RelA/p65 inhibition prevents tendon adhesion by modulating inflammation, cell proliferation, and apoptosis

Abstract

Peritendinous tissue fibrosis which leads to poor tendon function is a worldwide clinical problem; however, its mechanism remains unclear. Transcription factor RelA/p65, an important subunit in the NF-κB complex, is known to have a critical role in many fibrotic diseases. Here, we show that RelA/p65 functions as a core fibrogenic regulator in tendon adhesion and that its inhibition exerts an anti-fibrogenic effect on peritendinous adhesion. We detected the upregulation of the NF-κB pathway in human tendon adhesion using a gene chip microarray assay and revealed the overexpression of p65 and extracellular matrix (ECM) proteins Collagen I, Collagen III, and α-smooth muscle actin (α-SMA) in human fibrotic tissues by immunohistochemistry and western blotting. We also found that in a rat model of tendon injury, p65 expression correlated with tendon adhesion, whereas its inhibition by small interfering (si)RNA prevented fibrous tissue formation and inflammatory reaction as evidenced by macroscopic, biomechanical, histological, immunohistochemical, and western blotting analyses. Furthermore, in cultured fibroblasts, p65-siRNA, p65-specific inhibitor, Helenalin and JSH23 suppressed cell proliferation and promoted apoptosis, whereas inhibiting the mRNA and protein expression of ECM components and cyclo-oxygenase-2, an inflammatory factor involved in tendon adhesion. Our findings indicate that p65 has a critical role in peritendinous tissue fibrosis and suggest that p65 knockdown may be a promising therapeutic approach to prevent tendon adhesion.

Tendon Cell Deletion of IKKβ/NF-κB Drives Functionally Deficient Tendon Healing and Altered Cell Survival Signaling In Vivo

“…The present study demonstrates that inhibition of canonical NF-κB signaling in Scx Lin fibroblast populations in vivo is not biomechanically beneficial to flexor tendon healing and drives a pro-fibrotic response, suggesting that canonical NF-κB signaling may impact enthesis and flexor tendon healing in different ways. It has previously been shown that global overactivation of canonical NF-κB signaling drives increased matrix formation( 13 ) while global inhibition of canonical NF-κB decreases matrix deposition and adhesion formation( 12 ). There are several possibilities that could explain this discrepancy.…”

“…Canonical NF-κB signaling has recently been implicated in scar-mediated tendon healing. Canonical NF-κB signaling activation is elevated in fibrotic human tendon tissue compared to healthy human tendon controls( 12 ). In addition, global overactivation of canonical NF-κB signaling in a mouse model of acute flexor tendon injury and repair drives deposition of collagen matrix at the repair and increases the presence of alpha smooth muscle actin (αSMA+) myofibroblasts and F4/80+ macrophages, two cell types that jointly drive scar tissue formation( 2, 13 ).…”

“…In addition, global overactivation of canonical NF-κB signaling in a mouse model of acute flexor tendon injury and repair drives deposition of collagen matrix at the repair and increases the presence of alpha smooth muscle actin (αSMA+) myofibroblasts and F4/80+ macrophages, two cell types that jointly drive scar tissue formation( 2, 13 ). Global inhibition of canonical NF-κB signaling via either p65-directed siRNA or a variety of inhibitors resulted in decreased scar/adhesion formation, diminished αSMA+ myofibroblast presence, and decreased collagen production( 12 ). Additionally, cultured tendon fibroblasts treated with p65 inhibitors exhibited decreased proliferation and increased apoptosis, suggesting that canonical NF-κB signaling alters tendon cell survival( 12 ).…”

“…Global inhibition of canonical NF-κB signaling via either p65-directed siRNA or a variety of inhibitors resulted in decreased scar/adhesion formation, diminished αSMA+ myofibroblast presence, and decreased collagen production( 12 ). Additionally, cultured tendon fibroblasts treated with p65 inhibitors exhibited decreased proliferation and increased apoptosis, suggesting that canonical NF-κB signaling alters tendon cell survival( 12 ). While informative, these studies do not examine the specific roles of NF-κB-activating cell populations during tendon healing in vivo.…”

Hydroxycamptothecin Prevents Fibrotic Pathways in Fibroblasts In Vitro

“…Western blot was performed as previously described (22). Cells were lysed in radioimmunoprecipitation buffer, and protein concentration was measured using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, IL).…”