Abstract
Purpose:
The Myocardin-related transcription factor/ Serum response factor (MRTF/ SRF) pathway is a paradigm of how important gene transcription is in conjunctival fibrosis. Recent studies have also shown that matrix stiffness plays a key role in fibrosis, and new biomaterials with different substrate stiffness are currently being developed for tube implants in glaucoma. We hypothesised that matrix stiffness would be an important modulator of the MRTF/ SRF pathway and genes associated with conjunctival fibrosis.
Methods:
The localisation of MRTF-A was characterised in human Tenon’s fibroblasts using immunofluorescence and hydrogels of 0.5 kPa (soft matrix) and 50 kPa (stiff matrix). The effect of matrix stiffness on the expression of serum response factor and key fibrotic genes, ACTA2, CTGF, and MYL9, was measured using real-time qPCR. All mRNA values were normalised relative to that of GAPDH, and each experiment was carried out as independent triplicates (n=3) under different conditions. Statistical analysis was performed using the Student’s t-test to calculate statistically significant differences and individual p values. Western blotting was also used to measure the expression of SRF, ACTA2, and MYL9 proteins using different matrix stiffness.
Results:
Human Tenon’s fibroblasts (HTFs) showed marked changes in cell morphology when plated on soft matrix compared to stiff matrix. MRTF-A localisation in HTFs was nuclear on stiff matrix (50 kPa), but redistributed into the cytoplasm on soft matrix (0.5 kPa). The expression of serum response factor and key fibrotic genes, ACTA2, CTGF, and MYL9, were all significantly increased when HTFs were plated on stiff matrix compared to soft matrix (p<0.05). The expression of SRF, ACTA2, and MYL9 proteins were also increased in HTFs with stiff matrix compared to soft matrix.
Conclusions:
Human Tenon’s fibroblasts are highly sensitive to mechanotransduction. There is a significant increase in the expression of serum response factor and other key fibrotic genes in the conjunctiva with stiff matrix (50 kPa) compared to soft matrix (0.5 kPa). These data suggest that biomaterials with low substrate stiffness will tend to be less fibrogenic, which is important when designing materials for glaucoma tube implants and other drug delivery devices in the eye.