- Email: [email protected]
Fibulin_4 function in zebrafish
S20
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
488 labeled avidin in cell culture revealed an extracellular matrix pattern that co-localized with fibrillin I. Thus the C-terminus is capable of targeting to the matrix. We are currently using the antibody to the C-terminus and the biotin lableled C-terminal peptide for localization studies in tissue. doi:10.1016/j.matbio.2006.08.055
33 MAGP-1 and hemostasis C.C. Werneck, C.P. Vicente, T.J. Broekelmann, J.S. Weinberg, R.A. Pierce, D.M. Tollefsen, R.P. Mecham Washington University School of Medicine, Saint Louis, MO 63110, United States Microfibril-associated glycoprotein-1 (MAGP-1), a small extracellular glycoprotein (∼31KD), is one of the major components of elastin-containing microfibrils present along the arterial wall. Ross et al. have showed that microfibrils are capable of supporting platelet adhesion under dynamic shear conditions suggesting that microfibrillar components might be involved in hemostasis. To verify if MAGP-1 plays a role in this process we submitted MAGP-1KO mice to a photochemical injury thrombosis assay. In this assay, the endothelium from carotid is denuded leading to thrombus formation and vessel occlusion. The occlusion time obtained for MAGP1KO mice was almost double that of wild-type siblings. The coagulation cascade pathways, platelet number and function are normal. We were not able to detect MAGP-1 inside the platelets and MAGP-1 is incapable of activating platelets by itself or modulating their response in the presence of known agonists. MAGP-1 injection in deficient mice 15 minutes before starting the photochemical assay rescues the normal occlusion time. During earlier steps of thrombus formation, von Willebrand factor (vWF) has an important role binding to the exposed sub-endothelial components making platelets slow down, bind and become activated. We showed that vWF interacts with MAGP-1. Our current hypothesis is that MAGP-1 becomes exposed at the moment of the injury and might be working as an anchor, binding vWF and making the thrombus firm and compact at the site of injury. The mechanisms through which MAGP-1 are involved in thrombus formation are still under investigation. doi:10.1016/j.matbio.2006.08.056
34 ECM protein fibulin-5 and development of atherosclerosis S. Hacker a, P. Boucher b, J. Herz b, H. Yanagisawa
a
a
Molecular Biology, UT Southwestern Medical Center, Dallas, TX, United States b Molecular Genetics, UT Southwestern Medical Center, Dallas, TX, United States Atherosclerosis is a complex process involving endothelial cell dysfunction, production of superoxides, and activation of vascular smooth muscle cells (SMCs). The migration and proliferation of SMC and influx of macrophages and cholesterol molecules produces inflammation and damage to the elastic laminae. We have previously reported that mice deficient for the fibulin-5 gene (fbln5−/−) develop disorganized elastic laminae and fbln5−/− vessels display an exaggerated remodeling response to mechanical injury. We hypothesized that fbln5−/− mice would develop more severe atherosclerotic lesions due to the loss of intact elastic laminae. Fbln5−/− mice were mated to low-density lipoprotein receptor-null (ldlr−/−) mice and the progression of atherosclerotic lesions was compared between ldlr−/− and fbln5−/−; ldlr−/− (DKO) mice. Although we observed a significant increase in plasma cholesterol level in DKO mice after 8 weeks of high cholesterol diet, no difference was observed after 16 weeks of diet. Unexpectedly, whole-mount sudan IV staining revealed that DKO mice developed less severe atherosclerotic lesions compared to the ldlr−/− mice. Modest activation of the PDGFß receptormediated signaling pathway was observed in both genotypes. MMP2 activity was comparable between ldlr−/− and DKO mice. In contrast, MMP9 activity and SOD3 levels were altered in DKO mice. These data suggest that elastic laminae do not play a protective role in development of atherosclerosis, however, the cellular function of fibulin-5 needs to be further examined. doi:10.1016/j.matbio.2006.08.057
