Fibronectin Fibers Research Articles

Strain-dependent glutathionylation of fibronectin fibers impacts mechano-chemical behavior and primes an integrin switch

The extracellular matrix (ECM) is a protein polymer network that physically supports cells within a tissue. It acts as an important physical and biochemical stimulus directing cell behaviors. For fibronectin (Fn), a predominant component of the ECM, these physical and biochemical activities are inextricably linked as physical forces trigger conformational changes that impact its biochemical activity. Here, we analyze whether oxidative post-translational modifications, specifically glutathionylation, alter Fn’s mechano-chemical characteristics through stretch-dependent protein modification. ECM post-translational modifications represent a potential for time- or stimulus-dependent changes in ECM structure-function relationships that could persist over time with potentially significant impacts on cell and tissue behaviors. In this study, we show evidence that glutathionylation of Fn ECM fibers is stretch-dependent and alters Fn fiber mechanical properties with implications on the selectivity of engaging integrin receptors. These data demonstrate the existence of multimodal post-translational modification mechanisms within the ECM with high relevance to the microenvironmental regulation of downstream cell behaviors.

Diagnosis of nascent fibrosis: Interest of marking the relaxed form of fibronectin fibres

Fibrosis is a pathological and irreversible state of excessively synthesized extracellular matrix fibres induced by abnormal tissue scarring. Effective diagnostic tools for early management of patients with fibrosis are missing. Here, I discuss the recent advances in the quantification of Fibronectin fibres and the interest of novel biologics for diagnosis.

Salmonella-driven intestinal edema in mice is characterized by tensed fibronectin fibers.

Intestinal edema is a common manifestation of numerous gastrointestinal diseases and is characterized by the accumulation of fluid in the interstitial space of the intestinal wall. Technical advances in laser capture microdissection and low-biomass proteomics now allow us to specifically characterize the intestinal edema proteome. Using advanced proteomics, we identify peptides derived from antimicrobial factors with high signal intensity, but also highlight major contributions from the blood clotting system, extracellular matrix (ECM) and protease-protease inhibitor networks. The ECM is a complex fibrillar network of macromolecules that provides structural and mechanical support to the intestinal tissue. One abundant component of the ECM observed in Salmonella-driven intestinal edema is the glycoprotein fibronectin, recognized for its structure-function interplay regulated by mechanical forces. Using mechanosensitive staining of fibronectin fibers reveals that they are tensed in the edema, despite the high abundance of proteases able to cleave fibronectin. In contrast, fibronectin fibers increasingly relax in other cecal tissue areas as the infection progresses. Co-staining for fibrin(ogen) indicates the formation of a provisional matrix in the edema, similar to what is observed in response to skin injury, while collagen staining reveals a sparse and disrupted collagen fiber network. These observations plus the absence of low tensional fibronectin fibers and the additional finding of a high number of protease inhibitors in the edema proteome could indicate a critical role of stretched fibronectin fibers in maintaining tissue integrity in the severely inflamed cecum. Understanding these processes may also provide valuable functional diagnostic markers of intestinal disease progression in the future.

Transglutaminase 2 has higher affinity for relaxed than for stretched fibronectin fibers

Transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal β-sandwich and C-terminal β-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2’s C-terminal β-barrels that interacts with FN regions located outside of the canonical gelatin binding domain, specifically FNI2 and FNIII14–15. Combining XL-MS distance restraints with molecular docking revealed the mechano-regulated binding mechanism between TG2 and modules FNI7–9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how forces regulate cell adhesion, spreading, migration, phenotype modulation, depending on the tensional state of ECM fibers. Data are available via ProteomeXchange with identifier PXD043976.

