Biglycan has a major role in maintenance of mature tendon mechanics.

Decorin and biglycan are two small leucine-rich proteoglycans (SLRPs) that regulate collagen fibrillogenesis and extracellular matrix assembly in tendon. The objective of this study was to determine the individual roles of these molecules in maintaining the structural and mechanical properties of tendon during homeostasis in mature mice. We hypothesized that knockdown of decorin in mature tendons would result in detrimental changes to tendon structure and mechanics while knockdown of biglycan would have a minor effect on these parameters. To achieve this objective, we created tamoxifen-inducible mouse knockdown models targeting decorin or biglycan inactivation. This enables the evaluation of the roles of these SLRPs in mature tendon without the abnormal tendon development caused by conventional knockout models. Contrary to our hypothesis, knockdown of decorin resulted in minor alterations to tendon structure and no changes to mechanics while knockdown of biglycan resulted in broad changes to tendon structure and mechanics. Specifically, knockdown of biglycan resulted in reduced insertion modulus, maximum stress, dynamic modulus, stress relaxation, and increased collagen fiber realignment during loading. Knockdown of decorin and biglycan produced similar changes to tendon microstructure by increasing the collagen fibril diameter relative to wild-type controls. Biglycan knockdown also decreased the cell nuclear aspect ratio, indicating a more spindle-like nuclear shape. Overall, the extensive changes to tendon structure and mechanics after knockdown of biglycan, but not decorin, provides evidence that biglycan plays a major role in the maintenance of tendon structure and mechanics in mature mice during homeostasis.

Identification of proteomic signatures associated with COPD frequent exacerbators.

AIMS: Acute exacerbation is a major event that alters the natural course of chronic obstructive pulmonary disease (COPD), and recurrent exacerbation results in worse clinical outcomes and greater economic consequences. While some patients suffer frequent exacerbations, others experience no exacerbations; this study was designed to detect proteins that were differentially abundant in COPD frequent exacerbators and assess whether those expression profiles are unique among COPD patients. MAIN METHODS: Tandem mass tag labeled quantitative proteomics combined with two-dimensional liquid chromatography-tandem mass spectrometry was used to detect the changes in the lung proteome in COPD frequent exacerbators and infrequent exacerbators. A series of bioinformatics analyses were performed to screen potential signatures of COPD frequent exacerbations. The accuracy of proteomic results was further verified by western blot studies. KEY FINDINGS: Compared with infrequent exacerbators, 23 proteins in the lung tissues from frequent exacerbators showed significant degrees of differential expression; combined bioinformatics analyses of proteome indicated that the immune network for IgA production and the phenylalanine metabolism pathway were associated with frequent exacerbations. The Western blot analysis confirmed the expression pattern of three significantly regulated proteins (HLA-DQA1, pIgR and biglycan). SIGNIFICANCE: These findings indicate that immune response might play a key role in the pathophysiological mechanisms of COPD frequent exacerbations. Our results make a crucial contribution to the search for a comprehensive understanding of potential pathophysiological mechanisms associated with the frequent exacerbations of COPD, and might provide guidance for treating frequent exacerbations.

Class I and Class II small leucine-rich proteoglycans in human cutaneous pacinian corpuscles.

Pacinian corpuscles are onion bulb-like multilayered mechanoreceptors that consist of a complicated structure of axon terminals, Schwann related cells (inner core), endoneural related cells (intermediate layer) and perineurial related cells (outer core-capsule). The cells forming those compartments are continuous and share the properties of that covering the nerve fibers. Small leucine-rich proteoglycans are major proteoglycans of the extracellular matrix and regulate collagen fibrillogenesis, cell signalling pathways and extracellular matrix assembly. Here we used immunohistochemistry to investigate the distribution of class I (biglycan, decorin, asporin, ECM2 and ECMX) and class II (fibromodulin, lumican, prolargin, keratocan and osteoadherin) small leucine-rich proteoglycans in human cutaneous Pacinian corpuscles. The distribution of these compounds was: the inner core express decorin, biglycan, lumican, fibromodulin, osteoadherin; the intermediate layer display immunoreactivity for osteoadherin; the outer core biglycan, decorin, lumican, fibromodulin and osteoadherin; and the capsule contains biglycan, decorin, fibromodulin, and lumican. Asporin, prolargin and keratocan were undetectable. These results complement our knowledge about the distribution of small leucine-rich proteoglycans in human Pacinian corpuscles, and help to understand the composition of the extracellular matrix in these sensory formations.

Contaminants in commercial preparations of 'purified' small leucine-rich proteoglycans may distort mechanistic studies.

The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial 'decorin' and 'biglycan' were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of 'decorin' and 'biglycan' on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers' funds and time.

The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc.

