Abstract
Purpose :
Preclinical and clinical studies have shown that human corneal stromal stem cells (CSSC) prevent corneal scarring and regenerate transparent stromal tissue. CSSC treatment blocked neutrophil infiltration into the injured stroma via the paracrine action of extracellular vesicles (EV). This study investigated the mechanisms by which CSSC EV suppress corneal inflammation and fibrosis.
Methods :
Conditioned media of primary human CSSC cultures were collected for EV isolation. Total RNA library of EV fractions was analyzed by RNAseq and microRNAs (miRs) specific to CSSC of high regenerative potential were identified. Target gene search and enriched pathway analyses from single or combinations of miR were studied by TargetScan, miRDB, and DAVID bioinformatics. Transfection of miR having statistical significance in association to inflammatory, fibrosis, and immune pathways were examined by (1) mouse macrophage (RAW264.7) for M1/M2 phenotype switch assay; and (2) the fibrosis assay of human corneal stromal fibroblasts after TGFβ1 and ascorbate treatment.
Results :
A total of 17 miRs were significantly enriched in EVs of human CSSC having high regenerative potential and scar reduction in mouse corneal wound model. Using target gene search and pathway analyses, 9 miR combinations were predicted to be significantly associated with signaling cascades of tissue inflammatory and fibrosis responses, as well as tissue regeneration (P<10-6). Transfection of miR mimics to RAW cells showed that 4 groups suppressed M1 phenotype (reduced pro-inflammatory iNOS and MCP1 expression) after lipopolysaccharide (50 ng/ml) treatment. Among them, 1 group was shown to revert pre-M1 RAW to M2 phenotype (increased anti-inflammatory Arg1 and Ptgs1 expression), similar to interleukin-4 (20 ng/ml) treatment. These miRs also downregulated the fibrosis gene expression (αSMA and tenascin C) of human CSSC treated by TGFβ1 (10 ng/ml) and ascorbate.
Conclusions :
Specific miRs produced by CSSC and delivered via EVs exerted anti-inflammatory and anti-fibrotic activities. Further in vivo study will demonstrate their therapeutic efficiency in preventing corneal scarring and regenerating transparent corneal tissues after injury.
This is a 2021 ARVO Annual Meeting abstract.