Fibronectin Organization Research Articles

Synthesis and biological evaluation of selective Pepstatin based trifluoromethylated inhibitors of Cathepsin D

Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin.

Fine-Tuning Regulation of Surface Mobility by Acrylate Copolymers and Its Effect on Cell Adhesion and Differentiation.

Fibronectin (FN) mediates cell-material interactions during events such as tissue repair, and therefore the biomimetic modeling of this protein in vitro benefits regeneration. The nature of the interface is crucial in determining cell adhesion, morphology, and differentiation. Poly(ethyl acrylate) (PEA) spontaneously organizes FN into biological nanonetworks, resulting in exceptional bone regeneration in animal models. Spontaneous network organization of FN is also observed in poly(buthyl acrylate) (PBA) substrates that have higher surface mobility than PEA. C2C12 myoblasts differentiate efficiently on PEA and PBA substrates. In this study, we investigate if intermediate surface mobilities between PEA and PBA induce cell differentiation more efficiently than PEA. A family of P(EA-co-BA) copolymers were synthesized in the entire range of compositions to finely tune surface mobility between PEA and PBA. Surface characterization demonstrates that FN mobility steadily increased with the PBA content. All compositions allowed the biological organization of FN with similar exposure of cell binding domains. C2C12 myoblasts adhered well in all the materials, with higher focal adhesions in PEA and PBA. The increase of the interfacial mobility had an impact in cell adhesion by increasing the number of FAs per cell. In addition, cell differentiation decreased proportionally with surface mobility, from PEA to PBA.

Interplay between integrins and cadherins to control bone differentiation upon BMP-2 stimulation.

Introduction: Upon BMP-2 stimulation, the osteoblastic lineage commitment in C2C12 myoblasts is associated with a microenvironmental change that occurs over several days. How does BMP-2 operate a switch in adhesive machinery to adapt to the new microenvironment and to drive bone cell fate is not well understood. Here, we addressed this question for BMP-2 delivered either in solution or physically bound of a biomimetic film, to mimic its presentation to cells via the extracellular matrix (ECM). Methods: Biommetics films were prepared using a recently developed automated method that enable high content studies of cellular processes. Comparative gene expressions were done using RNA sequencing from the encyclopedia of the regulatory elements (ENCODE). Gene expressions of transcription factors, beta chain (1, 3, 5) integrins and cadherins (M, N, and Cad11) were studied using quantitative PCR. ECM proteins and adhesion receptor expressions were also quantified by Western blots and dot blots. Their spatial organization in and around cells was studied using immuno-stainings. The individual effect of each receptor on osteogenic transcription factors and alkaline phosphatase expression were studied using silencing RNA of each integrin and cadherin receptor. The organization of fibronectin was studied using immuno-staining and quantitative microscopic analysis. Results: Our findings highlight a switch of integrin and cadherin expression during muscle to bone transdifferentiation upon BMP-2 stimulation. This switch occurs no matter the presentation mode, for BMP-2 presented in solution or via the biomimetic film. While C2C12 muscle cells express M-cadherin and Laminin-specific integrins, the BMP-2-induced transdifferentiation into bone cells is associated with an increase in the expression of cadherin-11 and collagen-specific integrins. Biomimetic films presenting matrix-bound BMP-2 enable the revelation of specific roles of the adhesive receptors depending on the transcription factor. Discussion: While β3 integrin and cadherin-11 work in concert to control early pSMAD1,5,9 signaling, β1 integrin and Cadherin-11 control RunX2, ALP activity and fibronectin organization around the cells. In contrast, while β1 integrin is also important for osterix transcriptional activity, Cadherin-11 and β5 integrin act as negative osterix regulators. In addition, β5 integrin negatively regulates RunX2. Our results show that biomimetic films can be used to delinate the specific events associated with BMP-2-mediated muscle to bone transdifferentiation. Our study reveals how integrins and cadherins work together, while exerting distinct functions to drive osteogenic programming. Different sets of integrins and cadherins have complementary mechanical roles during the time window of this transdifferentiation.

