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Dieter P. Reinhardt - One of the best experts on this subject based on the ideXlab platform.
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Fibrillin-1 and Fibrillin-1-derived asprosin in adipose tissue function and metabolic disorders
Journal of Cell Communication and Signaling, 2020Co-Authors: Muthu L. Muthu, Dieter P. ReinhardtAbstract:The extracellular matrix microenvironment of adipose tissue is of critical importance for the differentiation, remodeling and function of adipocytes. Fibrillin-1 is one of the main components of microfibrils and a key player in this process. Furin processing of proFibrillin-1 results in mature Fibrillin-1 and releases the C-terminal propeptide as a circulating hunger hormone, asprosin. Mutations in the Fibrillin-1 gene lead to adipose tissue dysfunction and causes Marfan syndrome, marfanoid progeroid lipodystrophy syndrome, and neonatal progeroid syndrome. Increased TGF-β signaling, altered mechanical properties and impaired adipogenesis are potential causes of adipose tissue dysfunction, mediated through deficient microfibrils. Circulating asprosin on the other hand is secreted primarily by white adipose tissue under fasting conditions and in obesity. It increases hepatic glucose production and drives insulin secretion and appetite stimulation through inter-organ cross talk. This review discusses the metabolic consequences of Fibrillin-1 and Fibrillin-1-derived asprosin in pathological conditions. Understanding the dynamic role of Fibrillin-1 in the adipose tissue milieu and of circulating asprosin in the body can provide novel mechanistic insights into how Fibrillin-1 may contribute to metabolic syndrome. This could lead to new management regimens of patients with metabolic disease.
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the Fibrillin 1 rgd integrin binding site regulates gene expression and cell function through micrornas
Journal of Molecular Biology, 2019Co-Authors: Karina A Zeyer, Rongmo Zhang, Heena Kumra, Amani Hassan, Dieter P. ReinhardtAbstract:Abstract Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. Fibrillin-1 contains one evolutionarily conserved RGD sequence that mediates cell–matrix interactions through cell-surface integrins. Here, we present a novel paradigm how extracellular Fibrillin-1 controls cellular function through integrin-mediated microRNA regulation. Comparative mRNA studies by global microarray analysis identified growth factor activity, actin binding and integrin binding as the most important functional groups that are regulated upon Fibrillin-1 binding to dermal fibroblasts. Many of these mRNAs are targets of miRNAs that were identified when RNA from the Fibrillin-1-ligated fibroblasts was analyzed by a miRNA microarray. The expression profile was specific to Fibrillin-1 since interaction with fibronectin displayed a partially distinct profile. The importance of selected miRNAs for the regulation of the identified mRNAs was suggested by bioinformatics prediction and the interactions between miRNAs and mRNAs were experimentally validated. Functionally, we show that miR-503 controls p-Smad2-dependent TGF-β signaling, and that miR-612 and miR-3185 are involved in the focal adhesion formation regulated by Fibrillin-1. In conclusion, we demonstrate that Fibrillin-1 interaction with fibroblasts regulates miRNA expression profiles which in turn control critical cell functions.
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engineered mutations in Fibrillin 1 leading to marfan syndrome act at the protein cellular and organismal levels
Mutation Research-reviews in Mutation Research, 2015Co-Authors: Karina A Zeyer, Dieter P. ReinhardtAbstract:Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the Fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in Fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant Fibrillin-1 fragments. Those mutations affect Fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant Fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render Fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse Fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated Fibrillin-1.
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Fibrillin-1 and alpha8 integrin are co-expressed in the glomerulus and interact to convey adhesion of mesangial cells.
