Academic literature on the topic 'Cellular fibronectin EDA domain'
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Journal articles on the topic "Cellular fibronectin EDA domain"
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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.
Dissertations / Theses on the topic "Cellular fibronectin EDA domain"
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Neuhaus, Jochen, Birgit Schröppel, Martin Dass, Hans Zimmermann, Hartwig Wolburg, Petra Fallier-Becker, Thomas Gevaert, Claus J. Burkhardt, Do Hoang Minh, and Jens-Uwe Stolzenburg. "3D-electron microscopic characterization of interstitial cells in the human bladder upper lamina propria." Universitätsklinikum Leipzig, 2017. https://ul.qucosa.de/id/qucosa%3A15544.
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1) Aims
To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts.
2) Methods
Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders.
3) Results
3View-SEM image stacks as large as 59µm x 59µm x 17µm (xyz) at a resolution of 16nm x 16nm x 50 nm and high resolution (5nm x 5nm x 10nm) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium sheet-like morphology. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria.
4) Conclusions
Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.
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Moretti, Federico Andrea. "FN-EDA domain knock-in and knock-out mice, as a model for the study of the hepatic fibronectin (FN) functions." Thesis, Open University, 2007. http://oro.open.ac.uk/54926/.
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Fibronectin (FN) is a multidomain glycoprotein found in plasma and tissue extracellular matrices (ECM). Plasma FN (pFN) is produced and secreted into the bloodstream by the liver. Liver ECM-FN and pFN are characte,rized by the absence of the EDA domain, one of the three FN exons undergoing alternative splicing. However, in pathological situations such as wound healing, the liver produces and accumulates huge amounts of EDA+FN in sinusoidal ECM. In order to study the functions of the FN isoforms produced by the liver, we used two mouse strains devoid of EDA-exon alternative splicing, which constitutively include (EDA+/+ strain) or exclude (EDA-/- strain) the EDA domain. The EDA+/+ strain is characterized by an important decrease of FN levels both in plasma and tissues. Here, we show that the atypycal presence of the EDA domain in hepatocytes affects pFN secretion into the bloodstream and we demonstrate that a high proportion of tissue ECM-FN is supplied by plasma. In fact, removal of the EDA exon only from the liver, by hepatocyt'-specific deletion of the EDA exon, restores normal levels of FN in both plasma and tissues of the EDA+/+ animals. This finding not only suggests that plasma behaves as a sort of FN reservoir for tissue maintenance but also shows for the first time that the pFN contribution to the ECM of tissues is roughly equal to that produced locally. The EDA-/- strain was used to study the role of the EDA domain in the onset of liver fibrosis. In two different liver fibrosis models, we observed that EDA-/- animals develop attenuated fibrosis in comparison to control mice. The important role of the EDA domain in the activation and proliferation of the hepatic stellate cells (HSCs), these are the key cells responsible for the ECM protein overproduction in the fibrotic processes, is demonstrated for the first time in vivo. EDA-/- livers treated to induce fibrosis, present a striking reduction in the number of myofibroblasts or activated HSCs, suggesting that the EDA domain could be a potential target for the treatment of liver fibrosis.
Conference papers on the topic "Cellular fibronectin EDA domain"
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Uzan, G., A. Lajmanovich, M. H. Prandini, Ph Frachet, A. Duperray, and G. Marguerie. "MOLECULAR CLONING OF PLATELET GPIIb FROM HEL CELLS AND HUMAN MEGAKARYOCYTES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643960.
