Abstract
Purpose :
Endothelial colony forming cells (ECFCs) are endothelial progenitor cells with neurovasculotrophic effects achieved via paracrine action. ECFCs, however, are not functionally homogenous. ECFCs with high expression of CD44 hyaluronan receptor (CD44hi) rescue the oxygen-induced retinopathy (OIR) model of ischemic retinopathy as well as rd10 mice with retinal neurodegeneration; ECFCs with low expression of CD44 (CD44lo) do not. Extracellular vesicles from CD44hi ECFCs (EVshi) recapitulate this effect while vesicles from CD44lo ECFCs (EVslo) are ineffective. Here, we investigate the role of EVshi in mediating the paracrine rescue effects of CD44hi ECFCs and compare their intravesicular small RNA cargo of EVshi and EVslo using an -omics based approach to identify microRNA (miR) mediators of their trophic effects.
Methods :
Sorted CD44hi ECFCs treated with neutral sphingomyelinase inhibitor GW4869, used to inhibit exosome biogenesis/release, were intravitreally injected into OIR mice. DICER1 knockdown ECFCs (ECFCs-shDICER1) and a control scramble RNA cell line (ECFCs-scrRNA) were generated using lentiviral transfection. Sorted CD44hi ECFCs-shDICER1 and CD44hi ECFCs-scrRNA and their EVs were intravitreally injected into OIR mice. Small RNA of EVshi and EVslo were sequenced with Next Generation Sequencing to identify differentially expressed intravesicular miRs. miRs that validated well on RT-qPCR and control scramble miR were injected into OIR.
Results :
CD44hi ECFCs rescued OIR and these effects were attenuated after GW4869 treatment. CD44hi ECFCs-shDICER1 and their EVs were significantly less effective in rescuing OIR than CD44hi ECFCs-scrRNA and their EVs, respectively. Small RNA sequencing of EVshi identified 9 upregulated and 10 downregulated miRs in comparison to EVslo. On RT-qPCR, 7 of the 9 upregulated miRs validated well. All miRs were functionally tested in OIR and 3 candidate miRs fully rescued the model.
Conclusions :
CD44hi ECFCs shed vesicles containing neurovasculotrophic miRs that may contribute to the efficacy of EVshi treatment in mouse models of ischemic/neurodegenerative retinopathy.
This is a 2021 ARVO Annual Meeting abstract.