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Abstract
Purpose
To investigate the effects of high glucose (HG) and dexamethasone (DEX) on the survival and permeability of trabecular meshwork cells (HTMC), and associated changes in tight junctions.
Methods
Primary cultured HTMC were exposed to 5 μM low glucose (LG) or 25 μM HG with or without 1.0 μM DEX for 3 days. The permeability of the HTMC monolayer was assessed using carboxyfluorescein or transendothelial electrical resistance (TEER). Gene and protein expressions of claudin-5 and occludin were assessed with reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively.
Results
HG was significantly associated with greater HTMC monolayer permeability compared to LG by both the carboxyfluorescein permeability test and TEER (p = 0.022, 0.028). HG also decreased claudin-5 and occludin mRNA expression, respectively (7.5%, 12.9%). DEX abolished HG-induced increased permeability, and increased the protein expression of claudin-5 and occludin, respectively (p = 0.015, 0.012).
Conclusions
In HTMCs, DEX reversed HG-induced permeability increase. DEX increased tight junction molecules claudin-5 and occludin. Thus, DEX-induced changes in junctional proteins could be another mechanism of increased resistance through the trabecular meshwork and may result in steroid-induced glaucoma.
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Figure 1.
Effects of low glucose (5 μM, LG) and high glucose (25 μM, HG) with or without co-exposure of dexamethasone (1.0 μM, DEX) on the survival of trabecular meshwork cells. HG showed decreased the cellular survival significantly compared to LG. With co-exposure to DEX, HG also decreased the cellular survival compared to LG (*,**; p < 0.05).
Figure 2.
Effects of low glucose (5 μM, LG) and high glucose (25 μM, HG) with or without co-exposure of dexamethasone (1.0 μM, DEX) on the permeability of carboxyfluorescin through the trabecular meshwork cell monolayer. Exposure to HG showed a greater permeability of carboxyfluorescin significantly compared with LG (*p = 0.022). The permeability of LG and HG was significantly decreased when co-exposed to DEX, respectively (**p = 0.023, 0.048). Carboxyfluorescin intensity of outer chamber normalized to the mean value obtained using non-exposed control (permeability 100%).
Figure 3.
Effects of low glucose (5 μM, LG) and high glucose (25 μM, HG) with or without co-exposure of dexamethasone (1.0 μM, DEX) on the transepithelial electrical resistance of the trabecular meshwork cell monolayer. HG showed a smaller resistance significantly compared to LG (p = 0.028). Co-exposure to HG with DEX showed a greater resistance significantly compared to HG (**p = 0.045).
Figure 4.
Effects of low glucose (5 μM, LG) and high glucose (25 μM, HG) with or without co-exposure of dexamethasone (1.0 μM, DEX) on the expression of claudin-5 mRNA in trabecular meshwork cells. Exposure to HG showed a smaller expression of claudin-5 mRNA compared to LG. Expression of occludin mRNA was increased significantly by adding to DEX to LG (*p < 0.05). p-actin was used as internal standard.
Figure 5.
Effects of low glucose (5 μM, LG) and high glucose (25 μM, HG) with or without co-exposure of dexamethasone (1.0 μM, DEX) on the expression of occludin mRNA in trabecular meshwork cells. Exposure to HG showed a smaller expression of occludin mRNA significantly compared to LG. Expression of occludin mRNA was increased significantly by adding to DEX to LG (*p < 0.05). p-actin was used as internal standard.
Figure 6.
Effects of low glucose (5 μM, LG) and high glucose (25 μM, HG) with or without co-exposure of dexamethasone (1.0 μM, DEX) on the expression of claudin-5 in trabecular meshwork cells by wetern blot. Exposure to HG showed a smaller expression of claudin-5 compared to LG significantly. In both LG or HG, addition of DEX increased claudin-5 expression significantly (*p < 0.05). GAPDH was used as internal standard.
Figure 7.
Effects of low glucose (5 μM, LG) and high glucose (25 μM, HG) with or without co-exposure of dexamethasones (1.0 μM, DEX) on the expression of occludin in trabecular meshwork cells by wetern blot. In both LG or HG, addition of DEX increased occludin expression significantly (*p < 0.05). GAPDH was used as internal standard.
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