Connective Tissue Proteoglycans Research Articles

Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines—THP-1 and Mono Mac 6—Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation

Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.

Differential gene expression in skin RNA of horses affected with degenerative suspensory ligament desmitis

BackgroundEquine degenerative suspensory ligament desmitis (DSLD) is a systemic connective tissue disorder first identified in Peruvian Paso horses but afflicting other horse breeds as well. Inappropriate accumulation of proteoglycans in connective tissues, most prominently in tendons and ligaments, leads to progressive and debilitating lameness and pain. It is largely unknown what drives the overproduction of proteoglycans, but our previous studies suggest involvement of bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGFβ) family, impacting synthesis of proteoglycans. To identify potential players in pathogenesis of DSLD a new approach utilizing next generation sequencing was undertaken.MethodsNext generation sequencing was performed using RNA extracted from skin biopsies of six control Peruvian Pasos and six horses with DSLD (4 Peruvian Pasos and 2 warmbloods). The CuffDiff result sets were validated with algorithms used to run them. This was based on the determined false discovery rates derived from the P values adjusted for multiple testing for any given result.ResultsBioinformatics analysis of transcriptomes revealed differential expression of over 1500 genes, including increased expression of genes for several growth factors (most prominently BMP2, FGF5, CTGF, many members of the EGF family), and mediators of signaling (Fos, Myc, MAPK system), and keratins. Two genes encoding for enzymes involved in synthesis of hyaluronan were also overexpressed. Gene expression was decreased for protein cores of many proteoglycans, several growth factors, most collagens, and many peptides with immune function.ConclusionsThe overexpression of BMP2 correlates well with our previous data. However, the decrease in expression of numerous proteoglycans was unexpected. A mutation in a gene of a less characterized proteoglycan and/or glycosyltransferase with subsequent increased production of hyaluronan and/or a proteoglycan(s) undetected in our study could account for the systemic proteoglycan deposition. Decreased collagen gene expression indicates abnormal connective tissue metabolism. The increased expression of keratin genes and FGF5 supports reports of skin abnormalities in DSLD. Underexpression of immune function genes corresponds with lack of inflammation in DSLD tissues. Finally, though the proteoglycan and/or glycosaminoglycan abundant in DSLD has not been identified, we validated our previous data, including overexpression of BMP2, and systemic nature of DSLD due to disturbed metabolism of the extracellular matrix.

Structure of decorin binding protein B from Borrelia burgdorferi and its interactions with glycosaminoglycans

Decorin-binding proteins (DBPs), DBPA and DBPB, are surface lipoproteins on Borrelia burgdorferi, the causative agent of Lyme disease. DBPs bind to the connective tissue proteoglycan decorin and facilitate tissue colonization by the bacterium. Although structural and biochemical properties of DBPA are well understood, little is known about DBPB. In current work, we determined the solution structure of DBPB from strain B31 of B. burgdorferi and characterized its interactions with glycosaminoglycans (GAGs). Our structure shows that DBPB adopts the same topology as DBPA, but possesses a much shorter terminal helix, resulting in a longer unstructured C-terminal tail, which is also rich in basic amino acids. Characterization of DBPB–GAG interactions reveals that, despite similar GAG affinities of DBPA and DBPB, the primary GAG-binding sites in DBPB are different from DBPA. In particular, our results indicate that lysines in the C-terminus of DBPB are vital to DBPB's ability to bind GAGs whereas C-terminal tail for DBPA from strain B31 only plays a minor role in facilitating GAG bindings. Furthermore, the traditional GAG-binding pocket important to DBPA–GAG interactions is only secondary to DBPB's GAG-binding ability.

Molecular and histologic evaluation of idiopathic hyperextension of the metacarpophalangeal and metatarsophalangeal joints in adult llamas

To determine the molecular and histologic characteristics of hyperextension of the metacarpophalangeal and metatarsophalangeal joints in adult llamas. 12 adult llamas (6 with bilateral hyperextension of the metacarpophalangeal or metatarsophalangeal joints [affected] and 6 age- and sex-matched clinically normal control llamas). Llamas were euthanized, and specimens of superficial digital flexor tendon, deep digital flexor tendon, and suspensory ligament were obtained from 4 areas and snap frozen in liquid nitrogen or suspended in neutral-buffered 10% formalin. Histologic evaluation of collagen fiber orientation, elastin content, and proteoglycan content was performed by use of Masson trichrome, picrosirius red, Verhoeff, and Alcian blue stains. Total RNA was isolated from frozen suspensory ligament specimens. Gene expression of collagen types I and III, lysyl oxidase, and matrix metalloproteinase-13 was evaluated with a real-time quantitative reverse transcriptase PCR assay. Gene expression of collagen types I and III, lysyl oxidase, and matrix metalloproteinase-13 in suspensory ligaments was similar between affected and control llamas. Collagen orientation and elastin content of the flexor tendons and suspensory ligaments were also similar between the groups. Proteoglycan content was low in most specimens but was focally increased in discrete lesions of suspensory ligaments in 2 affected and 2 control llamas. Hyperextension of the metacarpophalangeal or metatarsophalangeal joints in llamas did not appear to be caused by degeneration or inflammation of the suspensory ligament. Although focal proteoglycan accumulation existed in the suspensory ligaments of 2 affected llamas, widespread abnormal connective tissue proteoglycan accumulation was not found.

