epidermal-growth-factor and Corneal-Neovascularization

Histone deacetylases (HDACs) regulate gene transcription by modifying the acetylation level of histone and nonhistone proteins. In this study, we examined the effect of largazole, an inhibitor of class I HDACs, on inflammatory corneal angiogenesis. In a mouse model of alkali-induced corneal neovascularization (CNV), topical application of largazole to the injured corneas attenuated CNV. In addition, in vivo treatment with largazole down-regulated the expression of the pro-angiogenic factors VEGF, b-FGF, TGFβ1 and EGF but up-regulated the expression of the anti-angiogenic factors Thrombospondin-1 (Tsp-1), Tsp-2 and ADAMTS-1 in the injured corneas. Furthermore, largazole inhibited the expression of pro-angiogenic factors, migration, proliferation and tube formation by human microvascular endothelial cells (HEMC-1) in vitro. These data indicate that largazole has therapeutic potential for angiogenesis-associated diseases.

Topics: ADAM Proteins; Animals; Anti-Inflammatory Agents; Cell Line; Cell Movement; Cell Proliferation; Corneal Neovascularization; Depsipeptides; Endothelial Cells; Epidermal Growth Factor; Female; Fibroblast Growth Factors; Histone Deacetylase Inhibitors; Humans; Mice, Inbred BALB C; RNA, Messenger; Thiazoles; Thrombospondin 1; Thrombospondins; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Epidermal growth factor (EGF) is used to treat alkali-burned corneas. However, EGF-induced corneal angiogenesis, which is currently untreatable, is a side effect of this therapy. We therefore explored the role of the intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) in EGF-induced angiogenesis and tested whether KCa3.1 blockade can suppress EGF-induced corneal angiogenesis. The proliferation, migration and tube formation of HUVECs (human umbilical vein endothelial cells) in response to EGF, the MEK inhibitor PD98059 and the KCa3.1 inhibitor TRAM-34 were analyzed in vitro via MTT, cell counting, scratch and tube formation assays. The protein and mRNA levels of KCa3.1, phosphorylated-ERK (P-ERK), total-ERK (T-ERK), cyclin-dependent kinase 4 (CDK4), vimentin and MMP-2 were assessed via western blotting and RT-PCR. KCa3.1 and vimentin expression were also detected through immunofluorescence staining. Flow cytometry was performed to examine the cell cycle. Further, an in vivo murine alkali-burned cornea model was developed and treated with EGF and TRAM-34 eye drops to analyze the effect of these treatments on corneal healing and angiogenesis. The corneas were also analyzed by histological staining. The in vitro results showed that EGF induces the upregulation of KCa3.1 and P-ERK in HUVECs and that this upregulation is suppressed by PD98059. EGF stimulates proliferation, migration and tube formation in HUVECs, and this effect can be suppressed by TRAM-34. TRAM-34 also arrests HUVECs in the G1 phase of the cell cycle and downregulates CDK4, vimentin and MMP-2 in these cells. The in vivo results indicated that TRAM-34 suppresses EGF-induced corneal angiogenesis without affecting EGF-induced corneal wound healing. In summary, the upregulation of KCa3.1 may be crucial for EGF-induced angiogenesis through the MAPK/ERK signaling pathway. Thus, KCa3.1 may be a potential target for the treatment of EGF-induced corneal angiogenesis.

Topics: Animals; Blotting, Western; Burns, Chemical; Cell Movement; Cell Proliferation; Cornea; Corneal Neovascularization; Cyclin-Dependent Kinase 4; Disease Models, Animal; Epidermal Growth Factor; Eye Burns; Flavonoids; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Hydroxide; Up-Regulation; Vimentin; Wound Healing

