Fibronectin Fragments Research Articles

Fibronectin fragment-induced expression of matrix metalloproteinases is mediated by MyD88-dependent TLR-2 signaling pathway in human chondrocytes.

IntroductionFibronectin fragments (FN-fs) are increased in the cartilage of patients with osteoarthritis (OA) and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. We investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage catabolism via the Toll-like receptor (TLR)-2 signaling pathway in human chondrocytes.MethodsSmall interfering RNA was used to knock down TLR-2 and myeloid differentiation factor 88 (MyD88). TLR-2 was overexpressed in chondrocytes transfected with a TLR-2 expression plasmid. The expression levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were analyzed using quantitative real-time reverse transcription polymerase chain reactions, immunoblotting, or enzyme-linked immunosorbent assay. The effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways was investigated by immunoblotting.ResultsTLR-2, TLR-3, TLR-4, and TLR-5 mRNA were significantly overexpressed in OA cartilage compared with normal cartilage, whereas no significant difference of TLR-1 mRNA expression was found. 29-kDa FN-f significantly increased TLR-2 expression in human chondrocytes in a dose- and time-dependent manner. Knockdown of TLR-2 or MyD88, the latter a downstream adaptor of TLR-2, significantly inhibited 29-kDa FN-f-induced MMP production at the mRNA and protein levels. Conversely, TLR-2 overexpression led to enhanced MMP production by 29-kDa FN-f. In addition, TLR-2 knockdown apparently inhibited 29-kDa FN-f-mediated activation of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and p38, but not of c-Jun N-terminal kinase or extracellular signal-regulated kinase. Exposure to synovial fluid (SF) from affected joints of patients with OA elevated MMP-1, MMP-3, and MMP-13 expression markedly in primary chondrocytes without reducing cell viability. However, TLR-2 knockdown in chondrocytes significantly suppressed SF-induced MMP induction.ConclusionsOur data demonstrate that the MyD88-dependent TLR-2 signaling pathway may be responsible for 29-kDa FN-f-mediated cartilage catabolic responses. Our results will enhance understanding of cartilage catabolic mechanisms driven by cartilage degradation products, including FN-f. The modulation of TLR-2 signaling activated by damage-associated molecular patterns, including 29-kDa FN-f, is a potential therapeutic strategy for the prevention of cartilage degradation in OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0833-9) contains supplementary material, which is available to authorized users.

Bioactive compounds immobilized on Ti and TiNbHf: AFM-based investigations of biofunctionalization efficiency and cell adhesion

Implant materials require optimal biointegration, including strong and stable cell-material interactions from the early stages of implantation. Ti-based alloys with low elastic modulus are attracting a lot of interest for avoiding stress shielding, but their osseointegration potential is still very low. In this study, we report on how cell adhesion is influenced by linear RGD, cyclic RGD, and recombinant fibronectin fragment III8–10 coated on titanium versus a novel low-modulus TiNbHf alloy. The bioactive molecules were either physisorbed or covalently coupled to the substrates and their conformation on the surfaces was investigated with atomic force microscopy (AFM). The influence of the different bioactive coatings on the adhesion of rat mesenchymal stem cells was evaluated using cell culture assays and quantitatively analyzed at the single cell level by AFM-based single-cell force spectroscopy. Our results show that bioactive moieties, particularly fibronectin fragment III8–10, improve cell adhesion on titanium and TiNbHf and that the covalent tethering of such molecules provides the most promising strategy to biofunctionalize these materials. Therefore, the use of recombinant protein fragments is of high importance for improving the osseointegration potential of implant materials.

Platelet-Rich Plasma Attenuates 30-kDa Fibronectin Fragment–Induced Chemokine and Matrix Metalloproteinase Expression by Meniscocytes and Articular Chondrocytes

