Chondroitin sulfate proteoglycan (CSPG) inhibits neurite outgrowth of various neuronal cell types, and CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK pathway. Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and have been shown to differentiate into neuron-like cells by co-treatment with the ROCK inhibitor Y27632 and the hypoxia condition mimicking agent CoCl2. In this study, we addressed the hypothesis that a ROCK inhibitor might be beneficial to regenerate neurons during stem cell therapy by preventing transplanted MSCs from inhibition by CSPG in damaged tissues. Indeed, dose-dependent inhibition by CSPG pretreatment was observed during morphological changes of Wharton's jelly-derived MSCs (WJ-MSCs) induced by Y27632 alone. The formation of neurite-like structures was significantly inhibited when WJ-MSCs were pre-treated with CSPG before induction under Y27632 plus CoCl2 conditions, and pretreatment with a protein kinase C inhibitor reversed such inhibition. However, CSPG treatment resulted in no significant inhibition of the WJ-MSC morphological changes into neuron-like cells after initiating induction by Y27632 plus CoCl2. No marked changes were detected in expression levels of neuronal markers induced by Y27632 plus CoCl2 upon CSPG treatment. CSPG also blocked the morphological changes of human bone marrow-derived MSCs into neuron-like cells under other neuronal induction condition without the ROCK inhibitor, and Y27632 pre-treatment blocked the inhibitory effect of CSPG. These results suggest that a ROCK inhibitor can be efficiently used in stem cell therapy for neuronal induction by avoiding hindrance from CSPG.
Anoxia ; Chondroitin Sulfate Proteoglycans* ; Chondroitin Sulfates* ; Chondroitin* ; Humans ; Neurites ; Neurons ; Protein Kinase C ; Stem Cells

STUDY DESIGN: Examination of hyaluronidase-4 (Hyal-4) expression in a rat spinal cord hemisection model. PURPOSE: To determine the status of Hyal-4 expression after hemisection of the spinal cord, and the relationship between its expression and that of chondroitin sulfate proteoglycans (CSPGs). OVERVIEW OF LITERATURE: CSPGs are expressed at the site of spinal cord injury and inhibit axon regeneration. Administration of exogenous chrondroitinase ABC (ChABC), derived from bacteria, digested CSPGs and promoted axonal regrowth. Using a rat hemisection model, we have demonstrated peak CSPGs levels at by 3 weeks after injury but then decreased spontaneously. Could there be an endogenous enzyme similar to ChABC in the spinal cord? It has been suggested that Hyal-4 is involved in CSPG degradation. METHODS: A rat hemisection model was prepared and spinal cord frozen sections were prepared at 4 days and 1, 2, 3, 4, 5, and 6 weeks post-cordotomy and stained for CSPGs and Hyal-4 and subjected to Western blotting. RESULTS: CSPGs appeared at the injury site at 4 days after hemisection, reached a peak after 3 weeks, and then decreased. Hyal-4 was observed around the injury site from 4 days after cordotomy and increased until after 5-6 weeks. Double staining showed Hyal-4 around CSPGs. Western blotting identified a band corresponding to Hyal-4 from 4 days after hemisection. CONCLUSIONS: Hyal-4 was expressed in a rat hemisection model in areas surrounding CSPGs, and as its peak was delayed compared with that of CSPGs. These results suggest the involvement of Hyal-4 in the digestion of CSPGs.
Animals ; Axons ; Bacteria ; Blotting, Western ; Chondroitin Sulfate Proteoglycans ; Cordotomy ; Digestion ; Frozen Sections ; Hyaluronoglucosaminidase ; Rats* ; Regeneration ; Spinal Cord Injuries ; Spinal Cord*

