Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells

In this study, we examined the effects of shark cartilage extract on the attachment and spreading properties and the focal adhesion structure of cultured bovine pulmonary artery endothelial cells. Treatment with cartilage extract resulted in cell detachment from the substratum. Immunofluorescence staining of those treated cells that remained attached showed that, instead of being present in both central and peripheral focal adhesions as in control cells, both integrin alpha(v)beta(3) and vinculin were found only in peripheral focal adhesion and thinner actin filament bundles were seen. In addition to causing cell detachment, cartilage extract partially inhibited the initial adherence of the cells to the substratum in a dose-dependent manner. Integrin alpha(v)beta(3) and vinculin staining of these cells also showed a peripheral focal adhesion distribution pattern. Vitronectin induced cell spreading in the absence of serum, but was blocked by simultaneous incubation with cartilage extract, which was shown to inhibit both integrin alpha(v)beta(3) and vinculin recruitment to focal adhesion and the formation of stress fibers. Dot binding assays showed that these inhibitory effects on cell attachment and spreading were not due to direct binding of cartilage extract components to integrin alpha(v)beta(3) or vitronectin. Shark cartilage chondroitin sulfate had no inhibitory effect on either cell attachment or spreading of endothelial cells. These results show that the inhibitory effects of cartilage extract on cell attachment and spreading are mediated by modification of the organization of focal adhesion proteins.     

Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study

Introduction

The effect of low-intensity pulsed ultrasound (LIPUS) on cell growth was examined in three-dimensional-cultured chondrocytes with a collagen sponge. To elucidate the mechanisms underlying the mechanical activation of chondrocytes, intracellular signaling pathways through the Ras/mitogen-activated protein kinase (MAPK) and the integrin/phosphatidylinositol 3 kinase (PI3K)/Akt pathways as well as proteins involved in proliferation of chondrocytes were examined in LIPUS-treated chondrocytes.

Methods

Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly sacrificed pigs. Isolated chondrocytes mixed with collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. Experimental cells were cultured with daily 20-minute exposures to LIPUS. The chondrocytes proliferated and a collagenous matrix was formed on the surface of the sponge. Cell counting, histological examinations, immunohistochemical analyses and western blotting analysis were performed.

Results

The rate of chondrocyte proliferation was slightly but significantly higher in the LIPUS group in comparison with the control group during the 2-week culture period. Western blot analysis showed intense staining of type-IX collagen, cyclin B₁ and cyclin D₁, phosphorylated focal adhesion kinase, and phosphorylated Akt in the LIPUS group in comparison with the control group. No differences were detected, however, in the MAPK, phosphorylated MAPK and type-II collagen levels.

Conclusion

LIPUS promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a three-dimensional culture using a collagen sponge. In addition, the anabolic LIPUS signal transduction to the nucleus via the integrin/phosphatidylinositol 3-OH kinase/Akt pathway rather than the integrin/MAPK pathway was generally associated with cell proliferation.     

Adenoviral transduction is more efficient in alginate-derived chondrocytes than in monolayer chondrocytes

Gene transfer into cultured chondrocytes by using adenoviral vectors has potential applications in treating cartilage disorders. The present study was undertaken to compare and optimize two chondrocyte culture conditions for adenoviral transduction efficacy by using primary human articular chondrocytes cultivated either directly in a monolayer condition or as outgrowths from alginate-stored chondrocyte cultures. Isolated primary chondrocytes from human articular cartilage were either immediately transduced with an EGFP (enhanced green fluorescent protein)-gene-bearing adenoviral vector (1,000 and 3,000 virus particles/cell) or cultured in alginate before transduction. Immunohistochemistry and flow cytometric analysis were employed to determine the expression of extracellular matrix proteins and of the αvβ5 integrin receptor involved in adenoviral cell entry. Monolayer chondrocytes exhibited moderate transduction rates (mean 22.2% and 46.9% EGFP-positive cells at 1,000 and 3,000 virus particles/cell by 72 h post-transduction), whereas alginate-derived chondrocytes revealed significantly higher transduction efficacies (95.7% and 99%). Both monolayer and alginate-derived chondrocytes expressed αvβ5 integrin, type II collagen and cartilage proteoglycans. The mean fluorescence intensity of type II collagen was significantly higher in the alginate-derived chondrocytes, whereas that of αvβ5 integrin was higher in the monolayer chondrocytes. Our results indicate that transduction efficacy is independent of αvβ5 integrin expression levels in chondrocytes. Moreover, adenoviral transduction of alginate-derived chondrocytes is more efficient than that for monolayer chondrocytes and may be a suitable tool to achieve sufficient numbers of transduced and differentiated chondrocytes for experimental applications and cartilage repair. Dr. Gundula Schulze-Tanzil is supported by a grant awarded by the Rahel Hirsh Foundation from the Charité Medical Schools Berlin. The study was supported by a grant from the Deutsche Arthrosehilfe e.V.     

