Plasticity of Blood- and Lymphatic Endothelial Cells and Marker Identification

The distinction between lymphatic and blood vessels is biologically fundamental. Here we wanted to rigorously analyze the universal applicability of vascular markers and characteristics of the two widely used vascular model systems human microvascular endothelial cell line-1 (HMEC-1) and telomerase-immortalized microvascular endothelial cell line (TIME). Therefore we studied the protein expression and functional properties of the endothelial cell lines HMEC-1 and TIME by flow cytometry and in vitro flow assays. We then performed microarray analyses of the gene expression in these two cell lines and compared them to primary endothelial cells. Using bioinformatics we then defined 39 new, more universal, endothelial-type specific markers from 47 primary endothelial microarray datasets and validated them using immunohistochemistry with normal and pathological tissues. We surprisingly found that both HMEC-1 and TIME are hybrid blood- and lymphatic cells. In addition, we discovered great discrepancies in the previous identifications of blood- and lymphatic endothelium-specific genes. Hence we identified and validated new, universally applicable vascular markers. Summarizing, the hybrid blood-lymphatic endothelial phenotype of HMEC-1 and TIME is indicative of plasticity in the gene expression of immortalized endothelial cell lines. Moreover, we identified new, stable, vessel-type specific markers for blood- and lymphatic endothelium, useful for basic research and clinical diagnostics.淋巴管与血管的区分在生物学中具有基础性意义。本研究旨在严谨分析血管标志物的普适适用性，以及两种广泛使用的血管模型系统——人微血管内皮细胞系-1（human microvascular endothelial cell line-1, HMEC-1）与端粒酶永生化微血管内皮细胞系（telomerase-immortalized microvascular endothelial cell line, TIME）——的特性。为此，本研究通过流式细胞术与体外流动实验，分析了HMEC-1与TIME这两种内皮细胞系的蛋白表达与功能特性。随后，我们对这两种细胞系开展了基因表达芯片分析，并将结果与原代内皮细胞进行比对。借助生物信息学方法，我们从47个原代内皮细胞基因芯片数据集中筛选出39种全新、普适性更强的内皮细胞类型特异性标志物，并通过免疫组织化学实验结合正常与病变组织对这些标志物进行了验证。本研究意外发现，HMEC-1与TIME均为兼具血管与淋巴管特性的杂合细胞。此外，我们还发现此前对血管内皮与淋巴管内皮特异性基因的鉴定存在显著偏差。基于此，我们鉴定并验证了全新的普适性血管标志物。综上，HMEC-1与TIME所具有的杂合血管-淋巴管内皮表型，提示永生化内皮细胞系的基因表达具有可塑性。此外，本研究还鉴定出针对血管内皮与淋巴管内皮的全新、稳定的血管类型特异性标志物，可用于基础研究与临床诊断。