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Biomedical subjects

F Ruggiero

Publications and source records attributed to F Ruggiero.

At least 19 recordsLinked to original sources
PubMed1999

Chronic hepatitis C virus infections in leukemia survivors: prevalence, viral load, and severity of liver disease.

The natural history of chronic hepatitis C (HCV) infections in long-term leukemia survivors has not been well characterized. We studied the prevalence of HCV infections, measured HCV RNA levels, and evaluated the severity of liver disease in patients with leukemia who achieved long-term remissions after intensive chemotherapy or bone marrow transplantation (BMT). HCV antibody tests were performed by the enzyme-linked immunosorbent assay (ELISA) and positive tests confirmed by the recombinant immunoblot assay (RIBA). HCV RNA levels were measured by the branched DNA (bDNA) assay. Seventy-five leukemia survivors with 25 or more blood donor exposures were identified. Nine (12%) were anti-HCV positive. All were infected before 1992 when second generation HCV screening tests were implemented. Mean HCV RNA levels were 10.3 x10(6) eq/mL versus 3.2 x 10(6) eq/mL (P =.056) in a control group of 20 anti-HCV positive immunocompetent individuals of comparable age who were infected twice as long (17.8 +/- 6.5 years v 9.0 +/- 4.4 years in leukemia survivors, P =.001). Liver biopsies were performed on six of the nine anti-HCV positive leukemia survivors. All showed at least moderate portal inflammation and half had evidence of bridging fibrosis. We conclude that viral loads in anti-HCV positive leukemia survivors are markedly higher than in immunocompetent controls. Our results suggest that long-term leukemia survivors with chronic HCV may have more rapidly progressive liver disease than has been previously recognized.

Adult↗
PubMed1998

Molecular features of the collagen V heparin binding site.

A heparin binding region is known to be present within the triple helical part of the alpha1(V) chain. Here we show that a recombinant alpha1(V) fragment (Ile824 to Pro950), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual alpha1(V) chains and HepV are eluted at identical NaCl concentrations (0.35 M) from a heparin-Sepharose column, and this binding can be inhibited specifically by the addition of free heparin or heparan sulfate. In contrast, a shorter 23-residue synthetic peptide, containing the putative heparin binding site in HepV, fails to bind heparin. Interestingly, HepV promotes cell attachment, and HepV-mediated adhesion is inhibited specifically by heparin or heparan sulfate, indicating that this region might behave as an adhesive binding site. The same site is equally functional on triple helical molecules as shown by heparin-gold labeling. However, the affinities for heparin of each of the collagen V molecular forms tested are different and increase with the number of alpha1(V) chains incorporated in the molecules. Molecular modeling of a sequence encompassing the putative HepV binding sequence region shows that all of the basic residues cluster on one side of the helical face. A highly positively charged ring around the molecule is thus particularly evident for the alpha1(V) homotrimer. This could strengthen its interaction with the anionic heparin molecules. We propose that a single heparin binding site is involved in heparin-related glycosaminoglycans-collagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues.

Amino Acid Sequence↗
PubMed1997

Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing.

Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human alpha1 chain of collagen V using an episomal vector. High yields (15 microgram/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the alpha1(V) collagen is correctly folded into a stable triple helix as shown by electron microscopy and pepsin resistance. Circular dichroism data confirm the triple-helix conformation and indicate a melting temperature of 37.5 degrees C for the recombinant homotrimer. The major secreted form is a 250-kDa polypeptide (alpha1FL). N-terminal sequencing and collagenase digestion indicate that alpha1FL retains the complete N-propeptide but lacks the C-propeptide. However, alpha1FL might undergo a further N-terminal trimming into a form (alpha1TH) corresponding to the main triple-helix domain plus the major part of the NC2 domain. This processing is different from the one of the heterotrimeric (alpha1(V))2alpha2(V) and could have some physiological relevance. Analysis of cell homogenates indicates the presence of a 280-kDa polypeptide that is disulfide-linked through its C-terminal globular domain. This C-propeptide is rapidly cleaved after secretion in the medium, giving the first evidence of a C-terminal processing of recombinant fibrillar collagens. Rotary shadowing observations not only confirm the presence of a globular domain at the N-terminal end of the molecule but reveal the presence of a kink within the triple helix in a region poor in iminoacids. This region could represent a target for proteases. Together with the thermal stability data, these results might explain the low amount of (alpha1(V))3 recovered from tissues.

