High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements.

Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.

Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina.

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

Synthesis of 13 C-labelled sulfated N-acetyl-d-lactosamines to aid in the diagnosis of mucopolysaccharidosis diseases.

Morquio A syndrome is an autosomal mucopolysaccharide storage disorder that leads to accumulation of keratan sulfate. Diagnosis of this disease can be aided by measuring the levels of keratan sulfate in the urine. This requires the liquid chromatography tandem mass spectrometry (LCMS/MS) measurement of sulfated N-acetyl-d-lactosamines in the urine after cleavage of the keratan sulfate with keratanase II. Quantification requires isotopically-labelled internal standards. The synthesis of these 13 C6 -labelled standards from 13 C6 -galactose and N-acetylglucosamine is described. The required protected disaccharide is prepared utilising a regioselective, high yielding ß-galactosylation of a partially protected glucosamine acceptor and an inverse addition protocol. Subsequent synthesis of the 13 C6 -labelled mono and disulfated N-acetyllactosamines was achieved in five and eight steps, respectively, from this intermediate to provide internal standards for the LCMS/MS quantification of keratan sulfate in urine.

Quantitative analysis of glycosaminoglycans, chondroitin/dermatan sulfate, hyaluronic acid, heparan sulfate, and keratan sulfate by liquid chromatography-electrospray ionization-tandem mass spectrometry.

We developed a method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive "GAGomic" analysis of biological tissues.

Expression of lumican in hidroacanthoma simplex and clonal-type seborrheic keratosis as a potent differential diagnostic marker.

Lumican, a member of the small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. The lumican expression correlates with pathological conditions and the growth and metastasis of various malignancies. In cutaneous neoplasms, the lumican expression is lower in advanced-stage malignant melanomas that invade the dermis than in early-stage melanomas. Furthermore, we have recently reported that the expression pattern of lumican is different from that of actinic keratosis and the Bowen disease. Lumican is positive in the poroid cells of intraepidermal sweat ducts; therefore, we examined the expression patterns of lumican in acanthotic-type seborrheic keratosis and Pinkus-type poroma followed by clonal-type seborrheic keratosis and hidroacanthoma simplex. The neoplastic cells of acanthotic-type seborrheic keratosis exhibited positive immunostaining in only 1 of 31 cases (3.23%), whereas the poroid cells of Pinkus-type poroma exhibited positive immunoreactivity in 26 of 28 patients (92.8%). In the hidroacanthoma simplex cases, lumican was expressed in poroid cells forming intraepidermal nests in 22 of 28 patients (78.6%), whereas the neoplastic cells in most cases of clonal-type seborrheic keratosis were negative for lumican. In some seborrheic keratosis cases that were positive for lumican in neoplastic cells, lumican was observed in squamoid cells but not in basaloid cells. Therefore, it is necessary to evaluate the immunoreactivity of lumican in seborrheic keratosis and in basaloid cells. These findings suggest that lumican is a potent differential diagnostic marker that distinguishes hidroacanthoma simplex from clonal-type seborrheic keratosis.

Expression of Podocalyxin Potentially Decorated With Low-sulfated Keratan Sulfate in Human Testicular Embryonal Carcinoma.

SummaryWe previously demonstrated that among various histological types of human testicular germinal cell tumors (GCTs), embryonal carcinoma (EC) preferentially expresses low-sulfated keratan sulfate (KS) consisting of repeating N-acetyllactosamine (LacNAc) disaccharide units composed of galactose and 6-O-sulfated N-acetylglucosamine (GlcNAc), which is recognized by the R-10G antibody. Recently, we generated another anti-low-sulfated KS monoclonal antibody, 294-1B1. Immunohistochemical analysis of testicular GCTs (n=83) revealed that the low-sulfated KS recognized by 294-1B1 is also preferentially expressed in EC but minimally in other GCT histological types. Moreover, immunolabeling with R-10G and 294-1B1 antibodies was resistant to peptide-N-glycosidase F digestion, and EC was not stained with the MECA-79 antibody, indicating that low-sulfated KS expressed in EC contains mucin-type core 2 O-glycans carrying GlcNAc-6-O-sulfated oligo-LacNAc. Double immunofluorescence staining showed that R-10G and 294-1B1 antibody signals colocalized with those for podocalyxin (PODXL). Furthermore, western blot analysis of recombinant human PODXL•IgG fusion proteins secreted from low-sulfated KS-expressing human embryonic kidney 293T cells revealed that PODXL functions as a core protein for low-sulfated KS. Taken together, these findings strongly suggest that the PODXL glycoform decorated with low-sulfated KS is preferentially expressed in human testicular EC and may therefore serve as a diagnostic marker for this malignancy.

