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This manuscript describes the functional properties of the exosomes released from melanoma cells. It details the characteristics of the tumor antigen chondroitin sulfate proteoglycan 4 (CSPG4), which is used as a marker to separate exosomes released by melanoma cells from exosomes released by nonmalignant cells. The results are discussed in view of the potential role of melanoma cell-derived exosomes in the escape of malignant cells from the host's immune system.

The brain changes in volume and composition with normal aging. Cellular components of the brain are supported by an extracellular matrix (ECM) comprised largely of hyaluronan (HA) and HA-associated members of the lectican family of chondroitin sulfate proteoglycans (CSPGs). We examined regional differences in microvascular density, neuronal and glial markers, and accumulation of HA and CSPGs in mouse brains during normal aging. The cortex, hippocampus, dentate gyrus, and cerebellum of young (4 months), middle-aged (14 months), and aged (24-26 months) brains were analyzed. Microvascular density decreased in cerebral cortex and cerebellum with age. There were no detectable differences in neuronal density. There was an increase in astrocytes in the hippocampus with aging. HA accumulation was higher in aged brain relative to young brain in the cerebral cortex and cerebellum, but not in other regions examined. In contrast, CSPGs did not change with aging in any of the brain regions examined. HA and CSPGs colocalized with a subset of neuronal cell bodies and astrocytes, and at the microvasculature. Differences in accumulation of ECM in the aging brain, in the setting of decreased microvascular density and/or increased glial activation, might contribute to age-related regional differences in vulnerability to injury and ischemia.

The human auditory brainstem, especially the cochlear nucleus (CN) and the superior olivary complex (SOC) are characterized by a high density of neurons associated with perineuronal nets (PNs). PNs build a specific form of extracellular matrix surrounding the neuronal somata, proximal dendrites and axon initial segments. They restrict synaptic plasticity and control high-frequency synaptic activity, a prominent characteristic of neurons of the auditory brainstem. The distribution of PNs within the auditory brainstem has been investigated in a number of mammalian species. However, much less is known regarding PNs in the human auditory brainstem. The present study aimed at the immunohistochemical identification of PNs in the cochlear nucleus (CN) and superior olivary complex (SOC) in the human brainstem. We focused on the complex nature and molecular variability of PNs in the CN and SOC by using specific antibodies against the main PN components (aggrecan, brevican, neurocan and hyaluronan and proteoglycan link protein 1). Virtually all subnuclei within the ventral CN and SOC were found to be associated with PNs. Direct comparison between gerbil and human yielded similar fine structure of PNs and confirmed the typical tight interdigitation of PNs with synaptic terminals in both species. Noticeably, an elaborate combination of immunohistochemical labelings clearly supports the still debated existence of the medial nucleus of trapezoid body (MNTB) in the human brain. In conclusion, the present study demonstrates that PNs form a prominent extracellular structure on CN and SOC neurons in the human brain, potentially stabilizing synaptic contacts, which is in agreement with many other mammalian species., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Neuron-glial antigen 2 (NG2, also known as CSPG4) and hyaluronic acid receptor CD44 are chondroitin sulphate proteoglycans actively involved in brain development and its malignant transformation. Here, we aimed to compare prognostic significances of NG2, CD44 and Ki-67 expression in glioblastoma multiforme patients. Totally, 45 tissue samples and 83 paraffin-embedded tissues for 75 patients were analysed. The prognostic values of the genes were analysed using Kaplan-Meier survival curves. Grade III gliomas showed 2-fold difference in NG2 expression between anaplastic astrocytoma and oligoastrocytoma (10.1 ± 3.5 and 25.5 ± 14.5, respectively). For grade IV gliomas, upregulated NG2 expression (21.0 ± 6.8) was associated with poor glioblastoma multiforme prognosis (overall survival < 12 months) compared with glioblastoma multiforme patients with good prognosis (4.4 ± 3.2; overall survival > 12 months). Multivariate survival analysis using Cox proportional hazards model confirmed that high NG2 expression was associated with low survival of the patients (hazard ratio: 3.43; 95% confidence interval: 1.18-9.93; p = 0.02), whereas age (hazard ratio: 1.02; 95% confidence interval: 0.96-1.09; p = 0.42), tumour resection (hazard ratio: 1.03; 95% confidence interval: 0.98-1.08; p = 0.25) and sex (hazard ratio: 0.62; 95% confidence interval: 0.21-1.86; p = 0.40) did not show significant association with prognosis. Although the positive correlation was shown for NG2 and CD44 expression in the glioblastomas (Pearson coefficient = 0.954), Kaplan-Meier and multivariate survival analyses did not revealed a significant association of the increased CD44 expression (hazard ratio: 2.18; 95% confidence interval: 0.50-9.43; p = 0.30) or high Ki-67 proliferation index (hazard ratio: 1.10; 95% confidence interval: 1.02-1.20; p = 0.02) with the disease prognosis. The results suggest that upregulation of NG2/CSPG4 rather than changes in CD44 or Ki-67 expression is associated with low overall survival in glioblastoma multiforme patients, supporting NG2/CSPG4 as a potential prognostic marker in glioblastoma.

Objective: Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis., Study Design: Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both., Results: Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects., Conclusion: These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users., Implications: Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding., (Copyright © 2017. Published by Elsevier Inc.)

Charge impurities and polar molecules on the surface of dielectric substrates has long been a critical obstacle to using graphene for its niche applications that involve graphene's high mobility and high sensitivity nature. Self-assembled monolayers (SAMs) have been found to effectively reduce the impact of long-range scatterings induced by the external charges. Yet, demonstrations of scalable device applications using the SAMs technique remains missing due to the difficulties in the device fabrication arising from the strong surface tension of the modified dielectric environment. Here, we use patterned SAM arrays to build graphene electronic devices with transport channels confined on the modified areas. For high-mobility applications, both rigid and flexible radio-frequency graphene field-effect transistors (G-FETs) were demonstrated, with extrinsic cutoff frequency and maximum oscillation frequency enhanced by a factor of ~2 on SiO2/Si substrates. For high sensitivity applications, G-FETs were functionalized by monoclonal antibodies specific to cancer biomarker chondroitin sulfate proteoglycan 4, enabling its detection at a concentration of 0.01 fM, five orders of magnitude lower than that detectable by a conventional colorimetric assay. These devices can be very useful in the early diagnosis and monitoring of a malignant disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells., (© 2016 The Histochemical Society.)

Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors (i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a "screenable" assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis.

Aim: To investigate whether neuron-glial antigen 2 (NG2) could be an effective prognostic marker in hepatocellular carcinoma (HCC)., Methods: NG2 expression was semi-quantitatively scored from the immunohistochemistry (IHC) data based on the number of positive cells and the staining intensity. A total of 132 HCC specimens and 96 adjacent noncancerous tissue samples were analyzed by IHC for NG2 protein expression. To confirm the NG2 expression levels observed by IHC, we measured NG2 expression in 30 randomly selected tumor and adjacent noncancerous tissue samples by quantitative real-time polymerase chain reaction and Western blot. The correlations between NG2 protein expression and the clinicopathological features of HCC patients were analyzed using the χ (2) test. To assess the prognostic value of NG2 for HCC, the association between NG2 expression and survival was analyzed using the Kaplan-Meier method with the log-rank test. To further evaluate the prognostic value of NG2 expression, a Cox multivariate proportional hazards regression analysis was performed with all the variables to derive risk estimates related to disease-free and overall survival and to control for confounders., Results: High NG2 expression was observed in significantly more primary tumor samples (63.6%; 84/132) compared with the adjacent noncancerous tissue samples (28.1%; 27/96) (P < 0.0001). Moreover, high NG2 protein expression was closely associated with tumor differentiation (χ (2) = 9.436, P = 0.0089), recurrence (χ (2) = 5.769, P = 0.0163), tumor-node-metastasis (TNM) stage (χ (2) = 8.976, P = 0.0027), and invasion (χ (2) = 5.476, P = 0.0193). However, no significant relationship was observed between NG2 protein expression in HCC and other parameters, such as age, sex, tumor size, serum alpha fetoprotein (AFP), tumor number, or tumor capsule. The log-rank test indicated a significant difference in the overall survival of HCC patients with high NG2 expression compared with those with low NG2 expression (29.2% vs 9.5%, P < 0.001). Moreover, NG2 expression in HCC tissue significantly correlated with disease-free survival (15.2% vs 6.7%, P < 0.001). Multivariate analysis showed that NG2 expression (HR = 2.035, P = 0.002), serum AFP (HR = 1.903, P = 0.003), TNM stage (HR = 2.039, P = 0.001), and portal vein invasion (HR = 1.938, P = 0.002) were independent prognostic indicators for OS in HCC patients. Furthermore, NG2 expression (HR = 1.974, P = 0.003), serum AFP (HR = 1.767, P = 0.008), TNM stage (HR = 2.078, P = 0.001), tumor capsule (HR = 0.652, P = 0.045), and portal vein invasion (HR = 1.941, P = 0.002) were independent prognostic indicators for DFS in HCC patients., Conclusion: The up-regulation of NG2 is associated with poor prognosis in HCC. Therefore, NG2 could be useful as an additional prognostic marker to increase the resolution of traditional approaches.

The expression and assembly of the extracellular matrix are profoundly associated with adaptive and pathological responses of the temporomandibular joint (TMJ). To better understand the adaptive responses of the TMJ disc to mechanical loading, we examined the expression of 2 modular proteoglycans and 10 small leucine-rich proteoglycans (SLRPs) at the mRNA and protein levels and determined the contents of proteoglycan-related glycosaminoglycans (GAGs) in rat TMJ discs in response to altered mechanical loading caused by an incisal bite plane. One hundred thirty 7-week-old male Wistar rats were assigned to control and bite plane groups. TMJ disc thickness and the intensity of toluidine blue staining of metachromasia increased in the posterior band after 2 weeks of wearing the bite plane. GAG content increased significantly in the bite plane group after 2 weeks. Quantitative real-time RT-PCR (reverse transcription polymerase chain reaction) analysis indicated that biglycan and chondroadherin mRNA levels increased after 2 weeks and that the level of decorin mRNA increased at 4 weeks. Versican mRNA levels increased after 3 weeks, particularly for the V0 and V1 versican isoforms, which carry more GAG attachment sites than do the V2 and V3 isoforms. Western analysis demonstrated a corresponding increase in the levels of versican, biglycan, and decorin core proteins at 4 weeks in the bite plane group. These results indicate that mechanical loading differentially influences proteoglycan mRNA expression and protein accumulation in the TMJ disc. The change in proteoglycan mRNA and protein levels may lead to the modulation of matrix-matrix and cell-matrix interactions and has important biological significance for adaptation to complicated biomechanical requirements and for tissue maintenance in the TMJ disc., (© International & American Associations for Dental Research 2014.)

Introduction: Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression., Methods: Multiplexed high throughput selected reaction monitoring (SRM) assays were developed to measure tryptic peptides representative of 23 proteins in matched serum and synovial fluid samples from late OA subjects at the time of joint replacement. Subsequently plasma samples from the baseline visit of 173 subjects in an observational OA cohort were tested by SRM for peptides from nine of these proteins: afamin, clusterin, cartilage oligomeric matrix protein, hepatocyte growth factor, kallistatin, insulin-like growth factor binding protein, acid labile subunit, lubricin, lumican, and pigment epithelium-derived factor. Linear regression was used to determine the association between the peptide biomarker level at baseline and change in joint space width (ΔJSW) from baseline to 30 months, adjusting for age and sex., Results: In the matched cohort, 17 proteins could be identified in synovial fluid and 16 proteins were detected in serum. For the progression cohort, the average age was 62 and average ΔJSW over 30 months was 0.68 mm. A high correlation between different peptides from individual proteins was observed, indicating our assays correctly measured their target proteins. Peptides representative of clusterin, lumican and lubricin showed statistically significant associations with joint space narrowing after adjustment for age and sex. Partial R2 values showed clusterin FMETVAEK and lubricin LVEVNPK peptide biomarkers explains about 2 to 3% of the variability of ΔJSW, similar to that explained by age. A biomarker score combining normalized data for both lubricin and clusterin peptides increased the model R2 to 0.079., Conclusions: Our results suggest that when combined, levels of peptides representative of clusterin and lubricin in plasma are as predictive of OA progression as age. Replication of these findings in other prospective OA cohorts is planned.

