INTRODUCTION Spontaneous tendon rupture and chronic tendon pain are common events in orthopaedic practice, although the underlying pathological processes are not well understood. Most research on tendon injuries has focused on a description of the condition and its management, rather than on the underlying cellular and molecular mechanisms. A genetic factor has been hypothesized in several tendinopathies that could explain why there is an increased risk of contralateral rupture of the Achilles tendon in subjects with a previous rupture.4 The interaction between a particular genetic pattern and the various intrinsic and extrinsic factors affecting tendon healing may develop a tendinopathy. 16 The tenocytes respond to exercise, mechanical strain and injury modifying the synthesis and degradation of the tendon extracellular matrix. 3, 25 This review will highlight the gene expression and the protein analysis of tendon and matrix-degrading enzymes implied in tendon healing and injuries. MOLECULAR STRUCTURE AND EXTRACELLULAR MATRIX TURNOVER OF TENDON Tendon is a poorly vascularized, highly specialized, connective tissue with a low metabolism and few cells. The extracellular matrix (ECM) consists of 70% water and 30% dry mass and it is constituted by a complex network of molecules interacting with each other, including collagen fibers, mainly represented by collagen type I (65 to 80% of dry mass), glycosaminoglycans (GAGs), proteoglycans (PGs), glycoproteins, and other noncollagenous proteins. 24 Collagen is arranged in hierarchical levels of increasing complexity, beginning with tropocollagen, a triple-helix polypeptide chain, which unites into fibrils; fibers (primary bundles); fascicles (secondary bundles); tertiary bundles; and the tendon itself. 24 Collagen molecules are surrounded by proteoglycans that are hydrophilic glycoproteins. They are involved in collagen fibrillogenesis and give resistance to compression and tensile stresses. Proteoglycans consist of a core protein with one or more covalently attached glycosaminoglycan chains. These glycosaminoglycan (GAG) chains are long, linear carbohydrate polymers made of repeated dysaccharides units that are negatively charged due to the occurrence of sulphate groups. Decorin and versican are tendon proteoglycans, while aggrecan and biglycan are specific of cartilage. Tenocytes lie in the ECM and contribute to its homeostasis. 24 The molecular structure and the metabolism of ECM is finely regulated, and involves several molecules. Matrix metalloproteases (MMPs) are involved in remodelling of the extracellular matrix of tendons, and the various MMPs can be up- or down-regulated in tendinopathy. 15 There are 23 MMPs in human subdivided into four main groups: collagenases, which cleave native collagen types I, II, and III; gelatinases, which cleave denatured collagens and type IV collagen; stromelysins, which degrade proteoglycans, fibronectin, casein, collagen types III, IV, and V; membrane type MMPs. 22 Proteoglycans are primarily degraded by enzymes of the ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) that consists of 19 different molecules. 10 The ADAMTS that regulate PGs turnover are known as ‘aggrecanases’, which include ADAMTS1, ADAMTS4, ADAMTS5, ADAMTS8 and ADAMTS9, although precisely which enzyme is involved in the turnover of tendon proteoglycans is currently unknown. 20 The activity of MMPs is inhibited reversibly by tissue inhibitors of metalloproteinases (TIMPs). 18 There are four types of TIMP: TIMP1, TIMP2, TIMP3, and TIMP4. 18 The balance between the activities of MMPs and TIMPs regulates tendon remodelling, and an imbalance produces collagen disturbances in tendons. 7 The mechanism of activation of MMPs is poorly understood. Their precise role in tendinopathy is still unclear, and it is conceivable that MMPs play a role in tendinopathies. 15 FROM GENE EXPRESSION TO TENDINOPATHY AND TENDON RUPTURE Several authors have observed an high percentage of degeneration in tendon rupture. 1, 2, 11 Although the role of inflammation is still debated, there is evidence of the absence of inflammatory cells in or around the tendon. 13 It is supposed that tendinosis is the cause but not the result of rupture. The cause of tendinosis has been classically described as an overuse phenomenon in which microscopic tendon fiber damage occurs, followed by catabolic response, mechanobiological understimulation and tendon weakening. In this way a vicious circle is started which will result in tendon rupture.3 Pattern, magnitude and duration of loading influence the regulation of the tenocyte remodelling response and then the synthesis of specific metalloproteinase involved in the turnover of the tendon or others metalloproteinase leading to degeneration of the tissue. 