Subtractive transcriptomics:: Establishing polarity drives in vitro human endothelial morphogenesis
Subtractive transcriptomics:: Establishing polarity drives in vitro human endothelial morphogenesis
复制标题
DOI：
10.1158/0008-5472.can-05-3294
复制
发表时间：
2006-04-15
期刊：
影响因子：
11.2
通讯作者：
Rodi, DJ
中科院分区：
文献类型：
--
作者：
Glesne, DA;Zhang, W;Rodi, DJ
虽然成熟的正常和肿瘤来源的毛细血管的研究已经导致这些结构的表征在表型水平上，较少是已知的关于细胞组装成人毛细血管的内皮细胞的初始分子线索。在这里，我们采用了一种新的组合微环境操作和微阵列数据过滤在狭窄划定的时间数据系列，以确定形态发生组件除了增殖组件，作为汇集的人微血管来源的内皮细胞诱导形成毛细血管样结构在体外的小鼠肿瘤来源的基质。使用这种消减转录组学方法鉴定的217个形态发生特异性基因大多独立于目前用作异常血管生成的治疗靶点的血管生成蛋白。定量实时PCR用于验证这些转录本的20%。增殖和管形成细胞的免疫荧光分析验证了在蛋白质水平上的形态发生特异性表达模式的16个217个基因产物确定。在管形成内皮细胞中选择性上调的转录物揭示了主要与细胞内运输、引导迁移、细胞骨架重组、细胞粘附和增殖抑制相关的基因的时间表达模式。这些数据表明，建立和维持极性的基因的顺序上调发生在迁移和形态发生的体外人内皮细胞进行tubulogenesis;其中一些可能是有效的新的抗血管生成药物的目标。
Although investigations of mature normal and tumor-derived capillaries have resulted in characterization of these structures at the phenotypic level, less is known regarding the initial molecular cues for cellular assembly of endothelial cells into human capillaries. Here, we employ a novel combination of microenvironmental manipulation and microarray data filtration over narrowly delineated temporal data series to identify the morphogenesis component apart from the proliferation component, as pooled human microvascular-derived endothelial cells are induced to form capillary-like structures in vitro in a murine tumor-derived matrix. The 217 morphogenesis-specific genes identified using this subtractive transcriptomics approach are mostly independent of the angiogenic proteins currently used as therapeutic targets for aberrant angiogenesis. Quantitative real-time PCR was used to validate 20% of these transcripts. Immunofluorescent analysis of proliferating and tube-forming cells validates at the protein level the morphogenesis-specific expression pattern of 16 of the 217 gene products identified. The transcripts that are selectively up-regulated in tube-forming endothelial cells reveal a temporal expression pattern of genes primarily associated with intracellular trafficking, guided migration, cytoskeletal reorganization, cellular adhesion, and proliferation inhibition. These data show that a sequential upregulation of genes that establish and maintain polarity occurs during migration and morphogenesis of in vitro human endothelial cells undergoing tubulogenesis; some of which may well be effective as novel antiangiogenic drug targets.