Nanoparticles traversing the extracellular matrix induce biophysical perturbation of fibronectin depicted by surface chemistry.

While the filtering and accumulation effects of the extracellular matrix (ECM) on nanoparticles (NPs) have been experimentally observed, the detailed interactions between NPs and specific biomolecules within the ECM remain poorly understood and pose challenges for in vivo molecular-level investigations. Herein, we adopt molecular dynamics simulations to elucidate the impacts of methyl-, hydroxy-, amine-, and carboxyl-modified gold NPs on the cell-binding domains of fibronectin (Fn), an indispensable component of the ECM for cell attachment and signaling. Simulation results show that NPs can specifically bind to distinct Fn domains, and the strength of these interactions depends on the physicochemical properties of NPs. NP-NH3+ exhibits the highest affinity to domains rich in acidic residues, leading to strong electrostatic interactions that induce severe deformation, potentially disrupting the normal functioning of Fn. NP-CH3 and NP-COO- selectively occupy the RGD/PHSRN motifs, which may hinder their recognition by integrins on the cell surface. Additionally, NPs can disrupt the dimerization of Fn through competing for residues at the dimer interface or by diminishing the shape complementarity between dimerized proteins. The mechanical stretching of Fn, crucial for ECM fibrillogenesis, is suppressed by NPs due to their local rigidifying effect. These results provide valuable molecular-level insights into the impacts of various NPs on the ECM, holding significant implications for advancing nanomedicine and nanosafety evaluation.

Fn-HMGB1 Adsorption Behavior Initiates Early Immune Recognition and Subsequent Osteoinduction of Biomaterials.

Implantable biomaterials are widely used in bone tissue engineering, but little is still known about how they initiate early immune recognition and the initial dynamics. Herein, the early immune recognition and subsequent osteoinduction of biphasic calcium phosphate (BCP) after implantation to the protein adsorption behavior is attributed. By liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, the biomaterial-related molecular patterns (BAMPs) formed after BCP implantation are mapped, dominated by the highly expressed extracellular matrix protein fibronectin (Fn) and the high mobility group box 1 (HMGB1). Molecular dynamics simulations show that Fn has the ability to bind more readily to the BCP surface than HMGB1. The preferential binding of Fn provides a higher adsorption energy for HMGB1. Furthermore, multiple hydrogen bonding sites between HMGB1 and Fn are demonstrated using a molecular docking approach. Ultimately, the formation of BAMPs through HMGB1 antagonist glycyrrhizic acid (GA), resulting in impaired immune recognition of myeloid differentiation factor 88 (MYD88) mediated dendritic cells (DCs) and macrophages (Mφs), as well as failed osteoinduction processes is obstructed. This study introduces a mechanism for early immune recognition of implant materials based on protein adsorption, providing perspectives for future design and application of tissue engineering materials.

CAR-T Therapy Targets Extra Domain B of Fibronectin Positive Solid Tumor Cells.

BACKGROUND: CAR-T cell immunotherapy has achieved remarkable success in malignant B-cell malignancies, but progress in solid tumors is slow, and one of the key reasons is the lack of ideal targets. Cancer-specific extra domain B of fibronectin (EDB-FN) is widely upregulated in solid tumors and expressed at low levels in normal tissues. Many imaging and targeted cancer therapies based on EDB-FN targets have been developed and tested in clinical trials, making EDB-FN an ideal target for immunotherapy. METHODS: We constructed two EDB-FN-targeted CAR-Ts based on the peptide APT0 and the single-chain antibody CGS2 in a lentiviral infection manner for the first time. Luciferase cytotoxicity assay to assess CAR-T killing of tumor cells. An enzyme-linked immunosorbent assay was used to detect the release of the cytokine IFN-γ. Fluorescence imaging to evaluate the dynamics of CAR-T cell and tumor cell coculture. Knockdown assays were used to validate the target specificity of CAR-T cells. RESULTS: In this research, two CAR-Ts targeting EDB-FN, APT0 CAR-T, and CGS2 CAR-T, were constructed. In vitro, both CAR-T cells produced broad-spectrum killing of multiple EDB-FN-positive solid tumor cell lines and were accompanied by cytokine IFN-γ release. Regarding safety, the two CAR-T cells did not affect T cells' normal growth and proliferation and were not toxic to HEK-293T human embryonic kidney epithelial cells. CONCLUSION: APT0 CAR-T and CGS2 CAR-T cells are two new CAR-Ts targeting EDB-FN. Both CAR-T cells can successfully identify and specifically kill various EDB-FN-positive solid tumor cells with potential clinical applications.