Ascorbic Acid↗
PubMed1997

Laminin 5 binds the NC-1 domain of type VII collagen.

Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

Antigens, Surface↗
PubMed1997

The membrane-spanning proteoglycan NG2 binds to collagens V and VI through the central nonglobular domain of its core protein.

NG2 is a membrane-spanning proteoglycan with a primary structure unique among cell surface or extracellular matrix proteins. To characterize the interaction between NG2 and extracellular matrix proteins, we have used a eukaryotic expression system to produce and purify several recombinant fragments covering not only the entire ectodomain of NG2 but also distinct subdomains of the molecule. Using a solid phase binding assay with various extracellular matrix proteins, we have identified two main ligands for NG2, namely, collagens V and VI. Consistent with previous models of glycosaminoglycan attachment, roughly 50% of the recombinant NG2 fragments containing the central domain have chondroitin sulfate chains attached to the protein core. These glycosaminoglycan chains are not directly involved in collagen binding, since chondroitinase-treated fragments exhibit an unimpaired ability to bind to both collagens. Using more restricted recombinant fragments of NG2, we mapped the binding site for both collagens to the central domain of NG2. Electron microscopy after rotary shadowing of native NG2 molecules indicates that this extended nonglobular domain provides a flexible connection joining the two N- and C-terminal globular regions of NG2. Rotary shadowing of mixtures of NG2 and collagen V or VI confirms a direct interaction between the molecules and indicates that the collagens align with the central region of NG2, giving the appearance of a rod between the N- and C-terminal globules.

Antigens↗
PubMed1996

Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V.

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.

Animals↗
PubMed1996

Human corneal fibrillogenesis. Collagen V structural analysis and fibrillar assembly by stromal fibroblasts in culture.

PURPOSE: The stroma of the developing cornea is a highly organized extracellular matrix formed essentially by uniform, small-diameter collagen fibrils with constant interfibrillar spacing. Unlike the fibrillogenesis of chicken cornea, the assembly and maturation of human corneal fibrils have been poorly investigated. In the current study, the authors aimed to ascertain the heterotypic organization (collagens I and V) of the human corneal fibrils at the supramolecular level. To gain more insight into the molecular structure of collagen V, its cellular source, and its role in fibrillogenesis, the authors used cultured human corneal fibroblasts. METHODS: The structure of human corneal stroma after brief homogenization of the tissue was analyzed by immunogold labeling using specific polyclonal antibodies and rotary shadowing. Biochemical, electron microscopic, and immunolabeling approaches were used to investigate the collagen fibril formation and the extracellular matrix synthesis using human corneal fibroblasts grown in culture as a model system. RESULTS: The authors showed that in human corneal stroma, collagen I is distributed uniformly along the striated fibrils, in contrast to collagen V, which could be identified only at sites at which the fibrils partially were disrupted. Rotary shadowing observations of the homogenate revealed that collagen VI, a major component of the human cornea, was associated closely with the collagen fibril surface. Corneal fibroblasts synthesize and deposit a collagenous matrix with fibrils resembling those of the human cornea in appearance and collagen composition. Biochemical data indicate that a high concentration (20% to 30%) of collagen V is synthesized by stromal fibroblasts and that collagen V molecules are processed similarly to matrix forms in which the extension peptides are retained on the molecules. CONCLUSIONS: The heterotypic nature (collagens I and V) of human corneal fibrils was determined. Results indicate that human corneal fibroblasts synthesize the major collagen types in human cornea (collagens I, V, and VI) and express all the posttranslational equipment for correct collagen molecular assembly and processing in a manner that closely resembles the situation in situ, offering the opportunity for more detailed study of this process, which is essential for optical transparency.

Adult↗
PubMed1995

Analysis of collagen synthesis and assembly in culture by immortalized mouse chondrocytes in the presence or absence of alpha 1(IX) collagen chains.

