A novel Erythropoietin-derived peptide regulates Endothelial Progenitor Cell (EPC) mediated retinal vasorepair

Abstract

Purpose: Intravitreally delivered endothelial progenitor cells (EPCs) can incorporate into the resident vasculature and become directly involved in vasoregeneration of the ischemic retina. Recent animal-based studies have demonstrated that exogenous Erythropoietin (EPO) therapy can evoke vascular regeneration in the diabetic retina. This may be due to mobilisation of EPC sub-classes and an enhancement of their reparative potential. This study has dissected the signalling events downstream of EPO and a unique peptide (pHBSP), based on the Helix-B domain of EPO, which is proposed to signal through the so-called Tissue Protective Receptor (TPR). We also determined how this multifunction protein alters EPC-mediated repair in the ischaemic retina.

Methods: Signal transduction events in whole cell lysates from umbilical cord blood-derived EPCs stimulated acutely with EPO or pHBSP were investigated by western blot. The Matrigel tubulogenesis and wound healing scratch assays were used to establish the in vitro angiogenic properties of EPO and pHBSP. Intravitreal or intra-carotid delivery of EPCs in the oxygen induced retinopathy (OIR) model at post-natal day 13 (P13) was carried out in the presence and absence of EPO or pHBSP.

Results: TPR expression, as evidenced by expression of the EPO-R and CD131 receptor subunits, was shown in EPCs by qRTPCR and western blot. The PI3K/Akt pathway was upregulated within 30 minutes in response to pHBSP and EPO in EPCs. pHBSP had a positive effect on EPCs in the scratch assay but no significant effect on tube formation. EPCs delivered both locally and by intracarotid administration to mice subjected to OIR showed incorporation into the retinal vasculature. Cell delivery by both routes markedly reduced the area of ischemic retina at P17. EPO-treatment of mice lead to an increased reticulocyte count in treated mice although pHBSP had no effect on this parameter. Systemic EPO treatment or cell pre-exposure prior to delivery evoked an enhancement of EPC vasoreparative function in OIR.

Conclusions: EPCs can be delivered directly to the vitreous or systemically resulting in incorporation of the cells into the resident vasculature. In a cell therapy regime involving EPCs, EPO or pHBSP appears to enhance retinal vascular repair possibly by activation of pro-survival pathways (Akt).

Keywords: 700 retinal neovascularization • 721 stem cells