Effects of particulates and lipids on the hydraulic conductivity of Matrigel

The hydraulic conductivity of a connective tissue is determined both by the fine ultrastructure of the extracellular matrix and the effects of larger particles in the interstitial space. In this study, we explored this relationship by examining the effects of 30- or 90-nm-diameter latex nanospheres or low-density lipoproteins (LDL) on the hydraulic conductivity of Matrigel, a basement membrane matrix. The hydraulic conductivity of Matrigel with latex nanospheres or LDL particles added at 4.8% weight fraction was measured and compared with the hydraulic conductivity of Matrigel alone. The LDL-derived lipids in the gel were visualized by transmission electron microscopy and were seen to have aggregated into particles up to 500 nm in size. The addition of these materials to the medium markedly decreased its hydraulic conductivity, with the LDL-derived lipids having a much larger effect than did the latex nanospheres. Debye-Brinkman theory was used to predict the effect of addition of particles to the hydraulic conductivity of the medium. The theoretical predictions matched well with the results from adding latex nanospheres to the medium. However, LDL decreased hydraulic conductivity much more than was predicted by the theory. The validation of the theoretical model for rigid particles embedded in extracellular matrix suggests that it could be used to make predictions about the influence of particulates (e.g., collagen, elastin, cells) on the hydraulic conductivity of the fine filamentous matrix (the proteoglycans) in connective tissues. In addition, the larger-than-predicted effects of lipidlike particles on hydraulic conductivity may magnify the pathology associated with lipid accumulation, such as in Bruch's membrane of the retina during macular degeneration and the blood vessel wall in atherosclerosis.

A novel keratanase‐generated keratan sulfate antibody and its application to structural analysis of skeletal and corneal keratan sulfate

Introduction The objective of this study was to make monoclonal antibodies specific for keratanase‐generated neoepitopes in keratan sulfate (KS) and to use them along with existing KS monoclonal antibodies (e.g. 5D4, IB4) to investigate KS sulfation pattern motifs in connective tissue proteoglycans during development, ageing and disease.Methods Bovine nasal cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of GAG‐peptide fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS peptides from CS attachment domain fragments. Fractions were analysed by Western blotting for positive immunoreactivity for KS, then pooled and keratanase digested to generate ‘KS stub’ antigens. Immunization and fusions were carried out as previously described (Caterson et al. 1983; Hughes et al. 1992). Screenings involved the use of a range of antigens; including keratanase vs. keratanase II‐digested bovine cartilage aggrecan and bovine corneal KS‐PGs. A new monoclonal antibody, BKS‐I, was identified that specifically recognized a keratanase‐generated neoepitope on both skeletal and corneal KS. This novel monoclonal antibody was used along with existing KS monoclonal antibodies 5D4 and 1B4 to investigate KS structure.Results and discussion Bovine trypsin‐generated aggrecan KS‐peptides were chondroitinase ABC treated and either keratanase or keratanase II treated. The digests were run on SDS‐PAGE and immunolocated with monoclonal antibody 5D4 (that recognizes linear disulfated N‐acetyl lactosamine disaccharide‐containing segments in KS) and the new ‘KS‐stub’ monoclonal antibody BKS‐I. Our results indicated that there was reduced monoclonal antibody 5D4 immunostaining after keratanase pretreatment. However, keratanase II digestion completely removed all 5D4 structural epitopes. In contrast, BKS‐I showed no immunostaining on the untreated KS‐peptides but strong staining on keratanase treated samples and no staining after keratanase II digestion. Similar patterns of immunoreactivity were observed with Western blot analysis of untreated, keratanase treated and keratanase II treated corneal KS‐PGs.Conclusion These data indicate that monoclonal antibody BKS‐I recognizes a nonreducing terminal neoepitope‐containing sulfated N‐acetylglucosamine adjacent to a nonsulfated lactosamine disaccharide. We also conclude that skeletal KS must have a structure with four possible variations opposed to the generic structures, proposed as being made of disulfated disaccharides at the nonreducing end, followed by a series of monosulfated disaccharides at the middle and nonsulfated disaccharides nearer the linkage region. 5D4 staining, observed after keratanase digestion, indicates that there must be a minimum structure of a pentasulfated hexasaccharide remaining on the KS chain ‘stubs’ near the linkage region of skeletal and corneal KS. The BKS‐I monoclonal antibody can be used to demonstrate differential substitution of KS GAG chains in the CS attachment region of cartilage aggrecan with ageing. It has also proven useful for immunohistochemical analyses identifying the sites of KS–PG association with collagen lamellae of cornea.