Relationship between pre-radiographic cartilage damage following anterior cruciate ligament injury and biomarkers of cartilage turnover in clinical practice: a cross-sectional observational study.

OBJECTIVE: To determine whether differences in synovial fluid (SF) biomarkers of collagen and proteoglycan turnover are associated with pre-radiographic damage to articular cartilage and menisci following anterior cruciate ligament (ACL) injury and are of clinical value. METHOD: SF samples from ACL injured knees of 108 patients were obtained when damage to cartilages and menisci was evaluated arthroscopically. Concentrations of SF collagenase-generated cleavage neoepitope of type II collagen (C2C) were determined using ELISA and aggrecan-derived disaccharides of chondroitin-4-sulfate (Δdi-C4S), chondroitin-6-sulfate (Δdi-C6S), and keratan sulfate (KS), were measured in SF by High performance liquid chromatography (HPLC). RESULTS: Radiographic examination failed to detect any intra-articular degenerative changes. The number of high-grade cartilage lesions was positively associated with age, duration after injury and the level of C2C, and negatively with the level of KS. There was no association between the number of high-grade cartilage and meniscal lesions. Multivariable logistic regression revealed significant associations of increased C2C (adjusted Odds ratio (OR) of the upper quartile to remainder of 2.49, 95% Confidence interval (CI) = 0.85-7.27) and decreased KS (adjusted OR of the lower quartile to the remainder of 3.32, 95% CI = 1.19-9.24) with the presence of three or more high-grade cartilage lesions, independent of age and duration after injury. The combined impact of increased C2C and decreased KS was 22.8 (95% CI = 1.95-265.9), far exceeding the impact of each independent biomarker. CONCLUSION: Combinations of the C2C and KS as described here may offer greater ability to identify patients with early pre-radiographic high-grade cartilage damage compared to single clinical or biomarker parameters.

Lumican as a novel marker for differential diagnosis of Bowen disease and actinic keratosis.

Lumican, a member of the small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. Lumican expression correlates with pathological conditions, including skin fragility, corneal opacification, and corneal and cardiac wound healing. Lumican is overexpressed in tumor cells, including in the breast, colorectal, neuroendocrine cell, uterine cervical, and pancreatic cancers. Lumican expression also correlates with the growth and metastasis of various malignancies. For example, lumican expression is lower in the dermis of malignant melanoma cases than in early-stage melanomas. However, the expression patterns and roles of lumican in nonmelanoma skin cancer have not been elucidated. In this study, we used immunohistochemistry and in situ hybridization to examine the expression patterns of lumican in normal skin, Bowen disease, and actinic keratosis. In normal skin, lumican was expressed in the collagen fibers in the dermis, acrosyringium, follicular epithelium, and sebocytes but not in epidermal keratinocytes. In Bowen disease, lumican was expressed in 34 (91.8%) of 37 patients. Notably, all cases of actinic keratosis were negative for lumican. These findings suggest that lumican plays an important role in the pathogenesis of Bowen disease and actinic keratosis and might be useful as an adjunct to the diagnosis for subtypes of 2 diseases: bowenoid actinic keratosis and Bowen disease in sun-exposed areas.

Lumican expression in pancreatic ductal adenocarcinoma.