21 Tendinopathy and tendon rupture represent two different clinical and molecular scenarios. Riley et al. 23 indicated that the amount of α1 (III) collagen in the human rotator cuff tendinosis increased as a result of degeneration and laceration and that a long-term excess of this collagen species in the human supraspinatus tendon might have unfavourable biomechanical consequences. This report suggests that production of collagen III may have an adverse effect on the tendon healing process. We have recently demonstrated that in human Achilles tendon rupture there is a significant difference in collagen I gene expression between ruptured and healthy tendon areas of the same tendon.12 The up regulation of collagen I gene testified the attempt of cells to synthesised new matrix. In normal Achilles tendon, decorin is the most highly-expressed proteoglycan while versican is higher than aggrecan. In painful tendinopathy both aggrecan and biglycan gene expression are increased compared with normal tendon samples, but levels of versican and decorin mRNA are not significantly changed. 6 This pattern resembles fibrocartilaginous regions of tendon and may reflect an altered mechanical environment at the site of the lesion. In ruptured tendons the levels of aggrecan, biglycan and versican mRNA are not changed compared with normal tendon samples, but decorin mRNA decreased markedly. 6 Main limits of these studies are the use, as control group, of tendons from other patients and the lack of analysis at protein level. It is known that an important interindividual variability can exist, related to several factors, such as use of drugs, smoke or post-mortem tissue hypoxia that can influence the tendon metabolism. 15, 21 Moreover, it is demonstrated that an apparently healthy area, also at ultrasound intraoperative examination, can be abnormal at morphological analysis. 1 A possible solution to this limit is to adopt as control group a sample harvested from the same patient in an apparently macroscopic healthy area. We found in human Achilles tendon rupture a significant increased of decorin and versican gene expression in ruptured area compared to healthy area of the same tendon. We observed presence of high amount of hyaluronan and not sulphured disaccharides in both area. 12 However, this finding is unusual for tendons. The amount of chondroitin sulfurated, testifying the presence of GAGs typical of decorin and versican, were higher in healthy areas compared to ruptured areas. The presence of big amount of hyaluronan and not sulphured disaccharides in healthy area demonstrated that this area is not normal. 6 Upregulation of decorin and versican is indicative of the repair attempt carried out by the tenocytes in the ruptured area. The low amount of GAGs in the ruptured area indicates that the catabolic processes prevail over the synthetic activity. 12 Tissue growth, remodelling, adaptation and repair depend by several proteolitic enzymes and the MMPs play a pivotal role. The MMPs differ in their activities and precisely which enzymes are implicated in the physiological and pathological turnover of tendon is still to be clarified. MMP3 plays a major role in regulation of tendon ECM degradation and tissue remodelling. An increased expression of MMP3 may be necessary for appropriate tissue remodelling and prevention of tendinopathic changes. 9 MMP3 and TIMP1, TIMP2, TIMP3 and TIMP4 are downregulated in tendinopathic tendons while TIMP1 and TIMP2 are upregulated in acute tears. 12, 15 Comparing a ruptured area to a healthy area of the same Achilles tendons we found no difference in gene expression of MMP9 while MMP2 gene expression was higher in ruptured area. However, MMP9 enzyme was more active in ruptured area compared to healthy area while no difference was observed in MMP2 activities. The activity of MMP9 and the absence of the real-time RT-PCR signal indicates that MMP9 is produced elsewhere from the tendon, and the molecule is probably secreted in the area by leucocytes. 14 MMP9 is a marker of overused tendon and it is typically observed after an intensive exercise in the circulation. 