http://informahealthcare.com/cts ISSN: 0300-8207 (print), 1607-8438 (electronic) Connect Tissue Res, 2014; 55(5–6): 397–402 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03008207.2014.959119
ORIGINAL RESEARCH
Distribution and expression of type VI collagen and elastic fibers in human rotator cuff tendon tears Dipti Thakkar1, Tyler M. Grant2, Osnat Hakimi1, and Andrew J. Carr1 Connect Tissue Res Downloaded from informahealthcare.com by University of Colorado Libraries on 12/25/14 For personal use only.
1
Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK and 2Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, UK Abstract
Keywords
There is increasing evidence for a progressive extracellular matrix change in rotator cuff disease progression. Directly surrounding the cell is the pericellular matrix, where assembly of matrix aggregates typically occurs making it critical in the response of tendon cells to pathological conditions. Studies in animal models have identified type VI collagen, fibrillin-1 and elastin to be located in the pericellular matrix of tendon and contribute in maintaining the structural and biomechanical integrity of tendon. However, there have been no reports on the localization of these proteins in human tendon biopsies. This study aimed to characterize the distribution of these ECM components in human rotator cuffs and gain greater insight into the relationship of pathology to tear size by analyzing the distribution and expression profiles of these ECM components. Confocal microscopy confirmed the localization of these structural molecules in the pericellular matrix of the human rotator cuff. Tendon degeneration led to an increased visibility of these components with a significant disorganization in the distribution of type VI collagen. At the genetic level, an increase in tear size was linked to an increased transcription of type VI collagen and fibrillin-1 with no significant alteration in the elastin levels. This is the first study to confirm the localization of type VI collagen, elastin and fibrillin-1 in the pericellular region of human supraspinatus tendon and assesses the effect of tendon degeneration on these structures, thus providing a useful insight into the composition of human rotator cuff tears which can be instrumental in predicting disease prognosis.
Elastin, fibrillin-1, pericellular matrix, rotator cuff tear, tendon, Type VI collagen
Introduction Degeneration and tears of the supraspinatus tendon in the rotator cuff, resulting in impaired shoulder function, are very common and account for 30–70% of all shoulder pain (1,2). Although improvements have been made to surgical repair techniques and postoperative care, the rate of re-rupture remains as high as 75% (3–5). There is evidence that the progression of tears is characterized by cellular and extracellular matrix (ECM) changes including reduced cell number, decreased vascularization and poor matrix organization (3). However, there remains a fundamental lack of understanding of the mechanisms that contribute to tendon degeneration. Tendon is a dense connective tissue primarily composed of type I collagen fibrils that are capable of transmitting large tensile loads from muscles to bones. Tenocytes are distributed longitudinally in the ECM and are arranged in arrays of cells (Figure 1). It has been shown that there is an intermediate matrix that directly surrounds cells called the pericellular Correspondence: Dr. Dipti Thakkar, Botnar Research Centre, Oxford University Institute of Musculoskeletal Sciences, Windmill Road, Oxford OX3 7HE, UK. Tel: +44-1865-223411. E-mail: thakkar. [email protected]
History Received 27 April 2014 Revised 19 August 2014 Accepted 22 August 2014 Published online 22 September 2014
matrix (PCM), which consists of a unique composition of structural molecules. Although the composition and function of the PCM is poorly understood, it has been suggested that the PCM serves important biochemical and biomechanical functions (6,7), which likely contribute to tendon health and disease. Studies on animal models have suggested that type VI collagen and elastic fibers are important components of the tendon PCM and contribute in maintaining the structural and biomechanical integrity of tendon (8–13). However, there are no studies of the localization of these structural molecules in human rotator cuff tendon or the effect of degeneration on their expression. Type VI collagen has a non-fibrillar organization and is ubiquitously distributed in the ECM of tendons, ligaments, muscles, skin, cornea and cartilage (14). Type VI collagen has been detected in both the compressional and tensional regions of various animal tendons (15) with its distribution localized in the PCM surrounding tendon cells (10,16). Type VI collagen has been shown to provide both structural integrity and act as a key regulator of matrix signals due to its cell– matrix and matrix–matrix interactions (17–19). Elastic fibers are composed of an elastin core surrounded by a sheath of fibrillin microfibrils, which act as a scaffold for
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Figure 1. The compartmental organization of tendon, depicting tenocytes aligned within the PCM, surrounded by crimped collagen fibrils.
tropoelastin deposition (20–22). Researchers have suggested that elastic fibers serve three main functions: (i) impart mechanical properties to tissue including elastic recoil and resilience (23,24), (ii) regulate the activity of the TGF-b family of growth factors (25,26) and (iii) influence cell-regulatory functions such as cell migration, survival and differentiation (27,28). The distribution of elastic fibers has been linked to elastic recoil in tissue subjected to large deformations (29). A recent study of bovine flexor tendon has demonstrated that elastic fibers are highly localized around tenocytes and between collagen fascicles, and have been proposed to contribute to the PCM and the endotenon sheath as a protective layer during sustained periods of loading (30). Alterations in pre-existing molecular components within tissue, such as proteoglycans and collagens, play a crucial role in rotator cuff tear pathology (31,32). The ratio of type III to type I collagen is an important indicator of tendon healing potential, with higher expression of type III collagen during injury associated with improved prognosis (33,34). Subsequently, we analyzed the expression of type I and type III collagens in our samples to better understand the pathology of our tissue samples and comprehend the alterations in expression of type VI collagen and elastic fibers associated with rotator cuff tear, being analyzed for the first time. This study aimed to gain greater insight into the relationship of pathology to tear size by analyzing the distribution and expression profiles of pre-existing ECM molecules, type VI collagen and elastic fibers in normal, small and massive rotator cuff tears. Our hypotheses were that (I) type VI collagen, elastin and fibrillin-1 were localized in the pericellular region of the human supraspinatus tendon, and that (ii) their distribution and gene expression would be significantly affected by tendon tears. Confocal microscopy and quantitative polymerase chain reaction (qPCR) were used to identify the distribution and expression of type VI collagen and elastic fibers in healthy and torn rotator cuff tendons.
