A Novel Profiling System of Endothelial Gene Expression in Angiogenic Retinal Vessels

Abstract

Purpose: : Deregulated retinal angiogenesis directly cause vision loss in many ocular diseases, such as diabetic retinopathy and retinopathy of prematurity. To identify endothelial-specific genes expressed in angiogenic retinal vessels, we conducted global transcriptional profiling in endothelial cells (ECs) sorted from living retinal vessels.

Methods: : Utilizing fluorescence-activated cell sorting (FACS), we isolated ECs from angiogenic and quiescent retinal vessels in neonatal and adult Tie2-GFP transgenic mice, respectively. Total RNAs extracted from purified retinal ECs were amplified and used for microarray analyses. Differentially expressed genes in distinct cell fractions were statistically analyzed with Significance Analysis of Microarrays and validated with the eXintegrator software. Bioinformatic analyses for functional annotations were performed with NIH-DAVID. Whole-mount in situ hybridization was performed with digoxigenin (DIG)-labeled RNA probes and anti-DIG antibodies conjugated with alkaline-phosphatase.

Results: : We successfully sorted ECs representing 0.1% of entire cell populations in living mouse retinas. In neonatal retinas, 1623 genes were statistically upregulated in EC fractions compared to non-endothelial fractions. The Gene Ontology annotations correlated the functions of a considerable number of the endothelial-specific gene products with cell motility and cytoskeletal arrangements. Exploiting the eXintegrator’s pattern-matching function, we further determined angiogenic endothelial genes whose expression levels were downregulated in quiescent vessels of adult retinas. These genes displayed spatially unique distribution in ECs of arteries, veins, and capillaries of the developing retinal vasculature.

Conclusions: : Our FACS-array transcriptional profiling system will contribute not only to the understanding of the pathogenesis of retinal diseases, but also to the discovery of novel drug targets to treat deregulated retinal angiogenesis.

Keywords: retinal neovascularization • gene microarray • flow cytometry