Troponin is a potential regulator for actomyosin interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.     

Rat mesangial cell-matrix interactions in culture

The glomerular mesangium contains fibronectin (FN), laminin, and collagen IV, but it remains unclear whether these matrix proteins affect mesangial cellular functions. The present experiments were designed to test whether cell-matrix interactions could affect some functions of mesangial cells. Cultured rat mesangial cells synthesized a cellular form of FN that was both secreted and incorporated into an extensive, fibrillar pericellular matrix. This FN matrix was increased in high-density cultures and was more developed in human mesangial cells. Rat mesangial cells in vitro displayed a marked capacity to incorporate exogenous FN into a pericellular matrix, demonstrating that accumulations of FN in the mesangial matrix could result from endogenous and/or exogenous sources. Rat mesangial cells also expressed RGD-sensitive integrin receptors for FN, laminin, and collagens I and IV that promoted cell adhesion and that directed differential changes in morphology. Indirect evidence suggested the existence of other mesangial binding sites for extracellular matrix proteins. FN and collagen IV also stimulated modest increases in [3H]thymidine uptake and cell number by quiescent cells. Taken together, these results suggest that cultured mesangial cells present a model system for studying the regulation of cell-matrix interactions in the mesangium.     

Disruption of epithelial cell-matrix interactions induces apoptosis

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Programmed cell death (apoptosis) is crucial for maintaining appropriate cell number and tissue organization. It was therefore of interest to determine whether cell- matrix interactions affect apoptosis. The present report demonstrates that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix. We have termed this phenomenon "anoikis." Overexpression of bcl-2 protected cells against anoikis. Cellular sensitivity to anoikis was apparently regulated: (a) anoikis did not occur in normal fibroblasts; (b) it was abrogated in epithelial cells by transformation with v-Ha-ras, v-src, or treatment with phorbol ester; (c) sensitivity to anoikis was conferred upon HT1080 cells or v-Ha-ras-transformed MDCK cells by reverse- transformation with adenovirus E1a; (d) anoikis in MDCK cells was alleviated by the motility factor, scatter factor. The results suggest that the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.     

Direct,dynamic assessment of cell-matrix interactions inside fibrillar collagen lattices

Cell mechanical behavior has traditionally been studied using 2-D planar elastic substrates. The goal of this study was to directly assess cell-matrix mechanical interactions inside more physiologic 3-D collagen matrices. Rabbit corneal fibroblasts transfected to express GFP-zyxin were plated at low density inside 100 micro m-thick type I collagen matrices. 3-D datasets of isolated cells were acquired at 1-3-min intervals for up to 5 h using fluorescent and Nomarski DIC imaging. Unlike cells on 2-D substrates, cells inside the collagen matrices had a bipolar morphology with thin pseudopodial processes, and without lamellipodia. The organization of the collagen fibrils surrounding each cell was clearly visualized using DIC. Using time-lapse color overlays of GFP and DIC images, displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized. During pseudopodial extension, new focal adhesions often formed in a line along collagen fibrils in front of the cell, while existing adhesions moved backward. This process generated tractional forces as indicated by the pulling in of collagen fibrils in front of the cell. Meanwhile, adhesions on both the dorsal and ventral surface of the cell body generally moved forward, resulting in contractile shortening along the pseudopodia and localized extracellular matrix (ECM) compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation, and ECM relaxation. This experimental model allows direct, dynamic assessment of cell-matrix interactions inside a 3-D fibrillar ECM. The data suggest that adhesions organize along actin-based contractile elements that are much less complex than the network of actin filaments that mechanically links lamellar adhesions on 2-D substrates.     

Stacked stem cell sheets enhance cell-matrix interactions

Cell sheet engineering has enabled the production of confluent cell sheets stacked together for use as a cardiac patch to increase cell survival rate and engraftment after transplantation, thereby providing a promising strategy for high density stem cell delivery for cardiac repair. One key challenge in using cell sheet technology is the difficulty of cell sheet handling due to its weak mechanical properties. A single-layer cell sheet is generally very fragile and tends to break or clump during harvest. Effective transfer and stacking methods are needed to move cell sheet technology into widespread clinical applications. In this study, we developed a simple and effective micropipette based method to aid cell sheet transfer and stacking. The cell viability after transfer was tested and multi-layer stem cell sheets were fabricated using the developed method. Furthermore, we examined the interactions between stacked stem cell sheets and fibrin matrix. Our results have shown that the preserved ECM associated with the detached cell sheet greatly facilitates its adherence to fibrin matrix and enhances the cell sheet-matrix interactions. Accelerated fibrin degradation caused by attached cell sheets was also observed.     

