Endothelial cell microparticles act as centers of matrix metalloproteinsase-2 (MMP-2) activation and vascular matrix remodeling

Endothelial cell (EC)-derived microparticles (MPs) are small membrane vesicles associated with various vascular pathologies. Here, we investigated the role of MPs in matrix remodeling by analyzing their interactions with the extracellular matrix. MPs were shown to bind preferentially to surfaces coated with matrix molecules, and MPs bound fibronectin via integrin α(V) . MPs isolated from EC-conditioned medium (Sup) were significantly enriched for matrix-altering proteases, including matrix metalloproteinases (MMPs). MPs lacked the MMP inhibitors TIMP-1 and TIMP-2 found in the Sup and, while Sup strongly inhibited MMP activities but MPs did not. In fact, MPs were shown to bind and activate both endogenous and exogenous proMMP-2. Taken together, these results indicate that MPs interact with extracellular matrices, where they localize and activate MMP-2 to modify the surrounding matrix molecules. These findings provide insights into the cellular mechanisms of vascular matrix remodeling and identify new targets of vascular pathologies.     

Characterization of heparin-binding site of tissue transglutaminase: its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca(2+)-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 (202)KFLKNAGRDCSRRSSPVYVGR(222). We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.     

The plant extracellular transglutaminase: what mammal analogues tell

The extracellular transglutaminases (TGs) in eukaryotes are responsible for the post-translational modification of proteins through different reactions, cross-linking being the best known. In higher plants, extracellular TG appears to be involved in roles similar to those performed by the mammalian counterparties. Since TGs are pleiotropic enzymes, to fully understand the role of plant enzymes it is possible to compare them with animal TGs, the most studied being TG of type 2 (TG2). The extracellular form of TG2 stabilizes the matrix and modulates the interaction of the integrin-fibronectin receptor, causing the adhesion of cells to the extracellular matrix; TG2 plays a role also in the pathogenicity. Extracellular TGs have also been identified in the cell wall of fungi, such as Candida and Saccharomyces, where they cross-link structural glycoproteins, and in Phytophthora, where they are involved in pathogenicity; in the alga Chlamydomonas, TGs link polyamines to glycoproteins thereby favouring the strengthening of cell wall. In higher plants, TG localized in the cell wall of flower petals appears to be involved in the structural reinforcement as well as senescence and cell death of the flower corolla. In the pollen cell wall an extracellular TG co-localizes with substrates and cross-linked products; it is required for the apical growth of pollen tubes. The pollen TG is also secreted into the extracellular matrix possibly allowing the migration of pollen tubes during fertilisation. Although pollen TGs seem to be secreted via vesicles transported along actin filaments, a different mechanism from the classical ER-Golgi pathway is possible, similar to TG2.     

Transglutaminase-2 differently regulates cartilage destruction and osteophyte formation in a surgical model of osteoarthritis

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodeling. Transglutaminases catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues and modifies the extracellular matrix. Increased transglutaminases-mediated activity is reported in osteoarthritis, but the relative contribution of transglutaminases-2 (TG2) is uncertain. We describe TG2 expression in human femoral osteoarthritis and in wild-type and homozygous TG2 knockout mice after surgically-induced knee joint instability. Increased TG2 levels were observed in human and wild-type murine osteoarthritic cartilage compared to the respective controls. Histomorphometrical but not X-ray investigation documented in osteoarthritic TG2 knockout mice reduced cartilage destruction and an increased osteophyte formation compared to wild-type mice. These differences were associated with increased TGFβ-1 expression. In addition to confirming its important role in osteoarthritis development, our results demonstrated that TG2 expression differently influences cartilage destruction and bone remodeling, suggesting new targeted TG2-related therapeutic strategies.     

Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications

This review summarises the functions of the enzyme tissue transglutaminase (TG2) in the extracellular matrix (ECM) both as a matrix stabiliser through its protein cross-linking activity and as an important cell adhesion protein involved in cell survival. The contribution of extracellular TG2 to the pathology of important diseases such as cancer and fibrosis are discussed with a view to the potential importance of TG2 as a therapeutic target. The medical applications of TG2 are further expanded by detailing the use of transglutaminase cross-linking in the development of novel biocompatible biomaterials for use in soft and hard tissue repair.     

