Showing papers on "Keratan sulfate published in 2004"

Multipotent Stem Cells in Human Corneal Stroma

Abstract: Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neurofilament protein, and beta-tubulin II. These results demonstrate the presence of a population of cells in the human corneal stroma expressing stem cell markers and exhibiting multipotent differentiation potential. These appear to be the first human cells identified with keratocyte progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanisms of corneal development, differentiation, and wound healing. These cells may be a resource for bioengineering of corneal stroma and for cell-based therapeutics.

Analysis of cartilage biomarkers in the early phases of canine experimental osteoarthritis.

Abstract: Objective To study 3 body fluids for changes in the levels of 5 biomarkers of cartilage metabolism during the early phases of experimental osteoarthritis (OA). Methods Twenty skeletally mature mixed-breed canines underwent unilateral surgical transection of the anterior cruciate ligament. Samples of joint fluid, serum, and urine were obtained preoperatively and just before necropsy (3 weeks or 12 weeks postoperatively). Biomarkers included 2 markers of cartilage matrix synthesis/turnover (aggrecan 846 epitope and C-propeptide of type II collagen) and 3 markers of cartilage degradation (keratan sulfate proteoglycan epitope, the collagenase-generated cleavage epitope of type II collagen [Col2-3/4Clong mono, or CIIC], and crosslinked peptides from the C-telopeptide domain of type II collagen [Col2CTx]). Significant changes in the levels of these biomarkers were determined by paired analyses. Results Joint pathology was more severe in the 12-week group compared with the 3-week group. In joint fluid, due to limited volume, only Col2-3/4Clong mono and Col2CTx were measured. Significant elevations in the levels of both of these markers were observed in experimental joints in both the 3-week group and the 12-week group. In serum, the level of aggrecan 846 epitope was elevated at both 3 weeks and 12 weeks, the level of Col2-3/4Clong mono was elevated at 12 weeks, and the level of Col2CTx was elevated at both 3 weeks and 12 weeks. In urine, the level of Col2-3/4Clong mono was elevated at 12 weeks after surgery. Conclusion Levels of biomarkers of intact aggrecan proteoglycan (aggrecan 846 epitope) and type II collagen degradation (Col2-3/4Clong mono and Col2CTx) were elevated early after unilateral stifle joint injury, suggesting that these markers are sensitive and specific for early cartilage changes associated with isolated joint injury in this established model of experimental OA.

Cleavage of lumican by membrane-type matrix metalloproteinase-1 abrogates this proteoglycan-mediated suppression of tumor cell colony formation in soft agar.

Abstract: The small leucine-rich proteoglycan lumican was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane-type matrix metalloproteinase-1 (MT1-MMP). Coexpression of MT1-MMP with lumican in HEK293T cells reduced the concentration of lumican secreted into culture medium, and this reduction was abolished by addition of the MMP inhibitor BB94. Lumican protein from bovine cornea and recombinant lumican core protein fused to glutathione S-transferase was shown to be cleaved at multiple sites by recombinant MT1-MMP. Transient expression of lumican in HEK293 cells induced expression of tumor suppressor gene product p21/Waf-1, which was abrogated by the coexpression of MT1-MMP concomitant with a reduction in lumican concentration in culture medium. Stable expression of lumican in HeLa cells induced expression of p21 and reduction of colony formation in soft agar, which were both abolished by the expression of MT1-MMP. HT1080 fibrosarcoma cells stably transfected with the lumican cDNA (HT1080/Lum), which express endogenous MT1-MMP, secreted moderate levels of lumican; however, treatment of HT1080/Lum cells with BB94 resulted in accumulation of lumican in culture medium. The expression levels of p21 in HT1080/Lum were proportional to the concentration of secreted lumican and showed reverse corelation with colony formation in soft agar. These results suggest that MT1-MMP abrogates lumican-mediated suppression of tumor cell colony formation in soft agar by degrading this proteoglycan, which down-regulates it through the induction of p21.

Interaction of lumican with aggrecan in the aging human sclera.

