The Experts below are selected from a list of 25308 Experts worldwide ranked by ideXlab platform
Robert P Mecham - One of the best experts on this subject based on the ideXlab platform.
-
functionally distinct tendons from Elastin haploinsufficient mice exhibit mild stiffening and tendon specific structural alteration
Journal of Biomechanical Engineering-transactions of The Asme, 2017Co-Authors: Jeremy D Eekhoff, Robert P Mecham, Austin J. Cocciolone, Jessica E. Wagenseil, Fei Fang, Lindsey G Kahan, Gabriela M Espinosa, Spencer P LakeAbstract:Elastic fibers are present in low quantities in tendon, where they are located both within fascicles near tenocytes and more broadly in the interfascicular matrix (IFM). While elastic fibers have long been known to be significant in the mechanics of Elastin-rich tissue (i.e., vasculature, skin, lungs), recent studies have suggested a mechanical role for elastic fibers in tendons that is dependent on specific tendon function. However, the exact contribution of Elastin to properties of different types of tendons (e.g., positional, energy-storing) remains unknown. Therefore, this study purposed to evaluate the role of Elastin in the mechanical properties and collagen alignment of functionally distinct supraspinatus tendons (SSTs) and Achilles tendons (ATs) from Elastin haploinsufficient (HET) and wild type (WT) mice. Despite the significant decrease in Elastin in HET tendons, a slight increase in linear stiffness of both tendons was the only significant mechanical effect of Elastin haploinsufficiency. Additionally, there were significant changes in collagen nanostructure and subtle alteration to collagen alignment in the AT but not the SST. Hence, Elastin may play only a minor role in tendon mechanical properties. Alternatively, larger changes to tendon mechanics may have been mitigated by developmental compensation of HET tendons and/or the role of elastic fibers may be less prominent in smaller mouse tendons compared to the larger bovine and human tendons evaluated in previous studies. Further research will be necessary to fully elucidate the influence of various elastic fiber components on structure-function relationships in functionally distinct tendons.
-
Fibulin-4 is essential for maintaining arterial wall integrity in conduit but not muscular arteries
Science advances, 2017Co-Authors: Carmen M. Halabi, Thomas J. Broekelmann, Michelle Lin, Vivian Lee, Mon-li Chu, Robert P MechamAbstract:Homozygous or compound heterozygous mutations in fibulin-4 (FBLN4) lead to autosomal recessive cutis laxa type 1B (ARCL1B), a multisystem disorder characterized by significant cardiovascular abnormalities, including abnormal Elastin assembly, arterial tortuosity, and aortic aneurysms. We sought to determine the consequences of a human disease–causing mutation in FBLN4 (E57K) on the cardiovascular system and vascular elastic fibers in a mouse model of ARCL1B. Fbln4E57K/E57K mice were hypertensive and developed arterial elongation, tortuosity, and ascending aortic aneurysms. Smooth muscle cell organization within the arterial wall of large conducting vessels was abnormal, and elastic fibers were fragmented and had a moth-eaten appearance. In contrast, vessel wall structure and elastic fiber integrity were normal in resistance/muscular arteries (renal, mesenteric, and saphenous). Elastin cross-linking and total Elastin content were unchanged in large or small arteries, whereas elastic fiber architecture was abnormal in large vessels. While the E57K mutation did not affect Fbln4 mRNA levels, FBLN4 protein was lower in the ascending aorta of mutant animals compared to wild-type arteries but equivalent in mesenteric arteries. We found a differential role of FBLN4 in elastic fiber assembly, where it functions mainly in large conduit arteries. These results suggest that Elastin assembly has different requirements depending on vessel type. Normal levels of Elastin cross-links in mutant tissue call into question FBLN4’s suggested role in mediating lysyl oxidase–Elastin interactions. Future studies investigating tissue-specific elastic fiber assembly may lead to novel therapeutic interventions for ARCL1B and other disorders of elastic fiber assembly.