14, 17 Conversely, tenocytes produce MMP2. In fact, the high level of MMP2 expression with low MMP2 activity testifies a recent change in tenocytes activity induced by rupture. 5 These findings suggest that MMP-9, as MMP13, participates only in collagen degradation, whereas MMP-2, as MMP3 and MMP14, participates in both collagen degradation and collagen remodelling.19 In tendinopathic patellar tendons an upregulation of MMP1 gene and a suppressed expression of TIMP1 were recorded. 8 This lack of TIMP1 in tendinopathic tendon perhaps causes a shift in the delicate balance in favour of greater collagenase activity, which would suggest that tendinopathy may be a disorder in healing of tendon with abnormal cellular responses to injury or repetitive stress. The high gene expression of TIMPs, seen in acute tendon rupture, could be considered a tissue reaction to overproduction of MMPs, in an effort to reduce their catalytic activity on the tendon matrix. 5, 12 In fact, TIMP-1 is not present in normal tendons, but, after acute tears of the supraspinatus tendon, in an animal model, it is expressed at the tendon edges for 2 weeks. 5 TIMP 2 is not only involved in MMPs and in particular MMP2 inhibition, but acts as a docking element, helping MMP2 activation by the membrane type-1 matrix metalloprotease (MT1-MMP). TIMP 2 drives the pro-MMP-2 to MT1-MMP that cleaves the pro-peptide definitively activating MMP2. 26 This finding indicate that concomitant stimulation and inhibition of ECM degradation occurs during exercise suggesting that TIMPs regulate ECM degradation. CONCLUSION Several authors have demonstrated that ECM is not a static and inert component of tendon. The ECM is a dynamic structure constantly remodelled, with rates of turnover depending of loading forces. The balance between the activities of MMPs and TIMPs is responsible for normal tendon remodelling. Alteration of MMP and TIMP expression from basal levels leads to alteration of tendon homoeostasis with progressive degeneration and weakening. Further studies are required to clarify the complexity of the relation between the different MMPs and their inhibitors in the pathogenesis of tendinopathy and tendon rupture in order to develop specific therapeutic strategies in these patients.

Gene and protein expression in tendon problems.

RONGA, MARIO
2009-01-01

Abstract

INTRODUCTION Spontaneous tendon rupture and chronic tendon pain are common events in orthopaedic practice, although the underlying pathological processes are not well understood. Most research on tendon injuries has focused on a description of the condition and its management, rather than on the underlying cellular and molecular mechanisms. A genetic factor has been hypothesized in several tendinopathies that could explain why there is an increased risk of contralateral rupture of the Achilles tendon in subjects with a previous rupture.4 The interaction between a particular genetic pattern and the various intrinsic and extrinsic factors affecting tendon healing may develop a tendinopathy. 16 The tenocytes respond to exercise, mechanical strain and injury modifying the synthesis and degradation of the tendon extracellular matrix. 3, 25 This review will highlight the gene expression and the protein analysis of tendon and matrix-degrading enzymes implied in tendon healing and injuries. MOLECULAR STRUCTURE AND EXTRACELLULAR MATRIX TURNOVER OF TENDON Tendon is a poorly vascularized, highly specialized, connective tissue with a low metabolism and few cells. The extracellular matrix (ECM) consists of 70% water and 30% dry mass and it is constituted by a complex network of molecules interacting with each other, including collagen fibers, mainly represented by collagen type I (65 to 80% of dry mass), glycosaminoglycans (GAGs), proteoglycans (PGs), glycoproteins, and other noncollagenous proteins. 24 Collagen is arranged in hierarchical levels of increasing complexity, beginning with tropocollagen, a triple-helix polypeptide chain, which unites into fibrils; fibers (primary bundles); fascicles (secondary bundles); tertiary bundles; and the tendon itself. 24 Collagen molecules are surrounded by proteoglycans that are hydrophilic glycoproteins. They are involved in collagen fibrillogenesis and give resistance to compression and tensile stresses. Proteoglycans consist of a core protein with one or more covalently attached glycosaminoglycan chains. These glycosaminoglycan (GAG) chains are long, linear carbohydrate polymers made of repeated dysaccharides units that are negatively charged due to the occurrence of sulphate groups. Decorin and versican are tendon proteoglycans, while aggrecan and biglycan are specific of cartilage. Tenocytes lie in the ECM and contribute to its homeostasis. 