Inhibition Of Corneal Neovascularization Research Articles

Corneal neovascularization is inhibited with nucleolin-binding aptamer, AS1411

Corneal neovascularization (CNV) is a common sight-threatening pathology that can be induced by a variety of inflammatory and angiogenic stimuli. Current CNV treatments include anti-inflammatory drugs and antibody-based inhibitors of vascular endothelial growth factor (VEGF). However, these are not always effective and novel therapeutic approaches are needed. Previous work has indicated a role for nucleolin (NCL) in VEGF-mediated neoangiogenesis in a suture-induced CNV model. The major goal for this current study is to test the effect of AS1411, a NCL-binding DNA aptamer that has reached human clinical trials, on neovascularization in a murine model of VEGF-mediated CNV. Our results show that topical administration of AS1411 can significantly inhibit corneal neovascularization in this model. Mechanistic studies indicate that AS1411 reduces the VEGF-stimulated proliferation, migration, and tube formation of primary cells obtained from human limbus stroma (HLSC). AS1411 treatment also significantly reduced VEGF-stimulated induction of miR-21 and miR-221 in HLSC, suggesting a role for these pro-angiogenic miRNAs in mediating the effects of AS1411 in this system. In sum, this new research further supports a role for NCL in the molecular etiology of CNV and identifies AS1411 as a potential anti-angiogenic CNV treatment that works by a novel mechanism of action.

Evaluation of pigment epithelium-derived factor concentration in equine amniotic membrane homogenate and its in-vitro vascular endothelial growth factor inhibition effect in tears of dogs with vascularized ulcerative keratitis.

Background:Corneal neovascularization can result from many pathological processes affecting the ocular surface leading to disturbances and opacifications that reduce corneal clarity and may impact vision. In veterinary medicine, the use of topical corticosteroid is contraindicated in the presence of ulcerative keratitis, and there is sparse research regarding safe medical alternatives to inhibit corneal neovascularization in dogs to improve visual outcome.Aim:To investigate the pigment epithelium-derived factor (PEDF) concentration in equine amniotic membrane homogenate (EAMH) and its in-vitro vascular endothelial growth factor (VEGF) inhibition in tears of dogs with vascularized ulcerative keratitis.Methods:Homogenates from 10 equine amniotic membranes (AM) were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of equine PEDF and VEGF. Forty tear samples were collected from both eyes of dogs diagnosed with vascularized ulcerative keratitis, and 50 samples from healthy dogs. Samples from affected eyes were allocated to G1 – affected undiluted tears; G2 – affected tears diluted with phosphate-buffer solution; G3 – affected tears treated with low-concentrated EAMH; and G4 – affected tears treated with high-concentrated EAMH. Tears from the unaffected contralateral eyes were composed in G5, while G6 was composed by tears from healthy dogs (control). The presence and levels of VEGF were evaluated in all groups by Western blot and ELISA.Results:The PEDF:VEGF ratio in EAMH was 110:1. An increase in VEGF levels was observed in tears from eyes with vascularized corneal ulcers (G1) as well as in contralateral tears (G5), compared to normal dogs (G6). High-concentrated EAMH provided a greater decrease in VEGF levels in-vitro compared to low-concentrated EAMH.Conclusion:EAMHs exhibited high concentrations of PEDF in comparison to VEGF and were able to partially decrease VEGF levels in tears of dogs with vascularized ulcers, in-vitro. Our results suggest that VEGF concentration is elevated in tears of dogs with active vascularized ulcerative keratitis in both affected and contralateral eyes compared to that of healthy dogs.