Fibronectin: structure, functions, clinical significance (review)

Plasma fibronectin is a high molecular weight adhesive glycoprotein. There are two types of fibronectin: plasma (soluble) and cellular derived (insoluble). Electron microscopy revealed two types of structural organization of fibronectin: compact and expanded. In solution, fibronectin has a compact conformation, and after binding to certain substrates (collagen, fibrin, heparin), it is expanded. Plasma fibronectin is one of the main opsonins of blood plasma in relation to the “targets” of phagocytosis of a predominantly non-bacterial nature, as well as to some types of bacteria. For the treatment of septic processes, as well as respiratory distress syndrome of adults with severe fibronectin deficiency, plasma cryoprecipitate is used – a donor plasma preparation containing a large amount of plasma fibronectin (more than 2 mg/ml). It was proposed to replenish the level of fibronectin in patients with sepsis and other conditions that cause plasma fibronectin deficiency with the help of donor freshly frozen plasma. Transfusion of large volumes of freshly frozen plasma (up to 1000–1500 ml) to patients effectively eliminates the deficiency of plasma fibronectin. The concentration of plasma fibronectin in the blood significantly decreases after the addition of severe infectious processes to hematological diseases, as well as acute DIC syndrome. Extracorporeal methods of blood purification – selective plasmapheresis – have been developed to correct immunocomplex and fibronectin-complex pathology. Two variants of selective plasmapheresis have been proposed: the method of heparinocryoprecipitation of plasma proteins and the method of heparinocryofractionation. In 1987, a plasma heparin precipitate was proposed as a source of fibronectin for the treatment of patients with trophic skin lesions. In 1992, a new method was proposed for obtaining blood preparations with a high concentration of plasma fibronectin from patients themselves (heparin cryofractionation). Autofibronectin preparations obtained by such methods are effective in the local treatment of trophic ulcers in 90–93% of cases. The proposed drugs are safe against infection of patients with infectious diseases transmitted through the blood.

Characterization of plasmatic proteins adsorption on poly(styrene sodium sulfonate) functionalized silicone surfaces

Proteins adsorption occurs spontaneously on biomaterial upon insertion within the body. The resulting protein layer influences biomaterial biocompatibility through enhanced bio-integration or, on the contrary, adverse reactions. Furthermore, upon adsorption, proteins can undergo modifications of their structure and, ultimately, their physicochemical properties and activity. Hence, the understanding of protein adsorption on implanted materials appears essential, as exemplified by silicone breast prostheses that might lead to serious health issues.Surface modifications with a bioactive polymer, poly(styrene sodium sulfonate)-polyNaSS, on a hydrophobic silicone surface that composes breast implants, have been successfully performed under UV irradiation by a radical surface polymerization. This strategy enhances cell biocompatibility and antibacterial features. Although detailed insights related to the mechanism are still scarce, polyNaSS is supposed to promote changes in the conformation and/or orientation of adsorbed plasma proteins, reducing the odd for a biofilm to form.The present work addresses more in-depth structural investigations of the adsorbed state of two plasma proteins: Bovine Serum Albumin (BSA), as a model protein, and fibronectin (FN), for its role in cell adhesion. Using Atomic force microscopy (AFM), we report that polyNaSS showed no significant impact on the BSA structure conversely to the FN one. However, imaging findings with AFM clearly outlined a change in the structural organization of FN, going from a nano fibrillar assembly with an average length of 130 nm to a globular one when the surface was grafted. Thus, it is highlighted that polyNaSS interacts specifically with FN. In addition, cell spreading assay of L929 fibroblasts on FN-coated surfaces with optical microscopy indicated no significant impact of the change in FN structure upon fibroblasts adhesion, which displayed active elongated shapes. The present features are crucial for understanding the cell adhesion mechanism induced by surface modification.

Micellar Hyaluronidase and Spiperone as a Potential Treatment for Pulmonary Fibrosis.

Concentration of hyaluronic acid (HA) in the lungs increases in idiopathic pulmonary fibrosis (IPF). HA is involved in the organization of fibrin, fibronectin, and collagen. HA has been proposed to be a biomarker of fibrosis and a potential target for antifibrotic therapy. Hyaluronidase (HD) breaks down HA into fragments, but is a subject of rapid hydrolysis. A conjugate of poloxamer hyaluronidase (pHD) was prepared using protein immobilization with ionizing radiation. In a model of bleomycin-induced pulmonary fibrosis, pHD decreased the level of tissue IL-1β and TGF-β, prevented the infiltration of the lung parenchyma by CD16+ cells, and reduced perivascular and peribronchial inflammation. Simultaneously, a decrease in the concentrations of HA, hydroxyproline, collagen 1, total soluble collagen, and the area of connective tissue in the lungs was observed. The effects of pHD were significantly stronger compared to native HD which can be attributed to the higher stability of pHD. Additional spiperone administration increased the anti-inflammatory and antifibrotic effects of pHD and accelerated the regeneration of the damaged lung. The potentiating effects of spiperone can be explained by the disruption of the dopamine-induced mobilization and migration of fibroblast progenitor cells into the lungs and differentiation of lung mesenchymal stem cells (MSC) into cells of stromal lines. Thus, a combination of pHD and spiperone may represent a promising approach for the treatment of IPF and lung regeneration.

