Extracellular vesicles shed from endothelial colony forming cells (ECFCs) with high expression of CD44 are paracrine mediators of neurovasculotrophic retinal repair

Abstract

Purpose : ECFCs are a subset of endothelial progenitor cells that promote vascular growth and stability. ECFCs with high expression of the hyaluronan receptor CD44 (CD44^hi) rescue the oxygen-induced retinopathy (OIR) model and rd10 mice via paracrine action while ECFCs with low CD44 expression (CD44^lo) have little effect. In this study, we describe an ECFC extracellular vesicle (EV)-mediated paracrine mechanism that recapitulates the enhanced neuro- and vasculotrophic effects of CD44^hi cells in the OIR and rd10 model.

Methods : EVs were harvested from media conditioned by CD44^hi and CD44^lo umbilical cord blood-derived ECFCs via a series of ultrafiltration and size exclusion chromatography prior to injection into the OIR or rd10 model. Therapeutic EVs from CD44^hi ECFCs and ineffective CD44^lo EVs were subject to small RNA sequencing to identify intravesicular cargo that may mediate this effect.

Results : In OIR, CD44^hi EVs from all 3 of 3 umbilical cord blood donors significantly reduced the area of retinal neovascularization and vaso-obliteration relative to CD44^lo EVs. Depletion of EVs from the sample via ultracentrifugation ameliorated this effect. Dose response experiments indicated CD44^hi EV samples derived our novel isolation protocol retained efficacy even after 10,000-fold dilution of the sample. In rd10 mice, injection of CD44^hi EVs significantly reduced vessel loss in the intermediate and deep vascular plexuses, decreased apoptosis in the outer nuclear layer (ONL), preserved ONL thickness, and improved retinal function on electroretinography relative to CD44^lo EV, vehicle, or untreated controls. Small RNA sequencing analyses identified agents that may mediate the therapeutic effect of CD44^hi EVs.

Conclusions : By comparing the bioactive cargo of EVs from therapeutic CD44^hi ECFCs in comparison to ineffective CD44^lo EVs, our “-omics” based approach may be used to identify microRNAs that promote the CD44^hi EV-mediated rescue effect in the OIR and rd10 model. EVs may be a highly effective drug delivery vehicle targeting multiple cell types with multiple mechanisms simultaneously while circumventing the risks of tumorigenesis associated with intact cell-based therapeutics.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.