We have previously shown that SV40 large T oncogene is able to induce mouse chondrocyte proliferation without loss of expression of types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. The cell line obtained (termed MC 615) also expressed some type I collagen in monolayer and we have investigated if anchorage-independent conditions could inhibit type I collagen synthesis and promote hypertrophy and type X collagen synthesis. The MC 615 cells were grown in agarose in the presence of serum, and GAG accumulation, DNA content, and matrix synthesis rates were monitored after incubation with [35S]sulfate and [3H]- or [14C]proline. SDS-PAGE analysis of pepsin-extracted samples showed that type I collagen was still synthesized by the MC 615 cells, from the beginning of the culture and at low or high density. Type II collagen synthesis was demonstrated by immunoblotting, but type X collagen synthesis was not detected, indicating that the MC 615 chondrocytes immortalized by large T were still blocked in their maturation pathway. The cells were also grown over agarose and electron microscopy (E. M.) analysis of the cell aggregates showed an extracellular matrix rich in proteoglycans and in type II-containing collagen fibrils. To gain insight into the role of type IX collagen in cartilage collagen assembly and/or matrix organization, we also immortalized embryonic chondrocytes isolated from mice lacking alpha 1 (IX) collagen and obtained a clone termed 4KO 91. As found for the MC 615 cells, the 4KO 91 cells synthesized type II collagen as demonstrated by Western blotting and some type I collagen identified by the presence of alpha 2(I) chains after electrophoretic analysis of pepsin-digested collagen chains. E. M. analysis of the extracellular matrices synthesized by the two cell lines revealed differences in collagen structure and organization. In the absence of alpha 1 (IX) collagen chains, the collagen fibrils seemed to fuse laterally, suggesting that collagen IX acts as a "spacer" between fibrils, to keep them apart.

Animals↗
PubMed1995

Questioning the clinical significance of upper gastrointestinal cytomegalovirus disease following heart transplantation.

We performed a retrospective review of patients who underwent esophagogastroduodenoscopy after heart transplantation to determine the clinical setting in which upper gastrointestinal cytomegalovirus disease is identified. No gastrointestinal cytomegalovirus disease was found prior to transplant 51 and this period (from transplant 1 to 50) correspond to a time when significantly fewer esophagogastroduodenoscopies included biopsy. Patients in whom cytomegalovirus was identified were more likely to have been CMV seronegative and to have received a heart from a seropositive donor (60% vs 20%, P = 0.029). In addition, patients with cytomegalovirus used aspirin more commonly (90% vs 31%, P = 0.001), and underwent esophagogastroduodenoscopy earlier after transplantation (123d vs 652d, P = 0.029). We conclude that factors that increase the use of esophagogastroduodenoscopy and biopsy in the early transplant period increase the likelihood of identifying cytomegalovirus in gastrointestinal tissue. However, the clinical course and significance of cytomegalovirus identified in the upper gastrointestinal tract in heart transplant patients may be difficult to discern.

Antibodies, Viral↗
PubMed1995

Another look at collagen V and XI molecules.

The fibrillar collagens are the most abundant proteins of extracellular matrices. Among them, collagens V and XI are quantitatively minor components which participate in the formation of the fibrillar collagen network. Since these collagens were discovered, studies have demonstrated that they may play a fundamental role in the control of fibrillogenesis, probably by forming a core within the fibrils. Another characteristic of these collagens is the partial retention of their N-propeptide extensions in tissue forms, an unusual observation in comparison to the other known fibrillar collagens. The tissue locations of collagens V and XI are different, but their structural and biological properties seem to be closely related. It has been shown that their primary structures are highly conserved at both the gene and protein levels, and that these conserved features are the bases of their similar biological properties. In particular, they are both resistant to mammalian collagenases, and surprisingly sensitive to trypsin treatment. Collagens V and XI are usually buried within the major collagen fibrils, although they have both cell adhesion and heparin binding sites which could be of crucial importance in physiological processes such as development and wound healing. It has became evident that several molecules are in fact heterotypic associations of chains from both collagens V and XI, demonstrating that these two collagens are not distinct types but a single type which can be called collagen V/XI.