Methods Tissue preparation Human supraspinatus tendon specimens were obtained intraoperatively from the edge of chronic tears that were present for41 year duration. The specimens were divided into
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two groups based on their tear size: small/medium (53 cm, n ¼ 3) and large/massive (43 cm, n ¼ 3) tears. Three subscapularis tendon specimens were used as normal controls. Normal tissue was obtained from hemiarthroplasties and stabilization patients with no history of rotator cuff problems, and normal tendon appearance intraoperatively. Tissue was obtained from equal number of male and female samples (three males and three females) aged 41–71 years (average age of 56 ± 15 years) and compared to age-matched normal controls. All tendon samples were obtained from the Oxford Musculoskeletal BioBank and were collected with informed donor consent in full compliance with national and institutional ethical requirements, as well as the UK Human Tissue Act and the Declaration of Helsinki. All tissue samples were snap frozen upon collection and stored at 80 C. Each specimen was sectioned into two samples and used for immunohistological or gene expression analysis. Immunohistochemistry The tissue samples were immediately snap frozen in liquid nitrogen and then stored in 80 C freezer in polypropylene tubes. Following removal, the frozen specimens were embedded in OCT compound (Sakura Finetek USA Inc., Torrance, CA) and sectioned using a microtome–cryostat. Frozen specimens were cut into 10 mm thick sections in the longitudinal direction. The sections were mounted on adhesion microscope slides (VWR International Ltd, Leicestershire, UK) and stored at 20 C until further histological analysis. Immunostaining procedures were conducted at room temperature, unless otherwise indicated. Phosphate-buffered saline (PBS) (Invitrogen, Paisley, UK) was used for washing between each incubation and treatment. Sections were blocked with 10% normal goat serum (Millipore, Watford, UK) for 30 min before incubating them in the primary antibody (Table 1) overnight at 4 C. Sections were then incubated with secondary antibody for 30 min. Finally, the samples were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) (1:1000 dilution; Sigma-Aldrich, Dorset, UK) for 10 min and were then mounted using Vectashield mounting media (Vector Laboratories, Inc., Burlingame, CA) and imaged using confocal microscopy. The primary antibody was eliminated for negative controls and run parallel with each experiment to test for non-specific staining. Confocal microscopy An LSM 710 inverted laser-scanning microscope (Zeiss, Cambridge, UK) was used to analyze immunostained specimens. The emission spectra corresponding to the secondary antibodies were collected using three digital band-pass filters (red, green and blue). A 40 oil immersion lens (axial resolution 0.4 mm) was used to capture images over five frames to reduce background noise. The images were captured using Zen 2010 (Zeiss, UK) and analyzed in Imaris 7.4.1 (Bitplane, South Windsor, CT). The images presented in this article are representative of 15 images of each tear group, comprising of three biological and five technical replicates.
DOI: 10.3109/03008207.2014.959119
Type VI collagen and elastic fibers in human rotator cuff tear
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Table 1. Primary and secondary antibodies used for immunohistological detection of type VI collagen, fibrillin-1 and elastin.
Type VI collagen Fibrillin-1
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Elastin
Primary antibody
Secondary antibody
Anti-collagen type VI raised in mouse, 1:300 (Millipore, UK; catalog no. MAB1944) Anti-fibrillin-1 raised in mouse, 1:300 (Millipore, UK; catalog no. MAB2499) Anti-elastin raised in mouse, 1:300 (Abcam, Cambridge, UK; catalog no. ab9519)
Rhodamine-conjugated goat anti-mouse IgG 1:50 (Millipore, UK; catalog no. AP124R) Rhodamine-conjugated goat anti-mouse IgG 1:50 (Millipore, UK; catalog no. AP124R) Rhodamine-conjugated goat anti-mouse IgG 1:50 (Millipore, UK; catalog no. AP124R)
[catalog no. for type VI collagen: QT00084343, elastin: QT00034594, fibrillin-1: QT00024507, glyceraldehyde-3phosphate dehydrogenase (GAPDH): QT01192646, type I collagen: QT00072058 and type III collagen: QT00072058; QuantiTect, Manchester, UK]. Absolute quantification was performed using standard curve method on the ViiAÔ 7 RealTime PCR System (Foster City, CA). The PCR cycling conditions were: 95 C at 10 min for one cycle, then 40 cycles of amplification for 30 s at 95 C, 30 s at 60 C and 30 s at 72 C followed by a thermal melt profile for amplicon identification. Standard curve for each gene was generated using cDNA from normal subscapularis tendon. The threshold crossing (Ct) values were normalized to GAPDH which was used as the endogenous control gene, selected after running a panel of potential housekeeping gene candidates (data not shown). Quantitative comparison of expression levels were performed using ViiAÔ software v1.1 (Applied Biosystems, Foster City, CA). No-template control was included to indicate that there was no contamination in real-time PCR reagents. Figure 2. Type VI collagen distribution in human subscapularis tendon depicting close interaction with tendon cells. Arrowheads highlight arrays of cells where type VI collagen (red) is distributed in the PCM with viable cells stained with DAPI (blue) Scale bar: 100 mm.
