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》

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 《-》

Platelet-activating factor (PAF) induces corneal neovascularization and upregulates VEGF expression in endothelial cells.

Platelet-activating factor (PAF) is a potent proinflammatory mediator that accumulates in the cornea after injury and induces the expression of genes related to inflammation and wound healing. The current study was conducted to investigate the direct effect of PAF on corneal neovascularization and on the expression of angiogenic growth factors in vascular endothelial cells. Pellets containing carbamyl-PAF (cPAF) were implanted in corneas of wild-type or PAF-receptor (PAF-R)-knockout mice, and the progression of angiogenesis was monitored by microscope. In some experiments, mice were treated with a daily intraperitoneal injection of the PAF-R antagonist LAU8080. Migration assays of human umbilical cord vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) were performed in a Boyden chamber after addition of various concentrations of cPAF or bovine fibroblast growth factor (FGF-2). Cell proliferation was assessed by fluorescence-binding assay in the presence of cPAF or FGF-2 for 8 days. Vascular endothelial growth factor (VEGF) and FGF-2 expression was studied by RT-PCR and Northern- and Western-blot analyses in cells stimulated with cPAF at different concentrations and for different times. Six days after cPAF pellet implantation, there were new vessels growing from the limbus to the center of the cornea. The PAF-induced neovascularization was significantly reduced in PAF-R-knockout mice and in mice treated with the PAF antagonist. cPAF added to the lower well of the Boyden chamber produced a dose-dependent migration of HUVECs and HMVECs that was inhibited in cells preincubated with LAU8080 or with a VEGF-blocking antibody. In contrast, cPAF did not stimulate proliferation of endothelial cells. cPAF induced VEGF mRNA and protein expression but not FGF-2 expression in HUVECs and HMVECs. PAF stimulates corneal neovascularization by a receptor-mediated mechanism. Induction of VEGF expression and stimulation of vascular endothelial cell migration are initial events in PAF-promoted corneal angiogenesis.

Ma X ，Ottino P ，Bazan HE ，Bazan NG ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

[Inhibition of growth and metastasis of hepatocellular carcinoma by rapamycin: experiment with mice].

To investigate the effects of rapamycin (RPM) in inhibiting the growth and metastasis of hepatocellular carcinoma (HCC). Human HCC cells of the line MHCC97H with a high potential of metastasis were divided into 3 groups to be cultured with cyclosporine A (CsA) 100 ng/ml, RPM 10 ng/ml, or CsA + RPM for 48 hours. Flow cytometry was used to examine the apoptosis and cell cycle MTT method was used to examine the effect of RMP on the proliferation of the MHCC97H cells. RT-PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1alpha (HIF-1alpha), and transforming growth factor b (TGFb). Another MHCC97H cells were cultured in complete medium without RPM for 48 hours, then the protein expression of VEGF in the supernatant was detected by ELISA. Twenty-eight nude LCI-D20 mice were inoculated with human HCC cells and then divided into 4 groups to be fed with CsA (25 mg/kg), RPM (2 mg/kg), CsA + RPM, and normal saline (0.2 ml, as control group) for 35 days. Then the mice were killed to take the weight of inoculated tumor, measure the blood drug concentration, calculate the lung metastasis rate and number of metastatic foci, and observe pathology of the lung. CsA showed no effect on the cycle of the MHCC97H cells. The MHCC97H cells of the RPM and CsA + RPM groups arrested at the stage G(0)/G(1) (both P = 0.000). MMT method also showed that the proliferation of the MHCC97H cells in the RPM and CsA + RPM groups were blocked (P = 0.003 and P = 0.002). However, CsA did not influence the proliferation of the MHCC97H cells. Flow cytometry showed that RPM did not promote the apoptosis of the MHCC97H cells. RT-PCR showed that RPM down-regulated the mRNA expression of VEGF and HIF-1alpha (both P < 0.05), however, did not influence the mRNA expression of bFGF, TGFb, and TGFb. The VEGF protein level in the supernatant of the culture fluid of MHCC97H cells of the RPM group was (890.3 +/- 25.1) pg/ml, significantly lower than that of the control group, (1583.7 +/- 17.3) pg/ml (P = 0.000). The tumor inhibiting rate of the RPM group was 63.7%, not significantly different from that of the RPM + CsA group (80.9%, P = 1.000). The metastatic rate of the CsA and control groups were both 100% with a higher number of metastatic tumors in the CsA group (P = 0.046). RPM significantly inhibits the growth and metastasis of HCC. RPM-based immunosuppressive regimen may be of value in HCC patients receiving liver transplantation.

Wang Z ，Fan J ，Zhou J ，Wu ZQ ，Qiu SJ ，Yu Y ，Huang XW ，Tang ZY ... -  《-》

Inhibitory effect of canstatin in alkali burn-induced corneal neovascularization.

To examine the effect of recombinant canstatin protein on the corneal neovascularization (CorNV) in an alkaline burn-induced CorNV model. This study involved 50 C57BL/6 mice. CorNV was induced by an alkaline burn of the corneas with 1 N NaOH under general anesthesia. Beginning 24 h after CorNV induction, recombinant canstatin protein was administered intraperitoneally at 5 or 10 mg/kg body weight once a day for up to 14 days. CorNV was evaluated by slit-lamp microscopy. Growth factors and cytokines relating to neovascularization and inflammation in the corneas were evaluated by real-time polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry or ELISA. Recombinant canstatin protein significantly inhibited CorNV. Compared to the untreated or PBS-treated CorNV group, expression of vascular endothelial growth factor (VEGF) markedly decreased in the canstatin-treated group as detected by various methods. Western blotting and RT-PCR showed that the canstatin treatment inhibited the expression of hypoxia-inducible factor and VEGF. Day 7 revealed the greatest changes: ELISA assay showed that TNF-α also significantly decreased in canstatin-treated corneas. Recombinant canstatin protein suppressed experimental CorNV, suggesting that canstatin may serve as a useful angiogenic inhibitor for the treatment of neovascularization-related corneal diseases.

Wang Y ，Yin H ，Chen P ，Xie L ，Wang Y ... -  《-》