Fibronectin Peptide Research Articles

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1shared epitope alleles

IntroductionFibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.MethodsCitrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.ResultsFive citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).ConclusionsFibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.

Targeting focal adhesion assembly by ethoxyfagaronine prevents lymphoblastic cell adhesion to fibronectin.

Background: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag), a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V). Methods: Phosphorylation of fak and pyk2 were evaluated by immunoblotting. Labelled proteins were localized by confocal microscopy. PI 3-kinase activity was evaluated by in vitro kinase assay. Results: Subtoxic concentration of etxfag reduced L1210 cell adhesion to FN/V dependently of β1 integrin engagement. Etxfag impaired FN-dependent formation of β1 clustering without modifying β1 expression at the cell membrane. This was accompanied by a decrease of focal adhesion number, a diminution of fak and pyk2 phosphorylation at Tyr-576, Tyr-861 and Tyr-579, respectively leading to their dissociations from β1 integrin and inhibition of PI 3-kinase activity. Etxfag also induced a cell retraction accompanied by a redistribution of phosphorylated fak and pyk2 in the perinuclear region and lipid raft relocalization. Conclusion: Through its anti-adhesive potential, etxfag, combined with conventional cytotoxic drugs could be potentially designed as a new anti-leukemic drug.

Targeting Focal Adhesion Assembly by Ethoxyfagaronine Prevents Lymphoblastic Cell Adhesion to Fibronectin

Background: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag), a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V).Methods: Phosphorylation of fak and pyk2 were evaluated by immunoblotting. Labelled proteins were localized by confocal microscopy. PI 3-kinase activity was evaluated byin vitrokinase assay.Results: Subtoxic concentration of etxfag reduced L1210 cell adhesion to FN/V dependently of β1 integrin engagement. Etxfag impaired FN-dependent formation of β1 clustering without modifying β1 expression at the cell membrane. This was accompanied by a decrease of focal adhesion number, a diminution of fak and pyk2 phosphorylation at Tyr-576, Tyr-861 and Tyr-579, respectively leading to their dissociations from β1 integrin and inhibition of PI 3-kinase activity. Etxfag also induced a cell retraction accompanied by a redistribution of phosphorylated fak and pyk2 in the perinuclear region and lipid raft relocalization.Conclusion: Through its anti-adhesive potential, etxfag, combined with conventional cytotoxic drugs could be potentially designed as a new anti-leukemic drug.

Towards Defining An Optimal Conditioning Regimen for Stem Cell Gene Therapy In Fanconi Anemia.