Cell Adhesion & Migration, 2014Co-Authors: Ines Marek, Dieter P. Reinhardt, Beate Bieritz, Karl F. Hilgers, Gudrun Volkert, Wolfgang Rascher, Andrea HartnerAbstract:Fibrillin-1 is a microfibrillar extracellular matrix protein that was described to be a ligand for α8 integrin. α8 integrin is a matrix receptor specifically expressed in mesangial and smooth muscle cells of the kidney. In previous studies we detected glomerular expression of Fibrillin-1. Moreover, Fibrillin-1 promoted adhesion, migration, and proliferation of mesangial cells. We hypothesized that Fibrillin-1 and α8 integrin might interact in the glomerulus, and thus, regulate mesangial cell properties. Our studies showed that Fibrillin-1 and α8 integrin colocalize in the glomerular mesangium. Induction of experimental glomerulonephritis led to an increase of both Fibrillin-1 and α8 integrin expression. In vitro studies revealed that mesangial cells deficient for α8 integrin adhere weaker to Fibrillin-1 and migrate more easily on Fibrillin-1 than wild-type mesangial cells. Baseline proliferation on Fibrillin-1 is higher in α8 integrin-deficient mesangial cells, but the induction of proliferation is not di...
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Early Fibrillin-1 assembly monitored through a modifiable recombinant cell approach.
Biomacromolecules, 2014Co-Authors: Dirk Hubmacher, Lynn Y. Sakai, Eric Bergeron, Christine Fagotto-kaufmann, Dieter P. ReinhardtAbstract:: Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in Fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant Fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant Fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant Fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant Fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect Fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length Fibrillin-1 assembly system that allows for rapid analysis of critical roles in Fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of Fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.
Lynn Y. Sakai - One of the best experts on this subject based on the ideXlab platform.
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Fibrillin‐1 in the Vasculature: In Vivo Accumulation of eGFP‐Tagged Fibrillin‐1 in a Knockin Mouse Model
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, 2019Co-Authors: Noe L. Charbonneau, Eric J Carlson, Sara F Tufa, Elise C Manalo, Valerie M Carlberg, Douglas R. Keene, Lynn Y. SakaiAbstract:: Immunolocalization studies have shown that Fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for Fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of Fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which Fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of Fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which Fibrillin-1 green fluorescence was detectable. Other arteries gained detectable Fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which Fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and Fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that Fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific Fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated Fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.
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characterization of metabolic health in mouse models of Fibrillin 1 perturbation
Matrix Biology, 2016Co-Authors: Tezin A Walji, Lynn Y. Sakai, Sarah E Turecamo, Antea J Demarsilis, Robert P Mecham, Clarissa S CraftAbstract:Mutations in the microfibrillar protein Fibrillin-1 or the absence of its binding partner microfibril-associated glycoprotein (MAGP1) lead to increased TGFβ signaling due to an inability to sequester latent or active forms of TGFβ, respectively. Mouse models of excess TGFβ signaling display increased adiposity and predisposition to type-2 diabetes. It is therefore interesting that individuals with Marfan syndrome, a disease in which Fibrillin-1 mutation leads to aberrant TGFβ signaling, typically present with extreme fat hypoplasia. The goal of this project was to characterize multiple Fibrillin-1 mutant mouse strains to understand how Fibrillin-1 contributes to metabolic health. The results of this study demonstrate that Fibrillin-1 contributes little to lipid storage and metabolic homeostasis, which is in contrast to the obesity and metabolic changes associated with MAGP1 deficiency. MAGP1 but not Fibrillin-1 mutant mice had elevated TGFβ signaling in their adipose tissue, which is consistent with the difference in obesity phenotypes. However, Fibrillin-1 mutant strains and MAGP1-deficient mice all exhibit increased bone length and reduced bone mineralization which are characteristic of Marfan syndrome. Our findings suggest that Marfan-associated adipocyte hypoplasia is likely not due to microfibril-associated changes in adipose tissue, and provide evidence that MAGP1 may function independently of Fibrillin in some tissues.
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Early Fibrillin-1 assembly monitored through a modifiable recombinant cell approach.
Biomacromolecules, 2014Co-Authors: Dirk Hubmacher, Lynn Y. Sakai, Eric Bergeron, Christine Fagotto-kaufmann, Dieter P. ReinhardtAbstract:: Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in Fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant Fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant Fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant Fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant Fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect Fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length Fibrillin-1 assembly system that allows for rapid analysis of critical roles in Fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of Fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.
