The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
David F Holmes - One of the best experts on this subject based on the ideXlab platform.
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Collagen Fibril assembly and function
Current Topics in Developmental Biology, 2018Co-Authors: David F Holmes, Tobias Starborg, Yinhui Lu, Karl E KadlerAbstract:Abstract Collagen Fibrils are the major mechanical component in the extracellular matrix of a broad range of multicellular animals from echinoderms to vertebrates where they provide a stable framework for tissues. They form the key tension-resisting element of a complex fiber-composite system that has a tissue-specific hierarchical structure linked to mechanical demands. Remarkably, these tissues are self-maintaining and avoid fatigue failure over the lifetime of the animal. Collagen Fibrils can assemble spontaneously from purified solutions of Collagen molecules. In developing tissues, however, in addition to the intrinsic self-assembly properties, there is cellular machinery that regulates Fibril nucleation, spatial orientation, and Fibril size, according to the tissue and stage of development. The intricate mechanisms underlying the generation of a Collagen Fibril network of defined architecture and mechanical properties are now becoming apparent. Impairment of this system leads ultimately to mechanical failure or tissue fibrosis.
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using transmission electron microscopy and 3view to determine Collagen Fibril size and three dimensional organization
Nature Protocols, 2013Co-Authors: Tobias Starborg, Yinhui Lu, David F Holmes, Nicholas S Kalson, Alexander A Mironov, Timothy F CootesAbstract:Using transmission electron microscopy and 3View to determine Collagen Fibril size and three-dimensional organization
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bimodal Collagen Fibril diameter distributions direct age related variations in tendon resilience and resistance to rupture
Journal of Applied Physiology, 2012Co-Authors: David F Holmes, Yinhui Lu, Karl E Kadler, Peter P Purslow, Daniel Bechet, Tim J WessAbstract:Scaling relationships have been formulated to investigate the influence of Collagen Fibril diameter (D) on age-related variations in the strain energy density of tendon. Transmission electron microscopy was used to quantify D in tail tendon from 1.7- to 35.3-mo-old (C57BL/6) male mice. Frequency histograms of D for all age groups were modeled as two normally distributed subpopulations with smaller (DD1) and larger (DD2) mean Ds, respectively. Both DD1 and DD2 increase from 1.6 to 4.0 mo but decrease thereafter. From tensile tests to rupture, two strain energy densities were calculated: 1) uE [from initial loading until the yield stress (σY)], which contributes primarily to tendon resilience, and 2) uF [from σY through the maximum stress (σU) until rupture], which relates primarily to resistance of the tendons to rupture. As measured by the normalized strain energy densities uE/σY and uF/σU, both the resilience and resistance to rupture increase with increasing age and peak at 23.0 and 4.0 mo, respectively, before decreasing thereafter. Multiple regression analysis reveals that increases in uE/σY (resilience energy) are associated with decreases in DD1 and increases in DD2, whereas uF/σU (rupture energy) is associated with increases in DD1 alone. These findings support a model where age-related variations in tendon resilience and resistance to rupture can be directed by subtle changes in the bimodal distribution of Ds.
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ageing changes in the tensile properties of tendons influence of Collagen Fibril volume fraction
Journal of Biomechanical Engineering-transactions of The Asme, 2008Co-Authors: David F Holmes, Karl E Kadler, H Y Lu, S Richardson, Peter P Purslow, Tim J WessAbstract:Connective tissues are biological composites comprising of Collagen Fibrils embedded in (and reinforcing) the hydrated proteoglycan-rich (PG) gel within the extracellular matrices (ECMs). Age-related changes to the mechanical properties of tissues are often associated with changes to the structure of the ECM, namely, Fibril diameter. However, quantitative attempts to correlate Fibril diameter to mechanical properties have yielded inconclusive evidence. Here, we described a novel approach that was based on the rule of mixtures for fiber composites to evaluate the dependence of age-related changes in tendon tensile strength (sigma) and stiffness (E) on the Collagen Fibril cross-sectional area fraction (rho), which is related to the Fibril volume fraction. Tail tendons from C57BL6 mice from age groups 1.6-35.3 months old were stretched to failure to determine sigma and E. Parallel measurements of rho as a function of age were made using transmission electron microscopy. Mathematical models (rule of mixtures) of Fibrils reinforcing a PG gel in tendons were used to investigate the influence of rho on ageing changes in sigma and E. The magnitudes of sigma, E, and rho increased rapidly from 1.6 months to 4.0 months (P-values 0.05); this trend continued for E and rho (P-values >0.05) from 29.0 months to 35.3 months, but not for sigma, which decreased gradually (P-values <0.05). Linear regression analysis revealed that age-related changes in sigma and E correlated positively to rho (P-values <0.05). Collagen Fibril cross-sectional area fraction rho is a significant predictor of ageing changes in sigma and E in the tail tendons of C57BL6 mice.