Perineuronal nets (PNNs) are reticular structures resulting from the aggregation of extracellular matrix (ECM) molecules around the cell body and proximal neurite of specific population of neurons in the central nervous system (CNS). Since the first description of PNNs by Camillo Golgi in 1883, the molecular composition, developmental formation and potential functions of these specialized extracellular matrix structures have only been intensively studied over the last few decades. The main components of PNNs are hyaluronan (HA), chondroitin sulfate proteoglycans (CSPGs) of the lectican family, link proteins and tenascin-R. PNNs appear late in neural development, inversely correlating with the level of neural plasticity. PNNs have long been hypothesized to play a role in stabilizing the extracellular milieu, which secures the characteristic features of enveloped neurons and protects them from the influence of malicious agents. Aberrant PNN signaling can lead to CNS dysfunctions like epilepsy, stroke and Alzheimer's disease. On the other hand, PNNs create a barrier which constrains the neural plasticity and counteracts the regeneration after nerve injury. Digestion of PNNs with chondroitinase ABC accelerates functional recovery from the spinal cord injury and restores activity-dependent mechanisms for modifying neuronal connections in the adult animals, indicating that PNN is an important regulator of neural plasticity. Here, we review recent progress in the studies on the formation of PNNs during early development and the identification of CSPG receptor - an essential molecular component of PNN signaling, along with a discussion on their unique regulatory roles in neural plasticity.
Animals ; Central Nervous System ; physiology ; physiopathology ; Chondroitin Sulfate Proteoglycans ; Extracellular Matrix ; physiology ; Humans ; Neuronal Plasticity ; Neurons ; Receptors, Cell Surface ; physiology

PURPOSE: To determine the effect of methylprednisolone (MP) and riluzole administration on axonal growth after spinal cord injury (SCI) in rats. MATERIALS AND METHODS: Three Sprague Dawley rats (SD rat) served as controls (average 24 weeks of age) and 24 SCI SD rats scoring below 7 points on on Basso, Beattie, and Bresnahan open field test served as test subjects (total 27 SD rats; mean weight 581 g, range=427-613 g). Test subjects were divided into two groups of 12 subjects each. Group I was injected with saline (1 ml/kg) and group II was injected with MP (300 mg/kg) and riluzole (5 mg/kg) intraperitoneally. Four SD rats were sacrificed in each group at the following time points after SCI: days 1, 4, and 7. We completed behavioral testing, immunohistochemical staining and RT-PCR for chondroitin sulfate proteoglycans (CSPG), and microarrays for c-JUN, ATF-2, p53, and Elk-1. RESULTS: On behavioral testing, group II showed superior results at only day 4 after SCI (p<0.05). On RT-PCR for CSPG, optical densities were 2.06 (ratio=Group I/Group II) and 2.11 at days 4 and 7, respectively. Microarray showed that lower expression of c-JUN in group II during the entire period (p< 0.05). ATF-2 showed lower expression in group II at days 4 and 7 (p<0.05). p53 showed lower expression in group I at day 1 (p<0.05). Elk-1 showed lower expression in group I at day 1 (p<0.05) and in group II at day 7 (p<0.05). CONCLUSION: Simultaneous administration of MP and riluzole led to various changes in the MAPK pathway, and decreased CSPG. Therefore, this method has a protective effect on axonal regeneration after SCI in an SD rat model.
Animals ; Axons ; Chondroitin Sulfate Proteoglycans ; Methylprednisolone ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Riluzole ; Spinal Cord ; Spinal Cord Injuries