Background: Proteolytic fragments of fibronectin have catabolic effects on cartilage and menisci. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions, but it is unknown whether PRP influences fibronectin fragment (FN-f) procatabolic activity. Hypotheses: The procatabolic activity of FN-f on meniscocytes and articular chondrocytes is attenuated by cotreatment with PRP. Study Design: Controlled laboratory study. Methods: Human meniscocytes were treated with FN-f (30 kDa) with or without PRP coincubation, and gene expression was analyzed by complementary DNA microarray analysis. Validation of altered expression of known and novel chemokine and protease genes was undertaken by real-time polymerase chain reaction (RT-PCR) in articular chondrocytes and meniscocytes. Chemokine release was assayed by enzyme-linked immunosorbent assay, and intracellular pathway signaling was evaluated by Western immunoblotting. Results: Microarray analysis and RT-PCR showed increased expression of matrix metalloproteinase (MMP)1, MMP2, MMP3, MMP9, MMP13, interleukin (IL)–6, IL-8 (CXCL8), CCL5, CCL20, and CXCL10 chemokines in meniscocytes after treatment with FN-f. Upregulation of these genes was significantly attenuated by PRP. Similar results were seen with articular chondrocytes, although no changes in MMP2 or MMP9 levels were identified. PRP-induced suppression of gene expression was associated with activation of Akt and p44/p42. Conclusion: PRP treatment attenuates the 30-kDa FN-f–induced expression of a range of proinflammatory chemokines and MMPs, including IL-8, IL-6, CCL20, CCL5, CXCL10, MMP1, MMP3, and MMP13, by both meniscocytes and articular chondrocytes. Clinical Relevance: These observations provide support for the use and further trials of PRP in management of cartilage and meniscal injuries.

Abstract 2378: The interaction of epithelial α5β1 integrin and stromal fibronectin is a candidate seed-and-soil mechanism in prostate cancer and bone metastases

Abstract Background: Morbidity and mortality from prostate cancer relates principally to its unique proclivity to disseminate with near-exclusivity within the hematopoietic bone marrow microenvironment. Prostate cancer cells compete for the bone marrow niche with CD45+ hematopoietic stem cells (Shiozawa, 2011). Several lines of evidence have implicated mesenchymal stem cells (MSCs) as key architects of the hematopoietic niche (Nakamura-Ishizu, 2013). The molecular mechanisms that determine the tropism of prostate cancer cells to the MSC-regulated niche are undetermined. We hypothesized that specific tropic interactions between human MSCs and prostate cancer cells contribute toward the efficient colonization of the bone marrow. Methods and Results: Bone-derived prostate cancer cells (PC-3) exhibit a strong migratory response when placed in co-culture with human MSCs. Serum-free conditioned media from MSCs similarly induces adhesion, migration and invasion of PC-3 cells, but not proliferation. Heat-inactivation and trypsin digest suggested the bioactive principle was a protein. Size-fractionation by spin-columns determined the bioactivity to reside in the >100kD fraction. Size-exclusion chromatography (SEC) recovered peak activity in a 440kD fraction containing thrombospondin-1 (TSP1). TSP1 put-back assays did not reproduce bioactivity. Further purification of the TSP-1 containing high-molecular weight fraction of the MSC secretome with heparin-affinity chromatography recovered bioactivity with highly restricted bands on polyacrylamide gel electrophoresis, determined by mass spectroscopy to be fragments of fibronectin (FN). Put-back experiments with FN recovered bioactivity. Monospecific antibodies to FN blocked adhesion. Neutralizing monoclonal antibodies to FN receptors, α5 and β1 integrins, but not α4, αv, α6 or β3 integrins blocked the adhesion and migration of prostate cancer cells cells to the MSC secretome and FN. Conclusions: The affinity of bone-derived human prostate cancer cells to the MSC secretome is mediated by fibronectin acting via the α5 and β1 integrins. Human prostate cancer cells isolated from clinical bone marrow specimens express α5β1 integrin in high-frequency (Putz, 1999) and fibronectin is richly expressed in the bone microenvironment (Van der Welde-Zimmermann 1997). The FN-α5β1 interaction requires translational study as a candidate molecular target for therapy, specific to prostate cancer and hematopoietic marrow interaction, the lethal phenotype of the disease. Citation Format: Raghav Joshi, Edi Goihberg, Monika Pilichowska, Paul Mathew. The interaction of epithelial α5β1 integrin and stromal fibronectin is a candidate seed-and-soil mechanism in prostate cancer and bone metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2378. doi:10.1158/1538-7445.AM2015-2378

Abstract 3137: A novel expansion method for functional natural killer cells and its clinical application