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stem tem studies of Collagen Fibril assembly
Micron, 2001Co-Authors: David F Holmes, H K Graham, JOHN ANDREW TROTTER, Karl E KadlerAbstract:Quantitative scanning transmission electron microscopy (STEM), implemented on a conventional transmission electron microscope with STEM-attachment, has been a primary tool in our laboratory for the quantitative analysis of Collagen Fibril assembly in vivo and in vitro. Using this technique, a precise measurement of mass per unit length can be made at regular intervals along a Fibril to generate an axial mass distribution (AMD). This in turn allows the number of Collagen molecules to be calculated for every transverse section of the Fibril along its entire length. All Fibrils show a near-linear AMD in their tip regions. Only Fibrils formed in tissue environments, however, show a characteristic abrupt change in mass slope along their tips. It appears that this tip growth characteristic is common to Fibrils from evolutionarily diverse systems including vertebrate tendon and the mutable tissues of the echinoderms. Computer models of Collagen Fibril assembly have now been developed based on interpretation of the STEM data. Two alternative models have so far been generated for Fibril growth by accretion; one is based on diffusion limited aggregation (DLA) and the other based on an interface-limited growth mechanism. Inter-Fibrillar fusion can also contribute to the growth of Fibrils in vertebrate tissues and STEM data indicates the presence of a tight regulation in this process. These models are fundamental for the hypotheses regarding how cells synthesise and spatially organise an extracellular matrix (ECM), rich in Collagen Fibrils.
Jess G Snedeker - One of the best experts on this subject based on the ideXlab platform.
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advanced glycation end products reduce Collagen molecular sliding to affect Collagen Fibril damage mechanisms but not stiffness
PLOS ONE, 2014Co-Authors: Gion Fessel, Yufei Li, Vincent E G Diederich, Manuel Guizarsicairos, Philipp Schneider, David R Sell, Vincent M Monnier, Jess G SnedekerAbstract:Advanced glycation end-products (AGE) contribute to age-related connective tissue damage and functional deficit. The documented association between AGE formation on Collagens and the correlated progressive stiffening of tissues has widely been presumed causative, despite the lack of mechanistic understanding. The present study investigates precisely how AGEs affect mechanical function of the Collagen Fibril – the supramolecular functional load-bearing unit within most tissues. We employed synchrotron small-angle X-ray scattering (SAXS) and carefully controlled mechanical testing after introducing AGEs in explants of rat-tail tendon using the metabolite methylglyoxal (MGO). Mass spectrometry and Collagen fluorescence verified substantial formation of AGEs by the treatment. Associated mechanical changes of the tissue (increased stiffness and failure strength, decreased stress relaxation) were consistent with reports from the literature. SAXS analysis revealed clear changes in molecular deformation within MGO treated Fibrils. Underlying the associated increase in tissue strength, we infer from the data that MGO modified Collagen Fibrils supported higher loads to failure by maintaining an intact quarter-staggered conformation to nearly twice the level of Fibril strain in controls. This apparent increase in Fibril failure resistance was characterized by reduced side-by-side sliding of Collagen molecules within Fibrils, reflecting lateral molecular interconnectivity by AGEs. Surprisingly, no change in maximum Fibril modulus (2.5 GPa) accompanied the changes in Fibril failure behavior, strongly contradicting the widespread assumption that tissue stiffening in ageing and diabetes is directly related to AGE increased Fibril stiffness. We conclude that AGEs can alter physiologically relevant failure behavior of Collagen Fibrils, but that tissue level changes in stiffness likely occur at higher levels of tissue architecture.
