Background Delta-like 4 （DLL4） is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Not...Background Delta-like 4 （DLL4） is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization （CNV） is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV. Methods We utilized chemical hypoxia induced by 200 pmol/L CoOl2 to observe the expression of DLL4 in choroid-retinal endothelial cells （RF/6A cells）, which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA （siRNA）, mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used： one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated. Results The results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplexl targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplexl, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA （P 〈0.05）. Conclusions DLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.展开更多
目的:观察血管生成素-1/重组质粒(pEGFP/Ang-1)转染的大鼠骨髓间充质干细胞(BMSCs)对高浓度葡萄糖损伤猴脉络膜-视网膜内皮细胞(RF/6A)的保护作用。方法:以pEGFP/Ang-1转染BMSCs,倒置荧光显微镜下观察增强型绿色荧光蛋白的表达,再利用Tr...目的:观察血管生成素-1/重组质粒(pEGFP/Ang-1)转染的大鼠骨髓间充质干细胞(BMSCs)对高浓度葡萄糖损伤猴脉络膜-视网膜内皮细胞(RF/6A)的保护作用。方法:以pEGFP/Ang-1转染BMSCs,倒置荧光显微镜下观察增强型绿色荧光蛋白的表达,再利用Transwell模型,将转染的BMSCs与RF/6A共培养于高浓度葡萄糖培养基中。3d后MTT法检测RF/6A活力,Western blot检测磷酸化蛋白激酶B(phosphorylated protein kinase B,P-PKB)的表达,从而探讨转染pEGFP/Ang-1的BMSCs对高浓度葡萄糖培养中的RF/6A的保护作用。结果:成功转染pEGFP/Ang-1的BMSCs可见增强型绿色荧光蛋白表达,与转染pEGFP/Ang-1的BMSCs共培养的RF/6A细胞活力及P-PKB的表达均高于未转染组(均P<0.01),与对照组无明显差别(均P>0.05)。结论:质粒pEGFP/Ang-1转染的BMSCs对高糖环境中的RF/6A具有保护作用,其机制可能与P-PKB表达上调有关。展开更多
OBJECTIVE:To investigate the effects of a modified Dahuang Zhechong Pill(MDZP) on the angiogene sis of rhesus choroid-retina endothelial(RF/6A cells and its preliminary mechanism.METHODS:A 3-(4,5-dimethylthiazol-2-yl)...OBJECTIVE:To investigate the effects of a modified Dahuang Zhechong Pill(MDZP) on the angiogene sis of rhesus choroid-retina endothelial(RF/6A cells and its preliminary mechanism.METHODS:A 3-(4,5-dimethylthiazol-2-yl)-5-(3-car boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazol ium(MTS) method was used to assess the effect o a MDZP on RF/6A cell proliferation induced by vas cular endothelial growth factor(VEGF).Transwell in serts were used to assess the effect of the MDZP on RF/6A cell migration.Matrigel was used to asses the effect of the MDZP on the tube formation of RF 6A cells.Western blotting and quantitative re al-time reverse transcription polymerase chain reac tion(RT-PCR) were used to detect the protein and mRNA expression,respectively,of VEGF and matri metalloproteinase-2(MMP-2) in RF/6A cells treatedwith the MDZP.RESULTS:RF/6A cell proliferation induced by VEGF was inhibited by 0.2 mg/mL MDZP.At 0,12.5,25 and 50 mg/mL MDZP,the number of cells that migrated through Transwell membranes was 73.33± 4.51,61.33±4.04,28.67±6.66 and 17.67±4.16,respectively,and the number of tubes formed in Matrigel was 20.33±0.58,13.33±1.53,11.00±1.00 and 1.33±0.58,respectively.At 100 and 200 mg/mL MDZP,the protein and mRNA expression of VEGF and MMP-2 were inhibited in RF/6A cells.At 400 mg/mL MDZP,the expression of VEGF mRNA and MMP-2 protein were inhibited in RF/6A cells.CONCLUSIONS:MDZP inhibits the angiogenesis of RF/6A cells via the suppression of proliferation,migration and tube formation of RF/6A cells.Inhibition of the protein and mRNA expression of VEGF and MMP-2 in RF/6A cells may be an important mechanism.展开更多
基金This research was supported by grants fi-om the National Natural Science Foundation of China （No. 30901640）, New Teacher Foundation of China Science and Technology Development Center of Education Ministry （No. 20090142120068）, Natural Science Foundation of Hubei Provincial Government （No. 2008CDB155） and Chenguang Program of Wuhan Science and Technology Bureau （No. 200950431170）.