-
Implications for Mouse Models of Human Disease
2015Co-Authors: Human Elastin Gene, Russell H Knutsen, Eiichi Hirano, Hideki Sugitani, Christopher H. Ciliberto, Robert P MechamAbstract:Abstract—Diseases linked to the Elastin gene arise from loss-of-function mutations leading to protein insufficiency (supravalvular aortic stenosis) or from missense mutations that alter the properties of the Elastin protein (dominant cutis laxa). Modeling these diseases in mice is problematic because of structural differences between the human and mouse genes. To address this problem, we developed a humanized Elastin mouse with Elastin production being controlled by the human Elastin gene in a bacterial artificial chromosome. The temporal and spatial expression pattern of the human transgene mirrors the endogenous murine gene, and the human gene accurately recapitulates the alternative-splicing pattern found in humans. Human Elastin protein interacts with mouse Elastin to form functional elastic fibers and when expressed in the Elastin haploinsufficient background reverses the hypertension and cardiovascular changes associated with that phenotype. Elastin from the human transgene also rescues the perinatal lethality associated with the null phenotype. The results of this study confirm that reestablishing normal Elastin levels is a logical objective for treating diseases of Elastin insufficiency such as supravalvular aortic stenosis. This study also illustrates how differences in gene structure and alternative splicing present unique problems for modeling human diseases in mice. (Circ Res. 2007;101:523-531.) Key Words: Elastin supravalvular aortic stenosis vascular disease transgenic mice Mutations within the Elastin gene lead to several elasti-nopathies in humans that affect large blood vessels, th
-
Oxidative and Nitrosative Modifications of TropoElastin Prevent Elastic Fiber Assembly in Vitro
The Journal of biological chemistry, 2010Co-Authors: Kamal Akhtar, Robert P Mecham, Barry Starcher, Thomas J. Broekelmann, Ming Miao, Fred W. Keeley, Richard A. Pierce, Tracy L. Adair-kirkAbstract:Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. TropoElastin (TE), the precursor of Elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of Elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO−) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO− and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.
-
discrete contributions of elastic fiber components to arterial development and mechanical compliance
Arteriosclerosis Thrombosis and Vascular Biology, 2009Co-Authors: Luca Carta, Elaine C Davis, Robert P Mecham, Boubacar Mariko, Barry Starcher, Jessica E. Wagenseil, Russell H Knutsen, Gilles Faury, Francesco RamirezAbstract:Objective— Even though Elastin and fibrillin-1 are the major structural components of elastic fibers, mutations in Elastin and fibrillin-1 lead to narrowing of large arteries in supravascular aortic stenosis and dilation of the ascending aorta in Marfan syndrome, respectively. A genetic approach was therefore used here to distinguish the differential contributions of Elastin and fibrillin-1 to arterial development and compliance. Methods and Results— Key parameters of cardiovascular function were compared among adult mice haploinsufficient for Elastin ( Eln +/−), fibrillin-1 ( Fbn1 +/−), or both proteins ( dHet ). Physiological and morphological comparisons correlate Elastin haploinsufficiency with increased blood pressure and vessel length and tortuosity in dHet mice, and fibrillin-1 haploinsufficiency with increased aortic diameter in the same mutant animals. Mechanical tests confirm that Elastin and fibrillin-1 impart elastic recoil and tensile strength to the aortic wall, respectively. Additional ex vivo analyses demonstrate additive and overlapping contributions of Elastin and fibrillin-1 to the material properties of vascular tissues. Lastly, light and electron microscopy evidence implicates fibrillin-1 in the hypertension-promoted remodeling of the Elastin-deficient aorta. Conclusions— These results demonstrate that Elastin and fibrillin-1 have both differential and complementary roles in arterial wall formation and function, and advance our knowledge of the structural determinants of vascular physiology and disease.
Barry Starcher - One of the best experts on this subject based on the ideXlab platform.