PURPOSE: To investigate the characteristics of cultured rabbit corneal keratocytes in vitro and evaluate the possibility of differentiation of mesenchymal stem cells to keratocytes using the keratocyte conditioned medium (KCM). METHODS: Isolated keratocytes were seeded on the stromal side of amniotic membranes (AM) or plastic dishes, and morphologic changes were evaluated. Rabbit mesenchymal stem cells were cultured on AM with alpha-MEM (minimum essential medium alpha) and KCM. The gene expression patterns of specific keratocyte markers (keratocan, lumican, and aldehyde dehydrogenase family, member A1 (ALDH1A1)) of cultured cells were evaluated by RT-PCR. RESULTS: Keratocytes on AM showed dendritic morphology with slow proliferation in contrast, cells on dishes were stellate in shape with fast proliferation. Cultured keratocytes on AM maintained the expression of keratocan, lumican and ALDH1A1 while keratocytes on plastic dishes steadily lost their keratocyte marker gene expression. Additionally, mesenchymal stem cells cultured with KCM on AM induced expression of keratocan and ALDH1A1. CONCLUSIONS: Keratocytes cultured on AM stromal matrix maintained their characteristic morphology and marker gene expression. Morphology changes and marker gene expressions of mesenchymal stem cells suggest an ability to differentiate into keratocytes when grown on AM with KCM.
Aldehyde Dehydrogenase ; Amnion ; Cells, Cultured ; Chondroitin Sulfate Proteoglycans ; Corneal Keratocytes ; Culture Media, Conditioned ; Gene Expression ; Humans ; Keratan Sulfate ; Mesenchymal Stromal Cells ; Organic Chemicals ; Plastics ; Seeds

Orientia tsutsugamushi is obligate intracellular bacterium that grows within the cytoplasm of the eukaryotic host cells. Therefore capability of the attachment, entry into the host cell and intracellular survival should be critical process for oriential infection. In this study we investigated the cellular invasion mechanism of Orientia tsutsugamushi and the role of transmembrane heparan sulfate proteoglycan, which binds diverse components at the cellular microenvironment and is implicated as host cell receptors for a variety of microbial pathogens. First of all Orientia tsutsugamushi can invade a wide range of nonprofessional phagocytic cells including fibroblast, epithelial cells a#nd endothelial cells of various host species, including B and T lymphocytes. Thus, it was postulated that the attachment of O. tsutsugamushi requires the recognition of ubiquitous surface structures of many kinds of host cells. Treatments with heparan sulfate and heparin inhibited the infection of Orientia tsutsugamushi in dose-dependent manner for L cell, mouse fibroblast, whereas other glycosaminoglycans such as chondroitin sulfate had no effect. Collectively, these findings provide strong evidence that initial interaction with heparan sulfate proteoglycan is required for the oriential invasion into host cells.
Animals ; Cellular Microenvironment ; Chondroitin Sulfates ; Cytoplasm ; Endothelial Cells ; Epithelial Cells ; Eukaryotic Cells* ; Fibroblasts ; Glycosaminoglycans ; Heparan Sulfate Proteoglycans ; Heparin ; Heparitin Sulfate ; Mice ; Orientia tsutsugamushi* ; Phagocytes ; T-Lymphocytes

OBJECTIVE: To develop an in vitro model analogous to the environment of traumatic spinal cord injury (SCI), the authors evaluated change of astrogliosis following treatments with kainate and/or scratch, and degree of neurite outgrowth after treatment with a kainate inhibitor. METHODS: Astrocytes were obtained from the rat spinal cord. Then, 99% of the cells were confirmed to be GFAP-positive astrocytes. For chemical injury, the cells were treated with kainate at different concentrations (10, 50 or 100 µM). For mechanical injury, two kinds of uniform scratches were made using a plastic pipette tip by removing strips of cells. For combined injury (S/K), scratch and kainate were provided. Cord neurons from rat embryos were plated onto culture plates immediately after the three kinds of injuries and some cultures were treated with a kainate inhibitor. RESULTS: Astro-gliosis (glial fibrillary acidic protein [GFAP], vimentin, chondroitin sulfate proteoglycan [CSPG], rho-associated protein kinase [ROCK], and ephrin type-A receptor 4 [EphA4]) was most prominent after treatment with 50 µM kainate and extensive scratch injury in terms of single arm (p<0.001) and in the S/K-induced injury model in view of single or combination (p<0.001). Neurite outgrowth in the seeded spinal cord (β-III tubulin) was the least in the S/K-induced injury model (p<0.001) and this inhibition was reversed by the kainate inhibitor (p<0.001). CONCLUSION: The current in vitro model combining scratch and kainate induced glial scarring and inhibitory molecules and restricted neurite outgrowth very strongly than either the mechanically or chemically-induced injury model; hence, it may be a useful tool for research on SCI.
Animals ; Arm ; Astrocytes ; Chondroitin Sulfate Proteoglycans ; Cicatrix* ; Embryonic Structures ; In Vitro Techniques ; Kainic Acid* ; Neurites ; Neuroglia ; Neurons ; Plastics ; Protein Kinases ; Rats ; Spinal Cord Injuries ; Spinal Cord* ; Vimentin

