Korean J Ophthalmol.  2020 Apr;34(2):97-105. 10.3341/kjo.2019.0124.

Effect of Minoxidil on Trabecular Outflow via the Paracellular Pathway

Affiliations
  • 1Cheil Eye Hospital, Daegu, Korea.
  • 2Department of Ophthalmology, Daegu Catholic University School of Medicine, Daegu, Korea.

Abstract

Purpose
To investigate the pathway and effects of minoxidil on trabecular outflow in cultured human trabecular meshwork (TM) cells.
Methods
After exposing primarily cultured TM cells to 0, 10, 50, or 100 µM minoxidil sulfate (MS), trabecular outflow was assessed by measuring TM cell monolayer permeability to carboxyfluorescein and transepithelial electrical resistance. To assess the pathway of permeability changes, caveolin-1, occludin, and claudin-5 levels were measured via western blot. Generation of reactive oxygen species (ROS) was measured using the dichlorofluorescein diacetate assay. To assess the involvement of nitric oxide (NO) in minoxidil-induced permeability increase, the degrees of endothelial nitric oxide synthase mRNA expression and NO production were measured with reverse transcription polymerase chain reaction and Griess assays, respectively. Permeability was also measured with co-exposure to 50 µM N-acetyl cysteine.
Results
MS significantly increased TM cell monolayer permeability (p < 0.05) and decreased transepithelial electrical resistance (p < 0.05). MS decreased the degree of endothelial nitric oxide synthase mRNA expression but did not affect NO production. MS decreased occludin and claudin-5 levels but did not affect caveolin-1 level. MS at 100 µM increased the generation of ROS, and MS-induced permeability increase was attenuated after co-exposure to 50 µM N-acetyl cysteine.
Conclusions
Minoxidil may preferentially increase trabecular permeability via a paracellular pathway by downregulation of tight junction proteins. This minoxidil-induced permeability through the TM may be mediated by generation of ROS.

Keyword

Minoxidil; Permeability; Reactive oxygen species; Trabecular meshwork

Figure

  • Fig. 1 Effect of minoxidil sulfate (MS) on the survival of cultured human trabecular meshwork cells. MS did not affect the survival of trabecular meshwork cells compared to non-exposed controls (all p > 0.05).

  • Fig. 2 Effect of minoxidil sulfate (MS) on the transepithelial electrical resistance (TEER) of the trabecular meshwork cell monolayer. (A) Exposure to 10, 50, or 100 µM MS significantly decreased TEER compared with non-exposed controls (*p < 0.05). (B) Exposure to 10 µM MS significantly decreased TEER in a time-dependent manner (*p < 0.05).

  • Fig. 3 Effect of minoxidil sulfate (MS) on trabecular meshwork cell monolayer permeability. Exposure to 10, 50, or 100 µM MS significantly increased permeability of the trabecular meshwork (*p < 0.05). Carboxyfluorescein intensity of the outer chamber was normalized to the mean value obtained with a non-exposed control (permeability 100%).

  • Fig. 4 Effect of minoxidil sulfate (MS) on the production of nitric oxide. Exposure to MS did not affect the production of nitric oxide compared to non-exposed controls (all p > 0.05).

  • Fig. 5 Effect of minoxidil sulfate (MS) on the expression of endothelial nitric oxide synthase mRNA measured with reverse transcription polymerase chain reaction in trabecular meshwork cells. Exposure to 50 or 100 µM MS significantly decreased the expression of endothelial nitric oxide synthase mRNA compared to non-exposed controls (*p < 0.05).

  • Fig. 6 Effect of minoxidil sulfate (MS) on the level of caveolin-1 protein measured with western blot. Exposure to MS did not affect caveolin-1 level compared to non-exposed controls (all p > 0.05).

  • Fig. 7 Effect of minoxidil sulfate (MS) on the level of occludin protein measured with western blot. Exposure to 10, 50, or 100 µM MS significantly decreased occludin level (*p < 0.05).

  • Fig. 8 Effect of minoxidil sulfate (MS) on the level of claudin-5 protein measured with western blot. Exposure to 50 or 100 µM MS significantly decreased claudin-5 level (*p < 0.05).

  • Fig. 9 Effect of minoxidil sulfate on generation of reactive oxygen species measured using the dichlorofluorescein diacetate assay. Exposure to 100 µM minoxidil sulfate significantly increased generation of reactive oxygen species after 1 and 2 hours of exposure (*p < 0.05).

  • Fig. 10 Effect of antioxidant on the minoxidil (M) sulfate-induced permeability increase measured using the carboxyfluorescein assay. Co-exposure to 50 µM N-acetyl cysteine (NAC) significantly decreased permeability compared to exposure to M sulfate alone at each concentration (µM) (*p < 0.05). Carboxyfluorescein intensity of the outer chamber was normalized to the mean value obtained using a non-exposed control (permeability 100%).


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