A serologically assessed neo-epitope biomarker of cellular fibronectin degradation is related to pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by excessive extracellular matrix (ECM) remodeling, herein ECM degradation. Fibronectin (FN) is an important component of the ECM that is produced by multiple cell types, including fibroblasts. Extra domain B (EDB) is specific for a cellular FN isoform which is found in the ECM. We sought to develop a non-invasive test to investigate whether matrix metalloproteinase 8 (MMP-8) degradation of EDB in cellular FN results in a specific protein fragment that can be assessed serologically and if levels relate to pulmonary fibrosis. METHOD: Cellular FN was cleaved in vitro by MMP-8 and a protein fragment was identified by mass spectrometry. A monoclonal antibody (mAb) was generated, targeting a neo-epitope originating from EDB in cellular FN. Utilizing this mAb, a neo-epitope specific enzyme-linked immunosorbent assay (FN-EDB) was developed and technically validated. Serum FN-EDB was assessed in an IPF cohort (n = 98), registered at clinicaltrials.gov (NCT02818712), and in healthy controls (n = 35). RESULTS: The FN-EDB assay had high specificity for the MMP-8 degraded neo-epitope and was technically robust. FN-EDB serum levels were not influenced by age, sex, ethnicity, or BMI. Moreover, FN-EDB serum levels were significantly higher in IPF patients (median 31.38 [IQR 25.79-46.84] ng/mL) as compared to healthy controls (median 28.05 [IQR 21.58-33.88] ng/mL, p = 0.023). CONCLUSION: We developed the neo-epitope specific FN-EDB assay, a competitive ELISA, as a tool for serological assessment of MMP-8 mediated degradation of EDB in cellular FN. This study indicates that degradation of EDB in cellular FN is elevated in IPF and warrants further investigation.

Engineered Biomimetic Fibrillar Fibronectin Matrices Regulate Cell Adhesion Initiation, Migration, and Proliferation via α5ß1 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 α5ß1 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.

Molecular basis of conformational changes and mechanics of integrins.

Integrin, as a mechanotransducer, establishes the mechanical reciprocity between the extracellular matrix (ECM) and cells at integrin-mediated adhesion sites. This study used steered molecular dynamics (SMD) simulations to investigate the mechanical responses of integrin αvß3 with and without 10th type III fibronectin (FnIII10) binding for tensile, bending and torsional loading conditions. The ligand-binding integrin confirmed the integrin activation during equilibration and altered the integrin dynamics by changing the interface interaction between ß-tail, hybrid and epidermal growth factor domains during initial tensile loading. The tensile deformation in integrin molecules indicated that fibronectin ligand binding modulates its mechanical responses in the folded and unfolded conformation states. The bending deformation responses of extended integrin models reveal the change in behaviour of integrin molecules in the presence of Mn2+ ion and ligand based on the application of force in the folding and unfolding directions of integrin. Furthermore, these SMD simulation results were used to predict the mechanical properties of integrin underlying the mechanism of integrin-based adhesion. The evaluation of integrin mechanics provides new insights into understanding the mechanotransmission (force transmission) between cells and ECM and contributes to developing an accurate model for integrin-mediated adhesion. This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

Nucleation of fibronectin fibril assembly requires binding between heparin and the 13th type III module of fibronectin.

Fibronectin (FN), a critical component of the extracellular matrix, is assembled into fibrils through a cell-mediated process. Heparan sulfate (HS) binds to the III13 module of FN, and fibroblasts lacking this glycosaminoglycan exhibit reduced FN fibril assembly. To determine if HS depends on III13 to control FN assembly, we deleted both III13 alleles in NIH 3T3 cells using the CRISPR-Cas9 system. ΔIII13 cells assembled fewer FN matrix fibrils and less DOC-insoluble FN matrix than wildtype cells. Little if any mutant FN matrix was assembled when purified ΔIII13 FN was provided to Chinese hamster ovary (CHO) cells, showing that lack of III13 caused the deficiency in assembly by ΔIII13 cells. Addition of heparin promoted the assembly of wildtype FN by CHO cells, but it had no effect on the assembly of ΔIII13 FN. Furthermore, heparin binding stabilized the folded conformation of III13 and prevented it from self-associating with increasing temperature suggesting that stabilization by HS/heparin binding might regulate interactions between III13 and other FN modules. This effect would be particularly important at matrix assembly sites where our data show that ΔIII13 cells require both exogenous wildtype FN and heparin in the culture medium to maximize assembly site formation. Our results show that heparin-promoted growth of fibril nucleation sites is dependent on III13. We conclude that HS/heparin binds to III13 to promote and control the nucleation and development of FN fibrils.