24 The molecular structure and the metabolism of ECM is finely regulated, and involves several molecules. Matrix metalloproteases (MMPs) are involved in remodelling of the extracellular matrix of tendons, and the various MMPs can be up- or down-regulated in tendinopathy. 15 There are 23 MMPs in human subdivided into four main groups: collagenases, which cleave native collagen types I, II, and III; gelatinases, which cleave denatured collagens and type IV collagen; stromelysins, which degrade proteoglycans, fibronectin, casein, collagen types III, IV, and V; membrane type MMPs. 22 Proteoglycans are primarily degraded by enzymes of the ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) that consists of 19 different molecules. 10 The ADAMTS that regulate PGs turnover are known as ‘aggrecanases’, which include ADAMTS1, ADAMTS4, ADAMTS5, ADAMTS8 and ADAMTS9, although precisely which enzyme is involved in the turnover of tendon proteoglycans is currently unknown. 20 The activity of MMPs is inhibited reversibly by tissue inhibitors of metalloproteinases (TIMPs). 18 There are four types of TIMP: TIMP1, TIMP2, TIMP3, and TIMP4. 18 The balance between the activities of MMPs and TIMPs regulates tendon remodelling, and an imbalance produces collagen disturbances in tendons. 7 The mechanism of activation of MMPs is poorly understood. Their precise role in tendinopathy is still unclear, and it is conceivable that MMPs play a role in tendinopathies. 15 FROM GENE EXPRESSION TO TENDINOPATHY AND TENDON RUPTURE Several authors have observed an high percentage of degeneration in tendon rupture. 1, 2, 11 Although the role of inflammation is still debated, there is evidence of the absence of inflammatory cells in or around the tendon. 13 It is supposed that tendinosis is the cause but not the result of rupture. The cause of tendinosis has been classically described as an overuse phenomenon in which microscopic tendon fiber damage occurs, followed by catabolic response, mechanobiological understimulation and tendon weakening. In this way a vicious circle is started which will result in tendon rupture.3 Pattern, magnitude and duration of loading influence the regulation of the tenocyte remodelling response and then the synthesis of specific metalloproteinase involved in the turnover of the tendon or others metalloproteinase leading to degeneration of the tissue. 21 Tendinopathy and tendon rupture represent two different clinical and molecular scenarios. Riley et al. 23 indicated that the amount of α1 (III) collagen in the human rotator cuff tendinosis increased as a result of degeneration and laceration and that a long-term excess of this collagen species in the human supraspinatus tendon might have unfavourable biomechanical consequences. This report suggests that production of collagen III may have an adverse effect on the tendon healing process. We have recently demonstrated that in human Achilles tendon rupture there is a significant difference in collagen I gene expression between ruptured and healthy tendon areas of the same tendon.12 The up regulation of collagen I gene testified the attempt of cells to synthesised new matrix. In normal Achilles tendon, decorin is the most highly-expressed proteoglycan while versican is higher than aggrecan. In painful tendinopathy both aggrecan and biglycan gene expression are increased compared with normal tendon samples, but levels of versican and decorin mRNA are not significantly changed. 6 This pattern resembles fibrocartilaginous regions of tendon and may reflect an altered mechanical environment at the site of the lesion. In ruptured tendons the levels of aggrecan, biglycan and versican mRNA are not changed compared with normal tendon samples, but decorin mRNA decreased markedly. 6 Main limits of these studies are the use, as control group, of tendons from other patients and the lack of analysis at protein level. It is known that an important interindividual variability can exist, related to several factors, such as use of drugs, smoke or post-mortem tissue hypoxia that can influence the tendon metabolism. 15, 21 Moreover, it is demonstrated that an apparently healthy area, also at ultrasound intraoperative examination, can be abnormal at morphological analysis. 