Preparation and analysis of synthetic multicomponent extracellular matrix

CHAPTER 6

Preparation and Analysis of Synthetic Multicomponent Extracellular Matrix K i m S. M i d w o o d , * I w o n a W i e r z b i c k a - P a t y n o w s k i , * a n d J e a n E. S c h w a r z b a u e r Department of Molecular Biology Princeton University Princeton, New Jersey 08544

I. Introduction to the E C M I1. Development of Synthetic Matrices A. The Provisional Matrix B. Cell-Assernbled E C M III. Discussion A. Synthetic Provisional Matrix B. Cell-Derived E C M References

I. I n t r o d u c t i o n t o t h e E C M All cells are surrounded by an extracellular matrix (ECM) which dynamically regulates cellular functions including adhesion, migration, growth, and differentiation. It also provides a stable structural support for cells and a framework for tissue architecture. Fibronectin (FN) is a multifunctional adhesive glycoprotein which is a major component of most matrices (Hynes, 1990; Mosher, 1989). Many cell types make FN, including fibroblasts, endothelial cells, myoblasts, and astrocytes, and incorporate this cellular FN into the ECM. The plasma form of FN (pFN) is synthesized by hepatocytes and released into the blood. The primary differences between pFN and cellular FN arise by alternative splicing (Schwarzbauer, 1991a). The composition of the ECM varies from tissue to tissue and undergoes continuous assembly and remodeling throughout the lifetime of an organism. Therefore, FN must be able to interact with different combinations of cells, *These authors contributed equally to this manuscript. M E T H O D S IN CELL BIOLOGY, VOL. 69 Copyright 20{)2, Elsevier Science (USA). All rights reserved. 0091 679X/02 $35 00

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collagens, proteoglycans, and other macromolecules. FN does this through functional domains for binding to fibrin, heparin, cells, collagen/gelatin, and FN itself. The matrix provides the cell with environmental information through interactions with a number of different types of cell-surface receptor, including integrins and proteoglycans. The integrins are a large family of related dimeric receptor complexes that are composed of or and fl subunits (Hynes, 1992). Proteoglycans are a diverse set of macromolecules defined by the posttranslational addition of glycosaminoglycan side chains such as chondroitin or heparan sulfate (Lander, 1998). Integrins and transmembrane proteoglycans bind to a number of ligands within the ECM and on the surfaces of other cells. They also interact with cytoplasmic and cytoskeletal signaling proteins. To address matrix assembly and function, a variety of experimental systems have been developed. One such system is based on the provisional matrix of the wound bed; another uses a cell-associated matrix. The provisional matrix is synthesized in response to tissue injury and blood vessel damage. It is a covalenfly cross-linked network consisting predominantly of fibrin and pFN, which is remodeled over time to regain normal tissue structure and function (Clark, 1996). The provisional matrix is formed as the culmination of a series of enzymatic reactions. Upon vascular endothelial damage, blood comes into contact with subendothelial structures and other exposed injured tissues. This initiates a chain of reactions that ultimately results in the cleavage of the inactive precursor enzyme prothrombin, which is present in circulating blood, to active thrombin. Activated thrombin acts on soluble fibrinogen and, by cleaving off small fibrinopeptides, converts it to fbrin. This reveals previously cryptic self-assembly sites in the newly formed fibrin that allow spontaneous polymerization to occur. Following polymerization, factor XIII (transglutaminase) catalyzes the formation of intermolecular covalent cross-links in a calcium-dependent reaction. Factor XIII is present in plasma and is activated upon cleavage by thrombin. Factor XIII also catalyzes the formation of covalent cross-links between pFN and fibrin to form heterodimers and large molecular weight polymers, pFN is covalently cross-linked to lysine residues in the carboxy termini of fibrin ot chains using glutamine residues in the amino terminus ofFN (Mosher, 1989). This leaves other FN domains free to interact with cell surface receptors, such as integrins and proteoglycans, and other matrix proteins, including collagen and tenascin. This cascade of reactions results in the formation of a stable, semirigid, three-dimensional matrix. The immediate function of the provisional matrix is to fill the wound and maintain the integrity of the vascular system. It then initiates the wound healing process by providing a substratum that supports cell adhesion and migration into the injured tissues. The development of matrices that recapitulate the provisional matrix cell autonomously, in vitro, was first described by Mosher (1975). Cell-mediated matrix formation is another model commonly used to study matrix function. Fibroblasts assemble the ECM into an extensive fibrillar network with FN as a major structural and functional component. FN matrices are vital to vertebrate development and wound healing, and modulate tumorigenesis. FN interacts with cells to control cell adhesion, cytoskeletal organization, and intracellular signaling (Hynes, 1990; Mosher, 1993). It is secreted as a disulfide-bonded dimer with subunits of 230-270 kDa and is