Characterization of fibronectin properties by integrated micro-fluidic experiments and fluid-structure interaction simulations.

Fibronectin (Fn) has been observed to assemble in the extracellular matrix (ECM) of cell culture and stretch in response to the external force. The alteration of molecule domain functions generally follows the extension of Fn. Several researchers have investigated fibronectin extensively in molecular architecture and conformation structure. However, the bulk material behavior of the Fn in the ECM has not been fully depicted at the cell scale, and many studies have ignored physiological conditions. Conversely, microfluidic techniques that explore cellular properties based on cell deformation and adhesion have emerged as a powerful and effective platform to study cell rheological transformation in a physiological environment. However, directly quantifying properties from microfluidic measurements remains a challenge. Therefore, it is an efficient way to combine experimental measurements with a robust and reliable numerical framework to calibrate the mechanical stress distribution in the test sample. In this paper, we present a monolithic Lagrangian fluid-structure interaction (FSI) approach within the Optimal Transportation Meshfree (OTM) framework that enables the investigation of the adherent Red Blood Cell (RBC) interacting with fluid and overcomes the drawbacks of the traditional computational tools such as the mesh entanglement and interface tracking, etc. This study aims to assess the material properties of the RBC and Fn fiber by calibrating the numerical predictions to experimental measurements. Moreover, a physical-based constitutive model will be proposed to describe the bulk behavior of the Fn fiber inflow, and the rate-dependent deformation and separation of the Fn fiber will be discussed.

Highly stable fibronectin-mimetic-peptide-based supramolecular hydrogel to accelerate corneal wound healing.

Without timely treatment, poor wound healing in corneal injuries can seriously impair vision and lead to blindness. Thus, it is vital to develop a therapeutic strategy to accelerate corneal re-epithelialization. The conjugation of self-assembled motifs with a fibronectin-mimetic peptide sequence (PHSRN) drastically improves the chemical stability of PHSRN against protease hydrolysis and minimally affects its biological activity to promote the migration of corneal epithelial cells. The optimized Nap-FFPHSRN self-assembled into bioactive supramolecular hydrogels increases cell motility by remolding F-actin and boosts the tight junction of the corneal epithelium by increasing the expression of zonula occludens-1 (ZO-1). An in vivo experiment showed that a Nap-FFPHSRN hydrogel provided extended precorneal retention with good ocular tolerance after topical instillation. An animal model of corneal scrape showed that a single daily dose of Nap-FFPHSRN hydrogel had a superior therapeutic effect in facilitating corneal re-epithelialization with complete morphological and architectural recovery. With a rational approach to mimic bioactive proteins, this study presents a new strategy to demonstrate the potential of peptide-based supramolecular hydrogels for use in clinical treatment of corneal injury. STATEMENT OF SIGNIFICANCE: Here we systematically investigate the self-assembly behavior and chemical stability of designed peptide amphiphiles (Nap-FPHRSN, Nap-FFPHSRN and Nap-FFFPHSRN). The introduction of self-assembled motifs (Nap-F, Nap-FF and Nap-FFF) drastically enhances the chemical stability of fibronectin-mimetic peptide (PHSRN). Moreover, topical instillation of Nap-FFPHSRN hydrogel once daily, exhibits a better in vivo effect than PHSRN and the same in vivo effect as fibronectin, both of which are instilled three times daily, for promoting full morphological and architectural recovery after corneal re-epithelialization. As a rational design of conjugating bioactive peptides with self-assembled motifs to mimic bioactive proteins, this work may lead to a new approach that improves the in vivo therapeutic effect for treating corneal injury in clinic settings.

Characterisation of collagen type I matrices for pathophysiologically relevant spatial cancer cell cultures.