RNA extraction and cDNA synthesis The frozen tissue specimens were lysed in 1 ml of a tissue lyser, TRIzolÕ (Sigma-Aldrich) using the gentleMACS tissue lyser (Miltenyi Biotec, Surrey, UK). Following homogenization, the samples were centrifuged at 12 000 g for 10 min at 4 C. Tissue was incubated at room temperature and 200 ml of chloroform was added, vigorously shaken for 15 s and centrifuged at 12 000 g for 15 min at 4 C. The upper aqueous phase (50% of the total volume) was transferred to a separate tube. RNA was precipitated using 100% isopropanaol for 10 min followed by 10-min centrifugation at 12 000 g at 4 C. The RNA pellet was then washed with 1 ml of 70% ethanol. Finally, the extracted RNA was dissolved in RNase-free water and eluted. One microgram of RNA was converted to complementary DNA (cDNA) using the First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) following the manufacturer’s protocol. Gene expression by real-time qPCR Aliquots of cDNA were amplified in a total volume 20 ml containing primers and Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Validated human primers were purchased from Qiagen (Chatsworth, CA) and were designed to cross introns to decrease any non-specific amplification due to potential genomic DNA contamination
Statistical analysis This study included three biological and three technical replicates. The mean values of the technical replicates were calculated and this mean value of each biological sample was used for the statistical analysis (n ¼ 3). Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA). All values were expressed as mean ± standard deviation for three independent experiments. One-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post hoc test was used to test the difference in expression levels of the PCM components between the three diseased groups. Values of p50.05 were considered to be statistically significant.
Results Immunohistochemistry and confocal microscopy indicated that in normal subscapularis tendon, type VI collagen was localized in the pericellular region of tendon and was in close association with tendon cells. Type VI collagen formed a dense mesh around groups of cells and fibrillin-1 was observed as longitudinally orientated fibers in the vicinity of cells (Figure 2). Both type VI collagen and fibrillin-1 fibers appeared to unite several cells into one array unit (Figure 3). Elastin fibers were sparsely distributed within the tissue, compared to fibrillin-1 and type VI collagen, which were more abundant. Negative controls indicated that non-specific staining did not occur in all specimens (Figure 3). Tendon tissue from large/massive rotator cuff tears was characterized by loss of structure and orientation of type VI
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Figure 3. Organization of type VI collagen, elastin and fibrillin-1 in human rotator cuff. All three proteins were distributed along arrays of tenocytes. Type VI collagen formed a dense distribution around groups of cells and was disorganized in the large tear. Elastin and fibrillin-1 were observed as longitudinally orientated fibers in the vicinity of cells. Negative control for Type VI collagen, elastin and fibrillin-1 with viable cells stained with DAPI (blue) indicated that non-specific staining did not occur. Scale bar: 25 mm.
Figure 4. Expression levels of (A) major collagens type I and type III (B) PCM components type VI collagen, elastin and fibrillin-1in healthy and torn rotator cuff tears (data values are mean ± standard error of three independent experiments (*p50.05, **p50.01, ***p50.001).
collagen. Fibrillin-1 and elastin did not show a significant alteration in orientation among the different tear groups. However, the visibility of these fibers increased in large tears. The influence of increased tear size on the transcriptional levels of type I, III and VI collagens in addition to elastin and fibrillin-1 was examined using qPCR (Figure 4). At the transcription level, the mRNA expression of all the measured structural molecules, except elastin, increased in the degenerated tendons compared to the healthy controls. Additionally,
mRNA levels of type III collagen were higher in small/medium tears than large/massive tear this ratio was reversed, with a significant decrease in the expression of type III collagen compared to that in the small and medium tears. The expression of type VI collagen and fibrillin-1 significantly increased with tear size. The level of the fibrillin-1 increased slightly with damage and the level of type V1 collagen increased 50-folds between normal and large/massive tear size. Elastin did not show a statistically significant change at the mRNA levels between groups. The mean fold change in
Type VI collagen and elastic fibers in human rotator cuff tear
DOI: 10.3109/03008207.2014.959119
Table 2. Mean fold change in the expression of type VI collagen, fibrillin-1 and elastin in different tear groups and healthy rotator cuff tear. Mean fold change differences
Type VI collagen
Elastin
Fibrillin 1
Normal Small/medium tear Large/massive tear
1 57.58 145.34
1 0.72 5.03
1 1.87 7.80
the expression of type VI collagen, fibrillin-1 and elastin in different tear groups and healthy rotator cuff tear have been shown in Table 2.
