Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway.

OBJECTIVE: To investigate the effects of emodin on alkali burn-induced corneal inflammation and neovascularization. METHODS: The ability of emodin to target vascular endothelial growth factor receptor 2 (VEGFR2) was predicted by molecular docking. The effects of emodin on the invasion, migration, and proliferation of human umbilical vein endothelial cells (HUVEC) were determined by cell counting kit-8, Transwell, and tube formation assays. Analysis of apoptosis was performed by flow cytometry. CD31 levels were examined by immunofluorescence. The abundance and phosphorylation state of VEGFR2, protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and P38 were examined by immunoblot analysis. Corneal alkali burn was performed on 40 mice. Animals were divided randomly into two groups, and the alkali-burned eyes were then treated with drops of either 10 µM emodin or phosphate buffered saline (PBS) four times a day. Slit-lamp microscopy was used to evaluate inflammation and corneal neovascularization (CNV) in all eyes on Days 0, 7, 10, and 14. The mice were killed humanely 14 d after the alkali burn, and their corneas were removed and preserved at －80 ℃ until histological study or protein extraction. RESULTS: Molecular docking confirmed that emodin was able to target VEGFR2. The findings revealed that emodin decreased the invasion, migration, angiogenesis, and proliferation of HUVEC in a dose-dependent manner. In mice, emodin suppressed corneal inflammatory cell infiltration and inhibited the development of corneal neovascularization induced by alkali burn. Compared to those of the PBS-treated group, lower VEGFR2 expression and CD31 levels were found in the emodin-treated group. Emodin dramatically decreased the expression of VEGFR2, p-VEGFR2, p-Akt, p-STAT3, and p-P38 in VEGF-treated HUVEC. CONCLUSION: This study provides a new avenue for evaluating the molecular mechanisms underlying corneal inflammation and neovascularization. Emodin might be a promising new therapeutic option for corneal alkali burns.

Long-Term Benefits of Tear Exchangeable Limbal-Rigid Contact Lens Wear Therapy in Stevens-Johnson Syndrome Cases.

OBJECTIVES: To evaluate the long-term benefits of tear-exchangeable, limbal-rigid contact lens (CL) wear therapy in patients with Stevens-Johnson syndrome (SJS)-associated ocular sequelae. METHODS: This retrospective study evaluated 50 eyes of 41 SJS patients (15 men and 26 women) who underwent limbal-rigid CL wear therapy for more than 2 years post fitting. Ocular sequelae (i.e., conjunctival hyperemia, corneal neovascularization, and upper tarsus scarring) before fitting and at 3 months, 6 months, 12 months, and annually after initiating CL wear therapy were evaluated and then graded on a severity score (range: 0-3, maximum score: 3). Moreover, visual acuity (VA) at immediately post initiating CL wear therapy was evaluated. RESULTS: The mean follow-up period was 4.3±1.1 years. Compared with before fitting, the mean conjunctival hyperemia score improved from 1.14 to 0.86 at 3 months of CL wear therapy ( P <0.01) and was maintained thereafter; the mean corneal neovascularization score improved from 2.10 to 1.98 at 3 months of CL wear therapy, with no deterioration of the score observed in all cases at the final follow-up examination, and mean VA (log of minimum angle of resolution) improved from 1.60 to 1.04 at immediately post initiating CL wear therapy ( P <0.01). CONCLUSIONS: Limbal-rigid CL wear therapy can provide long-term ocular surface stabilization and improved VA in SJS patients.

Sorafenib-loaded nanostructured lipid carriers for topical ocular therapy of corneal neovascularization: development, in-vitro and in vivo study.