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Platelet GP IIb-IIIa is an heterodimer which functions as a receptor for fibrinogen, fibronectin and Von Willebrand factor and is implicated in platelet adhesive reactions. To study the structure function relationship of this glycoprotein, a recombinant DNA approach was initiated. cDNA expression libraries were constructed in » gtll vector, from erythro-leukemia cells (HEL) and megakaryocytes mRNA. The human megakaryocytes were isolated from patients with chronic myeloid leukemia. The HEL library was initially screened with polyclonal antibodies anti GPIIb IIIa. One clone, λIIbI, containing a 1.65 kbp insert reacted with a panel of different polyclonal antibodies anti GPIIb IIIa and a monoclonal antibody anti GPIIb. To further characterize this clone the synthesis of the fusion protein was induced by IPTG. The bacterial protein was then blotted onto nitro cellulose and incubated with antisera anti GPIIb-IIIa. Antibodies that specifically bound with the fusion protein were eluted and tested on platelet membrane extracts. The selected antibodies produced a positive signal at the GPIIb position similar to the signal produced by the monoclonal antibody anti GPIIb on the same membrane extract. Finally on western blotting, a protein of Mr= 170kD reacted with the monoclonal antibody anti GPIIb. λIIbI insert was used to screen the megakaryocyte library and 3 clones, λIIb2,λIIb3 and λIIb4 were isolated. The size of HEL cells and megakaryocytes GPIIb mRNA was estimated by northern blotting. Only one species of 3.9 kb was identified in both cells. The four different clones accounted for 50% of the coding sequence of this mRNA.Sequencing of these cDNAs indicated that the plasmatic domain of GPIIb contains a cystein rich region. The sequence of these clones will allow the study of the adhesines genetic diversity in different cellular systems.
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Hawiger, J. "PLATELET RECEPTOR RECOGNITION DOMAINS AND THEIR SYNTHETIC PEPTIDE ANALOGS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643726.
Full textAbstract:
Adhesive molecules and their receptorsplay an essential role in hemostasis and thrombosis. Platelet thrombi are formed through the interaction of cell adhesion molecules (CAMs) with intercellular adhesion molecules (IAMs)and substrate adhesion molecules (SAMs). Platelet CAMs encompass membrane glycoproteins lb, lib, Ilia,and possibly la and IV, which constitutemembrane receptors for IAMs(e.g., fibrinogen) and for SAMs encompassingvon Willebrand Factor (vWF), fibronectin, vitronectin, collagen, and thrcmbospondin. Receptorfunction of platelet CAMs can be specific,i.e., only one adhesive protein among IAMs and SAMs is selected forbinding as exemplified by GPIb and vWF. Alternatively,more than one adhesive protein can interact with platelet CAMs comprising the GPIIb/IIIa complex.This common adhesive receptor mechanism switched on by thrombin, ADP, phorbol ester or ionophore A23187 is turned off by a rise in intraplatelet cyclic AMP which provides a negative control.Fibrinogen, the most abundant adhesiveprotein in plasma, interacts with platelet CAMs via receptor recognition domains on gamma and alpha chains. Pinpointing platelet receptor recognition domain to a carboxy-terminal segment of the gamma chain encompassing residues 400-411gave rise to a series of synthetic peptide analogs which do not interfere with themetabolic pathways of platelets but blockbinding of I fibrinogen to its receptors on stimulated platelets, inhibit their aggregation in vitro, and formation of a platelet thrombus in vivo. The alpha chain of human fibrinogen contains the sequenceRGD (residues 95-97 and 572-574). Synthetpeptide analogs of the RGD sequence, which constitute the "cell adhesion site" of fibronectin, also inhibit binding of 125I-fibrinogen to stimulated platelets. However, these synthetic peptides are not "specific" for fibrinogen chains because thealpha chain of human fibrinogen which hasnosequence homology with gamma 400-411 is prevented by a peptide gamma 400-411 from interaction with platelet receptors. Viceversa, the human gamma chain is blocked by tetrapeptide RGDS not expressed in the human gamma chain. Interaction of human vWF with human platelets is blocked by synthetic peptide analogs of gamma 400-411 (not present in vWF)and of RGD sequence (present in