-
Chronic administration of minoxidil protects elastic fibers and stimulates their neosynthesis with improvement of the aorta mechanics in mice
Cellular Signalling, 2019Co-Authors: Wassim Fhayli, Jeanpierre Andrieu, Barry Starcher, Mariepaule Jacob, Marjorie Boyer, Zeinab Ghandour, Eric Estève, Gilles FauryAbstract:Arterial wall elastic fibers, made of 90% Elastin, are arranged into elastic lamellae which are responsible for the resilience and elastic properties of the large arteries (aorta and its proximal branches). Elastin is synthesized only in early life and adolescence mainly by the vascular smooth muscles cells (VSMC) through the cross-linking of its soluble precursor, tropoElastin. In normal aging, the elastic fibers become fragmented and the mechanical load is transferred to collagen fibers, which are 100-1000 times stiffer than elastic fibers. Minoxidil, an ATP-dependent K+ channel opener, has been shown to stimulate Elastin expression in vitro, and in vivo in the aorta of male aged mice and young adult hypertensive rats. Here, we have studied the effect of a 3-month chronic oral treatment with minoxidil (120 mg/L in drinking water) on the abdominal aorta structure and function in adult (6-month-old) and aged (24-month-old) male and female mice. Our results show that minoxidil treatment preserves elastic lamellae integrity at both ages, which is accompanied by the formation of newly synthesized elastic fibers in aged mice. This leads to a generally decreased pulse pressure and a significant improvement of the arterial biomechanical properties in female mice, which present an increased distensibility and a decreased rigidity of the aorta. Our studies show that minoxidil treatment reversed some of the major adverse effects of arterial aging in mice and could be an interesting anti-arterial aging agent, also potentially usable for female-targeted therapies.
-
studies of human pancreatic elastase treatment of rabbit and human vein rings to predict human therapeutic doses
Pharmacology Research & Perspectives, 2016Co-Authors: Steven K Burke, Barry Starcher, David Bunton, Karen Bingham, Emma Moss, Kimberly S Bland, Marco D Wong, Nicholas F FrananoAbstract:Vascular tissue contains abundant elastic fibers that contribute to vessel elasticity. Vonapanitase (formerly PRT-201) is a recombinant human chymotrypsin-like elastase family member 1 (CELA1) shown to cleave the Elastin component of elastic fibers, resulting in increased vessel diameter. The purpose of these current studies was to determine vein diameter, wall thickness, Elastin content, and vonapanitase potency in veins used in a model of arteriovenous fistula (AVF) and in patients undergoing AVF creation for hemodialysis access to guide dose selection for human trials. Rabbit linguofacial, maxillary, and external jugular veins, and human basilic and upper and lower arm cephalic veins were dissected postmortem and sectioned into 2 mm length rings. Rings were incubated in vonapanitase at 37°C at varying concentrations and times. Elastin content was estimated histologically and by quantifying desmosine, a protein cross-link unique to Elastin. Rabbit veins were substantially thinner and contained less Elastin than human veins. In human veins, Elastin content was greatest in basilic and least in lower arm cephalic. Vonapanitase removed Elastin in a time- and concentration-dependent manner in all vein types. A lower concentration of vonapanitase was required to remove Elastin from rabbit relative to human veins. In summary, vonapanitase reduced the Elastin content of rabbit and human veins but did so at a lower concentration in the rabbit veins. Rabbit models may overestimate the potency of vonapanitase in humans. These results indicate that human dose selection should be guided by human vein ring experiments.
