Provisional matrix deposition within an impaired collagen network
Fibrin network deposition during inflammation
Pegah Seddigh
Degree project inapplied biotechnology, Master ofScience (2years), 2011 Examensarbete itillämpad bioteknik 45 hp tillmasterexamen, 2011
Biology Education Centre and Department ofbiochemistry and microbiology (IMBIM), Uppsala
Index
Summary 2
List of abbreviations 3
1. Introduction
1.1. Loose connective tissue
1.2. Extra cellular matrix (ECM) of loose connective tissue 1.3. Collagen (CN-I)
1.4. Integrin family 1.5. Signaling
1.6. Platelet derived growth factor (PDGF) 1.7. PDGF receptor (PDGF-R)
1.8. Interstitial fluid pressure (IFP) 1.9. Cytoskeleton
1.10. Cell contraction
1.11. Inflammation and fibrosis 1.12 Fibrinogen
1.13. Tumor microenvironment 1.14. Aim
4 4 4 5 6 6 7 7 8 8 8 9 9 10 2. Results
2.1. Cell-mediated gel contraction
2.2. Effect of cyclic RGD peptide on Cell-mediated contraction of composite gels
2.3. Binding of biotinylated fibrinogen to CN-I coating
2.4. The effect of CNE on the Binding of the biotinylated fibrinogen to CN-I coating
2.5. The effect of CNE on composite gel contraction
2.6. Scanning electron microscopy images of collagen and composite gel
11 12 13 13 14 15
3. Discussion 16
4. Material and method 4.1. Cells and cell cultures
4.2. Collagen gel contraction (CGC)
4.3. Fibrin collagen gel contraction (FCGC) 4.4. Solid phase assay
4.4.1. CNE addition as a third protein 4.5. Electron Microscopy (EM)
17 17 18 18 18 18
5. Acknowledgement 19
6. References 20
Summary
Loose connective tissue fills the interstitial space and is Consisting of cells and extracellular matrix proteins (ECM). Collagen-I is the main component of the ECM.
Water and nutrients inside the capillaries flow out to interstitial space to feed the tissues. Interstitial fluid pressure (IFP) is the pressure of the fluid in interstitum. In normal tissues this pressure is slightly negative. This negativity is needed for the outflow of the nutrients from capillaries to tissues. Collagen binding integrins like α2β1 are the active integrins for regulating this pressure. These integrins are bound to the actin cytoskeleton from one end and to collagen-I fibers with the other end. They transmit signals from cytoskeleton to ECM and vice versa. Migration, spreading, proliferation and cell mediated matrix contraction are the main cellular responds to integrins. Within an impaired collagen network during pathological conditions such as inflammation or fibrosis the IFP alters. For example during inflammation the IFP is lowered compare to physiological conditions, while in fibrotic conditions such as tumor stroma the IFP is elevated. This elevated pressure in tumor stroma can act as a barrier against drug delivery.
Normalization of altered IFP in pathological conditions can happen in αVβ3 integrin dependent manner while collagen binding integrins are impaired. αVβ3 integrins can bind to those ECM proteins, which posses the exposed RGD sequence like fibrinogen, fibronectin, vitronectin and periostin. Signal transduction via αVβ3 integrin can induce cell spreading, cell migration and proliferation.
In order to investigate the effect of αVβ3 ligand deposition within an impaired collagen matrix, porcine aortic endothelial (PAE) cells were cultivated in
fibrin/collagen gels. These gels provide a three-dimensional cell culture condition for the cells. The gels can be made of only collagen fibers or in the case of composite gels combination of collagen fiber and another ECM protein such as fibrin.
In collagen gels, collagen binding integrins are the only active integrins, while in composite gels in which fibrin is added to the collagen gels, αVβ3 integrins can be part of cell-matrix interactions which resulted in an additional contraction comparing to the gels containing of only collagen.
It is shown that during pathological conditions, such as inflammation, the interactions between the cells and the collagen fibers are impaired. Thereby the interstitial
pressure is lowered. Stimulation of cells with PDGF can normalize the lowered IFP only through the function of αVβ3 integrins. Deposition of fibrin fibers induced additional cell-mediated matrix contraction. In order to check whether binding of fibrin fibers to collagen fibers is important in fibrin-induced contraction, binding of fibrinogen to collagen was investigated. Fibrinogen binds to collagen and its binding site is one of the two, exposed binding sites of collagen fibers in vivo. This binding site is known as matricellular binding site of collagen fibers. CNE, which is a
bacterial protein, binds to this site and it inhibits the binding of fibrinogen to collagen as well as fibrin-mediated contraction.