BACKGROUND/AIMS: The present study was aimed to investigate lumican expression in and correlation with severity of pancreatic ductal adenocarcinoma (PDA). METHODOLOGY: We assessed mRNA expression and protein localization (using immunohistochemistry) in PDA samples collected from 260 patients. Additionally, we compared lumican expression with expression of Ki-67, VEGF and mutated p53 proteins, which are markers of cancer progression. RESULTS: Expression levels of lumican mRNA and protein in cancer tissue were significantly higher than those in tumor-adjacent tissue (t=5.69, p<0.05). The stromal expression of lumican in poorly differentiated cases was significantly higher at stage T4 than stage T2-3 (χ²=21.06, p<0.05); similarly, the stromal expression of lumican was significantly higher in TNM stage III-IV than in stage I-Il (χ²=17.01, p<0.05). Additionally, expression of Ki67 was higher in poorly differentiated cases than in highly-moderately differentiated cases (χ²=13.06, p<0.05). Finally, in highly-moderately differentiated samples, stromal expression of lumican was negatively correlated with expression of Ki-67, VEGF and mutated P53 (p<0.05). CONCLUSIONS: Lumican expression is higher in pancreatic ductal adenocarcinoma than in tumor-adjacent tissue, and the correlation of lumican expression with TNM stage in poorly differentiated samples, in contrast with its negative correlation with expression of Ki-67, VEGF and mutated P53 mutation in highly-moderately differentiated samples.

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle.

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.

Glycosaminoglycans in the accessory sex glands, testes and seminal plasma of alpaca and ram.

The viscous nature of alpaca semen limits its use in cryopreservation and other assisted reproductive technologies. The cause and source of this viscosity is unknown although it has been postulated, but never proven, that glycosaminoglycans (GAGs) secreted by the bulbourethral gland are responsible. The present study investigated the concentration and composition of GAGs in alpaca seminal plasma, testes, bulbourethral gland and prostate gland and compared them to those in the ram to determine the relationship between seminal plasma GAGs and viscosity and to identify the source of seminal plasma GAGs. Alpaca seminal plasma contained more GAGs than ram (P<0.001) and the predominant GAG, keratan sulfate, was correlated with viscosity (P=0.05, R(2)=0.2635). The alpaca bulbourethral gland contained most GAGs compared with prostate or testis (P<0.001). In the ram, the prostate contained most GAGs. These findings suggest that GAGs, particularly keratan sulfate, may be the cause of seminal plasma viscosity in alpacas, and that the seminal plasma GAGs originate from the bulbourethral gland.

Identification and characterization of proteins in amniotic fluid that are differentially expressed before and after antenatal corticosteroid administration.

OBJECTIVE: We sought to examine changes in the intraamniotic proteomic environment after the administration of antenatal corticosteroids to women with impending preterm delivery. STUDY DESIGN: Amniotic fluid samples were collected at the time of clinically indicated amniocentesis before and within 7 days of administration of antenatal corticosteroids for impending preterm delivery (n = 12). Proteins differentially expressed before and after corticosteroids were identified by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. They were isolated, characterized, and quantified by fast protein liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digestion, immunodepletion assays, enzyme-linked immunosorbent assay, and electrospray ionization tandem mass spectrometry. RESULTS: Five protein peaks of interest were identified and characterized, all of which were significantly decreased after antenatal corticosteroid administration. These included 2 isoforms of transthyretin, albumin, prothrombin fragment 2, and lumican. CONCLUSION: Four proteins, identified and characterized in amniotic fluid, were differentially expressed with antenatal corticosteroid administration. These data may provide additional insight into the molecular mechanisms by which antenatal corticosteroids prevent neonatal complications.

Immunophenotypes of macular corneal dystrophy in India and correlation with mutations in CHST6.

PURPOSE: To determine the immunophenotypes of macular corneal dystrophy (MCD) in Indian patients and to correlate them with mutations in the carbohydrate 6-sulfotransferase (CHST6) gene. METHODS: Sixty-four patients from 53 families with MCD that were previously screened for mutations in CHST6 were included in an immunophenotype analysis. Antigenic keratan sulfate (AgKS) in serum as well as corneal tissue was evaluated in 31 families. Only cornea was evaluated in 11 families, and only serum was evaluated in 11 families. AgKS was detected in formalin-fixed, paraffin-embedded corneal sections by immunohistochemistry and in serum by ELISA using a monoclonal antibody against sulfated forms of KS in patients with MCD as well as normal controls. RESULTS: Analysis of corneal and/or serum AgKS disclosed MCD type I (27 families), MCD type IA (5 families), and MCD type II (3 families) in the cases studied. An additional 10 families were either MCD type I or MCD type IA since only serum AgKS data were available. Seven families manifested atypical immunophenotypes since the corneal AgKS expression was either of MCD type I or MCD type IA, but serum AgKS levels ranged from 19 ng/ml to 388 ng/ml. More than one immunophenotype was detected amongst siblings in two families. Each immunophenotype was associated with mutational heterogeneity in CHST6. CONCLUSIONS: MCD type I was the predominant immunophenotype in the Indian population studied followed by MCD type IA and then MCD type II. We detected further immunophenotypic heterogeneity by finding atypical patterns of AgKS reactivity in a subset of families. There were no simple correlations between immunophenotypes and specific mutations in CHST6, suggesting that factors other than CHST6 mutations may be contributing to the immunophenotypes in MCD.

