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




Akoto, Theresa
Cai, Jingwen
Nicholas, Sarah
McCord, Hayden
Estes, Amy J.
Xu, Hongyan
Karamichos, Dimitrios
Liu, Yutao


0000-0002-8761-3824 (Karamichos, Dimitrios)

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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 TGFbeta1 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 TGFbeta1 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, TGFbeta1 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 TGFbeta1 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 TGFbeta1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFbeta1, thirteen were responsive to CMS, and six were responsive to TGFbeta1 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. TGFbeta1-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 TGFbeta1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFbeta1 treatment under CMS, suggesting a potential role of TGFbeta1 and biomechanical stretch in KC development.



Akoto, T., Cai, J., Nicholas, S., McCord, H., Estes, A. J., Xu, H., Karamichos, D., & Liu, Y. (2023). Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus-A Transcriptomic Profiling Study. International journal of molecular sciences, 24(8), 7437. https://doi.org/10.3390/ijms24087437


© 2023 by the authors.


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