Cancer Research United Kingdom Tumour Microcirculation Group, Department of Oncology, School of Medicine, University of Sheffield, Sheffield, United Kingdom.
PLoS One. 2012;7(5):e35231. doi: 10.1371/journal.pone.0035231. Epub 2012 May 2.
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.
人类血管内皮生长因子-A（VEGF-A）基因的剪接已被报道产生血管生成（VEGFxxx）和抗血管生成（VEGFxxxb）异构体。在大鼠和小鼠中也报道了相应的 VEGFxxxb 异构体。我们检查了在体外和体内生长的表达所有或个别 VEGF 异构体（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进行了比较。重要的是，VEGF164 和 VEGF188 表达的纤维肉瘤的基因构建，其中外显子 7 融合到常规外显子 8 中，排除了 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。我们使用 RT-PCR 表明，设计用于同时扩增 VEGFxxx 和 VEGFxxxb 异构体的引物仅在两种物种中扩增 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 是报道的人足细胞中 VEGFxxxb 剪接选择的激活剂。VEGF164/120 异源双链体物种被鉴定为 PCR 伪影，特别是在小鼠中。VEGFxxxb 异构体特异性 PCR 确实在小鼠和人类组织中扩增了推定的 VEGFxxxb 物种，但出乎意料的是，在 VEGF188 和 VEGF164 纤维肉瘤细胞和肿瘤中也扩增了这些物种，在这些细胞和肿瘤中，无法产生真正的 VEGFxxxb 异构体。此外，只有使用跨越 8b/7 或 8b/5 剪接接头超过 5 个碱基的反向引物进行扩增时，才会一致地生成这些产物。退火到 VEGFxxx 转录本的引物和扩增外显子 8b 引物“尾部”解释了 VEGFxxxb 产物的人为产生，因为当使用从用于生成表达单个 VEGF 异构体的转基因小鼠的 VEGF164/VEGF188“敲入”载体获得的 cDNA 进行 PCR 反应时，也会生成相同的产物。总之，我们的结果突出了使用当前方法检测 VEGFxxxb 异构体时与数据解释相关的重要陷阱，并证明抗血管生成异构体在小鼠或人类组织中并不常见表达。
