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Deformation of collagen triple helix in brittle bone under stress
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
193 The subdermal model: New insights in aortic valve calcification F. Ortolani a, A. Bonetti a, M. Contin a, M. Spina b, M. Marchini a a
Department of Medical Morphological Research, University of Udine, Udine, Italy b Department of Experimental Biomedical Sciences, University of Padua, Padua, Italy Although "in vivo" subdermal model is very usefully to predict tissue propensity to calcification, the inherent calcification mechanism is unclear and the reliability of this "in vivo" simulation is still debated. Samples were obtained from (i) native aortic valve leaflets excised from pig (AVLs), (ii) AVLs undergone pre-implantation treatment alone, including fixation in 0.6% glutaraldehyde under nitrogen atmosphere, (pre-TAVLs), and (iii) AVLs implanted into rat subcutis for 2 days, 2 weeks, and 6 weeks (SI-AVLs). TUNEL-reactivity was found for pre-T-AVLs and all SI-AVLs, and ultrastructural apoptosisrelated features were observed. Immunoreactivity was found for Annexin-V, with diffuse intracytoplasmic distribution, for AVLs, pre-T-AVLs and 2d-SI-AVLs, and pericellular localization, for 2w- and 6w-SI-AVLs. Ultrastructural colocalization was evident between Annexin-V (immunogold labeling) and endogenous acidic lipids clustering at cell surfaces (modified pre-embedding reactions with cuprolinic blue) and acting as apatite nucleators (additional post-embedding von Kossa silver staining). These data indicate that hypoxic/anoxic conditions characterizing the pre-T-step prime uncomplete, apoptotic cell death and a distinct cascade of cell reaction/degradation steps leading to initial apatite precipitation. New parameters are now available for more proper comparison between the experimental calcification occuring in the subdermal model and physiological or pathological calcific processes. doi:10.1016/j.matbio.2006.08.216
194 Unique matrix structure in the rER cisternae of PSACH chondrocytes T.M. Merritt a , R. Bick a, B.J. Poindexter a, J.L. Alcorn a, J.T. Hecht a,b a
University of Texas Medical School at Houston, Houston, TX, United States b Shriners Hospital for Children, Houston, TX, United States Mutations in cartilage oligomeric matrix protein (COMP) causes pseudoachondroplasia (PSACH). Since COMP exists as a
S79
homopentamer, one subunit of the COMP complex can be recognized as abnormal and accumulates in expanded rER cisternae. Type IX collagen and matrilin 3 (MATN3), also accumulate in the rER cisternae of PSACH chondrocytes but it is not known how mutant COMP, type IX collagen and MATN3 interact with the retained COMP. These studies focus on defining the organization of these retained proteins using fluorescence deconvolution microscopy. A unique matrix organization was identified in which type II procollagen is found as a central core surrounded by a protein network composed of mutant COMP, type IX collagen and MATN3. This pattern of retained matrix organization was consistent in multiple cisternae from a single chondrocyte and was observed in chondrocytes with different COMP mutations indicating a common pattern of interaction. This suggests that stalling of mutant COMP and an interaction between mutant COMP- type II procollagen could be the initiating events that causes the assembly of matrix in the rER. The presence of type II procollagen in the core of the matrix structure has not previously been reported. The existence of this intracellular matrix explains the relative deficiency of COMP, type IX collagen and MATN3 in the extracellular matrix and possibly why the material is not readily cleared from the rER. Altogether, these results suggest that mutant COMP initiates and perhaps catalyses intracellular matrix assembly. doi:10.1016/j.matbio.2006.08.217
195 Deformation of collagen triple helix in brittle bone under stress E.L. Mertz a, D. McBride b, S. Lekin a b
a
NICHD, NIH, Bethesda, MD, United States University of Maryland, MD, United States
Mechanical testing of mouse model of osteogenesis imperfecta (OI) with G610C mutation in a2-chain of type I collagen was previously shown to exhibit mild bone brittleness. In this work we analyzed potential chemical, structural and dynamic origins of such defect. We used newly developed cell for infrared micro-spectroscopy allowing studying biological tissues in physiological and non-physiological environments and yielding quality and amount of spectral information substantially exceeding those of the previous designs. First, we analyzed more than twenty spectral characteristics of chemical mineral composition, collagen content, absolute and relative alignment of collagen vs. mineral in cortical layer of femur bones at physiological conditions. This analysis indicated no significant chemical and structural differences between homozygous mutant and normal mice. Then, we analyzed response of collagen fibrils in tendon and cortical bone to lateral compression caused by mechanical, thermal or dehydration stresses. These stresses resulted in similar vibrational frequency
