Corneal epithelial and neuronal interactions: Role in nerve regeneration and wound healing
Final Report Abstract
In the cornea, on-going interactions between the corneal epithelial cells, stromal keratocytes and trigeminal sensory neurons result to the release of various growth factors that play prominent roles during wound repair. This study aims to unravel molecular cues involved in these cell-cell interactions along with their role in nerve regeneration during wound healing. In response to the injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and contribute to the wound repair. Using antibody microarrays (AM), we have profiled differentially expressed cell surface markers and cytokines to study the influence of the conditioned medium collected from the activated SFs (SFCM) along with substance P (SP) on corneal epithelial cell function. AM analysis data revealed enhanced expression of the ITGB1 in epithelial cells upon the addition of SP whereas SFCM induced expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. We also observed activation of MAPK signaling, increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT)-regulating transcription factors Slug and ZEB1 expression were also enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with up-regulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing. Bone morphogenetic proteins (BMPs) are unique multi-functional potent growth factors of the TGF-β superfamily. Treatment of corneal epithelial cells with SP and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 revealed activation of TGF-β signaling and cell cycle pathways. BMP7 stimulation studies show activation of MAPK cascades proteins, differences in the expression of claudin, ZEB1 along with phosphorylation levels of cofilin. Wound healing assay shows increased motility and migration of BMP7 treated cells. An elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed in corneal epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. Based on our transcriptome analysis data and the results, we conclude that BMP7 contributes to EMT-like responses and plays a role equivalent to TGF-β in the course of corneal wound healing.
Publications
- (2018) Role of Bone Morphogenetic Protein 7 (BMP7) in the Modulation of Corneal Stromal and Epithelial Cell Functions. International journal of molecular sciences 19 (5) 1415
Kowtharapu, Bhavani S.; Prakasam, Ruby Kala; Murín, Radovan; Koczan, Dirk; Stahnke, Thomas; Wree, Andreas; Jünemann, Anselm G. M.; Stachs, Oliver
(See online at https://doi.org/10.3390/ijms19051415) - Quantitative assessment of central and limbal epithelium after long-term wear of soft contact lenses and in patients with dry eyes: a pilot study. Eye (Lond). 2016 Jul;30(7):979-86
Prakasam RK, Kowtharapu BS, Falke K, Winter K, Diedrich D, Glass A, Jünemann A, Guthoff RF, Stachs O
(See online at https://doi.org/10.1038/eye.2016.58) - Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining. Eye (Lond). 2017 Mar;31(3):481-490
Kowtharapu BS, Winter K, Marfurt C, Allgeier S, Köhler B, Hovakimyan M, Stahnke T, Wree A, Stachs O, Guthoff RF
(See online at https://doi.org/10.1038/eye.2016.220) - Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation. J Microsc. 2017 Jan;265(1):21-26
Kowtharapu BS, Marfurt C, Hovakimyan M, Will F, Richter H, Wree A, Stachs O, Guthoff RF
(See online at https://doi.org/10.1111/jmi.12457) - Role of corneal stromal cells on epithelial cell function during wound healing. Int J Mol Sci. 2018 Feb 4;19(2)
Kowtharapu BS, Murín R, Jünemann AGM, Stachs O
(See online at https://doi.org/10.3390/ijms19020464)
