Matrix Biology Plus最新文献

Bovine forelimb flexor and extensor tendons serve as a model for examining high stress, energy storing and low stress, positional tendons, respectively. Previous research has shown structural differences between the collagen fibrils of these tissues. The nanoscale collagen fibrils of flexor tendons are smaller in size, more heavily crosslinked, and respond differently to mechanical loading. Meanwhile, energy storing tendons undergo less collagen turnover compared to positional tendons and are more commonly injured. These observations raise the question of whether collagen fibril structure influences the collagen degradation processes necessary for remodelling. Atomic force microscopy was used to image dry collagen fibrils before and after 5-hour exposure to matrix metalloproteinase-1 (MMP-1) to detect changes in fibril size. Collagen fibrils from three tissue types were studied: bovine superficial digital flexor tendons, matched-pair bovine lateral digital extensor tendons, and rat tail tendons. Compared to control fibrils exposed only to buffer, a significant decrease in fibril cross-sectional area (CSA) following MMP-1 exposure was observed for bovine extensor and rat tail fibrils, with larger fibrils experiencing a greater magnitude of CSA decrease in both fibril types. Fibrils from bovine flexor tendons, on the other hand, showed no decrease in CSA when exposed to MMP-1. The result did not appear to be linked to the small size of flexor fibrils, as equivalently sized extensor fibrils were readily degraded by the enzyme. Increased proteolytic resistance of collagen fibrils from high stress tendons may help to explain the longevity of collagen within these tissues in vivo.

The healthy skeletal muscle extracellular matrix (ECM) has several functions including providing structural integrity to myofibers, enabling lateral force transmission, and contributing to overall passive mechanical properties. In diseases such as Duchenne Muscular dystrophy, there is accumulation of ECM materials, primarily collagen, which results in fibrosis. Previous studies have shown that fibrotic muscle is often stiffer than healthy muscle, in part due to the increased number and altered architecture of collagen fibers within the ECM. This would imply that the fibrotic matrix is stiffer than the healthy matrix. However, while previous studies have attempted to quantify the extracellular contribution to passive stiffness in muscle, the outcomes are dependent on the type of method used. Thus, the goals of this study were to compare the stiffness of healthy and fibrotic muscle ECM and to demonstrate the efficacy of two methods for quantifying extracellular-based stiffness in muscle, namely decellularization and collagenase digestion. These methods have been demonstrated to remove the muscle fibers or ablate collagen fiber integrity, respectively, while maintaining the contents of the extracellular matrix. Using these methods in conjunction with mechanical testing on wildtype and D2.mdx mice, we found that a majority of passive stiffness in the diaphragm is dependent on the ECM, and the D2.mdx diaphragm ECM is resistant to digestion by bacterial collagenase. We propose that this resistance is due to the increased collagen cross-links and collagen packing density in the ECM of the D2.mdx diaphragm. Taken altogether, while we did not find increased stiffness of the fibrotic ECM, we did observe that the D2.mdx diaphragm conveyed resistance against collagenase digestion. These findings demonstrate how different methods for measuring ECM-based stiffness each have their own limitations and can produce different results.

Type II collagen is the major fibrillar collagen in cartilage. It is synthesized in the form of precursors (procollagens) containing N- and C-terminal propeptides. The two main isoforms of type II procollagen protein are type IIA and type IIB procollagens, generated in a developmentally regulated manner by differential splicing of the primary gene transcript. Isoform IIA contains exon 2 and is produced mainly by chondroprogenitor cells while isoform IIB lacks exon 2 and is produced by differentiated chondrocytes. Thus, expression of IIA and IIB isoforms are reliable markers for identifying the differentiation status of chondrocytes but their biological function in the context of skeletal development is still not yet fully understood. Specific antibodies against IIA and IIB procollagen isoforms are already available. In this study, a synthetic peptide spanning the junction between exon 1 and exon 3 of the murine sequence was used as an immunogen to generate a novel rabbit polyclonal antibody directed against procollagen IIB. Characterization of this antibody by Western-blotting analysis of murine cartilage extracts and ELISA tests demonstrated its specificity to the type IIB isoform. Furthermore, by immunohistochemical studies, this antibody allowed the detection of procollagen IIB in embryonic cartilage as well as in articular cartilage and growth plate of young adult mice. Interestingly, this is the first antibody that has allowed the detection of procollagen IIB at both the intra- and extracellular level. This antibody therefore represents an interesting new tool for monitoring the spatial and temporal distribution of IIB isoforms in skeletal tissues of mouse models and for tracking the trafficking and processing of type IIB procollagen.