S80
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
shifts and reorientation of collagen backbone groups. The results suggest that deformations of a major fraction of triple helical backbone contribute to relaxation of lateral stress in bone. Largely abnormal deformations observed in mutant fibrils indicated that this abnormality may be responsible for mutant bone brittleness. Although tendon fibrils of this particular mutant are composed of 30% mutant 2a1a2(G610C) and 70% “normal” 3a1 collagen molecules, purely homotrimeric 3a1 fibers from OIM mice revealed nearly normal deformations, suggesting that that treatments affecting fraction of 3a1 molecules can modulate bone brittleness in G610C mice. doi:10.1016/j.matbio.2006.08.218
196 Integrin and non-integrin mediated signaling mechanisms of α1(IV)NC1 S. Akulapalli a, D. Cosgrove b, C.M. Overall c, C. Shekhar Boosani a a
Cell Signaling and Angiogenesis Laboratory, Department of Genetics, Boys Town National Research Hospital, Omaha, NE 68131, United States b Gene Expression Laboratory, Department of Genetics, Boys Town National Research Hospital Omaha, NE 68131, United States c The UBC Center for Blood Research, CIHR Group in Matrix Dynamics, University of British Columbia, Vancouver, Canada V6T 1Z3 The proteolytic cleavage fragments of vascular basement membrane (VBM) were found to inhibit the endothelial cell proliferation, migration, tube formations and their corresponding neutralizing antibodies reversed these inhibitory effects. α1 (IV)NC1, which is efficiently generated from VBM or Col Type IV by matrix metalloproteinase 2. In endothelial cells, recombinant human α1(IV)NC1 binds to α1β1 integrin and inhibits focal adhesion kinase/c-Raf/MAPK pathway, affecting HIF-1α and VEGF expression. α1(IV)NC1 influences MMP-2 activation in a non-cell specific way by forming a tight complex with the catalytic domain causing inhibition of endothelial and tumor cell invasion in reconstituted basement membrane. Consistent with this, tumor and matrigel studies in mice deficient in α1 integrin shows inhibition of tumor growth at higher dose of α1(IV)NC1 treatment. In addition α1(IV)NC1 was also found to regulate TIMP-2, MT1-MMP and elastase expressions. Collectively our in vitro and in vivo data is the first evidence describing that proteases generated fragments of VBM have dual function in integrin and non-integrin mediated suppression of tumor angiogenesis. doi:10.1016/j.matbio.2006.08.219
197 Nidogen functions with multiple adhesion/guidance receptors S. Kang, J.M. Kramer Dept. of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, United States Null mutants for the basement membrane (BM) component nidogen in C. elegans, nid-1(0), have defects in synaptic organization/transmission and axon guidance. A screen for mutations with enhanced phenotypes in nid-1(0) background identified mutations in several matrix receptors and downstream effectors that result in synthetic lethality. These include Slit, netrin and semaphorin guidance receptors, the matrix receptors integrin and dystroglycan, and downstream effectors GEF, Rac and Ena. Penetrance of lethality varies (15–100%), but always results from embryonic epithelial adhesion defects. Nervous system defects of the various single mutants are also enhanced in the nid-1(0) background. We propose that nidogen subtly affects signaling through each of these receptors and severe defects only occur when nidogen and one of the receptors are missing. We have generated GFP::NID-1 protein that localizes normally to BMs and rescues mutant phenotypes. BM localization requires the G3 domain and its laminin-binding residues, but not G1 or G2 domains. Non-laminin binding NID1 accumulates in extracellular space during embryogenesis, but rapidly disappears later indicating a post-embryonic uptake/ degradation process. Rescue of epithelial adhesion and axon guidance defects requires laminin-binding G3 and the G1 domain, but deletion of G2 has little effect on phenotypic rescue. Thus, the G1 domain has a critical role in nidogen function, although no binding interactions have been identified. Over-expression of laminin-binding G3 alone dominantly causes lethality and axon defects, suggesting that it may interfere with these receptors and/or their ligands. doi:10.1016/j.matbio.2006.08.220
198 Laminin-nidogen interaction in the induction of AChR clustering S. Smirnov a, D. Harrison a, K. McKee a, U. Mayer b, P.D. Yurchenco a a
Robert Wood Johnson Medical School, Piscataway, NJ, 08854 b School of Biological Sciences, Univ. of East Anglia, Norwich NR4 7TJ, UK Aneural clustering of the acetylcholine receptor (AChR) is an early post-synaptic step of neuromuscular junction assembly.
