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Ex Vivo Corneal Organ Culture Model for Wound Healing Studies
Published on: February 15, 2019
Temporal and spatial distribution of TGF-beta isoforms and signaling intermediates in corneal regenerative wound
Man-I L Huh1, Yongmin Chang, Jae-Chang Jung
1Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, Korea.
Abstract:
The present study analyzed the temporal and spatial expression of TGF-beta isoforms and activated pSmad2 and p38MAPK during epithelial debridement wound repair, using chick cornea by immunohistochemistry. Normal corneas showed low-level TGF-betas staining. Following wounding, TGF-beta1 expression was strong in the Bowman's layer (BL). TGF-beta3 expression was confined to basal cells in the regenerating and unwounded regions, and was not detected in migrating epithelial, stromal or endothelial cells. In addition, TGF-beta3 treatment stimulated the proliferation of cultured epithelial cells. Our present findings seem to suggest that the TGF-beta3 signal may be required for epithelial cell proliferation. TGF-beta2 expression was strong in migrating and proliferating epithelial cells, many active migrating fibroblasts at the wound edge, endothelial cells and Descemet's membrane (DM). Although both nuclear pSmad2 and p38MAPK staining was observed in many basal epithelial cells, pSmad2 positive cells were co-localized with PCNA positive cells. Therefore, it seems likely that the pSmad2 signal may affect epithelial cell proliferation in healing corneas. Both pSmad2 and p38MAPK expression were also observed in endothelial cells. Interestingly, many active fibroblasts over the whole stroma in early wound healing at day 2 expressed nuclear pSmad2, but little if any cytoplasmic p38MAPK. Collectively, temporal/spatial up-regulation and distribution of the three TGF-beta isoforms, as well as concerted activation of both Smad2 and p38MAPK, appears to be a key aspect of regenerative corneal wound healing in the chick.
Insights
Transforming growth factor-beta (TGF-beta) isoforms and signaling pathways are crucial for corneal wound healing. This study reveals TGF-beta3 promotes epithelial cell proliferation, while TGF-beta2 is involved in fibroblast and endothelial cell activity.
Area of Science:
- Ophthalmology
- Cell Biology
- Regenerative Medicine
Background:
- Corneal wound healing is a complex process involving epithelial regeneration.
- Transforming growth factor-beta (TGF-beta) signaling pathways play a critical role in tissue repair.
- Understanding the specific roles of TGF-beta isoforms and downstream signaling molecules is essential for optimizing corneal healing.
Purpose of the Study:
- To investigate the temporal and spatial expression of TGF-beta isoforms (TGF-beta1, TGF-beta2, TGF-beta3) during epithelial debridement wound repair in the chick cornea.
- To analyze the activation patterns of key signaling molecules, pSmad2 and p38 mitogen-activated protein kinase (MAPK), in corneal wound healing.
- To elucidate the potential roles of TGF-beta isoforms and their signaling pathways in promoting corneal epithelial cell proliferation and overall wound regeneration.
Main Methods:
- Immunohistochemistry was employed to examine the expression and localization of TGF-beta isoforms, activated pSmad2, and p38MAPK in chick corneas following debridement wounding.
- Primary corneal epithelial cells were cultured and treated with TGF-beta3 to assess its effect on cell proliferation.
- Co-localization studies with PCNA (proliferating cell nuclear antigen) were performed to correlate pSmad2 activation with epithelial cell proliferation.
Main Results:
- Normal corneas exhibited low TGF-beta staining. Post-wounding, TGF-beta1 was prominent in the Bowman's layer.
- TGF-beta3 was localized to basal epithelial cells and stimulated epithelial cell proliferation in vitro.
- TGF-beta2 showed strong expression in migrating epithelial cells, fibroblasts, endothelial cells, and Descemet's membrane.
- Both nuclear pSmad2 and p38MAPK were detected in basal epithelial cells, with pSmad2 co-localizing with PCNA.
- Activated pSmad2 and p38MAPK were also observed in endothelial cells. Fibroblasts showed nuclear pSmad2 expression early in wound healing.
Conclusions:
- TGF-beta3 signaling appears essential for stimulating corneal epithelial cell proliferation during wound repair.
- TGF-beta2 plays a role in the migration and proliferation of epithelial cells and fibroblasts, as well as endothelial cell activity.
- The concerted activation of Smad2 and p38MAPK pathways, alongside the specific distribution of TGF-beta isoforms, is a critical component of regenerative corneal wound healing in this model.
- These findings highlight the intricate molecular mechanisms underlying corneal regeneration and suggest potential therapeutic targets for enhancing healing outcomes.
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