Abstract: PURPOSE. Lumican is a keratan sulfate proteoglycan originally identified in cornea, but present in a variety of connective tissues where it presumably regulates collagen fibril formation and organization. The present study was designed to describe the chemical nature of lumican core protein in the aging human sclera. METHODS. Western blot analyses, immunohistochemistry, and immunoaffinity chromatography were used to detect and purify the lumican core protein from tissue extracts from human donors 6 to 89 years of age. Treatment of lumican with chondroitinase ABC, keratanase-I and -II, and/or endo--galactosidase was used to determine the degree of glycosylation of the lumican core protein. RESULTS. Lumican was present in the human sclera as a 70- to 80-kDa core protein with short unsulfated lactosaminoglycan side chains. In addition, on Western blots, a larger 200-kDa species was apparent that was immunologically related to lumican. This high-molecular-weight material increased in scleral extracts with increasing age. The complex was most abundant in unreduced samples, and approximately two thirds of the 70- to 80-kDa lumican core protein was released from the complex on reduction of the scleral extract. Further characterization of the 200-kDa lumican-immunopurified complex indicated that aggrecan (the cartilage proteoglycan) was covalently associated with lumican. CONCLUSIONS. Reducible and nonreducible lumican-aggrecan interactions occur in the scleral extracellular matrix and result in the formation of high-molecular-weight complexes that increase with age. These results represent the first report demonstrating lumican-aggrecan interactions and suggest they may play a role in age-related scleral extracellular matrix changes. (Invest Ophthalmol Vis Sci. 2004;45:3849‐3856) DOI:

Keratan sulfate proteoglycan phosphacan regulates mossy fiber outgrowth and regeneration.

Abstract: We have examined the role of chondroitin sulfate proteoglycans (CSPGs) and keratan sulfate proteoglycans (KSPGs) in directing mossy fiber (MF) outgrowth and regeneration in rat hippocampal slice cultures. MFs normally exhibit a very specific innervation pattern that is restricted to the stratum lucidum (SL). In addition, MFs in hippocampal slice cultures will regenerate this specific innervation pattern after transection. CSPGs are one of the best characterized inhibitory axon guidance molecules in the CNS and are widely expressed in all areas of the hippocampus except SL. KSPGs are also widely expressed in the hippocampus, but their role in axon outgrowth has not been extensively studied in the CNS where phosphacan is the only protein that appears to contain KS-GAGs. Cultured hippocampal slices were treated with either chondroitin ABC lyase or keratanases to reduce the inhibitory axon guidance properties of CS and KS proteoglycans, respectively. The ability of transected MFs to regenerate their normal innervation pattern after digestion of CS and KS-GAGS sugars with these enzymes was examined. Only keratanase treatment resulted in misrouting of MFs. Identifying the mechanism by which keratanase produced MF misrouting is complicated by the presence of splice variants of the phosphacan gene that include the extracellular form of phosphacan and the transmembrane receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). Both forms of phosphacan are made by astrocytes, suggesting that keratanase alters MF outgrowth by modifying astrocyte function.

Pig chondrocyte xenoimplants for human chondral defect repair: an in vitro model.

Abstract: The objective of this study was to evaluate the use of cultured porcine chondrocyte xenotransplantation for the repair of human chondral defects. Two-millimeter-diameter defects were drilled into explants of femoral cartilage from healthy adult donors. No cells were implanted in the chondral defects of the control group, while pig chondrocytes from normal femoral cartilage were deposited into the treated chondral defects. Cartilage explants were cultured for 4, 8, and 12 weeks. Tissue sections were processed for standard histologic staining and immunostaining with monoclonal antibodies against types I and II collagen, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. The porcine origin of chondrocytes was confirmed using a specific pig monoclonal anti-CD46. Repair was only observed in the cell-treated defects. Mono- or bilayers of cells were detected after 4 culture weeks on the bottom of the defects, while after 8-12 weeks a repair tissue filled near 30-40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline-like region. This hypercellular region showed excellent bonding with the native cartilage, cells were located in numerous lacunae, and a high content of proteoglycans as indicated by an intense toluidine blue stain was observed. The repaired tissue showed positive immunostaining for both type I and II collagen, as well as chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. Positive staining for porcine anti-CD46 was localized exclusively in the neo-synthesized tissue. We conclude that xenotransplantation of pig chondrocytes can repair, in an in vitro model, defects in human articular cartilage.