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assembled into matrix fibrils through a stepwise process (Schwarzbauer and Sechler, 1999). Initiation of assembly depends on the interactions between the cell-binding domain of FN and ot5/31 integrin receptor. These interactions induce receptor clustering and promote FN self-association. Fibril elongation is then propagated by continual addition of FN dimers to growing multimers. As fibrils mature, they are gradually converted into a detergent-insoluble high molecular weight form yielding stable matrix. Matrix assembly modulates cell growth and activates a number of intracellular signal transduction cascades. The ot5fll integrin receptor is a major receptor for matrix assembly (Ruoslahti, 1991); however, three other integrins, allbfl3, ce4fll, and oevfl3 (Sechler et al., 2000; Wennerberg et al., 1996; Wu et al., 1995b) can substitute for c~5flI to support fibril formation. Sechler (Sechler et al., 1996) developed a matrix assembly system using cell lines lacking an endogenous FN matrix but which are capable of assembling exogenously provided FN. This system has been used to study the involvement of different FN domains in the fibril assembly process.

II. D e v e l o p m e n t o f Synthetic Matrices A. The Provisional Matrix In this section we describe how to reconstitute the final stages of the blood-clotting cascade using purified components to form a three-dimensional fibrin-FN matrix.

1, Advantages Three-dimensional matrix substrates reflect a physiologically relevant environment in which to analyze cell behavior. This overcomes the limitations of presenting cells with a single protein as a substratum in a planar organization. Since the provisional matrix is synthesized using purified components, it is easy to control the composition of the matrix and the proportion in which the matrix proteins are presented to cells. This system also allows for the use of recombinant FNs. The provisional matrix interacts with and regulates the behavior of many cells in vivo, including fibroblasts, blood platelets, endothelial cells, and smooth muscle cells (Clark, 1996). The formation of this matrix is not cell mediated; therefore it is possible to analyze the response of many different cells types, with known repertoires of receptors, to the matrix.

2. Disadvantages By reducing the ingredients to include only the simplest necessary components, we can produce a matrix that enables the dissection of the function of a single protein. However, since not all components present at sites of wound healing are included, preparation of synthetic matrices of this kind in vitro may not recapitulate all of the functions of native matrix.

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3. Protocols a. Fibrinogen Preparation Lyophilized human fibrinogen (98 % clottable, American Diagnostica Inc., Greenwich, CT) is reconstituted in 0.15 M NaC1, 10 mM Tris-HC1, pH 7.4. These preps contain 15-20/zg endogenous human pFN per mg fibrinogen. Elimination of contaminating FN is achieved by batch incubation with gelatin-agarose beads for 1 h at room temperature, after which time the sample is centrifuged to pellet the beads. The supernatant should be collected carefully and recentrifuged to ensure that all agarose is removed. The fibrinogen is then aliquoted, stored at -85°C, and thawed immediately before use. FN specifically binds to the gelatin (denatured collagen) using its collagen-binding domain. Removal of FN can be monitored by SDS-PAGE and Western blotting with anti-human pFN monoclonal antibodies. This procedure results in 100-fold depletion of FN to 0.15 #g per mg fibrinogen (Corbett et al., 1996; Wilson and Schwarzbauer, 1992) and is important to enable precise control of the concentration and type of FN in the synthetic matrices. This treatment eliminates the activity of endogenous factor XIII in the fibrinogen preps; therefore purified human factor XIII must be added to achieve levels of matrix crosslinking equivalent to untreated fibrinogen. b. In Vitro Synthesis of Provisional Matrices Bovine thrombin (96% clottable, Sigma Chemical Co., St. Louis, MO) is reconstituted in distilled water, aliquoted, stored at -20°C, and thawed immediately before use. Rat pFN is purified from freshly drawn rat plasma by gelatin agarose chromatography (Corbett et al., 1996). Matrix components are mixed together in physiological proportions. A 10:1 mass ratio of fibrin : pFN (600/zg/ml fibrin and 60/zg/ml pFN) is incubated with 10 #g/ml human coagulation factor XIII (Calbiochem-Novabiochem Corp., La Jolla, CA), 50 mM CaC12, 0.15 M NaC1, 0.05 M Tris HC1, pH 7.5, in 0.1 to 1.5 ml volumes. The mixtures are vortexed and kept on ice for 10 rain. Two U/ml thrombin are added to start the reaction, then the mixture is rapidly pipetted into 48-well nontissue culture dishes or onto glass coverslips and allowed to incubate at 4°C overnight to allow maximum cross-linking (see Fig. 1). To inhibit fibrinolysis, 1/zg/ml aprotinin can be added to matrices intended for long time point experiments. Fibrin circulates in the blood at 3000/zg/ml and pFN at 300/zg/mi (Clark, 1996). These higher physiological concentrations can be used in the formation of the matrix. Alternatively, a mass ratio of 20:1 fibrin : pFN can be used with identical results to ratios of 10:1 to allow more conservative usage of matrix proteins (Wenk et al., 2000). After polymerization, this matrix can be used for cell adhesion experiments. c. Inclusion of Other Proteins Full-length native pFN can be replaced with recombinant FNs to enable analysis of the specific effect of different domains of FN on matrix formation and cell phenotype (Corbett et al., 1997; Corbett and Schwarzbauer, 1999; Wilson and Schwarzbauer, 1992). This synthetic matrix can also be built upon by the addition of relevant proteins to reconstitute increasingly physiological matrices. Other proteins that contact the provisional