Sorafenib (SRB), a multikinase inhibitor, is effective in reducing experimental corneal neovascularization (CNV) after oral administration; however, its therapeutic use in ocular surface disorders is restricted due to poor solubility and limited bioavailability. This study aimed to develop and optimize SRB-loaded nanostructured lipid carriers (SRB-NLCs) for topical ocular delivery by a central composite design response surface methodology (CCD-RSM). It was spherical and uniform in morphology with an average particle size of 111.87 ± 0.93 nm and a narrow size distribution. The in vitro drug release from the released SRB-NLC formulation was well fitted to Korsmeyer Peppas release kinetics. The cell counting kit-8 (CCK-8) cell viability assay demonstrated that SRB-NLC was not obviously cytotoxic to human corneal epithelial cells (HCECs). An in vivo ocular irritation test showed that SRB-NLC was well tolerated by rabbit eyes. Ocular pharmacokinetics revealed 6.79-fold and 1.24-fold increase in the area under concentration-time curves (AUC0-12h) over 12 h in rabbit cornea and conjunctiva, respectively, treated with one dose of SRB-NLC compared with those treated with SRB suspension. Moreover, SRB-NLC (0.05% SRB) and dexamethasone (0.025%) similarly suppressed corneal neovascularization in mice. In conclusion, the optimized SRB-NLC formulation demonstrated excellent physicochemical properties and good tolerance, sustained release, and enhanced ocular bioavailability. It is safe and potentially effective for the treatment of corneal neovascularization.

Cannabis and the Cornea.

Purpose: While cannabis has the potential to reduce corneal pain, cannabinoids might induce side effects. This review article examines the effects of cannabinoids on the cornea. As more states and countries consider the legalization of adult cannabis use, health-care providers will need to identify ocular effects of cannabis consumption.Methods: Studies included in this review examined the connection between cannabis and the cornea, more specifically anti-nociceptive and anti-inflammatory actions of cannabinoids. NCBI Databases from 1781 up to December 2019 were consulted.Results: Five studies examined corneal dysfunctions caused by cannabis consumption (opacification, decreased endothelial cell density). Twelve studies observed a reduction in corneal pain and inflammation (less lymphocytes, decreased corneal neovascularization, increased cell proliferation and migration).Conclusion: More than half of the studies examined the therapeutic effects of cannabinoids on the cornea. As the field is still young, more studies should be conducted to develop safe cannabinoid treatments for corneal diseases.

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 ELISA 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 ELISA 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 ELISA 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.

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.

Inhibition of COX-2, mPGES-1 and CYP4A by isoliquiritigenin blocks the angiogenic Akt signaling in glioma through ceRNA effect of miR-194-5p and lncRNA NEAT1.

BACKGROUND: Arachidonic acid (AA) metabolic enzymes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1) and cytochrome P450 (CYP) 4A11 play important roles in glioma angiogenesis. Thus, there is an urgent need to identify the underlying mechanisms and develop strategies to overcome them. METHODS: A homology model of human CYP4A11 was constructed using SYBYL-X 2.0. Structure-based virtual screening against COX-2, mPGES-1 and CYP4A11was performed using the Surflex-Dock of the SYBYL suite. The candidates were further evaluated their antiangiogenic activities in a zebrafish embryo and rabbit corneal angiogenesis model. Laser doppler analysis was used to measure tumor perfusion. The expression of CD31 and α-SMA was measured by immunofluorescence. Western blot was used to measure the expression of HIF-1, Akt and p-Akt. The gene expression of FGF-2, G-CSF, PDGF, TGF-ß, Tie-2, VEGF, lncRNA NEAT1 and miR-194-5p were determined using qPCR. The production of FGF-2, TGF-ß and VEGF were analyzed using ELISA. Bioinformatic analysis and luciferase reporter assays confirmed the interaction between lncRNA NEAT1 and miR-194-5p. RESULTS: The nearly 36,043 compounds from the Traditional Chinese Medicine (TCM) database were screened against COX-2, mPGES-1 and CYP4A11 3D models, and the 17 top flavonoids were identified. In zebrafish screening, isoliquiritigenin (ISL) exhibited the most potent antiangiogenic activities with the EC50 values of 5.9 µM. Conversely, the antiangiogenic effects of ISL in the zebrafish and rabbit corneal models were partly reversed by 20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE2). ISL normalized glioma vasculature and improved the efficacy of temozolomide therapy in the rat C6 glioma model. Inhibition of COX-2, mPGES-1 and CYP4A by ISL decreased FGF-2, TGF-ß and VEGF production in the C6 and U87 glioma cells with p-Akt downregulation, which was reversed by Akt overexpression. Furthermore, ISL downregulated lncRNA NEAT1 but upregulated miR-194-5p in the U87 glioma cell. Importantly, lncRNA NEAT1 overexpression reversed ISL-mediated increase in miR-194-5p expression, and thereby attenuated FGF-2, TGF-ß and VEGF production. CONCLUSIONS: Reprogramming COX-2, mPGES-1 and CYP4A mediated-AA metabolism in glioma by flavonoid ISL inhibits the angiogenic Akt- FGF-2/TGF-ß/VEGF signaling through ceRNA effect of miR-194-5p and lncRNA NEAT1, and may serve as a novel therapeutic strategy for human glioma.