Enhancement of cell adhesion and spreading by a cartilage-specific noncollagenous protein, cartilage matrix protein (CMP/Matrilin-1), via integrin alpha1beta1

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.     

Focal adhesion kinase: protein interactions and cellular functions

Integrin-mediated cell adhesion to extracellular matrix (ECM) plays important roles in a variety of biological processes. Recent studies suggested that integrins mediate signal transduction across the plasma membrane via activating several intracellular signaling pathways. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been shown to be a major mediator of integrin signal transduction pathways. Upon activation by integrins, FAK undergoes autophosphorylation as well as associations with several other intracellular signaling molecules. These interactions in the signaling pathways have been shown to regulation a variety of cellular functions such as cell spreading, migration, cell proliferation, apoptosis and cell survival. Recent progress in the understanding of FAK interactions with other proteins in the regulation of these cellular functions will be discussed in this review.     

应力作用下软骨细胞信号传导的研究进展

研究显示应力刺激对软骨细胞生长及基质代谢具重要作用。软骨正常结构形态以及应力下的软骨细胞形态和基质代谢的变化是力-生物信号转化的基础,信号分子及信号通路则是应力信号传导的核心,二者是对软骨细胞应力下信号传导过程深入了解不可或缺的信息组成,了解应力对软骨细胞的作用方式及作用机制有助于软骨相关疾病诊治、组织工程等领域的研究,本文就这两个方面研究进展做一综述。     

Integrins and chondrocyte–matrix interactions in articular cartilage

The integrin family of cell adhesion receptors plays a major role in mediating interactions between cells and the extracellular matrix. Normal adult articular chondrocytes express α1β1, α3β1, α5β1, α10β1, αVβ1, αVβ3, and αVβ5 integrins, while chondrocytes from osteoarthritic tissue also express α2β1, α4β1, α6β1. These integrins bind a host of cartilage extracellular matrix (ECM) proteins, most notably fibronectin and collagen types II and VI, which provide signals that regulate cell proliferation, survival, differentiation, and matrix remodeling. By initiating signals in response to mechanical forces, chondrocyte integrins also serve as mechanotransducers. When the cartilage matrix is damaged in osteoarthritis, fragments of fibronectin are generated that signal through the α5β1 integrin to activate a pro-inflammatory and pro-catabolic response which, if left unchecked, could contribute to progressive matrix degradation. The cell signaling pathways activated in response to excessive mechanical signals and to fibronectin fragments are being unraveled and may represent useful therapeutic targets for slowing or stopping progressive matrix destruction in arthritis.     

NMDA receptor expression and roles in human articular chondrocyte mechanotransduction

Mechanical forces influence articular cartilage structure by regulating chondrocyte activity. Mechanical stimulation results in activation of an alpha5beta1 integrin dependent intracellular signal cascade involving focal adhesion kinase and protein kinase C, triggering the release of interleukin-4 from the cell. In normal HAC the response to physiological mechanical stimulation is characterised by increased levels of aggrecan mRNA and a decrease in levels of mRNA for matrix metalloproteinase 3 (MMP-3), the net result of which would be to maintain and optimise cartilage structure and function. This protective/anabolic response is not seen when chondrocytes from osteoarthritic cartilage are subjected to an identical mechanical stimulation regime. Following the observation that the neurotransmitter substance P is involved in chondrocyte mechanotransduction the present study was undertaken to establish potential roles for glutamate receptors in the control of chondrocyte mechanical responses. Using immunohistochemistry and RTPCR normal and OA chondrocytes are shown to express NR1 and NR2a subunits of the NMDA receptor. Addition of NMDA receptor agonists to chondrocytes in primary culture resulted in changes in membrane potential consistent with expression of functional receptors. NMDA receptor antagonists inhibited the hyperpolarisation response of normal chondrocytes to mechanical stimulation but had no effect on the depolarisation response of osteoarthritic chondrocytes to mechanical stimulation. These studies indicate that at least one subset of the NMDA receptor family of molecules is expressed in cartilage and may have important modulatory effects on mechanotransduction and cellular responses following mechanical stimulation. Indeed the results suggest that there is an alteration of NMDA receptor signalling in OA chondrocytes, which may be critical in the abnormal response of OA chondrocytes to mechanical stimulation. Thus NMDA receptors appear to be involved in the regulation of human articular chondrocyte responses to mechanical stimulation, and in OA, mechanotransduction pathways may be modified as a result of altered activation and function of these receptors.     