All together and according to presented data, deposition of fibrin fibers within the impaired collagen matrix during inflammation can be the mechanism through which PDGF can normalize the lowered IFP. Even though further investigations on PDGF- induced fibrin deposition are required.
List of abbreviations CT Connective tissue
LCT Loose Connective tissue DCT Dense Connective Tissue ECM Extracellular matrix protein CN Collagen
CN-I Collagen type one
PDGF Platelet Derived Growth Factor
PDGF-R Platelet Derived Growth Factor Receptor IFP Interstitial Fluid Pressure
F-actin Filamentous actin G-actin Globular actin MLC Myosin Light Chain FPA Fibrinopeptide
TAF Tumor Associated Fibroblasts PAE Porcine Aortic Endothelial CGC Collagen Gel Contraction FCGC Fibrin/Collagen Gel Contration RGD Arginine-Glysine-Aspartic acid EM Electron Microscopy
SEM Scanning Electron Microscopy TEM Transmission Electron Microscopy DMEM Dulbecco´s Modified Eagle Medium FBS Fetal Bovine Serum
BSA Bovine Serum Albumin
1. Introduction
1.1. Loose connective tissue
Muscle, nervous, epithelial and connective tissues are the four major types of tissues in vertebrates. Loose connective tissue (LCT) and dense connective tissue (DCT) are two subtypes of connective tissue (CT). LCT is spread all over the body. Under the skin, it covers all the muscles, bones and nerves. The main difference between these two subtypes is in their amount of fibrillar components.
LCT lays down a mechanical support for the cells of other tissues. LCT is also noticed as interstitium, which rests in interstitial space. LCT is consisting of extracellular matrix (ECM) proteins and cells.
Collagen type-I (CN-I), elastic fibers, reticular fibers, small amount of salt and water are LCT substances. CN-I is the most abundant protein in LCT among all the other ECM proteins. CN-I is an important element for cell adhesion. In LCT fibroblasts bind to CN-I fibers through their collagen binding integrins. This binding can result in the balance of cell-matrix tension, Cell-mediated ECM contraction, and cell migration depending on the number of cells, collagen density and matrix restraint.
Fibroblasts are the main cells in LCT. Other than fibroblasts, there are other cells in LCT like macrophages, mast cells, lymphocytes, eosinophilic cells and plasma cells.(Grinnell 2008)
1.2. ECM of loose connective tissue
ECM forms interstitial matrix and the basement membrane. ECM proteins form huge and complicated fibrilar structures, which are folded separately. Cells bind to different binding domains of these fibers through their integrins
Cells bind to different binding domains of these fibers through their integrins, which triggers different signaling cascades. ECM has different elements with different structures. These structures provide a framework in which fibroblast cells function and generate tensions that give physical supports to the other tissues.(Hynes 2009)
1.3. Collagen
Collagen (CN) is a family of proteins with 29 known members. CN fibers are tape- shaped, collarless and wavy strands with 1 to 100 nm thickness(Ushiki 2002).
Human and all vertebrates have CN-I with the largest amount, comparing to all other types of matricellular proteins, in their body. Almost 90% of the CN in human body is type I. It is manufactured in endoplasmic reticulum and it can be detected mostly in ECM, skin, tendons, teeth, and bones. Different human diseases are related to mutations in this type of CN. Vascular disorders and osteogenesis imperfecta are some familiar examples.CN-I includes two procollagen chains, α1 and α2. These two chains are encoded with different genes. Two α1 and one, α2 chains are the
components of a procollagen type-I monomer. This monomer is a triple helix which specific proteinases cut their globular ends before they aggregate. Five, 67-70 nm
long, monomers combine in a quarter-staggered shape and form a micro fibril, which is a CN fiber subunit(Sweeney, Orgel et al. 2008).
CN fibrils are cylindrical with a diameter of 10-500 nm(Ushiki 2002). They bind together through their C and N terminals and they form a CN fiber. Less than 10 nm thick proteoglycan filaments are visible on collagen fibril bundles, which binds adjacent collagen fibrils(Fleischmajer, Fisher et al. 1991). CN fibers have a banding pattern called D periods (Sweeney, Orgel et al. 2008), because it contains neighboring overlap and gap zones repeatedly.(Ushiki 2002) These overlaps and gap zones expose as dark and light zones. It has been shown that proteoglycans play a role in adjusting such structures. Some investigations showed that D periods have different lengths according to the tissue and the organ(Marchini, Morocutti et al. 1986).