Osthole: A Traditional Chinese Medicine for Ocular Anti-Angiogenic Therapy

Purpose: Osthole is an agent isolated from Cnidium monnieri (L.) Cusson and has been used to treat several disorders. Corneal neovascularization is a sight-threatening condition associated with several inflammatory or infectious ocular disorders. In this study, we investigated the anti-angiogenic effects of osthole on corneal neovascularization and the underlying mechanism. Methods: MTT assay, HE staining, and calcein-AM/propidium iodide staining was conducted to detect the toxicity of osthole in vitro and in vivo. Corneal neovascularization of ICR mice was induced by alkali burn and observed by a slit lamp microscopy on day 7 after alkali injury. EdU assay, Ki67 immunofluorescence assay, Transwell migration assay, and Matrigel assay were conducted to investigate the role of osthole in endothelial angiogenic effects in vitro. Western blots were conducted to investigate the anti-angiogenic mechanism of osthole in corneal neovascularization. Results: Administration of osthole ranging from 0.05 to 25 µM had no detectable cytotoxicity or tissue toxicity in vivo and in vitro. Topical administration of osthole inhibited corneal neovascularization induced by alkali burn. Osthole decreased the proliferation, migration, and tube-formation of endothelial cells induced by VEGF. Osthole inhibited endothelial angiogenic functions through blocking the phosphorylation of ERK1/2, JNK, and p38. Conclusion: Our study provides evidence that osthole is a promising drug for the treatment of corneal neovascularization.

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.

PEDF Reduces the Severity of Herpetic Simplex Keratitis in Mice.

The purpose of this study was to explore the effects of pigment epithelium derived factor (PEDF) and PEDF-derived peptides Mer44 and Mer34 on the severity of herpetic simplex keratitis (HSK) in mice. Adult C57BL/6 mice were infected ocularly with the herpes simplex virus type 1 (HSV-1, McKrae strain) and injected subconjunctivally with PEDF, Mer44, or Mer34. Corneal nerve degeneration, neovascularization, sensitivity, neutrophils, macrophages and CD4+ T-cell infiltration, virus contents, and expressions of VEGF, PEDF, and proinflammatory factors were evaluated during acute period. The direct inhibitory effect of PEDF on HSV-1 replication was further evaluated in cultured monkey Vero cells. Following HSV-1 infection, corneal PEDF expression decreased at 3 and 7 days postinfection (dpi) but increased at 15 dpi, and returned to the similar level of normal mice at 45 dpi, which was accompanied with the progress of corneal nerve degeneration and neovascularization. Exogenous PEDF application attenuated corneal nerve degeneration and neovascularization and improved the impaired corneal sensitivity. Moreover, PEDF attenuated the neutrophils, but not macrophage or CD4+ T-cell infiltration, with the reduced expressions of IL-1β, IL-6, TNF-α, and VEGF. In addition, PEDF inhibited the replication of HSV-1 both in vitro and in mice. Mer44 attenuated corneal nerve degeneration more significantly than Mer34, whereas Mer34 inhibited corneal neovascularization. PEDF and its derived peptides reduce the severity of herpetic simplex keratitis in mice, representing the potential therapeutic approach to control HSK lesions.

Xanthatin inhibits corneal neovascularization by inhibiting the VEGFR2‑mediated STAT3/PI3K/Akt signaling pathway.