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thoracic aortic aneurysm frequency and dissection are associated with Fibrillin 1 fragment concentrations in circulation
Circulation Research, 2013Co-Authors: Lynn M Marshall, Noe L. Charbonneau, Eric J Carlson, Jean P Omalley, Caryn K Snyder, Susan J Hayflick, Joseph S Coselli, Scott A Lemaire, Lynn Y. SakaiAbstract:Rationale:Mutations in Fibrillin-1 are associated with thoracic aortic aneurysm (TAA) in Marfan syndrome. Genome-wide association studies also implicate Fibrillin-1 in sporadic TAA. Fragmentation of the aortic elastic lamellae is characteristic of TAA. Objective:Immunoassays were generated to test whether circulating fragments of Fibrillin-1, or other microfibril fragments, are associated with TAA and dissection. Methods and Results:Plasma samples were obtained from 1265 patients with aortic aneurysm or dissection and from 125 control subjects. Concentrations of Fibrillin-1, Fibrillin-2, and fibulin-4 were measured with novel immunoassays. One hundred and seventy-four patients (13%) had aneurysms with only abdominal aortic involvement (abdominal aortic aneurysm), and 1091 (86%) had TAA. Of those with TAA, 300 patients (27%) had chronic dissection and 109 (10%) had acute or subacute dissection. Associations of fragment concentrations with TAA (versus abdominal aortic aneurysm) or with dissection (versus no...
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adamts10 protein interacts with Fibrillin 1 and promotes its deposition in extracellular matrix of cultured fibroblasts
Journal of Biological Chemistry, 2011Co-Authors: Wendy E Kutz, Lynn Y. Sakai, Douglas R. Keene, Lauren W Wang, Hannah L Bader, Alana K Majors, Kazushi Iwata, Elias I Traboulsi, Suneel S ApteAbstract:Autosomal recessive and autosomal dominant forms of Weill-Marchesani syndrome, an inherited connective tissue disorder, are caused by mutations in ADAMTS10 (encoding a secreted metalloprotease) and FBN1 (encoding Fibrillin-1, which forms tissue microfibrils), respectively, yet they are clinically indistinguishable. This genetic connection prompted investigation of a potential functional relationship between ADAMTS10 and Fibrillin-1. Specifically, Fibrillin-1 was investigated as a potential ADAMTS10 binding partner and substrate, and the role of ADAMTS10 in influencing microfibril biogenesis was addressed. Using ligand affinity blotting and surface plasmon resonance, recombinant ADAMTS10 was found to bind to Fibrillin-1 with a high degree of specificity and with high affinity. Two sites of ADAMTS10 binding to Fibrillin-1 were identified, one toward the N terminus and another in the C-terminal half of Fibrillin-1. Confocal microscopy and immunoelectron microscopy localized ADAMTS10 to Fibrillin-1-containing microfibrils in human tissues. Furin-activated ADAMTS10 could cleave Fibrillin-1, but innate resistance of ADAMTS10 zymogen to propeptide excision by furin was observed, suggesting that, unless activated, ADAMTS10 is an inefficient Fibrillinase. To investigate the role of ADAMTS10 in microfibril biogenesis, fetal bovine nuchal ligament cells were cultured in the presence or absence of ADAMTS10. Exogenously added ADAMTS10 led to accelerated Fibrillin-1 microfibril biogenesis. Conversely, fibroblasts obtained from a Weill-Marchesani syndrome patient with ADAMTS10 mutations deposited Fibrillin-1 microfibrils sparsely compared with unaffected control cells. Taken together, these findings suggest that ADAMTS10 participates in microfibril biogenesis rather than in Fibrillin-1 turnover.
Cay M Kielty - One of the best experts on this subject based on the ideXlab platform.