35 Fibulin-4 function in zebrafish A.B. Maxfield a, V. Hucthagowder a, J.D. Gitlin a, E.M. Joseph b, Z. Urban a a
Departments of Pediatrics and Genetics, Washington University School of Medicine, St. Louis, MO 63110, United States b Massachusetts General Hospital, Charlestown, MA 02129, United States Recently, a homozygous mutation in fibulin-4 has been linked to a severe connective-tissue disease. To elucidate the molecular mechanisms of this novel disease, a zebrafish model organism was chosen for study. In silico analysis of the zebrafish genome demonstrated that human FBLN4 has two paralogues in the zebrafish genome, fbln4a and fbln4b. Whole mount in situ hybrization analysis of zebrafish fbln4a and fbln4b displays a dynamic expression pattern during embryogenesis associated with the segmentation of the lateral mesoderm. Expression of fibulin 4a is then detectable in the bulbus arteriosus (outflow tract)
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
of the developing heart around 101 hours post fertilization (hpf). Zebrafish embryos injected with morpholinos designed to knockdown the expression fbln4a and fbln4b during development where dramatically shorter than uninjected controls, displayed delayed development of segmental vasculature and circulation, developed thinner notochords as well as disorganized ventral mesenchyme at 24 hours post injection. At five days post injection, juvenile zebrafish injected with fbln4a specific morpholinos did not develop a swim bladder whereas juvenile fish injected with the fbln4b specific morpholino developed swimbladders but showed impaired gut development. These results indicate that fibulin-4 is critical for multiple developmental processes including segmentation and vasculogenesis. Disruption of early mesodermal differentiation may lead to a complex connective tissue disease in patients with fibulin-4 mutations. doi:10.1016/j.matbio.2006.08.058
36 Evidence for elastic fiber repair in emphysema R.A. Pierce a, J.C. Woods a, G. Patterson a, J.D. Cooper b, J.C. Hogg c a
Washington University School of Medicine, St. Louis, MO 63130, United States b University of British Columbia, Vancouver, Canada c University of Pennsylvania, Philadelphia, PA, United States
Elastic fibers are key targets for degradation in the pathogenesis in emphysema. To explore the hypothesis that there is attempted repair of damage to elastic fibers in human emphysema, specimens of explanted end-stage COPD lungs from transplant recipients (n=10) and control lungs (n=10) were studied for elastic fiber density, quantification and localization of ELN mRNA, and expression of two elastic fiber microfibrillar genes, MFAP2 and emilin-1. Elastin mRNA expression in severe emphysema lungs was upregulated up to 40-fold over levels in control lungs and localized to alveolar myofibroblasts. Elastic fibers were more abundant as a proportion of total tissue in emphysema specimens and there was no selective loss of alveolar myofibroblasts with severe disease. Expression of MFAP2 was induced coordinately with ELN mRNA in emphysema, whereas expression of emilin-1, a negative regulator of TGF-β signaling, was independent of ELN mRNA expression levels. Together these data establish that there is ongoing robust alveolar elastin expression from abundant alveolar myofibroblasts in end-stage emphysema. Expression of some but not all elastic fiber components is coordinated with elastin expression. These findings support the hypothesis that effectiveness of repair of elastic fibers during chronic inflammation from cigarette smoke may contribute to the variable susceptibility to this disease. doi:10.1016/j.matbio.2006.08.059
S21
37 Interactions between LTBPs and fibrillins in vitro and in vivo R.N. Ono a, N.L. Charbonneau a, J. Alvarez a, F. Ramirez b, L.Y. Sakai a a
