Klein Diana, Demory Alexandra, Peyre Francis, Kroll Jens, Augustin Hellmut G, Helfrich Wijnand, Kzhyshkowska Julia, Schledzewski Kai, Arnold Bernd, Goerdt Sergij
Department of Dermatology, Venereology, and Allergology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany.
Hepatology. 2008 Mar;47(3):1018-31. doi: 10.1002/hep.22084.
The mechanisms regulating the growth and differentiation of hepatic sinusoidal endothelial cells (HSECs) are not well defined. Because Wnt signaling has become increasingly important in developmental processes such as vascular and hepatic differentiation, we analyzed HSEC-specific Wnt signaling in detail. Using highly pure HSECs isolated by a newly developed protocol selecting against nonsinusoidal hepatic endothelial cells, we comparatively screened the multiple components of the Wnt pathway for differential expression in HSECs and lung microvascular endothelial cells (LMECs) via reverse-transcription polymerase chain reaction (RT-PCR). As confirmed via quantitative RT-PCR and northern and western blotting experiments, Wnt2 (and less so Wnt transporter wls/evi) and Wnt coreceptor Ryk were overexpressed by HSECs, whereas Wnt inhibitory factor (WIF) was strongly overexpressed by LMECs. Exogenous Wnt2 superinduced proliferation of HSECs (P < 0.05). The Wnt inhibitor secreted frizzled-related protein 1 (sFRP1) (P < 0.005) and transfection of HSECs with Wnt2 small interfering RNA (siRNA) reduced proliferation of HSECs. These effects were rescued by exogenous Wnt2. Tube formation of HSECs on matrigel was strongly inhibited by Wnt inhibitors sFRP1 and WIF (P < 0.0005). Wnt signaling in HSECs activated the canonical pathway inducing nuclear translocation of beta-catenin. GST (glutathione transferase) pull-down and co-immunoprecipitation assays showed Fzd4 to be a novel Wnt2 receptor in HSECs. Gene profiling identified vascular endothelial growth factor receptor-2 (VEGFR-2) as a target of Wnt2 signaling in HSECs. Inhibition of Wnt signaling down-regulated VEGFR-2 messenger RNA and protein. Wnt2 siRNA knock-down confirmed Wnt2 specificity of VEGFR-2 regulation in HSECs.
Wnt2 is an autocrine growth and differentiation factor specific for HSECs that synergizes with the VEGF signaling pathway to exert its effects.
调节肝窦内皮细胞（HSEC）生长和分化的机制尚未完全明确。由于Wnt信号通路在血管和肝脏分化等发育过程中变得越来越重要，我们详细分析了HSEC特异性Wnt信号通路。使用通过新开发的针对非窦状肝内皮细胞的方案分离出的高度纯化的HSEC，我们通过逆转录聚合酶链反应（RT-PCR）比较筛选了Wnt通路的多个组分在HSEC和肺微血管内皮细胞（LMEC）中的差异表达。通过定量RT-PCR以及Northern和Western印迹实验证实，HSEC中Wnt2（以及较少的Wnt转运体wls/evi）和Wnt共受体Ryk过表达, 而Wnt抑制因子（WIF）在LMEC中强烈过表达。外源性Wnt2超诱导HSEC增殖（P <0.05）。Wnt抑制剂分泌型卷曲相关蛋白1（sFRP1）（P <0.005）以及用Wnt2小干扰RNA（siRNA）转染HSEC可降低HSEC的增殖。这些作用可被外源性Wnt2挽救。基质胶上HSEC的管腔形成受到Wnt抑制剂sFRP1和WIF的强烈抑制（P <0.0005）。HSEC中的Wnt信号激活了诱导β-连环蛋白核转位的经典通路。谷胱甘肽转移酶（GST）下拉和免疫共沉淀实验表明Fzd4是HSEC中一种新的Wnt2受体。基因谱分析确定血管内皮生长因子受体2（VEGFR-2）是HSEC中Wnt2信号通路的一个靶点。抑制Wnt信号可下调VEGFR-2信使RNA和蛋白质水平。Wnt2 siRNA敲低证实了HSEC中VEGFR-2调节的Wnt2特异性。
Wnt2是一种对HSEC特异的自分泌生长和分化因子，它与VEGF信号通路协同发挥作用。