The dynamic structure of the pericellular matrix on living cells.

Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b-HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan-aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.

Keratan sulfate-containing proteoglycans in sheep brain with particular reference to phosphacan and synaptic vesicle proteoglycan isoforms.

Proteoglycans (PGs) are widely expressed in all areas of the brain. In this study, the keratan sulfate-containing PGs (KS-PGs) from cerebrum (CB), cerebellum (CL) and brainstem (BS) of young sheep brain were isolated, purified and characterized. The amount of KS-PGs in CL was significantly lower than that in CB and BS. KS-PGs were characterized by increased extent of glycosylation and heterogeneity of KS chains in CL. Western blot analyses demonstrated the presence of the KS-PGs phosphacan, SV2A and SV2B isoforms of synaptic vesicle proteoglycan in all three areas of the young sheep brain. Phosphacan predominated in BS and CB, showing significant molecular heterogeneity. SV2A and SV2B were found in two forms of high and low molecular sizes according to their extent of glycosylation in sheep brain. SV2A predominated in CL, where forms with very high molecular sizes were detected. Immunohistochemical examination revealed that SV2A was localized in the extracellular matrix of both gray and white matter. In contrast, phosphacan and SV2B were mainly localized in the white matter in all brain regions. The results of the present study demonstrated that KS-PGs are present in the three areas of the sheep brain, showing significant variations in their content, structure and localization among the distinct areas. These differences may be important for the physiology of the brain.

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.

Increased expression of highly sulfated keratan sulfate synthesized in malignant astrocytic tumors.

Keratan sulfate (KS) proteoglycans are expressed on a subpopulation of microglia in normal adult brain. We previously showed the up-regulated expression of KS in one of glioblastoma cell lines using anti-KS antibody (5D4). However, it has not been clarified whether KS is expressed in brain tumors and is involved in their malignancy. In this study, 54 astrocytic tumors were investigated about KS-expression using Western-blot with 5D4. In six of 14 anaplastic astrocytomas (43%) and 23 of 34 glioblastomas (68%), KS was detected by 5D4. KS was hardly detected by 5D4 in diffuse astrocytoma, suggesting that KS-expression is significantly expressed in malignant astrocytic tumors. In immunohistochemistry, KS is highly expressed in cell surface of malignant astrocytic tumors. Taken together, KS might be associated with the malignancy of astrocytic tumors, and be useful for a prognostic factor of astrocytic tumors.

Keratocyte phenotype is enhanced in the absence of attachment to the substratum.

PURPOSE: Keratocytes, mesenchymal cells populating the corneal stroma, secrete the unique transparent connective tissue of the cornea as well as opaque scar tissue after injury. Previous studies identified factors mediating keratocyte phenotype in vitro, particularly the expression of the keratan sulfate proteoglycans, which are essential for vision. Whereas earlier work emphasized effects of cytokines, the current study examines the effects of substratum attachment on keratocyte phenotype. METHODS: Primary keratocytes from collagenase digestion of bovine corneas were cultured on tissue-culture plastic or on poly (2-hydroxyethylmethacrylate)(polyHEMA)-coated, non-adhesive surfaces. Secreted proteoglycans from culture media and cell-associated proteins were characterized using western blotting or isotopic labeling. Gene expression was characterized with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Secreted matrix was examined with immunostaining. RESULTS: We observed that virtually all primary keratocytes participate in the formation of spheroidal aggregates, remaining viable for at least four weeks in vitro. Spheroid keratocytes secrete more keratan sulfate and keratocan than attached cells in the same culture medium. In spheroids, keratocytes accumulate substantial matrix in intercellular spaces, including keratan sulfate, lumican, keratocan, and collagens V and VI. The unattached cells undergo limited cell division and do not differentiate into myofibroblasts in response to transforming growth factor beta (TGFbeta), which is based on the expression of extra domain A (EDA) fibronectin and alpha-smooth muscle actin. Similarly, the platelet derived growth factor, a cytokine initiating the fibroblastic phenotype in attached keratocytes, had a limited effect on the spheroid-associated keratocytes. Ascorbate-2-phosphate was the only agent stimulating keratan sulfate secretion in the spheroid keratocytes. CONCLUSIONS: These results provide a new paradigm for understanding signals that regulate extracellular matrix secretion. For primary keratocytes, the alteration of the cellular environment in terms of cell-cell and cell-matrix interactions mediates and can override signals from soluble cytokines in influencing matrix expression and also in adopting other aspects of the fibroblastic and myofibroblastic phenotypes found in healing wounds.

