Gene Expression Changes Under Mechanical Stretching In Rat Retinal M(u|")ller Cells

The retina is subjected to tractional forces in various conditions, such as pathological myopia, posterior vitreous detachment, proliferative vitreoretinopathy, and so on. Such stretching forces played an important role in the mechanisms of these diseases.As the predominant glial element in the sensory retina, Muller cells are responsible for the homeostatic and metabolic support of retinal neurons and play an active role in almost all forms of retinal injury and disease. A recent study has confirmed that they are sensitive and responsive to tissue stretching. However, the cellular and molecular effects of mechanical stretching of the retina are relatively unexplored, and therefore, further research is required in this regard.In this study, we aim to test the hypothesis that the effect of constant mechanical stretching on MMP-2 expression in Muller cells, and to investigate the genome regulation of Muller cells under mechanical stretching in detail. This may provide clues to understanding the molecular mechanisms that would account for many retinal diseases in which the retina is often subjected to mechanical forces.Part IConstant mechanical stretching induces MMP-2expression in rat retinal glial (Muller) cellsPurpose:To investigate whether mechanical stretching enhances the expression of MMP-2 in Muller cells, which might be involved in retinal remodeling.Methods:Isolated rat retina Muller cells were seeded onto flexible bottom culture plates, and subjected to a constant stretch regimen of 15% equibiaxial stretch for 24h. Nonstretched cells served as controls. Intracellular MMP-2 mRNA and protein levels were measured by real-time PCR and western blot respectively. Extracellular MMP-2 protein levels were determined by western blot and ELISA analysis, and its activity was assessed by reverse zymography. Results:Mechanical stretching of Muller cells introduced significant increase in intracellular MMP-2 mRNA and protein levels after 24h. Extracellular secretory MMP-2 protein expression and activity were also enhanced. Conclusions:Constant mechanical stretching induces MMP-2 expression in rat retina Muller cells and enhances its extracellular activity. These results suggests a possible novel molecular mechanism that would count for retinal remolding in many ocular diseases in which the retina is often over-stretched, such as degenerative axial myopia and proliferative diabetic retinopathy.Part â…¡Gene Expression Changes under Cyclic Mechanical Stretching in Rat Retinal Muller CellsPurpose:To identify patterns of gene expression changes induced by cyclic mechanical stretching in Muller cells.Methods:Rat Muller cells were seeded onto flexible bottom culture plates and subjected to a cyclic stretching regimen of 15% equibiaxial stretching foul and 24 h. RNA was extracted and amplified, labeled, and hybridized to rat genome microarrays. The expression profiles were analyzed using GeneSpring software, and gene ontology analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to select, annotate, and visualize genes by function and pathway. The selected genes of interest were further validated by Quantitative Real-time PCR (qPCR).Results:Microarray data analysis showed that at 1 and 24 h, the expression of 532 and 991 genes in the Muller cells significantly (t-test, p,0.05) differed between the mechanically stretched and unstretched groups. Of these genes,56 genes at 1 h and 62 genes at 24 h showed more than a twofold change in expression. Several genes related to response to stimulus (e.g., Egr2, IL6), cell proliferation (e.g., Areg, Atf3), tissue remodeling (e.g., PVR, Loxl2), and vasculogenesis (e.g., Epha2, Nrn1) were selected and validated by qPCR. KEGG pathway analysis showed significant changes in MAPK signaling at both time points.Conclusions:Cyclic mechanical strain induces extensive changes in the gene expression in Muller cells through multiple molecular pathways. These results indicate the complex mechanoresponsive nature of Muller cells, and they provide novel insights into possible molecular mechanisms that would account for many retinal diseases in which the retina is often subjected to mechanical forces, such as pathological myopia and proliferative vitreoretinopathy.