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Andrews, Jonathan P., Jaana Marttala, Edward Macarak, Joel Rosenbloom, and Jouni Uitto. "Keloid Pathogenesis: Potential Role of Cellular Fibronectin with the EDA Domain." Journal of Investigative Dermatology 135, no. 7 (July 2015): 1921–24. http://dx.doi.org/10.1038/jid.2015.50.
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Dhanesha, Nirav, Manish Jain, Prakash Doddapattar, Anetta Undas, and Anil K. Chauhan. "Cellular Fibronectin Promotes Deep Vein Thrombosis in Obese Mice." Blood 136, Supplement 1 (November 5, 2020): 19–20. http://dx.doi.org/10.1182/blood-2020-141441.
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Objective: Obesity is a significant risk factor for deep vein thrombosis (DVT). The mechanisms of increased DVT in preexisting comorbid condition of obesity remain poorly understood. Cellular fibronectin containing extra domain A (Fn-EDA), an endogenous ligand for toll-like-receptor 4 (TLR4), is known to contribute to thrombo-inflammation in the experimental models. However, the role of Fn-EDA in modulation of venous thrombosis in context of obesity is not elucidated yet. Approach and Results: We found that cellular Fn-EDA levels were significantly elevated in plasma of venous thromboembolism (VTE) patients that positively correlated with body mass index (BMI). To investigate whether Fn-EDA promotes venous thrombosis in obese condition, WT and Fn-EDA-/- mice were either fed a control or high-fat diet (HF-diet) for 12-weeks. DVT was induced by inferior vena cava stenosis and evaluated after 48 hours. We found that HF diet-fed WT mice exhibited increased DVT susceptibility compared with control diet-fed WT mice. In contrast, HF-fed Fn-EDA-/- mice exhibited significantly reduced thrombus weight and decreased incidence (%) of DVT compared with HF-fed WT mice that was concomitant with improved blood flow, reduced neutrophil content and citrullinated histone H3-positive cells (a marker of NETosis) in IVC thrombus. Exogenous Fn-EDA potentiated NETosis in neutrophils stimulated with thrombin-activated platelets via TLR4. Genetic deletion of TLR4 in Fn-EDAfl/fl mice, which constitutively express Fn-EDA, reduced DVT compared with Fn-EDAfl/fl mice. Conclusion: These results demonstrate a previously unknown role of Fn-EDA in the modulation of DVT, which may be an important mechanism promoting DVT in the setting of obesity. Figure Disclosures No relevant conflicts of interest to declare.
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Prakash, Prem, Paresh P. Kulkarni, Steven R. Lentz, and Anil K. Chauhan. "Cellular fibronectin containing extra domain A promotes arterial thrombosis in mice through platelet Toll-like receptor 4." Blood 125, no. 20 (May 14, 2015): 3164–72. http://dx.doi.org/10.1182/blood-2014-10-608653.
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Chauhan, Anil, Mohammad M. Khan, Chintan Gandhi, Neelam Chauhan, Asgar Zaheer, David G. Motto, and Steven R. Lentz. "EDA-Containing Fibronectin Aggravates Ischemic Brain Injury In Mice." Blood 116, no. 21 (November 19, 2010): 330. http://dx.doi.org/10.1182/blood.v116.21.330.330.
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Abstract Abstract 330 Background: Fibronectin (FN) is a dimeric glycoprotein that plays an important role in several cellular processes, such as embryogenesis, malignancy, hemostasis, wound healing and maintenance of tissue integrity. FN is a ligand for many members of the integrin family (e.g. αIIbβ3, α5β1, α4β1, α9β1, αvβ3 and αvβ5) and also binds to thrombosis-related proteins including heparin, collagen and fibrin. FN generates protein diversity as a consequence of alternative processing of a single primary transcript. Two forms of FN exist; soluble plasma FN (pFN), which lacks the alternatively-spliced Extra Domain A (EDA); and insoluble cellular FN (cFN), which contains EDA. FN containing EDA (EDA+FN) is normally absent in plasma of human and mouse but EDA+FN has been found in patients with vascular injury secondary to vasculitis, sepsis, acute major trauma or ischemic stroke. We tested the hypothesis that elevated levels of plasma EDA+FN increase brain injury in an experimental model of ischemic stroke in mice. Model and Method: We used two genetically modified mouse strains: EDA+/+ mice contain optimized spliced sites at both splicing junctions of the EDA exon and constitutively express only EDA+FN, whereas EDA-/- mice contain an EDA-null allele of the EDA exon and express only FN lacking EDA. Control EDAwt/wt mice contain the wild-type FN allele. Transient focal cerebral ischemia was induced by 60 minutes of occlusion of the right middle cerebral artery with a 7.0 siliconized filament in male mice (8-10 weeks in age). Mice were anesthetized with 1–1.5% isoflurane mixed with medical air. Body temperature was maintained at 37°C ± 1.0 using a heating pad. Laser Doppler flowmetry was used to confirm induction of ischemia and reperfusion. At 23 hours after MCAO, mice were evaluated for neurological deficits as a functional outcome and were sacrificed for quantification of infarct volume. For morphometric measurement eight 1 mm coronal sections were stained with 2% triphenyl-2, 3, 4-tetrazolium-chloride (TTC). Sections were digitalized and infarct areas were measured blindly using NIS elements. Result: In EDA+/+ mice the percentage of infarct volume (mean ± SEM: 37.25 ± 4.11, n= 12,) in the ipsilateral (ischemic) hemisphere was increased by approximately two-fold compared to EDA wt/wt mice (mean ± SEM: 22.33 ± 3.39, n=11; P< 0.05, ANOVA) or EDA-/- mice (mean ± SEM: 21.72 ± 2.94, n=9). Regional cerebral blood flow during ischemia was not different among groups as assessed by laser Doppler flowmetry. The percentage increase in infarct volume in the EDA+/+ mice correlated well with severe neurological deficits (motor-deficit assessed by a four-point neurological score scale) compared to EDA wt/wt or EDA-/- mice. Because both thrombosis and inflammation contributes to brain injury during ischemic stroke, we investigated the time to form an occlusive thrombus in ferric-chloride carotid artery injury model by intravital microscopy. EDA+/+ mice demonstrated significantly faster time to occlusion (mean ± SEM: 12.35 ± 1.51 n=12,) compared to EDAwt/wt (Mean ± SEM: 17.27 ± 1.72 min, n=13, P<0.05, ANOVA) or EDA-/- (Mean ± SEM: 15.61 ± 1.76, n=11) mice. Additionally, the inflammatory response in the ischemic region was increased by two fold in EDA+/+ mice compared to EDA wt/wt and EDA-/- mice as sensed by myeloperoxidase activity and immunohistochemical analysis of neutrophils. Conclusion: EDA-containing FN is pro-thrombotic and pro-inflammatory, and aggravates ischemic brain injury in an experimental model of stroke in mice. The presence of EDA+FN in plasma may be a risk factor for vascular injury secondary to ischemic stroke. Disclosures: No relevant conflicts of interest to declare.
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Malara, Alessandro, Cristian Gruppi, Andrés Muro, Luigi De Marco, and Alessandra Balduini. "EDA-Fibronectin Is Dispensable for An Efficient Bone Marrow Thrombopoiesis, but Affects Platelet Size in Mice." Blood 118, no. 21 (November 18, 2011): 2388. http://dx.doi.org/10.1182/blood.v118.21.2388.2388.