Abstract Background: Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. But, it is difficult to generate the sufficient scale and purity of NK cells, and reliable methods to produce large number of functional NK cells have not been established yet. We have developed novel clinical-grade NK cells expansion method to produce the high purity, large scale and functional NK cells using a combination of recombinant human fibronectin fragment (RetroNectin®) -induced T-cells (RN-T cells), OK-432 and IL-2. We subsequently conducted a Phase 1 clinical study to evaluate the safety and efficacy of this NK cell therapy. In this paper, we address the characteristics of the NK cells elicited by this method and the results of immune monitoring in the clinical trial. Methods: To confirm the significance of RN-T cells as stimulator, we compared the stimulation effects on NK cells between RN-T cells and aCD3-T cells, which stimulated by anti-CD3 mAb only. Next, we analyzed expanded NK cells from PBMCs obtained from 31 cancer patients to verify the efficacy of this method. In the Phase 1 trial, patients with unresectable digestive cancer were enrolled. They received weekly intravenous administration of autologous NK cells elicited by the novel method three times to assess the safety of the number of adoptive cells at 0.5 × 109(cohort1), 1 × 109(cohort 2) and 2 × 109 cells (cohort 3) per dose. The phenotype and cytotoxicity of expanded NK cells were analyzed. For immune monitoring, cytotoxicity of PBMCs and whole blood cytokine levels were examined following NK cell infusion. Results: The stimulation by RN-T cells could induce preferable NK cell proliferation rather than the one by aCD3-T. As results of 31 cancer patients, 688±76-fold expansion was achieved in this system with PBMCs, and the NK cell populations were highly purified (84.7±3.6%) and highly expressed functional markers such as NKG2D (97.3±0.6%) and CD16 (96.8±0.7%). In the Phase 1 clinical trial, no NK cell infusion related severe or unexpected toxicities were observed. The response rate and the disease control rate in 10 per protocol patients were 0% and 50.0%, respectively. Although no clinical responses were observed, the cytotoxicity of PBMCs against K-562 targets increased in most patients (80%). The average of cytotoxic activity of PBMCs increased more than two times after the NK cell infusion. Conclusion: Although no patients receiving the NK cell therapy alone experienced a clinical response, adoptive immunotherapy of the NK cells elicited by the novel method is expected to exert considerable ADCC activity in vivo because of their high expression of CD16. Now, we are conducting clinical trial in which we explore the combination of this novel NK therapy with IgG1 antibodies such as trastuzumab and cetuximab. We also address the ongoing clinical trial in this paper. Citation Format: Takeshi Ishikawa, Naoyuki Sakamoto, Tetsuya Okayama, Kaname Oka, Satoshi Kokura, Mitsuko Ideno, Akiko Kato, Tatsuji Enoki, Masanari Kitagawa, Junichi Mineno, Tomoyo Yasuda, Toshifumi Doi, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. A novel expansion method for functional natural killer cells and its clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2015-3137

Simple coating with fibronectin fragment enhances stainless steel screw osseointegration in healthy and osteoporotic rats

Metal implants are widely used to provide structural support and stability in current surgical treatments for bone fractures, spinal fusions, and joint arthroplasties as well as craniofacial and dental applications. Early implant-bone mechanical fixation is an important requirement for the successful performance of such implants. However, adequate osseointegration has been difficult to achieve especially in challenging disease states like osteoporosis due to reduced bone mass and strength. Here, we present a simple coating strategy based on passive adsorption of FN7-10, a recombinant fragment of human fibronectin encompassing the major cell adhesive, integrin-binding site, onto 316-grade stainless steel (SS). FN7-10 coating on SS surfaces promoted α5β1 integrin-dependent adhesion and osteogenic differentiation of human mesenchymal stem cells. FN7-10-coated SS screws increased bone-implant mechanical fixation compared to uncoated screws by 30% and 45% at 1 and 3 months, respectively, in healthy rats. Importantly, FN7-10 coating significantly enhanced bone-screw fixation by 57% and 32% at 1 and 3 months, respectively, and bone-implant ingrowth by 30% at 3 months compared to uncoated screws in osteoporotic rats. These coatings are easy to apply intra-operatively, even to implants with complex geometries and structures, facilitating the potential for rapid translation to clinical settings.