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immobilisation of a Fibrillin 1 fragment enhances the biocompatibility of ptfe
Colloids and Surfaces B: Biointerfaces, 2014Co-Authors: Hamid Hajian, Cay M Kielty, Anthony S. Weiss, Steven G. Wise, Alexey Kondyurin, Anna Waterhouse, Louise L. Dunn, Young Yu, Marcela M.m. Bilek, Paul G BannonAbstract:Current vascular biomaterials exhibit poor biocompatibility characterised by failure to promote endothelialisation, predisposition to neoinitmal hyperplasia and excessive thrombogenicity. Fibrillin-1, a major constituent of microfibrils is associated with elastic fibres in the arterial wall. Fibrillin-1 binds to endothelial cells through an RGD cell adhesion motif in the fourth TB module. The RGD motif is present in PF8, a recombinant Fibrillin-1 fragment. We investigated the potential of PF8 to improve the biocompatibility of PTFE. PF8 enhanced endothelial cell attachment and cell proliferation to a greater extent than fibronectin (p < 0.01). PF8 immobilised on PTFE using plasma immersion ion implantation (PIII), retained these favourable cell interactive properties, again promoting endothelial cell attachment and proliferation. The thrombogenicity of covalently bound PF8 on PTFE was assessed in both static and dynamic conditions. In static conditions, uncoated PIII treated PTFE was more thrombogenic than untreated PTFE, while PF8 coating reduced thrombogenicity. Under flow, there was no difference in the thrombogenicity of PF8 coated PTFE and untreated PTFE. Immobilised PF8 shows a striking ability to promote attachment and growth of endothelial cells on PTFE, while providing a non-thrombogenic surface. These features make PF8 a promising candidate to improve the biocompatibility of current synthetic vascular grafts.
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Immobilisation of a Fibrillin-1 fragment enhances the biocompatibility of PTFE.
Colloids and Surfaces B: Biointerfaces, 2014Co-Authors: Hamid Hajian, Cay M Kielty, Anthony S. Weiss, Steven G. Wise, Alexey Kondyurin, Anna Waterhouse, Louise L. Dunn, Young Yu, Marcela M.m. BilekAbstract:Current vascular biomaterials exhibit poor biocompatibility characterised by failure to promote endothelialisation, predisposition to neoinitmal hyperplasia and excessive thrombogenicity. Fibrillin-1, a major constituent of microfibrils is associated with elastic fibres in the arterial wall. Fibrillin-1 binds to endothelial cells through an RGD cell adhesion motif in the fourth TB module. The RGD motif is present in PF8, a recombinant Fibrillin-1 fragment. We investigated the potential of PF8 to improve the biocompatibility of PTFE. PF8 enhanced endothelial cell attachment and cell proliferation to a greater extent than fibronectin (p
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heparan sulfate regulates Fibrillin 1 n and c terminal interactions
Journal of Biological Chemistry, 2008Co-Authors: Stuart A Cain, Adrian Shuttleworth, Andrew K Baldwin, Yashithra Mahalingam, Bertrand Raynal, Thomas A Jowitt, John R Couchman, Cay M KieltyAbstract:Abstract Fibrillin-1 N- and C-terminal heparin binding sites have been characterized. An unprocessed monomeric N-terminal fragment (PF1) induced a very high heparin binding response, indicating heparin-mediated multimerization. Using PF1 deletion and short fragments, a heparin binding site was localized within the domain encoded by exon 7 after the first hybrid domain. Rodent embryonic fibroblasts adhered to PF1 and deletion fragments, and, when cells were plated on Fibrillin-1 or fibronectin Arg-Gly-Asp cell-binding fragments, cells showed heparin-dependent spreading and focal contact formation in response to soluble PF1. Within domains encoded by exons 59–62 near the Fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal Fibrillin-1 interactions. Thus, Fibrillin-1 N-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls Fibrillin-1 interactions.
