Abstract
Purpose :
Corneal opacity stands as a leading cause of global blindness, traditionally addressed through penetrating and lamellar keratoplasties with donor corneal tissues. However, the challenges of a limited global supply, immune response, and long-term graft rejection underscore the need for alternative therapies. Corneal stromal stem cells (CSSCs) present a promising avenue, with their paracrine action facilitated by extracellular vesicles (EVs), showing an efficacy in inhibiting fibrosis and opacities. This study explored the anti-scarring potential of CSSC-derived EVs containing transforming growth factor (TGF)-β3 mRNA in a mouse model of corneal stromal injury.
Methods :
Donor CSSCs were cultured with conditioned media from pro-inflammatory M1 mouse macrophages (RAW264.7). Cellular and EV expression of human TGF-β1, 2, and 3 were examined by qPCR. In vitro fibrosis assay was conducted using primary corneal stromal fibroblasts (SFs) and keratocytes (CSKs) treated with and without CSSC-derived EVs. Six batches of CSSC-EVs (107 particles) were applied separately to mouse corneas (n=60) with anterior stromal injury. Corneal changes were examined with anterior segment optical coherent tomography and serial images were 3D-reconstructed for opacity density and volume analyses using Fiji and a customized Python-based tool. Corneas were harvested for inflammatory and fibrosis marker expression by qPCR and whole-mount immunostaining examined under confocal microscopy. Statistical analysis was conducted using one-way ANOVA.
Results :
Stimulation of CSSCs with M1 RAW cells upregulated TGFβ3 and CSSC-EV cargo had an upregulated TGFβ3 mRNA transcript levels. CSK and SF incubated with CSSC-derived EVs exhibited anti-fibrotic properties and reduced fibroblast-to-myofibroblast differentiation. Topical application of CSSC-EVs to injured mouse corneas suppressed corneal opacities with reduced opacity volume (P=0.0095) and density (P=0.0135), when compared with sham controls. The EV-treated mouse corneas had reduced expression of fibrosis markers (fibronectin, SPARC, tenascin C, collagen 3a1, and α-smooth muscle actin).
Conclusions :
Our findings highlight the potential of EV therapeutics as a cell-free strategy for corneal wound healing and opacity inhibition. EV quality can be evaluated by TGFb3 mRNA transcript levels in EV cargo prior to in vivo applications.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.