Hydroxycamptothecin and Substratum Stiffness Synergistically Regulate Fibrosis of Human Corneal Fibroblasts

Corneal fibrosis is a common outcome of inappropriate repair associated with trauma or ocular infection. Altered biomechanical properties with increased corneal stiffness is a feature of fibrosis that cause corneal opacities, resulting in severe visual impairment and even blindness. The present study aims to determine the effect of hydroxycamptothecin (HCPT) and matrix stiffness on transforming growth factor-β1 (TGF-β1)-induced fibrotic processes in human corneal fibroblasts (HTK cells). HTK cells were cultured on substrates with different stiffnesses (“soft”, ∼261 kPa; “stiff”, ∼2.5 × 10 3 kPa) and on tissue culture plastic (TCP, ∼10 6 kPa) and simultaneously treated with or without 1 μg/mL HCPT and 10 ng/mL TGF-β1. We found that HCPT induced decreased cell viability and antiproliferative effects on HTK cells. TGF-β1-induced expression of fibrosis-related genes ( FN1, ACTA2 ) was reduced if the cells were simultaneously treated with HCPT. Substrate stiffness did not affect the expression of fibrosis-related genes. The TGF-β1 induced expression of FN1 on both soft and stiff substrates was reduced if cells were simultaneously treated with HCPT. However, this trend was not seen for ACTA2, i.e., the TGF-β1 induced expression of ACTA2 was not reduced by simultaneous treatment of HCPT in either soft or stiff substrate. Instead, HCPT treatment in the presence of TGF-β1 resulted in increased gene expression of keratocyte phenotype makers ( LUM, KERA, AQP1, CHTS6 ) on both substrate stiffnesses. In addition, the protein expression of keratocyte phenotype makers LUM and ALDH3 was increased in HTK cells simultaneously treated with TGF-β1 and HCPT on stiff substrate as compared to control, i.e., without HCPT. In conclusion, we found that HCPT can reduce TGF-β1-induced fibrosis and promote the keratocyte phenotype in a substrate stiffness dependent manner. Thus, HCPT stimulation might be an approach to stimulate keratocytes in the appropriate healing stage to avoid or reverse fibrosis and achieve more optimal corneal wound healing.