AbstractAbstract 1478One of the major hurdles for gene therapy (GT) for inherited bone marrow (BM) disorders, including Fanconi anemia (FA), is poor engraftment of transduced cells. Because FA cells are hypersensitive to cross-linking agents and thus FA patients are very sensitive to chemotherapy with alkylating agents and irradiation, conditioning with these agents has been a concern. Although when used at a lower dose, cyclophosphamide appears to be well tolerated in the transplant setting. Here we have evaluated whether a commonly used drug for conditioning in stem cell transplantation, cyclophosphamide, could be replaced by non alkylating agents, fludarabine (F) or cytarabine (A), and have compared the results to no preparation or cyclophosphamide (Cy)-only preparation in the FancA-/- mutant mouse. We performed two types of experiments: (1) transplant of heterozygous FancA+/− mouse BM cells transduced with a lentiviral vector encoding green fluorescence protein (RRLsincPPT-PGK-GFP) into homozygous FancA-/- recipients, and (2) transplant of homozygous FancA-/- deficient cells transduced with the lentiviral vector RRLsincPPT-PGK-FA encoding FancA, which has been developed for a clinical trial of GT for Fanconi anemia, into homozygous FancA-/- recipients. In both experiments, female donor BM cells were transduced at an MOI of 10 with G-CSF, SCF, Flt3 ligand, and TPO at 100 ng/ml each, on recombinant fibronectin peptide CH296. Experiment 1 compared Cont (no chemotherapy), A+F (A-300 mg/kg dailyX3, F-200 mg/kg daily X3), A (1000 mg/kg), and Cy (120 mg/kg). The Cy group displayed the most efficient engraftment with 3/5 mice exhibiting circulating GFP+ white blood cells (WBCs) by day +35, compared to 2/5 mice in the other groups, whereas for the Cont and A+F group, marking was <1% of WBCs. All mice received post-transplant Cy (120mg/kg) on day +45 as a method of inducing selection of FancA +/− cells. Following selection, WBC marking increased across all groups, with the highest in the Cy group at 77.4%, 24.1% and 77% GFP+ WBCs in the 3 engrafted mice. Methylcellulose colonies grew from BM from all mice, with many more large colonies, >2mm reflecting more primitive multipotent colony forming cells (CFCs), observed for the A+F or A groups (p=0.0006 and p=0.0051, respectively). Mice that exhibited high level marking by flow cytometry for GFP and colony PCR for presence of provirus, also exhibited more CFCs in 10 or 20 nM mitomycin C (MMC). Genotyping PCR confirmed that colonies positive for provirus were heterozygous, and cytogenetic analysis of BM from male recipient mice confirmed male:female chimerism. Importantly, no cytogenetic abnormalities were observed in mice treated with Cy. Based on these results, the following groups were included in experiment 2: Cont (no chemotherapy), A (1000 mg/kg), and Cy (120 mg/kg). In this experiment, all marking was determined by PCR for proviral sequences, as the vector developed for clinical use does not contain GFP. By day +35, 3/5 mice in the control group, 3/5 mice in the A group and 4/5 mice in the Cy group were evaluable for marking. All four mice in the Cy group displayed efficient engraftment of gene-corrected cells (WBC marking range of 0.14 to 0.53 provirus copies per cell), while no mice in the control group and only one mouse in the A group displayed detectable marking (0.05 provirus copies per cell on day +22). As in experiment 1, following selection, WBC marking increased across all groups, with the Cy group again displaying the most prominent marking (5/5 mice, range of 0.42 to 1.3 provirus copies per cell). Colony plating efficiency from BM at day +72 was equivalent across all groups in 0 nM MMC; however, in 20 nM MMC, there was a significant difference between the Cy group (44.6 ± 28.3 colonies/plate) as compared to the Cont (4.8 ± 1.7, p=0.014) or A (5.4 ± 6, p=0.031) groups. Similarly, for day +72 BM cells assessed in liquid culture at 96 hr, the Cy group had the highest cell numbers for 0, 10 and 20 nM MMC, as compared to the Cont (p=0.0002 for 20 nM MMC) or A group (p=0.0015 for 20 nM MMC). Colony PCR confirmed BM marking in transplanted mice and corresponded with MMC sensitivity and male:female chimerism. Conclusion: Cyclophosphamide is a superior preparative regimen, compared to fludarabine or cytarabine, with respect to engraftment of lentivirus-transduced cells and optimal functional correction in FancA-deficient mice. Disclosures:No relevant conflicts of interest to declare.

Novel bioactive scaffolds with fibronectin recognition nanosites based on molecular imprinting technology

AbstractBiomimetic materials for application in the field of tissue engineering are usually obtained through covalent bonding between the polymer backbone and the bioactive molecules. A totally new approach, proposed for the first time by our research group, for the creation of advanced synthetic support structures for cell adhesion and proliferation is represented by molecular imprinting (MI) technology. In this article, we describe the synthesis and characterization of molecularly imprinted polymers with recognition properties toward a fibronectin peptide sequence and their application as functionalization structures. Polymers, in the form of densely fused microgel particles, were obtained by precipitation polymerization. The imprinted particles showed good performance in terms of recognition capacity and quantitative rebinding; moreover, the epitope effect was observed, with the particles able to recognize and rebind not only the specific peptide sequence but also a larger fibronectin fragment. The cytotoxicity tests showed normal vitality in C2C12 myoblasts cultured in a medium that was put in contact with the imprinted particles. Therefore, imprinted particles were used to functionalize synthetic polymeric films by deposition on their surface. The deposition of the imprinted particles did not alter their specific recognition and rebinding behavior. The most remarkable result was obtained by the biological characterization: in fact, the functionalized materials appeared able to promote cell adhesion and proliferation. These results are very promising and suggest that MI can be used as an innovative functionalization technique to prepare bioactive scaffolds with an effective capacity for improving tissue regeneration. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34+ cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor (SCF), and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC exhibited increased colony formation compared to 21% oxygen without NAC (P < 0.03), demonstrated increased resistance to mitomycin C compared to green fluorescent protein (GFP )-transduced controls (P < 0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