Corneal neovascularization: updates on pathophysiology, investigations & management.

Objective. Corneal neovascularization is a sight-threatening condition affecting more than 1.4 million people per year. Left untreated, it can lead to tissue scarring, oedema, lipid deposition, and persistent inflammation that may significantly affect visual prognosis and quality of life. The aim was to review the recent evidence relating to the pathophysiology, investigations and management of corneal neovascularization. Methods. Literature review of prospective and retrospective studies, clinical trials and animal models relating to the pathophysiology, investigation and management of corneal neovascularization. Results. Corneal neovascularization is characterized by the invasion of new blood vessels into the cornea caused by an imbalance between angiogenic and antiangiogenic factors that preserve corneal transparency as a result of various ocular insults and hypoxic injuries. Risk factors that have been implicated in the pathogenesis of the disease include contact lens wear, ocular surface disease, trauma, previous surgery and herpes. The results highlighted the current and future management modalities of corneal neovascularization, which includes corneal transplantation, laser - phototherapy, injections and topical treatment. Conclusion. The future of corneal neovascularization is promising and this paper discusses the upcoming revolution in local gene therapy. Abbreviations. HSK = herpes stromal keratitis, VEGF = vascular endothelial growth factor, VEGFR-1 = VEGF Receptor-1, FGF = Fibroblast growth factor, PDGF = Platelet-derived growth factor, IL-6 = interleukin-6, IL-7 = interleukin-7, IL-8 = interleukin-8, IRS-1 = insulin receptor substrate-1.

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-1ß) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2, Il-1ß, 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-1ß, 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.

Investigation the effect of Hypericum perforatum on corneal alkali burns.

Purpose: To investigate the effect of Hypericum perforatum on corneal alkali burn. Methods: We studied 45 250 g weighing, 4 months old Wistar albino rats. Alkaline burns were performed in the corneas of all experimental animals with 2 mol/L NaOH after general anaesthesia. Rats were divided into five groups according to the subsequent process applied to them: group 1 was the topical Hypericum perforatum group, group 2 was the topical pure olive oil group, group 3 was the oral Hypericum perforatum group, group 4 was the oral pure olive oil group, and group 5 was the control untreated group. Rats were sacrificed under general anaesthesia on the 14 day. The rate of corneal inflammation, neovascularization, fibroblastic activity, and cluster of differentiation 31 (CD31) staining was investigated. Result: There were 45 rats at the beginning of the study. One rat in groups 1, 2, and 3 died during the study; therefore, 42 rats could be evaluated. There were 8 rats in group 1, 8 rats in group 2, 8 rats in group 3, and 9 rats in group 4. We found less corneal neovascularization (CNV), inflammation, and fibroblastic activity in group 1 and group 2 than in the other groups (p ˂ 0.001 for all parameters). CNV, inflammation, fibroblastic activity, and CD31 staining rates were lower in group 1 than in group 2 (p ˂ 0.001 for all parameters). There was no difference between groups 3, 4, and 5 (respectively, p = 0.436, 0.634, and 0.750). Conclusions: We found that both topical Hypericum perforatum oily extract and olive oil have anti-inflammatory, anti-angiogenic, and anti-fibroblastic effects when applied after corneal alkali burns in rat corneas. Further studies should be conducted in this field.

Comparison of the Effects of Subconjunctival Injections of Bevacizumab and Interferon Alpha-2a on Corneal Angiogenesis in a Rat Model.

