AIM: To develop an in vitro three-dimensional (3-D) angiogenesis system to analyse the capillary sprouts induced in response to the concentration ranges of basic fibroblast growth factor (bFGF) and vascular endothelia...AIM: To develop an in vitro three-dimensional (3-D) angiogenesis system to analyse the capillary sprouts induced in response to the concentration ranges of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and to quantify their synergistic activity.METHODS: Microcarriers (MCs) coated with human microvascular endothelial cells (HMVECs) were embedded in fibrin gel and cultured in 24-well plates with assay media. The growth factors bFGF, or VEGF, or both were added to the system. The wells (n = 8/group) were digitally photographed and the average length of capillary-like sprouts (ALS) from each microcarrier was quantitated.RESULTS: In aprotinin-stabilized fibrin matrix, human microvascular endothelial cells on the MCs invaded fibrin,forming sprouts and capillary networks with lumina. The angiogenic effects of bFGF or VEGF were dose-clependent in bhe range from 10 to 40 ng/mL. At d 1, 10 ng/mL of bFGF and VEGF induced angiogenesis with an ALS of 32.13±16.6 μm and 43.75±27.92 μm, respectively, which were significantly higher than that of the control (5.88±4.45 μm, P<0.01),and the differences became more significant as the time increased. In addition, the combination of 10 ng/mL of bFGF and VEGF each induced a more significant effect than the summed effects of bFGF (10 ng/mL) alone and VEGF (10 ng/mL) alone when analyzed using SPSS system for general linear model (GLM) (P= 0.011), and bhat also exceeded the effects by 20 ng/mL of either bFGF or VEGF.CONCLUSION: A microcarrier-based in vitro threedimensional angiogenesis model can be developed in fibrin.It offers a unique system for quantitative analysis of angiogenesis. Both bFGF and VEGF exert their angiogenic effects on HMVECs synergistically and in a dose-dependent manner.展开更多
AIM:To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogeness system with affymetrix oligonucleotide array. METHOD...AIM:To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogeness system with affymetrix oligonucleotide array. METHODS: A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed. RESULTS: A total of 1 961 genes were up- or downreg-ulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, OOCL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis. CONCLUSION: The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.展开更多
文摘AIM: To develop an in vitro three-dimensional (3-D) angiogenesis system to analyse the capillary sprouts induced in response to the concentration ranges of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and to quantify their synergistic activity.METHODS: Microcarriers (MCs) coated with human microvascular endothelial cells (HMVECs) were embedded in fibrin gel and cultured in 24-well plates with assay media. The growth factors bFGF, or VEGF, or both were added to the system. The wells (n = 8/group) were digitally photographed and the average length of capillary-like sprouts (ALS) from each microcarrier was quantitated.RESULTS: In aprotinin-stabilized fibrin matrix, human microvascular endothelial cells on the MCs invaded fibrin,forming sprouts and capillary networks with lumina. The angiogenic effects of bFGF or VEGF were dose-clependent in bhe range from 10 to 40 ng/mL. At d 1, 10 ng/mL of bFGF and VEGF induced angiogenesis with an ALS of 32.13±16.6 μm and 43.75±27.92 μm, respectively, which were significantly higher than that of the control (5.88±4.45 μm, P<0.01),and the differences became more significant as the time increased. In addition, the combination of 10 ng/mL of bFGF and VEGF each induced a more significant effect than the summed effects of bFGF (10 ng/mL) alone and VEGF (10 ng/mL) alone when analyzed using SPSS system for general linear model (GLM) (P= 0.011), and bhat also exceeded the effects by 20 ng/mL of either bFGF or VEGF.CONCLUSION: A microcarrier-based in vitro threedimensional angiogenesis model can be developed in fibrin.It offers a unique system for quantitative analysis of angiogenesis. Both bFGF and VEGF exert their angiogenic effects on HMVECs synergistically and in a dose-dependent manner.
文摘AIM:To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogeness system with affymetrix oligonucleotide array. METHODS: A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed. RESULTS: A total of 1 961 genes were up- or downreg-ulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, OOCL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis. CONCLUSION: The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.