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Abstract Abstract 2388 BACKGROUND: Extracellular matrix proteins in the bone marrow concur to the generation of niche microenvironment that regulates stem cells differentiation into mature megakaryocytes (MK) and platelet release in the bloodstream. Among them, fibronectin (FN) has been involved in the regulation of MK maturation and proplatelet release. FN is a multimeric glycoprotein playing important roles in cell adhesion, migration, growth and differentiation. FN gene generates protein diversity as a consequence of alternative processing of a single transcript at three different sites: the Extra Domain A (EDA), the Extra Domain B and the type III connecting segment (IIICS). The EDA is absent from soluble plasma FN but included in the cellular form of FN deposited as insoluble fibrils within tissues. We have recently demonstrated that human cord blood derived MKs express EDA+ FN, fibronectins are important in mediating MK collagen-dependent behavior and MKs are involved in the process of FN fibrillogenesis within bone marrow environment. Further, circulating platelets contain a small amount of MK-derived EDA fibronectin and EDA+ FN has prothrombotic activity in mice. However, the role of FN and its variants in normal thrombopoiesis is still unraveled. METHODS: Mouse strains constitutively including (EDA+/+) or excluding (EDA−/−) the EDA domain in all tissues and plasma were used. Primary mouse megakaryocytes were differentiated in vitro from bone marrow and used for RNA isolation, immunofluorescence, fibrinogen adhesion assay and flow cytometry analysis. Femurs of 8 months old mice were fixed, decalcified and frozen sections of 8 microns were stained in immunoistochemistry. RESULTS: Modification of EDA+ FN expression in all mouse strains was demonstrated in murine MKs by RT-PCR and immunofluorescence analysis. The presence or the absence of EDA+ FN in murine MKs did not affect platelet count (WT 1462±294 vs EDA+/+ 1273±131 vs EDA−/− 1365±207 103/ml), while both mutant strains presented a higher Mean Platelet Volume (MPV) (WT 7,7±0,3 vs EDA+/+ 8,1±0,2 vs EDA−/− 8,2±0,3 fL n=12 for wt, n=11 for +/+ and −/−; p<0.01 for +/+ and −/− vs WT, respectively; no differences between +/+ and −/−). The Platelet Distribution Width (PDW) was also different between WT and mutants. Further, preliminary results on MK bone marrow content demonstrated similar results in all genotypes (WT 1,29% vs EDA+/+ 1,33% vs EDA−/− 1,36%). MKs differentiated in vitro, from bone marrow haemopoietic progenitors, showed a comparable ploidy levels and ability to extend proplatelets on fibrinogen (WT 3,72% vs EDA+/+ 4,6% vs EDA−/− 3,12%) in all the different genotypes. Bone marrow histology revealed similar architecture after ematoxylin and eosin stain, similar FN distribution in immunofluorescence and no signs of fibrosis independently of EDA inclusion as demonstrated by Masson's trichrome stain. DISCUSSION: Although EDA+ FN expression has been demonstrated in human and murine MKs, mouse strains that can't undergo regulated splicing of the EDA exon showed no signs of altered thrombopoiesis, but differences in peripheral platelet dimension. Therefore EDA+ FN may play a role as a platelet dimension determinant during their release: this is an unknown function that requires further investigations. Disclosures: No relevant conflicts of interest to declare.
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McNitt, Dudley H., Livingston Van De Water, Daniela Marasco, Rita Berisio, and Slawomir Lukomski. "Streptococcal Collagen-like Protein 1 Binds Wound Fibronectin: Implications in Pathogen Targeting." Current Medicinal Chemistry 26, no. 11 (June 28, 2019): 1933–45. http://dx.doi.org/10.2174/0929867325666180831165704.
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Group A Streptococcus (GAS) infections are responsible for significant morbidity and mortality worldwide. The outlook for an effective global vaccine is reduced because of significant antigenic variation among GAS strains worldwide. Other challenges in GAS therapy include the lack of common access to antibiotics in developing countries, as well as allergy to and treatment failures with penicillin and increasing erythromycin resistance in the industrialized world. At the portal of entry, GAS binds to newly deposited extracellular matrix, which is rich in cellular fibronectin isoforms with extra domain A (EDA, also termed EIIIA) via the surface adhesin, the streptococcal collagen-like protein 1 (Scl1). Recombinant Scl1 constructs, derived from diverse GAS strains, bind the EDA loop segment situated between the C and C’ β-strands. Despite the sequence diversity in Scl1 proteins, multiple sequence alignments and secondary structure predictions of Scl1 variants, as well as crystallography and homology modeling studies, point to a conserved mechanism of Scl1-EDA binding. We propose that targeting this interaction may prevent the progression of infection. A synthetic cyclic peptide, derived from the EDA C-C’ loop, binds to recombinant Scl1 with a micromolar dissociation constant. This review highlights the current concept of EDA binding to Scl1 and provides incentives to exploit this binding to treat GAS infections and wound colonization.
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Malara, Alessandro, Cristian Gruppi, Vittorio Abbonante, Daniele Cattaneo, Luigi De Marco, Alessandra Iurlo, Umberto Gianelli, et al. "EDA Fibronectin-TLR4 Axis Sustains Megakaryocyte Expansion and Inflammation during Bone Marrow Fibrosis Progression." Blood 132, Supplement 1 (November 29, 2018): 1781. http://dx.doi.org/10.1182/blood-2018-99-116454.
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Abstract Fibronectin (FN) is a glycoprotein of approximately 220 kDa, whose mRNA has three alternative splicing sites (termed EDA, EDB and IIICS) that allow 20 different isoforms of FN mRNA. Circulating plasma FN lacks both EDA and EDB segments and is a soluble form secreted by the hepatocytes, while cellular FN contains variable proportions of EDA and EDB segments and is organized as fibrils in the tissue matrix. FN containing EDA segment (EDA FN) presents unique biochemical properties as compared to the isoform lacking this domain, specifically: 1) pro-fibrotic, as the EDA-containing isoform becomes predominantly expressed during wound healing to sustain myofibroblast differentiation; 2) pro-inflammatory, as inclusion of EDA domain activates Toll Like Receptor 4 (TLR4), involved in the defense of the innate immune response following recognition of pathogens associated molecular patterns resulting in NF-κβ-dependent cytokine release; 3) pro-thrombotic, as EDA FN was shown to promote agonist-induced platelet aggregation and thrombus formation in vivo through TLR4 dependent mechanisms. EDA FN is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We identified the up-regulation, at both molecular and protein levels, of EDA FN in an experimental mouse model of BM fibrosis treated with supra-pharmacological doses of the thrombopoietin (TPO) mimetic Romiplostim (TPOhigh). Higher expression levels of EDA FN were detected in endothelial and stromal cells after inducing experimental fibrosis in wild type (WT) mice as compared to untreated controls. To unravel the role of EDA segment on the progression of BM fibrosis in vivo, we exploited two unique transgenic mouse models that present an aberrant splicing of the EDA exon. Mice with constitutive inclusion of EDA exon (EIIIA+/+), in tissues, were more prone to BM fibrosis development as compared to knockout mice (EIIIA-/-). Upon fibrosis induction with TPO mimetic treatment, EIIIA+/+ mice presented extensive reticulin deposition, thrombocytosis, splenomegaly and increased extra-medullary hematopoiesis. Applying numerous in vitro methods, we demonstrated that EDA FN as opposed to plasma FN lacking the EDA domain, induces megakaryocyte (Mk) proliferation in a TPO-independent fashion through engagement of TLR4 and activation of STAT5 and ERK 1/2 signalling pathways. Additionally, in vitro activation of EDA FN/TLR4 axis on Mks resulted in lipopolysaccharide-like responses, such as NF-kb activation, the release of pro-fibrotic Interleukin-6 (IL-6), and no anti-fibrotic TNF-a. Pharmacological inhibition of TLR4 in TPOhigh and EIIIA+/+/TPOhigh mice or TLR4 deletion in TPOhigh mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis and splenomegaly. Finally, developing a novel ELISA assay, we analysed samples from patients affected by primary myelofibrosis (PMF). PMF is one of the Philadelphia-negative Myeloproliferative Neoplasms (MPNs), a group of heterogeneous clonal disorders affecting the hematopoietic stem cell. MPNs are characterised by neoplastic proliferation of the myeloid lineage leading to an abnormal increase of platelet count in essential thrombocythemia (ET), red blood cells in polycythemia vera (PV), or Mks with BM fibrosis in primary myelofibrosis (PMF). To date, three major driver mutations have been identified affecting, respectively, the TPO receptor gene (MPL), the intracellular Janus Kinase 2 gene (JAK2) and the latest discovered mutations affecting the endoplasmic reticulum (ER) molecular chaperone calreticulin gene (CALR). All of these mutations induce a permanent activation of the JAK/STAT signalling pathways. We found that the EDA FN is increased in plasma and BM biopsies of PMF patients, as compared to healthy controls and ET patients, correlating with the fibrotic phase. In conclusion, we identified EDA FN/TLR4 as a new pathological axis that sustains Mk expansion and inflammation during BM fibrosis progression. EDA FN and TLR4 targeting has very promising potential for new therapeutic approaches in PMF patients with high phase BM fibrosis. Disclosures No relevant conflicts of interest to declare.
