Discussion
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.
Methods
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.
Results
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.