Shriners Hospital for Children, Oregon Health and Science University, Portland, OR 97239, United States b Child Health Institute of New Jersey, New Brunswick, NJ 08901, United States Previously published results from our lab demonstrated that LTBPs interact with fibrillin-1 in vitro and are associated with fibrillin microfibrils in tissues (1). We showed that the last three C-terminal domains in LTBPs contain the fibrillin-1 binding site, and that the domains present in the recombinant fibrillin-1 peptide called rF38 (EGF2/EGF3/hybrid1/cbEGF1) contains the LTBP binding site. New data more precisely define the LTBP binding site in fibrillin-1. Our goal with these studies is to mutate this site in mice in order to test whether loss of this binding site destabilizes large latent TGFβ complexes and leads to aberrant TGFββ signaling. Weaker binding between LTBPs and fibrillin2 was previously reported (1). In new studies using better antibody detection reagents, we have found that LTBPs interact equally well with fibrillin-1 and fibrillin-2, indicating that our previous results were likely due to poorer detection of bound fibrillin-2. Immunofluorescence of Fbn1 null mice demonstrates localization of LTBPs in fetal tissues, suggesting that fibrillin-2 can stabilize LTBPs in the absence of fibrillin-1. However, in postnatal Fbn1 null mice, LTBP staining is gradually lost in various tissues, showing that fibrillin-1 is required to stabilize LTBPs in postnatal tissues. 1. Isogai, Z., Ono, R.N., Ushiro, S., Keene, D.R., Chen, Y., Mazzieri, R., Charbonneau, N.L., Reinhardt, D.P., Rifkin, D.B., and Sakai, L.Y. Latent Transforming Growth Factor β-binding Protein 1 Interacts with Fibrillin and Is a Microfibril-Associated Protein. J. Biol. Chem. 278:2750-2757, 2003. doi:10.1016/j.matbio.2006.08.060
38 FGF-2 and transcription factor expression in adipose stem cells J.T. Rich, A. McAlinden, L. Sandell Washington University, St. Louis, MO, United States Human adipose-derived stem cells (hADSCs) offer great potential for tissue engineering of bone, cartilage, and adipose. However, variability in proliferation and early senescence limit their use. These effects may be due to varying levels of growth factors in serum. Therefore, we investigated the effect of FGF-2
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
488 labeled avidin in cell culture revealed an extracellular matrix pattern that co-localized with fibrillin I. Thus the C-terminus is capable of targeting to the matrix. We are currently using the antibody to the C-terminus and the biotin lableled C-terminal peptide for localization studies in tissue. doi:10.1016/j.matbio.2006.08.055
33 MAGP-1 and hemostasis C.C. Werneck, C.P. Vicente, T.J. Broekelmann, J.S. Weinberg, R.A. Pierce, D.M. Tollefsen, R.P. Mecham Washington University School of Medicine, Saint Louis, MO 63110, United States Microfibril-associated glycoprotein-1 (MAGP-1), a small extracellular glycoprotein (∼31KD), is one of the major components of elastin-containing microfibrils present along the arterial wall. Ross et al. have showed that microfibrils are capable of supporting platelet adhesion under dynamic shear conditions suggesting that microfibrillar components might be involved in hemostasis. To verify if MAGP-1 plays a role in this process we submitted MAGP-1KO mice to a photochemical injury thrombosis assay. In this assay, the endothelium from carotid is denuded leading to thrombus formation and vessel occlusion. The occlusion time obtained for MAGP1KO mice was almost double that of wild-type siblings. The coagulation cascade pathways, platelet number and function are normal. We were not able to detect MAGP-1 inside the platelets and MAGP-1 is incapable of activating platelets by itself or modulating their response in the presence of known agonists. MAGP-1 injection in deficient mice 15 minutes before starting the photochemical assay rescues the normal occlusion time. During earlier steps of thrombus formation, von Willebrand factor (vWF) has an important role binding to the exposed sub-endothelial components making platelets slow down, bind and become activated. We showed that vWF interacts with MAGP-1. Our current hypothesis is that MAGP-1 becomes exposed at the moment of the injury and might be working as an anchor, binding vWF and making the thrombus firm and compact at the site of injury. The mechanisms through which MAGP-1 are involved in thrombus formation are still under investigation. doi:10.1016/j.matbio.2006.08.056