-
Analysis of Dermal Elastic Fibers in the Absence of Fibulin-5 Reveals Potential Roles for Fibulin-5 in Elastic Fiber Assembly
2015Co-Authors: Jiwon Choi, Barry Starcher, Hiromi Yanagisawa, Andreas Bergdahl, Qian Zheng, Elaine C DavisAbstract:Fibulin-5 is a 66 kDa modular, extracellular matrix protein that localizes to elastic fibers. Although in vitro protein-protein binding studies have shown that fibulin-5 binds many proteins involved in elastic fiber formation, the specific role of fibulin-5 in elastogenesis remains unclear. To provide a more detailed analysis of elastic fiber assembly in the absence of fibulin-5, the dermis of wild-type and fibulin-5 gene knockout (Fbln5−/−) mice was examined with electron microscopy (EM). Although light microscopy showed apparently normal elastic fibers near the hair follicles and the absence of elastic fibers in the intervening dermis of the Fbln5−/ − mouse, EM revealed the presence of aberrantly assembled elastic fibers in both locales. Instead of the Elastin being incorporated into the microfibrillar scaffold, the Elastin appeared as globules juxtaposed to the microfibrils. Desmosine analysis showed significantly lower levels of mature cross-linked Elastin in the the Fbln5−/ − dermis, however, gene expression levels for tropoElastin and fibrillin-1, the major elastic fiber components, were unaffected. Based on these results, the nature of tropoElastin cross-linking was investigated using domain specific antibodies to lysyl oxidase like-1 (LOXL-1). Immunolocalization with an antibody to the N-terminal pro-peptide, which is cleaved to generate the active enzyme, revealed abundant staining in the Fbln5−/ − dermis and no staining in the wild-type dermis. Overall, these results suggest two previously unrecognized functions for fibulin-5 in elastogenesis; first, to limit the extent of aggregation of tropoElastin monomers and/or coacervates and aid in the incorporation of Elastin into the microfibril bundles, and second, to potentially assist in the activation of LOXL-1
-
fibrillin 1 genetic deficiency leads to pathological ageing of arteries in mice
The Journal of Pathology, 2011Co-Authors: Boubacar Mariko, Mylene Pezet, Jeanpierre Andrieu, Stéphanie Bouillot, Philippe Huber, Daniela Quaglino, Barry Starcher, Mariepaule Jacob, Brigitte Escoubet, 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.
-
Oxidative and Nitrosative Modifications of TropoElastin Prevent Elastic Fiber Assembly in Vitro
The Journal of biological chemistry, 2010Co-Authors: Kamal Akhtar, Robert P Mecham, Barry Starcher, Thomas J. Broekelmann, Ming Miao, Fred W. Keeley, Richard A. Pierce, Tracy L. Adair-kirkAbstract:Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. TropoElastin (TE), the precursor of Elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of Elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO−) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO− and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.
Tomoyuki Nakamura - One of the best experts on this subject based on the ideXlab platform.
-
latent tgf β binding protein 2 binds to dance fibulin 5 and regulates elastic fiber assembly
The EMBO Journal, 2007Co-Authors: Maretoshi Hirai, Masahito Horiguchi, Tetsuya Ohbayashi, Toru Kita, Kenneth R Chien, Tomoyuki NakamuraAbstract:Elastic fibers play the principal roles in providing elasticity and integrity to various types of human organs, such as the arteries, lung, and skin. However, the molecular mechanism of elastic fiber assembly that leads to deposition and crosslinking of Elastin along microfibrils remains largely unknown. We have previously shown that developing arteries and neural crest EGF-like protein (DANCE) (also designated fibulin-5) is essential for elastogenesis by studying DANCE-deficient mice. Here, we report the identification of latent transforming growth factor-β-binding protein 2 (LTBP-2), an elastic fiber-associating protein whose function in elastogenesis is not clear, as a DANCE-binding protein. Elastogenesis assays using human skin fibroblasts reveal that fibrillar deposition of DANCE and Elastin is largely dependent on fibrillin-1 microfibrils. However, downregulation of LTBP-2 induces fibrillin-1-independent fibrillar deposition of DANCE and Elastin. Moreover, recombinant LTBP-2 promotes deposition of DANCE onto fibrillin-1 microfibrils. These results suggest a novel regulatory mechanism of elastic fiber assembly in which LTBP-2 regulates targeting of DANCE on suitable microfibrils to form elastic fibers.
