Do anti-angiogenic VEGF (VEGFxxxb) isoforms exist? A cautionary tale.
Do anti-angiogenic VEGF (VEGFxxxb) isoforms exist? A cautionary tale.
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是否存在抗血管生成VEGF（VEGFXXXB）？一个警示性的故事。
DOI：
10.1371/journal.pone.0035231
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Kanthou C
中科院分区：
文献类型：
--
作者：
Harris S;Craze M;Newton J;Fisher M;Shima DT;Tozer GM;Kanthou C
据报道，人血管内皮生长因子-A (VEGF-A) 基因的剪接可产生血管生成 (VEGFxxx) 和抗血管生成 (VEGFxxxb) 亚型。在大鼠和小鼠中也报道了相应的 VEGFxxxb 同工型。我们检测了在体外和体内生长的表达所有或单个 VEGF 同工型（VEGF120、164 或 188）的小鼠纤维肉瘤细胞系中的 VEGFxxxb 表达，并将结果与正常小鼠和人类组织的结果进行了比较。重要的是，表达 VEGF164 和 VEGF188 的纤维肉瘤（其中外显子 7 与常规外显子 8 融合）的基因构建阻止了 VEGFxxxb 剪接的发生。因此，这两种纤维肉瘤细胞系提供了内源性阴性对照。使用 RT-PCR，我们发现设计用于同时扩增 VEGFxxx 和 VEGFxxxb 同工型的引物仅扩增了两个物种中的 VEGFxxx 变体。此外，当用 TGFβ-1 处理小鼠足细胞时，仅产生 VEGFxxx 物种，TGFβ-1 是据报道的人类足细胞 VEGFxxxb 剪接选择的激活剂。 VEGF164/120 异源双链体物种被鉴定为 PCR 假象，特别是在小鼠中。 VEGFxxxb 同工型特异性 PCR 确实在小鼠和人体组织中扩增了假定的 VEGFxxxb 种类，但出乎意料的是，也在 VEGF188 和 VEGF164 纤维肉瘤细胞和肿瘤中扩增，在这些细胞和肿瘤中，无法发生剪接以产生真正的 VEGFxxxb 同工型。此外，这些产物只能使用跨越 8b/7 或 8b/5 剪接点超过 5 个碱基的反向引物一致地生成。引物退火至 VEGFxxx 转录本和外显子 8b 引物“尾部”的扩增解释了 VEGFxxxb 产物的人为生成，因为当使用来自 VEGF164/VEGF188“敲入”载体的 cDNA 进行 PCR 反应时，会生成相同的产物，该载体用于生成表达单一 VEGF 同种型的转基因小鼠，从该小鼠中产生纤维肉瘤 线路被开发出来。总的来说，我们的结果强调了与使用当前方法检测 VEGFxxxb 同工型相关的数据解释中的重要缺陷，并证明抗血管生成同工型通常在小鼠或人体组织中不表达。
Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues.
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影响因子：
3.6
作者：
Artac, Robin A.;McFee, Renee M.;Cupp, Andrea S.
通讯作者：
Cupp, Andrea S.
DOI：
10.1083/jcb.200409115
发表时间：
2005-05-23
期刊：
The Journal of cell biology
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作者：
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