A novel keratanase‐generated keratan sulphate antibody and its applications

Introduction The sequencing of the genome has provided us with important information regarding the primary structure of many matrix proteins. This in turn has lead to advances in studies of the functions of post‐translational modifications on connective tissue proteoglycans (PGs). Changes in GAG structure with ageing and disease have been well documented (Thonar et al. 1986; Brown et al. 1998). However, little is known about the exact sites of and differential substitution of GAGs on the aggrecan core protein and how these substitutions facilitate normal function or the changes seen with disease. The CS : KS ratio of substitution change significantly, with KS levels increasing with age and decreasing with the onset of disease. Objective was to produce monoclonal antibody (MAb) reagents to keratanase (k'ase) generated stub epitopes, that could be used to help identify, characterize and quantify sites of KS substitution on PGs, providing the potential to determine how the arrangement of such substitutions change with development, ageing and pathology.Methods Bovine Nasal Cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of glycosaminoglycan (GAG) fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS from CS fragments. Fractions collected were analysed by SDS‐PAGE and Western blotting. Fractions positive for KS were pooled and kinase digested to expose the KS stub antigens. Immunization and fusions were carried out as previously described (Nieduszynski et al. 1990). Initial screenings were carried out using ELISA. Briefly, 96‐well microtitre plates were coated with the immunizing antigen overnight at 37 °C. The plates were then blocked prior to the addition of hybridoma media for 1–2 h at 37 °C. Binding was detected using an alkaline phosphatase‐conjugated secondary antibody for 1 h at 37 °C prior to the addition of the substrate. Positive wells were further screened by ELISA and SDS‐PAGE using the immunizing antigen, chondroitinase‐digested BNC and an A1D1 BNC preparation to establish the kinase stub specificity of the hybridomas. Further screenings by Western blotting was carried out on positive hybridomas selected. Antigens used included keratanase‐digested bovine corneal KS‐PGs, keratanase‐II‐digested KS‐PGs and a nonkeratanase‐digested corneal KS‐PG sample.Results Screening: Screening identified two positive hybridomas, B‐KS‐I and B‐KS‐II, which were specific for kinase‐generated KS stub. On screening, these antigens showed reactivity specifically for kinase‐digested BNC abc core, with no reactivity to the nonkinased linear KS GAG epitopes. Reactivity to kinase‐digested corneal KS‐PGs indicated that the MAbs generated were indeed to a stub structure in the KS chain and not to some linkage region epitope, amino acid sequence or oligosaccharide present on the core protein.Application: Immunohistochemistry utilizing B‐KS‐I was used to localize KS in a range of tissues along side anti‐KS 5D4. In human articular cartilage engineered grafts, labelling showed B‐KS‐I and 5D4 to have broadly overlapping labelling patterns for KS; however, label for B‐KS‐I had a much more restricted and subtle tissue distribution than that of antibody 5D4.Discussion These new KS stub MAbs have potential to be used in many different areas of research. They may be used in analysis of trypsin‐digested purified aggrecan from cattle joints of different ages to determine sites of KS substitution, which remain common or change with development and ageing. They may also be used in analysis of cartilage explant culture metabolites to assess KS substitution on the aggrecan fragments generated after stimulation of these cultures with cytokines such as IL‐1 or TNF‐α. Collectively it will provide important new information on the changing pattern of KS substitution in connective tissue PGs with development, ageing and the onset of pathology.