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Moretti, Federico A., Anil K. Chauhan, Alessandra Iaconcig, Fabiola Porro, Francisco E. Baralle, and Andrés F. Muro. "A Major Fraction of Fibronectin Present in the Extracellular Matrix of Tissues Is Plasma-derived." Journal of Biological Chemistry 282, no. 38 (July 19, 2007): 28057–62. http://dx.doi.org/10.1074/jbc.m611315200.
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The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA+FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA+FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues.
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Rizk, T. A., R. A. Rebres, P. A. Vincent, W. E. Charash, P. J. McKeown-Longo, E. P. Lewis, T. P. Brien, F. L. Minnear, J. B. Fortune, and T. M. Saba. "Delayed elevation of ED1-cellular fibronectin in plasma following postsurgical bacteremia." American Journal of Physiology-Lung Cellular and Molecular Physiology 266, no. 6 (June 1, 1994): L689—L697. http://dx.doi.org/10.1152/ajplung.1994.266.6.l689.
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Fibronectin (Fn) exists in both a soluble form in plasma and lymph as well as an insoluble form in the extracellular matrix. Matrix-localized cellular fibronectin (cFn) contains extra domains (ED1 and/or ED2) not found in plasma Fn (pFn). Very little (< 1-2%) ED1-containing cFn exists in normal blood, and its rapid release into plasma and/or lymph is believed to reflect acute vascular injury. We used a polyclonal antibody to sheep pFn and a monoclonal antibody to ED1 domain of cFn to measure both pFn and ED1-cFn in relationship to lung lymph flow (QL), lung lymph-to-plasma (L/P) total protein concentration ratio, and lung protein clearance (LPC). Unanesthetized sheep (n = 7) were injected intravenously with Pseudomonas aeruginosa (5 x 10(8)) at both 2 and 7 days following surgical preparation of a lung lymph fistula. After both bacterial challenges, we observed an early increase in QL and a small decline in the L/P ratio (0-2 h), reflecting increased fluid filtration in the presence of an intact vascular barrier. This was followed by a further increase (P < 0.05) in QL; an elevation in the L/P ratio; and a marked (P < 0.05) increase in LPC over 3-6 h, indicative of an increase in lung endothelial protein permeability. Before the first bacterial infusion, ED1-cFn in plasma was 9.97 micrograms/ml or approximately 2% of the total Fn antigen in plasma; whereas ED1-cFn in lung lymph was 6-8% of total lymph Fn.(ABSTRACT TRUNCATED AT 250 WORDS)
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Malara, Alessandro, Cristian Gruppi, Margherita Massa, Vittorio Rosti, Giovanni Barosi, and Alessandra Balduini. "Plasma EDA Fibronectin in Primary Myelofibrosis Correlates with Bone Marrow Fibrosis and Predicts Splenomegaly." Blood 134, Supplement_1 (November 13, 2019): 1688. http://dx.doi.org/10.1182/blood-2019-130735.
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Introduction: Primary myelofibrosis (PMF) is a Philadelphia chromosome negative myeloproliferative neoplasm with adverse prognosis characterized by bone marrow (BM) fibrosis and extramedullary hematopoiesis. Fibronectin (FN) is an extracellular matrix glycoprotein that plays vital roles during tissue repair and regeneration. It exists in different forms. Plasma FN is synthesized by hepatocytes and secreted into the blood plasma, where circulates at a concentration of 300-600 μg/ml in a soluble, compact form. Differently, cellular FN is synthesized by several cell types, such as fibroblasts, endothelial cells, chondrocytes and myocytes. The alternative splicing of EDA and EDB and more complex splicing of the V domain, during transcription of FN1 gene, allows different isoforms of FN to be expressed in a tissue-dependent and temporally regulated manner. Very low levels (1.3-3 μg/ml) of FN containing EDA and/or EDB are present in plasma. Although its function is not well understood, EDA containing FN (EDA-FN) is known to agonize Toll like receptor 4 (TLR4), resulting in NF-κβ-dependent cytokine release; to induce myofibroblast differentiation during wound healing; and to increase agonist-induced platelet aggregation and thrombus formation in vivo. We previously showed that EDA-FN levels are increased in plasma and BM biopsies of PMF patients. Mechanistically, BM EDA-FN sustains megakaryocyte proliferation through TLR4 binding and confer a pro-inflammatory phenotype to cell niches promoting fibrosis progression in Romiplostim-treated mice. In this work we measured the plasma levels of EDA-FN in 104 well characterized patients with PMF to determine whether elevated levels of EDA-FN predict the occurrence of disease-related events. Methods: Plasma circulating EDA FN was measured with an enzyme linked immunosorbent assay developed at the University of Pavia, by our group. We obtained plasma EDA-FN concentration values and health care data of persons with PMF from the data-base of the Centre for the Study of Myelofibrosis at the IRCCS Policlinico S. Matteo Foundation in Pavia. We sequentially excluded persons treated with disease-modifying drugs at any time before or on the date of base-cohort entry, and those who had been splenectomized or had received a stem cell transplant. We also excluded persons with acute inflammatory diseases, autoimmune diseases, other neoplasms, and severe liver or renal dysfunction. For this study we selected everyone giving written informed consent and the study was approved by the local Ethic Committee. Immunofluorescence was performed on spleen sections from PMF patients who underwent splenectomy either because of anemia or symptomatic splenomegaly, or both; and healthy controls that were splenectomized following traumatic lesion of the spleen. Data were analyzed using STATISTICA software. Results: A homozygous JAK2V617F genotype was the major determinant of elevated plasma EDA-FN. Elevated EDA-FN levels were associated with anemia, increased levels of high-sensitivity C-reactive protein, BM fibrosis and splanchnic vein thrombosis at diagnosis. We interpreted these associations as reflecting the role EDA-FN plays in tissue remodeling, inflammation and vascular injury. Interestingly, EDA-FN levels resulted also associated with spleen size, and elevated levels of EDA-FN at diagnosis predicted large splenomegaly (more than 10 cm from the left costal margin) outcome. The evidence that plasma EDA-FN levels were not associated with the CD34+ hematopoietic stem cells mobilization, drove us to hypothesize that EDA-FN could reflect spleen endothelial cell activation and/or neoangiogenesis. Immunofluorescence analysis of spleen specimens from PMF patients and healthy controls revealed that high levels of EDA-FN were present in pathological spleens in strong association with endothelial neoangiogenesis. Conclusions: Quantification of EDA-FN level in PMF strongly correlates with BM fibrosis and may be the first marker of an altered spleen microvasculature that contributes to splenomegaly. Understanding the role of this FN isoform in PMF would be useful for testing new mechanisms of disease progression and new hypotheses about the treatment of splenomegaly in PMF. Disclosures No relevant conflicts of interest to declare.
