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.

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.

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.

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.

Curcumin inhibits angiogenesis by up-regulation of microRNA-1275 and microRNA-1246: a promising therapy for treatment of corneal neovascularization.

OBJECTIVE: Curcumin (capable of inhibiting angiogenic growth of human umbilical vein endothelial cells [HUVECs]), can be employed in vitro as a model of pathogenesis of corneal neovascularization (CRNV). The aim of this study was to explore regulatory mechanisms of microRNA (miR) levels after curcumin treatment. MATERIALS AND METHODS: Expression profiles of miRs in curcumin-treated HUVECs were investigated by miR microassay. Specific mimics and inhibitors of miR-1275 or miR-1246 were transfected into HUVECs. Then, their target genes, vascular endothelial growth factor B (VEGFB) and nuclear transcription factor kappa B acting protein (NKAP) were detected by quantitative real-time PCR, Western blotting assay or immunofluorescence assay. Cell proliferation and cell cycle parameters were measured with the help of CCK-8 assay and flow cytometry. RESULTS: MiR-1275 and miR-1246 expression levels were up-regulated by curcumin. Administration of the specific mimics and inhibitors of the two miRs led to significant changes in expression of VEGFB and NKAP as well as the indicators related to angiogenesis. Anti-angiogenic effect of curcumin depended on expression patterns of the two miRs in that inhibition of either miR interfered with the effect of curcumin. Furthermore, overexpression of NKAP interrupted effects of curcumin on the cells. CONCLUSION: Collectively, our findings demonstrate that curcumin inhibited HUVEC proliferation by up-regulation of miR-1275 and miR-1246.

Therapeutic effects of a novel PIGF-1 derived peptide, ZY-1, on corneal neovascularization in vitro and in vivo.

Corneal neovascularization (NV) is one of the major sight-threatening pathological changes caused by corneal diseases. Current therapeutics generate various adverse effects. Small peptides derived from endogenous protein display certain advantages. This study aims to evaluate the anti-angiogenic effect and molecular mechanism of a novel peptide ZY-1, derived from placental growth factor-1 (PlGF-1), on corneal NV by topical administration, and to investigate its safety profile after long-term treatment. CCK-8 assay and tube formation assay were used to evaluate the effect of ZY-1 on human umbilical vein endothelial cells (HUVECs). The anti-angiogenic effect of topical ZY-1 was estimated in a rat model of alkali burn induced corneal NV. The safety profile of topical ZY-1 was analyzed by CCK-8 assay, tear film break-up time (BUT), and histological examination. Firstly, we found that ZY-1 co-localized with membrane vascular epithelial growth factor receptor-1 (VEGFR-1) and effectively inhibited VEGF/PlGF-1 induced proliferation and tube formation of HUVECs. The topical ZY-1 administration efficiently inhibited alkali-burn induced corneal NV, while it did not show any significant effect on human corneal epithelial cell (HCEC) proliferation, as well as the functionality and morphology of cornea and conjunctiva. Our findings suggested that topical administration of ZY-1 could effectively and safely inhibit corneal NV partially through competing for VEGFR-1 binding, and it would be a promising alternative for ocular topical anti-angiogenic therapy.

[Neovascularization corneal regression in patients treated with photodynamic therapy with verteporfin]. / Regresión en la neovascularización corneal en pacientes tratados con terapia fotodinámica con verteporfirina.

BACKGROUND: Corneal neovascularization is a vision-threatening condition usually associated with inflammatory or infectious disorders of the ocular surface. One current treatment is photodynamic therapy, which uses a photosensitizer to occlude the vessel, is successfully produced microvascular thrombosis with minimal damage to surrounding normal tissue. The aim of this article is to quantitatively determine the percentage of regression of corneal neovascularization experienced by patients treated with photodynamic therapy with verteporfin. METHODS: A before and after treatment; experimental, analytical, prospective and longitudinal. RESULTS: Of the 25 new vessels analyzed, 8 glasses (32 %) had total occlusion one month after, 15 vessels (60 %) had a partial occlusion in the range of 15.3 to 85.1 %, and 2 vessels (8 %) worsening in corneal vascularization. The mean area of corneal neovascularization decreased significantly a 70 % from 0.147 ± 0.118 mm2 to 0.045 ± 0.046 mm2 (p < 0.0005) after photodynamic therapy. No side efects were reported. CONCLUSIONS: Photodynamic therapy with verteporfin is a safe and effective method of reducing corneal neovascularization and can be used to inhibit angiogenesis in the cornea.

