Pre-treatment of Endothelial Colony Forming Cells (ECFCs) with a novel anti-oxidant enhances their vasoregenerative potential.

Abstract

Purpose : ECFCs have considerable potential as therapeutic cells to achieve vascular regeneration in the ischaemic retina and choroid. Whilst ECFCs have proven reparative efficacy, we sought to determine if their function could be enhanced prior to delivery into the pro-inflammatory and pro-oxidative microenvironment which characterises ischaemic tissues. This study has evaluated the potential for a novel synthetic flavonoid, ELN/41 or Proxison™ (7-decyl-3-hydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone) to enhance ECFC function.

Methods : ECFCs isolated from umbilical cord blood, were pre-treated with 5 µM ELN/41. ELN/41 auto-fluoresces green and the localisation of the agent was assessed via confocal microscopy . ECFCs were exposed to 10 µM hydrogen peroxide (H₂O₂) to simulate a pro-oxidative environment while other cells were treated with ELN/41 or the vehicle (veh) alone. Clonogenic ECFC assays were assessed over 5-6 days and colonies enumerated. ECFC barrier formation was assessed via Xcelligence. Stable ECFC monolayers were treated with thrombin and permeability response quantified. Integration of control and ELN/41 treated ECFCs into choroidal explants from P8 C57BL/6J mice were assessed for angiogenic response over 5 days. Results were analysed by one way ANOVA.

Results : ELN/41 localised to ECFC mitochondria. The clonogenic capacity of ECFCs treated with ELN/41 was significantly increased compared to those without treatment (p=<0.001). ELN/41 treatment improved migration of ECFCs in H₂O₂, whilst non-treated ECFCs showed decreased wound closure after 6hrs versus pre-treated cells (p=<0.001) and veh control (p=<0.001). H₂O₂ exposed ECFCs had impaired barrier properties compared to those pre-treated with ELN/41 (p=<0.01) and veh controls (p=<0.001). ECFCs integrated with, and significantly enhanced the angiogenic sprouting of choroidal explants compared to those without ECFCs (p=<0.001). Explants treated with H₂O₂ had decreased angiogenesis versus control explants (p=<0.01) and explants in co-culture with ELN/41 treated cells.

Conclusions : While ECFCs possess endogenous anti-oxidant capacity, these cells show diminished function when exposed to a pro-oxidant insult. Priming ECFCs with ELN/41 imparts a potent protective effect and suggests potential for enhancing the reparative function in the context of cell therapy into ischaemic retina and choroid.

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