beta-Integrin-collagen interaction reduces chondrocyte apoptosis.

We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte-matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin beta1 suggest that the collagen-chondrocyte interactions are mediated through integrin beta1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte-matrix network.     

Aggrecan and link protein affect cell adhesion to culture plates and to type II collagen

Cartilage is a hypocellular tissue in which a balance of matrix molecules, especially aggrecan and link protein, play a critical role in maintaining structural integrity. To study the role of aggrecan and link protein in mediating cell activities, we have stably expressed them in NIH/3T3 fibroblasts and observed the effect on cell-substratum interactions. Overexpression of either protein destabilized the cell-substratum interaction. However, when both were co-expressed, the interaction between cell and substratum was less impaired. Similar results were obtained on type II collagen-coated plates. The addition of exogenous gene products into fibroblast cell lines and chondrocyte culture had the same effect as expression of the genes. The addition of exogenous hyaluronan to the growth medium or treatment of cells with hyaluronidase also decreased cell adhesion, indicating that hyaluronan also plays a role in the cell-substratum adhesion. The presence of aggrecan seems to increase the amount of link protein on the cell surface. Chondrocytes expressing high concentrations of aggrecan and link protein were maintained within a matrix network and were able to survive in suspended culture. Imbalances in aggrecan or link protein concentrations, or degradation of hyaluronan, disrupted the network and caused the chondrocytes to aggregate or adhere to the plates.     

Structure and function of aggrecan

INTRODUCTIONDegenerative joint disease is a leading sourceof morbidity resulting in significant social and eco-nomic impact. One to 5% of the population underthe age of 45 and 15-85% of oIder individuals sufferfrom some fOrm of degenerative joint disease, mainlyosteoarthritis. Osteoarthritis is characterized by theslow progressive deterioration of articular carti-1age[1, 2]. Current therapeutic regimens addressmainly pain but not degeneration. A better under-standing of the distinct micr…     

A neocartilage ideal for extracellular matrix macromolecule immunolocalization

A neocartilage construct readily amenable to microscopy and biomechanical studies is described. Porcine articular cartilage was digested with a mixture of dispase and collagenase for chondrons or pronase and collagenase for chondrocytes. Chondrons or chondrocytes plated in 96-well plates were fixed and immunolabeled in situ for fluorescence microscopy at days 4 and 11. Collagen types I and II, aggrecan, and MMP-13 expression was assayed by semiquantitative RT-PCR. Cell numbers were analyzed by MTT assay. Chondrons and chondrocytes produced neocartilage that could be handled with minimal tearing on day 3 and none on day 11. Some cell division occurred between days 4 and 7. In both cultures, chondrocytes were surrounded by a thin rim of type VI collagen and osteopontin. Type II collagen, keratan sulfate, and tenascin were abundant throughout. At day 3, cells were rounded but by day 11 flattened cells were visible in the substratum. Continued synthesis of aggrecan and type II collagen mRNA indicated maintenance of the chondrocyte phenotype. The neocartilage was easy to immunolabel in situ without the need for sectioning, and individual cells were readily observed by microscopy. The versatility of these constructs makes them ideal for microscopy and for biomechanical studies.     

Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix

Initial assembly of extracellular matrix occurs within a zone immediately adjacent to the chondrocyte cell surface termed the cell- associated or pericellular matrix. Assembly within the pericellular matrix compartment requires specific cell-matrix interactions to occur, that are mediated via membrane receptors. The focus of this study is to elucidate the mechanisms of assembly and retention of the cartilage pericellular matrix proteoglycan aggregates important for matrix organization. Assembly of newly synthesized chondrocyte pericellular matrices was inhibited by the addition to hyaluronan hexasaccharides, competitive inhibitors of the binding of hyaluronan to its cell surface receptor. Fully assembled chondrocyte pericellular matrices were displaced using hyaluronan hexasaccharides as well. When exogenous hyaluronan was added to matrix-free chondrocytes in combination with aggrecan, a pericellular matrix equivalent in size to an endogenous matrix formed within 30 min of incubation. Addition of hyaluronan and aggrecan to glutaraldehyde-fixed chondrocytes resulted in matrix assembly comparable to live chondrocytes. These matrices could be inhibited from assembling by the addition of excess hyaluronan hexasaccharides or displaced once assembled by subsequent incubation with hyaluronan hexasaccharides. The results indicate that the aggrecanrich chondrocyte pericellular matrix is not only on a scaffolding of hyaluronan, but actually anchored to the cell surface via the interaction between hyaluronan and hyaluronan receptors.     

Link protein has greater affinity for versican than aggrecan

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.     

Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

Hyaluronidase and CD44 hyaluronan receptor expression in squamous cell laryngeal carcinoma

Squamous cell laryngeal carcinoma undergoes significant structural-related modifications of the extracellular matrix components (ECM), the most characteristics being the presence of degraded collagen, aggrecan and hyaluronan. We examined the presence of hyaluronidase and of the cellular hyaluronan receptor CD44 during the various stages of cancer. ECM components were extracted by using PBS, 4 M GdnHCl and 4 M GdnHCl-0.1% Triton-X 100 sequentially and hyaluronidase and CD44 analyzed by zymography and immunochemistry techniques. Total RNA was also extracted and the mRNA of the various hyaluronidases and of CD44 was analyzed after amplification with RT-PCR. Hyaluronidase was detected as a double band of 45 and 55 kDa molecular mass, only in cancer samples. The analysis of mRNA indicated an aberrant expression of PH-20, the testicular-type hyaluronidase, at late stages of cancer and an overexpression of HYAL1 only at stage IV. In addition, CD44 was identified in two protein bands of 80 and 64 kDa in cancer samples. The analysis of mRNA showed that hyaluronan receptor was expressed in a stage-related order. Thus, it could be suggested that in laryngeal squamous cell carcinoma, cancer cells migrated and proliferated under the influence of small molecular mass hyaluronan, by expressing increased amounts of its receptor.     

Perlecan,the multidomain HS-proteoglycan of basement membranes,is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2- to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cell-associated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation.     

Type VI collagen microfibrils: evidence for a structural association with hyaluronan

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.     

Hyaluronan is essential for the expansion of the cranial base growth plates

Exquisite control of chondrocyte function in the zone of hypertrophy results in expansive growth of cartilaginous growth plates, and is a prerequisite for normal skeletal lengthening. We hypothesize that hyaluronan-mediated hydrostatic pressure causes lacunae expansion in the zone of hypertrophy; an important mechanism in cartilaginous growth plate and associated skeletal expansion. The role of hyaluronan and CD44 in this mechanism was studied using organ culture of the bipolar cranial base synchondroses. Hyaluronan was present in the hypertrophic zones, pericellular to the hypertrophic chondrocytes, while no hyaluronan was detected in the resting, proliferating and maturing zones. This localization of hyaluronan was associated with increased lacunae size, suggesting that chondrocytes deposit and retain pericellular hyaluronan as they mature. In comparison, Toluidine Blue staining was associated with the territorial matrix. Hyaluronidase, the hyaluronan-degrading enzyme, and CD44, the receptor for hyaluronan which also participates in the uptake and degradation of hyaluronan, were co-localized within the zone of ossification. This pattern of expression suggests that cells in the early zone of ossification internalize and degrade hyaluronan through a CD44-mediated mechanism. Treatment of the cultured segments with either Streptomyces hyaluronidase or hyaluronan hexasaccharides inhibited lacunae expansion. These observations demonstrate that hyaluronan-mediated mechanisms play an important role in controlling normal skeletal lengthening.     

Direct quantification of the rupture force of single hyaluronan/hyaluronan binding protein bonds

The non-covalent bond between aggrecan and hyaluronan is critical for maintaining the normal structure and function of the extracellular matrix in articular cartilage. The failure of this bond can cause the loss of aggrecan and destruction of the extracellular matrix of articular cartilage. In this study, the rupture force of the single bond between hyaluronan and hyaluronan binding protein - the complex of the hyaluronan binding region of aggrecan and link protein - was directly measured with a nanomechanical testing system as 40+/-11 pN. The results were compared to a theoretical prediction based on a smart version of the Monte Carlo simulation.     

Hyaluronan digestion and synthesis in an experimental model of metastatic tumour

To approach the question of hyaluronan catabolism in tumours, we have selected the cancer cell line H460M, a highly metastatic cell line in the nude mouse. H460M cells release hyaluronidase in culture media at a high rate of 57 pU/cell/h, without producing hyaluronan. Hyaluronidase was measured in the H460M cell culture medium at the optimum pH 3.8, and was not found above pH 4.5, with the enzyme-linked sorbent assay technique and zymography. Tritiated hyaluronan was digested at pH 3.8 by cells or cell membranes as shown by gel permeation chromatography, but no activity was recorded at pH 7 with this technique. Hyaluronan was digested in culture medium by tumour slices, prepared from tumours developed in nude mice grafted with H460M cells, showing that hyaluronan could be digested in complex tissue at physiological pH. Culture of tumour slices with tritiated acetate resulted in the accumulation within 2 days of radioactive macromolecules in the culture medium. The radioactive macromolecular material was mostly digested by Streptomyces hyaluronidase, showing that hyaluronan was its main component and that hyaluronan synthesis occurred together with its digestion. These results demonstrate that the membrane-associated hyaluronidase of H460M cells can act in vivo, and that hyaluronan, which is synthesised by the tumour stroma, can be made soluble and reduced to a smaller size by tumour cells before being internalised and further digested.     