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tendon glycosaminoglycan proteoglycan sidechains promote Collagen Fibril sliding afm observations at the nanoscale
Journal of Biomechanics, 2013Co-Authors: S Rigozzi, Ralph Muller, Jess G Snedeker, Andreas StemmerAbstract:The extracellular matrix of tendon is mainly composed of discontinuous Type-I Collagen Fibrils and small leucine rich proteoglycans (PG). Macroscopic tendon behaviors like stiffness and strength are determined by the ultrastructural arrangement of these components. When a tendon is submitted to load, the Collagen Fibrils both elongate and slide relative to their neighboring Fibrils. The role of PG glycosaminoglycan (GAG) sidechains in mediating inter-Fibril load sharing remains controversial, with competing structure–function theories suggesting that PGs may mechanically couple neighboring Collagen Fibrils (cross-linking them to facilitate Fibril stretch) or alternatively isolating them (promoting Fibril gliding). In this study, we sought to clarify the functional role of GAGs in tensile tendon mechanics by directly investigating the mechanical response of individual Collagen Fibrils within their Collagen network in both native and GAG depleted tendons. A control group of Achilles tendons from adult mice was compared with tendons in which GAGs were enzymatically depleted using chondroitinase ABC. Tendons were loaded to specific target strains, chemically fixed under constant load, and later sectioned for morphological analysis by an atomic force microscope (AFM). Increases in periodic banding of the Collagen Fibrils (D-period) or decreases in Fibril diameter was considered to be representative of Collagen Fibril elongation and the mechanical contribution of GAGs at the ultrascale was quantified on this basis. At high levels of applied tendon strain (10%), GAG depleted tendons showed increased Collagen stretch (less Fibril sliding). We conclude that the hydrophilic GAGs seem thus not to act as mechanical crosslinks but rather act to promote Collagen Fibril sliding under tension.
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Collagen Fibril morphology and mechanical properties of the achilles tendon in two inbred mouse strains
Journal of Anatomy, 2010Co-Authors: S Rigozzi, Ralph Muller, Jess G SnedekerAbstract:The relationship between Collagen Fibril morphology and the functional behavior of tendon tissue has been investigated in numerous experimental studies. Several of these studies suggest that larger Fibril radius is a primary determinant of higher tendon stiffness and strength; others have shown that factors apart from Fibril radius (such as Fibril-Fibril interactions) may be critical to improved tendon strength. In the present study, we investigate these factors in two inbred mouse strains that are widely used in skeletal structure-function research: C57BL/6J (B6) and C3H/HeJ (C3H). The aim was to establish a quantitative baseline that will allow one to assess how regulation of tendon extracellular matrix architecture affects tensile mechanical properties. We specifically focused on Collagen Fibril structure and glycosaminoglycan (GAG) content--the two primary constituents of tendon by dry weight--and their potential functional interactions. For this purpose, Achilles tendons from both groups were tested to failure in tension. Tendon Collagen morphology was analyzed from transmission electron microscopy images of tendon sections perpendicular to the longitudinal axis. Our results showed that the two inbred strains are macroscopically similar, but C3H mice have a higher elastic modulus (P < 0.05). Structurally, C3H mice showed a larger Collagen Fibril radius compared to B6 mice (96 +/- 7 nm and 80 +/- 10 nm respectively). Tendons from C3H mice also showed smaller specific Fibril surface (0.015 +/- 0.001 nm nm(-2) vs. 0.017 +/- 0.003 nm nm(-2) in the B6 tendons, P < 0.05), and accordingly a lower concentration of GAGs (0.60 +/- 0.07 microg mg(-1) vs. 0.83 +/- 0.11 microg mg(-1), P < 0.05). As in other studies of tendon structure and function, larger Collagen Fibril radius appears to be associated with stiffer tendon, but this functional difference could also be attributed to reduced potential surface area exchange between Fibrils and the surrounding proteoglycan-rich matrix, in which the hydrophilic GAG side chains may promote inter-Fibril sliding. This study provides an architectural and functional baseline for a comparative murine model that can be used to investigate the genetic regulation of tendon biomechanics.
Karl E Kadler - One of the best experts on this subject based on the ideXlab platform.