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\ Fibrinogen and fibronectin in solution

Soluble fibrin clot Ca2+ Factor Xilla

Covalently cross-linked provisional matrix Fig. 1 Provisional matrix formation. The events common to provisional matrix formation both in vivo and in vitro are depicted. Fibrinogen (thin lines), the soluble precursor of fibrin, is mixed with FN. Polymerization is initiated by the addition of thrombin to the system, which cleaves fibrinogen to form fibrin. The resulting monomeric fibrin spontaneouslypolymerizes to form a clot. In the presence of Ca2+, factor XllIa covalently cross-links fibrin to itself and FN (thick gray lines), producing high molecular weight polymers. These matrices provide a physiologicallyrelevant three-dimensional substrate for cell adhesion.

matrix in vivo, for example collagen and tenascin-C, can be incorporated into the matrix simply by adding them into the matrix mixture along with the basic components (Wenk et al., 2000). Those that contain binding domains for fibrin or F N will be incorporated into the matrix via interactions with target proteins. Proteins that contain neither type of site will be incorporated into the matrix by being trapped into the network o f fibrin-FN fibrils. After polymerization, the provisional matrix can be treated prior to the addition

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To incorporate cells into the three-dimensional matrix, cells are added to the matrix proteins before the addition of thrombin (Corbett and Schwarzbauer, 1999). Once thrombin is added, the mixture should be incubated at 37°C for 30 min. The cells become evenly distributed within the matrix as it polymerizes. Matrix contraction can be studied using this synthetic three-dimensional matrix in a well-characterized assay (Corbett et aL, 1997; Corbett and Schwarzbauer, 1999). After polymerization, the cell-matrix mixture is released from the sides of the dish and the ability of cells to contract the matrix is assessed by measuring the area of the matrix over time. The extent of contraction is dependent on matrix composition and cell type. For example, fibrin matrices will contract up to 60% over a 4-h time course, while fibrin matrices containing FN will contract 80% over the same time course (Corbett and Schwarzbauer, 1999). The concentration of cells added to achieve optimum cell density varies depending on the cell type; for example, for maximal contraction NIH3T3 fibroblasts are used at 1 x 106/ml and CHO cells at 2 × 106/ml. e. Immunofluorescence