-
targeted disruption of fibulin 4 abolishes elastogenesis and causes perinatal lethality in mice
Molecular and Cellular Biology, 2006Co-Authors: Precious J Mclaughlin, Robert P Mecham, Barry Starcher, Thomas J. Broekelmann, Qiuyun Chen, Brett J Stanton, Alan D Marmorstein, Brian S Mckay, Tomoyuki NakamuraAbstract:Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4 / mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4 / embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4 / mice do not develop intact elasticfibers but contain irregular Elastin aggregates. Electron microscopy revealed that the Elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that Elastin cross-links in fibulin-4 / tissues were largely diminished. However, expression of tropoElastin or lysyl oxidase mRNA was unaffected in fibulin-4 / mice. In addition, fibulin-4 strongly interacts with tropoElastin and colocalizes with elastic fibers in culture. These results demonstrate that
-
fibulin 5 dance is essential for elastogenesis in vivo
Nature, 2002Co-Authors: Tomoyuki Nakamura, Aleksander Hinek, Pilar Ruiz Lozano, Yasuhiro Ikeda, Yoshitaka Iwanaga, Susumu Minamisawa, Chingfeng Cheng, Kazuhiro Kobuke, Nancy D Dalton, Yoshikazu TakadaAbstract:The elastic fibre system has a principal role in the structure and function of various types of organs that require elasticity, such as large arteries, lung and skin1,2. Although elastic fibres are known to be composed of microfibril proteins (for example, fibrillins and latent transforming growth factor (TGF)-β-binding proteins) and polymerized Elastin, the mechanism of their assembly and development is not well understood. Here we report that fibulin-5 (also known as DANCE), a recently discovered integrin ligand3, is an essential determinant of elastic fibre organization. fibulin-5-/- mice generated by gene targeting exhibit a severely disorganized elastic fibre system throughout the body. fibulin-5-/- mice survive to adulthood, but have a tortuous aorta with loss of compliance, severe emphysema, and loose skin (cutis laxa). These tissues contain fragmented Elastin without an increase of elastase activity, indicating defective development of elastic fibres. Fibulin-5 interacts directly with elastic fibres in vitro, and serves as a ligand for cell surface integrins αvβ3, αvβ5 and α9β1 through its amino-terminal domain. Thus, fibulin-5 may provide anchorage of elastic fibres to cells, thereby acting to stabilize and organize elastic fibres in the skin, lung and vasculature.
Elaine C Davis - One of the best experts on this subject based on the ideXlab platform.
-
Analysis of Dermal Elastic Fibers in the Absence of Fibulin-5 Reveals Potential Roles for Fibulin-5 in Elastic Fiber Assembly
2015Co-Authors: Jiwon Choi, Barry Starcher, Hiromi Yanagisawa, Andreas Bergdahl, Qian Zheng, Elaine C DavisAbstract:Fibulin-5 is a 66 kDa modular, extracellular matrix protein that localizes to elastic fibers. Although in vitro protein-protein binding studies have shown that fibulin-5 binds many proteins involved in elastic fiber formation, the specific role of fibulin-5 in elastogenesis remains unclear. To provide a more detailed analysis of elastic fiber assembly in the absence of fibulin-5, the dermis of wild-type and fibulin-5 gene knockout (Fbln5−/−) mice was examined with electron microscopy (EM). Although light microscopy showed apparently normal elastic fibers near the hair follicles and the absence of elastic fibers in the intervening dermis of the Fbln5−/ − mouse, EM revealed the presence of aberrantly assembled elastic fibers in both locales. Instead of the Elastin being incorporated into the microfibrillar scaffold, the Elastin appeared as globules juxtaposed to the microfibrils. Desmosine analysis showed significantly lower levels of mature cross-linked Elastin in the the Fbln5−/ − dermis, however, gene expression levels for tropoElastin and fibrillin-1, the major elastic fiber components, were unaffected. Based on these results, the nature of tropoElastin cross-linking was investigated using domain specific antibodies to lysyl oxidase like-1 (LOXL-1). Immunolocalization with an antibody to the N-terminal pro-peptide, which is cleaved to generate the active enzyme, revealed abundant staining in the Fbln5−/ − dermis and no staining in the wild-type dermis. Overall, these results suggest two previously unrecognized functions for fibulin-5 in elastogenesis; first, to limit the extent of aggregation of tropoElastin monomers and/or coacervates and aid in the incorporation of Elastin into the microfibril bundles, and second, to potentially assist in the activation of LOXL-1
-
in vitro elastogenesis instructing human vascular smooth muscle cells to generate an elastic fiber containing extracellular matrix scaffold
Biomedical Materials, 2015Co-Authors: Elaine C Davis, Svenja Hinderer, Nian Shen, Leajeanne Ringuette, Jan Hansmann, Dieter P Reinhardt, Sara Y Brucker, Katja SchenkelaylandAbstract:Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)Elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased Elastin expression followed by Elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, Elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and Elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.
