Endothelial Progenitor Cell Interactions With Retinal Vascular Endothelial Cell Monolayers and in a 3-D Model of Retinal Angiogenesis

Abstract

Purpose:: Endothelial progenitor cells have been shown to engage in angiogenesis (AG) in various tissues including the retina. However, little is known of the intimate associations of EPCs with retinal vascular endothelial cells (RVEC). This study was undertaken to examine the interactions of EPCs with monolayers of RVEC and the ability of EPCs to engage in an in vitro AG model in Matrigel 3-D culture .

Methods:: Human EPCs were isolated from peripheral blood and characterised by staining for VEGFR2, CD34, CD31, UEA-1 lectin and uptake of acLDL-DiI. 1-week culures of DiI-labelled EPCs were added to confluent monolayers of bovine RVEC and EPCs from 1 and 3 week cultures were added to the secondary (2nd) layer of a duplex Matrigel model of sprouting AG. RVEC were plated in 30µl spots of 50% Matrigel and formed tubular networks by 24 hours. A 2nd Matrigel layer containing the EPCs was then superimposed on the primary cultures; controls had only Matrigel in the 2nd layer. At times up to 48 hours the cultures were fixed in 4%PFA and stained with Alexa-488-B4 isolectin. Some monolayers were stained with anti-ZO1 to outline the tight junctions. All stained cultures were examined by confocal microscopy.

Results:: The chimeric nature of the cultures did not appear to inhibit interaction between the bovine RVEC and human EPCs, which were able to attach to uninjured monolayers. After 48 hours, confocal microscopy revealed that large numbers of DiI-labelled (red fluorescent) EPCs had crossed the monolayers and lay in sub-endothelial pockets. The majority of EPCs at such sites showed little physical contact with the overlying endothelium, as revealed by optical sectioning, and ZO1 staining showed no discontinuity of the tight junctions. Rarely, EPCs were found to be incorporated within the monolayer. The presence of 1-week EPCs in the 2nd layer of the Matrigel cultures stimulated profuse angiogenic sprouting from the primary RVEC networks, yet despite close association with the sprouts, no incorporation was observed. In contrast, EPC from 3-week cultures stimulated AG and incorporated within sprouts, including the tip cells which showed filopodia labelled with DiI. Interestingly, in these cultures EPCs could be seen to communicate directly with RVEC via nanotubes over 30µm long.

Conclusions:: This study has revealed that EPCs from both short and long term cultures are able to stimulate retinal angiogenesis in vitro but only those from long-term cultures show significant incorporation in vascular structures.

Keywords: retina • vascular cells • microscopy: confocal/tunneling