Secondary Sprouting From Retinal Endothelial Cells Involves Endothelial Precursors

Abstract

Abstract: : Purpose: To determine whether secondary sprouting colonies derived from retinal endothelial cells (ECs) contain endothelial cell precursors (ECPs). Methods: 1) Isolated secondary sprouting colonies (SSCs) were subject to repeated cycles of culturing under differentiative (without basement membrane matrix, BMM) or expansive (BMM +/- PDGF) conditions to determine their sustained ability to generate mature ECs. 2) SSCs were embedded in OCT freezing medium, cryosectioned and stained with various antibodies to ECP markers. 3) mRNA derived from mature EC monolayers as well as SSCs was analyzed by RT-PCR using ECP marker-specific primers. 4) High telomerase activity has been correlated with ECPs but not with differentiated ECs. Therefore, the TRAP assay was used to determine telomerase activity in differentiated and secondary sprouting EC cultures with or without PDGF. Results: When transferred to differentiative culture conditions, secondary sprouting colonies gave rise to monolayers phenotypically consistent with mature ECs. These monolayers behaved as the original cultures when replated on BMM, forming capillary-like tubes and subsequent secondary sprouts. This process could be repeated at least five times, indicating that SSCs contained cells that were able to generate progenitor-like cells as well as mature ECs. RT-PCR and immunohistochemical analysis confirmed that the expression pattern of ECP markers in the SSCs was consistent with that of ECPs. Furthermore, cells derived from the differentiated monolayers did not express most ECP markers and was positive for markers of mature ECs. High telomerase activity was detected in the SSCs but not in the differentiated monolayers. It was increased by PDGF treatment of the SSCs but not of mature ECs. Conclusions: Endothelial cells in secondary sprouting colonies demonstrated behaviors, morphology and biomarkers consistent with that of endothelial cell precursors. Their existence in retinal endothelial cell cultures and their ability to respond to growth factors known to be increased and diabetic retinopathy make the secondary sprouting phenomenon a novel assay. It may also explain how retinal capillaries are able to regenerate after cellular injury produced by pathological conditions such as diabetic retinopathy.

Keywords: vascular cells • growth factors/growth factor receptors • retinal neovascularization