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Sample records for collagen fragmentation promotes

  1. Collagen Fragmentation Promotes Oxidative Stress and Elevates Matrix Metalloproteinase-1 in Fibroblasts in Aged Human Skin

    PubMed Central

    Fisher, Gary J.; Quan, Taihao; Purohit, Trupta; Shao, Yuan; Cho, Moon Kyun; He, Tianyuan; Varani, James; Kang, Sewon; Voorhees, John J.

    2009-01-01

    Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and α2β1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension, as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and α2β1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ10 significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging. PMID:19116368

  2. Unusual Fragmentation Pathways in Collagen Glycopeptides

    NASA Astrophysics Data System (ADS)

    Perdivara, Irina; Perera, Lalith; Sricholpech, Marnisa; Terajima, Masahiko; Pleshko, Nancy; Yamauchi, Mitsuo; Tomer, Kenneth B.

    2013-07-01

    Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids -(X—Y—Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides (i.e., in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed). The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways—amide bond and glycosidic bond cleavage—are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e., Arg, Lys, HyK, and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides.

  3. Unusual fragmentation pathways in collagen glycopeptides

    PubMed Central

    Perdivara, Irina; Perera, Lalith; Sricholpech, Marnisa; Terajima, Masahiko; Pleshko, Nancy; Yamauchi, Mitsuo; Tomer, Kenneth B.

    2013-01-01

    Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids –(X—Y—Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides, i.e. in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed. The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways – amide bond and glycosidic bond cleavage – are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e. Arg, Lys, HyK and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations, to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides. PMID:23633013

  4. Type I Collagen and Collagen Mimetics as Angiogenesis Promoting Superpolymers

    SciTech Connect

    Twardowski, T.; Fertala, A.; Orgel, J.P.R.O.; San Antonio, J.D.

    2008-07-18

    Angiogenesis, the development of blood vessels from the pre-existing vasculature, is a key component of embryogenesis and tissue regeneration. Angiogenesis also drives pathologies such as tumor growth and metastasis, and hemangioma development in newborns. On the other hand, promotion of angiogenesis is needed in tissues with vascular insufficiencies, and in bioengineering, to endow tissue substitutes with appropriate microvasculatures. Therefore, much research has focused on defining mechanisms of angiogenesis, and identifying pro- and anti-angiogenic molecules. Type I collagen, the most abundant protein in humans, potently stimulates angiogenesis in vitro and in vivo. Crucial to its angiogenic activity appears to be ligation and possibly clustering of endothelial cell (EC) surface {alpha}1{beta}1/{alpha}2{beta}1 integrin receptors by the GFPGER502-507 sequence of the collagen fibril. However, additional aspects of collagen structure and function that may modulate its angiogenic properties are discussed. Moreover, type I collagen and fibrin, another angiogenic polymer, share several structural features. These observations suggest strategies for creating 'angiogenic superpolymers', including: modifying type I collagen to influence its biological half-life, immunogenicity, and integrin binding capacity; genetically engineering fibrillar collagens to include additional integrin binding sites or angiogenic determinants, and remove unnecessary or deleterious sequences without compromising fibril integrity; and exploring the suitability of poly(ortho ester), PEG-lysine copolymer, tubulin, and cholesteric cuticle as collagen mimetics, and suggesting means of modifying them to display ideal angiogenic properties. The collagenous and collagen mimetic angiogenic superpolymers described here may someday prove useful for many applications in tissue engineering and human medicine.

  5. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

    PubMed Central

    Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2016-01-01

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins. PMID:26883295

  6. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

    PubMed

    Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2016-02-17

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

  7. Exposure to Mimivirus Collagen Promotes Arthritis

    PubMed Central

    Shah, Nikunj; Hülsmeier, Andreas J.; Hochhold, Nina; Neidhart, Michel; Gay, Steffen

    2014-01-01

    Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens. PMID:24173233

  8. 7S Fragment of Type IV Collagen as a Serum Marker of Canine Liver Fibrosis.

    PubMed

    Glińska-Suchocka, K; Orłowska, A; Kubiak, K; Spużak, J; Jankowski, M

    2016-09-01

    The aim of this study was to assess whether the serum levels of the 7S fragment of type IV collagen may aid in diagnosing liver fibrosis in dogs. The study was carried out on 20 dogs with liver disease. Serum levels of the 7S fragment of type IV collagen were measured in all dogs. The analysis showed that healthy dogs and dogs with type 1, 2 and 3 liver fibrosis had low serum concentrations of the 7S fragment of type IV collagen compared to dogs with type 4 liver fibrosis. The study revealed that the assessment of serum levels of the 7S fragment of type IV collagen is useful in the diagnosis of advanced liver fibrosis and cirrhosis.

  9. Heparin fragments modulate the collagen phenotype of fibroblasts from radiation-induced subcutaneous fibrosis

    SciTech Connect

    el Nabout, R.; Martin, M.; Remy, J.; Robert, L.; Lafuma, C. )

    1989-10-01

    Acute local gamma irradiation of porcine skin induces, as in human skin, an extensive and mutilating sclerosis characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of the macromolecular components of the extracellular matrix. Collagen synthesis, content, and types were studied in the presence of heparin fragments (100 micrograms/10(6) cells) in the culture medium, by measuring the incorporation of the radiolabeled precursor (3H)proline into confluent primary cultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. Enhancement in collagen biosynthesis and deposition and preferential increase in collagen type III synthesis were observed in fibrotic fibroblast cultures when compared to those in normal dermis fibroblasts. The total collagen synthesis and the rate of collagen hydroxylation appear unmodified by heparin fragments both in normal and in fibrotic fibroblast cultures. But heparin fragments induce a 10- and 2-fold decrease, respectively, in collagen type III and type V syntheses by fibrosis fibroblasts. As only minor effects upon collagen type III and V are observed in cultures of normal dermis fibroblasts, these results highly suggest that heparin fragments are capable of specifically modulating the collagen phenotype of fibroblasts derived from radiation-induced dermis fibrosis and thus are able to regulate the fibrotic process.

  10. Plasma clot-promoting effect of collagen in relation to collagen-platelet interaction

    SciTech Connect

    Gentry, P.A.; Schneider, M.D.; Miller, J.K.

    1981-01-01

    The hemostatic function of several acid-soluble collagen preparations and a fibrillar-form collagen preparation have been compared. Pepsin-treated acid-soluble collagen isolated from burro and horse aortic tissue and acid-soluble colagen isolated from human umbilical cord readily promoted platelet aggregation, but failed to activate the coagulation mechanism even after prolonged incubation with plasma at 37 C. By contrast, fibrillar-form collagen isolated from burro aorta was both an efficient stimulant for the induction of platelet aggregation and a potent clot-promoting agent. Similar results were found for all the collagen preparations irrespective of whether the studies were conducted with sheep or with burro plasma. Heat denaturation studies showed that the hemostatic functon of the fibrillar-form colagen was dependent on an intact tirple-helical structure.

  11. A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy.

    PubMed

    Liang, Hui; Li, Xiaoran; Chen, Bing; Wang, Bin; Zhao, Yannan; Zhuang, Yan; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2015-07-10

    Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) antibody fragment for targeting the collagen-rich ECM in tumors. The single chain fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Collagen-derived matricryptins promote inhibitory nerve terminal formation in the developing neocortex

    PubMed Central

    Su, Jianmin; Chen, Jiang; Lippold, Kumiko; Monavarfeshani, Aboozar; Carrillo, Gabriela Lizana; Jenkins, Rachel

    2016-01-01

    Inhibitory synapses comprise only ∼20% of the total synapses in the mammalian brain but play essential roles in controlling neuronal activity. In fact, perturbing inhibitory synapses is associated with complex brain disorders, such as schizophrenia and epilepsy. Although many types of inhibitory synapses exist, these disorders have been strongly linked to defects in inhibitory synapses formed by Parvalbumin-expressing interneurons. Here, we discovered a novel role for an unconventional collagen—collagen XIX—in the formation of Parvalbumin+ inhibitory synapses. Loss of this collagen results not only in decreased inhibitory synapse number, but also in the acquisition of schizophrenia-related behaviors. Mechanistically, these studies reveal that a proteolytically released fragment of this collagen, termed a matricryptin, promotes the assembly of inhibitory nerve terminals through integrin receptors. Collectively, these studies not only identify roles for collagen-derived matricryptins in cortical circuit formation, but they also reveal a novel paracrine mechanism that regulates the assembly of these synapses. PMID:26975851

  13. A graphene oxide-based FRET sensor for rapid and specific detection of unfolded collagen fragments.

    PubMed

    Sun, Xiuxia; Fan, Jun; Zhang, Yuping; Chen, Hongli; Zhao, Yongqing; Xiao, Jianxi

    2016-05-15

    The unstructured collagen species plays a critical role in a variety of important biological processes as well as pathological conditions. In order to develop novel diagnosis and therapies for collagen-related diseases, it is essential to construct simple and efficient methods to detect unfolded collagen fragments. We therefore have designed a FITC-labeled collagen mimic triple helical peptide, whose adsorption on the surface of GO effectively quenches its fluorescence. The newly constructed GO/FITC-GPO complex specifically detects unstructured collagen fragments, but not fully folded triple helix species. The detection shows a clear preference for the collagen targets with complementary GPO-rich sequences. The conformation-sensitive, sequence-specific GO-based approach can be applied as an efficient biosensor for rapid detection of unfolded collagen fragments at nM level, and may have great potential in drug screening for inhibitors of unfolded collagen. It may provide a prototype to develop GO-based assays to detect other important unstructured proteins involved in diseases.

  14. Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death.

    PubMed Central

    Nilsson, I M; Patti, J M; Bremell, T; Höök, M; Tarkowski, A

    1998-01-01

    Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Morbidity and mortality due to infections such as sepsis, osteomyelitis, septic arthritis, and invasive endocarditis remain high despite the use of antibiotics. The emergence of antibiotic resistant super bugs mandates that alternative strategies for the prevention and treatment of S. aureus infections are developed. We investigated the ability of vaccination with a recombinant fragment of the S. aureus collagen adhesin to protect mice against sepsis-induced death. Actively immunized NMRI mice were intravenously inoculated with the S. aureus clinical isolate strain Phillips. 14 d after inoculation, mortality in the collagen adhesin-vaccinated group was only 13%, compared with 87% in the control antigen immunized group (P < 0.001). To determine if the protective effect was antibody mediated, we passively immunized naive mice with collagen adhesin-specific antibodies. Similar to the active immunization strategy, passive transfer of collagen adhesin-specific antibodies protected mice against sepsis-induced death. In vitro experiments indicated that S. aureus opsonized with sera from collagen adhesin immunized mice promoted phagocytic uptake and enhanced intracellular killing compared with bacteria opsonized with sera from control animals. These results indicate that the collagen adhesin is a viable target in the development of immunotherapeutics against S. aureus. PMID:9637697

  15. Collagen fibril diameter and alignment promote the quiescent keratocyte phenotype

    PubMed Central

    Muthusubramaniam, Lalitha; Peng, Lily; Zaitseva, Tatiana; Paukshto, Michael; Martin, George R.; Desai, Tejal

    2011-01-01

    In this study, we investigated how matrix nanotopography affects corneal fibroblast phenotype and matrix synthesis. To this end, corneal fibroblasts isolated from bovine corneas were grown on collagen nanofiber scaffolds of different diameters and alignment – 30 nm aligned fibrils (30A), 300 nm or larger aligned fibrils (300A), and 30 nm nonaligned fibrils (30NA) in comparison to collagen coated flat glass substrates (FC). Cell morphology was visualized using confocal microscopy. Quantitative PCR was used to measure expression levels of six target genes: the corneal crystallin - transketolase (TKT), the myofibroblast marker - α-smooth muscle actin (SMA), and four matrix proteins - collagen 1 (COL1), collagen 3 (COL3), fibronectin (FN) and biglycan. It was found that SMA expression was down-regulated and TKT expression was increased on all three collagen nanofiber substrates, compared to the FC control substrates. However, COL3 and biglycan expression was also significantly increased on 300A, compared to the FC substrates. Thus matrix nanotopography down-regulates the fibrotic phenotype, promotes formation of the quiescent keratocyte phenotype and influences matrix synthesis. These results have significant implications for the engineering of corneal replacements and for promoting regenerative healing of the cornea after disease and/or injury. PMID:22213336

  16. Promoter and transcription of type X collagen gene in broiler chickens with tibial dyschondroplasia.

    PubMed

    Zhang, X; McDaniel, G R; Giambrone, J J; Smith, E

    1996-06-01

    Type X collagen is produced exclusively in hypertrophic chondrocytes of the growth plate of the proximal tibiotarsus and is believed to play an important role during normal development from chondrogenesis to osteogenesis. Chondrocytes of chickens with tibial dyschondroplasia (TD) fail to attain full hypertrophy and the amount of type X collagen, being a marker of hypertrophy, is likely to be reduced. It is not clear whether transcriptional regulation is functional for expression of the type X collagen gene in TD birds. Nucleotide sequence of the type X collagen gene promoter was determined by sequencing PCR-based DNA clones. Nucleotide identity of this fragment between the normal and TD carriers was 97.6%. Both normal and TD birds were similar in a putative transcription start site, the site of TATAA box, and neither had a CCAAT box. However, there were two gaps in TD carriers, four gaps in normals, and five nucleotide substitution sites. By rapid amplification of cDNA ends by PCR (RACE-PCR), transcription of the gene was assessed using total RNA and mRNA from both normal chondrocytes and TD lesions at 3 and 4 wk of age. The RACE-PCR product for type X collagen mRNA was detectable in both normal and TD birds at two stages. No difference was found between them. This result does not support the hypothesis that transcriptional regulation of type X collagen gene is important in TD development of chickens. Variations in the promoter region did not affect transcription of type X collagen gene in TD carrier chickens.

  17. Mitochondrial fragmentation caused by phenanthroline promotes mitophagy.

    PubMed

    Park, So Jung; Shin, Ji Hyun; Kim, Eun Sung; Jo, Yoon Kyung; Kim, Jung Ho; Hwang, Jung Jin; Kim, Jin Cheon; Cho, Dong-Hyung

    2012-12-14

    Mitochondrial dynamics and mitophagy are thought to be important events for the quality control of mitochondria and mitochondria-associated diseases. To identify novel mitophagy modulators, we developed a cell-based screening system and selected 1,10-phenanthroline (Phen) as a target molecule. Phen treatment highly induced mitochondrial fragmentation and mitochondrial dysfunctions in a Drp1 dependent manner. Phen treatment also increased autophagy. Moreover, prolonged exposure of Phen increased mitochondria clearance through mitophagy. Phen-mediated loss of mitochondrial mass was more reduced in ATG5 deficient cells than in wild type cells. In addition, down-regulation of Drp1 decreased autophagy activation, suggesting that mitochondrial fission is involved in Phen-mediated mitophagy. Thus, our results demonstrate that the disruption of mitochondrial dynamics and mitochondrial dysfunctions provokes mitophagy in Phen-treated cells.

  18. Thermochemical Fragment Energy Method for Biomolecules: Application to a Collagen Model Peptide.

    PubMed

    Suárez, Ernesto; Díaz, Natalia; Suárez, Dimas

    2009-06-09

    Herein, we first review different methodologies that have been proposed for computing the quantum mechanical (QM) energy and other molecular properties of large systems through a linear combination of subsystem (fragment) energies, which can be computed using conventional QM packages. Particularly, we emphasize the similarities among the different methods that can be considered as variants of the multibody expansion technique. Nevertheless, on the basis of thermochemical arguments, we propose yet another variant of the fragment energy methods, which could be useful for, and readily applicable to, biomolecules using either QM or hybrid quantum mechanical/molecular mechanics methods. The proposed computational scheme is applied to investigate the stability of a triple-helical collagen model peptide. To better address the actual applicability of the fragment QM method and to properly compare with experimental data, we compute average energies by carrying out single-point fragment QM calculations on structures generated by a classical molecular dynamics simulation. The QM calculations are done using a density functional level of theory combined with an implicit solvent model. Other free-energy terms such as attractive dispersion interactions or thermal contributions are included using molecular mechanics. The importance of correcting both the intermolecular and intramolecular basis set superposition error (BSSE) in the QM calculations is also discussed in detail. On the basis of the favorable comparison of our fragment-based energies with experimental data and former theoretical results, we conclude that the fragment QM energy strategy could be an interesting addition to the multimethod toolbox for biomolecular simulations in order to investigate those situations (e.g., interactions with metal clusters) that are beyond the range of applicability of common molecular mechanics methods.

  19. Collagen Fragments Inhibit Hyaluronan Synthesis in Skin Fibroblasts in Response to Ultraviolet B (UVB)

    PubMed Central

    Röck, Katharina; Grandoch, Maria; Majora, Marc; Krutmann, Jean; Fischer, Jens W.

    2011-01-01

    UVB irradiation causes characteristic features of skin aging including remodeling of the dermal extracellular matrix. A key feature during this process is the up-regulation of matrix metalloproteinases and cleavage of collagen. Hyaluronic acid (HA), a major component of the dermal matrix, decreases after chronic UVB exposure. However, the factors that govern the decline of HA synthesis during the course of actinic aging are largely unknown. The aim of the present study was to explore whether collagen degradation causes inhibition of HA synthesis in human skin fibroblasts. After treatment of fibroblasts with collagen fragments (CF) in vitro, resolution of the actin cytoskeleton and inhibition of HA secretion occurred because of specific down-regulation of hyaluronan synthase 2 (HAS2) expression. The αvβ3-agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to αvβ3 abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by αvβ3-blocking antibodies. The key role of ERK signaling was shown by reduced nuclear accumulation of phosphoERK and of ELK-1 phosphorylation in response to CF. In addition, the ERK inhibitor PD98059 reduced HAS2 expression. Also, UVB irradiation of fibroblasts caused down-regulation of HAS2, which was sensitive to matrix metalloproteinase inhibitors and to αvβ3-blocking antibodies. In conclusion, these data suggest that CF activate αvβ3-integrins and in turn inhibit Rho kinase (ROCK) signaling and nuclear translocation of phosphoERK, resulting in reduced HAS2 expression. Therefore, a novel mechanism is presented how proteolytic collagen cleavage may inhibit HA synthesis in dermal fibroblasts during extrinsic skin aging. PMID:21454612

  20. Expression of catalytically active Matrix Metalloproteinase-1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

    PubMed Central

    Xia, Wei; Hammerberg, Craig; Li, Yong; He, Tianyuan; Quan, Taihao; Voorhees, John J; Fisher, Gary J

    2013-01-01

    Summary Increased expression of matrix metalloproteinase-1 (MMP-1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP-1-mediated collagen fibril fragmentation is a key driver of age-related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP-1 mutant (MMP-1 V94G) in adult human skin in organ culture and fibroblasts in three dimensional collagen lattice cultures. Expression of MMP-1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP-1 V94G in dermal fibroblasts cultured in three-dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP-1 siRNA-mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP-1 V94G-fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF-β pathway, and reduced collagen production. These results support the concept that MMP-1-mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts, and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age-related decline of fibroblast function. PMID:23601157

  1. Differential effects of parathyroid hormone fragments on collagen gene expression in chondrocytes

    PubMed Central

    1996-01-01

    The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule. To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system of fetal bovine and human chondrocytes. Expression of alpha1 (I), alpha1 (II), alpha1 (III), and alpha1 (X) mRNA was investigated by in situ hybridization and quantified by Northern blot analysis. NH2- terminal and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in alpha1 (II) mRNA in proliferating chondrocytes. In addition, the COOH-terminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on alpha1 (II) and alpha1 (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage. PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell. PMID:8922395

  2. In vivo tumor targeting and imaging with engineered trivalent antibody fragments containing collagen-derived sequences.

    PubMed

    Cuesta, Angel M; Sánchez-Martín, David; Sanz, Laura; Bonet, Jaume; Compte, Marta; Kremer, Leonor; Blanco, Francisco J; Oliva, Baldomero; Alvarez-Vallina, Luis

    2009-01-01

    There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed "trimerbody", comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting.

  3. In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences

    PubMed Central

    Cuesta, Ángel M.; Sánchez-Martín, David; Sanz, Laura; Bonet, Jaume; Compte, Marta; Kremer, Leonor; Blanco, Francisco J.; Oliva, Baldomero; Álvarez-Vallina, Luis

    2009-01-01

    There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed “trimerbody”, comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting. PMID:19401768

  4. Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications.

    PubMed

    Chan, Elsa C; Kuo, Shyh-Ming; Kong, Anne M; Morrison, Wayne A; Dusting, Gregory J; Mitchell, Geraldine M; Lim, Shiang Y; Liu, Guei-Sheung

    2016-01-01

    Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.

  5. Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis.

    PubMed

    Orgel, J P R O; Antipova, O; Sagi, I; Bitler, A; Qiu, D; Wang, R; Xu, Y; San Antonio, J D

    2011-02-01

    Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the "master control region." Moreover, the collagen's most exposed aspect contains its most stable part-the C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of "cryptic" sequences poised to promote hemostasis and cell-collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.

  6. Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis

    SciTech Connect

    Orgel, J.P.; Antipova, O.; Sagi, I.; Bitler, A.; Qiu, D.; Wang, R.; Xu, Y.; San Antonio, J.D.

    2011-12-14

    Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the 'master control region.' Moreover, the collagen's most exposed aspect contains its most stable part - the C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of 'cryptic' sequences poised to promote hemostasis and cell - collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.

  7. Chitosan-Coated Collagen Membranes Promote Chondrocyte Adhesion, Growth, and Interleukin-6 Secretion

    PubMed Central

    Mighri, Nabila; Mao, Jifu; Mighri, Frej; Ajji, Abdallah; Rouabhia, Mahmoud

    2015-01-01

    Designing scaffolds made from natural polymers may be highly attractive for tissue engineering strategies. We sought to produce and characterize chitosan-coated collagen membranes and to assess their efficacy in promoting chondrocyte adhesion, growth, and cytokine secretion. Porous collagen membranes were placed in chitosan solutions then crosslinked with glutaraldehyde vapor. Fourier transform infrared (FTIR) analyses showed elevated absorption at 1655 cm−1 of the carbon–nitrogen (N=C) bonds formed by the reaction between the (NH2) of the chitosan and the (C=O) of the glutaraldehyde. A significant peak in the amide II region revealed a significant deacetylation of the chitosan. Scanning electron microscopy (SEM) images of the chitosan-coated membranes exhibited surface variations, with pore size ranging from 20 to 50 μm. X-ray photoelectron spectroscopy (XPS) revealed a decreased C–C groups and an increased C–N/C–O groups due to the reaction between the carbon from the collagen and the NH2 from the chitosan. Increased rigidity of these membranes was also observed when comparing the chitosan-coated and uncoated membranes at dried conditions. However, under wet conditions, the chitosan coated collagen membranes showed lower rigidity as compared to dried conditions. Of great interest, the glutaraldehyde-crosslinked chitosan-coated collagen membranes promoted chondrocyte adhesion, growth, and interleukin (IL)-6 secretion. Overall results confirm the feasibility of using designed chitosan-coated collagen membranes in future applications, such as cartilage repair. PMID:28793669

  8. Promotion of wound collagen formation in normal and diabetic mice by quadrol.

    PubMed

    Bhide, M V; Dunphy, M J; Mirkopulos, N; Smith, D J

    1988-01-01

    The rate of collagen deposition in implanted polytetrafluoroethylene (PTFE) tubing in non-diabetic and streptozotocin-induced (STZ) diabetic mice was measured during 14 days post-wounding. At the time of implantation, test groups received injections of either Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine], glucan, or buffer in an area adjacent to the wound site. The accumulation of collagen in the implants of Quadrol-treated non-diabetic animals was more than 200% above control on days 8 to 11 and was 50% above control on day 14. In Quadrol-treated STZ-diabetic mice, the collagen accumulation gradually increased from 50% above control on day 8 to 200% above control on day 14. Treatment with glucan increased the collagen accumulation in normal mice 200 to 300% above control from days 8 to 11 respectively and then 30% above control on day 14. Collagen accumulation in the implants of the glucan-treated STZ-diabetic mice was similar to the control group. These results indicate that Quadrol promotes in vivo collagen synthesis and that Quadrol may be effective as a stimulator of wound healing in diabetic and non-diabetic animals.

  9. A novel native collagen dressing with advantageous properties to promote physiological wound healing.

    PubMed

    Wiegand, C; Buhren, B A; Bünemann, E; Schrumpf, H; Homey, B; Frykberg, R G; Lurie, F; Gerber, P A

    2016-12-02

    Chronic hard-to-heal wounds generate high costs and resource use in western health systems and are the focus of intense efforts to improve healing outcomes. Here, we introduce a novel native collagen (90 %):alginate (10 %) wound dressing and compare it with the established oxidised dressings Method: Matrices were analysed by atomic force microscopy (AMF), scanning electron microscopy (SEM), and immunoelectron microscopy for collagen types I, III and V. Viability assays were performed with NIH-3T3 fibroblasts. Matrix metalloproteinase (MMP) binding was analysed, and the effect of the wound dressings on platelet-derived growth factor B homodimer (PDGF-BB) was investigated. Unlike oxidised regenerated cellulose (ORC)/collagen matrix and ovine forestomach matrix (OFM), the three-dimensional structure of the native collagen matrix (NCM) was found to be analogous to intact, native, dermal collagen. Fibroblasts seeded on the NCM showed exponential growth whereas in ORC/collagen matrix or OFM, very low rates of proliferation were observed after 7 days. MMP sequestration was effective and significant in the NCM. In addition, the NCM was able to significantly stabilise PDGF-BB in vitro. We hypothesise that the observed microstructure of the NCM allows for an effective binding of MMPs and a stabilisation and protection of growth factors and also promotes the ingrowth of dermal fibroblasts, potentially supporting the re commencement of healing in previously recalcitrant wounds. This work was supported by BSN Medical, Hamburg, Germany.

  10. Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Plopper, George E.

    2004-01-01

    The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity (fibronectin>collagen I≥collagen IV≥vitronectin>laminin-1) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells. PMID:15123885

  11. Breast Tumor-Generated Type 1 Collagen Breakdown Fragments Act as Matrikines to Drive Osteolysis

    DTIC Science & Technology

    2009-09-01

    triplicate. A dose - response curve will be determined using different concentrations of matrikine fragments. Cathepsin K, PTHrP, TNF-alpha, GM...in triplicate. A dose - response curve will be determined using different concentrations of matrikine fragments. Cathepsin K, PTHrP, TNF-alpha, GM

  12. Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling

    PubMed Central

    Arora, Pamma D.; Wang, Yongqiang; Bresnick, Anne; Janmey, Paul A.; McCulloch, Christopher A.

    2015-01-01

    We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1–6 capped actin filaments efficiently, whereas FliI GLD 2–6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1–6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2–6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2–6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1–6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts. PMID:25877872

  13. Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate.

    PubMed

    Feyen, J H; Petersen, D N; Kream, B E

    1988-04-01

    We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

  14. Collagen nerve conduits promote enhanced axonal regeneration, schwann cell association, and neovascularization compared to silicone conduits.

    PubMed

    Kemp, Stephen W P; Syed, Shahbaz; Walsh, Walsh; Zochodne, Douglas W; Midha, Rajiv

    2009-08-01

    Peripheral nerve regeneration within guidance conduits involves a critical association between regenerating axons, Schwann cells (SCs), and neovascularization. However, it is currently unknown if there is a greater association between these factors in nonpermeable versus semipermeable nerve guide conduits. We therefore examined this collaboration in both silicone- and collagen-based nerve conduits in both 5- and 10-mm-injury gaps in rat sciatic nerves. Results indicate that collagen conduits promoted enhanced axonal and SC regeneration and association when compared to silicone conduits in the shorter 5-mm-gap model. In addition, collagen tubes displayed enhanced neovascularization over silicone conduits, suggesting that these three factors are intimately related in successful peripheral nerve regeneration. At later time points (1- and 2-month analysis) in a 10-mm-gap model, collagen tubes displayed enhanced axonal regeneration, myelination, and vascularization when compared to silicone-based conduits. Results from these studies suggest that regenerating cables within collagen-based conduits are revascularized earlier and more completely, which in turn enhances peripheral nerve regeneration through these nerve guides as compared to silicone conduits.

  15. Transforming Growth Factor (TGF)-β Promotes de Novo Serine Synthesis for Collagen Production.

    PubMed

    Nigdelioglu, Recep; Hamanaka, Robert B; Meliton, Angelo Y; O'Leary, Erin; Witt, Leah J; Cho, Takugo; Sun, Kaitlyn; Bonham, Catherine; Wu, David; Woods, Parker S; Husain, Aliya N; Wolfgeher, Don; Dulin, Nickolai O; Chandel, Navdeep S; Mutlu, Gökhan M

    2016-12-30

    TGF-β promotes excessive collagen deposition in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). The amino acid composition of collagen is unique due to its high (33%) glycine content. Here, we report that TGF-β induces expression of glycolytic genes and increases glycolytic flux. TGF-β also induces the expression of the enzymes of the de novo serine synthesis pathway (phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)) and de novo glycine synthesis (serine hydroxymethyltransferase 2 (SHMT2)). Studies in fibroblasts with genetic attenuation of PHGDH or SHMT2 and pharmacologic inhibition of PHGDH showed that these enzymes are required for collagen synthesis. Furthermore, metabolic labeling experiments demonstrated carbon from glucose incorporated into collagen. Lungs from humans with IPF demonstrated increased expression of PHGDH and SHMT2. These results indicate that the de novo serine synthesis pathway is necessary for TGF-β-induced collagen production and suggest that this pathway may be a therapeutic target for treatment of fibrotic diseases including IPF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Measurement of matrix metalloproteinase 9-mediated collagen type III degradation fragment as a marker of skin fibrosis.

    PubMed

    Vassiliadis, Efstathios; Veidal, Sanne Skovgård; Barascuk, Natasha; Mullick, Jhinuk Basu; Clausen, Rikke Elgaard; Larsen, Lise; Simonsen, Henrik; Larsen, Dorthe Vang; Bay-Jensen, Anne-Christine; Segovia-Silvestre, Toni; Leeming, Diana Julie; Karsdal, Morten A

    2011-03-29

    The current study utilized a Bleomycin-induced model of skin fibrosis to investigate the neo-epitope CO3-610 (KNGETGPQGP), a fragment of collagen III released during matrix metalloproteinase-9 (MMP9) degradation of the protein, we have previously described as a novel biomarker for liver fibrosis. The aim was to investigate CO3-610 levels in another well characterised model of fibrosis, to better describe the biomarker in relation to additional fibrotic pathologies. Skin fibrosis was induced by daily injections of Bleomycin to a total of 52 female C3 H mice, while control mice (n = 28) were treated with phosphate buffered saline (PBS), for 2, 4, 6 or 8 weeks. Skin fibrosis was evaluated using Visiopharm software on Sirius-red stained skin sections. Urine ELISA assays and creatinine corrections were performed to measure CO3-610 levels. CO3-610 levels were significantly higher in Bleomycin-treated vs. PBS-treated mice at each time point of termination. The mean increases were: 59.2%, P < 0.0008, at 2 weeks; 113.5%, P < 0.001, at 4 weeks; 136.8%, P < 0.0001 at 6 weeks; 157.2%, P < 0.0001 at 8 weeks). PBS-treated mice showed a non-significant increase in CO3-610 levels (mean increase for weeks 2-8 = 1.7%, P = 0.789) CO3-610 levels assayed in urine were statistically significantly correlated with Western blot analysis showing increased skin fibrosis (P < 0.0001, r = 0.65). Increased levels in mouse urine of the MMP-9 mediated collagen III degradation fragment CO3-610 were correlated with skin fibrosis progression, suggesting that CO3-610 may be a potential positive biomarker to study the pathogenesis of skin fibrosis in mice.

  17. The electron-impact promoted fragmentation of aurone epoxides.

    NASA Technical Reports Server (NTRS)

    Brady, B. A.; O'Sullivan, W. I.; Duffield, A. M.

    1972-01-01

    The mass spectra of six aurone epoxides have been rationalized with the aid of high resolution mass spectrometry and metastable ion evidence. These compounds fragment in a well defined manner and mechanisms are proposed for the formation of their characteristic ions. Some similarity was observed between the mass spectra of 6-methoxyaurone epoxide (II), 4-hydroxy-7-methoxy-3-phenylcoumarin (VII) and 7-methoxyflavonol (IX). The possibility that VII and IX are intermediates in the fragmentation of epoxide II is discussed. Thermal rearrangement of aurone epoxide II was shown to yield the corresponding flavonol IX and coumarin VII.

  18. uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion.

    PubMed

    Engelholm, Lars H; List, Karin; Netzel-Arnett, Sarah; Cukierman, Edna; Mitola, David J; Aaronson, Hannah; Kjøller, Lars; Larsen, Jørgen K; Yamada, Kenneth M; Strickland, Dudley K; Holmbeck, Kenn; Danø, Keld; Birkedal-Hansen, Henning; Behrendt, Niels; Bugge, Thomas H

    2003-03-31

    The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

  19. ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth.