The effects of enrofloxacin on decorin and glycosaminoglycans in avian tendon cell cultures

Abstract: Tendonitis and tendon rupture have been reported to occur during or following therapy with fluoroquinolone antibiotics. Though the pathogenesis is unknown, several studies suggest that fluoroquinolone antibiotics alter proteoglycan content in soft tissues, including tendons, and thereby alter collagen fibrillogenesis. To better understand the mechanism of action of fluoroquinolones, we studied the effects of enrofloxacin, a widely used fluoroquinolone in veterinary medicine, on avian tendon cell cultures established from gastrocnemius tendons from 18-day-old chicken embryos. We found that cell proliferation was progressively inhibited with increasing concentrations of enrofloxacin. This was accompanied by changes in morphology, extracellular matrix content and collagen fibril formation as detected by electron microscopy. We also observed a 35% decrease in the content of total monosaccharides in enrofloxacin-treated cells. The ratio of individual monosaccharides was also altered in enrofloxacin-treated cells. Enrofloxacin also induced the synthesis of small amounts of keratan sulfate in tendon cells. Moreover we observed enrofloxacin-induced changes in glycosylation of decorin, the most abundant tendon proteoglycan, resulting in the emergence of multiple lower molecular bands that were identifiable as decorin after chondroitinase ABC and N-glycanase treatment of extracts from enrofloxacin-treated cells. Medium conditioned by enrofloxacin-treated cells contained less decorin than did medium conditioned by control cells. We hypothesize that enrofloxacin induces either changes in the number of N-linked oligosaccharides attached to the core protein of decorin or changes in decorin degradation process. In conclusion, our data suggest that enrofloxacin affects cell proliferation and extracellular matrix through changes in glycosylation.

Modeling glycosaminoglycans—hyaluronan, chondroitin, chondroitin sulfate A, chondroitin sulfate C and keratan sulfate

Abstract: Molecular modeling of glycosaminoglycans, hyaluronan, chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate and keratan sulfate is presented. Semiempirical quantum mechanical calculations, PM3 method, have been used to locate the minimal energy regions in the conformational space around the β 1→3 and β 1→4 glycosidic linkages. The global minima obtained for each of the glycosaminoglycans correspond to a compact conformation across the β 1→3 linkage and to the standard extended form across the β 1→4 linkage. The higher minima obtained around the β 1→3 linkage reproduces the crystallographic data for hyaluronan. A complex of hyaluronan and hydrated chromium ion has been modeled as well (ZINDO method). The complexation at the β 1→3 linkage resulted in an extended conformation.

Purification of the Keratan Sulfate proteoglycan expressed in prostatic secretory cells.

Abstract: Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation.

Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumican.

Abstract: BACKGROUND. Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation. METHODS. Anion exchange and affinity chromatography were used to purify KSPG from human prostate tissue. Enzymatic deglycosylation was used to remove keratan sulfate (KS). The core protein was isolated using 2D gel electrophoresis, digested in-gel with trypsin, and identified by peptide mass fingerprinting (PMF). RESULTS. The purified proteoglycan was detected as a broad smear on Western blots with an apparent molecular weight of 65-95 kDa. The KS moiety was susceptible to digestion with keratanase 11 and peptide N-glycosidase F defining it as highly sulfated and N-linked to the core protein. The core protein was identified, following deglycosylation and PMF, as lumican and subsequently confirmed by Western blotting using an anti-lumican antibody. CONCLUSIONS. The KSPG associated with PSGs in normal prostate epithelium is lumican. While the role of lumican in extracellular matrix is well established, its function in the prostate secretory process is not known. It's potential to facilitate packaging of polyamines in PSGs, to act as a tumor suppressor and to mark the early stages of malignant transformation warrant further investigation. (C) 2003 Wiley-Liss, Inc.

Immunocytochemistry of keratan sulfate proteoglycan and dermatan sulfate proteoglycan in porcine tooth-germ dentin.

Abstract: Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated their distribution in order to evaluate the role of proteoglycan in dentinogenesis. Specimens of porcine tooth-germ dentin and erupted teeth were the materials on which antibodies to keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30–70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future. Both antibodies reacted positively along these fibrils. In contrast, along the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10–300 nm in diameter were noted occasionally in dentinal tubules whose odontoblastic processes had disappeared and these heterogeneous fibrils were negative for both antibodies. Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification front. It is further suggested that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Therefore, keratan sulfate proteoglycan and dermatan sulfate proteoglycan were thought to be involved in both calcification and matrix formation.