For cell adhesion on or culture within three-dimensional matrices, cells are released from tissue culture dishes using 0.2 mg/ml EDTA in PBS, washed with PBS, and resuspended in 0.025 M Hepes, pH 7.4, 0.13 M NaC1. TPCK trypsin (Sigma) may also be used to harvest ceils from tissues culture dishes at a concentration of 0.1 mg/ml in Versene (Life technologies/Gibco-BRL). Cells are then washed in 0.5 mg/ml soybean trypsin inhibitor (Sigma) and resuspended in 0.025 M Hepes, pH 7.4, 0.13 M NaC1. Cells within three-dimensional matrices can be stained with fluorescently labeled antibodies (Fig. 2). The cell-matrix mixture is fixed with 3.7% formaldehyde for 15 min at room temperature, permeabilized with ice-cold acetone for 5 min at -20°C, then incubated with primary or secondary antibody in 2% ovalbumin (Sigma) in PBS at 37°C for 1 h. The cell-matrix mixture is then removed from the 48-well dish and mounted onto slides with SlowFade Light Antifade Kit (Molecular Probes Inc., Eugene, OR). To eliminate matrix thickness as a variable in the analysis of cell behavior, the provisional matrix can be carefully removed from the dish/coverslip by aspiration, leaving behind a visible fibrillar matrix. Cells respond identically to this fibrillar matrix as to a threedimensional matrix (Corbett etal., 1996). Matrices are prepared as before except cells are not added to the matrix components. Immediately after the addition of thrombin at 2 U/ml, the mixture is pipetted onto a glass coverslip (Fisher Scientific). After polymerization the matrix is aspirated, and the remaining matrix substratum blocked with 1% BSA in PBS. Cells are allowed to adhere to matrix-coated glass coverslips, then washed with PBS, fixed for 15 min at room temperature with 3.7% formaldehyde in PBS, and permeabilized for 15 rain at room temperature with 0.5% NP-40 (Calbiochem) in PBS. Cells are

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F i g . 2 Immunofluorescent staining of cells within a three-dimensional matrix. Cells were cultured for 18 h in a three-dimensional matrix consisting of fibrin and FN. After this time, cells were fixed and the actin cytoskeleton visualized using rhodamine-labeled phalloidin. Scale bar = 20 tzm. (See Color Plate.)

incubated with primary or secondary antibody in 2% ovalbumin (Sigma) in PBS at 37°C for 1 h. Coverslips are mounted with SlowFade Light Antifade Kit (Molecular Probes). Cells adherent to two-dimensional matrices can also be lysed using RIPA buffer (50 mM Tris-HC1, pH 7.5, 150 mM NaC1, 1% NP-40, 0.25% sodium deoxycholate, I mM PMSF, 1 mM NaVO4, 1 mM EDTA, 50 mg/ml leupeptin, 0.5% aprotinin) on ice for 15 min (Kanner et al., 1989). The cells are then scraped with a rubber policeman and the lysate collected and centrifuged for 10 min at 4°C. The cell lysates can be used in biochemical analyses such as immunoprecipitations to characterize levels of protein expression or activation.

4. Controls and Troubleshooting One variable that affects cell behavior on this synthetic matrix is fibrin-FN crosslinking. The degree of cross-linking of matrix components can be monitored by adding an equal volume of solubilization buffer (8 M urea, 2% SDS, 2% 2-mercaptoethanol, 0.16 MTris HC1, pH 6.8) to the matrix for 10 rain at 100°C. Separation and identification of cross-linked products is performed by SDS-PAGE. Fibrin polymers can be detected on a 7% gel, visualized by silver staining, and pFN on a 5% gel, detected by Western blots probed with anti-FN monoclonal antibodies.

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B. C e l l - A s s e m b l e d E C M

In this section we describe de novo matrix assembly by cells that do not produce an endogenous FN matrix. The mouse pituitary cell line AtT-20, which does not produce either the o~5 integrin subunit or endogenous FN, and Chinese hamster ovary (CHO)-K1 cells, which express low levels of hamster o~5 integrin, were transfected with human a5 cDNA (Sechler etal., 1996). The resulting transfected cells (AtT-20ot5 and CHOot5) produced no or negligible endogenous FN, respectively, but both could assemble a fibrillar matrix using exogenous FN supplied in the culture medium.

1. Advantages This matrix assembly system is not complicated by the presence of normal endogenous FN. It allows for the control of timing, rate, and amount of matrix assembly. One can plate the cells overnight to ensure appropriate adhesion and spreading, add known types and concentrations of FN, and follow the progression of fibril formation at specific time points after addition of FN. In vivo matrices are composed of more than one protein. The described method allows for the use of a mixture of different ECM proteins to create a more physiological composition of the matrix. Cells can be transfected with different types of receptors or combinations of receptors. One can also use different recombinant FNs to test for involvement of FN domains in the process of matrix assembly. A major advantage of this system is the ability to dissect the independent assembly of recombinant FNs from the earliest stages of matrix assembly.

