Fibronectin extra domain a limits liver dysfunction and protects mice during acute inflammation☆
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ABSTRACT: Background and aim
The primary transcript of fibronectin (FN) undergoes alternative splicing to generate different isoforms, including FN containing the Extra Domain A (FN_EDA+), whose expression is regulated spatially and temporarily during developmental and disease conditions including acute inflammation. The role of FN_EDA+ during sepsis, however, remains elusive. Methods
Mice constitutively express the EDA domain of fibronectin (EDA+/+); lacking the FN EDA domain (EDA−/−) or with a conditional ablation of EDA + inclusion only in liver produced FN (alb-CRE+EDA floxed mice) thus expressing normal plasma FN were used. Systemic inflammation and sepsis were induced by either LPS injection (70 mg/kg) or by cecal ligation and puncture (CLP) Neutrophils isolated from septic patients were tested for neutrophil binding ability. Results
We observed that EDA+/+ were protected toward sepsis as compared to EDA−/− mice. Also alb-CRE+EDA floxed mice presented reduced survival, thus indicating a key role for EDA in protecting toward sepsis. This phenotype was associated with improved liver and spleen inflammatory profile. Ex vivo experiments showed that neutrophils bind to a larger extent to an FN_EDA + coated surface as compared to FN, thus potentially limiting their over-reactivity. Conclusions
Our study demonstrates that the inclusion of the EDA domain in fibronectin dampens the nflammatoryi consequences of sepsis. Highlights • FN1 and FN_EDA are highly expressed in the liver of obese human.• FN_EDA+ in plasma increases survival during severe sepsis-related inflammation.• FN_EDA + promote neutrophils adherence and migration.
SUBMITTER: Venu V
PROVIDER: S-EPMC10242638 | biostudies-literature | 2023 May
REPOSITORIES: biostudies-literature
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Project description:Background and purposeThe 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 human pathological conditions associated with inflammation including ischemic stroke. It remains unknown whether EDA(+)-FN contributes to stroke pathogenesis or is simply an associative marker. Several in vitro studies suggest that EDA(+)-FN can activate Toll-like receptor 4, an innate immune receptor that triggers proinflammatory responses. We undertook a genetic approach in mice to investigate the ability of EDA(+)-FN to mediate inflammatory brain damage in a focal cerebral ischemia/reperfusion injury model.MethodsWe used genetically modified EDA(+/+) mice, which constitutively express EDA(+)-FN. Extent of injury, neurological outcome, and inflammatory mechanisms were assessed after 1-hour cerebral ischemia/23-hour reperfusion injury and compared with wild-type mice.ResultsWe found that EDA(+/+) mice developed significantly larger infarcts and severe neurological deficits that were associated with significant increased neutrophil and macrophage infiltration as quantitated by immunohistochemistry. Additionally, we found upregulation of nuclear factor-κB, cyclo-oxygenase-2, and inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the EDA(+/+) mice compared with wild-type mice. Interestingly, increased brain injury and neurological deficits were largely abrogated in EDA(+/+) mice by treatment with a specific Toll-like receptor 4 inhibitor.ConclusionsThese findings provide the first evidence that EDA(+)-FN promotes inflammatory brain injury after ischemic stroke and suggest that the elevated levels of plasma EDA(+)-FN observed in chronic inflammatory conditions could worsen injury and outcome in patients after acute stroke.
| S-EPMC3335936 | biostudies-literature
Project description: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.
| S-EPMC7027995 | biostudies-literature
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| S-EPMC5065221 | biostudies-literature
Project description:BackgroundThe fibronectin-splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans but markedly elevated in patients with comorbid conditions, including diabetes mellitus and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe(-/-)) mouse.Methods and resultsIn a transient cerebral ischemia/reperfusion injury model, Apoe(-/-) mice expressing fibronectin deficient in EDA (Fn-EDA(-/-)Apoe(-/-) mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 versus Apoe(-/-) mice). Concomitantly, intracerebral thrombosis [assessed by fibrin(ogen) deposition] and postischemic inflammation (phospho-nuclear factor-κB p65, phospho-IκB kinase α/β, interleukin 1β, and tumor necrosis factor-α) within lesions of Fn-EDA(-/-)Apoe(-/-) mice were markedly decreased (P<0.05 versus Apoe(-/-) mice). In an FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA(-/-)Apoe(-/-) mice (P<0.05 versus Apoe(-/-) mice). Genetic ablation of TLR4 improved stroke outcome in Apoe(-/-) mice (P<0.05) but had no effect on stroke outcome in Fn-EDA(-/-)Apoe(-/-) mice. Bone marrow transplantation experiments revealed that nonhematopoietic cell-derived Fn-EDA exacerbates stroke through Toll-like receptor-4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe(-/-) mouse 15 minutes after reperfusion significantly improved stroke outcome.ConclusionsHypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.
