Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.Connective tissues are characterized by an abundant extracellular matrix in which a wide variety of different proteins and proteoglycans assemble into multimolecular complexes, often in the form of networks. The fibrillar collagens are major components (for review, see Ref. 1) and, in cartilage, collagen II forms cross-striated fibrils in association with collagen IX and XI (2). Collagen VI is another member of the collagen family that distinguishes itself by containing large globular domains at its N and C termini (3-6). The molecule consists of three genetically distinct ␣-chains, ␣1(VI), ␣2(VI), and ␣3(VI). The N-terminal globular region is composed of nine or ten von Willebrand factor (vWF) 1 A-like domains derived from the ␣3-chain. Collagen VI molecules associate laterally in an antiparallel fashion into dimers that are stabilized by disulfide bridges (3, 4, 7). The dimers aggregate further into tetramers that are secreted into the extracellular matrix (7), where they join end to end into microfibrils. These subsequently form characteristic thin beaded filaments that are found in a variety of tissues (3,8,9). The formation of microfibrils was recently shown to depend on the N5 vWFA-like domain of ␣3(VI) (10). In addition to the collagens, the large hyaluronan-binding proteoglycan, aggrecan is a major constituent of the cartilage extracellular matrix. The aggrecan core protein has a molecular weight of ϳ220 kDa (11) These major constituents provide the basic organization of the extracellular matrix, while other molecules modulate its assembly and structure. The matrilins are a family of oligomeric matrix proteins containing common structural motifs such as vWFA-like domains, epidermal growth factor-like EGF modules and coiled-coil regions (reviewed in Ref. 18). Matrilin-1 (also known as cartilage matrix protein, CMP), and matrilin-3 are abundant in ca...

The binding of native biglycan and decorin to pepsinextracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore TM 2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K D ) of ϳ30 nM. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the ␣2(VI) chain probably plays a role in the interaction.Collagens are a large family of extracellular structural proteins made up of three ␣ chains that are intracellularly associated and folded into specific structures including characteristic triple helical domains (1). The major class, recognized as the fibril-forming collagens, contains molecules with one large uninterrupted triple helical domain (for review, see Refs. 1 and 2). Other members of the collagen family have one or more non-triple helical domains, which may constitute the major part of the protein. Most of these collagens do not form prominent lateral aggregates in a manner similar to that of the fibril-forming collagens. Instead, they form complex aggregates together with other matrix macromolecules. Collagen VI is one example, forming multimolecular filamentous beaded structures after secretion from the cell (for review, see Ref. 3). This collagen is composed of three different peptide chains (␣1(VI), ␣2(VI), and ␣3(VI)), which form the basic unit consisting of a relatively short triple helical domain flanked by two large multidomain globular regions (4). These are composed primarily of repeating units of von Willebrand type A domains (5). Collagen VI assembles intracellularly into antiparallel, overlapping dimers that then align and form tetramers (6). These structures are stabilized by disulfide bonds. Secreted tetramers assemble extracellularly in a characteristic end-to-end fashion into thin (3-10 nm) beaded filaments with a periodicity of about 100 nm (7-9). Further supramolecular assembly includes lateral associations of the beaded filaments into microfibrils (8, 9).Collagen VI is ubiquitous. It can be found intermingled with fibril-forming collagens and is often enriched in the pericellular matrix (for review, see Refs. 3 and 10). Decreased amounts of secreted collagen VI resulting from mutations in COL6A1 have been shown in Bethlem myopathy (11, 12), a dominantly inherited disorder characterized by progressive muscle weakness and wasting....