Building a better infarct: Modulation of collagen cross-linking to increase infarct stiffness and reduce left ventricular dilation post-myocardial infarction

Matrix metalloproteinase-9 (MMP-9) deletion attenuates collagen accumulation and dilation of the left ventricle (LV) post-myocardial infarction (MI); however the biomechanical mechanisms underlying the improved outcome are poorly understood.The aim of this study was to determine the mechanisms whereby MMP-9 deletion alters collagen network composition and assembly in the LV post-MI to modulate the mechanical properties of myocardial scar tissue. Adult C57BL/6J wild-type (WT; n=88) and MMP-9 null (MMP-9−/−; n=92) mice of both sexes underwent permanent coronary artery ligation and were compared to day 0 controls (n=42). At day 7 post-MI, WT LVs displayed a 3-fold increase in end-diastolic volume, while MMP-9−/− showed only a 2-fold increase (p<0.05). Biaxial mechanical testing revealed that MMP-9−/− infarcts were stiffer than WT infarcts, as indicated by a 1.3-fold reduction in predicted in vivo circumferential stretch (p<0.05). Paradoxically, MMP-9−/− infarcts had a 1.8-fold reduction in collagen deposition (p<0.05). This apparent contradiction was explained by a 3.1-fold increase in lysyl oxidase (p<0.05) in MMP-9−/− infarcts, indicating that MMP-9 deletion increased collagen cross-linking activity. Furthermore, MMP-9 deletion led to a 3.0-fold increase in bone morphogenetic protein-1, the metalloproteinase that cleaves pro-collagen and pro-lysyl oxidase (p<0.05) and reduced fibronectin fragmentation by 49% (p<0.05) to enhance lysyl oxidase activity. We conclude that MMP-9 deletion increases infarct stiffness and prevents LV dilation by reducing collagen degradation and facilitating collagen assembly and cross-linking through preservation of the fibronectin network and activation of lysyl oxidase.

Interaction Between Fibronectin Fragments and Immunoglobulin G in Gingival Crevicular Fluid of Patients with Periodontal Disease/ Kompleksi fibronektinskih fragmenata i imunoglobulina G u gingivalnoj tečnosti osoba obolelih od parodontopatije

SUMMARYIntroduction Fibronectin (FN) can interact with immunoglobulin G (IgG) molecules affecting the process of physiological elimination and causing abnormal deposition of immune complexes. The aim of the study was to analyze interaction between FN fragments and IgG molecules with different glycosylation profiles in gingival crevicular fluid (GCF) of patients with periodontal disease and healthy controls.Material and Methods The study included 30 patients with moderate and advanced periodontitis and 22 healthy subjects. IgG and FN content in GCF were measured as well as the presence of FN and galactose expression on IgG molecules.Results IgG content in GCF was five times higher in patients with moderate (p&lt;0.01) and eight time higher in patients with advanced periodontitis (p&lt;0.001) compared to healthy subjects. Also, hypogalactosylated forms of IgG were found in higher concentration in GCF of patients with advanced periodontitis compared to moderate periodontitis and healthy subjects (p&lt;0.05). FN fragments of molecular mass 48 - 53 kDa were the most commonly found fragments in all three groups. Furthermore, in patients with advanced periodontitis, fibronectin fragments were attached to IgG molecules.Conclusion IgG and FN fragments form complexes in GCF in patients with periodontal disease and healthy subjects

Interrogating protonated/deuterated fibronectin fragment layers adsorbed to titania by neutron reflectivity and their concomitant control over cell adhesion

The fibronectin fragment, 9th-10th-type III domains (FIII9-10), mediates cell attachment and spreading and is commonly investigated as a bioadhesive interface for implant materials such as titania (TiO2). How the extent of the cell attachment-spreading response is related to the nature of the adsorbed protein layer is largely unknown. Here, the layer thickness and surface fraction of two FIII9-10 mutants (both protonated and deuterated) adsorbed to TiO2 were determined over concentrations used in cell adhesion assays. Unexpectedly, the isotopic forms had different adsorption behaviours. At solution concentrations of 10 mg l(-1), the surface fraction of the less conformationally stable mutant (FIII9'10) was 42% for the deuterated form and 19% for the protonated form (fitted to the same monolayer thickness). Similarly, the surface fraction of the more stable mutant (FIII9'10-H2P) was 34% and 18% for the deuterated and protonated forms, respectively. All proteins showed a transition from monolayer to bilayer between 30 and 100 mg l(-1), with the protein longitudinal orientation moving away from the plane of the TiO2 surface at high concentrations. Baby hamster kidney cells adherent to TiO2 surfaces coated with the proteins (100 mg l(-1)) showed a strong spreading response, irrespective of protein conformational stability. After surface washing, FIII9'10 and FIII9'10-H2P bilayer surface fractions were 30/25% and 42/39% for the lower/upper layers, respectively, implying that the cell spreading response requires only a partial protein surface fraction. Thus, we can use neutron reflectivity to inform the coating process for generating bioadhesive TiO2 surfaces.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities

We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage.