| S-EPMC4674336 | biostudies-literature
Project description:Glucose-induced changes of artery anatomy and function account for diabetic vascular complications, which heavily impact disease morbidity and mortality. Since fibronectin containing extra domain A (EDA + FN) is increased in diabetic vessels and participates to vascular remodeling, we wanted to elucidate whether and how EDA + FN is implicated in diabetes-induced endothelial dysfunction using isometric-tension recording in a murine model of diabetes. In thoracic aortas of EDA-/-, EDA+/+ (constitutively lacking and expressing EDA + FN respectively), and of wild-type mice (EDAwt/wt), streptozotocin (STZ)-induced diabetes impaired endothelial vasodilation to acetylcholine, irrespective of genotype. However STZ + EDA-/- mice exhibited increased endothelial dysfunction compared with STZ + EDA+/+ and with STZ + EDAwt/wt. Analysis of the underlying mechanisms revealed that STZ + EDA-/- mice show increased oxidative stress as demonstrated by enhanced aortic superoxide anion, nitrotyrosine levels and expression of NADPH oxidase NOX4 and TGF-β1, the last two being reverted by treatment with the antioxidant n-acetylcysteine. In contrast, NOX1 expression and antioxidant potential were similar in aortas from the three genotypes. Interestingly, reduced eNOS expression in STZ + EDA+/+ vessels is counteracted by increased eNOS coupling and function. Although EDA + FN participates to vascular remodelling, these findings show that it plays a crucial role in limiting diabetic endothelial dysfunction by preventing vascular oxidative stress.
| S-EPMC5126581 | biostudies-literature
Project description:ObjectiveCellular fibronectin containing extra domain A (EDA(+)-FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA(+)-FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA(+)-FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E-deficient (Apoe(-/-)) mouse.Approach and resultsThe extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA(-/-)Apoe(-/-) mice (which lack EDA(+)-FN), EDA(fl/fl)Apoe(-/-) mice (which constitutively express EDA(+)-FN), and control Apoe(-/-) mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA(fl/fl)Apoe(-/-) mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA(-/-)Apoe(-/-) mice exhibited reduced atherosclerotic lesions (P<0.05 versus Apoe(-/-), n=10-12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA(fl/fl)Apoe(-/-) mice (P<0.05) but had no effect on atherosclerotic lesions in EDA(-/-)Apoe(-/-) mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/NFκB p65, tumor necrosis factor-α, and interleukin-1β) in bone marrow-derived macrophages from EDA(-/-)Apoe(-/-) mice but not from EDA(-/-)TLR4(-/-)Apoe(-/-) mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA(+)-FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques.ConclusionsOur findings reveal that TLR4 signaling contributes to EDA(+)-FN-mediated exacerbation of atherosclerosis. We suggest that EDA(+)-FN could be a therapeutic target in atherosclerosis.
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Project description:Aging is an important risk factor for cardiovascular diseases, and aging-related cardiac dysfunction serves as a major determinant of morbidity and mortality in elderly populations. Our previous study has identified fibronectin type III domain-containing 5 (FNDC5) and its cleaved form, irisin, as the cardioprotectant against doxorubicin-induced cardiomyopathy. Herein, aging or matched young mice were overexpressed with FNDC5 by adeno-associated virus serotype 9 (AAV9) vectors, or subcutaneously infused with irisin to uncover the role of FNDC5 in aging-related cardiac dysfunction. To verify the involvement of nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) and AMP-activated protein kinase α (AMPKα), Nlrp3 or Ampkα2 global knockout mice were used. Besides, young mice were injected with AAV9-FNDC5 and maintained for 12 months to determine the preventive effect of FNDC5. Moreover, neonatal rat cardiomyocytes were stimulated with tumor necrosis factor-α (TNF-α) to examine the role of FNDC5 in vitro. We found that FNDC5 was downregulated in aging hearts. Cardiac-specific overexpression of FNDC5 or irisin infusion significantly suppressed NLRP3 inflammasome and cardiac inflammation, thereby attenuating aging-related cardiac remodeling and dysfunction. In addition, irisin treatment also inhibited cellular senescence in TNF-α-stimulated cardiomyocytes in vitro. Mechanistically, FNDC5 activated AMPKα through blocking the lysosomal degradation of glucagon-like peptide-1 receptor. More importantly, FNDC5 gene transfer in early life could delay the onset of cardiac dysfunction during aging process. We prove that FNDC5 improves aging-related cardiac dysfunction by activating AMPKα, and it might be a promising therapeutic target to support cardiovascular health in elderly populations.
