Senescence in cultured trabecular meshwork cells

Yukari Yamazaki

Hiroshi Matsunaga

Maki Nishikawa

Akira Ando

Koji Okuda, Department of Paediatrics, Kansai Medical University, Moriguchi, Osaka, Japan

Seiji Ito, Department 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; matsunag@takii.kmu.ac.jp

Yukari Yamazaki, Hiroshi Matsunaga, Maki Nishikawa, Akira Ando, Shiho Kaneko, Mitsumasa Wada, Miyo Matsumura, Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, JapanKoji Okuda, Department of Paediatrics, Kansai Medical University, Moriguchi, Osaka, JapanSeiji Ito, Department of Medical Chemistry, Kansai Medical University, Moriguchi, Osaka, JapanCorrespondence to: Dr H Matsunaga
Department of Ophthalmology, Kansai Medical University, 10–15 Fumizono‐cho, Moriguchi, Osaka 570‐8507, Japan; matsunag@takii.kmu.ac.jp

Accepted 2007 Jan 1.

Abstract

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 ^{P‐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.

Abstract

Normal diploid cells have a finite proliferative life span and finally enter a non‐dividing state termed senescence.¹ Senescent cells are unable to duplicate themselves and are accompanied with altered gene expression, at least when cultured in vitro.² It has been suggested that replicative senescence might be involved in the pathophysiology of age‐related disorders, such as progeria and Werner's syndrome, as well as related atherothrombotic diseases.³⁴⁵ Primary open‐angle glaucoma (POAG) is an optic neuropathy associated with abnormally increased intraocular pressure that can lead to blindness, especially in elderly patients, and age‐ and disease‐related losses of trabecular meshwork (TM) cells have been reported in patients with POAG.⁶⁷ It has also been suggested that a loss of TM cells, followed by substitution with extracellular matrix, might contribute to an increased resistance to aqueous outflow in those patients, resulting in an increase in intraocular pressure.⁸⁹ Thus, ageing is thought to have a relationship with the pathophysiology of POAG. Our previous study results showed that aged retinal pigment epithelial (RPE) cells exhibited characteristics of cellular senescence and suggested that senescent RPE cells could be involved in the pathogenesis of age‐related macular degeneration.¹⁰ TM cells are derived from the embryonic neural crest,¹¹ and are known to have phagocytic¹² and migratory¹³ abilities similar to RPE cells. Therefore, we speculated that TM cells also exhibit cellular senescence, which may be involved in the pathophysiology of POAG, as it was reported previously that levels of type VI collagen, thrombospondin and fibronectin were increased in aged TM cells.¹⁴ In this study, we investigated whether cellular senescence occurred in cultured TM cells.

Acknowledgements

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.

Acknowledgements

Abbreviations

PDL - population doubling level

POAG - primary open‐angle glaucoma

LDL - low‐density lipoprotein

TM - trabecular meshwork

TRF - terminal restriction fragment

RPE cells - retinal pigment epithelial cells

Abbreviations

Footnotes

Competing interests: None.

Footnotes

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