The Effect Of Matrix Stiffness On The Biological Function Of Retinal Pigment Epithelial Cells And Its Mechanism

BackgroundEpithelial-mesenchymal transition（EMT）is the process by which the epithelial cells are transformed into mesenchyme cells through a specific procedure,thereby acquiring the ability to migrate and invade.It has been shown that the pathogenesis of retinal fibrosis diseases such as age-related macular degeneration and proliferative vitreoretinopathy involves EMT in the retinal pigment epithelium（RPE）cells.However,the initiation mechanism of EMT in RPE cells has not been fully elucidated.Recently,the role of extracellular matrix（ECM）stiffness in regulating cellular biological functions has received extensive attention.Retinal ECM stiffness is altered in physiological and pathological conditions.It has been shown that the stiffness of Bruch membranes can increase by at least one order of magnitude with age.ECM stiffness has been shown to affect RPE cell phagocytosis and expression of inflammation-inducing factors.However,whether matrix stiffness regulates the EMT process in RPE cells remains to be confirmed by the study.Therefore,in this study,we propose to simulate in vivo retinal ECM stiffness changes by constructing polyacrylamide hydrogels with different stiffnesses and to investigate whether matrix stiffness can promote the EMT process in RPE cells and to explore the molecular mechanisms behind it using morphological,functional phenotypic and molecular biological studies.MethodsIn this study,polyacrylamide hydrogels with different stiffnesses were successfully prepared.The actual elastic modulus of each group of hydrogels were5.45±0.62 k Pa,10.89±1.39 k Pa,20.91±1.34 k Pa and 41.07±1.40 k Pa respectively,which are consistent with the theoretical elastic modulus.The height of the largest protrusion on the surface of the hydrogel covered with Col-I was 2.60 nm,which had a negligible effect on ARPE-19 cells with a size of μm.There was no difference in the effects of the hydrogel extracts on the proliferation activity of ARPE-19 cells among the groups,which proves the safety of the hydrogels.As the substrate stiffness increases,ARPE-19 cells transformed from spherical to spindle shape,with increased mean cell area and mean cell aspect ratio,which proves that substrate stiffness promoted the transition from epithelial to mesenchymal cell morphology.We found that enhanced F-actin cytoskeleton assembly and increased stress fiber formation were accompanied by increased substrate stiffness.The F-actin cytoskeleton showed diffuse distribution in the low stiffness group（5k Pa）,failing to form fiber bundles or only forming short fiber bundles at the cell edge;in the high stiffness group（40k Pa）,it showed a thick and long fiber network,and the formed stress fibers spanned the entire cell,which demonstrates that substrate stiffness can promote the assembly of F-actin cytoskeleton and the formation of stress fiber.The total cell area per unit area of ARPE-19 cells increased with increasing matrix stiffness,which proves that substrate stiffness promotes proliferation of ARPE-19 cells.The relative migration area of ARPE-19 cells increased with increasing substrate stiffness,which proves that substrate stiffness promotes migration of ARPE-19 cells.Finally,we found that the expression levels of epithelial marker proteins（E-cadherin and ZO-1）were decreased in ARPE-19 cells and the expression levels of mesenchymal marker proteins（vimentin）were increased along with increased substrate stiffness.ResultsThe increase of substrate stiffness promoted ARPE-19 cells to transform from spherical to spindle shape,promoted the assembly of F-actin cytoskeleton in ARPE-19 cells,and promoted ARPE-19 cell proliferation and migration process.Increased substrate stiffness down-regulated E-Cadherin and ZO-1 expression levels and upregulated vimentin expression levels.ConclusionsIncreased substrate stiffness promoted proliferation and migration of ARPE-19 cells,alters cell morphology and cytoskeleton assembly,inhibit the expression of epithelialization protein（E-Caherin and ZO-1）,and promote the expression of interstitial protein（vimentin）,namely,increased substrate stiffness induces EMT in ARPE-19 cells.