Adenoid cystic carcinoma is malignant tumor in salivary gland, and its behavior is very invasive. Of all malignant tumor adenoid cystic carcinoma is occured in frequency of 4.4% in major salivary gland, and 1.29% in minor salivary gland. Histopathologically, adenoid cystic carcinoma is characterized by a cribriform appearance, and tubular form and solid nest type tumor can be seen. The tumor cell structure composed of modified myoepithelial cell, and basaloid cell. Extracellular matrix of this tumor cell contains variable ground substance with basement membrane component. Basement membrane matrix composed of collagen fibers, glycoproteins, proteoglycans, and its function is well known that it participate in differentiation, proliferation, and growth of tumor cell. Basement membrane molecule is essential for invasion of peripheral nerve, blood vessel, skeletal muscle in tumor cell of adenoid cystic carcinoma. In many studies, the tumor cell of adenoid cystic carcinoma containing modified myoepithelial cell participate in synthesis of proteoglycan. In this study, tissue sample of adenoid cystic carcinoma of human salivary gland were obtained from 15 surgical specimen, and all specimen were routinely fixed in 10% formalin and embedded. Serial 4-micrometer thick sections were cut from paraffin blocks. the histopathologic evaluation was done with light microscopy. And, the immunohistochemical staining, characteristics of glycosaminoglycan were observed. For biochemical analysis of glycosaminoglycan, isolation of crude glycosaminoglycan from tumor tissue and Western bolt analysis were carried out. With transmission electomicroscopy, tumor cell were observed. Biologic behavior of adenoid cystic carcinoma was observed with distribution and expression of basement membrane of glycosaminoglycan in tumor cells, The results obtained were as follows: 1. In immunohistochemical study, chondroitin sulfate is postively stained in tumor cell and interstitial space, dermatan sulfate is weakly stained in ductal cell. But keratan sulfate is negatively stained. 2. In immunohistochemical study, heparan sulfate is strong positive stained in tumor cell and basement membrane, especially in invasion area to peripheral nerve tissue. 3. In transmission electromicroscpic view, the tumor cells are composed modifed myoepithelial cells, and contains many microvilli and rough endoplasmic reticulum. 4. In Western blot analysis, the expression of glycosaminoglycan is expressed mostly in heparan sulfate. From the results obtained in this study, tumor cell of adenoid cystic carcinoma is composed modified myoepithelial cell, and glycosaminoglycan of basement membrane molecule of heparan sulfate and chondroitin sulfate mostly participate in the development and invasiveness of adenoid cystic carcinoma by immunohistochemical study and western blot analysis.
Adenoids* ; Basement Membrane ; Blood Vessels ; Blotting, Western ; Carcinoma, Adenoid Cystic* ; Chondroitin Sulfates ; Collagen ; Dermatan Sulfate ; Endoplasmic Reticulum, Rough ; Extracellular Matrix ; Formaldehyde ; Glycoproteins ; Heparitin Sulfate ; Humans ; Keratan Sulfate ; Microscopy ; Microvilli ; Muscle, Skeletal ; Paraffin ; Peripheral Nerves ; Proteoglycans ; Salivary Glands ; Salivary Glands, Minor

