Soft Tissue Injury and Repair Group

Our research projects are directed toward understanding the biochemistry, cell biology and molecular pathology of tendon, and the effects of various substances on tendon cell metabolism.

We are investigating what happens to the tendon to make it weak and/or painful, and what causes the change in tendon cell activity that is thought to underlie degenerative tendon disease.

Degenerate tendon typically shows the following features:

cell rounding increased cellularity increased amounts of matrix preoteoglycan

Immunohistochemical staining of a degenerate tendon with H&E and Alcian Blue. Proteoglycans are blue.

The following investigations are taking place:

Matrix degrading enzymes and tendon disease

The role of ADAM-12 in tendinopathy

The role of MMP-23 in tendinopathy

Neuropeptides and tendon pain

Fluoroquinolone antibiotics and tendon pain

Exercise, mechanical strain and their effects on tendon

Studies of Dupuytrens disease

Publications

Matrix-degrading enzymes and tendon disease

Our laboratory recently became the first to complete a comprehensive analysis of the many different ‘metalloproteinase’ enzymes expressed in normal and diseased human Achilles tendon.

These enzymes, of which there are at least 42 different types, are capable of breaking down the proteins which make up the bulk of the tendon tissue. These enzymes can also affect the activity of cells in the tendon and are thought likely to have some role in chronic tendon problems.

We used a technique known as realtime RT-PCR to measure the levels of a substance known as messenger RNA (mRNA). Each enzyme has its own mRNA, which is produced when the enzyme is made by the cell.

Our study showed that many metalloproteinase enzymes are produced by cells in normal tendon, and these are thought to be required for the health of the tissue. In painful and ruptured tendons, the pattern of enzyme expression is different, with several enzymes showing increased expression and others showing reduced expression.

Our challenge now is to try and prove the connection between the expression of particular enzymes and the development of tendon pain or rupture. We have identified a few enzymes that show the largest changes in expression, and are beginning studies to try and understand their precise role in tendon.

Two metalloproteinase enzymes chosen for further study are ADAM-12 and MMP-23. We are hopeful that treatments may in the future be directed against these enzymes, creating drugs that can be used to treat painful tendons.

The role of ADAM-12 tendinopathy

The project is based on our novel and potentially important discovery that the expression of ADAM-12 is substantially increased in painful Achilles tendinopathy compared to normal tendon.

Our hypothesis is that ADAM-12 may be implicated in some of the cellular changes commonly found in tendinopathy, such as the increased cell proliferation, cell migration, cell rounding and ‘fibrocartilaginous transformation’.

The specific aims of this project are to:

1. Investigate the expression of ADAM-12 in normal and pathological human tendon
2. Investigate the regulation of ADAM-12 expression by human tenocytes in culture
3. Investigate the role of ADAM-12 and its effect on tendon cell activity in vitro

The purposes of this project are to confirm and extend our preliminary data, to understand why and how ADAM-12 expression is increased in painful tendinopathy and to ascertain its potential role in the regulation of tendon cell activity.

These data will add to our knowledge of the mechanisms of tendon degeneration, potentially leading to novel anti-proteolytic therapeutic strategies for chronic tendinopathy.

Project funded the Arthritis Research Campaign

The role of MMP-23 in tendinopathy

The project is based on the novel discovery that the expression of MMP-23 is substantially increased in painful Achilles tendinopathy compared to normal tendons.

Our hypothesis is that MMP-23 will degrade the tendon ECM to exacerbate the disease process.

MMP-23 may also be implicated in the ‘fibrocartilaginous transformation’ that is associated with chronic tendinopathy.

The specific aims of this project are to:

1. Investigate the expression of MMP-23 in normal and pathological human tendon
2. Investigate the regulation of MMP-23 expression by human tenocytes in culture
3. Express recombinant MMP-23 for detailed studies of the enzyme

The purpose of this project is to understand why and how MMP-23 expression is increased in painful tendinopathy and to ascertain its potential role in tendon ECM catabolism and/or the regulation of tendon cell activity.

These data will add to our knowledge of the mechanisms of tendon degeneration, potentially leading to novel anti-proteolytic therapeutic strategies for tendinopathy.

