The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiological functions. The most abundant family is the collagens with more than 20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramolecular processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutical use of members of this protein family discussed in other chapters of this issue. D

Objective. Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo.Methods. During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology.Results. The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content.Conclusion. An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.

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BMP = bone morphogenetic protein; COL2A = type IIA procollagen; COL2B = type IIB procollagen; FGF = fibroblast growth factor; IGF = insulinlike growth factor; IL = interleukin; MMP = matrix metalloproteinase; NO = nitric oxide; NOS = nitric oxide synthase; OA = osteoarthritis; TGF = transforming growth factor; TIMP = tissue inhibitor of metalloproteinases; TNF = tumor necrosis factor. Available online http://arthritis-research.com/content/3/2/107 Introduction Osteoarthritis (OA) involves the entire synovial joint, encompassing the cartilage, synovium, and underlying bone. The cells in each of these tissues have independent capacities to initiate and respond to injury in the joint, ultimately resulting in degeneration of cartilage. It is generally believed that degeneration of cartilage in OA is characterized by two phases: a biosynthetic phase, during which the cells resident in cartilage, the chondrocytes, attempt to repair the damaged extracellular matrix; and a degradative phase, in which the activity of enzymes produced by the chondrocytes digests the matrix, matrix synthesis is inhibited, and the consequent erosion of the cartilage is accelerated [1][2][3][4]. New techniques of molecular biology have provided invaluable insights into the function of cells during the onset and perpetuation of OA. Analysis of mRNA levels in cartilage chondrocytes remaining even at joint replacement provided a surprise: the cells are not metabolically inert, but are actively synthesizing cartilage proteins. The proteins synthesized by OA chondrocytes are structural and functional macromolecules, and degradative enzymes. In addition, the areas of cellular activity and inactivity are now known to be regional. Unfortunately, at some point the biosynthetic anabolic activity is unable to keep pace with the degradative catabolic activity, and degeneration of the tissue results. Influences of cytokines and growth factorsIn normal adult cartilage, chondrocytes synthesize matrix components very slowly. During development, however, biosynthesis is stimulated by a variety of anabolic AbstractThe reaction patterns of chondrocytes in osteoarthritis can be summarized in five categories:(1) proliferation and cell death (apoptosis); changes in (2) synthetic activity and (3) degradation; (4) phenotypic modulation of the articular chondrocytes; and (5) formation of osteophytes. In osteoarthritis, the primary responses are reinitiation of synthesis of cartilage macromolecules, the initiation of synthesis of types IIA and III procollagens as markers of a more primitive phenotype, and synthesis of active proteolytic enzymes. Reversion to a fibroblast-like phenotype, known as 'dedifferentiation', does not appear to be an important component. Proliferation plays a role in forming characteristic chondrocyte clusters near the surface, while apoptosis probably occurs primarily in the calcified cartilage.

Objective. Osteoarthritic (OA) cartilage destruction depends on collagen-and aggrecan-degrading proteases such as collagenases (MMP-1 and MMP-13), stromelysin (MMP-3), MMP-14, as well as the so-called aggrecanases (ADAM-TS4 and ADAM-TS5). In this study, we tried to clarify whether these proteases are expressed in vivo in human normal and OA cartilage (and whether they are up-regulated or down-regulated during the disease process) and in interleukin-1␤ (IL-1␤)-stimulated chondrocytes in vitro.Methods. Quantitative polymerase chain reaction assays were developed and performed on RNA isolated directly from normal and degenerative cartilage tissue as well as from primary human articular chondrocytes cultured with and without IL-1␤.Results. In vivo, MMP-1 was detectable only at very low levels in any condition. MMP-13 expression was low in normal and early degenerative cartilage but was strongly up-regulated in late-stage OA specimens. MMP-1 and MMP-13 were expressed much higher in vitro than in vivo and were up-regulated by IL-1␤. Among all proteases, MMP-3 was by far the most strongly expressed, although it was strongly downregulated in late-stage OA specimens. Expression of MMP-3 was higher in vitro than in vivo and was up-regulated by IL-1␤. ADAM-TS5 and MMP-14 were expressed in all sample groups. Expression of ADAM-TS4 was very low in vivo and was induced in vitro after stimulation by IL-1␤.Conclusion. Our expression data clearly support MMP-13 as the major collagenase in OA cartilage. The most strongly expressed aggrecanase was ADAM-TS5. ADAM-TS4 was expressed only at a very low level in normal cartilage and was only slightly up-regulated in OA cartilage, casting doubt on this enzyme being the relevant aggrecanase of articular cartilage. Results of our study show that expression of many enzymes is significantly different in vitro and in vivo and suggest that IL-1␤ stimulation of articular chondrocytes might not be a good model for the matrix catabolism in OA cartilage.Osteoarthritic (OA) cartilage degeneration as well as cartilage destruction in rheumatoid arthritis depend, at least to a significant degree, on enzymatic degradation of matrix components. Two types of molecules, collagen fibrils and the proteoglycan aggrecan, represent the major targets in terms of enzymatic activity and functional loss of the cartilage matrix as a result of damage to these molecules. Whereas degradation of collagen fibrils leads to matrix instability with tissue swelling, degradation of proteoglycans leads to cartilage softening and loss of fixed charges (1), both of which are classic features of cartilage destruction.Catabolism of both types of molecules is supposed to involve different types of enzymes, largely from 2 protease families: matrix metalloproteinases (MMPs) and the "A disintegrin and metalloproteinases with thrombospondin type 1 motif" (ADAM-TS). The initial degradation of collagen fibrils (within the triple-helical region) depends on cleavage at the collagenase site, for which there exist 2 major candidate enzymes...

Congenital nephrotic syndrome (CNS) is clinically and genetically heterogeneous, with mutations in WT1, NPHS1 and NPHS2 accounting for part of cases. We recently delineated a new autosomal recessive entity comprising CNS with diffuse mesangial sclerosis and distinct ocular anomalies with microcoria as the leading clinical feature (Pierson syndrome). On the basis of homozygosity mapping to markers on chromosome 3p14-p22, we identified homozygous or compound heterozygous mutations of LAMB2 in patients from five unrelated families. Most disease-associated alleles were truncating mutations. Using immunohistochemistry and western blotting we could demonstrate that the respective LAMB2 mutations lead to loss of laminin beta2 expression in kidney and other tissues studied. Laminin beta2 is known to be abundantly expressed in the glomerular basement membrane (GBM) where it is thought to play a key role in anchoring as well as differentiation of podocyte foot processes. Lamb2 knockout mice were reported to exhibit congenital nephrosis in association with anomalies of retina and neuromuscular junctions. By studying ocular laminin beta2 expression in unaffected controls, we detected the strongest expression in the intraocular muscles corresponding well to the characteristic hypoplasia of ciliary and pupillary muscles observed in patients. Moreover, we present first clinical evidence of severe impairment of vision and neurodevelopment due to LAMB2 defects. Our current data suggest that human laminin beta2 deficiency is consistently and specifically associated with this particular oculorenal syndrome. In addition, components of the molecular interface between GBM and podocyte foot processes come in the focus as potential candidates for isolated and syndromic CNS.

This paper is an overview of the in vitro and in vivo studies published on the influence of oxygen and derived reactive species on chondrocyte aging, metabolic function, and the chondrogenic phenotype. It shows, that oxygen and ROS play a crucial role in the control of cartilage homeostasis and that at this time, the exact role of "oxidative stress" in cartilage degradation still remains questionable.

Objective. Despite many research efforts in recent decades, the major pathogenetic mechanisms of osteoarthritis (OA), including gene alterations occurring during OA cartilage degeneration, are poorly understood, and there is no disease-modifying treatment approach. The present study was therefore initiated in order to identify differentially expressed disease-related genes and potential therapeutic targets.Methods. This investigation consisted of a large gene expression profiling study performed based on 78 normal and disease samples, using a custom-made complementary DNA array covering >4,000 genes.Results. Many differentially expressed genes were identified, including the expected up-regulation of anabolic and catabolic matrix genes. In particular, the down-regulation of important oxidative defense genes, i.e., the genes for superoxide dismutases 2 and 3 and glutathione peroxidase 3, was prominent. This indicates that continuous oxidative stress to the cells and the matrix is one major underlying pathogenetic mechanism in OA. Also, genes that are involved in the phenotypic stability of cells, a feature that is greatly reduced in OA cartilage, appeared to be suppressed.Conclusion. Our findings provide a reference data set on gene alterations in OA cartilage and, importantly, indicate major mechanisms underlying central cell biologic alterations that occur during the OA disease process. These results identify molecular targets that can be further investigated in the search for therapeutic interventions.