Although most work has focused on resolution of collagen ECM, fibrosis resolution involves changes to several ECM proteins. The purpose of the current study was twofold: 1) to examine the role of MMP12 and elastin; and 2) to investigate the changes in degraded proteins in plasma (i.e., the “degradome”) in a preclinical model of fibrosis resolution. Fibrosis was induced by 4 weeks carbon tetrachloride (CCl₄) exposure, and recovery was monitored for an additional 4 weeks. Some mice were treated with daily MMP12 inhibitor (MMP408) during the resolution phase. Liver injury and fibrosis was monitored by clinical chemistry, histology and gene expression. The release of degraded ECM peptides in the plasma was analyzed using by 1D-LC-MS/MS, coupled with PEAKS Studio (v10) peptide identification. Hepatic fibrosis and liver injury rapidly resolved in this mouse model. However, some collagen fibrils were still present 28d after cessation of CCl₄. Despite this persistent collagen presence, expression of canonical markers of fibrosis were also normalized. The inhibition of MMP12 dramatically delayed fibrosis resolution under these conditions. LC-MS/MS analysis identified that several proteins were being degraded even at late stages of fibrosis resolution. Calpains 1/2 were identified as potential new proteases involved in fibrosis resolution. CONCLUSION. The results of this study indicate that remodeling of the liver during recovery from fibrosis is a complex and highly coordinated process that extends well beyond the degradation of the collagenous scar. These results also indicate that analysis of the plasma degradome may yield new insight into the mechanisms of fibrosis recovery, and by extension, new “theragnostic” targets. Lastly, a novel potential role for calpain activation in the degradation and turnover of proteins was identified.

Glycans are one of the fundamental biopolymers encountered in living systems. Compared to polynucleotide and polypeptide biosynthesis, polysaccharide biosynthesis is a uniquely combinatorial process to which interdependent enzymes with seemingly broad specificities contribute. The resulting intracellular cell surface, and secreted glycans play key roles in health and disease, from embryogenesis to cancer progression. The study and modulation of glycans in cell and organismal biology is aided by small molecule inhibitors of the enzymes involved in glycan biosynthesis. In this review, we survey the arsenal of currently available inhibitors, focusing on agents which have been independently validated in diverse systems. We highlight the utility of these inhibitors and drawbacks to their use, emphasizing the need for innovation for basic research as well as for therapeutic applications.

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling.

Lysyl oxidase-like 1 encoded by the LOXL1 gene is a member of the lysyl oxidase family of enzymes that are important in the maintenance of extracellular matrix (ECM)-rich tissue. LOXL1 is important for proper elastic fiber formation and mice lacking LOXL1 (Loxl1^−/−) exhibit systemic elastic fiber disorders, such as pelvic organ prolapse, a phenotype associated with exfoliation syndrome (XFS) in humans. Patients with XFS have a significant risk of developing exfoliation glaucoma (XFG), a severe form of glaucoma, which is a neurodegenerative condition leading to irreversible blindness if not detected and treated in a timely fashion. Although Loxl1^−/− mice have been used extensively to investigate mechanisms of pelvic organ prolapse, studies of eyes in those mice are limited and some showed inconsistent ocular phenotypes. In this study we demonstrate that Loxl1^−/− mice have significant anterior segment biometric abnormalities which recapitulate some human XFS features. We then focused on the peripapillary sclera (PPS), a critical structure for maintaining optic nerve health. We discovered quantitative and qualitive changes in ultrastructure of PPS, such as reduced elastic fibers, enlarged collagen fibrils, and transformed collagen lamella organization detected by transmission electron microscopy (TEM). Importantly, these changes corelate with altered tissue biomechanics detected by Atomic Force Microscopy (AFM) of PPS in mice. Together, our results support a crucial role for LOXL1 in ocular tissue structure and biomechanics, and Loxl1^−/− mice could be a valuable resource for understanding the role of scleral tissue biomechanics in ocular disease.

Collagen XII is a fibril-associated collagen with interrupted triple helices (FACIT). This non-fibrillar collagen is a homotrimer composed of three α1(XII) chains assembled into a collagenous molecule with a C terminal collagenous domain and a large N terminal non-collagenous domain. During tendon development and growth, collagen XII is broadly expressed throughout the extracellular matrix and enriched pericellularly around tenocytes. Tendons in a global Col12a1^-/- knockout model demonstrated disrupted fibril and fiber structure and disordered tenocyte organization, highlighting the critical regulatory roles of collagen XII in determining tendon structure and function. However, muscle and bone also are affected in the collagen XII knockout model. Therefore, secondary effects on tendon due to involvement of bone and muscle may occur in the global knockout. The global knockout does not allow the definition of intrinsic mechanisms involving collagen XII in tendon versus extrinsic roles involving muscle and bone. To address this limitation, we created and characterized a conditional Col12a1-null mouse model to permit the spatial and temporal manipulation of Col12a1 expression. Collagen XII knockout was targeted to tendons by breeding conditional Col12a1^flox/flox mice with Scleraxis-Cre (Scx-Cre) mice to yield a tendon-specific Col12a1-null mouse line, Col12a1^Δten/Δten. Both mRNA and protein expression in Col12a1^Δten/Δten mice decreased to near baseline levels in flexor digitorum longus tendons (FDL). Collagen XII immuno-localization revealed an absence of reactivity in the tendon proper, but there was reactivity in the cells of the surrounding peritenon. This supports a targeted knockout in tenocytes while peritenon cells from a non-tendon lineage were not targeted and retained collagen XII expression. The tendon-targeted, Col12a1^Δten/Δten mice had significantly reduced forelimb grip strength, altered gait and a significant decrease in biomechanical properties. While the observed decrease in tendon modulus suggests that differences in tendon material properties in the absence of Col12a1 expression underlie the functional deficiencies. Together, these findings suggest an intrinsic role for collagen XII critical for development of a functional tendon.