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Discussion Assembly of matrix aggregates typically occurs within 20 mm of the cell surface (35) making the PCM an important region in the complex architecture of tendons and critical in the response of tendon cells to pathological conditions. As mentioned above, recent studies on animal models have shown that type VI collagen and elastic fibers are localized in the PCM region of tendon and are in intimate association with tendon cells (8–13,30). Our results are consistent with studies in animal models and confirm the presence of type VI collagen, elastin and fibrillin-1 in the PCM of the human supraspinatus tendon for the first time. Moreover, unlike cartilage, where the PCM surrounds individual cells (10,36), the current study suggests that tendon PCM forms a contiguous array uniting several tenocytes along its length. This observation highlights the unique structure of tendon and leads us to speculate that tenocytes may respond to biochemical and biomechanical signals as longitudinal units rather than individual cells. Of particular significance are mechanobiological mechanisms, which have been shown to be significantly altered by the mechanical properties of the PCM in other tissues (6,7). Our results suggest that tendon tears alter the structure and mechanics of the cell microenvironment, which may be key to understanding tissue disease and regeneration. It was found that the contribution of type VI collagen and fibrillin-1 was greater than elastin, which had a sparse distribution. Qualitative evaluation of images from torn supraspinatus tendon indicated that the organization of type VI collagen was significantly disrupted in large tissue tears. This may be indicative of an enhanced robustness or turnover associated with type VI collagen in tendon compared to elastic fibers, perhaps with significance to cellular microenvironment. The distribution of elastin did not appear to be significantly different in torn samples, which may be attributed to the unique ability of elastin to withstand deformations of 100% of its initial length (23). Large/massive tears have a significantly different expression profile and decreased reparative potential compared to small tears (37). The ratio of type III to type I collagen is considered a sensitive marker for tendon injury, with higher levels of type III collagen indicating improved healing potential for the tear (33,34). The transcription levels of type I and III collagen in our study were consistent with the literature, with higher type III collagen in the small/medium tear, suggesting that there is a greater potential for healing
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within this group. Interestingly, it was found that catastrophic tendon tears, with respect to their size, are characterized by elevated PCM molecule expression. The transcription levels of type VI collagen were significantly altered in the torn tendon with a 50-fold difference observed between normal and small/medium tears, and a further increase in the large/ massive tears. The expression of fibrillin-1 also significantly increased from small/medium to large/massive tears. The consequential effect of significant increase of type VI collagen and fibrillin-1 transcription levels is currently unknown. Since the expression increases in both pathological groups, it is harder to speculate on its exact function. It may be an adaptive mechanism of cells to restore tissue integrity in their immediate environment as an early injury response. Moreover, it could be an indication of altered cell differentiation as a result of mechanical changes in the tissue. Numerous in vitro studies have shown that the addition of ECM molecules to cell medium has a significant effect on the differentiation state of the cells (38). During compressive load the accumulation of type VI collagen has been shown to be a good marker of fibrocartilage differentiation (11). The high transcriptional rate of type VI collagen could be a potential indication of altered cell differentiation, resulting in tissue of altered physiology. Interestingly, although the expression of elastin did not increase with tissue tear size, fibrillin-1 was significantly increased. Given that elastin and fibrillin-1 both contribute to the composition of elastic fibers this may be indicative of the underlying mechanisms governing elastogenesis. Elastic fibers are formed by first laying down a scaffold of microfibrils, which are mainly composed of fibrillin-1 and fibrillin-2. Next, elastin is deposited into the center of the structure forming a mature elastic fiber. When tendon is torn, the tenoyctes may attempt to create fibrillinrich scaffolds for production of elastic fibers, but fail when depositing elastin. Torn specimens for this study were collected from supraspinatus tendon whereas the control tissue was collected from subscapularis specimens due to the difficulty in obtaining normal human supraspinatus controls. Interpretations of the gene expressions should therefore weigh the functional and structural differences between these two tendons, which may have affected the gene profile. A lack of access to healthy supraspinatus tendon tissue has been a known limitation in studying rotator cuff tears. Using age-matched subscapularis instead is a common practice in the field, and has been used in many studies (3,39–42). In this study, the subscapularis, used as the control sample, confirms the presence of type VI collagen, elastin and fibrillin-1 in the PCM of the human rotator cuff for the first time. The changes observed among the diseased groups are statistically different, reflecting a true change in profile under pathological conditions in the supraspinatus. Although the sample size is limited this is the first study to confirm the localization of these minor structural molecules in the pericellular region of the human supraspinatus tendon and show that degeneration affects their expression and orientation causing, significant disruption to the type VI collagen networks in immediate proximity of cells. The alteration in the transcription levels of type VI collagen with increasing tear size, as seen in this study, provides the starting
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point for further investigations of the role of this PCM component in healing and disease. Larger samples that show the distribution of these molecules in context to the other histological structures along with colocalization immunostaining and quantitative analysis would provide useful insight into the mechanisms that contribute to tendon degeneration and its significance in rotator cuff disease prognosis.
Declaration of interest
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The work was supported by the National Institute of Health and Research (NIHR), UK.