    PubMed

    Rath, Nicola; Morton, Jennifer P; Julian, Linda; Helbig, Lena; Kadir, Shereen; McGhee, Ewan J; Anderson, Kurt I; Kalna, Gabriela; Mullin, Margaret; Pinho, Andreia V; Rooman, Ilse; Samuel, Michael S; Olson, Michael F

    2017-02-01

    Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a Kras(G12D)/p53(R172H) mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three-dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK-induced genes that facilitate extracellular matrix remodeling, with greatest fold-changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13 MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three-dimensional contexts. Treatment of Kras(G12D)/p53(R172H) PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor-associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.

  20. Biomimetic LBL structured nanofibrous matrices assembled by chitosan/collagen for promoting wound healing.

    PubMed

    Huang, Rong; Li, Wangzhou; Lv, Xiaoxing; Lei, Zhanjun; Bian, Yongqian; Deng, Hongbing; Wang, Hongjun; Li, Jinqing; Li, Xueyong

    2015-06-01

    This paper reports the fabrication of biomimetic nanofibrous matrices via co-electrospinning of polycaprolactone (PCL)/cellulose acetate (CA) and layer-by-layer self-assembly (LBL) of positively charged chitosan (CS) and negatively charged Type Ⅰ collagen on the nanofibrous matrix. FE-SEM images indicate that the average fiber diameter increased from 392 to 541 nm when the coating bilayers varied from 5 to 20.5. Besides, the excellent biocompatibility and enhanced attachment and spreading of normal human dermal fibroblasts (NHDFs) of prepared nanofibrous mats are confirmed by MTT and SEM results. Furthermore, the LBL structured (CS/collagen)n nanofibrous mats greatly improve the cell migration in vitro, promote re-epithelialization and vascularization in vivo, and up-regulate the expression of collagen Ⅳ and α-tubulin, as well as the Integrin β1 and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. The levels of expressed protein are significantly enhanced with increasing coating bilayers via immunohistochemistry and western blotting analyses. Collectively, these results suggest that the LBL structured biomimetic nanofibrous matrices may enhance cell migration and further promote the skin regeneration by up-regulating the secretion of ECM protein and triggering Integrin/FAK signaling pathway, which demonstrate the potential use of the nanofibrous mats to rapidly restore the structural and functional properties of wounded skin.

  1. Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis.

    PubMed

    Veidal, Sanne S; Karsdal, Morten A; Nawrocki, Arkadiusz; Larsen, Martin R; Dai, Yueqin; Zheng, Qinlong; Hägglund, Per; Vainer, Ben; Skjøt-Arkil, Helene; Leeming, Diana J

    2011-10-05

    Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens by proteases produces small fragments, so-called neoepitopes, which are released systemically. Technologies investigating MMP-generated fragments of collagens may provide more useful information than traditional serological assays that crudely measure total protein. In the present study, we developed an ELISA for the quantification of a neoepitope generated by MMP degradation of type IV collagen and evaluated the association of this neoepitope with liver fibrosis in two animal models. Type IV collagen was degraded in vitro by a variety of proteases. Mass spectrometric analysis revealed more than 200 different degradation fragments. A specific peptide sequence, 1438'GTPSVDHGFL'1447 (CO4-MMP), in the α1 chain of type IV collagen generated by MMP-9 was selected for ELISA development. ELISA was used to determine serum levels of the CO4-MMP neoepitope in two rat models of liver fibrosis: inhalation of carbon tetrachloride (CCl4) and bile duct ligation (BDL). The levels were correlated to histological findings using Sirius red staining. A technically robust assay was produced that is specific to the type IV degradation fragment, GTPSVDHGFL. CO4-MMP serum levels increased significantly in all BDL groups compared to baseline, with a maximum increase of 248% seen two weeks after BDL. There were no changes in CO4-MMP levels in sham-operated rats. In the CCl4 model, levels of CO4-MMP were significantly elevated at weeks 12, 16 and 20 compared to baseline levels, with a maximum increase of 88% after 20 weeks. CO4-MMP levels correlated to Sirius red staining results. This ELISA is the first assay developed for assessment of proteolytic degraded type IV collagen, which, by enabling

  2. Metal-Promoted Assembly of Two Collagen Mimetic Peptides into a Biofunctional "Spiraled Horn" Scaffold.

    PubMed

    Strauss, Kevin; Chmielewski, Jean

    2016-10-17

    Biofunctional scaffolds for the delivery of living cells are of the utmost importance for regenerative medicine. Herein, a novel, robust "spiraled horn" scaffold was elucidated through the Co(2+)-promoted hierarchical assembly of two collagen mimetic peptides, NCoH and HisCol. Each "horn" displayed a periodic banding pattern with band lengths corresponding to the length of the collagen peptide triple helix. Strand exchange between the two peptide trimers resulted in failure to form this intricate morphology, lending support to a precise metal-ligand-based mechanism of assembly. Little change occurred to the observed morphology when the Co(2+) concentration was varied from 0.5 to 4.0 mM, and the scaffold was found to be fully formed within two minutes of exposure to the metal ion. The horned network also displayed biological functionality by binding to a His-tagged fluorophore and associating with cells.

  3. Nitrated fatty acids reverse pulmonary fibrosis by dedifferentiating myofibroblasts and promoting collagen uptake by alveolar macrophages.

    PubMed

    Reddy, Aravind T; Lakshmi, Sowmya P; Zhang, Yingze; Reddy, Raju C

    2014-12-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disease, thought to be largely transforming growth factor β (TGFβ) driven, for which there is no effective therapy. We assessed the potential benefits in IPF of nitrated fatty acids (NFAs), which are unique endogenous agonists of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor that exhibits wound-healing and antifibrotic properties potentially useful for IPF therapy. We found that pulmonary PPARγ is down-regulated in patients with IPF. In vitro, knockdown or knockout of PPARγ expression in isolated human and mouse lung fibroblasts induced a profibrotic phenotype, whereas treating human fibroblasts with NFAs up-regulated PPARγ and blocked TGFβ signaling and actions. NFAs also converted TGFβ to inactive monomers in cell-free solution, suggesting an additional mechanism through which they may inhibit TGFβ. In vivo, treating mice bearing experimental pulmonary fibrosis with NFAs reduced disease severity. Also, NFAs up-regulated the collagen-targeting factor milk fat globule-EGF factor 8 (MFG-E8), stimulated collagen uptake and degradation by alveolar macrophages, and promoted myofibroblast dedifferentiation. Moreover, treating mice with established pulmonary fibrosis using NFAs reversed their existing myofibroblast differentiation and collagen deposition. These findings raise the prospect of treating IPF with NFAs to halt and perhaps even reverse the progress of IPF. © FASEB.

  4. rFN/Cad-11-modified collagen type II biomimetic interface promotes the adhesion and chondrogenic differentiation of mesenchymal stem cells.

    PubMed

    Dong, Shiwu; Guo, Hongfeng; Zhang, Yuan; Li, Zhengsheng; Kang, Fei; Yang, Bo; Kang, Xia; Wen, Can; Yan, Yanfei; Jiang, Bo; Fan, Yujiang

    2013-11-01

    Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7-10 of fibronectin module III (heterophilic motif) and extracellular domains 1-2 of cadherin-11 (rFN/Cad-11) (homophilic motif), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo.

  5. rFN/Cad-11-Modified Collagen Type II Biomimetic Interface Promotes the Adhesion and Chondrogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Guo, Hongfeng; Zhang, Yuan; Li, Zhengsheng; Kang, Fei; Yang, Bo; Kang, Xia; Wen, Can; Yan, Yanfei; Jiang, Bo; Fan, Yujiang

    2013-01-01

    Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7–10 of fibronectin module III (heterophilic motif ) and extracellular domains 1–2 of cadherin-11 (rFN/Cad-11) (homophilic motif ), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo. PMID:23919505

  6. PDGF-metronidazole-encapsulated nanofibrous functional layers on collagen membrane promote alveolar ridge regeneration

    PubMed Central

    Ho, Ming-Hua; Chang, Hao-Chieh; Chang, Yu-Chia; Claudia, Jeiannete; Lin, Tzu-Chiao; Chang, Po-Chun

    2017-01-01

    This study aimed to develop a functionally graded membrane (FGM) to prevent infection and promote tissue regeneration. Poly(l-lactide-co-d,l-lactide) encapsulating platelet-derived growth factor (PDLLA-PDGF) or metronidazole (PDLLA-MTZ) was electrospun to form a nanofibrous layer on the inner or outer surface of a clinically available collagen membrane, respectively. The membrane was characterized for the morphology, molecule release profile, in vitro and in vivo biocompatibility, and preclinical efficiency for alveolar ridge regeneration. The PDLLA-MTZ and PDLLA-PDGF nanofibers were 800–900 nm in diameter, and the thicknesses of the functional layers were 20–30 μm, with sustained molecule release over 28 days. All of the membranes tested were compatible with cell survival in vitro and showed good tissue integration with minimal fibrous capsule formation or inflammation. Cell proliferation was especially prominent on the PDLLA-PDGF layer in vivo. On the alveolar ridge, all FGMs reduced wound dehiscence compared with the control collagen membrane, and the FGM with PDLLA-PDGF promoted osteogenesis significantly. In conclusion, the FGMs with PDLLA-PDGF and PDLLA-MTZ showed high biocompatibility and facilitated wound healing compared with conventional membrane, and the FGM with PDLLA-PDGF enhanced alveolar ridge regeneration in vivo. The design represents a beneficial modification, which may be easily adapted for future clinical use. PMID:28831251

  7. PDGF-metronidazole-encapsulated nanofibrous functional layers on collagen membrane promote alveolar ridge regeneration.

    PubMed

    Ho, Ming-Hua; Chang, Hao-Chieh; Chang, Yu-Chia; Claudia, Jeiannete; Lin, Tzu-Chiao; Chang, Po-Chun

    2017-01-01

    This study aimed to develop a functionally graded membrane (FGM) to prevent infection and promote tissue regeneration. Poly(l-lactide-co-d,l-lactide) encapsulating platelet-derived growth factor (PDLLA-PDGF) or metronidazole (PDLLA-MTZ) was electrospun to form a nanofibrous layer on the inner or outer surface of a clinically available collagen membrane, respectively. The membrane was characterized for the morphology, molecule release profile, in vitro and in vivo biocompatibility, and preclinical efficiency for alveolar ridge regeneration. The PDLLA-MTZ and PDLLA-PDGF nanofibers were 800-900 nm in diameter, and the thicknesses of the functional layers were 20-30 μm, with sustained molecule release over 28 days. All of the membranes tested were compatible with cell survival in vitro and showed good tissue integration with minimal fibrous capsule formation or inflammation. Cell proliferation was especially prominent on the PDLLA-PDGF layer in vivo. On the alveolar ridge, all FGMs reduced wound dehiscence compared with the control collagen membrane, and the FGM with PDLLA-PDGF promoted osteogenesis significantly. In conclusion, the FGMs with PDLLA-PDGF and PDLLA-MTZ showed high biocompatibility and facilitated wound healing compared with conventional membrane, and the FGM with PDLLA-PDGF enhanced alveolar ridge regeneration in vivo. The design represents a beneficial modification, which may be easily adapted for future clinical use.

  8. Preparation of chitosan-collagen-alginate composite dressing and its promoting effects on wound healing.

    PubMed

    Xie, Haixia; Chen, Xiuli; Shen, Xianrong; He, Ying; Chen, Wei; Luo, Qun; Ge, Weihong; Yuan, Weihong; Tang, Xue; Hou, Dengyong; Jiang, Dingwen; Wang, Qingrong; Liu, Yuming; Liu, Qiong; Li, Kexian

    2017-08-30

    The present study aimed to prepare a composite dressing composed of collagen, chitosan, and alginate, which may promote wound healing and prevent from seawater immersion. Chitosan-collagen-alginate (CCA) cushion was prepared by paintcoat and freeze-drying, and it was attached to a polyurethane to compose CCA composite dressing. The swelling, porosity, degradation, and mechanical properties of CCA cushion were evaluated. The effects on wound healing and seawater prevention of CCA composite dressing were tested by rat wound model. Preliminary biosecurity was tested by cytotoxicity and hemocompatibility. The results revealed that CCA cushion had good water absorption and mechanical properties. A higher wound healing ratio was observed in CCA composite dressing treated rats than in gauze or chitosan treated ones. On the fifth day, the healing rates of CCA composite dressing, gauze, and chitosan were 48.49%±1.07%, 28.02%±6.4%, and 38.97%±8.53%, respectively. More fibroblast and intact re-epithelialization were observed in histological images of CCA composite dressing treated rats, and the expressions of EGF, bFGF, TGF-β, and CD31 increased significantly. CCA composite dressing showed no significant cytotoxicity, and favorable hemocompatibility. These results suggested that CCA composite dressing could prevent against seawater immersion and promote wound healing while having a good biosecurity. Copyright © 2017. Published by Elsevier B.V.

  9. Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice

    PubMed Central

    1996-01-01

    To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out. This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath. (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase. (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines. The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence

  10. PDGFRα signalling promotes fibrogenic responses in collagen-producing cells in Duchenne muscular dystrophy.

    PubMed

    Ieronimakis, Nicholas; Hays, Aislinn; Prasad, Amalthiya; Janebodin, Kajohnkiart; Duffield, Jeremy S; Reyes, Morayma

    2016-12-01

    Fibrosis is a characteristic of Duchenne muscular dystrophy (DMD), yet the cellular and molecular mechanisms responsible for DMD fibrosis are poorly understood. Utilizing the Collagen1a1-GFP transgene to identify cells producing Collagen-I matrix in wild-type mice exposed to toxic injury or those mutated at the dystrophin gene locus (mdx) as a model of DMD, we studied mechanisms of skeletal muscle injury/repair and fibrosis. PDGFRα is restricted to Sca1+, CD45- mesenchymal progenitors. Fate-mapping experiments using inducible CreER/LoxP somatic recombination indicate that these progenitors expand in injury or DMD to become PDGFRα+, Col1a1-GFP+ matrix-forming fibroblasts, whereas muscle fibres do not become fibroblasts but are an important source of the PDGFRα ligand, PDGF-AA. While in toxin injury/repair of muscle PDGFRα, signalling is transiently up-regulated during the regenerative phase in the DMD model and in human DMD it is chronically overactivated. Conditional expression of the constitutively active PDGFRα D842V mutation in Collagen-I+ fibroblasts, during injury/repair, hindered the repair phase and instead promoted fibrosis. In DMD, treatment of mdx mice with crenolanib, a highly selective PDGFRα/β tyrosine kinase inhibitor, reduced fibrosis, improved muscle strength, and was associated with decreased activity of Src, a downstream effector of PDGFRα signalling. These observations are consistent with a model in which PDGFRα activation of mesenchymal progenitors normally regulates repair of the injured muscle, but in DMD persistent and excessive activation of this pathway directly drives fibrosis and hinders repair. The PDGFRα pathway is a potential new target for treatment of progressive DMD. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society

  11. Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

    PubMed

    Schofield, Desmond M; Templar, Alex; Newton, Joseph; Nesbeth, Darren N

    2016-07-08

    Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016.

  12. NF-κB accumulation associated with COL1A1 transactivators defects during chronological aging represses type I collagen expression through a -112/-61-bp region of the COL1A1 promoter in human skin fibroblasts.

    PubMed

    Bigot, Nicolas; Beauchef, Gallic; Hervieu, Magalie; Oddos, Thierry; Demoor, Magali; Boumediene, Karim; Galéra, Philippe

    2012-10-01

    The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

  13. Epithelial derived CTGF promotes breast tumor progression via inducing EMT and collagen I fibers deposition

    PubMed Central

    Zhao, Zhen; Sheng, Jianting; Wang, Jiang; Liu, Jiyong; Cui, Kemi; Chang, Jenny; Zhao, Hong; Wong, Stephen

    2015-01-01

    Interactions among tumor cells, stromal cells, and extracellular matrix compositions are mediated through cytokines during tumor progression. Our analysis of 132 known cytokines and growth factors in published clinical breast cohorts and our 84 patient-derived xenograft models revealed that the elevated connective tissue growth factor (CTGF) in tumor epithelial cells significantly correlated with poor clinical prognosis and outcomes. CTGF was able to induce tumor cell epithelial-mesenchymal transition (EMT), and promote stroma deposition of collagen I fibers to stimulate tumor growth and metastasis. This process was mediated through CTGF-tumor necrosis factor receptor I (TNFR1)-IκB autocrine signaling. Drug treatments targeting CTGF, TNFR1, and IκB signaling each prohibited the EMT and tumor progression. PMID:26318291

  14. Review on Labisia pumila (Kacip Fatimah): bioactive phytochemicals and skin collagen synthesis promoting herb.

    PubMed

    Chua, Lee Suan; Lee, Sze Yean; Abdullah, Norhanisah; Sarmidi, Mohamad Roji

    2012-12-01

    Labisia pumila is a traditional herb widely used as post-partum medication for centuries. Recently, extensive researches have been carried out on the phytochemical identification, biological and toxicological studies for the herb. Phytochemicals found in the herbal extract showed high antioxidant properties, which were essential for various pharmacological activities. The significant findings are anti-estrogenic deficiency and -immunodeficiency diseases. Another finding that has considerable impact on natural product research is the contribution of L. pumila in promoting skin collagen synthesis. The performance of the herb as anti-aging agent due to natural aging process and accelerated by UV radiation was reviewed critically. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    NASA Astrophysics Data System (ADS)

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-03-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  16. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    PubMed Central

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-01-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS – which was released from scaffolds quickly – significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA. PMID:28256634

  17. Glutaminolysis Promotes Collagen Translation and Stability via α-ketoglutarate Mediated mTOR Activation and Proline Hydroxylation.

    PubMed

    Ge, Jing; Cui, Huachun; Xie, Na; Banerjee, Sami; Guo, Sijia; Dubey, Shubham; Barnes, Stephen; Liu, Gang

    2017-10-11

    Glutaminolysis is the metabolic process of glutamine, of which aberration has been implicated in several pathogeneses. While we and others recently found a diversity of metabolic dysregulations in organ fibrosis, it is unknown if glutaminolysis regulates the pro-fibrotic activities of myofibroblasts, the primary effector in this pathology. In this study, we found that lung myofibroblasts demonstrated significantly augmented glutaminolysis that was mediated by elevated Glutaminase 1 (Gls1). Inhibition of glutaminolysis by specific Gls1 inhibitors CB-839 and BPTES as well as Gls1 siRNA blunted the expression of collagens, but not that of Fibronectin, Elastin or myofibroblastic marker smooth muscle actin α (SMA-α). We found that glutaminolysis enhanced collagen translation and stability, which were mediated by glutaminolysis dependent mammalian target of rapamycin complex 1 (mTORC1) activation and collagen proline hydroxylation, respectively. Furthermore, we found that level of glutaminolytic end product, α-ketoglutarate (α-KG), was increased in myofibroblasts. Similar to glutaminolysis, α-KG activated mTORC1 and promoted expression of collagens, but not that of Fibronectin, Elastin, or SMA-α. α-KG also remarkably inhibited collagen degradation in fibroblasts. Taken together, our studies identified a previously unrecognized mechanism by which a major metabolic program regulates the exuberant production of collagens in myofibroblasts and suggest glutaminolysis being a novel therapeutic target for treating organ fibrosis, including idiopathic pulmonary fibrosis (IPF).

  18. Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells

    PubMed Central

    Chen, Jennifer Y.; Newcomb, Benjamin; Zhou, Chan; Pondick, Joshua V.; Ghoshal, Sarani; York, Samuel R.; Motola, Daniel L.; Coant, Nicolas; Yi, Jae Kyo; Mao, Cungui; Tanabe, Kenneth K.; Bronova, Irina; Berdyshev, Evgeny V.; Fuchs, Bryan C.; Hannun, Yusuf; Chung, Raymond T.; Mullen, Alan C.

    2017-01-01

    Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide. PMID:28322247

  19. Pyrazinamide potential effects on male rats DNA fragmentation, bone type I collagen amino acid composition, reproductive capability and posterity antenatal and postnatal development.

    PubMed

    Bondarenko, Larysa B; Shayakhmetova, Ganna M; Byshovets, Taisiya F; Kovalenko, Valentina M

    2012-01-01

    Current therapeutic regimens with first-line antitubercular agents are associated with a high rate of adverse effects which can lead to therapeutic failure. Understanding the nature and the severity of these effects is important for treatment optimization. The aim of present study was to investigate pyrazinamide potential effects on male rats DNA fragmentation, amino acid composition of bone type I collagen, reproductive capability and their posterity antenatal and postnatal development. Wistar albino male rats (160-200 g b.w.) were divided into three groups: I--received pyrazinamide per os at a dose of 1000 mg/kg b.w./day, II--at a dose of 2000 mg/kg b.w./day, in both groups it was given for 60 days; III--control. After 60 days of the experiment, rats of the experimental (groups I and II) and control groups were mated with intact virgin females. The amino acids contents of male rat bone type I collagens were determined using amino acid analyzer, epididymis and testis DNA fragmentation--electrophoretically; posterity antenatal development indices and postnatal development--by standard procedures. The study of pyrazinamide effects (administered in different doses) on males bone type I collagen amino acid contents and testis DNA fragmentation demonstrated the presence of dose-dependent pyrazinamide-mediated quantitative and qualitative changes in male rat reproductive organs DNA and extracellular matrix proteins in comparison with control. Changes in nucleic acids and proteins structure were accompanied by alterations in processes of fertilization (with intact females), embryogenesis and by lowering of posterity survival.

  20. Fish scale-derived collagen patch promotes growth of blood and lymphatic vessels in vivo.

    PubMed

    Wang, Jun Kit; Yeo, Kim Pin; Chun, Yong Yao; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Angeli, Véronique; Choong, Cleo

    2017-09-06

    In this study, Type I collagen was extracted from fish scales as a potential alternative source of collagen for tissue engineering applications. Since unmodified collagen typically has poor mechanical and degradation stability both in vitro and in vivo, additional methylation modification and 1,4-butanediol diglycidyl ether (BDE) crosslinking steps were used to improve the physicochemical properties of fish scale-derived collagen. Subsequently, in vivo studies using a murine model demonstrated the biocompatibility of the different fish scale-derived collagen patches. In general, favorable integration of the collagen patches to the surrounding tissues, with good infiltration of cells, blood vessels (BVs) and lymphatic vessels (LVs) were observed under growth factor-free conditions. Interestingly, significantly higher (p < 0.05) number of LVs was found to be more abundant around collagen patches with methylation modification and BDE crosslinking. Overall, we have demonstrated the potential application of fish scale-derived collagen as a promising scaffolding material for various biomedical applications. Currently the most common sources of collagen are of bovine and porcine origins, although the industrial use of collagen obtained from non-mammalian species is growing in importance, particularly since they have a lower risk of disease transmission and are not subjected to any cultural or religious constraints. However, unmodified collagen typically has poor mechanical and degradation stability both in vitro and in vivo. Hence, in this study, Type I collagen was successfully extracted from fish scales and chemically modified and crosslinked. In vitro studies showed overall improvement in the physicochemical properties of the material, whilst in vivo implantation studies showed improvements in the growth of blood and lymphatic host vessels in the vicinity of the implants. Copyright © 2017. Published by Elsevier Ltd.

  1. Cryptic collagen IV promotes cell migration and adhesion in myeloid leukemia.

    PubMed

    Favreau, Amanda J; Vary, Calvin P H; Brooks, Peter C; Sathyanarayana, Pradeep

    2014-04-01

    Previously, we showed that discoidin domain receptor 1 (DDR1), a class of collagen-activated receptor tyrosine kinase (RTK) was highly upregulated on bone marrow (BM)-derived CD33+ leukemic blasts of acute myeloid leukemia (AML) patients. Herein as DDR1 is a class of collagen-activated RTK, we attempt to understand the role of native and remodeled collagen IV in BM microenvironment and its functional significance in leukemic cells. Exposure to denatured collagen IV significantly increased the migration and adhesion of K562 cells, which also resulted in increased activation of DDR1 and AKT. Further, levels of MMP9 were increased in conditioned media (CM) of denatured collagen IV exposed cells. Mass spectrometric liquid chromatography/tandem mass spectrometry QSTAR proteomic analysis revealed exclusive presence of Secretogranin 3 and InaD-like protein in the denatured collagen IV CM. Importantly, BM samples of AML patients exhibited increased levels of remodeled collagen IV compared to native as analyzed via anti-HUIV26 antibody. Taken together, for the first time, we demonstrate that remodeled collagen IV is a potent activator of DDR1 and AKT that also modulates both migration and adhesion of myeloid leukemia cells. Additionally, high levels of the HUIV26 cryptic collagen IV epitope are expressed in BM of AML patients. Further understanding of this phenomenon may lead to the development of therapeutic agents that directly modulate the BM microenvironment and attenuate leukemogenesis.

  2. Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing.

    PubMed

    Fortunati, Dario; Chau, David Yi San; Wang, Zhuo; Collighan, Russell John; Griffin, Martin

    2014-07-01

    Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.

  3. Type I pro-collagen promoting and anti-collagenase activities of Phyllanthus emblica extract in mouse fibroblasts.

    PubMed

    Chanvorachote, Pithi; Pongrakhananon, Varisa; Luanpitpong, Sudjit; Chanvorachote, Boontarika; Wannachaiyasit, Sumalee; Nimmannit, Ubonthip

    2009-01-01

    As part of an ongoing search for the novel pharmacological activities of Phyllanthus emblica, the present study has shown its type I collagen promoting and anti-collagenase effects on primary mouse fibroblast cells. At a concentration of 0.1 mg/ml, emblica extract significantly increased the type I pro-collagen level up to 1.65-fold, and 6.78-fold greater than that of an untreated control, determined by immunocytochemistry and Western blot analysis, respectively. Emblica extract caused an approximately 7.75-fold greater type I pro-collagen induction compared to the known herbal collagen enhancer asiaticoside at the same treatment concentration (0.1 mg/ml). Moreover, emblica extract inhibited collagenase activity in a dose-dependent manner. Maximal inhibition was observed (78.67 +/- 3.51%) at a concentration of 1 mg/ml. In summary, emblica extract has a promising pharmacological effect that benefits collagen synthesis and protects against its degradation and could be used as a natural anti-aging ingredient.

  4. Hyperbaric Oxygen Promotes Proximal Bone Regeneration and Organized Collagen Composition during Digit Regeneration

    PubMed Central

    Sammarco, Mimi C.; Simkin, Jennifer; Cammack, Alexander J.; Fassler, Danielle; Gossmann, Alexej; Marrero, Luis; Lacey, Michelle; Van Meter, Keith; Muneoka, Ken

    2015-01-01

    Oxygen is critical for optimal bone regeneration. While axolotls and salamanders have retained the ability to regenerate whole limbs, mammalian regeneration is restricted to the distal tip of the digit (P3) in mice, primates, and humans. Our previous study revealed the oxygen microenvironment during regeneration is dynamic and temporally influential in building and degrading bone. Given that regeneration is dependent on a dynamic and changing oxygen environment, a better understanding of the effects of oxygen during wounding, scarring, and regeneration, and better ways to artificially generate both hypoxic and oxygen replete microenvironments are essential to promote regeneration beyond wounding or scarring. To explore the influence of increased oxygen on digit regeneration in vivo daily treatments of hyperbaric oxygen were administered to mice during all phases of the entire regenerative process. Micro-Computed Tomography (μCT) and histological analysis showed that the daily application of hyperbaric oxygen elicited the same enhanced bone degradation response as two individual pulses of oxygen applied during the blastema phase. We expand past these findings to show histologically that the continuous application of hyperbaric oxygen during digit regeneration results in delayed blastema formation at a much more proximal location after amputation, and the deposition of better organized collagen fibers during bone formation. The application of sustained hyperbaric oxygen also delays wound closure and enhances bone degradation after digit amputation. Thus, hyperbaric oxygen shows the potential for positive influential control on the various phases of an epimorphic regenerative response. PMID:26452224

  5. Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing.

    PubMed

    Gilmartin, Daniel J; Soon, Allyson; Thrasivoulou, Christopher; Phillips, Anthony R J; Jayasinghe, Suwan N; Becker, David L

    2016-07-01

    Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F-127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Hyperbaric Oxygen Promotes Proximal Bone Regeneration and Organized Collagen Composition during Digit Regeneration.

    PubMed

    Sammarco, Mimi C; Simkin, Jennifer; Cammack, Alexander J; Fassler, Danielle; Gossmann, Alexej; Marrero, Luis; Lacey, Michelle; Van Meter, Keith; Muneoka, Ken

    2015-01-01

    Oxygen is critical for optimal bone regeneration. While axolotls and salamanders have retained the ability to regenerate whole limbs, mammalian regeneration is restricted to the distal tip of the digit (P3) in mice, primates, and humans. Our previous study revealed the oxygen microenvironment during regeneration is dynamic and temporally influential in building and degrading bone. Given that regeneration is dependent on a dynamic and changing oxygen environment, a better understanding of the effects of oxygen during wounding, scarring, and regeneration, and better ways to artificially generate both hypoxic and oxygen replete microenvironments are essential to promote regeneration beyond wounding or scarring. To explore the influence of increased oxygen on digit regeneration in vivo daily treatments of hyperbaric oxygen were administered to mice during all phases of the entire regenerative process. Micro-Computed Tomography (μCT) and histological analysis showed that the daily application of hyperbaric oxygen elicited the same enhanced bone degradation response as two individual pulses of oxygen applied during the blastema phase. We expand past these findings to show histologically that the continuous application of hyperbaric oxygen during digit regeneration results in delayed blastema formation at a much more proximal location after amputation, and the deposition of better organized collagen fibers during bone formation. The application of sustained hyperbaric oxygen also delays wound closure and enhances bone degradation after digit amputation. Thus, hyperbaric oxygen shows the potential for positive influential control on the various phases of an epimorphic regenerative response.

  7. Fucoidan is the active component of fucus vesiculosus that promotes contraction of fibroblast-populated collagen gels.

    PubMed

    Fujimura, T; Shibuya, Y; Moriwaki, S; Tsukahara, K; Kitahara, T; Sano, T; Nishizawa, Y; Takema, Y

    2000-10-01

    The fibroblast-populated collagen gel culture method has been evaluated as a dermal model of wound contraction and granulation in tissues during the wound healing process and as an in vitro model of dermal tissue. We previously reported that an extract of Fucus vesiculosus promoted fibroblast-populated collagen gel contraction and that the promotion of the gel contraction was due to the increased expression of integrin alpha2beta1 on the surface of the fibroblasts. In this study, we investigated the active component of the extract of this alga using extraction and fractionation techniques. Water extraction of the alga was followed by precipitation with excess ethanol and then gel filtration with the boundary molecular weight of 30,000. The high molecular weight fraction obtained from gel filtration was fractionated by ion exchange chromatography on diethylaminoethyl cellulose column to give active fractions that have more polar properties. These polar, high molecular weight fractions which contained molecules with fucose and sulfate groups showed significant gel contraction-promoting activity and integrin expression-enhancing activity, and were estimated to be the sulfated-polysaccharide fucoidan. Commercially available fucoidan showed similar activities to the above-described fraction of this alga. Although it remains necessary to precisely identify the specific active component, the above results indicate that fucoidan is the active component which promotes collagen gel contraction, and also indicate the possibility that it dose so by enhancing the integrin alpha2beta1 expression.

  8. Transgenic mice with a mutated collagen promoter display normal response during bleomycin-induced fibrosis and possess neurological abnormalities.

    PubMed

    Stoddart, J H; Ladd, D; Bronson, R T; Harmon, M; Jaworski, J; Pritzker, C; Lausen, N; Smith, B D

    2000-02-01

    We have previously identified a potential TGF-beta activation element (TAE) in the rat collagen alpha1(I) promoter at -1624 upstream of the transcriptional start site [Ritzenthaler et al., 1991, 1993]. To determine the importance of the TAE in vivo, we produced transgenic mice carrying 3.6 kb of the rat collagen alpha1(I) promoter linked to the reporter gene chloramphenicol acetyl transferase with and without site-directed mutations that eliminate DNA-protein binding at the TAE site. Tissue-specific expression of the reporter gene in transgenic mice with the mutated collagen promoter was similar to that of transgenic mice with the normal promoter in two genetic backgrounds as judged by in situ hybridization, reporter assays, and immunochemistry. Endotracheal instillation of bleomycin induces lung fibrosis, mediated in part by TGF-beta. Earlier studies indicated that expression of wild-type collagen-reporter gene was upregulated in transgenic mice lungs in response to endotracheal instillation of bleomycin. A similar level of reporter gene upregulation was observed in transgenic mice carrying the mutation in the TAE. Two lines of transgenic mice carrying the mutated promoter construct displayed unexpected neurological abnormalities. In the FVB genetic background, there was a higher than normal incidence of mortality, spontaneous seizures, and an inability to nurture offspring. Histological evidence demonstrated clear abnormalities, including disorderly arrangement of neurons in the hippocampus and significant laminar cortical necrosis in the cerebrum in animals after seizures. In the C57Bl/6 background, there was a high incidence of severe communicating hydrocephalus, early runting, and increased mortality similar to that in transgenic animals with astroglial overexpression of TGF-beta. These animals provide an interesting model system to investigate molecular mechanisms responsible for seizures and hydrocephalus. Copyright 2000 Wiley-Liss, Inc.