vWF).These synthetic peptides inhibite "common" receptor pathwaystimulated with ADP, thrombin, or phorbolester, but they do not interfere with binding of 125I-vWF via a "specific" pathvoy induced with ristocetin and involving GPIb.The design of synthetic peptide analogs which inhibit platelet receptors for adhesive molecules includes the following considerations: ligand specificity (is thepeptide inhibitory toward binding of one or more adhesive molecules?),cell speciicity (is the peptide specific for platelets or does it perturb the adhesive properties of other cells, e.g.,endothelium?);the hydrophilic character; protection against degradation by peptidases; and a sufficiently long half-life to achieve platelet inhibitory potency in vivo without overloading the blood with excessive amounts of peptide.This is accomplished by constructing a peptide-albumin conjugate with ahalf-life extended at least 30 times.Whenpeptides are modeled with predominantly hydrophilic or hydrophobic residues, only the hydrophilic peptide remained active to block the platelet receptor. This agreed with the general observation that sequences on adhesive molecules that are knownto interact with cellular receptors have a hydrophilic rather than a hydrophobic character. Furthermore, changing the charge of synthetic peptides toward the negative reduced the reactivity, whereas introducing additional arginine residues enhanced the reactivity toward platelet receptors. Localization of the functionally important binding domain in the flexible segment of an adhesive protein increases the likelihood that the synthetic peptide will assume the conformation mimicking such a domain in the native adhesive protein. Structure-function studies of the receptor recognition domains on adhesive molecules led to development of a new class of platelet inhibitors acting at the membranereceptors responsible for anchoring of platelets to the vessel wall and linking them to each other.
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Charo, I. F., L. A. Fitzgerald, D. Meyer, L. S. Bekeart, and D. R. Phillips. "PLATELET GLYCOPROTEIN IIb-IIIa-LIKE PROTEINS MEDIATE ENDOTHELIAL CELL ATTACHMENT TO ADHESIVE MATRIX PROTEINS AND ARE UP-REGULATED BY PHORBOL ESTERS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642816.
Full textAbstract:
Human endothelial cells (EC) express glycoproteins that are similar to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa), the platelet receptor for adhesive proteins. Although GP IIb—IIIa is abundant in both platelets and EC, its only known function is to mediate platelet aggregation. The present study tests the hypotheses that EC attachment to adhesive proteins in the extracellular matrix is mediated by the GP IIb-IIIa-1ike proteins. Endothelial cells attached well to glass slides that were previously coated with adhesive proteins, but not albumin. To determine whether GP IIb-IIIa was involved, EC adherence was measured in the presence and absence of a GP IIb-IIIa monoclonal antibody (7E3) which inhibits fibrinogen (Fg) binding to platelets. The attachment of EC to Fg and von Willebrand factor (vWf), but not fibronectin (Fn) coated slides, was completely inhibited by 7E3. Attachment to vitronectin was partially inhibited. In contrast, EC attachment to Fn was specifically inhibited by a Fn-receptor antibody. Endothelial cell adherence to vWf was also inhibited by a monoclonal antibody (Mab9) against the GP IIb-IIIa binding domain of vWf, but not by antibodies agains.t other portions of vWf. We have further found that 7E3 disrupts monolayers of endothelial cells by detaching the cells from their extracellular matrix. EC incubated in phorbol myris-tate.acetate (PMA) increase in size and appear more tightly adherent to their extracellular matrix. To determine if PMA increases synthesis of cellular receptors for matrix proteins, we have used cDNA probes to measure the mRNA levels of the large subunit of the Fn-receptor (FnRα) and GP IIIa in EC. After a 4 hour incubation in the presence of PMA (10 nM), there was a 2-fold increase in the mRNA levels of both FnRα and GP IIIa, as well as increased cell spreading on the matrix. We conclude: i) the GP Ilb-IIIa complex in EC is a surface receptor for specific adhesive proteins, and is distinct from the FnR, and ii) both GP IIIa and FnRα synthesis are increased by PMA, which causes a concomittant change in cell morphology.