Studies have shown that there are grooves and ridges, repeatedly, on the surface of the CN fibrils, which is due to the dark and light zones(Gross and Schmitt 1948;
Marchini, Beltramello et al. 1984).
CN fibers bind together and make large bundles of CN in LCT(Orberg, Klein et al.
1982).
Basically the fibers in the ECM are divided into two groups. The collagen fibers, which act as a backbone for other tissues and cells and the elastin fibers(Ushiki 2002).
CN-I is the main component of ECM. Some investigators believe that the thickness of CN fibers change when they bind to other ECM proteins(Hulmes, Kadler et al. 1989).
CN-I is an important element for cell adhesion. Cells can bind to it through their collagen binding integrins, directly or indirectly(Hynes 2009).
1.4. Integrin family
Integrins are cellular transmembrane receptors, which have several domains. Three main domains of integrins are the ligand binding domain, the transmembrane domain and the cytoplasmic domain by which integrins mediate cell-cell, cell- ECM and cell- pathogen interactions(Luo, Carman et al. 2007).
The integration of the ECM and cytoskeleton is the reason for naming these molecules as integrins.
Integrins are heterodimeric glycoproteins, which consist of two subunits, α and β.
There are 18 α and 8 β subunits in vertebrates, which make 24 αβ complexes. The α and β subunits are noncovalently bound together. Integrins transfer signals
bidirectional, from outside to inside the cell and vice versa.
Investigations on the topology of the integrins showed that they have a globular ligand-binding N terminal, which is bound to the C terminal legs via their transmembrane domain. There is a seven bladed β-propeller domain on the N- terminal of the α subunit. The α subunit contains a domain called inserted domain, which is the ligand-binding domain in integrins. The other name for this 200 amino acid domain is von Willebrand factor A domain. There is a homologous domain to α I domain in β subunit. All α subunits contain four or five extracellular domains, one transmembrane and one cytoplasmic domain. All β subunits contain eight
extracellular domains, one Von Willebrand factor A and one cytoplasmic domain with different lengths(Du, Chang et al. 2010). Basically integrins are bound to their ligands in ECM via their β subunit end(Ramsay, Marshall et al. 2007).
Integrins are multi specific. According to their conformational state they bind to the targeted ligand. Most integrins can be subdivided into three main groups based on their compositions, β1 integrins, β2 integrins and αv integrins. β1 integrins can bind
to ECM proteins like CN, fibronectin and laminin. And αv integrins can bind RGD sequence, which is present in many ECM proteins like fibronectin, vitronectin, periostin and fibrinogen(Wipff and Hinz 2008).
1.5. Signaling
Integrins bind the certain ECM protein based on their specific binding affinity to the ECM members. After binding of integrins to their ligands, the signaling process starts with some conformational changes of the integrin. Due to their activation there are many different integrin-dependent adhesions and a big number of proteins are taking part in such adhesions. All together, they deliver different structures and functions.
Focal adhesions are, the best known integrin-based adhesions. Focal adhesions are elongated structures in which, integrins bind to their ligands via one end in ECM, with the other end they bind to cytoskeletal components(Winograd-Katz, Brunner et al. 2011). This binding results in signal transduction. This leads to cell responses, like contraction, migration and spreading.
Based on the surrounding situations, integrins can stimulate either cell survival or apoptosis. Integrins, consistently, look for ligands in ECM. The ligated integrins are the ones, which transfer survival signals to the cell. In this way, inappropriate cells enter apoptosis step, which is useful for cell integrity(Desgrosellier and Cheresh 2010).
It is shown that during physiological conditions, collagen binding integrins including α1β1, α2β1, α10β1 and α11β1are playing the main role in the maintenance of IFP.
While in inflamed tissues these integrins are impaired of signaling, normalization of lowered IFP can only happen after stimulation of cells with PDGF in αvβ3 integrin- dependent fashion.(Heldin, Rubin et al. 2004)
Thereby, in this case platelet derived growth factor (PDGF) has a synergistic activity with αvβ3 integrin(Woodard, Garcia-Cardena et al. 1998).
1.6. Platelet derived growth factor (PDGF)
Growth factors (GF) are molecules, which stimulate cell growth, proliferation and differentiation. The uncontrolled activity and gene expression of the GFs leads to generation of abnormal cell conformations like cancer cells(Srinivasan, Kapoor et al.