Introducción: la neovascularización corneal es una condición que amenaza la visión, por lo general, se asocia a trastornos inflamatorios o infecciosos de la superficie ocular. Uno de los tratamientos actuales es la terapia fotodinámica, el uso de un fotosensibilizador para ocluir los vasos ha producido con éxito la trombosis microvascular con un daño mínimo al tejido normal circundante. El objetivo de este artículo es determinar cuantitativamente el porcentaje de regresión en la neovascularización corneal que presentan los pacientes tratados con terapia fotodinámica con verteporfirina. Métodos: estudio de tratamiento de antes y después; experimental, analítico, prospectivo y longitudinal. Resultados: de los 25 neovasos analizados, 8 vasos (32 %) presentaron al mes una oclusión total del 100 %, 15 vasos (60 %) una oclusión parcial en el rango de 15.3 al 85.1 %, y 2 vasos (8 %) empeoramiento en la vascularización corneal. El promedio del área de neovascularización corneal disminuyó significativamente en un 70 % de 0.147 ± 0.118 mm2 a 0.045 ± 0.046 mm2, (p < 0.0005) posterior a la terapia fotodinámica. No se reportaron efectos adversos. Conclusiones: la terapia fotodinámica con verteporfina es un procedimiento seguro y efectivo para reducir la neovascularización de la córnea y puede utilizarse para inhibir la angiogénesis en la córnea.

[Vascular morphological and microdensity changes of corneal neovascularization induced by topical bevacizumab and sunitinib in an animal model]. / Cambios en la morfología y la microdensidad neovascular corneal inducidos tras la administración tópica de bevacizumab y sunitinib en un modelo animal.

OBJECTIVE: To evaluate the effects of topical bevacizumab and topical sunitinib on vascular microdensity and morphology of corneal neovascularization (NV). METHODS: A total of 33 rabbits were distributed into 3 groups: group 1 (control; n=11): saline; group 2 (n=11): bevacizumab 5mg/ml; and group 3 (n=11): sunitinib 0.5mg/ml. A corneal NV model was used, based on sutures in the right eye of each rabbit. Each treatment was administered topically 3 times daily for 14 days. Corneas were then processed for the study of vascular microdensity (6 eyes) and vascular morphology analysis (5 eyes) using enzymatic staining histological techniques RESULTS: The vascular response in group 3 was limited to small-sized tree formations with various vascular axes compared with the extensive, lush and directional corneal NV of group 1 and 2. In the histological sections near the limb, there were no differences in vascular microdensity studies between the three groups. However, the mean sectional area of vessels (MSAV) in group 3 was 41.88% lower than in group 1 and 19.19% lower than in group 2. In distal sections, there were no differences between groups 1 and 2. However, group 3 was characterized by absence of vessels. CONCLUSIONS: Bevacizumab produced no changes in the morphology of the vessels or the vascular microdensity. Sunitinib reduced the size of the new vessels and induced changes in the vascular tree.

Anti-angiogenic effects of purine inhibitors of cyclin dependent kinases.

Small molecular inhibitors of Cyclin dependent kinases (Cdks) are currently being developed as anticancer therapeutics due to their antiproliferative properties. The purine Cdk specific inhibitor (R)-roscovitine (seliciclib, CYC202) represents one of the most promising of these compounds. It is currently evaluated in clinical trials concerning cancer therapy. Recently, we have shown that roscovitine exerts potent antiangiogenic effects and elucidated Cdk5 as a new player in angiogenesis. These findings introduce Cdk5 as novel target for antiangiogenic therapy, and Cdk5 inhibitors as an attractive therapeutic approach. Here, we present the antiangiogenic profile of 15 derivatives of roscovitine in vitro and in vivo and provide structure activity relationships of the roscovitine analogs. The (S)-isomer LGR561 and the respective (R)- and (S)-isomers LGR848 and LGR849 strongly inhibited proliferation and cell cycle progression, induced cell death, and reduced migration of endothelial cells in vitro. In comparison to roscovitine, these compounds showed an increased potency to inhibit Cdk2, Cdk5, Cdk7, and Cdk9. By analyzing the effects of LGR561, LGR848, and LGR849 on endothelial cell tube formation, mouse aortic ring sprouting, angiogenesis in the chick chorioallantoic membrane, and neovessel formation in the mouse cornea, we elucidate the two (S)-isomers LGR561 and LGR849 as highly potent inhibitors of angiogenesis. This study provides first information on how to modify roscovitine to develop Cdk inhibitors with increased antiangiogenic activity and suggests the application of existing and the development of new Cdk inhibitors to inhibit both, cancer cell proliferation and angiogenesis.