Alkali burn is one of the main causes of corneal injury. The inflammation and neovascularization caused by alkali burns aggravate corneal damage, resulting in loss of vision. The aim of the present study was to evaluate the efficacy of xanthatin in the treatment of alkali burn-induced inflammation and neovascularization. A CCK-8 assay was used to detect the effects of different concentrations of xanthatin on the proliferation of human umbilical vein endothelial cells (HUVECs). The effects of xanthatin on the migration of HUVECs and the ability of lumen formation were examined using a scratch test and lumen formation assay, respectively. A total of 60 Sprague-Dawley rats were randomly divided into two groups to establish a corneal alkali burn model, and were treated with PBS and xanthatin eye drops four times a day. A slit lamp microscope recorded changes of the cornea at 0, 4, 7, 10 and 14 days, and the inflammatory indices of the cornea and the neovascular area were evaluated. The expression levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in the cornea under different treatment conditions were detected using immunofluorescence and western blot analysis. In order to investigate the mechanism of xanthatin on the inhibition of inflammation and neovascularization, HUVECs were treated with xanthatin and PBS following VEGF treatment. The subcellular localization of signal transducer and activator of transcription 3 (STAT3) was detected using immunofluorescence. The expression levels of VEGF receptor 2 (VEGFR2), STAT3, phosphoinositide 3-kinase (PI3K) and Akt were detected using western blot analysis. The results revealed that xanthatin inhibited the proliferation of HUVECs in a concentration-dependent manner. The migration ability and lumen-forming ability of the HUVECs were also inhibited by xanthatin. Slit lamp microscopy showed that the inflammatory index and the area of neovascularization in the xanthatin-treated group were significantly reduced, compared with those in the PBS treatment group. The xanthatin treatment group exhibited a lower protein expression level of VEGF and increased protein expression level of PEDF, compared with the PBS treatment group. In the VEGF-treated HUVECs, xanthatin significantly decreased the expression levels of p-VEGFR2, phosphorylated (p-)STAT3, p-PI3K and p-Akt. In conclusion, the present study confirmed that xanthatin inhibited corneal neovascularization and inflammation in the alkali burn model, elucidating the underlying mechanisms involved in its protective effects. Therefore, xanthatin may be a novel drug for the treatment of corneal alkali burn.

Rhamnazin attenuates inflammation and inhibits alkali burn-induced corneal neovascularization in rats.

The purpose of our study was to determine whether rhamnazin inhibits corneal neovascularization in the rat alkali burn model, and alleviates the inflammatory response of the cornea. Rhamnazin inhibited the proliferation of HUVEC cells in a dose-dependent manner, and it also inhibited the migration and luminal formation of HUVEC cells. 20 μM rhamnazin eye drops were applied to an animal model of corneal alkali burn neovascularization 4 times a day for 14 days. The corneal neovascularization in the rhamnazin group was obviously less than that in the PBS control group. In the rhamnazin group, the inflammatory index of the cornea decreased gradually over time, whereas the inflammatory index of the PBS group decreased only slightly with time. The corneal CNV area in the PBS group was significantly larger than that in the rhamnazin group. The expression level of VEGF protein of the rhamnazin group was lower than that in the PBS group, and the expression level of PEDF was significantly higher than that of the PBS group. Rhamnazin downregulated the expression of VEGFR2 protein and decreased the expression levels of p-STAT3, p-MAPK and p-Akt proteins. This study provides a new idea for the study of the molecular mechanism of corneal neovascularization.

Suppression of corneal neovascularization by curcumin via inhibition of Wnt/β-catenin pathway activation.

To investigate whether curcumin suppressed corneal neovascularization (CNV) formation via inhibiting activation of Wnt/β-catenin pathway. Suture-induced CNV was established on Sprague-Dawley (SD) rats. Curcumin were daily administrated by subconjunctival injection. Phosphorylation of low-density lipoprotein receptor-related protein 6 (LRP6) and nuclear accumulation of β-catenin, two indicators of activated Wnt/β-catenin pathway, were determined by Western-blot analysis in subconfluent/proliferating human microvascular endothelial cells (HMEC) and neovascularized corneas. Wnt3a conditioned medium (WCM) were harvested from Wnt3a expressing cells. WCM-induced cell proliferation and endothelial tubular formation capacity was measured by MTT assay and Matrigel assay, respectively. Phosphorylation of LRP6 and nuclear accumulation of β-catenin was significantly increased in subconfluent/proliferating endothelial cells. Activation of Wnt/β-catenin pathway by WCM markedly promotes HMEC proliferation and tubular formation. Curcumin inhibited LRP6 phosphorylation and nuclear accumulation of β-catenin. In addition, curcumin attenuated WCM-induced HMEC proliferation and disrupted tubular structure of endothelial cells on Matrigel. Meanwhile curcumin suppressed suture-induced CNV and inhibited LRP6 phosphorylation as well as β-catenin accumulation in SD rats. Taken together, activation of Wnt/β-catenin pathway could be involved in endothelial proliferation during suture-induced CNV formation and curcumin attenuated CNV formation via inhibition of Wnt/β-catenin pathway activation.

