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Hiromi Yanagisawa - One of the best experts on this subject based on the ideXlab platform.

  • dysregulated assembly of elastic fibers in Fibulin 5 knockout mice results in a tendon specific increase in elastic modulus
    Journal of The Mechanical Behavior of Biomedical Materials, 2021
    Co-Authors: Jeremy D Eekhoff, Hiromi Yanagisawa, Heiko Steenbock, Ian M Berke, Jurgen Brinckmann, Jessica E Wagenseil, Spencer P Lake
    Abstract:

    Elastic fiber assembly is coordinated in part by Fibulin-5, a matricellular protein. When Fibulin-5 is not available to guide elastogenesis, elastin forms into disconnected globules instead of the dense elastic fiber core found in healthy tissues. Despite the growing evidence for a significant role of elastic fibers in tendon mechanics and the clinical relevance to cutis laxa, a human disease which can be caused by a mutation in the gene encoding Fibulin-5, it is unknown how malformed elastic fibers affect tendon function. Therefore, this study investigated the effects of dysregulated elastic fiber assembly in tendons from Fibulin-5 knockout mice in comparison to wild-type controls. Due to evidence for a more prominent role of elastic fibers in tendons with higher functional demands, both the energy-storing Achilles tendon and the more positional tibialis anterior tendon were evaluated. The linear modulus of knockout Achilles tendons was increased compared to controls, yet there was no discernible change in mechanical properties of the tibialis anterior tendon across genotypes. Transmission electron microscopy confirmed the presence of malformed elastic fibers in knockout tendons while no other changes to tendon composition or structure were found. The mechanism behind the increase in linear modulus in Fibulin-5 knockout Achilles tendons may be greater collagen engagement due to decreased regulation of strain-induced structural reorganization. These findings support the theory of a significant, functionally distinct role of elastic fibers in tendon mechanics.

  • dysregulation of Fibulin 5 and matrix metalloproteases in epithelial ovarian cancer
    Oncotarget, 2018
    Co-Authors: Dustin B Manders, Hiromi Yanagisawa, Patrick W Keller, Hari Annavarapu Kishore, Adi F Gazdar, Jun Tsunezumi, Jayanthi S Lea, R A Word
    Abstract:

    Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility. Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC). FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary. Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression. FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC. We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5. MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC. Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors. This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment. Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells. Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

  • reduced biaxial contractility in the descending thoracic aorta of Fibulin 5 deficient mice
    Journal of Biomechanical Engineering-transactions of The Asme, 2016
    Co-Authors: Hiromi Yanagisawa, Saeil Murtada, Jacopo Ferruzzi, Jay D Humphrey
    Abstract:

    The precise role of smooth muscle cell contractility in elastic arteries remains unclear, but accumulating evidence suggests that smooth muscle dysfunction plays an important role in the development of thoracic aortic aneurysms and dissections (TAADs). Given the increasing availability of mouse models of these conditions, there is a special opportunity to study roles of contractility ex vivo in intact vessels subjected to different mechanical loads. In parallel, of course, there is a similar need to study smooth muscle contractility in models that do not predispose to TAADs, particularly in cases where disease might be expected. Multiple mouse models having compromised glycoproteins that normally associate with elastin to form medial elastic fibers present with TAADs, yet those with Fibulin-5 deficiency do not. In this paper, we show that deletion of the Fibulin-5 gene results in a significantly diminished contractility of the thoracic aorta in response to potassium loading despite otherwise preserved characteristic active behaviors, including axial force generation and rates of contraction and relaxation. Interestingly, this diminished response manifests around an altered passive state that is defined primarily by a reduced in vivo axial stretch. Given this significant coupling between passive and active properties, a lack of significant changes in passive material stiffness may help to offset the diminished contractility and thereby protect the wall from detrimental mechanosensing and its sequelae.