Glycosaminoglycans in the pericellular matrix of chondrons and chondrocytes.

This is the first study to quantitate and profile the glycosaminoglycan (GAG) composition of the pericellular matrix (PCM) of chondrons and chondrocytes using the highly sensitive technique; fluorophore-assisted carbohydrate electrophoresis (FACE). Bovine articular chondrocytes and chondrons were isolated enzymatically. High cell yield and viability were obtained for both preparations. Chondrons had strong immunofluorescent labeling for keratan sulphate and chondroitin-6 sulphate but no labeling for hyaluronan. We compared the immunofluorescent data with FACE. The quantities of total keratan sulphate were determined to be 0.013 +/- 0.002 pg cell(-1) and 0.032 +/- 0.003 pg cell(-1) in the chondrocyte and chondron preparations, respectively. Four internal keratan sulphate sugars were detected (gal beta 1,4glcNAc6S, gal6S beta 1,4glcNAc6S, glcNAc beta 1,3gal and glcNAc6S beta 1,3gal) for both preparations but they were present at significantly higher concentrations in chondron preparations (P < 0.01). Total chondroitin sulphate (CS) was determined to be 0.054 +/- 0.004 pg cell(-1) and 0.077 +/- 0.005 pg cell(-1) for chondrocyte and chondron preparations, respectively. Unsulphated CS disaccharide levels were similar but chondrons had significantly more chondroitin-4 sulphated disaccharides and chondroitin-6 sulphated disaccharides (P < 0.05). Hyaluronan acid was present at low concentrations (0.010 +/- 0.001 pg cell(-1)) in both chondrocytes and chondrons. In this study, enzyme digestion coupled with FACE separation revealed new information about the differences in GAGs from isolated chondrocyte and chondron preparations. Further investigation of the differences in GAGs from chondrocytes and chondrons from different zones of articular cartilage may be useful for tissue engineering approaches.

Localization of small leucine-rich proteoglycans and transforming growth factor-beta in human oral mucosal wound healing.

Wound healing in oral mucosa is fast and results in little scar formation as compared with skin. The biological mechanisms underlying this property are poorly understood but may provide valuable information about the factors that promote wound regeneration. Small leucine-rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are extracellular matrix molecules that regulate collagen fibrillogenesis, inhibit transforming growth factor-beta (TGF-beta) activity and reduce scarring. In the present study, we analyzed accumulation of SLRPs and TGF-beta during non-scarring human oral mucosal wound healing. Biopsies were collected from healthy volunteers from unwounded tissue and from standardized experimental wounds 3-60 days postwounding. Localization of SLRPs, TGF-beta1 and TGF-beta3 was analyzed by immunohistochemical staining and quantitated by image analysis. Double immunostaining was used to study localization of SLRPs or active TGF-beta in distinct cells. Decorin, biglycan, fibromodulin, and TGF-beta isoforms showed significantly increased accumulation in the wound extracellular matrix and distinct wound cells while the abundance of lumican in the extracellular matrix was strongly reduced during wound healing. Localization and abundance of fibromodulin, lumican, and TGF-beta isoforms was also spatiotemporally regulated in the wound epithelium. The findings suggest that SLRPs regulate wound reepithelialization and connective tissue regeneration during oral mucosal wound healing.