D—CXL treatment for 30 min, 15 min, and 45 min administered immediately after injury, respectively; and Groups E and F—CXL treatment for 30 min administered 1 day and 3 days after injury, respectively. CNV area, corneal edema, and corneal epithelial defects were observed on days 4, 7, 10, and 14 after injury. Western blot was used to detect expression of the vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-2 (MMP-9), and tissue inhibitor of metalloproteinases 1 (TIMP-1) at 7 and 14 days after injury. Results CXL treatment decreased CNV and corneal edema in all groups compared to Group A. On day 7, MMP-9 expression was significantly increased in all CXL treatment groups, and TIMP-1 was upregulated in Groups D and F compared to Group A. In addition, VEGF, MMP-2, MMP-9, and TIMP-1 expression were increased in Group A on day 14 after injury. Conclusions Our results indicate that riboflavin-ultraviolet-A-induced cross-linking (corneal collagen cross-linking, CXL) significantly inhibits alkali burn-induced CNV in rabbits, possibly through downregulating VEGF, MMP-2, MMP-9, and TIMP-1 expression.

In vivo biodistribution of topical low molecular weight heparin-taurocholate in a neovascularized mouse cornea.

To investigate the ocular biodistribution and clearance of topically administered 7-taurocholic acid conjugated low-molecular weight heparin (LHT7) in a neovascularized mouse cornea using an in vivo optical imaging system. A total of 10 eyes of 6 to 8-week-old BALB/c mice were analyzed. Corneal neovascularization (CoNV) was induced in the inferior cornea (IC) of each animal by penetrating the stroma with two interrupted sutures. The development of CoNV was verified after one week and the area of each neovascularized region was measured. A near-infrared fluorescent probe of 20 µmol/L Cy5.5 labeled LHT7 (LHT7-Cy5.5) in 0.02 mL solution was topically instilled onto the cornea in the experimental group (n=5). Free-Cy5.5 of 20 µmol/L in 0.02 mL was instilled in the control group (n=5). In vivo optical images were obtained before instillation and 5min, 2, 4, and 6h after instillation. The intensities were separately measured at the superior cornea (SC) and the IC. The mean CoNV areas were 1.97±0.17 mm2 and 1.92±0.96 mm2 in the experimental and control groups, respectively (P=0.832). The SC remained normal in all 10 subject animals. The IC intensity of the LHT7-Cy5.5 was greater than the SC intensity at 5min (P=0.038), 2h (P=0.041), and 4h (P=0.041) after application. The IC intensity fell to less than half of its initial value (42.9%±8.6%) at 6h in the experimental group. In the control mice, here were no significant differences in the free-Cy5.5 intensity between the IC and SC. Topically administered LHT7 shows a high biodistribution in CoNV areas for 4h and should be reapplied accordingly to maintain its effects. In vivo optical imaging can be a useful tool for evaluating the ocular biodistribution of a drug in an animal model.

Comparison of the inhibitory effect of different doses of subconjunctival bevacizumab application in an experimental model of corneal neovascularization.

To evaluate the inhibitory effect of subconjunctival bevacizumab as single- and multiple-dose application, and compare their effects on corneal neovascularization in a rat model. Thirty adult Sprague-Dawley rats were used in this experimental study. The central cornea of the rats was cauterized chemically. The rats were randomly enrolled into three groups. All groups received subconjunctival injections. In Group 1 (control group, n=10), 0.05 mL 0.9% NaCl solution was injected on the first day. In Group 2 (single-dose group, n=10), 0.05 mL bevacizumab (1.25 mg) was injected on the first day. In Group 3 (multiple-dose group, n=10), four doses of 0.05 mL bevacizumab (1.25 mg) were injected on the first, third, fifth and seventh day. Slit-lamp examination of all rats was performed at the third and ninth day. Digital images of the corneas were taken and analyzed using image analysis software to calculate corneal neovascularization area. All rats were sacrificed on the tenth day. In corneal sections, the number of blood vessels, state of inflammation and collagen formation was evaluated histopathologically. In Group 3, corneal edema grades were significantly lower than Group 1 and Group 2 (P=0.02, and P=0.035, respectively). The mean percentage of neovascularized corneal area in Group 3 was significantly lower than Group 2 (P=0.005). On histopathological examination, Group 2 and Group 3 showed significantly less number of blood vessels than Group 1 (P=0.005, and P=0.001, respectively). Additionally, Group 3 showed significantly less number of blood vessels compared to Group 2 (P=0.019). Inflammation and edema grades were significantly lower in Group 3 compared to Group 1 (P=0.001). Subconjunctival bevacizumab injection is effective in inhibition of newly formed corneal neovascularization. The multiple-dose bevacizumab treatment seems to be more effective compared to single-dose treatment.