INTRODUCTION: In the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP). METHODS: We used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines. RESULTS: We detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion. CONCLUSIONS: The CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

Achilles tendons from decorin- and biglycan-null mouse models have inferior mechanical and structural properties predicted by an image-based empirical damage model.

Achilles tendons are a common source of pain and injury, and their pathology may originate from aberrant structure function relationships. Small leucine rich proteoglycans (SLRPs) influence mechanical and structural properties in a tendon-specific manner. However, their roles in the Achilles tendon have not been defined. The objective of this study was to evaluate the mechanical and structural differences observed in mouse Achilles tendons lacking class I SLRPs; either decorin or biglycan. In addition, empirical modeling techniques based on mechanical and image-based measures were employed. Achilles tendons from decorin-null (Dcn(-/-)) and biglycan-null (Bgn(-/-)) C57BL/6 female mice (N=102) were used. Each tendon underwent a dynamic mechanical testing protocol including simultaneous polarized light image capture to evaluate both structural and mechanical properties of each Achilles tendon. An empirical damage model was adapted for application to genetic variation and for use with image based structural properties to predict tendon dynamic mechanical properties. We found that Achilles tendons lacking decorin and biglycan had inferior mechanical and structural properties that were age dependent; and that simple empirical models, based on previously described damage models, were predictive of Achilles tendon dynamic modulus in both decorin- and biglycan-null mice.

Disruption of Tgfbr2 in odontoblasts leads to aberrant pulp calcification.

Transforming growth factor ß (TGF-ß) signaling has been implicated in dentin formation and repair; however, the molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-ß signaling in dentin formation, we analyzed odontoblast-specific Tgfbr2 conditional knockout mice. The mutant mice had aberrant teeth with thin dysplastic dentin and pulpal obliteration, similar to teeth from human patients with dentinogenesis imperfecta type II and dentin dysplasia. In mutant, the odontoblasts lost their cellular polarity, and matrix secretion was disrupted after mantle dentin formation. As a consequence, the amount of predentin decreased significantly, and an ectopic fibrous matrix was formed below the odontoblast layer. This matrix gradually calcified and obliterated the pulp chamber with increasing age. Immunohistochemistry revealed decreased expression of alkaline phosphatase in mutant odontoblasts. In mutant dentin, Dsp expression was reduced, but Dmp1 expression increased significantly. Collagen type I, biglycan, and Dsp were expressed in the ectopic matrix. These results suggest that loss of responsiveness to TGF-ß in odontoblasts results in impaired matrix formation and pulpal obliteration. Our study indicates that TGF-ß signaling plays an important role in dentin formation and pulp protection. Furthermore, our findings may provide new insight into possible mechanisms underlying human hereditary dentin disorders and reparative dentin formation.

Proteoglycan expression is influenced by mechanical load in TMJ discs.

The expression and assembly of the extracellular matrix are profoundly associated with adaptive and pathological responses of the temporomandibular joint (TMJ). To better understand the adaptive responses of the TMJ disc to mechanical loading, we examined the expression of 2 modular proteoglycans and 10 small leucine-rich proteoglycans (SLRPs) at the mRNA and protein levels and determined the contents of proteoglycan-related glycosaminoglycans (GAGs) in rat TMJ discs in response to altered mechanical loading caused by an incisal bite plane. One hundred thirty 7-week-old male Wistar rats were assigned to control and bite plane groups. TMJ disc thickness and the intensity of toluidine blue staining of metachromasia increased in the posterior band after 2 weeks of wearing the bite plane. GAG content increased significantly in the bite plane group after 2 weeks. Quantitative real-time RT-PCR (reverse transcription polymerase chain reaction) analysis indicated that biglycan and chondroadherin mRNA levels increased after 2 weeks and that the level of decorin mRNA increased at 4 weeks. Versican mRNA levels increased after 3 weeks, particularly for the V0 and V1 versican isoforms, which carry more GAG attachment sites than do the V2 and V3 isoforms. Western analysis demonstrated a corresponding increase in the levels of versican, biglycan, and decorin core proteins at 4 weeks in the bite plane group. These results indicate that mechanical loading differentially influences proteoglycan mRNA expression and protein accumulation in the TMJ disc. The change in proteoglycan mRNA and protein levels may lead to the modulation of matrix-matrix and cell-matrix interactions and has important biological significance for adaptation to complicated biomechanical requirements and for tissue maintenance in the TMJ disc.

High expression of biglycan is associated with poor prognosis in patients with esophageal squamous cell carcinoma.

