Induction of the senescence phenotype in equine tendon derived cells by dexamethasone

Jul 1, 2022, 12:20 PM
10m
Room: S3 B

Room: S3 B

Speaker

Smith, Roger K.W. (Royal Veterinary College)

Description

"Introduction
Tendon injuries in humans are a major healthcare concern but they also occur spontaneously in other species including horses. The superficial digital flexor tendon (SDFT) of the horse is a highly relevant model for studying tendinopathies because it is a functional homologue of the Achilles tendon with similarities in aetiopathogenesis and associated risk factors (1,2). Previous studies with cyclical loading of tendon explants have revealed that injured SDFTs produced high levels of pro-inflammatory cytokines and 10-1000-fold overexpression of matrix metalloproteinases with impaired resolving mechanisms, especially in older horses (3). In combination, these markers are hallmarks of senescent cells. Senescent cells are generated by many pathways (both cell division dependent and independent) all of which produce viable cells that (a) overproduce pro-inflammatory cytokines collectively known as the Senescence Associated Secretory Phenotype (SASP) and (b) actively remodel matrix, all of which are implicated in age-related degenerative changes that predispose to tendinopathies. Corticosteroids frequently used clinically to treat tendinopathies initially block the inflammatory component of tenocyte senescence but also may cause therapy-induced senescence and interfere with tendon repair, contributing to the high re-injury rate (4). The aim of this study was therefore to use an in vitro model to investigate the effects of dexamethasone on senescence.

Methodology
Tendon derived cells (TDCs), isolated from the superficial digital flexor tendon (SDFT) of adult horses (11 years old), were cultured in DMEM (supplemented with 10% fetal bovine serum and 1% P/S) in a humidified incubator with 5% CO2 at 37°C. TDCs were treated with 1 and 10µM dexamethasone for 48 hours and then cultured in medium without dexamethasone for a further 24 and 72h. The effect of dexamethasone on cell viability was measured by the MTT assay at 24 h. Senescence was confirmed in treated cells by cytochemical analysis with EdU (Click-iT Plus EdU Cell Proliferation Kit, Fisher) and Ki-67 (Rabbit monoclonal to Ki67, Abcam).

Results
The viability of TDCs was not affected by 1 and 10 µM doses of dexamethasone treatment. However, dexamethasone at both concentrations inhibited cell proliferation and induced cell cycle arrest. Senescence in TDCs was confirmed by reduced expression of proliferative-associated protein (Ki-67) and reduced DNA synthesis (EdU incorporation). Exposure to dexamethasone at both doses for 48 hours rendered more than 50% of the tenocytes senescent.

Conclusion
Dexamethasone at clinically relevant doses induced growth arrest in equine tenocyte cells. These data provide a mechanistic explanation for potential adverse effects of using corticosteroids for the treatment of tendinopathies. The model will enable investigations of novel candidate molecules that can slow or stop the degenerative process by inhibiting senescence in vitro, which could be used to improve the clinical benefits of corticosteroids via its anti-inflammatory effects.

References
1.Lui PP, Maffulli N, Rolf C, et al. Scand J Med Sci Sports 2011;21:3-17.
2.Patterson-Kane JC, Rich T. ILAR J 2014;55:86-99.
3.Hosaka Y, Kirisawa R, Yamamoto E, Ueda H, Iwai H, Takehana K. L. J Vet Med Sci. 2002;64(10):945-7.
4.Poulson RC, Watts AC, Murphy RJ, Snelling SJ, Carr AJ, Hulley PA. Ann Rheum Dis. 2014;73(7):1405-13
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