  9. Type IV collagen aggregates promote keratinocyte proliferation and formation of epidermal layer in human skin equivalents.

    PubMed

    Matsuura-Hachiya, Yuko; Arai, Koji Y; Muraguchi, Taichi; Sasaki, Tasuku; Nishiyama, Toshio

    2017-03-07

    Type IV collagen isolated from lens capsule without enzymatic treatment is known to form a gel under physiological condition and influences cellular activities. In case of human keratinocytes, the suppression of proliferation on reconstituted type IV collagen gels was reported in monolayer culture. In this study, we examined effects of type IV collagen isolated from porcine lens capsule on epidermal formation in human skin equivalents. Type IV collagen aggregates were prepared under the culture condition and the aggregates suppressed keratinocyte proliferation in monolayer culture as well as the culture on the gels. In human skin equivalents type IV collagen aggregates were reconstituted on the surface of contracted collagen gels containing human dermal fibroblasts and the keratinocytes were then cultured on the aggregates for 14 days. Interestingly, in human skin equivalents with type IV collagen aggregates, the BrdU-positive keratinocytes were increased and the thickness of the epidermal layer was around twice than that of control culture. Epidermal differentiation markers were expressed in the upper layer of the epidermis and the defined deposition of human basement membrane components were increased at the dermal-epidermal junction. These results indicate that the type IV collagen aggregates stimulate the proliferation of basal keratinocytes and improve the stratification of epidermal layers in human skin equivalents. This article is protected by copyright. All rights reserved.

  10. A fragment of alpha-actinin promotes monocyte/macrophage maturation in vitro.

    PubMed

    Luikart, S; Wahl, D; Hinkel, T; Masri, M; Oegema, T

    1999-02-01

    Conditioned media (CM) from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix contains a factor that induces macrophage-like maturation of HL-60 cells. This factor was purified from the CM of HL-60 cells grown on bone marrow stroma by ammonium sulfate precipitation, then sequential chromatography on DEAE, affi-gel blue affinity, gel exclusion, and wheat germ affinity columns, followed by C-4 reverse phase HPLC, and SDS-PAGE. The maturation promoting activity of the CM was identified in a single 31 kD protein. Amino acid sequence analysis of four internal tryptic peptides of this protein confirmed significant homology with amino acid residues 48-60, 138-147, 215-220, and 221-236 of human cytoskeletal alpha-actinin. An immunoaffinity purified rabbit polyclonal anti-chicken alpha-actinin inhibited the activity of HL-60 conditioned media. A 27 kD amino-terminal fragment of alpha-actinin produced by thermolysin digestion of chicken gizzard alpha-actinin, but not intact alpha-actinin, had maturation promoting activity on several cell types, including blood monocytes, as measured by lysozyme secretion and tartrate-resistant acid phosphatase staining. We conclude that an extracellular alpha-actinin fragment can promote monocyte/macrophage maturation. This represents the first example of a fragment of a cytoskeletal component, which may be released during tissue remodeling and repair, playing a role in phagocyte maturation.

  11. Lactase gene promoter fragments mediate differential spatial and temporal expression patterns in transgenic mice.

    PubMed

    Wang, Zhi; Maravelias, Charalambos; Sibley, Eric

    2006-04-01

    Lactase gene expression is spatiotemporally regulated during mammalian gut development. We hypothesize that distinct DNA control regions specify appropriate spatial and temporal patterning of lactase gene expression. In order to define regions of the lactase promoter involved in mediating intestine-specific and spatiotemporal restricted expression, transgenic mice harboring 100 bp, 1.3- and 2.0- kb fragments of the 5' flanking region of the rat lactase gene cloned upstream of a luciferase reporter were characterized. The 100-bp lactase promoter-reporter transgenic mouse line expressed maximal luciferase activity in the intestine with a posterior shift in spatial restriction and ectopic expression in the stomach and lung. The temporal pattern of expression mediated by the 1.3-kb promoter?reporter transgene increases with postnatal maturation in contrast with the postnatal decline mediated by the 2.0-kb promoter-reporter transgene and the endogenous lactase gene. The differential transgene expression patterns mediated by the lactase promoter fragments suggests that intestine-specific spatial and temporal control elements reside in distinct regions of the DNA sequences upstream of the lactase gene transcription start-site.

  12. Oleanane-type triterpene saponins with collagen synthesis-promoting activity from the flowers of Bellis perennis.

    PubMed

    Morikawa, Toshio; Ninomiya, Kiyofumi; Takamori, Yasunobu; Nishida, Eriko; Yasue, Misato; Hayakawa, Takao; Muraoka, Osamu; Li, Xuezheng; Nakamura, Seikou; Yoshikawa, Masayuki; Matsuda, Hisashi

    2015-08-01

    The methanol extract from Bellis perennis (Asteraceae) flowers was found to promote collagen synthesis in normal human dermal fibroblasts (NHDFs). Seven oleanane-type triterpene saponins, perennisosides XIII-XIX, and two known saponins, bellissaponins BS5 and BS9, were isolated from the methanol extract. The structures were determined based on chemical and physicochemical data, and confirmed using previously isolated related compounds as references. Among the isolates, including 19 previously reported saponins, perennisosides XVIII, I, II, VII, IX, and XI, asterbatanoside D, bernardioside B2, and bellissaponins BS5 and BS9 significantly promoted collagen synthesis at 3-30μM without cytotoxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition.

    PubMed

    Aikio, Mari; Elamaa, Harri; Vicente, David; Izzi, Valerio; Kaur, Inderjeet; Seppinen, Lotta; Speedy, Helen E; Kaminska, Dorota; Kuusisto, Sanna; Sormunen, Raija; Heljasvaara, Ritva; Jones, Emma L; Muilu, Mikko; Jauhiainen, Matti; Pihlajamäki, Jussi; Savolainen, Markku J; Shoulders, Carol C; Pihlajaniemi, Taina

    2014-07-29

    Collagen XVIII is an evolutionary conserved ubiquitously expressed basement membrane proteoglycan produced in three isoforms via two promoters (P). Here, we assess the function of the N-terminal, domain of unknown function/frizzled-like sequences unique to medium/long collagen XVIII by creating P-specific null mice. P2-null mice, which only produce short collagen XVIII, developed reduced bulk-adiposity, hepatic steatosis, and hypertriglyceridemia. These abnormalities did not develop in P1-null mice, which produce medium/long collagen XVIII. White adipose tissue samples from P2-null mice contain larger reserves of a cell population enriched in early adipocyte progenitors; however, their embryonic fibroblasts had ∼ 50% lower adipocyte differentiation potential. Differentiating 3T3-L1 fibroblasts into mature adipocytes produced striking increases in P2 gene-products and dramatic falls in P1-transcribed mRNA, whereas Wnt3a-induced dedifferentiation of mature adipocytes produced reciprocal changes in P1 and P2 transcript levels. P2-derived gene-products containing frizzled-like sequences bound the potent adipogenic inhibitor, Wnt10b, in vitro. Previously, we have shown that these same sequences bind Wnt3a, inhibiting Wnt3a-mediated signaling. P2-transcript levels in visceral fat were positively correlated with serum free fatty acid levels, suggesting that collagen α1 (XVIII) expression contributes to regulation of adipose tissue metabolism in visceral obesity. Medium/long collagen XVIII is deposited in the Space of Disse, and interaction between hepatic apolipoprotein E and this proteoglycan is lost in P2-null mice. These results describe a previously unidentified extracellular matrix-directed mechanism contributing to the control of the multistep adipogenic program that determines the number of precursors committing to adipocyte differentiation, the maintenance of the differentiated state, and the physiological consequences of its impairment on ectopic fat

  14. Collagenase-resistant collagen promotes mouse aging and vascular cell senescence.

    PubMed

    Vafaie, Faran; Yin, Hao; O'Neil, Caroline; Nong, Zengxuan; Watson, Alanna; Arpino, John-Michael; Chu, Michael W A; Wayne Holdsworth, David; Gros, Robert; Pickering, J Geoffrey

    2014-02-01

    Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1(r/r) ) that yields collagenase-resistant type I collagen. Compared with wild-type littermates, Col1a1(r/r) mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1(r/r) mice were susceptible to stress-induced senescence, displaying senescence-associated ß-galactosidase (SA-ßGal) activity and upregulated p16(INK4A) in response to angiotensin II infusion. To elucidate the basis of this pro-aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1(r/r) or wild-type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress-induced SA-ßGal activity, p16(INK4A) expression, and p21(CIP1) expression. The pro-senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3-blocking antibody increased senescence of SMCs on wild-type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging-related diseases. © 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  15. Effect of growth-promoting technologies on Longissimus lumborum muscle fiber morphometrics, collagen solubility, and cooked meat tenderness.

    PubMed

    Ebarb, S M; Drouillard, J S; Maddock-Carlin, K R; Phelps, K J; Vaughn, M A; Burnett, D D; Van Bibber-Krueger, C L; Paulk, C B; Grieger, D M; Gonzalez, J M

    2016-02-01

    The objective of the study was to examine the effect of growth-promoting technologies (GP) on Longissimus lumborum steak tenderness, muscle fiber cross-sectional area (CSA), and collagen solubility. Crossbred feedlot heifers ( = 33; initial BW 464 ± 6 kg) were blocked by BW and assigned to 1 of 3 treatments: no GP (CON; = 11); implant, no zilpaterol hydrochloride (IMP; = 11); implant and zilpaterol hydrochloride (COMBO; = 11). Heifers assigned to receive an implant were administered Component TE-200 on d 0 of the study, and the COMBO group received 8.3 mg/kg DM of zilpaterol hydrochloride for the final 21 d of feeding with a 3 d withdrawal period. Following harvest, strip loins were collected and fabricated into 4 roasts and aged for 3, 14, 21, or 35 d postmortem. Fiber type was determined by immunohistochemistry. After aging, objective tenderness and collagen solubility were measured. There was a treatment × day of aging (DOA) interaction for Warner-Bratzler shear force (WBSF; < 0.01). At d 3 of aging, IMP and COMBO steaks had greater WBSF than CON steaks ( < 0.01). By d 14 of aging, the WBSF of IMP steaks was not different ( = 0.21) than CON steaks, but COMBO steaks had greater shear values than steaks of other treatments ( < 0.02). The COMBO steaks only remained tougher ( = 0.04) than the CON steaks following 35 DOA. Compared to CON muscles, IMP and COMBO type I and IIX muscle fibers were larger ( < 0.03). Treatment, DOA, or the two-way interactions did not impact measures of total and insoluble collagen ( > 0.31). Soluble collagen amount tended to be affected ( 0.06) by a treatment × DOA interaction which was due to COMBO muscle having more soluble collagen than the other 2 treatments on d 21 of aging ( < 0.02). Correlation analysis indicated that type I, IIA, and IIX fiber CSA are positively correlated with WBSF at d 3 and 14 of aging ( < 0.01), but only type IIX fibers are correlated at d 21 and 35 of aging ( < 0.03). At these time periods, total and

  16. Oligomer-targeting with a conformational antibody fragment promotes toxicity in Aβ-expressing flies

    PubMed Central

    2014-01-01

    Introduction The self-assembly of Aβ peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer’s disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments. Results We show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aβ-expressing Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aβ(1–40) and not with those expressing Aβ(1–42) or the arctic variant of Aβ(1–42) This finding is consistent with the binding preference of KW1 for Aβ(1–40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation. Conclusions While our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents. PMID:24725347

  17. Cyclophilin A Aggravates Collagen-Induced Arthritis via Promoting Classically Activated Macrophages.

    PubMed

    Dongsheng, Zhai; Zhiguang, Fu; Junfeng, Jia; Zifan, Lu; Li, Wang

    2017-07-29

    Activated macrophages exhibiting diverse phenotypes and various functions contribute to the pathogenesis or amelioration of different diseases like cancer, inflammation, and infectious and autoimmune diseases. However, the mechanisms of macrophage polarization in inflamed joint and its effects on rheumatoid arthritis (RA) are still not clarified. This study is designed to explore the effects of cyclophilin A (CypA) on macrophage polarization and describe the underlying mechanisms. Collagen-induced arthritis (CIA) was employed to address the pro-arthritic effects of CypA. Flow cytometry was performed to investigate the populations of M1 and M2 macrophages in synovial tissues of the mice. Knockdown or overexpression of CypA macrophage cells was used to study the functions of CypA on macrophage polarization. Western blot was carried out to examine the potential signaling pathways. We found that CypA aggravated the severity of CIA in mice, as assessed by the increase of clinical score of inflammation, cartilage damage, and bone erosion. Moreover, the level of cytokines, such as IL-6, IL-1β, and IL-17, and the number of pro-inflammatory macrophages in synovial fluid were significantly elevated. In accordance with our observation, CypA dysregulation could actually influence the M1 macrophages polarization and pro-inflammatory cytokines production. Further mechanism study disclosed that CypA could regulate the transcriptional activity of NF-κB, the pivotal transcriptional factor regulating M1 polarization, dependent of its PPIase activity. Our findings provide evidence that PPIase CypA promoted macrophages polarization toward pro-inflammatory M1 phenotype via transcriptional activating NF-κB, thus leading to aggravated arthritis.

  18. Collagen with simvastatin promotes cell metabolism in osteoblast-like SaOS-2 cells.

    PubMed

    Suthanthiran, Thanga Kumaran; Elavarasu, Sugumari; Naveen, Devisree; Nagarathinam, Umamaheswari; Arun, K V; Srinivasan, N

    2012-08-01

    Simvastatin (SMV) is one of the cholesterol-lowering pharmacological drugs. Recent studies demonstrate that it has a bone stimulatory effect. The present study was designed to investigate the effect of SMV along with collagen membrane on osteoblast-like SaOS-2 cells and also to standardize the dosage of SMV to be incorporated into the collagen membrane to achieve regeneration. SMV at doses of 0.5, 1, 1.5, and 2 mg was incorporated into the collagen membrane and cell metabolism was assessed by (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) (MTT) assay for 24 h. SMV enhanced cell metabolism dose dependently at 24-h time and the maximum effect was obtained at a concentration of 1.5 mg of SMV. These results indicate that collagen with 1.5 mg SMV exhibits positive effect on cell metabolism of human osteoblast-like SaOS-2 cells.

  19. Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters.

    PubMed

    Jathoul, Amit P; Holley, Jane L; Garmory, Helen S

    2004-09-28

    DNA vaccines which expressed the Hc fragment of the Clostridium botulinum type F neurotoxin (BoNT/F Hc) fused to a signal peptide downstream of four different eukaryotic promoters were prepared. Subsequently, the immunogenicity of the DNA vaccines and protection afforded in mice against challenge with 10(4) MLD of type F botulinum toxin was evaluated. The DNA vaccine containing the human ubiquitin gene (UbC) promoter induced the highest BoNT/F Hc-specific antibody concentration following two intramuscular immunisations and afforded 90% protection against challenge. The results from this study indicate that the selection of promoter used in DNA vaccination studies may be of importance in designing optimised vaccines.

  20. Hyaluronic Acid is Overexpressed in Fibrotic Lung Tissue and Promotes Collagen Expression

    DTIC Science & Technology

    2008-04-01

    cause of morbidity and mortality in scleroderma . The overexpression of collagen is accompanied by the overexpression of other extracellular matrix...7 Appendices…………………………………………………………………………… 7 3 INTRODUCTION Systemic scleroderma is a debilitating disease...excessive accumulation of extracellular matrix [ECM] proteins, particularly collagen I) is the major cause of morbidity and mortality in scleroderma . The

  1. Novel Vanadium-Loaded Ordered Collagen Scaffold Promotes Osteochondral Differentiation of Bone Marrow Progenitor Cells

    PubMed Central

    Cortizo, Ana M.; Ruderman, Graciela; Mazzini, Flavia N.; Molinuevo, M. Silvina; Mogilner, Ines G.

    2016-01-01

    Bone and cartilage regeneration can be improved by designing a functionalized biomaterial that includes bioactive drugs in a biocompatible and biodegradable scaffold. Based on our previous studies, we designed a vanadium-loaded collagen scaffold for osteochondral tissue engineering. Collagen-vanadium loaded scaffolds were characterized by SEM, FTIR, and permeability studies. Rat bone marrow progenitor cells were plated on collagen or vanadium-loaded membranes to evaluate differences in cell attachment, growth and osteogenic or chondrocytic differentiation. The potential cytotoxicity of the scaffolds was assessed by the MTT assay and by evaluation of morphological changes in cultured RAW 264.7 macrophages. Our results show that loading of VOAsc did not alter the grooved ordered structure of the collagen membrane although it increased membrane permeability, suggesting a more open structure. The VOAsc was released to the media, suggesting diffusion-controlled drug release. Vanadium-loaded membranes proved to be a better substratum than C0 for all evaluated aspects of BMPC biocompatibility (adhesion, growth, and osteoblastic and chondrocytic differentiation). In addition, there was no detectable effect of collagen or vanadium-loaded scaffolds on macrophage viability or cytotoxicity. Based on these findings, we have developed a new ordered collagen scaffold loaded with VOAsc that shows potential for osteochondral tissue engineering. PMID:27293438

  2. Heparin promotes fibril formation by the N-terminal fragment of amyloidogenic apolipoprotein A-I.

    PubMed

    Mikawa, Shiho; Mizuguchi, Chiharu; Nishitsuji, Kazuchika; Baba, Teruhiko; Shigenaga, Akira; Shimanouchi, Toshinori; Sakashita, Naomi; Otaka, Akira; Akaji, Kenichi; Saito, Hiroyuki

    2016-10-01

    Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N-terminal 1-83 fragment of human apolipoprotein A-I (apoA-I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes β-transition and fibril formation of the highly amyloidogenic region spanning residues 44-65 and colocalizes with fibrils formed by the W50R variant. These results suggest the possible role of glycosaminoglycans in fibril formation by amyloidogenic apoA-I variants. © 2016 Federation of European Biochemical Societies.

  3. Metal-Promoted Assembly of Two Collagen Mimetic Peptides into a Biofunctional “Spiraled Horn” Scaffold

    PubMed Central

    Strauss, Kevin; Chmielewski, Jean

    2016-01-01

    Biofunctional scaffolds for the delivery of living cells are of the utmost importance for regenerative medicine. Herein, a novel, robust “spiraled horn” scaffold was elucidated through the Co2+-promoted hierarchical assembly of two collagen mimetic peptides, NCoH and HisCol. Each “horn” displayed a periodic banding pattern with band lengths corresponding to the length of the collagen peptide triple helix. Strand exchange between the two peptide trimers resulted in failure to form this intricate morphology, lending support to a precise metal-ligand-based mechanism of assembly. Little change occurred to the observed morphology when the Co2+ concentration was varied from 0.5 to 4.0 mM, and the scaffold was found to be fully formed within two minutes of exposure to the metal ion. The horned network also displayed biological functionality by binding to a His-tagged fluorophore and associating with cells. PMID:28773959

  4. The promoting effects of geniposidic acid and aucubin in Eucommia ulmoides Oliver leaves on collagen synthesis.

    PubMed

    Li, Y; Sato, T; Metori, K; Koike, K; Che, Q M; Takahashi, S

    1998-12-01

    We have reported that collagen synthesis was stimulated by the administration of a hot water extract from the leaves of Eucommia ulmoides OLIVER, Eucommiaceae (Du-Zhong leaves) in false aged model rats. In this paper, we set out to examine the compounds in Du-Zhong leaves that stimulated collagen synthesis in false aged model rats. In experiment 1, a methanol extract of Du-Zhong leaves also stimulated collagen synthesis in aged model rats. An acetone fraction was derived from the methanol extract by silica gel chromatography in experiment 2. The acetone fraction mainly contained iridoides mono-glycosides such as geniposidic acid and aucubin. The administration of geniposidic acid or aucubin stimulated collagen synthesis in aged model rats in experiments 3 and 4 (significance (p<0.05)). The reported pharmacological effects of Du-Zhong leaves, including healing organs and strengthening bone and muscle, are closely related to collagen metabolism. It appears that geniposidic acid and aucubin are the actual compounds in Du-Zhong which caused the effect in our experiments.

  5. Lack of collagen VI promotes neurodegeneration by impairing autophagy and inducing apoptosis during aging

    PubMed Central

    Castagnaro, Silvia; Gregorio, Ilaria; Bonaldo, Paolo

    2016-01-01

    Collagen VI is an extracellular matrix (ECM) protein with a broad distribution in different tissues and mostly deposited at the close periphery of the cell surface. Previous studies revealed that collagen VI protects neurons from the toxicity of amyloid-βpeptides and from UV-induced damage. However, the physiological role of this protein in the central nervous system (CNS) remains unknown. Here, we established primary neural cultures from murine cortex and hippocampus, and carried out in vitro and in vivo studies in wild-type and collagen VI null (Col6a1−/−) mice. Col6a1−/− neural cultures displayed an increased incidence of spontaneous apoptosis and higher vulnerability to oxidative stress, accompanied by altered regulation of autophagy with increased p62 protein levels and decreased LC3 lipidation. Analysis of brain sections confirmed increased apoptosis and abnormal regulation of autophagy in the CNS of collagen VI-deficient animals. To investigate the in vivo physiological consequences of these CNS defects, we carried out functional studies and found that motor and memory task performances were impaired in aged Col6a1−/− mice. These findings indicate that lack of collagen VI leads to spontaneous apoptosis and defective autophagy in neural cells, and point at a protective role for this ECM protein in the CNS during physiological aging. PMID:27060109

  6. Collagen peptide-based biomaterials for protein delivery and peptide-promoted self-assembly of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Ernenwein, Dawn M.

    2011-12-01

    Bottom-up self-assembly of peptides has driven the research progress for the following two projects: protein delivery vehicles of collagen microflorettes and the assembly of gold nanoparticles with coiled-coil peptides. Collagen is the most abundant protein in the mammals yet due to immunogenic responses, batch-to-batch variability and lack of sequence modifications, synthetic collagen has been designed to self-assemble into native collagen-like structures. In particular with this research, metal binding ligands were incorporated on the termini of collagen-like peptides to generate micron-sized particles, microflorettes. The over-arching goal of the first research project is to engineer MRI-active microflorettes, loaded with His-tagged growth factors with differential release rates while bound to stem cells that can be implemented toward regenerative cell-based therapies. His-tagged proteins, such as green fluorescent protein, have successfully been incorporated on the surface and throughout the microflorettes. Protein release was monitored under physiological conditions and was related to particle degradation. In human plasma full release was obtained within six days. Stability of the microflorettes under physiological conditions was also examined for the development of a therapeutically relevant delivery agent. Additionally, MRI active microflorettes have been generated through the incorporation of a gadolinium binding ligand, DOTA within the collagen-based peptide sequence. To probe peptide-promoted self-assemblies of gold nanoparticles (GNPs) by non-covalent, charge complementary interactions, a highly anionic coiled-coil peptide was designed and synthesized. Upon formation of peptide-GNP interactions, the hydrophobic domain of the coiled-coil were shown to promote the self-assembly of peptide-GNPs clustering. Hydrophobic forces were found to play an important role in the assembly process, as a peptide with an equally overall negative charge, but lacking an

  7. Prevention of liver fibrosis by triple helix-forming oligodeoxyribonucleotides targeted to the promoter region of type I collagen gene.

    PubMed

    Koilan, Subramaniyan; Hamilton, David; Baburyan, Narina; Padala, Mythili K; Weber, Karl T; Guntaka, Ramareddy V

    2010-10-01

    Hepatic fibrosis leading to cirrhosis remains a global health problem. The most common etiologies are alcoholism and viral infections. Liver fibrosis is associated with major changes in both quantity and composition of extracellular matix and leads to disorganization of the liver architecture and irreversible damage to the liver function. As of now there is no effective therapy to control fibrosis. The end product of fibrosis is abnormal synthesis and accumulation of type I collagen in the extracellular matrix, which is produced by activated stellate or Ito cells in the damaged liver. Therefore, inhibition of transcription of type I collagen should in principle inhibit its production and accumulation in liver. Normally, DNA exists in a duplex form. However, under some circumstances, DNA can assume triple helical (triplex) structures. Intermolecular triplexes, formed by the addition of a sequence-specific third strand to the major groove of the duplex DNA, have the potential to serve as selective gene regulators. Earlier, we demonstrated efficient triplex formation between the exogenously added triplex-forming oligodeoxyribonucleotides (TFOs) and a specific sequence in the promoter region of the COL1A1 gene. In this study we used a rat model of liver fibrosis, induced by dimethylnitrosamine, to test whether these TFOs prevent liver fibrosis. Our results indicate that both the 25-mer and 18-mer TFOs, specific for the upstream nucleotide sequence from -141 to -165 (relative to the transcription start site) in the 5' end of collagen gene promoter, effectively prevented accumulation of liver collagen and fibrosis. We also observed improvement in liver function tests. However, mutations in the TFO that eliminated formation of triplexes are ineffective in preventing fibrosis. We believe that these TFOs can be used as potential antifibrotic therapeutic molecules.

  8. Association of collagen with calcium phosphate promoted osteogenic responses of osteoblast-like MG63 cells.

    PubMed

    Hong, Yoon Jung; Chun, Jae-Sig; Lee, Woo-Kul

    2011-04-01

    In this investigation, the effects of the association of the collagen (COLL) molecules with the calcium phosphate (CaP) film were examined with respect to both the physicochemical properties of the CaP films and the osteoblast responses, such as the adhesion, proliferation, differentiation, and mineralization. The COLL pre-adsorbed CaP film (CaPA) exhibited significant changes in the surface morphology compared to the COLL incorporated CaP film (CaPC). The adhesions of the osteoblast-like MG63 cells were similar on the CaPC or CaPA films. However, the proliferation of the MG63 cells on CaPC was comparable to CaP but considerably different than CaPA. The differentiation of the MG63 cells was greatly improved on CaPC and CaPA compared to CaP and more pronounced on CaPA. The presence of COLL within or on the CaP films significantly modulated the expression of the phenotypic genes, including osteopontin (OPN), alkaline phosphatase (ALP), and the transforming growth factor-β (TGF-β). The expression patterns of these genes elucidated that COLL that was present within or on the CaP film supported the osteoblast proliferation and differentiation. These positive effects were stronger for CaPA than CaPC. The bone-like nodules formed on all of the specimens. However, the mineralization of CaPC and CaPA was significantly higher than CaP, indicating that the association of CaP with COLL promoted the mineral deposition. Therefore, the association of the COLL molecules with the CaP film induced positive effects on the biomineralization. Overall, the incorporation of COLL efficiently enhanced the osteoblast responses of CaP. This system can be utilized in a drug delivery system using calcium phosphate. Although the incorporation effects were slightly higher for the osteoblast responses of CaPA than CaPC, CaPC can be used when the longer drug release times are desirable.

  9. AN APOLIPOPROTEIN E4 FRAGMENT CAN PROMOTE INTRACELLULAR ACCUMULATION OF AMYLOID PEPTIDE BETA 42

    PubMed Central

    Dafnis, Ioannis; Stratikos, Efstratios; Tzinia, Athina; Tsilibary, Effie C.; Zannis, Vassilis I.; Chroni, Angeliki

    2010-01-01

    Apolipoprotein E (apoE) plays a crucial role in lipid transport in circulation and the brain. The apoE4 isoform is a major risk factor for Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than other apoE isoforms and apoE4 fragments have been found in brains of AD patients. These apoE4 fragments have been hypothesized to be involved in the pathogenesis of AD, although the mechanism is not clear. In this study we examined the effect of lipid-free apoE4 on amyloid precursor protein (APP) processing and Aβ40 and Aβ42 levels in human neuroblastoma SK-N-SH cells. We discovered that a specific apoE4 fragment, apoE4[Δ(166-299)], can promote the cellular uptake of extracellular Aβ40 and Aβ42 either generated after APP transfection or added exogenously. A longer length fragment, apoE4[Δ(186-299)], or full-length apoE4 failed to elicit this effect. ApoE4[Δ(166-299)] effected a 20% reduction of cellular sphingomyelin levels, as well as changes in cellular membrane micro-fluidity. Following uptake, approximately 50% of Aβ42 remained within the cell for at least 24h, and led to increased formation of reactive oxygen species. Overall, our findings suggest a direct link between two early events in the pathogenesis of AD, apoE4 proteolysis and intraneuronal presence of Aβ. PMID:20412390

  10. A 470 bp WAP-promoter fragment confers lactation independent, progesterone regulated mammary-specific gene expression in transgenic mice.

    PubMed

    Lipnik, Karoline; Petznek, Helga; Renner-Müller, Ingrid; Egerbacher, Monika; Url, Angelika; Salmons, Brian; Günzburg, Walter H; Hohenadl, Christine

    2005-04-01

    The ability of a 470 bp sub-fragment of the murine whey acidic protein (WAP) promoter in the context of a retroviral expression plasmid to direct gene expression to mammary epithelial cells was analysed in a number of independent transgenic mouse lines. In contrast to previous findings with the genuine 2.5 kb promoter fragment, our studies revealed a highly mammary gland-specific expression detectable only in non-lactating animals. This suggested a mainly progesterone-regulated activity of the short fragment. Therefore, transgene expression was examined in the progesterone-determined estrous cycle and during pregnancy. In accordance with in vitro data from stably transfected cell lines, in both situations expression was upregulated at stages associated with high progesterone levels. Taken together these data provide deeper insight into WAP-promoter regulation and stress the usefulness of the shortened fragment for a lactation independent mammary-targeted expression.

  11. Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition.

    PubMed

    Xiong, Gaofeng; Deng, Lei; Zhu, Jieqing; Rychahou, Piotr G; Xu, Ren

    2014-01-02

    Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated. Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis. Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and

  12. Overexpression of HMGA2-LPP fusion transcripts promotes expression of the {alpha} 2 type XI collagen gene

    SciTech Connect

    Kubo, Takahiro; Matsui, Yoshito . E-mail: ymatsui@sb4.so-net.ne.jp; Goto, Tomohiro; Yukata, Kiminori; Yasui, Natsuo

    2006-02-10

    In a subset of human lipomas, a specific t (3; 12) chromosome translocation gives rise to HMGA2-LPP fusion protein, containing the amino (N)-terminal DNA binding domains of HMGA2 fused to the carboxyl (C)-terminal LIM domains of LPP. In addition to its role in adipogenesis, several observations suggest that HMGA2-LPP is linked to chondrogenesis. Here, we analyzed whether HMGA2-LPP promotes chondrogenic differentiation, a marker of which is transactivation of the {alpha} 2 type XI collagen gene (Col11a2). Real-time PCR analysis showed that HMGA2-LPP and COL11A2 were co-expressed. Luciferase assay demonstrated that either of HMGA2-LPP, wild-type HMGA2 or the N-terminal HMGA2 transactivated the Col11a2 promoter in HeLa cells, while the C-terminal LPP did not. RT-PCR analysis revealed that HMGA2-LPP transcripts in lipomas with the fusion were 591-fold of full-length HMGA2 transcripts in lipomas without the fusion. These results indicate that in vivo overexpression of HMGA2-LPP promotes chondrogenesis by upregulating cartilage-specific collagen gene expression through the N-terminal DNA binding domains.

  13. Collagen-nanofiber hydrogel composites promote contact guidance of human lymphatic microvascular endothelial cells and directed capillary tube formation.

    PubMed

    Laco, Filip; Grant, M Helen; Black, Richard A

    2013-06-01

    Collagen and fibronectin matrices are known to stimulate migration of microvascular endothelial cells and the process of tubulogenesis, but the physical, chemical, and topographical cues for directed vessel formation have yet to be determined. In this study, growth, migration, elongation, and tube formation of human lymphatic microvascular endothelial cells (LECs) were investigated on electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic-co-D-lactic acid) (PLDL) nanofiber-coated substrates, and correlated with fiber density and diameter. Directed migration of LECs was observed in the presence of aligned nanofibers, whereas random fiber alignment slowed down migration and growth of LECs. Cell guidance was significantly enhanced in the presence of more hydrophobic PLDL polymer nanofibers compared to PLGA (10:90). Subsequent experiments with tube-forming assays reveal the ability of resorbable hydrophobic nanofibers >300 nm in diameter to promote cell guidance in collagen gels without direct cell-fiber contact, in contrast to the previously reported contact-guidance phenomena. Our results show that endothelial cell guidance is possible within nanofiber/collagen-gel constructs that mimic the native extracellular matrix in terms of size and orientation of fibrillar components.

  14. Suppression of microRNA-29 Expression by TGF-β1 Promotes Collagen Expression and Renal Fibrosis

    PubMed Central

    Wang, Bo; Komers, Radko; Carew, Rosemarie; Winbanks, Catherine E.; Xu, Bei; Herman-Edelstein, Michal; Koh, Philip; Thomas, Merlin; Jandeleit-Dahm, Karin; Gregorevic, Paul; Cooper, Mark E.