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18. Kielty CM, Whittaker SP, Grant ME, Shuttleworth CA. Type VI collagen microfibrils: evidence for a structural association with hyaluronan. J Cell Biol 1992;118:979–90. 19. Kuo HJ, Maslen CL, Keene DR, Glanville RW. Type VI collagen anchors endothelial basement membranes by interacting with type IV collagen. J Biol Chem 1997;272:26522–9. 20. Kielty CM. Elastic fibres in health and disease. Expert Rev Mol Med 2006;8:1–23. 21. Kielty CM, Shuttleworth CA. Fibrillin-containing microfibrils: structure and function in health and disease. Int J Biochem Cell Biol 1995;27:747–60. 22. Rosenbloom J, Abrams WR, Mecham R. Extracellularmatrix 4: the elastic fibre. FASEB J 1993;7:1208–18. 23. Butler DL, Grood ES, Noyes FR, Zernicke RF. Biomechanics of ligaments and tendons. Exerc Sport Sci Rev 1978;6:125–81. 24. Eriksen TA, Wright DM, Purslow PP, Duance VC. Role of Ca2+ for the mechanical properties of fibrillin. Proteins 2001;45:90–5. 25. Charbonneau NL, Ono RN, Corson GM, Keene DR, Sakai LY. Fine tuning of growth factor signals depends on fibrillin microfibril networks. Birth Defects Res C Embryo Today 2004;72:37–50. 26. Feng XH, Derynck R. Specificity and versatility in TGF-beta signaling through Smads. Annu Rev Cell Dev Biol 2005;21: 659–93. 27. Ito S, Ishimaru S, Wilson SE. Inhibitory effect of type 1 collagen gel containing alpha-elastin on proliferation and migration of vascular smooth muscle and endothelial cells. Cardiovasc Surg 1997;5:176–83. 28. Wendel DP, Taylor DG, Albertine KH, Keating MT, Li DY. Impaired distal airway development in mice lacking elastin. Am J Respir Cell Mol Biol 2000;23:320–6. 29. Ritty TM, Ditsios K, Starcher BC. Distribution of the elastic fibre and associated proteins in flexor tendon reflects function. Anat Rec 2002;268:430–40. 30. Grant TM, Thompson MS, Urban J, Yu J. Elastic fibres are broadly distributed in tendon and highly localized around tenocytes. J Anat 2013;222:573–9. 31. Chaudhury S, Dicko C, Burgess M, Vollrath F, Carr AJ. Fourier transform infrared spectroscopic analysis of normal and torn rotator-cuff tendons. J Bone Joint Surg Br 2011;93:370–7. 32. Chaudhury S, Holland C, Porter D, Tirlapur UK, Vollrath F, Carr AJ. Torn human rotator cuff tendons have reduced collagen thermal properties on differential scanning calorimetry. J Orthop Res 2011; 29:1938–43. 33. Williams IF, McCullagh KG, Silver IA. The distribution of types I and III collagen and fibronectin in the healing equine tendon. Connect Tissue Res 1984;12:211–17. 34. Takahasih S, Nakajima M, Kobayashi M, Wakabayashi I, Miyakoshi N, Minagawa H, Itoi E. Effect of recombinant basic fibroblast growth factor (bFGF) on fibroblast-like cells from human rotator cuff tendon. Tohoku J Exp Med 2002;198:207–14. 35. Trelstad RL, Silver FH. Matrix assembly. In: Hay ED, ed. Cell biology of extracellular matrix. New York: Plenum Press; 1981. 36. Poole CA, Ayad S, Gilbert RT. Chondrons from articular cartilage V: immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy. J Cell Sci 1992;103:1101–10. 37. Schmutz S, Fuchs T, Regenfelder F, Steinmann P, Zumstein M, Fuchs B. Expression of atrophy mRNA relates to tendon tear size in supraspinatus muscle. Clin Orthop Relat Res 2009;467:457–64. 38. Adams JC, Watt FM. Regulation of development and differentiation by the extracellular matrix. Development 1993;117:1183–98. 39. Castagna A1, Cesari E1, Garofalo R, Gigante A, Conti M, Markopoulos N, Maffulli N. Matrix metalloproteases and their inhibitors are altered in torn rotator cuff tendons, but also in the macroscopically and histologically intact portion of those tendons. Muscles Ligaments Tendons J 2013;3:132–8. 40. Millar NL, Reilly JH, Kerr SC, Campbell AL, Little KJ, Leach WJ, Rooney BP, Murrell GA, McInnes IB. Hypoxia: a critical regulator of early human tendinopathy. Ann Rheum Dis 2012;71:302–10. 41. Lundgreen K, Lian OB, Engebretsen L, Scott A. Tenocyte apoptosis in the torn rotator cuff: a primary or secondary pathological event? Br J Sports Med 2011;45:1035–9. 42. Millar NL, Wei AQ, Molloy TJ, Bonar F, Murrell GA. Heat shock protein and apoptosis in supraspinatus tendinopathy. Clin Orthop Relat Res 2008;466:1569–76.
ORIGINAL RESEARCH
Distribution and expression of type VI collagen and elastic fibers in human rotator cuff tendon tears Dipti Thakkar1, Tyler M. Grant2, Osnat Hakimi1, and Andrew J. Carr1 Connect Tissue Res Downloaded from informahealthcare.com by University of Colorado Libraries on 12/25/14 For personal use only.
1
Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK and 2Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, UK Abstract
Keywords
There is increasing evidence for a progressive extracellular matrix change in rotator cuff disease progression. Directly surrounding the cell is the pericellular matrix, where assembly of matrix aggregates typically occurs making it critical in the response of tendon cells to pathological conditions. Studies in animal models have identified type VI collagen, fibrillin-1 and elastin to be located in the pericellular matrix of tendon and contribute in maintaining the structural and biomechanical integrity of tendon. However, there have been no reports on the localization of these proteins in human tendon biopsies. This study aimed to characterize the distribution of these ECM components in human rotator cuffs and gain greater insight into the relationship of pathology to tear size by analyzing the distribution and expression profiles of these ECM components. Confocal microscopy confirmed the localization of these structural molecules in the pericellular matrix of the human rotator cuff. Tendon degeneration led to an increased visibility of these components with a significant disorganization in the distribution of type VI collagen. At the genetic level, an increase in tear size was linked to an increased transcription of type VI collagen and fibrillin-1 with no significant alteration in the elastin levels. This is the first study to confirm the localization of type VI collagen, elastin and fibrillin-1 in the pericellular region of human supraspinatus tendon and assesses the effect of tendon degeneration on these structures, thus providing a useful insight into the composition of human rotator cuff tears which can be instrumental in predicting disease prognosis.
Elastin, fibrillin-1, pericellular matrix, rotator cuff tear, tendon, Type VI collagen
Introduction Degeneration and tears of the supraspinatus tendon in the rotator cuff, resulting in impaired shoulder function, are very common and account for 30–70% of all shoulder pain (1,2). Although improvements have been made to surgical repair techniques and postoperative care, the rate of re-rupture remains as high as 75% (3–5). There is evidence that the progression of tears is characterized by cellular and extracellular matrix (ECM) changes including reduced cell number, decreased vascularization and poor matrix organization (3). However, there remains a fundamental lack of understanding of the mechanisms that contribute to tendon degeneration. Tendon is a dense connective tissue primarily composed of type I collagen fibrils that are capable of transmitting large tensile loads from muscles to bones. Tenocytes are distributed longitudinally in the ECM and are arranged in arrays of cells (Figure 1). It has been shown that there is an intermediate matrix that directly surrounds cells called the pericellular Correspondence: Dr. Dipti Thakkar, Botnar Research Centre, Oxford University Institute of Musculoskeletal Sciences, Windmill Road, Oxford OX3 7HE, UK. Tel: +44-1865-223411. E-mail: thakkar. [email protected]
History Received 27 April 2014 Revised 19 August 2014 Accepted 22 August 2014 Published online 22 September 2014
matrix (PCM), which consists of a unique composition of structural molecules. Although the composition and function of the PCM is poorly understood, it has been suggested that the PCM serves important biochemical and biomechanical functions (6,7), which likely contribute to tendon health and disease. Studies on animal models have suggested that type VI collagen and elastic fibers are important components of the tendon PCM and contribute in maintaining the structural and biomechanical integrity of tendon (8–13). However, there are no studies of the localization of these structural molecules in human rotator cuff tendon or the effect of degeneration on their expression. Type VI collagen has a non-fibrillar organization and is ubiquitously distributed in the ECM of tendons, ligaments, muscles, skin, cornea and cartilage (14). Type VI collagen has been detected in both the compressional and tensional regions of various animal tendons (15) with its distribution localized in the PCM surrounding tendon cells (10,16). Type VI collagen has been shown to provide both structural integrity and act as a key regulator of matrix signals due to its cell– matrix and matrix–matrix interactions (17–19). Elastic fibers are composed of an elastin core surrounded by a sheath of fibrillin microfibrils, which act as a scaffold for
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Figure 1. The compartmental organization of tendon, depicting tenocytes aligned within the PCM, surrounded by crimped collagen fibrils.
