Liver sinusoidal endothelium: a microenvironment-dependent differentiation program in rat including the novel junctional protein liver endothelial differentiation-associated protein-1. Rattus norvegicus

Liver sinusoidal endothelium (LSEC) is a prime example for organ-specific microvascular differentiation and functions. Disease-associated capillarization of LSEC in vivo and dedifferentiation of LSEC in vitro indicate the importance of the hepatic microenvironment. To identify the LSEC-specific molecular differentiation program in the rat, we used a two-sided gene expression profiling approach comparing LSEC freshly isolated ex vivo with both lung microvascular endothelial cells (LMEC) and with LSEC cultured for 42h. The LSEC signature consisted of 48 genes both down-regulated in LMEC and in LSEC upon culture (FC>7 in at least one comparison); qRT-PCR confirmation of these genes included numerous family members and signalling pathway-associated molecules. The LSEC differentiation program comprised distinct sets of growth (wnt2, Fzd4, 5, 9, wls, VEGFR1, 2, 3, Nrp2) and transcription factors (Gata4, Lmo3, Tcfec, Maf) as well as endocytosis-related (Stabilin-1/2, Lyve1 and Ehd3) and cytoskeleton-associated molecules (Rnd3/RhoE). Specific gene induction in cultured LSEC versus freshly isolated LSEC as well as LMEC (Esm-1, Aatf) and up-regulation of gene expression to LMEC levels (CXCR4, Apelin) confirmed true transdifferentiation of LSEC in vitro. In addition, our analysis identified a novel 26 kDa single-pass transmembrane protein, liver endothelial differentiation-associated protein (Leda)-1, that was selectively expressed in all liver endothelial cells and preferentially localized to the abluminal cell surface. Upon forced over-expression in MDCK cells, Leda-1 was sorted baso-laterally to E-cadherin-positive adherens junctions suggesting functional involvement in cell adhesion and polarity. Conclusion: Comparative microvascular analysis in rat identified a hepatic microenvironment-dependent LSEC-specific differentiation program including the novel junctional molecule Leda-1. Overall design: Highly pure liver sinusoidal endothelial cells (LSEC) and lung microvascular endothelial cells (LMEC) were isolated by enzymatic digestion, density gradient centrifugation and MACS sorting and subjected to RNA extraction and affymetrix hybridization without any culturing step (LSEC_0h, LMEC_0h). LSEC were also plated on collagen coated dishes and RNA extraction and Affymetrix hybridization was carried out after 2h and 42h in culture (LSEC_2h, LSEC_42h).肝窦内皮细胞（liver sinusoidal endothelium, LSEC）是器官特异性微血管分化与功能的典型范例。体内LSEC发生的疾病相关性毛细血管化，以及体外LSEC的去分化现象，均凸显了肝脏微环境的重要性。为明确大鼠体内LSEC特异性的分子分化程序，本研究采用双组对照基因表达谱分析策略，将新鲜离体分离的LSEC分别与肺微血管内皮细胞（lung microvascular endothelial cells, LMEC）以及体外培养42小时的LSEC进行对比。LSEC特征基因集包含48个基因，这些基因在LMEC中以及培养后的LSEC中均呈现下调趋势（至少在一组对比中折叠变化倍数>7）；针对上述基因的实时定量聚合酶链反应（qRT-PCR）验证结果显示，其中包含大量基因家族成员及信号通路相关分子。LSEC的分化程序包含多类功能基因：生长相关基因（wnt2、Fzd4、5、9、wls、VEGFR1、2、3、Nrp2）、转录因子（Gata4、Lmo3、Tcfec、Maf）、内吞作用相关分子（Stabilin-1/2、Lyve1及Ehd3）以及细胞骨架相关分子（Rnd3/RhoE）。培养后的LSEC相较于新鲜离体LSEC及LMEC所出现的特异性基因诱导（Esm-1、Aatf），以及基因表达上调至LMEC水平的现象（CXCR4、Apelin），均证实了体外培养过程中LSEC确实发生了转分化。此外，本研究通过分析发现了一种全新的26 kDa单次跨膜蛋白——肝内皮分化相关蛋白（liver endothelial differentiation-associated protein, Leda）-1，该蛋白仅在所有肝内皮细胞中特异性表达，且优先定位于细胞的腔外侧表面。当在MDCK细胞中强制过表达Leda-1时，该蛋白会被分选至与E-钙粘蛋白阳性黏着连接共定位的基底外侧区域，这提示其可能参与细胞黏附与细胞极性调控。结论：针对大鼠的微血管对比分析明确了依赖于肝脏微环境的LSEC特异性分化程序，其中包含全新的连接分子Leda-1。实验整体设计：通过酶消化、密度梯度离心及磁激活细胞分选（MACS）技术分离得到高纯度的肝窦内皮细胞（LSEC）与肺微血管内皮细胞（LMEC），未经任何体外培养步骤直接进行RNA提取及Affymetrix基因芯片杂交（对应样本为LSEC_0h、LMEC_0h）；同时将LSEC接种于包被胶原蛋白的培养皿中，分别在体外培养2小时及42小时后进行RNA提取与Affymetrix基因芯片杂交（对应样本为LSEC_2h、LSEC_42h）。