  • pelvic organ support in animals with partial loss of Fibulin 5 in the vaginal wall
    PLOS ONE, 2016
    Co-Authors: Kathleen Chin, Hiromi Yanagisawa, Ignacio T. Montoya, Haolin Shi, Cecilia K Wieslander, Sunil Balgobin, Ann R Word
    Abstract:

    Compromise of elastic fiber integrity in connective tissues of the pelvic floor is most likely acquired through aging, childbirth-associated injury, and genetic susceptibility. Mouse models of pelvic organ prolapse demonstrate systemic deficiencies in proteins that affect elastogenesis. Prolapse, however, does not occur until several months after birth and is thereby acquired with age or after parturition. To determine the impact of compromised levels of Fibulin-5 (Fbln5) during adulthood on pelvic organ support after parturition and elastase-induced injury, tissue-specific conditional knockout (cKO) mice were generated in which doxycycline (dox) treatment results in deletion of Fbln5 in cells that utilize the smooth muscle α actin promoter-driven reverse tetracycline transactivator and tetracycline responsive element-Cre recombinase (i.e., Fbln5f/f/SMA++-rtTA/Cre+, cKO). Fbln5 was decreased significantly in the vagina of cKO mice compared with dox-treated wild type or controls (Fbln5f/f/SMA++-rtTA/Cre-/-). In controls, perineal body length (PBL) and bulge increased significantly after delivery but declined to baseline values within 6-8 weeks. Although overt prolapse did not occur in cKO animals, these transient increases in PBL postpartum were amplified and, unlike controls, parturition-induced increases in PBL (and bulge) did not recover to baseline but remained significantly increased for 12 wks. This lack of recovery from parturition was associated with increased MMP-9 and nondetectable levels of Fbln5 in the postpartum vagina. This predisposition to prolapse was accentuated by injection of elastase into the vaginal wall in which overt prolapse occurred in cKO animals, but rarely in controls. Taken together, our model system in which Fbln5 is conditionally knock-downed in stromal cells of the pelvic floor results in animals that undergo normal elastogenesis during development but lose Fbln5 as adults. The results indicate that vaginal Fibulin-5 during development is crucial for baseline pelvic organ support and is also important for protection and recovery from parturition- and elastase-induced prolapse.

  • Analysis of Dermal Elastic Fibers in the Absence of Fibulin-5 Reveals Potential Roles for Fibulin-5 in Elastic Fiber Assembly
    2015
    Co-Authors: Jiwon Choi, Barry Starcher, Hiromi Yanagisawa, Andreas Bergdahl, Qian Zheng, Elaine C Davis
    Abstract:

    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

Ann R Word - One of the best experts on this subject based on the ideXlab platform.

Elaine C Davis - One of the best experts on this subject based on the ideXlab platform.

  • Fibulin 5 regulates angiopoietin 1 tie 2 receptor signaling in endothelial cells
    PLOS ONE, 2016
    Co-Authors: Wilson Ventura Chan, Kerstin Tiedemann, Elaine C Davis, Hodan Ismail, Dominique Mayaki, Veronica Sanchez, Sabah N A Hussain
    Abstract:

    Background Fibulin-5 is an extracellular matrix glycoprotein that plays critical roles in vasculogenesis and embryonic development. Deletion of Fibulin-5 in mice results in enhanced skin vascularization and upregulation of the angiogenesis factor angiopoietin-1 (Ang-1), suggesting that Fibulin-5 functions as an angiogenesis inhibitor. In this study, we investigate the inhibitory effects of Fibulin-5 on Ang-1/TIE-2 receptor pathway signaling and cell survival in human endothelial cells. Methodology/Principal Findings Recombinant wild-type and RGE-mutant Fibulin-5 proteins were generated through stable transfection of HEK293 and CHO cells, respectively. In vitro solid phase binding assays using pure proteins revealed that wild-type Fibulin-5 does not bind to Ang-1 or TIE-2 proteins but strongly binds to heparin. Binding assays using human umbilical vein endothelial cells (HUVECs) indicated that wild-type Fibulin-5 strongly binds to cells but RGE-mutant Fibulin-5, which is incapable of binding to integrins, does not. Pre-incubation of HUVECs for 1 hr with Fibulin-5 significantly increased caspase 3/7 activity, ERK1/2 phosphorylation, and expressions of the transcription factor early growth response 1 (EGR1) and the dual-specificity phosphatase 5 (DUSP5). Fibulin-5 also strongly attenuated Ang-1-induced TIE-2 and AKT phosphorylation, decreased Ang-1-induced expressions of the transcription factors Inhibitor of DNA Binding 1 (ID1) and Kruppel-like Factor 2 (KLF2), and reversed the inhibitory effect of Ang-1 on serum deprivation-induced cytotoxicity and caspase 3/7 activity. Conclusion/Significance We conclude that Fibulin-5 strongly binds to the endothelial cell surface through heparin-sulfate proteoglycans and possibly integrins and that it exerts strong anti-angiogenic effects by reducing endothelial cell viability and interfering with the signaling pathways of the Ang-1/TIE-2 receptor axis.