Funded by a project grant from Action Medical Research

Neuropeptides and tendon pain

There is usually no inflammation in chronic painful tendons, so it is a mystery what is causing the pain and degeneration. The pain may be caused by stretching the damaged tendon, although little is known about how the tendon is damaged or the pain is generated.

We are investigating the hypothesis that the ingrowth of nerves is a major factor in painful tendons. There are a variety of different types of nerve, and these respond to different sensations such as movement, chemical stimulation and tissue damage.

Studies to date have shown that nerve fibres are relatively more abundant in painful tendons compared to normal tissues. There is also some evidence to suggest that some of the substances produced by nerves, known as neuropeptides, may have an effect on the enzymes that are damaging the tendon.

We hope that drugs which prevent the damaging effects of neuropeptides could in the future be used in the treatment of tendinopathy.

Project funded the Arthritis Research Campaign

Fluoroquinolone antibiotics and tendon pain

Fluoroquinolone antibiotics such as ciprofloxacin induce tendon pain and rupture in a small proportion of patients. We are investigating the hypothesis that this side-effect may result from alterations in the balance of synthesis and breakdown of the tendon extracellular matrix

Our studies of tendon cells in culture have shown that unstimulated cells expressed undetectable levels of the enzymes MMP-1 and MMP-3, and substantial levels of MMP-2. The inflammatory mediator IL-1b induced a substantial output of both MMP-1 and MMP-3, reflecting increases (typically 100 fold) in mRNA, but had only minor effects on MMP-2 expression and output.

Preincubation of the cells with ciprofloxacin enhanced IL-1b stimulated MMP-1 and MMP-3 output, reflecting a similar effect on mRNA expression. However, ciprofloxacin decreased the expression (and output of) MMP-13 and there were no significant effects on MMP-2 and MMP-9.

We conclude that ciprofloxacin can selectively enhance MMP expression in tendon-derived cells, and that such effects might compromise tendon microstructure and integrity.

In subsequent work we have investigated the mechanism of this effect. We have shown that ciprofloxacin does not affect the major signalling pathways of IL-1. Although there is a partial inhibitory effect on IL-1b-stimulated prostaglandin synthesis, we have gone on to show that this does not account for the effect on MMP expression. These studies are still in progress.

Project funded the Arthritis Research Campaign

Exercise, mechanical strain and their effects on tendon

Exercise is generally good for the joint, helping to maintain mobility and muscle strength, even in patients with arthritis. Tendons, which transfer force from muscle to bone, are able to respond to increased physical demands by becoming stronger, and too little exercise can lead to weakness.

However, too much exercise - or ‘overuse’ - can damage tendons, causing chronic pain and injury. Thus it is important to understand how tendons respond to changing physical demands, particularly in the elderly.

In collaboration with Professor Michael Kjaer, Denmark, and Professor Albert Banes, USA, we are beginning to investigate how human tendon cells and tissues respond to different levels of exercise.

This study will improve our understanding of the cellular and molecular processes that are required to maintain a healthy tendon, and how these processes are altered in disease.

Studies of Dupuytren’s disease

Dupuytren’s disease, a common and debilitating disease affecting the hand, may be caused by an abnormal tissue response to vibration or repeated stretching. In affected individuals, there is a production of dense cords of tissue which contract and limit movement of the fingers.

Cells within the tissues of the palm (the palmar fascia) are able to respond to mechanical loading, and there is a continual process of remodelling that is normally in balance. Pilot studies have shown that cells from patients with Dupuytren’s disease respond differently to mechanical strain compared to normal cells and this may lead to the excessive production of extracellular matrix.

To investigate this process in more detail we have established collaboration with Dr Ian Clark, Reader in Cell Biology, University of East Anglia, Norwich.

Equipment grants from the Royal Society, Action Arthritis, The FH Muirhead Charitable Trust and the Arthritis Research Campaign have enabled us to purchase a special item of equipment, The Flexercell Tissue Train Cell Culture system.

This equipment will be used to apply physical load to cells in culture, and to investigate the downstream effects on the expression of extracellular matrix and matrix-degrading enzymes. The aim is to determine how repetitive strain affects the cells within the palmar fascia and how this may lead to Dupuytren’s disease.