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IKEGAYA, NAOKI, TATSUO YAMAMOTO, AKIHIRO TAKESHITA, TAKUYA WATANABE, KATSUHIKO YONEMURA, TAKEHIKO MIYAJI, KAZUHISA OHISHI, MITSUYOSHI FURUHASHI, YUKITAKA MARUYAMA, and AKIRA HISHIDA. "Elevated Erythropoietin Receptor and Transforming Growth Factor-β1 Expression in Stenotic Arteriovenous Fistulae Used for Hemodialysis." Journal of the American Society of Nephrology 11, no. 5 (May 2000): 928–35. http://dx.doi.org/10.1681/asn.v115928.
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Abstract. The issue of whether recombinant human erythropoietin (rhEPO) increases thrombosis of arteriovenous (AV) fistulae used for hemodialysis remains unclear. Thrombosis often occurs at stenotic segments of fistulae where there is marked intimal hyperplasia and extracellular matrix accumulation. Increased expression of transforming growth factor-β1 (TGF-β1) has been shown to be involved in the development of atherosclerotic lesions by promoting intimal hyperplasia and extracellular matrix accumulation. To clarify the role of rhEPO in the development of stenosis of AV fistulae, this study examined expression of the erythropoietin receptor (EPO-R), TGF-β1, plasminogen activator inhibitor type 1 (PAI-1), cellular fibronectin containing an extra domain A (EDA+), and TGF-β1 mRNA, and assessed in situ rhEPO binding in tissue specimens from seven cutaneous veins and eight patent and seven stenosed portions of AV fistulae of patients undergoing dialysis. Prominent intimal hyperplasia was evident in the stenosed segments. Significant elevation in expression of EPO-R and TGF-β1 was noted in patent AV fistulae compared to the cutaneous veins. Significant enhancement of EPO-R and TGF-β expression was detected in the stenotic fistulae. Fibronectin EDA+ and PAI-1 expression was increased in intimal hyperplasia compared to patent fistulae and cutaneous veins. Elevated EPO-R expression was further confirmed by in situ binding of biotin-labeled rhEPO in stenosed tissue specimens. It is hypothesized that increased rhEPO binding due to elevated EPO-R expression contributes to the development of AV fistula stenosis caused by intimal hyperplasia and extracellular matrix accumulation in response to increased TGF-β1 expression in patients receiving hemodialysis.
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Venu, Vivek Krishna Pulakazhi, Patrizia Uboldi, Ashish Dhyani, Alessandro Patrini, Roberta Baetta, Nicola Ferri, Alberto Corsini, Andrés F. Muro, Alberico Luigi Catapano, and Giuseppe Danilo Norata. "Fibronectin extra domain A stabilises atherosclerotic plaques in apolipoprotein E and in LDL-receptor-deficient mice." Thrombosis and Haemostasis 114, no. 07 (2015): 186–97. http://dx.doi.org/10.1160/th14-09-0790.
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SummaryThe primary transcript of fibronectin undergoes alternative splicing in the cassette-type EDA and EDB exons and in the IIICs segment to generate different protein isoforms. Human carotid atherosclerotic plaques with a more stable phenotype are enriched with EDA containing fibronectin (FN-EDA). The aim of this study was to investigate the role of EDA containing fibronectin during atherogenesis. Mice constitutively expressing or lacking the EDA domain of fibronectin (EDA+/+ or EDA-/-) were crossed with ApoE-/- or LDL-R-/- mice and fed with a western type diet for 12 weeks. Lack of FN-EDA resulted in reduced atherosclerosis and in a plaque phenotype characterised by decreased calponin positive VSMC’s (-15 %) and increased macrophages (+20 %). This was paralleled by increased MMP2, MMP9, and reduced TIMP2, collagen 1A1, 1A2 and 3A1 gene expression compared to that of wild-type and EDA+/+ mice. In vitro, VSMCs and macrophages isolated from EDA-/- mice showed increased MMPs expression and activity compared to wild-type or EDA+/+ mice. Albumin-Cre recombinase/ EDA+/+/ApoE-/- mice, which produce EDA containing FN only in peripheral tissues, presented an extension, a composition and a gene expression pattern in the atherosclerotic lesions similar to that of controls. The inclusion of EDA in FN results in larger atherosclerotic plaques compared to mice lacking EDA but with a more favourable phenotype in two animals models of atherosclerosis. This effect depends on the EDA-containing fibronectin produced by cells in the vasculature but not in the liver. These observations set the stage for investigating the properties of circulating EDA containing FN in improving plaque stability.
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Chauhan, Anil K., Janka Kisucka, Maria R. Cozzi, Meghan T. Walsh, Federico A. Moretti, Monica Battiston, Mario Mazzucato, et al. "Prothrombotic Effects of Fibronectin Isoforms Containing the EDA Domain." Arteriosclerosis, Thrombosis, and Vascular Biology 28, no. 2 (February 2008): 296–301. http://dx.doi.org/10.1161/atvbaha.107.149146.
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Gandhi, Chintan, Mohammad Moshahid Khan, and Anil K. Chauhan. "Fibronectin Isoform Containing the Alternatively-Spliced Extra Domain A in Circulation Accelerates the Development of Atherosclerosis in Mice." Blood 118, no. 21 (November 18, 2011): 2205. http://dx.doi.org/10.1182/blood.v118.21.2205.2205.
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Abstract Abstract 2205 Background and Objective: The fibronectin isoform containing the alternatively-spliced extra domain A (EDA+-FN) is normally absent from the circulation, but plasma levels of EDA+-FN can become markedly elevated in several pathological conditions including atherosclerosis. It remains unclear in humans whether these elevated levels of EDA+-FN are actively contributing to disease pathogenesis, or rather simply serving as a marker associated with vascular stress and/or injury. Several in vitro studies suggest that EDA+-FN can activate toll-like receptor 4 (TLR4), an innate immune receptor that triggers pro-inflammatory responses We hypothesize that presence of EDA+-FN in plasma promotes inflammation and accelerates atherosclerotic plaque formation. Model and Method: We generated EDA+/+/ApoE−/− mice, which contain optimized spliced sites at both splicing junctions of the EDA exon and constitutively express only EDA+-FN, and EDA−/−/ApoE−/− mice, which contain an EDA-null allele of the EDA exon and express only FN lacking EDA. ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain. Results: We report that atherosclerotic plaque (% of total aorta) formation in the aorta of EDA+/+/ApoE−/− mice was increased by two-fold compared to control ApoE−/− mice (P<0.0001). Deletion of the alternatively spliced EDA domain in the ApoE−/− mice (EDA−/−/ApoE−/−) significantly reduced atherosclerotic plaque formation in the aorta (P<0.05) compared to ApoE−/− mice. Total cholesterol and triglycerides levels were similar in ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− mice. Similarly, atherosclerotic plaque formation was significantly increased in the aortic sinus of EDA+/+/ApoE−/− mice, intermediate in control ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice (P<0.05). Additionally, we found that macrophage content, as analyzed by immunohistochemistry, was significantly elevated in the aortic root lesions of EDA+/+/ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice compared to ApoE−/− mice (P<0.05). Moreover, EDA+-FN did not affect the sex-dependent regulation of atherosclerosis in ApoE−/− mice. Future experiments using EDA+/+/ApoE−/−/TLR4−/− are under progress to determine whether EDA+-FN exacerbate atherosclerosis via upregulating TLR4 signaling. Conclusions: Our findings reveal that EDA+-FN is pro-inflammatory and promotes atherosclerotic lesion formation and that monitoring plasma EDA+-FN might have prognostic value in patients at high risk for atherosclerosis. Disclosures: No relevant conflicts of interest to declare.