2005). GFs are mostly proteins. They are divided into different groups based on their structures and evolutionary origin. PDGF as its name shows, is a growth factor, which was thought to be originated only from platelets but later many cells were found to secrete PDGF (Heldin and Westermark 1999; Uutela, Taulu et al. 2001; Italiano, Richardson et al. 2008). It is one of the growth factors that plays important role in tissue remodeling and fibrosis. PDGF is a homo- and heterodimeric glycoprotein. The two chains are bound together via disulfide bond. There are four PDGF polypeptide chains, A, B, C and D, which make 5 dimeric PDGF isomers. PDGF-A and B chains form hetero and homodimers whereas PDGF-C and D chains only assemble as homodimers. AA, BB, AB, CC and DD are the five dimers. PDGF basically has been isolated from PDGF-AB, which has the largest number among all the
others(Hammacher, Hellman et al. 1988). They are all synthesized in endoplasmic reticulum. mesenchymal cells like fibroblasts and smooth muscle cells can mainly produce them(Shimokado, Raines et al. 1985; Paulsson, Hammacher et al. 1987).
There is no priority for the production of PDGF A or B for the cells, which can produce both. This shows that this production happens randomly(Hammacher, Nister et al. 1988).
1.7. PDGF receptor (PDGF-R)
PDGF-R is a member of tyrosine kinase family. Binding of PDGF to the receptor induces receptor dimerization and further cytosolic/intrinsic activation of the receptor.
Since PDGF-R has 2 different isoforms, ligand binding to PDGF-R can lead to formation of 3 following dimerized receptors: αα, ββ and αβ(Fredriksson, Li et al.
2004). PDGF-R is a transmembrane protein consisting of 3 distinct domains.
Extracellular domain that contains five immunoglobin-like domains from which, the three outer domains are specific for ligand binding. The other two domains are the middle domain and the intracellular domain, which is a tyrosine kinase domain.
Receptor dimerization leads to autophosphorylation of tyrosine residues of
intracellular domain(Claesson-Welsh, Eriksson et al. 1989; Heidaran, Pierce et al.
1990).
For termination of the signaling downstream of PDGF either the whole complex of the PDGF-R and PDGF diminishes or the PDGF-R returns to plasma membrane after separation from the PDGF molecule(Heldin and Westermark 1999).
1.8. Interstitial fluid pressure (IFP)
The interstitium is one sixth of the human body volume. Water and molecules are transported from capillaries to tissues through interstitial space. The out flow of water and molecules from capillaries to tissues, have an important role in tissue
homeostasis. This transcapillary flow is possible because the hydrostatic pressure of the interstitial fluid during physiological conditions is always slightly negative. In normal tissues the IFP is strongly regulated through the function of collagen binding integrins, which in turn regulates the exchange of solutes between plasma and the tissue. Fibroblast cells are the main cells of LCT in regulation of IFP. They generate cellular tensions on fibers of ECM, mainly CN-I fibers, through their integrins. These cellular tensions are the main physical forces, which on one hand give support to the other tissues and on the other hand they compact the whole interstitium, which leads to IFP maintenance.
In tumor stroma IFP is higher than the LCT during physiological condition. Fibrosis, contraction of the interstitial matrix and vessel abnormalities are some factors that assist the increase of IFP in tumors. This Elevated IFP acts as a barrier for the transcapillary flow, which in turn impairs the drug delivery within the tumor tissue.
During anaphylaxis, burn injuries and inflammation IFP is lowered, which can lead to edema formation. One of the known mechanisms to normalize the lowered IFP is PDGF-induced fibroblast-mediated contraction of ECM through function of αVβ3 integrins(Heldin, Rubin et al. 2004).
1.9. Cytoskeleton
Cytoskeleton is the cell skeleton. It is mainly involved in the cell motility and intracellular transport. It is composed of three different groups of proteins,
microfilaments (actin), microtubuls and intermediate filaments(Pollard and Borisy 2003). Filamentous actin (F-actin) is the most abundant component of the
cytoskeleton in eukaryotes. Actin can be found in the cytosol mainly in two forms, F- actin or globular actin (G-actin). There are many actin-binding proteins that are involved in polymerization, depolymerization of actin as well as stabilization of F- actin. Some of these actin proteins also act as G-actin carrier(Pellegrin and Mellor 2007).
1.10. Cell contraction
The contraction of the eukaryotic cells is mainly depended on the combination of the two main extra and intracellular interactions, the interactions between the ECM proteins and integrins as the extracellular interactions and the turnover of F-actins as intracellular interaction.
The intracellular interaction during cell contraction is mainly mediated by myosin.
Myosin has an ATPase activity, which increases by miyosin light chain (MLC) phosphorylation. The increase of the ATPase activity triggers the actomyosin contraction, which results in cell body contraction(Pellegrin and Mellor 2007).