Transglutaminase 2 as a novel activator of LRP6/β-catenin signaling

The β-catenin signaling axis is critical for normal embryonic development and tissue homeostasis in adults. We have previously shown that extracellular enzyme transglutaminase 2 (TG2) activates β-catenin signaling in vascular smooth muscle cells (VSMCs). In this study, we provide several lines of evidence that TG2 functions as an activating ligand of the LRP5/6 receptors. Specifically, we show that TG2 synergizes with LRP6 in the activation of β-catenin-dependent gene expression in Cos-7 cells. Interfering with the LRP5/6 receptors attenuates TG2-induced activation of β-catenin in Cos-7 cells. Further, we show that TG2 binds directly to the extracellular domain of LRP6, which is also able to act as a substrate for TG2-mediated protein cross-linking. Furthermore, inhibitors of TG2 protein cross-linking quench the observed TG2-induced β-catenin activation, implicating protein cross-linking as a novel regulatory mechanism for this pathway. Together, our findings identify and characterize a new activating ligand of the LRP5/6 receptors and uncover a novel activity of TG2 as an agonist of β-catenin signaling, contributing to the understanding of diverse developmental events and pathological conditions in which transglutaminase and β-catenin signaling are implicated.     

The redox state of transglutaminase 2 controls arterial remodeling

While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall.     

Detection of Transglutaminase 2 conformational changes in living cell

Transglutaminase 2 (TG2) is a ubiquitous Ca(2+)-dependent protein cross-linking enzyme that is implicated in a variety of biological disorders. In in vitro experiments when Ca(2+) concentration was increased TG2 changed its conformation and was able to cross-link other proteins via formation of an isopeptide bond. However the mechanisms that regulate TG2 transamidation activity in cells are still unknown. In this study we have developed FRET-based method for monitoring TG2 conformation changes and, probably, cross-linking activity in living cells. Using this approach we have showed that a significant amount of TG2 within the cell is accumulated in perinuclear endosomes and has a cross-linking inactive conformation, while TG2 that is located beneath the cell membrane has a transamidation active conformation. After the induction of apoptosis cytoplasmic TG2 changed its conformation and activates while, TG2 in endosomes retained transamidation inactive conformation even at late stages of apoptosis.     

Transglutaminase 2 regulates early chondrogenesis and glycosaminoglycan synthesis

The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-β activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering.     

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and {alpha}5{beta}1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and β1 integrin co-signaling pathway. By using α5 null cells, β1 integrin functional blocking antibody, and a α5β1 integrin targeting peptide A5-1, we demonstrate that the α5 and β1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCα is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.     

Microparticles and acute lung injury

The pathophysiology of acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), is characterized by increased vascular and epithelial permeability, hypercoagulation and hypofibrinolysis, inflammation, and immune modulation. These detrimental changes are orchestrated by cross talk between a complex network of cells, mediators, and signaling pathways. A rapidly growing number of studies have reported the appearance of distinct populations of microparticles (MPs) in both the vascular and alveolar compartments in animal models of ALI/ARDS or respective patient populations, where they may serve as diagnostic and prognostic biomarkers. MPs are small cytosolic vesicles with an intact lipid bilayer that can be released by a variety of vascular, parenchymal, or blood cells and that contain membrane and cytosolic proteins, organelles, lipids, and RNA supplied from and characteristic for their respective parental cells. Owing to this endowment, MPs can effectively interact with other cell types via fusion, receptor-mediated interaction, uptake, or mediator release, thereby acting as intrinsic stimulators, modulators, or even attenuators in a variety of disease processes. This review summarizes current knowledge on the formation and potential functional role of different MPs in inflammatory diseases with a specific focus on ALI/ARDS. ALI has been associated with the formation of MPs from such diverse cellular origins as platelets, neutrophils, monocytes, lymphocytes, red blood cells, and endothelial and epithelial cells. Because of their considerable heterogeneity in terms of origin and functional properties, MPs may contribute via both harmful and beneficial effects to the characteristic pathological features of ALI/ARDS. A better understanding of the formation, function, and relevance of MPs may give rise to new promising therapeutic strategies to modulate coagulation, inflammation, endothelial function, and permeability either through removal or inhibition of "detrimental" MPs or through administration or stimulation of "favorable" MPs.     

Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis

Introduction

The microenvironment surrounding inflamed synovium leads to the activation of fibroblast-like synoviocytes (FLSs), which are important contributors to cartilage destruction in rheumatoid arthritic (RA) joints. Transglutaminase 2 (TG2), an enzyme involved in extracellular matrix (ECM) cross-linking and remodeling, is activated by inflammatory signals. This study was undertaken to assess the potential contribution of TG2 to FLS-induced cartilage degradation.

Methods

Transglutaminase (TGase) activity and collagen degradation were assessed with the immunohistochemistry of control, collagen-induced arthritic (CIA) or TG2 knockdown (shRNA)-treated joint tissues. TGase activity in control (C-FLS) and arthritic (A-FLS) rat FLSs was measured by in situ 5-(biotinamido)-pentylamine incorporation. Invadopodia formation and functions were measured in rat FLSs and cells from normal (control; C-FLS) and RA patients (RA-FLS) by in situ ECM degradation. Immunoblotting, enzyme-linked immunosorbent assay (ELISA), and p3TP-Lux reporter assays were used to assess transforming growth factor-β (TGF-β) production and activation.

Results

TG2 and TGase activity were associated with cartilage degradation in CIA joints. In contrast, TGase activity and cartilage degradation were reduced in joints by TG2 knockdown. A-FLSs displayed higher TGase activity and TG2 expression in ECM than did C-FLSs. TG2 knockdown or TGase inhibition resulted in reduced invadopodia formation in rat and human arthritic FLSs. In contrast, increased invadopodia formation was noted in response to TGase activity induced by TGF-β, dithiothreitol (DTT), or TG2 overexpression. TG2-induced increases in invadopodia formation were blocked by TGF-β neutralization or inhibition of TGF-βR1.

Conclusions

TG2, through its TGase activity, is required for ECM degradation in arthritic FLS and CIA joints. Our findings provide a potential target to prevent cartilage degradation in RA.     

Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell‐derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non‐MP secreted factors (Sup) were isolated from serum‐free medium conditioned by human microvascular ECs (HMEC‐1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP‐containing MPs were isolated from cells transduced with CMV‐GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP‐MPs, but not free GFP. Thus, only MP‐associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP‐1, MMP‐3, CCL‐2/MCP‐1 and IL‐6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF‐κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms.     

External GTP-bound transglutaminase 2 is a molecular switch for chondrocyte hypertrophic differentiation and calcification

Chondrocyte maturation to hypertrophy, associated with up-regulated transglutaminase 2 (TG2) expression, mediates not only physiologic growth plate mineralization but also pathologic matrix calcification and dys-regulated matrix repair in osteoarthritic articular cartilage. TG2-/- mouse chondrocytes demonstrate markedly inhibited progression to hypertrophic differentiation in response to both retinoic acid and the chemokine CXCL1. Here, our objectives were to test if up-regulated TG2 alone is sufficient to promote chondrocyte hypertrophic differentiation and to identify TG2 molecular determinants and potential downstream signals involved. TG2 activities, regulated by nucleotides and calcium, include cross-linking of cartilage matrix proteins, binding of fibronectin, and hydrolysis of GTP and ATP. Following transfection of TG2 site-directed mutants into chondrocytic cells, we observed that wild type TG2, and TG catalytic site and fibronectin-binding mutants promoted type X collagen expression and matrix calcification consistent with chondrocyte hypertrophic differentiation. In contrast, transfected mutants of TG2 GTP binding (K173L) and externalization (Y274A) sites did not stimulate chondrocyte hypertrophy. Recombinant TG2 treatment of bovine cartilage explants demonstrated that extracellular TG2 induced hypertrophy more robustly in the GTP-bound state, confirming an essential role of TG2 GTP binding. Finally, TG2 treatment induced type X collagen in a beta1 integrin-mediated manner, associated with rapid phosphorylation of both Rac1 and p38 kinases that were inhibited by mutation of the TG2 GTP binding site. In conclusion, externalized GTP-bound TG2 serves as a molecular switch for differentiation of chondrocytes to a hypertrophic, calcifying phenotype in a manner that does not require either TG2 transamidation activity or fibronectin binding.     