Cysteine-mediated redox regulation of cell signaling in chondrocytes stimulated with fibronectin fragments

Objective Oxidative posttranslational modifications of intracellular proteins can potentially regulate signaling pathways relevant to cartilage destruction in arthritis. In this study, oxidation of cysteine residues to form sulfenic acid (S-sulfenylation) was examined in osteoarthritic (OA) chondrocytes and investigated in normal chondrocytes as a mechanism by which fragments of fibronectin (FN-f) stimulate chondrocyte catabolic signaling. Methods Chondrocytes isolated from OA and normal human articular cartilage were analyzed using analogs of dimedone that specifically and irreversibly react with protein S-sulfenylated cysteines. Global S-sulfenylation was measured in cell lysates with and without FN-f stimulation by immunoblotting and in fixed cells by confocal microscopy. S-sulfenylation in specific proteins was identified by mass spectroscopy and confirmed by immunoblotting. Src activity was measured in live cells using a fluorescence resonance energy transfer biosensor. Results Proteins in chondrocytes isolated from OA cartilage were found to have elevated basal levels of S-sulfenylation relative to those of chondrocytes from normal cartilage. Treatment of normal chondrocytes with FN-f induced increased levels of S-sulfenylation in multiple proteins, including the tyrosine kinase Src. FN-f treatment also increased the levels of Src activity. Pretreatment with dimedone to alter S-sulfenylation function or with Src kinase inhibitors inhibited FN-f–induced production of matrix metalloproteinase 13. Conclusion These results demonstrate for the first time the presence of oxidative posttranslational modification of proteins in human articular chondrocytes by S-sulfenylation. Due to the ability to regulate the activity of a number of cell signaling pathways, including catabolic mediators induced by fibronectin fragments, S-sulfenylation may contribute to cartilage destruction in OA and warrants further investigation.

Granzyme B Mediates Both Direct and Indirect Cleavage of Extracellular Matrix in Skin After Chronic Low‐Dose Ultraviolet Light Irradiation

Extracellular matrix (ECM) degradation is a hallmark of tissue aging, as well as many other chronic inflammatory diseases that can lead to a loss of function. Granzyme B (GzmB), a serine protease that is expressed by a variety cells, has been shown to accumulate in the extracellular space during chronic inflammation and cleave a number of proteins. Using a chronic low‐grade UV‐irradiation murine model, we hypothesized that GzmB contributes to skin damage and ECM degradation through processes involving both direct ECM cleavage and indirect ECM cleavage through the induction of other proteinases. Wild‐type and GzmB‐knockout (KO) mice were repeatedly exposed to minimal erythemal doses of solar simulated UV‐irradiation for up to 20 weeks. GzmB expression was significantly increased in wild‐type UV‐treated skin compared to non‐irradiated controls. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density. GzmB‐KO mice were also protective against the loss of fibronectin (FN) in vivo, and FN fragments were released from the cell‐derived matrix of cultured fibroblasts after GzmB treatment. GzmB‐mediated FN fragments increased the release of metalloproteinase (MMP)‐1 and MMP‐3 from fibroblasts. Furthermore, GzmB cleavage of decorin predisposed collagen fibrils more susceptible to attack by MMP‐1. Finally, GzmB reduced the barrier function of a monolayer of keratinocytes in vitro as measured by electrical impedance. Collectively, these findings indicate a significant role for GzmB in ECM degradation, which may have implications in many aged‐related dermatological indications.