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Fibrillin 1 regulates the bioavailability of tgfβ1
Journal of Cell Biology, 2007Co-Authors: Cay M Kielty, Adrian Shuttleworth, Amanda Morgan, Shazia S Chaudhry, Stuart A Cain, Sarah L DallasAbstract:We have discovered that Fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) β1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFβ signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a Fibrillin-1 sequence encoded by exons 44–49 releases endogenous TGFβ1, thereby stimulating TGFβ receptor–mediated Smad2 signaling. This altered TGFβ1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFβ1 or its receptors. Mass spectrometry revealed that a Fibrillin-1 fragment containing the TGFβ1-releasing sequence specifically associates with full-length Fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal Fibrillin-1, thereby inhibiting the association of C-terminal latent TGFβ-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal Fibrillin-1. By releasing LLC from microfibrils, the Fibrillin-1 sequence encoded by exons 44–49 can contribute to MFS and related diseases.
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Fibrillin 1 interactions with heparin implications for microfibril and elastic fiber assembly
Journal of Biological Chemistry, 2005Co-Authors: Stuart A Cain, Adrian Shuttleworth, Anthony S. Weiss, Amanda Morgan, Clair Baldock, John Thomas Gallagher, Cay M KieltyAbstract:Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate Fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on Fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the Fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with Fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four Fibrillin-1 sites. Heparin did not inhibit Fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the Fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central Fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.
Francesco Ramirez - One of the best experts on this subject based on the ideXlab platform.
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The influence of Fibrillin‐1 and physical activity upon tendon tissue morphology and mechanical properties in mice
Physiological Reports, 2019Co-Authors: Peter H T Tran, Francesco Ramirez, Tanja Skrba, Elisabeth Wondimu, Giuseppina Galatioto, Rene B Svensson, Annesofie T. Olesen, Abigail L. Mackey, S. Peter Magnusson, Michael KjaerAbstract:: Fibrillin-1 mutations cause pathological changes in connective tissue that constitute the complex phenotype of Marfan syndrome. In this study, we used Fibrillin-1 hypomorphic and haploinsufficient mice (Fbn1mgr/mgR and Fbn1+/- mice, respectively) to investigate the impact of Fibrillin-1 deficiency alone or in combination with regular physical activity on tendon tissue morphology and mechanical properties. Morphological and biomechanical analyses revealed that Fbn1mgr/mgR but not Fbn1+/- mice displayed smaller tendons with physical properties that were unremarkable when normalized to tendon size. Fbn1mgR/mgR mice (n = 43) Fbn1+/- mice (n = 27) and wild-type mice (WT, n = 25) were randomly assigned to either control cage conditions (n = 54) or to a running on a running wheel for 4 weeks (n = 41). Both Fibrillin-1-deficient mice ran voluntarily on the running wheel in a manner similar to WT mice (3-4 km/24 h). Regular exercise did not mitigate aneurysm progression in Fbn1mgR/mgR mice (P
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Fibrillin 1 microfibrils influence adult bone marrow hematopoiesis
Matrix Biology, 2016Co-Authors: Silvia Smaldone, Carolina L Bigarella, Maria Del Solar, Saghi Ghaffari, Francesco RamirezAbstract:Abstract We have recently demonstrated that Fibrillin-1 assemblies regulate the fate of skeletal stem cells (aka, mesenchymal stem cells [MSCs]) by modulating TGFβ activity within the microenvironment of adult bone marrow niches. Since MSCs can also influence hematopoietic stem cell (HSC) activities, here we investigated adult hematopoiesis in mice with Cre-mediated inactivation of the Fibrillin-1 ( Fbn1 ) gene in the mesenchyme of the forming limbs ( Fbn1 Prx1 − / − mice). Analyses of 3-month-old Fbn1 Prx1 − / − mice revealed a statistically significant increase of circulating red blood cells, which a differentiation assay correlated with augmented erythropoiesis. This finding, together with evidence of Fibrillin-1 deposition in erythroblastic niches, supported the notion that this extracellular matrix protein normally restricts differentiation of erythroid progenitors. Whereas flow cytometry measurements identified a decreased HSC frequency in mutant relative to wild type mice, no appreciable differences were noted with regard to the relative abundance and differentiation potential of myeloid progenitor cells. Together these findings implied that Fibrillin-1 normally promotes HSC expansion but does not influence cell lineage commitment. Since local TGFβ hyperactivity has been associated with abnormal osteogenesis in Fbn1 Prx1 − / − mice, 1-month-old mutant and wild type animals were systemically treated for 8 weeks with either a pan-TGF-β-neutralizing antibody or an antibody of the same IgG1 isotype. The distinct outcomes of these pharmacological interventions strongly suggest that Fibrillin-1 differentially modulates TGFβ activity in HSC vs. erythroid niches.