文摘Background Delta-like 4 （DLL4） is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization （CNV） is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV. Methods We utilized chemical hypoxia induced by 200 pmol/L CoOl2 to observe the expression of DLL4 in choroid-retinal endothelial cells （RF/6A cells）, which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA （siRNA）, mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used： one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated. Results The results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplexl targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplexl, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA （P 〈0.05）. Conclusions DLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.
文摘目的:观察血管生成素-1/重组质粒(pEGFP/Ang-1)转染的大鼠骨髓间充质干细胞(BMSCs)对高浓度葡萄糖损伤猴脉络膜-视网膜内皮细胞(RF/6A)的保护作用。方法:以pEGFP/Ang-1转染BMSCs,倒置荧光显微镜下观察增强型绿色荧光蛋白的表达,再利用Transwell模型,将转染的BMSCs与RF/6A共培养于高浓度葡萄糖培养基中。3d后MTT法检测RF/6A活力,Western blot检测磷酸化蛋白激酶B(phosphorylated protein kinase B,P-PKB)的表达,从而探讨转染pEGFP/Ang-1的BMSCs对高浓度葡萄糖培养中的RF/6A的保护作用。结果:成功转染pEGFP/Ang-1的BMSCs可见增强型绿色荧光蛋白表达,与转染pEGFP/Ang-1的BMSCs共培养的RF/6A细胞活力及P-PKB的表达均高于未转染组(均P<0.01),与对照组无明显差别(均P>0.05)。结论:质粒pEGFP/Ang-1转染的BMSCs对高糖环境中的RF/6A具有保护作用,其机制可能与P-PKB表达上调有关。
基金Supported by the Scientific Research Fund of the Shanghai Bureau of Public Health for Traditional Chinese Medicine,China (No. 2006ZD01)
文摘OBJECTIVE:To investigate the effects of a modified Dahuang Zhechong Pill(MDZP) on the angiogene sis of rhesus choroid-retina endothelial(RF/6A cells and its preliminary mechanism.METHODS:A 3-(4,5-dimethylthiazol-2-yl)-5-(3-car boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazol ium(MTS) method was used to assess the effect o a MDZP on RF/6A cell proliferation induced by vas cular endothelial growth factor(VEGF).Transwell in serts were used to assess the effect of the MDZP on RF/6A cell migration.Matrigel was used to asses the effect of the MDZP on the tube formation of RF 6A cells.Western blotting and quantitative re al-time reverse transcription polymerase chain reac tion(RT-PCR) were used to detect the protein and mRNA expression,respectively,of VEGF and matri metalloproteinase-2(MMP-2) in RF/6A cells treatedwith the MDZP.RESULTS:RF/6A cell proliferation induced by VEGF was inhibited by 0.2 mg/mL MDZP.At 0,12.5,25 and 50 mg/mL MDZP,the number of cells that migrated through Transwell membranes was 73.33± 4.51,61.33±4.04,28.67±6.66 and 17.67±4.16,respectively,and the number of tubes formed in Matrigel was 20.33±0.58,13.33±1.53,11.00±1.00 and 1.33±0.58,respectively.At 100 and 200 mg/mL MDZP,the protein and mRNA expression of VEGF and MMP-2 were inhibited in RF/6A cells.At 400 mg/mL MDZP,the expression of VEGF mRNA and MMP-2 protein were inhibited in RF/6A cells.CONCLUSIONS:MDZP inhibits the angiogenesis of RF/6A cells via the suppression of proliferation,migration and tube formation of RF/6A cells.Inhibition of the protein and mRNA expression of VEGF and MMP-2 in RF/6A cells may be an important mechanism.
