TGF-ß1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair.

Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, Transforming Growth Factor Beta 1(TGF-ß1), but the effects of TGF-ß1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-ß1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-ß1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-ß1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-ß1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-ß1 on protease activity were completely abolished in tendon cells from homozygous plasminogen activator inhibitor 1 (PAI-1) knockout (KO) mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-ß1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries.

Serpine1 Knockdown Enhances MMP Activity after Flexor Tendon Injury in Mice: Implications for Adhesions Therapy.

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Adhesions have been associated with TGF-ß1, which causes upregulation of PAI-1, a master suppressor of protease activity, including matrix metalloproteinases (MMP). In the present study, the effects of inhibiting PAI-1 in murine zone II flexor tendon injury were evaluated utilizing knockout (KO) mice and local nanoparticle-mediated siRNA delivery. In the PAI-1 KO murine model, reduced adherence of injured tendon to surrounding subcutaneous tissue and accelerated recovery of normal biomechanical properties compared to wild type controls were observed. Furthermore, MMP activity was significantly increased in the injured tendons of the PAI-1 KO mice, which could explain their reduced adhesions and accelerated remodeling. These data demonstrate that PAI-1 mediates fibrotic adhesions in injured flexor tendons by suppressing MMP activity. In vitro siRNA delivery to silence Serpine1 expression after treatment with TGF-ß1 increased MMP activity. Nanoparticle-mediated delivery of siRNA targeting Serpine1 in injured flexor tendons significantly reduced target gene expression and subsequently increased MMP activity. Collectively, the data demonstrate that PAI-1 can be a druggable target for treating adhesions and accelerating the remodeling of flexor tendon injuries.

Parathyroid hormone 1-34 enhances extracellular matrix deposition and organization during flexor tendon repair.

Parathyroid hormone (PTH) 1-34 is known to enhance fracture healing. Tendon repair is analogous to bone healing in its dependence on the proliferation and differentiation of mesenchymal stem cells, matrix formation, and tissue remodeling.(1,2,3) We hypothesized that PTH 1-34 enhances tendon healing in a flexor digitorum longus (FDL) tendon repair model. C57Bl/6J mice were treated with either intraperitoneal PTH 1-34 or vehicle-control (PBS). Tendons were harvested at 3-28 days for histology, gene expression, and biomechanical testing. The metatarsophalangeal joint range of motion was reduced 1.5-2-fold in PTH 1-34 mice compared to control mice. The gliding coefficient, a measure of adhesion formation, was 2-3.5-fold higher in PTH 1-34 mice. At 14 days post-repair, the tensile strength was twofold higher in PTH 1-34 specimens, but at 28 days there were no differences. PTH 1-34 mice had increased fibrous tissue deposition that correlated with elevated expression of collagens and fibronectin as seen on quantitative PCR. PTH 1-34 accelerated the deposition of reparative tissue but increased adhesion formation.

Gene expression analysis of the pleiotropic effects of TGF-ß1 in an in vitro model of flexor tendon healing.

Flexor tendon injuries are among the most challenging problems for hand surgeons and tissue engineers alike. Not only do flexor tendon injuries heal with poor mechanical strength, they can also form debilitating adhesions that may permanently impair hand function. While TGF-ß1 is a necessary factor for regaining tendon strength, it is associated with scar and adhesion formation in the flexor tendons and other tissues as well as fibrotic diseases. The pleiotropic effects of TGF-ß1 on tendon cells and tissue have not been characterized in detail. The goal of the present study was to identify the targets through which the effects of TGF-ß1 on tendon healing could be altered. To accomplish this, we treated flexor tendon tenocytes cultured in pinned collagen gels with 1, 10 or 100 ng/mL of TGF-ß1 and measured gel contraction and gene expression using RT-PCR up to 48 hours after treatment. Specifically, we studied the effects of TGF-ß1 on the expression of collagens, fibronectin, proteoglycans, MMPs, MMP inhibitors, and the neotendon transcription factors, Scleraxis and Mohawk. Area contraction of the gels was not dose-dependent with the TGF-ß1 concentrations tested. We observed dose-dependent downregulation of MMP-16 (MT3-MMP) and decorin, and upregulation of biglycan, collagen V, collagen XII, PAI-1, Scleraxis, and Mohawk by TGF-ß1. Inter-gene analyses were also performed to further characterize the expression of ECM and MMP genes in the tenocyte-seeded collagen gels. These analyses illustrate that TGF-ß1 tilts the balance of gene expression in favor of ECM synthesis rather than the matrix-remodeling MMPs, a possible means by which TGF-ß1 promotes adhesion formation.