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

BackgroundAs in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses.MethodsWe used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis.ResultsIn silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis.ConclusionsOur results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

Extracellular matrix remodelling in dental pulp tissue of carious human teeth through the prism of single-cell RNA sequencing

Carious lesions are bacteria-caused destructions of the mineralised dental tissues, marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue. While major molecular players in tooth decay have been uncovered during the past years, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. In this study we used single-cell RNA sequencing analysis, supplemented with immunostaining, to generate a comprehensive single-cell atlas of the pulp of carious human teeth. Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the pulp of healthy human teeth. Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters. These changes include the upregulation of genes encoding extracellular matrix (ECM) components, including COL1A1 and Fibronectin (FN1), and the enrichment of the fibroblast cluster with myofibroblasts. The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses. Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues, compared to the healthy ones. The present data demonstrate molecular, cellular and biomechanical alterations in the pulp of human carious teeth, indicative of extensive ECM remodelling, reminiscent of fibrosis observed in other organs. This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.

How the mechanobiology orchestrates the iterative and reciprocal ECM-cell cross-talk that drives microtissue growth.

Controlled tissue growth is essential for multicellular life and requires tight spatiotemporal control over cell proliferation and differentiation until reaching homeostasis. As cells synthesize and remodel extracellular matrix, tissue growth processes can only be understood if the reciprocal feedback between cells and their environment is revealed. Using de novo-grown microtissues, we identified crucial actors of the mechanoregulated events, which iteratively orchestrate a sharp transition from tissue growth to maturation, requiring a myofibroblast-to-fibroblast transition. Cellular decision-making occurs when fibronectin fiber tension switches from highly stretched to relaxed, and it requires the transiently up-regulated appearance of tenascin-C and tissue transglutaminase, matrix metalloprotease activity, as well as a switch from α5β1 to α2β1 integrin engagement and epidermal growth factor receptor signaling. As myofibroblasts are associated with wound healing and inflammatory or fibrotic diseases, crucial knowledge needed to advance regenerative strategies or to counter fibrosis and cancer progression has been gained.

Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilization of Bioemulsions Enabling Adherent Cell Expansion.

Bioemulsions are attractive platforms for the scalable expansion of adherent cells and stem cells. In these systems, cell adhesion is enabled by the assembly of protein nanosheets that display high interfacial shear moduli and elasticity. However, to date, most successful systems reported to support cell adhesion at liquid substrates have been based on coassemblies of protein and reactive cosurfactants, which limit the translation of bioemulsions. In this report, we describe the design of protein nanosheets based on two globular proteins, bovine serum albumin (BSA) and β-lactoglobulin (BLG), biofunctionalized with RGDSP peptides to enable cell adhesion. The interfacial mechanics of BSA and BLG assemblies at fluorinated liquid-water interfaces is studied by interfacial shear rheology, with and without cosurfactant acyl chloride. Conformational changes associated with globular protein assembly are studied by circular dichroism and protein densities at fluorinated interfaces are evaluated via surface plasmon resonance. Biofunctionalization mediated by sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is studied by fluorescence microscopy. On the basis of the relatively high elasticities observed in the case of BLG nanosheets, even in the absence of cosurfactant, the adhesion and proliferation of mesenchymal stem cells and human embryonic kidney (HEK) cells on bioemulsions stabilized by RGD-functionalized protein nanosheets is studied. To account for the high cell spreading and proliferation observed at these interfaces, despite initial moderate interfacial elasticities, the deposition of fibronectin fibers at the surface of corresponding microdroplets is characterized by immunostaining and confocal microscopy. These results demonstrate the feasibility of achieving high cell proliferation on bioemulsions with protein nanosheets assembled without cosurfactants and establish strategies for rational design of scaffolding proteins enabling the stabilization of interfaces with strong shear mechanics and elasticity, as well as bioactive and cell adhesive properties. Such protein nanosheets and bioemulsions are proposed to enable the development of new generations of bioreactors for the scale up of cell manufacturing.