Effectiveness of photodynamic therapy with verteporfin combined with intrastromal bevacizumab for corneal neovascularization in Stevens-Johnson syndrome.

To investigate the effectiveness of combined photodynamic therapy with verteporfin and intrastromal injection of bevacizumab for the treatment of corneal neovascularization in patients with Stevens-Johnson syndrome (SJS). Eight eyes of eight patients with SJS having corneal neovascularization who were refractory to 1% prednisolone instillation received photodynamic therapy with verteporfin (6mg/m2) combined with intrastromal bevacizumab injection (2.5mg/0.1mL). Best-corrected visual acuity and intraocular pressure were assessed, and slit-lamp biomicroscopic examination was performed before treatment and at 1week and every month. A chronic ocular manifestation score was assigned based on the involvement area or the severity before treatment. The cumulative length of corneal blood vessels and area of corneal neovascularization were measured by anterior segment photographs before and after treatment. At 3 and 6months after treatment, all eyes showed regression of corneal neovascularization. Complete regression was achieved in five eyes (62.5%) and partial regression in three eyes (37.5%). Among five patients who were followed up for more than 1year, two eyes maintained complete regression and one eye maintained partial regression at 1year. However, two eyes with severe chronic ocular manifestation showed revascularization. Combined photodynamic therapy with intrastromal bevacizumab injection can effectively inhibit corneal neovascularization in patients with SJS. However, patients with severe chronic ocular manifestation may exhibit revascularization.

Epithelium-derived miR-204 inhibits corneal neovascularization

MicroRNA-204 (miR-204) is highly expressed in cornea, here we explored the role and mechanism of miR-204 in corneal neovascularization (CNV). Mouse CNV was induced by intrastromal placement of suture in BALB/c mice with the subconjunctival injection of miR-204 agomir or negative control. Human primary limbal epithelial cells (LECs) and immortalized microvascular endothelial cells (HMECs) were used to evaluate the expression changes and anti-angiogenic effects of miR-204 under biomechanical stress (BS). The expression and localization of miR-204, vascular endothelial growth factor (VEGF) and their receptors were detected by quantitative real-time PCR, in situ hybridization, immunohistochemistry and Western blot. The results showed that miR-204 expression was mainly localized in epithelium and down-expressed in vascularized cornea. Subconjunctival injection of miR-204 agomir inhibited CNV and reduced the expression of VEGF and VEGF receptor 2. Similarly, miR-204 overexpression attenuated the increased expression of VEGF by biomechanical stress in LECs, and suppressed the proliferation, migration, and tube formation of HMECs. These novel findings indicate that epithelium-derived miR-204 inhibits suture-induced CNV through regulating VEGF and VEGF receptor 2.

Topical dimethyl sulfoxide inhibits corneal neovascularization and stimulates corneal repair in rabbits following acid burn

Neovascularization of the cornea is characterized by the growth of blood vessels caused by imbalances between angiogenic and anti-angiogenic factors. We investigated whether the expression of Vascular endothelial growth factor (VEGF), Vascular endothelial growth factor receptor (VEGF), Vascular endothelial growth inhibitor (VEGI) receptors, as well as topical drug treatments, participate in regulating corneal neovascularization after corneal damage and remodeling. We used 72 mature male New Zealand rabbits. Corneal burns were induced by hydrofluoric acid under general anesthesia. The rabbits then were treated with indomethacin or dimethyl sulfoxide (DMSO). The animals were euthanized on days 2, 7 and 14 after injury. Each cornea was fixed with 10% neutral formalin. On days 2, 7 and 14, VEGF, flk1/KDR and flt1/fms were strongly expressed in the epithelial, stromal and inflammatory cells, but not in the corneal endothelial cells. On day 7, newly formed blood vessels were observed growing toward the center of the cornea. In the control, indomethacin treated, DMSO–treated, and indomethacin + DMSO–treated animals, VEGI, VEGF, and the receptors, flk1/KDR, flt1/fms and flt4, were expressed at different densities in the neovascular regions. This was particularly evident in the indomethacin- and indomethacin + DMSO–treated groups on days 7 and 14, compared to day 2. Treatment with VEGF and DMSO stimulated repair of corneal damage. We suggest that VEGI in the endothelial cells of neovascularized cornea may act as a signaling protein that promotes balance between cell proliferation and apoptosis. Topical administration of DMSO inhibited corneal neovascularization more effectively than indomethacin.