Rheumatoid arthritis: regulation of synovial inflammation

Rheumatoid arthritis (RA) is a systemic, inflammatory autoimmune disorder that presents as a symmetric polyarthritis associated with swelling and pain in multiple joints, often initially occurring in the joints of the hands and feet. Articular inflammation causes activation and proliferation of the synovial lining, expression of inflammatory cytokines, chemokine-mediated recruitment of additional inflammatory cells, as well as B cell activation with autoantibody production. A vicious cycle of altered cytokine and signal transduction pathways and inhibition of programmed cell death contribute to synoviocyte and osteoclast mediated cartilage and bone destruction. A combination of targeted interventions at various stages in the pathogenesis of RA will likely be required to control symptoms in certain patients with this complex and potentially disabling disease. The regulation of rheumatoid synovial inflammation will be reviewed, followed by a brief summary of the therapeutic implications of these advances, including strategies targeting key cytokines, signal transduction molecules, co-stimulatory molecules, B cells, chemokines, and adhesion molecules.     

Altered expression of integrins in RSV-transformed chick epiphyseal chondrocytes

Chondrocytes have been shown to express both in vivo and in vitro a number of integrins of the beta1-, beta3- and beta5-subfamilies (Biorheology 37 (2000) 109). Normal and v-Src-transformed chick epiphyseal chondrocytes (CEC) display different adhesion properties. While normal CEC with time in culture tends to increase their adhesion to the substrate by organizing focal adhesions and actin stress fibers, v-Src-transformed chondrocytes display a refractile morphology and disorganization of actin cytoskeleton. We wondered whether the reduced adhesion and spreading of v-Src-transformed chondrocytes could be ascribed to changes in integrin expression and/or function. Integrin expression by normal CEC is studied and compared to v-Src-transformed chick chondrocytes, using monoclonal and polyclonal antibodies to integrins alpha- and beta-chains. We show the presence of alpha1-, alpha3-, alphav-, alpha6-, beta1- and beta3-chains on CEC, with very low levels of alpha2- and alpha5-chains. Alphav chain associates with multiple beta subunits in normal and transformed chondrocytes. With the exception of alpha1- and alpha2-chains, the levels of the integrin chains analyzed are higher in transformed chondrocytes as compared with normal chondrocytes. In spite of the increased levels of integrin expression, transformed chondrocytes exhibit loss of focal adhesion and actin stress fibers and low adhesion activity on several extracellular matrix constituents. These observations raise the possibility that, in addition to its effects on global pattern of integrin expression, v-Src can influence integrin function in chondrocytes.     

Mechanical responses and integrin associated protein expression by human ankle chondrocytes