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Collagen Fibril assembly and function
Current Topics in Developmental Biology, 2018Co-Authors: David F Holmes, Tobias Starborg, Yinhui Lu, Karl E KadlerAbstract:Abstract Collagen Fibrils are the major mechanical component in the extracellular matrix of a broad range of multicellular animals from echinoderms to vertebrates where they provide a stable framework for tissues. They form the key tension-resisting element of a complex fiber-composite system that has a tissue-specific hierarchical structure linked to mechanical demands. Remarkably, these tissues are self-maintaining and avoid fatigue failure over the lifetime of the animal. Collagen Fibrils can assemble spontaneously from purified solutions of Collagen molecules. In developing tissues, however, in addition to the intrinsic self-assembly properties, there is cellular machinery that regulates Fibril nucleation, spatial orientation, and Fibril size, according to the tissue and stage of development. The intricate mechanisms underlying the generation of a Collagen Fibril network of defined architecture and mechanical properties are now becoming apparent. Impairment of this system leads ultimately to mechanical failure or tissue fibrosis.
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bimodal Collagen Fibril diameter distributions direct age related variations in tendon resilience and resistance to rupture
Journal of Applied Physiology, 2012Co-Authors: David F Holmes, Yinhui Lu, Karl E Kadler, Peter P Purslow, Daniel Bechet, Tim J WessAbstract:Scaling relationships have been formulated to investigate the influence of Collagen Fibril diameter (D) on age-related variations in the strain energy density of tendon. Transmission electron microscopy was used to quantify D in tail tendon from 1.7- to 35.3-mo-old (C57BL/6) male mice. Frequency histograms of D for all age groups were modeled as two normally distributed subpopulations with smaller (DD1) and larger (DD2) mean Ds, respectively. Both DD1 and DD2 increase from 1.6 to 4.0 mo but decrease thereafter. From tensile tests to rupture, two strain energy densities were calculated: 1) uE [from initial loading until the yield stress (σY)], which contributes primarily to tendon resilience, and 2) uF [from σY through the maximum stress (σU) until rupture], which relates primarily to resistance of the tendons to rupture. As measured by the normalized strain energy densities uE/σY and uF/σU, both the resilience and resistance to rupture increase with increasing age and peak at 23.0 and 4.0 mo, respectively, before decreasing thereafter. Multiple regression analysis reveals that increases in uE/σY (resilience energy) are associated with decreases in DD1 and increases in DD2, whereas uF/σU (rupture energy) is associated with increases in DD1 alone. These findings support a model where age-related variations in tendon resilience and resistance to rupture can be directed by subtle changes in the bimodal distribution of Ds.
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ageing changes in the tensile properties of tendons influence of Collagen Fibril volume fraction
Journal of Biomechanical Engineering-transactions of The Asme, 2008Co-Authors: David F Holmes, Karl E Kadler, H Y Lu, S Richardson, Peter P Purslow, Tim J WessAbstract:Connective tissues are biological composites comprising of Collagen Fibrils embedded in (and reinforcing) the hydrated proteoglycan-rich (PG) gel within the extracellular matrices (ECMs). Age-related changes to the mechanical properties of tissues are often associated with changes to the structure of the ECM, namely, Fibril diameter. However, quantitative attempts to correlate Fibril diameter to mechanical properties have yielded inconclusive evidence. Here, we described a novel approach that was based on the rule of mixtures for fiber composites to evaluate the dependence of age-related changes in tendon tensile strength (sigma) and stiffness (E) on the Collagen Fibril cross-sectional area fraction (rho), which is related to the Fibril volume fraction. Tail tendons from C57BL6 mice from age groups 1.6-35.3 months old were stretched to failure to determine sigma and E. Parallel measurements of rho as a function of age were made using transmission electron microscopy. Mathematical models (rule of mixtures) of Fibrils reinforcing a PG gel in tendons were used to investigate the influence of rho on ageing changes in sigma and E. The magnitudes of sigma, E, and rho increased rapidly from 1.6 months to 4.0 months (P-values 0.05); this trend continued for E and rho (P-values >0.05) from 29.0 months to 35.3 months, but not for sigma, which decreased gradually (P-values <0.05). Linear regression analysis revealed that age-related changes in sigma and E correlated positively to rho (P-values <0.05). Collagen Fibril cross-sectional area fraction rho is a significant predictor of ageing changes in sigma and E in the tail tendons of C57BL6 mice.