Animals↗
PubMed1994

Interactions between cells and collagen V molecules or single chains involve distinct mechanisms.

Acid-soluble and pepsin-treated collagen V were prepared from fetal human bones or human placenta, respectively, to be tested for potential cell adhesion promoting activity. Out of 14 different collagen I-adhering cell lines, 10 showed distinct adhesion to collagen V. In all cases adhesion was followed by spreading. The activities of intact and pepsin-solubilized collagen V were similar, suggesting that the cell binding sites are restricted to the triple-helical domain of the molecules. Cell adhesion was also induced by the unfolded form of collagen V and after separation of the alpha chains by heparin affinity chromatography. Isolated alpha 2(V) chains, rich in RGD sequences, were more efficient than isolated alpha 1(V) chains. However, cell adhesion to native or denatured collagen V did not proceed by the same molecular mechanisms as shown by cell adhesion inhibition experiments. Cell adhesion to native collagen V was insensitive to the presence of RGD-containing synthetic peptides while adhesion to denatured collagen V was inhibited by the peptides. Furthermore, the results strongly suggested a major role for alpha 1 beta 1 and alpha 2 beta 1 integrins in the RGD-independent cell adhesion to native collagen V. These data indicate that collagen V is a specific adhesive substrate for different cell types. It also suggests that distinct sets of RGD-dependent and RGD-independent receptors mediate cell attachment to unfolded and native collagen V, respectively. This mechanism is shared by at least the interstitial collagens I and VI, which supports the hypothesis that when included in the triple-helical conformation of collagens, RGD sequences are either not accessible to cells or exhibit specific conformations recognized by different integrins.

Actins↗
PubMed1994

Identification and characterisation of a novel repetitive antigen from Onchocerca spp.

A novel repetitive antigen from the cattle parasite Onchocerca gibsoni was shown to be recognised by sera from humans infected with Onchocerca volvulus, Wuchereria bancroftii or Brugia malayi. The O. gibsoni protein was produced in a recombinant form, and antibodies raised to this protein used to screen cDNA libraries for O. volvulus. A series of clones were isolated which encoded repetitive regions very similar to those in O. gibsoni, but interspersed between these were longer repeating units which we have not so far found in O. gibsoni. The repetitive antigen was shown to be of high molecular weight and present only in the insoluble (membrane) fraction of O. gibsoni microfilariae. Immunofluorescence techniques demonstrated that the antigen was associated both with muscle and with specific membrane layers, including a peripheral layer which corresponds to either the outer hypodermis or an inner region of the cuticle in adult female O. gibsoni. In many respects, the proteins encoded by the O. gibsoni and O. volvulus cDNA clones resembled repetitive antigens from several distantly related eukaryotic parasites, and a possible common role in immune evasion is discussed.

Amino Acid Sequence↗
PubMed1993

Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density.

Fetal bovine chondrocytes isolated from the resting zone of epiphyseal cartilage were maintained in high-density culture for 4 weeks. From Day 2 in culture, the chondrocytes deposited an extracellular matrix composed of Types II, IX, and XI collagen. Types IX and XI collagen were restricted to the pericellular domain from Day 5. By 2 weeks the entire cell layer stained for antibodies to Type II and IX collagens. Type XI could be demonstrated throughout the cell layer by pepsinization of the sections. Results from both rotary shadowing and immunochemistry showed that the fibrils formed in culture were heterotypic, with Type IX collagen arranged along the surface and with Type XI collagen buried in Type II fibrils. Nonspecific Type VI collagen and the glycoproteins tenascin and fibrillin, previously described in cartilaginous tissue, were identified by their ultrastructural characteristics in the cell layer homogenate. Although the cells presented morphological characteristics of chondrocytes and still expressed cartilage-specific collagens, the appearance of Type I collagen in the culture cell layer after 4 weeks of culture demonstrates a partial dedifferentiation of the chondrocytes. The culture system described in this report provides an interesting tool for maintaining chondrocytes in a cartilage-like matrix to study the influence of different physical and chemical factors on the expression and differentiation of the cells.

Animals↗
PubMed1991

Structure of the basement membrane of corneal epithelium: quick-freeze, deep-etch comparative study of networks deposited in culture and during development.