Infiltrating CD8+ T cells and M2 macrophages are retained in tumor matrix tracks enriched in low tension fibronectin fibers

Tracks rich in matrix and cells, as described in several cancer types, have immunosuppressive functions and separate tumor nests and stroma, yet their origin is unknown. Immunostainings of cryosections from mouse breast tumors show that these tracks are bordered by an endothelial-like basement membrane, filled with fibers of collagen adjacent to tenascin-C (TNC) and low-tension fibronectin (Fn) fibers. While present in early-stage tumors and maturing with time, tracks still form under TNC KO conditions, however, host (not tumor cell)-derived TNC is important for track maturation. Tumor infiltrating leukocytes (mostly M2 macrophages and CD8+ T cells) are retained in tracks of early-stage tumors. Following track maturation, retained tumor infiltrating leukocyte (TIL) numbers get reduced and more CD8+ TIL enter the tumor nests in the absence of TNC. As these tracks are enriched with platelets and fibrinogen and have a demarcating endothelial-like basement membrane often adjacent to endothelial cells, this suggests a role of blood vessels in the formation of these tracks. The Fn fiber tension probe FnBPA5 colocalizes with TNC and immune cells in the tracks and shows decreased binding in tracks lacking TNC. Consequently, FnBPA5 can serve as probe for tumor matrix tracks that have immune suppressive properties.

A spatial statistical framework for the parametric study of fiber networks: Application to fibronectin deposition by normal and activated fibroblasts.

Due to the complex architectural diversity of biological networks, there is an increasing need to complement statistical analyses with a qualitative and local description of their spatial properties. One such network is the extracellular matrix (ECM), a biological scaffold for which changes in its spatial organization significantly impact tissue functions in health and disease. Quantifying variations in the fibrillar architecture of major ECM proteins should considerably advance our understanding of the link between tissue structure and function. Inspired by the analysis of functional magnetic resonance imaging (fMRI) images, we propose a novel statistical analysis approach embedded into a machine learning paradigm, to measure and detect local variations of meaningful ECM parameters. We show that parametric maps representing fiber length and pore directionality can be analyzed within the proposed framework to differentiate among various tissue states. The parametric maps are derived from graph-based representations that reflect the network architecture of fibronectin (FN) fibers in a normal, or disease-mimicking in vitro setting. Such tools can potentially lead to a better characterization of dynamic matrix networks within fibrotic tumor microenvironments and contribute to the development of better imaging modalities for monitoring their remodeling and normalization following therapeutic intervention.

Microstructure-based engineering of soft biological materials

Aortic disease (AoD) is a leading cause of mortality in developed countries. Two of the most common forms of AoD are aneurysm (widening) and dissection (tear in inner wall). Aneurysm and dissection often associate with bicuspid aortic valve (BAV) instead of the normal tricuspid aortic valve, and BAV aneurysms of ascending thoracic aorta have the tendency to bulge asymmetrically towards the greater curvature of aorta. Multiphoton microscopy can help us image collagen and elastin fibres, which are considered as main load-bearing constituents of the aortic wall, in order to investigate potential role of fibre microstructure in ascending thoracic aortic aneurysm or dissection. Regional differences in fibre microstructure may be driven by distinct mechanisms of vascular remodelling, and, combined with mechanical tests, could improve our understanding of the biomechanical mechanisms of aortic aneurysm and dissection potential. Should we wish to investigate the effect of microstructure in soft tissue formation and organ development, we would have to consider a rapidly growing process. In that process, the cells are the main load-bearing components, which cooperate to produce tissue-level forces that shape tissue formation. Our understanding of this phenomenon, called mechanotransduction, has advanced significantly over the past years, to the point where it is now clear that nearly every biological process is modulated by how these forces are decoded intracellularly. It is therefore important to create our own fluorescently-labeled matrix that could integrate into the tissue and enable tracking of these forces in-vivo. A new 3D optical nanomechanical biosensor (NMBS) based on fluorescent fibronectin fibres was developed based on integrated photolithography and micro-contact printing technology. NMBS was successfully validated under uniaxial tensile test of biologically relevant materials for microscopic vs. macroscopic mechanical strains. In the future, biomimetic 3D scaffolds could be fabricated by assembly of 2D fibre constructs based on the NMBS technology, in order to analyse the effect of selected set of load-bearing microstructural components on both mechanical and functional response of soft biological materials.