A Fibronectin Peptide‐Coupled Biopolymer Nanofibrous Matrix to Speed Up Initial Cellular Events

AbstractDegradable polymer nanofibers produced by electrospinning are attractive for use in cell culture and tissue repair. However, the hydrophobicity and initial poor cell adhesion of synthetic polymers have limited their use in tissue regeneration. Herein, the surface of a poly(lactide‐co‐caprolactone) Arg‐Gly‐Asp sequence of nanofiber was tailored with a fibronectin peptide (FN10), which was designed to retain the central cell‐binding domain. The electrospun nanofibers are first treated with an alkaline solution to reveal the carboxyl groups on the surface, which is followed by coupling with an FN10 solution in conjunction with a carbodiimide‐based agent. Peptide coupling occurs effectively with saturation within 1 h, and the coupled peptide maintains its stability for several days. The peptide‐coupled nanofibers show significant improvements in initial cell adhesion and spreading compared with the untreated one, confirming the role of the FN10 peptide in the initial cell events. This methodology may be useful in tailoring the surface of polymeric nanofibers with biomolecules targeted for specific tissue responses.

The neuroprotective effects of fibronectin mats and fibronectin peptides following spinal cord injury in the rat

We have shown previously that mats made from the glycoprotein fibronectin are permissive for axonal growth when implanted into the injured spinal cord. Recent evidence has indicated that fibronectin and its peptides also have neuroprotective effects in the CNS. We have therefore examined the neuroprotective effects of fibronectin applied to a spinal cord injury site. Adult rats with fibronectin mats implanted into a spinal cord lesion cavity had decreased apoptosis in the intact adjoining spinal cord tissue at 1 and 3 days post-injury compared to rats that had gelfoam implanted into the lesion cavity. Rats with fibronectin mat implants also showed enhanced hindlimb locomotor performance for the first 3 weeks post-surgery compared to control animals. To further examine the neuroprotective potential of fibronectin following spinal cord injury, we examined the effects of placing fibronectin mats over the site of a spinal cord hemisection or of delivering a solution derived from a dissolved fibronectin mat. The effects of these treatments were compared with control animals and animals that were treated with a fibronectin peptide (PRARIY) that has been shown to decrease secondary damage in a rodent model of cerebral ischemia. Results showed that both types of fibronectin mat treatment resulted in decreased lesion size, apoptosis, and axonal damage within the first week post-injury compared to control animals and were comparable in their neuroprotective efficacy to treatment with the fibronectin peptide. The results of the current study indicate that fibronectin based biomaterials have neuroprotective effects following spinal cord injury, in addition to their previously reported ability to promote axonal regeneration.

Anastellin, the Angiostatic Fibronectin Peptide, Is a Selective Inhibitor of Lysophospholipid Signaling

