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Pericellular Matrix (pericellular + matrix)

Distribution by Scientific Domains

Medical Sciences	87%

Selected Abstracts

Direct and indirect manipulation of the MEK-ERK pathway regulates the formation of a pericellular HA-dependent matrix by chick articular surface cells without modifying CD44 expresssion

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004
Edward R. Bastow
Introduction Recent evidence suggests that hyaluronan (HA) facilitates the mechano-dependent joint cavity-forming process through the elaboration and retention of a HA-rich pericellular matrix in the developing joint interzone (IZ). The presumptive joint IZ phenotype shows a capacity to bind and synthesize HA and also exhibits elevated activated ERK, prior to synovial joint cavity formation (Lamb et al. 2001; Edwards et al. 1994; Dowthwaite et al. 1998). We have found that immobilization, which induces embryonic joint fusion with loss of the joint IZ phenotype, also reduces ERK activity levels in the IZ. As the signalling events regulating the synthesis and binding of HA have yet to be determined, we hypothesize that ERK activation plays a pivotal role in determining the presumptive joint IZ phenotype through HA synthetic and binding capacity. Materials and methods Chick articular surface (AS) cells were harvested from proximal tibiotarsal joints of embryos by collagenase digestion. Pericellular coat formation was assessed using the erythrocyte exclusion assay and cell-coat area ratios determined. ERK activity was modulated by transient transfection of GFP constructs of constitutively active (CA-) or dominant negative (DN-) forms of MEK, the direct upstream regulator of ERK or by treatment with the MEK inhibitor PD98059 (50 µm). ERK activation was monitored by immunochemistry. CD44 expression and ERK activation in PD98059-treated cells were monitored by immunoblotting and medium HA concentrations by ELISA. Results AS cells form large pericellular coats that are lost following hyaluronidase treatment and thus dependent upon HA for their construction. Treatment with PD98059 significantly reduced pericellular coat formation after 6 h. In parallel, we confirmed that PD98059 diminished active ERK expression without modifying overall levels of ERK, suggesting that the elaboration of large HA-pericellular coats is dependent upon MEK's activation of ERK. Western blot analysis of PD98059-treated cells showed that loss of pericellular coats was not, however, associated with any decreased levels of the cell surface HA receptor CD44. Although treatment with PD98059 did not change medium HA concentration after short times of exposure, at times (up to 6 h) during which coat loss was evident, prolonged treatment over 24 h significantly decreased medium HA concentration. Consistent with a role for ERK in pericellular coat formation, transfection with DN-MEK diminished, while CA-MEK increased, both active ERK expression and coat formation efficiency. We also found that, commensurate with this modification in coat forming efficiency, cells expressing DN-MEK exhibited a significant reduction in labelling of free HA on the cell surface. Discussion These studies extend our recent work to indicate that: (i) direct modulation of ERK activation by transfection with its endogenous upstream regulator modifies cell surface-associated HA (ii) PD98059-induced blockade of ERK activation restricts medium HA release and (iii) ERK-mediated changes in pericellular coat elaboration are independent of changes in cellular CD44 expression. These findings suggest an intimate relationship between ERK activation and the formation/retention of HA-rich pericellular matrices in vitro and highlight the role for ERK activation in regulating joint line-related differentiation. [source]

Cartilage Tissue Engineering With Demineralized Bone Matrix Gelatin and Fibrin Glue Hybrid Scaffold: An In Vitro Study

ARTIFICIAL ORGANS, Issue 2 2010
Zheng-Hui Wang
Abstract To develop a cartilage-like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte,fibrin suspension, which was polymerized by submerging the constructs into thrombin,calcium chloride solution. Engineered cartilage-like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene-encoded cartilage-specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering. [source]

Hyaluronan synthase-3 is upregulated in metastatic colon carcinoma cells and manipulation of expression alters matrix retention and cellular growth