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stem tem studies of Collagen Fibril assembly
Micron, 2001Co-Authors: David F Holmes, H K Graham, JOHN ANDREW TROTTER, Karl E KadlerAbstract:Quantitative scanning transmission electron microscopy (STEM), implemented on a conventional transmission electron microscope with STEM-attachment, has been a primary tool in our laboratory for the quantitative analysis of Collagen Fibril assembly in vivo and in vitro. Using this technique, a precise measurement of mass per unit length can be made at regular intervals along a Fibril to generate an axial mass distribution (AMD). This in turn allows the number of Collagen molecules to be calculated for every transverse section of the Fibril along its entire length. All Fibrils show a near-linear AMD in their tip regions. Only Fibrils formed in tissue environments, however, show a characteristic abrupt change in mass slope along their tips. It appears that this tip growth characteristic is common to Fibrils from evolutionarily diverse systems including vertebrate tendon and the mutable tissues of the echinoderms. Computer models of Collagen Fibril assembly have now been developed based on interpretation of the STEM data. Two alternative models have so far been generated for Fibril growth by accretion; one is based on diffusion limited aggregation (DLA) and the other based on an interface-limited growth mechanism. Inter-Fibrillar fusion can also contribute to the growth of Fibrils in vertebrate tissues and STEM data indicates the presence of a tight regulation in this process. These models are fundamental for the hypotheses regarding how cells synthesise and spatially organise an extracellular matrix (ECM), rich in Collagen Fibrils.
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STEM/TEM studies of Collagen Fibril assembly
Micron, 2001Co-Authors: David F Holmes, H K Graham, JOHN ANDREW TROTTER, Karl E KadlerAbstract:Quantitative scanning transmission electron microscopy (STEM), implemented on a conventional transmission electron microscope with STEM-attachment, has been a primary tool in our laboratory for the quantitative analysis of Collagen Fibril assembly in vivo and in vitro. Using this technique, a precise measurement of mass per unit length can be made at regular intervals along a Fibril to generate an axial mass distribution (AMD). This in turn allows the number of Collagen molecules to be calculated for every transverse section of the Fibril along its entire length. All Fibrils show a near-linear AMD in their tip regions. Only Fibrils formed in tissue environments, however, show a characteristic abrupt change in mass slope along their tips. It appears that this tip growth characteristic is common to Fibrils from evolutionarily diverse systems including vertebrate tendon and the mutable tissues of the echinoderms. Computer models of Collagen Fibril assembly have now been developed based on interpretation of the STEM data. Two alternative models have so far been generated for Fibril growth by accretion; one is based on diffusion limited aggregation (DLA) and the other based on an interface-limited growth mechanism. Inter-Fibrillar fusion can also contribute to the growth of Fibrils in vertebrate tissues and STEM data indicates the presence of a tight regulation in this process. These models are fundamental for the hypotheses regarding how cells synthesise and spatially organise an extracellular matrix (ECM), rich in Collagen Fibrils.
Frank Zaucke - One of the best experts on this subject based on the ideXlab platform.
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combined role of type ix Collagen and cartilage oligomeric matrix protein in cartilage matrix assembly cartilage oligomeric matrix protein counteracts type ix Collagen induced limitation of cartilage Collagen Fibril growth in mouse chondrocyte cultur
Arthritis & Rheumatism, 2009Co-Authors: Katrin Blumbach, Y M Bastiaansenjenniskens, J Degroot, Mats Paulsson, G J V M Van Osch, Frank ZauckeAbstract:Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the Collagen Fibril-associated proteins type IX Collagen and cartilage oligomeric matrix protein (COMP) during cartilage matrix formation. Methods. Primary chondrocytes from mice deficient in type IX Collagen and COMP (double-deficient) were cultured in monolayer or alginate beads. Anchorage of matrix proteins, proteoglycan and Collagen content, Collagen crosslinks, matrix metalloproteinase activity, and mechanical properties of the matrix were measured. Electron microscopy was used to study the formation of Fibrillar structures. Results. In cartilage lacking both type IX Collagen and COMP, matrilin 3 showed decreased matrix anchorage. Less matrilin 3 was deposited in the matrix of double-deficient chondrocytes, while larger amounts were secreted into the medium. Proteoglycans were less well retained in the matrix formed in alginate cultures, while Collagen deposition was not significantly affected. Electron microscopy revealed similar cartilage Collagen Fibril diameters in the cultures of double-deficient and wild-type chondrocytes. In contrast, a larger Fibril diameter was observed in the matrix of chondrocytes deficient in only type IX Collagen. Conclusion. Our results show that type IX Collagen and COMP are involved in matrix assembly by mediating the anchorage and regulating the distribution of other matrix macromolecules such as proteoglycans and matrilins and have counteracting effects on Collagen Fibril growth. Loss of type IX Collagen and COMP leads to matrix aberrations that may make cartilage more susceptible to degeneration. © 2009, American College of Rheumatology.