-
alternative splicing and tissue specific Elastin misassembly act as biological modifiers of human Elastin gene frameshift mutations associated with dominant cutis laxa
Journal of Biological Chemistry, 2012Co-Authors: Hideki Sugitani, Elaine C Davis, Jessica E. Wagenseil, Adrian Shifren, Zsolt Urban, Russell H Knutsen, Eiichi Hirano, Christopher H. Ciliberto, Beth A Kozel, Thomas J. BroekelmannAbstract:Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the Elastin gene has undergone significant changes in the primate lineage, modeling Elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of Elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human Elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant Elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic Elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoElastin in elastic fiber assembly and suggest tissue-specific differences in the Elastin assembly pathway.
-
discrete contributions of elastic fiber components to arterial development and mechanical compliance
Arteriosclerosis Thrombosis and Vascular Biology, 2009Co-Authors: Luca Carta, Elaine C Davis, Robert P Mecham, Boubacar Mariko, Barry Starcher, Jessica E. Wagenseil, Russell H Knutsen, Gilles Faury, Francesco RamirezAbstract:Objective— Even though Elastin and fibrillin-1 are the major structural components of elastic fibers, mutations in Elastin and fibrillin-1 lead to narrowing of large arteries in supravascular aortic stenosis and dilation of the ascending aorta in Marfan syndrome, respectively. A genetic approach was therefore used here to distinguish the differential contributions of Elastin and fibrillin-1 to arterial development and compliance. Methods and Results— Key parameters of cardiovascular function were compared among adult mice haploinsufficient for Elastin ( Eln +/−), fibrillin-1 ( Fbn1 +/−), or both proteins ( dHet ). Physiological and morphological comparisons correlate Elastin haploinsufficiency with increased blood pressure and vessel length and tortuosity in dHet mice, and fibrillin-1 haploinsufficiency with increased aortic diameter in the same mutant animals. Mechanical tests confirm that Elastin and fibrillin-1 impart elastic recoil and tensile strength to the aortic wall, respectively. Additional ex vivo analyses demonstrate additive and overlapping contributions of Elastin and fibrillin-1 to the material properties of vascular tissues. Lastly, light and electron microscopy evidence implicates fibrillin-1 in the hypertension-promoted remodeling of the Elastin-deficient aorta. Conclusions— These results demonstrate that Elastin and fibrillin-1 have both differential and complementary roles in arterial wall formation and function, and advance our knowledge of the structural determinants of vascular physiology and disease.