A full understanding of the key regulators controlling periodontal development and homeostasis is necessary for the design of improved periodontal regenerative therapies. Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules suggested to regulate collagen organization and cell signaling. Mice with double-deficiency of 2 SLRPs, fibromodulin and biglycan (dKO), acquire skeletal abnormalities, but their roles in regulating the periodontium remain undefined and were the focus of our studies. Transmission electron microscopy studies showed abnormal collagen fibrils in the periodontal ligament (PDL) and altered remodeling of alveolar bone in dKO mice. Immunohistochemistry (IHC) revealed increased staining of SLRPs (asporin, lumican, and decorin) and dentin matrix protein-1 (DMP1, a mechanosensory/osteocyte marker), while osteoblast markers, bone sialoprotein and osteopontin, remained unchanged. Disruption of homeostasis was further evidenced by increased expression of receptor-activator of nuclear factor-κB ligand (RANKL) and elevated numbers of osteoclasts, especially noted around the alveolar bone of molars (buccal side) and incisors. Polymerase chain reaction (PCR) array revealed hyperactive transforming growth factors beta/bone morphogenetic protein (TGFβ/BMP) signaling in dKO PDL tissues, which was further confirmed by elevated expression of phosphorylated Smad5 (p-Smad5) by IHC in dKO PDL. These studies highlight the importance of SLRPs in maintaining periodontal homeostasis through regulation of TGFβ/BMP signaling, matrix turnover, and collagen organization., (© International & American Associations for Dental Research.)

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.

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.

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.

Deficits in parvalbumin containing interneurons are a consistent observation in animal models and schizophrenia patients. These neurons are surrounded by chondroitin sulfate proteoglycans, forming perineuronal nets, thought to support the high firing frequencies observed in these neurons. A loss of perineuronal nets has been observed post mortem in human schizophrenia patients, however, whether this contributes to the symptoms of schizophrenia is not known. Here we directly examine the effects of chondroitinase ABC degradation of ventral hippocampal (vHipp) perineuronal nets, and demonstrate that this results in an enhanced hippocampal activity and significant increase in dopamine neuron population activity. In addition, chondroitinase-treated rats display an augmented locomotor response to amphetamine, consistent with the enhanced response to psychomotor stimulants observed in schizophrenia patients. Taken together, these data demonstrate that a loss of vHipp perineuronal nets is sufficient, in and of itself, to induce aberrant hippocampal and dopamine system function consistent with that observed in rodent models and schizophrenia patients.

Hormonal regulation of the dermal collagenous extracellular matrix has a key role in maintaining proper tissue homeostasis. However, the factors and pathways involved in this process are not fully defined. This study investigated the role of estrogen receptors (ERs) in the regulation of collagen biosynthesis in mice lacking either ERα or ERβ. Collagen content was significantly increased in the skin of ERα(-/-) mice, as measured by acetic acid extraction and the hydroxyproline assay, and correlated with the decreased levels of matrix metalloproteinase (MMP)-15 and elevated collagen production by ERα(-/-) fibroblasts. In contrast, collagen content was decreased in the skin of ERβ(-/-) mice, despite markedly increased collagen production by ERβ(-/-) fibroblasts. However, expression of several MMPs, including MMP-8 and -15, was significantly elevated, suggesting increased degradation of dermal collagen. Furthermore, ERβ(-/-) mice were characterized by significantly reduced levels of small leucine proteoglycans, lumican (Lum), and decorin (Dcn), leading to defects in collagen fibrillogenesis and possibly less stable collagen fibrils. ERα(-/-) mice also exhibited fibrils with irregular structure and size, which correlated with increased levels of Lum and Dcn. Together, these results demonstrate distinct functions of ERs in the regulation of collagen biosynthesis in mouse skin in vivo.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population., (Copyright © 2012 International Society for Advancement of Cytometry.)

Changes in expression of neurorepair and neuroregenerative factors were examined after transient cerebral ischemia in relation to the effects of tissue plasminogen activator (tPA) and the free radical scavenger edaravone. Physiological saline or edaravone was injected twice during 90 min of transient middle cerebral artery occlusion (tMCAO) in rats, followed by the same saline or tPA at reperfusion. Sizes of the infarct and protein factors relating to neurorepair and neuroregeneration were examined at 4d after tMCAO. The protein factors examined were: a chondroitin sulfate proteoglycan neurocan, semaphorin type 3A (Sema3A), a myelin-associated glycoprotein receptor (Nogo receptor, Nogo-R), a synaptic regenerative factor (growth associated protein-43, GAP43), and a chemotropic factor netrin receptor (deleted in colorectal cancer, DCC). Two groups treated by edaravone only or edaravone plus tPA showed a reduction in infarct volume compared to the two groups treated by vehicle only or vehicle plus tPA. Immunohistochemistry and western blot analyses indicated that protein expression of neurocan, Sema3A, Nogo-R, GAP43, and DCC was decreased with tPA, but recovered with edaravone. Additive edaravone prevented the reductions of these five proteins induced by tPA. The present study demonstrates for the first time that exogenous tPA reduced protein factors involved in inhibiting and promoting axonal growth, but that edaravone ameliorated such damage in brain repair after acute ischemia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).

Perineuronal nets (PNNs) are specialized assemblies of chondroitin sulfate proteoglycans (CSPGs) in the central nervous system that form a lattice-like covering over the cell body, primary dendrites and initial axon segment of select neuronal populations. PNNs appear to play significant roles in development of the central nervous system, neuronal protection, synaptic plasticity and local ion homeostasis. In seven human brainstems (average age=81 years), we have utilized Wisteria floribunda (WFA) histochemistry and immunocytochemistry for CSPG to map the distribution of PNNs within the nuclei of the human superior olivary complex (SOC). Within the SOC, the majority of net-bearing neurons are situated in the most medially situated nuclei, especially the superior paraolivary nucleus and medial nucleus of the trapezoid body. Net-bearing neurons are consistently found in the ventral nucleus of the trapezoid body and posterior periolivary nucleus, but to a lesser extent in the lateral nucleus of the trapezoid body. Finally, perineuronal nets are typically absent from the lateral and medial superior olives., (2010 Elsevier B.V. All rights reserved.)