tropoelastin deposition (20–22). Researchers have suggested that elastic fibers serve three main functions: (i) impart mechanical properties to tissue including elastic recoil and resilience (23,24), (ii) regulate the activity of the TGF-b family of growth factors (25,26) and (iii) influence cell-regulatory functions such as cell migration, survival and differentiation (27,28). The distribution of elastic fibers has been linked to elastic recoil in tissue subjected to large deformations (29). A recent study of bovine flexor tendon has demonstrated that elastic fibers are highly localized around tenocytes and between collagen fascicles, and have been proposed to contribute to the PCM and the endotenon sheath as a protective layer during sustained periods of loading (30). Alterations in pre-existing molecular components within tissue, such as proteoglycans and collagens, play a crucial role in rotator cuff tear pathology (31,32). The ratio of type III to type I collagen is an important indicator of tendon healing potential, with higher expression of type III collagen during injury associated with improved prognosis (33,34). Subsequently, we analyzed the expression of type I and type III collagens in our samples to better understand the pathology of our tissue samples and comprehend the alterations in expression of type VI collagen and elastic fibers associated with rotator cuff tear, being analyzed for the first time. This study aimed to gain greater insight into the relationship of pathology to tear size by analyzing the distribution and expression profiles of pre-existing ECM molecules, type VI collagen and elastic fibers in normal, small and massive rotator cuff tears. Our hypotheses were that (I) type VI collagen, elastin and fibrillin-1 were localized in the pericellular region of the human supraspinatus tendon, and that (ii) their distribution and gene expression would be significantly affected by tendon tears. Confocal microscopy and quantitative polymerase chain reaction (qPCR) were used to identify the distribution and expression of type VI collagen and elastic fibers in healthy and torn rotator cuff tendons.
Methods Tissue preparation Human supraspinatus tendon specimens were obtained intraoperatively from the edge of chronic tears that were present for41 year duration. The specimens were divided into
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two groups based on their tear size: small/medium (53 cm, n ¼ 3) and large/massive (43 cm, n ¼ 3) tears. Three subscapularis tendon specimens were used as normal controls. Normal tissue was obtained from hemiarthroplasties and stabilization patients with no history of rotator cuff problems, and normal tendon appearance intraoperatively. Tissue was obtained from equal number of male and female samples (three males and three females) aged 41–71 years (average age of 56 ± 15 years) and compared to age-matched normal controls. All tendon samples were obtained from the Oxford Musculoskeletal BioBank and were collected with informed donor consent in full compliance with national and institutional ethical requirements, as well as the UK Human Tissue Act and the Declaration of Helsinki. All tissue samples were snap frozen upon collection and stored at 80 C. Each specimen was sectioned into two samples and used for immunohistological or gene expression analysis. Immunohistochemistry The tissue samples were immediately snap frozen in liquid nitrogen and then stored in 80 C freezer in polypropylene tubes. Following removal, the frozen specimens were embedded in OCT compound (Sakura Finetek USA Inc., Torrance, CA) and sectioned using a microtome–cryostat. Frozen specimens were cut into 10 mm thick sections in the longitudinal direction. The sections were mounted on adhesion microscope slides (VWR International Ltd, Leicestershire, UK) and stored at 20 C until further histological analysis. Immunostaining procedures were conducted at room temperature, unless otherwise indicated. Phosphate-buffered saline (PBS) (Invitrogen, Paisley, UK) was used for washing between each incubation and treatment. Sections were blocked with 10% normal goat serum (Millipore, Watford, UK) for 30 min before incubating them in the primary antibody (Table 1) overnight at 4 C. Sections were then incubated with secondary antibody for 30 min. Finally, the samples were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) (1:1000 dilution; Sigma-Aldrich, Dorset, UK) for 10 min and were then mounted using Vectashield mounting media (Vector Laboratories, Inc., Burlingame, CA) and imaged using confocal microscopy. The primary antibody was eliminated for negative controls and run parallel with each experiment to test for non-specific staining. Confocal microscopy An LSM 710 inverted laser-scanning microscope (Zeiss, Cambridge, UK) was used to analyze immunostained specimens. The emission spectra corresponding to the secondary antibodies were collected using three digital band-pass filters (red, green and blue). A 40 oil immersion lens (axial resolution 0.4 mm) was used to capture images over five frames to reduce background noise. The images were captured using Zen 2010 (Zeiss, UK) and analyzed in Imaris 7.4.1 (Bitplane, South Windsor, CT). The images presented in this article are representative of 15 images of each tear group, comprising of three biological and five technical replicates.
DOI: 10.3109/03008207.2014.959119
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Table 1. Primary and secondary antibodies used for immunohistological detection of type VI collagen, fibrillin-1 and elastin.