-
molecular analysis of fibulin 5 function during de novo synthesis of elastic fibers
Molecular and Cellular Biology, 2007Co-Authors: Qian Zheng, Elaine C Davis, Barry Starcher, James A Richardson, Robert D Gerard, Hiromi YanagisawaAbstract:Elastic fibers contribute to the structural support of tissues and to the regulation of cellular behavior. Mice deficient for the fibulin-5 gene (fbln5(-/-)) were used to further elucidate the molecular mechanism of elastic fiber assembly. Major elastic fiber components were present in the skin of fbln5(-/-) mice despite a dramatic reduction of mature elastic fibers. We found that fibulin-5 preferentially bound the monomeric form of Elastin through N-terminal and C-terminal Elastin-binding regions and to a preexisting matrix scaffold through calcium-binding epidermal growth factor (EGF)-like (CB-EGF) domains. We further showed that adenovirus-mediated gene transfer of fbln5 was sufficient to regenerate elastic fibers and increase elastic fiber-cell connections in vivo. A mutant fibulin-5 lacking the first 28 amino acids of the first CB-EGF domain, however, was unable to rescue elastic fiber defects. Fibulin-5 thus serves as an adaptor molecule between monomeric Elastin and the matrix scaffold to aid in elastic fiber assembly. These results also support the potential use of fibulin-5 as a therapeutic agent for the treatment of Elastinopathies.
Aleksander Hinek - One of the best experts on this subject based on the ideXlab platform.
-
changes in Elastin Elastin binding protein and versican in alveoli in chronic obstructive pulmonary disease
Respiratory Research, 2008Co-Authors: Mervyn J Merrilees, Aleksander Hinek, Pamela S T Ching, Brent W Beaumont, Thomas N Wight, Peter N BlackAbstract:Background COPD is characterised by loss of alveolar elastic fibers and by lack of effective repair. Elastic fibers are assembled at cell surfaces by Elastin binding protein (EBP), a molecular chaperone whose function can be reversibility inhibited by chondroitin sulphate of matrix proteoglycans such as versican. This study aimed to determine if alveoli of patients with mild to moderate COPD contained increased amounts of versican and a corresponding decrease in EBP, and if these changes were correlated with decreases in Elastin and FEV1.
-
ellagic and tannic acids protect newly synthesized elastic fibers from premature enzymatic degradation in dermal fibroblast cultures
Journal of Investigative Dermatology, 2006Co-Authors: Felipe Jimenez, Thomas F Mitts, Kela Liu, Yanting Wang, Aleksander HinekAbstract:Progressive proteolytic degradation of cutaneous elastic fibers, that cannot be adequately replaced or repaired by adult dermal fibroblasts, constitutes a major feature of aging skin. Our present investigations, employing monolayer cultures of human dermal fibroblasts and organ cultures of skin biopsies, were aimed at testing whether the hydrophilic tannic acid (TA) and lipophilic ellagic acid (EA) would protect dermal Elastin from exogenous and endogenous enzymatic degradation. Results from both culture systems indicated that dermal fibroblasts, maintained with TA or EA, deposit significantly more elastic fibers than untreated control cultures despite the fact that neither polyphenol enhanced transcription of Elastin mRNA or cellular proliferation. Results of a pulse and chase experiment showed that pretreatment with both polyphenols enhanced biostability of tropoElastin and newly deposited Elastin. Results of in vitro assays indicated that both polyphenols bound to purified Elastin and significantly decreased its proteolytic degradation by elastolytic enzymes belonging to the serine proteinase, cysteine proteinase, and metallo-proteinase families. Importantly, both polyphenols also synergistically enhanced elastogenesis induced by selected elastogenic compounds in cultures of dermal fibroblasts. We propose that EA and TA may be useful for preventing proteolytic degradation of existing dermal elastic fibers and for enhancing more efficient elastogenesis in aged skin.