Basement Membrane Regulates Fibronectin Organization Using Sliding Focal Adhesions Driven by a Contractile Winch.

We have discovered that basement membrane and its major components can induce rapid, strikingly robust fibronectin organization. In this new matrix assembly mechanism, α5β1 integrin-based focal adhesions slide actively on the underlying matrix toward the ventral cell center through the dynamic shortening of myosin IIA-associated actin stress fibers to drive rapid fibronectin fibrillogenesis distal to the adhesion. This mechanism contrasts with classical fibronectin assembly based on stable or fixed-position focal adhesions containing αVβ3 integrins plus α5β1 integrin translocation into proximal fibrillar adhesions. On basement membrane components, these sliding focal adhesions contain standard focal adhesion constituents but completely lack classical αVβ3 integrins. Instead, peripheral α3β1 or α2β1 adhesions mediate initial cell attachment but over time are switched to α5β1 integrin-based sliding focal adhesions to assemble fibronectin matrix. This basement-membrane-triggered mechanism produces rapid fibronectin fibrillogenesis, providing a mechanistic explanation for the well-known widespread accumulation of fibronectin at many organ basement membranes.

Label-Free Imaging of Fibronectin Adsorption at Poly-(l-lysine)-Decorated Liquid Crystal Droplets

Interactions and subsequent structural organization of fibronectin (FibN) (major component of an extracellular matrix) molecules in contact with chemically modified surfaces form an essential basis to the understanding of the regulation of matrix assembly in pathological states and to the development of new biomedical devices. Here, using poly-l-lysine (PLL)-coated liquid crystal (LC) droplets, we present a simple but fruitful advance of an experimental system that can report label-free imaging of adsorption of FibN at the LC–aqueous interface. We observed that the interfacial intermolecular interactions of anionic FibN molecules and cationic PLL residues is accompanied by a director configuration transition of the LC droplets from radial to bipolar/preradial, giving a distinct optical output. Specifically, it is found that the ordering transitions of LC can be effectively tuned by blocking the anionic sites of FibN with divalent cations (Ca2+). Combination of fluorescence measurements, circular dichroism...

Functionalization of PLLA with Polymer Brushes to Trigger the Assembly of Fibronectin into Nanonetworks.

Poly-l-lactic acid (PLLA) has been used as a biodegradable polymer for many years; the key characteristics of this polymer make it a versatile and useful resource for regenerative medicine. However, it is not inherently bioactive. Thus, here, a novel process is presented to functionalize PLLA surfaces with poly(ethyl acrylate) (PEA) brushes to provide biological functionality through PEA's ability to induce spontaneous organization of the extracellular matrix component fibronectin (FN) into physiological-like nanofibrils. This process allows control of surface biofunctionality while maintaining PLLA bulk properties (i.e., degradation profile, mechanical strength). The new approach is based on surface-initiated atomic transfer radical polymerization, which achieves a molecularly thin coating of PEA on top of the underlying PLLA. Beside surface characterization via atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle to measure PEA grafting, the biological activity of this surface modification is investigated. PEA brushes trigger FN organization into nanofibrils, which retain their ability to enhance adhesion and differentiation of C2C12 cells. The results demonstrate the potential of this technology to engineer controlled microenvironments to tune cell fate via biologically active surface modification of an otherwise bioinert biodegradable polymer, gaining wide use in tissue engineering applications.

Nanoscale Coatings for Ultralow Dose BMP-2-Driven Regeneration of Critical-Sized Bone Defects.

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Some existing regenerative approaches rely on high doses of growth factors, such as bone morphogenetic protein‐2 (BMP‐2) in bone regeneration, which can cause serious side effects. An ultralow‐dose growth factor technology is described yielding high bioactivity based on a simple polymer, poly(ethyl acrylate) (PEA), and mechanisms to drive stem cell differentiation and bone regeneration in a critical‐sized murine defect model with translation to a clinical veterinary setting are reported. This material‐based technology triggers spontaneous fibronectin organization and stimulates growth factor signalling, enabling synergistic integrin and BMP‐2 receptor activation in mesenchymal stem cells. To translate this technology, plasma‐polymerized PEA is used on 2D and 3D substrates to enhance cell signalling in vitro, showing the complete healing of a critical‐sized bone injury in mice in vivo. Efficacy is demonstrated in a Münsterländer dog with a nonhealing humerus fracture, establishing the clinical translation of advanced ultralow‐dose growth factor treatment.

