The Impaired Wound Healing Process Is a Major Factor in Remodeling of the Corneal Epithelium in Adult and Adolescent Patients With Keratoconus

Abstract

Purpose Keratoconus (KTCN) is the most common corneal ectasia, characterized by pathological cone formation. Here, to provide an insight into the remodeling of the corneal epithelium (CE) during the course of the disease, we evaluated topographic regions of the CE of adult and adolescent patients with KTCN. Methods The CE samples from 17 adult and 6 adolescent patients with KTCN, and 5 control CE samples were obtained during the CXL and PRK procedures, respectively. Three topographic regions, central , middle , and peripheral , were separated toward RNA sequencing and MALDI-TOF/TOF Tandem Mass Spectrometry. Data from transcriptomic and proteomic investigations were consolidated with the morphological and clinical findings. Results The critical elements of the wound healing process, epithelial–mesenchymal transition, cell–cell communications, and cell–extracellular matrix interactions were altered in the particular corneal topographic regions. Abnormalities in pathways of neutrophils degranulation, extracellular matrix processing, apical junctions, IL, and IFN signaling were revealed to cooperatively disorganize the epithelial healing. Deregulation of the epithelial healing, G2M checkpoints, apoptosis, and DNA repair pathways in the middle CE topographic region in KTCN explains the presence of morphological changes in the corresponding doughnut pattern (a thin cone center surrounded by a thickened annulus). Despite similar morphological characteristics of CE samples in adolescents and adults with KTCN, their transcriptomic features were different. Values of the posterior corneal elevation differentiated adults with KTCN from adolescents with KTCN and correlated with the expression of TCHP , SPATA13 , CNOT3 , WNK1 , TGFB2 , and KRT12 genes. Conclusions Identified molecular, morphological, and clinical features indicate the effect of impaired wound healing on corneal remodeling in KTCN CE.

Sequence variants contributing to dysregulated inflammatory responses across keratoconic cone surface in adolescent patients with keratoconus

“…Previously, as elements of innate immune responses, the expression of TLR2 and TLR4 has been studied by flow cytometry in corneal epithelial cells and blood samples of KTCN patients and their relatives (16,84,85), and significant upregulation has been shown, indicating a potential of the TLR2 and TLR4 proteins to be the KTCN biomarkers. However, no sequence variants in the TLRs were recognized here as well as in our current gene and protein expression assessment (13). Instead, we recognized sequence variants of other innate immune system elements (e.g., MUC5AC, as mucins consist the first-line defense).…”

“…Our study performed in the designated three topographic regions of the CE, central, middle, and peripheral, enabled the conclusion that there is no genomic heterogeneity across the corneal surface in KTCN. However, in a comprehensive morphological, transcriptomic, and proteomic evaluation performed in the same corneal samples, we identified the variability shaping the distinct features of CE zones (13). Continuing the topographic aspects of the CE, we have considered features of mosaicism to be involved in the process of remodeling of the KTCN CE.…”

“…In the morphological observations of the CE (including confocal and immunofluorescence microscopy), the decreased cell density and their elongated shape, disruptions in basal integrity, increased number of apoptotic cells, and depositions of iron particles have been previously described (7,10,11). Moreover, some molecular proteomic and transcriptomic findings showed disruption of elements of Wnt signaling (e.g., WNT10A) (12), cell-cell communications (e.g., CDH13) (13), pro-inflammatory cytokines (e.g., IL6) (14), matrix metalloproteinases (e.g., MMP9) (15), and innate immune system (e.g., TLR2 and TLR4) (16).…”

“…Simultaneously, the transcriptomic and proteomic assessments as well as morphological evaluation in the same CE samples were performed, as described in detail elsewhere (13). All the data of the mentioned investigation were considered in final interpretation of the study outcomes.…”

Differentially expressed microRNAs targeting genes in key pathways in keratoconus

“…Moreover, in our continuous study on additional corneal samples derived again from unrelated Polish patients with KTCN, we have identified both coding and non-coding sequence variation in genes that are involved, among others, in ECM and Wnt signaling ( Jaskiewicz et al, 2023a ). Cell-ECM interactions and ECM processing, being the critical elements of the wound healing process, were altered in corneal topographic regions of corneal epithelium from 23 patients with KTCN ( Jaskiewicz et al, 2023b ). Furthermore, we identified changes in DNA methylation level in WNT3 and WNT5A genes encoding Wnt ligands, which might be an explanation for the Wnt signaling pathway dysregulation in KTCN ( Kabza et al, 2019 ).…”