Angiogenesis is regulated by integrin-dependent cell adhesion and the activation of specific cell surface receptors on vascular endothelial cells by angiogenic factors. Lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are bioactive lysophospholipids that activate G protein-coupled receptors that stimulate phosphatidylinositol 3-kinase (PI3K), Ras, and Rho effector pathways involved in vascular cell survival, proliferation, adhesion, and migration. Previous studies have shown that anastellin, a fragment of the first type III module of fibronectin, functions as an antiangiogenic peptide suppressing tumor growth and metastasis. We have previously shown that anastellin blocks serum-dependent proliferation of microvessel endothelial cells (MVEC) by affecting extracellular signal-regulated kinase (ERK)-dependent G(1)-S transition. However, the mechanism by which anastellin regulates endothelial cell function remains unclear. In the present study, we mapped several lysophospholipid-mediated signaling pathways in MVEC and examined the effects of anastellin on LPA- and S1P-induced MVEC proliferation, migration, and cytoskeletal organization. Both LPA and S1P activated PI3K, Ras/ERK, and Rho/Rho kinase pathways, leading to migration, G(1)-S cell cycle progression, and stress fiber formation, respectively. Stimulation of proliferation by LPA/S1P occurred through a G(i)-dependent Ras/ERK pathway, which was independent of growth factor receptors and PI3K and Rho/Rho kinase signaling. Although LPA and S1P activated both PI3K/Akt and Ras/ERK signaling through G(i), anastellin inhibited only the Ras/ERK pathway. Stress fiber formation in response to LPA was dependent on Rho/Rho kinase but independent of G(i) and unaffected by anastellin. These results suggest that lysophospholipid mediators of G(i) activation leading to PI3K/Akt and Ras/ERK signaling bifurcate downstream of G(i) and that anastellin selectively inhibits the Ras/ERK arm of the pathway.

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Fibroblast growth factor (FGF) is an important modulator of cell growth and differentiation of various cells including neuron. Cells need to adhere specifically to cellular and extracellular components of their environment to carry out diverse physiological functions. Here, we examined whether fibronectin (FN) and FGF can cooperate for neuronal adhesion and neurite outgrowth. Using recombinant FN peptide (FNIII9–10), we found that FNIII9–10-mediated adhesion promotes the effect of FGF1 on neurite outgrowth of PC12 cells, while FGF1 enhances the FNIII9–10-mediated neuronal adhesion of PC12 cells. This collaboration of FNIII9–10 and FGF1 was the result of the sustained activation of extracellular signal-regulated kinase (ERK)-type MAP kinase. Finally, the synergistic activity of FGF1 and FN was inhibited by PD98059, an MEK inhibitor. Taken together, these findings indicate that FN-mediated signaling can collaborate with FGFRs signaling for neurite outgrowth through selective activation of ERK-type MAP kinase in PC12 cells, and suggest that FN and FGF act in concert to regulate cell differentiation in the nervous system.

Inhibition of Angiogenesis by a Tenascin-C Peptide which Is Capable of Activating Beta1-Integrins

In addition to humoral angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), integrin-mediated adhesion of vascular endothelial cells to the extracellular matrix plays an important role in neovascularization. We recently found that TNIIIA2, a peptide derived from tenascin-C, induces functional activation of beta1 integrins. Here we investigated the effect of TNIIIA2 on vascular endothelial cell migration and proliferation, key processes for angiogenesis. TNIIIA2 was shown to activate beta1-integrins on human dermal microvascular endothelial cells (HDMEC). HDMEC adhered to fibronectin mainly via integrin alpha5beta1 and their haptotactic migration on that substrate was inhibited by TNIIIA2, in concomitant with a marked inhibition of Rac activation. TNIIIA2-treatment unaffected autophosphorylation of focal adhesion kinase (FAK), but induced its physical association with phospho-paxillin (Tyr118), suggesting the FAK/paxillin-dependent negative regulation of Rac activation. HDMEC proliferation on the fibronectin substrate was also inhibited by TNIIIA2-treatment, and this was accompanied either by an increase in the population of G 0/G1 cells and, conversely, a decrease in the population of S and G2/M cells or by dephosphorylation/inactivation of MAP-kinase (ERK1/2). Inhibited HDMEC migration and proliferation were both restored by pretreating the cells with a fibronectin peptide, FNIII14, which is capable of inactivating beta1-integrins. The chorioallantoic membrane assay demonstrated an antiangiogenic effect of TNIIIA2 in vivo. Thus, TNIIIA2 appears to negatively regulate angiogenesis by inhibiting migration and proliferation of endothelial cells. The ability to activate beta1-integrins may be responsible for the antiangiogenic effect of TNIIIA2, although it cannot be excluded the possibility that an additional mechanism(s) may play a role.

