Fibrillar Fibronectin Research Articles

Low shear-induced fibrillar fibronectin: comparative analyses of morphologies and cellular effects on bovine aortic endothelial cell adhesion and proliferation

Wall shear stress (WSS) is a critical factor in vascular biology, and both high and low WSS are implicated in atherosclerosis. Fibronectin (FN) is a key extracellular matrix protein that plays an important role in cell activities. Under high shear stress, plasma FN undergoes fibrillogenesis; however, its behavior under low shear stress remains unclear. This study aimed to investigate the formation ofin vitrocell-free fibrillar FN (FFN) under low shear rate conditions and its effect on bovine aortic endothelial cell behavior. FN (500µg ml-1) was perfused through slide chambers at three flow rates (0.16 ml h-1, 0.25 ml h-1, and 0.48 ml h-1), corresponding to low shear rates of 0.35 s-1, 0.55 s-1, and 1.05 s-1, respectively, for 4 h at room temperature. The formed FN matrices were observed using fluorescence microscopy and scanning electron microscopy. Under low shear rates, distinct FN matrix structures were observed. FFN0.48 formed immense fibrils with smooth surfaces, FFN0.25 formed a matrix with a rough surface, and FFN16 exhibited nodular structures. FFN0.25 supported cell activities to a greater extent than native FN and other FFN surfaces. Our study suggests that abnormally low shear conditions impact FN structure and function and enhance the understanding of FN fibrillogenesis in vascular biology, particularly in atherosclerosis.

Engineering fibronectin-templated multi-component fibrillar extracellular matrices to modulate tissue-specific cell response

Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.

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.

Engineered Biomimetic Fibrillar Fibronectin Matrices Regulate Cell Adhesion Initiation, Migration, and Proliferation via α5β1 Integrin and Syndecan-4 Crosstalk.

Cells regulate adhesion to the fibrillar extracellular matrix (ECM) of which fibronectin is an essential component. However, most studies characterize cell adhesion to globular fibronectin substrates at time scales long after cells polarize and migrate. To overcome this limitation, a simple and scalable method to engineer biomimetic 3D fibrillar fibronectin matrices is introduced and how they are sensed by fibroblasts from the onset of attachment is characterized. Compared to globular fibronectin substrates, fibroblasts accelerate adhesion initiation and strengthening within seconds to fibrillar fibronectin matrices via α5β1 integrin and syndecan-4. This regulation, which additionally accelerates on stiffened fibrillar matrices, involves actin polymerization, actomyosin contraction, and the cytoplasmic proteins paxillin, focal adhesion kinase, and phosphoinositide 3-kinase. Furthermore, this immediate sensing and adhesion of fibroblast to fibrillar fibronectin guides migration speed, persistency, and proliferation range from hours to weeks. The findings highlight that fibrillar fibronectin matrices, compared to widely-used globular fibronectin, trigger short- and long-term cell decisions very differently and urge the use of such matrices to better understand in vivo interactions of cells and ECMs. The engineered fibronectin matrices, which can be printed onto non-biological surfaces without loss of function, open avenues for various cell biological, tissue engineering and medical applications.

Engineered extracellular matrices facilitate brain organoids from human pluripotent stem cells.

Brain organoids are miniaturized in vitro brain models generated from pluripotent stem cells, which resemble full-sized brain more closely than conventional two-dimensional cell cultures. Although brain organoids mimic the human brain's cell-to-cell network interactions, they generally fail to faithfully recapitulate cell-to-matrix interactions. Here, an engineered framework, called an engineered extracellular matrix (EECM), was developed to provide support and cell-to-matrix interactions to developing brain organoids. We generated brain organoids using EECMs comprised of human fibrillar fibronectin supported by a highly porous polymer scaffold. The resultant brain organoids were characterized by immunofluorescence microscopy, transcriptomics, and proteomics of the cerebrospinal fluid (CSF) compartment. The interstitial matrix-mimicking EECM enhanced neurogenesis, glial maturation, and neuronal diversity from human embryonic stem cells versus conventional protein matrix (Matrigel). Additionally, EECMs supported long-term culture, which promoted large-volume organoids containing over 250 μL of CSF. Proteomics analysis of the CSF found it superseded previous brain organoids in protein diversity, as indicated by 280 proteins spanning 500 gene ontology pathways shared with adult CSF. Engineered EECM matrices represent a major advancement in neural engineering as they have the potential to significantly enhance the structural, cellular, and functional diversity that can be achieved in advanced brain models.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