Biglycan (BGN), an extracellular matrix component, has been reported to play a crucial role in the tumor progression of various cancers. However, the relation between the expression of BGN and clinical prognosis has not been studied yet. We therefore carry out the present study to elucidate the role of BGN in predicting outcomes of patients with esophageal squamous cell carcinoma (ESCC). In this study, the expression of BGN in 170 cases of ESCC tissues and matched 46 adjacent non-tumorous tissues was measured by quantitative real-time PCR and immunohistochemistry. Upregulation of BGN occurred in approximately 60% of primary ESCCs compared with their non-tumor counterparts. In addition, high expression of BGN was significantly associated with clinical stage (P = 0.009), tumor invasion (P = 0.006) and lymph node metastasis (P = 0.046). The 5-year disease-specific survival (DSS) in high expression of BGN group is poorer than that in low level expression group (36.8% VS 57.4%, P = 0.006). Stratified analysis according to the pathological stage revealed its discernibility on DSS was only pronounced in patients with advanced clinical stage (P = 0.010). Cox multivariate analysis revealed that pathologic N category (P < 0.001; hazard ratio, 2.482, 95% CI, 1.576-3.909) and BGN expression (P = 0.019; hazard ratio, 1.713, 95% CI, 1.092-2.688) were two independent prognostic factors. The findings of the present study provide evidence that BGN represents a potential novel prognostic biomarker for resected ESCC patients in advanced clinical stage.

Immunohistochemical expression of biglycan and decorin in the pulp tissue of human primary teeth during resorption.

Primary teeth are interesting models that can be used to study physiological and pathological processes involving cells and extracellular matrices in hard and soft tissues. This study investigated the expression and distribution of biglycan and decorin-the non-collagenous components of the extracellular matrix-in primary teeth tissue, during physiological root resorption. Thirty healthy human primary teeth were grouped together according to root length: Group I - two-thirds root length, Group II - one-third root length, and Group III - teeth with no root. The streptavidin-biotin-peroxidase immunohistochemical method was used with antibodies against the previously named antigens. The proteoglycans studied were found in the pulp and dentin extracellular matrix in all groups without any differences in the proteins, among the groups. Biglycan was observed mainly in predentin and in pulp connective tissue in the resorption area. In addition, decorin was observed mainly in pulp connective tissue, but near the resorption area. Biglycan and decorin were distributed differentially in the dental tissues. The present immunohistocytochemical data, combined with previously reported data, suggest that these proteoglycans could be involved in regulating the physiological resorption process in healthy primary teeth.

Immunohistochemical expression of biglycan and decorin in the pulp tissue of human primary teeth during resorption

Primary teeth are interesting models that can be used to study physiological and pathological processes involving cells and extracellular matrices in hard and soft tissues. This study investigated the expression and distribution of biglycan and decorin-the non-collagenous components of the extracellular matrix-in primary teeth tissue, during physiological root resorption. Thirty healthy human primary teeth were grouped together according to root length: Group I - two-thirds root length, Group II - one-third root length, and Group III - teeth with no root. The streptavidin-biotin-peroxidase immunohistochemical method was used with antibodies against the previously named antigens. The proteoglycans studied were found in the pulp and dentin extracellular matrix in all groups without any differences in the proteins, among the groups. Biglycan was observed mainly in predentin and in pulp connective tissue in the resorption area. In addition, decorin was observed mainly in pulp connective tissue, but near the resorption area. Biglycan and decorin were distributed differentially in the dental tissues. The present immunohistocytochemical data, combined with previously reported data, suggest that these proteoglycans could be involved in regulating the physiological resorption process in healthy primary teeth.

Matrix-assisted laser desorption ionization-imaging mass spectrometry: a new methodology to study human osteoarthritic cartilage.

OBJECTIVE: Information about the distribution of proteins and the modulation that they undergo in the different phases of rheumatic pathologies is essential to understanding the development of these diseases. We undertook this study to demonstrate the utility of mass spectrometry (MS)-based molecular imaging for studying the spatial distribution of different components in human articular cartilage sections. METHODS: We compared the distribution of peptides and proteins in human control and osteoarthritic (OA) cartilage. Human control and OA cartilage slices were cut and deposited on conductive slides. After tryptic digestion, we performed matrix-assisted laser desorption ionization-imaging MS (MALDI-IMS) experiments in a MALDI-quadrupole time-of-flight mass spectrometer. Protein identification was undertaken with a combination of multivariate statistical methods and Mascot protein database queries. Hematoxylin and eosin staining and immunohistochemistry were performed to validate the results. RESULTS: We created maps of peptide distributions at 150-µm raster size from control and OA human cartilage. Proteins such as biglycan, prolargin, decorin, and aggrecan core protein were identified and localized. Specific protein markers for cartilage oligomeric matrix protein and fibronectin were found exclusively in OA cartilage samples. Their distribution displayed a stronger intensity in the deep area than in the superficial area. New tentative OA markers were found in the deep area of the OA cartilage. CONCLUSION: MALDI-IMS identifies and localizes disease-specific peptides and proteins in cartilage. All the OA-related peptides and proteins detected display a stronger intensity in the deep cartilage. MS-based molecular imaging is demonstrated to be an innovative method for studying OA pathology.