193 The subdermal model: New insights in aortic valve calcification F. Ortolani a, A. Bonetti a, M. Contin a, M. Spina b, M. Marchini a a
Department of Medical Morphological Research, University of Udine, Udine, Italy b Department of Experimental Biomedical Sciences, University of Padua, Padua, Italy Although "in vivo" subdermal model is very usefully to predict tissue propensity to calcification, the inherent calcification mechanism is unclear and the reliability of this "in vivo" simulation is still debated. Samples were obtained from (i) native aortic valve leaflets excised from pig (AVLs), (ii) AVLs undergone pre-implantation treatment alone, including fixation in 0.6% glutaraldehyde under nitrogen atmosphere, (pre-TAVLs), and (iii) AVLs implanted into rat subcutis for 2 days, 2 weeks, and 6 weeks (SI-AVLs). TUNEL-reactivity was found for pre-T-AVLs and all SI-AVLs, and ultrastructural apoptosisrelated features were observed. Immunoreactivity was found for Annexin-V, with diffuse intracytoplasmic distribution, for AVLs, pre-T-AVLs and 2d-SI-AVLs, and pericellular localization, for 2w- and 6w-SI-AVLs. Ultrastructural colocalization was evident between Annexin-V (immunogold labeling) and endogenous acidic lipids clustering at cell surfaces (modified pre-embedding reactions with cuprolinic blue) and acting as apatite nucleators (additional post-embedding von Kossa silver staining). These data indicate that hypoxic/anoxic conditions characterizing the pre-T-step prime uncomplete, apoptotic cell death and a distinct cascade of cell reaction/degradation steps leading to initial apatite precipitation. New parameters are now available for more proper comparison between the experimental calcification occuring in the subdermal model and physiological or pathological calcific processes. doi:10.1016/j.matbio.2006.08.216
194 Unique matrix structure in the rER cisternae of PSACH chondrocytes T.M. Merritt a , R. Bick a, B.J. Poindexter a, J.L. Alcorn a, J.T. Hecht a,b a
University of Texas Medical School at Houston, Houston, TX, United States b Shriners Hospital for Children, Houston, TX, United States Mutations in cartilage oligomeric matrix protein (COMP) causes pseudoachondroplasia (PSACH). Since COMP exists as a
S79
homopentamer, one subunit of the COMP complex can be recognized as abnormal and accumulates in expanded rER cisternae. Type IX collagen and matrilin 3 (MATN3), also accumulate in the rER cisternae of PSACH chondrocytes but it is not known how mutant COMP, type IX collagen and MATN3 interact with the retained COMP. These studies focus on defining the organization of these retained proteins using fluorescence deconvolution microscopy. A unique matrix organization was identified in which type II procollagen is found as a central core surrounded by a protein network composed of mutant COMP, type IX collagen and MATN3. This pattern of retained matrix organization was consistent in multiple cisternae from a single chondrocyte and was observed in chondrocytes with different COMP mutations indicating a common pattern of interaction. This suggests that stalling of mutant COMP and an interaction between mutant COMP- type II procollagen could be the initiating events that causes the assembly of matrix in the rER. The presence of type II procollagen in the core of the matrix structure has not previously been reported. The existence of this intracellular matrix explains the relative deficiency of COMP, type IX collagen and MATN3 in the extracellular matrix and possibly why the material is not readily cleared from the rER. Altogether, these results suggest that mutant COMP initiates and perhaps catalyses intracellular matrix assembly. doi:10.1016/j.matbio.2006.08.217
195 Deformation of collagen triple helix in brittle bone under stress E.L. Mertz a, D. McBride b, S. Lekin a b
a
NICHD, NIH, Bethesda, MD, United States University of Maryland, MD, United States
Mechanical testing of mouse model of osteogenesis imperfecta (OI) with G610C mutation in a2-chain of type I collagen was previously shown to exhibit mild bone brittleness. In this work we analyzed potential chemical, structural and dynamic origins of such defect. We used newly developed cell for infrared micro-spectroscopy allowing studying biological tissues in physiological and non-physiological environments and yielding quality and amount of spectral information substantially exceeding those of the previous designs. First, we analyzed more than twenty spectral characteristics of chemical mineral composition, collagen content, absolute and relative alignment of collagen vs. mineral in cortical layer of femur bones at physiological conditions. This analysis indicated no significant chemical and structural differences between homozygous mutant and normal mice. Then, we analyzed response of collagen fibrils in tendon and cortical bone to lateral compression caused by mechanical, thermal or dehydration stresses. These stresses resulted in similar vibrational frequency