Metabolic, biochemical and biomechanical differences between ankle and knee joint cartilage and chondrocytes including resistance to the effects of catabolic cytokines and fibronectin fragments may be relevant to differences in prevalence of OA in these joints. Although there is increasing information available on how chondrocytes from knee and hip joint cartilage recognise and respond to mechanical stimuli, knowledge of mechanotransduction in ankle joint chondrocytes is limited. This study was undertaken to (i) establish whether the response of normal ankle joint derived chondrocytes to mechanical stimulation in vitro was similar to that of normal and osteoarthritic knee joint derived chondrocytes and (ii) to investigate whether these chondrocytes showed differences in expression of integrin associated regulatory and signalling molecules. Unlike normal knee joint chondrocytes, ankle joint chondrocytes did not show an increase in relative levels of aggrecan mRNA when mechanically stimulated. No obvious change in protein tyrosine phosphorylation was seen in ankle chondrocytes subsequent to mechanical stimulation but these cells expressed elevated levels of tyrosine phosphorylated proteins at rest when compared to normal knee joint chondrocytes. Ankle joint chondrocytes showed an increase in protein kinase B phosphorylation following 1 min 0.33 Hz stimulation which was inhibited by the presence of antibodies to alpha5beta1 integrin. Ankle joint chondrocytes appeared to show significant differences in levels of the integrin-associated proteins CD98, CD147 and galectin 3, PKCgamma and differences in responses to glutamate were seen. Chondrocytes from ankle and knee joint cartilage respond differently to 0.33 Hz mechanical stimulation. This may be related to modified integrin-dependent mechanotransduction as a result of changes in expression of integrin regulatory molecules such as CD98 or differential expression and function of downstream components of the mechanotransduction pathway such as PKC or NMDA receptors.     

Adhesion-mediated signal transduction in human articular chondrocytes: the influence of biomaterial chemistry and tenascin-C

Chondrocyte 'dedifferentiation' involves the switching of the cell phenotype to one that no longer secretes extracellular matrix found in normal cartilage and occurs frequently during chondrocyte expansion in culture. It is also characterized by the differential expression of receptors and intracellular proteins that are involved in signal transduction pathways, including those associated with cell shape and actin microfilament organization. The objective of this study was to examine the modulation of chondrocyte phenotype by cultivation on polymer substrates containing poly(ethylene glycol) (PEG). We observed differential arrangement of actin organization in articular chondrocytes, depending on PEG length. When cultivated on 300 g/mol PEG substrates at day 19, chondrocytes had lost intracellular markers characteristic of the differentiated phenotype, including type II collagen and protein kinase C (PKC). On these surfaces, chondrocytes also expressed focal adhesion and signaling proteins indicative of cell attachment, spreading, and FA turnover, including RhoA, focal adhesion kinase, and vinculin. The switch to a dedifferentiated chondrocyte phenotype correlated with integrin expression. Conversely, the expression of CD44 receptors coincided with chondrogenic characteristics, suggesting that binding via these receptors could play a role in maintaining the differentiated phenotype on such substrates. These effects can be similar to those of compounds that interfere in intracellular signaling pathways and can be utilized to engineer cellular response.     

Actin cytoskeletal association of cytohesin-1 is regulated by specific phosphorylation of its carboxyl-terminal polybasic domain.

Cell adhesion mediated by integrin receptors is controlled by intracellular signal transduction cascades. Cytohesin-1 is an integrin-binding protein and guanine nucleotide exchange factor that activates binding of the leukocyte integrin leukocyte function antigen-1 to its ligand, intercellular adhesion molecule 1. Cytohesin-1 bears a carboxyl-terminal pleckstrin homology domain that aids in reversible membrane recruitment and functional regulation of the protein. Although phosphoinositide-dependent membrane attachment of cytohesin-1 is mediated primarily by the pleckstrin homology domain, this function is further strengthened by a short carboxyl-terminal polybasic amino acid sequence. We show here that a serine/threonine motif within the short polybasic stretch of cytohesin-1 is phosphorylated by purified protein kinase C delta in vitro. Furthermore, the respective residues are also found to be phosphorylated after phorbol ester stimulation in vivo. Biochemical and functional analyses show that phosphorylated cytohesin-1 is able to tightly associate with the actin cytoskeleton, and we further demonstrate that phosphorylation of the protein is required for maximal leukocyte function antigen-1-mediated adhesion of Jurkat cells to intercellular adhesion molecule 1. These data suggest that both phosphatidylinositol 3-kinase and protein kinase C-dependent intracellular pathways that stimulate beta(2)-integrin-mediated adhesion of T lymphocytes converge on cytohesin-1 as functional integrator.     