PUBLICATIONS

Research papers

1. Jones GC, Corps AN, Pennington CJ, Clark IM, Edwards DR, Bradley MM, Hazleman BL, Riley GP. Expression profiling of metalloproteinases and tissue inhibitors of metalloproteinases in normal and degenerate human Achilles tendon. Arthritis Rheum. Accepted for publication.
2. Corps AN, Harrall RL, Curry VA, Hazleman BL, Riley GP. Contrasting effects of fluoroquinolone antibiotics on the expression of the collagenases, matrix metalloproteinases (MMP)-1 and -13, in human tendon-derived cells. Rheumatology Advance Access published on September 7, 2005. doi:10.1093/rheumatology/kei087
3. Corps AN, Robinson AHN, Movin T, Costa M, Hazleman BL, Riley GP. Increased expression of aggrecan and biglycan mRNA in Achilles tendinopathy Rheumatology Advance Access published on October 11, 2005. doi:10.1093/rheumatology/kei152
4. Corps AN, Curry VA, Buttle D, Hazleman BL and Riley GP. Inhibition of interleukin-1β-stimulated collagenase and stromelysin expression in human tendon fibroblasts by epigallocatechin gallate ester. Matrix Biol. 2004;23:163-9.
5. Corps AN, Robinson AHN, Movin T, Costa ML, Ireland DC, Hazleman BL and Riley GP. Versican splice variant messenger RNA expression in normal human Achilles tendon and tendinopathies. Rheumatology 2004;43:969-72.
6. Corps AN, Curry V, Dutt, D, Harrall RL, Hazleman BL and Riley GP. Ciprofloxacin reduces the stimulation of prostaglandin E2 output by human tendon-derived cells. Rheumatology 2003.42(1):1306-10.
7. Corps AN, Harrall RL, Curry V, Fenwick SA, Hazleman BL and Riley GP. Ciprofloxacin enhances the stimulation of matrix metalloproteinase 3 expression by interleukin-1b in human tendon derived cells. Arthritis Rheum. 2002;46(11):3034-40.
8. Riley GP, Curry V, DeGroot J, van El B, Verzijl N, TeKoppele JM, Hazleman BL, and Bank RA. Matrix metalloproteinase activities and their relationship with collagen remodelling in tendon pathology. Matrix Biology 2002;21(2):185-95.
9. Fenwick SA, Curry V, Harrall RL, Hazleman BL, Hackney R and Riley GP. Expression of transforming growth factor-beta isoforms and their receptors in chronic tendinosis. J Anatomy 2001;199:231-40.
10. Ireland D, Curry V, Holloway G, Hackney R, Hazleman BL. and Riley GP. Multiple changes in gene expression in chronic human Achilles tendinopathy. Matrix Biology 2001;20:159-69.
11. Fenwick SA, Curry V, Harrall RL, Hazleman BL, Hackney R and Riley GP. Expression of transforming growth factor-beta isoforms and their receptors in chronic tendinosis. J Anatomy 2001;199:231-40.
12. Fenwick SA, Clements S, Hazleman B. and Riley GP. A one-step plate based method for total collagen analysis from cell culture. Biotechniques 2001; 30(5): 1010-1014.
13. Riley GP, Cox M, Harrall RL, Clements S and Hazleman BL. Inhibition of tendon cell proliferation and glycosaminoglycan synthesis by non-steroidal anti-inflammatory drugs in vitro. J Hand Surgery 2001; 26B: 224-8.
14. Roberts PJ, Riley GP, Morgan GP, Miller R, Hunter JO and Middleton SJ. Physiological expression of inducible nitric oxide synthase (iNOS) in human colon. J. Clin.Path. 2001;54:293-7.
15. Bank RA, TeKoppele, J.M, Oostingh G, Hazleman BL and Riley GP. Lysylhydroxylation and non-reducible cross-links of human supraspinatus tendon collagen: changes with age and in chronic rotator cuff tendinitis. Ann. Rheum. Dis. 1999;58(1):35-41.
16. Cawston TE, Curry VA, Summers CA, Clark IM, Riley GP, Life PF, Spaull JR, Goldring MB, Koshy PJT, Rowan AD and Shingleton WD. The role of oncostatin M in animal and human connective tissue collagen turnover and its localisation within the rheumatoid joint. Arthritis and Rheumatism. 1998; 41(10):1760-71.
17. Riley GP, Harrall RL, Cawston TE, Hazleman BL. and Mackie EJ. Tenascin C and human tendon degeneration. Am. J. Pathol. 1996; 149(3):933-943. 18. Riley GP, Harrall RL, Constant CR, Cawston TE and Hazleman BL. The prevalence and possible pathological significance of calcium phosphate salt accumulation in tendon matrix degeneration. Ann. Rheum Dis. 1996; 55(2): 109-115
18. Riley GP, Harrall RL, Watson PG, Cawston TE and Hazleman BL. Collagenase (MMP-1) and TIMP-1 in destructive corneal disease associated with rheumatoid arthritis. Eye. 1995; 9:703-718.
19. Dalton S, Cawston TE, Riley GP, Bayley IJL and Hazleman BL. Human shoulder tendon biopsy samples in organ culture produce procollagenase and tissue inhibitor of metalloproteinases. Ann. Rheum.Dis. 1995; 54:571-7.
20. Riley GP, Harrall RL, Constant CR, Chard MD, Cawston TE and Hazleman BL. Tendon degeneration and chronic shoulder pain: changes in the collagen composition of the human rotator cuff tendons in rotator cuff tendinitis. Ann. Rheum. Dis. 1994;53:359-66.
21. Riley GP, Harrall RL, Constant CR, Chard MD, Cawston TE and Hazleman BL. Glycosaminoglycans of human rotator cuff tendons: changes with age and in chronic rotator cuff tendinitis. Ann. Rheum. Dis. 1994; 53: 367-76.
22. Chard MD, Cawston TE, Riley GP, Gresham GA, Hazleman BL. Rotator cuff degeneration and lateral epicondylitis - a comparative histological study. Ann. Rheum. Dis. 1994;53:30-4.