2. Disadvantages In a defined or reconstituted system cells will not necessarily have access to all naturally occurring ECM proteins. The composition of ECM is complex and not fully known; thus the absence of some proteins may affect matrix structure or assembly.

3. Protocols a. Cells and Proteins

AtT-20~5 cells are grown in a 50:50 mixture of Ham's F12 and DMEM supplemented with 20 mM Hepes, pH 7.4, 4 mM L-glutamine, 10% fetal calf serum (FCS) (Hyclone Labs, Logan, UT), and Geneticin (Life Technologies/Gibco-BRL). CHOo~5 cells are cultured in DMEM medium containing 10% FCS, 2 mM L-glutamine, 1% nonessential amino acids, and 100 tzg/ml Geneticin. In a typical experiment, 1.5 x 105 ceils per well are plated on a 24-well dish in 500/zl of medium. To avoid traces of FN present in serum, one can use medium containing FN depleted serum. FN depleted serum can be prepared by passing FCS over a gelatin-agarose column twice (Engvall and Ruoslahti, 1977). The cells are allowed to adhere and spread (for a few hours or overnight) and then incubated with 25-50/.tg/ml of pFN added to the medium for different amounts of time. The extent of matrix assembly can be modulated by increasing the amount of

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FN added. Adding 25-50/~g/ml of FN results in formation of an extensive FN matrix. At concentrations below 25/zg/ml, a sparser pattern of FN fibrils is observed, with no apparent matrix formation below 5/zg/ml. Native FN can be purified from human, rat or bovine plasma by gelatin-agarose affinity chromatography (Engvall and Ruoslahti, 1977). Recombinant FNs are expressed using the baculovirus insect cell expression system (Aguirre et al.. 1994; Sechler et al., 1996).

b. lmmunofluorescence Staining Cells are plated on glass coverslips in 24-well dish or Lab Tek Chamber Slides (Nunc Inc., Naperville, IL). Spread cells are then incubated with FN. After the desired incubation time, cells are washed with PBS + 0.5 mMMgC12 and then fixed with fresh formaldehyde solution (3.7% in PBS) for 15 rain at room temperature Coverslips are incubated in a moist chamber with primary anti-fibronectin antibody followed by incubation with fluorescently labeled secondary antibody, both diluted in 2% ovalbumin in PBS at 37°C for 30 min. Each step is proceeded by several gentle washes with PBS. Finally, coverslips are mounted with FluoroGuard Antifade Reagent (Bio-Rad, Hercules, CA). Fibrils are visualized with a Nikon Optiphot microscope with epifluorescence (Fig. 3A).

A

FN

1 hr

6 hrs

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1 3 6 8 24 1 3 I

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Fig. 3 Time course of FN fibril formation. (A) Analysis by immunofluorescence staining. CHO~5 cells were incubated with 50/zg/ml of rat pFN. At indicated times, the cells were fixed, stained with rat-specific monoclonal antibody IC3, and visualized with fluorescently labeled secondary antibody. (B) Analysis of DOC-soluble and DOC-insoluble material. Cells were lysed with DOC lysis buffer. Fractions (3 Izg/lane) were separated by 5% SDS-PAGE and FN was detected with rat-specific monoclonal antibody IC3 and ECL reagents. (See Color Plate.)

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c. Isolation and Detection of DOC-Soluble and -Insoluble Material

