Korean J Ophthalmol.  2017 Oct;31(5):452-459. 10.3341/kjo.2017.0020.

Comparative Study of the Effects of Trabecular Meshwork Outflow Drugs on the Permeability and Nitric Oxide Production in Trabecular Meshwork Cells

• Kim JW

• KMID: 2390289
• DOI: http://doi.org/10.3341/kjo.2017.0020

Abstract

PURPOSE
To compare the effects of the barrier function in human trabecular meshwork (TM) cells monolayer and the production of nitric oxide (NO) between trabecular outflow drugs, Rho-associated kinase (ROCK) inhibitors, adenosine, and statin.
METHODS
Primary cultured TM cells were exposed to 10 or 25 µM Y-27632, 0.1 or 1 µM N6-cyclohexyladenosine (CHA), or 15 or 30 µM simvastatin for 24 hours. NO production and expression of endothelial nitric oxide synthase mRNA were measured by Griess assay and reverse transcription polymerase chain reaction, respectively. Barrier functions of the TM cell monolayer were measured by carboxyfluorescein and trans-endothelial electrical resistance. The expression of matrix metalloproteinase-2 mRNA was assessed with reverse transcription polymerase chain reaction.
RESULTS
In TM cells, treatment with each drug increased endothelial nitric oxide synthase mRNA expression. Treatment with 25 µM Y-27632 and 1.0 µM CHA increased NO production significantly (p = 0.035 and p = 0.043, respectively). Treatment with each drug increased the permeability (all p = 0.001) and decreased the trans-endothelial electron resistance of the TM cell monolayer. Treatment with 0.1 µM and 1.0 µM CHA significantly increased matrix metalloproteinase-2 mRNA expression, but simvastatin inhibited its expression.
CONCLUSIONS
Since treatment with ROCK inhibitor more greatly increased NO production and permeability than did adenosine or statin, ROCK inhibitor seems to be more effective for lowering intraocular pressure.

Keyword

Adenosine; Permeability; Rho-associated kinase inhibitor; Statin; Trabecular meshwork

MeSH Terms

Adenosine
Electric Impedance
Humans
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Intraocular Pressure
Matrix Metalloproteinase 2
Nitric Oxide Synthase Type III
Nitric Oxide*
Permeability*
Polymerase Chain Reaction
Reverse Transcription
rho-Associated Kinases
RNA, Messenger
Simvastatin
Trabecular Meshwork*
Adenosine
Matrix Metalloproteinase 2
Nitric Oxide
Nitric Oxide Synthase Type III
RNA, Messenger
Simvastatin
rho-Associated Kinases

Figure

• 

  Fig. 1 Effects of trabecular outflow drugs on the survival of trabecular meshwork cells. Treatment with each drug did not significantly affect survival compared to nonexposed controls (p > 0.05). Y = Y-27632; CHA = N6-cyclohexyladenosine; SS = simvastatin.

• 

  Fig. 2 Effects of trabecular outflow drugs on the production of nitric oxide in trabecular meshwork cells after exposure for 24 hours. Treatment with 25 µM Y-27632 or 1.0 µM CHA significantly increased the production of nitric oxide in a dose-dependent manner. Y = Y-27632; CHA = N6-cyclohexyladenosine; SS = simvastatin. *p = 0.035; †p = 0.043.

• 

  Fig. 3 Effects of trabecular outflow drugs on the expression of endothelial nitric oxide synthase µRNA in trabecular meshwork cells. Treatment with each drug significantly increased the expression of endothelial nitric oxide synthase mRNA (*p < 0.05). b-actin was used as an internal standard. Y = Y-27632; CHA = N6-cyclohexyladenosine; SS = simvastatin.

• 

  Fig. 4 Effects of trabecular outflow drugs on the permeability of carboxyfluorescein through the trabecular meshwork cell monolayer. Treatment with 10 µM or 25 µM Y-27632, 1.0 µM CHA, or 15 µM or 30 µM simvastatin significantly increased the permeability of carboxyfluorescein compared with non-exposed controls (*p< 0.05). Carboxyfluorescein intensity of the outer chamber normalized to the mean value of non-exposed controls (permeability 100%). Y = Y-27632; CHA = N6-cyclohexyladenosine; SS = simvastatin.

• 

  Fig. 5 Effects of trabecular outflow drugs on the trans-endothelial electrical resistance of the trabecular meshwork cell monolayer. Treatment with Y-27632, 1.0 µM CHA, or simvastatin significantly increased the resistance compared with nonexposed controls (*p < 0.05). Y = Y-27632; CHA = N6-cyclohexyladenosine; SS = simvastatin.

• 

  Fig. 6 Effects of trabecular outflow d rugs on the expressin of matrix metalloproteinases-2 (MMP-2) mRNA in trabecular meshwork cells. Treatment with 0.1 µM or 1.0 µM CHA significantly increased the expression of MMP-2 mRNA (*p < 0.05). Simvastatain did not affect the expression of MMP-2. b-actin was used as an internal standard. Y = Y-27632; CHA = N6-cyclohexyladenosine; SS = simvastatin.

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