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M.P. Fautsch, A.M. Vrabel, C.K. Bahler, D.H. Johnson; Aqueous humor supplementation enhances cultured trabecular cell protein expression profiles . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4564.
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Purpose: The two models currently used to study molecular and cellular aspects of the human aqueous outflow pathway are trabecular cell monolayer culture and perfusion organ culture of the anterior segment. These systems use either minimal media or media containing various serum concentrations. We examined protein expression in these two culture models following media supplementation with aqueous humor. Methods: Trabecular monolayer cells were grown in 1) DMEM, 2) DMEM supplemented with ascorbic acid, 3) 10% fetal bovine serum (FBS), 4) 50% human aqueous humor, or 5) heat–denatured 50% aqueous humor. Western analysis was performed to measure protein expression of myocilin, PEDF, and TIMP–1 in cell media collected from trabecular cells at various timepoints over 21 days. Trabecular cell lysates were analyzed for vimentin and ß–actin expression. In perfusion organ culture, protein expression of selected molecules in effluent and trabecular meshwork cell lysates were compared with cultured human anterior segments maintained in either standard minimal media or minimal media supplemented with 50% porcine aqueous humor. Overall protein expression was analyzed by 2–dimensional electrophoresis. Cell morphology was analyzed by light microscopy. Results: Expression of myocilin and PEDF was increased in both culture systems when supplemented with aqueous humor. Supplementation with ascorbic acid or heat–denatured 50% human aqueous humor failed to increase myocilin expression. TIMP–1 and vimentin were increased in perfusion organ culture and maintained in trabecular monolayer cultures while ß–actin was maintained in both culture systems following aqueous humor supplementation. Overall protein expression in trabecular meshwork cell lysates isolated from cultured anterior segments following supplementation with aqueous humor was similar to fresh, non–cultured trabecular meshworks. Trabecular monolayer cells supplemented with 50% aqueous humor showed larger, broader–appearing cells when compared to spindle–shaped cells grown in 10% FBS. In cultured anterior segments, 50% aqueous humor maintained a normal trabecular meshwork appearance and cell number. Conclusions: Addition of aqueous humor to the standard culture medium in either organ perfusion culture or trabecular cell monolayer culture, triggers significant changes in intracellular and extracellular protein expression. Aqueous humor supplementation (or components of aqueous humor) may be necessary to maintain cultured trabecular cell protein expression so that it more closely resembles fresh, non–cultured trabecular cells.
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