Progenitor cell combination normalizes pericyte ensheathment in the oxygen-induced retinopathy (OIR) model

Abstract

Purpose : We asked if intravitreal administration of CD34⁺ cells (bone marrow-derived) and/or endothelial colony forming cells (ECFCs) (vascular wall-derived) would promote vascular stabilization in the OIR model.

Methods : Cells were injected at postnatal day 5 (P5). On P7 pups were placed in 75% oxygen for 5 days and returned to room air on P12. Pups received a single intravitreal injection of saline or cells (human CD34⁺ isolated from peripheral blood, 10,000 cells; ECFCs - cord blood derived and expanded in culture, 100,000 cells; combination CD34⁺+ ECFCs, 110,000 cells). Experimental groups differed based on the euthanasia time (P5/P12, P5/P17). Retina wholemounts were stained with collagen IV antibodies to assess vaso-obliteration (VO) and neovascularization (NV) and to allow pericyte enumeration based on morphology. Retinas from all 4 cohorts were used for analysis by Reverse Phase Protein Array (RPPA).

Results : Maximum VO was seen in the saline and CD34⁺ pups of the P5/P12 group (Saline=22.3±0.4%; CD34⁺=23.4±0.8%, p=0.1), while ECFCs and CD34⁺/ECFCs (Combo) showed significant reduction (ECFCs=18.4±1.3%, p<0.05; Combo=20.0±0.7%, p<0.05). P5-injected ECFCs incorporated into and stimulated the Deep Vascular Plexus (DVP) formation by P12, whereas CD34⁺ cells did not. The combination of both cell types further enhanced DVP formation. Injection of either cell type alone at P5 resulted in NV levels similar to saline controls at P17; however, the combination of cells prevented NV. Pericyte ensheathment assessed at P17 showed in normoxia pups 5 ± 0.47 pericytes/100 µm; OIR and saline 3±0.31 pericytes/100 µm; OIR and ECFCs 4±0.40 pericytes/100 µm (p=0.39 compared to OIR and saline); OIR and combo 5±0.56 pericytes/100 µm (p=0.01 compared to OIR and saline). Proteomic analysis of P5/P17 retinas demonstrated that either cell type alone or the combo affects expression of proteins involved in remodeling. However, only the combination of both cell types increased the cytoskeletal reorganization/pericyte stabilization & ensheathment proteins, FAK (OIR ↑; restored by CD34⁺, ECFCs & CD34⁺/ECFCs), PDGFR-b (OIR ↑; restored partially by CD34⁺/ECFCs) and PKC-α-pS657(OIR ↓; restored only by CD34⁺/ECFCs).

Conclusions : Only the cell combination of CD34⁺ cells and ECFCs orchestrates normalization of vessel maturation, vessel stabilization and pericyte ensheathment in the OIR model.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.