AB1032 Sigma-AldrichAnti-Versican Antibody, a.a. 535-598 of mouse versican

It is not known how extensively a localised flexor tendon injury affects the entire tendon. This study examined the extent of and relationship between histopathologic and gene expression changes in equine superficial digital flexor tendon after a surgical injury. One forelimb tendon was hemi-transected in six horses, and in three other horses, one tendon underwent a sham operation. After euthanasia at six weeks, transected and control (sham and non-operated contralateral) tendons were regionally sampled (medial and lateral halves each divided into six 3 cm regions) for histologic (scoring and immunohistochemistry) and gene expression (real time PCR) analysis of extracellular matrix changes. The histopathology score was significantly higher in transected tendons compared to control tendons in all regions except for the most distal (P ≤ 0.03) with no differences between overstressed (medial) and stress-deprived (lateral) tendon halves. Proteoglycan scores were increased by transection in all but the most proximal region (P less than 0.02), with increased immunostaining for aggrecan, biglycan and versican. After correcting for location within the tendon, gene expression for aggrecan, versican, biglycan, lumican, collagen types I, II and III, MMP14 and TIMP1 was increased in transected tendons compared with control tendons (P less than 0.02) and decreased for ADAMTS4, MMP3 and TIMP3 (P less than 0.001). Aggrecan, biglycan, fibromodulin, and collagen types I and III expression positively correlated with all histopathology scores (P less than 0.001), whereas lumican, ADAMTS4 and MMP14 expression positively correlated only with collagen fiber malalignment (P less than 0.001). In summary, histologic and associated gene expression changes were significant and widespread six weeks after injury to the equine SDFT, suggesting rapid and active development of tendinopathy throughout the entire length of the tendon. These extensive changes distant to the focal injury may contribute to poor functional outcomes and re-injury in clinical cases. Our data suggest that successful treatments of focal injuries will need to address pathology in the entire tendon, and that better methods to monitor the development and resolution of tendinopathy are required.

The present study examined the effect of ADAMTS5 (TS5) knockout on the properties of murine flexor digitorum longus (FDL) and Achilles tendons. FDL and Achilles tendons were analyzed using biomechanical testing, histology, and immunohistochemistry; further characterization of FDL tendons was conducted using transmission electron microscopy (collagen fibril ultrastructure), SDS-PAGE (collagen content and type), fluorescence-assisted carbohydrate electrophoresis for chondroitin sulfate and hyaluronan, and Western blotting for aggrecan, versican, and decorin abundance and distribution. FDL tendons of TS5(-/-) mice showed a 33% larger cross-sectional area, increased collagen fibril area fraction, and decreased material properties relative to those of wild type mice. In TS5(-/-) mice, aggrecan accumulated in the pericellular matrix of tendon fibroblasts. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in TS5(-/-) and wild type mice; however, the TS5(-/-) tendons exhibited a higher tensile modulus and a weakened enthesis. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of ADAMTS5 in tendon is to remove pericellular and interfibrillar aggrecan to maintain the molecular architecture responsible for normal tissue function. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:620-626, 2012.

To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses.Laminae from the forelimb hooves of 8 healthy horses.Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization.Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae.Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.

Chondroitin sulfate proteoglycans (CSPGs) play a pivotal role in many neuronal growth mechanisms including axon guidance and the modulation of repair processes following injury to the spinal cord or brain. Many actions of CSPGs in the central nervous system (CNS) are governed by the specific sulfation pattern on the glycosaminoglycan (GAG) chains attached to CSPG core proteins. To elucidate the role of CSPGs and sulfated GAG chains following traumatic brain injury (TBI), controlled cortical impact injury of mild to moderate severity was performed over the left sensory motor cortex in mice. Using immunoblotting and immunostaining, we found that TBI resulted in an increase in the CSPGs neurocan and NG2 expression in a tight band surrounding the injury core, which overlapped with the presence of 4-sulfated CS GAGs but not with 6-sulfated GAGs. This increase was observed as early as 7 days post injury (dpi), and persisted for up to 28 dpi. Labeling with markers against microglia/macrophages, NG2+ cells, fibroblasts, and astrocytes showed that these cells were all localized in the area, suggesting multiple origins of chondroitin-4-sulfate increase. TBI also caused a decrease in the expression of aggrecan and phosphacan in the pericontusional cortex with a concomitant reduction in the number of perineuronal nets. In summary, we describe a dual response in CSPGs whereby they may be actively involved in complex repair processes following TBI.

ADAMTS5 has been implicated in the degradation of cartilage aggrecan in human osteoarthritis. Here, we describe a novel role for the enzyme in the regulation of TGFβ1 signaling in dermal fibroblasts both in vivo and in vitro. Adamts5(-/-) mice, generated by deletion of exon 2, exhibit impaired contraction and dermal collagen deposition in an excisional wound healing model. This was accompanied by accumulation in the dermal layer of cell aggregates and fibroblastic cells surrounded by a pericellular matrix enriched in full-length aggrecan. Adamts5(-/-) wounds exhibit low expression (relative to wild type) of collagen type I and type III but show a persistently elevated expression of tgfbRII and alk1. Aggrecan deposition and impaired dermal repair in Adamts5(-/-) mice are both dependent on CD44, and Cd44(-/-)/Adamts5(-/-) mice display robust activation of TGFβ receptor II and collagen type III expression and the dermal regeneration seen in WT mice. TGFβ1 treatment of newborn fibroblasts from wild type mice results in Smad2/3 phosphorylation, whereas cells from Adamts5(-/-) mice phosphorylate Smad1/5/8. The altered TGFβ1 response in the Adamts5(-/-) cells is dependent on the presence of aggrecan and expression of CD44, because Cd44(-/-)/Adamts5(-/-) cells respond like WT cells. We propose that ADAMTS5 deficiency in fibrous tissues results in a poor repair response due to the accumulation of aggrecan in the pericellular matrix of fibroblast progenitor cells, which prevents their transition to mature fibroblasts. Thus, the capacity of ADAMTS5 to modulate critical tissue repair signaling events suggests a unique role for this enzyme, which sets it apart from other members of the ADAMTS family of proteases.