BACKGROUND AND OBJECTIVE: Corneal neovascularization (CNV) is a vision-threatening condition arising from various corneal diseases. The aim of this study is to compare the effectiveness of bevacizumab and interferon alpha-2a (IFNα-2a) treatment on corneal neovascularization. MATERIALS AND METHODS: Twenty-four Wistar albino rats were used in this study. After cauterization of the cornea with a silver nitrate applicator stick, the control group received 0.1 mL saline solution, the second group received 0.1 mL IFNα-2a (IFNα-2a, 6 million international units [MIU]/0.5 mL), and the third group received 2.5 mg bevacizumab by subconjunctival injection. An additional injection was administered to each group on the fourth day. After one week, the corneal neovascularization rate and the longest neovascular sprout length were determined. RESULTS: The neovascularization rate (saline 0.65 ± 0.05; IFNα-2a 0.62 ± 0.07; bevacizumab 0.42 ± 0.11) with bevacizumab was significantly lower, more than those with IFNα-2a and saline (p < 0.001 and p < 0.001). The longest neovascular sprout length (saline, 4.00 ± 0.6 mm; IFNα-2a, 3.63 ± 0.52 mm; bevacizumab, 2.81 ± 0.65 mm) with bevacizumab was significantly shorter than those with saline and IFNα-2a (p = 0.001 and p = 0.012). CONCLUSIONS: Subconjunctival IFNα-2a has limited efficacy in the treatment of corneal neovascularization.

Tetramethylpyrazine in a Murine Alkali-Burn Model Blocks NFκB/NRF-1/CXCR4-Signaling-Induced Corneal Neovascularization.

Purpose: Tetramethylpyrazine (TMP) is the active ingredient extracted from the Chinese herb Chuanxiong. The purpose of our study was to identify the mechanism of therapeutic TMP suppression of pathologic chemokine receptor 4 (CXCR4) transcription. Methods: C57BL/6J mice with alkali-burned corneas were treated with either TMP eye drops (1.5 mg/mL) or PBS. Corneal neovascularization (CNV) was measured and a clinical assessment was made by slit lamp microscopy. Expression of CXCR4 and the transcription factors nuclear respiratory factor-1 (NRF-1), nuclear factor kappa B (NFκB), forkhead box C1, and yin yang 1 were tracked by real-time RT-PCR and immunofluorescence staining of murine corneas. Western blot, real-time PCR, and immunofluorescence evaluated expression of related genes in human umbilical vein endothelial cells (HUVECs) after 200-µmol/L TMP treatment. In addition, plasmid transfection and chromatin immunoprecipitation assays elucidated the relationship among NRF-1, NFκB, and CXCR4. Results: Corneas treated with TMP had smaller areas of neovascularization and scored better in clinical assessments. Injured corneas showed significantly elevated expressions of NRF-1, NFκB, and CXCR4 that were normalized in vivo by TMP treatment. Similarly, in HUVECs in vitro, TMP decreased expression of NRF-1, NFκB, and CXCR4. Overexpression of NFκB or NRF-1 raised the expression of CXCR4 in HUVECs, but not synergistically. Chromatin immunoprecipitation assays detected only NRF-1 bound to the CXCR4 promoter region, suggesting NFκB controls CXCR4 expression by upregulating NRF-1. Together, our data suggest TMP downregulates CXCR4 by repressing NRF-1 expression in CNV, likely indirectly by downregulating NFκB. Conclusions: Our results implicate a novel mechanism wherein TMP inhibits neovascularization via an NFκB/NRF-1/CXCR4 circuit.

Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.

VEGF-induced neovascularization plays a pivotal role in corneal neovascularization (CoNV). The current study investigated the potential effect of ginsenoside Rh2 (GRh2) on neovascularization. In HUVECs, pretreatment with GRh2 largely attenuated VEGF-induced cell proliferation, migration, and vessel-like tube formation in vitro. At the molecular level, GRh2 disrupted VEGF-induced VEGF receptor 2 (VEGFR2)-Grb-2-associated binder 1 (Gab1) association in HUVECs, causing inactivation of downstream AKT and ERK signaling. Gab1 knockdown (by targeted short hairpin RNA) similarly inhibited HUVEC proliferation and migration. Notably, GRh2 was ineffective against VEGF in Gab1-silenced HUVECs. In a mouse cornea alkali burn model, GRh2 eyedrops inhibited alkali-induced neovascularization and inflammatory cell infiltrations in the cornea. Furthermore, alkali-induced corneal expression of mRNAs/long noncoding RNAs in cornea were largely attenuated by GRh2. Overall, GRh2 inhibits VEGF-induced angiogenic effect via inhibiting VEGFR2-Gab1 signaling in vitro. It also alleviates angiogenic and inflammatory responses in alkali burn-treated mouse corneas.-Zhang, X.-P., Li, K.-R., Yu, Q., Yao, M.-D., Ge, H.-M., Li, X.-M., Jiang, Q., Yao, J., Cao, C. Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.