  • Fibulin-5 Regulates Angiopoietin-1/Tie-2 Receptor Signaling in Endothelial Cells - Fig 1
    2016
    Co-Authors: Wilson Chan, Kerstin Tiedemann, Elaine C Davis, Hodan Ismail, Dominique Mayaki, Veronica Sanchez, Sabah N A Hussain
    Abstract:

    A: Solid-phase binding assays showing the interactions between Ang-1, TIE-2, and Fibulin-5. Tropoelastin was used as a positive control. Ang-1, TIE-2, and tropoelastin were used as immobilized ligands and Fibulin-5 as the soluble ligand. Fibulin-5 showed a strong binding for tropoelastin but no significant binding was observed to Ang-1 or TIE-2. N = 5 per group. B: Solid-phase binding assays showing the interactions between Ang-1, TIE-2, and Fibulin-5. TIE-2 and Fibulin-5 were used as immobilized ligands and Ang-1 protein as the soluble ligand. Ang-1 showed a strong binding to TIE-2 but no significant binding to Fibulin-5. N = 5 per group.

  • Analysis of Dermal Elastic Fibers in the Absence of Fibulin-5 Reveals Potential Roles for Fibulin-5 in Elastic Fiber Assembly
    2015
    Co-Authors: Jiwon Choi, Barry Starcher, Hiromi Yanagisawa, Andreas Bergdahl, Qian Zheng, Elaine C Davis
    Abstract:

    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

  • function of latent tgfβ binding protein 4 and Fibulin 5 in elastogenesis and lung development
    Journal of Cellular Physiology, 2015
    Co-Authors: Branka Dabovic, Elaine C Davis, Ian B Robertson, Lior Zilberberg, Melinda Vassallo, Daniel B Rifkin
    Abstract:

    Mice deficient in Latent TGFβ Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFβ2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGFβ1 or TGFβ3 did not improve lung septation indicating that the TGFβ isoform elevated in Ltbp4S(-/-) lungs is TGFβ2. Expression of a form of Ltbp4 that could not bind latent TGFβ did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFβ complexes. Therefore, the change in TGFβ-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S(-/-) mutation with a Fibulin-5 null mutant in Fbln5(-/-) ;Ltbp4S(-/-) mice improves the lung septation compared to Ltbp4S(-/-) lungs. Large globular elastin aggregates characteristic for Ltbp4S(-/-) lungs do not form in Fbln5(-/-) ;Ltbp4S(-/-) lungs and EM studies showed that elastic fibers in Fbln5(-/-) ;Ltbp4S(-/-) lungs resemble those found in Fbln5(-/-) mice. These results are consistent with a role for TGFβ2 in lung septation and for Ltbp4 in regulating Fibulin-5 dependent elastic fiber assembly.

  • extracellular matrix proteases contribute to progression of pelvic organ prolapse in mice and humans
    Journal of Clinical Investigation, 2011
    Co-Authors: Madhusudhan Budatha, Elaine C Davis, Barry Starcher, Ann R Word, Qian Zheng, Shelby L. Chapman, Shayzreen M Roshanravan, Cecilia Weislander, Hiromi Yanagisawa
    Abstract:

    Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that Fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, Fibulin-5 inhibited the β1 integrin-dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5RGE/RGE) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to Fibulin-5 knockouts (Fbln5-/-), Fbln5RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with β-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5-/- mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre- and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women.

Tomoyuki Nakamura - One of the best experts on this subject based on the ideXlab platform.