Different Conformations of Fibronectin Fibrils Formed Under Cell-Free Condition in Vitro display Distinct Effects on Cell Adhesion and Spreading

Introduction: Insoluble fibrils are the active form of fibronectin (FN) with exposed functional domains capable of interacting with a variety of biomolecules and cells to support many cellular processes. Recent studies show that the formation of FN fibrils can occur in cell-free systems. Currently, the characteristic of FN fibrils and their potential in wound healing application are not fully understood. Our project aims at (i) using urea to induce the formation of FN fibrils with different conformations and (ii) to characterize their conformation-dependent effect on cellular adhesion and spreading.Method: To induce FN fibrillogenesis, native plasma FN at various initial concentrations (0.25, 0.5, 0.75 and 1 mg/ml) was treated by dialysis in urea at concentrations of 1, 2, and 4 M for 16h at room temperature followed by dialysis in PBS buffer (to remove urea). Morphological analysis of formed FN fibrils was conducted bylight microscopy and scanning electron microscopy (SEM). To assess biological effects of FN fibrils on cells in relationto their morphologies, platelet and fibroblast cell adhesion assays were performed. Specifically, platelet rich plasma (5x107 platelets/ml) or fibroblasts (L929, 104 cells/ml) were incubated on surfaces pre-coated with 0.25 and 0.75 mg/ml native plasma FN or FN fibrils for 30 min at 370C.Results: Our cell-free FN fibrillogenesis experiments in vitro indicated that the optimal concentrations of FN and urea were 1 mg/ml and 2 M, respectively, to induce formation of FN fibrils with various conformations. FN only formed minuscule fibrils at 1 M urea while there was no fibril formation at 4 M urea. In contrast, FN fibrils with various conformations ranging from fibrillar FN to non-fibrillar FN (i.e., nodular FN aggregates) were formed when using 2 M urea. Fibril formation was shown to be dependent on the FN concentration. FN at low concentrations (0.25 or 0.5 mg/ml) formed a few insoluble minuscule fibrils or nodular FN aggregates with variation in size ranging from 10 to 50 µm. FN at higher concentrations (0.75 or 1 mg/ml) formed much more nodular FN and fibrillar FN with diameters mostly ranging from 10 to 30 µm that tended to interlace into the fibrillar matrix. SEM analysis documented morphological variations of fibrils formed under different conditions. For example, at low FN concentration (0.25 mg/ml) small fibrils with a smooth surface were formed. By contrast, larger fibrils with attached nodular FN and a resulting rough surface were formed at high FN concentration (≥ 0.75 mg/ml). Adhesion assays indicated that platelets adhered approximately 6 times more firmly on FN fibrils than on native plasma FN (n=3, p < 0.05).On immobilized native FN, adherent platelets were evenly distributed or formed small clusters, while fibrillar FN induced platelet accumulation on the matrix. Fibroblasts adhered both on non-fibrillar FN and matrix of small fibrils (diameter in range of 10-30 µm) but did not interact to larger fibrillar bundles (diameter > 292 µm). Investigations of FN-cell interaction by SEM revealed a structure-dependent effect of FN on fibroblast morphology after adhesion. Specifically, cells remained in spherical shape and separately adhered on nodular FN while extending their shape in multiple directions on the rough surface of FN fibrils.Conclusion: Treatment of native plasma FN by urea at a concentration of 2 M induces irreversible formation of heterogeneous FN fibrils, displaying a rough surface due to attachment of FN nodules. The conformational and compositional changes of the FN matrix can lead to firm adhesion of platelets and fibroblasts and may thus provide a future tool to improve wound healing upon local application. DisclosuresNo relevant conflicts of interest to declare.