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., (Copyright 2010 Mosby, Inc. All rights reserved.)

Objectives: Ineffective surgical and radiotherapy of glioblastoma is mainly due to its intensive infiltrating behavior. Contrarily, brain metastases of anaplastic carcinomas are well-circumscribed intracerebral lesions that can be easily exstirpated in most cases. The molecules of the extracellular matrix (ECM) play a pivotal role in the peritumoral infiltration. In this study the mRNA expression of the ECM components was investigated in two types of intracerebral malignoma with different invasion activity. Our aim was to identify the ECM molecules that are responsible for the different intensity of peritumoral infiltration of tumors from different origin., Methods: The mRNA expression of twenty-three ECM molecules was determined by quantitative reverse transcriptase polymerase chain reaction. Four pieces of glioblastoma and four pieces of intracerebral lung adenocarcinoma metastasis from neurosurgical operation were investigated. Immunohistochemical investigations were performed in case of five molecules., Results: The mRNA expression of nine molecules (brevican, neurocan, neuroglycan-C, syndecan-1,2,4, tenascin-C, versican and matrix-metalloproteinase-[MMP]2) differed significantly by comparison of the two tumor types. By immunohistochemistry, neurocan, syndecan, versican and MMP-2 showed alteration in staining intensity according to the mRNA expression, while MMP-9 showed higher staining intensity in the metastatic tumor., Conclusions: The identified molecules can play an important role in the different infiltration activity of tumors from different origin. Thus these ECM-components could serve as targets for anti-invasion therapy in the future.

Study Design: Variation in abundance and structure of chondroadherin (CHAD) were studied in the extracellular matrix (ECM) of scoliotic and normal human discs., Objective: To determine whether CHAD abundance and fragmentation vary between different sides of the scoliotic disc and between scoliotic and normal discs., Summary of Background Data: Scoliosis involves curvature of the spine including wedging of the intervertebral discs (IVDs), resulting in altered mechanical loading, which can influence cell metabolism and matrix structure in the IVD. A protein such as CHAD that can influence both cell metabolism and ECM organization could influence disc pathology in scoliosis., Methods: IVDs were obtained from patients with scoliosis and from normal individuals. A proteomic analysis was performed to identify molecules that exhibit side-specific variations in abundance. In addition, changes in the abundance and fragmentation of CHAD and other members of the leucine-rich repeat protein family were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Aggrecan fragmentation was used as an indicator of proteinase action., Results: The relative amount of CHAD was consistently lower on the concave side of the discs in all patients studied. In addition, proteolytic degradation of CHAD occurred in some patients with scoliosis, but not in normal IVDs. The presence of aggrecan fragments provided evidence for both aggrecanase and metalloproteinase activity in the scoliotic discs although no side-specific difference was found. Other members of the leucine-rich repeat family of proteins did not show evidence of the same consistent variation in abundance between the 2 sides and did not show signs of degradation., Conclusion: As CHAD can interact with both the ECM and the cells, it can provide a mechanism for regulating cell metabolism and ECM structure, and so play a role in promoting matrix homeostasis. Thus, changes in CHAD abundance or structure could be associated with the pathologic changes occurring in the scoliotic IVD.

A specialised form of extracellular matrix consisting of large aggregating chondroitin sulphate proteoglycans connected to hyaluronan and tenascins, as main components, is termed perineuronal nets. These perineuronal nets surround subpopulations of neurons in many vertebrates including man. In this study we investigated the distribution and the postnatal development of perineuronal nets in the brain of the domestic chicken using immunohistochemical, lectin-histochemical and biochemical methods. Perineuronal nets could be identified very early, already on the first postnatal day throughout various regions and nuclei in chicken fore- and midbrains, most expressively in nidopallium, hyperpallium, lateral striatum, globus pallidus and mesopallium. These mostly delicate, scanty structures around the cell bodies of neurons thicken and complete during the first 2 weeks, however, differ in shape and clearness of contours from the mature form of perineuronal nets found in the adult, 3 year old animals. Perineuronal nets frequently co-localized with the potassium channel subunit Kv3.1b characteristic for fast spiking neurons but remained unrevealed around cholinergic or monoaminergic neurons. The early appearance of perineuronal nets in the precocial birds' brain is probably due to the rapid establishment of neuronal morphology and function which is required for the immediate functional and behavioural performance of chicken.

The evolutionarily conserved cohesin proteins Smc1, Smc3, Rad21 (Mcd1), and Scc3 function in the cohesin complex that provides the basis for chromosome cohesion and is involved in gene regulation. Understanding how these proteins link together the genome requires the use of whole-genome approaches to study the molecular mechanisms of these essential proteins. While chromatin immunoprecipitation followed by DNA microarray (ChIP-chip) studies have provided a snapshot in time of where these proteins associate with various genomes, the cohesin proteins are dynamic in their localization and interactions on chromatin. Study of the dynamic nature of these proteins requires approaches such as live cell imaging. We present evidence from fluorescence loss in photobleaching (FLIP) experiments in budding yeast that the decay constant of each cohesin subunit is approximately 60-90 s in interphase. The decay constant on chromatin increases from G(1) to S phase to metaphase, consistent with the interaction with chromatin becoming more stable once chromosomes are cohered. A small population of Smc3 at a position consistent with centromeric location has a longer decay constant than bulk Smc3. The characterization of the interaction of cohesin with chromatin, in terms of both its position and its dynamics, may be key to understanding how this protein complex contributes to chromosome segregation and gene regulation.

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.