Topical treatment of corneal alkali burns with Gly-thymosin β4 solutions and in situ hydrogels via inhibiting corneal neovascularization and improving corneal epidermal recovery in experimental rabbits

AimCorneal alkali burns are a severe disease and commonly encountered in the emergent clinic. A rapid medical treatment for the burn is very important. Gly-thymosin β4 (Gly-Tβ4) is a biomimic derivative of natural thymosin β4. The aim of this study is to evaluate the corneal recovery effects of Gly-Tβ4 topical therapy on alkali burns in rabbit corneas. MethodsRabbit alkali burns were induced with NaOH-contained filter paper. Phosphate-buffered solutions at pH 7.0, Gly-Tβ4 solutions, blank in situ hydrogels, and Gly-Tβ4in situ hydrogels were dropped on the burned corneas. The treatments were continued for 14 days. Conjunctiva hyperemia, corneal edema, intraeye extravasation, hemorrhaging, corneal neovascularization (CNV), and corneal opacity were observed. Corneal immunohistochemistry and histopathology were performed. ResultsGly-Tβ4 solutions led to a lower corneal burn index than the other regimens. Hydrogels may stimulate the burned corneas due to the direct contact of them, and prevent the rapid release of Gly-Tβ4. Gly-Tβ4 significantly inhibited CNV according to the images of the corneas, CNV areas, and CD31 expression. Furthermore, Gly-Tβ4 improved corneal epidermal recovery according to the histopathological result. ConclusionGly-Tβ4 solutions are a promising formulation for topical treatment of corneal alkali burns.

A novel peptide specifically binding to VEGF receptor suppresses angiogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis through binding to VEGF receptors (VEGFRs). Here we report a novel peptide designated HRHTKQRHTALH (peptide HRH), which was isolated from the Ph.D. -12 phage display library using VEGFR-Fc fusion protein as the bait. This peptide was found to dose-dependently inhibit the proliferation of human umbilical vein endothelial cells stimulated by VEGF. The anti-angiogenesis effect of the HRH peptide was further confirmed in vivo using the chick chorioallantoic membrane assay, which was also dose-dependent. Besides, peptide HRH was proved to inhibit corneal neovascularization in an alkali-burnt rat corneal model and a suture-induced rat corneal model. Taken together, these findings suggest that the HRH peptide can inhibit angiogenesis both in vitro and in vivo. Consequently, the HRHTKQRHTALH peptide might be a promising lead peptide for the development of potential angiogenic inhibitors.

Anti-angiogenic effect of hexahydrocurcumin in rat corneal neovascularization.

This study was to investigate the anti-angiogenic effect of hexahydrocurcumin (HHC) to evaluate gene (p-basic fibroblast growth factor (bFGF)-SAINT-18 & p-vascular endothelial growth factor (VEGF)-SAINT-18 complex)-induced corneal neovascularization (CorNV) in rats. CorNV was induced in 24 eyes of 24 rats. Four groups (Group A: 0μg, B: 0.01μg, C: 0.1μg, and D: 1μg) of HHC were prepared and implanted into the rat subconjunctival substantia propria 1.5mm from the limbus at temporal side. The 1μg of p-bFGF-SAINT-18 & p-VEGF-SAINT-18 complex were prepared and implanted into the rat corneal stroma 1.5mm from the limbus at the same side. Inhibition of CorNV was observed and quantified from day 1 to day 60. bFGF and VEGF protein expression were analyzed by biomicroscopic examination, western blot analysis, and immunohistochemistry. Subconjunctival injection by 1μg HHC successfully inhibited gene-induced CorNV in rats. bFGF and VEGF protein expression were reduced after 6days. Meanwhile, the reduction of HLA-DR expression was detected. Our study showed that the HHC might provide an important anti-angiogenesis factor to inhibit CorNV development at the corneal experimental angiogenesis model.

