The impact of substrate stiffness on morphological, transcriptional and functional aspects in RPE.
Lasse WolframClara GimpelMelanie SchwämmleSimon J ClarkDaniel BöhringerGünther SchlunckPublished in: Scientific reports (2024)
Alterations in the structure and composition of Bruch's membrane (BrM) and loss of retinal pigment epithelial (RPE) cells are associated with various ocular diseases, notably age-related macular degeneration (AMD) as well as several inherited retinal diseases (IRDs). We explored the influence of stiffness as a major BrM characteristic on the RPE transcriptome and morphology. ARPE-19 cells were plated on soft ( E = 30 kPa ) or stiff ( E = 80 kPa ) polyacrylamide gels (PA gels) or standard tissue culture plastic (TCP). Next-generation sequencing (NGS) data on differentially expressed small RNAs (sRNAs) and messenger RNAs (mRNAs) were validated by qPCR, immunofluorescence or western blotting. The microRNA (miRNA) fraction of sRNAs grew with substrate stiffness and distinct miRNAs such as miR-204 or miR-222 were differentially expressed. mRNA targets of differentially expressed miRNAs were stably expressed, suggesting a homeostatic effect of miRNAs. mRNA transcription patterns were substrate stiffness-dependent, including components of Wnt/beta-catenin signaling, Microphthalmia-Associated Transcription Factor (MITF) and Dicer. These findings highlight the relevance of mechanical properties of the extracellular matrix (ECM) in cell culture experiments, especially those focusing on ECM-related diseases, such as AMD.
Keyphrases
- extracellular matrix
- age related macular degeneration
- transcription factor
- induced apoptosis
- cell proliferation
- cell cycle arrest
- long non coding rna
- gene expression
- endoplasmic reticulum stress
- amino acid
- signaling pathway
- cell death
- binding protein
- dna binding
- diabetic retinopathy
- oxidative stress
- genome wide
- rna seq
- heat shock protein