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K. E. Keller, J. M. Bradley, T. S. Acott; Expression and Alternative Splicing of Extracellular Matrix Genes in Normal and Glaucomatous Eyes Subject to Increased Pressure in Perfusion Culture. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1611.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that mRNA levels of certain extracellular matrix (ECM) gene are modified in glaucoma trabecular meshwork (TM) cells as compared to normal human TM cells. In addition, ECM gene levels and alternate splice forms were modified by cells in culture that were subject to mechanical stretch. Here, ECM gene levels and isoforms are compared and contrasted in normal and glaucomatous eyes subject to constant or increased pressure in anterior segment perfusion culture.
Human donor eye pairs were perfused with serum-free medium at a constant pressure of 8.8 mm Hg, giving a flow rate of 1-6 µl/min, for approximately 24 hours. One eye of each pair was then subject to a doubling of pressure for a further 24-48 hours. The TMs were then removed and total RNA was isolated using Trizol. Quantitative RT-PCR was used to assess mRNA levels and splicing of certain ECM genes with gene-specific primers. Some anterior segments were formalin-fixed, embedded in paraffin and evaluated by immunofluorescence and confocal microscopy.
In normal human eyes subjected to increased pressure, mRNA expression of syndecan-2, collagen XII and myocilin were increased compared to non-stretched control eyes. In similar experiments with glaucomatous eyes, however, syndecan-2 and collagen XII mRNAs were decreased, while that of myocilin was unchanged. Versican mRNA levels decreased in both normal and glaucomatous eyes subjected to increased pressure. V1 and V2 versican isoforms were also decreased. A previously described isoform of collagen XII, which lacks exons 29 and 30, was detected in normal eyes subject to increased pressure. Microscopic analysis of anterior segments showed abundant immunostaining of collagen XII and versican in the juxtacanalicular region of the TM.
Normal human eyes, when subject to increased pressure, modified expression of ECM genes in a manner similar to that found previously when cultured TM cells were mechanically stretched. However, glaucomatous eyes responded differently and decreased mRNA expression of syndecan-2 and collagen XII. These results suggest that glaucomatous TM cells do not respond in a similar manner to normal human eyes in response to increased intraocular pressure.
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