  • stage specific roles of Fibulin 5 during oxidative stress induced renal carcinogenesis in rats
    Free Radical Research, 2011
    Co-Authors: Hiroki Ohara, Tomoyuki Nakamura, Shinya Akatsuka, Hirotaka Nagai, Yuting Liu, Li Jiang, Yasumasa Okazaki, Yoriko Yamashita, Shinya Toyokuni
    Abstract:

    AbstractBy using a rat model of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA), this study performed genome-wide analysis to identify target genes during carcinogenesis. It screened for genes with decreased expression in RCCs, with simultaneous loss of heterozygosity, eventually to focus on the Fibulin-5 (fbln5) gene. Oxidative damage via Fe-NTA markedly increased Fbln5 in the proximal tubules. RCCs presented lower levels of Fbln5. However, a fraction of RCCs presenting pulmonary metastasis revealed significantly higher levels of Fbln5 than those without metastasis, accompanied by immunopositivity of RCC cells and myofibroblast proliferation. Experiments revealed that RCC cell lines showed lower expression of fbln5 than its non-transformed counterpart NRK52E, but that fbln5 transfection to RCC cell lines changed neither proliferation nor migration/invasion. The data suggest that Fbln5 plays a role not only in the tissue repair and remodelling after renal tubular oxidative damage b...

  • latent tgf β binding protein 2 binds to dance Fibulin 5 and regulates elastic fiber assembly
    The EMBO Journal, 2007
    Co-Authors: Maretoshi Hirai, Masahito Horiguchi, Tetsuya Ohbayashi, Toru Kita, Kenneth R Chien, Tomoyuki Nakamura
    Abstract:

    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.

  • reduced secretion of Fibulin 5 in age related macular degeneration and cutis laxa
    Human Mutation, 2006
    Co-Authors: Caroline Ridley, Tomoyuki Nakamura, Edwin M Stone, Andrew J Lotery, Richard Jones, Dominique C Baas, Caroline C W Klaver, A J Luff, Helen Griffiths
    Abstract:

    Age-related macular degeneration (ARMD) is the leading cause of irreversible visual loss in the Western world, affecting approximately 25 million people worldwide. The pathogenesis is complex and missense mutations in FBLN5 have been reported in association with ARMD. We have investigated the role of Fibulin 5 in ARMD by completing the first European study of the gene FBLN5 in ARMD (using 2 European cohorts of 805 ARMD patients and 279 controls) and by determining the functional effects of the missense mutations on Fibulin 5 expression. We also correlated the FBLN5 genotype with the ARMD phenotype. We found two novel sequence changes in ARMD patients that were absent in controls and expressed these and the other nine reported FBLN5 mutations associated with ARMD and two associated with the autosomal recessive disease cutis laxa. Fibulin 5 secretion was significantly reduced (P<0.001) for four ARMD (p.G412E, p.G267S, p.I169 T, and p.Q124P) and two cutis laxa (p.S227P, p.C217R) mutations. These results suggest that some missense mutations associated with ARMD lead to decreased Fibulin 5 secretion with a possible corresponding reduction in elastinogenesis. This study confirms the previous work identifying an association between FBLN5 mutations and ARMD and for the first time suggests a functional mechanism by which these mutations can lead to ARMD. It further demonstrates that FBLN5 mutations can be associated with different phenotypes of ARMD (not limited to the previously described cuticular drusen type). Such knowledge may ultimately lead to the development of novel therapies for this common disease.

  • Fibulin 5 dance is essential for elastogenesis in vivo
    Nature, 2002
    Co-Authors: Tomoyuki Nakamura, Aleksander Hinek, Pilar Ruiz Lozano, Yasuhiro Ikeda, Yoshitaka Iwanaga, Susumu Minamisawa, Chingfeng Cheng, Kazuhiro Kobuke, Nancy D Dalton, Yoshikazu Takada
    Abstract:

    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.

Cay M. Kielty - One of the best experts on this subject based on the ideXlab platform.