Cells are plated in a 24-well dish without coverslips and incubated with FN. After the desired incubation time, cells are gently washed with cold PBS buffer and then lysed with 200/zl of deoxycholate (DOC) lysis buffer (2% DOC, 0.02 M Tris-HC1, pH 8.8). Two mM PMSF, 2 mM EDTA, 2 mM iodoacetic acid, and 2 mM N-ethylmaleimide are added as protease inhibitors. Other inhibitors can be substituted. Cells are scraped with a rubber policeman and transferred with a 26G needle to tubes. To shear the DNA, the lysate should be passed through the needle 5 times. DOC-insoluble material is isolated by centrifugation at 14,000 rpm for 15 rain at 4°C, and then solubilized in 25 tzl of 1% SDS, 25 mM Tris-HC1, pH 8.0, plus protease inhibitors. Total protein concentration can be determined using BCA Protein Assay (Pierce, Rockford, IL). Equal aliquots or equal amounts of total protein of DOC-soluble and insoluble material is electrophoresed on a 5% polyacrylamide SDS gel nonreduced or reduced with 0.1 M DTT. Separated proteins are transferred to nitrocellulose (Sartorius Corp., Bohemia, NY) for immunodetection. Membranes are blocked overnight in buffer A (25 mM Tris-HC1, pH 7.5, 150 mM NaC1, 0.1% Tween-20) at room temperature followed by 1 h incubation with anti-fibronectin antibody in buffer A and 1 h incubation with secondary antibody in buffer A. Each incubation is followed by extensive washes with buffer A (3 times for 10 rain). Finally, immunoblots are developed with chemiluminescence reagents (Pierce) (Fig. 3B). To quantitate the amount of DOC-soluble and insoluble material, equal amounts of total protein are separated by SDS-PAGE and transferred to nitrocellulose. After blocking overnight with 5% BSA in TBS buffer (50 mM Tris-HC1 pH 7.5, 200 mM NaC1), the membrane is incubated with anti-fibronectin antibody in blocking buffer for 1 h and then with 1/zg/ml of unconjugated secondary antibody in blocking buffer for I h. Each incubation is followed by extensive washes with TBS. Approximately 6/zCi 125I-protein A (specific activity l0 mCi/mg, ICN Biomedicals Inc., Irvine, CA) in 10 ml of blocking buffer is then used in a 1-h incubation. After extensive washes with buffer A (until the background is minimal), the blot is exposed to a phosphor storage screen and analyzed using a Molecular Dynamics Phosphorlmager (Sunnyville, CA). All of these steps are performed at room temperature. d. Time Course

One can follow the matrix assembly process using both immunofluorescence staining and analysis of D0C lysates by incubating the cells with FN for different lengths of time. In order to keep the cell number similar for each time point, it is suggested to add FN first to the longest time point, and then accordingly to the next ones. All incubations should finish at the same time. e. Serum Starvation

In some applications, such as analysis of protein phosphorylation or cell cycle progression, it is necessary to keep the ceils in serum-free medium. CHOot5 cells can be serum starved for up to 24 h to enrich for a population of cells in Go. Matrix assembly can then be followed by addition of FN in serum-free medium.

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4. Controls and Troubleshooting In experiments that test matrix assembly using recombinant FNs, one should always include a control of native FN purified from plasma. When analyzing a new cell type, it is advisable to compare FN matrix assembly between test ceils and fihroblasts, a cell type that normally produces FN matrix. Fibroblasts use endogenously produced FN for the assembly; however, they are also capable of assembling exogenously provided FN. Thus the same amounts and types of FN can be added to parallel cultures and assembly monitored microscopically and biochemically. Cell density is an important variable in determining the amount of fibril formation. Sparse ceils form short fibrils, mainly between the cell surface and the substratum. Cell cultures with many ceils in contact will assemble a denser matrix with fibrils extending between and over neighboring cells. When comparing different matrices, one should make sure that the cell density in the observed fields is comparable.

III. D i s c u s s i o n A. Synthetic Provisional Matrix The three-dimensional provisional matrix is an excellent model for deciphering the molecular events that regulate tissue injury and wound healing. Its applications are not limited to these events, however, and matrices of this kind can also be used to examine other processes which depend on a three-dimensional framework. Studies have focused on the organization of matrix architecture (Weisel, 1996) and how specific variations in the composition of the fibrin-FN matrix can be used as a mechanism to control cell behavior at sites of tissue repair (Wenk et al., 2000). Using recombinant protein technology, it has also been determined precisely which domains of matrix proteins mediate specific cellular effects. For example, the alternatively spliced V region of FN is required for efficient incorporation into a fibrin matrix (Wilson and Schwarzbauer, 1992), and covalent cross-linking of fibrin and FN is needed for maximum cell adhesion to the matrix (Corbett et al., 1997). Three-dimensional synthetic matrices have also been used to study different stages of tissue injury including initial cell attachment to the matrix, migration through the matrix, and contraction of the matrix (Clark, 1996). For example, wound contraction has been implicated in the pathology of organ scarring in a variety of fibrotic diseases. Disruptions in the regulation of contraction can lead to undesirable cosmetic scarring; and body deformation and loss of joint motion have been observed in cases where contraction persists after wound closure. Contraction has been studied by culturing cells within a three-dimensional matrix and determining the ability of cells to exert force on and contract the matrix. Recent work has demonstrated that integrins are required to communicate signals from three-dimensional matrices to the cell, causing Rho activation and focal adhesion and stress fiber formation, which lead to contraction of the matrix (Corbett and Schwarzbauer, 1999; Grinnell, 2000; Hocking et al., 2000; 'fee et aL, 1998; Midwood, unpublished observations).