The basement membranes elaborated by corneal epithelium in the chick embryo and in culture conditions have been studied by quick-freezing and deep-etching methods. Electron microscope observations of en face unidirectional platinum shadow castings revealed a polygonal network comparable to the type IV collagen network described in human amniotic basement membrane and EHS mouse tumor matrix. The material deposited in culture contained type type IV collagen, as demonstrated by immunofluorescence labeling using anti-type IV collagen antibodies and formed delicate networks. Fine filaments and granules composing this network were interpreted respectively as linear and globular NC1 domains of the type IV collagen molecule. These loose networks were considered as first steps in basement membrane assembly. Staggered superimposition of comparable networks could lead to the dense network organization as observed for basement membranes in situ. These observations showed that the basement membrane of the chick embryo corneal epithelium is also organized in a complex polygonal framework that is preserved even when secreted in culture conditions.

Animals↗
PubMed1990

Extracellular matrix production by embryonic epithelium cultured on type IV collagen. Deposition of a primary corneal stroma-like structure containing large irregular type I fibrils without type II collagen.

The corneal stroma of the chick embryo is deposited in two steps. The primary stroma is laid down by the corneal epithelium and it contains type I, type II and type IX collagens. Its formation is subsequent to the presumptive epithelial cells' migration onto the lens capsule (which is rich in type IV collagen). The secondary, ultimate stroma is synthesized by fibroblasts which, on day 5 of development, invade the swollen primary stroma. It is composed of a matrix of thin (25 nm), regular fibrils containing type I and type V collagens. We found that a chick corneal epithelium isolated from either a 6-day or a 14-day embryo was able to produce, in vitro, stroma-containing type I collagen fibrils. However, the amount of collagen deposited and its organization were highly dependent on the substratum used. Plastic or purified bovine type I collagen substrata led to the release of very few fibrils. Purified human type IV collagen induced the production of an abundant matrix made of large irregular collagen fibrils. When compared to native corneal stroma, there were two aspects in which this matrix differed: (1) it contained only type I collagen, as shown by indirect immunofluorescence, and (2) there were numerous large, irregular fibrils of about 100 to 130 nm in diameter. In conclusion, it is suggested that purified type IV collagen substitutes, in part, for the basement membrane and allows the production of a corneal stroma-like matrix by an embryonic corneal epithelium in culture. This production is possible even with a 14-day epithelium which, in vivo, is no more involved in the synthesis of the stroma collagens. Moreover, the regulatory effect of type II collagen, previously suggested by in vivo observations, may be confirmed in this in vitro system by the appearance of large fibrils in the newly deposited stroma that are made only by type I collagen.

Animals↗
PubMed1990

Proteoglycan core protein and type II collagen gene expressions are not correlated with cell shape changes during low density chondrocyte cultures.

Chondrocytes isolated from chicken embryo sterna were cultivated in low density monolayer cultures to induce their dedifferentiation. At different stages of the long-term cultures, changes in expression of a cartilage-specific sulfated proteoglycan and cartilage-characteristic type II collagen have been examined and related to the shape change of cells using in situ hybridization and immunocytochemistry. At the beginning of the culture, all cells exhibit a round shape and express the cartilage phenotype. Then, during the course of the culture, chondrocytes flatten and become fibroblast-like, but this morphological modification does not start for all the cells at the same time. Interestingly, the loss of cartilage proteoglycan or type II collagen expression did not occur for all polygonal or fibroblast-like cells. Moreover, we observed a variability in the steady state levels of RNA or protein accumulation among chondrocytes exhibiting a similar shape, as judged by the intensity of hybridization signal or immunofluorescence over the cells. These observations support the hypothesis that the shape change does not have a causative role in the chondrocyte phenotype expression, but is rather a secondary effect of the dedifferentiation process. Furthermore, the disappearance of hybridizable core protein or type II collagen mRNA during the dedifferentiation process was coincident with the disappearance of the proteins for which they code as detected by immunohistochemical staining. This suggest that core protein and type II collagen gene expressions are controlled primarily at the transcriptional level in long-term chondrocyte cultures.

Aggrecans↗