    2012-01-01

    Synthesis and deposition of extracellular matrix (ECM) within the glomerulus and interstitium characterizes renal fibrosis, but the mechanisms underlying this process are incompletely understood. The profibrotic cytokine TGF-β1 modulates the expression of certain microRNAs (miRNAs), suggesting that miRNAs may have a role in the pathogenesis of renal fibrosis. Here, we exposed proximal tubular cells, primary mesangial cells, and podocytes to TGF-β1 to examine its effect on miRNAs and subsequent collagen synthesis. TGF-β1 reduced expression of the miR-29a/b/c/family, which targets collagen gene expression, and increased expression of ECM proteins. In both resting and TGF-β1–treated cells, ectopic expression of miR-29 repressed the expression of collagens I and IV at both the mRNA and protein levels by targeting the 3′untranslated region of these genes. Furthermore, we observed low levels of miR-29 in three models of renal fibrosis representing early and advanced stages of disease. Administration of the Rho-associated kinase inhibitor fasudil prevented renal fibrosis and restored expression of miR-29. Taken together, these data suggest that TGF-β1 inhibits expression of the miR-29 family, thereby promoting expression of ECM components. Pharmacologic modulation of these miRNAs may have therapeutic potential for progressive renal fibrosis. PMID:22095944

  15. Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene.

    PubMed

    Mukhopadhyay, K; Lefebvre, V; Zhou, G; Garofalo, S; Kimura, J H; de Crombrugghe, B

    1995-11-17

    We show that a new rat chondrosarcoma (RCS) cell line established in long-term culture from the Swarm tumor displayed a stable differentiated chondrocyte-like phenotype. Indeed, these cells produced the collagen types II, IX, and XI and alcian blue-stainable cartilage-specific proteoglycans, but no type I or type III collagen. To functionally characterize their chondrocytic nature, the cells were stably transfected with a type II collagen/beta geo chimeric gene which confers essentially perfect chondrocyte-specific expression in transgenic mice. RCS cells expressed both beta-galactosidase and G418 resistance, in comparison with similarly transfected 10T1/2 and NIH/3T3 fibroblasts which did not. These cells were then used to perform a systematic deletion analysis of the first intron of the mouse type II collagen gene (Col2a1) using transient expression experiments to determine which segments stimulated expression of a luciferase reporter gene in RCS cells but not in 10T1/2 fibroblasts. Cloning of two tandem copies of a 156-base pair (bp) intron 1 fragment (+2188 to +2343) in a construction containing a 314-bp Col2a1 promoter caused an almost 200-fold increase in promoter activity in RCS cells but no increase in 10T1/2 cells. DNase I footprint analysis over this 156-bp fragment revealed two adjacent protected regions, FP1 and FP2, located in the 3'-half of this segment, but no differences were seen with nuclear extracts of RCS cells and 10T1/2 fibroblasts. Deletion of FP2 to leave a 119-bp segment decreased enhancer activity by severalfold, but RCS cell specificity was maintained. Further deletions indicated that sequences both in the 5' part of the 119-bp fragment and in FP1 were needed simultaneously for RCS cell-specific enhancer activity. A series of deletions in the promoter region of the mouse Col2a1 gene progressively reduced activity when these promoters were tested by themselves in transient expression experiments. However, these promoter deletions were all

  16. Type I collagen promotes epithelial-mesenchymal transition through ILK-dependent activation of NF-κB and LEF-1

    PubMed Central

    Medici, Damian; Nawshad, Ali

    2010-01-01

    Collagen I has been shown to promote epithelial-mesenchymal transition (EMT), a critical process of embryonic development and disease progression. However, little is known about the signaling mechanisms by which collagen I induces this cellular transformation. Here we show that collagen I causes ILK-dependent phosphorylation of IκB and subsequent nuclear translocation of active NF-κB, which in turn promotes increased expression of the Snail and LEF-1 transcription factors. ILK also causes inhibitory phosphorylation of GSK-3β, a kinase that prevents functional activation of both Snail and LEF-1. These transcription factors alter expression of epithelial and mesenchymal markers to initiate EMT and stimulate cell migration. These data provide a foundation for understanding the mechanisms by which collagen I stimulates EMT and identify potential therapeutic targets for suppressing this transition in pathological conditions. PMID:20018240

  17. A modified collagen gel dressing promotes angiogenesis in a preclinical swine model of chronic ischemic wounds.

    PubMed

    Elgharably, Haytham; Ganesh, Kasturi; Dickerson, Jennifer; Khanna, Savita; Abas, Motaz; Ghatak, Piya Das; Dixit, Sriteja; Bergdall, Valerie; Roy, Sashwati; Sen, Chandan K

    2014-01-01

    We recently performed proteomic characterization of a modified collagen gel (MCG) dressing and reported promising effects of the gel in healing full-thickness excisional wounds. In this work, we test the translational relevance of our aforesaid findings by testing the dressing in a swine model of chronic ischemic wounds recently reported by our laboratory. Full-thickness excisional wounds were established in the center of bipedicle ischemic skin flaps on the backs of animals. Ischemia was verified by laser Doppler imaging, and MCG was applied to the test group of wounds. Seven days post wounding, macrophage recruitment to the wound was significantly higher in MCG-treated ischemic wounds. In vitro, MCG up-regulated expression of Mrc-1 (a reparative M2 macrophage marker) and induced the expression of anti-inflammatory cytokine interleukin (IL)-10 and of fibroblast growth factor-basic (β-FGF). An increased expression of CCR2, an M2 macrophage marker, was noted in the macrophages from MCG treated wounds. Furthermore, analyses of wound tissues 7 days post wounding showed up-regulation of transforming growth factor-β, vascular endothelial growth factor, von Willebrand's factor, and collagen type I expression in MCG-treated ischemic wounds. At 21 days post wounding, MCG-treated ischemic wounds displayed higher abundance of proliferating endothelial cells that formed mature vascular structures and increased blood flow to the wound. Fibroblast count was markedly higher in MCG-treated ischemic wound-edge tissue. In addition, MCG-treated wound-edge tissues displayed higher abundance of mature collagen with increased collagen type I : III deposition. Taken together, MCG helped mount a more robust inflammatory response that resolved in a timely manner, followed by an enhanced proliferative phase, angiogenic outcome, and postwound tissue remodeling. Findings of the current study warrant clinical testing of MCG in a setting of ischemic chronic wounds.

  18. Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue.

    PubMed

    Roberts, Meredith A; Tran, Dominic; Coulombe, Kareen L K; Razumova, Maria; Regnier, Michael; Murry, Charles E; Zheng, Ying

    2016-04-01

    Cardiac tissue engineering is a strategy to replace damaged contractile tissue and model cardiac diseases to discover therapies. Current cardiac and vascular engineering approaches independently create aligned contractile tissue or perfusable vasculature, but a combined vascularized cardiac tissue remains to be achieved. Here, we sought to incorporate a patterned microvasculature into engineered heart tissue, which balances the competing demands from cardiomyocytes to contract the matrix versus the vascular lumens that need structural support. Low-density collagen hydrogels (1.25 mg/mL) permit human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to form a dense contractile tissue but cannot support a patterned microvasculature. Conversely, high collagen concentrations (density ≥6 mg/mL) support a patterned microvasculature, but the hESC-CMs lack cell-cell contact, limiting their electrical communication, structural maturation, and tissue-level contractile function. When cocultured with matrix remodeling stromal cells, however, hESC-CMs structurally mature and form anisotropic constructs in high-density collagen. Remodeling requires the stromal cells to be in proximity with hESC-CMs. In addition, cocultured cardiac constructs in dense collagen generate measurable active contractions (on the order of 0.1 mN/mm(2)) and can be paced up to 2 Hz. Patterned microvascular networks in these high-density cocultured cardiac constructs remain patent through 2 weeks of culture, and hESC-CMs show electrical synchronization. The ability to maintain microstructural control within engineered heart tissue enables generation of more complex features, such as cellular alignment and a vasculature. Successful incorporation of these features paves the way for the use of large scale engineered tissues for myocardial regeneration and cardiac disease modeling.

  19. A Naturally Occurring HER2 Carboxy-Terminal Fragment Promotes Mammary Tumor Growth and Metastasis▿ †

    PubMed Central

    Pedersen, Kim; Angelini, Pier-Davide; Laos, Sirle; Bach-Faig, Alba; Cunningham, Matthew P.; Ferrer-Ramón, Cristina; Luque-García, Antonio; García-Castillo, Jesús; Parra-Palau, Josep Lluis; Scaltriti, Maurizio; y Cajal, Santiago Ramón; Baselga, José; Arribas, Joaquín

    2009-01-01

    HER2 is a tyrosine kinase receptor causally involved in cancer. A subgroup of breast cancer patients with particularly poor clinical outcomes expresses a heterogeneous collection of HER2 carboxy-terminal fragments (CTFs). However, since the CTFs lack the extracellular domain that drives dimerization and subsequent activation of full-length HER2, they are in principle expected to be inactive. Here we show that at low expression levels one of these fragments, 611-CTF, activated multiple signaling pathways because of its unanticipated ability to constitutively homodimerize. A transcriptomic analysis revealed that 611-CTF specifically controlled the expression of genes that we found to be correlated with poor prognosis in breast cancer. Among the 611-CTF-regulated genes were several that have previously been linked to metastasis, including those for MET, EPHA2, matrix metalloproteinase 1, interleukin 11, angiopoietin-like 4, and different integrins. It is thought that transgenic mice overexpressing HER2 in the mammary glands develop tumors only after acquisition of activating mutations in the transgene. In contrast, we show that expression of 611-CTF led to development of aggressive and invasive mammary tumors without the need for mutations. These results demonstrate that 611-CTF is a potent oncogene capable of promoting mammary tumor progression and metastasis. PMID:19364815

  20. Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal.

    PubMed

    Jalili, Reza B; Moeen Rezakhanlou, Alireza; Hosseini-Tabatabaei, Azadeh; Ao, Ziliang; Warnock, Garth L; Ghahary, Aziz

    2011-07-01

    Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.

  1. Red blood cell membrane fragments but not intact red blood cells promote calcium oxalate monohydrate crystal growth and aggregation.

    PubMed

    Chutipongtanate, Somchai; Thongboonkerd, Visith

    2010-08-01

    Cell membranes are thought to promote calcium oxalate kidney stone formation but to our knowledge the modulating effect of red blood cell membranes on calcium oxalate crystals has not been previously investigated. Thus, we examined the effects of red blood cell membrane fragments on calcium oxalate monohydrate and calcium oxalate dihydrate crystal growth and aggregation. Calcium oxalate monohydrate and calcium oxalate dihydrate crystals were treated with red blood cell membrane fragments or intact red blood cells from a healthy donor. Phase contrast microscopy was performed to evaluate crystal morphology and aggregation. We used ImageMaster 2D Platinum software to evaluate crystal size and spectrophotometric oxalate depletion assay to monitor crystal growth. Red blood cell membrane fragments had significant promoting activity on calcium oxalate monohydrate crystal growth with an approximately 75% increase in size and aggregation with an approximately 2.5-fold increase in aggregate number compared to the control without membrane fragments or cells. Approximately 50% of calcium oxalate monohydrate crystals were adhered by red blood cell membrane fragments. Intact red blood cells had no significant effect on calcium oxalate monohydrate crystal growth or aggregation but they could transform calcium oxalate monohydrate to calcium oxalate dihydrate crystals. Red blood cell membrane fragments and intact red blood cells had no effect on calcium oxalate dihydrate crystals. The promoting activity of red blood cell membrane fragments on calcium oxalate monohydrate crystal growth was successfully confirmed by spectrophotometric oxalate depletion assay. To our knowledge our data provide the first direct evidence that red blood cell membrane fragments are a promoting factor for calcium oxalate monohydrate crystal growth and aggregation. Thus, they may aggravate calcium oxalate stone formation. Copyright (c) 2010 American Urological Association Education and Research, Inc

  2. In Vitro Oxidation of Collagen Promotes the Formation of Advanced Oxidation Protein Products and the Activation of Human Neutrophils.

    PubMed

    Bochi, Guilherme Vargas; Torbitz, Vanessa Dorneles; de Campos, Luízi Prestes; Sangoi, Manuela Borges; Fernandes, Natieli Flores; Gomes, Patrícia; Moretto, Maria Beatriz; Barbisan, Fernanda; da Cruz, Ivana Beatrice Mânica; Moresco, Rafael Noal

    2016-04-01

    The accumulation of advanced oxidation protein products (AOPPs) has been linked to several pathological conditions. Here, we investigated collagen as a potential source for AOPP formation and determined the effects of hypochlorous acid (HOCl)-treated collagen (collagen-AOPPs) on human neutrophil activity. We also assessed whether alpha-tocopherol could counteract these effects. Exposure to HOCl increased the levels of collagen-AOPPs. Collagen-AOPPs also stimulated the production of AOPPs, nitric oxide (NO), superoxide radicals (O2(-)), and HOCl by neutrophils. Collagen-AOPPs induced apoptosis and decreased the number of viable cells. Alpha-tocopherol prevented the formation of collagen-AOPPs, strongly inhibited the collagen-AOPP-induced production of O2(-) and HOCl, and increased the viability of neutrophils. Our results suggest that collagen is an important protein that interacts with HOCl to form AOPPs, and consequently, collagen-AOPP formation is related to human neutrophil activation and cell death.

  3. Looping Mediated Interaction between the Promoter and 3′ UTR Regulates Type II Collagen Expression in Chondrocytes

    PubMed Central

    Jash, Arijita; Yun, Kangsun; Sahoo, Anupama; So, Jae-Seon; Im, Sin-Hyeog

    2012-01-01

    Type II collagen is the major component of articular cartilage and is mainly synthesized by chondrocytes. Repeated sub-culturing of primary chondrocytes leads to reduction of type II collagen gene (Col2a1) expression, which mimics the process of chondrocyte dedifferentiation. Although the functional importance of Col2a1 expression has been extensively investigated, mechanism of transcriptional regulation during chondrocyte dedifferentiation is still unclear. In this study, we have investigated the crosstalk between cis-acting DNA element and transcription factor on Col2a1 expression in primary chondrocytes. Bioinformatic analysis revealed the potential regulatory regions in the Col2a1 genomic locus. Among them, promoter and 3′ untranslated region (UTR) showed highly accessible chromatin architecture with enriched recruitment of active chromatin markers in primary chondrocytes. 3′ UTR has a potent enhancer function which recruits Lef1 (Lymphoid enhancer binding factor 1) transcription factor, leading to juxtaposition of the 3′ UTR with the promoter through gene looping resulting in up-regulation of Col2a1 gene transcription. Knock-down of endogenous Lef1 level significantly reduced the gene looping and subsequently down-regulated Col2a1 expression. However, these regulatory loci become inaccessible due to condensed chromatin architecture as chondrocytes dedifferentiate which was accompanied by a reduction of gene looping and down-regulation of Col2a1 expression. Our results indicate that Lef1 mediated looping between promoter and 3′ UTR under the permissive chromatin architecture upregulates Col2a1 expression in primary chondrocytes. PMID:22815835

  4. SCF promotes dental pulp progenitor migration, neovascularization, and collagen remodeling - potential applications as a homing factor in dental pulp regeneration.

    PubMed

    Pan, Shuang; Dangaria, Smit; Gopinathan, Gokul; Yan, Xiulin; Lu, Xuanyu; Kolokythas, Antonia; Niu, Yumei; Luan, Xianghong

    2013-10-01

    Stem cell factor (SCF) is a powerful chemokine that binds to the c-Kit receptor CD117 and has shown promise as a homing agent capable of progenitor cell recruitment. In the present study we have documented high levels of both SCF and its receptor c-Kit in differentiating dental pulp (DP) cells and in the sub-odontoblastic layer of Höhl. In vitro studies using human DP progenitors revealed a significant increase in cell proliferation after100 nM SCF application, explained by a 2-fold upregulation in cyclin D3 and FGF2 cell cycle regulators, and a 7-fold increase in CDK4 expression. DP cell migration in the presence of SCF was up-regulated 2.7-fold after a 24 h culture period, and this effect was accompanied by cytoskeletal rearrangement, a 1.5-fold increase in polymeric F-actin over G-actin, and a 1.8-fold increase in RhoA expression. Explaining the signaling effect of SCF on DP migration, PI3K/Akt and MEK/ERK pathway inhibitors were demonstrated to significantly reduce DP cell migration, while SCF alone doubled the number of migrated cells. ERK and AKT phosphorylation were dramatically upregulated already 3-5 min after SCF addition to the culture medium and declined thereafter, classifying SCF as a fast acting chemokine. When applied as an agent to promote tissue regeneration in subcutaneously implanted collagen sponges, SCF resulted in a 7-fold increase in the cell number in the implanted tissue construct, a more than 9-fold increase in capillaries, as well as collagen sponge remodeling and collagen fiber neogenesis. Together, these studies demonstrate the suitability of SCF as a potent aid in the regeneration of dental pulp and other mesenchymal tissues, capable of inducing cell homing, angiogenesis, and tissue remodeling.

  5. Pulmonary microvascular endothelial cells from bleomycin-induced rats promote the transformation and collagen synthesis of fibroblasts.

    PubMed

    Yin, Qian; Nan, Hai-Yan; Zhang, Wen-Hu; Yan, Lin-Feng; Cui, Guang-Bin; Huang, Xiao-Feng; Wei, Jing-Guo

    2011-08-01

    Accumulation and activation of myofibroblasts are the hallmark of progressive pulmonary fibrosis, and the resident fibroblasts are the major source of myofibroblasts. However, the key factors involved in the transformation of fibroblasts are unknown. Pulmonary microvascular endothelial cells (PMVECs), major effector cells against pathogenesis in early stages of the disease, can secrete cytokines to induce the differentiation of mesenchymal cells. We speculated that PMVECs could secrete pro-fibrotic cytokines and promote the transformation of fibroblasts into myofibroblasts. Accordingly, we established a co-culture system with PMVECs and fibroblasts to examine the specific transformation and collagen synthesis of the co-cultured fibroblasts by FACS and Western blot, prior to and after treatment with neutralizing antibodies against transforming growth factor-beta1 (TGF-β1) and connective tissue growth factor (CTGF). We also analyzed expression of TGF-β1 and CTGF in PMVECs. The synthesis and secretion of TGF-β1 and CTGF protein were up-regulated in PMVECs isolated from bleomycin (BLM)-treated rats, most prominently at 7 days post-instillation. We showed that the PMVECs isolated from BLM-induced rats could induce the transformation of normal fibroblasts and their secretion of collagen I, which was inhibited by both neutralizing anti-TGF-β1 and anti-CTGF antibodies. Therefore, up-regulation of TGF-β1 and CTGF in PMVECs plays an important role in activation, transformation, and collagen synthesis of fibroblasts; in particular, these effects in PMVECs are likely to be the key factors for activation and stimulation of static fibroblasts in lung interstitium in early stages of pulmonary fibrosis disease. Copyright © 2010 Wiley-Liss, Inc.

  6. G-CSF loaded nanofiber/nanoparticle composite coated with collagen promotes wound healing in vivo.

    PubMed

    Tanha, Shima; Rafiee-Tehrani, Morteza; Abdollahi, Mohamad; Vakilian, Saeid; Esmaili, Zahra; Naraghi, Zahra Safaei; Seyedjafari, Ehsan; Javar, Hamid Akbari

    2017-10-01

    Sustained release of functional growth factors can be considered as a beneficial methodology for wound healing. In this study, recombinant human granulocyte colony-stimulating factor (G-CSF)-loaded chitosan nanoparticles were incorporated in Poly(ε-caprolactone) (PCL) nanofibers, followed by surface coating with collagen type I. Physical and mechanical properties of the PCL nanofibers containing G-CSF loaded chitosan nanoparticles PCL/NP(G-CSF) and in vivo performance for wound healing were investigated. G-CSF structural stability was evaluated through SDS_PAGE, reversed phase (RP) HPLC and size-exclusion chromatography, as well as circular dichroism. Nanofiber/nanoparticle composite scaffold was demonstrated to have appropriate mechanical properties as a wound dresser and a sustained release of functional G-CSF. The PCL/NP(G-CSF) scaffold showed a suitable proliferation and well-adherent morphology of stem cells. In vivo study and histopathological evaluation outcome revealed that skin regeneration was dramatically accelerated under PCL/NP(G-CSF) as compared with control groups. Superior fibroblast maturation, enhanced collagen deposition and minimum inflammatory cells were also the beneficial properties of PCL/NP(G-CSF) over the commercial dressing. The synergistic effect of extracellular matrix-mimicking nanofibrous membrane and G-CSF could develop a suitable supportive substrate in order to extensive utilization for the healing of skin wounds. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2830-2842, 2017. © 2017 Wiley Periodicals, Inc.

  7. Modification of mature non-reducible collagen cross-link concentrations in bovine m. gluteus medius and semitendinosus with steer age at slaughter, breed cross and growth promotants.

    PubMed

    Roy, B C; Sedgewick, G; Aalhus, J L; Basarab, J A; Bruce, H L

    2015-12-01

    Increased meat toughness with animal age has been attributed to mature trivalent collagen cross-link formation. Intramuscular trivalent collagen cross-link content may be decreased by reducing animal age at slaughter and/or inducing muscle re-modeling with growth promotants. This hypothesis was tested in m. gluteus medius (GM) and m. semitendinosus (ST) from 112 beef steers finished at either 12 to 13 (rapid growth) or 18 to 20 (slow growth) months of age. Hereford-Aberdeen Angus (HAA) or Charolais-Red Angus (CRA) steers were randomly assigned to receive implants (IMP), ractopamine (RAC), both IMP and RAC, or none (control). RAC decreased pyridinoline (mol/mol collagen) and IMP increased Ehrlich chromogen (EC) (mol/mol collagen) in the GM. In the ST, RAC increased EC (mol/mol collagen) but decreased EC (nmol/g raw muscle) in slow growing CRA steers. Also, IMP increased ST pyridinoline (nmol/g raw muscle) of slow-growing HAA steers. Results indicated alteration of perimysium collagen cross-links content in muscle in response to growth promotants.

  8. MSC-seeded dense collagen scaffolds with a bolus dose of VEGF promote healing of large bone defects.

    PubMed

    Gao, C; Harvey, E J; Chua, M; Chen, B P; Jiang, F; Liu, Y; Li, A; Wang, H; Henderson, J E

    2013-10-13

    The functional repair of large skeletal defects remains a significant challenge to orthopaedic surgeons due to the lack of effective strategies to promote bone regeneration, particularly in the elderly. This study investigated the potential use of bone marrow derived mesenchymal stromal cells (MSC) in a dense collagen scaffold with a bolus dose of vascular endothelial growth factor (VEGF) to repair a defect in the femoral diaphysis of mice. MSC isolated from bone marrow of 4-month-old donor mice were seeded in type I collagen gels that were then compressed to form scaffolds with a fibrillar density similar to osteoid. The cells remained metabolically active in scaffolds incubated in vitro for up to 15 days and differentiated into osteoblasts that deposited calcium-phosphate mineral into the scaffold, which was quantified using micro-computed tomographic (micro-CT) imaging. When implanted in a 1 mm x 3 mm unicortical defect the MSC-loaded scaffolds were rapidly mineralised and integrated into host bone with administration of 10 ng of recombinant VEGF injected into the femoral canal at 4 days postoperative. Empty scaffolds and MSC-seeded scaffolds implanted in defects that did not receive a bolus dose of VEGF did not mineralise or integrate with native bone. The approach with MSC, hydrogels and a biologic factor already approved for human use warrants further pre-clinical investigation with a large animal model.

  9. Promotion of fibroblast adhesion by triple-helical peptide models of type I collagen-derived sequences.

    PubMed

    Grab, B; Miles, A J; Furcht, L T; Fields, G B

    1996-05-24

    The dissection of the activities mediated by type I collagen requires an approach by which the influence of triple-helical conformation can be evaluated. The alpha 1 beta 1 and alpha 2 beta 1 integrin binding sites within type I collagen are dependent upon triple-helical conformation and contained within residues 14-822 from alpha 1(I). Seven alpha 1(I)-derived triple-helical peptides (THPs) were synthesized based on charge clustering (alpha 1(I)256-270, alpha 1(I)385-396, alpha 1(I)406-417, alpha 1(I)415-423, alpha 1(I)448-456, alpha 1(I)496-507, and alpha 1(I)526-537). Three additional THPs were synthesized (alpha 1(I)85-96, alpha 1(I)433-441, and alpha 1(I)772-786) based on previously described or proposed activities (Kleinman, H. K., McGoodwin, E.B., Martin, G. R., Klebe, R. J., Fietzek, P. P., and Wooley, D. E. (1978) J. Biol. Chem. 253, 5642-5646; Staatz, W. D., Foik, K. F., Zutter, M. M., Adams, S. P., Rodriquez, B. A., and Santoro, S. A. (1991) J. Biol. Chem. 266, 7363-7367; San Antonio, J. D., Lander, A. D., Karnovsky, M. J., and Slayter, H. S. (1994) J. Cell Biol. 125, 1179-1188). Of the ten THPs, alpha 1(I)772-786 THP had the greatest activity, with half-maximal normal dermal fibroblast adhesion occurring at a peptide concentration of 1.6 microM. Triple-helicity was essential for activity of this sequence, as the non-triple-helical peptide analog (alpha 1(I)772-786 SSP) exhibited considerably lower levels of cell adhesion promotion even at peptide concentrations as high as 100 microM. Within the sequence itself, residues 784-786 (Gly-Leu-Hyp) were important for cellular recognition, as the alpha 1(I)772-783 THP had greatly reduced cell adhesion activity compared with alpha 1(I)772-786 THP. Preliminary studies indicate that the beta 1 integrin subunit mediates fibroblast adhesion to alpha 1(I)772-786 THP. The identification of fibroblast integrin binding sites within type I collagen may have important implications for understanding collagen metabolism.

  10. Linear ordered collagen scaffolds loaded with collagen-binding neurotrophin-3 promote axonal regeneration and partial functional recovery after complete spinal cord transection.

    PubMed

    Fan, Juan; Xiao, Zhifeng; Zhang, Hongtian; Chen, Bing; Tang, Guoqiang; Hou, Xianglin; Ding, Wenyong; Wang, Bin; Zhang, Peng; Dai, Jianwu; Xu, Ruxiang

    2010-09-01

    Neurotrophin-3 (NT3) is an important neurotrophic factor for spinal cord injury (SCI) repair. However, constant exchange of cerebrospinal fluid often decreases the effective dosage of NT3 at the targeted injury site. In the present study, a recombinant collagen-binding NT3 (CBD-NT3), consisting of a collagen-binding domain (CBD) and native NT3, was constructed. Linear rat-tail collagen (LRTC) was used as a physical carrier for CBD-NT3 to construct a LRTC/C3 system. The collagen-binding ability of CBD-NT3 was verified, and the bioactivity of CBD-NT3 was assayed with neurite outgrowth of dorsal root ganglia (DRG) explants and DRG cells in vitro. After complete spinal cord transection in rats, LRTC/CBD-NT3 or the LRTC/NT3 system was transplanted into the injury site. Hindlimb locomotion recovery was closely observed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale and the grid walk test. Significant improvement was observed in the LRTC/CBD-NT3 group. The results of regenerating nerve fiber and anterograde tracing of biotinylated dextran amine (BDA)-labeled corticospinal tract (CST) fibers demonstrated axonal regeneration of LRTC/CBD-NT3 in the injured spinal cord. Serotonin fiber regrowth also illustrated the effectiveness of LRTC/CBD-NT3. Thus, collagen-binding NT3 with LRTC may provide an effective method for treating SCI.

  11. Synthesis of α-collagen fragments and research of their influence on the degree of hydration of a model of epidermis

    PubMed Central

    Grobelna, Beata; Maćkiewicz, Zbigniew

    2013-01-01

    Introduction In recent years the interest into areas of science, such as cosmetology, dermatology, pharmacology or aesthetic medicine has increased significantly. Scientists are more frequently looking for ingredients that affect the skin's condition and slow down the aging process. Practically every year, the scientists discover a number of new chemical substances (both natural and synthetic) that can be potentially used to manufacture cosmetics. Aim To evaluate the influence of selected peptides derived from α-collagen fragments on the degree of hydration of a model of epidermis isolated from a pig. Material and methods The synthesis of selected cosmetic oligopeptides were performed manually, on the solid medium, using procedure of SPPS (solid phase peptide synthesis). Following components: aqua, carbomer, glycerine, phenonip, D-panthenol, dimethicone and triethanolamine were used to prepare a reference hydrogel masks. Both the number of components and the composition of hydrogels have been developed individually for the purposes of this research. For this study the skin from a domestic pig was used. The degree of the skin hydration was measured with the SKINTEST plus camera, which uses the latest semiconductor technology. Results During the study the absorption of hydrogels with peptides was faster than that of the reference hydrogel mask. The combination of hydrophilic properties of the peptide with hydrophobic properties of Palm enabled receiving an amphiphilic structure. Such molecules are considered to be able to penetrate the corneum barrier with the greatest ease. Conclusions The results showed that the modified compounds have contributed to water retention in the cells, thereby increasing the degree of hydration of the biological material. PMID:24278040

  12. TGF-β1 promotes linear invadosome formation in hepatocellular carcinoma cells, through DDR1 up-regulation and collagen I cross-linking.

    PubMed

    Ezzoukhry, Zakaria; Henriet, Elodie; Piquet, Léo; Boyé, Kevin; Bioulac-Sage, Paulette; Balabaud, Charles; Couchy, Gabrielle; Zucman-Rossi, Jessica; Moreau, Violaine; Saltel, Frédéric

    2016-11-01

    Transforming growth factor-β1 (TGF-β1) is an important player in chronic liver diseases inducing fibrogenesis and hepatocellular carcinoma (HCC) development. TGF-β1 promotes pleiotropic modifications at the cellular and matrix microenvironment levels. TGF-β1 was described to enhance production of type I collagen and its associated cross-linking enzyme, the lysyl oxidase-like2 (LOXL2). In addition, TGF-β1 and type I collagen are potent inducers of invadosomes. Indeed, type I collagen fibers induce the formation of active linear invadosomes through the discoidin domain receptor 1 (DDR1). The goal of our study was to address the role of TGF-β1 in collagen cross-linking and its impact on the formation of linear invadosomes in liver cancer cells. We first report a significant correlation between expressions of TGF-β1, and type I collagen, LOXL2, DDR1 and MT1-MMP in human HCCs. We demonstrate that TGF-β1 promotes a Smad4-dependent up-regulation of DDR1, together with LOXL2, in cultured HCC cells. Moreover, we show that LOXL2-induced collagen cross-linking enhances linear invadosome formation. Altogether, our data demonstrate that TGF-β1 favors linear invadosome formation through the expressions of both the inducers, such as collagen and LOXL2, and the components such as DDR1 and MT1-MMP of linear invadosomes in cancer cells. Meanwhile, our data uncover a new TGF-β1-dependent regulation of DDR1 expression.

  13. Transformed MDCK cells secrete elevated MMP1 that generates LAMA5 fragments promoting endothelial cell angiogenesis

    PubMed Central

    Gopal, Shashi K.; Greening, David W.; Zhu, Hong-Jian; Simpson, Richard J.; Mathias, Rommel A.

    2016-01-01

    Epithelial-mesenchymal transition (EMT) enhances the migration and invasion of cancer cells, and is regulated by various molecular mechanisms including extracellular matrix metalloproteinase (MMP) activity. Previously, we reported transformation of epithelial Madin-Darby canine kidney (MDCK) cells with oncogenic H-Ras (21D1 cells) induces EMT, and significantly elevates MMP1 expression. To explore the biological significance, in this study we characterized 21D1 cells with knocked-down MMP1 expression (21D1−MMP1). MMP1 silencing diminished 21D1 cell migration, invasion and anchorage-independent growth in vitro. Additionally, 21D1−MMP1 cells displayed reduced tumour volume when grown as in vivo subcutaneous xenografts in mice. Depletion of MMP1 lowered the ability of the cellular secretome (extracellular culture medium) to influence recipient cell behaviour. For example, supplementation with 21D1 secretome elevated cell migration of recipient fibroblasts, and enhanced endothelial cell angiogenesis (vessel length and branching). By contrast, 21D1−MMP1 secretome was less potent in both functional assays. We reveal laminin subunit alpha-5 (LAMA5) as a novel biological substrate of MMP1, that generates internal and C-terminal proteolytic fragments in 21D1 secretome. Furthermore, antibody-based inhibition of integrin αvβ3 on endothelial cells nullified the angiogenic capability of 21D1 secretome. Therefore, we report this as a new VEGF-independent mechanism that oncogenic cells may employ to promote tumour angiogenesis. PMID:27324842

  14. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Effects of acetaldehyde on nuclear protein binding to the nuclear factor I consensus sequence in the alpha 2(I) collagen promoter.

