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Abstract：
BACKGROUND: Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs.
OBJECTIVE: The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined.
METHODS: Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored.
RESULTS: Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis.
CONCLUSIONS: Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.
OBJECTIVE: The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined.
METHODS: Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored.
RESULTS: Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis.
CONCLUSIONS: Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.
摘要：
背景：血管生成行为已被证明在内皮细胞（EC）中具有很高的通用性，考虑到其可重复性，这引起了血管生成体外测定的问题。研究内皮细胞血管生成潜能的影响因素是必不可少的。
目的：本研究旨在分析敲低磷酸三糖异构酶（TPI）对体外血管生成的影响，同时对波形蛋白（VIM）和腺苷甲硫氨酸合成酶同工型2（MAT2A）表达的影响。此外，TPI的天然表达谱，VIM和MAT2A在体外血管生成进程中停止了检测。
方法:将两批人真皮微血管EC培养50天并刺激以进行血管生成。进行shRNA介导的TPI敲低。在种植期间,检测到时间依赖性的形态学变化,并将其应用于EC分期,作为量化体外血管生成的先决条件.此外,监测所有蛋白质的mRNA和蛋白质水平。
结果：与天然细胞相反，敲低细胞不能进入血管生成的晚期阶段,主要表现为VIM下调和MAT2A表达上调.在体外血管生成过程中,天然细胞增加其TPI表达并降低其VIM表达。对于MAT2A，在血管生成开始和结束时观察到最高表达。
结论：敲低TPI会引起VIM和MAT2A的表达变化以及体外血管生成的减速，表明TPI代表血管生成蛋白。天然表达谱导致VIM在开始阶段主要相关的假设，在体外血管生成的整个过程中，MAT2A在开始和晚期阶段以及TPI。
目的：本研究旨在分析敲低磷酸三糖异构酶（TPI）对体外血管生成的影响，同时对波形蛋白（VIM）和腺苷甲硫氨酸合成酶同工型2（MAT2A）表达的影响。此外，TPI的天然表达谱，VIM和MAT2A在体外血管生成进程中停止了检测。
方法:将两批人真皮微血管EC培养50天并刺激以进行血管生成。进行shRNA介导的TPI敲低。在种植期间,检测到时间依赖性的形态学变化,并将其应用于EC分期,作为量化体外血管生成的先决条件.此外,监测所有蛋白质的mRNA和蛋白质水平。
结果：与天然细胞相反，敲低细胞不能进入血管生成的晚期阶段,主要表现为VIM下调和MAT2A表达上调.在体外血管生成过程中,天然细胞增加其TPI表达并降低其VIM表达。对于MAT2A，在血管生成开始和结束时观察到最高表达。
结论：敲低TPI会引起VIM和MAT2A的表达变化以及体外血管生成的减速，表明TPI代表血管生成蛋白。天然表达谱导致VIM在开始阶段主要相关的假设，在体外血管生成的整个过程中，MAT2A在开始和晚期阶段以及TPI。