Anti-angiogenic effect of hexahydrocurcumin in rat corneal neovascularization.

AIM: 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. METHODS: CorNV was induced in 24 eyes of 24 rats. Four groups (Group A: 0 µg, B: 0.01 µg, C: 0.1 µg, and D: 1 µg) of HHC were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus at temporal side. The 1 µg of p-bFGF-SAINT-18 & p-VEGF-SAINT-18 complex were prepared and implanted into the rat corneal stroma 1.5 mm 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. RESULTS: Subconjunctival injection by 1 µg HHC successfully inhibited gene-induced CorNV in rats. bFGF and VEGF protein expression were reduced after 6 days. Meanwhile, the reduction of HLA-DR expression was detected. CONCLUSIONS: Our study showed that the HHC might provide an important anti-angiogenesis factor to inhibit CorNV development at the corneal experimental angiogenesis model.

AAV vector-meditated expression of HLA-G reduces injury-induced corneal vascularization, immune cell infiltration, and fibrosis.

Over 1.5 million individuals suffer from cornea vascularization due to genetic and/or environmental factors, compromising visual acuity and often resulting in blindness. Current treatments of corneal vascularization are limited in efficacy and elicit undesirable effects including, ironically, vision loss. To develop a safe and effective therapy for corneal vascularization, adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by human leukocyte antigen G (HLA-G), was investigated. Self-complementary AAV cassettes containing codon optimized HLA-G1 (transmembrane) or HLA-G5 (soluble) isoforms were validated in vitro. Then, following a corneal intrastromal injection, AAV vector transduction kinetics, using a chimeric AAV capsid, were determined in rabbits. One week following corneal trauma, a single intrastromal injection of scAAV8G9-optHLA-G1 + G5 prevented corneal vascularization, inhibited trauma-induced T-lymphocyte infiltration (some of which were CD8+), and dramatically reduced myofibroblast formation compared to control treated eyes. Biodistribution analyses suggested AAV vectors persisted only in the trauma-induced corneas; however, a neutralizing antibody response to the vector capsid was observed inconsistently. The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to safely and effectively treat corneal vascularization and inhibit fibrosis while alluding to broader roles in ocular surface immunity and allogenic organ transplantation.

Therapeutic effects of zerumbone in an alkali-burned corneal wound healing model.

Cornea is an avascular transparent tissue. Ocular trauma caused by a corneal alkali burn induces corneal neovascularization (CNV), inflammation, and fibrosis, leading to vision loss. The purpose of this study was to examine the effects of Zerumbone (ZER) on corneal wound healing caused by alkali burns in mice. CNV was induced by alkali-burn injury in BALB/C female mice. Topical ZER (three times per day, 3µl each time, at concentrations of 5, 15, and 30µM) was applied to treat alkali-burned mouse corneas for 14 consecutive days. Histopathologically, ZER treatment suppressed alkali burn-induced CNV and decreased corneal epithelial defects induced by alkali burns. Corneal tissue treated with ZER showed reduced mRNA levels of pro-angiogenic genes, including vascular endothelial growth factor, matrix metalloproteinase-2 and 9, and pro-fibrotic factors such as alpha smooth muscle actin and transforming growth factor-1 and 2. Immunohistochemical analysis demonstrated that the infiltration of F4/80 and/or CCR2 positive cells was significantly decreased in ZER-treated corneas. ZER markedly inhibited the mRNA and protein levels of monocyte chemoattractant protein-1 (MCP-1) in human corneal fibroblasts and murine peritoneal macrophages. Immunoblot analysis revealed that ZER decreased the activation of signal transducer and activator of transcription 3 (STAT3), with consequent reduction of MCP-1 production by these cells. In conclusion, topical administration of ZER accelerated corneal wound healing by inhibition of STAT3 and MCP-1 production.