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Cell migration into three-dimensional fibrin matrices has been used as a model for tumor metastasis and invasion. The ability of cells to cross barrier matrices gives an indication of the potency of tumor growth and spread. This type of system also allows analysis of the molecular basis of tumor progression, including identification of cell surface receptors used for attachment to and invasion into tissues (Svee et aL, 1996) and enzymes used to proteolytically forge paths through matrices for tumor cells and supporting vascular systems (Weidner et aL, 1993). Migration of cells is also important in the inflammatory response, and neutrophil migration through fibrin gels has been used as a model for events occurring in early inflammation (Schnyder et al., 1999). The relationship between the migration of macrophages into three-dimensional fibrin matrices to the composition of the matrix, for example, fibrin concentration, glycosaminoglycan content, or the degree of cross-linking, has also been studied (Ciano et al., 1986; Lanir et al., 1988). Matrix degradation is an essential part of wound repair. Multiple cell types must migrate through the matrix scaffolding, and migration is dependent on the activity of the fibrinolytic and proteolytic enzymes. Proteolysis has been analyzed in vitro using synthetic matrices. Migration of human keratinocytes into fibrin matrices occurs through tunnels of digested fibrin created by periceUular fibrinolysis. Formation of the tunnels requires that plasminogen activator be localized on the advancing surface of the keratinocyte (Ronfard and Barrandon, 2001). Fibrin-based matrices have also been used in the study of angiogenesis: the formation of new blood vessels. The angiogenic response of human microvascular endothelial cells can be analyzed by seeding cells on top of a three-dimensional fibrin matrix, resulting in the in-growth of capillary-like tubular structures into the matrix (van Hinsbergh et al., 2001). Similarly, endothelial cells resuspended in fibrin matrices form intracellular vacuoles that coalesce into lumenal structures, which process is regulated by integrins (Bayless et al., 2000). Tissue engineering combines cell biology, biomaterials science, and surgery, with a view to achieving tissue and organ replacement therapies using the patient's own cells. Fibrin matrices are commonly used as three-dimensional in vitro culture systems, consisting of single or multiple different cell populations, to develop vital tissue transplants before these preformed tissues are implanted into test subjects. Fibrin is an excellent biocompafible, biodegradable scaffold for cell anchorage, proliferation, and differentiation. It allows uniform cell distribution and quick tissue development, with none of the immunogenic effects of traditional scaffolds, which exhibit toxic degradation and inflammatory reactions (Ye et al., 2000). Tissues cultured in fibrin matrices have resulted in successful joint cartilage regeneration. Stable in vivo transplants which produce typical morphological tissue structure have been introduced into mice and rabbits (Sittinger et al., 1999). Treatment of defects in dogs with exogenous fibrin clots promotes fibrocartilaginous repair and stimulates the regeneration of tissue with a normal histological appearance. Such therapy may be used in the arthroscopic treatment of injury in an effort to improve postoperative outcome (Arnoczky et al., 1988). The attachment of endothelial cells after angioplasty can be greatly improved with fibrin glue matrix, resulting in a significant reduction of

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restenosis in atherosclerotic rabbits (Kipshidze et al., 2000). Other applications include the long-term regeneration of human epidermis on third-degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix (Ronfard et al., 2000). Fibrin gels modified with covalently bound heparin-binding peptides have been studied as a therapeutic agent to enhance peripheral nerve regeneration by stimulating neurite extension (Sakiyama et al., 1999). The use of three-dimensional matrices in tissue engineering has progressed to the development of designer matrices. Similarly, in many fields manipulation of matrix composition and molecular interactions has been used to develop agents for in vivo use. In this way, analyses done with synthetic multicomponent provisional matrices encompass a wide range of biological and therapeutic applications.