Aligned Networks of Engineered Fibrillar Fibronectin Guide Cellular Orientation and Motility

The extracellular matrix (ECM) influences biological processes associated with tissue development and disease progression. However, robust cell‐free techniques to control fiber alignment of naturally derived ECM proteins, such as fibronectin (Fn), remain elusive. It is demonstrated that controlled hydrodynamics of Fn solutions at the air/fluid interface of porous tessellated polymer scaffolds (TPSs) generates suspended 3D fibrillar networks with alignment across multiple length scales (&lt;1, 1–20 μm, extended to &gt;1 mm). The direction of the fluid flow and the architecture of the polymeric supports influence protein solution flow profiles and, subsequently, the alignment of insoluble Fn fibrils. Aligned networks of fibrillar Fn characteristically alter fibroblast phenotype, indicated by increased directional orientation, enhanced nuclear and cytoskeletal polarity, and highly anisotropic and persistent cell motility when compared with nonaligned 3D networks and 2D substrates. Engineered extracellular matrices (EECMs) establish a critically needed tool for both fundamental and applied cell biology studies, with potential applications in diverse areas such as cancer biology and regenerative medicine.

Fibronectin fibril alignment is established upon initiation of extracellular matrix assembly.

The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the functioning of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned meshwork matrix form matrix assembly sites around the cell periphery, but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked, since we discovered that transforming growth factor beta (TGF-β1) enhances matrix assembly site polarity and assembly of aligned fibrils independent of cell elongation. We conclude that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites that form during the initial stages of cell–FN interactions.

Morphological dependent effect of cell-free formed supramolecular fibronectin on cellular activities.

Fibrillar fibronectin (FFN), an active form of fibronectin (FN), plays important roles in various cellular processes. Our goal is to investigate effect of FFN morphology on cellular behaviors. Plasma FN at two concentrations was cross-linked into FFN by dialysis against 2M urea followed by morphological analysis under Scanning Electron Microscopy. To evaluate effect of FFN morphology, fibroblasts were cultured on FN or different FFNs. Fibroblast behaviors including adhesion, spreading, and migration were evaluated. Our data showed that FN fibrillogenesis was dependent on FN concentration. At high concentrations (0.75mg/mL), large FFN approximately 2.167+0.875µm in diameter were formed with attached nodular structures and rough surface. In contrast, smooth surface FFN fibrils with diameter of 1.886+0.412µm were formed from FN at 0.25mg/mL. Cellular assays revealed morphological dependent biological effects of different FFNs. Fibroblast separately adhered to native FN and remained spherical while on FFN, cells attached with higher quantity and showed spreading morphology. A synergistic ligand interaction of integrin α5β1 and αvβ3 was observed in cell adhering on FFN. Cell migration results showed that large FFN decreased migration rate while small FFN did not. Taken together, our data draws new attention towards controlling biological function of FN by its fibrillar structure.

High-Throughput Magnetic Actuation Platform for Evaluating the Effect of Mechanical Force on 3D Tumor Microenvironment.

Accurately replicating and analyzing cellular responses to mechanical cues is vital for exploring metastatic disease progression. However, many of the existing in vitro platforms for applying mechanical stimulation seed cells on synthetic substrates. To better recapitulate physiological conditions, a novel actuating platform is developed with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using a physiologically-relevant substrate. Suspending fibrillar fibronectin across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that is not reliant on a synthetic substrate. This platform enables the culturing and analysis of various cell types in an environment that mimics the dynamic stretching of lung tissue during normal respiration. Metabolic activity, YAP activation, and morphology of breast cancer cells are analyzed within one week of cyclic stretching or static culture. Further, matrix degradation is significantly reduced in breast cancer cell lines with metastatic potential after actuation. These new findings demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases.

Oxidative cross-linking of fibronectin confers protease resistance and inhibits cellular migration.