Extracellular Matrix Fibronectin Attenuates PDGF‐Induced Intracellular Calcium Release

The extracellular matrix (ECM) has structural and signaling roles within tissues. Fibronectin is an ECM protein that is secreted by fibroblasts in a protomeric form and assembled into a fibrillar matrix by a cell‐mediated process termed fibronectin matrix assembly. ECM proteins affect growth factor signaling. Platelet derived growth factor (PDGF) is a major mitogen for fibroblasts that promotes proliferation and migration. The objective of this study was to determine the effect of ECM fibronectin on the response of fibroblasts to PDGF. The response of cells to PDGF was quantified by measuring intracellular calcium release in Fluo‐4 loaded cells. Calcium release was measured in fibronectin‐null mouse embryonic fibroblasts, allowing for control over the amount of fibronectin to which cells were exposed and the initiation of fibronectin matrix assembly. Results showed that in the absence of fibronectin, addition of 50 ng/mL PDGF to cells transiently increased intracellular calcium concentration. In contrast, incubation of cells with 25 nM fibronectin for 20 hr significantly attenuated the PDGF‐induced increase in calcium release. Fibronectin‐mediated attenuation of PDGF‐induced calcium release was blocked by an inhibitor of fibronectin matrix assembly, indicating that the ECM form of fibronectin is required for fibronectin‐mediated attenuation of PDGF signaling. A fibronectin fragment engineered to mimic the ECM form of fibronectin also attenuated PDGF‐induced calcium release. Attenuation of PDGF‐induced calcium release was not due to downregulation of PDGF receptor β expression. This study demonstrates that fibronectin matrix assembly attenuates PDGF‐induced intracellular calcium release by an as‐yet unidentified mechanism.Supported by: NIH/NHLBI: T32 HL066988

Proteolysis of decellularized extracellular matrices results in loss of fibronectin and cell binding activity

Excessive inflammation in the chronic wound bed is believed to result in increased fibronectin (FN) proteolysis and poor tissue repair. However, FN fragments can prime the immune response and result in higher protease levels. The reciprocity between FN proteolysis and inflammation makes it challenging to determine the specific contribution of FN proteolysis in the extracellular matrix (ECM) on tissue responses. We studied the impact of proteolysis of decellularized extracellular matrices (dECMs) obtained from NIH 3T3 mouse fibroblasts on FN level and activity. The dECMs were treated with α chymotrypsin and proteolysis was stopped at different time points. The protease solution was obtained, the remaining dECM was scrapped and examined by immunoblotting and Bicinchoninic Acid assays. Fibronectin was 9.4 ± 1.8% of the total protein content in the dECM but was more susceptible to proteolysis. After 15 min of protease treatment there was a 67.6% and 11.1% decrease in FN and total protein, respectively, in the dECMs. Fibronectin fragments were present both in the proteolysis solution and in the dECM. Cell adhesion, spreading and actin extensions on dECMs decreased with increasing proteolysis time. Interestingly, the solutions obtained after proteolysis of the dECMs supported cell adhesion and spreading in a time dependent manner, thus demonstrating the presence of FN cell binding activity in the protease solution of dECMs. This study demonstrates the susceptibility of FN in the ECM to proteolysis and the resulting loss of cell adhesion due to the decrease of FN activity and places weight on bioengineering strategies to stabilize FN against proteolysis.

Bivalent Ligation of the Collagen-binding Modules of Fibronectin by SFS, a Non-anchored Bacterial Protein of Streptococcus equi

SFS is a non-anchored protein of Streptococcus equi subspecies equi that causes upper respiratory infection in horses. SFS has been shown to bind to fibronectin (FN) and block interaction of FN with type I collagen. We have characterized interactions of a recombinant 60-mer polypeptide, R1R2, with FN. R1R2 contains two copies of collagen-like 19-residue repeats. Experiments utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies located the binding site to (8-9)FNI modules of the gelatin-binding domain. Fluorescence polarization and competitive enzyme-linked assays demonstrated that R1R2 binds preferentially to compact dimeric FN rather than monomeric constructs containing (8-9)FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind (2-5)FNI in addition to (8-9)FNI, R1R2 did not cause conformational extension of FN as assessed by a conformationally sensitive antibody. Equilibrium and stopped-flow binding assays and size exclusion chromatography were compatible with a two-step binding reaction in which each of the repeats of R1R2 interacts with one of the subunits of dimeric FN, resulting in a stable complex with a slow koff. In addition to not binding to type I collagen, the R1R2·FN complex incorporated less efficiently into extracellular matrix than free FN. Thus, R1R2 binds to FN utilizing features of compact soluble FN and in doing so interferes with the organization of the extracellular matrix. A similar bivalent binding strategy may underlie the collagen-FN interaction.

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation.

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases.

