TGF-β1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair

Abstract

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-β1), but the effects of TGF-β1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-β1 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-β1 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-β1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-β1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-β1 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-β1 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.

Figure 1. TGF-β1 significantly upregulated PAI-1 gene and protein levels

(A) Relative Serpine1 (PAI-1) gene expression increased 2–6 fold with TGF-β1 treatment in all groups except those supplemented with tPA (n=5–9 from 2 separate experiments). In the presence of tPA, TGF-β1 increased Serpine1 expression by as much as 70%, but was not significantly different than untreated controls. (B) PAI-1 protein levels in culture media, on the other hand, were significantly elevated by 12- to 25-fold in all groups treated with TGF-β1, including those supplemented with plasminogen and tPA. n=12 from 4 separate experiments.

Figure 2. Effects of TGF-β1 on Plat (tPA) and Plau (uPA) expression

(A) Gene expression analysis with RT-PCR showed that Plat (tPA) was significantly downregulated 81% by TGF-β1 in the absence of plasminogen and tPA, but this effect was reduced in the presence of those two factors and did not reach significance. (B) Plau (uPA) was not significantly affected by TGF-β1 alone, but different combinations of TGF-β1, plasminogen, and tPA significantly reduced Plau expression by 47–55%. n=5–9 samples per treatment per time point from two separate experiments.

Figure 3. TGF-β1 reduced Plasminogen conversion to Plasmin

(A) Plasminogen in culture media was assessed by ELISA, and showed that tPA supplementation caused a depletion of 85% of plasminogen when compared to non-tPA supplemented control media. TGF-β1 reduced plasminogen depletion to only 40% of controls. (B) Plasmin levels were assessed with a fluorogenic substrate assay. Plasmin activity was minimal in groups that were not treated with exogenous Plasminogen. Addition of Plasminogen caused a 5-fold increase in Plasmin activity (not significant), but this increase was completely reversed by treatment with TGF-β1. Addition of tPA caused a 150-fold increase in plasmin activity over plasminogen -and tPA-free media, but TGF-β1 reduced plasmin activity by 84% in the presence of plasminogen and tPA. n=9 samples from three separate experiments.

Figure 4. Plasmin-mediated MMP-2 activity was suppressed by TGF-β1

(A) Gene expression analysis with RT-PCR showed that Mmp2 was not significantly regulated by TGF-β1 in the presence or absence of plasminogen and tPA. (B) Representative gelatin zymogram that was used to assess pro- and active MMP-2 in media after treating samples for 48 h as indicated below panel D. (C) Pro-MMP-2 was not significantly different among any of the treatments. (D) TGF-β1 reduced active MMP-2 by 90% in the presence of plasminogen alone, however this was not significant. In the presence of plasminogen and tPA, TGF-β1 reduced active MMP-2 by 71% (p<0.0001). n=8 samples from three separate experiments. (E) Representative zymogram from samples treated with plasminogen and tPA, as well as combinations of TGF-β1 and tranexamic acid (plasmin inhibitor). (F) Pro-MMP-2 was lower in groups treated with tranexamic acid. (G) Tranexamic acid reduced active MMP-2 by 87%, which was a reduction similar to the 78% reduction seen with TGF-β1 treatment. n=3 samples from one experiment.

Figure 5. TGF-β1 suppresses tPA, plasmin and MMP activity in wild type, but not PAI-1 KO tendon cells

(A) tPA activity in culture media collected from the samples after 48 hours of treatment with or without TGF-β1 was measured with a fluorescent substrate (FRET) assay. TGF-β1 suppressed tPA activity by 81% in wild type cells (p<0.05), but did not suppress tPA activity in tendon cells null for PAI-1. (B) Plasmin activity measured with a FRET substrate also showed that TGF-β1 suppressed plasmin activity by 96% in wild type cells (p<0.05), but not in tendon cells null for PAI-1. (C) MMP activity measured with a FRET substrate showed that TGF-β1 suppressed MMP activity by 59% in media collected from wild type cells (p<0.0001), but did not suppress MMP activity in media from tendon cells null for PAI-1. n=6 samples from two separate experiments. Significance for tPA and plasmin activity was determined using Dunn’s post test to correct for multiple comparisons.

Figure 6. Proposed interactions of TGF-β1 with the plasminogen activation system and MMPs

MMPs may be activated from their “Pro” state by cleavage of the inhibitory pro-peptide by plasmin. However, plasmin must itself first be activated from its pro-form, “plasminogen,” via either tissue- or urokinase-type Plasminogen Activators (tPA and uPA, respectively). Both tPA and uPA are inhibited by PAI-1, which is upregulated by TGF-β1 in a number of tissues and diseases. Thus, TGF-β1 may inhibit both plasmin and MMP activity through PAI-1.