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Manabe, Ri-ichiroh, Naoko Oh-e, Toshinaga Maeda, Tomohiko Fukuda, and Kiyotoshi Sekiguchi. "Modulation of Cell-adhesive Activity of Fibronectin by the Alternatively Spliced EDA Segment." Journal of Cell Biology 139, no. 1 (October 6, 1997): 295–307. http://dx.doi.org/10.1083/jcb.139.1.295.
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Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA− FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin α5 and β1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin α5β1. In support of this conclusion, purified integrin α5β1 bound more avidly to EDA+ FN than to EDA− FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA− FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin α5β1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180° at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.
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Lerat, H., JC Lissitzky, JW Singer, A. Keating, P. Herve, and P. Charbord. "Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures." Blood 82, no. 5 (September 1, 1993): 1480–92. http://dx.doi.org/10.1182/blood.v82.5.1480.1480.
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Abstract Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.
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Lerat, H., JC Lissitzky, JW Singer, A. Keating, P. Herve, and P. Charbord. "Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures." Blood 82, no. 5 (September 1, 1993): 1480–92. http://dx.doi.org/10.1182/blood.v82.5.1480.bloodjournal8251480.
Full textAbstract:
Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.
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Lemańska-Perek, Anna, Dorota Krzyżanowska-Gołąb, Tomasz Skalec, and Barbara Adamik. "Plasma and Cellular Forms of Fibronectin as Prognostic Markers in Sepsis." Mediators of Inflammation 2020 (August 1, 2020): 1–9. http://dx.doi.org/10.1155/2020/8364247.
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Background. There is a pressing need for specific prognostic markers that could be used to monitor the severity of sepsis. The aims of our study were to investigate changes in the expression of different molecular forms of fibronectin in sepsis and to assess their relationship to the clinical severity and mortality of patients. Material and Methods. Forms of fibronectin: plasma (pFN), cellular (EDA-FN), FN-fibrin complexes, and fibronectin fragments were analyzed in 71 sepsis patients (survivors and nonsurvivors) and in the control by ELISA and immunoblotting. Results. The baseline pFN concentration of patients with sepsis was significantly lower than in the control (133.0 mg/L vs. 231.2 mg/L) (P<0.001), and in nonsurvivors, it was lower than in survivors (106.0 mg/L vs. 152.8 mg/L) (P=0.004). The baseline EDA-FN was significantly elevated in both sepsis groups (survivors: 6.7 mg/L; nonsurvivors: 9.4 mg/L) compared to the control (1.4 mg/L) (P<0.001). It should be noted that among patients with more severe sepsis, the EDA-FN level was higher in nonsurvivors than in survivors. Furthermore, molecular FN-fibrin complexes as well as FN fragments occurred much more frequently in nonsurvivors than in survivors. Conclusion. The study showed that in sepsis, changes in plasmatic and cellular form of fibronectin were associated with the severity of sepsis and may be useful predictors of outcome.
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Du, Hansong, Yu Wang, Zhengfeng Zhang, Jing Yang, Jie Zhang, and Ying Zhang. "Fibronectin Overexpression Modulates Formation of Macrophage Foam Cells by Activating SREBP2 Involved in Endoplasmic Reticulum Stress." Cellular Physiology and Biochemistry 36, no. 5 (2015): 1821–34. http://dx.doi.org/10.1159/000430153.
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Aims: To explore the explicit role of fibronectin (FN) isforms in atherosclerotic lesions and the underlying mechanisms. Methods and Results: Inducible stable expression was performed, and similar results were observed between EDA+FN (FN containing EDA domain) and EDA-FN (FN devoid of EDA domain). FN isforms could trigger endoplasmic reticulum (ER) stress, thereby leading to lipid accumulation in cultured Raw264.7 cells. FN isforms-induced gene expression and lipid accumulation were inhibited by a chemical chaperone 4-phenyl butyric acid (PBA) or by overexpression of the ER chaperone, GRP78/BiP, demonstrating a direct role of ER stress in activation of cholesterol/triglyceride biosynthesis. Moreover, activation of the sterol regulatory element binding protein-2 (SREBP2) was found to be downstream of ER stress, and this activation was affirmed to account for the intracellular accumulation of cholesterol using RNAi technique. Conclusion: our study suggests that enhanced FN in lesions facilitates foam cell formation due to dysregulation of the endogenous sterol response pathway by activation of ER stress, and confirms that EDA+FN has no more pro-atherogenic role than EDA-FN in triggering ER stress.
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Malara, Alessandro, Cristian Gruppi, Vittorio Abbonante, Daniele Cattaneo, Luigi De Marco, Margherita Massa, Alessandra Iurlo, et al. "EDA fibronectin–TLR4 axis sustains megakaryocyte expansion and inflammation in bone marrow fibrosis." Journal of Experimental Medicine 216, no. 3 (February 7, 2019): 587–604. http://dx.doi.org/10.1084/jem.20181074.
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The fibronectin EDA isoform (EDA FN) is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We found that mice constitutively expressing the EDA domain (EIIIA+/+), but not EDA knockout mice, are more prone to develop BM fibrosis upon treatment with the thrombopoietin (TPO) mimetic romiplostim (TPOhigh). Mechanistically, EDA FN binds to TLR4 and sustains progenitor cell proliferation and megakaryopoiesis in a TPO-independent fashion, inducing LPS-like responses, such as NF-κB activation and release of profibrotic IL-6. Pharmacological inhibition of TLR4 or TLR4 deletion in TPOhigh mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis, and splenomegaly. Finally, developing a novel ELISA assay, we analyzed samples from patients affected by primary myelofibrosis (PMF), a well-known pathological situation caused by altered TPO signaling, and found that the EDA FN is increased in plasma and BM biopsies of PMF patients as compared with healthy controls, correlating with fibrotic phase.
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Rizk, T., R. Rebres, P. Vincent, E. Lewis, P. McKeown-Longo, and T. M. Saba. "ED1-containing cellular fibronectin release into lung lymph during lung vascular injury with postoperative bacteremia." American Journal of Physiology-Lung Cellular and Molecular Physiology 264, no. 1 (January 1, 1993): L66—L73. http://dx.doi.org/10.1152/ajplung.1993.264.1.l66.
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Fibronectin (Fn) exists in both a soluble and insoluble form. Soluble Fn in plasma and lymph is an opsonic molecule that enhances phagocytic host defense. Insoluble Fn in the subendothelial and extracellular matrix is an adhesive molecule that mediates cell adhesion to substratum. The extracellular matrix of tissues such as the lung contains a mixture of both plasma-derived fibronectin (pFn) as well as locally synthesized cellular fibronectin (cFn). cFn is antigenically related to pFn, but cFn has extra domains (ED1 and ED2) that do not exist in liver synthesized pFn. The purpose of this study was to determine whether ED1-Fn was released into lung lymph before an increase in lung vascular permeability following postoperative bacteremia. Male sheep (n = 8) with surgically prepared lung lymph fistulae were infused intravenously with a sublethal dose (5 x 10(8)) of Pseudomonas aeruginosa 2 days following surgery. Lymph flow (QL), lymph-to-plasma (L/P) total protein ratio, lung protein clearance (QL x L/P), and hemodynamics were measured over 48 h following bacterial challenge. The lymph and plasma ED1-Fn concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using a murine monoclonal antibody specific to the ED1 region of human cFn. There was a rapid rise of ED1-Fn flux in lung lymph which was evident 60 min after the start of bacterial infusion, resulting in a maximum three- to fourfold increase (P < 0.05) in this parameter. In contrast, the ED1-Fn concentration in plasma before bacterial infusion was less than lung lymph and it did not increase over the initial 6 h following bacterial infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
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Kumazaki, T., Y. Mitsui, K. Hamada, H. Sumida, and M. Nishiyama. "Detection of alternative splicing of fibronectin mRNA in a single cell." Journal of Cell Science 112, no. 10 (May 15, 1999): 1449–53. http://dx.doi.org/10.1242/jcs.112.10.1449.