INTERNATIONAL JOURNAL OF CANCER, Issue 5 2003
Kelli M. Bullard
Abstract HA is a glycosaminoglycan that is synthesized on the inner surface of the plasma membrane and secreted into the pericellular matrix. HA and its biosynthetic enzymes (HAS1, HAS2 and HAS3) are thought to participate in tumor growth and cancer progression. In our study, colon carcinoma cells isolated from a lymph node metastasis (SW620) produced more pericellular HA and expressed higher levels of HAS3 mRNA compared to cells isolated from a primary colon carcinoma (SW480). To assess functionality, HAS3 expression in SW620 cells was inhibited by transfection with an asHAS3 construct. Decreased HA secretion and cell-surface retention by asHAS3 transfectants were confirmed using competitive binding and particle exclusion assays. Anchorage-independent growth, a correlate of tumor growth in vivo, was assessed by colony formation in soft agar. SW620 cells stably transfected with asHAS3 demonstrated significant growth inhibition, as evidenced by fewer colonies and smaller colony area than either SW620 cells or cells transfected with vector alone. Addition of exogenous HA restored growth in asHAS3 transfectants. Thus, we demonstrate that pericellular HA secretion and retention and HAS3 expression are increased in metastatic colon carcinoma cells relative to cells derived from a primary tumor. Inhibition of HAS3 expression in these cells decreased the pericellular HA matrix and inhibited anchorage-independent growth. These data suggest that HA and HAS3 function in the growth and progression of colon carcinoma. © 2003 Wiley-Liss, Inc. [source]

Direct and indirect manipulation of the MEK-ERK pathway regulates the formation of a pericellular HA-dependent matrix by chick articular surface cells without modifying CD44 expresssion

Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytes

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2003
Wen-Ning Qi
Abstract The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(,) neoepitope of chondroitin-6- sulfate, 3B3(+) chondroitinase-generatedepitope of chondroitin-6-sulfate, and tenascin-C expression. TGF-,1 stimulated the production of 3B3(+), 5D4, and tenascin-C in a dose-dependent manner and decreased 3B3(,) levels. Following the addition of exogenous type II collagen, 3B3(,) increased and tenascin-C decreased but did not change the direction of TGF-,1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF-,1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF-,1 regulation of the articular cartilage physiology. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source]

Developmental and osteoarthritic changes in Col6a1 -knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrix

ARTHRITIS & RHEUMATISM, Issue 3 2009
Leonidas G. Alexopoulos
Objective Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the "pericellular matrix" (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading. Methods Col6a1,/, mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1-month-old Col6a1,/,, Col6a1+/,, and Col6a1+/+ mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique. Results In Col6a1,/, and Col6a1+/, mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild-type controls. With age, Col6a1,/, mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD. Conclusion These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1,/, -derived joint laxity, can lead to progression of OA. [source]

Matrix homeostasis in aging normal human ankle cartilage

ARTHRITIS & RHEUMATISM, Issue 11 2002
Matthias Aurich
Objective To study age-related (as opposed to arthritis-related) changes in collagen and proteoglycan turnover. Methods Macroscopically nondegenerate normal ankle cartilage obtained from 30 donors (ages 16,75 years) was processed for in situ hybridization to detect messenger RNA (mRNA) of type IIB collagen (CIIB); antibodies to the C-propeptide of type II collagen (CPII), to the type II collagen (CII) collagenase-generated cleavage neoepitope (Col2-3/4Cshort), and to the CII denaturation product (Col2-3/4m) were used for immunohistochemistry analysis and immunoassay. In addition, immunoblotting was used to detect the 4 collagenases. Assays were also performed to detect glycosaminoglycan (GAG) content and the 846 epitope of aggrecan. Results There were no significant changes in CII, GAG, and the content of the 846 epitope after the age of 30 years. Both mRNA for CIIB and the CPII were present in all zones, and CPII content did not change significantly with age. While the collagenase-cleaved CII showed a trend to increase with age, the denatured collagen did not. However, the molar ratio of cleaved versus denatured collagen was positively correlated with age. All 4 collagenases were detectable in the ankle cartilage but showed no identifiable changes in content with age. Conclusion Synthesis and degradation of CII is associated with the pericellular matrix and is maintained at a steady state throughout life. The contents of CII and proteoglycan did not change. There was a significant reduction in the denaturation of CII with age, relative to collagenase-mediated cleavage. These observations reveal that, in aging of the intact ankle articular cartilage, there is no evidence of molecular degenerative changes of the kind observed in osteoarthritis, thereby distinguishing aging from the osteoarthritis process. [source]