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.     

Homocysteine enhances clot-promoting activity of endothelial cells via phosphatidylserine externalization and microparticles formation

Total elevated plasma homocysteine (Hcy) is a risk factor for thromboembolism. Vascular endothelium is important to regulate coagulation, but the impact of Hcy on the clot-promoting activity (CPA) of endothelial cells has not been fully understood. In our study, human umbilical vein endothelial cells (HUVECs) were treated with Hcy (8, 20, 80, 200, 800?μmol/L) for 24?h. Annexin V was utilized to detect phosphatidylserine (PS) externalization and endothelial microparticles (MPs) formation. CPA was assessed by recalcification time and purified clotting complex tests. We found that Hcy enhanced the externalized PS and consequent CPA of HUVECs in a dose-dependent fashion, effect of Hcy had statistical significance at 800?μmol/L. In addition, Hcy also increased the shedding of procoagulant endothelial MPs. Blocking of PS with 128?nmol/L annexin V reduced approximately 70% CPA of HUVECs and endothelial MPs, but human anti-tissue factor antibody had little inhibitive effect. Our results showed that Hcy increased CPA of HUVECs via PS externalization and MPs release. Our present study has implications for hyperhomocysteinemia-related hypercoagulability.     

The role of TG2 in ECV304-related vasculogenic mimicry

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry.     

Fabrication of oxygen-carrying microparticles functionalized with liver ECM-proteins to improve phenotypic three-dimensional in vitro liver assembly,function, and responses

Oxygen and extracellular matrix (ECM)-derived biopolymers play vital roles in regulating many cellular functions in both the healthy and diseased liver. This study highlights the significance of synergistically tuning the internal microenvironment of three-dimensional (3D) cell aggregates composed of hepatocyte-like cells from the HepG2 human hepatocellular carcinoma cell line and hepatic stellate cells (HSCs) from the LX-2 cell line to enhance oxygen availability and phenotypic ECM ligand presentation for promoting the native metabolic functions of the human liver. First, fluorinated (PFC) chitosan microparticles (MPs) were generated with a microfluidic chip, then their oxygen transport properties were studied using a custom ruthenium-based oxygen sensing approach. Next, to allow for integrin engagements the surfaces of these MPs were functionalized using liver ECM proteins including fibronectin, laminin-111, laminin-511, and laminin-521, then they were used to assemble composite spheriods along with HepG2 cells and HSCs. After in vitro culture, liver-specific functions and cell adhesion patterns were compared between groups and cells showed enhanced liver phenotypic responses to laminin-511 and 521 as evidenced via enhanced E-cadherin and vinculin expression, as well as albumin and urea secretion. Furthermore, hepatocytes and HSCs exhibited more pronounced phenotypic arrangements when cocultured with laminin-511 and 521 modified MPs providing clear evidence that specific ECM proteins have distinctive roles in the phenotypic regulation of liver cells in engineering 3D spheroids. This study advances efforts to create more physiologically relevant organ models allowing for well-defined conditions and phenotypic cell signaling which together improve the relevance of 3D spheroid and organoid models.     

Fibulin-2 Is a Driver of Malignant Progression in Lung Adenocarcinoma

The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-of-function experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wild-type littermates, implying that malignant progression was dependent specifically upon tumor cell-derived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen.     

Synthetic extracellular matrices for in situ tissue engineering

Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed.