Fibronectin Fibers Research Articles

636 Mechano-sensing and inflammatory signalling in normal and keloid dermal fibroblasts

Keloid scar formation is associated with aberrant inflammatory signalling however the underlying causes remain poorly understood. Additionally, keloid scars have been shown to be 10-50 times stiffer than normal skin and often form in areas of the body where skin tension is high. As a result, it is thought that the formation of keloid scars may be influenced by mechanical forces. The aim of this study was to investigate the direct effects of tissue stiffness on the behaviour of normal (NF) and keloid fibroblasts (KF) and explore the potential cross-talk with inflammatory signalling pathways. First, we characterised the stiffness dependent responses within an immortalised fibroblast line, HCA2, when cultured onto collagen-coated polyacrylamide hydrogels mimicking the stiffness of normal skin (8kPa) and keloid skin (70-214kPa). HCA2 cells showed stiffness dependent changes in cell spreading, F-actin intensity, and focal adhesion (FA) assembly. In addition, phospho-SMAD2 activation following stimulation with TGF-β was higher on stiff gels. Secondly, we extended this technique to study primary NFs and KFs from three different donors. KFs were more spread, exhibited more FAs and more intense F-actin stress fibres than NFs, particularly on the stiff hydrogels. Additionally, both cell types demonstrated a stiffness dependent response in phospho-SMAD2 when stimulated with TGF-β. Finally, after culturing both KFs and NFs on the hydrogels for 10 days we studied the cell derived matrix of each cell type. Analysis revealed that substrate stiffness regulates extracellular matrix (ECM) assembly with increased total fibronectin deposited on the stiff substrates. ECM on the stiff substrates showed smaller but more densely packed fibronectin fibres, and the ECM assembled by the KFs showed thicker and more aligned fibronectin fibres compared to NFs. Collectively, these results provide further insight into the mechanical regulation of dermal fibroblast behaviour showing a distinctive difference between KFs and NFs. The increased stress fibre and FA formation suggest that KFs are more contractile and mechano-sensitive than NFs

Fibronectin fiber creep under constant force loading

Viscoelasticity is a fundamental property of virtually all biological materials, and proteinaceous, fibrous materials that constitute the extracellular matrix (ECM) are no exception. Viscoelasticity may be particularly important in the ECM since cells can apply mechanical stress resulting from cell contractility over very long periods of time. However, measurements of ECM fiber response to long-term constant force loading are scarce, despite the increasing recognition that mechanical strain regulates the biological function of some ECM fibers. We developed a dual micropipette system that applies constant force to single fibers for up to 8 h. We utilized this system to study the time dependent response of fibronectin (Fn) fibers to constant force, as Fn fibers exhibit tremendous extensibility before mechanical failure as well as strain dependent alterations in biological properties. These data demonstrate the Fn fibers continue to stretch under constant force loading for at least 8 h and that this long-term creep results in plastic deformation of Fn fibers, in contrast to elastic deformation of Fn fibers under short-term, but fast loading rate extension. These data demonstrate that physiologically-relevant loading may impart mechanical features to Fn fibers by switching them into an extended state that may have altered biological functions. Statement of significanceMeasurements of extracellular matrix (ECM) fiber response to constant force loading are scarce, so we developed a novel technique for applying constant force to single ECM fibers. We used this technique to measure constant force creep of fibronectin fibers since these fibers have been shown to be mechanotransducers whose functions can be altered by mechanical strain. We found that fibronectin fibers creep under constant force loading for the duration of the experiment and that this creep behavior resembles a power law. Furthermore, we found that constant force creep results in plastic deformation of the fibers, which suggests that the mechanobiological switching of fibronectin can only occur once after long-term loading.

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.

