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

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. 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. 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. Curcumin may be useful as an angiogenic inhibitor in the treatment of corneal diseases that show neovascularization.

Bian F ，Zhang MC ，Zhu Y 《-》

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

To examine the effect of rapamycin on the proliferation and the migration of human umbilical vein endothelial cells (HUVECs) and on the corneal neovascularization in the corneal alkaline burn murine model. HUVEC proliferation, migration, and apoptosis were examined after treatment with rapamycin. The effect of rapamycin on the mRNA expression of FK506 binding protein (FKBP)-12 and mammalian target of rapamycin (mTOR) was also evaluated in vitro. Corneal neovascularization was induced in vivo by an alkaline burn of the cornea with 1 N NaOH on BALB/c mice. Rapamycin was given intraperitoneally at 2 mg/kg body weight once a day for 12 days after the corneal alkaline burn. Growth factors and cytokines related with neovascularization and inflammation were evaluated in the corneal tissue and the peripheral blood by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The corneal neovascularization was evaluated by a slit lamp biomicroscopy. Rapamycin at the concentration of 1000 ng/mL for >48 hours' exposure significantly inhibited the growth of HUVECs. The double chamber assay showed that rapamycin dramatically inhibited the migration of HUVECs at concentrations of 10 and 100 ng/mL and that these concentrations did not affect endothelial cell growth. When TUNEL assays were performed, the number of apoptotic cells increased 1.9-, 2.1-, and 2.6-fold compared with the control at 10, 100, and 1000 ng/mL, respectively, of rapamycin at 48 hours of exposure. RT-PCR showed that the expression of mTOR was suppressed in the HUVECs after rapamycin treatment; however, FKBP-12 expression was not affected. Among the angiogenic factors, gene expression of substance P and hypoxia inducible factor (HIF)-1 alpha was inhibited by rapamycin earlier (1-3 days), with vascular endothelial growth factor (VEGFR)-1 gene expression being suppressed for the first 7 days in the corneal tissue. The protein level of substance P and vascular endothelial growth factor (VEGF) was significantly decreased--more in mice treated with rapamycin than the control mice--as shown by ELISA assay of peripheral blood. Furthermore, rapamycin significantly inhibited corneal neovascularization in the alkaline-burned cornea. Rapamycin strongly inhibited HUVEC migration at doses that did not cause cytotoxicity and apoptosis in this in vitro model. Rapamycin also suppressed corneal neovascularization, possibly by inhibiting proinflammatory cytokines, as shown by the in vivo study. Therefore, rapamycin may be useful as an angiogenic regulator in the treatment of corneal diseases that manifest with neovascularization.

Kwon YS ，Hong HS ，Kim JC ，Shin JS ，Son Y ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

The effect of curcumin on corneal neovascularization in rabbit eyes.

To investigate the anti-angiogenic effect of topical curcumin on corneal neovascularization in a rabbit model. One week after suturing, six eyes were treated with balanced salt solution (BSS) (group A), and six eyes were treated with curcumin 40, 80, or 160 micromol/L (groups B, C, and D, respectively), topically two times a day. After one week, light microscopy was used to analyze corneal neovascularization. The concentration of vascular endothelial growth factor (VEGF) mRNA in the corneal tissue was measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the activation of NF-kappaB was examined by immunofluorescent staining. Seven days after treatment, the sizes of the neovascularized areas were significantly reduced in groups B (50.1% +/- 6.7%), C (43.2% +/- 8.1%), and D (29.5% +/- 7.8%) compared with group A (69.5% +/- 1.5%) (p < 0.05). The corneal VEGF mRNA levels were significantly lower in groups C and D than they were in group A (p < 0.05). Immunofluorescent staining showed that phospho-NF-kappaB staining of the corneal tissue was weaker in group C than it was in groups A and B. Topical application of curcumin was useful in reducing experimental corneal neovascularization and can be used to inhibit angiogenesis in the cornea.

Kim JS ，Choi JS ，Chung SK 《-》

Plasminogen kringle 5 inhibits alkali-burn-induced corneal neovascularization.

Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. The purpose of the present study was to evaluate the therapeutic effect of K5 on alkali-burn-induced corneal neovascularization (NV) and to investigate its mechanism of action. Corneal NV was induced in rabbits by NaOH. The rabbits received eye drops containing K5 or vehicle alone, four times per day. Corneal NV and inflammation were monitored every other day with a slit lamp microscope, and the length of the vessels in the cornea and the area of NV were measured. Vascular endothelial growth factor (VEGF) was determined by immunohistochemical and Western blot analyses. The TUNEL assay was used to assess the apoptosis of endothelial cells. The effects of K5 on primary bovine aortic endothelial cells (BAECs) were determined by MTT assay, flow cytometry, transmission electron microscopy, and DNA fragmentation assay. Alkali-burn-induced progressive corneal NV and inflammation in the cornea. K5 delayed the onset of corneal NV (P < 0.05) and decreased NV areas (P < 0.05) in a dose-dependent manner. K5 treatment, after the formation of corneal NV, induced regression of newly formatted vessels in the cornea. K5 decreased the inflammatory index in the corneas at different time points after the alkali burn. Corneal VEGF levels were reduced by K5 treatment. K5 inhibits proliferation and induces apoptosis in BAECs. Topical application of K5 may have therapeutic potential for the chemical burn-induced corneal NV and inflammation. The inhibitory effect of K5 on corneal NV may be by downregulation of VEGF expression.

Zhang Z ，Ma JX ，Gao G ，Li C ，Luo L ，Zhang M ，Yang W ，Jiang A ，Kuang W ，Xu L ，Chen J ，Liu Z ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

The expression of nuclear factor kappa B in inflammation-induced rat corneal neovascularization.

To investigate the involvement of the nuclear-transfactor-kappa B (NF-kappa B) in the rat model of inflammation-induced corneal neovascularization (CNV). The CNV model in Sprague-Dawley rats was induced by alkaline cauterization of the central cornea. The corneas were examined by a slit lamp microscope. NF-kappa B was assayed by Western blot. Vascular endothelial growth factor (VEGF) protein was evaluated by immunohistochemistry. VEGF mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). Morphologically, the CNV was shown on the second day after cautery. In corneas after cautery, NF-kappa B protein increased 6 hours after cautery, peaked 4 days after cautery, and decreased to near baseline by day 14. VEGF protein and mRNA increased gradually in the early stage after cautery, reached the highest level on the fourth day, and then decreased to near baseline slowly after 7 days. The activated NF-kappa B was up-regulated in the early stage of CNV of rat induced by cauterization, which suggests it may participate in the pathogenesis of wound healing, inflammation, and neovascularization processes in the cornea.

Zhang MC ，Wang Y ，Yang Y 《OCULAR IMMUNOLOGY AND INFLAMMATION》