-
fibulin 5 dance is essential for elastogenesis in vivo
Nature, 2002Co-Authors: Tomoyuki Nakamura, Aleksander Hinek, Pilar Ruiz Lozano, Yasuhiro Ikeda, Yoshitaka Iwanaga, Susumu Minamisawa, Chingfeng Cheng, Kazuhiro Kobuke, Nancy D Dalton, Yoshikazu TakadaAbstract:The elastic fibre system has a principal role in the structure and function of various types of organs that require elasticity, such as large arteries, lung and skin1,2. Although elastic fibres are known to be composed of microfibril proteins (for example, fibrillins and latent transforming growth factor (TGF)-β-binding proteins) and polymerized Elastin, the mechanism of their assembly and development is not well understood. Here we report that fibulin-5 (also known as DANCE), a recently discovered integrin ligand3, is an essential determinant of elastic fibre organization. fibulin-5-/- mice generated by gene targeting exhibit a severely disorganized elastic fibre system throughout the body. fibulin-5-/- mice survive to adulthood, but have a tortuous aorta with loss of compliance, severe emphysema, and loose skin (cutis laxa). These tissues contain fragmented Elastin without an increase of elastase activity, indicating defective development of elastic fibres. Fibulin-5 interacts directly with elastic fibres in vitro, and serves as a ligand for cell surface integrins αvβ3, αvβ5 and α9β1 through its amino-terminal domain. Thus, fibulin-5 may provide anchorage of elastic fibres to cells, thereby acting to stabilize and organize elastic fibres in the skin, lung and vasculature.
-
impaired elastogenesis in hurler disease dermatan sulfate accumulation linked to deficiency in Elastin binding protein and elastic fiber assembly
American Journal of Pathology, 2000Co-Authors: Aleksander Hinek, Sarah E WilsonAbstract:Hurler disease resulting from a deficiency in α-l-iduronidase, which causes an accumulation of dermatan sulfate and heparan sulfate glycosaminoglycans, is characterized by connective tissue and skeletal deformations, cardiomyopathy, cardiac valve defects, and progressive coronary artery stenosis. In this report, we present evidence that accumulation of dermatan sulfate but not heparan sulfate moieties is linked to impaired elastic fiber assembly that, in turn, contributes substantially to the development of the clinical phenotype in Hurler disease. Our data suggest that dermatan sulfate-bearing moieties bind to and cause functional inactivation of the 67-kd Elastin-binding protein, a molecular chaperone for tropoElastin, which normally facilitates its secretion and assembly into elastic fibers. We demonstrate that, in contrast to normal skin fibroblasts and cells from Sanfilippo disease, which accumulate heparan sulfate, Hurler fibroblasts show reduced expression of Elastin-binding protein and do not assemble elastic fibers, despite an adequate synthesis of tropoElastin and sufficient production of a microfibrillar scaffold of elastic fibers. Because cultured Hurler fibroblasts proliferate more quickly than their normal counterparts and the addition of exogenous insoluble Elastin reduces their proliferation, we suggest that cell contacts with insoluble Elastin play an important role in controlling their proliferation.
-
uvb irradiation stimulates deposition of new elastic fibers by modified epithelial cells surrounding the hair follicles and sebaceous glands in mice
Journal of Investigative Dermatology, 1999Co-Authors: Barry Starcher, Richard Pierce, Aleksander HinekAbstract:UVB irradiation stimulates the synthesis of Elastin in the skin of humans and experimental animals. In this study we localized the site and the cells that are responsible for the synthesis of murine dermal elastic fibers. SKH-1 hairless mice were irradiated with UVB and the skin removed for light microscopy, electron microscopy, in situ hybridization, immunohistochemistry, and biochemical studies. In response to chronic low doses of UVB there was an initial moderate increase in tropoElastin mRNA in the papillary dermis. By contrast, there was a continuous marked elevation of collagen α1(I) message localizing to sites of inflammatory cell influx throughout the upper and lower dermis. After 25 wk of UV irradiation there was a 2-fold increase in skin Elastin, yet total collagen remained unchanged. Serial desmosine analysis from en face sections indicated the increase in Elastin content was due to dermal elastic fibers, an increase in the size and number of the dermal cysts, and an increase in subpanniculus elastic fibers. Elastin stains of en face sections suggested that the elastic fibers in the upper dermis were exclusively derived from cells lining the epithelial root sheath and sebaceous glands. In response to UV irradiation, the elastic fibers increased in number and size, wrapping around these structures and aligning in both directions as long fibers parallel to the body axis. Electron micrographs indicated that modified epithelial cells in close proximity to the flattened epithelial cells that encircled the root sheath and sebaceous glands were the source of the elastic fibers.