Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study

Abstract

Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFβ1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFβ1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70–90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFβ1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFβ1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFβ1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFβ1, thirteen were responsive to CMS, and six were responsive to TGFβ1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFβ1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFβ1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFβ1 treatment under CMS, suggesting a potential role of TGFβ1 and biomechanical stretch in KC development.

Differentially expressed microRNAs targeting genes in key pathways in keratoconus

“…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 ). Several other studies support our results ( Khaled et al, 2018 ; Xu L. et al, 2022 ; Dou et al, 2022 ; Akoto et al, 2023 ), including Bykhovskaya et al claiming that the development of KTCN results in deregulation of gene expression of ECM and adhesion molecules ( Bykhovskaya et al, 2016 ). The result highlighting the pathway of response to mechanical stimuli is consistent with the existing literature and our current studies on environmental factors and habits of patients with KTCN, which have pointed to repetitive mechanical corneal injuries caused by eye rubbing as a factor contributing to the development of KTCN, potentially through the alteration of gene expression in corneal epithelium ( Najmi et al, 2019 ; Jaskiewicz et al, 2023c ).…”

Longitudinal Changes of Cornea Volume Measured by Means of Anterior Segment-Optical Coherence Tomography in Patients with Stable and Progressive Keratoconus

“…This alteration leads to loss of corneal rigidity and, consequently, corneal protrusion and thinning [10,11]. It has been proposed that several factors, including localized changes in proteolitic enzyme activity and pro-inflammatory citokines and a reduced production of collagen I, are associated with breaks in Bowman's layer and may simultaneously also promote the unraveling of collagen fibrils, lamellar slippage and the disinsertion of lamellae from their anchoring sites in Bowman's layer [12][13][14][15][16]. When comparing keratoconus with normal corneas, the collagen fibrillar mass is unevenly distributed, particularly at the apex of the cone, and the organization of the stromal lamellae is drastically altered [12].…”