The actin-myosin drift in the cell results in the movement of the complex of the ECM protein and integrin. This movement leads to the contraction of the ECM protein.
Collagen is the ECM protein that mainly is involved in this system(Dallon and Ehrlich 2008).
1.11. Inflammation and fibrosis
Wound formation and body infection by bacterial or viral attack faces a biological response called inflammation. Redness, swelling, pain and heat are the related
symptoms to inflammation. Inflammation occurs in the body with the aim of recovery of the damaged or infected tissue.
Inflammatory cells like mast cells, macrophages and leukocytes stimulate
vasodilatation by secreting vasodilators like prostaglandins which loweres the IFP in these tissues rapidly. This early response to inflammation is called edema.
Inflammation and edema lower the IFP. Fibrosis develops by extreme deposition of CN-I. Fibrosis is the reaction of a never healing injured tissue to chronic
inflammation, which is irreversible. Fibrosis can happen both in external and internal organs(Shih, Garside et al. 2010).
1.12. Fibrinogen
Fibrinogen is a plasma protein with the molecular weight of 340 kDa and length of 45 nm. This protein is composed of two sets of polypeptide chains. Each chain is
comprised of three chains, α, β and γ. Each fibrinogen molecule has a central domain, which is named E domain and two outer domains, which are called D domains. α, β and γ chains are bound together in the N-terminal of the E domain with 5 disulfide bridges(Henschen, Lottspeich et al. 1983; Hoeprich and Doolittle 1983).
Fibrinogen plays an important role in coagulation process and wound healing, cellular and matrix interactions and inflammatory responses(Dempfle and Mosesson 2003).
Plasma factor XIII binds to fibrinogen and fibrinogen is a carrier for circulating factor XIII(Siebenlist, Meh et al. 1996).
Each fibrinogen has a sequence termed fibrinopeptide A (FPA) at the N-terminal of the α chain. Thrombin, which is a serine protease enzyme, triggers polymerization of fibrinogen to fibrin by cutting this FPA sequence. This is the spot when fibrin
aggregation starts. This assembly is due to exposure of polymerization site after FPA separation(Liu, Koehn et al. 1985). Thrombin binds to a high affinity thrombin-
binding site in fibrinogen through a site, which is specified for fibrinogen recognition.
This binding site is called exosite(Fenton, Olson et al. 1988).
Fig.1. SEM image of Fibrin fibers, 50 ug/ml Fibrinogen, 1:1000 Thrombin
1.13. Tumor microenvironment
Perpetuated exposure of the inflamed tissue to the body immune respond can result in formation of cancer(Coussens and Werb 2002). Solid tumor tissues are some times considered as never healing wounds. They are very similar to acute inflammation and fibrosis. Same cells, growth factors and processes are found in solid tumor tissues as in inflammations.
Angiogenesis is one of the tumor microenvironment characteristics through which tumor tissue produces new blood vessels. All the active tissues need to get oxygen
and nutrients and they also need to expel the wastes. Tumor tissues even need more nutrients like glucose(Kroemer and Pouyssegur 2008). Therefore they produce new vessels. This new vessels appear as a consequence of the continuous activity of the growth factors within tumor microenvironment. This, usually, leads to find immature blood vessels in tumor tissues, which have poor functions.
Although the blood vessels in tumor tissues are leaky the IFP is higher than the IFP in the normal tissues. The reason for this phenomenon is the compact collagen network in ECM due to fibrosis(Alexandrakis, Brown et al. 2004). The ECM in tumor tissue is composed of altered collagen fibrils compared to normal tissue(Eyden and
Tzaphlidou 2001). They have different diameters and they are usually thicker than collagen fibrils in a normal tissue(Hull and Warfel 1986).
In a tumor interstitium there are also fibroblasts. The fibroblasts in tumor tissue are called tumor-associated fibroblasts (TAFs). Tumor cells not only activate their own growth but also they stimulate growth in other cells in the microenvironment by secretion of a large amount of growth factors(Van Ginderachter, Movahedi et al.
2006).
1.14. Aim
The general aim of this thesis was study on the effect of αvβ3 ligand proteins in cell- mediated matrix contraction. More specifically, Investigation on the mechanism by which, αvβ3 integrin participates in normalization of the lowered IFP in inflammation and injured tissues.
2. Results
2.1. Cell-mediated gel contraction
The effect of fibrin fibers on cell-mediated matrix contraction was investigated by studying cell-mediated contraction of composite gels consisting CN-I and fibrinogen.
Addition of fibrin to CN-I gels induced additional contraction compare to gels consisting only CN-I. This additional matrix contraction was dependent on the function of αVβ3 integrins (Fig.2.).