Specific cues provided to cells by the extracellular matrix (ECM) are determined by its composition. Except of collagens other naturally occurring ECM components should be considered in designing 3D models of diseases. We used spectrophotometric and rheological measurements and confocal imaging to characterise collagen matrices of human origin that can be modified by clinically relevant ECM components. pH of the neutralising solution, but not incubation of solidified collagen matrices in serum-free culture medium with pH 5.0-9.0 affected distribution of collagen fibres. Admixture of fibronectin or tenascin-C influenced assembly kinetics and resulted in slight increase in the Young's moduli of the matrices, indicating their incorporation into the collagen matrices. Co-localization of fibronectin with collagen fibres was confirmed by fluorescence imaging. Various cell types relevant for tumour tissue were able to proliferate within the matrices suggesting that they can be used to study role of ECM components in cancer in spatial models.

A computational study on strontium ion modified hydroxyapatite-fibronectin interactions.

Protein adsorption is the first key step in cell-material interactions. The initial phase of such an adsorption process can only be probed using modelling approaches like molecular dynamics (MD) simulations. Despite a large number of studies on the adsorption behaviour of proteins on different biomaterials including calcium phosphates (CaP), little attention has been paid towards the quantitative assessment of the effects of various physicochemical influencers like surface modification, pH, and ionic strength. In the case of doped CaPs, surface modification through isomorphic substitution of foreign ions inside the apatite structure is of particular interest in the context of protein-HA interactions, as it is widely used to tailor the biological response of HA. Given this background, we present here the molecular-level understanding of the fibronectin (FN) adsorption mechanism and kinetics on a Sr2+-doped hydroxyapatite, HA, (001) surface at 300 K by means of all-atom molecular dynamics simulations. Electrostatic interactions involved in the adsorption of FN on HA were found to be significantly modified due to Sr2+ doping into the apatite lattice. In harmony with the published experimental observations, the Sr-doped surfaces were found to better support FN adhesion compared to pure HA, with 10 mol% Sr-doped HA exhibiting the best FN adsorption. The observed altered adsorption behaviour of FN on Sr-doped HA was correlated with the Hofmeister effect. Moreover, the non-monotonous trend of the FN-material interaction energy can be attributed to the spatial rearrangement of the functional groups (PO43-, OH-) in the apatite crystal. Sr2+ ions also influence the stability of the secondary structure of FN, as observed from the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis. The presence of Sr2+ enhances the flexibility of specific residues (residue nos. 20-44, 74-88) of the FN module. Rupture forces to disentangle FN from the biomaterial surface, obtained from steered molecular dynamics (SMD) simulations, were found to corroborate well with the results of equilibrium MD simulations. One particular observation is that the availability of an RGD motif (Arginine-Glycine-aspartate sequence, which interacts with cell surface receptor integrin to form a focal adhesion complex) for the interaction with cell surface receptor integrin is not significantly influenced by Sr2+ substitution.

Dynamics of integrin α5ß1, fibronectin, and their complex reveal sites of interaction and conformational change.

Integrin α5ß1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5ß1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5ß1-binding affinities and stabilities. We also interrogated binding of the α5ß1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two ß-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit ß-propeller domain for the Fn9 synergy site and in the ß1-subunit ßI domain for Fn10 based on decreases in α5ß1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5ß1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5ß1 binding to Fn and dynamics associated with this interaction.

A Novel Cell Delivery System Exploiting Synergy between Fresh Titanium and Fibronectin.

Delivering and retaining cells in areas of interest is an ongoing challenge in tissue engineering. Here we introduce a novel approach to fabricate osteoblast-loaded titanium suitable for cell delivery for bone integration, regeneration, and engineering. We hypothesized that titanium age influences the efficiency of protein adsorption and cell loading onto titanium surfaces. Fresh (newly machined) and 1-month-old (aged) commercial grade 4 titanium disks were prepared. Fresh titanium surfaces were hydrophilic, whereas aged surfaces were hydrophobic. Twice the amount of type 1 collagen and fibronectin adsorbed to fresh titanium surfaces than aged titanium surfaces after a short incubation period of three hours, and 2.5-times more fibronectin than collagen adsorbed regardless of titanium age. Rat bone marrow-derived osteoblasts were incubated on protein-adsorbed titanium surfaces for three hours, and osteoblast loading was most efficient on fresh titanium adsorbed with fibronectin. The number of osteoblasts loaded using this synergy between fresh titanium and fibronectin was nine times greater than that on aged titanium with no protein adsorption. The loaded cells were confirmed to be firmly attached and functional. The number of loaded cells was strongly correlated with the amount of protein adsorbed regardless of the protein type, with fibronectin simply more efficiently adsorbed on titanium surfaces than collagen. The role of surface hydrophilicity of fresh titanium surfaces in increasing protein adsorption or cell loading was unclear. The hydrophilicity of protein-adsorbed titanium increased with the amount of protein but was not the primary determinant of cell loading. In conclusion, the osteoblast loading efficiency was dependent on the age of the titanium and the amount of protein adsorption. In addition, the efficiency of protein adsorption was specific to the protein, with fibronectin being much more efficient than collagen. This is a novel strategy to effectively deliver osteoblasts ex vivo and in vivo using titanium as a vehicle.

