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Rac1 Activity Maintains the Human Tenocyte Phenotype
Rowena McBeath, MD, PhD1; A. Lee Osterman, MD1; Andrzej Fertala, PhD2; (1)The Philadelphia Hand Center, Thomas Jefferson University, (2)Thomas Jefferson University
The Philadelphia Hand Center, Thomas Jefferson University, Philadelphia, PA, USA
When cultured in vitro, human tenocytes undergo ‘dedifferentiation’, a process by which they change shape – lose their normal elongated phenotype – and stop producing collagen I1. Previous work has demonstrated the involvement of the Rho GTPases, including Rac1, in human connective tissue cell differentiation2. We hypothesized that the process of human tenocyte dedifferentiation is caused by loss of activity of Rac1GTPase.
Human tenocytes were dedifferentiated via in vitro culture at low cell density. Pharmacologic inhibition of Rac1 activity in human tenocytes resulted in dedifferentiation, cell shape changes and decreased collagen I production. When Rac1 activity was controlled using adenoviral infection of constitutively active Rac1, the tenocyte phenotype was regained, and collagen I production was increased. These findings show that increased Rac1 activity underlies the maintenance of the normal human tenocyte phenotype in vitro.
Cell culture Human tenocytes were harvested from tendons of individuals undergoing finger revision amputation using IRB-approved protocols. The Rac1 GTPase inhibitor NSC23766 were purchased from Calbiochem.
Virus Adenovirus carrying constitutively active Rac1 (Rac1(+)) was purchased from CellBioLabs, expanded, and used at MOI resulting in 95% infection.
Human tenocyte dedifferentiation occurs with in vitro passage
Human tenocytes were plated at 5k cells/cm2 and passaged in vitro. Tenocytes from passage 0, passage 2 and passage 8 were harvested after one week in culture. There is a change in tenocyte phenotype from elongated to polygonal, accompanied by decreased collagen I expression, with time in culture at low density.
Inhibition of Rac1 activity causes human tenocyte dedifferentiation
Human tenocytes were treated with the Rac1 inhibitor NSC23766. The normal tenocyte phenotype, and ability to produce collage I, is lost with Rac1 inhibition.
Rac1 Activity maintains the human tenocyte phenotype in vitro
Human dedifferentiated tenocytes were infected with constitutively active Rac1, and harvested after one week in culture. The human tenocyte phenotype, and collagen I produciton, is regained with Rac1 activation in vitro.
We have observed the dedifferentiation of human tenocytes when plated at low density in culture. We have reproduced this phenotype via Rac1 inhibition, and have discovered that the normal tenocyte phenotype can be potentiated in dedifferentiated tenocytes by increasing Rac1 activity. Knowledge of this molecular mechanism lends insight into strategies to improve tendon tissue engineering, repair and regeneration.
OREF and ASSH
1Schulze-Tanzil et al. (2004) 122: 219-228
2McBeath et al. (2004) 6(4): 483-495
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