Combination therapy of an anticancer drug with the FNIII14 peptide of fibronectin effectively overcomes cell adhesion-mediated drug resistance of acute myelogenous leukemia

We investigated whether FNIII14, a 22-mer peptide derived from fibronectin (FN) that potently impairs interaction of FN with beta1-integrin, could overcome cell adhesion-mediated drug resistance (CAM-DR) induced by very late antigen (VLA)-4-to-FN interaction in acute myelogenous leukemia (AML). Two AML cell lines, U937 cells and HL-60 cells, and fresh leukemic cells from six AML patients with high alpha4-integrin expression exhibited CAM-DR to cytosine arabinoside (Ara C) through VLA-4-to-FN interaction, while fresh leukemic cells from two AML patients with low alpha4-integrin expression did not display CAM-DR to Ara C. FNIII14 impaired VLA-4-to-FN interaction and restored sensitivity to Ara C in the CAM-DR leukemic cells. In these CAM-DR leukemic cells, upregulation of Bcl-2, which was induced through the focal adhesion kinase/Akt signal pathway upon VLA-4-to-FN interaction, was inhibited by FNIII14 treatment. In a mouse model of minimal residual disease (MRD) in bone marrow, 100% survival was achieved by combining FNIII14 with Ara C, whereas Ara C alone prolonged survival only slightly. The myelosuppression induced by Ara C was not augmented by the combination of FNIII14 in mouse experiments. Thus, the combination of anticancer drugs and FNIII14 holds promise to eradicate MRD in bone marrow after chemotherapy.

Plasminogen Activation–Induced Pericellular Fibronectin Proteolysis Promotes Fibroblast Apoptosis

Apoptosis of fibroblasts/myofibroblasts is a critical event in the resolution of tissue repair responses; however, mechanisms for the regulation of (myo)fibroblast apoptosis/survival remain unclear. In this study, we demonstrate counter-regulatory interactions between the plasminogen activation system and transforming growth factor-beta1 (TGF-beta1) in the control of fibroblast apoptosis. Plasmin treatment induced fibroblast apoptosis in a time- and dose-dependent manner in association with proteolytic degradation of extracellular matrix proteins, as detected by the release of soluble fibronectin peptides. Plasminogen, which was activated to plasmin by fibroblasts, also induced fibronectin proteolysis and fibroblast apoptosis, both of which were blocked by alpha2-antiplasmin but not by inhibition of matrix metalloproteinase activity. TGF-beta1 protected fibroblasts from apoptosis induced by plasminogen but not from apoptosis induced by exogenous plasmin. The protection from plasminogen-induced apoptosis conferred by TGF-beta1 is associated with the up-regulation of plasminogen activator-1 (PAI-1) expression and inhibition of plasminogen activation. Moreover, lung fibroblasts from mice genetically deficient in PAI-1 lose the protective effect of TGF-beta1 against plasminogen-induced apoptosis. These findings support a novel role for the plasminogen activation system in the regulation of fibroblast apoptosis and a potential role of TGF-beta1/PAI-1 in promoting (myo)fibroblast survival in chronic fibrotic disorders.

The FN13 peptide inhibits human tumor cells invasion through the modulation of αvβ3 integrins organization and the inactivation of ILK pathway

We report the effect of the stable expression of a 13 amino acid human fibronectin (FN) peptide (FN13) on the organization of the FN extracellular matrix (ECM) and of FN integrin receptors (FNRs), in relationship with the inhibition of cellular invasion, in three FN-ECM defective human tumor-derived cell lines: SK-Hep1C3, hepatoma, ACN, neuroblastoma, and SK-OV-3, ovary carcinoma. All these cell lines stably expressing the FN13 peptide, organized an FN-ECM, disorganized αvβ1 integrins and inactivated the ILK pathway, with the loss of secretion of MMP-9. This was associated with the inhibition of cell invasion in Matrigel matrix only in SK-Hep1C3 and ACN, but not in SK-OV-3 cells. Analysis of the integrin receptors organization showed that the FN13 expressing cells SK-Hep1C3 and ACN organized αvβ3 integrins, whereas SK-OV-3 organized αvβ5 dimers. The functional block of αvβ5 integrins, with an inactivating anti-αvβ5 antibody, led to the induction of αvβ3 integrins also in SK-OV-3 cells, and to the inhibition of cell invasion. These data show that in the human tumor cells studied FN13 inhibits the in vitro invasion through the dissociation of αvβ1 dimers, leading to ILK pathway inactivation, only when the organization of αvβ3 integrins is induced in the plasma membrane.