Engineering of CD19-CAR T Cells from Non-Hodgkin Lymphoma Patients in a Closed System in Combination with Retronectin/OKT3 Stimulation

Background; Adoptive immunotherapy with chimeric antigen receptor (CAR) gene transduced CD19-CAR T cells, which are engineered to express extracellular single-chain immunoglobulin variable fragments to CD19, linked to cytoplasmic T cell activation domains including CD3-ζ, showed remarkable therapeutic benefits toward CD19+ B cell malignancies including acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma (B-NHL). For clinical setting, the phenotype of manufactured CAR T cells is an important factor; especially less differentiated T cells are anticipated to provide a long-lasting immune reconstitution. Furthermore, in order to avoid the risk of technical error and contamination during T cell manufacturing process, a closed system needs to be established. In this study, we addressed these issues and have established a novel CD19-CAR T cell manufacturing method from a small amount of blood in a closed system for clinical trial to treat patients with B-NHL.Methods; Peripheral blood was obtained from B-NHL patient volunteers and healthy donor volunteers who gave their written informed consents. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of blood using Ficoll-Paque PREMIUM density gradient centrifugation. PBMCs were stimulated in a plastic bag pre-coated with anti-CD3 monoclonal antibody (OKT3) and recombinant fibronectin fragment (RetroNectin®; RN). Following four days of stimulation, stimulated T cells were transferred into a 215 cm2 plastic bag pre-loaded with SFG-1928z retroviral vector (Brentjens et al., Clin Cancer Res. 2007) onto RN-coated substratum with low-temperature shaking (RBV-LTS method; Dodo et al., PLoS ONE, 2014). After one hour incubation, the bag was flipped over to facilitate more efficient utilization of the retroviral vector adsorbed on both top and bottom surfaces of the bag and further incubated. On Day 5, the transduction procedure was repeated, and the cells were transferred into 640 cm2 plastic bags until Day 10-14 for expansion. Closed system liquid handling was managed in all processes of manipulating T cells for stimulation, transduction, expansion and final product formulation.Results; We have previously reported that the fold expansion of T cells under stimulation with RN together with OKT3 enhanced cell proliferation while preserving the naïve phenotype of T cells in comparison to stimulation with OKT3 alone or OKT3 and anti-CD28 monoclonal antibody co-stimulation. Although B-NHL patients’ T cells showed much lower fold expansion compared to healthy donors’ T cells, 4/7 patients’ CAR T cells reached their target dose of 1 x 106 cells/kg from 30 ml of blood on Day 10. For the other 3 patients, 70-150 ml blood was estimated to be required to reach their target dose. The delay in proliferations was marked in B-NHL patients’ T cells compared to healthy donors’ T cells by Day 5, but B-NHL patients’ T cells represented significant catch-up growth, which was superior to healthy donor T cells during Day 7-14. Gene transfer efficiency of patients’ T cells (N = 7, 17.9 ± 4.7%) was equivalent to that of healthy donors’ T cells (N = 5, 22.2 ± 5.3%), and CAR T cells showed potent anti-tumor reactivity with cytokine productions against CD19 positive Raji cells in vitro. Comparing to CD3/CD28 beads stimulation method, RN/OKT3 stimulation method showed equivalent expansion. Furthermore, RN/OKT3 stimulated T cells preserved higher proportion of CD8+/CCR7+/CD45RA+/CD62L+ naïve phenotype T cells (43.6% in healthy donor and 30.9% in patient donor) compared to CD3/CD28 beads stimulated T cells (22.8% in healthy donor and 11.7% in patient donor).Conclusions; With our novel closed system manufacturing method utilizing RN/OKT3 stimulation combined with RBV-LTS transduction from a small amount of blood of B-NHL patients, we are able to manufacture a sufficient number of CAR T cells maintaining higher proportion of naïve phenotype, which is expected to improve the efficacy of adoptive immunotherapy. In our cell manufacturing, patients are not required to undergo leukapheresis, which is more invasive to patients. Based on our results, we employed our novel manufacturing method for the phase I/II clinical trial to treat patients with relapsed/refractory CD19+ B-NHL (clinicaltrials.gov, identifier; NCT02134262). DisclosuresChono:Takara Bio Inc.: Employment. Tahara:Takara Bio Inc.: Employment. Nukaya:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Membership on an entity’s Board of Directors or advisory committees. Tsukahara:Takara Bio Inc.: Research Funding. Ohmine:Takara Bio Inc.: Research Funding. Ozawa:Takara Bio Inc.: Research Funding. Takesako:Takara Bio Inc.: Membership on an entity’s Board of Directors or advisory committees.