The oxidation of tyrosine residues to generate o,o'-dityrosine cross-links in extracellular proteins is necessary for the proper function of the extracellular matrix (ECM) in various contexts in invertebrates. Tyrosine oxidation is also required for the biosynthesis of thyroid hormone in vertebrates, and there is evidence for oxidative cross-linking reactions occurring in extracellular proteins secreted by myofibroblasts. The ECM protein fibronectin circulates in the blood as a globular protein that dimerizes through disulfide bridges generated by cysteine oxidation. We found that cellular (fibrillar) fibronectin on the surface of transforming growth factor-β1 (TGF-β1)-activated human myofibroblasts underwent multimerization by o,o'-dityrosine cross-linking under reducing conditions that disrupt disulfide bridges, but soluble fibronectin did not. This reaction on tyrosine residues required both the TGF-β1-dependent production of hydrogen peroxide and the presence of myeloperoxidase (MPO) derived from inflammatory cells, which are active participants in wound healing and fibrogenic processes. Oxidative cross-linking of matrix fibronectin attenuated both epithelial and fibroblast migration and conferred resistance to proteolysis by multiple proteases. The abundance of circulating o,o'-dityrosine-modified fibronectin was increased in a murine model of lung fibrosis and in human subjects with interstitial lung disease compared to that in control healthy subjects. These studies indicate that tyrosine can undergo stable, covalent linkages in fibrillar fibronectin under inflammatory conditions and that this modification affects the migratory behavior of cells on such modified matrices, suggesting that this modification may play a role in both physiologic and pathophysiologic tissue repair.

Peroxynitrous acid (ONOOH) modifies the structure of anastellin and influences its capacity to polymerize fibronectin

Anastellin (AN), a fragment of the first type III module in fibronectin (FN), initiates formation of superfibronectin, a polymer which resembles the native cell-derived fibrillar FN found in the extracellular matrix of many tissues, but which displays remarkably different functional properties. Here we demonstrate that exposure of AN to the biologically-important inflammatory oxidant, peroxynitrous acid (ONOOH), either as a bolus or formed at low levels in a time-dependent manner from SIN-1, impairs the capability of AN to polymerize FN. In contrast, exposure of FN to ONOOH does not seem to affect superfibronectin formation to the same extent. This oxidant-induced loss-of-function in AN occurs in a dose-dependent manner, and correlates with structural perturbations, loss of the amino acid tyrosine and tryptophan, and dose-dependent formation of modified amino acid side-chains (3-nitrotyrosine, di-tyrosine and 6-nitrotryptophan). Reagent ONOOH also induces formation of oligomeric species which decrease in the presence of bicarbonate, whereas SIN-1 mainly generates dimers. Modifications were detected at sub-stoichiometric (0.1-fold), or greater, molar excesses of oxidant compared to AN. These species have been localized to specific sites by peptide mass mapping. With high levels of oxidant (>100 times molar excess), ONOOH also induces unfolding of the beta-sheet structure of AN, thermal destabilization, and formation of high molecular mass aggregates. These results have important implications for the understanding of FN fibrillogenesis in vivo, and indicates that AN is highly sensitive to pathophysiological levels of oxidants such as ONOOH.

Morphologies of Fibronectin Fibrils Formed under Shear Conditions and Their Cellular Adhesiveness Properties

Fibrillar fibronectin (FFN) is a biological active form of FN which form linear and branched meshwork around cells and support cellular activities. Previous studies have demonstrated that shear stress can induce cell-free FN fibrillogenesis. In this study, we further examined the effect of shear stress conditions on morphology of formed FFN and preliminarily looked for relationship between FFN’s morphology and cell adhesion. Plasma FN at 50 µg/ml was perfused through channel slides at shear rates of 500 s-1 or 4000 s-1. Our results showed that there were four FFN structures formed: (1) FN nodules, (2) fibril in different sizes (3) with or without nodule attachment, and (4) fibrillar matrix. At 4000 s-1, FFN fibrils was formed within the first 10 min and reached the highest surface coverage only after 20 min. In contrast, FFN formation was significant more slowly at 500 s-1 at which only FN nodules and small fibrils were formed. Platelets bound on thin layer of FN and rarely found on large FN fibrils. In contrast, fibroblast stretched their shape on platform of FFN matrix and bound actively to all types of FFNs. Taken together, our data suggests a morphological dependent biological activity of FFN.

A Matricryptic Conformation of the Integrin-Binding Domain of Fibronectin Regulates Platelet-Derived Growth Factor-Induced Intracellular Calcium Release.