Antiadhesive Sites Present in the Fibronectin Type III-Like Repeats of Human Plasma Fibronectin

We have found that fibronectin (FN) has a functional cryptic site opposing cell adhesion to extracellular matrix (ECM): a synthetic FN peptide derived from the 14th FN type III-like (FN-III) repeat, termed peptide FNIII14, inhibits cell adhesion to the FN without binding to beta1 integrins. This antiadhesive activity of peptide FNIII14 depends on its C-terminal amino acid sequence YTIYVIAL. A 50-kDa membrane protein (p50) has been detected as a specific binding protein of peptide FNIII14. Here we showed that antiadhesive activity of peptide FNIII14 was depedent upon the presence of p50 on cell surfaces. Furthermore, we found that there exists a sequence, analogous to the YTIYVIAL, in the 10th FN-III repeat of the FN molecule and that a FN peptide containing this analogous sequence, termed peptide FNIII10, inhibited cell adhesion to the FN. Peptide FNIII10 appeared to share p50 with peptide FNIII14 in expressing the antiadhesive activity. As a physiological consequence of decreased adhesion, peptides FNIII10 and FNIII14 accelerated the anoikis-like apoptosis of normal fibroblasts by down-regulating Bcl-2 expression through blocking the FAK/PI3K/Akt signaling pathway. Thus, the YTIYVIAL-related sequences of the FN molecule may be involved in cell regulation by modulating negatively cell adhesion to the ECM, in which p50 probably serves as a membrane receptor.

Combination Therapy of Anti-Cancer Drug with FNIII14 Peptide of Fibronectin Effectively Overcome Cell Adhesion Mediated-Drug Resistance of Acute Myelogenous Leukemia.

We have previously reported that in acute myelogenous leukemia (AML), drug resistance is induced by the attachment of α4β1-integrin (very late antigen (VLA)-4) on leukemic cells (AML cells) to fibronectin (FN) on bone marrow (BM) stromal cells, and this interaction is crucial in BM minimal residual disease (MRD) and prognosis after chemotherapy (Matsunaga T et al, Nature Med 2003, 9, 1158–1165). We also reported a successful overcome of drug resistance in human AML cells transplanted mice with the use of anti-VLA-4 antibody. However, a drawback of progressive multifocal leukoencephalopathy by anti-VLA-4 antibody administration was reported in multiple sclerosis and Chron's disease patients. Therefore, in this study, we examined whether FNIII14, a 22-mer peptide derived from FN which was reportedly potent to block the interaction between FN and VLA-4, VLA-5 or αvβ3-integrin (Fukai F et al. Clin Cancer Res 2002, 8, 2455–2462), can overcome cell adhesion mediated drug resistance (CAM-DR) induced by VLA-4/FN interaction in AML cells. By in vitro culturing system, FNIII14 dose dependently inhibited adhesion of two AML cell lines (U937 cells, HL-60 cells) and fresh AML cells from patients to FN, and significantly circumvented CAM-DR of these AML cells to cytosine arabinoside (Ara C). The mechanism for this circumvention of CAM-DR involved conformational inactivation of β1-integrin as revealed by FACS through inside out inhibitory signal from complex FNIII14/its 50kDa receptor resulting in down regulation of FAK/Akt/Bcl-2 signal which is responsible for VLA-4/FN induced CAM-DR. In a mouse model of MRD, a 100% survival rate was achieved by combining FNIII14 and Ara C, in contrast to a slight prolongation of survival by Ara C alone. These results indicate that combination treatment of anticancer drugs and FNIII14 is the potent modality for eradication of MRD in BM, and should improve the prognosis of AML.