Fibronectin Functions as a Selective Agonist for Distinct Toll-like Receptors in Triple-Negative Breast Cancer.

The microenvironment of tumors is characterized by structural changes in the fibronectin matrix, which include increased deposition of the EDA isoform of fibronectin and the unfolding of the fibronectin Type III domains. The impact of these structural changes on tumor progression is not well understood. The fibronectin EDA (FnEDA) domain and the partially unfolded first Type III domain of fibronectin (FnIII-1c) have been identified as endogenous damage-associated molecular pattern molecules (DAMPs), which induce innate immune responses by serving as agonists for Toll-Like Receptors (TLRs). Using two triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, we show that FnEDA and FnIII-1c induce the pro-tumorigenic cytokine, IL-8, by serving as agonists for TLR5 and TLR2, the canonical receptors for bacterial flagellin and lipoprotein, respectively. We also find that FnIII-1c is not recognized by MDA-MB-468 cells but is recognized by MDA-MB-231 cells, suggesting a cell type rather than ligand specific utilization of TLRs. As IL-8 plays a major role in the progression of TNBC, these studies suggest that tumor-induced structural changes in the fibronectin matrix promote an inflammatory microenvironment conducive to metastatic progression.

Recent Progress on the Role of Fibronectin in Tumor Stromal Immunity and Immunotherapy.

As a major component of the stromal microenvironment of various solid tumors, the extracellular matrix (ECM) has attracted increasing attention in cancer-related studies. ECM in the tumor stroma not only provides an external barrier and framework for tumor cell adhesion and movement, but also acts as an active regulator that modulates the tumor microenvironment, including stromal immunity. Fibronectin (Fn), as a core component of the ECM, plays a key role in the assembly and remodeling of the ECM. Hence, understanding the role of Fn in the modulation of tumor stromal immunity is of great importance for cancer immunotherapy. Hence, in-depth studies on the underlying mechanisms of Fn in tumors are urgently needed to clarify the current understanding and issues and to identify new and specific targets for effective diagnosis and treatment purposes. In this review, we summarize the structure and role of Fn, its potent derivatives in tumor stromal immunity, and their biological effects and mechanisms in tumor development. In addition, we discuss the novel applications of Fn in tumor treatment. Therefore, this review can provide prospective insight into Fn immunotherapeutic applications in tumor treatment.

Myeloid immune checkpoint ILT3/LILRB4/gp49B can co-tether fibronectin with integrin on macrophages.

LILRB4 (B4, also known as ILT3/CD85k) is an immune checkpoint of myeloid lineage cells, albeit its mode of function remains obscure. Our recent identification of a common ligand for both human B4 and its murine ortholog gp49B as the fibronectin (FN) N-terminal 30 kDa domain poses the question of how B4/gp49B regulate cellular activity upon recognition of FN in the plasma and/or the extracellular matrix. Since FN in the extracellular matrix is tethered by FN-binding integrins, we hypothesized that B4/gp49B would tether FN in cooperation with integrins on the cell surface, thus they should be in close vicinity to integrins spatially. This scenario suggests a mode of function of B4/gp49B by which the FN-induced signal is regulated. The FN pull-down complex was found to contain gp49B and integrin ß 1 in bone marrow-derived macrophages. The confocal fluorescent signals of the three molecules on the intrinsically FN-tethering macrophages were correlated to each other. When FN-poor macrophages adhered to culture plates, the gp49-integrin ß 1 signal correlation increased at the focal adhesion, supporting the notion that gp49B and integrin ß 1 become spatially closer to each other there. Adherence of RAW264.7 and THP-1 cells to immobilized FN induced phosphorylation of spleen tyrosine kinase, whose level was augmented under B4/gp49B deficiency. Thus, we concluded that B4/gp49B can co-tether FN in cooperation with integrin in the cis configuration on the same cell, forming a B4/gp49B-FN-integrin triplet as a regulatory unit of a focal adhesion-dependent pro-inflammatory signal in macrophages.

