Influence of cyclical mechanical strain on extracellular matrix gene expression in human lamina cribrosa cells in vitro.
OBJECTIVE
The mechanical effect of raised intraocular pressure is a recognised stimulus for optic neuropathy in primary open angle glaucoma (POAG). Characteristic extracellular matrix (ECM) remodelling accompanies axonal damage in the lamina cribrosa (LC) of the optic nerve head in POAG. Glial cells in the lamina cribrosa may play a role in this process but the precise cellular responses to mechanical forces in this region are unknown. The authors examined global gene expression profiles in lamina cribrosa cells exposed to cyclical mechanical stretch, with an emphasis on ECM genes.


METHODS
Glial fibrillary acid protein negative primary LC cells were generated from the optic nerve head tissue of three normal human donors. Confluent cell passages (n=4) were exposed to 15% stretch at 1 Hz or static conditions for 24 h using the Flexercell system. Gene expression was assessed using Affymetrix U133A microarrays with pooled RNA. 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 experiment using quantitative real-time PCR and protein synthesis was measured using ELISA and immunohistochemistry.


RESULTS
Compared with static controls, 805 genes were upregulated and 644 were downregulated by +/-1.5 fold in stretched LC cells. Gene ontologies included ECM, cell proliferation, growth factor activity, and signal transduction. Differentially expressed ECM genes included elastin, collagens (IV, VI, VIII, IX), thrombospondin 1, perlecan, and lysl oxidase. Quantitative PCR demonstrated that the expression of TGF-beta2, BMP-7, elastin, collagen VI, biglycan, versican, EMMPRIN, VEGF, and thrombomodulin were reproducible and consistent with the microarray data. VEGF and TGF-beta2 protein levels were also significantly (p<0.05) increased in stretched cell media supernatants. Immunohistochemistry demonstrated increased EMMPRIN (an extracellular matrix metalloproteinase inducer) protein in human POAG optic nerve head tissue compared to nonglaucomatous controls.


CONCLUSIONS
These findings demonstrate that LC cells respond to mechanical stimuli in vitro by transcription of several components and modulators of the ECM. Some of the upregulated ECM genes identified are novel in the context of glaucomatous optic neuropathy (biglycan, versican, EMMPRIN, and BMP-7). The LC cell may represent both an important pro-fibrotic cell type in the optic nerve head and an attractive target for novel therapeutic intervention in POAG.
Kirwan
Ruaidhrí P
RP
Conway Institute of Biomolecular and Biomedical Research University College Dublin, Dublin, Ireland. ruaidhri.kirwan@ucd.ie
Fenerty
Cecilia H
CH
Crean
John
J
Wordinger
Robert J
RJ
Clark
Abbot F
AF
O'Brien
Colm J
CJ
D002478:Cells, Cultured; D003238:Connective Tissue / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D009898:Optic Disk / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D015854:Up-Regulation
D016326:Extracellular Matrix Proteins; D005136:Eye Proteins