Fig.2. PAE-Rβwt mediated contraction of Collagen gel, +/-PDGF-BB and Fibrin/collagen gel, +/- PDGF-BB. PAE-Rβwt cells were cultivated both in collagen and fibrin/collagen gels. Cells in fibrin/collagen gel showed higher contraction compare to collagen gels. PAE-Rβwt cells have both collagen binding integrins and αVβ3 integrins. The amount of the contraction of the gel in collagen gel contraction shows the activity of the collagen binding integrins in the cell. The amount of the
contraction of the gel in fibrin/collagen gel shows the activity of the both integrins. Collagen is the ligand for collagen binding integrin and fibrinogen is the ligand for αVβ3 integrn. The extra contraction of the fibrin/collagen gel comparing collagen gel is the pure activity of αVβ3 integrin bound to fibrinogen.
2.2. Effect of cyclic RGD peptide on Cell-mediated contraction of composite gels
In order to confirm that fibrin-induced cell-mediated matrix contraction is depending on the function of αVβ3 integrin, the effect of cyclic RGD (cRGD) peptide in
contraction of composite gels was investigated. cRGD is a specific inhibitor for αVβ3 integrins. Treatment of cells with cRGD inhibited the fibrin-induced contraction of gels via cells (Fig 3).
Fig.3. PAE-Rβwt mediated contraction of composite gels. This contraction is αVβ3 dependent. Cyclic RGD blocked αVβ3 integrin. Therefore the binding of the fibrinogen to αVβ3 integrin was inhibited. In consequence of this event fibrin/collagen gel lost its extra contraction.
2.3. Binding of fibrinogen to CN-I coating
The first step during investigation on the effect of deposition of fibrin fibers network within collagen network was studying the binding of fibrinogen to CN-I. In order to do that, binding of biotinylated soluble fibrinogen to the coating of CN-I was
performed by solid phase assy. Biotinylated fibrinogen bound to CN-I coating in the concentration-dependent manner. Based on the saturation curve, the KD of this binding was 100 nM (Fig.4.)
Fig.4. The binding of fibrinogen to collagen coating (10 ug/ml) in a dose dependent manner, (Kd≈100 nM), Fibrinogen is 340 KDa
2.4. The effect of CNE on the Binding of the biotinylated fibrinogen to CN-I coating
After finding the binding affinity of fibrinogen to CN-I, in order to investigate the binding site of fibrinogen on CN-I, a bacterial protein called CNE was used. CNE binds to a specific binding site on collagen fibers and this site is one of the two exposed binding sites on collagen fibers in vivo. This means that CNE can compete with any protein that binds to the same site on collagen fibers.
Adding CNE to CN-I and fibrinogen surprisingly caused an inhibition in the binding of the biotinylated fibrinogen to CN-I. This inhibition proved that fibrinogen and CNE are bound on the same site, on collagen-I molecule (Fig.5).
Fig.5. The inhibition of the binding of fibrinogen (25 ug/ml) to CN-I coat (10 ug/ml) with CNE
2.5. The effect of CNE on composite gel contraction
In order to investigate the importance of binding of fibrin fibers to CN-I fibers in fibrin-induced contraction of composite gels, CNE, the bacterial protein that inhibited the binding of fibrinogen to CN-I was used. In order to study this contraction in more specific way, C2C12 cells that are lacking collagen binding integrins were used.
Thereby these cells in order to contract the composite gel can only use fibrin fibers through their αVβ3 integrins (Fig 6). Treatment of these cells with CNE protein inhibited the cell-mediated compaction of composite gels via C2C12 cells. In this case the weight of gels after 24 hours in different conditions was measured which
correlates with the magnitude of contraction (compaction) of composite gels (Fig.7).
Fig.6. C2C12 fibrin/collagen gel contraction
CNE inhibits the compaction of the fibrin/collagen gel contraction. The more CNE in the gel results in heavier gels. So the process is dose dependent.
Fig.7. CNE inhibits the weight loss of the fibrin/collagen gels, which means it inhibits the c2c12 contraction.
2.6. Scanning electron microscopy images of collagen and composite gels
Scanning electron microscopy analysis of composite gels was performed in ordered the study the fibrilar structure of composite gels containing CN-I and fibrin compare to CN-I gels. As it is shown in figure 8 panels B, fibrin fibers are bound to CN-I fibers and form a network within CN-I fibers network.
A B
Fig.8.A: collagen, B: Fibrin/collagen. Collagen-I (1,2 mg/ml) Fibrinogen (50 µg/ml). Fibrinogen binds collagen-I.
