Abstract
Purpose :
Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in primary human corneal (HCF) and KC fibroblasts (HKC) combined with TGFβ1 treatment under cyclic mechanical stretch (CMS) mimicking the pathophysiological condition in KC.
Methods :
HCF (n=4) and HKC (n=4) cells 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 hrs) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in a total of 48 HCF/HKC samples (100bp PE, 70-90 million reads/sample) followed by a bioinformatics analysis using our established pipeline with PartekFlow 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. We used the PANTHER classification system and the DAVID bioinformatics resources to identify significantly enriched pathways (FDR≤0.05).
Results :
Our multi-factorial ANOVA analyses identified 479 DEGs in HKCs vs. HCFs justified by the TGFβ1 treatment and CMS status. Among these DEGs, 199 KC-altered genes were responsive to TGFβ1, 13 were responsive to CMS, and six were responsive to TGFβ1 and CMS together. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in many KC-relevant functions, including but not limited to degradation of extracellular matrix, 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 (OBSCN, CLU, HDAC5, AK4, ITGA10 and F2RL1) have been reported to be involved in cytoskeletal organization, apoptosis, activation and myofibroblastic differentiation of corneal stromal cells, oxidative stress, collagen binding and inflammatory response, respectively. Some KC-altered genes such as CLU and F2RL1 were identified to be responsive to both TGFβ1 and CMS.
Conclusions :
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.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.