34 ECM protein fibulin-5 and development of atherosclerosis S. Hacker a, P. Boucher b, J. Herz b, H. Yanagisawa
a
a
Molecular Biology, UT Southwestern Medical Center, Dallas, TX, United States b Molecular Genetics, UT Southwestern Medical Center, Dallas, TX, United States Atherosclerosis is a complex process involving endothelial cell dysfunction, production of superoxides, and activation of vascular smooth muscle cells (SMCs). The migration and proliferation of SMC and influx of macrophages and cholesterol molecules produces inflammation and damage to the elastic laminae. We have previously reported that mice deficient for the fibulin-5 gene (fbln5−/−) develop disorganized elastic laminae and fbln5−/− vessels display an exaggerated remodeling response to mechanical injury. We hypothesized that fbln5−/− mice would develop more severe atherosclerotic lesions due to the loss of intact elastic laminae. Fbln5−/− mice were mated to low-density lipoprotein receptor-null (ldlr−/−) mice and the progression of atherosclerotic lesions was compared between ldlr−/− and fbln5−/−; ldlr−/− (DKO) mice. Although we observed a significant increase in plasma cholesterol level in DKO mice after 8 weeks of high cholesterol diet, no difference was observed after 16 weeks of diet. Unexpectedly, whole-mount sudan IV staining revealed that DKO mice developed less severe atherosclerotic lesions compared to the ldlr−/− mice. Modest activation of the PDGFß receptormediated signaling pathway was observed in both genotypes. MMP2 activity was comparable between ldlr−/− and DKO mice. In contrast, MMP9 activity and SOD3 levels were altered in DKO mice. These data suggest that elastic laminae do not play a protective role in development of atherosclerosis, however, the cellular function of fibulin-5 needs to be further examined. doi:10.1016/j.matbio.2006.08.057
35 Fibulin-4 function in zebrafish A.B. Maxfield a, V. Hucthagowder a, J.D. Gitlin a, E.M. Joseph b, Z. Urban a a
Departments of Pediatrics and Genetics, Washington University School of Medicine, St. Louis, MO 63110, United States b Massachusetts General Hospital, Charlestown, MA 02129, United States Recently, a homozygous mutation in fibulin-4 has been linked to a severe connective-tissue disease. To elucidate the molecular mechanisms of this novel disease, a zebrafish model organism was chosen for study. In silico analysis of the zebrafish genome demonstrated that human FBLN4 has two paralogues in the zebrafish genome, fbln4a and fbln4b. Whole mount in situ hybrization analysis of zebrafish fbln4a and fbln4b displays a dynamic expression pattern during embryogenesis associated with the segmentation of the lateral mesoderm. Expression of fibulin 4a is then detectable in the bulbus arteriosus (outflow tract)
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
of the developing heart around 101 hours post fertilization (hpf). Zebrafish embryos injected with morpholinos designed to knockdown the expression fbln4a and fbln4b during development where dramatically shorter than uninjected controls, displayed delayed development of segmental vasculature and circulation, developed thinner notochords as well as disorganized ventral mesenchyme at 24 hours post injection. At five days post injection, juvenile zebrafish injected with fbln4a specific morpholinos did not develop a swim bladder whereas juvenile fish injected with the fbln4b specific morpholino developed swimbladders but showed impaired gut development. These results indicate that fibulin-4 is critical for multiple developmental processes including segmentation and vasculogenesis. Disruption of early mesodermal differentiation may lead to a complex connective tissue disease in patients with fibulin-4 mutations. doi:10.1016/j.matbio.2006.08.058
36 Evidence for elastic fiber repair in emphysema R.A. Pierce a, J.C. Woods a, G. Patterson a, J.D. Cooper b, J.C. Hogg c a