Exacerbated corneal inflammation and neovascularization in the HO-2 null mice is ameliorated by biliverdin.

Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. An 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827,600+/-129,000 vs. 294,500+/-57,510; p<0.05), neovessels measured by vital microscopy (21.91+/-1.05 vs. 12.77+/-1.55 mm; p<0.001) 4 days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.

Inhibitory effect of curcumin on corneal neovascularization in vitro and in vivo.

PURPOSE: To examine the effect of curcumin on the proliferation and the migration of human umbilical vein endothelial cells (HUVECs) and on the corneal neovascularization in the corneal alkaline burn rat model. METHODS: HUVEC proliferation, migration, and apoptosis were examined after treatment with various concentrations of curcumin. The effect of curcumin on the activation of nuclear factor-kappaB (NF-kappaB) was measured by an electrophoretic mobility shift assay (EMSA) in vivo. Corneal neovascularization was induced in vivo by an alkaline burn of the cornea in Sprague-Dawley rats. After topical drug treatments with curcumin, vascular endothelial growth factor (VEGF) was evaluated in the corneal tissue by reverse transcription polymerase chain reaction and by immunohistochemistry. Corneal neovascularization was evaluated by slit-lamp biomicroscopy. RESULTS: Curcumin at a concentration of 40 micromol/l for 24 h significantly inhibited the growth of HUVECs. The Boyden microchamber assay showed that curcumin dramatically inhibited the migration of HUVECs at a concentration of 40 micromol/l. When TUNEL assays were performed, the number of apoptotic cells increased after treated with curcumin. The EMSA revealed that curcumin inhibits the activation of NF-kappaB in HUVECs. The expression of VEGF in the corneal tissues was inhibited by curcumin on days 7 and 14 after alkaline burn. CONCLUSIONS: Curcumin may be useful as an angiogenic inhibitor in the treatment of corneal diseases that show neovascularization.

The effects of the subconjunctival injection of bevacizumab (Avastin) on angiogenesis in the rat cornea.

The purpose of this study was to evaluate the effects of the use of the subconjunctival injection of bevacizumab (Avastin) on angiogenesis in the rat cornea. Corneas of 20 Wistar male rats were cauterized with silver nitrate crystal. Animals were divided in four groups: control group (GC) that received subconjunctivally 0.02 ml of 0.9% saline solution on the day of the lesion; group GO that received subconjunctivally 0.02 ml of bevacizumab just after the lesion; group G3 that received bevacizumab on day 3 and group G5 that received bevacizumab on day 5 after lesion. Animals were euthanized on day 7. The newly formed vessels were quantified after China Ink perfusion and photographs were obtained and analyzed in a computerized system (Image Pro-Plus(R)). In the control group, neovascularization covered 53.56% +/- 15.11 (mean +/- SD) of the corneal surface, compared with 35.57% +/- 18.80 (mean +/- SD) in the G0 group, 30.60%+/-11.82 (mean+/-SD) in the G3 and 35.86%+/-0.07 (mean+/-SD) in the G5. The results showed an inhibition of angiogenesis when the control group was compared with all treated groups. These results suggest that subconjunctival injection of bevacizumab is able to inhibit corneal angiogenesis independently of the day of treatment.

The effects of the subconjunctival injection of bevacizumab (Avastin®) on angiogenesis in the rat cornea

The purpose of this study was to evaluate the effects of the use of the subconjunctival injection of bevacizumab (Avastin®) on angiogenesis in the rat cornea. Corneas of 20 Wistar male rats were cauterized with silver nitrate crystal. Animals were divided in four groups: control group (GC) that received subconjunctivally 0.02 ml of 0.9 percent saline solution on the day of the lesion; group GO that received subconjunctivally 0.02 ml of bevacizumab just after the lesion; group G3 that received bevacizumab on day 3 and group G5 that received bevacizumab on day 5 after lesion. Animals were euthanized on day 7. The newly formed vessels were quantified after China Ink perfusion and photographs were obtained and analyzed in a computerized system (Image Pro-Plus®). In the control group, neovascularization covered 53.56 percent ± 15.11 (mean ± SD) of the corneal surface, compared with 35.57 percent ± 18.80 (mean ± SD) in the G0 group, 30.60 percent±11.82 (mean±SD) in the G3 and 35.86 percent±0.07 (mean±SD) in the G5. The results showed an inhibition of angiogenesis when the control group was compared with all treated groups. These results suggest that subconjunctival injection of bevacizumab is able to inhibit corneal angiogenesis independently of the day of treatment.