Abstract 15972: Fibronectin is Essential for Mesendoderm Induction During Cardiac Differentiation of Human Pluripotent Stem Cells

Extracellular matrix (ECM) plays important roles in development, and we have previously demonstrated a matrix sandwich protocol for efficient cardiac differentiation of human pluripotent stem cells (hPSCs). The purpose of the present study was to identify the key ECM proteins required to initiate cardiogenic differentiation of hPSCs and define their mechanism of action. We tested purified ECM proteins including human laminin (LN111, LN521), fibronectin (FN) and collagen IV in the matrix sandwich protocol, and found FN promoted efficient cardiac differentiation. To determine if FN is essential for the early stages of cardiogenesis we tested three FN blockers: FUD peptide that binds the N-terminal 70-kDa fragment of FN, anti-human FN antibody clone10 which recognizes FN domain III2, and anti-human FN antibody clone 3E3 that binds on the cell attachment domain. All three blockers showed concentration-dependent inhibition of cardiac differentiation of hPSCs when added at day 0 for 24 hours, measured by flow cytometry for cTnT+ cells in the Matrigel/Matrigel (bottom/overlay) and FN/FN sandwich culture as well as in the small molecule GiWi protocol. Furthermore, clone10 showed significant inhibition of cardiac differentiation even in the absence of adding exogenous FN due to the robust endogenous expression of FN by the hPSCs. Gene expression analysis with qRT-PCR demonstrated that blocking FN by clone10 caused a significant suppression of gene expression related to: 1) epithelial to mesenchymal transition, e.g. SNAIL1, SNAIL2, GSC, VIM, CDH2 and FN1; 2) Mesendoderm e.g. T, MIXL1, SOX17 and precardiac mesoderm of MESP1; 3) Cardiac transcription factors ISL1 and NKX2.5. To investigate the cellular signaling activated by FN, we tested a blocking antibody (clone P5D2) to the major FN receptor, integrin β1, and found that P5D2 produced a concentration-dependent inhibition of cardiogenesis in multiple hPSC lines. Furthermore, the integrin-linked kinase (ILK) inhibitor, Cpd22, inhibited cardiac differentiation in a concentration-dependent fashion as well when added on day 0. We conclude that FN, acting via integrin β1-activated ILK signaling, is essential to mesendoderm formation enabling cardiogenesis of hPSCs.

Presentation of fibronectin fragments using affinity protein interactions for enhanced retention and function

We present a protein immobilization system, based on the Src Homology 3 (SH3) affinity domain, allowing for a transient interaction between a fibronectin ligand and a biomaterial surface. This strategy leads to enhanced retention of the fibronectin fragment over adsorbed fibronectin, and increased cellular proliferation and motility over either covalently immobilized or adsorbed fibronectin. The results indicate that intermediate affinity protein immobilization could provide benefits for tissue engineering beyond the traditional immobilization techniques, adsorption or covalent attachment.

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

BBK32 is a fibronectin (FN)-binding protein expressed on the cell surface of Borrelia burgdorferi, the causative agent of Lyme disease. There is conflicting information about where and how BBK32 interacts with FN. We have characterized interactions of a recombinant 86-mer polypeptide, "Bbk32," comprising the unstructured FN-binding region of BBK32. Competitive enzyme-linked assays utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies showed that Bbk32 binding involves both the fibrin-binding and the gelatin-binding domains of the 70-kDa N-terminal region (FN70K). Crystallographic and NMR analyses of smaller Bbk32 peptides complexed, respectively, with (2-3)FNI and (8-9)FNI, demonstrated that binding occurs by β-strand addition. Isothermal titration calorimetry indicated that Bbk32 binds to isolated FN70K more tightly than to intact FN. In a competitive enzyme-linked binding assay, complex formation with Bbk32 enhanced binding of FN with mAbIII-10 to the (10)FNIII module. Thus, Bbk32 binds to multiple FN type 1 modules of the FN70K region by a tandem β-zipper mechanism, and in doing so increases accessibility of FNIII modules that interact with other ligands. The similarity in the FN-binding mechanism of BBK32 and previously studied streptococcal proteins suggests that the binding and associated conformational change of FN play a role in infection.