Oxygen Ion Implantation Improving Cell Adhesion on Titanium Surfaces through Increased Attraction of Fibronectin PHSRN Domain.

Mechanistic understanding of fibronectin (FN) adsorption which determines cell adhesion on cell-implant interfaces is significant for improving the osteoconduction and soft-tissue healing of implants. Here, it is shown that the adsorption behavior of FN on the titanium oxide surface (TiO2 ) is highly relative to its Pro-His-Ser-Arg-Asn (PHSRN) peptide. FN lacking PHSRN fails to bind to surfaces, resulting in inhibited cell adhesion and spreading. Molecular dynamics simulation shows higher affinity and greater adsorption energy of PHSRN peptide with TiO2 surface due to the stronger hydrogen bonds formed by the serine and arginine residues with O ion of the substrate. Finally, by increasing O content in TiO2 surfaces through O ion-beam implantation, improving the cell adhesion, cell differentiation, and the subsequent biomineralization on titanium implant is realized. This study reveals the vital role of PHSRN in FN-mediated cell adhesion on implant surfaces, providing a promising new target for further tissue integration and implant success.

Model for surface-dependent factor XII activation: the roles of factor XII heavy chain domains.

Factor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged surfaces such as polymeric orthophosphate. FXII is composed of noncatalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation but restricts activation when FXII is not surface bound. From the N terminus, the heavy chain contains fibronectin type 2 (FN2), epidermal growth factor-1 (EGF1), fibronectin type 1 (FN1), EGF2, and kringle (KNG) domains and a proline-rich region. It shares this organization with its homolog, pro-hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains in FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog ε-aminocaproic acid. A model is proposed in which an ε-aminocaproic acid-sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.

Magnetic resonance molecular imaging of extradomain B fibronectin enables detection of pancreatic ductal adenocarcinoma metastasis.

Extradomain-B Fibronectin (EDB-FN) is an oncomarker that can be visualized with magnetic resonance molecular imaging (MRMI) to detect pancreatic ductal adenocarcinoma (PDAC) metastasis. In this study, we sought to assess the expression of EDB-FN in clinical samples of PDAC and to evaluate MRMI of PDAC metastasis with an EDB-FN-specific gadolinium-based contrast agent (MT218) in an orthotopic KPC-GFP-Luc mouse model. EDB-FN expression was evaluated in PDAC tissue samples through immunohistochemistry. RNA-Seq data obtained from the GEPIA2 project was evaluated to demonstrate EDB-FN expression in large patient cohorts. FLASH-3D MRI at 3 T of the KPC-GFP-Luc metastasis model was performed following injection of MT218. Tumor enhancement in MR images was correlated to postmortem distribution of KPC-GFP-Luc tumors using fluorescent and bright-field cryo-imaging and anatomical landmarks. EDB-FN immunohistochemical staining scores of human metastatic tumor stroma, (2.17 ± 0.271), metastatic tumor parenchyma (2.08 ± 0.229), primary tumor stroma (1.61 ± 0.26), and primary tumor parenchyma (1.61 ± 0.12) were significantly (p < 0.0001) higher than normal pancreas stroma (0.14 ± 0.10) and normal pancreas parenchyma (0.14 ± 0.14). EDB-FN mRNA expression in tumors is 4.98 log2(TPM + 1) and 0.18 log2(TPM + 1) in normal tissue (p < 0.01). A mouse model of EDB-FN rich PDAC metastasis exhibited T1-weighted contrast to noise (CNR) changes of 21.80 ± 4.34 in perimetastatic regions and 8.38 ± 0.79 in metastatic regions identified through cryo-imaging, significantly higher (p < 0.05) than CNR changes found in normal liver (-6.43 ± 0.92), mesentery (2.24 ± 0.92), spleen (-3.06 ± 2.38) and intestine (1.08 ± 2.15). We conclude that EDB-FN is overexpressed in metastatic and primary PDAC tumors and MRMI with MT218 enables the detection of metastatic and perimetastatic tissues.

Engineered protein-small molecule conjugates empower selective enzyme inhibition.

Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.