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Fibrillin 1 genetic deficiency leads to pathological ageing of arteries in mice
The Journal of Pathology, 2011Co-Authors: Boubacar Mariko, Mylene Pezet, Brigitte Escoubet, Stephanie Bouillot, Jeanpierre Andrieu, Barry Starcher, Daniela Quaglino, Mariepaule Jacob, Philippe Huber, Francesco RamirezAbstract:Fibrillin-1, the major component of extracellular microfibrils that associate with insoluble elastin in elastic fibres, is mainly synthesized during development and postnatal growth and is believed to guide elastogenesis. Mutations in the Fibrillin-1 gene cause Marfan syndrome, a multisystem disorder characterized by aortic aneurysms and dissections. The recent finding that early deficiency of elastin modifies vascular ageing has raised the possibility that Fibrillin-1 deficiency could also contribute to late-onset pathology of vascular remodelling. To address this question, we examined cardiovascular function in 3-week-old, 6-month-old, and 24-month-old mice that are heterozygous for a hypomorphic structural mutation of Fibrillin-1 (Fbn1 mice). Our results indicate that Fbn1 mice, particularly those that are 24 months old, are slightly more hypotensive than wild-type littermates. Additionally, aneurysm and aortic insufficiency were more frequently observed in ageing Fbn1 mice than in the wild-type counterparts. We also noted substantial fragmentation and decreased number of elastic lamellae in the aortic wall of Fbn1 mice, which were correlated with an increase in aortic stiffness, a decrease in vasoreactivity, altered expression of elastic fibre-related genes, including Fibrillin-1 and elastin, and a decrease in the relative ratio between tissue elastin and collagen. Collectively, our findings suggest that the heterozygous mgΔ mutation accelerates some aspects of vascular ageing and eventually leads to aortic manifestations resembling those of Marfan syndrome. Importantly, our data also indicate that vascular abnormalities in Fbn1 mice are opposite to those induced by elastin haploinsufficiency during ageing that affect blood pressure, vascular dimensions, and number of elastic lamellae. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Material and mechanical properties of bones deficient for Fibrillin-1 or Fibrillin-2 microfibrils.
Matrix Biology, 2011Co-Authors: Emilio Arteaga-solis, Lee Sui-arteaga, Mitchell B. Schaffler, Karl J. Jepsen, Nancy Pleshko, Francesco RamirezAbstract:The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of Fibrillin-1 or Fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in Fibrillin-1 or Fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that Fibrillin-1 plays a predominantly greater role than Fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that Fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than Fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by Fibrillin proteins. Our study therefore advance our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in Fibrillin-1 or -2.