Effect of valproic acid on functional bleb morphology in a rabbit model of minimally invasive surgery

PurposeTo determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS).MethodsNine New Zealand white rabbits were subjected...

The Responsiveness of Thymic Stromal Cells to semaphorin-3A

ABSTRACT Background The thymus is responsible for thymocyte differentiation into immunocompetent T lymphocytes. Different cell types in the thymic microenvironment actively cooperate in this process, interacting with the developing thymocytes through soluble factors, extracellular matrix (ECM) molecules, and receptors. In addition, this microenvironment can be influenced by several factors, such as semaphorin-3A (Sema3A), which is a multifunctional protein involved in cell migration. We evaluated the Sema3A effects on the cellular parameters and functional features of thymic stromal cells. Methods Thymic stromal cells were obtained by enzymatic digestion of the murine thymus. These cells were treated with Sema3A and evaluated as follows: cell morphology by scanning electron microscope, F-actin cytoskeleton and deposition of ECM molecules by fluorescence microscopy, and adhesion assays with freshly obtained thymocytes. Results The obtained thymic stroma was composed of 67% of thymic epithelial cells (TECs), and 90% of the TECs were positive for the Sema3A receptor neuropilin-1. These cells secreted CXCL12, IL-7 and extended thymocyte survival. Sema3A changed the morphology of thymic stromal cells and promoted F-actin reorganization. In addition, the fibronectin fibers were reoriented, and the laminin production was increased in Sema3A-treated thymic stromal cells. In the adhesion assays, there was an increase in the number of adhered thymocytes when thymic stromal cells were pretreated with Sema3A. Conclusion Our data strongly suggest the active participation of Sema3A in thymic physiology, highlighting its role as an immunomodulatory molecule. This may provide important knowledge for understanding the interactions of thymic cells.

Interstitial changes after reperfused myocardial infarction in swine: morphometric and genetic analysis

BackgroundFollowing myocardial infarction (MI), we aimed to characterize morphometric and genetic changes in extracellular matrix (ECM) components from ischemia onset until late phases after coronary reperfusion in necrotic and salvaged myocardium.ResultsSwine were divided into one control (n = 5) and three MI groups: 90-min of ischemia without reperfusion, or followed by 1-week or 1-month reperfusion (n = 5 per group). In samples from the necrotic and salvaged areas, ECM components were morphometrically quantified and mRNA levels of factors involved in ECM remodeling were evaluated. After 90-min of ischemia, fibronectin, laminin, and elastic fibers content as well as upregulated mRNA expression of tissue inhibitors of metalloproteinases (TIMP)1, TIMP2, TIMP3 and connective tissue growth factor increased in the necrotic and salvaged myocardium. In both reperfused MI groups, collagen-I, collagen-III, elastic fibers, glycosaminoglycans, laminin, and fibronectin levels heightened in the necrotic but not the salvaged myocardium. Moreover, mRNA expression of TIMP1, TIMP2 and TIMP3, as well as metalloproteinase-2 and metalloproteinase-9 heightened in the necrotic but not in the salvaged myocardium.ConclusionsMatrix remodeling starts after ischemia onset in both necrotic and salvaged myocardium. Even if ECM composition from the salvaged myocardium was altered after severe ischemia, ECM makes a full recovery to normal composition after reperfusion. Therefore, rapid coronary reperfusion is essential not only to save cardiomyocytes but also to preserve matrix, thus avoiding impaired left ventricular remodeling.

CD4+ T Cell Interstitial Migration Controlled by Fibronectin in the Inflamed Skin.