Traumatic injury to the CNS results in peri-wound sprouting without significant axonal growth beyond the lesion edge. We have previously demonstrated that dopaminergic sprouting in the injured striatum follows an increasing gradient of BDNF and GDNF expression, with sprouting ceasing at the point of maximal factor expression. Progressively more complicated associations of sprouting fibers with increasingly activated microglia and macrophages suggest these factors are localized to the cell surface. To establish whether an increased concentration of immobilized BDNF and GDNF could stimulate axonal growth beyond the lesion edge, both factors were covalently attached to 10 microm polycarbonate microspheres. These spheres were implanted into the site of striatal injury 1 week after lesioning. A profusion of axons grew from the region of the lesion edge across the surface of the spheres. Some axons traversed the entire site of injury. Ultrastructural examination demonstrated the juxtaposition of regenerating axons to the surface of implanted spheres. CSPG immunostaining demonstrated that, in animals implanted with neurotrophin-microspheres, axonal growth was induced beyond the area of maximal CSPG reactivity. Surprisingly however, CSPG production at the wound edge was greater in control animals than those implanted with neurotrophin-microspheres. Overall, we show that axonal growth can be encouraged beyond the wound edge by an elevated concentration of immobilized trophic factors. This growth occurs despite the presence of inhibitory CSPGs at the lesion edge. Axonal growth appears to be stimulated mainly via the direct effects of neurotrophins. However, there also appears to be an indirect mechanism whereby neurotrophins reduce the synthesis of CSPG at the wound edge, making the peri-wound environment more permissive.

Background: Proteoglycan (PG) in the extracellular matrix (ECM) of the central nervous system (CNS) may act as a barrier for neurite elongation in a growth tract, and regulate other characteristics collectively defined as structural neural plasticity. Proteolytic cleavage of PGs appears to alter the environment to one favoring plasticity and growth. Brevican belongs to the lectican family of aggregating, chondroitin sulfate (CS)-bearing PGs, and it modulates neurite outgrowth and synaptogenesis. Several ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are glutamyl-endopeptidases that proteolytically cleave brevican. The purpose of this study was to localize regions of adult CNS that contain a proteolytic-derived fragment of brevican which bears the ADAMTS-cleaved neoepitope sequence. These regions were compared to areas of Wisteria floribunda agglutin (WFA) reactivity, a common reagent used to detect "perineuronal nets" (PNNs) of intact matrix and a marker which is thought to label regions of relative neural stability., Results: WFA reactivity was found primarily as PNNs, whereas brevican and the ADAMTS-cleaved fragment of brevican were more broadly distributed in neuropil, and in particular regions localized to PNNs. One example is hippocampus where the ADAMTS-cleaved brevican fragment is found surrounding pyramidal neurons, in neuropil of stratum oriens/radiatum and the lacunosum moleculare. The fragment was less abundant in the molecular layer of the dentate gyrus. Mostly PNNs of scattered interneurons along the pyramidal layer were identified by WFA. In lateral thalamus, the reticular thalamic nucleus stained abundantly with WFA whereas ventral posterior nuclei were markedly immunopositive for ADAMTS-cleaved brevican. Using Western blotting techniques, no common species were reactive for brevican and WFA., Conclusion: In general, a marked discordance was observed in the regional localization between WFA and brevican or the ADAMTS-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.

To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4(+) T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4(+) Th cells from the blood of a healthy donor-recognizing peptide MCSP(693-709) on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4(+) T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.

In the present study, the amounts and the fine structural characteristics of chondroitin sulphate proteoglycans (CSPGs) present in sheep and goat vitreous gels were determined. The results showed that in both examined species hyaluronan was the predominant glycosaminoglycan (GAG), whereas CSPGs were present in minor amounts. CSPGs were identified as versican and collagen IX with versican being the predominant PG type. Fine structural characterization indicated that the CS chains of versican in both mammalian species were of smaller size than those found in collagen IX. The difference in the sulphation pattern of CS chains between versican and collagen IX was also of particular interest. The results indicated that the predominant disaccharide type in CS side chains of versican and collagen IX from both sheep and goat vitreous gels was the 4-sulphated disaccharide. CS chains of versican were found to be richer in 4-sulphated disaccharide units than those in collagen IX, which also contained a significant proportion of non-sulphated disaccharides. These findings showed that, firstly, the CS content and the hydrodynamic size of the CS chain and, secondly, the sulphation pattern of CS chains from versican and collagen IX in both sheep and goat vitreous gels are PG type-dependent., ((c) 2006 John Wiley & Sons, Ltd.)

During endometrial differentiation the extracellular matrix (ECM) changes dramatically to prepare for implantation of the embryo. However, the genes regulating the ECM build-up in the uterine endometrium during early pregnancy are not well known. Using the PCR-select cDNA subtraction method, dermatopontin was identified in the uterus of a pregnant mouse on day 4 of gestation. Dermatopontin mRNA increased dramatically on day 3, and was at its highest level at the time of implantation. Administration of RU 486 significantly inhibited mRNA expression by day 4 of gestation, but ICI 182,780 did not. Progesterone markedly induced dermatopontin expression in ovariectomized uteri within 4 h of administration, whereas estrogen had little effect. In silico analysis revealed progesterone receptor binding sites in the dermatopontin promoter region. Decidualization did not induce expression of dermatopontin; instead dermatopontin mRNA became strongly localized at the interimplantation site. In situ hybridization revealed that expression gradually decreased in the luminal epithelial cells as pregnancy progressed, whereas it increased in the stromal cells. The pattern of localization and the changes of intensity of dermatopontin mRNA coincided with those of collagen. Collectively, these results strongly suggest that dermatopontin expression is steroid-dependent. They also suggest that, at the time of implantation, dermatopontin expression is primarily regulated spatio-temporally by progesterone via progesterone receptors, and is modulated by the decidual response during implantation. Dermatopontin may be one of the regulators used to remodel the uterine ECM for pregnancy.

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times., ((c) 2006 Wiley-Liss, Inc.)

Objective: Fibronectin fragments are present at high concentrations in the cartilage of patients with rheumatoid arthritis and patients with osteoarthritis (OA) and have been shown to promote cartilage catabolism in human cartilage cultures, suggesting that fibronectin fragments participate in the initiation and progression of arthritic disease. This study was undertaken to 1) identify the major fibronectin fragments in human OA cartilage and confirm their ability to elicit cartilage catabolism, 2) identify the cleavage sites in fibronectin and generate the corresponding neoepitope antibodies, and 3) explore the utility of fibronectin neoepitopes as biomarkers., Methods: Fibronectin fragments were purified from human OA cartilage using affinity chromatography; their N-termini were then identified by sequencing. Bovine nasal cartilage was treated with affinity-purified fibronectin fragments and assayed for aggrecan breakdown by monitoring the release of glycosaminoglycans and the aggrecan neoepitope 1771AGEG. Fibronectin neoepitopes were detected by Western blotting in cytokine-treated media of human cartilage explants, and by immunohistochemical analyses of human OA cartilage., Results: Multiple fibronectin fragments were isolated from human OA cartilage, and all contained the N-terminus 272VYQP. These fragments induced aggrecanase-mediated cartilage catabolism in bovine cartilage explants. Fibronectin fragments with the N-terminus 272VYQP and fragments with the C-terminus VRAA271 were detected following cytokine treatment of human cartilage extracts. These neoepitopes localized with areas of aggrecan loss in OA cartilage., Conclusion: Human OA cartilage contains fibronectin fragments with catabolic activity and a major cleavage site within fibronectin. This study is the first to characterize fibronectin neoepitopes in OA cartilage, suggesting that they may represent a novel biomarker of arthritis.

Objectives: Previously, we demonstrated rapid changes in temporomandibular joint (TMJ) disk gene expression during monolayer expansion. This study's objective was to investigate the ability of pellet culture and growth factors to rescue TMJ disk gene expression changes., Design: Temporomandibular joint disk cells were isolated from mature porcine tissue and passaged up to five times. At each passage, 300 000 cells were placed in a monolayer or pellet culture environment before being exposed to transforming growth factor-beta 3 (TGF-beta3) (5 ng/ml), TGF-beta1 (5 ng/ml), and insulin-like growth factor I (IGF-I) (10 ng/ml)., Outcome Measure: After 24 h, gene expression was analyzed via reverse transcriptase-polymerase chain reaction (RT-PCR)., Results: Pelleting was detrimental to TMJ disk gene expression, marked by gene expression decreases in collagen type I (5.5-fold), aggrecan (1.4-fold), decorin (0.73-fold), and biglycan (0.73-fold) relative to monolayer cultures. IGF-I, TGF-beta1, and TGF-beta3 demonstrated limited ability to rescue TMJ disk gene expression in the pellet culture. In monolayer, TGF-beta3 and TGF-beta1 increased decorin and biglycan gene expression relative to passaged controls. Collagen type I expression, the TMJ disk's primary matrix constituent, was highest in TGF-beta3 cultures; however, differences were not statistically significant., Conclusion: These results indicate that pellet cultures are a poor choice for TMJ disk tissue engineering, and the effects of TGF-beta1, TGF-beta3, and IGF-I on TMJ disk gene expression are minimal relative to passaging and pelleting effects.

Dermatopontin is a tyrosine-rich acidic extracellular matrix protein of 22 kDa with possible functions in cell-matrix interactions and matrix assembly. We have previously isolated mRNAs expressed in hormone-refractory, but not in hormone-sensitive, mouse mammary cancer by comparing the mRNAs expressed in either tumor. A partial mRNA sequence isolated was later proven to be a part of mouse dermatopontin mRNA sequence. Transfectants of mouse dermatopontin cDNA into PC-3 human prostate cancer cells enhanced tumor growth when those were implanted subcutaneously in nude mice compared with the controls. Those transfectants showed a prominent stroma compared with the controls. Localization of the targets of a fusion protein of mouse dermatopontin and alkaline phosphatase was in the stroma of the PC-3 tumor tissues, but not in the tumor cells themselves. Additionally, we have established mouse dermatopontin transgenic mice under the control of the rat probasin promoter. The prostatic dorsal lobes of dermatopontin transgenic mouse showed prostate intra-epithelial neoplasia at the age of 11 months, but the control littermates did not. Epithelium of other prostatic lobes was not markedly different from that of the controls. In conclusion, dermatopontin may be involved in the pathogenesis and growth of the prostate cancer.

We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.

The cartilage tissue has a limited self-regenerative capacity. Tissue-engineering represents a promising trend for cartilage repair. The present study was aimed to develop a biomaterial formulation by combining fragments of chitosan hydrogel with isolated rabbit or human chondrocytes. We first reported the properties of the constructs elaborated with rabbit chondrocytes and pure chitosan physical hydrogels with defined molecular weight, acetylation degree and polymer concentration. Morphological data showed that chondrocytes were not penetrating the hydrogels but tightly bound to the surface of the fragments and spontaneously formed aggregates of combined cell/chitosan. A significant amount of neo-formed cartilage-like extracellular matrix (ECM) was first accumulated in-between cells and hydrogel fragments and furthermore was widely distributed within the neo-construct. The optimal biological response was obtained with hydrogel fragments concentrated at 1.5% (w/w) of polymer made from a chitosan with a degree of acetylation between 30 and 40%. Such hydrogels were then mixed with human chondrocytes. The phenotype of the cells was analyzed by using chondrocytic (mRNA expression of mature type II collagen and aggrecan as well as secretion of proteoglycans of high molecular weight) and non chondrocytic (mRNA expression of immature type II collagen and type I collagen) molecular markers. As compared with human chondrocytes cultured without chitosan hydrogel which rapidly dedifferentiated in primary culture, cells mixed with chitosan rapidly loose the expression of type I and immature type II collagen while they expressed mature type II collagen and aggrecan. In these conditions, chondrocytes maintained their phenotype for as long as 45 days, thus forming cartilage-like nodules. Taken together, these data suggest that a chitosan hydrogel does not work as a scaffold, but could be considered as a decoy of cartilage ECM components, thus favoring the binding of chondrocytes to chitosan. Such a biological response could be described by the concept of reverse encapsulation.

Increased intra-carpal-tunnel pressure due to swelling of the flexor tenosynovium is the most probable pathological mechanism of idiopathic carpal tunnel syndrome (CTS). To clarify the role of tenascin-C and PG-M/versican, which have often been found to be involved in tissue remodeling and vascular stenosis in the pathogenesis of CTS, we histologically and biochemically examined the production of extracellular matrix in the flexor tenosynovium from 40 idiopathic CTS patients. Tenascin-C was temporarily expressed in the vessel wall, synovial lining and fibrous tissue, with expression regulated differently in each tissue. Tenascin-C expression by vessels correlated with disease duration and appeared to be involved in vascular lesion pathology. Morphometric analysis showed that tenascin-C expression by small arteries is correlated with PG-M/versican expression in surrounding connective tissue. PG-M/versican was also present at the neointima of severely narrowed vessels. Although tenascin-C expression by synovial lining and connective tissue shows marked regional variation and seems inconsistent, in vitro examination suggested that tenascin-C production by these tissues is regulated in response to mechanical strain on the flexor tenosynovium.

Biomarkers of skeletal turnover, such as the synthesis and degradation of extracellular matrix molecules in specific tissues, offer the opportunity to gain new insights into spinal pathology and treatment. The creation, use, and interpretation of these analytical body-fluid measures of process (rather than outcome) require a clear understanding of the nature of the molecules and events being measured. This review provides examples of how protein and carbohydrate assays of biomarkers can be used to measure the contribution from the intervertebral discs and vertebrae of the spine. With regard to spinal degeneration and ankylosing spondylitis, these investigations are providing important new information, in weeks rather than years, on the response of patients to treatment.

Objective: Articular chondrocyte senescence is responsible, at least in part, for the increased incidence of osteoarthritis (OA) with increased age. Recently, it was suggested that caveolin 1, a 21-24-kd membrane protein, participates in premature cellular senescence. Caveolin 1 is the principal structural component of caveolae, vesicular invaginations of the plasma membrane. This study was undertaken to investigate whether the catabolic factors oxidative stress and interleukin-1beta (IL-1beta) induce features of premature senescence of articular chondrocytes through up-regulation of caveolin 1 expression., Methods: Caveolin 1 expression was investigated in human OA cartilage by real-time polymerase chain reaction and in rat OA cartilage by immunohistologic analysis. We studied whether IL-1beta and H2O2 induce caveolin 1 expression in OA chondrocytes and analyzed the relationship between cellular senescent phenotypes and caveolin 1 expression in human chondrocytes., Results: In human and rat OA articular cartilage, caveolin 1 positivity was associated with cartilage degeneration. Both IL-1beta and H2O2 up-regulated caveolin 1 messenger RNA and protein levels, and both treatments induced marked expression of senescent phenotypes: altered cellular morphology, cell growth arrest, telomere erosion, and specific senescence-associated beta-galactosidase activity. Caveolin 1 overexpression induced p38 MAPK activation and impaired the ability of chondrocytes to produce type II collagen and aggrecan. In contrast, down-regulation of caveolin 1 with antisense oligonucleotide significantly inhibited the features of chondrocyte senescence induced by catabolic factors. Caveolin 1 induction and stresses with both IL-1beta and H2O2 up-regulated p53 and p21 and down-regulated phosphorylated retinoblastoma (Rb), suggesting that the p53/p21/Rb phosphorylation pathway, as well as prolonged p38 MAPK activation, may mediate the features of chondrocyte senescence induced by stress., Conclusion: Our findings suggest that IL-1beta and oxidative stress induce features of premature senescence in OA chondrocytes, mediated, at least in part, by stress-induced caveolin 1 expression. This indicates that caveolin 1 plays a role in the pathogenesis of OA via promotion of chondrocyte down-regulation.

To investigate the origin and postnatal changes of mouse mandibular angular cartilage, in situ hybridization for cartilaginous marker proteins, histochemistry for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), and bromodeoxyuridine (BrDU) analyses were performed. Chondrocytes of the mandibular angular cartilage were derived from ALP-positive progenitor cells and first detected at embryonic day (E) 15.5. Newly formed chondrocytes rapidly differentiated into hypertrophic chondrocytes and hypertrophic cell zone rapidly extended in subsequent a few days. During this period, bone sialoprotein mRNA was more widely expressed than osteopontin mRNA in cartilage. Endochondral bone formation started at E 17.5 with the resorption of the bone collar by osteoclasts. These characteristics were consistent with those of the condylar cartilage, although developmental process was 0.5-1.5 day delayed relative to the condylar cartilage. During the postnatal period, contrast to the condylar cartilage, the angular cartilage constantly decreased in volume with advancing age. Reduction of proliferating activity estimated by BrDU incorporation accounts for this phenomenon. We demonstrate new structural features of the mandibular angular cartilage that may contribute to a coming research for the secondary cartilage.

Objective: The vascular extracellular matrix (ECM) can affect smooth muscle cell (SMC) adhesion, migration and proliferation-events that are important during the atherosclerotic process. Fibulin-2 is a member of the ECM protein family of fibulins and has been found to cross-link versican/hyaluronan complexes, an ECM network that has been suggested to be important during tissue repair. In this study we have analysed the presence of fibulin-2 in two different models of murine vascular lesions. We have also examined how the fibulin-2/versican network influences SMC migration., Methods: Presence of fibulin-2 was analysed by immunohistochemistry in atherosclerotic aortas and in mechanically injured carotid arteries from mice. Fibulin-2 protein levels were also studied by Western blotting during rat aortic SMC phenotypic modulation in vitro. The importance of a fibulin-2/versican interaction for SMC migration was studied in the presence of two inhibiting peptides (FN III 3-5 and aggrecan C-type lectin-like domain)., Results: Fibulin-2 is expressed in SMC rich regions of atherosclerotic lesions where it colocalises with versican and hyaluronan. It is also present in injury-induced vascular lesions and is upregulated during SMC phenotypic modulation in cell culture. Moreover, treatments with peptides that block the interaction between versican and fibulin-2 inhibit SMC migration in vitro., Conclusions: Fibulin-2 can be produced by SMC as a response to injury and may participate in the ECM organisation that regulates SMC migration during vessel wall repair.

Objective: Injury to the anterior cruciate ligament (ACL) frequently leads to post-traumatic osteoarthritis (OA). In this study we determined whether early degenerative changes characteristic of idiopathic OA are induced in articular cartilage following ACL injury., Methods: A small sample of femoral articular cartilage was removed at surgery, as part of ACL reconstruction, from a total of 50 patients with ACL injuries. Of these, 28 underwent surgery less than 1 year post-injury. Control cartilages were obtained from the same site from 21 persons at autopsy. All cartilages were examined for molecular changes. The content of type II collagen, its cleavage by collagenases and its denaturation were determined by immunoassay. The total content of glycosaminoglycan (GAG), which is principally aggrecan, was measured colorimetrically. Data were expressed per unit DNA (GAG and collagen content) or as a percentage of total collagen cleaved or denatured. Other cartilages from the same site (8 controls, 12 less than 1 year and 8 more than 1 year post-injury) were frozen sectioned and examined histologically to determine by Mankin grading cartilage degeneration., Results: Histological analyses revealed that control subjects exhibited staining for proteoglycan, which was reduced in some patients following ACL rupture. Degeneration of the articular surface was sometimes observed 1 year after ACL rupture. Although the Mankin grade increased with time after rupture these changes were not significant. Immunoassays, however, revealed an increase in GAG content within 1 year which was maintained after 1 year although no longer significant. No changes in total type II collagen content were observed during the period of study. However, there were significant increases in the denaturation and cleavage of type II collagen less than and more than 1 year post-ACL rupture. Total type II collagen content was directly correlated with GAG content in all three groups, with the significance being weakest at more than 1 year. After 1 year an inverse correlation was observed between total type II collagen content and collagen cleavage as well as denaturation., Conclusions: These observations reveal that joint instability resulting from ACL injury rapidly results in degenerative changes characteristic of those seen in idiopathic OA at arthroplasty and in experimental OA following ACL surgery. These changes may contribute to the development of post-traumatic OA that is commonly observed following ACL injury. The observations support and extend conclusions from other studies on human and animal articular cartilage and synovial fluids post-ACL injury that have revealed a rapid onset of damage to type II collagen and an initial increase in proteoglycan content characteristic of experimental OA post-ACL injury. This study provides direct evidence for the rapid development of degenerative changes characteristic of OA following ACL injury.

Objective: To identify the major aggrecanase- and matrix metalloproteinase (MMP)-generated aggrecan fragments in human osteoarthritis (OA) synovial fluid and in human OA joint cartilage., Method: Aggrecan fragments were prepared by CsCl gradient centrifugation. Fragment distributions were compared with aggrecanase-1 (ADAMTS-4) and MMP-3 digested human aggrecan by analysis with neoepitope antibodies and an anti-G1 domain antibody, using Western immuno-blots., Results: The overall fragment pattern of OA synovial fluid aggrecan was similar to the fragment pattern of cartilage aggrecan cleaved in vitro by ADAMTS-4. However, multiple glycosaminoglycan (GAG) containing aggrecanase and MMP-generated aggrecan fragments were identified in OA synovial fluid and some of these fragments were produced by the action of both types of proteinases. The synovial fluid content of large size aggrecan fragments with (374)ARGS- and (342)FFGV- N-terminals was about 107 and 40 pmoles per ml, respectively, out of a total concentration of aggrecan fragments of about 185 pmoles per ml. OA synovial fluid contained insignificant amounts of the G1-IPEN(341) fragment as compared to the G1-TEGE(373) fragment, while OA cartilage contained significant amounts of both fragments. OA cartilage contained several GAG-containing aggrecan fragments with N-terminals of G1- or (342)FFGV- but no fragments with an N-terminal of (374)ARGS-., Conclusions: The overall pattern of aggrecan fragments in human OA synovial fluid and cartilage supports an important role for aggrecanase in aggrecan degradation. However, the fragment patterns and their differential distribution between cartilage and synovial fluid are consistent with the existence of at least two proteolytic pathways for aggrecan degradation in human OA, generating both (342)FFGV- and (374)ARGS-fragments.

Unlabelled: Joint instability was believed to be the main cause of osteoarthritis following non-fracture articular trauma. However, sudden high impact load through articular cartilage onto subchondral bone may also cause osteoarthritic changes., Objective: We asked whether early osteoarthritic changes following transarticular impact may be depicted using immunofluorescence on unfixed cryosections to contribute to a more detailed understanding of degenerative processes of joint impaction., Design: Transarticular impacts were applied to patellofemoral joints of 12 skeletally mature beagle dogs (age: 15-16 months) using a drop tower. Biopsies of impact areas were sampled after 6 months and processed for standard light microscopy on formalin-fixed sections and for immunofluorescence for collagen type I (col I), type II (col II) and aggrecan (AC) on unfixed cryosections. Gross morphology and immunofluorescence on cryosections were documented using a semi-quantitative scaling system, compared to healthy controls and to standard light microscopy., Results: Four biopsies showed almost entirely fibrocartilaginous morphology, four appeared to be of preserved hyaline morphology with only minor signs of fibrocartilaginous remodelling and four showed preserved hyaline appearance. We found decrease in col II and AC expression in highly degenerative specimens as well as increase of col I expression. Increased col I expression in the pericellular matrix could even be depicted in specimens with intact hyaline morphology., Discussion/conclusion: Observations suggest that joint impaction causes early osteoarthritic changes after 6 months. Collagen network disruption seems to lead to AC loss, although other researchers found isolated AC loss without denaturation of col II using immunofluorescence in formalin-fixed specimens. This is the first study on effects of transarticular impact using immunofluorescence on unfixed cryosections.

Aim: Chondroblastoma and clear cell chondrosarcoma are uncommon skeletal neoplasms that have a strong tendency to involve the epiphysis of long bones. They also share some overlapping histological features. Thus, it can be difficult both radiographically and histologically to distinguish these neoplasms. So far there are no immunohistochemical markers available that have been shown to be helpful in differentiating these neoplasms., Methods: In our study of a series of clear cell chondrosarcomas (n = 15) and chondroblastomas (n = 35), S100, vimentin, aggrecan and collagen type II were detected by immunohistochemistry., Results: We detected immunohistochemical evidence of type II collagen within both the extracellular matrix-rich (chondroid) and matrix-poor areas in all 15 cases of clear cell chondrosarcoma. In contrast, immunohistochemical analysis failed to show staining of collagen type II in any of the 35 chondroblastomas. Other markers, including S100 protein, vimentin and aggrecan proteoglycan were tested in parallel and found to be focally positive in both neoplasms., Conclusion: Therefore, our data show that in cases when clear cell chondrosarcoma and chondroblastoma pose a diagnostic challenge, the presence of type II collagen in the extracellular tumour matrix significantly supports the diagnosis of clear cell chondrosarcoma and aids in distinguishing it from chondroblastoma.