Coordinate regulation of IL-1beta and MMP-13 in rat tendons following subrupture fatigue damage

Abstract

Mechanical overloading is a major causative factor of tendinopathy; however, its underlying mechanisms are unclear. We hypothesized mechanical overloading would damage tendons and alter genes associated with tendinopathy in a load-dependent manner. To test this hypothesis, we fatigue loaded rat patellar tendons in vivo and measured expression of the matrix-degrading enzyme MMP-13 and the inflammatory cytokine IL-1beta. We also examined these responses in cultured tenocytes exposed to intermittent hydrostatic pressure in vitro. Additionally, we hypothesized load-induced changes in tenocyte MMP-13 expression would be dependent on expression of IL-1beta. In vivo fatigue loading at 1.7% strain caused overt microstructural damage and upregulated expression of MMP-13 and IL-1beta, while 0.6% strain produced only minor changes in matrix microstructure and downregulated expression of both MMP-13 and IL-1beta. Loading of cultured tenocytes at 2.5 and 7.5 MPa produced comparable changes in expression to those of in vivo tendon loading. Blocking IL-1beta expression with siRNA suppressed load-induced both MMP-13 mRNA expression and activity. The data suggest fatigue loading alters expression of MMP-13 and IL-1beta in tendons in vivo and tenocytes in vitro in a load-dependent manner. The data also suggest MMP-13 is regulated by both IL-1beta-dependent and IL-1beta-independent pathways.

Fig. 1A–C

Mechanical loading causes microstructural damage in rat patellar tendon in a loading intensity dependent manner. (A) Second harmonic generation microscopic images show a control tendon characterized by intact, aligned, densely packed fibers with no patterns of disruption. (B) In 0.6% strain loaded tendons, localized transverse patterns of kinked fiber deformation were evident with no fiber rupture. (C) Tendons loaded to 1.7% exhibited, in addition to kinked fiber deformation, longitudinal separation among the fibers. Field of view = 400 μm.

Fig. 2A–C

Fatigue loading of patellar tendons in vivo alters expression of MMP-13 and IL-1β. (A) IL-1β and MMP-13 mRNA were suppressed by low-level strain (0.6%) loading, but upregulated by higher strain (1.7%) loading. Changes in (B) MMP-13 and IL-1β protein and (C) MMP-13 activity due to loading were similar to those seen in (A) for mRNA. * denotes significant difference from the appropriate sham-operated group (p ≤ 0.001 based on ANOVA and Bonferroni post-hoc test).

Fig. 3A–C

Intermittent hydrostatic pressure alters expression and activity of MMP-13, and expression of IL-1β by tenocytes in vitro. Expression of MMP-13 and IL-1β (A) mRNA, (B) protein, and (C) MMP-13 enzyme activity were downregulated at 1.0, 2.5, and 5.0 MPa while they were upregulated at 7.5 MPa in response to intermittent hydrostatic pressure loading. Differences from control are indicated by * (p ≤ 0.001) and # (p = 0.003).

Fig. 4

MMP-13 expression in cultured tenocytes is partially dependent on IL-1β. Tenocytes were either transfected with si-IL-1β RNA or a control scrambled siRNA, or left untransfected prior to culture in the presence or absence of intermittent hydrostatic pressure (7.5 MPa), then assayed for MMP-13 mRNA expression. Significance levels are presented for differences between the treatment groups indicated by horizontal bars.