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in vivo studies of mutant Fibrillin 1 microfibrils
Journal of Biological Chemistry, 2010Co-Authors: Noe L. Charbonneau, Francesco Ramirez, Gerhard Sengle, Eric J Carlson, Sara F Tufa, Elise C Manalo, Valerie M Carlberg, D R Keene, Lynn Y. SakaiAbstract:In humans, mutations in Fibrillin-1 result in a variety of genetic disorders with distinct clinical phenotypes. While most of the known mutations in Fibrillin-1 cause Marfan syndrome, a number of other mutations lead to clinical features unrelated to Marfan syndrome. Pathogenesis of Marfan syndrome is currently thought to be driven by mechanisms due to haploinsufficiency of wild-type Fibrillin-1. However, haploinsufficiency-driven mechanisms cannot explain the distinct phenotypes found in other Fibrillinopathies. To test the hypothesis that mutations in Fibrillin-1 cause disorders through primary effects on microfibril structure, two different mutations were generated in Fbn1 in mice. One mutation leads to a truncated Fibrillin-1 molecule that is tagged with green fluorescent protein, allowing visualization of mutant Fibrillin-1 incorporated into microfibrils. In heterozygosity, these mutant mice demonstrate progressive fragmentation of the aortic elastic lamellae and also display fragmentation of microfibrils in other tissues. Fibrillin-2 epitopes are also progressively revealed in these mice, suggesting that Fibrillin-2 immunoreactivity can serve as a marker for microfibril degradation. In contrast, a second mutation (in-frame deletion of the first hybrid domain) in Fibrillin-1 results in stable microfibrils, demonstrating that Fibrillin-1 molecules are not required to be in perfect register for microfibril structure and function and that the first hybrid domain is dispensable for microfibril assembly. Taken together, these results suggest that perturbation of microfibril structure may underlie one of the major features of the Marfan syndrome: fragmentation of aortic elastic lamellae.
Douglas R. Keene - One of the best experts on this subject based on the ideXlab platform.
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Fibrillin‐1 in the Vasculature: In Vivo Accumulation of eGFP‐Tagged Fibrillin‐1 in a Knockin Mouse Model
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, 2019Co-Authors: Noe L. Charbonneau, Eric J Carlson, Sara F Tufa, Elise C Manalo, Valerie M Carlberg, Douglas R. Keene, Lynn Y. SakaiAbstract:: Immunolocalization studies have shown that Fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for Fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of Fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which Fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of Fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which Fibrillin-1 green fluorescence was detectable. Other arteries gained detectable Fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which Fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and Fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that Fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific Fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated Fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.
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microenvironmental regulation by Fibrillin 1
PLOS Genetics, 2012Co-Authors: Gerhard Sengle, Eric J Carlson, Sara F Tufa, Douglas R. Keene, Ko Tsutsui, Noe L. CharbonneauAbstract:Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for Fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that Fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in Fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving Fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the Fibrillin microfibril scaffold can influence local tissue microenvironments and link Fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a Fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.
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adamts10 protein interacts with Fibrillin 1 and promotes its deposition in extracellular matrix of cultured fibroblasts
Journal of Biological Chemistry, 2011Co-Authors: Wendy E Kutz, Lynn Y. Sakai, Douglas R. Keene, Lauren W Wang, Hannah L Bader, Alana K Majors, Kazushi Iwata, Elias I Traboulsi, Suneel S ApteAbstract:Autosomal recessive and autosomal dominant forms of Weill-Marchesani syndrome, an inherited connective tissue disorder, are caused by mutations in ADAMTS10 (encoding a secreted metalloprotease) and FBN1 (encoding Fibrillin-1, which forms tissue microfibrils), respectively, yet they are clinically indistinguishable. This genetic connection prompted investigation of a potential functional relationship between ADAMTS10 and Fibrillin-1. Specifically, Fibrillin-1 was investigated as a potential ADAMTS10 binding partner and substrate, and the role of ADAMTS10 in influencing microfibril biogenesis was addressed. Using ligand affinity blotting and surface plasmon resonance, recombinant ADAMTS10 was found to bind to Fibrillin-1 with a high degree of specificity and with high affinity. Two sites of ADAMTS10 binding to Fibrillin-1 were identified, one toward the N terminus and another in the C-terminal half of Fibrillin-1. Confocal microscopy and immunoelectron microscopy localized ADAMTS10 to Fibrillin-1-containing microfibrils in human tissues. Furin-activated ADAMTS10 could cleave Fibrillin-1, but innate resistance of ADAMTS10 zymogen to propeptide excision by furin was observed, suggesting that, unless activated, ADAMTS10 is an inefficient Fibrillinase. To investigate the role of ADAMTS10 in microfibril biogenesis, fetal bovine nuchal ligament cells were cultured in the presence or absence of ADAMTS10. Exogenously added ADAMTS10 led to accelerated Fibrillin-1 microfibril biogenesis. Conversely, fibroblasts obtained from a Weill-Marchesani syndrome patient with ADAMTS10 mutations deposited Fibrillin-1 microfibrils sparsely compared with unaffected control cells. Taken together, these findings suggest that ADAMTS10 participates in microfibril biogenesis rather than in Fibrillin-1 turnover.