O objetivo deste estudo foi avaliar os efeitos da aplicação subconjuntival de bevacizumab (Avastin®) na angiogênese corneal em ratos. Vinte ratos Wistar, machos, foram submetidos a cauterização química com cristal de nitrato de prata. Os animais foram divididos em 4 grupos: O grupo controle (GC), recebeu injeção de 0,02 ml de solução fisiológica pela via subconjuntival no momento da lesão. O grupo G0 recebeu 0,02 ml de bevacizumab (Avastin®) imediatamente depois da lesão. O grupo G3 recebeu 0,02 ml de bevacizumab no terceiro dia após a lesão.O grupo G5 recebeu 0,02 ml de bevacizumab no quinto dia após a lesão. Os animais foram eutanasiados 7 dias após a cauterização. Os vasos neoformados foram quantificados após preenchimento do leito vascular com Tinta da China e imagens foram obtidas e analisadas em sistema computadorizado (Image Pro-Plus®). No grupo controle a neovascularização ocupou 53,56 por cento ± 15,11 (média ± DP) da superfície corneal comparando a 35,57 por cento ± 18,80 no grupo G0, 30,60 por cento ± 11,82 (média ± DP) no G3 e 35,86 por cento ± 0,07 (média ± DP) no G5. Os resultados mostram uma inibição da angiogênese quando se compara GC com os grupos tratados. Os resultados sugerem que a injeção subconjuntival de Bevacizumab é capaz de inibir a angiogênese corneal independentemente do dia de aplicação.

Pfaffia paniculata (Brazilian ginseng) methanolic extract reduces angiogenesis in mice.

Pfaffia paniculata (Brazilian ginseng) roots have been indicated for the treatment of several diseases. Our studies have shown that P. paniculata roots present antineoplastic effects and cancer chemopreventive activity in a mouse hepatocarcinogenesis model. The purpose of this study was to investigate the effects of the Brazilian ginseng on corneal angiogenesis in mice. We first conducted a toxicological study employing 250, 500, or 1000mg/kg/day of the methanolic extract of P. paniculata roots by gavage to BALB/c mice. Animals did not lose weight during the treatment nor presented histopathological alterations. The effect of this root on angiogenesis in the cornea of BALB/c mice was then assessed. Male mice were treated, by gavage, once a day, with doses of 250, 500, or 1000mg/kg of methanolic extract of P. paniculata powdered root for 10 days; filtered water was used as control. Corneal cauterization was accomplished by the contact of a silver nitrate crystal on the central area of the cornea, in the 5th day of treatment with P. paniculata, which continued thereafter; the animals were euthanized on the 6th day after cauterization. Newly formed blood vessels were filled with India ink, and the corneas were routinely processed. Blood vessels were quantified in an image analysis system. A smaller total area of neovascularization in the mouse cornea was observed in animals treated with 1000mg/kg of the methanolic extract of P. paniculata. These results indicate an antiangiogenic effect of this extract. The mechanisms of this antiangiogenic activity of P. paniculata should be further investigated.

Inhibition of experimental corneal neovascularisation by bevacizumab (Avastin).

AIM: To evaluate the effect of topically administered bevacizumab (Avastin) on experimental corneal neovascularisation in rats. METHODS: Silver nitrate sticks (75% silver nitrate, 25% potassium nitrate) were used to perform chemical cauterisation on the corneas of 16 eyes from 16 male Long Evans rats. For the following 7 days, the 10 eyes in the treatment group were instilled with bevacizumab 4 mg/ml drops twice daily, whereas the 6 eyes in the control group received placebo (normal saline drops twice daily). Digital photographs of the cornea were analysed to determine the area of cornea covered by neovascularisation as a percentage of the total corneal area. RESULTS: In the bevacizumab-treated eyes, neovascularisation covered, on average, 38.2% (15.5%) (mean (SD)) of the corneal surface compared with 63.5% (5.0%) in the control group (p<0.02, Mann-Whitney U test). CONCLUSION: Topically administered bevacizumab (Avastin) at a concentration of 4 mg/ml limits corneal neovascularisation following chemical injury in the male Long Evans rat model.