Abstract B42: The cell adhesion molecule, L1CAM, is important for the dissemination and metastasis of fallopian tube precursor lesions

Abstract One of the hallmarks during the progression of high-grade serous ovarian cancer (HGSOC) is the dissemination and metastasis of cells from fallopian tube precursor lesions, namely serous tubal in situ carcinoma (STIC), to other sites of the fallopian tube, ovary, or peritoneal cavity. We, and others, have previously demonstrated that HGSOC originates from benign-appearing lesions in the fallopian tube, harboring TP53 mutations (P53 signatures), that progress into STIC lesions, and show an increase in DNA damage and proliferation. While studies have analyzed those precursor lesions at the distal end of the fallopian tube, little is known about the mechanism that triggers the dissemination of cells from fallopian tube precursor lesions to other sites. The L1 cell adhesion molecule (L1CAM, CD171) has been shown to play an important role in many types of cancer, including ovarian carcinoma. In vitro studies have shown that L1CAM enhances proliferation, cell migration, epithelial-to-mesenchymal transition (EMT), and chemoresistance in ovarian carcinoma cells. In HGSOC, high expression of L1CAM is associated with poor overall survival and confers chemoresistance in this setting. It was further shown that L1CAM expression and the presence of its soluble form in ovarian carcinoma patient ascites correlates with shorter patient survival. Current knowledge is limited on the role of L1CAM during the initiation or the early stages of HGSOC. Therefore, we investigated precursor STIC lesions for L1CAM expression. We found expression of L1CAM in most of the analyzed STIC lesions, but also in scattered normal fallopian epithelial cells. Interestingly, the expression in STIC lesions was not homogenous but showed a gradient with its lowest expression in cells attached to the basement membrane and its highest expression in pseudo-stratified cells that appear to be in the process of detaching. To investigate whether L1CAM contributes to detachment, we used the CRISPR/CAS9 system to knock down L1CAM in the ovarian cancer cell line OVCAR8. Importantly, the knockdown of L1CAM decreased the ability of cells to grow and survive under anchorage-independent culture conditions and to build 3-dimensional spheres. Conversely, overexpression of L1CAM in primary fallopian tube cells led to a strong increase in their ability to form spheres. We found that the expression of L1CAM in ovarian cancer and primary fallopian tube cell lines led to an increase in integrin alpha-5 and integrin beta-1 expression. This ultimately leads to an increased recruitment and organization of fibronectin and assembly of a basement membrane under anchorage-independent culture conditions. These data indicate that in precursor lesions L1CAM expression may be necessary for the tumor to disseminate and metastasize to other sites of the fallopian tube, ovary, or peritoneal cavity. Finally, we found a strong link between the noncanonical WNT pathway and L1CAM expression, which might explain the high L1CAM expression at the apical side of the STIC lesions. However, further studies must be performed to analyze which factors regulate the expression gradient of L1CAM and whether the inhibition of those factors or L1CAM itself delay the onset of the malignant disease. Citation Format: Kai Doberstein, Marcin Iwanicki, Mina Fogel, Peter Altevogt, Ronny Drapkin. The cell adhesion molecule, L1CAM, is important for the dissemination and metastasis of fallopian tube precursor lesions. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B42.

CD93 promotes β1 integrin activation and fibronectin fibrillogenesis during tumor angiogenesis.

Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is upregulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates β1 integrin signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytic cleavage. The CD93-MMRN2 complex was required for activation of β1 integrin, phosphorylation of focal adhesion kinase (FAK), and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of β1 integrin and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.

Engineered Coatings for Titanium Implants To Present Ultralow Doses of BMP-7.