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adamtsl 6 is a novel extracellular matrix protein that binds to Fibrillin 1 and promotes Fibrillin 1 fibril formation
Journal of Biological Chemistry, 2010Co-Authors: Ko Tsutsui, Lynn Y. Sakai, Gerhard Sengle, Douglas R. Keene, Ri Ichiroh Manabe, Tomiko Yamada, Itsuko Nakano, Yasuko Oguri, Kiyotoshi SekiguchiAbstract:Abstract ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs)-like (ADAMTSL) proteins, a subgroup of the ADAMTS superfamily, share several domains with ADAMTS proteinases, including thrombospondin type I repeats, a cysteine-rich domain, and an ADAMTS spacer, but lack a catalytic domain. We identified two new members of ADAMTSL proteins, ADAMTSL-6α and -6β, that differ in their N-terminal amino acid sequences but have common C-terminal regions. When transfected into MG63 osteosarcoma cells, both isoforms were secreted and deposited into pericellular matrices, although ADAMTSL-6α, in contrast to -6β, was barely detectable in the conditioned medium. Immunolabeling at the light and electron microscopic levels showed their close association with Fibrillin-1-rich microfibrils in elastic connective tissues. Surface plasmon resonance analyses demonstrated that ADAMTSL-6β binds to the N-terminal half of Fibrillin-1 with a dissociation constant of ∼80 nm. When MG63 cells were transfected or exogenously supplemented with ADAMTSL-6, Fibrillin-1 matrix assembly was promoted in the early but not the late stage of the assembly process. Furthermore, ADAMTSL-6 transgenic mice exhibited excessive Fibrillin-1 fibril formation in tissues where ADAMTSL-6 was overexpressed. All together, these results indicated that ADAMTSL-6 is a novel microfibril-associated protein that binds directly to Fibrillin-1 and promotes Fibrillin-1 matrix assembly.
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versican interacts with Fibrillin 1 and links extracellular microfibrils to other connective tissue networks
Journal of Biological Chemistry, 2002Co-Authors: Zenzo Isogai, Dieter P. Reinhardt, Douglas R. Keene, Anders Aspberg, Lynn Y. SakaiAbstract:Fibrillin-containing microfibrils are polymeric structures that are difficult to extract from connective tissues. Proteolytic digestion of tissues has been utilized to release microfibrils for study. Few of the molecules that connect microfibrils to other elements in the matrix have been identified. In this study, electron microscopic immunolocalization of anti-versican antibodies in tissues and in extracted microfibrils demonstrated that the C-terminal region of versican is found associated with Fibrillin microfibrils. Extraction of microfibrils followed by treatment of microfibrils under dissociating conditions suggested that the versican C terminus is covalently bound to microfibrils. Binding assays using recombinant Fibrillin-1 polypeptides and recombinant lectican lectin domains indicated that the versican lectin domain binds to specific Fibrillin-1 polypeptides. The versican lectin domain also bound to molecules comigrating with authentic Fibrillin-1 monomers in anassay using cell culture medium. In assays using microfibrils, the versican lectin domain demonstrated preferential binding compared with other lecticans. Binding was calcium-dependent. The binding site for versican in microfibrils is most likely within a region of Fibrillin-1 between calcium-binding epidermal growth factor-like domains 11 and 21. Human mutations irk this region can result in severe forms of the Marfan syndrome ("neonatal" Marfan syndrome). The connection between versican and Fibrillin microfibrils may be functionally significant, particularly in cardiovascular tissues.