BACKGROUND: The extracellular matrix constitutes the dermis supporting an overlying epidermal lining. A small portion of the cell component includes fibroblasts and mast cells, a large amount of fibrous tissue as struct ural proteins, intra-and extracellular adhesive molecules, and extracellular macromolecules, glyccsarninoglycan(GAG) and proteoglycan make up the dermal connective tissue. GAGs, polysacchavide chains secreted by fibroblasts, are the main macromolecules of the extracellular matrix. GAG chains occupy large amounts of extracellular space and form hydrated gels. The various manifestations observed during the cutaneous intrinsic aging process reflect the age-related changes of dermal connective tissue. OBJECTIVE: This study was directed to observe the age-related changes of GAG in human abdominal skin without the influences of photoaeging. MaTERIALS AND METHODS: The subjects involved in this study were grouped according to the age span of 10 years. A minimum of 3 skin samples were included in each age group. After permission, three to five grams of skin samples without known underlying skin diseases were obtained from the pat,ients who were subjected to open abdominal surgery under general anesthesia. GAGs were extracted and purified by repeated cetylpyridium chloride-ethanol precipitation from each skin sample. The qualitative and quantitative analysis of GAGs was carried out with the action of specific GAG degradirig enzymes. RESULTS: The amount of GAGs per gram skin tissue decreased slowly until the 4th decade and thereafter. A statistically- significant reduction in the amount of GAGs was noted between the age groups of under 10 and that of 51-60. This pattern of decline was similar with tte change of the hyaluronic acid content in gram skin tissue. The content of chondroitin sulfate and heparan sulfate also decreased as a function of ageing. The compositional ratio of GAGs showed no age related changes. The water content measured by acetone dehydration was reduced to a significant level up to the age group of over 30 years. CONCLUSION: These results imply that the wrinkling and laxity of skin during the chronological ageing process reflect the age-related reduction of GAGs and the water content of the skin.
Acetone ; Adhesives ; Aging ; Anesthesia, General ; Cellular Structures ; Cetylpyridinium ; Chondroitin Sulfates ; Connective Tissue ; Dehydration ; Dermis ; Extracellular Matrix ; Extracellular Space ; Fibroblasts ; Gels ; Heparitin Sulfate ; Humans* ; Hyaluronic Acid ; Mast Cells ; Proteoglycans ; Skin Diseases ; Skin* ; Water

OBJECTIVEto study the role of structural maintenance of chromosome (SMC)1, SMC3, Separase and Securin in tumorgenesis that contact with coal tar pitch.

METHODSthe BEAS-2B cells was induced by coal tar pitch smoke extracts to form malignant transformation cell model in vitro. The gene expression levels of mRNA were assessed by real-time quantitative RT-PCR, and the protein expression variation were determined by cell culture overslip of immunohistochemical methods.

RESULTSin malignant transformation cells, the mRNA and the protein expression level of SMC1 gene was not statistically significantly different compared with the BEAS-2B group and DMSO group (P > 0.05); SMC3 and Separase was increased and Securin was decreased (P < 0.05), while the difference between other two control groups was not significant (P > 0.05).

CONCLUSIONSthe up expression level of SMC3 and Separase and the down expression level of Securin are involved in the process that evolves into malignant transformation in bronchial epithelial cells BEAS-2B induced by coal tar pitch smoke extracts.

Bronchi ; cytology ; Cell Cycle Proteins ; metabolism ; Cell Line ; Cell Line, Transformed ; cytology ; drug effects ; Chondroitin Sulfate Proteoglycans ; metabolism ; Chromosomal Proteins, Non-Histone ; metabolism ; Coal Tar ; toxicity ; Endopeptidases ; metabolism ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Membrane Proteins ; metabolism ; Separase ; Sister Chromatid Exchange ; Smoke ; adverse effects