David E. Birk - One of the best experts on this subject based on the ideXlab platform.
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decorin and biglycan are necessary for maintaining Collagen Fibril structure fiber realignment and mechanical properties of mature tendons
Matrix Biology, 2017Co-Authors: Kelsey A Robinson, Renato V. Iozzo, Sheila M Adams, Carrie E Barnum, Stephanie N Weiss, Julianne Huegel, Snehal S Shetye, Daniel Saez, Louis J Soslowsky, David E. BirkAbstract:Abstract The small leucine-rich proteoglycans (SLRPs), decorin and biglycan, are key regulators of Collagen Fibril and matrix assembly. The goal of this work was to elucidate the roles of decorin and biglycan in tendon homeostasis. Our central hypothesis is that decorin and biglycan expression in the mature tendon would be critical for the maintenance of the structural and mechanical properties of healthy tendons. Defining the function(s) of these SLRPs in tendon homeostasis requires that effects in the mature tendon be isolated from their influence on development. Thus, we generated an inducible knockout mouse model that permits genetic ablation of decorin and biglycan expression in the mature tendon, while maintaining normal expression during development. Decorin and biglycan expression were knocked out in the mature patellar tendon with the subsequent turnover of endogenous SLRPs deposited prior to induction. The acute absence of SLRP expression was associated with changes in Fibril structure with a general shift to larger diameter Fibrils in the compound knockout tendons, together with Fibril diameter heterogeneity. In addition, tendon mechanical properties were altered. Compared to wild-type controls, acute ablation of both genes resulted in failure of the tendon at lower loads, decreased stiffness, a trend towards decreased dynamic modulus, as well as a significant increase in percent relaxation and tissue viscosity. Collagen fiber realignment was also increased with a delayed and slower in response to load in the absence of expression. These structural and functional changes in response to an acute loss of decorin and biglycan expression in the mature tendon demonstrate a significant role for these SLRPs in adult tendon homeostasis.
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fibulin 4 e57k knock in mice recapitulate cutaneous vascular and skeletal defects of recessive cutis laxa 1b with both elastic fiber and Collagen Fibril abnormalities
Journal of Biological Chemistry, 2015Co-Authors: Olga Igoucheva, Takako Sasaki, Vitali Alexeev, Carmen M Halabi, Sheila M Adams, Ivan Stoilov, Machiko Arita, Adele Donahue, Robert P Mecham, David E. BirkAbstract:Abstract Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced, but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in crosslinking of elastin and Collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific crosslink compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant Collagen Fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I Collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular crosslinks, which could contribute to the Collagen Fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating Collagen Fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.
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type v Collagen controls the initiation of Collagen Fibril assembly
Journal of Biological Chemistry, 2004Co-Authors: Richard J Wenstrup, Jane B Florer, Sheila M Bell, Eric W Brunskill, Inna Chervoneva, David E. BirkAbstract:Abstract Vertebrate Collagen Fibrils are heterotypically composed of a quantitatively major and minor Fibril Collagen. In non-cartilaginous tissues, type I Collagen accounts for the majority of the Collagen mass, and Collagen type V, the functions of which are poorly understood, is a minor component. Type V Collagen has been implicated in the regulation of Fibril diameter, and we reported recently preliminary evidence that type V Collagen is required for Collagen Fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113–124). The purpose of this study was to define the roles of type V Collagen in the regulation of Collagen Fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-α1(V) chains were created by homologous recombination. The col5a1–/– animals die in early embryogenesis, at approximately embryonic day 10. The type V Collagen-deficient mice demonstrate a virtual lack of Collagen Fibril formation. In contrast, the col5a1+/– animals are viable. The reduced type V Collagen content is associated with a 50% reduction in Fibril number and dermal Collagen content. In addition, relatively normal, cylindrical Fibrils are assembled with a second population of large, structurally abnormal Collagen Fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V Collagen in the regulation of Fibrillogenesis. The complete dependence of Fibril formation on type V Collagen is indicative of the critical role of the latter in early Fibril initiation. In addition, this Fibril Collagen is important in the determination of Fibril structure and matrix organization.