A Novel Peptide Derived From Tissue-Type Plasminogen Activator Potently Inhibits Angiogenesis and Corneal Neovascularization.

The recombinant protein TK1-2, which consists of two kringle domains of tissue-type plasminogen activator (t-PA), inhibits angiogenesis and tumor growth. ɪn this study, we examined the anti-angiogenic activities of peptides derived from kringle 2 domain of t-PA to identify the functional core sequence. Seven peptides were constructed from the kringle 2 sequence, based on the structure and characteristics of amino acid residues, and were analyzed for their inhibitory effects on endothelial cells (ECs). Among them, TP-7 (derived from a β-sheet motif) potently inhibited proliferation, tube formation, and migration of ECs in a dose-dependent manner, whereas truncation of 3-9 amino acid residues from either N or C terminus of TP-7 abrogated its inhibitory effects on ECs. TP-7 also potently inhibited angiogenesis in a Matrigel plug assay in vivo. Moreover, TP-7 dose-dependently suppressed corneal neovascularization induced by an acute chemical burn in a rat model. At the molecular level, TP-7 inhibited VEGF- or bFGF-induced phosphorylation of FAK and ERK1/2 and drastically disrupted VEGF- or bFGF-induced formation of stress fibers and focal adhesion complexes. In addition, TP-7 markedly suppressed attachment and spreading of ECs on a collagen type I or fibronectin matrix. Adhesion of ECs to immobilized TP-7 increased dose-dependently, which was disrupted strongly by pretreatment with soluble TP-7 and slightly by an integrin α2β1-blocking antibody. These results suggest that TP-7 is a potent anti-angiogenic peptide in part affecting the integrin α2β1-dependent pathway and that it can be used for treatment of corneal neovascularization by targeting VEGF and non-VEGF pathways. J. Cell. Biochem. 118: 1132-1143, 2017. © 2016 Wiley Periodicals, Inc.

Effects of topical and subconjunctival use of bevacizumab on corneal neovascularization in rabbits’ eyes

To evaluate and compare the effects of topical application and subconjunctival injection of bevacizumab on corneal neovascularization (CNV) in rabbits' eyes after chemical burning of the cornea. The animals were randomly distributed into four groups of five animals. In one group, the drug was instilled, while in another, it was administered by subconjunctival injection. The two procedures using bevacizumab were compared with instillation and subconjunctival injection of saline solution (S). Neovascularization was evaluated according to the size of the invasion area of new blood vessels and through computerized analysis of this area. The data were analyzed using the Kruskal-Wallis test followed by Dunn's test for two-by-two comparison of the groups, to assess the external examination of CNV. Analysis of variance was used to assess the area of CNV. P<0.05 was considered statistically significant. Assessing both the external examination and the invasion area of neovessels on the 5th and 10th days, there was a clear difference between the groups. The group to which saline solution had been applied showed higher scores for CNV, as well as increases in the invasion area of neovessels. Two-by-two comparison of groups revealed no significant differences. However, an analysis of the factors involved (injection vs. instillation and bevacizumab vs. saline solution) showed that injection did not differ from instillation, but that bevacizumab differed from saline solution. Bevacizumab showed an inhibitory effect on CNV in rabbits' eyes after chemical burning of the cornea. There was no difference between the topical or subconjunctival administration of bevacizumab in the inhibition of CNV.

Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD) peptide–hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

Novel application assigned to toluquinol: inhibition of lymphangiogenesis by interfering with VEGF-C/VEGFR-3 signalling pathway.

