Abstract
Purpose :
Keratoconus (KC) represents the most prevalent ectatic corneal disease influenced by corneal biomechanics, yet its pathogenesis remains elusive. This study aims to develop an in vitro KC disease model with modifiable stromal stiffness to explore the effects of reduced stromal stiffness on KC pathogenesis.
Methods :
The KC disease group (L group) was created using a low matrix stiffness collagen hydrogel containing human corneal stromal cells (HCSCs), while a high matrix stiffness group (H group) was established using a plastic compression method. Additionally, a cross-linking treatment model was applied to the L group using corneal collagen cross-linking (CXL) (Fig 1A). Initial testing included assessing the matrix stiffness of H/L/CXL groups. Subsequently, utilizing bulk RNA-seq, proteomics, and qPCR, we investigated the differential expression of genes related to immune, oxidative, and antioxidant processes in HCSCs under varying stromal stiffness conditions. Reactive oxygen species (ROS) pressure testing on the H/L group was conducted using H2O2. The differences in ROS production between the H and L groups were also examined. The main antioxidant factor, ALDH3A1, was detected through immunofluorescence staining. Furthermore, ELISA was employed to analyze IL-6 and TNF-α levels.
Results :
Matrix stiffness analysis revealed significantly lower stromal stiffness in the L group compared to the H and CXL groups (Fig 1B). Bulk RNA-seq, proteomics, and qPCR analysis demonstrated a notable increase in the expression of immune and oxidative stress-related factors in the L group, while collagen-related factors and antioxidant-related factors exhibited a significant decrease (Fig 1C-D). The L group exhibited a remarkable increase in ROS production without H2O2 intervention (Fig 1E). ALDH3A1 showed a reduction in the L group compared to the H and CXL groups (Fig 1F). ELISA results indicated a significant increase in IL-6 and TNF-α levels in the L group (Fig 1G).
Conclusions :
This study successfully established an in vitro KC model with adjustable stromal stiffness, revealing that low matrix stiffness can replicate the pathogenesis of KC in vitro and can be mitigated through CXL treatment.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.