Type VI collagen Fibrillin-1
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Elastin
Primary antibody
Secondary antibody
Anti-collagen type VI raised in mouse, 1:300 (Millipore, UK; catalog no. MAB1944) Anti-fibrillin-1 raised in mouse, 1:300 (Millipore, UK; catalog no. MAB2499) Anti-elastin raised in mouse, 1:300 (Abcam, Cambridge, UK; catalog no. ab9519)
Rhodamine-conjugated goat anti-mouse IgG 1:50 (Millipore, UK; catalog no. AP124R) Rhodamine-conjugated goat anti-mouse IgG 1:50 (Millipore, UK; catalog no. AP124R) Rhodamine-conjugated goat anti-mouse IgG 1:50 (Millipore, UK; catalog no. AP124R)
[catalog no. for type VI collagen: QT00084343, elastin: QT00034594, fibrillin-1: QT00024507, glyceraldehyde-3phosphate dehydrogenase (GAPDH): QT01192646, type I collagen: QT00072058 and type III collagen: QT00072058; QuantiTect, Manchester, UK]. Absolute quantification was performed using standard curve method on the ViiAÔ 7 RealTime PCR System (Foster City, CA). The PCR cycling conditions were: 95 C at 10 min for one cycle, then 40 cycles of amplification for 30 s at 95 C, 30 s at 60 C and 30 s at 72 C followed by a thermal melt profile for amplicon identification. Standard curve for each gene was generated using cDNA from normal subscapularis tendon. The threshold crossing (Ct) values were normalized to GAPDH which was used as the endogenous control gene, selected after running a panel of potential housekeeping gene candidates (data not shown). Quantitative comparison of expression levels were performed using ViiAÔ software v1.1 (Applied Biosystems, Foster City, CA). No-template control was included to indicate that there was no contamination in real-time PCR reagents. Figure 2. Type VI collagen distribution in human subscapularis tendon depicting close interaction with tendon cells. Arrowheads highlight arrays of cells where type VI collagen (red) is distributed in the PCM with viable cells stained with DAPI (blue) Scale bar: 100 mm.
RNA extraction and cDNA synthesis The frozen tissue specimens were lysed in 1 ml of a tissue lyser, TRIzolÕ (Sigma-Aldrich) using the gentleMACS tissue lyser (Miltenyi Biotec, Surrey, UK). Following homogenization, the samples were centrifuged at 12 000 g for 10 min at 4 C. Tissue was incubated at room temperature and 200 ml of chloroform was added, vigorously shaken for 15 s and centrifuged at 12 000 g for 15 min at 4 C. The upper aqueous phase (50% of the total volume) was transferred to a separate tube. RNA was precipitated using 100% isopropanaol for 10 min followed by 10-min centrifugation at 12 000 g at 4 C. The RNA pellet was then washed with 1 ml of 70% ethanol. Finally, the extracted RNA was dissolved in RNase-free water and eluted. One microgram of RNA was converted to complementary DNA (cDNA) using the First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) following the manufacturer’s protocol. Gene expression by real-time qPCR Aliquots of cDNA were amplified in a total volume 20 ml containing primers and Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Validated human primers were purchased from Qiagen (Chatsworth, CA) and were designed to cross introns to decrease any non-specific amplification due to potential genomic DNA contamination
Statistical analysis This study included three biological and three technical replicates. The mean values of the technical replicates were calculated and this mean value of each biological sample was used for the statistical analysis (n ¼ 3). Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA). All values were expressed as mean ± standard deviation for three independent experiments. One-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post hoc test was used to test the difference in expression levels of the PCM components between the three diseased groups. Values of p50.05 were considered to be statistically significant.
Results Immunohistochemistry and confocal microscopy indicated that in normal subscapularis tendon, type VI collagen was localized in the pericellular region of tendon and was in close association with tendon cells. Type VI collagen formed a dense mesh around groups of cells and fibrillin-1 was observed as longitudinally orientated fibers in the vicinity of cells (Figure 2). Both type VI collagen and fibrillin-1 fibers appeared to unite several cells into one array unit (Figure 3). Elastin fibers were sparsely distributed within the tissue, compared to fibrillin-1 and type VI collagen, which were more abundant. Negative controls indicated that non-specific staining did not occur in all specimens (Figure 3). Tendon tissue from large/massive rotator cuff tears was characterized by loss of structure and orientation of type VI
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Figure 3. Organization of type VI collagen, elastin and fibrillin-1 in human rotator cuff. All three proteins were distributed along arrays of tenocytes. Type VI collagen formed a dense distribution around groups of cells and was disorganized in the large tear. Elastin and fibrillin-1 were observed as longitudinally orientated fibers in the vicinity of cells. Negative control for Type VI collagen, elastin and fibrillin-1 with viable cells stained with DAPI (blue) indicated that non-specific staining did not occur. Scale bar: 25 mm.
Figure 4. Expression levels of (A) major collagens type I and type III (B) PCM components type VI collagen, elastin and fibrillin-1in healthy and torn rotator cuff tears (data values are mean ± standard error of three independent experiments (*p50.05, **p50.01, ***p50.001).
collagen. Fibrillin-1 and elastin did not show a significant alteration in orientation among the different tear groups. However, the visibility of these fibers increased in large tears. The influence of increased tear size on the transcriptional levels of type I, III and VI collagens in addition to elastin and fibrillin-1 was examined using qPCR (Figure 4). At the transcription level, the mRNA expression of all the measured structural molecules, except elastin, increased in the degenerated tendons compared to the healthy controls. Additionally,
mRNA levels of type III collagen were higher in small/medium tears than large/massive tear this ratio was reversed, with a significant decrease in the expression of type III collagen compared to that in the small and medium tears. The expression of type VI collagen and fibrillin-1 significantly increased with tear size. The level of the fibrillin-1 increased slightly with damage and the level of type V1 collagen increased 50-folds between normal and large/massive tear size. Elastin did not show a statistically significant change at the mRNA levels between groups. The mean fold change in
Type VI collagen and elastic fibers in human rotator cuff tear
DOI: 10.3109/03008207.2014.959119
Table 2. Mean fold change in the expression of type VI collagen, fibrillin-1 and elastin in different tear groups and healthy rotator cuff tear. Mean fold change differences
Type VI collagen
Elastin
Fibrillin 1
Normal Small/medium tear Large/massive tear
1 57.58 145.34
1 0.72 5.03
1 1.87 7.80
the expression of type VI collagen, fibrillin-1 and elastin in different tear groups and healthy rotator cuff tear have been shown in Table 2.
