Analyses of blood outgrowth endothelial cells reveal an endothelial HOX gene signature in human beings

Abstract

Endothelial cells have a remarkable ability for subspecialisation, adapting to the needs of a variety of vascular beds. It is not known how much of this subspecialisation is related to developmental programming versus the environment in which the endothelial cell finds itself. In transcriptomic studies of endothelial cells we have noted a hierarchy of HOX gene expression which predicts endothelial cell specification and fate. HOX genes are well described as master-regulators of positional identity, predominately in the developing embryo.Initial studies in human cells compared blood outgrowth endothelial cells (BOECs), a circulation-derived endothelial progenitor cell, with mature adult pulmonary artery endothelial cells (PAECs). We confirmed the endothelial phenotype of BOECs with a combination of traditional cell surface markers, electron microscopy, vacuolisation and network formation, ligand stimulation studies, leucocyte transmigration assays, and transcript expression profiling.In microarray analysis of mRNA transcripts from BOECs and PAECs only 0·005% of genes were differentially expressed. Developmental processes dominated these differentially regulated genes when analysed by gene ontology. In particular we identify a BOEC HOX gene signature, particularly in the B and D HOX gene clusters. These differences in HOX gene expression were confirmed by quantitative PCR. Furthermore, in analyses of three independent datasets of microarrays from 56 adult cell and tissue arrays (both human and mouse), HOX genes discriminated endothelial cells from other cell types. According to these analyses, HOX gene expression clustered endothelial cells into hierarchies based on their anatomical location. In particular the HOXD cluster, identified as highly expressed in BOECs, was observed in microvascular endothelial cells and in angiogenesis. Adult and embryonic tissue staining of HOXD proteins confirmed this pattern of expression.Since microvascular cells have been shown to be capable of repopulating the entire endothelial hierarchy, they have been posited as an angiogenic progenitor niche. Together these observations suggest a specific HOX signature involved in angiogenesis, endothelial cell differentiation and fate. FundingWellcome Trust.