Lymphangiogenesis is an important biological process associated with the pathogenesis of several diseases, including metastatic dissemination, graft rejection, lymphoedema and other inflammatory disorders. The development of new drugs that block lymphangiogenesis has become a promising therapeutic strategy. In this study, we investigated the ability of toluquinol, a 2-methyl-hydroquinone isolated from the culture broth of the marine fungus Penicillium sp. HL-85-ALS5-R004, to inhibit lymphangiogenesis in vitro, ex vivo and in vivo. We used human lymphatic endothelial cells (LECs) to analyse the effect of toluquinol in 2D and 3D in vitro cultures and in the ex vivo mouse lymphatic ring assay. For in vivo approaches, the transgenic Fli1:eGFPy1 zebrafish, mouse ear sponges and cornea models were used. Western blotting and apoptosis analyses were carried out to search for drug targets. Toluquinol inhibited LEC proliferation, migration, tubulogenesis and sprouting of new lymphatic vessels. Furthermore, toluquinol induced apoptosis of LECs after 14 h of treatment in vitro, blocked the development of the thoracic duct in zebrafish and reduced the VEGF-C-induced lymphatic vessel formation and corneal neovascularization in mice. Mechanistically, we demonstrated that this drug attenuates VEGF-C-induced VEGFR-3 phosphorylation in a dose-dependent manner and suppresses the phosphorylation of Akt and ERK1/2. Based on these findings, we propose toluquinol as a new candidate with pharmacological potential for the treatment of lymphangiogenesis-related pathologies. Notably, its ability to suppress corneal neovascularization paves the way for applications in vascular ocular pathologies.

33. Advancing a State of the Art Gene Therapy Called OXB-202 That Resists Corneal Rejection in High Risk Patients

Due to both the avascularity of the cornea and the relatively immune-privileged status of the eye corneal transplantation is one of the most successful clinical transplant procedures. However in high risk patients, which account for >20% of the 100,000 transplants carried out worldwide each year, the rejection rate is high due to vascularization of the recipient corneal bed. In some of these patients the prognosis is extremely poor, with grafts failing at an accelerating rate to the point where patients are no longer considered suitable for further transplants and are left blind, despite an otherwise normally functioning visual system. The main reason for graft failure is irreversible immunological rejection and it is therefore unsurprising that neovascularization (both pre-and post-grafting) is a significant risk factor for subsequent graft failure. Neovascularization is thus an attractive target to prevent corneal graft rejection. OXB-202 (previously known as EncorStat®) is a human donor cornea modified prior to transplant by ex vivo genetic modification with genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin. This is achieved using a lentiviral vector derived from the Equine Infectious Anaemia Virus (EIAV) called pONYK1EiA, which subsequently prevents rejection by suppressing neovascularization. Previously we have shown that rabbit corneas treated with pONYK1EiA substantially suppress corneal neovascularization, opacity and subsequent rejection in rabbit models of cornea graft rejection (Parker et al, 2014). We will present data from a 3-month GLP toxicology and biodistribution safety study of pONYK1EiA modified rabbit corneas in a rabbit corneal transplant model. In particular, the GLP study has been designed to include a number of high content in-life assessments that include regular slitlamp ophthalmic examinations, evaluation of corneal thickness and endothelial cell density using pachymetry and specular microscopy respectively and intraocular pressure measurements. We will present a summary of these data to show that there are no safety issues with pONYK1EiA modified corneas. The GLP safety study data to be presented supports the evaluation of OXB-202 corneas in a First-in-Man trial. The toxicology study, GMP manufactures and clinical development of OXB-202 has been supported by the UK Technology Strategy Board (Innovate UK).

Retracted: Inhibition of Corneal Neovascularization by Hydrazinocurcumin

This article previously published in Volume 15 Issue 2 of this journal in February 2016 has been retracted in line with the guidelines from the Committee on Publication Ethics (COPE, http://publicationethics.org/resources/guidelines) Retracted: Zhan W, Zhu J, Zhang Y. Inhibition of corneal neovascularization by hydrazinecurcumin. Trop J Pharm Res 2016; 15(2):349-354 doi: http://dx.doi.org/10.4314/tjpr.v15i2.18