I ntrabody‐based strategies for inhibition of vascular endothelial growth factor receptor‐2: effects on apoptosis, cell growth, and angiogenesis-猫眼课题宝

抑制血管内皮生长因子受体 2 的体内策略：对细胞凋亡、细胞生长和血管生成的影响

DOI：

10.1096/fj.02-0942fje

发表时间：

2003

期刊：

The FASEB Journal

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Y. Y. Wheeler;T. Kute;M. Willingham;Si;D. Sane

血管内皮生长因子是一种血管内皮细胞特异性有丝分裂原，是重要的肿瘤血管生成生长因子。血管内皮细胞上的主要受体是KDR。我们假设体内可以结合新合成的KDR并阻止受体转运到细胞表面，从而抑制重要的血管内皮生长因子的作用。我们用p3S5-HAK或tet-off腺病毒系统(Ad-Hak)表达了抗KDR的单链抗体(P3S5)，包括内质网(ER)保留信号(KDEL)和内质网(ER)保留信号(KDEL)。质粒介导的体内表达显著降低了KDR的表达(从82.5±12.5%的细胞减少到27.9±13.6%的细胞；P&lt；0.01)和胸腺嘧啶核苷的掺入。Ad-HAK感染后90%的人脐静脉内皮细胞(HUVECs)在体内表达，感染后48h出现明显的细胞凋亡(80%)。体内对这些作用是必不可少的，通过用多西环素抑制其表达或通过表达无关基因(LacZ，GFP)来证实。细胞死亡依赖于KDR，因为Ad-Hak感染极少量或不含KDR的细胞株对细胞存活率几乎没有影响。感染的人脐静脉内皮细胞不能在Engelbreth Holm-Spot(EHS)肿瘤凝胶基质上形成管状细胞。这些结果证明了开发一种基于体内的策略来阻止血管生成和防止肿瘤生长的潜力。

VEGF, an endothelial‐specific mitogen, is an important tumor angiogenesis growth factor. The major receptor for VEGF on endothelial cells is KDR. We hypothesized that an intrabody could bind newly synthesized KDR and block receptor transport to the cell surface, thereby inhibiting important VEGF effects. We expressed a single chain antibody (p3S5) to KDR with or without the endoplasmic reticulum (ER) retention signal (KDEL), using either a plasmid (p3S5‐HAK) or a tet‐off adenoviral system (Ad‐HAK). Plasmid‐mediated expression of the tethered intrabody significantly reduced KDR expression (from 82.5±12.5% to 27.9±13.6% of cells; P<0.01) and thymidine incorporation in successfully transfected cells. Ad‐HAK infection resulted in intrabody expression in >90% of human umbilical vein endothelial cells (HUVECs), producing marked (80%) apoptosis at 48 h postinfection. The intrabody was essential for these effects, as confirmed by inhibiting its expression with doxycycline or by expressing irrelevant genes (lacZ, GFP). Cell death was dependent on KDR, because Ad‐HAK infection of cell lines with minimal or no KDR had little effect on cell viability. Infected HUVECs were unable to form tubes on Engelbreth Holm‐Swarm (EHS) tumor gel matrix. These results demonstrate the potential for development of an intrabody‐based strategy to block angiogenesis and prevent tumor growth.