Actin filament and microtubule-centered cytoskeletal abnormalities are associated with intracellular structural disorder and cellular softening in keratoconus stromal fibroblasts.

Keratoconus (KC) is characterized by progressive corneal thinning and biomechanical weakening, however, the cellular mechanobiological basis underlying stromal softening remains poorly understood. In this study, we investigated actin filament and microtubule-centered cytoskeletal abnormalities and their associations with intracellular structural disorder and cellular mechanics in primary stromal fibroblasts derived from corneas with KC and normal corneas. Confocal microscopy revealed disrupted actin filament orientation and altered microtubule coherency in KC stromal fibroblasts. Quantitative phase imaging demonstrated significantly increased intracellular disorder strength in KC (t = 5.597, p < 0.001), while atomic force microscopy revealed a reduced Young's modulus (t = 11.206, p < 0.001). Notably, disorder strength showed a strong negative correlation with cellular stiffness (r = -0.904, p < 0.001), indicating a close correlation between intracellular structural disorganization and cellular softening. Furthermore, KC stromal fibroblasts exhibited coordinated reductions in actin, tubulin, vimentin, and lamin A/C expression at both the mRNA and protein levels, suggesting broad alterations in cytoplasmic and nucleo-cytoskeletal components. To evaluate the functional contribution of cytoskeletal integrity to cellular mechanics, normal stromal fibroblasts were treated with cytochalasin D or nocodazole to disrupt actin filaments or microtubules, respectively. Both interventions reduced Young's modulus and were accompanied by increased MMP2 and MMP9 expression and decreased COL1A expression, suggesting changes in ECM-remodeling-related protein expression following cytoskeletal perturbation. Collectively, these findings provide multimodal cellular mechanobiological evidence demonstrating associations among cytoskeletal abnormalities, intracellular structural disorder, and altered cellular mechanics in KC, expanding the understanding of KC pathogenesis beyond collagen and extracellular matrix abnormalities.