To investigate the inhibitory effect of subconjunctival application of VEGF antibodies bevacizumab, ranibizumab, pegaptanib, and HER2 antibody trastuzumab on corneal neovascularization in a rat model of experimental corneal neovascularization.. Thirty male Wistar albino rats were included in the study. A chemical burn was induced in central cornea of one eye of the rats by a 75% silver nitrate and 25% potassium nitrate stick. Rats were randomly divided into five groups so that each group contained 6 subjects. Right after the chemical burn, 0.1 ml serum physiologic was injected subconjuctivally in control group (group 1). 1.25 mg/0.05 ml bevacizumab was injected in group 2; 1.2 mg/0.1 ml trastuzumab was injected in group 3; 0.5 mg/0.05 ml ranibizumab was injected in group-4; and 0.3 mg/0.1 ml pegaptanib was injected in group 5. On the 8th day of the experiment, rat corneas were photographed by digital photo-camera. Later, eyes of the sacrificed rats were enucleated and corneal speciements were histopathologically analyzed. The percentages of neovascularization on corneal photographs were examined with digital image analysis.. The percentage of corneal neovascularization in all treatment groups was found to be significantly lower than the control group (p < 0.05). Bevacizumab was found to be more effective than all other agents (p < 0.05). While the degree of inflammation and vascularization in bevacizumab and trastuzumab groups were significantly lower than the control group (p < 0.05), the difference was not significant in ranibizumab and pegaptanib groups (p > 0.05). In all treatment groups, fibroblast intensity was significantly lower than the control group. In terms of corneal thickness, no significant difference was observed between treatment and control groups (p > 0.05).. Bevacizumab, ranibizumab, pegaptanib, and trastuzumab were found effective for the inhibition of corneal NV. In our study we detected that the most effective agent was bevacizumab.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal, Humanized; Aptamers, Nucleotide; Bevacizumab; Conjunctiva; Corneal Neovascularization; Disease Models, Animal; Epidermal Growth Factor; Follow-Up Studies; Injections; Male; Ranibizumab; Rats; Rats, Wistar; Receptor, ErbB-2; Trastuzumab; Treatment Outcome; Vascular Endothelial Growth Factor A

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

Topics: Angiogenesis Inhibitors; Animals; Cell Movement; Chickens; Chorioallantoic Membrane; Collagen; Corneal Neovascularization; Drug Combinations; Endothelium, Vascular; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Heme Oxygenase-1; Humans; Laminin; Matrix Metalloproteinases; Mice; Molecular Mimicry; Phosphorylation; Prolactin; Proteoglycans; Rats; Rats, Sprague-Dawley; Ribonuclease, Pancreatic; Umbilical Veins; Vascular Endothelial Growth Factor A

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB-EGF when introduced subcutaneously into nude mice. HB-EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti-vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor-alpha (TGF-alpha) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT-PCR analysis demonstrated that VEGF165 mRNA levels were increased in vascular SMC 4-10-fold by 0.35-2 nM of HB-EGF, respectively. Our data suggest that HB-EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.

Topics: Animals; Antibodies; Cattle; Cell Communication; Cell Movement; Cells, Cultured; Cornea; Corneal Neovascularization; Endothelial Growth Factors; Endothelium, Vascular; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Lymphokines; Magnetic Resonance Imaging; Mice; Mice, Nude; Muscle, Smooth, Vascular; Neovascularization, Physiologic; Rabbits; RNA, Messenger; Skin; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

With a view toward possible future clinical application, we investigated whether recombinant human epidermal growth factor (hEGF) could induce corneal neovascularization. Ethylene-vinyl-acetate copolymer slow-release pellets containing either 250 ng, 500 ng or 1 microgram of hEGF or 250 ng of bovine serum albumin (BSA) were implanted into rabbit corneal stroma, and the corneas were examined by slitlamp biomicroscope for 3 weeks. The results indicated that less than 1 microgram of hEGF per pellet did not induce neovascularization in the cornea. However, when pellets containing 250 ng of basic fibroblast growth factor (bFGF) were implanted, corneal neovascularization toward these pellets occurred within 10 days. The same phenomenon occurred toward the 250 ng hEGF pellets embedded in the ipsilateral or contralateral cornea of the same animal, but was significantly less severe. When a pellet containing 250 ng bFGF was intramuscularly implanted in the animal's back, similar neovascularization was observed toward the pellets containing hEGF implanted in the same animal's cornea, but not toward implanted pellets containing BSA. These results suggest that less than 1 microgram of hEGF cannot initiate corneal angiogenesis, but can promote new vessel formation if the limbal vasculature is activated by a sprouting signal from a certain substance as trace amounts of bFGF.

Topics: Animals; Corneal Neovascularization; Corneal Stroma; Delayed-Action Preparations; Epidermal Growth Factor; Fibroblast Growth Factor 2; Rabbits; Recombinant Proteins