  • structural effects of Fibulin 5 missense mutations associated with age related macular degeneration and cutis laxa
    Investigative Ophthalmology & Visual Science, 2010
    Co-Authors: Richard Jones, Caroline Ridley, Ming Chuan Wang, Thomas A Jowitt, Marjorie Howard, Nicoletta Bobola, Tao Wang, Paul N Bishop, Cay M. Kielty
    Abstract:

    PURPOSE: AMD has a complex etiology with environmental and genetic risk factors. Ten Fibulin 5 sequence variants have been associated with AMD and two other Fibulin 5 mutations cause autosomal-recessive cutis laxa. Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Biophysical techniques were used to detect structural changes in the Fibulin 5 mutants and to determine whether changes are predictive of pathogenicity. METHODS: Native PAGE, nonreduced SDS-PAGE, size-exclusion column multiangle laser light scattering, sedimentation velocity, and circular dichroism (CD) were used to investigate the mobility, hydrodynamic radii, folding, and oligomeric states of the Fibulin 5 mutants in the absence and presence of Ca(2+). RESULTS: CD showed that all mutants are folded, although perturbations to secondary structure contents were detected. Both cutis laxa mutants increased dimerization. Most other mutants slightly increased self-association in the absence of Ca(2+) but this was also demonstrated by G202R, a polymorphism detected in a control individual. The AMD-associated mutant G412E showed lower-than-expected mobility during native-PAGE, the largest hydrodynamic radius for the monomer form and the highest levels of aggregation in both the absence and presence of Ca(2+). CONCLUSIONS: The results identified structural differences for the disease-causing cutis laxa mutants and for one AMD variant (G412E), suggesting that this may also be pathogenic. Although the other AMD-associated mutants showed no gross structural differences, they cannot be excluded as pathogenic by differences outside the scope of this study-for example, disruption of heterointeractions.

  • the role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention and the inhibition of smooth muscle cell recruitment
    Biomaterials, 2007
    Co-Authors: Matthew R Williamson, Adrian Shuttleworth, Cay M. Kielty, Ann E Canfield, R A Black
    Abstract:

    The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, Fibulin-5 and tropoelastin. On fibrillin-1 or Fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or Fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and Fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

  • cell matrix biology in vascular tissue engineering
    Journal of Anatomy, 2006
    Co-Authors: Simon Stephan, Amanda Lomas, Cay M. Kielty, Stephen Ball, Matthew R Williamson, Daniel V Bax, Adrian C Shuttleworth
    Abstract:

    We are developing biocompatible small-calibre vascular substitutes based on polymeric scaffolds that incorporate cell-matrix signals to enhance vascular cell attachment and function. Our graft scaffold comprises an outer electrostatically spun porous polyurethane layer seeded with smooth muscle cells, and a luminal polycaprolactone layer for endothelial cell attachment. Vascular cell adhesion properties of three vascular elastic fibre molecules, tropoelastin, fibrillin-1 and Fibulin-5, have been defined, and adhesion fragments optimized. These fragments are being used to coat the scaffolds to enhance luminal endothelial cell attachment, and to regulate smooth muscle cell attachment and function. Tropoelastin-based cell seeding materials are also being developed. In this way, vascular cell-matrix biology is enhancing graft design.

  • Fibulin 5 interacts with fibrillin 1 molecules and microfibrils
    Biochemical Journal, 2005
    Co-Authors: Lyle J Freeman, Nigel Hodson, Kieran T Mellody, Anthony S. Weiss, Amanda Lomas, Adrian Shuttleworth, Michael J. Sherratt, Cay M. Kielty
    Abstract:

    Fibulin-5 plays an important role in elastic fibre formation in vivo. We have investigated the molecular interactions between Fibulin-5 and components of fibrillin-rich microfibrils which form a template for elastin. Fibulin-5 interacted in a dose-dependent manner with a fibrillin-1 N-terminal sequence and with tropoelastin, but not with MAGP-1 (microfibril-associated glycoprotein-1) or decorin. Fibulin-5 did not inhibit interactions between fibrillin-1 N- and C-terminal fragments, or fibrillin-1 interactions with tropoelastin. Fibulin-5 may provide a link between tropoelastin and microfibrils in the pericellular space during elastic fibre assembly.