Stacked stem cell sheets enhance cell-matrix interactions

Cell sheet engineering has enabled the production of confluent cell sheets stacked together for use as a cardiac patch to increase cell survival rate and engraftment after transplantation, thereby providing a promising strategy for high density stem cell delivery for cardiac repair. One key challenge in using cell sheet technology is the difficulty of cell sheet handling due to its weak mechanical properties. A single-layer cell sheet is generally very fragile and tends to break or clump during harvest. Effective transfer and stacking methods are needed to move cell sheet technology into widespread clinical applications. In this study, we developed a simple and effective micropipette based method to aid cell sheet transfer and stacking. The cell viability after transfer was tested and multi-layer stem cell sheets were fabricated using the developed method. Furthermore, we examined the interactions between stacked stem cell sheets and fibrin matrix. Our results have shown that the preserved ECM associated with the detached cell sheet greatly facilitates its adherence to fibrin matrix and enhances the cell sheet-matrix interactions. Accelerated fibrin degradation caused by attached cell sheets was also observed.     

Decorin regulates endothelial cell-matrix interactions during angiogenesis

Interactions between endothelial cells and the surrounding extracellular matrix are continuously adapted during angiogenesis, from early sprouting through to lumen formation and vessel maturation. Regulated control of these interactions is crucial to sustain normal responses in this rapidly changing environment, and dysfunctional endothelial cell behaviour results in angiogenic disorders. The proteoglycan decorin, an extracellular matrix component, is upregulated during angiogenesis. While it was shown previously that the absence of decorin leads to dysregulated angiogenesis in vivo, the molecular mechanisms were not clear. These abnormal endothelial cell responses have been attributed to indirect effects of decorin; however, our recent data provides evidence that decorin directly regulates endothelial cell-matrix interactions. This data will be discussed in conjunction with findings from previous studies, to better understand the role of this proteoglycan in angiogenesis.Key words: decorin, angiogenesis, motility, α2β1 integrin, insulin-like growth factor I receptor, Rac GTPaseLed by appropriate cues, the vascular system undergoes postnatal remodelling (angiogenesis), to maintain tissue homeostasis. Thus while much of the mature endothelium is quiescent, locally activated endothelial cells re-enter the cell cycle, and assume a motile phenotype essential for sprouting and neo-vessel formation. Concomitantly, the surrounding extracellular matrix (ECM) is significantly altered through de novo protein expression, deposition of plasma components and protease-mediated degradation. The latter liberates cryptic binding sites and sequestered growth factors in addition to intact and degraded ECM components, which themselves possess pro- and anti-angiogenic signalling properties. For supported blood flow, endothelium quiescence and integrity is re-established, and the ECM is organized into mature, cross-linked networks. In short, endothelial cells regulate ECM synthesis, assembly and turnover while the structure and composition of ECM in turn influences cellular phenotype. The ECM therefore, plays a critical role in control of endothelial cell behaviour during angiogenesis.Decorin is a member of the small leucine-rich repeat proteoglycan (SLRP) family, which was first discovered ‘decorating’ collagen I fibrils and was subsequently shown to regulate fibrillogenesis.^1,2 Both the protein core and the single, covalently attached glycosaminoglycan (GAG) moieties of decorin are involved in this function, the relevance of which is demonstrated by the phenotype of the decorin null mouse, which exhibits loose, fragile skin due to dysregulated fibrillogenesis.² Interestingly, a role for decorin in postnatal angiogenesis was also revealed by studies in the decorin null background. Corneal neoangiogenesis was reduced.³ Conversely, neo-angiogenesis was enhanced during dermal wound healing, although surprisingly this led to delayed wound closure.⁴ In this case, skin fragility due to the absence of decorin may have hindered wound closure, despite an increased blood supply. It is apparent however, that decorin plays a role in inflammation-associated angiogenesis. Indeed, endothelial cells undergoing angiogenic morphogenesis in this environment express decorin, while quiescent endothelial cells do not,^3–6 indicating that decorin modulates endothelial cell behaviour specifically during inflammatory-associated remodelling of the vascular system.To understand decorin effects on angiogenic morphogenesis within a minimalist environment, various in vitro models of angiogenesis have been employed (6 Similarly, decorin expression enhanced tube formation on matrigel,⁸ but in other studies utilising this substrate was found to either have no influence⁹ or to inhibit tubulogenesis induced by growth factors.¹⁰ In yet another study, decorin inhibited tube formation when presented as a substrate prior to addition of collagen I.⁷ These contrasting observations may reflect the importance of the micro-environment within which decorin is presented. Alternatively, controversial results could result from different sources of decorin since cell types differ in their post-translational modifications of the GAG moiety. Hence, varying length or sulfation patterns of GAG chains may account for different biological activities of decorin. Discrepancies can also be explained as artefacts due to different purification protocols, such as when denaturing conditions are used to extract decorin from tissue. Taken together however, these observations suggest that decorin is neither a pro- nor an anti-angiogenic factor per se, but rather a regulator of angiogenesis, dependent on local cues for different activities. Further, that decorin is capable of both enhancing and inhibiting tubulogenesis may suggest a role in balancing vessel regression versus persistence. Immature vessels have a period of plasticity prior to maturation, during which they can be remodelled, and either regress, or given the appropriate signals, proceed to maturity.¹¹ As a modulator of tube formation, it is tempting to speculate that decorin could influence the switch from immature to mature vessels, favouring one or the other in conjunction with signals from the local environment.