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Discussion Assembly of matrix aggregates typically occurs within 20 mm of the cell surface (35) making the PCM an important region in the complex architecture of tendons and critical in the response of tendon cells to pathological conditions. As mentioned above, recent studies on animal models have shown that type VI collagen and elastic fibers are localized in the PCM region of tendon and are in intimate association with tendon cells (8–13,30). Our results are consistent with studies in animal models and confirm the presence of type VI collagen, elastin and fibrillin-1 in the PCM of the human supraspinatus tendon for the first time. Moreover, unlike cartilage, where the PCM surrounds individual cells (10,36), the current study suggests that tendon PCM forms a contiguous array uniting several tenocytes along its length. This observation highlights the unique structure of tendon and leads us to speculate that tenocytes may respond to biochemical and biomechanical signals as longitudinal units rather than individual cells. Of particular significance are mechanobiological mechanisms, which have been shown to be significantly altered by the mechanical properties of the PCM in other tissues (6,7). Our results suggest that tendon tears alter the structure and mechanics of the cell microenvironment, which may be key to understanding tissue disease and regeneration. It was found that the contribution of type VI collagen and fibrillin-1 was greater than elastin, which had a sparse distribution. Qualitative evaluation of images from torn supraspinatus tendon indicated that the organization of type VI collagen was significantly disrupted in large tissue tears. This may be indicative of an enhanced robustness or turnover associated with type VI collagen in tendon compared to elastic fibers, perhaps with significance to cellular microenvironment. The distribution of elastin did not appear to be significantly different in torn samples, which may be attributed to the unique ability of elastin to withstand deformations of 100% of its initial length (23). Large/massive tears have a significantly different expression profile and decreased reparative potential compared to small tears (37). The ratio of type III to type I collagen is considered a sensitive marker for tendon injury, with higher levels of type III collagen indicating improved healing potential for the tear (33,34). The transcription levels of type I and III collagen in our study were consistent with the literature, with higher type III collagen in the small/medium tear, suggesting that there is a greater potential for healing
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within this group. Interestingly, it was found that catastrophic tendon tears, with respect to their size, are characterized by elevated PCM molecule expression. The transcription levels of type VI collagen were significantly altered in the torn tendon with a 50-fold difference observed between normal and small/medium tears, and a further increase in the large/ massive tears. The expression of fibrillin-1 also significantly increased from small/medium to large/massive tears. The consequential effect of significant increase of type VI collagen and fibrillin-1 transcription levels is currently unknown. Since the expression increases in both pathological groups, it is harder to speculate on its exact function. It may be an adaptive mechanism of cells to restore tissue integrity in their immediate environment as an early injury response. Moreover, it could be an indication of altered cell differentiation as a result of mechanical changes in the tissue. Numerous in vitro studies have shown that the addition of ECM molecules to cell medium has a significant effect on the differentiation state of the cells (38). During compressive load the accumulation of type VI collagen has been shown to be a good marker of fibrocartilage differentiation (11). The high transcriptional rate of type VI collagen could be a potential indication of altered cell differentiation, resulting in tissue of altered physiology. Interestingly, although the expression of elastin did not increase with tissue tear size, fibrillin-1 was significantly increased. Given that elastin and fibrillin-1 both contribute to the composition of elastic fibers this may be indicative of the underlying mechanisms governing elastogenesis. Elastic fibers are formed by first laying down a scaffold of microfibrils, which are mainly composed of fibrillin-1 and fibrillin-2. Next, elastin is deposited into the center of the structure forming a mature elastic fiber. When tendon is torn, the tenoyctes may attempt to create fibrillinrich scaffolds for production of elastic fibers, but fail when depositing elastin. Torn specimens for this study were collected from supraspinatus tendon whereas the control tissue was collected from subscapularis specimens due to the difficulty in obtaining normal human supraspinatus controls. Interpretations of the gene expressions should therefore weigh the functional and structural differences between these two tendons, which may have affected the gene profile. A lack of access to healthy supraspinatus tendon tissue has been a known limitation in studying rotator cuff tears. Using age-matched subscapularis instead is a common practice in the field, and has been used in many studies (3,39–42). In this study, the subscapularis, used as the control sample, confirms the presence of type VI collagen, elastin and fibrillin-1 in the PCM of the human rotator cuff for the first time. The changes observed among the diseased groups are statistically different, reflecting a true change in profile under pathological conditions in the supraspinatus. Although the sample size is limited this is the first study to confirm the localization of these minor structural molecules in the pericellular region of the human supraspinatus tendon and show that degeneration affects their expression and orientation causing, significant disruption to the type VI collagen networks in immediate proximity of cells. The alteration in the transcription levels of type VI collagen with increasing tear size, as seen in this study, provides the starting
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point for further investigations of the role of this PCM component in healing and disease. Larger samples that show the distribution of these molecules in context to the other histological structures along with colocalization immunostaining and quantitative analysis would provide useful insight into the mechanisms that contribute to tendon degeneration and its significance in rotator cuff disease prognosis.
Declaration of interest
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The work was supported by the National Institute of Health and Research (NIHR), UK.
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