Elsevier

Matrix Biology

Volume 21, Issue 2, March 2002, Pages 185-195
Matrix Biology

Matrix metalloproteinase activities and their relationship with collagen remodelling in tendon pathology

https://doi.org/10.1016/S0945-053X(01)00196-2Get rights and content

Abstract

Our aim was to correlate the activity of matrix metalloproteinases (MMPs) with denaturation and the turnover of collagen in normal and pathological human tendons. MMPs were extracted from ruptured supraspinatus tendons (n=10), macroscopically normal (‘control’) supraspinatus tendons (n=29) and normal short head of biceps brachii tendons (n=24). Enzyme activity was measured using fluorogenic substrates selective for MMP-1, MMP-3 and enzymes with gelatinolytic activity (MMP-2, MMP-9 and MMP-13). Collagen denaturation was determined by α-chymotrypsin digestion. Protein turnover was determined by measuring the percentage of d-aspartic acid (% d-Asp). Zymography was conducted to identity specific gelatinases. MMP-1 activity was higher in ruptured supraspinatus compared to control supraspinatus and normal biceps brachii tendons (70.9, 26.4 and 11.5 fmol/mg tendon, respectively; P<0.001). Gelatinolytic and MMP-3 activities were lower in normal biceps brachii and ruptured supraspinatus compared to control supraspinatus (gelatinase: 0.18, 0.23 and 0.82 RFU/s/mg tendon respectively; P<0.001; MMP-3: 9.0, 8.6 and 55 fmol/mg tendon, respectively; P<0.001). Most gelatinase activity was shown to be MMP-2 by zymography. Denatured collagen was increased in ruptured supraspinatus compared to control supraspinatus (20.4% and 9.9%, respectively; P<0.001). The % d-Asp content increased linearly with age in normal biceps brachii but not in control supraspinatus and was significantly lower in ruptured supraspinatus compared to age-matched control tendons (0.33 and 1.09% d-Asp, respectively; P<0.01). We conclude that the short head of biceps brachii tendons show little protein turnover, whereas control supraspinatus tendons show relatively high turnover mediated by the activity of MMP-2, MMP-3 and MMP-1. This activity is thought to represent a repair or maintenance function that may be associated with an underlying degenerative process caused by a history of repeated injury and/or mechanical strain. After tendon rupture, there was increased activity of MMP-1, reduced activity of MMP-2 and MMP-3, increased turnover and further deterioration in the quality of the collagen network. Tendon degeneration is shown to be an active, cell-mediated process that may result from a failure to regulate specific MMP activities in response to repeated injury or mechanical strain.

Introduction

Lesions of the supraspinatus tendon in the rotator cuff are extremely common and are major causes of chronic shoulder pain (Chard and Hazleman, 1987, Leadbetter, 1992). We have previously shown that the collagen network is qualitatively and quantitatively different in ruptured supraspinatus tendons: there was a reduced collagen content, an increased proportion of type III collagen relative to type I collagen, and higher levels of hydroxylysine and the collagen cross-links hydroxylysylpyridinoline and lysylpyridinoline compared to normal supraspinatus (Riley et al., 1994a, Bank et al., 1999). Similar changes have also been observed in equine tendons affected by chronic tendinopathy, consistent with the hypothesis that changes in the collagen network are an integral part of tendon pathology (Birch et al., 1998). The changes seen in pathological tendons have been attributed to a deposition of newly synthesised collagen molecules showing a different profile of post-translational modifications (Riley et al., 1994a, Bank et al., 1999). The altered collagen content in degenerate and ruptured tendons implies that the mature collagen network is at least partially removed by proteinases and this may predispose to tendon rupture.

Collagen type I, the major constituent of the tendon matrix, is normally resistant to proteolytic cleavage (Matrisian, 1990). Collagenases, enzymes of the matrix metalloproteinase (MMP) family, are among the relatively few enzymes capable of cleaving intact fibrillar collagen. MMPs with collagenase activity include MMP-1 (EC 3.4.24.7), MMP-2 (EC 3.4.24.24), MMP-8 (EC 3.4.24.34), MMP-13 (MEROPS ID M10.013) and MMP-14 (MEROPS ID M10.014) (Nagase and Woessner, 1999, Aimes and Quigley, 1995). Cleavage occurs at a single locus in the collagen triple helix, creating 34 and 14 fragments which can then be further degraded by a variety of proteinases including the gelatinases, MMP-2 and MMP-9 (EC 3.4.24.35). Although collagenases play an important role in rheumatological disorders associated with cartilage collagen turnover and pathology (Shlopov et al., 1997, Konttinen et al., 1998), the role of collagenolytic enzymes in degenerative tendinopathy has not been thoroughly investigated.