Study of conformational properties of a biologically active peptide of fibronectin by circular dichroism, NMR and molecular dynamics simulation

Circular dichroism (CD), and NMR spectra have been recorded and molecular dynamics (MD) simulations have been performed in water and water-trifluoroethanol (TFE) mixed solvent for a synthetic biologically active 13-amino-acid fragment of human fibronectin and two related peptides. The CD results are interpreted on the basis of statistical analyses of MD trajectories and of ensuing calculations of CD spectra based on Schellman's matrix method. It is observed that the peptide conformation is quite variable in water and loses its mobility with the addition of TFE. (1)H-NOE data were found to be consistent with the most abundant calculated conformation.

Tropoelastin Interacts with Cell-surface Glycosaminoglycans via Its COOH-terminal Domain

Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of tropoelastin responsible for the adhesive activity.

Synthetic fibronectin peptide exerts neuroprotective effects on transient focal brain ischemia in rats

Leukocytes have been investigated during the past decade for their roles in secondary tissue damage after ischemia/reperfusion injury. Peptide PRARIY, a synthetic fibronectin peptide, has shown an anti-adhesion effect in in vitro studies. Previous studies have demonstrated that anti-adhesion agents lead to reductions in apoptosis. The purpose of the present study was to determine whether the peptide PRARIY displays anti-inflammatory, anti-apoptotic, and neuroprotective effects following transient focal brain ischemia in rats. Twenty-six male Sprague–Dawley rats (300–350 g) were randomly divided into three groups: phosphate-buffered saline (PBS) controls, PRARI controls, and PRARIY treatments. The right middle cerebral artery was transiently occluded using a 4-0 nylon suture. One hour later, the occluder was withdrawn, and reperfusion was maintained for 48 h. Immediately after reperfusion, the peptides (20 mg/kg, dissolved in PBS) and the same volume of PBS were continuously infused through the right external carotid artery using an osmotic minipump for 24 h. Neurological deficits were examined at 3, 24, and 48 h after ischemia. Forty-eight hours after reperfusion, the rats were sacrificed for determining infarction size, leukocyte infiltration, and apoptosis in the ischemia area. Unexpectedly, PRARIY did not influence leukocyte infiltration. However, PRARIY-treated rats showed significantly functional outcome, reduction of infarction size, decrease of TUNEL positive cells, and increase of Bcl-2 (anti-apoptotic protein) positive cells in the ischemic areas when compared to the controls. These data indicate that the peptide PRARIY exerts its neuroprotective effects via supporting neural cell survival rather than anti-leukocyte recruitment following brain ischemia/reperfusion injury.

Effect of magnetic field on the fibronectin adsorption, cell attachment and proliferation on titanium surface

We studied the effect of various static magnetic fields (SMFs) on the adsorption of specific recombinant fibronectin (FN) peptide (hFNIII9-10) on the titanium surface. Furthermore, the responses of human osteosarcoma TE-85 cells in the SMF were observed. Various magnetic fields--1, 2, 3, 5, 7, 10 mT--were established by controlling the distance from Nd-Fe-B magnet to the disks. For FN adsorption experiment, machined titanium disks were incubated in 1 microM hFNIII9-10 at 37 degrees C overnight under magnetic field. The adsorbed hFNIII9-10 was measured as optical density (OD). For attachment study, TE-85 cells were incubated for 2 h on the hFNIII9-10 coated machined titanium disks and OD values were measured. As for proliferation study, titanium disks were incubated for 48 h after washing unattached cells in 2 h. The amount of proliferated TE-85 cell was also measured as OD value. Attachments of TE-85 cells under various intensities of magnetic field were observed using a scanning electron microscope. The amount of adsorbed hFNIII9-10 showed no significant difference between control (0 mT) and six experimental groups (1, 2, 3, 5, 7, 10 mT). However, TE-85 cells attached significantly higher in groups of 1, 2, 5, 10 mT than in control group (P=0). Cell attachment in groups of 3, 7 mT showed no significant difference with that of control group. TE-85 cells were observed to attach through filopodia. Especially in 1 mT, flattened cells were predominant. In proliferation assay, 1 mT stimulated TE-85 cells showed significantly higher proliferation than those in 2, 3 and 7 mT (P=0). Magnetic fields under 10 mT did not influence FN adsorption on the titanium surface. However, a significant effect was found on cell attachment and proliferation.