Platelet-derived growth factor (PDGF) signaling is dysregulated in a wide variety of diseases, making PDGF an attractive therapeutic target. However, PDGF also affects numerous signaling cascades essential for tissue homeostasis, limiting the development of PDGF-based therapies that lack adverse side-effects. Recent studies showed that fibroblast-mediated assembly of extracellular matrix (ECM) fibronectin fibrils attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact. In the present study, a series of recombinant fibronectin-derived fusion proteins were used to localize the sequences in fibronectin that are responsible for this inhibition. Results demonstrate that attenuation of PDGF-induced intracellular calcium release by the fibronectin matrix mimetic, FNIII1H,8-10 requires α5β1 integrin ligation, but is not dependent upon the matricryptic, heparin-binding site of FNIII1. Intact cell-binding fibronectin fragments were also unable to attenuate PDGF-induced intracellular calcium release. In contrast, a novel integrin-binding fragment that adopts an extended and aligned conformational state, inhibited both PI3K activation and intracellular calcium release in response to PDGF. Taken together, these studies provide evidence that attenuation of PDGF-induced intracellular calcium release by fibronectin is mediated by a novel conformation of the α5β1 integrin-binding, FNIII9-10 modules, that is expressed by fibrillar fibronectin.

Engineered Fibrillar Fibronectin Networks as Three-Dimensional Tissue Scaffolds.

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre-coatings in an attempt to create a cell-supporting environment in both two- and three-dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM-constituting fibrillar protein networks naturally deposited by cells. Here, the cell-free and scalable synthesis of freely suspended and mechanically robust three-dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three-phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial-to-mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre-clinical diagnostics.

Abstract 825: Attenuation of Cardiac Fibrosis by Scleraxis Gene Deletion Improves Pressure Overload-Induced Cardiac Remodeling

Cardiac fibrosis is a significant independent risk factor for heart failure with increasing incidence. The fibrotic myocardium shows increased arrhythmogenesis, and poor pumping and relaxation due to greater tissue stiffness. A critical step in this process is the conversion of fibroblasts to myofibroblasts, which are responsible for excessive extracellular matrix (ECM) production; limiting this conversion may reduce fibrosis and restore cardiac function. We previously reported that the transcription factor scleraxis, following mechanical stretch or TGFβ signaling, is both sufficient and necessary to convert fibroblasts to myofibroblasts by direct transcriptional control of myofibroblast genes including collagens, α-smooth muscle actin and fibronectin. In a pressure overload transverse aorta constriction (TAC) mouse model analyzed by echocardiography, we found that fibroblast-specific scleraxis gene deletion prior to TAC using a tamoxifen-inducible TCF21-Cre/loxP approach attenuated both systolic (LV ejection fraction, fractional shortening) and diastolic (early and late filling velocity) dysfunction, as well as chamber dilation, despite persistent hypertrophy. Functional improvement was matched by an almost complete attenuation of cardiac fibrosis (Masson’s trichrome; qPCR and western blots for fibrillar collagens and ED-A fibronectin). Scleraxis deletion also prevented induction of the myofibroblast marker periostin, suggesting a failure of scleraxis-null fibroblasts to convert to myofibroblasts. We next tested if scleraxis deletion 4 weeks post-TAC could reverse subsequent remodeling at 8 weeks post-TAC. Adverse remodeling occurred in all animals 4 weeks post-TAC (prior to scleraxis deletion), but cardiac function and chamber dimensions subsequently declined further in scleraxis-intact animals, while scleraxis-deleted animals showed preserved or improved cardiac function and morphology. Our results demonstrate that scleraxis is required for the initiation and progression of cardiac fibrosis, and that reducing fibrosis alone improves cardiac performance and morphology even in the presence of persistent pressure overload. Scleraxis is thus an important target for anti-fibrotic therapy development.

Fibrillar fibronectin plays a key role as nucleator of collagen I polymerization during macromolecular crowding-enhanced matrix assembly.

