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E. Lee, B. T. Gabelt, D. M. Peters, C. R. Brandt, S. K. Bhattacharya, P. L. Kaufman; Effect of Cochlin Transgene Expression by Adenovirus on Cultured Human Trabecular Meshwork Cells and Outflow Facility in Organ Cultured Anterior Segments. Invest. Ophthalmol. Vis. Sci. 2009;50(13):818.
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© ARVO (1962-2015); The Authors (2016-present)
Cochlin protein has been identified in human glaucomatous trabecular meshwork tissue but not in controls and was found to be associated with TGF-β induced intraocular pressure (IOP) elevation in organ cultured anterior segments. The current study determined the effects of cochlin (Coch) transgene expression on cultured human trabecular meshwork (HTM) cells and outflow facility (OF) in monkey organ cultured anterior segments (MOCAS).
An adenoviral (Ad) vector expressing both cochlin and green fluorescent protein (GFP) was used to transduce cultured HTM cells (28 and 2.8 MOI). Expression of the Coch transgene in transduced HTM cells and its culture media were verified by Western blot analysis and immunofluorescence detection 5 days after transduction. Both cynomolgus and rhesus MOCAS were used to test the effect of Ad vectors (2.8E10 VP) on OF. Transduced MOCAS were embedded in EPON-812 and their morphology evaluated by light microscopy.
Western blot analysis showed a dose-dependent expression of cochlin in transduced cells and its culture media. There was no noticeable morphologic change in transduced cells compared to non-transduced control cells. In MOCAS, cochlin expression was detectable in media by 3 days after transduction. There was no difference in baseline OF between MOCAS transduced by Ad-Coch-GFP and Ad-GFP (0.46 ± 0.05 and 0.62 ± 0.09 ul/min/mmHg, respectively, paired analysis, n=9, p>0.05). Reduction in OF was detected after 3 days (39% decrease, p<0.001) and was maintained for the 12-day duration of the study (69% decrease, p<0.005). IOP was significantly elevated in segments injected with Ad-Coch-GFP when compared to control segments injected with Ad-GFP (12-day post transduction, 134% increase, p<0.05). Light microscopy revealed normal angle structures for transduced segments.
Ad vector delivery of the Coch transgene resulted in cochlin expression in HTM cells and MOCAS. Cochlin expression was effective in decreasing OF and increasing IOP in MOCAS suggesting possible involvement of cochlin in IOP elevation. These results also suggest that Coch gene delivery may be used to develop a glaucoma model.
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