3.Discussion
In the present study the possible mechanisms through which PDGF normalizes the lowered IFP at inflammatory sites was investigated. This normalization process is αVβ3-dependent. It was hypothesized that PDGF-induced secretion of αVβ3-ligands within the impaired matrix can be part of this normalization mechanism. Since fibrin deposition is one of the early events during inflammation, the aim of this particular project was to investigate on the effect of fibrin deposition on cell-mediated matrix contraction.
Deposition of fibrin within CN-I gels triggered additional cell-mediated contraction of composite gels compare to gels containing only CN-I (fig.2). This contraction was dependent on the function of αVβ3 integrins. cRGD peptide which is a specific inhibitor of αVβ3 integrins blocked the additional contraction of composite gels (fig.3).
The next step was to investigate whether fibrin and CN-I fibers interact or not and if these interactions are involved in fibrin-induced contraction of composite gels.
Protein-protein binding studies showed that fibrinogen binds to CN-I with the Kd of 100 nM (fig.4 ). In LCT the collagen fibers are covered with collagen binding
proteoglycans except two sites on the fibers that are always exposed. One of these two sites is the binding site for collagen binding integrins and the other one is the binding site for a number of matricellular proteins such as fibronectin. Fibrinogen also bind to the exposed site of CN-I fibers which is the binding site for certain matricellular proteins. CNE is a bacterial protein that has a binding affinity to this site of CN-I fibers. Addition of CNE inhibited the binding of fibrinogen to CN-I (fig.5 ). This indicates that fibrinogen also binds to the exposed site on CN-I fibers, which is a target for certain number of matricellular proteins such as fibronectin.
The next step was to investigate whether the binding of fibrin to CN-I fibers is involved in fibrin-induced contraction of composite gels. Treatment of cells with CNE inhibited the additional compaction of gels caused by fibrin (fig.7).
Altogether, this study suggests a possible mechanism through which lowered IFP at the inflammatory sites can be normalized. Deposition of fibrin as one of the early events during inflammation can be involved in stimulation of the fibroblast-mediated contraction of impaired matrix at the inflammatory sites. The stimulation of
contraction can lead to increase in IFP and normalization of the lowered IFP. During this normalization process, fibrin matrix, play as a provisional matrix within the impaired matrix of CN-I. This provisional matrix can be targeted for cellular tensions generated by fibroblast that are involved in the regulation of IFP. However, further investigations on the link between PDGF-R stimulation and deposition of fibrin or any other provisional matrix within the impaired matrix is required.
4. Materials and Methods
4.1. Cells and cell cultures
C212 cells are mouse myofibroblast. This cell line was provided by Dr.Anna Starzinski-Powitz(Goethe-University, Frankfurt, Germany). Dulbecco´s Modified Eagle Medium (DMEM) with Glutamax (Gibco-BRL life Technologies, Gaitherburg, MD) is the proper medium for this cell type. 10% FBS( fetal bovine serum) was added to the medium as the nutrition material and 0.1% gentamicin (Gibco) was added to protect the medium from bacterial growth.
L3 cells are C2C12 cells that have been transfected by α2 subunit (were kindly provided by proffessor Stephan Johansson). The appropriate medium for this cell line is exactly the same as C2C12 cells.
Porcine aortic endothelial (PAE) cells that were transfected with human PDGF- receptor-β were kindly donated by Dr. Carl Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala Branch). For this cell line Ham´s F12 medium (Gibco-BRL life Technologies, Gaithersburg, MD) supplemented with 10% FBS and 0.1%
gentamicin was used.
During the cell culture, all the cell lines were incubated at 37°C and 5% CO2
4.2. Collagen gel contraction
Collagen gel contraction (CGC) is a method to investigate the cells in a three- dimensional situation. Fibroblast cells cultivated in these gels can compact the gels through their collagen binding integrins(Bell, Ivarsson et al. 1979). These integrins are consisting of α1, α2, α10 or α11 together with β1 subunit. (Gullberg, Tingstrom et al. 1990). This model can be used in order to study cellular behavior during processes such as wound healing and tissue homeostasis. In this method cells are cultivated in free-floating gels in which the culture conditions resembles the situations that cells have in real tissues. In this method, if cells have the right integrins and functioning contractile machinery, they will compact the gel in the course of time. This
contraction can be monitored and the result can be presented either as the amount of reduction in the gel area or reduction of gel weight. In both case the result will resemble the compaction of the whole gel.
