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. 2022 Sep 16;22(1):368.
doi: 10.1186/s12886-022-02592-8.

Mechanobiological responses of astrocytes in optic nerve head due to biaxial stretch

Affiliations

Mechanobiological responses of astrocytes in optic nerve head due to biaxial stretch

Zhiwen Li et al. BMC Ophthalmol. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Cell identification results: The results of cell identification showed these cells were astrocytes through positive staining for GFAP, vimentin and Connexin43(CX43). Negative staining for Aquaporin 4 (AQP4), OSP and F4/80 was also shown. Scale bar: 100 μm
Fig. 2
Fig. 2
Cytoskeleton changes before and after stretch: (A) Cells in the control group were arranged irregularly. (B) and (C) were cytoskeleton changes after 10% and 17% stretch, in which the astrocyte cytoskeleton was arranged in an orderly manner and the cell shape was fusiform
Fig. 3
Fig. 3
Cell apoptosis before and after stretch: (A) 10% control group, (B) 10% stretch group, (C) 17% control group, (D) 17% stretch group. (E) The result of apoptosis rate. Error bars indicate standard error of the mean (SEM); there was no statistically significant difference between control and stretch group in both groups
Fig. 4
Fig. 4
Astrocytes proliferation of different groups: (A, B, C, D) EDU labeled astrocytes in 10 and 17% groups. (E, F, G, H) DAPI labeled astrocytes in 10 and 17% groups. (I, J, K, L) Merged images showed double-labeled astrocytes in all the four groups. (N) Cell proliferation rate decreased in both the 10 and 17% stretch groups. Error bars indicate standard error of the mean (SEM); **P < 0.01 and indicates statistical significance
Fig. 5
Fig. 5
GO of differentially expressed proteins in 10% stretch group (A) and 17% stretch group (B)
Fig. 6
Fig. 6
Differential proteins from GO and KEGG enrichment analysis, where CA is control group; EA is 10% stretch group; EB is 17% stretch group
Fig. 7
Fig. 7
Western blot analysis of specific protein expression (n = 3). (A) Colored bands of GFAP, LRP6, THBS1 and CD81; (B) Expression levels of LRP6, THBS1 and CD81 gradually decreased with the increase of the stretch amplitude, while the expression level of GFAP gradually increased with the increase of the stretch amplitude. Error bars indicate standard error of the mean (SEM); *P < 0.05 and **P < 0.01 indicate statistical significance

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