S80
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
shifts and reorientation of collagen backbone groups. The results suggest that deformations of a major fraction of triple helical backbone contribute to relaxation of lateral stress in bone. Largely abnormal deformations observed in mutant fibrils indicated that this abnormality may be responsible for mutant bone brittleness. Although tendon fibrils of this particular mutant are composed of 30% mutant 2a1a2(G610C) and 70% “normal” 3a1 collagen molecules, purely homotrimeric 3a1 fibers from OIM mice revealed nearly normal deformations, suggesting that that treatments affecting fraction of 3a1 molecules can modulate bone brittleness in G610C mice. doi:10.1016/j.matbio.2006.08.218
196 Integrin and non-integrin mediated signaling mechanisms of α1(IV)NC1 S. Akulapalli a, D. Cosgrove b, C.M. Overall c, C. Shekhar Boosani a a
Cell Signaling and Angiogenesis Laboratory, Department of Genetics, Boys Town National Research Hospital, Omaha, NE 68131, United States b Gene Expression Laboratory, Department of Genetics, Boys Town National Research Hospital Omaha, NE 68131, United States c The UBC Center for Blood Research, CIHR Group in Matrix Dynamics, University of British Columbia, Vancouver, Canada V6T 1Z3 The proteolytic cleavage fragments of vascular basement membrane (VBM) were found to inhibit the endothelial cell proliferation, migration, tube formations and their corresponding neutralizing antibodies reversed these inhibitory effects. α1 (IV)NC1, which is efficiently generated from VBM or Col Type IV by matrix metalloproteinase 2. In endothelial cells, recombinant human α1(IV)NC1 binds to α1β1 integrin and inhibits focal adhesion kinase/c-Raf/MAPK pathway, affecting HIF-1α and VEGF expression. α1(IV)NC1 influences MMP-2 activation in a non-cell specific way by forming a tight complex with the catalytic domain causing inhibition of endothelial and tumor cell invasion in reconstituted basement membrane. Consistent with this, tumor and matrigel studies in mice deficient in α1 integrin shows inhibition of tumor growth at higher dose of α1(IV)NC1 treatment. In addition α1(IV)NC1 was also found to regulate TIMP-2, MT1-MMP and elastase expressions. Collectively our in vitro and in vivo data is the first evidence describing that proteases generated fragments of VBM have dual function in integrin and non-integrin mediated suppression of tumor angiogenesis. doi:10.1016/j.matbio.2006.08.219
197 Nidogen functions with multiple adhesion/guidance receptors S. Kang, J.M. Kramer Dept. of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, United States Null mutants for the basement membrane (BM) component nidogen in C. elegans, nid-1(0), have defects in synaptic organization/transmission and axon guidance. A screen for mutations with enhanced phenotypes in nid-1(0) background identified mutations in several matrix receptors and downstream effectors that result in synthetic lethality. These include Slit, netrin and semaphorin guidance receptors, the matrix receptors integrin and dystroglycan, and downstream effectors GEF, Rac and Ena. Penetrance of lethality varies (15–100%), but always results from embryonic epithelial adhesion defects. Nervous system defects of the various single mutants are also enhanced in the nid-1(0) background. We propose that nidogen subtly affects signaling through each of these receptors and severe defects only occur when nidogen and one of the receptors are missing. We have generated GFP::NID-1 protein that localizes normally to BMs and rescues mutant phenotypes. BM localization requires the G3 domain and its laminin-binding residues, but not G1 or G2 domains. Non-laminin binding NID1 accumulates in extracellular space during embryogenesis, but rapidly disappears later indicating a post-embryonic uptake/ degradation process. Rescue of epithelial adhesion and axon guidance defects requires laminin-binding G3 and the G1 domain, but deletion of G2 has little effect on phenotypic rescue. Thus, the G1 domain has a critical role in nidogen function, although no binding interactions have been identified. Over-expression of laminin-binding G3 alone dominantly causes lethality and axon defects, suggesting that it may interfere with these receptors and/or their ligands. doi:10.1016/j.matbio.2006.08.220
198 Laminin-nidogen interaction in the induction of AChR clustering S. Smirnov a, D. Harrison a, K. McKee a, U. Mayer b, P.D. Yurchenco a a
Robert Wood Johnson Medical School, Piscataway, NJ, 08854 b School of Biological Sciences, Univ. of East Anglia, Norwich NR4 7TJ, UK Aneural clustering of the acetylcholine receptor (AChR) is an early post-synaptic step of neuromuscular junction assembly.