Reviews, editorials and book chapters

1. Riley G. Gene expression and matrix turnover in overused and damaged tendons. Scand.J.Med.Sci. Sport. 2005;15:241-251.
2. Jones G, Riley G. ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis. Arthritis Research & Therapy 2005;7:160-9.
3. Riley G. Chronic tendon pathology: molecular basis and therapeutic implications. Expert Reviews in Molecular Medicine 2005; 7(5):1-25.
4. Riley GP. Tendon and ligament biochemistry and pathology. In: Hazleman BL, Riley G, Speed C, Eds. Soft tissue rheumatology. Oxford: Oxford University Press 2004; 20-53.
5. Riley GP. The pathogenesis of tendinopathy. A molecular perspective. Rheumatology 2004 43:131-142.
6. Riley GP. The molecular pathology of tendon. In: Pandalai SG, Ed. Recent research developments in human pathology. Volume 1 part II. Trivandrum: Transworld Research Networks. 2003; 371-401.
7. Fenwick SA, Hazleman BL, and Riley GP. The vasculature and its role in the damaged and healing tendon. Arthritis Research. 2002;4(4):252-60.
8. Riley GP. Ageing and Pathology of the Rotator Cuff. In ' The Biology of the Synovial Joint' Eds Archer, Benjamin, Caterson and Ralphs. Harwood Academic Publishers. 1999.
9. Cawston TE, Riley GP and Hazleman BL. Tendons and soft tissue rheumatism. Great outback or great opportunity? Editorial. Ann. Rheum Dis. 1996; 55(1):1-3.
10. Riley GP, Harrall RL, Constant CR, Chard MD, Cawston TE and Hazleman BL. Tendon degeneration and chronic shoulder pain: changes in the collagen composition of the human rotator cuff tendons in rotator cuff tendinitis. Focus on Rheumatology. 1995; 2:36-39.

Letters

1. Fenwick SA, Curry V, Harrall RL, Hazleman BL, Hackney R and Riley GP. Endochondral ossification in achilles and patella tendinopathy. Rheumatology. 2002;41 474-476.
2. Riley GP, Goddard MJ and Hazleman BL. Histopathological assessment and pathological significance of matrix degeneration in supraspinatus tendons. Rheumatology 2001; 40: 229-230.
3. Riley GP, Fenwick SA and Hazleman BL. Fluoroquinolones and tendinopathy – some observations on tendon matrix metabolism and the possible mechanism. Am J Sports Med. 2001; 29(2):262-3.

Books co-edited/co-authored

1. Soft tissue rheumatology. Hazleman BL, Riley G, Speed C, Eds. Oxford: Oxford University Press 2004

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