Senescence in cultured trabecular meshwork cells

Yamazaki, Yukari
Matsunaga, Hiroshi
Nishikawa, Maki
Ando, Akira
Kaneko, Shiho
Okuda, Koji
Wada, Mitsumasa
Ito, Seiji
Matsumura, Miyo

¹Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan
²Department of Paediatrics, Kansai Medical University, Moriguchi, Osaka, Japan
³Department of Medical Chemistry, Kansai Medical University, Moriguchi, Osaka, Japan

Correspondence to: Dr H Matsunaga Department of Ophthalmology, Kansai Medical University, 10–15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan; [email protected]

Accepted 01 January 2007

This work was supported in part by a Grant-in-Aid for Young Scientists (B) (number 17791262) from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from the Science Research Promotion Fund of the Japan Private School Promotion Foundation.

Published Online First 10 January 2007

Competing interests: None.

British Journal of Ophthalmology 91(6):p 808-811, June 2007.

Background:

It has been suggested that replicative senescence might be involved in the pathophysiology of age-related diseases.

Aim:

To study the process of senescence in trabecular meshwork (TM) cells.

Methods:

Porcine TM tissues were obtained and placed in primary cultures with Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium. After 2–3 weeks, migrated and proliferated TM cells were trypsinised and cultured in serial passages, and identified with fluorescein-labelled low-density lipoprotein (DiI-Ac-LDL), a marker of TM cells. Staining for senescence-related β-galactosidase activity was performed at population doubling level (PDL) 2, 8 and 16 at pH 6. Terminal restriction fragment (TRF) length was examined by Southern blot analysis using a ³²P-labelled telomere-specific sequence (TTAGGG)₃ at each PDL.

Results:

DiI-Ac-LDL staining revealed that most (nearly 100%) of the cells in the culture were TM cells, which were flattened in shape and positive for senescence-related β-galactosidase staining at PDL 16. Reduction of TRF length as a function of population doubling was also shown.

Conclusions:

TM cells exhibited characteristics of senescence at PDL 16 in vitro. The results demonstrated that cellular senescence may be related to the pathophysiology of primary open-angle glaucoma.