In many pathological conditions, there is an imbalance between the synthesis and degradation of the matrix, leading to net tissue degradation. The important role of MMPs in connective tissue turnover is generally accepted: an increase in net MMP activity is likely to indicate matrix degradation, which may represent part of the remodelling process in wound healing. Although numerous reports have described increased levels of MMPs in pathological matrix turnover, most studies have focused on MMP gene expression levels (as determined by RT-PCR) or on total amounts of MMPs (as determined by immunological methods or zymography) rather then on net enzyme activity. The latter is a much more important parameter, as MMPs present in the inactive pro-form or complexed to inhibitors such as TIMPs (Tissue Inhibitors of Matrix Metalloproteinases) do not show proteolytic activity. In addition, only a very few studies (mainly in cartilage) have investigated the relationship between the amount of damaged extracellular matrix components present in situ and net enzyme activity of specific MMPs (Billinghurst et al., 2000, Dahlberg et al., 2000, Mort et al., 1993). Furthermore, we are not aware of any study investigating the relationship between active MMP levels and the molecular age of the collagen network.

In this study, fluorogenic substrates were used to monitor net MMP activity in tendon extracts. This type of substrate consists of a short amino acid sequence, recognised by MMPs, to which a fluorophore is attached. The fluorescence is quenched by an absorbing moiety (quencher) also coupled to the peptide. Upon cleavage, quenching is lost, and an increase in fluorescence is measured proportional to the amount of hydrolysed substrate (Beekman et al., 1996, Beekman et al., 1997, Beekman et al., 1999). We have designed fluorogenic substrates using fluorescein/Dabcyl as the fluorophore/quencher combination (Beekman et al., 1999); these substrates are approximately 100–200 times more sensitive than previously used substrates in which EDANS/Dabcyl acted as the fluorophore/quencher combination (Beekman et al., 1996, Beekman et al., 1998, Beekman et al., 1999, Brama et al., 1998, Brama et al., 2000). We have developed formats in which it is possible to determine MMP-1 (collagenase 1) activity, MMP-3 (stromelysin 1) activity or gelatinolytic (MMP-2, MMP-9 and MMP-13) activity. Zymography was carried out on the same extracts to determine the nature of the gelatinolytic activity. The amount of damaged (denatured) collagen molecules present in situ was quantified with a recently developed protocol.

Pentosidine, an advanced glycation end-product (AGE), has been used as a marker of the biological age of the collagen network in a wide range of tissues such as dura mater (Sell and Monnier, 1989, Monnier et al., 1992), skin (Monnier et al., 1992, Sell and Monnier, 1990, Sell et al., 1993), lens-proteins (Dyer et al., 1991) and cartilage (Bank et al., 1998, Verzijl et al., 2000a). It accumulates in a linear fashion with age in the short head of biceps brachii, a tendon that is rarely affected by pathology (Bank et al., 1999). However, in the normal supraspinatus tendon this relationship is not observed and there is less pentosidine in older tendons than would be expected for the age of the tendon (Bank et al., 1999). In addition, significantly lower pentosidine levels were found in ruptured supraspinatus tendons compared to age-matched normal supraspinatus. The most likely explanation is increased remodelling of the collagen network, with mature collagen being degraded and replaced with newly synthesised collagen (Bank et al., 1999). However, although collagen turnover is a key determinant of pentosidine levels (Verzijl et al., 2000b), the extent of glycation of long-lived tissue proteins also depends on the tissue glucose concentration and oxidative stress (Sell and Monnier, 1989). As we know little about these conditions in biceps brachii tendon and supraspinatus tendons, a more reliable measurement of the biological age of the collagen network is required. Consequently, in this study, the molecular age of matrix protein was determined by measuring the percentage d-aspartic acid (% d-Asp). This measure of protein residence time is based on the relatively fast racemisation of aspartic acid from the l-form (in which it is built into proteins) into the d-form (Helfman and Bada, 1975, Masters and Bada, 1977, Pfeiffer et al., 1995).