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Pre-fibronectin mRNA is subject to alternative splicing at three sites, EDA, EDB and IIICS. We analyzed the alternative splicing of fibronectin mRNA in a single cell. Reverse transcription-polymerase chain reaction analyses showed cells that produced a single form of mRNA at each one of these sites as well as cells that produced multiple forms at a given site: for example, some cells produced either the EDA(+) or EDA(-) form of the mRNA and other cells produced both forms. About 80% of the cells produced both (+) and (-) forms of the mRNA at the EDA and EDB sites, and the remaining cells contained either the (+) or (-) form. Five forms of fibronectin mRNA can result from alternative splicing at the IIICS site. Complex combinations of alternative splicing products were observed among the individual cells: there were ten different combinations of mRNA isoforms with respect to the IIICS site. Statistically significant changes in alternative splicing at the IIICS site were observed during cellular senescence.
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Chorawala, Mehul, Prem Prakash, Prakash Doddapattar, Manish Jain, Nirav Dhanesha, and Anil Chauhan. "Deletion of Extra Domain A of Fibronectin Reduces Acute Myocardial Ischaemia/Reperfusion Injury in Hyperlipidaemic Mice by Limiting Thrombo-Inflammation." Thrombosis and Haemostasis 118, no. 08 (June 30, 2018): 1450–60. http://dx.doi.org/10.1055/s-0038-1661353.
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Background Fibronectin splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for Toll-like receptor 4 (TLR4), is present in negligible amounts in the plasma of healthy humans, but markedly elevated in patients with co-morbid conditions including diabetes and hyperlipidaemia, which are risk factors for myocardial infarction (MI). Very little is known about the role of Fn-EDA in the pathophysiology of acute MI under these co-morbid conditions. Materials and Methods We determined the role of Fn-EDA in myocardial ischaemia/reperfusion (I/R) injury in the hyperlipidaemic apolipoprotein E-deficient (ApoE−/−) mice. Infarct size, plasma cardiac troponin I (cTnI) levels, intravascular thrombosis (CD41-positive), neutrophil infiltration (Ly6 B.2-positive), neutrophil extracellular traps (citrullinated H3-positive) and myocyte apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive) were assessed in myocardial I/R injury model (1-hour ischaemia/23 hours of reperfusion). Results Irrespective of gender, Fn-EDA−/−ApoE−/− mice exhibited smaller infarct size and decreased cTnI levels concomitant with reduced post-ischaemic intra-vascular thrombi, neutrophils influx, neutrophil extracellular traps and myocyte apoptosis (p < 0.05 vs. ApoE−/− mice). Genetic deletion of TLR4 attenuated myocardial I/R injury in ApoE−/− mice (p < 0.05 vs. ApoE−/− mice), but did not further reduce in Fn-EDA−/− ApoE−/− mice suggesting that Fn-EDA requires TLR4 to mediate myocardial I/R injury. Bone marrow transplantation experiments revealed that Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the haematopoietic cells. Infusion of a specific inhibitor of Fn-EDA, 15 minutes post-reperfusion, into ApoE−/− mice attenuated myocardial I/R injury. Conclusion Fn-EDA exacerbates TLR4-dependent myocardial I/R injury by promoting post-ischaemic thrombo-inflammatory response. Targeting Fn-EDA may reduce cardiac damage following coronary artery re-canalization after acute MI.
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Lopez-Mejia, Isabel Cristina, Marion De Toledo, Flavio Della Seta, Patrick Fafet, Cosette Rebouissou, Virginie Deleuze, Jean Marie Blanchard, Christian Jorgensen, Jamal Tazi, and Marie-Luce Vignais. "Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion." Molecular Biology of the Cell 24, no. 20 (October 15, 2013): 3164–76. http://dx.doi.org/10.1091/mbc.e13-03-0142.
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Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA–) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.
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Muro, Andrés F., Anil K. Chauhan, Srecko Gajovic, Alessandra Iaconcig, Fabiola Porro, Giorgio Stanta, and Francisco E. Baralle. "Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan." Journal of Cell Biology 162, no. 1 (July 7, 2003): 149–60. http://dx.doi.org/10.1083/jcb.200212079.
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Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP–mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.
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Zhang, Lin, Hongyu Yan, Yifan Tai, Yueming Xue, Yongzhen Wei, Kai Wang, Qiang Zhao, Shufang Wang, Deling Kong, and Adam C. Midgley. "Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity." International Journal of Molecular Sciences 22, no. 4 (February 4, 2021): 1575. http://dx.doi.org/10.3390/ijms22041575.
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Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α4β1 and α4β7. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C′ loop binding cleft within integrins α4β1 and α4β7. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C′ loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α4β1-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C′ loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.
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Ou, Juanjuan, Jia Deng, Xing Wei, Ganfeng Xie, Rongbin Zhou, Liqing Yu, and Houjie Liang. "Fibronectin extra domain A (EDA) sustains CD133+/CD44+ subpopulation of colorectal cancer cells." Stem Cell Research 11, no. 2 (September 2013): 820–33. http://dx.doi.org/10.1016/j.scr.2013.05.009.
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Széll, Márta, Zsuzsanna Bata-Csörgő, Andrea Koreck, Andor Pivarcsi, Hilda Polyánka, Csilla Szeg, Magdolna Gaál, Attila Dobozy, and Lajos Kemény. "Proliferating Keratinocytes Are Putative Sources of the Psoriasis Susceptibility-Related EDA+(Extra Domain A of Fibronectin) Oncofetal Fibronectin." Journal of Investigative Dermatology 123, no. 3 (September 2004): 537–46. http://dx.doi.org/10.1111/j.0022-202x.2004.23224.x.
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Maurer, Eric, Mathieu Schaff, Nicolas Receveur, Catherine Bourdon, Luc Mercier, Bernhard Nieswandt, Christophe Dubois, et al. "Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity." Thrombosis and Haemostasis 114, no. 12 (2015): 1175–88. http://dx.doi.org/10.1160/th14-11-0958.
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SummaryThe ability of cellular fibronectin, found in the vessel wall in a fibrillar conformation, to regulate platelet functions and trigger thrombus formation remains largely unknown. In this study, we evaluated how parietal cellular fibronectin can modulate platelet responses under flow conditions. A fibrillar network was formed by mechanically stretching immobilised dimeric cellular fibronectin. Perfusion of anticoagulated whole blood over this surface resulted in efficient platelet adhesion and thrombus growth. The initial steps of platelet adhesion and activation, as evidenced by filopodia extension and an increase in intracellular calcium levels (419 ± 29 nmol/l), were dependent on integrins α5β1 and αIIbβ3. Subsequent thrombus growth was mediated by these integrins together with the GPIb-V-IX complex, GPVI and Toll-like receptor 4. The involvement of Toll-like receptor 4 could be conveyed via its binding to the EDA region of cellular fibronectin. Upon thrombus formation, the platelets became procoagulant and generated fibrin as revealed by video-microscopy. This work provides evidence that fibrillar cellular fibronectin is a strong thrombogenic surface which supports efficient platelet adhesion, activation, aggregation and procoagulant activity through the interplay of a series of receptors including integrins α5β1 and αIIbβ3, the GPIb-V-IX complex, GPVI and Toll-like receptor 4.
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Xia, Ping, and Lloyd A. Culp. "Adhesion Activity in Fibronectin's Alternatively Spliced Domain EDa (EIIIA): Complementarity to Plasma Fibronectin Functions." Experimental Cell Research 217, no. 2 (April 1995): 517–27. http://dx.doi.org/10.1006/excr.1995.1117.
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Sens, C., N. Kawelke, and I. Nakchbandi⁎. "A fibronectin isoform containing the extra domain A (EDA) increases osteoblast differentiation and function." Bone 50 (May 2012): S74—S75. http://dx.doi.org/10.1016/j.bone.2012.02.213.
