Background: Elevated intraocular pressure (IOP) is the main risk factor for glaucoma, which might cause the activation of astrocytes in optic nerve head. To determine the effect of mechanical stretch on the astrocytes, we investigated the changes in cell phenotype, proteins of interest and signaling pathways under biaxial stretch.
Method: The cultured astrocytes in rat optic nerve head were stretched biaxially by 10 and 17% for 24 h, respectively. Then, we detected the morphology, proliferation and apoptosis of the stretched cells, and performed proteomics analysis. Protein expression was analyzed by Isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. Proteins of interest and signaling pathways were screened using Gene Ontology enrichment analysis and pathway enrichment analysis, and the results were verified by western blot and the gene-chip data from Gene Expression Omnibus (GEO) database.
Result: The results showed that rearrangement of the actin cytoskeleton in response to stimulation by mechanical stress and proliferation rate of astrocytes decreased under 10 and 17% stretch condition, while there was no significant difference on the apoptosis rate of astrocytes in both groups. In the iTRAQ quantitative experiment, there were 141 differential proteins in the 10% stretch group and 140 differential proteins in the 17% stretch group. These proteins include low-density lipoprotein receptor-related protein (LRP6), caspase recruitment domain family, member 10 (CARD10), thrombospondin 1 (THBS1) and tetraspanin (CD81). The western blot results of LRP6, THBS1 and CD81 were consistent with that of iTRAQ experiment. ANTXR2 and CARD10 were both differentially expressed in the mass spectrometry results and GEO database. We also screened out the signaling pathways associated with astrocyte activation, including Wnt/β-catenin pathway, NF-κB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, ECM-receptor interaction, and transforming growth factor-β (TGF-β) signaling pathway.
Conclusion: Mechanical stimulation can induce changes in cell phenotype, some proteins and signaling pathways, which might be associated with astrocyte activation. These proteins and signaling pathways may help us have a better understanding on the activation of astrocytes and the role astrocyte activation played in glaucomatous optic neuropathy.
Keywords: Astrocytes; Biaxial stretch; Mechanobiological responses; Optic nerve head; Proteomics.
© 2022. The Author(s).