Effect of Tranilast on Collagen Synthesis and TGF–Beta2 Expression of Cultured Human Lamina Cribrosa Astrocytes

Abstract

Purpose: : To study the effect of tranilast on collagen synthesis and transforming growth factor–ß₂ (TGF–ß₂) expression of cultured human lamina cribrosa astrocytes.

Methods: : Primary cultures of human lamina cribrosa cells were generated from four normal donors. Only astrocytes cultures which were glial fibrillary acidic protein positive were used for further experiments. Human lamina cribrosa astrocytes of passage three were plated in each well of a 96–well disk and grown to confluence. Tranilast was added into the fresh incubation medium to final concentrations of 0 µg/ml (control), 12.5 µg/ml, 25 µg/ml or 50 µg/ml 24 hours after confluence. Four cultures were used at each concentration. 48 hours after incubation, collagen synthesis of the cultured human lamina cribrosa astrocytes was quantified by measuring the amount of the ³H–proline incorporated into the cells by a scintillation counter. In addition, the relative levels of expression of TGF–ß₂ were examined by using semi–quantitative reverse transcription polymerase chain reaction (RT–PCR) with glyceraldehyde–3–phosphate dehydrogenase (G3PDH) as an internal control.

Results: : Compared with the control group, incorporation of ³H–proline into human lamina cribrosa astrocytes were significantly decreased by tranilast at concentrations of 12.5 µg/ml (p<0.05), 25 µg/ml (p<0.05) and 50 µg/ml (p<0.01) in a dose–dependent manner. And there was statistical significant difference between the ratio of TGF–ß₂/G3PDH PCR products signal intensities in the experimental groups treated with tranilast at 25 µg/ml (p<0.01) and 50 µg/ml (p<0.01) and that of the control group. 12.5 µg/ml tranilast did not affect the ratio significantly.

Conclusions: : Our current study demonstrated that tranilast inhibits the collagen synthesis and TGF–ß₂ mRNA expression in cultured human lamina cribrosa astrocytes. In view of the involvement of excess collagen and TGF–ß₂ in the remodelling of the extracellular matrix in the optic nerve head of primary open–angle glaucoma, our results indicate that tranilast may be a potential therapeutic agent in the treatment of this blinding disorder.

Keywords: lamina cribrosa • drug toxicity/drug effects