Table 1

Summary of the key functions of decorin in controlling cell behaviour

Decorin regulates endothelial cell-matrix interactions during angiogenesis

Interactions between endothelial cells and the surrounding extracellular matrix are continuously adapted during angiogenesis, from early sprouting through to lumen formation and vessel maturation. Regulated control of these interactions is crucial to sustain normal responses in this rapidly changing environment, and dysfunctional endothelial cell behaviour results in angiogenic disorders. The proteoglycan decorin, an extracellular matrix component, is upregulated during angiogenesis. While it was shown previously that the absence of decorin leads to dysregulated angiogenesis in vivo, the molecular mechanisms were not clear. These abnormal endothelial cell responses have been attributed to indirect effects of decorin; however, our recent data provides evidence that decorin directly regulates endothelial cell-matrix interactions. This data will be discussed in conjunction with findings from previous studies, to better understand the role of this proteoglycan in angiogenesis.     

ADAMs: modulators of cell-cell and cell-matrix interactions

ADAMs contain adhesive and metalloprotease domains. As major ectodomain sheddases, they release a variety of cell-surface proteins, including growth factors, cytokines, cell adhesion molecules and receptors. ADAMs can also cleave and remodel components of the extracellular matrix. Hence, ADAMs are emerging as key modulators of cell-cell and cell-matrix interactions. Important questions, including if and how ADAM adhesive domains promote ADAM protease function, are currently being addressed.     

Three-dimensional traction force microscopy: a new tool for quantifying cell-matrix interactions

The interactions between biochemical processes and mechanical signaling play important roles during various cellular processes such as wound healing, embryogenesis, metastasis, and cell migration. While traditional traction force measurements have provided quantitative information about cell matrix interactions in two dimensions, recent studies have shown significant differences in the behavior and morphology of cells when placed in three-dimensional environments. Hence new quantitative experimental techniques are needed to accurately determine cell traction forces in three dimensions. Recently, two approaches both based on laser scanning confocal microscopy have emerged to address this need. This study highlights the details, implementation and advantages of such a three-dimensional imaging methodology with the capability to compute cellular traction forces dynamically during cell migration and locomotion. An application of this newly developed three-dimensional traction force microscopy (3D TFM) technique to single cell migration studies of 3T3 fibroblasts is presented to show that this methodology offers a new quantitative vantage point to investigate the three-dimensional nature of cell-ECM interactions.     

Drosophila PS2 integrin mediates RGD-dependent cell-matrix interactions.

Integrins are a family of transmembrane glycoproteins that mediate cell-matrix and cell-cell interactions. We have transfected cultured Drosophila cells with genes that express the Drosophila PS2 integrin. We demonstrate that this integrin is expressed on the surface of the cells and can mediate cell spreading on an undefined component of fetal calf serum or on the purified vertebrate matrix molecules vitronectin and fibronectin. Additionally, PS2 integrin can cause cell spreading on RGD peptide. The spreading on matrix components or RGD peptide can be inhibited by soluble RGD peptide and is dependent on divalent cations.     

Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation

The latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR alpha 4 beta 1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis.     

The effect of advanced glycation end-product formation upon cell-matrix interactions.

The formation of advanced glycation end-products plays a central role in the progressive deterioration of tissues with age, a process that is accelerated in diabetes. Collagen in addition to providing structure and tensile strength to tissues also provides a dynamic matrix for cells to interact with, and due to its long-lived nature is particularly susceptible to modification with age and disease. We have recently identified methylglyoxal as a key intermediate in this process, reacting predominantly with arginine residues to form imidazolone compounds. We therefore postulated that modification of RGD sequences in collagen with methylglyoxal would interfere with crucial cell-matrix interactions. To investigate this concept we studied the interaction of two cell lines, MG63 and HT1080, with collagen modified to varying degrees with respect to arginine. Adhesion and subsequent spreading of both cell lines was significantly decreased by minimal methylglyoxal modification leading to the conclusion that such modification of collagen severely inhibits cell matrix interactions, most likely via the loss of specific arginine residues involved in integrin mediated cell attachment. This is the first demonstration that methylglyoxal modification of collagen can affect cell-matrix interactions and introduces a possible mechanism by which some of the deleterious changes in tissues with age and disease are occurring.     

Disruption of cell-matrix interactions by heparin enhances mesenchymal progenitor adipocyte differentiation

Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0. 1 μg/ml when applied before day 3 during the induction course. Heparin's effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.