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Kropf, J., and A. M. Gressner. "Two sensitive time-resolved fluoroimmunoassays for cellular fibronectin." Clinical Chemistry 41, no. 9 (September 1, 1995): 1283–87. http://dx.doi.org/10.1093/clinchem/41.9.1283.
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Abstract Two sensitive sandwich-type immunoassays for determination of cellular fibronectin (cFN) in cell culture supernatants and in human plasma were developed. Both assays used a monoclonal antibody with specificity against the EDA sequence, which is characteristic for the cellular form of human and rat FN. Assay 1 involves binding of FN on gelatin-coated microwells followed by reaction with the anti-cFN antibody, whereas in assay 2 the anti-cFN antibody was immobilized first and detection was with an anti-FN antiserum. Time-resolved fluorescence spectrophotometry with measurement of an Eu3+ chelator after dissociation of the solid-phase complexes with urea/sodium dodecyl sulfate in the presence of excess Eu3+ was the detection method. The detection limit of the new assays was between 2.6 and 4.0 micrograms/L cFN. In serial dilution of human plasma samples, parallelism with the calibration curve was obtained over the whole measuring range (12-1000 micrograms/L) with assay 2, whereas assay 1 had deviations of the dilution curves at concentrations > or = 100 micrograms/L. The between-run CVs for assays 1 and 2 were 11.4% and 7.2%, respectively, at a concentration of 200 micrograms/L (median value of 18 experiments). Respective within-series CVs of 4.3% and 4.7% were obtained at the same concentration. The recovery of added cFN from human plasma was between 90% and 96%.
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Arribillaga, Laura, Maika Durantez, Teresa Lozano, Francesc Rudilla, Federico Rehberger, Noelia Casares, Lorea Villanueva, et al. "A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell ResponsesIn Vivo." BioMed Research International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/864720.
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The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with aKd~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβby TLR4-expressing cells, as well as the production of TNF-αby the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.
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Khankan, Rima, Noelynn Oliver, Shikun He, Stephen J. Ryan, and David R. Hinton. "Regulation of Fibronectin-EDA through CTGF Domain–Specific Interactions with TGFβ2 and Its Receptor TGFβRII." Investigative Opthalmology & Visual Science 52, no. 8 (July 7, 2011): 5068. http://dx.doi.org/10.1167/iovs.11-7191.
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Shihab, F. S., T. Yamamoto, C. C. Nast, A. H. Cohen, N. A. Noble, L. I. Gold, and W. A. Border. "Transforming growth factor-beta and matrix protein expression in acute and chronic rejection of human renal allografts." Journal of the American Society of Nephrology 6, no. 2 (August 1995): 286–94. http://dx.doi.org/10.1681/asn.v62286.
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Because the increased tissue expression of TGF-beta underlies fibrosis in many diseases, it was hypothesized that sustained elevated transforming growth factor (TGF)-beta overexpression might be responsible for fibrosis in chronic rejection of the renal allograft. To test this hypothesis, biopsies were obtained from 5 patients with acute rejection, 5 patients with chronic rejection, 10 normal individuals, and 10 patients with kidney disease. The tissues were examined by immunofluorescence for the three TGF-beta isoforms (1, 2, and 3) and the two matrix proteins induced by TGF-beta that serve as markers of fibrosis: fibronectin extradomain A positive (EDA+) and plasminogen activator inhibitor-1 (PAI-1). The tubulointerstitium from all cases of acute rejection and chronic rejection showed highly significant increases in immunostaining for the three TGF-beta isoforms (P < 0.001), fibronectin EDA+ (P < 0.005), and PAI-1 (P < 0.001). In the glomeruli, only TGF-beta 1 expression achieved statistical significance (P < 0.005) in acute rejection, whereas in chronic rejection, all three TGF-beta isoforms (p < 0.001) in addition to fibronectin EDA+ (p < 0.001) and PAI-1 (p < 0.001) were elevated. There was both cellular and matrix staining of the TGF-beta isoforms. In striking contrast, control kidney tissues were negative or only weakly positive. Because TGF-beta was present both in acute and in chronic rejection but not in control tissues and because acute rejection episodes are a good predictor for chronic rejection, these results suggest that TGF-beta may play a role in the pathogenesis of fibrosis in chronic rejection.
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Shinde, Arti V., Rhiannon Kelsh, John H. Peters, Kiyotoshi Sekiguchi, Livingston Van De Water, and Paula J. McKeown-Longo. "The α4β1 integrin and the EDA domain of fibronectin regulate a profibrotic phenotype in dermal fibroblasts." Matrix Biology 41 (January 2015): 26–35. http://dx.doi.org/10.1016/j.matbio.2014.11.004.
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Bootz, Franziska, Anja Sophie Schmid, and Dario Neri. "Alternatively Spliced EDA Domain of Fibronectin Is a Target for Pharmacodelivery Applications in Inflammatory Bowel Disease." Inflammatory Bowel Diseases 21, no. 8 (August 2015): 1908–17. http://dx.doi.org/10.1097/mib.0000000000000440.
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Ou, Juan-Juan, Feng Wu, and Hou-Jie Liang. "Colorectal tumor derived fibronectin alternatively spliced EDA domain exserts lymphangiogenic effect on human lymphatic endothelial cells." Cancer Biology & Therapy 9, no. 3 (February 2010): 186–91. http://dx.doi.org/10.4161/cbt.9.3.10651.
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Zent, Joshua, and Lian-Wang Guo. "Signaling Mechanisms of Myofibroblastic Activation: Outside-in and Inside-Out." Cellular Physiology and Biochemistry 49, no. 3 (2018): 848–68. http://dx.doi.org/10.1159/000493217.
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Myofibroblasts are central mediators of fibrosis. Typically derived from resident fibroblasts, myofibroblasts represent a heterogeneous population of cells that are principally defined by acquired contractile function and high synthetic ability to produce extracellular matrix (ECM). Current literature sheds new light on the critical role of ECM signaling coupled with mechanotransduction in driving myofibroblastic activation. In particular, transforming growth factor β1 (TGF-β1) and extra domain A containing fibronectin (EDA-FN) are thought to be the primary ECM signaling mediators that form and also induce positive feedback loops. The outside-in and inside-out signaling circuits are transmitted and integrated by TGF-β receptors and integrins at the cell membrane, ultimately perpetuating the abundance and activities of TGF-β1 and EDA-FN in the ECM. In this review, we highlight these conceptual advances in understanding myofibroblastic activation, in hope of revealing its therapeutic anti-fibrotic implications.
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Kratz, Ewa M., Marcin Wójtowicz, Magdalena Przybysz, Ricardo Faundez, and Iwona Kątnik-Prastowska. "Human seminal fibronectin fragmentation patterns and their domain immunoreactivities in leucocytospermic patients." Reproduction, Fertility and Development 26, no. 7 (2014): 1044. http://dx.doi.org/10.1071/rd13049.
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The aim of the work was to analyse fibronectin (FN) domain immunoreactivities and profiles of FN fragmentation in seminal plasmas of fertile normozoospermic and infertile leucocytospermic male patients. ELISA with domain-specific monoclonal antibodies and immunoblotting were used in these measurements. Immunoblotting of normal and leucocytospermic seminal plasmas revealed the presence of twelve FN bands of ~70–196 kDa with nearly identical FN profiles under reducing and non-reducing conditions. The epitopes of the cell-, fibrin-, collagen-binding FN domains and the extra domain A (EDA) FN segment retained the ability to bind their specific monoclonal antibodies, whereas the fibrin–heparin domain (N-terminal end) and the area around the disulfide bridges (C-terminal end) of the FN polypeptide did not show any reactivities with their respective specific antibodies. The mean values of cell- (338.4 ± 138.4 and 398.3 ± 310 mg L–1), fibrin- (79.1 ± 38.5 and 145.2 ± 188.8 mg L–1) and collagen-binding (19 ± 19.8 and 50.9 ± 73.4 mg L–1) FN domain immunoreactivities and the relative amount of EDAFN did not show any significant differences between the normal and leucocytospermic groups. The high values of standard deviations for the FN domain immunoreactivities in the leucocytospermic group probably results from different aetiology of leucocytospermia. The profile of FN fragmentation and alterations of FN domain immunoreactivities in seminal plasma may influence their engagement in the fertilisation process. The analysis of seminal FN molecular status would be helpful for selecting the highest quality spermatozoa for use in assisted reproduction techniques.
