Influence of cyclical mechanical strain on extracellular matrixgene expression in human lamina cribrosa cells in vitro

[摘要] Purpose: The mechanical effect of raised intraocular pressure is arecognised stimulus for optic neuropathy in primary open angle glaucoma(POAG). Characteristic extracellular matrix (ECM) remodellingaccompanies axonal damage in the lamina cribrosa (LC) of the optic nervehead in POAG. Glial cells in the lamina cribrosa may play a role in thisprocess but the precise cellular responses to mechanical forces in thisregion are unknown. The authors examined global gene expression profilesin lamina cribrosa cells exposed to cyclical mechanical stretch, with anemphasis on ECM genes.Methods: Glial fibrillary acid protein negative primary LC cellswere generated from the optic nerve head tissue of three normal humandonors. Confluent cell passages (n=4) were exposed to 15% stretch at 1Hz or static conditions for 24 h using the Flexercell system. Geneexpression was assessed using Affymetrix U133A microarrays with pooledRNA. Expression levels were normalized using robust multi-chip average(RMA). Expression data was annotated using NIH DAVID software.ECM-related gene expression was validated in an independent experimentusing quantitative real-time PCR and protein synthesis was measuredusing ELISA and immunohistochemistry.Results: Compared with static controls, 805 genes were upregulatedand 644 were downregulated by ±1.5 fold in stretched LC cells. Geneontologies included ECM, cell proliferation, growth factor activity, andsignal transduction. Differentially expressed ECM genes includedelastin, collagens (IV, VI, VIII, IX), thrombospondin 1, perlecan, andlysl oxidase. Quantitative PCR demonstrated that the expression ofTGF-β2, BMP-7, elastin, collagen VI, biglycan, versican, EMMPRIN,VEGF, and thrombomodulin were reproducible and consistent with themicroarray data. VEGF and TGF-β2 protein levels were alsosignificantly (p<0.05) increased in stretched cell mediasupernatants. Immunohistochemistry demonstrated increased EMMPRIN (anextracellular matrix metalloproteinase inducer) protein in human POAGoptic nerve head tissue compared to nonglaucomatous controls.Conclusions: These findings demonstrate that LC cells respond tomechanical stimuli in vitro by transcription of several components andmodulators of the ECM. Some of the upregulated ECM genes identified arenovel in the context of glaucomatous optic neuropathy (biglycan,versican, EMMPRIN, and BMP-7). The LC cell may represent both animportant pro-fibrotic cell type in the optic nerve head and anattractive target for novel therapeutic intervention in POAG.

[学科分类] 生物化学/生物物理