Genetically engineered chondrocytes overexpressing elastin improve cell retention and chondrogenesis in a three-dimensional GelMA culture system

Elastic cartilage possesses many elastic fibers and has a high degree of elasticity. However, insufficient elastic fiber production remains unsolved in elastic cartilage tissue engineering. Exogenous elastin is difficult to degrade and violates cell proliferation and migration during cartilage regeneration. Moreover, exogenous elastic fibers are difficult to assemble with endogenous extracellular matrix components. We produced genetically engineered chondrocytes overexpressing elastin to boost endogenous elastic fiber production. After identifying that genetic manipulation hardly impacted the cell viability and chondrogenesis of chondrocytes, we co-cultured genetically engineered chondrocytes with untreated chondrocytes in a three-dimensional gelatin methacryloyl (GelMA) system. In vitro study showed that the co-culture system produced more elastic fibers and increased cell retention, resulting in strengthened mechanics than the control system with untreated chondrocytes. Moreover, in vivo implantation revealed that the co-culture GelMA system greatly resisted host tissue invasion by promoting elastic fiber production and cartilage tissue regeneration compared with the control system. In summary, our study indicated that genetically engineered chondrocytes overexpressing elastin are efficient and safe for promoting elastic fiber production and cartilage regeneration in elastic cartilage tissue engineering.     

Akt-dependent cell size regulation by the adhesion molecule on glia occurs independently of phosphatidylinositol 3-kinase and Rheb signaling

The role of cell adhesion molecules in mediating interactions with neighboring cells and the extracellular matrix has long been appreciated. More recently, these molecules have been shown to modulate intracellular signal transduction cascades critical for cell growth and proliferation. Expression of adhesion molecule on glia (AMOG) is downregulated in human and mouse gliomas, suggesting that AMOG may be important for growth regulation in the brain. In this report, we examined the role of AMOG expression on cell growth and intracellular signal transduction. We show that AMOG does not negatively regulate cell growth in vitro or in vivo. Instead, expression of AMOG in AMOG-deficient cells results in a dramatic increase in cell size associated with protein kinase B/Akt hyperactivation, which occurs independent of phosphatidylinositol 3-kinase activation. AMOG-mediated Akt phosphorylation specifically activates the mTOR/p70S6 kinase pathway previously implicated in cell size regulation, but it does not depend on tuberous sclerosis complex/Ras homolog enriched in brain (Rheb) signaling. These data support a novel role for a glial adhesion molecule in cell size regulation through selective activation of the Akt/mTOR/S6K signal transduction pathway.     

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.     

Effect of hyaluronic acid on human chondrocyte cell lines from articular cartilage

The effects of hyaluronic acid (HA) derivative on the proliferation and metabolism of human chondrocytes were examined. Cells were obtained from cartilage from metatarsal phalangeal joints of 20 adult humans (aged 22-63) and from femoral knee condyles of 10 subjects (aged 22-77). Chondrocytes isolated by collagenase/Dnase digestion were cultured with addition of different doses of HA for 4 weeks. Morphological studies demonstrated that HA enhanced the adhesion of cells to substrate; HA-treated chondrocytes proliferated better than chondrocytes cultured in HA-free medium. This study shows that HA improves in vitro substrate adhesion ability and proliferative activity of human cartilage cells and that the response to the treatment varies on an individual basis.     

Pleiotrophin inhibits chondrocyte proliferation and stimulates proteoglycan synthesis in mature bovine cartilage

Pleiotrophin (PTN) is a secreted heparin-binding, developmentally regulated protein that is found in abundance in fetal, but not mature, cartilage. SDS-page and glycosaminoglycan (GAG) analysis of sulfate-radiolabeled proteoglycans isolated from the medium of mature cultured chondrocytes treated with PTN showed a threefold increase in the levels of proteoglycan synthesis. In contrast, in cultures of fetal chondrocytes, no changes in proteoglycan synthesis were observed. Thymidine incorporation experiments showed a dose-dependent decrease in proliferation of treated cells compared with control cultures, suggesting that pleiotrophin had an inhibitory effect on growth of chondrocytes. Neither FGF or heparin reversed the inhibitory effect of PTN. Capillary electrophoresis of chondroitinase ABC-digested proteoglycans isolated from mature chondrocytes showed 2-4-fold increases in the amounts of the 4S- and 6S-substituted GAG chains for the PTN-treated chondrocytes. Northern analysis showed a twofold upregulation in the mRNA levels of biglycan and collagen type II, but no difference in the message levels for decorin and aggrecan. These results establish that PTN inhibits cell proliferation, while stimulating the synthesis of proteoglycans in mature chondrocytes in vitro, suggesting that PTN may act directly or indirectly to regulate growth and proteoglycan synthesis in the developing matrix of fetal cartilage.