    PubMed

    Anania, F A; Potter, J J; Rennie-Tankersley, L; Mezey, E

    1995-06-01

    Acetaldehyde has been shown to increase collagen production in cultured rat myofibroblastlike cells and to activate the mouse alpha 2(I) collagen promoter in transfected NIH 3T3 cells. Nuclear factor I (NF-I), a CCAAT binding transcription factor, is known to bind and activate the alpha 1(I) and alpha 2(I) collagen genes. Activation of the alpha 2(I) collagen promoter was not observed when the NF-I binding site of the promoter was deleted. In this study, we determined if acetaldehyde influences the binding of NF-I to the alpha 2(I) collagen promoter. Nuclear proteins extracted from NIH 3T3 cells, or myofibroblastlike cells, 36 hours after the addition of acetaldehyde (200 mumol/L) in serum-free media showed increased binding to the consensus sequence of the NF-I binding site by DNase I protection analysis and by electrophoretic mobility shift assay (EMSA) as compared with control nuclear extracts that were not exposed to acetaldehyde. Furthermore, nuclear proteins extracted from myofibroblastlike cells that had been previously exposed to acetaldehyde had a marked increase in NF-I protein, as shown by Western blot with NF-I antibodies. Antisera to NF-I resulted in a slow migrating DNA-protein-antibody complex (supershift) on EMSA. However, the NF-I antibody did not supershift all the DNA-protein complexes, and the supershift band was not increased with nuclear proteins from acetaldehyde-treated cells despite the increased binding of these nuclear protein preparations to the NF-I oligo. Therefore, nuclear proteins, in addition to NF-I, bind to the NF-I consensus sequence and may have their binding altered by acetaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Etanercept Promotes Bone Formation via Suppression of Dickkopf-1 Expression in Rats with Collagen-Induced Arthritis

    PubMed Central

    Tanida, Atsushi; Kishimoto, Yuji; Okano, Toru; Hagino, Hiroshi

    2013-01-01

    Background Various clinical reports suggest etanercept (ETN) has some efficacy in bone formation in rheumatoid arthritis (RA). To examine this effect, we investigated the gene expression of cytokines relevant to osteoblast/osteoclast differentiation, and evaluated histomorphometric findings in mature rats with collagen-induced arthritis (CIA). Methods Total RNA was extracted from knee joints with CIA after ETN or placebo administration. Subsequently, realtime-PCR was carried out to quantify the mRNAs encoding Wnt-1, Dickkopf-1 (DKK-1), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegelin (OPG) and TNF (tumor necrosis factor)-alpha. In histomorphometric analysis, the infiltrating pannus volume and pannus surface, and the following items in contact with pannus surface were measured: osteoclast number, osteoid surface, osteoid volume and labeling surface. These were evaluated in the distal femur with CIA with or without ETN administration. Results TNF-alpha, RANKL and OPG mRNA expressions, linked to osteoclastogenesis, were not significantly different with or without ETN administration. ETN administration significantly increased Wnt-1 mRNA expression, the osteoblast promoter, and decreased DKK-1 mRNA expression, the Wnt signal inhibitor. In histomorphometric analysis, pannus volume, pannus surface and osteoclast number, parameters of bone destruction, were not significantly different among groups. Osteoid volume, osteoid surface and labeling surface, parameters of bone formation, increased significantly with ETN administration. Conclusion Our results suggest that ETN suppresses DDK-1 expression, and, as a result, Wnt expression is promoted and osteoblastogenesis becomes more active, independent of the regulation of osteoclast activity. Marked bone formation is attributed to the fact that ETN directly promotes osteoblastogenesis, not as a result of suppressing osteoclastogenesis. PMID:24031147

  17. Nutrient enrichment promotes survival and dispersal of drifting fragments in an invasive tropical macroalga

    NASA Astrophysics Data System (ADS)

    Vermeij, M. J. A.; Dailer, M. L.; Smith, C. M.

    2009-06-01

    The effect of nutrient availability on growth, survival, and photosynthetic performance of drifting fragments of the invasive red alga Hypnea musciformis was studied in Maui (Hawaii), where this species smothers native reef communities and forms localized blooms. H. musciformis does not sexually reproduce in Hawaii and drifting fragments represent the only pathway by which H. musciformis can disperse and invade new areas. Growth rates decreased with age and approached zero when fragments aged 32 days. Increased nutrient availability did not result in increased relative growth rates during this period. In contrast to growth, photosynthetic performance remained unaffected through time and showed no clear relationship with nutrient availability. Increased nutrient availability increased fragment survival and fragments survived for >2 months in the high nutrient treatment (3.0 μmolPO4 + 30.0 μmolNH4). This indicates that increased nutrient availability increases the dispersal potential of H. musciformis. Low growth rates of drifting Hypnea fragments increased recruitment success since attachment success of this epiphytic species decreased with increasing fragment size. H. musciformis thus uses resources for survival and maintenance rather than growth, resulting in long competency periods and optimal recruitment, which likely contribute to its success as an invader of Hawaiian reef communities.

  18. Engineered collagen hydrogels for the sustained release of biomolecules and imaging agents: promoting the growth of human gingival cells.

    PubMed

    Choi, Jonghoon; Park, Hoyoung; Kim, Taeho; Jeong, Yoon; Oh, Myoung Hwan; Hyeon, Taeghwan; Gilad, Assaf A; Lee, Kwan Hyi

    2014-01-01

    We present here the in vitro release profiles of either fluorescently labeled biomolecules or computed tomography contrast nanoagents from engineered collagen hydrogels under physiological conditions. The collagen constructs were designed as potential biocompatible inserts into wounded human gingiva. The collagen hydrogels were fabricated under a variety of conditions in order to optimize the release profile of biomolecules and nanoparticles for the desired duration and amount. The collagen constructs containing biomolecules/nanoconstructs were incubated under physiological conditions (ie, 37°C and 5% CO2) for 24 hours, and the release profile was tuned from 20% to 70% of initially loaded materials by varying the gelation conditions of the collagen constructs. The amounts of released biomolecules and nanoparticles were quantified respectively by measuring the intensity of fluorescence and X-ray scattering. The collagen hydrogel we fabricated may serve as an efficient platform for the controlled release of biomolecules and imaging agents in human gingiva to facilitate the regeneration of oral tissues.

  19. Modifications on collagen structures promoted by 1,4-dioxane improve thermal and biological properties of bovine pericardium as a biomaterial.

    PubMed

    Forti, Fábio L; Goissis, Gilberto; Plepis, Ana M G

    2006-01-01

    Collagen is a widely used raw material for biomaterial manufacture, which generally depends on chemical modifications of this fibrillar protein with cross-linking agents to improve biocompatibility and mechanical properties. However, cross-linking reduces the natural properties of collagen, such as low immune response, low toxicity as well as the ability to promote cellular growth and attachment. In this work, the modifications promoted by 1,4-dioxane solvent on the collagen present in native bovine pericardium (NBP) matrix routinely used in bioprosthesis manufacture, with or without subsequent cross-linking by glutaraldehyde, has been studied. The structural changes of NBP evaluated by scanning electron microscopy show that 1,4-dioxane induces a more homogeneous material by increasing aggregation of collagen fibers, while transmission scanning electron microscopy shows that natural collagen fibril arrangement, integrity, and the D-periodicity pattern are maintained by solvent treatments. Measurements of thermal stability and resistance to collagenase enzymatic digestion of NBP matrices treated with 1,4-dioxane show an increase in melting temperature and decrease in biodegradability, as compared to native pericardium. Cross-linking with glutaraldehyde improves all the analyzed NBP properties, which are not impaired by previous treatment with 1,4-dioxane. Histological evaluation of NBP submitted to 1,4-dioxane treatment shows lower lipid and cell contents and improvement in other morphologic characteristics compared to native pericardium. Altogether, these results suggest the use of 1,4-dioxane organic solvent as an alternative non-cross-linking treatment for direct utilization on rich collagen matrices, resulting in materials with improved biocompatibility and physicochemical properties suitable for tissue engineering.

  20. Functionalized Collagen Scaffold Neutralizing the Myelin-Inhibitory Molecules Promoted Neurites Outgrowth in Vitro and Facilitated Spinal Cord Regeneration in Vivo.

    PubMed

    Li, Xing; Han, Jin; Zhao, Yannan; Ding, Wenyong; Wei, Jianshu; Han, Sufang; Shang, Xianping; Wang, Bin; Chen, Bing; Xiao, Zhifeng; Dai, Jianwu

    2015-07-01

    Research has demonstrated that many myelin-associated inhibitory molecules jointly contribute to the failure of adult spinal cord regeneration. Therapies comprehensively targeting the multiple inhibitory nature of the injured spinal cord are being concerned. Here, two collagen-binding proteins, CBD-EphA4LBD and CBD-PlexinB1LBD, were constructed, respectively, to neutralize the axon guidance molecules ephrinB3 and sema4D that inhibit the regeneration of nerve fibers. The two neutralizing proteins have proven their ability to specifically bind collagen and to continuously release from collagen scaffolds. They could also promote neurites outgrowth of cerebellar granular neurons and dorsal root ganglion neurons in vitro. Subsequently, the functionalized collagen scaffolds by physically absorbing NEP1-40 and immobilizing CBD-EphA4LBD and CBD-PlexinB1LBD were transplanted into a rat T10 complete spinal cord transection model. Our results showed that rats that received the treatment of transplanting the functionalized collagen scaffold exhibited great advantage on axonal regeneration and locomotion recovery after spinal cord injury.

  1. Identification of a seed coat-specific promoter fragment from the Arabidopsis MUCILAGE-MODIFIED4 gene.

    PubMed

    Dean, Gillian H; Jin, Zhaoqing; Shi, Lin; Esfandiari, Elahe; McGee, Robert; Nabata, Kylie; Lee, Tiffany; Kunst, Ljerka; Western, Tamara L; Haughn, George W

    2017-07-20

    The Arabidopsis seed coat-specific promoter fragment described is an important tool for basic and applied research in Brassicaceae species. During differentiation, the epidermal cells of the Arabidopsis seed coat produce and secrete large quantities of mucilage. On hydration of mature seeds, this mucilage becomes easily accessible as it is extruded to form a tightly attached halo at the seed surface. Mucilage is composed mainly of pectin, and also contains the key cell wall components cellulose, hemicellulose, and proteins, making it a valuable model for studying numerous aspects of cell wall biology. Seed coat-specific promoters are an important tool that can be used to assess the effects of expressing biosynthetic enzymes and diverse cell wall-modifying proteins on mucilage structure and function. Additionally, they can be used for production of easily accessible recombinant proteins of commercial interest. The MUCILAGE-MODIFIED4 (MUM4) gene is expressed in a wide variety of plant tissues and is strongly up-regulated in the seed coat during mucilage synthesis, implying the presence of a seed coat-specific region in its promoter. Promoter deletion analysis facilitated isolation of a 308 base pair sequence (MUM4 0.3Pro ) that directs reporter gene expression in the seed coat cells of both Arabidopsis and Camelina sativa, and is regulated by the same transcription factor cascade as endogenous MUM4. Therefore, MUM4 0.3Pro is a promoter fragment that serves as a new tool for seed coat biology research.

  2. Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments

    PubMed Central

    Watanabe, Sumiyo; Ogasawara, Toru; Tamura, Yoshifuru; Saito, Taku; Ikeda, Toshiyuki; Suzuki, Nobuchika; Shimosawa, Tatsuo; Shibata, Shigeru; Chung, Ung-il; Nangaku, Masaomi; Uchida, Shunya

    2017-01-01

    Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. “adenovirus driven NPT2a-EGFP and endogenous NHE3 protein”, “adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein” and “adenovirus driven AQP2-EGFP and endogenous AQP2 protein”. Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and

  3. Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.

    PubMed

    Watanabe, Sumiyo; Ogasawara, Toru; Tamura, Yoshifuru; Saito, Taku; Ikeda, Toshiyuki; Suzuki, Nobuchika; Shimosawa, Tatsuo; Shibata, Shigeru; Chung, Ung-Il; Nangaku, Masaomi; Uchida, Shunya

    2017-01-01

    Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene

  4. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    SciTech Connect

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    2014-10-24

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

  5. Sp7/Osterix induces the mouse pro-α2(I) collagen gene (Col1a2) expression via the proximal promoter in osteoblastic cells.

    PubMed

    Yano, Hiroyuki; Hamanaka, Ryoji; Nakamura-Ota, Miki; Adachi, Sawako; Zhang, Juan Juan; Matsuo, Noritaka; Yoshioka, Hidekatsu

    2014-09-26

    Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amounts of other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process in which many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-rich sequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 gene containing multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site specifically decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its specific siRNA showed that this G/C-rich sequence is responsible for the specific expression in osteoblastic cells. Consistent with these data, Sp7/Osterix bound to the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.

  6. Characterization of the BM88 promoter and identification of an 88 bp fragment sufficient to drive neurone-specific expression.

    PubMed

    Papadodima, Olga; Sergaki, Maritina; Hurel, Catherine; Mamalaki, Avgi; Matsas, Rebecca

    2005-10-01

    BM88 is a neurone-specific protein implicated in cell cycle exit and differentiation of neuronal precursors. It is widely expressed in terminally differentiated neurones but also in neuronal progenitors, albeit in lower levels. Thus BM88 expression shows a tight correlation with the progression of progenitor cells towards neuronal differentiation. Here we report the genomic organization and proximal promoter characterization of the human and mouse BM88 genes. Both promoters lie in a CpG island, are TATA-less and have multiple transcription start sites. Deletion analysis performed on the human BM88 gene revealed an 88 bp minimal promoter fragment that is preferentially active in neural cells. Importantly, this minimal promoter is sufficient to confer specific transcriptional activity in primary neurones, but not in glial cells. Within the promoter region there are four functional Sp1-binding sites. Simultaneous mutations to all four Sp1 sites results in complete loss of promoter activity. Transactivation experiments revealed that Sp1 directly activates the BM88 promoter while activation also occurs in the presence of neurogenin-1. Characterization of the promoter elements that control neurone-specific and developmental expression of BM88 should contribute to the elucidation of the transcriptional networks that regulate the transition from a proliferative neural progenitor to a post-mitotic neurone.

  7. Sp1 upregulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes.

    PubMed

    Watanabe, Keijirou; Hida, Mariko; Sasaki, Takako; Yano, Hiroyuki; Kawano, Kenji; Yoshioka, Hidekatsu; Matsuo, Noritaka

    2016-02-01

    Type XI collagen is a cartilage-specific extracellular matrix, and is important for collagen fibril formation and skeletal morphogenesis. We have previously reported that NF-Y regulated the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes (Hida et. al. In Vitro Cell. Dev. Biol. Anim. 2014). However, the mechanism of the Col11a1 gene regulation in chondrocytes has not been fully elucidated. In this study, we further characterized the proximal promoter activity of the mouse Col11a1 gene in chondrocytes. Cell transfection experiments with deletion and mutation constructs indicated that the downstream region of the NF-Y binding site (-116 to +1) is also necessary to regulate the proximal promoter activity of the mouse Col11a1 gene. This minimal promoter region has no TATA box and GC-rich sequence; we therefore examined whether the GC-rich sequence (-96 to -67) is necessary for the transcription regulation of the Col11a1 gene. Luciferase assays using a series of mutation constructs exhibited that the GC-rich sequence is a critical element of Col11a1 promoter activity in chondrocytes. Moreover, in silico analysis of this region suggested that one of the most effective candidates was transcription factor Sp1. Consistent with the prediction, overexpression of Sp1 significantly increased the promoter activity. Furthermore, knockdown of Sp1 expression by siRNA transfection suppressed the proximal promoter activity and the expression of endogenous transcript of the mouse Col11a1 gene. Taken together, these results indicate that the transcription factor Sp1 upregulates the proximal promoter activity of the mouse Col11a1 gene in chondrocytes.

  8. S-adenosylmethionine blocks collagen I production by preventing transforming growth factor-beta induction of the COL1A2 promoter.

    PubMed

    Nieto, Natalia; Cederbaum, Arthur I

    2005-09-02

    To study the anti-fibrogenic mechanisms of S-adenosylmethionine (AdoMet), transgenic mice harboring the -17 kb to +54 bp of the collagen alpha2 (I) promoter (COL1A2) cloned upstream from the beta-gal reporter gene were injected with carbon tetrachloride (CCl4) to induce fibrosis and coadministered either AdoMet or saline. Control groups received AdoMet or mineral oil. AdoMet lowered the pathology in CCl4-treated mice as shown by transaminase levels, hematoxylin and eosin, Masson's trichrome staining, and collagen I expression. beta-Galactosidase activity indicated activation of the COL1A2 promoter in stellate cells from CCl4-treated mice and repression of such activation by AdoMet. Lipid peroxidation, transforming growth factor-beta (TGFbeta) expression, and decreases in glutathione levels were prevented by AdoMet. Incubation of primary stellate cells with AdoMet down-regulated basal and TGFbeta-induced collagen I and alpha-smooth muscle actin proteins. AdoMet metabolites down-regulated collagen I protein and mRNA levels. AdoMet repressed basal and TGFbeta-induced reporter activity in stellate cells transfected with COL1A2 promoter deletion constructs. AdoMet blocked TGFbeta induction of the -378 bp region of the COL1A2 promoter and prevented the phosphorylation of extracellular signal-regulated kinase 1/2 and the binding of Sp1 to the TGFbeta-responsive element. These observations unveil a novel mechanism by which AdoMet could ameliorate liver fibrosis.

  9. In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation.

    PubMed

    Cicaré, J; Caille, A; Zumoffen, C; Ghersevich, S; Bahamondes, L; Munuce, M J

    2015-10-01

    The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham's F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen-free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity. © 2014 Blackwell Verlag GmbH.

  10. TURBULENT DISKS ARE NEVER STABLE: FRAGMENTATION AND TURBULENCE-PROMOTED PLANET FORMATION

    SciTech Connect

    Hopkins, Philip F.; Christiansen, Jessie L.

    2013-10-10

    A fundamental assumption in our understanding of disks is that when the Toomre Q >> 1, the disk is stable against fragmentation into self-gravitating objects (and so cannot form planets via direct collapse). But if disks are turbulent, this neglects a spectrum of stochastic density fluctuations that can produce rare, high-density mass concentrations. Here, we use a recently developed analytic framework to predict the statistics of these fluctuations, i.e., the rate of fragmentation and mass spectrum of fragments formed in a turbulent Keplerian disk. Turbulent disks are never completely stable: we calculate the (always finite) probability of forming self-gravitating structures via stochastic turbulent density fluctuations in such disks. Modest sub-sonic turbulence above Mach number M∼0.1 can produce a few stochastic fragmentation or 'direct collapse' events over ∼Myr timescales, even if Q >> 1 and cooling is slow (t{sub cool} >> t{sub orbit}). In transsonic turbulence this extends to Q ∼ 100. We derive the true Q-criterion needed to suppress such events, which scales exponentially with Mach number. We specify to turbulence driven by magneto-rotational instability, convection, or spiral waves and derive equivalent criteria in terms of Q and the cooling time. Cooling times ∼> 50 t{sub dyn} may be required to completely suppress fragmentation. These gravo-turbulent events produce mass spectra peaked near ∼(Q M{sub disk}/M{sub *}){sup 2} M{sub disk} (rocky-to-giant planet masses, increasing with distance from the star). We apply this to protoplanetary disk models and show that even minimum-mass solar nebulae could experience stochastic collapse events, provided a source of turbulence.

  11. Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis.

    PubMed

    Butoi, E; Gan, A M; Tucureanu, M M; Stan, D; Macarie, R D; Constantinescu, C; Calin, M; Simionescu, M; Manduteanu, I

    2016-07-01

    Coronary atherosclerosis complicated by plaque disruption and thrombosis is a critical event in myocardial infarction and stroke, the major causes of cardiovascular death. In atherogenesis, macrophages (MAC) and smooth muscle cells (SMC) are key actors; they synthesize matrix components and numerous factors involved in the process. Here, we design experiments to investigate whether SMC-MAC communication induces changes in ECM protein composition and/or neo-angiogenesis. Cell to cell communication was achieved using trans-well chambers, where SMCs were grown in the upper chamber and differentiated MAC in the bottom chamber for 24 or 72h. We found that cross-talk between MAC and SMC during co-culture: (i) significantly decreased the expression of ECM proteins (collagen I, III, elastin) in SMC; (ii) increased the expression and activity of metalloprotease MMP-9 and expression of collagenase MMP-1, in both MAC and SMC; (iii) augmented the secretion of soluble VEGF in the conditioned media of cell co-culture and VEGF gene expression in both cell types, compared with control cells. Moreover, the conditioned media collected from MAC-SMC co-culture promoted endothelial cell tube formation in Matrigel, signifying an increased angiogenic effect. In addition, the MAC-SMC communication led to an increase in inflammatory IL-1β and TLR-2, which could be responsible for cellular signaling. In conclusion, MAC-SMC communication affects factors and molecules that could alter ECM composition and neo-angiogenesis, features that could directly dictate the progression of atheroma towards the vulnerable plaque. Targeting the MAC-SMC cross-talk may represent a novel therapeutic strategy to slow-down or retard the plaque progression. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Analysis of the promoter region and the N-propeptide domain of the human pro alpha 2(I) collagen gene.

    PubMed Central

    Dickson, L A; de Wet, W; Di Liberto, M; Weil, D; Ramirez, F

    1985-01-01

    We have located the exon coding for the start site of transcription of the human pro alpha 2(I) collagen gene. Comparison with the homologous region of other fibrillar collagen genes has confirmed the existence of a consensus sequence (CATGTCTA-n-TAGACATG) capable of forming a hairpin secondary structure possibly involved in the regulation of collagen biosynthesis. Sequence comparison of the chromosomal regions at the 5' end of the pro alpha 1(I) and pro alpha 2(I) collagen genes failed to identify unique DNA elements potentially mediating common regulatory signals. Sequencing of four exons coding for the N-terminal propeptide has determined most of its structure and it has implied the existence of smaller coding units similar to the 11 and 18 bp exons originally described in the avian gene. Images PMID:4011429

  13. Synergistic intrafibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to promote the regeneration of bone defects.

    PubMed

    Wang, Yao; Van Manh, Ngo; Wang, Haorong; Zhong, Xue; Zhang, Xu; Li, Changyi

    2016-01-01

    The mineralization of collagen scaffolds can improve their mechanical properties and biocompatibility, thereby providing an appropriate microenvironment for bone regeneration. The primary purpose of the present study is to fabricate a synergistically intra- and extrafibrillar mineralized collagen scaffold, which has many advantages in terms of biocompatibility, biomechanical properties, and further osteogenic potential. In this study, mineralized collagen scaffolds were fabricated using a traditional mineralization method (ie, immersed in simulated body fluid) as a control group and using a biomimetic method based on the polymer-induced liquid precursor process as an experimental group. In the polymer-induced liquid precursor process, a negatively charged polymer, carboxymethyl chitosan (CMC), was used to stabilize amorphous calcium phosphate (ACP) to form nanocomplexes of CMC/ACP. Collagen scaffolds mineralized based on the polymer-induced liquid precursor process were in gel form such that nanocomplexes of CMC/ACP can easily be drawn into the interstices of the collagen fibrils. Scanning electron microscopy and transmission electron microscopy were used to examine the porous micromorphology and synergistic mineralization pattern of the collagen scaffolds. Compared with simulated body fluid, nanocomplexes of CMC/ACP significantly increased the modulus of the collagen scaffolds. The results of in vitro experiments showed that the cell count and differentiated degrees in the experimental group were higher than those in the control group. Histological staining and micro-computed tomography showed that the amount of new bone regenerated in the experimental group was larger than that in the control group. The biomimetic mineralization will assist us in fabricating a novel collagen scaffold for clinical applications.

  14. Synergistic intrafibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to promote the regeneration of bone defects

    PubMed Central

    Wang, Yao; Van Manh, Ngo; Wang, Haorong; Zhong, Xue; Zhang, Xu; Li, Changyi

    2016-01-01

    The mineralization of collagen scaffolds can improve their mechanical properties and biocompatibility, thereby providing an appropriate microenvironment for bone regeneration. The primary purpose of the present study is to fabricate a synergistically intra- and extrafibrillar mineralized collagen scaffold, which has many advantages in terms of biocompatibility, biomechanical properties, and further osteogenic potential. In this study, mineralized collagen scaffolds were fabricated using a traditional mineralization method (ie, immersed in simulated body fluid) as a control group and using a biomimetic method based on the polymer-induced liquid precursor process as an experimental group. In the polymer-induced liquid precursor process, a negatively charged polymer, carboxymethyl chitosan (CMC), was used to stabilize amorphous calcium phosphate (ACP) to form nanocomplexes of CMC/ACP. Collagen scaffolds mineralized based on the polymer-induced liquid precursor process were in gel form such that nanocomplexes of CMC/ACP can easily be drawn into the interstices of the collagen fibrils. Scanning electron microscopy and transmission electron microscopy were used to examine the porous micromorphology and synergistic mineralization pattern of the collagen scaffolds. Compared with simulated body fluid, nanocomplexes of CMC/ACP significantly increased the modulus of the collagen scaffolds. The results of in vitro experiments showed that the cell count and differentiated degrees in the experimental group were higher than those in the control group. Histological staining and micro-computed tomography showed that the amount of new bone regenerated in the experimental group was larger than that in the control group. The biomimetic mineralization will assist us in fabricating a novel collagen scaffold for clinical applications. PMID:27274235

  15. Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling.

    PubMed

    Barascuk, Natasha; Vassiliadis, Efstathios; Larsen, Lise; Wang, Jianxia; Zheng, Qinlong; Xing, Rui; Cao, Yu; Crespo, Christine; Lapret, Isabelle; Sabatini, Massimo; Villeneuve, Nicole; Vilaine, Jean-Paul; Rasmussen, Lars Melholt; Register, Thomas C; Karsdal, Morten A

    2011-07-01

    Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine. A monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice. The lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p<0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice. We have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. Engineered collagen hydrogels for the sustained release of biomolecules and imaging agents: promoting the growth of human gingival cells

    PubMed Central

    Choi, Jonghoon; Park, Hoyoung; Kim, Taeho; Jeong, Yoon; Oh, Myoung Hwan; Hyeon, Taeghwan; Gilad, Assaf A; Lee, Kwan Hyi

    2014-01-01

    We present here the in vitro release profiles of either fluorescently labeled biomolecules or computed tomography contrast nanoagents from engineered collagen hydrogels under physiological conditions. The collagen constructs were designed as potential biocompatible inserts into wounded human gingiva. The collagen hydrogels were fabricated under a variety of conditions in order to optimize the release profile of biomolecules and nanoparticles for the desired duration and amount. The collagen constructs containing biomolecules/nanoconstructs were incubated under physiological conditions (ie, 37°C and 5% CO2) for 24 hours, and the release profile was tuned from 20% to 70% of initially loaded materials by varying the gelation conditions of the collagen constructs. The amounts of released biomolecules and nanoparticles were quantified respectively by measuring the intensity of fluorescence and X-ray scattering. The collagen hydrogel we fabricated may serve as an efficient platform for the controlled release of biomolecules and imaging agents in human gingiva to facilitate the regeneration of oral tissues. PMID:25429215

  17. Human pancreatic stellate cells modulate 3D collagen alignment to promote the migration of pancreatic ductal adenocarcinoma cells.

    PubMed

    Drifka, Cole R; Loeffler, Agnes G; Esquibel, Corinne R; Weber, Sharon M; Eliceiri, Kevin W; Kao, W John

    2016-12-01

    A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the ability for cancer cells to aggressively infiltrate and navigate through a dense stroma during the metastatic process. Key features of the PDAC stroma include an abundant population of activated pancreatic stellate cells (PSCs) and highly aligned collagen fibers; however, important questions remain regarding how collagen becomes aligned and what the biological manifestations are. To better understand how PSCs, aligned collagen, and PDAC cells might cooperate during the transition to invasion, we utilized a microchannel-based in vitro tumor model and advanced imaging technologies to recreate and examine in vivo-like heterotypic interactions. We found that PSCs participate in a collaborative process with cancer cells by orchestrating the alignment of collagen fibers that, in turn, are permissive to enhanced cell migration. Additionally, direct contact between PSCs, collagen, and PDAC cells is critical to invasion and co-migration of both cell types. This suggests PSCs may accompany and assist in navigating PDAC cells through the stromal terrain. Together, our data provides a new role for PSCs in stimulating the metastatic process and underscores the importance of collagen alignment in cancer progression.

  18. Habitat destruction, fragmentation, and disturbance promote invasion by habitat generalists in a multispecies metapopulation.

    PubMed

    Marvier, Michelle; Kareiva, Peter; Neubert, Michael G

    2004-08-01

    Species invasions are extremely common and are vastly outpacing the ability of resource agencies to address each invasion, one species at a time. Management actions that target the whole landscape or ecosystem may provide more cost-effective protection against the establishment of invasive species than a species-by-species approach. To explore what ecosystem-level actions might effectively reduce invasions, we developed a multispecies, multihabitat metapopulation model. We assume that species that successfully establish themselves outside their native range tend to be habitat generalists and that a tradeoff exists between competitive ability and habitat breadth, such that habitat specialists are competitively superior to habitat generalists. In this model, habitat destruction, fragmentation, and short-term disturbances all favor invasion by habitat generalists, despite the inferior competitive abilities of generalist species. Our model results illustrate that providing relatively undisturbed habitat and preventing further habitat degradation and fragmentation can provide a highly cost-effective defense against invasive species.

  19. Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells.

    PubMed

    Mittag, Falk; Falkenberg, Eva-Maria; Janczyk, Alexandra; Götze, Marco; Felka, Tino; Aicher, Wilhelm K; Kluba, Torsten

    2012-11-02

    Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications.

  20. Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells

    PubMed Central

    Mittag, Falk; Falkenberg, Eva-Maria; Janczyk, Alexandra; Götze, Marco; Felka, Tino; Aicher, Wilhelm K.; Kluba, Torsten

    2012-01-01

    Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1–5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications. PMID:23589764

  1. Interactions between Amyloid-β and Tau Fragments Promote Aberrant Aggregates: Implications for Amyloid Toxicity

    PubMed Central

    2015-01-01

    We have investigated at the oligomeric level interactions between Aβ(25–35) and Tau(273–284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer’s disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273–284) shows a high affinity to form heteroligomers with existing Aβ(25–35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25–35) or Tau(273–284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25–35) fibrils. The incorporation of some Tau into β-rich Aβ(25–35) oligomers reduces the aggregation propensity of Aβ(25–35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25–35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves. PMID:25153942

  2. Interactions between amyloid-β and Tau fragments promote aberrant aggregates: implications for amyloid toxicity.

    PubMed

    Do, Thanh D; Economou, Nicholas J; Chamas, Ali; Buratto, Steven K; Shea, Joan-Emma; Bowers, Michael T

    2014-09-25

    We have investigated at the oligomeric level interactions between Aβ(25-35) and Tau(273-284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer's disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273-284) shows a high affinity to form heteroligomers with existing Aβ(25-35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25-35) or Tau(273-284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25-35) fibrils. The incorporation of some Tau into β-rich Aβ(25-35) oligomers reduces the aggregation propensity of Aβ(25-35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25-35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves.

  3. Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts.

    PubMed

    Lerner, Chad A; Rutagarama, Pierrot; Ahmad, Tanveer; Sundar, Isaac K; Elder, Alison; Rahman, Irfan

    2016-09-02

    Oxidants or nanoparticles have recently been identified as constituents of aerosols released from various styles of electronic cigarettes (E-cigs). Cells in the lung may be directly exposed to these constituents and harbor reactive properties capable of incurring acute cell injury. Our results show mitochondria are sensitive to both E-cig aerosols and aerosol containing copper nanoparticles when exposed to human lung fibroblasts (HFL-1) using an Air-Liquid Interface culture system, evident by elevated levels of mitochondrial ROS (mtROS). Increased mtROS after aerosol exposure is associated with reduced stability of OxPhos electron transport chain (ETC) complex IV subunit and nuclear DNA fragmentation. Increased levels of IL-8 and IL-6 in HFL-1 conditioned media were also observed. These findings reveal both mitochondrial, genotoxic, and inflammatory stresses are features of direct cell exposure to E-cig aerosols which are ensued by inflammatory duress, raising a concern on deleterious effect of vaping.

  4. Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts

    SciTech Connect

    Lerner, Chad A.; Rutagarama, Pierrot; Ahmad, Tanveer; Sundar, Isaac K.; Elder, Alison; Rahman, Irfan

    2016-09-02

    Oxidants or nanoparticles have recently been identified as constituents of aerosols released from various styles of electronic cigarettes (E-cigs). Cells in the lung may be directly exposed to these constituents and harbor reactive properties capable of incurring acute cell injury. Our results show mitochondria are sensitive to both E-cig aerosols and aerosol containing copper nanoparticles when exposed to human lung fibroblasts (HFL-1) using an Air-Liquid Interface culture system, evident by elevated levels of mitochondrial ROS (mtROS). Increased mtROS after aerosol exposure is associated with reduced stability of OxPhos electron transport chain (ETC) complex IV subunit and nuclear DNA fragmentation. Increased levels of IL-8 and IL-6 in HFL-1 conditioned media were also observed. These findings reveal both mitochondrial, genotoxic, and inflammatory stresses are features of direct cell exposure to E-cig aerosols which are ensued by inflammatory duress, raising a concern on deleterious effect of vaping. - Graphical abstract: Oxidants and possibly reactive properties of metal particles in E-cig aerosols impart mitochondrial oxidative stress and DNA damage. These biological effects accompany inflammatory response which may raise concern regarding long term E-cig use. Mitochondria may be particularly sensitive to reactive properties of E-cig aerosols in addition to the potential for them to induce genotoxic stress by generating increased ROS. - Highlights: • Mitochondria are sensitive to both E-cig aerosols and metal nanoparticles. • Increased mtROS by E-cig aerosol is associated with disrupted mitochondrial energy. • E-cig causes nuclear DNA fragmentation. • E-cig aerosols induce pro-inflammatory response in human fibroblasts.