Arguments for an increasing differentiation towards fibrocartilaginous components in midportion Achilles tendinopathy.

PURPOSE: The objective of this study was to investigate the fibrocartilaginous differentiation occurring in midportion Achilles tendinopathy. METHODS: Tendon samples were retrospectively collected from 23 patients, who had undergone surgery for midportion Achilles tendinopathy resistant to conservative treatment. Based on histological scores, the biopts were subdivided into three categories: a light, moderate and severe histopathological stage. Throughout these stages, immunohistochemical staining was performed against biglycan, aggrecan and collagen type II, components characteristic for fibrocartilage. Staining of these components was evaluated using a semi-quantitative scoring method. RESULTS: The immunohistochemical scores of biglycan and aggrecan were statistically significant between the histopathological stages (P < 0.001). The immunohistochemical scores were positively correlated with the increasing histopathological stages [Spearman's correlation coefficient = 0.93 for biglycan and 0.78 for aggrecan (P < 0.001)]. Staining for collagen type II remained negative throughout these stages. CONCLUSION: Immunohistochemical staining of the fibrocartilaginous components biglycan and aggrecan showed a progressive increase, correlated with a further evolved histopathological stage. This observation gave arguments for an increased differentiation towards fibrocartilaginous components at protein level in midportion Achilles tendinopathy.

Excessive Wnt/ß-catenin signaling disturbs tooth-root formation.

BACKGROUND AND OBJECTIVE: Wingless-type MMTV integration site family (Wnt)/ß-catenin signaling plays an essential role in cellular differentiation and matrix formation during skeletal development. However, little is known about its role in tooth-root formation. In a previous study, we found excessive formation of dentin and cementum in mice with constitutive ß-catenin stabilization in the dental mesenchyme. In the present study we analyzed the molar roots of these mice to investigate the role of Wnt/ß-catenin signaling in root formation in more detail. MATERIAL AND METHODS: We generated OC-Cre:Catnb(+/lox(ex3)) mice by intercrossing Catnb(+/lox(ex3)) and OC-Cre mice, and we analyzed their mandibular molars using radiography, histomorphometry and immunohistochemistry. RESULTS: OC-Cre:Catnb(+/lox(ex3)) mice showed impaired root formation. At the beginning of root formation in mutant molars, dental papilla cells did not show normal differentiation into odontoblasts; rather, they were prematurely differentiated and had a disorganized arrangement. Interestingly, SMAD family member 4 was upregulated in premature odontoblasts. In 4-wk-old mutant mice, molar roots were about half the length of those in their wild-type littermates. In contrast to excessively formed dentin in crown, root dentin was thin and hypomineralized in mutant mice. Biglycan and dentin sialophosphoprotein were downregulated in root dentin of mutant mice, whereas dentin matrix protein 1 and Dickkopf-related protein 1 were upregulated. Additionally, ectonucleotide pyrophosphatase/phosphodiesterase 1 was significantly downregulated in the cementoblasts of mutant molars. Finally, in the cementum of mutant mice, bone sialoprotein was downregulated but Dickkopf-related protein 2 was upregulated. CONCLUSION: These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays an important role in cell differentiation and matrix formation during root and cementum formation.

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle.

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.

Differential proteoglycan and hyaluronan distribution in calcified aortic valves.

BACKGROUND: While the prevalence of calcified aortic valve disease continues to rise and no pharmacological treatments exist, little is known regarding the pathogenesis of the disease. Proteoglycans and the glycosaminoglycan hyaluronan are involved in calcification in arteriosclerosis and their characterization in calcified aortic valves may lend insight into the pathogenesis of the disease. METHODS: Fourteen calcified aortic valves removed during valve replacement surgery were immunohistochemically stained for the proteoglycans decorin, biglycan, and versican, as well as the glycosaminoglycan hyaluronan. Staining intensity was evaluated in the following regions of interest: center of calcified nodule, edge of nodule, tissue directly surrounding the nodule; center and tissue surrounding small "prenodules"; and fibrosa layer of normal regions of the leaflet distanced from the nodule. RESULTS: Decorin, biglycan, and versican, as well as hyaluronan, were abundantly present immediately surrounding the calcified nodules, but minimally within the nodule itself. Expression of decorin and biglycan in and surrounding prenodules was greater than in the edge and center regions of mature nodules. The levels of expression of the proteoglycans and hyaluronan were highly correlated with one another in the different regions of the valve. CONCLUSIONS: The three proteoglycans and hyaluronan demonstrated distinctive localization relative to nodules within calcified aortic valves, where they likely mediate lipid retention, cell proliferation, and extracellular matrix remodeling, and motivate further study. Comparisons between expression of these components in mature nodules and prenodules suggest distinct roles for these components in nodule progression, especially in the tissues surrounding the nodules.