In this method, first the gel solution was made of 5 part double concentrated DMEM, 1 part HEPES buffer (0,1 M, pH 8) and 4 parts CN-I solution (3 mg/ml, pH 2). Then, after adjusting the density of cells in cell suspension solution, cells were added to the gel solution with the ratio of 1:9. Then 100 µl of final solution was casted to each well on 96-well plate. After 1,5 hours incubation of gels at 37 C, when gels were
polymerized, floating step was done. In this step, 100 µl of cell culture medium (DMEM) was added to each well in order to float the gels and avoid any tensions between gels and well that can act as counter force against cell-mediated contraction of gels.
4.3. Fibrin collagen gel contraction
Fibrin/ collagen gel contraction (FCGC) was performed in the same way as CGC, with two additional steps. First, after mixing cells in the collagen gel solution, fibrinogen was added to make the concentration of 50 µg/ml in the final volume of the solution. Second, in the floating solution thrombin, which is a serine protease involved in fibrin polymerization, was added.
4.4. Solid phase assay
This method is based on protein protein interactions. The binding of the fibrinogen to collagen was investigated in our study. Also the effect of the addition of the third protein was investigated. The third protein was CNE (kindly donated by professor Bengt Guss), which, is a bacterial protein. CNE has a binding site on collagen type I.
It has been isolated from Streptococcus equi, which causes a contagious respiratory disease in horses. CNE binds native fibrillar collagen.(Lannergard, Frykberg et al.
2003)
96 well plates has been coated with 10 µg/ml CN-I over night at 4°C. The plate has been blocked with the solution of 2% bovine serum albomine (BSA) for another 24 hours. 120 ul of hydroxy succinidine biotin ester (1mg/ml in DMSO) was added to 1 ml Fibrinogen with the concentration of 1 mg/ml. Fibrinogen was diluted in 0.5%
BSA in PBS. The mixture of the fibrinogen and biotin was incubated in cold room, over night. 1.38 ml 0.5% BSA in PBS was added to mixture to make the total volume of 2.5 ml. In order to elute the sample PD-10 column was used. The column was equilibrated with 25 ml PBS solution (1:500 azide and 0.5% BSA in PBS). The sample was eluted with 3 ml of the PBS solution.Different concentrations of the Biotynilated fibrinogen was added to 96-well plate and incubated in 37°C for 2 hours.
The plate was washed with 0.05 % tween in PBS 3 times and dried carefully. Avidin, as an enzyme which binds to Biotin, was added to the plate (1:500 in 0.5%
BSA,0.05% tween in PBS). The plate was in the room temperature for 2 hours. After a step of 3 times washing with 0.05 % tween in PBS the substrate of the enzyme (p- nitrophenyl phosphatase 0.6 mg/ml in ethanolamine solution,pH:9.8) was added to the dried plate. 15-30 minutes incubation in 37°C leads to an enzymatic interaction and yellow color in the plate. Optical density (OD 405) was measured.
4.4.1.CNE addition as a third protein
In order to investigate the binding of fibrinogen to CN-I in the presence of CNE, CNE was added to the blocked and 10 ug/ml CN-I coated plated. After 2 hours incubation in 37°C the plate was dried and fibrinogen was added to the plate. The rest of the experiment was done as it has been explained above.
4.5. Electron Microscopy (EM)
Electron microscope is a microscope that uses electron as energy source to magnify the sample image. The resolving power in electron microscope is much higher than
light microscope. There are two types of electron microscope: transmission electron microscope (TEM) which produces images by passing through very thin pieces of the sample and scanning electron microscope (SEM), which produces images of the sample’s surface.
Collagen gels and composite gels were made through the method, which has been explained. The gels were fixed with 0.15 M cacodylate buffer pH 7.4 with 2.5% fresh gluteraldehyde. Gels were kept in dark and 4°C in the fixing buffer over night. After overnight incubation, samples were washed 3 times with 3 times wash with
cacodylate buffer (0.15M, pH 7.4) each time samples were incubated in the washing solution for 10 minutes. Then the samples were dehydrated by gradient of alcohol (70%, 95% and 100%) 3 times each and each time for 10 minutes. Critical point drying, which was performed by electron microscopy staff, was the last step of the sample preparation for electron microscopy.
Acknowledgements
First I would like to thank my supervisors Prof. Kristofer Rubin and Vahid Reyhani for having me in the group and their kind guidance and patience throughout this project.
I also thank the whole IMBIM department and specially B9:3 corridor members for the very friendly environment.
Furthermore, I want to thank my coordinator Prof. Staffan Svärd and my friends for being on my side in all situations.
Last but not least, I thank my family, specially mum and dad for their everyday love and support.
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