  5. Classical complement pathway component C1q: purification of human C1q, isolation of C1q collagen-like and globular head fragments and production of recombinant C1q-derivatives. Functional characterization.

    PubMed

    Kojouharova, Mihaela

    2014-01-01

    The classical complement pathway (CCP) activation is a multimolecular complex, composed of three subcomponents namely C1q, C1r, and C1s. C1q is the recognition subunit of this complex and its binding to the specific targets leads to the formation of active C1, which in turn activates the CCP in an immunoglobulin-dependent or -independent manner. C1q is a hexameric glycoprotein composed of 18 polypeptide chains of three different types (A, B, and C), organized in two fragments-collagen-like (CLR) and globular head (gC1q) possessing different functional activity. The contemporary knowledge of the C1q structure allows the isolation and purification of a C1q molecule from serum by combination of different chromatography procedures including ion-exchange, size-exclusion, and affinity chromatography, as well as the isolation of CLR and gC1q by limited enzymatic hydrolysis of the native C1q molecule. In this chapter, we described methods for purification of human C1q and its CLR and gC1q fragments, as well as methods for their biochemical and functional characterization. The production and purification of recombinant C1q derivatives ghA, ghB, and ghC (globular fragments of the individual C1q chains) are also presented.

  6. The synthetic hydroxyproline-containing collagen analogue (Gly–Pro–Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro

    PubMed Central

    Freise, C.; Ruehl, M.; Erben, U.; Farndale, R. W.; Somasundaram, R.; Heimesaat, M. M.

    2012-01-01

    Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP–collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly–Pro–Hyp)10 – (GPO)10 – was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP–(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP–(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis. PMID:24688764

  7. The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro.

    PubMed

    Freise, C; Ruehl, M; Erben, U; Farndale, R W; Somasundaram, R; Heimesaat, M M

    2012-09-01

    Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp)10 - (GPO)10 - was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.

  8. Aromatic interactions promote self-association of collagen triple-helical peptides to higher-order structures.

    PubMed

    Kar, Karunakar; Ibrar, Sajjad; Nanda, Vikas; Getz, Todd M; Kunapuli, Satya P; Brodsky, Barbara

    2009-08-25

    Aromatic residues are relatively rare within the collagen triple helix, but they appear to play a specialized role in higher-order structure and function. The role of aromatic amino acids in the self-assembly of triple-helical peptides was investigated in terms of the kinetics of self-association, the nature of aggregated species formed, and the ability of these species to activate platelet aggregation. The presence of aromatic residues on both ends of a type IV collagen model peptide is observed to greatly accelerate the kinetics of self-association, decreasing the lag time and leading to insoluble, well-defined linear fibrils as well as small soluble aggregates. Both macroscopic visible aggregates and small multimolecular complexes in solution are capable of inducing platelet aggregation through the glycoprotein VI receptor on platelets. Proline-aromatic CH...pi interactions are often observed within globular proteins and in protein complexes, and examination of molecular packing in the crystal structure of the integrin binding collagen peptide shows Phe interacts with Pro/Hyp in a neighboring triple-helical molecule. An intermolecular interaction between aromatic amino acids and imino acids within the triple helix is also supported by the observed inhibitory effect of isolated Phe amino acids on the self-association of (Pro-Hyp-Gly)(10). Given the high fraction of Pro and Hyp residues on the surface of collagen molecules, it is likely that imino acid-aromatic CH...pi interactions are important in formation of higher-order structure. We suggest that the catalysis of type I collagen fibrillogenesis by nonhelical telopeptides is due to specific intermolecular CH...pi interactions between aromatic residues in the telopeptides and Pro/Hyp residues within the triple helix.

  9. IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance.

    PubMed

    Lubberts, Erik; van den Bersselaar, Liduine; Oppers-Walgreen, Birgitte; Schwarzenberger, Paul; Coenen-de Roo, Christina J J; Kolls, Jay K; Joosten, Leo A B; van den Berg, Wim B

    2003-03-01

    IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-kappa B ligand (RANKL)/receptor activator of NF-kappa B/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-kappa B, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.

  10. Characterization of low molecular weight fragments from gamma irradiated κ-carrageenan used as plant growth promoter

    NASA Astrophysics Data System (ADS)

    Abad, Lucille V.; Aurigue, Fernando B.; Relleve, Lorna S.; Montefalcon, Djowel Recto V.; Lopez, Girlie Eunice P.

    2016-01-01

    Radiation degraded κ-carrageenan (1% solution at absorbed doses of 20 kGy and 30 kGy) were tested for its plant growth promoter (PGP) effect on pechay plants under hydroponics condition. Results revealed that higher PGP effects were found in κ-carrageenan irradiated at an absorbed dose of 30 kGy. Mw of irradiated κ-carrageenan as measured by GPC were determined to be 7362 Da and 6762 Da for 20 kGy and 30 kGy, respectively. Fractionation of the irradiated κ-carrageenan (30 kGy) was done to separate different Mw fractions using Mw cut-off filters of 1 kDa, 3 kDa, and 5 kDa. The PGP effect of the different retentates showed that biological activity in plants followed the order of 5 kDa>3 kDa>1 kDa using hydroponics condition but the reverse was observed in the order of 1 kDa>3 kDa>5 kDa when absorbed in plants by foliar spraying. GPC chromatogram indicated at least three (3) low molecular weight (LMW) fragments from radiation modified κ-carrageenan solution with an Mw<2000 Da. A fragment has also been identified with an Mw of as low as 160 Da which was produced under acidic (un-neutralized) condition. This may be attributed to the formation of 5-hydroxymethylfurfural (5-HMF).

  11. Interleukin-35 (IL-35) inhibits proliferation and promotes apoptosis of fibroblast-like synoviocytes isolated from mice with collagen-induced arthritis.

    PubMed

    Li, Yunxia; Wu, Suqin; Li, Yuxuan; Jiang, Shenyi; Lin, Tiantian; Xia, Liping; Shen, Hui; Lu, Jing

    2016-09-01

    Rheumatoid arthritis (RA) is an inflammatory disorder of the joints that affects 0.5-1 % of adults. Excessive growth of the fibroblast-like synoviocytes (FLS) promotes hyperplasia of synovial tissues and causes its invasion into the bone and cartilage, which eventually causes deformity and dysfunction of affected joints. Interleukin 35 (IL-35) was shown to suppress the inflammatory responses to collagen-induced arthritis (CIA) via upregulation of T regulatory cells and suppression of T helper type 17 cells in a mouse model. To study the effects of IL-35 on the proliferation and apoptosis frequency of cultured FLS isolated from mice with CIA as well as to examine the effects of IL-35 on CIA in vivo. Thirty DBA/1 J mice, which are used as an animal model for RA, were divided randomly (ten mice per group) to a CIA group (collagen treatment), a CIA + IL-35 group (collagen and IL-35 treatments), and a control group (no treatment). Starting on the 24th day after collagen administration, IL-35 was injected intraperitoneally into mice of the CIA + IL-35 group once per day for 10 days. An arthritis index was calculated, and pathological analysis of synovial tissue was performed. FLS isolated from CIA mice were treated with various concentrations of IL-35 (12.5-100 ng/ml). The MTT assay was used to examine FLS proliferation, and apoptosis frequency of FLS was detected by flow cytometry. On day 24, the CIA mice began to exhibit arthritis symptoms, and the symptoms rapidly progressed with time. Treatment with IL-35 significantly alleviated arthritis symptoms and reduced the synovial tissue inflammation. In addition, IL-35 treatment inhibited proliferation and promoted apoptosis in cultured FLS from CIA mice in a dose-dependent manner. IL-35 could ameliorate the symptoms of arthritis in the CIA mouse model in vivo and inhibited FLS proliferation while promoting FLS apoptosis in vitro, thereby exhibited the potential in inhibiting the progression of RA.

  12. A Modified Collagen Gel Dressing Promotes Angiogenesis in a Pre-Clinical Swine Model of Chronic Ischemic Wounds

    PubMed Central

    Elgharably, Haytham; Ganesh, Kasturi; Dickerson, Jennifer; Khanna, Savita; Abas, Motaz; Ghatak, Piya Das; Dixit, Sriteja; Bergdall, Valerie; Roy, Sashwati; Sen, Chandan K.

    2015-01-01

    We recently performed proteomic characterization of a modified collagen gel (MCG) dressing and reported promising effects of the gel in healing full-thickness excisional wounds. In this work, we test the translational relevance of our aforesaid findings by testing the dressing in a swine model of chronic ischemic wounds recently reported by our laboratory. Full thickness excisional wounds were established in the center of bi- pedicle ischemic skin flaps on the backs of animals. Ischemia was verified by Laser Doppler imaging and MCG was applied to the test group of wounds. Seven days post- wounding, macrophage recruitment to the wound was significantly higher in MCG- treated ischemic wounds. In vitro, MCG up-regulated expression of Mrc-1 (a reparative M2 macrophage marker) and induced the expression of anti-inflammatory cytokine IL-10 and of β-FGF. An increased expression of CCR2, a M2 macrophage marker, was noted in the macrophages from MCG treated wounds. Furthermore, analyses of wound tissues 7 days post wounding showed up-regulation of TGF-β, VEGF, vWF, and collagen type I expression in MCG-treated ischemic wounds. At 21 days post-wounding, MCG-treated ischemic wounds displayed higher abundance of proliferating endothelial cells that formed mature vascular structures and increased blood flow to the wound. Fibroblast count was markedly higher in MCG-treated ischemic wound-edge tissue. In addition, MCG-treated wound-edge tissues displayed higher abundance of mature collagen with increased collagen type I:III deposition. Taken together, MCG helped mount a more robust inflammatory response which resolved in a timely manner, followed by an enhanced proliferative phase, angiogenic outcome and post-wound tissue remodeling. Findings of the current study warrant clinical testing of MCG in a setting of ischemic chronic wounds. PMID:25224310

  13. Characterization of EBV Promoters and Coding Regions by Sequencing PCR-Amplified DNA Fragments.

    PubMed

    Szenthe, Kalman; Bánáti, Ferenc

    2017-01-01

    DNA sequencing approaches originally developed in two directions, the chemical degradation method and the chain-termination method. The latter one became more widespread and a huge amount of sequencing data including whole genome sequences accumulated, based on the use of capillary sequencer systems and the application of a modified chain-termination method which proved to be relatively easy, fast, and reliable. In addition, relatively long, up to 1000 bp sequences could be obtained with a single read with high per-base accuracy. Although the recent appearance of next-generation DNA sequencing (NGS) technologies enabled high-throughput and low cost analysis of DNA, the modified chain-terminating methods are often applied in research until now. In the following, we shall present the application of capillary sequencing for the sequence characterization of viral genomes in case of partial and whole genome sequencing, and demonstrate it on the BARF1 promoter of Epstein Barr virus (EBV).

  14. Extract of passion fruit (Passiflora edulis) seed containing high amounts of piceatannol inhibits melanogenesis and promotes collagen synthesis.

    PubMed

    Matsui, Yuko; Sugiyama, Kenkichi; Kamei, Masanori; Takahashi, Toshio; Suzuki, Tamio; Katagata, Yohtaro; Ito, Tatsuhiko

    2010-10-27

    The effect of passion fruit, the fruit of Passiflora edulis , on melanin inhibition and collagen synthesis was studied using cultured human melanoma and fibroblast cells. Passion fruit was divided into three parts, rind (PF-R), pulp (PF-P), and seed (PF-S), and each part was extracted using 80% ethanol. The concentration of polyphenols was higher in PF-S than in PF-R or PF-P. Treatment of melanoma cells with PF-S led to inhibition of melanogenesis. In addition, the production of total soluble collagen was elevated in dermal fibroblast cells cultured in the presence of PF-S. PF-R and PF-P did not yield these effects. Furthermore, the removal of polyphenols from PF-S led to the abolishment of the effects described above. We discovered that piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene) is present in passion fruit seeds in large amounts and that this compound is the major component responsible for the PF-S effects observed on melanogenesis and collagen synthesis.

  15. Effect of a novel botanical agent Drynol Cibotin on human osteoblast cells and implications for osteoporosis: promotion of cell growth, calcium uptake and collagen production.

    PubMed

    Wegiel, Barbara; Persson, Jenny L

    2010-06-01

    Osteoporosis is a widespread problem afflicting millions of people. Drynol Cibotinis is a newly developed proprietary botanical combination of eight botanicals including Angelica sinensis, Glycine max, Wild yam, Ligustrum lucidum, Astragalus membranaceus, Cuscuta chinensis, Psoraleae corylifoliae, and Drynaria fortune. Each of the botanicals has been used in traditional Chinese medicine to treat osteoporosis. The effect of Drynol Cibotinis, with the specific combination of these anti-osteoporosis botanicals for promoting bone growth, was examined in this study. The effects of Drynol Cibotin on cell growth, apoptosis, cell spreading, calcium uptake and production of bone matrix proteins Collagen I and Laminin B2 on human osteoblast cells were assessed by BrdU incorporation, TUNEL assay, cell staining, intracellular Ca2+ measurement and Western blot analysis. The results showed that Drynol Cibotin significantly increased cell proliferation and inhibited apoptosis in osteoblasts (P < 0.01). In addition, Drynol Cibotin was found to promote cell spreading and greatly increase calcium uptake both instantaneously and in the long term (P < 0.01). Furthermore, Drynol Cibotin significantly increased production of two key extracellular matrix proteins in bone cells: Collagen I and Laminin B2. These results indicate that Drynol Cibotin alone or in combination with amino acids and vitamins may have prophylactic potentials in osteoporosis.

  16. Collagen-binding domains of gelatinase A and thrombospondin-derived peptides impede endocytic clearance of active gelatinase A and promote HT1080 fibrosarcoma cell invasion.

    PubMed

    Robinet, Arnaud; Emonard, Hervé; Banyai, Laszlo; Laronze, Jean-Yves; Patthy, Lazlo; Hornebeck, William; Bellon, Georges

    2008-02-13

    Gelatinase A (matrix metalloproteinase-2, MMP-2) binds to several proteins through its collagen-binding domains (CBDs). Surface plasmon resonance analysis revealed a strong interaction between CBD123 and thrombospondin-1 (TSP-1), with a K(D) value of 2x10(-9) M. CBD123, as well as individual domains, behave as competitive inhibitors of the TSP-1-directed endocytic clearance of active MMP-2, but not of its latent form, by HT1080 fibrosarcoma cells. Enhanced level of active MMP-2 in conditioned medium was associated to increased matrigel invasion. Similarly, GGWSHWSPWSS and GGWSHW peptides, as tryptophan-rich peptides within properdin-repeat motifs (TSRs) of TSP-1, promoted MMP-2 accumulation and cell invasiveness. Our data document the importance of TSP-1 in promoting MMP-2-mediated cancer cell invasion through interaction between CBDs of the enzyme and TSRs motifs of TSP-1.

  17. Dual therapeutic functions of F-5 fragment in burn wounds: preventing wound progression and promoting wound healing in pigs.

    PubMed

    Bhatia, Ayesha; O'Brien, Kathryn; Chen, Mei; Wong, Alex; Garner, Warren; Woodley, David T; Li, Wei

    2016-01-01

    Burn injuries are a leading cause of morbidity including prolonged hospitalization, disfigurement, and disability. Currently there is no Food and Drug Administration-approved burn therapeutics. A clinical distinction of burn injuries from other acute wounds is the event of the so-called secondary burn wound progression within the first week of the injury, in which a burn expands horizontally and vertically from its initial boundary to a larger area. Therefore, an effective therapeutics for burns should show dual abilities to prevent the burn wound progression and thereafter promote burn wound healing. Herein we report that topically applied F-5 fragment of heat shock protein-90α is a dual functional agent to promote burn wound healing in pigs. First, F-5 prevents burn wound progression by protecting the surrounding cells from undergoing heat-induced caspase 3 activation and apoptosis with increased Akt activation. Accordingly, F-5-treated burn and excision wounds show a marked decline in inflammation. Thereafter, F-5 accelerates burn wound healing by stimulating the keratinocyte migration-led reepithelialization, leading to wound closure. This study addresses a topical agent that is capable of preventing burn wound progression and accelerating burn wound healing.

  18. Dual therapeutic functions of F-5 fragment in burn wounds: preventing wound progression and promoting wound healing in pigs

    PubMed Central

    Bhatia, Ayesha; O’Brien, Kathryn; Chen, Mei; Wong, Alex; Garner, Warren; Woodley, David T.; Li, Wei

    2016-01-01

    Burn injuries are a leading cause of morbidity including prolonged hospitalization, disfigurement, and disability. Currently there is no Food and Drug Administration-approved burn therapeutics. A clinical distinction of burn injuries from other acute wounds is the event of the so-called secondary burn wound progression within the first week of the injury, in which a burn expands horizontally and vertically from its initial boundary to a larger area. Therefore, an effective therapeutics for burns should show dual abilities to prevent the burn wound progression and thereafter promote burn wound healing. Herein we report that topically applied F-5 fragment of heat shock protein-90α is a dual functional agent to promote burn wound healing in pigs. First, F-5 prevents burn wound progression by protecting the surrounding cells from undergoing heat-induced caspase 3 activation and apoptosis with increased Akt activation. Accordingly, F-5–treated burn and excision wounds show a marked decline in inflammation. Thereafter, F-5 accelerates burn wound healing by stimulating the keratinocyte migration-led reepithelialization, leading to wound closure. This study addresses a topical agent that is capable of preventing burn wound progression and accelerating burn wound healing. PMID:27382602

  19. Biased signaling favoring gi over β-arrestin promoted by an apelin fragment lacking the C-terminal phenylalanine.

    PubMed

    Ceraudo, Emilie; Galanth, Cécile; Carpentier, Eric; Banegas-Font, Inmaculada; Schonegge, Anne-Marie; Alvear-Perez, Rodrigo; Iturrioz, Xavier; Bouvier, Michel; Llorens-Cortes, Catherine

    2014-08-29

    Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Collagen XXIV (Col24α1) Promotes Osteoblastic Differentiation and Mineralization through TGF-β/Smads Signaling Pathway

    PubMed Central

    Wang, Weizhuo; Olson, Douglas; Liang, Gang; Franceschi, Renny T; Li, Chunyi; Wang, Bingyan; Wang, Shuen Shiuan; Yang, Shuying

    2012-01-01

    Collagen XXIV (Col24α1) is a recently discovered fibrillar collagen. It is known that mouse Col24α1 is predominantly expressed in the forming skeleton of the mouse embryo, as well as in the trabecular bone and periosteum of the newborn mouse. However, the role and mechanism of Col24α1 in osteoblast differentiation and mineralization remains unclear. By analyzing the expression pattern of Col24α1, we confirmed that it is primarily expressed in bone tissues, and this expression gradually increased concomitant with the progression of osteoblast differentiation. Through the use of a lentivirus vector-mediated interference system, silencing Col24α1 expression in MC3T3-E1 murine preosteoblastic cells resulted in significant inhibition of alkaline phosphatase (ALP) activity, cell mineralization, and the expression of osteoblast marker genes such as runt-related transcription factor 2 (Runx2), osteocalcin (OCN), ALP, and type I collagen (Col I). Subsequent overexpression not only rescued the deficiency in osteoblast differentiation from Col24α1 silenced cells, but also enhanced osteoblastic differentiation in control cells. We further revealed that Col24α1 interacts with integrin β3, and silencing Col24α1 up-regulated the expression of Smad7 during osteoblast differentiation while at the same time inhibiting the phosphorylation of the Smad2/3 complex. These results suggest that Col24α1 imparts some of its regulatory control on osteoblast differentiation and mineralization at least partially through interaction with integrin β3 and the transforming growth factor beta (TGF-β) /Smads signaling pathway. PMID:23139630

  1. Gelatin-methacrylamide gel loaded with microspheres to deliver GDNF in bilayer collagen conduit promoting sciatic nerve growth

    PubMed Central

    Zhuang, Hai; Bu, Shoushan; Hua, Lei; Darabi, Mohammad A; Cao, Xiaojian; Xing, Malcolm

    2016-01-01

    In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide®). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 μm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable

  2. Gelatin-methacrylamide gel loaded with microspheres to deliver GDNF in bilayer collagen conduit promoting sciatic nerve growth.

    PubMed

    Zhuang, Hai; Bu, Shoushan; Hua, Lei; Darabi, Mohammad A; Cao, Xiaojian; Xing, Malcolm

    2016-01-01

    In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide(®)). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 μm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable

  3. Inhibitor of PI3K/Akt Signaling Pathway Small Molecule Promotes Motor Neuron Differentiation of Human Endometrial Stem Cells Cultured on Electrospun Biocomposite Polycaprolactone/Collagen Scaffolds.

    PubMed

    Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Yazdankhah, Meysam; Ai, Jafar; Khakbiz, Mehrdad; Faghihi, Faezeh; Tajerian, Roksana; Bayat, Neda

    2017-05-01

    Small molecules as useful chemical tools can affect cell differentiation and even change cell fate. It is demonstrated that LY294002, a small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, can inhibit proliferation and promote neuronal differentiation of mesenchymal stem cells (MSCs). The purpose of this study was to investigate the differentiation effect of Ly294002 small molecule on the human endometrial stem cells (hEnSCs) into motor neuron-like cells on polycaprolactone (PCL)/collagen scaffolds. hEnSCs were cultured in a neurogenic inductive medium containing 1 μM LY294002 on the surface of PCL/collagen electrospun fibrous scaffolds. Cell attachment and viability of cells on scaffolds were characterized by scanning electron microscope (SEM) and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The expression of neuron-specific markers was assayed by real-time PCR and immunocytochemistry analysis after 15 days post induction. Results showed that attachment and differentiation of hEnSCs into motor neuron-like cells on the scaffolds with Ly294002 small molecule were higher than that of the cells on tissue culture plates as control group. In conclusion, PCL/collagen electrospun scaffolds with Ly294002 have potential for being used in neural tissue engineering because of its bioactive and three-dimensional structure which enhances viability and differentiation of hEnSCs into neurons through inhibition of the PI3K/Akt pathway. Thus, manipulation of this pathway by small molecules can enhance neural differentiation.

  4. TGF-β/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1α expression

    PubMed Central

    Hayashida, Tomoko; Hubchak, Susan C.; Hanna, Christian; Platanias, Leonidas C.; William Schnaper, H.

    2013-01-01

    Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-β-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-β in a TGF-β receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-β stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-β-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis. PMID:23761672

  5. TGF-β/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1α expression.

    PubMed

    Rozen-Zvi, Benaya; Hayashida, Tomoko; Hubchak, Susan C; Hanna, Christian; Platanias, Leonidas C; Schnaper, H William

    2013-08-15

    Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-β-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-β in a TGF-β receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-β stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-β-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis.

  6. Type I collagen promotes primary cilia growth through down-regulating HDAC6-mediated autophagy in confluent mouse embryo fibroblast 3T3-L1 cells.

    PubMed

    Xu, Qian; Liu, Weiwei; Liu, Xiaoling; Otkur, Wuxiyar; Hayashi, Toshihiko; Yamato, Masayuki; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Ikejima, Takashi

    2017-08-12

    Primary cilia are microtubule-based organelles that extend from nearly all vertebrate cells. Abnormal ciliogenesis and cilia length are suggested to be associated with hypertension and obesity as well as diseases such as Meckel-Gruber syndrome. Extracellular matrix (ECM), comprising cellular microenvironment, influences cell shape and proliferation. However, influence of ECM on cilia biogenesis has not been well studied. In this study we examined the effects of type I collagen (col I), the major component of ECM, on primary cilia growth. When cultured on collagen-coated dishes, confluent 3T3-L1 cells were found to exhibit fibroblast-like morphology, which was different from the cobblestone-like shape on non-coated dishes. The level of autophagy in the cells cultured on col I-coated dishes was attenuated compared with the cells cultured on non-coated dishes. The cilia of the cells cultured on col I-coated dishes became longer, accompanying increased expression of essential proteins for cilia assembly. Transfection of the siRNA targeting microtubule-associated protein light chain 3 (LC3) further enhanced the length of primary cilia, suggesting that col I positively regulated cilia growth through inhibition of autophagy. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy in our previous study on primary cilia, was down-regulated with col I. 3T3-L1 cells treated with the siRNA against HDAC6 reduced the autophagy level and enhanced collagen-induced cilia elongation, implying that HDAC6 was involved in mediating autophagy. In conclusion, col I promotes cilia growth through repressing the HDAC-autophagy pathway that can be involved in the interaction between primary cilia and col I. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. SCF promotes dental pulp progenitor migration, neovascularization, and collagen remodeling – potential applications as a homing factor in dental pulp regeneration

    PubMed Central

    Pan, Shuang; Dangaria, Smit; Gopinathan, Gokul; Yan, Xiulin; Lu, Xuanyu; Kolokythas, Antonia; Niu, Yumei; Luan, Xianghong

    2014-01-01

    Stem cell factor (SCF) is a powerful chemokine that binds to the c-Kit receptor CD117 and has shown promise as a homing agent capable of progenitor cell recruitment. In the present study we have documented high levels of both SCF and its receptor c-Kit in differentiating dental pulp (DP) cells and in the sub-odontoblastic layer of Höhl. In vitro studies using human DP progenitors revealed a significant increase in cell proliferation after100nM SCF application, explained by a 2-fold upregulation in cyclin D3 and FGF2 cell cycle regulators, and a 7-fold increase in CDK4 expression. DP cell migration in the presence of SCF was up-regulated 2.7-fold after a 24 hour culture period, and this effect was accompanied by cytoskeletal rearrangement, a 1.5-fold increase in polymeric F-actin over G-actin, and a 1.8-fold increase in RhoA expression. Explaining the signaling effect of SCF on DP migration, PI3K/Akt and MEK/ERK pathway inhibitors were demonstrated to significantly reduce DP cell migration, while SCF alone doubled the number of migrated cells. ERK and AKT phosphorylation were dramatically upregulated already 3-5 minutes after SCF addition to the culture medium and declined thereafter, classifying SCF as a fast acting chemokine. When applied as an agent to promote tissue regeneration in subcutaneously implanted collagen sponges, SCF resulted in a 7-fold increase in the cell number in the implanted tissue construct, a more than 9-fold increase in capillaries, as well as collagen sponge remodeling and collagen fiber neogenesis. Together, these studies demonstrate the suitability of SCF as a potent aid in the regeneration of dental pulp and other mesenchymal tissues, capable of inducing cell homing, angiogenesis, and tissue remodeling. PMID:23703692

  8. Hierarchical assembly of collagen peptide triple helices into curved disks and metal ion-promoted hollow spheres.

    PubMed

    Przybyla, David E; Rubert Pérez, Charles M; Gleaton, Jeremy; Nandwana, Vikas; Chmielewski, Jean

    2013-03-06

    A 27 amino acid collagen-based peptide (Hbyp3) was designed to radially display nine hydrophobic bipyridine moieties from a triple helical scaffold. Self-assembly of such functionalized triple helices led to the formation of micrometer-scaled disks with a curved morphology, presumably mediated by aromatic interactions, with a height that is in the range of the length of the triple helical peptide. Higher order assembly of these curved disks into micrometer-sized hollow spheres was accomplished through metal-ligand interactions between bipyridine groups of the disks and metal ions such as Fe(II), Co(II), Zn(II) and Cu(II). The thickness of the shell of these hollow spheres corresponds well with the thickness of the collagen peptide-based triple helix and the corresponding self-assembled disks. Addition of a metal ion chelator was found to reverse the assembly of the hollow spheres back to the curved disk structures. These data support the formation of the hollow spheres from the self-assembled disks of Hbyp3 upon addition of metal ions.

  9. Characterization of the mouse gene for the {alpha}1 chain of type XVIII collagen (Col18a1) reveals that the three variant N-terminal polypeptide forms are transcribed from two widely separated promoters

    SciTech Connect

    Rehn, M.; Hintikka, E.; Pihlajaniemi, T.

    1996-03-05

    The mouse gene for the {alpha}1 chain of type XVIII collagen (Col18a1) is more than 102 kb and consists of 43 exons. Type XVIII collagen transcripts encode polypeptides that differ with respect to three variant N-terminal noncollagenous domains that are 301 (NC1-301), 517 (NC1-517), or 764 (NC1-764) residues in length. Characterization of genomic clones revealed that the three variant NC1 domains result from the use of two alternative promoters, separated by a distance of 50 kb. The upstream promoter, promoter 1, directs the synthesis of the NC1-301 domain in conjunction with exons 1 and 2, whereas the downstream promoter, promoter 2, directs that of the NC1-517 and NC1-764 domains in conjunction with exon 3, with the latter two variants differing with respect to alternative splicing of the exon 3 sequences. Exons 4-9 encode a portion of the NC1 domain shared by all three polypeptide variants, and exons 9-43 encode the common collagenous and C-terminal noncollagenous sequences. The marked differences previously observed in the expression of variant type XVIII collagen transcripts in mouse tissues thus result from tissue-specific use of these two promoters. 45 refs., 4 figs., 2 tabs.

  10. Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells.

    PubMed

    Bellon, G; Chaqour, B; Antonicelli, F; Wegrowski, J; Claisse, D; Haye, B; Borel, J P

    1994-07-01

    In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the

  11. The One-Kilobase DNA Fragment Upstream of the ardC Actin Gene of Physarum polycephalum Is Both a Replicator and a Promoter

    PubMed Central

    Pierron, Gérard; Pallotta, Dominick; Bénard, Marianne

    1999-01-01

    The 1-kb DNA fragment upstream of the ardC actin gene of Physarum polycephalum promotes the transcription of a reporter gene either in a transient-plasmid assay or as an integrated copy in an ectopic position, defining this region as the transcriptional promoter of the ardC gene (PardC). Since we mapped an origin of replication activated at the onset of S phase within this same fragment, we examined the pattern of replication of a cassette containing the PardC promoter and the hygromycin phosphotransferase gene, hph, integrated into two different chromosomal sites. In both cases, we show by two-dimensional agarose gel electrophoresis that an efficient, early activated origin coincides with the ectopic PardC fragment. One of the integration sites was a normally late-replicating region. The presence of the ectopic origin converted this late-replicating domain into an early-replicating domain in which replication forks propagate with kinetics indistinguishable from those of the native PardC replicon. This is the first demonstration that initiation sites for DNA replication in Physarum correspond to cis-acting replicator sequences. This work also confirms the close proximity of a replication origin and a promoter, with both functions being located within the 1-kb proximal region of the ardC actin gene. A more precise location of the replication origin with respect to the transcriptional promoter must await the development of a functional autonomously replicating sequence assay in Physarum. PMID:10207074

  12. Le Fort I osteotomies using Bio-Oss® collagen to promote bony union: a prospective clinical split-mouth study.

    PubMed

    Rohner, D; Hailemariam, S; Hammer, B

    2013-05-01

    The purpose of this study was to evaluate whether a bone substitute can be used to promote bony union in patients undergoing maxillary advancement after Le Fort l osteotomy. Nine patients were treated bilaterally with Le Fort I osteotomies and maxillary advancements of 5mm or less. In each patient, one gap was grafted with the bone substitute Bio-Oss(®) Collagen (BOC). The contralateral site was left empty and served as control. After 6 months there were still empty gaps in the control sites of three patients, while in the grafted sites all gaps were completely filled with bone. The histomorphometric analysis performed with biopsies from the region of the original gap showed a similar amount of new bone in both groups, however, in the test group the mean overall amount of the mineralized fraction was higher compared to the control group (test site 65.0±6.2%, control site 38.9±32.6%). The bone substitute seemed to be a suitable material to promote bony union in Le Fort I osteotomies. Further studies are needed to analyse whether this technique is efficient in preventing relapse and promoting bony union in larger advancements. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  13. Bioengineered collagens

    PubMed Central

    Ramshaw, John AM; Werkmeister, Jerome A; Dumsday, Geoff J

    2014-01-01

    Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications. PMID:24717980

  14. Overexpression of Bcl(XL) in B cells promotes Th1 response and exacerbates collagen-induced arthritis.

    PubMed

    Zheng, Biao; Marinova, Ekaterina; Switzer, Kirsten; Wansley, Daniel; He, Hongxia; Bheekha-Escura, Roy; Behrens, Timothy W; Han, Shuhua

    2007-11-15

    B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, Bcl(XL), they significantly increased production of IFN-gamma and enhanced Th1 response. Consistently, Bcl-x(L) transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in Bcl(XL) transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of Bcl(XL) in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate Bcl(XL) expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if Bcl(XL) expression can be down-regulated in autoreactive B cells.

  15. A methylcellulose and collagen based temperature responsive hydrogel promotes encapsulated stem cell viability and proliferation in vitro.