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Vincent, P. A., R. A. Rebres, E. P. Lewis, V. Hurst, and T. M. Saba. "Release of ED1 fibronectin from matrix of perfused lungs after vascular injury is independent of protein synthesis." American Journal of Physiology-Lung Cellular and Molecular Physiology 265, no. 5 (November 1, 1993): L485—L492. http://dx.doi.org/10.1152/ajplung.1993.265.5.l485.
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Fibronectin (Fn) is an adhesive protein found in the plasma and extracellular tissue matrix. Locally synthesized tissue or cellular Fn (cFn) has extra domains (ED1 and ED2) not present in liver synthesized plasma Fn (pFn). In the lung, Fn is found in the endothelial and epithelial basement membranes, as well as in the interstitial matrix. Utilizing murine monoclonal antibodies to ED1 of cFn, we studied the release of total Fn as well as ED1-Fn into the plasma-free perfusate of the isolated perfused rabbit lung in relation to changes in lung weight due to fluid accumulation after oxidant (H2O2) challenge. Both parameters were also studied after addition of cycloheximide (20 micrograms/ml perfusate) to the perfusion medium to inhibit lung protein synthesis. After continuous H2O2 challenge (11 nmol.ml buffer-1.min-1), there was a 2.25 +/- 0.62 g increase in lung weight over 60 min. Measurement of 125I-labeled albumin clearance at 20 min after the start of H2O2 infusion confirmed an increase in lung endothelial protein permeability after H2O2 treatment. Fn antigen was released into the perfusate as early as 15 min after oxidant challenge. By 60 min, total perfusate Fn increased in H2O2-treated lungs (n = 6) to 2.10 +/- 0.48 micrograms/ml compared with only 0.35 +/- 0.09 micrograms/ml in normal control lungs (n = 5). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of nonreduced samples revealed that the Fn released consisted of primarily intact (440 kDa) Fn as well as Fn fragments. A rapid release of ED1-Fn paralleled the increased release of total Fn.(ABSTRACT TRUNCATED AT 250 WORDS)
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Blystone, SD, LK Weston, and JE Kaplan. "Fibronectin dependent macrophage fibrin binding." Blood 78, no. 11 (December 1, 1991): 2900–2907. http://dx.doi.org/10.1182/blood.v78.11.2900.2900.
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Abstract Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.
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Blystone, SD, LK Weston, and JE Kaplan. "Fibronectin dependent macrophage fibrin binding." Blood 78, no. 11 (December 1, 1991): 2900–2907. http://dx.doi.org/10.1182/blood.v78.11.2900.bloodjournal78112900.
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Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.
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Fournier, Henri-Noël, Sandra Dupé-Manet, Daniel Bouvard, Frédéric Luton, Simona Degani, Marc R. Block, Saverio Francesco Retta, and Corinne Albiges-Rizo. "Nuclear Translocation of Integrin Cytoplasmic Domain-associated Protein 1 Stimulates Cellular Proliferation." Molecular Biology of the Cell 16, no. 4 (April 2005): 1859–71. http://dx.doi.org/10.1091/mbc.e04-08-0744.
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Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the β1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of β1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a β1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.
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Franz, Marcus, Ingrid Hilger, Katja Grün, Susanne Kossatz, Petra Richter, Iver Petersen, Christian Jung, et al. "Selective imaging of chronic cardiac rejection using a human antibody specific to the alternatively spliced EDA domain of fibronectin." Journal of Heart and Lung Transplantation 32, no. 6 (June 2013): 641–50. http://dx.doi.org/10.1016/j.healun.2013.04.003.
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Christopher, R. A., S. R. Judge, P. A. Vincent, P. J. Higgins, and P. J. McKeown-Longo. "The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression." Journal of Cell Science 112, no. 19 (October 1, 1999): 3225–35. http://dx.doi.org/10.1242/jcs.112.19.3225.
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Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.
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Shinde, Arti V., Christopher Bystroff, Chunyu Wang, Mariette G. Vogelezang, Peter A. Vincent, Richard O. Hynes, and Livingston Van De Water. "Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities." Journal of Biological Chemistry 283, no. 5 (October 29, 2007): 2858–70. http://dx.doi.org/10.1074/jbc.m708306200.
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Richardson, T. P., V. Trinkaus-Randall, and M. A. Nugent. "Regulation of heparan sulfate proteoglycan nuclear localization by fibronectin." Journal of Cell Science 114, no. 9 (May 1, 2001): 1613–23. http://dx.doi.org/10.1242/jcs.114.9.1613.
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Heparan sulfate proteoglycans (HSPG) regulate multiple cellular processes and mediate the cellular uptake of numerous molecules. While heparan sulphate glycosaminoglycan chains are known to modulate receptor binding of several heparin-binding proteins, here we show that distinct extracellular matrices direct HSPG to the nucleus. We analyzed HSPG localization in primary corneal fibroblasts, cultured on fibronectin or collagen type I matrices, using confocal laser scanning microscopy and cell fractionation. Image analysis revealed that the nuclear localization of HSPG core proteins was greater when cells were cultured on fibronectin versus collagen. Matrices containing the heparin-binding domain of fibronectin, but not the integrin-activating domain, demonstrated increased nuclear staining of core proteins. Furthermore, activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited nuclear targeting of HSPG in cells on fibronectin, whereas inhibition of protein kinase C with Ro-31-8220 greatly enhanced nuclear localization of HSPG in cells on both collagen and fibronectin. We propose a matrix-dependent mechanism for nuclear localization of cell surface HSPG involving protein kinase C-mediated signaling. Nuclear localization of HSPG might play important roles in regulating nuclear function.
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Katnik-Prastowska, Iwona, Magdalena Przybysz, and Anna Chełmońska-Soyta. "Fibronectin fragments in human seminal plasma." Acta Biochimica Polonica 52, no. 2 (May 15, 2005): 557–60. http://dx.doi.org/10.18388/abp.2005_3473.
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The study has revealed the presence of fibronectin (FN) fragments and a lack of intact FN in 72 seminal plasma samples. The FN fragmentation was examined by immunoblotting with a monoclonal antibody specific to the central cellular FN domain and was confirmed with a monoclonal antibody directed to the C-terminal domain of FN. Nine FN fragments between 60 and 200 kDa and five fragments of 60-150 kDa were identified in seminal plasma samples of normozoospermic and of terato-, oligoterato-, and oligoasthenoterato-spermic groups, respectively. The relative amounts of the 60, 90 and 100 kDa FN fragments were 2-3 times higher in seminal plasmas with abnormal semen characteristics than in the normozoospermic group. The results suggest that seminal plasma FN fragments may contribute to fertilization and the analysis of FN fragmentation may have a diagnostic value in andrological investigations.
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Dhanesha, Nirav, Mehul R. Chorawala, Manish Jain, Abhinav Bhalla, Daniel Thedens, Manasa Nayak, Prakash Doddapattar, and Anil K. Chauhan. "Fn-EDA (Fibronectin Containing Extra Domain A) in the Plasma, but Not Endothelial Cells, Exacerbates Stroke Outcome by Promoting Thrombo-Inflammation." Stroke 50, no. 5 (May 2019): 1201–9. http://dx.doi.org/10.1161/strokeaha.118.023697.
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