Laboratory of Novel Therapeutic Targets, Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain.
Mol Cancer. 2010 Dec 31;9:320. doi: 10.1186/1476-4598-9-320.
Different isoforms of VEGF-A (mainly VEGF₁₂₁, VEGF₁₆₅ and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGF(xxx)b, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF₁₂₁/₁₆₅b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential.
Recombinant VEGF₁₂₁/₁₆₅b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF₁₆₅. Furthermore, treatment of endothelial cells with VEGF₁₂₁/₁₆₅b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF₁₆₅. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF₁₂₁/₁₆₅b isoforms. A549 and PC-3 cells overexpressing VEGF₁₂₁b or VEGF₁₆₅b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGF(xxx)b and total VEGF-A was found.
Our results demonstrate that VEGF₁₂₁/₁₆₅b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF₁₂₁/₁₆₅b-based therapies in patients.
不同的 VEGF-A 异构体（主要是 VEGF₁₂₁、VEGF₁₆₅ 和 VEGF189）已被证明在生成功能性肿瘤血管方面具有特殊的血管生成特性。最近，一类新的 VEGF-A 异构体被描述为通过选择性剪接产生的 VEGF(xxx)b。先前的研究表明，这些异构体可能抑制血管生成。在本工作中，我们在酵母毕赤酵母中产生了重组 VEGF₁₂₁/₁₆₅b 蛋白，并构建了载体来过表达这些异构体并评估它们的血管生成潜力。
在酵母或哺乳动物细胞中产生的重组 VEGF₁₂₁/₁₆₅b 蛋白激活了 VEGFR2 及其下游效应物 ERK1/2，尽管程度低于 VEGF₁₆₅。此外，与未处理的细胞相比，用 VEGF₁₂₁/₁₆₅b 处理内皮细胞会增加细胞增殖，尽管这种刺激低于 VEGF₁₆₅ 诱导的刺激。此外，体内血管生成实验证实了 VEGF₁₂₁/₁₆₅b 异构体的血管生成刺激作用。过表达 VEGF₁₂₁b 或 VEGF₁₆₅b 的 A549 和 PC-3 细胞（或携带空载体 PCDNA3.1，作为对照）并异种移植到裸鼠中，与对照组相比，肿瘤体积和血管生成增加。为了评估 VEGF(xxx)b 异构体在肿瘤组织中与健康组织中的表达是否存在差异，对乳腺癌组织微阵列进行了免疫组织化学分析。与正常乳房相比，浸润性导管癌中 VEGF(xxx)b 和总 VEGF-A 蛋白表达均显著增加（p <0.05）。发现 VEGF(xxx)b 和总 VEGF-A 之间存在显著正相关（r = 0.404，p = 0.033）。
我们的结果表明，VEGF₁₂₁/₁₆₅b 不是抗血管生成的，而是 VEGF-A 的弱血管生成异构体。此外，与非恶性乳腺组织相比，乳腺癌中 VEGF(xxx)b 异构体上调。在考虑 VEGF₁₂₁/₁₆₅b 为基础的治疗在患者中的可能应用时，应考虑这些结果。
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