Section snippets

d-Aspartic acid racemisation

The percentage d-Asp is a marker of the biological age of the protein network, and consequently, an indicator of the rate of matrix protein turnover. In biceps brachii tendons, a linear increase in % d-Asp levels was seen throughout the entire age range (R2=0.83, P<0.001; Fig. 1a). In the control supraspinatus tendon, this relationship was not apparent, particularly in older specimens (Fig. 1f). After the age of 60 years, a large scatter was observed, mainly because of the low levels in a

Discussion

Few studies have attempted to compare net MMP activity with indices of matrix turnover in connective tissue pathology, and little was previously known about the activity and expression of these enzymes in normal and degenerate tendons. In this study, we have shown that, similar to osteoarthritic cartilage, degeneration of the supraspinatus tendon is associated with increased collagen turnover, potentially mediated by several members of the matrix metalloproteinase family. We have also shown

Tendon specimens

Macroscopically normal supraspinatus tendons (n=29, age range 18–96 years) were obtained post-mortem from cadavers that had no previous history of shoulder lesions and were processed within 1 h of removal. Many of these specimens had previously showed microscopic evidence of degeneration (Chard et al., 1994, Riley et al., 2001) and were consequently designated as control tendons (as opposed to normal). Surgical specimens of supraspinatus (n=10; age range 55–80 years) were taken from patients

Acknowledgements

We wish to thank Mr Chris Constant, Consultant Orthopaedic Surgeon, Addenbrooke's Hospital, Cambridge, UK, for the provision of surgical tendon specimens. We also wish to acknowledge the contribution of Dr Mike Chard, now Consultant Rheumatologist, Worthing Hospital, UK, for his help in obtaining post-mortem specimens during his training at Addenbrooke's Hospital. Dr Graham Riley was supported by the Cambridge Arthritis Research Endeavour (CARE).

References (53)

  • D.G Dyer et al.

    Formation of pentosidine during nonenzymatic browning of proteins by glucose

    J. Biol. Chem.

    (1991)
  • D Ireland et al.

    Multiple change in gene expression in chronic human Acilles tendinopathy

    Matrix Biol.

    (2001)
  • Y.T Konttinen et al.

    New collagenolytic enzymes/cascade identified at the pannus–hard tissue junction in rheumatoid arthritis: destruction from above

    Matrix Biol.

    (1998)
  • W.B Leadbetter

    Cell-matrix response in tendon injury

    Clin. Sports. Med.

    (1992)
  • L.M Matrisian

    Metalloproteinases and their inhibitors in matrix remodelling

    TIG

    (1990)
  • J.S Mort et al.

    Direct evidence for active metalloproteinases mediating matrix degradation in interleukin 1-stimulated human articular cartilage

    Matrix Biol.

    (1993)
  • H Nagase et al.

    Matrix metalloproteinases

    J. Biol. Chem.

    (1999)
  • D.R Sell et al.

    Structure elucidation of a senescence cross-link from human extracellular matrix. Implication of pentoses in the aging process

    J. Biol. Chem.

    (1989)
  • N Verzijl et al.

    Effect of collagen turnover on the accumulation of advanced glycation end products

    J. Biol. Chem.

    (2000)
  • A.B Wysocki et al.

    Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9

    J. Invest. Dermatol.

    (1993)
  • R.A Bank et al.

    Ageing and zonal variation in post-translational modification of collagen in normal human articular cartilage. The age-related increase in non-enzymatic glycation affects biomechanical properties of cartilage

    Biochem. J.

    (1998)
  • R.A Bank et al.

    Lysylhydroxylation and non-reducible cross-linking of human supraspinatus tendon collagen: changes with age and in chronic rotator cuff tendinitis

    Ann. Rheum. Dis.

    (1999)
  • R.A Bank et al.

    The increased swelling and instantaneous deformation of osteoarthritic cartilage is highly correlated with collagen degradation

    Arthr. Rheum.

    (2000)
  • B Beekman et al.

    Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin

    Ann. N.Y. Acad. Sci.

    (1999)
  • R.C Billinghurst et al.

    Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and selective inhibition of type II collagen cleavage by collagenase

    Arthr. Rheum.

    (2000)
  • H.L Birch et al.

    Macroscopic ‘degeneration’ of equine superficial digital flexor tendon is accompanied by a change in extracellular matrix composition

    Equine Vet. J.

    (1998)
  • Cited by (0)

    View full text