    PubMed

    Payne, Christina; Dolan, Eimear B; O'Sullivan, Janice; Cryan, Sally-Ann; Kelly, Helena M

    2017-02-01

    With the number of stem cell-based therapies emerging on the increase, the need for novel and efficient delivery technologies to enable therapies to remain in damaged tissue and exert their therapeutic benefit for extended periods, has become a key requirement for their translation. Hydrogels, and in particular, thermoresponsive hydrogels, have the potential to act as such delivery systems. Thermoresponsive hydrogels, which are polymer solutions that transform into a gel upon a temperature increase, have a number of applications in the biomedical field due to their tendency to maintain a liquid state at room temperature, thereby enabling minimally invasive administration and a subsequent ability to form a robust gel upon heating to physiological temperature. However, various hurdles must be overcome to increase the clinical translation of hydrogels as a stem cell delivery system, with barriers including their low tensile strength and their inadequate support of cell viability and attachment. In order to address these issues, a methylcellulose based hydrogel was formulated in combination with collagen and beta glycerophosphate, and key development issues such as injectability and sterilisation processes were examined. The polymer solution underwent thermogelation at ~36 °C as determined by rheological analysis, and when gelled, was sufficiently robust to resist significant disintegration in the presence of phosphate buffered saline (PBS) while concomitantly allowing for diffusion of methylene blue dye solution into the gel. We demonstrate that human mesenchymal stem cells (hMSCs) encapsulated within the gel remained viable and showed raised levels of dsDNA at increasing time points, an indication of cell proliferation. Mechanical testing showed the "injectability", i.e. force required for delivery of the polymer solution through devices such as a syringe, needle or catheter. Sterilisation of the freeze-dried polymer wafer via gamma irradiation showed no adverse

  16. Poly (3-hydroxyalkanoates)-co-(6-hydroxyhexanoate) hydrogel promotes angiogenesis and collagen deposition during cutaneous wound healing in rats.

    PubMed

    Gumel, Ahmad Mohammed; Razaif-Mazinah, Mohd Rafais Mohd; Anis, Siti Nor Syairah; Annuar, Mohamad Suffian Mohamad

    2015-07-08

    Wound management and healing in several physiological or pathological conditions, particularly when comorbidities are involved, usually proves to be difficult. This presents complications leading to socio-economic and public health burdens. The accelerative wound healing potential of biocompatible poly(3-hydroxyalkanoates)-co-(6-hydroxyhexanoate) (PHA-PCL) composite hydrogel is reported herein. The biosynthesized PHA-PCL macromer was cross-linked with PEGMA to give a hydrogel. Twenty-four rats weighing 200-250 g each were randomly assigned to four groups of six rats. Rats in group I (negative control) were dressed with sterilized gum acacia paste in 10% normal saline while PEGMA-alone hydrogel (PH) was used to dress group II (secondary control) rats. Group III rats were dressed with PHAs-PCL cross-linked PEGMA hydrogel (PPH). For the positive control (group IV), the rats were dressed with Intrasite(®) gel. Biochemical, histomorphometric and immunohistomorphometric analyses revealed a significant difference in area closure and re-epithelialization on days 7 and 14 in PPH or Intrasite(®) gel groups compared to gum acacia or PEGMA-alone groups. Furthermore, wounds dressed with PPH or Intrasite(®) gel showed evident collagen deposition, enhanced fibrosis and extensively organized angiogenesis on day 14 compared to the negative control group. While improvement in wound healing of the PH dressed group could be observed, there was no significant difference between the negative control group and the PH dressed group in any of the tests. The findings suggested that topical application of PPH accelerated the rats' wound healing process by improving angiogenesis attributed to the increased microvessel density (MVD) and expressions of VEGF-A in tissue samples. Thus, PPH has been demonstrated to be effective in the treatment of cutaneous wounds in rats, and could be a potential novel agent in the management and acceleration of wound healing in humans and animals.

  17. Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 Pre-Osteoblasts

    PubMed Central

    Liu, JunLi; Zhang, Bing; Song, ShuJun; Ma, Ming; Si, ShaoYan; Wang, YiHu; Xu, BingXin; Feng, Kai; Wu, JiGong; Guo, YanChuan

    2014-01-01

    Objective Collagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells. Methods Mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0. Results Cell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days. Conclusions Bovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis. PMID:24926875

  18. Collagen triple helix repeat containing 1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation and motility.

    PubMed

    Tameda, Masahiko; Sugimoto, Kazushi; Shiraki, Katsuya; Yamamoto, Norihiko; Okamoto, Ryuji; Usui, Masanobu; Ito, Masaaki; Takei, Yoshiyuki; Nobori, Tsutomu; Kojima, Takahiro; Suzuki, Hideaki; Uchida, Masako; Uchida, Kazuhiko

    2014-08-01

    Although several therapeutic options are available for hepatocellular carcinoma (HCC), the outcome is still very poor. One reason is the complexity of signal transduction in the pathogenesis of HCC. The aim of this study was to identify new HCC-related genes and to investigate the functions of these genes in the pathogenesis and progression of HCC. Whole genomes of 15 surgically resected HCC specimens were examined for copy number alterations with comparative genomic hybridization. Gene expression was compared between HCC and normal liver tissues. The roles of the new genes in the progression of HCC were studied using cultured cell lines. Copy number gain in chromosome 8q was detected in 53% of HCC tissues examined. The gene that coded for collagen triple helix repeat containing 1 (CTHRC1), located at chromosome 8q22.3, was overexpressed in HCC compared with normal or liver cirrhosis tissues and identified as a new HCC-related gene. CTHRC1 deletion with short hairpin RNA significantly reduced proliferation, migration and invasion of HepG2 and Huh7 cells. In addition, mRNA of integrins β-2 and β-3 was downregulated, with deletion of CTHRC1 in these cells. Immunohistochemical staining on resected HCC tissues showing positive staining areas for CTHRC1 was significantly greater in poorly-differentiated HCC compared with well‑differentiated HCC. Moreover, some cases showed strong staining for CTHRC1 in invasive areas of HCC. CTHRC1 has the potential to be a new biomarker for the aggressive HCC, and to be a new therapeutic target in treating HCC.

  19. Enhanced hepatic uptake and bioactivity of type alpha1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol.

    PubMed

    Cheng, Kun; Ye, Zhaoyang; Guntaka, Ramareddy V; Mahato, Ram I

    2006-05-01

    A triplex-forming oligonucleotide (TFO) specific for type alpha1(I) collagen promoter is a promising candidate for treating liver fibrosis. Earlier, we determined the pharmacokinetics and biodistribution of TFO after systemic administration into normal and fibrotic rats. In this study, we conjugated cholesterol to the 3' end of the TFO via a disulfide bond and determined its cellular and nuclear uptake and bioactivity using HSC-T6 cell lines in vitro, followed by biodistribution at whole-body, organ (liver), and subcellular levels. Conjugation with cholesterol had little effect on the triplex-forming ability of the TFO with target duplex DNA, and the cellular uptake of (33)P-TFO-cholesterol (Chol) increased by 2- to approximately 4-fold. Real-time reverse transcriptase-polymerase chain reaction analysis after transfection of HSC-T6 cells with TFO-Chol or TFO indicated that TFO-Chol had higher inhibition on type alpha1(I) collagen primary transcript than naked TFO at low concentration (200 nM) but showed similar inhibition at higher concentration (500 and 1000 nM). There was increase in the inhibition on primary transcript with transfection time. The hepatic uptake of (33)P-TFO-Chol after systemic administration was 72.22% of the dose compared with 45.8% of (33)P-TFO. There was significant increase in the uptake of (33)P-TFO-Chol by hepatic stellate cells and hepatocytes. More importantly, the nuclear uptake of TFO-Chol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.

  20. Epidermal growth factor promotes a mesenchymal over an amoeboid motility of MDA-MB-231 cells embedded within a 3D collagen matrix

    NASA Astrophysics Data System (ADS)

    Geum, Dongil T.; Kim, Beum Jun; Chang, Audrey E.; Hall, Matthew S.; Wu, Mingming

    2016-01-01

    The receptor of epidermal growth factor (EGFR) critically regulates tumor cell invasion and is a potent therapeutic target for treatment of many types of cancers, including carcinomas and glioblastomas. It is known that EGF regulates cell motility when tumor cells are embedded within a 3D biomatrix. However, roles of EGF in modulating tumor cell motility phenotype are largely unknown. In this article, we report that EGF promotes a mesenchymal over an amoeboid motility phenotype using a malignant breast tumor cell line, MDA-MB-231, embedded within a 3D collagen matrix. Amoeboid cells are rounded in shape, while mesenchymal cells are elongated, and their migrations are governed by a distinctly different set of biomolecules. Using single cell tracking analysis, we also show that EGF promotes cell dissemination through a significant increase in cell persistence along with a moderate increase of speed. The increase of persistence is correlated with the increase of the percentage of the mesenchymal cells within the population. Our work reveals a novel role of microenvironmental cue, EGF, in modulating heterogeneity and plasticity of tumor cell motility phenotype. In addition, it suggests a potential visual cue for diagnosing invasive states of breast cancer cells. This work can be easily extended beyond breast cancer cells.

  1. Transplantation of hUC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury

    PubMed Central

    Li, Xing; Tan, Jun; Xiao, Zhifeng; Zhao, Yannan; Han, Sufang; Liu, Dingyang; Yin, Wen; Li, Jing; Li, Juan; Wanggou, Siyi; Chen, Bing; Ren, Caiping; Jiang, Xingjun; Dai, Jianwu

    2017-01-01

    Spinal cord injury (SCI) can lead to locomotor deficits, and the repair of chronic SCI is considered one of the most challenging clinical problems. Although extensive studies have evaluated treatments for acute SCI in small animals, comparatively fewer studies have been conducted on large-animal SCI in the chronic phase, which is more clinically relevant. Here, we used a collagen-based biomaterial, named the NeuroRegen scaffold, loaded with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in a canine chronic SCI model. To generate chronic SCI, the T8 spinal cord segment was removed by complete transection of the spinal cord. Two months later, glial scar tissue was removed and a NeuroRegen scaffold was transplanted into the lesion area. Functionalized NeuroRegen scaffold implantation promoted both locomotor recovery and endogenous neurogenesis in the lesion area. Moreover, some newly generated neurons successfully matured into 5-HT-positive neurons at 1 year post-injury. In addition, many regenerated axon fibers in the lesion area exhibited remyelination and synapse formation at 1 year post-injury in the functionalized NeuroRegen scaffold group. In conclusion, the NeuroRegen scaffold functionalized with hUC-MSCs is a promising potential therapeutic approach to chronic SCI that promotes neuronal regeneration, reduces glial scar formation, and ultimately improves locomotor recovery. PMID:28262732

  2. Activation of hageman factor by collagen

    PubMed Central

    Wilner, G. D.; Nossel, H. L.; LeRoy, E. C.

    1968-01-01

    Purified acid-soluble and insoluble human collagen accelerated the clotting of plateletpoor plasma in silicone-treated tubes. The clot-promoting effect did not appear to be due to thromboplastic activity since the collagen preparations did not activate factor X in the presence of factor VII and calcium. Instead, collagen appeared to accelerate clotting by activating Hageman factor (factor XII) on the basis of the following findings: collagen increased the clot-promoting activity of partially purified Hageman factor but exerted no further effect in the presence of kaolin, a known activator of Hageman factor; clot-promoting eluates were obtained from collagen exposed to normal, hemophilic, or PTC-deficient plasma but not from collagen exposed to Hageman or PTA-deficient plasma. The collagen molecule itself appeared to be required for the clot-promoting activity since digestion with collagenase or thermal denaturation at pH 2.5 (about 35°C) resulted in very marked reduction in clot-promoting activity. Since thermal denaturation is associated with transformation of collagen structure from triple helical to random coil form, it is suggested that the native form of collagen is essential for the ability to activate Hageman factor. Blockage of the free amino groups by treatment with nitrous acid or dinitrofluorobenzene only slightly reduced the clot-promoting activity of collagen. In contrast, since addition of cationic proteins to collagen markedly reduced pro-coagulant activity it is suggested that negatively charged sites on the collagen molecule are critical for Hageman factor activation. This suggestion is supported by the finding that pepsin treatment of collagen, which removes the predominantly negatively charged telopeptides, results in significant decrease in coagulant activity. Esterification of collagen, which neutralizes 80-90% of the free carboxyl groups, reduced coagulant activity by over 90% and it is suggested that the free carboxyl groups of glutamic and

  3. Collagen triple helix repeat containing 1 (CTHRC1) acts via ERK-dependent induction of MMP9 to promote invasion of colorectal cancer cells.

    PubMed

    Kim, Hee Cheol; Kim, Yong Sung; Oh, Hyun-Woo; Kim, Kwoneel; Oh, Sang-Seok; Kim, Jong-Tae; Kim, Bo Yeon; Lee, Seon-Jin; Choe, Yong-Kyung; Kim, Dong Hyeok; Kim, Seok-Hyung; Chae, Seoung Wan; Kim, Kwang Dong; Lee, Hee Gu

    2014-01-30

    Collagen triple helix repeat-containing 1 (CTHRC1) is known to be aberrantly upregulated in most human solid tumors, although the functional roles of CTHRC1 in colorectal cancer remain unclear. In this study, we investigated the occurrence of CTHRC1 upregulation and its role in vivo and in vitro. The expression profile and clinical importance of CTHRC1 were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses in normal and tumor patient samples. CTHRC1 was detectable in normal tissues, but also was highly expressed in tumor specimens. CTHRC1 upregulation was significantly associated with demethylation of the CTHRC1 promoter in colon cancer cell lines and tumor tissues. Clinicopathologic analyses showed that nodal status and expression of CTHRC1 (95% CI 0.999-3.984, p=0.05) were significant prognostic factors for disease-free survival. Promoter CpG methylation and hypermethylation status were measured by bisulfite sequencing and pyrosequencing analysis. Furthermore, we showed that overexpression of CTHRC1 in the SW480 and HT-29 cell lines increased invasiveness, an effect mediated by extracellular signal-regulated kinase (ERK)-dependent upregulation of matrix metalloproteinase 9 (MMP9). Consistent with this, we found that knockdown of CTHRC1 attenuated ERK activation and cancer cell invasivity. These results demonstrate that CTHRC1 expression is elevated in human colon cancer cell lines and clinical specimens, and promotes cancer cell invasivity through ERK-dependent induction of MMP9 expression. Our results further suggest that high levels of CTHRC1 expression are associated with poor clinical outcomes.

  4. New functional roles for non-collagenous domains of basement membrane collagens.

    PubMed

    Ortega, Nathalie; Werb, Zena

    2002-11-15

    Collagens IV, XV and XVIII are major components of various basement membranes. In addition to the collagen-specific triple helix, these collagens are characterized by the presence of several non-collagenous domains. It is clear now that these ubiquitous collagen molecules are involved in more subtle and sophisticated functions than just the molecular architecture of basement membranes, particularly in the context of extracellular matrix degradation. Degradation of the basement membrane collagens occurs during numerous physiological and pathological processes such as embryonic development or tumorigenesis and generates collagen fragments. These fragments are involved in the regulation of functions differing from those of their original intact molecules. The non-collagenous C-terminal fragment NC1 of collagen IV, XV and XVIII have been recently highlighted in the literature because of their potential in reducing angiogenesis and tumorigenesis, but it is clear that their biological functions are not limited to these processes. Proteolytic release of soluble NC1 fragments stimulates migration, proliferation, apoptosis or survival of different cell types and suppresses various morphogenetic events.

  5. The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific

    PubMed Central

    Dafnis, Ioannis; Argyri, Letta; Sagnou, Marina; Tzinia, Athina; Tsilibary, Effie C.; Stratikos, Efstratios; Chroni, Angeliki

    2016-01-01

    The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer’s disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients’ brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aβ42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment. PMID:27476701

  6. Surface study of collagen/poloxamine hydrogels by a 'deep freezing' ToF-SIMS approach.

    PubMed

    Sosnik, Alejandro; Sodhi, Rana N S; Brodersen, Peter M; Sefton, Michael V

    2006-04-01

    In order to determine the presence of collagen molecules at the surface of a collagen-modified poloxamine hydrogel (a semi-interpenetrating network), the surface composition was studied using Time-of-Flight Secondary Ion Mass Spectra (ToF-SIMS). Collagen was added to the poloxamine hydrogel (poloxamine is a commercially available four-arm poly(ethylene oxide)/poly(propylene oxide) block copolymer, PEO/PPO) to promote the attachment of endothelial or liver cells. X-ray photoelectron spectroscopy (XPS) of dry samples showed a sharp increase in the N content from 0.6% in a pure poloxamine hydrogel to 8.8% in the collagen-containing material. Afterwards, the surface was studied by a 'deep freezing' ToF-SIMS approach under progressive heating from -120 to -60 degrees C. The positive spectrum of collagen/poloxamine at -65 degrees C displayed distinct signals corresponding to different amino acid fragments such as CH4N+ (30 m/z, Gly), C3HN2+ (43 m/z, Arg), C2H6N+ (44 m/z, Ala) and C4H5N2+(81m/z, His) and others corresponding to the PEO and PPO blocks of poloxamine. In addition, the negative spectrum showed peaks at 26 m/z (CN-), 32 m/z (S-) and 42 m/z (CNO-) characteristic of fragments of the collagen molecule. Imaging experiments indicated the homogeneous distribution of the collagen on the surface. These results supported the use of ToF-SIMS for the surface characterization of hydrated hydrogels and confirmed the collagen presence as the means whereby cells attach to the modified poloxamine matrix.

  7. TGF-β2 promotes RPE cell invasion into a collagen gel by mediating urokinase-type plasminogen activator (uPA) expression.

    PubMed

    Sugioka, Koji; Kodama, Aya; Okada, Kiyotaka; Iwata, Mihoko; Yoshida, Koji; Kusaka, Shunji; Matsumoto, Chota; Kaji, Hiroshi; Shimomura, Yoshikazu

    2013-10-01

    Transforming growth factor-beta (TGF-β) is one of the main epithelial-mesenchymal transition (EMT)-inducing factors. In general, TGF-β-induced EMT promotes cell migration and invasion. TGF-β also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPAR's interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-β and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-β2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-β2 and/or the inhibitor of uPA (u-PA-STOP(®)) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-β2 or u-PA-STOP(®) suppressed cell proliferation. Combination treatment of TGF-β2 and u-PA-STOP(®) enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-β2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-β inhibitor SB431542 suppressed TGF-β2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-β2 alone or with TGF-β2 and u-PA-STOP(®). TGF-β2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-β2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with

  8. Type II collagen fragment HELIX-II is a marker for early cartilage lesions but does not predict the progression of cartilage destruction in human knee joint synovial fluid.

    PubMed

    Wei, Xiaochun; Yin, Kun; Li, Pengcui; Wang, Huan; Ding, Juan; Duan, Wangping; Wei, Lei

    2013-07-01

    To determine whether there is a direct correlation between the concentration of type II collagen fragment HELIX-II in synovial fluid and the severity of cartilage damage at the knee joint, 83 patients who had undergone knee arthroscopy or total knee replacement were enrolled in this study (49% women, mean ± SD age 49.5 ± 19). The content of HELIX-II in the synovial fluid samples was measured by enzyme-linked immunosorbent assay (ELISA). Cartilage damage at the knee joint was classified during arthroscopy or direct surgical observation, using the Outerbridge cartilage damage scoring system. The maximum damage score was defined as the highest score among the six areas of the knee joint, and the cumulative score was defined as the sum of the scores of the six areas of the knee joint. The intra-assay and inter-assay variations of the HELIX-II ELISA were lower than 13 and 15%, respectively. The level of HELIX-II in the severely damaged cartilage groups (cumulative scores = 11-24 or maximum score = 2-4) was much higher than in the slightly damaged cartilage groups (cumulative scores = 0-10 or maximum score = 0-1). The level of HELIX-II in cartilage from severely damaged cartilage groups was significantly higher than in the slightly damaged groups, but no significant difference was detected in the level of HELIX-II among the severely damaged cartilage sub-groups. There was a significant correlation between the HELIX-II concentration in the synovial fluid and the cumulative (r = 0.807) and maximum scores (r = 0.794). Thus, elevated HELIX-II level is correlated with early cartilage lesions, but does not have the sensitivity to predict the progression of severity of cartilage damage in the knee joint.

  9. Pulsed electromagnetic field (PEMF) promotes collagen fibre deposition associated with increased myofibroblast population in the early healing phase of diabetic wound.

    PubMed

    Choi, Ming-Chun; Cheung, Kwok-Kuen; Li, Xiaohui; Cheing, Gladys Lai-Ying

    2016-01-01

    The present study evaluated the effects of PEMF on collagen fibre deposition, collagen fibril alignment and collagen fibre orientation. The potential relationships between collagen fibre deposition and myofibroblast population in diabetic wound healing were also examined. Forty young male streptozotocin-induced diabetic Sprague-Dawley rats were randomly assigned to PEMF group or control group. 2 cm × 2 cm square wounds were made at their back. The PEMF group received daily exposure of PEMF to the wounds, while control group was handled in the same manner except that the PEMF device was not activated. Wound tissues harvested on post-wounding day 7, 10 and 14 were fixed, processed and sectioned. The abundance, fibril alignment and fibre orientation of type I collagen were quantified with picro-sirius polarization method and image analysis software (Nikon NIS Element AR). Myofibroblast population data were adopted from our previous study. Correlation between myofibroblast population and collagen fibre deposition was examined. There was significantly greater abundance of type I collagen fibre in the PEMF group than in the control on day 7 (P = .013), but not on day 10 or 14. No significant between-group differences were found in collagen fibril alignment and collagen fibre orientation at any measured time points. Positive correlation was found between collagen fibre deposition and myofibroblast population only on day 7 (r = .729, P = .007). In conclusion, PEMF can significantly increase collagen fibre in the early phase of diabetic wound healing, which is associated with the enhancement of myofibroblast population.

  10. Human adipose-derived mesenchymal stem cells seeded into a collagen-hydroxyapatite scaffold promote bone augmentation after implantation in the mouse.

    PubMed

    Calabrese, Giovanna; Giuffrida, Raffaella; Forte, Stefano; Fabbi, Claudia; Figallo, Elisa; Salvatorelli, Lucia; Memeo, Lorenzo; Parenti, Rosalba; Gulisano, Massimo; Gulino, Rosario

    2017-08-02

    Traumatic injury or surgical excision of diseased bone tissue usually require the reconstruction of large bone defects unable to heal spontaneously, especially in older individuals. This is a big challenge requiring the development of biomaterials mimicking the bone structure and capable of inducing the right commitment of cells seeded within the scaffold. In particular, given their properties and large availability, the human adipose-derived stem cells are considered as the better candidate for autologous cell transplantation. In order to evaluate the regenerative potential of these cells along with an osteoinductive biomaterial, we have used collagen/hydroxyapatite scaffolds to test ectopic bone formation after subcutaneous implantation in mice. The process was analysed both in vivo, by Fluorescent Molecular Tomography (FMT), and ex vivo, to evaluate the formation of bone and vascular structures. The results have shown that the biomaterial could itself be able of promoting differentiation of host cells and bone formation, probably by means of its intrinsic chemical and structural properties, namely the microenvironment. However, when charged with human mesenchymal stem cells, the ectopic bone formation within the scaffold was increased. We believe that these results represent an important advancement in the field of bone physiology, as well as in regenerative medicine.

  11. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro.

    PubMed

    Martens, Wendy; Sanen, Kathleen; Georgiou, Melanie; Struys, Tom; Bronckaers, Annelies; Ameloot, Marcel; Phillips, James; Lambrichts, Ivo

    2014-04-01

    In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury.

  12. A 1-kb bacteriophage lambda fragment functions as an insulator to effectively block enhancer-promoter interactions in Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    The 35S cauliflower mosaic virus (CaMV) promoter contains an enhancer element that is able to override the tissue-, organ- and developmental-stage specificity of nearby promoters. Consequently, the precise control of transgene expression in transgenic plants, which often contain the 35S CaMV promot...

  13. Anti-IgD antibody attenuates collagen-induced arthritis by selectively depleting mature B-cells and promoting immune tolerance.

    PubMed

    Nguyen, Tue G; Little, Christopher B; Yenson, Vanessa M; Jackson, Christopher J; McCracken, Sharon A; Warning, Julia; Stevens, Veronica; Gallery, Eileen G; Morris, Jonathan M

    2010-08-01

    Membrane (m)IgD forms a major part of B-cell receptor complexes. Its wider role in the immune system has been enigmatic. Stimulation of mIgD with an antibody (anti-IgD) can activate B-cells and elicit a broad immune response in vivo. Given the role of B-cells in autoimmune diseases and the profound impact of anti-IgD on B-cells, the potential effects of anti-IgD on autoimmune conditions are intriguing and yet to be explored. Here we report a novel therapeutic effect of anti-IgD in the collagen-induced arthritis (CIA) mouse model. Administration of anti-IgD at the onset of early clinical symptoms as a therapeutic intervention, but not as a prophylactic treatment, significantly ameliorates disease severity and joint pathology. Anti-IgD treatment selectively depletes mature B cells while it spares regulatory B-cell subsets. This results in a significant reduction of autoantibody levels but does not affect antibody responses to a T-cell-dependent antigen. Therapeutic treatment with anti-IgD increases the numbers of regulatory B-cells and regulatory T-cells whilst it augments adaptive Th1/Th2 responses in vivo. In human PBMC samples, anti-IgD also promotes adaptive Th1/Th2 responses and modulates the innate responses toward an anti-inflammatory Th2-biased response. Collectively, anti-IgD treatment may offer a selective approach to B-cell depletion that also promotes immune tolerance and anti-inflammatory tendencies without compromising the general adaptive B-cell and T-cell responses. The multiple mechanisms of action by anti-IgD treatment suggest a wider clinical application for a number of chronic inflammatory and autoimmune conditions.

  14. Highly purified collagen coating enhances tissue adherence and integration properties of monofilament polypropylene meshes.

    PubMed

    Siniscalchi, Rodrigo Teixeira; Melo, Marli; Palma, Paulo César Rodrigues; Dal Fabbro, Inácio Maria; Vidal, Benedicto de Campos; Riccetto, Cassio Luiz Zanettini

    2013-10-01

    Complications related to tissue integration of polypropylene implants used in the treatment of pelvic organ prolapse are relatively prevalent. Collagen, a biocompatible, less immunogenic material with modulating properties on the inflammatory process, may improve polypropylene integration. The objective was to study biomechanical and histological effects of monofilament polypropylene mesh coated with purified collagen gel. Forty rats were implanted with two fragments of polypropylene mesh in their abdominal walls (one on each side of the linea alba). One of the fragments had a collagen gel coating (group I) while the other one did not (group II). The animals were euthanized at 7, 14, 90, and 180 days after implantation and their abdominal walls were excised for analysis. The biomechanical study showed that mesh adherence to neighboring tissue increased significantly in group II (p < 0.05). Acute (p < 0.001) and chronic (p = 0.004) inflammatory responses as well as granulation tissue formation (p = 0.001) were less intense in group II at 7 and 14 days. Granulomatous inflammation and foreign body reaction was less significant at 7 days in group II (p = 0.029 and p < 0.001). The birefringence analysis showed higher mean brightness density in the late phase of implantation in group II meshes (p = 0.000). Polypropylene mesh coated with purified collagen gel increases adherence to tissue, promotes a less intense and lasting inflammatory response and triggers a greater organization and packing arrangement of collagen fibers in the late phase of implantation.

  15. Collagen I confers gamma radiation resistance.

    PubMed

    Azorin, E; González-Martínez, P R; Azorin, J

    2012-12-01

    The effect of collagen on the response of somatomammotroph tumor cells (GH3) to gamma, radiation therapy was studied in vitro. After incubating confluent GH3 cell monolayers in a serum-free, maintaining medium, either with or without collagen, the monolayers were irradiated with 137Cs, gamma radiation. Collagen reduces cell mortality via ERK1/2 activation, abolishing gamma radiation, cell death, and promotes cell invasion when acting in synergy with collagen and in association with the, MAPK/ERK1/2 signaling pathway activation. The presence of collagen in somatomammotroph tumors, confers resistance to radiation.

  16. Collagen for bone tissue regeneration.

    PubMed

    Ferreira, Ana Marina; Gentile, Piergiorgio; Chiono, Valeria; Ciardelli, Gianluca

    2012-09-01

    In the last decades, increased knowledge about the organization, structure and properties of collagen (particularly concerning interactions between cells and collagen-based materials) has inspired scientists and engineers to design innovative collagen-based biomaterials and to develop novel tissue-engineering products. The design of resorbable collagen-based medical implants requires understanding the tissue/organ anatomy and biological function as well as the role of collagen's physicochemical properties and structure in tissue/organ regeneration. Bone is a complex tissue that plays a critical role in diverse metabolic processes mediated by calcium delivery as well as in hematopoiesis whilst maintaining skeleton strength. A wide variety of collagen-based scaffolds have been proposed for different tissue engineering applications. These scaffolds are designed to promote a biological response, such as cell interaction, and to work as artificial biomimetic extracellular matrices that guide tissue regeneration. This paper critically reviews the current understanding of the complex hierarchical structure and properties of native collagen molecules, and describes the scientific challenge of manufacturing collagen-based materials with suitable properties and shapes for specific biomedical applications, with special emphasis on bone tissue engineering. The analysis of the state of the art in the field reveals the presence of innovative techniques for scaffold and material manufacturing that are currently opening the way to the preparation of biomimetic substrates that modulate cell interaction for improved substitution, restoration, retention or enhancement of bone tissue function.

  17. Collagenous gastritis.

    PubMed

    Jin, Xiaoyi; Koike, Tomoyuki; Chiba, Takashi; Kondo, Yutaka; Ara, Nobuyuki; Uno, Kaname; Asano, Naoki; Iijima, Katsunori; Imatani, Akira; Watanabe, Mika; Shirane, Akio; Shimosegawa, Tooru

    2013-09-01

    In the present paper, we report a case of rare collagenous gastritis. The patient was a 25-year-old man who had experienced nausea, abdominal distention and epigastralgia since 2005. Esophagogastroduodenoscopy (EGD) carried out at initial examination by the patient's local doctor revealed an extensively discolored depression from the upper gastric body to the lower gastric body, mainly including the greater curvature, accompanied by residual mucosa with multiple islands and nodularity with a cobblestone appearance. Initial biopsies sampled from the nodules and accompanying atrophic mucosa were diagnosed as chronic gastritis. In August, 2011, the patient was referred to Tohoku University Hospital for observation and treatment. EGD at our hospital showed the same findings as those by the patient's local doctor. Pathological findings included a membranous collagen band in the superficial layer area of the gastric mucosa, which led to a diagnosis of collagenous gastritis. Collagenous gastritis is an extremely rare disease, but it is important to recognize its characteristic endoscopic findings to make a diagnosis.

  18. Collagenous colitis.

    PubMed Central

    Kingham, J G; Levison, D A; Morson, B C; Dawson, A M

    1986-01-01

    Clinical and pathological aspects of six patients with collagenous colitis are presented. These patients have been observed for between four and 15 years and the evolution of the condition is documented in three (cases 1, 3 and 5). Management and possible pathogenetic mechanisms of this enigmatic condition are discussed. Images Fig. 1 Fig. 2 PMID:3699567

  19. Pyropia yezoensis peptide promotes collagen synthesis by activating the TGF-β/Smad signaling pathway in the human dermal fibroblast cell line Hs27

    PubMed Central

    Kim, Cho-Rong; Kim, Young-Min; Lee, Min-Kyeong; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2017-01-01

    Pyropia yezoensis (P. yezoensis) is a marine algae that exhibits antioxidant, anti-inflammatory, antitumor and anti-aging activities. In this study, we investigated the effects of the P. yezoensis peptide, PYP1-5, on collagen synthesis in the human dermal fibroblast cell line Hs27. Skin aging is related to reduced collagen production and the activities of multiple enzymes, including matrix metalloproteinases (MMPs), which degrade collagen structure in the dermis, and tissue inhibitor of tissue inhibitor of metalloproteinases (TIMPs), which inhibit the action of MMPs. While collagen synthesis is associated with a number of signaling pathways, we examined the increased collagen synthesis via the upregulation of the transforming growth factor-β (TGF-β)/Smad signaling pathway. Using MTS assay, we found that PYP1-5 did not affect cell viability. Moreover, we confirmed that PYP1-5 increased type 1 collagen expression using enzyme-linked immunosorbent assay (ELISA), western blot analysis and quantitative PCR. In addition, we identified changes in various enzymes, as well as the mechanisms behind the PYP1-5-induced collagen synthesis. PYP1-5 decreased the MMP-1 protein and mRNA levels, and increased the TIMP-1 and TIMP-2 protein and mRNA levels. In addition, PYP1-5 activated the TGF-β/Smad signaling pathway, which increased TGF-β1, p-Smad2 and p-Smad3 expression, while inhibiting Smad7, an inhibitor of the TGF-β/Smad pathway. Furthermore, PYP1-5 upregulated transcription factor specificity protein 1 (Sp1) expression, which is reportedly involved in type 1 collagen expression. These findings indicate that PYP1-5 activates the TGF-β/Smad signaling pathway, which subsequently induces collagen synthesis in Hs27 cells. PMID:27878236

  20. UV-Induced Triggering of a Biomechanical Initiation Switch Within Collagen Promotes Development of a Melanoma-Permissive Microenvironment in the Skin

    DTIC Science & Technology

    2011-09-01

    structure of ECM proteins may represent a mechanism to initiate triggering of a biomechanical switch that allows multiple cell types within the tissue...observed while little change in adhesion was observed to UVB irradiated collage type-I. These studies suggest that distinct cell types exhibit different...adhesive responses to UVA and UVB irradiated collagen type- I and -IV in vitro. Biomechanical switch -- Collagen structure -- Conformational change

  1. The intrinsically kinase-inactive EPHB6 receptor predisposes cancer cells to DR5-induced apoptosis by promoting mitochondrial fragmentation

    PubMed Central

    El Zawily, Amr M.; Toosi, Behzad M.; Freywald, Tanya; Indukuri, Vijaya V.; Vizeacoumar, Franco J.; Leary, Scot C.; Freywald, Andrew

    2016-01-01

    Death Receptor 5 (DR5) is a promising target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells. However, the therapeutic usefulness of DR5 agonists is currently limited by the frequent resistance of malignant tumours to its activation. The identification of molecular mechanisms that determine outcomes of DR5 action is therefore crucial for improving the efficiency of DR5-activating reagents in cancer treatment. Here, we provide evidence that an intrinsically kinase-inactive member of the Eph group of receptor tyrosine kinases, EPHB6, induces marked fragmentation of the mitochondrial network in breast cancer cells of triple-negative origin, lacking expression of the estrogen, progesterone and HER2 receptors. Remarkably, this response renders cancer cells more susceptible to DR5-mediated apoptosis. EPHB6 action in mitochondrial fragmentation proved to depend on its ability to activate the ERK-DRP1 pathway, which increases the frequency of organelle fission. Moreover, DRP1 activity is also essential to the EPHB6-mediated pro-apoptotic response that we observe in the context of DR5 activation. These findings provide the first description of a member of the receptor tyrosine kinase family capable of producing a pro-apoptotic effect through the activation of ERK-DRP1 signaling and subsequent mitochondrial fragmentation. Our observations are of potential practical importance, as they imply that DR5-activating therapeutic approaches should be applied in a more personalized manner to primarily treat EPHB6-expressing tumours. Finally, our findings also suggest that the EPHB6 receptor itself may represent a promising target for cancer therapy, since EPHB6 and DR5 co-activation should support more efficient elimination of cancer cells. PMID:27788485

  2. MET receptor variant R970C favors calpain-dependent generation of a fragment promoting epithelial cell scattering.

    PubMed

    Montagne, Rémi; Baranzelli, Anne; Muharram, Ghaffar; Catherine, Leroy; Lesaffre, Marie; Vinchent, Audrey; Kherrouche, Zoulika; Werkmeister, Elisabeth; Cortot, Alexis B; Tulasne, David

    2017-01-04

    The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

  3. [Will health promotion remain a utopia in a fragmented political system? The case of the Wallonia-Brussels Federation].

