Changes in matrix protein biochemistry and the expression of mRNA encoding matrix proteins and metalloproteinases in posterior tibialis tendinopathy.

OBJECTIVES: Adult-acquired flat foot secondary to a dysfunctional posterior tibialis tendon (PTT) is often treated by surgical transfer of the flexor digitorum longus tendon (FDLT). In this study, the authors compared normal PTT, stage II dysfunctional PTT and replacement FDLT, aiming to define changes in collagen modification, glycosaminoglycan (GAG) and the expression of matrix and metalloproteinase mRNA. METHODS: Normal PTTs were obtained from patients with no history of tendon problems. Samples of dysfunctional PTT and replacement FDLT tissue were obtained from patients undergoing surgical reconstruction. Tissue samples were analysed for total collagen and GAG, pentosidine and collagen cross-links. Total RNA was assayed for mRNA encoding matrix proteins and metalloproteinases, using real-time reverse transcription PCR. Differences between clinical groups were assessed using non-parametric statistics. RESULTS: Dysfunctional PTT contained higher levels of GAG and lower levels of pentosidine than normal PTT or FDLT. In contrast, collagen in FDLT contained fewer ketoimine and more aldimine cross-links than either normal or dysfunctional PTT. mRNA encoding types I and III collagens, aggrecan, biglycan, matrix metalloproteinase (MMP)-2, -13 and -23, and a disintegrin and metalloproteinase (ADAM)-12L each showed increased levels in dysfunctional PTT compared with either normal PTT or (except MMP-13) FDLT. In contrast, MMP-3 and ADAM with thrombospondin domain (ADAMTS)-5 mRNA were lower in both dysfunctional PTT and FDLT than in normal PTT, while ADAMTS-1 mRNA was lower in dysfunctional PTT than in FDLT. CONCLUSIONS: Stage II dysfunctional PTT shows biochemical and molecular changes consistent with a chronic remodelling of the extracellular matrix, rather than rupture, while the replacement FDLT resembles normal PTT in many, but not all, parameters.

The regulation of aggrecanase ADAMTS-4 expression in human Achilles tendon and tendon-derived cells.

Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-beta (TGF-beta) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-beta. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-beta-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-beta are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology.

Ciprofloxacin enhances the stimulation of matrix metalloproteinase 3 expression by interleukin-1beta in human tendon-derived cells. A potential mechanism of fluoroquinolone-induced tendinopathy.

OBJECTIVE: To determine whether the fluoroquinolone antibiotic ciprofloxacin, which can cause tendon pain and rupture in a proportion of treated patients, affects the expression of matrix metalloproteinases (MMPs) in human tendon-derived cells in culture. METHODS: Cell cultures were derived from 6 separate tendon explants, and were incubated in 6-well culture plates for 2 periods of 48 hours each, with ciprofloxacin (or DMSO in controls) and interleukin-1beta (IL-1beta), alone and in combination. Samples of supernatant medium from the second 48-hour incubation were assayed for MMPs 1, 2, and 3 by Western blotting. RNA was extracted from the cells and assayed for MMP messenger RNA (mRNA) by semiquantitative reverse transcription-polymerase chain reaction, with normalization for GAPDH mRNA. RESULTS: Unstimulated tendon cells expressed low or undetectable levels of MMP-1 and MMP-3, and substantial levels of MMP-2. IL-1beta induced a substantial output of both MMP-1 and MMP-3 into cell supernatants, reflecting increases (typically 100-fold) in MMP mRNA, but had only minor effects on MMP-2 expression. Ciprofloxacin had no detectable effect on MMP output in unstimulated cells. Preincubation with ciprofloxacin potentiated IL-1beta-stimulated MMP-3 output, reflecting a similar effect on MMP-3 mRNA expression. Ciprofloxacin also potentiated IL-1beta-stimulated MMP-1 mRNA expression, but did not potentiate the output of MMP-1, and had no significant effects on MMP-2 mRNA expression or output. CONCLUSION: Ciprofloxacin can selectively enhance MMP expression in tendon-derived cells. Such effects might compromise tendon microstructure and integrity.