Suppression of VEGF-induced angiogenesis and tumor growth by Eugenia jambolana, Musa paradisiaca, and Coccinia indica extracts.

CONTEXT: Abnormal angiogenesis and evasion of apoptosis are hallmarks of cancer. Accordingly, anti-angiogenic and pro-apoptotic therapies are effective strategies for cancer treatment. Medicinal plants, namely, Eugenia jambolana Lam. (Myrtaceae), Musa paradisiaca L. (Musaceae), and Coccinia indica Wight & Arn. (Cucurbitaceae), have not been greatly investigated for their anticancer potential. OBJECTIVE: We investigated the anti-angiogenic and pro-apoptotic efficacy of ethyl acetate (EA) and n-butanol (NB) extracts of E. jambolana (seeds), EA extracts of M. paradisiaca (roots) and C. indica (leaves) with respect to mammary neoplasia. MATERIALS AND METHODS: Effect of extracts (2-200 µg/mL) on cytotoxicity and MCF-7, MDA-MB-231 and endothelial cell (EC) proliferation and in vitro angiogenesis were evaluated by MTT, 3[H]thymidine uptake and EC tube formation assays, respectively. In vivo tumour proliferation, VEGF secretion and angiogenesis were assessed using the Ehrlich ascites tumour (EAT) model followed by rat corneal micro-pocket and chicken chorioallantoic membrane (CAM) assays. Apoptosis induction was assessed by morphological and cell cycle analysis. RESULTS: EA extracts of E. jambolana and M. paradisiaca exhibited the highest cytotoxicity (IC50 25 and 60 µg/mL), inhibited cell proliferation (up to 81%), and tube formation (83% and 76%). In vivo treatment reduced body weight (50%); cell number (16.5- and 14.7-fold), secreted VEGF (∼90%), neoangiogenesis in rat cornea (2.5- and 1.5-fold) and CAM (3- and 1.6-fold) besides EAT cells accumulation in sub-G1 phase (20% and 18.38%), respectively. DISCUSSION AND CONCLUSION: Considering the potent anti-angiogenic and pro-apoptotic properties, lead molecules from EA extracts of E. jambolana and M. paradisiaca can be developed into anticancer drugs.

Antiangiogenic activity of a bevacizumab-loaded polyurethane device in animal neovascularization models.

PURPOSE: To evaluate the antiangiogenic activity of bevacizumab-loaded polyurethane using two animal models of neovascularization. METHODS: The percentage of blood vessels was evaluated in a chicken chorioallantoic membrane model (n=42) and in the rabbit cornea (n=24) with neovascularization induced by alkali injury. In each model, the animals were randomly divided into the groups treated with the bevacizumab-loaded polyurethane device, phosphate-buffered-saline (negative control) and bevacizumab commercial solution (positive control). Clinical examination, as well as histopathological and immunohistochemical evaluation, were performed in the rabbit eyes. Microvascular density in hot spot areas was determined in semi-thin sections of corneal tissue by hematoxylin-eosin staining and factor VIII immunohistochemistry. Immunohistochemical analysis was also performed to evaluate VEGF expression. RESULTS: In the evaluated models, the use of bevacizumab (Avastin®) and the bevacizumab-loaded polyurethane device led to similar results with regard to inhibition of neovascularization. In the chorioallantoic membrane model, the bevacizumab-loaded polyurethane device reduced angiogenesis by 50.27% when compared to the negative control group. In the rabbit model of corneal neovascularization, the mean density of vessels/field was reduced by 46.87% on analysis of factor VIII immunohistochemistry photos in the bevacizumab-loaded polyurethane device group as compared to the negative control (PBS) sections. In both models, no significant difference could be identified between the bevacizumab-loaded polyurethane device and the positive control group, leading to similar results with regard to inhibition of neovascularization. CONCLUSIONS: The present study shows that the bevacizumab-loaded polyurethane device may release bevacizumab and inhibit neovascularization similarly to commercial bevacizumab solution in the short-term.

Triptolide Suppresses Alkali Burn-Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression.

Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti-inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical-induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real-time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn-induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose-dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn-induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348-1355, 2017. © 2017 Wiley Periodicals, Inc.