Washington University School of Medicine, St. Louis, MO 63130, United States b University of British Columbia, Vancouver, Canada c University of Pennsylvania, Philadelphia, PA, United States
Elastic fibers are key targets for degradation in the pathogenesis in emphysema. To explore the hypothesis that there is attempted repair of damage to elastic fibers in human emphysema, specimens of explanted end-stage COPD lungs from transplant recipients (n=10) and control lungs (n=10) were studied for elastic fiber density, quantification and localization of ELN mRNA, and expression of two elastic fiber microfibrillar genes, MFAP2 and emilin-1. Elastin mRNA expression in severe emphysema lungs was upregulated up to 40-fold over levels in control lungs and localized to alveolar myofibroblasts. Elastic fibers were more abundant as a proportion of total tissue in emphysema specimens and there was no selective loss of alveolar myofibroblasts with severe disease. Expression of MFAP2 was induced coordinately with ELN mRNA in emphysema, whereas expression of emilin-1, a negative regulator of TGF-β signaling, was independent of ELN mRNA expression levels. Together these data establish that there is ongoing robust alveolar elastin expression from abundant alveolar myofibroblasts in end-stage emphysema. Expression of some but not all elastic fiber components is coordinated with elastin expression. These findings support the hypothesis that effectiveness of repair of elastic fibers during chronic inflammation from cigarette smoke may contribute to the variable susceptibility to this disease. doi:10.1016/j.matbio.2006.08.059
S21
37 Interactions between LTBPs and fibrillins in vitro and in vivo R.N. Ono a, N.L. Charbonneau a, J. Alvarez a, F. Ramirez b, L.Y. Sakai a a
Shriners Hospital for Children, Oregon Health and Science University, Portland, OR 97239, United States b Child Health Institute of New Jersey, New Brunswick, NJ 08901, United States Previously published results from our lab demonstrated that LTBPs interact with fibrillin-1 in vitro and are associated with fibrillin microfibrils in tissues (1). We showed that the last three C-terminal domains in LTBPs contain the fibrillin-1 binding site, and that the domains present in the recombinant fibrillin-1 peptide called rF38 (EGF2/EGF3/hybrid1/cbEGF1) contains the LTBP binding site. New data more precisely define the LTBP binding site in fibrillin-1. Our goal with these studies is to mutate this site in mice in order to test whether loss of this binding site destabilizes large latent TGFβ complexes and leads to aberrant TGFββ signaling. Weaker binding between LTBPs and fibrillin2 was previously reported (1). In new studies using better antibody detection reagents, we have found that LTBPs interact equally well with fibrillin-1 and fibrillin-2, indicating that our previous results were likely due to poorer detection of bound fibrillin-2. Immunofluorescence of Fbn1 null mice demonstrates localization of LTBPs in fetal tissues, suggesting that fibrillin-2 can stabilize LTBPs in the absence of fibrillin-1. However, in postnatal Fbn1 null mice, LTBP staining is gradually lost in various tissues, showing that fibrillin-1 is required to stabilize LTBPs in postnatal tissues. 1. Isogai, Z., Ono, R.N., Ushiro, S., Keene, D.R., Chen, Y., Mazzieri, R., Charbonneau, N.L., Reinhardt, D.P., Rifkin, D.B., and Sakai, L.Y. Latent Transforming Growth Factor β-binding Protein 1 Interacts with Fibrillin and Is a Microfibril-Associated Protein. J. Biol. Chem. 278:2750-2757, 2003. doi:10.1016/j.matbio.2006.08.060
38 FGF-2 and transcription factor expression in adipose stem cells J.T. Rich, A. McAlinden, L. Sandell Washington University, St. Louis, MO, United States Human adipose-derived stem cells (hADSCs) offer great potential for tissue engineering of bone, cartilage, and adipose. However, variability in proliferation and early senescence limit their use. These effects may be due to varying levels of growth factors in serum. Therefore, we investigated the effect of FGF-2