Thomas Roberta A, Pietrzak Dana C, Scicchitano Marshall S, Thomas Heath C, McFarland David C, Frazier Kendall S
Department of Safety Assessment, GlaxoSmithKline, King of Prussia, PA 19406, USA.
J Pharmacol Toxicol Methods. 2009 Nov-Dec;60(3):263-74. doi: 10.1016/j.vascn.2009.06.002. Epub 2009 Jul 3.
There are currently few widely accepted noninvasive detection methods for drug-induced vascular damage. Circulating endothelial progenitor cell (EPC) enumeration in humans has recently gained attention as a potential biomarker of vascular injury/endothelial damage/dysfunction. The rat is commonly used in preclinical drug development toxicity testing and lacks consensus noninvasive methodologies for immunophenotypic identification of EPCs. Identification of immunophenotypic markers of EPCs in the rat would enable transfer of technologies used in human for potential development of biomarkers for vascular injury the rat. Therefore, the aim of this work was to develop methods to consistently identify a discreet population of EPCs from rat peripheral blood.
EPCs were identified phenotypically from rat blood using cell culture, immunolabeling, fluorescence microscopy, and flow cytometry. EPCs isolated using immunolabeling coupled with magnetic separation and flow cytometric cell sorting were characterized genotypically using mRNA analysis.
A modified colony forming unit (CFU)-Hill assay confirmed existence of immature EPCs in peripheral blood. Extended in vitro culture resulted in a morphology and immunophenotype consistent with mature endothelial cells as noted by positive staining for CD31, von Willebrand factor, rat endothelial cell antigen, and negative staining for smooth muscle cell alpha-actin. The majority of the cells identified as LDL+/CD11b/c(-) did not stain positively for either vWF or CD31. EPC populations isolated using magnetic separation and cell sorting were consistently positive for PECAM1, EDN1, FLK1, VWF, ITGAD, CCR1, IP30, and MMP2 mRNA expression. Cells identified as EPCs express cell-surface and gene expression markers consistent with endothelial cells and endothelial progenitor cell populations.
Vascular trauma induces transient mobilization of EPCs in humans and their enumeration and characterization have been proposed as a surrogate biomarker for assessment of vascular injury. Potential exists for using rat circulating EPCs as a surrogate sampling population for biomarker development in drug-related injury in preclinical toxicity studies. A prerequisite to biomarker development is the ability to consistently identify a discreet population of EPCs from peripheral rat blood. This work describes novel methods for isolation and validation of phenotypically and genotypically consistent populations of rat EPCs from peripheral blood. These methods are well suited for potential future use in validation of enumeration and/or biomarker development methods in the rat.
目前，药物诱导的血管损伤几乎没有被广泛接受的非侵入性检测方法。人类循环内皮祖细胞（EPC）计数作为血管损伤/内皮损伤/功能障碍的潜在生物标志物，最近受到了关注。大鼠常用于临床前药物开发毒性测试，并且在EPCs免疫表型鉴定方面缺乏一致的非侵入性方法。鉴定大鼠EPCs的免疫表型标志物将有助于将人类使用的技术转移，以开发用于大鼠血管损伤的生物标志物。因此，这项工作的目的是开发方法，以一致地从大鼠外周血中鉴定出离散的EPCs群体。
使用细胞培养、免疫标记、荧光显微镜和流式细胞术从大鼠血液中表型鉴定EPCs。使用免疫标记结合磁性分离和流式细胞术细胞分选分离的EPCs，通过mRNA分析进行基因型表征。
改良的集落形成单位（CFU）-希尔试验证实外周血中存在未成熟的EPCs。延长体外培养导致细胞形态和免疫表型与成熟内皮细胞一致，表现为CD31、血管性血友病因子、大鼠内皮细胞抗原染色阳性，平滑肌细胞α-肌动蛋白染色阴性。大多数被鉴定为LDL+/CD11b/c(-)的细胞，vWF或CD31染色均为阴性。使用磁性分离和细胞分选分离的EPCs群体，PECAM1、EDN1、FLK1、VWF、ITGAD、CCR1、IP30和MMP2 mRNA表达始终呈阳性。被鉴定为EPCs的细胞表达与内皮细胞和内皮祖细胞群体一致的细胞表面和基因表达标志物。
血管损伤在人类中会诱导EPCs的短暂动员，其计数和表征已被提议作为评估血管损伤的替代生物标志物。在临床前毒性研究中，使用大鼠循环EPCs作为药物相关损伤生物标志物开发的替代采样群体具有潜力。生物标志物开发的一个先决条件是能够一致地从大鼠外周血中鉴定出离散的EPCs群体。这项工作描述了从外周血中分离和验证表型和基因型一致的大鼠EPCs群体的新方法。这些方法非常适合未来在大鼠中验证计数和/或生物标志物开发方法的潜在应用。