A Syndrome of Joint Laxity and Impaired Tendon Integrity in Lumican- and Fibromodulin-deficient Mice

Lumican and fibromodulin regulate the assembly of collagens into higher order fibrils in connective tissues. Here, we show that mice deficient in both of these proteoglycans manifest several clinical features of Ehlers-Danlos syndrome. The Lum(-/-)Fmod(-/-) mice are smaller than their wild type littermates and display gait abnormality, joint laxity, and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis, and extreme tendon weakness are the likely causes for joint laxity in the double-nulls. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in the Lum(+/+)Fmod(-/-) mice, with further loss in stiffness in a Lum gene dose-dependent way. At the protein level, we show marked increase of lumican in Fmod(-/-) tendons, which may partially rescue the tendon phenotype in this genotype. These results establish fibromodulin as a key regulator and lumican as a modulator of tendon strength. A disproportionate increase in small diameter immature collagen fibrils and a lack of progression to mature, large diameter fibrils in the Fmod(-/-) background may constitute the underlying cause of tendon weakness and suggest that fibromodulin aids fibril maturation. This study demonstrates that the collagen fibril-modifying proteoglycans, lumican and fibromodulin, are candidate genes and key players in the pathogenesis of certain types of Ehlers-Danlos syndrome and other connective tissue disorders.

Purification of recombinant human decorin and its subdomains.

AbstractConventional approaches to purifying most connective tissue proteoglycans (PGs) generally involve treatments that consequently alter the binding properties of the purified PG (e.g., exposure to strong detergents or harsh denaturants such as guanidinium). Herein we describe a protocol that we use to isolate human decorin in which the PG remains functional in its binding to various ligands (1,2). This method utilizes a eukaryotic expression system that produces high quantities of recombinant human decorin as a soluble component of the transfected cell culture medium together with a combination of conventional ion-exchange and hydrophobic-interaction chromatography. Here we provide detailed instructions for (1) the production of conditioned medium containing secreted recombinant human decorin, (2) the initial ion-exchange chromatography step using DEAE-Sepharose, and (3) the final hydrophobic interaction chromatography step.KeywordsConditioned MediumHydrophobic Interaction ChromatographyRoller BottleHydrophobic Interaction Chromatography ColumnRoller Bottle CultureThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Molecular structure of the carbohydrate-protein linkage region fragments from connective-tissue proteoglycans

Molecular structure of the carbohydrate-protein linkage region fragments from connective-tissue proteoglycans

NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans.

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.

Symptomatic restenosis after carotid percutaneous transluminal angioplasty

She elected to have carotid endarterectomy, which was performed in October, 1997. Pathologically, the plaque was composed of dense connective tissue with calcification. A few macrophages were present. Newer connective tissue with loosely arranged collagen was superimposed on the older plaque material. These areas of new connective tissue were rich in connective tissue proteoglycans and had a characteristic arrangement of smooth-muscle cells in a storiform pattern. The appearances were characteristic of accelerated smoothmuscle proliferation (figure). The finding of smooth-muscle proliferation rather than recurrent atherosclerosis after restenosis is consistent with the hypothesis that this patient’s recurrent symptoms after carotid PTA resulted from haemodynamic rather than embolic mechanisms. The precipitation of the transient ischaemic attack by a hot bath strongly suggests that vasodilatation and relative hypotension decreased cerebral perfusion. Haemodynamic symptoms are uncommon in the cerebral circulation because of the collateral supply provided by the circle of Willis. In our patient, however, this mechanism is likely to have been deficient because her restenosis was severe and her contralateral internal carotid artery was occluded. Such severe impairment of collateral supply is uncommon and, assuming that most restenoses after carotid PTA have a similar pathology to our patient, may explain why recurrent symptoms are much rarer after carotid PTA than after coronary PTA.

Immunohistochemical localization of proteoglycans in bovine skeletal muscle and adipose connective tissues

Monoclonal antibodies were used to immunolocate proteoglycans in bovine skeletal muscle and adipose connective tissues. Intense stainings with the anti-decorin antibody were observed in the epimysium, perimysium and adipose connective tissues. Weak but positive immunostainings for chondroitin sulphate were also observed in these tissues. Key words: Proteoglycan, glycosaminoglycan, muscle, adipose tissue, beef

Structure and metabolism of some connective tissue proteoglycans

Structure and metabolism of some connective tissue proteoglycans

Expression of decorin in human tissues and cell lines and defined chromosomal assignment of the gene locus (DCN).

Earlier studies had shown that the expression of the gene coding for one eminent connective tissue proteoglycan, decorin (DCN), is deficient in the fibroblasts of 3 out of 15 Marfan patients (Pulkkinen et al., 1990). To obtain more information on the expression of this gene, various human tissues and cell lines were studied. High mRNA levels of decorin were detected in aorta, lung, skin, kidney, smooth muscle, and placenta, whereas significantly lower mRNA levels were found in the rest of the tissues analyzed. Two sizes of transcripts were observed in all tissues. The two transcripts of decorin most probably do not represent two different genes, since in situ hybridization gave only one strong signal, placing the gene in 12q21----q22. No tissue-specific differences in the two mRNA species of decorin were detected. This is in contrast to the gene of versican, another connective tissue proteoglycan gene, that was analyzed as a control; high expression of a longer transcript of the versican gene was found in brain and smooth muscle, whereas the shorter transcript was predominant in all other tissues studied. DCN was actively transcribed in cultured mesenchymal cells, whereas in cells of endothelial or epithelial origin, the transcription level was undetectable. These tissue- and cell type-specific variations in the expression of DCN may help to explain the complex phenotypic variation typical of individuals with Marfan syndrome.

Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1—-4)-O-beta-D-xylopyranosyl-(1—-0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans.

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.

Initial Characterization of small Proteoglycans Synthesized by Embryonic Chick Leg Muscle-Associated Connective Tissues

Muscle development in the embryonic chick involves the interfacing of two different cell types: myogenic cells forming the contractile network and connective tissue cells which wrap this network into discrete morphologies. Previous reports from this laboratory detailed the characterization of large extracellular matrix proteoglycans synthesized by skeletal muscle. This report describes the initial characterization of small muscle-associated connective tissue proteoglycans synthesized in embryonic chick leg in ovo and by embryonic leg muscle-associated secondary fibroblasts in vitro by CsCl equilibrium density gradient centrifugation. Analysis of the D1 gradient fractions from the in ovo and in vitro material revealed that the proteoglycans were of relatively small size. These molecules eluted from Sepharose CL-2B through a Kav range of 0.6-0.8. Analysis of alkaline borohydride-released glycosaminoglycan chains by Sepharose CL-6B, Sephadex G-25, and thin layer chromatography demonstrated a mixture of three proteoglycan families composed of chondroitin sulfate, dermatan sulfate, and heparan sulfate glycosaminoglycans.

MZ15, a monoclonal antibody recognizing keratan sulphate, stains chick tendon.

Biochemical, morphometric and electron histochemical methods have failed to demonstrate the presence in tendon of keratan sulphate, a common component of connective tissue proteoglycans. Using a monoclonal antibody specific for keratan sulphate, a positive localization of this molecule was made in the gastrocnemius tendon of stage 44 chicken embryos both at the light and electron microscopical levels.

Proliferation and connective tissue proteoglycan synthesis of cultured rat liver fat storing cells (Ito-cells) are not activated by ethanol and its metabolites

Proliferation and connective tissue proteoglycan synthesis of cultured rat liver fat storing cells (Ito-cells) are not activated by ethanol and its metabolites

Kupffer cell-mediated induction of synthesis and secretion of proteoglycans by rat liver fat-storing cells in culture

The effect of conditioned media from Kupffer cells of normal, D-galactosamine- and thioacetamide-treated rats on the synthesis of proteoglycans by rat liver fat-storing cells in culture was studied in order to elucidate some of the mechanisms initiating enhanced connective tissue proteoglycan synthesis in injured liver. The incorporation of [35S]sulfate and [3H]glucosamine into proteoglycans was 2.1-2.5 fold (P less than 0.005) stimulated by the additions of normal, D-galactosamine- and thioacetamide-exposed Kupffer cell media. The concentrations of hexuronic acid and amino sugars in the medium glycosaminoglycan fraction were enhanced 5-fold and 4.5-fold, respectively, if the fat-storing cells were cultured in the presence of normal Kupffer cell conditioned medium. Treatment of normal Kupffer cells in culture with zymosan and phorbol esters, but not the addition of lipopolysaccharide, enhanced further the proteoglycan synthesis-stimulating effect of normal (untreated) Kupffer cells. The pattern of newly formed [35S]sulfate-labeled proteoglycans was changed in the presence of Kupffer cell media, showing a strong fractional increase of chondroitin sulfate and a relative decrease of dermatan sulfate, but the fraction of heparan sulfate was almost unaffected. In absolute terms Kupffer cells stimulated the total (medium and cell fraction) synthesis of chondroitin sulfate 2.8-fold and that of dermatan sulfate 1.5-fold. Although the DNA content of fat-storing cell cultures was increased by incubation with Kupffer cell media, an enhancement of proteoglycan synthesis was also observed when related to the DNA content of the cultures. The stimulation of proteoglycan synthesis was not dependent on the induction of cell proliferation. Gel chromatography and beta-elimination of medium proteoglycans revealed no changes of the molecular weight distribution profile of native proteoglycans and glycosaminoglycan chains synthesized under the influence of the various Kupffer cell media. Activation of proteoglycan synthesis and secretion in fat-storing cells by Kupffer cell-derived factor(s) might be an important mechanism of their strong accumulation in the connective tissue of fibrotic livers.