S-Glutathionylation Influences the Property of Fibronectin

Fibronectin is a key extracellular matrix (ECM) protein that plays a role in cell growth, migration and differentiation through its engagement of cell surface receptors, known as integrins, in a spatiotemporal manner. Fibronectin is a mechanically sensitive protein displaying force-induced conformational changes that impact its multiple growth factor, integrin, and ECM protein binding domains. Fibronectin is a unique ECM protein in that it contains 4 free cysteine residues that are protected in cryptic sites, but that can be exposed under force. S-glutathionylation is a post-translational modification of cysteines that occurs in oxidative environments like the extracellular space during pathologies like cancer and fibrosis. Here, we show for the first time that fibronectin's cysteines can be glutathionylated when exposed either through chemical unfolding in its soluble form or mechanical unfolding in its fibrillar form (both on the single fiber and matrix level). Moreover, we proved that S-glutathionylation impacts the mechanical properties of fibronectin fibers. Specifically, our results reveal that glutathionylated fibronectin fibers are more extensible and less stiff compared to unmodified fibronectin. This is true for both single fibronectin fibers and fibronectin matrices. Thus S-glutathionylation of mechanically exposed cysteines on fibronectin may act as a mechanism of mechanical memory within fibronectin-rich ECMs, regulating subsequent mechanotransduction. One possible realism of this mechanism could be the enhancement fibronectin's integrin binding mechanoswitch due to fibronectin fibers being more easily stretched under lower force regimes.

Formation of Multi-Component Extracellular Matrix Protein Fibers

The extracellular matrix (ECM) consists of polymerized protein monomers that form a unique fibrous network providing stability and structural support to surrounding cells. We harnessed the fibrillogenesis mechanisms of naturally occurring ECM proteins to produce artificial fibers with a heterogeneous protein makeup. Using ECM proteins as fibril building blocks, we created uniquely structured multi-component ECM fibers. Sequential incubation of fibronectin (FN) and laminin (LAM) resulted in self-assembly into locally stacked fibers. In contrast, simultaneous incubation of FN with LAM or collagen (COL) produced molecularly stacked multi-component fibers because both proteins share a similar assembly mechanism or possess binding domains specific to each other. Sequential incubation of COL on FN fibers resulted in fibers with sandwiched layers because COL molecules bind to the external surface of FN fibers. By choosing proteins for incubation according to the interplay of their fibrillogenesis mechanisms and their binding domains (exposed when they unfold), we were able to create ECM protein fibers that have never before been observed.

Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin-7.

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (110TKSLEEN116 and the truncated 112SLEE115 in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI2) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies.

Novel peptide probes to assess the tensional state of fibronectin fibers in cancer

Transformations of extracellular matrix (ECM) accompany pathological tissue changes, yet how cell-ECM crosstalk drives these processes remains unknown as adequate tools to probe forces or mechanical strains in tissues are lacking. Here, we introduce a new nanoprobe to assess the mechanical strain of fibronectin (Fn) fibers in tissue, based on the bacterial Fn-binding peptide FnBPA5. FnBPA5 exhibits nM binding affinity to relaxed, but not stretched Fn fibers and is shown to exhibit strain-sensitive ECM binding in cell culture in a comparison with an established Fn-FRET probe. Staining of tumor tissue cryosections shows large regions of relaxed Fn fibers and injection of radiolabeled 111In-FnBPA5 in a prostate cancer mouse model reveals specific accumulation of 111In-FnBPA5 in tumor with prolonged retention compared to other organs. The herein presented approach enables to investigate how Fn fiber strain at the tissue level impacts cell signaling and pathological progression in different diseases.

Directional Matrix Nanotopography with Varied Sizes for Engineering Wound Healing.

Topographic features play a crucial role in the regulation of physiologically relevant cell and tissue functions. Here, an analysis of feature-size-dependent cell-nanoarchitecture interactions is reported using an array of scaffolds in the form of uniformly spaced ridge/groove structures for engineering wound healing. The ridge and groove widths of nanopatterns are varied from 300 to 800 nm and the nanotopography features are classified into three size ranges: dense (300-400 nm), intermediate (500-600 nm), and sparse (700-800 nm). On these matrices, fibroblasts demonstrate a biphasic trend of cell body and nucleus elongation showing the maximum at intermediate feature density, whereas maximum migration speed is observed at the dense case with monotonic decrease upon increasing feature size. The directional organization of cell-synthesized fibronectin fibers can be regulated differently via the nanotopographical features. In an in vitro wound healing model, the covering rate of cell-free regions is maximized on the dense nanotopography and decreased with increasing feature size, showing direct correlation with the trend of migration speed. It is demonstrated that the properties of repaired tissue matrices in the process of wound healing may be controlled via the feature-size-dependent cell-nanoarchitecture interactions, which can be an important consideration for designing tissue engineering scaffolds.

AMPK negatively regulates tensin-dependent integrin activity.

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.

Histological and Immunohistochemical Evaluation of the Efficacy of a New Cosmetic Formulation in the Treatment of Skin Photoaging

Objective. Mechanism of action of cosmetic products is not often studied. The aim of this study is to determine the histological, immunohistochemical, and clinical changes of a new cosmetic formulation. Methods. Prospective, single-blind, patient-controlled, randomized study in 10 volunteers with mild to moderate skin photoaging on the back of their hands. The product was applied on one hand and a standard cream on the other hand, twice a day for three months. Standardized photographs were taken on basal (T0) and final visit (T1) and skin biopsies were performed. Changes on histological and immunohistochemical markers were studied. Subjective clinical changes were determined. Results. After treatment, a 26.3% improvement on epidermal thickness was detected and a significant increase on collagens I and III, elastin, and fibronectin fibers was achieved (p < 0.05). As the expression of MMPs remained stable, this improvement of dermal matrix was attributed to the stimulation of their synthesis. A significant clinical improvement on the treated hand was obtained, compared to control hand. Conclusion. This new cosmetic product with combination of three registered technologies (IFC-CAF, WGC, and RetinSphere), focused on regenerating dermal matrix and activating proliferation of skin cells, has shown to be efficient in the reversion of skin photoaging.

Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. ‘compact and adsorbed to collagen’ versus ‘extended and fibrillar’ fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin’s contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of single fibronectin fibers enhanced the non-persistent migration of both Fnf/f and Fn-/- MEFs, the migration speed was increased for Fn-/- MEFs on plasma fibronectin fibers compared to Fnf/f MEFs. In contrast, the average speed was the same for all cells on collagen-coated Fn fibers. A Fn-FRET sensor revealed that fibronectin on average was more extended on the microtissue surface compared to fibronectin in the core. Gradients of collagen-to-fibronectin ratios and of the fraction of collagen-adsorbed to stretched fibrillar fibronectin conformations might thereby provide critical cell migration cues. This study highlights a dominant role for fibronectin in tissue morphogenesis and the development of tissue heterogeneities.

Microfluidic spinning of fibronectin fibers via silk-induced cross-linking

Event Abstract Back to Event Microfluidic spinning of fibronectin fibers via silk-induced cross-linking Matthew Jacobsen1*, Joyce Y. Wong1 and Michael L. Smith1 1 Boston University, Department of Biomedical Engineering, United States Introduction: Fibronectin (Fn) is an essential extracellular matrix protein that is assembled from its monomeric form in solution in the cytosol to an insoluble fibrous network as it is extruded by the cell into the extracellular space. This polymerization process is not well understood and difficult to recapitulate in a laboratory environment, even using alternate means such as electrospinning. Successful reconstitution methods for Fn fibers outside of the tissue environment rely on surface tension as a driving polymerization force[1],[2]. The limitations of this method are the length scale and quantity of Fn that can be obtained, such that scalability is low. In this work, we present a microfluidic technique that takes advantage of the polymerization method of silk fibroin (SF) in order to cross-link Fn into a continuous spin to improve fiber yields while not altering the biological effects of Fn. Materials and Methods: A tiered microfluidic device (Figure 1) designed for the spinning of pure SF solutions was adapted by modifying the spinning solution[3]. Concentrated solutions of SF (>70 mg/ml) and Fn (>3 mg/ml) were mixed at various ratios to create a range of protein densities in extruded fibers. Flow through the microfluidic device induces elongation of protein chains leading to hydrogen bonding into hierarchical secondary structures. Extrusion into an organic solvent dehydrates the assembled protein fiber to finalize assembly of protein strands in a mature fiber. Fibers were tested for cellular interactivity and structural effects and compared to Fn[1] and SF properties[3], respectively. Results and Discussion: Continuous extrusion was observed for fibers with mass ratios of Fn:SF ranging from 1:1600 to 1:10. Fiber diameters decreased proportionately from the low Fn:SF ratio (30-50 micron) to the high ratio (1-5 micron), shown in Figure 2. Fiber diameters of Fn fibers extruded using the surface tension methodologies range from hundreds of nanometers to 1-3 microns, demonstrating the shift from a predominantly silk-like fiber morphology to that closer resembling pure Fn fibers. Functional degrees of cellular adherence are observed in the spun fibers shown in Figure 3 even at the minimal level of Fn incorporation (1:1600, fluorescently labeled – red), which is not a capability of SF fibers alone. FTIR results demonstrate that the interaction of the SF and Fn solutions occurs at the secondary structure level. This is supported by the result obtained where SF solution was diluted to deprive it of sufficient protein strands to form physical cross-links and, therefore, no fiber. When Fn is added at minimal concentrations, protein fiber production resumes. Conclusion: These results demonstrate that this method for spinning Fn fibers allows for the creation of a physically cross-linked fiber with composite properties that vary depending on the mixing ratio of the spinning solution. This method provides a means to create large quantities of Fn fibers using a simple microfluidic approach that previously could not be achieved with existing methods.

Endothelial directed collective migration depends on substrate stiffness via localized myosin contractility and cell-matrix interactions

Macrovascular endothelial injury, which may be caused by percutaneous intervention, requires endothelial cell directed collective migration to restore an intact endothelial monolayer. While interventions are often performed in arteries stiffened by cardiovascular disease, the effect of substrate stiffness on endothelial cell collective migration has not been examined. We studied porcine aortic endothelial cell directed collective migration using a modified cage assay on 4, 14, and 50kPa collagen-coated polyacrylamide gels. Total cell migration distance was measured over time, as were nuclear alignment and nuclear:total β-catenin as measures of cell directedness and cell–cell junction integrity, respectively. In addition, fibronectin fibers were examined as a measure of extracellular matrix deposition and remodeling. We now show that endothelial cells collectively migrate farther on stiffer substrates by 24h. Cells were more directed in the migration direction on intermediate stiffness substrates from 12 to 24h, with an alignment peak 400–700µm back from the migratory interface. However, cells on the softest substrates had the highest cell–cell junction integrity. Cells on all substrates deposited fibronectin, however fibronectin fibers were most linear and aligned on the stiffer substrates. When Rho kinase (ROCK) was inhibited with Y27632, cells on soft substrates migrated farther and cells on both soft and stiff substrates were more directed. When α5 integrin was knocked down with siRNA, cells on stiffer substrates did not migrate as far and were less directed. These data suggest that ROCK-mediated myosin contractility inhibits endothelial cell collective migration on soft substrates, while cell-matrix interactions are critical to endothelial cell collective migration on stiff substrates.

Nanopore Diameters Tune Strain in Extruded Fibronectin Fibers.

Fibronectin is present in the extracellular matrix and can be assembled into nanofibers in vivo by undergoing conformational changes. Here, we present a novel approach to prepare fibronectin nanofibers under physiological conditions using an extrusion approach through nanoporous aluminum oxide membranes. This one-step process can prepare nanofiber bundles up to a millimeter in length and with uniform fiber diameters in the nanometer range. Most importantly, by using different pore diameters and protein concentrations in the extrusion process, we could induce varying lasting structural changes in the fibers, which were monitored by Förster resonance energy transfer and should impose different physiological functions.

Role of region-distinctive expression of Rac1 in regulating fibronectin arrangement during palatal shelf elevation.

Palatal shelf elevation is a crucial process in palate development, with the contribution of various factors. Disturbances in any factor during this process result in cleft palate. Prior to palatal shelf elevation starting from embryonic day 12.5, the Rac1 expression level in the bend region of the mid-palatal shelf progressively increases and the cell densities in the bend and groove regions gradually become higher than those in the middle region. The comparative decrease of cell density in the middle region is correlated with a gradual alteration of the arrangement of fibronectin (FN) fibers, whereas the bend and groove regions with higher cell densities maintain ring-like FN arrangements. Rac1 overexpression alters the fibrillar FN arrangement in the middle region to the ring-like arrangement by increasing cell density. This alteration is sufficient to induce the failure of palatal shelf elevation, ultimately leading to cleft palate. Furthermore, the inhibition of FN delays palatal shelf elevation. Thus, the spatiotemporal expression of Rac1 plays an impressive role in palatal shelf elevation by regulating FN arrangement within the palatal shelf.

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.

The ultrastructure of fibronectin fibers pulled from a protein monolayer at the air-liquid interface and the mechanism of the sheet-to-fiber transition

Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.

Glycated collagen decreased endothelial cell fibronectin alignment in response to cyclic stretch via interruption of actin alignment.

Hyperglycemia is a defining characteristic of diabetes, and uncontrolled blood glucose in diabetes is associated with accelerated cardiovascular disease. Chronic hyperglycemia glycates extracellular matrix (ECM) collagen, which can lead to endothelial cell dysfunction. In healthy conditions, endothelial cells respond to mechanical stimuli such as cyclic stretch (CS) by aligning their actin cytoskeleton. Other cell types, specifically fibroblasts, align their ECM in response to CS. We previously demonstrated that glycated collagen inhibits endothelial cell actin alignment in response to CS. The aim of this study was to determine the effect of glycated collagen on ECM remodeling and protein alignment in response to stretch. Porcine aortic endothelial cells (PAEC) seeded on native or glycated collagen coated elastic substrates were exposed to 10% CS. Cells on native collagen aligned subcellular fibronectin fibers in response to stretch, whereas cells on glycated collagen did not. The loss of fibronectin alignment was due to inhibited actin alignment in response to CS, since fibronectin alignment did not occur in cells on native collagen when actin alignment was inhibited with cytochalasin. Further, while ECM protein content did not change in cells on native or glycated collagen in response to CS, degradation activity decreased in cells on glycated collagen. Matrix metalloproteinase 2 (MMP-2) and membrane-associated type 1 matrix metalloproteinase (MT1-MMP) protein levels decreased, and therefore MMP-2 activity also decreased. These MMP changes may relate to c-Jun N-terminal kinase (Jnk) phosphorylation inhibition with CS, which has previously been linked to focal adhesion kinase (FAK). These data demonstrate the importance of endothelial cell actin tension in remodeling and aligning matrix proteins in response to mechanical stimuli, which is critical to vascular remodeling in health and disease.

Early fibrillin-1 assembly monitored through a modifiable recombinant cell approach.

Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length fibrillin-1 assembly system that allows for rapid analysis of critical roles in fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.

Mesenchymal Stem Cells Exploit Extracellular Matrix as Mechanotransducer

While stem cells can sense and respond to physical properties of their environment, the molecular aspects how physical information is translated into biochemical signals remain unknown. Here we show that human mesenchymal stem cells (hMSCs) harvest and assemble plasma fibronectin into their extracellular matrix (ECM) fibrils within 24 hours. hMSCs pro-actively pull on newly assembled fibronectin ECM fibrils, and the fibers are more stretched on rigid than on soft fibronectin-coated polyacrylamide gels. Culturing hMSCs on single stretched fibronectin fibers upregulates hMSC osteogenesis. Osteogenesis was increased when αvβ3 integrins were blocked on relaxed fibronectin fibers, and decreased when α5β1 integrins were blocked or when epidermal growth factor (EGF) receptor signaling was inhibited on stretched fibronectin fibers. This suggests that hMSCs utilize their own contractile forces to translate environmental cues into differential biochemical signals by stretching fibronectin fibrils. Mechanoregulation of fibronectin fibrils may thus serve as check point to regulate hMSC osteogenesis.