The extracellular matrix (ECM) is extensively remodeled during inflammation providing essential guidance cues for immune cell migration and signals for cell activation and survival. There is increasing interest in the therapeutic targeting of ECM to mitigate chronic inflammatory diseases and enhance access to the tumor microenvironment. T cells utilize the ECM as a scaffold for interstitial migration, dependent on T cell expression of matrix-binding integrins αVβ1/αVβ3 and tissue display of the respective RGD-containing ligands. The specific ECM components that control T cell migration are unclear. Fibronectin (FN), a canonical RGD-containing matrix component, is heavily upregulated in inflamed tissues and in vitro can serve as a substrate for leukocyte migration. However, limited by lack of tools to intravitally visualize and manipulate FN, the specific role of FN in effector T cell migration in vivo is unknown. Here, we utilize fluorescently-tagged FN to probe for FN deposition, and intravital multiphoton microscopy to visualize T cell migration relative to FN in the inflamed ear dermis. Th1 cells were found to migrate along FN fibers, with T cells appearing to actively push or pull against flexible FN fibers. To determine the importance of T cell interactions with FN, we used a specific inhibitor of FN polymerization, pUR4. Intradermal delivery of pUR4 (but not the control peptide) to the inflamed skin resulted in a local reduction in FN deposition. We also saw a striking attenuation of Th1 effector T cell movement at the pUR4 injection site, suggesting FN plays a key role in T cell interstitial migration. In mechanistic studies, pUR4 incubation with FN in vitro resulted in enhanced tethering of T cells to FN matrix, limiting productive migration. In vivo, such tethering led to increased Th1 accumulation in the inflamed dermis. Enhanced Th1 accumulation exacerbated inflammation with increased Th1 activation and IFNγ cytokine production. Thus, our studies highlight the importance of ECM FN fibrils for T cell migration in inflamed tissues and suggest that manipulating local levels of ECM FN may prove beneficial in promoting T cell accumulation in tissues and enhancing local immunity to infection or cancer.

Fibronectin fibers are highly tensed in healthy organs in contrast to tumors and virus-infected lymph nodes

The extracellular matrix (ECM) acts as reservoir for a plethora of growth factors and cytokines some of which are hypothesized to be regulated by ECM fiber tension. Yet, ECM fiber tension has never been mapped in healthy versus diseased organs. Using our recently developed tension nanoprobe derived from the bacterial adhesin FnBPA5, which preferentially binds to structurally relaxed fibronectin fibers, we discovered here that fibronectin fibers are kept under high tension in selected healthy mouse organs. In contrast, tumor tissues and virus-infected lymph nodes exhibited a significantly higher content of relaxed or proteolytically cleaved fibronectin fibers. This demonstrates for the first time that the tension of ECM fibers is significantly reduced upon pathological tissue transformations. This has wide implications, as the active stretching of fibronectin fibers adjusts critical cellular niche parameters and thereby tunes the reciprocal cell-ECM crosstalk. Mapping the tensional state of fibronectin fibers opens novel and unexpected diagnostic opportunities.

Collagen microarchitecture mechanically controls myofibroblast differentiation

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Replication Study: Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Fiering et al., 2015) that described how we intended to replicate selected experiments from the paper 'Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis' (Goetz et al., 2011). Here we report the results. Primary mouse embryonic fibroblasts (pMEFs) expressing caveolin 1 (Cav1WT) demonstrated increased extracellular matrix remodeling in vitro compared to Cav1 deficient (Cav1KO) pMEFs, similar to the original study (Goetz et al., 2011). In vivo, we found higher levels of intratumoral stroma remodeling, determined by fibronectin fiber orientation, in tumors from cancer cells co-injected with Cav1WT pMEFs compared to cancer cells only or cancer cells plus Cav1KO pMEFs, which were in the same direction as the original study (Supplemental Figure S7C; Goetz et al., 2011), but not statistically significant. Primary tumor growth was similar between conditions, like the original study (Supplemental Figure S7Ca; Goetz et al., 2011). We found metastatic burden was similar between Cav1WT and Cav1KO pMEFs, while the original study found increased metastases with Cav1WT (Figure 7C; Goetz et al., 2011); however, the duration of our in vivo experiments (45 days) were much shorter than in the study by Goetz et al. (2011) (75 days). This makes it difficult to interpret the difference between the studies as it is possible that the cells required more time to manifest the difference between treatments observed by Goetz et al. We also found a statistically significant negative correlation of intratumoral remodeling with metastatic burden, while the original study found a statistically significant positive correlation (Figure 7Cd; Goetz et al., 2011), but again there were differences between the studies in terms of the duration of the metastasis studies and the imaging approaches that could have impacted the outcomes. Finally, we report meta-analyses for each result.