The effect of link peptide on proteoglycan synthesis in equine articular cartilage

The basal rate of in vitro proteoglycan (PG) synthesis in explants of equine articular cartilage was subject to considerable variation in animals of the same age but was greater in younger than older animals. Synthesis of PGs in explant cultures was stimulated by a synthetic link peptide, identical in sequence to the N-terminus of the link protein (LP) of PG aggregates, in a similar manner to that demonstrated previously for human articular cartilage [Biochem. Soc. Trans. 25 (1997) 427; Arthritis Rheum. 41 (1998) 157]. Stimulation occurred in tissue from animals ranging from 1 to 30 years old but older animals required higher concentrations of peptide to produce a measurable response. Synthesis of PGs increased in a concentration-dependent manner and was paralleled by increases in the ability of aggrecan monomers to form aggregates with hyaluronan (HA). In addition to its effect on synthesis of PGs, link peptide also increased synthesis of both aggrecan and LP mRNA. Cartilage explant and chondrocyte cultures secreted small amounts of biologically active interleukin 1 (IL 1) and secretion of this cytokine was reduced considerably by the addition of link peptide. Reduction in the activity of this catabolic cytokine coupled with the increased synthesis of mRNA for aggrecan and link peptide may be the mechanism by which link peptide exerts its positive effect on the rate of PG synthesis in articular cartilage.     

The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain

We report here molecular cloning and expression analysis of the gene for a novel human brain link protein-1 (BRAL1) which is predominantly expressed in brain. The predicted open reading frame of human brain link protein-1 encoded a polypeptide of 340 amino acids containing three protein modules, the immunoglobulin-like fold and proteoglycan tandem repeat 1 and 2 domains, with an estimated mass of 38 kDa. The brain link protein-1 mRNA was exclusively present in brain. When analyzed during mouse development, it was detected solely in the adult brain. Concomitant expression pattern of mRNAs for brain link protein-1 and various lectican proteoglycans in brain suggests a possibility that brain link protein-1 functions to stabilize the binding between hyaluronan and brevican. The human BRAL1 gene contained 7 exons and spanned approximately 6 kb. The entire immunoglobulin-like fold was encoded by a single exon and the proteoglycan tandem repeat 1 and 2 domains were encoded by a single and two exons, respectively. The deduced amino acid sequence of human brain link protein-1 exhibited 45% identity with human cartilage link protein-1 (CRTL1), previously reported as link protein to stabilize aggregates of aggrecan and hyaluronan in cartilage. These results suggest that brain link protein-1 may have distinct function from cartilage link protein-1 and play specific roles, especially in the adult brain.     

Mammalian vitreous humor contains networks of hyaluronan molecules: electron microscopic analysis using the hyaluronan-binding region (G1) of aggrecan and link protein.

Vitreous humor from human, bovine, and chicken eyes was analyzed by rotary shadowing to characterize further the supramolecular organization of the gel-like matrix which forms this tissue. Extensive filamentous networks, distinct from collagen fibrils, were found in both human and bovine vitreous but not in chicken vitreous. The networks consisted of branching structures of various diameters, due to variable numbers of hyaluronan molecules being laterally associated with each other and apparently giving rise to a three-dimensional lattice. These networks could be decorated in a specific and regular manner by the hyaluronan-binding region called G1 purified from bovine nasal septum cartilage. The extent of decoration of hyaluronan was dependent on the relative concentration of G1. In the presence of an excess of G1 the networks were destabilized giving rise to individual unbranched hyaluronan chains of varying length that were saturated with G1. One or more globular proteins, as yet uncharacterized, were seen interacting with the hyaluronan networks, often at branch points. These proteins may serve to stabilize the three-dimensional structure of the matrix although highly ordered networks were also observed without globular proteins. Link protein, which also binds to hyaluronan, bound to the networks in a fashion clearly distinct from G1. Neither G1 nor link protein bound directly to human or bovine vitreous collagen fibrils. However, link protein did bind extensively to the glycosaminoglycan coat of chicken vitreous collagen fibrils described previously (D. W. Wright, and R. Mayne J. Ultrastruct. Mol. Struct. Res. 100, 224-234, 1988), while G1 did not. Digestion of the chicken vitreous collagen fibrils with Streptomyces hyaluronidase did not result in the removal of the glycosaminoglycan coat of the collagen fibrils nor did it affect the binding of G1 or link protein to the fibrils, indicating that hyaluronan is not a component of this structure. These studies demonstrate that proteins with specific binding properties can be used as probes to investigate the structure of the native vitreous humor gel from several species and suggest that this method potentially can be used for structural studies of other connective tissue matrices.