Abstract
Purpose :
Biomechanical factors may contribute to the pathogenesis of keratoconus (KC). We aimed to determine the protein expression alterations of primary human corneal fibroblasts (HCF) and KC fibroblasts (HKC) in response to cyclic mechanical stretch (CMS) to mimic the physiological condition in human corneas.
Methods :
HCF (n=3) and HKC (n=3) cells were cultured in 6-well plates with or without of 15% CMS (1 cycle/s for 24-hours) using a computer-controlled Flexcell FX-6000T Tension system. Extracted cellular protein (30µg) was digested with trypsin followed with liquid chromatography-mass spectrometry-based comparative proteomics using label-free spectral counting method. After protein database search, we normalized the peptide spectra matches (PSM) of each protein to the total PSM of each sample expressed as a percentage before comparison to compensate for possible variation due to sample loading. We performed the following comparisons: HKC-CMS vs HKC-no-CMS, HCF-CMS vs HCF-no-CMS, HKC-CMS vs HCF-CMS, and HKC-no-CMS vs HCF-no-CMS. We used two-tailed Student’s t-test to identify differentially expressed proteins (DEP) (p<0.05). Gene ontology (GO) enrichment analysis was done using the WebGestalt 2013 version.
Results :
We identified a total of 5370 proteins in all the samples. On average, 1717 proteins were identified per sample, of which 1241 proteins were detected in ≥6 samples and 696 proteins in all 12 samples. Differential analysis identified 74 and 65 CMS-responsive proteins in HCF and HKC cells respectively. 12 and 6 proteins were induced by CMS in HCF and HKC cells respectively. We identified 79 DEP in HKC-no-CMS vs HCF-no-CMS and 67 DEP in HKC-CMS vs HCF-CMS. GO analysis indicated the involvement of cell cycle regulation and interferon-mediated signaling in HCF response to CMS while HKC responds to CMS with the involvement of DNA damage response, insulin receptor binding, and oxidoreductase activity. KC-specific GO includes protein complex assembly/disassembly, hydrolase activity, and mitochondrial proteins without CMS, but includes wound healing and DNA damage response with CMS.
Conclusions :
For the first time, our biomechanical study has identified a differential response of HKC cells to CMS with proteins involved in wound healing, DNA damage response, and oxidoreductase activity, suggesting the potential role of biomechanical stretch in KC development.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.