1 A possible solution to this limit is to adopt as control group a sample harvested from the same patient in an apparently macroscopic healthy area. We found in human Achilles tendon rupture a significant increased of decorin and versican gene expression in ruptured area compared to healthy area of the same tendon. We observed presence of high amount of hyaluronan and not sulphured disaccharides in both area. 12 However, this finding is unusual for tendons. The amount of chondroitin sulfurated, testifying the presence of GAGs typical of decorin and versican, were higher in healthy areas compared to ruptured areas. The presence of big amount of hyaluronan and not sulphured disaccharides in healthy area demonstrated that this area is not normal. 6 Upregulation of decorin and versican is indicative of the repair attempt carried out by the tenocytes in the ruptured area. The low amount of GAGs in the ruptured area indicates that the catabolic processes prevail over the synthetic activity. 12 Tissue growth, remodelling, adaptation and repair depend by several proteolitic enzymes and the MMPs play a pivotal role. The MMPs differ in their activities and precisely which enzymes are implicated in the physiological and pathological turnover of tendon is still to be clarified. MMP3 plays a major role in regulation of tendon ECM degradation and tissue remodelling. An increased expression of MMP3 may be necessary for appropriate tissue remodelling and prevention of tendinopathic changes. 9 MMP3 and TIMP1, TIMP2, TIMP3 and TIMP4 are downregulated in tendinopathic tendons while TIMP1 and TIMP2 are upregulated in acute tears. 12, 15 Comparing a ruptured area to a healthy area of the same Achilles tendons we found no difference in gene expression of MMP9 while MMP2 gene expression was higher in ruptured area. However, MMP9 enzyme was more active in ruptured area compared to healthy area while no difference was observed in MMP2 activities. The activity of MMP9 and the absence of the real-time RT-PCR signal indicates that MMP9 is produced elsewhere from the tendon, and the molecule is probably secreted in the area by leucocytes. 14 MMP9 is a marker of overused tendon and it is typically observed after an intensive exercise in the circulation. 14, 17 Conversely, tenocytes produce MMP2. In fact, the high level of MMP2 expression with low MMP2 activity testifies a recent change in tenocytes activity induced by rupture. 5 These findings suggest that MMP-9, as MMP13, participates only in collagen degradation, whereas MMP-2, as MMP3 and MMP14, participates in both collagen degradation and collagen remodelling.19 In tendinopathic patellar tendons an upregulation of MMP1 gene and a suppressed expression of TIMP1 were recorded. 8 This lack of TIMP1 in tendinopathic tendon perhaps causes a shift in the delicate balance in favour of greater collagenase activity, which would suggest that tendinopathy may be a disorder in healing of tendon with abnormal cellular responses to injury or repetitive stress. The high gene expression of TIMPs, seen in acute tendon rupture, could be considered a tissue reaction to overproduction of MMPs, in an effort to reduce their catalytic activity on the tendon matrix. 5, 12 In fact, TIMP-1 is not present in normal tendons, but, after acute tears of the supraspinatus tendon, in an animal model, it is expressed at the tendon edges for 2 weeks. 5 TIMP 2 is not only involved in MMPs and in particular MMP2 inhibition, but acts as a docking element, helping MMP2 activation by the membrane type-1 matrix metalloprotease (MT1-MMP). TIMP 2 drives the pro-MMP-2 to MT1-MMP that cleaves the pro-peptide definitively activating MMP2. 26 This finding indicate that concomitant stimulation and inhibition of ECM degradation occurs during exercise suggesting that TIMPs regulate ECM degradation. CONCLUSION Several authors have demonstrated that ECM is not a static and inert component of tendon. The ECM is a dynamic structure constantly remodelled, with rates of turnover depending of loading forces. The balance between the activities of MMPs and TIMPs is responsible for normal tendon remodelling. Alteration of MMP and TIMP expression from basal levels leads to alteration of tendon homoeostasis with progressive degeneration and weakening. Further studies are required to clarify the complexity of the relation between the different MMPs and their inhibitors in the pathogenesis of tendinopathy and tendon rupture in order to develop specific therapeutic strategies in these patients.
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