Inhibitory activities of sulfated proteoglycans on chondroitin sulfate A-mediated cytoadherence of Plasmodium falciparum isolates from Thailand.

Abstract: Chondroitin sulfate A (CSA) is an important receptor for Plasmodium falciparum-infected erythrocytes in the placenta. To study the molecular interaction between parasitized erythrocytes (PE) to CSA, we performed in vitro cytoadherence inhibition assays of PE infected with wild and laboratory isolates of P. falciparum to CSA using various glycosaminoglycans (GAGs). Marked decrease in PE adhesion to immobilized CSA and CSA-expressed cells was achieved with soluble chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE) at low concentrations. The effect was dose dependent with the degree of inhibition exceeded that of soluble CSA in certain clinical isolates. The results suggested the influence of oversulfation of CS variant chains on PE adherence to CSA. Interestingly, PE of the tested wild isolates could adhere to immobilized CSD and CSE at different levels while PE of CSA-selected laboratory lines could not. Partial inhibitory activity was observed when chondroitin sulfate C (CSC), chondroitin sulfate B (CSB), and polyolpolysulfate were used even at high concentrations. Keratan sulfate, colominic acid, and Suramine were unable to inhibit PE adherence. Taken together, the results confirm that the 4-sulfate amino sugar moiety, as well as the basic disaccharide structure of N-acetylgalactosamine linked to glucuronic acid, may influence the degree of this molecular interaction. However, other sulfation patterns that could influence the interaction could not be overlooked, as in the case of CSD which contains 2-O-sulfation at glucuronic acid. Studies using pentosan polysulfate, an oversulfated molecule with a xylan backbone, as an inhibitor also showed a reduction of PE adherence of most isolates tested. Thus, only the sulfate content and pattern of this molecule could affect the adhesive interactions. In addition, difference in capacity of low molecular weight heparins to inhibit CSA-mediated PE cytoadherence of clinical isolates was also observed, thereby providing evidence on the heterogeneity in cytoadherence characteristics of maternal parasite isolates as well as their therapeutic potentials.

Gray hair-preventing/improving agent

Abstract: PROBLEM TO BE SOLVED: To provide a gray hair-preventing/improving agent exhibiting excellent gray hair-preventing and improving effect by externally applying the agent on the scalp. SOLUTION: This gray hair-preventing/improving agent comprises a sulfated polysaccharide and/or its salt excluding chitin-chitosan, preferably one or more kinds of substances selected from mucopolysaccharides selected from the group consisting of chondroitin, chondroitinsulfuric acid, chondroitinpolysulfuric acid, dermatan, dermatan sulfate, dermatan polysulfate, hyaluronic acid, heparin, heparan sulfate, keratan sulfate and keratan polysulfate and heparinoids which are sulfated polysaccharides. COPYRIGHT: (C)2005,JPO&NCIPI

Chapter 10 – Hyaluronan and Associated Proteins in the Visual System

Abstract: This chapter describes recent findings and roles of both hyaluronan and its binding proteins in the ocular physiological system, and also discusses them with regard to the pathogenesis of several ocular diseases. Hyaluronan synthase and hyaluronan are expressed in corneal epithelial cells. Hyaluronan stimulates corneal epithelial cell proliferation and migration, while others, such as chondroitin sulfate, keratan sulfate and heparan sulfate, do not increase epithelial migration. Hyaluronan enhances the formation of hemidesmosomes, and has a positive influence on the epithelial resurfacing during the healing phase in corneal wounds, with an increase of corneal hyaluronan occurring in the corneal healing region. Further, the process of corneal epithelial wound healing is modified by fibronectin or matrix metalloprotease (MMP). Versican, a large extracellular matrix proteoglycan, is distributed in corneal epithelium. In normal corneas, the hyaluronan binding protein CD44 is predominantly expressed on the membranes of basal epithelial cells, and its expression is closely correlated with corneal re-epithelialization. Hyaluronan, CD44 and fibronectin collectively play important roles in corneal epithelial wound healing. In recent years, significant advances have been made in understanding the roles of hyaluronan and its binding proteins in the field of visual science. Each ocular tissue physiologically expresses these molecules in a tissue-specific manner, and disordered expressions of these molecules are involved in the pathogenesis of ocular diseases.

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

Abstract: 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.