BACKGROUND： Vascular endothelial growth factor （VEGF） acts as ＂molecular bridge＂ following ischemic stroke to improve and restore blood supply and reduce infarction volume. Clinical studies have demonstrated the ...BACKGROUND： Vascular endothelial growth factor （VEGF） acts as ＂molecular bridge＂ following ischemic stroke to improve and restore blood supply and reduce infarction volume. Clinical studies have demonstrated the efficacy of Rhizoma Chuanxiong （Chuanxiong） in the treatment of ischemic cerebrovascular diseases. However, whether it promotes endogenous VEGF expression in ischemic stroke remains unknown. OBJECTIVE： To investigate the influence of Rhizoma Chuanxiong on VEGF production in vitro cultured human umbilical vein endothelial cells and on VEGF expression in ischemic cerebral tissues to explore its role in angiogenesis. DESIGN, TIME AND SETTING： In vitro basic comparison of traditional Chinese drug-containing serum pharmacology; in vivo randomized, controlled, animal experiment. This study was performed at the Medical Laboratory of West China Hospital, Sichuan University between December 2002 and April 2004. MATERIALS： Two Chinese rabbits were selected. One was intragastrically perfused with 5.8 g/kg Rhizoma Chuanxiong extract twice per day for three consecutive days to prepare Rhizoma Chuanxiong extract-containing serum. The remaining rabbit was intragastrically perfused with the same volume of normal saline twice per day for three consecutive days. Rhizoma Chuanxiong extract was provided by Beijing Traditional Chinese Medicine Research Institute, predominantly composed of ligustrazine, ligustilide, and ferulic acid. ChemiKineTM human VEGF Kit was purchased from Chemicon, USA; mouse anti-VEGF monoclonal antibody and biotin-goat anti-mouse IgG were purchased from Santa Cruz Biotechnology. Inc., USA. METHODS： （1） In vitro experiment： in vitro cultured human umbilical vein endothelial cells were separately incubated in rabbit serum with 10% Rhizoma Chuanxiong extract, normal medium without rabbit serum, and rabbit serum without Rhizoma Chuanxiong extract （blank control）. In addition, cells from the three groups were incubated under normoxia （5% CO2, 95% air） and hypoxia （1% 02, 5% CO2, 94% N2）, respectively, for 24 hours. （2） In vivo experiment： a total of 4/44 Sprague Dawley rats were selected for the sham-operated group （no occlusion）, and the remaining rats were used to establish a cerebral ischemiaJreperfusion model by suture occlusion. 32 animals with ischemia/reperfusion injury were randomly divided into treatment and model groups, with 16 rats in each group. Both groups were intraperitoneally infused with 0.58 g/kg Rhizoma Chuanxiong extract and normal saline two hours following reperfusion. The sham-operated group was administrated normal saline. Animals were treated with saline or Chuanxiong extracts （0.58 g/kg） twice per day for three consecutive days. MAIN OUTCOME MEASURES： In vitro experiment： VEGF concentration was detected in each group by enzyme-linked immunosorbent assay. In vivo experiment： behavioral alterations of rats were evaluated by neurological function scale; infarct volume was assessed by hematoxylin-eosin staining; VEGF protein expression in the infarct regions was determined by immunohistochemistry. RESULTS： （1） VEGF levels were similar between the three groups under normexic condition （P 〉 0.05）; while hypoxia induced VEGF production （P 〈 0.01 ）. VEGF levels in the drug-containing serum group were particularly higher compared with the other groups （P 〈 0.01）. （2） Compared with normal saline treatment, Rhizoma Chuanxiong extract significantly improved the neurological scale and reduced cerebral infarct volumes （P〈 0.05）. The percent of VEGF-positive cells was significantly greater than the model group （P 〈 0.05）. The sham-operated group exhibited normal neurological function, with no infarct focus. CONCLUSION： Rhizoma Chuanxiong extract-containing rabbit serum effectively promoted cultured VEGF production under hypoxia. Rhizoma Chuanxiong extract upregulated VEGF expression in the infarct region, improved neurological function, and reduced infarct size.展开更多
AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cho...AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.展开更多
Background： Endothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated...Background： Endothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated the effect of transplanting endothelial progenitor cells （EPCs） derived from human early fetal aortas in rat injured arteries. Methods： The carotid arterial endothelium of Sprague-Dawley rats was damaged by dilatation with a 1.5 F balloon catheter, and then EPCs derived from human early fetal aortas （〈14 weeks） were injected into the lumen of the injured artery in transplanted rats, with an equal volume of normal saline injected into control rats. Rats were sacrificed at 2 and 4 weeks after treatment and transplanted cells were identified by immunohistochemical staining with anti-human CD31 and anti-human mitochondria antibodies. Arterial cross-sections were analyzed by pathology, immunohistochemistry, and morphometry. Results： Green fluorescence-labeled EPCs could be seen in the endovascular surface of balloon-injured vessels after transplantation. The intimal area and intimal/medial area ratio were significantly smaller in the transplanted group than in the control （P 〈 0.05） and the residual lumen area was larger （P 〈 0.05）. After EPC transplantation, a complete vascular endothelial layer was formed, which was positive for human yon Willebrand factor after immunohistochemical staining, and immunohistochemical staining revealed many CD31- and mitochondria-positive cells in the re-endothelialized endothelium with EPC transplantation but not control treatment. Conclusion： EPCs derived from human early fetal aorta were successfully transplanted into injured vessels and might inhibit neointimal hyperplasia after vascular injury.展开更多
基金the Major State Basic Research Development Program of China,No.G1999054402
文摘BACKGROUND： Vascular endothelial growth factor （VEGF） acts as ＂molecular bridge＂ following ischemic stroke to improve and restore blood supply and reduce infarction volume. Clinical studies have demonstrated the efficacy of Rhizoma Chuanxiong （Chuanxiong） in the treatment of ischemic cerebrovascular diseases. However, whether it promotes endogenous VEGF expression in ischemic stroke remains unknown. OBJECTIVE： To investigate the influence of Rhizoma Chuanxiong on VEGF production in vitro cultured human umbilical vein endothelial cells and on VEGF expression in ischemic cerebral tissues to explore its role in angiogenesis. DESIGN, TIME AND SETTING： In vitro basic comparison of traditional Chinese drug-containing serum pharmacology; in vivo randomized, controlled, animal experiment. This study was performed at the Medical Laboratory of West China Hospital, Sichuan University between December 2002 and April 2004. MATERIALS： Two Chinese rabbits were selected. One was intragastrically perfused with 5.8 g/kg Rhizoma Chuanxiong extract twice per day for three consecutive days to prepare Rhizoma Chuanxiong extract-containing serum. The remaining rabbit was intragastrically perfused with the same volume of normal saline twice per day for three consecutive days. Rhizoma Chuanxiong extract was provided by Beijing Traditional Chinese Medicine Research Institute, predominantly composed of ligustrazine, ligustilide, and ferulic acid. ChemiKineTM human VEGF Kit was purchased from Chemicon, USA; mouse anti-VEGF monoclonal antibody and biotin-goat anti-mouse IgG were purchased from Santa Cruz Biotechnology. Inc., USA. METHODS： （1） In vitro experiment： in vitro cultured human umbilical vein endothelial cells were separately incubated in rabbit serum with 10% Rhizoma Chuanxiong extract, normal medium without rabbit serum, and rabbit serum without Rhizoma Chuanxiong extract （blank control）. In addition, cells from the three groups were incubated under normoxia （5% CO2, 95% air） and hypoxia （1% 02, 5% CO2, 94% N2）, respectively, for 24 hours. （2） In vivo experiment： a total of 4/44 Sprague Dawley rats were selected for the sham-operated group （no occlusion）, and the remaining rats were used to establish a cerebral ischemiaJreperfusion model by suture occlusion. 