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Collagen Fibril Formation in a Wound Healing Model
Journal of Structural Biology, 2002Co-Authors: Jacinta F. White, David E. Birk, Jerome A. Werkmeister, Ian A. Darby, Teresa Bisucci, John A. M. RamshawAbstract:Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of Collagen Fibril formation, growth, and organization is not yet fully understood. In this study we have examined Collagen Fibril formation in a wound healing model in which the newly formed Fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant Collagen formation at 14 and 28 days. Individual Collagen Fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact Fibrils. Examination by transmission electron microscopy showed that 80% of the intact Fibrils showed a single polarity reversal, with both ends of each Fibril comprising Collagen amino-terminal domains; the remaining Fibrils had no polarity reversal. All Fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled Fibrils contained both type I and III Collagens. These data indicate that this wound healing model provides a system in which Collagen Fibril formation can be readily followed. © 2002 Elsevier
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corneal opacity in lumican null mice defects in Collagen Fibril structure and packing in the posterior stroma
Investigative Ophthalmology & Visual Science, 2000Co-Authors: Shukti Chakravarti, Jonathan H Lass, Walter M Petroll, John R Hassell, James V Jester, J Paul, David E. BirkAbstract:It has long been recognized that Collagen architecture of the corneal stroma is crucially important in the ultimate transparency of the cornea.1 Collagen Fibrils in the stroma are maintained in the range of 20 to 40 nm and organized into a highly ordered, latticelike configuration. The highly ordered architecture of the corneal stroma is affected by multiple factors. Recent studies of several types of hereditary corneal dystrophies elucidated abnormal Collagen Fibril architecture of the corneal stroma. For example, corneal opacification is a clinical feature of Scheie’s syndrome or mucopolysaccharidosis (MPS) type I, a lysosomal storage disorder with an iduronidase A deficiency.2 In addition to featuring granular deposits, transmission electron microscopy of MPS I–affected corneas revealed the presence of thicker Collagen Fibrils and localized disorganization of the matrix.3,4 Macular corneal dystrophy, with deficiencies in keratan sulfate (KS) biosynthesis, also causes clouding of the cornea and similar disruptions in stromal Fibril structure and organization.5–8 In both cases, altered proteoglycan synthesis and composition are to be expected. Recently, a mouse model for corneal dystrophy was developed by targeted disruption of the lumican gene (lumtm1sc/lumtm1sc).9 The mutant mice had cloudy corneas and stromal Collagen Fibrils with increased diameter and altered structure. Lumican is a member of the leucine-rich proteoglycan (LRP) family.10 It is a major keratan sulfate proteoglycan of the corneal stroma as well as other Collagenous extracellular matrices (skin, cardiac valves, cartilage, and bone).11 Other LRP members include decorin, fibromodulin, biglycan, keratocan, osteoglycin, and epiphycan.12 Decorin, a chondroitin sulfate (CS) proteoglycan widely expressed during mouse embryonic development, is also a major component of the corneal stroma.13 Previous studies have shown that the core proteins of lumican, decorin, and other LRPs from tendons can delay spontaneous Collagen Fibril formation and inhibit the lateral growth of Fibrils in Fibrillogenesis assays in vitro.14–16 Also, the abnormal lateral growth of isolated corneal Fibrils stripped of their surface-associated macromolecules is prevented by the corneal proteoglycans.17 Recent gene-targeting studies of LRPs suggest a similar role for these proteoglycans in vivo. Thus, absence of lumican in our lumtm1sc/lumtm1sc mouse model of corneal dystrophy affected Collagen architecture of the cornea and skin with consequent corneal opacity and reduced dermal biomechanical tensile strength. In addition to lumican, gene-targeted null mutations in decorin and fibromodulin also led to abnormal Collagen Fibril architecture in skin and tendons.18,19 However, to date only the lumican-deficient mice have demonstrated a corneal phenotype. The purpose of the present study was to assess corneal opacification in the lumtm1sc/lumtm1sc mice and define its source in the corneal stroma by in vivo confocal microscopy. Parallel analyses of Collagen Fibril structure, Fibril packing, and organization in the lumican-deficient and wild-type control mice and lumican expression in the mature normal cornea indicate that lumican serves a key role in the establishment and maintenance of corneal transparency.