The ongoing research to improve the clinical outcome of titanium implants has resulted in the implemetation of multiple approches to deliver osteogenic growth factors accelerating and sustaining osseointegration. Here we show the presentation of human bone morphogenetic protein 7 (BMP-7) adsorbed to titanium discs coated with poly(ethyl acrylate) (PEA). We have previously shown that PEA promotes fibronectin organization into nanonetworks exposing integrin- and growth-factor-binding domains, allowing a synergistic interaction at the integrin/growth factor receptor level. Here, titanium discs were coated with PEA and fibronectin and then decorated with ng/mL doses of BMP-7. Human mesenchymal stem cells were used to investigate cellular responses on these functionalized microenvironments. Cell adhesion, proliferation, and mineralization, as well as osteogenic markers expression (osteopontin and osteocalcin) revealed the ability of the system to be more potent in osteodifferentiation of the mesenchymal cells than combinations of titanium and BMP-7 in absence of PEA coatings. This work represents a novel strategy to improve the biological activity of titanium implants with BMP-7.

Glaucomatous cell derived matrices differentially modulate non-glaucomatous trabecular meshwork cellular behavior

Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment. Statement of SignificanceExtracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.

Fibronectins: Structural-functional relationships

This review summarized current data on the structure of fibronectin (FN), a multifunctional glycoprotein of vertebrates. FN is not only a permanent component of the extracellular matrix (ECM) but also an important regulator of cell functions via transformation of the ECM composition and organization and/or interaction with receptor and other membranebound cell proteins. Multifunctionality of FN owes hierarchical relationships between its structuralfunctional determinants, which comprise the linear ones (FN peptide fragments), association zones (surface contacts between the FN molecule and a FN-associated protein) and functional domains (those binding fibrin, heparin, gelatin and integrins). The modular architectonic principle of FN organization is pivotal to intrinsic adaptation of this glycoprotein to changing microenvironmental conditions. We also discuss the issue of key stages of FN fibrillogenesis with a special focus on the molecular mechanisms that underlie polymerization of FN molecules.

Fibroblast-fibronectin patterning and network formation in 3D fibrin matrices

PurposeWe previously reported that fibroblasts migrating within 3-D collagen matrices move independently, whereas fibroblasts within 3-D fibrin matrices form an interconnected network. Similar networks have been identified previously during in vivo corneal wound healing. In this study, we investigate the role of fibronectin in mediating this mechanism of collective cell spreading, migration and patterning. MethodsTo assess cell spreading, corneal fibroblasts were plated within fibrillar collagen or fibrin matrices. To assess migration, compacted cell-populated collagen matrices were nested inside cell-free fibrin matrices. Constructs were cultured in serum-free media containing PDGF, with or without RGD peptide, anti-α5 or anti-fibronectin blocking antibodies. In some experiments, LifeAct and fluorescent fibronectin were used to allow dynamic assessment of cell-induced fibronectin reorganization. 3-D and 4-D imaging were used to assess cell mechanical behavior, connectivity, F-actin, α5 integrin and fibronectin organization. ResultsCorneal fibroblasts within 3-D fibrin matrices formed an interconnected network that was lined with cell-secreted fibronectin. Live cell imaging demonstrated that fibronectin tracks were formed at the leading edge of spreading and migrating cells. Furthermore, fibroblasts preferentially migrated through fibronectin tracks laid down by other cells. Interfering with cell-fibronectin binding with RGD, anti α5 integrin or anti fibronectin antibodies inhibited cell spreading and migration through fibrin, but did not affect cell behavior in collagen. ConclusionsIn this study, a novel mode of cell patterning was identified in which corneal fibroblasts secrete and attach to fibronectin via α5β1 integrin to facilitate spreading and migration within 3-D fibrin matrices, resulting in the formation of localized fibronectin tracks. Other cells use these fibronectin tracks as conduits, resulting in an interconnected cell-fibronectin network.

Engineered microenvironments for synergistic VEGF – Integrin signalling during vascularization

We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications.

Role of chemical crosslinking in material-driven assembly of fibronectin (nano)networks: 2D surfaces and 3D scaffolds

Poly(ethyl acrylate) (PEA) induces the formation of biomimetic fibronectin (FN) (nano)networks upon simple adsorption from solutions, a process referred to as material-driven FN fibrillogenesis. The ability of PEA to organize FN has been demonstrated in 2D and 2.5D environments, but not as yet in 3D scaffolds, which incorporate three-dimensionality and chemical crosslinkers that may influence its fibrillogenic potential. In this paper we show for the first time that while three-dimensionality does not interfere with PEA-induced FN fibrillogenesis, crosslinking does, and we determined the maximum amount of crosslinker that can be added to PEA to maintain FN fibrillogenesis. For this, we synthesised 2D substrates with different amounts of crosslinker (1–10% of ethylene glycol dimethacrylate) and studied the role of crosslinking in FN organization using AFM. The glass transition temperature was seen to increase with crosslinking density and, accordingly, polymer segmental mobility was reduced. The organization of FN after adsorption (formation of FN fibrils) and the availability of the FN cell-binding domain were found to be dependent on crosslinking density. Surface mobility was identified as a key parameter for FN supramolecular organization. PEA networks with up to 2% crosslinker organize the FN in a similar way to non-crosslinked PEA. Scaffolds prepared with 2% crosslinker also had FN (nano)networks assembled on their walls, showing PEA’s ability to induce FN fibrillogenesis in 3D environments as long as the amounts of crosslinker is low enough.