| S-EPMC8920441 | biostudies-literature
Project description:BackgroundElevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. Here, we investigate the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA+/+) that constitutively expresses only FN containing the EDA isoform.MethodsEyes from C57BL6/J (wild-type), B6.EDA+/+ (constitutively active EDA), B6.EDA-/- (EDA null) mice were processed for electron microscopy and consecutive images of the entire length of the TM and Schlemm's canal (SC) from anterior to posterior were collected and montaged into a single image. ECM accumulation, basement membrane length, and size and number of giant vacuoles were quantified by ImageJ analysis. Tlr4 and Iba1 expression in the TM and ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, ON damage assessed by PPD stain, and RGC loss quantified in RBPMS labeled retina flat mounts.ResultsUltrastructure analyses show the TM of B6.EDA+/+ mice have significantly increased accumulation of ECM between TM beams with few empty spaces compared to C57BL/6 J mice (p < 0.05). SC basement membrane is thicker and more continuous in B6.EDA+/+ mice compared to C57BL/6 J. No significant structural differences are detected in the TM of EDA null mice. Tlr4 and Iba1 expression is increased in the TM of B6.EDA+/+ mice compared to C57BL/6 J eyes (p < 0.05). IOP is significantly higher in B6.EDA+/+ mice compared to C57BL/6 J eyes (p < 0.001), and significant ON damage (p < 0.001) and RGC loss (p < 0.05) detected at 1 year of age. Tlr4 mRNA is expressed in mouse ONH cells, and is present in ganglion cell axons, microglia, and astrocytes. There is a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA+/+ mice compared to C57BL/6 J control eyes (p < 0.01).ConclusionsB6.EDA+/+ mice have increased ECM accumulation in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, loss of RGCs, and ONH damage. These data suggest B6.EDA+/+ mice recapitulate many aspects of glaucomatous damage.
| S-EPMC9137085 | biostudies-literature
Project description:ObjectiveFibronectin containing extra domain A (Fn-EDA) is an endogenous ligand of TLR4 (toll-like receptor 4) and is abundant in the extracellular matrix of advanced atherosclerotic lesions in human and mice. Irrespective of sex, deletion of Fn-EDA reduces early atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. However, the contribution of Fn-EDA in advanced atherosclerosis remains poorly characterized. We determined the contribution of Fn-EDA in advanced atherosclerotic lesions of aged (1-year-old) Apoe-/- mice.Approach and resultsPlaque composition was determined in the innominate artery, a plaque instability site that is known to mimic several histological features of vulnerable human plaques. Female Apoe-/-, Fn-EDA-/-Apoe-/-, TLR4-/-Apoe-/-, and Fn-EDA-/-TLR4-/-Apoe-/- mice were fed a high-fat Western diet for 44 weeks. Fn-EDA-/-Apoe-/- mice exhibited reduced plaque size characterized by smaller necrotic cores, thick fibrous caps containing abundant vascular smooth muscle cells and collagen, reduced CD68/MMP9 (matrix metalloproteinase 9)-positive content, less accumulation of MMP-cleaved extracellular matrix aggrecan, and decreased vascular smooth muscle cell and macrophage apoptosis (P<0.05 versus Apoe-/- mice). Together these findings suggest that Fn-EDA induces plaque destabilization. Deletion of TLR4 reduced histological features of plaque instability in Apoe-/- mice but did not further reduce features of plaque destabilization in Fn-EDA-/-Apoe-/- mice, suggesting that TLR4 may contribute to Fn-EDA-induced plaque destabilization. Fn-EDA potentiated TLR4-dependent MMP9 expression in bone marrow-derived macrophages, suggesting that macrophage TLR4 may contribute to Fn-EDA-mediated plaque instability.ConclusionsFn-EDA induces histological features of plaque instability in established lesions of aged Apoe-/- mice. The abundance of Fn-EDA in advanced atherosclerotic lesions may increase the risk of plaque destabilization.
| S-EPMC5823768 | biostudies-literature
Project description:BackgroundBacterial-induced inflammation instigates the destruction of hard and soft tissues surrounding teeth in periodontitis. In severe cases, the increased number and activity of osteoclasts induces the resorption of alveolar bones, ultimately leading to tooth loss. Because of their diverse chemical structures and bioactivities, natural compounds are often suggested to treat a wide variety of diseases, including inflammatory disorders.MethodsIn the present study, we demonstrated an inhibitory effect of gossypetin, a hexahydroxy flavone, on osteoclast differentiation and bone resorption using in vitro culture of osteoclasts from mouse bone marrow macrophage (BMM) precursors and in vivo model of ligature-induced periodontitis in mice.ResultsGossypetin significantly reduced the differentiation of osteoclasts from mouse BMM precursors in the presence of the receptor activator of nuclear factor κB ligand (RANKL). In vitro, gossypetin inhibited critical signaling events downstream of RANKL including the auto-amplification of nuclear factor of activated T-cells, cytoplasmic 1, Ca2+ oscillations, and the generation of reactive oxygen species. In a mouse ligature-induced periodontitis model, the administration of gossypetin significantly reduced osteoclastogenesis and alveolar bone resorption. Furthermore, gossypetin prevented the ligature-induced increase in macrophages and T cells and reduced the production of tumor necrosis factor-α and interleukin-6.ConclusionTaken together, these results show anti-osteoclastogenic and anti-inflammatory effects of gossypetin, suggesting the potential use of this natural compound in periodontitis.
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