Adaptive Liquid Interfacially Assembled Protein Nanosheets for Guiding Mesenchymal Stem Cell Fate.

There is a growing interest in the development of dynamic adaptive biomaterials for regulation of cellular functions. However, existing materials are limited to two-state switching of the presentation and removal of cell-adhesive bioactive motifs that cannot emulate the native extracellular matrix (ECM) in vivo with continuously adjustable characteristics. Here, tunable adaptive materials composed of a protein monolayer assembled at a liquid-liquid interface are demonstrated, which adapt dynamically to cell traction forces. An ultrastructure transition from protein monolayer to hierarchical fiber occurs through interfacial jamming. Elongated fibronectin fibers promote formation of elongated focal adhesion structures, increase focal adhesion kinase activation, and enhance neuronal differentiation of stem cells. Cell traction force results in spatial rearrangement of ECM proteins, which feeds back to alter stem cell fate. The reported biomimetic adaptive liquid interface enables dynamic control of stem cell behavior and has potential translational applications.

P863Morphometric analysis of the dynamic changes of the interstitium after reperfused myocardial infarction

Abstract Background The interstitial space is mainly composed by cells, fibers and gels of polysaccharides, which act as a compression buffer against the stress placed on the extracellular matrix (ECM). After myocardial infarction (MI), heart has to withstand higher mechanical stress due to injured cardiomyocytes. ECM composition notably influences the mechanical properties of the myocardium and participates in left ventricular remodeling. Purpose To characterize the myocardial ECM changes from ischemia onset until late phases after coronary reperfusion in a swine model of reperfused MI. Methods MI was induced in swine by transient 90-min coronary occlusion using angioplasty balloons. One control group and three MI groups were defined: 1) without reperfusion, 2) 1-week, and 3) 1-month reperfusion (n=5, each). Myocardial samples from the infarcted area were isolated and histologically staining to evaluate the presence of collagen type I, collagen type III, elastic fibers, and proteinglycans. Moreover, the presence of laminin, fibronectin, and secreted protein, acidic and rich in cysteine (SPARC) was determined by immunohistochemistry. Five independent photographs for each group and each stain were taken and the presence of the different components of the interstitium was morphometrically quantified. Indeed, the mRNA levels of metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), and transcription factors implicated in ECM remodeling were quantified. Results In the no-reperfusion group, an increase in fibronectin, laminin, and elastic fibers were observed, whereas no changes neither in the quantity nor in the organization of collagen-I and collagen-III fibers were detected. In the 1-week and 1-month reperfusion groups, an augmentation in the content of collagen-I, collagen-III, elastic fibers were found. These fibers displayed a more organized pattern compared to control tissue. The quantity of proteinglycans, laminin, fibronectin, and SPARC were also increased. These changes could be explained by a heightening in the mRNA expression of TIMP1, TIMP2, and TIMP3 from the ischemic period until chronic phases, whereas no changes were observed for TIMP4. MMP2 and MMP9 mRNA levels were boosted in late phases post-MI. When exploring the mRNA levels of transcription factors implicated in ECM remodeling, CTGF increased during the ischemic period, whereas TNF-α and TGF-β augmented after 1-week and 1-month post-reperfusion, respectively. Conclusions ECM remodeling starts after ischemia onset, probably aiming to protect cardiomyocytes from the ischemic damage. After reperfusion, the ECM evolves to constitute a fibrotic scar to maintain a proper cardiac function. These changes may be orchestrated by the fluctuation in genes specially implicated in ECM distribution. ECM changes might be accurately regulated since it could provoke an adverse left ventricular remodelling, consequently impairing patient prognosis. Acknowledgement/Funding This study was funded by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (PIE15/00013, PI17/01836, and CIBERCV16/11