The ability of the leucine-rich repeat (LRR) proteins biglycan, decorin, and chondroadherin to interact with collagen VI and influence its assembly to supramolecular structures was studied by electron microscopy and surface plasmon resonance measurements in the BIAcore 2000 system. Biglycan showed a unique ability to organize collagen VI into extensive hexagonal-like networks over a time period of only a few minutes. Only the intact molecule, substituted with two dermatan sulfate chains, had this capacity. Intact decorin, with one dermatan sulfate chain only, was considerably less efficient, and aggregates of organized collagen VI were found only after several hours. Chondroadherin without glycosaminoglycan substitutions did not induce any ordered collagen VI organization. However, all three related LRR proteins were shown to interact with collagen VI using electron microscopy and surface plasmon resonance. Biglycan and decorin were exclusively found close to the N-terminal parts of the collagen VI tetramers, whereas chondroadherin was shown to bind close to both the N-and C-terminal parts of collagen VI. In the formed hexagonal networks, biglycan was localized to the intra-network junctions of the collagen VI filaments. This was demonstrated by electron microscopy after negative staining of gold-labeled biglycan in aggregation experiments with collagen VI.Collagen VI is unique among the collagens in its molecular and fibrillar arrangement. The monomer consists of three genetically distinct peptide chains (␣ 1 (VI), ␣ 2 (VI), and ␣ 3 (VI)) that form a triple helical, central domain, flanked by two globular domains at the N and C termini. The N-terminal globular domain is larger than the C-terminal domain and consists almost exclusively of the ␣ 3 (VI) chain, which has nearly twice the mass of the ␣ 1 (VI) and ␣ 2 (VI) chains (1, 2). Collagen VI monomers assemble intracellularly into disulfide-bonded tetramers that are secreted into the extracellular matrix (3). There they assemble in a characteristic end-to-end fashion into thin (3-10 nm) beaded filaments as well as hexagonal networks (4 -6).Collagen VI has been identified in microfibrillar assemblies in the extracellular matrix of connective tissues as well as in netlike structures, broad banded "zebra" fibrils with a periodicity of 100 nm and as hexagonal networks (4, 7). When collagen VI is isolated by pepsin digestion, most of the N-and C-terminal domains are cleaved off, leaving the triple helical region with minor parts of the globular domains of the collagen VI molecules, which is still sufficient for forming the tetramer (6, 8).Collagen VI is present in most tissues. It is enriched close to cells and around basement membranes. It is associated with structures like blood vessels and nerves and found interspersed among interstitial collagen fibers (for reviews see Refs. 2 and 9). In Bethlem myopathy, a dominantly inherited disorder characterized by muscle weakness and wasting, decreased presence of secreted collagen VI due to mutations in COL6A1 has been r...

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to differentiate into various different cell lineages and can generate bone, cartilage and adipose tissue. MSCs are presently characterized using a broad range of different cell-surface markers that are not exclusive to MSCs and not sensitive to culture conditions or differentiation capacity. We show that the integrin subunits alpha10 and alpha11 of the collagen binding integrins alpha10beta1 and alpha11beta1 are expressed by human MSCs in monolayer cultures. We also demonstrate that the expression of alpha10 increases, while alpha1 and alpha11 decrease, during aggregate culture of MSCs in chondrogenic medium. Alpha10beta1 is expressed by chondrocytes in cartilage, whereas alpha11beta1 integrins are predominantly expressed by subsets of the fibroblastic lineage. In extensive monolayer cultures of MSCs, alpha10 expression is down-regulated. We show that this down-regulation is reversed by fibroblast growth factor-2 (FGF-2) treatment. Addition of FGF-2 to MSCs not only results in increased alpha10 expression, but also in decreased alpha11 expression. FGF-2 treatment of MSCs has been shown to keep the cells more multipotent and also induces cell proliferation and Sox-9 up-regulation. We demonstrate improved chondrogenecity as well as increased collagen-dependant migratory potential of FGF-2-treated MSCs having a high alpha10 expression. We also demonstrate expression of alpha10 and alpha11 integrin subunits in the endosteum and periosteum of mice, but very low or not detectable expression levels in freshly aspired human or mouse BM. We show that MSCs with high chondrogenic differentiation potential are highly alpha10 positive and propose alpha10 as a potential marker to predict the differentiation state of MSCs.

Aim: To investigate whether severe loss of activities of daily living (ADL) in asylum-seeking children is associated with physical disease or toxic influences and to describe the clinical course during the recovery process. Methods: A total of 29 asylum-seeking children with severe loss of ADL were regularly assessed by physical examinations, laboratory tests and a structured evaluation of their ADL status during rehabilitation. Results: A total of 12 children had previously recorded suicide attempts and 21 were recorded to have experienced traumatic events in their country of origin.The mean time from turning point to recovery was 6 months. Of the study participants, 22needed enteral feeding and 18 gained weight during recovery. All children had a pulse rate and systolic blood pressure within the normal range. No sign of intoxication or physical disease was identified in laboratory tests or clinical examinations, with the exception of one case of epilepsy.Conclusion: Physical disease, pharmacological sedation or anorexia nervosa was not considered to be a probable cause of the loss of ADL in these children. The high rate of psychosocial risk factors and the stressful event of being in an asylum-seeking process call for further investigation of psychosomatic mechanisms.