32 animals with ischemia/reperfusion injury were randomly divided into treatment and model groups, with 16 rats in each group. Both groups were intraperitoneally infused with 0.58 g/kg Rhizoma Chuanxiong extract and normal saline two hours following reperfusion. The sham-operated group was administrated normal saline. Animals were treated with saline or Chuanxiong extracts （0.58 g/kg） twice per day for three consecutive days. MAIN OUTCOME MEASURES： In vitro experiment： VEGF concentration was detected in each group by enzyme-linked immunosorbent assay. In vivo experiment： behavioral alterations of rats were evaluated by neurological function scale; infarct volume was assessed by hematoxylin-eosin staining; VEGF protein expression in the infarct regions was determined by immunohistochemistry. RESULTS： （1） VEGF levels were similar between the three groups under normexic condition （P 〉 0.05）; while hypoxia induced VEGF production （P 〈 0.01 ）. VEGF levels in the drug-containing serum group were particularly higher compared with the other groups （P 〈 0.01）. （2） Compared with normal saline treatment, Rhizoma Chuanxiong extract significantly improved the neurological scale and reduced cerebral infarct volumes （P〈 0.05）. The percent of VEGF-positive cells was significantly greater than the model group （P 〈 0.05）. The sham-operated group exhibited normal neurological function, with no infarct focus. CONCLUSION： Rhizoma Chuanxiong extract-containing rabbit serum effectively promoted cultured VEGF production under hypoxia. Rhizoma Chuanxiong extract upregulated VEGF expression in the infarct region, improved neurological function, and reduced infarct size.
基金Supported by the Austrian Science Fund,No.P20116-B13 and No.P22838-B13
文摘AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.
文摘Background： Endothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated the effect of transplanting endothelial progenitor cells （EPCs） derived from human early fetal aortas in rat injured arteries. Methods： The carotid arterial endothelium of Sprague-Dawley rats was damaged by dilatation with a 1.5 F balloon catheter, and then EPCs derived from human early fetal aortas （〈14 weeks） were injected into the lumen of the injured artery in transplanted rats, with an equal volume of normal saline injected into control rats. Rats were sacrificed at 2 and 4 weeks after treatment and transplanted cells were identified by immunohistochemical staining with anti-human CD31 and anti-human mitochondria antibodies. Arterial cross-sections were analyzed by pathology, immunohistochemistry, and morphometry. Results： Green fluorescence-labeled EPCs could be seen in the endovascular surface of balloon-injured vessels after transplantation. The intimal area and intimal/medial area ratio were significantly smaller in the transplanted group than in the control （P 〈 0.05） and the residual lumen area was larger （P 〈 0.05）. After EPC transplantation, a complete vascular endothelial layer was formed, which was positive for human yon Willebrand factor after immunohistochemical staining, and immunohistochemical staining revealed many CD31- and mitochondria-positive cells in the re-endothelialized endothelium with EPC transplantation but not control treatment. Conclusion： EPCs derived from human early fetal aorta were successfully transplanted into injured vessels and might inhibit neointimal hyperplasia after vascular injury.
文摘目的:探讨氧化低密度脂蛋白(ox-LDL)对人冠状动脉内皮细胞表达可诱导共刺激分子配体(ICOSL)的影响。方法:以人冠状动脉内皮细胞为研究对象,通过间接免疫荧光、逆转录聚合酶链反应(RT-PCR)和Western blot等方法,观察ICOSL在人冠状动脉内皮细胞的表达及ox-LDL的干预作用。结果:对照组和ox-LDL刺激组ICOSL mRNA的平均光密度OD值分别为0.071±0.035和0.186±0.044(n=6),ICOSL Western blot吸光度A值分别为10.88±1.53和16.03±4.08(n=6),ox-LDL(100mg/L)可增加ICOSL在人冠状动脉内皮细胞中mRNA和蛋白的表达,并具有统计学意义(P<0.05)。结论:ox-LDL可上调人冠状动脉内皮细胞表达ICOSL。