B. Cell-Derived ECM The deposition of FN into the ECM is an integrin-dependent, complex, and highly regulated process. Its involvement in many physiological and pathological processes encouraged scientists to study the assembly of FN matrices. Cell-assembled ECM systems have provided valuable insights into the role of matrix organization in the regulation of cell function. Different cell systems have been used to study how matrices are assembled. One of the first systems used fibroblasts, the cells that naturally assemble FN fibrils, to analyze the timing of incorporation of FN into existing matrix. Iodinated pFN or FN fragments such as the amino-terminal 70-kDa fragment were added to confluent fibroblasts that were surrounded by an endogenous FN matrix (McKeown-Longo and Mosher, 1983,1985). Incorporation of radiolabeled protein was followed over time, and this assay defined two pools of matrix FN. Pool I was soluble in buffered DOC and, over time, was converted into pool II, which was DOC-insoluble. The DOC-solubility assay became the standard for following FN assembly into matrix. A number of groups have combined this approach with inhibitory fragments, peptides, or antibodies to identify FN domains and receptors involved in the matrix assembly process (Chernousov et al., 1991; Hocking et al., 1996; McDonald et al., 1987; Morla and Ruoslahti, 1992). To avoid the complications inherent in using a cell system that produces an endogenous FN matrix, we have developed the CHO cell system described in this chapter (Sechler et al., 1996). CHOot5 cells have been used to identify FN domains involved and to understand the mechanism and regulation of the assembly process. Using CHOot5 cells and recombinant FNs containing or lacking specific sequences, it has been demonstrated that RGD-dependent interactions with ~5/~ 1 are essential for the initiation of matrix assembly (Sechler et al., 1996), that the cell-binding synergy site in FN is involved in ~5fl 1-mediated accumulation of FN matrix (Sechler et al., 1997), and that the first type III repeats play a regulatory role in conversion to DOC insolubility and in the rate of fibril formation (Sechler et al., 1996). Receptor requirements for FN assembly have also been dissected using CHO transfectants. CHO(B2) cells selected for deficiency in c~5 integrin expression have been particularly useful for these purposes (Schreiner et al., 1989). These cells require reintroduction

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of or5 by transfection in order to assemble FN matrix (Wu et al., 1993). CHO(B2) cells engineered to express different integrin receptors have been used to demonstrate that otlIb/33 and otvfl3 receptors provide alternative pathways for assembly ofFN (Wu et al., 1996, 1995b). Integrin subunits other than or5 can also be introduced into these cells. CHO B2 cells transfected with a 4 integrin receptor adhere, spread, and migrate on FN but do not assemble FN into fibrils (Wu et al., 1995a). Interestingly, however, CHO(B2)ot4 cells are able to assemble FN matrix after integrin stimulation with Mn 2+ or a fl 1-stimulatory antibody (Sechler et al., 2000). Cell lines other than CHO and AtT-20 have been adapted for use in analyzing de n o v o FN matrix assembly and the effects of matrix on cell behavior. Sottile et al. (1998) isolated a FN-null cell line and showed that it is capable of assembling exogenous FN by a mechanism similar to fibroblasts. Oncogenically transformed cells, which often express reduced levels of FN, other matrix proteins, and their receptors, are also useful models for analyzing the molecules and intracellular pathways involved in these processes (Brenner et al., 2000; Schwarzbauer, 1991b; Zhang et aL, 1997). Cells that depend on exogenous sources of FN have been very useful in determining the contributions of cytoskeletal organization to FN assembly as well as the effects of FN matrix on intracellular signaling and cell cycle progression. CHOt~5 cells show a rapid reorganization of actin into stress fibers, an accumulation of focal adhesion proteins, and activation of focal adhesion kinase (FAK) during assembly of native FN (Sechler and Schwarzbauer, 1997, 1998). A mutant recombinant FN lacking type III repeats 1-7 (FNAIIII_7) forms a structurally distinct fibrillar network. Cells assembling FNAIII1_7 show mainly cortical actin organization and reduced activation of FAK. Whereas native FN matrix stimulates cell growth (Mercurius and Moda, 1998; Sechler and Schwarzbauer, 1998; Sottile et aL, 1998), this mutant FN has the opposite effect and inhibits cell growth (Sechler and Schwarzbauer, 1998). These observations show that modification of matrix architecture has profound effects on cells and may provide a novel approach to control cell proliferation. The ECM has important effects on cell morphology, growth, and gene expression. Defects in matrix organization contribute to disease and developmental defects. Therefore, it is important to understand the mechanisms of ECM assembly and function as well as to decipher the compositions of matrices from different tissues. Using synthetic multicomponent matrices will allow us to draw a more complete picture of how cells are affected by their environment and will provide new insights into the control of cell phenotype by ECM interactions with cell surface receptors.

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