Macromolecular crowding is used by tissue engineers to accelerate extracellular matrix assembly in vitro, however, most mechanistic studies focus on the impact of crowding on collagen fiber assembly and largely ignore the highly abundant provisional matrix protein fibronectin. We show that the accelerated collagen I assembly as induced by the neutral crowding molecule Ficoll is regulated by cell access to fibronectin. Ficoll treatment leads to significant increases in the amount of surface adherent fibronectin, which can readily be harvested by cells to speed up fibrillogenesis. FRET studies reveal that Ficoll crowding also upregulates the total amount of fibronectin fibers in a low-tension state through upregulating fibronectin assembly. Since un-stretched fibronectin fibers have more collagen binding sites to nucleate the onset of collagen fibrillogenesis, our data suggest that the Ficoll-induced upregulation of low-tension fibronectin fibers contributes to enhanced collagen assembly in crowded conditions. In contrast, chemical cross-linking of fibronectin to the glass substrate prior to cell seeding prevents early force mediated fibronectin harvesting from the substrate and suppresses upregulation of collagen I assembly in the presence of Ficoll, even though the crowded environment is known to drive enzymatic cleavage of procollagen and collagen fiber formation. To show that our findings can be exploited for tissue engineering applications, we demonstrate that the addition of supplemental fibronectin in the form of an adsorbed coating markedly improves the speed of tissue formation under crowding conditions.

Autocrine Fibronectin Inhibits Breast Cancer Metastasis.

Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are linked to metastasis via their ability to increase invasiveness and enhance tumor-initiating capacity. Growth factors, cytokines, and chemotherapies present in the tumor microenvironment (TME) are capable of inducing EMT, but the role of the extracellular matrix (ECM) in this process remains poorly understood. Here, a novel tessellated three-dimensional (3D) polymer scaffolding is used to produce a fibrillar fibronectin matrix that induces an EMT-like event that includes phosphorylation of STAT3 and requires expression of β1 integrin. Consistent with these findings, analysis of the METABRIC dataset strongly links high-level fibronectin (FN) expression to decreased patient survival. In contrast, in vitro analysis of the MCF-10A progression series indicated that intracellular FN expression was associated with nonmetastatic cells. Therefore, differential bioluminescent imaging was used to track the metastasis of isogenic epithelial and mesenchymal cells within heterogeneous tumors. Interestingly, mesenchymal tumor cells do not produce a FN matrix and cannot complete the metastatic process, even when grown within a tumor containing epithelial cells. However, mesenchymal tumor cells form FN-containing cellular fibrils capable of supporting the growth and migration of metastatic-competent tumor cells. Importantly, depletion of FN allows mesenchymal tumor cells to regain epithelial characteristics and initiate in vivo tumor growth within a metastatic microenvironment.Implications: In contrast to the tumor-promoting functions of fibronectin within the ECM, these data suggest that autocrine fibronectin production inhibits the metastatic potential of mesenchymal tumor cells. Mol Cancer Res; 16(10); 1579-89. ©2018 AACR.

Abstract 621: Vascular Smooth Muscle EphA2 Signaling Regulates Fibrillar Adhesion Formation to Drive Fibronectin Deposition

During atherosclerosis, synthetic vascular smooth muscle cells drive development of the fibrous cap through extracellular matrix deposition. Deposition of the provisional matrix protein fibronectin appears to critically regulate fibrous cap development by promoting smooth muscle recruitment and by providing a critical scaffold for assembly of fibrillar collagens. We previously showed that deletion of the receptor tyrosine kinase EphA2 in ApoE knockout mice attenuated plaque size and progression, characterized by a reduction of both smooth muscle content and fibrosis, and we further demonstrated that EphA2 depletion attenuates smooth muscle fibronectin deposition in vitro. Despite this, the mechanisms by which EphA2 regulates smooth muscle matrix deposition remains unknown. Fibronectin fibrillogenesis involves both the formation of α5β1 integrin-rich fibrillar adhesions and contractility-dependent fibronectin unfolding. We now show EphA2 localizes to integrin adhesions and appears to regulate matrix deposition through multiple mechanisms. Knockdown of EphA2 reduces the formation of fibrillar adhesions characterized by a loss of α5β1 integrins and tensin, whereas EphA2 overexpression is sufficient to induce fibrillar adhesion formation in contractile smooth muscle cells. In addition, vascular smooth muscle cells show diminished collagen gel contraction following EphA2 knockdown, and EphA2 depletion significantly reduces smooth muscle myosin light chain phosphorylation, suggesting that EphA2 expression is required to generate the contractive forces required for matrix remodeling in synthetic smooth muscle cells. Together these data identify a novel role for EphA2 in the regulation of smooth muscle matrix deposition through effects on both integrin adhesion structure and cytoskeletal contractility.