Material-driven fibronectin assembly for high-efficiency presentation of growth factors.

Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness.

Fibronectin (nano)fibrils induced by materials: from 2D to 3D microenvironments

Event Abstract Back to Event Fibronectin (nano)fibrils induced by materials: from 2D to 3D microenvironments Laia León-Boigues1, Antonio Sánchez-Laosa1, Luis Gómez-Estrada2, José Luis Gómez Ribelles1, 3, Manuel Salmerón-Sánchez4, Gloria Gallego Ferrer1, 3 and Roser Sabater I Serra1 1 Universitat Politècnica de València, Center for Biomaterials and Tissue Engineering, Spain 2 Ikasia Technologies SL, Spain 3 Biomedical Research Networking Centre (CIBER), Spain 4 University of Glasgow, Division of Biomedical Engineering, School of Engineering, United Kingdom Introduction: Biomaterials play a key role in regenerative medicine, acting as a synthetic extracellular matrix. However, synthetic biomaterials, biologically inert, have to be functionalised to become bioactive. Adsorption of adhesive proteins, as fibronectin (FN), on the biomaterial surface is one of the strategies commonly used to enhance bioactivity[1],[2]. It have been shown that certain materials, such as poly(ethyl acrylate), PEA, are able to induce the organization of FN in a biomimetic way. A physiological-like FN nanonetwork is organised upon simple adsorption of FN from a protein solution as consequence of protein-material interactions[3]. The novelty of this work is to show the ability of PEA to induce the formation of fibronectin (nano)fibrils in 3D-like environments, as it has been shown only on 2D substrates. For this purpose, PEA scaffolds with interconnected pores larger than 300 µm were synthesised, to be afterwards 3D-coated with FN to promote vascularization and new bone formation[4]. The distribution of the adsorbed FN within the walls of the scaffold surface was studied by AFM. Experimental Methods: PEA scaffolds were obtained by radical polymerization (1 wt% of benzoyl peroxide initiator). 2 wt% ethylene glycol dimethacrylate, EGDMA, was used as crosslinker to improve PEA mechanical properties. Polyvinyl alcohol templates, obtained by rapid-prototyping, were used as porogen. The polymerization was carried out at 60 ºC for 24h. Then, the template was dissolved in water. 2D substrates (films) with 0 and 2 wt% EGDMA were also prepared to be used as 2D controls. Scaffold mechanical properties, morphology and porosity were studied by compression tests, SEM and a liquid displacement method, respectively. The distribution of FN (10 µg/mL PBS solution, 10 min) was investigated by AFM. Results and Discussion: PEA scaffolds show a homogeneous structure, with channels (ca. 350 µm of diameter) in orthogonal directions and distance between channels ca. 400 µm (Fig. 1); the overall porosity is ca. 50%. The elastic modulus is ~ 2.5 MPa. Figure 1. SEM images. Cross-section of PEA scaffolds (2 %wt EGDMA)) AFM images (Fig. 2) show FN distribution after adsorption on 3D substrates (Fig. 2c) and 2D uncrosslinked and crosskinked films (Fig. 2a and 2b respectively). Similar interconnected FN nanofibrils were found on both 2D surfaces, indicating that the process of material-driven FN assembly is unaffected by the crosslinking process. FN fibrils induced in 3D substrates show no significant differences with the ones induced on 2D PEA films. Figure 2. AFM phase (1 μm x 1 μm). a) film (0 wt% EGDMA), b) film (2 wt% EGDMA), c) scaffold (2 wt% EGDMA). Conclusion: We demonstrated that PEA is able to induce the formation of fibronectin (nano)fibrils in 3D environments. Bioactive scaffolds with homogeneous channels and suitable pore size for bone regeneration have been engineered. The support of the project MAT2012-38359-C03-01 (including the FEDER financial support) as well as CIBER is acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with the assistance from the European Regional Development Fund.