    PubMed

    Bantuelle, Martine

    2013-01-01

    In the French Community of Belgium (the Wallonia-Brussels Federation), the changing political landscape and the various laws relating to the roles of the federal state, communities and regions introduced since 1980 have had a significant impact on health policy. Since then, there have been significant developments in health education services and activities. In 1997, a government decree was issued to promote the concept of health promotion, to reform the existing system and to define policy priorities as part of a new five-year plan (1998-2003). Significant progress was made during this period as a result of the development of a global approach extending beyond the mere analysis of risk factors. The second five-year plan (2004-2008), aimed at combining preventive medicine and health promotion, resulted in the involvement of a wider range of actors and greater cross-sector collaboration. However, the sheer number of decision-making levels has been a major obstacle to popular participation and consultation. If the question of social and cultural accessibility is not seriously addressed, the focus on preventive medicine programs may prove to be detrimental to the development of an effective health promotion framework. The disconnect between the political vision and the reality of practice has had an adverse impact on health promotion. Health promotion professionals have repeatedly called for a third five-year plan involving all ministers and aimed at developing a cross-sector approach, at addressing the determinants of health, at promoting the active participation of local communities and at reducing social health inequalities. The concerns of health promotion practitioners were further exacerbated by the introduction of an external assessment process initiated by the Ministry of Health in 2010. The current concerns over the future of the Belgian state, the economic crisis and the impact of spending cuts have increased the sense of uncertainty. The upcoming elections

  4. Single chain fragment variable antibodies developed by using as target the 3rd fibronectin type III homologous repeat fragment of human neural cell adhesion molecule L1 promote cell migration and neuritogenesis.

    PubMed

    Tang, Dan-Yang; Yu, Yang; Zhao, Xuan-Jun; Schachner, Melitta; Zhao, Wei-Jiang

    2015-01-15

    L1CAM plays important roles during ontogeny, including promotion of neuronal cell migration and neuritogenesis, and stimulation of axonal outgrowth, fasciculation and myelination. These functions are at least partially exerted through a 16-mer amino acid sequence in the third fibronectin type III-like repeat of L1, which associates with several interaction partners, including integrins, other adhesion molecules and growth factor receptors. Here, using the Tomlinson I library for phage display, we obtained two single-chain variable fragment antibodies (scFvs) against this peptide sequence of human L1, hereafter called H3 peptide. Both scFvs recognize the H3 peptide and the extracellular domain of L1, as tested by enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunofluorescence staining of L1 expresssing cells. Furthermore, both scFvs reduce U-87 MG cell adhesion to fibronectin, while stimulating cell migration. Application of scFvs to human neuroblastoma SK-N-SH cells promote process outgrowth. Similar to triggering of endogenous L1 functions at the cell surface, both scFvs activate the signal transducers Erk and Src in these cells. Our results indicate that scFvs against a functionally pivotal domain in L1 trigger its regeneration-beneficial functions in vitro, encouraging thoughts on therapy of neurodegenerative diseases in the hope to ameliorate human nervous system diseases.

  5. Clinical uses of collagen shields.

    PubMed

    Poland, D E; Kaufman, H E

    1988-09-01

    Collagen shields immersed in tobramycin solution for one minute were applied to one eye each of 60 patients who had had cataract extraction, penetrating keratoplasty, or epikeratophakia or who had nonsurgical epithelial healing problems. The shields were well tolerated; one patient had the shield removed and one patient lost the shield in the early postoperative period. The surgical patients showed more rapid healing of epithelial defects after surgery with the use of the collagen shield. Patients with acute nonsurgical epithelial problems, such as contact lens abrasions and recurrent erosion, responded to the use of the collagen shield with improved healing. Patients with chronic epithelial defects responded poorly, presumably because underlying abnormalities in Bowman's layer prevented epithelial growth in the area of the defect. No infections were noted in any of the patients. The collagen shields appear to promote enhanced healing in patients with postsurgical and acute epithelial defects and to provide adequate antibiotic prophylaxis against infection in these vulnerable eyes.

  6. Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder.

    PubMed

    Miyake, Makito; Hori, Shunta; Morizawa, Yosuke; Tatsumi, Yoshihiro; Toritsuka, Michihiro; Ohnishi, Sayuri; Shimada, Keiji; Furuya, Hideki; Khadka, Vedbar S; Deng, Youping; Ohnishi, Kenta; Iida, Kota; Gotoh, Daisuke; Nakai, Yasushi; Inoue, Takeshi; Anai, Satoshi; Torimoto, Kazumasa; Aoki, Katsuya; Tanaka, Nobumichi; Konishi, Noboru; Fujimoto, Kiyohide

    2017-05-30

    Current knowledge of the molecular mechanism driving tumor budding is limited. Here, we focused on elucidating the detailed mechanism underlying tumor budding in urothelial cancer of the bladder. Invasive urothelial cancer was pathologically classified into three groups as follows: nodular, trabecular, and infiltrative (tumor budding). Pathohistological analysis of the orthotopic tumor model revealed that human urothelial cancer cell lines MGH-U3, UM-UC-14, and UM-UC-3 displayed typical nodular, trabecular, and infiltrative patterns, respectively. Based on the results of comprehensive gene expression analysis using microarray (25 K Human Oligo chip), we identified two collagens, COL4A1 and COL13A1, which may contribute to the formation of the infiltrative pattern. Visualization of protein interaction networks revealed that proteins associated with connective tissue disorders, epithelial-mesenchymal transition, growth hormone, and estrogen were pivotal factors in tumor cells. To evaluate the invasion pattern of tumor cells in vitro, 3-D collective cell invasion assay using Matrigel was performed. Invadopodial formation was evaluated using Gelatin Invadopodia Assay. Knockdown of collagens with siRNA led to dramatic changes in invasion patterns and a decrease in invasion capability through decreased invadopodia. The in vivo orthotopic experimental model of bladder tumors showed that intravesical treatment with siRNA targeting COL4A1 and COL13A1 inhibited the formation of the infiltrative pattern. COL4A1 and COL13A1 production by cancer cells plays a pivotal role in tumor invasion through the induction of tumor budding. Blocking of these collagens may be an attractive therapeutic approach for treatment of human urothelial cancer of the bladder.

  7. The human {alpha}2(XI) collagen gene (COL11A2): Completion of coding information, identification of the promoter sequence, and precise localization within the major histocompatibility complex reveal overlap with the KE5 gene

    SciTech Connect

    Lui, V.C.H.; Ng, Ling Jim; Sat, E.W.Y.; Cheah, K.S.E.

    1996-03-05

    Type XI collagen, a fibril-forming collagen, is important for the integrity and development of the skeleton because mutations in the genes encoding its consituent {alpha} chains have been found in some osteochondrodysplasias. We provide data that complete information for the coding sequence of human {alpha}2(XI) procollagen, with details of the promoter region and intron-exon organization at the 5{prime} and 3{prime} ends of the gene (COL11A2), including the transcription start and polyadenylation sites. COL11A2 is 30.5 kb with a minimum of 62 exons, differing from other reported fibrillar collagen genes because the amino propeptide is encoded by 14 not 5 to 8 exons. But exon numbers for the carboxy propeptide and 3{prime}-untranslated region are conserved. The promoter region of COL11A2 lacks a TATA box but is GC-rich with two potential SP1 binding sites. Mouse {alpha}2(XI) collagen mRNAs undergo complex alternative splicing involving three amino-terminal propeptide exons but only one of these has been reported for COL11A2. We have located these missing human exons and have identified splice signals that point to additional splice variants. We have precisely mapped COL11A2 within the major histocompatibility complex on chromosome 6. The retinoid X receptor {beta} (RXR{beta}) gene is located 1.1 kb upstream of COL11A2. KE5, previously thought to be a distinct transcribed gene sequence, was mapped within COL11A2 in the alternatively spliced region, raising the question whether KE5 and COL11A2 are separate genes. 37 refs., 7 figs., 1 tab.

  8. [Promoting of angiogenesis and osteogenesis in radial critical bone defect regions of rabbits with nano-hydroxyapatite/collagen/PLA scaffolds plus endothelial progenitor cells].

    PubMed

    Wu, Dong-jin; Hao, Ai-hua; Zhang, Cheng; Cui, Fu-zhai; Wang, Xiu-wen; Gao, Chun-zheng; Zhou, Cheng-jun; Liu, Jun-li; Qiao, Yong; Ma, Sheng-zhong

    2012-06-19

    To explore the roles of nano-hydroxyapatite/collagen/PLA (nHAC/PLA) plus endothelial progenitor cells (EPCs) in repairing segmental bone defects of rabbit radius and enhancing angiogenesis and new bone formation. EPCs isolated from New Zealand white rabbit bone marrow were cultured, identified and seeded into nHAC/PLA scaffolds. And the growth of EPCs in scaffolds was observed under scanning electron microscopy (SEM). Thirty-six were randomly divided into 3 groups to establish segmental bone defect models in radii. Two groups were implanted with EPCs/scaffolds constructs (group A, n = 16) and scaffolds alone (group B, n = 16) respectively. The remaining four rabbits were used as negative control (group C) and nothing was implanted. Animals were sacrificed at different timepoints and radii harvested to undergo radiological examination, histological examination and microvessle density test. These cells isolated from bone marrow were confirmed as EPCs. SEM showed that EPCs attached to the nHAC/PLA scaffolds, grew and proliferated well. Animal experiments revealed that radiological scores (5w: 2.25 ± 0.50 vs 1.00 ± 0.00; 10w: 2.75 ± 0.50 vs 1.75 ± 0.50; 15w: 4.25 ± 0.50 vs 3.0 ± 0.0; each P < 0.05), percentage of new bone formation area in bone defect regions (5w: 29.0% ± 3.5% vs 8.1% ± 0.8%; 10w: 63.4% ± 5.5% vs 16.6% ± 1.3%; 15w: 96.0% ± 4.3% vs 34.0% ± 6.6%; each P < 0.05) and microvessel density (2w: 13.5 ± 0.9 vs 4.3 ± 1.0; 5w:9.8 ± 0.7 vs 4.8 ± 0.3; 10w: 7.0 ± 0.4 vs 4.5 ± 0.4; each P < 0.05) in group A were significantly higher than those in group B. No new bone formation occurred in group C. The composite structure of EPCs-nHAC/PLA can enhance angiogenesis and new bone formation in segmental bone defects in rabbit radii. It may become a potential candidate of promoting revascularization of tissue engineering bone and repairing large bone defects.

  9. The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

    PubMed

    Clarke, Cassie J; Berg, Tracy J; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Kostaras, Eleftherios; Jones, J Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R; Norman, Jim C

    2016-03-21

    Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Teasing out the truth about collagen.

    PubMed

    Rennie, M J

    1999-11-15

    adaptation, rather quickly. Think about the stiffness and discomfort that accompanies un-accustomed exercise, which not only abates with time but ceases to occur once the exercise has become customary. What is happening to collagen protein turnover in these circumstances? One obvious way to study protein turnover, even of collagen, is to follow the incorporation of stable isotope markers such as proline into the tissue (although the breakdown is harder to quantify), but this is technically difficult and requires biopsy of the tissue in question. Another way is to follow the appearance in biological fluids of markers of collagen turnover. Since the propeptides which make collagen soluble are cleaved as collagen is deposited extracellularly, their concentration is an index of the rate of collagen synthesis; similarly when tropocollagen is degraded by extracellular proteases, specific N- and C-terminal fragments are released, the amount of which scales with the rate of collagen breakdown. These bits of collagen find their way into the blood. However, assaying them there introduces non-specificity and dilution, rendering interpretation difficult. The ideal would be to measure them in the extracellular fluid at the site of production. This of course is not easy in vivo. One of the delights of the paper by Langberg and colleagues in this issue of The Journal of Physiology (Langberg et al. 1999) is the sheer cheek with which the authors decided to use the microdialysis technique to do this. Microdialysis is a technique whereby a slowly perfused, thin-walled membranous tube is introduced into the extracellular space and the collected fluid assayed for molecules which have diffused into it. Until now the idea of using microdialysis to measure concentrations of molecules much bigger than 300 Da would be regarded as ludicrous. (ABSTRACT TRUNCATED)

  11. Structural insight for chain selection and stagger control in collagen

    PubMed Central

    Boudko, Sergei P.; Bächinger, Hans Peter

    2016-01-01

    Collagen plays a fundamental role in all known metazoans. In collagens three polypeptides form a unique triple-helical structure with a one-residue stagger to fit every third glycine residue in the inner core without disturbing the poly-proline type II helical conformation of each chain. There are homo- and hetero-trimeric types of collagen consisting of one, two or three distinct chains. Thus there must be mechanisms that control composition and stagger during collagen folding. Here, we uncover the structural basis for both chain selection and stagger formation of a collagen molecule. Three distinct chains (α1, α2 and α3) of the non-collagenous domain 2 (NC2) of type IX collagen are assembled to guide triple-helical sequences in the leading, middle and trailing positions. This unique domain opens the door for generating any fragment of collagen in its native composition and stagger. PMID:27897211

  12. Effect of Lung Surfactant Protein SP-C and SP-C-Promoted Membrane Fragmentation on Cholesterol Dynamics.

    PubMed

    Roldan, Nuria; Nyholm, Thomas K M; Slotte, J Peter; Pérez-Gil, Jesús; García-Álvarez, Begoña

    2016-10-18

    To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Fragmentation of oxygen-containing molecules via C-O bond cleavage promoted by coordination to niobium and tantalum pentahalides.

    PubMed

    Marchetti, Fabio; Pampaloni, Guido; Zacchini, Stefano

    2009-09-14

    The novel mu-oxo complexes NbOX(3)[kappa(2)-O(Me)CH(2)CO(2)Me]NbX(5) (X = Cl, ; X = Br, ), NbOCl(3)[kappa(2)-(MeO(2)C)CH[double bond, length as m-dash]CH(CO(2)Me)]NbCl(5) () and NbOCl(3)[kappa(2)-CH(2)(CO(2)Me)(2)]NbCl(5) () have been prepared in good yields by 1 : 1 molar ratio reactions of the halides NbX(5) (X = Cl, Br) with methoxymethyl acetate [MeOCH(2)CO(2)Me], dimethyl maleate [(MeO(2)C)CH[double bond, length as m-dash]CH(CO(2)Me)] and dimethyl malonate [CH(2)(CO(2)Me)(2)] respectively, in different experimental conditions. NMR studies have indicated that the oxo unit of , and forms via selective fragmentation (C-O bonds cleavage) of half equivalent of the organic material. The ionic complexes [NbX(4){O-O}(2)][NbX(6)] [X = Cl, O-O = MeOCH(2)CO(2)Me, ; X = Cl, O-O = (MeO(2)C)CH[double bond, length as m-dash]CH(CO(2)Me), ; X = Cl, O-O = CH(2)(CO(2)Me)(2), ] have been identified as intermediates of the reactions affording , and . The stable complexes [NbX(4){O-O}(2)][NbX(6)] [X = F, O-O = (MeO(2)C)CH[double bond, length as m-dash]CH(2)(CO(2)Me), ; X = Cl, O-O = (EtO(2)C)CH[double bond, length as m-dash]CH(2)(CO(2)Et), ; X = F, O-O = CH(2)(CO(2)Me)(2), ] and [NbCl(4){(EtO(2)C)CH[double bond, length as m-dash]CH(CO(2)Et)}][NbCl(6)] () have been prepared by reacting NbX(5) (X = F, Cl) with variable amounts of the appropriate diester. Alternatively, NbX(5) (X = F, Cl) react with diethyl fumarate in 2 : 1 molar ratio affording the dinuclear [NbX(5)](2){mu-kappa(2)-(EtO(2)C)CH[double bond, length as m-dash]CH(CO(2)Et)} (X = F, ; X = Cl, ), in high yields. The neutral, monomeric, compounds MX(5)(L) [L = HCO(2)Me, Et(2)NCO(2)Me, OMe(2), OEt(2), OMeCH(2)Cl, OMeCH(2)CH(2)Cl, OMeCH(2)CH(2)Br, O(CH(2)CH(2)Cl)(2), 1,4-dioxane] have been obtained upon addition of the appropriate O-donor to MX(5). Complexes NbX(5)(OMeCH(2)CH(2)Y) (X = Br, Y = Cl, ; X = Y = Br, ; X = I, Y = Br, ) slowly convert into the corresponding alkoxides [NbX(4)(OCH(2)CH(2)Y)](2) (X = Br, Y = Cl, ; X

  14. Calcium Binding Promotes Prion Protein Fragment 90–231 Conformational Change toward a Membrane Destabilizing and Cytotoxic Structure

    PubMed Central

    Corsaro, Alessandro; Tosatto, Alessio; Thellung, Stefano; Villa, Valentina; Schininà, M. Eugenia; Maras, Bruno; Galeno, Roberta; Scotti, Luca; Creati, Francesco; Marrone, Alessandro; Re, Nazzareno; Aceto, Antonio; Florio, Tullio; Mazzanti, Michele

    2012-01-01

    The pathological form of prion protein (PrPSc), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrPSc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90–231 (hPrP90–231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrPSc. In the present study we demonstrate that hPrP90–231, pre-incubated with 10 mM Ca++ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP90–231 bearing pathogenic mutations (D202N and E200K). We also report that Ca++ binding to hPrP90–231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP90–231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca++ binding to hPrP90–231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity. PMID:22811758

  15. A fragment substitution in the promoter of CsHDZIV11/CsGL3 is responsible for fruit spine density in cucumber (Cucumis sativus L.).

    PubMed

    Zhang, Haiyang; Wang, Lina; Zheng, Shuangshuang; Liu, Zezhou; Wu, Xiaoqin; Gao, Zhihui; Cao, Chenxing; Li, Qiang; Ren, Zhonghai

    2016-07-01

    The indel in the promoter of CsHDZIV11 co-segregates with fruit spine density and could be used for molecular breeding in cucumber. Fruit spine density is an important quality trait for marketing in cucumber (Cucumis sativus L.). However, the molecular basis of fruit spine density in cucumber remains unclear. In this study, we isolated a mutant, few spines 1 (fs1), from CNS2 (wild type, WT), a North China-type cucumber with a high density of fruit spines. Genetic analysis showed that fs1 was controlled by a single recessive Mendelian factor. Bulked segregant analysis combined with genome resequencing were used for mapping fs1 in the F2 population derived from a cross between the fs1 mutant and WT, and it was located on chromosome 6 through association analysis. To develop more polymorphic markers to locate fs1, another F2 population was constructed from the cross between fs1 and 'Chinese long' 9930. Then, fs1 was narrowed down to a 110.4-kb genomic region containing 25 annotated genes. A fragment substitution was identified in the promoter region of Csa6M514870 between fs1 and WT. This fragment in fs1 was also present in wild cucumber. Csa6M514870 encodes a PDF2-related protein, a homeodomain-leucine zipper IV transcription factor (CsHDZIV11/CsGL3) sharing high identity and similarity with proteins related to trichome formation or epidermal cell differentiation. Quantitative reverse-transcription PCR revealed a higher expression level of CsHDZIV11 in young fruits from fs1 compared to WT. A molecular marker based on this indel co-segregated with the spine density. This work provides a solid foundation not only for understanding the molecular mechanism of fruit spine density, but also for molecular breeding in cucumber.

  16. UV-Induced Triggering of a Biomechanical Initiation Switch Within Collagen Promotes Development of a Melanoma-Permissive Microenvironment in the Skin

    DTIC Science & Technology

    2014-11-01

    including neutrophils, macrophages and mast cells . Pre-treatment of mice with anti-HU177 epitope antibody inhibited UV-induced mast cell accumulation...Collagen 3). Tumor Growth 4). Inflammation 5). UV-Irradiation 6). Mast Cells 7). Macrophages 8). Fibroblasts 9). Angiogenesis 10). Cell ...examined the differential adhesive interactions of RAW macrophage cell line and a mast cell line (Appendix Figs 5 and 32). As was shown with melanoma

  17. Ormocomp-modified glass increases collagen binding and promotes the adherence and maturation of human embryonic stem cell-derived retinal pigment epithelial cells.

    PubMed

    Käpylä, Elli; Sorkio, Anni; Teymouri, Shokoufeh; Lahtonen, Kimmo; Vuori, Leena; Valden, Mika; Skottman, Heli; Kellomäki, Minna; Juuti-Uusitalo, Kati

    2014-12-09

    In in vitro live-cell imaging, it would be beneficial to grow and assess human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells on thin, transparent, rigid surfaces such as cover glasses. In this study, we assessed how the silanization of glass with 3-aminopropyltriethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (MAPTMS), or polymer-ceramic material Ormocomp affects the surface properties, protein binding, and maturation of hESC-RPE cells. The surface properties were studied by contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a protein binding assay. The cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV-coated untreated or silanized surfaces for 42 days. The Ormocomp treatment significantly increased the hydrophobicity and roughness of glass surfaces compared to the APTES and MAPTMS treatments. The XPS results indicated that the Ormocomp treatment changes the chemical composition of the glass surface by increasing the carbon content and the number of C-O/═O bonds. The protein-binding test confirmed that the Ormocomp-treated surfaces bound more collagen IV than did APTES- or MAPTMS-treated surfaces. All of the silane treatments increased the number of cells: after 42 days of culture, Ormocomp had 0.38, APTES had 0.16, MAPTMS had 0.19, and untreated glass had only 0.062, all presented as million cells cm(-2). There were no differences in cell numbers compared to smoother to rougher Ormocomp surfaces, suggesting that the surface chemistry and, more specifically, the collagen binding in combination with Ormocomp are beneficial to hESC-RPE cell culture. This study clearly demonstrates that Ormocomp treatment combined with collagen coating significantly increases hESC-RPE cell attachment compared to commonly used silanizing agents APTES and MAPTMS. Ormocomp silanization could thus enable the use of microscopic live cell imaging methods for h

  18. MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

    SciTech Connect

    Takino, Takahisa; Tsuge, Hisashi; Ozawa, Terumasa; Sato, Hiroshi

    2010-06-11

    Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21{sup WAF1} and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin {alpha}{sub v}{beta}{sub 3} were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.

  19. Recognition of a core fragment of Beauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential

    PubMed Central

    Wang, Zheng-Liang; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    To identify a suitable promoter for use in engineering fungal entomopathogens to improve heterologous gene expression and fungal biocontrol potential, a 1798 bp promoter (Phyd1) upstream of Beauveria bassiana class I hydrophobin gene (hyd1) was optimized by upstream truncation and site-directed mutation. A truncated 1290 bp fragment (Phyd1-t1) drove eGFP expression in B. bassiana much more efficiently than full-length Phyd1. Further truncating Phyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in Phyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein). Under Phyd1-t1 control, eGFP was expressed more abundantly in conidiogenic cells and conidia than in mycelia. Therefore, Phyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of PgpdA, a promoter widely used for gene expression in fungi. Consequently, BbHV8 showed much higher per os virulence to Spodoptera litura larvae than BbV28 in standardized bioassays with normal conidia for both cuticle penetration and ingestion or heat-killed conidia for ingestion only. Conclusively, Phyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides. PMID:22639846

  20. Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.

    PubMed

    Wang, Zheng-Liang; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    To identify a suitable promoter for use in engineering fungal entomopathogens to improve heterologous gene expression and fungal biocontrol potential, a 1798 bp promoter (P hyd1) upstream of Beauveria bassiana class I hydrophobin gene (hyd1) was optimized by upstream truncation and site-directed mutation. A truncated 1290 bp fragment (P hyd1-t1) drove eGFP expression in B. bassiana much more efficiently than full-length P hyd1. Further truncating P hyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in P hyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein). Under P hyd1-t1 control, eGFP was expressed more abundantly in conidiogenic cells and conidia than in mycelia. Therefore, P hyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of P gpdA, a promoter widely used for gene expression in fungi. Consequently, BbHV8 showed much higher per os virulence to Spodoptera litura larvae than BbV28 in standardized bioassays with normal conidia for both cuticle penetration and ingestion or heat-killed conidia for ingestion only. Conclusively, P hyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.

  1. Platelet-derived Growth-factor-releasing Aligned Collagen-nanoparticle Fibers Promote the Proliferation and Tenogenic Differentiation of Adipose-derived Stem Cells

    DTIC Science & Technology

    2013-11-27

    bundled by a silk suture to create one 3-D aligned collagen scaffold (Fig. 9) that should be able to fit a 5 mm Achilles tendon gap defect in rat. Third...meshes. Biomaterials 2006;27:5681–8. [16] Moffat KL, Kwei ASP, Spalazzi JP, Doty SB, Levine WN, Lu HH. Novel nanofiber - based scaffold for rotator cuff...scaffold for potential application in tendon/ligament tissue engineering. Tissue eng 2006;12:91–9. [18] Sahoo S, Toh SL, Goh JCH. PLGA nanofiber -coated silk

  2. UV-Induced Triggering of a Biomechanical Initiation Switch Within Collagen Promotes Development of a Melanoma-Permissive Microenvironment in the Skin

    DTIC Science & Technology

    2012-09-01

    human dermal fibroblasts, cell adhesion assays were carried out with collagen type-I that was not irradiated or irradiated with UVA over a dose... dermal fibroblasts to UVA -irradiated MatrigelTM. Given the differential impact of UVB-irradiated MatrigelTM had on cell adhesion, we examined the...or higher as compared to control. In addition, human dermal fibroblast adhesion was enhanced by nearly 40%, at a UVA dose of 5.0J/cm2 (figure 7B

  3. Sol-gel assisted fabrication of collagen hydrolysate composite scaffold: a novel therapeutic alternative to the traditional collagen scaffold.

    PubMed

    Ramadass, Satiesh Kumar; Perumal, Sathiamurthi; Gopinath, Arun; Nisal, Anuya; Subramanian, Saravanan; Madhan, Balaraman

    2014-09-10

    Collagen is one of the most widely used biomaterial for various biomedical applications. In this Research Article, we present a novel approach of using collagen hydrolysate, smaller fragments of collagen, as an alternative to traditionally used collagen scaffold. Collagen hydrolysate composite scaffold (CHCS) was fabricated with sol-gel transition procedure using tetraethoxysilane as the silica precursor. CHCS exhibits porous morphology with pore sizes varying between 380 and 780 μm. Incorporation of silica conferred CHCS with controlled biodegradation and better water uptake capacity. Notably, 3T3 fibroblast proliferation was seen to be significantly better under CHCS treatment when compared to treatment with collagen scaffold. Additionally, CHCS showed excellent antimicrobial activity against the wound pathogens Staphylococcus aureus, Bacillus subtilis, and Escherichia coli due to the inherited antimicrobial activity of collagen hydrolysate. In vivo wound healing experiments with full thickness excision wounds in rat model demonstrated that wounds treated with CHCS showed accelerated healing when compared to wounds treated with collagen scaffold. These findings indicate that the CHCS scaffold from collagen fragments would be an effective and affordable alternative to the traditionally used collagen structural biomaterials.

  4. Rapid biomimetic mineralization of collagen fibrils and combining with human umbilical cord mesenchymal stem cells for bone defects healing.

    PubMed

    Ye, Bihua; Luo, Xueshi; Li, Zhiwen; Zhuang, Caiping; Li, Lihua; Lu, Lu; Ding, Shan; Tian, Jinhuan; Zhou, Changren

    2016-11-01

    Collagen biomineralization is regulated by complicated interactions between the collagen matrix and non-collagenous extracellular proteins. Here, the use of sodium tripolyphosphate to simulate the templating functional motif of the C-terminal fragment of non-collagenous proteins is reported, and a low molecular weight polyacrylic acid served as a sequestration agent to stabilize amorphous calcium phosphate into nanoprecursors. Self-assembled collagen fibrils served as a fixed template for achieving rapid biomimetic mineralization in vitro. Results demonstrated that, during the mineralization process, intrafibrillar and extrafibrillar hydroxyapatite mineral with collagen fibrils formed and did so via bottom-up nanoparticle assembly based on the non-classical crystallization approach in the presence of these dual biomimetic functional analogues. In vitro human umbilical cord mesenchymal stem cell (hUCMSC) culture found that the mineralized scaffolds have a better cytocompatibility in terms of cell viability, adhesion, proliferation, and differentiation into osteoblasts. A rabbit femoral condyle defect model was established to confirm the ability of the n-HA/collagen scaffolds to facilitate bone regeneration and repair. The images of gross anatomy, MRI, CT and histomorphology taken 6 and 12weeks after surgery showed that the biomimetic mineralized collagen scaffolds with hUCMSCs can promote the healing speed of bone defects in vivo, and both of the scaffolds groups performing better than the bone defect control group. As new bone tissue formed, the scaffolds degraded and were gradually absorbed. All these results demonstrated that both of the scaffolds and cells have better histocompatibility. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. A 3.7 kb Fragment of the Mouse Scn10a Gene Promoter Directs Neural Crest But Not Placodal Lineage EGFP Expression in a Transgenic Animal

    PubMed Central

    Lu, Van B.; Ikeda, Stephen R.

    2015-01-01

    Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP-positive (mean diameter = 26.5 μm). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (∼10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8-immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons. PMID:25995484

  6. A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal.

    PubMed

    Lu, Van B; Ikeda, Stephen R; Puhl, Henry L

    2015-05-20

    Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP-positive (mean diameter = 26.5 μm). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (∼10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8-immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons.

  7. Collagen scaffolds loaded with collagen-binding NGF-beta accelerate ulcer healing.

    PubMed

    Sun, Wenjie; Lin, Hang; Chen, Bing; Zhao, Wenxue; Zhao, Yannan; Xiao, Zhifeng; Dai, Jianwu

    2010-03-01

    Studies have shown that exogenous nerve growth factor (NGF) accelerates ulcer healing, but the inefficient growth factor delivery system limits its clinical application. In this report, we found that the native human NGF-beta fused with a collagen-binding domain (CBD) could form a collagen-based NGF targeting delivery system, and the CBD-fused NGF-beta could bind to collagen membranes efficiently. Using the rabbit dermal ischemic ulcer model, we have found that this targeting delivery system maintains a higher concentration and stronger bioactivity of NGF-beta on the collagen membranes by promoting peripheral nerve growth. Furthermore, it enhances the rate of ulcer healing through accelerating the re-epithelialization of dermal ulcer wounds and the formation of capillary lumens within the newly formed tissue area. Thus, collagen membranes loaded with collagen-targeting human NGF-beta accelerate ulcer healing efficiently.

  8. Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model

    PubMed Central

    Chan, Barbara P.; Lo, Amy C. Y.

    2016-01-01

    Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. However, CAC ECT properties and performance in the eye are not well-understood. Previously, we reported a cultured 3D CAC system that supported the growth of GDNF-secreting HEK293 cells with sustainable GDNF delivery. Here, the system was further developed into an intravitreally injectable gel with 1x104 or 2x105 cells encapsulated in 2mg/ml type I collagen and 1% alginate. Gels with lower alginate concentration yielded higher initial cell viability but faster spheroid formation while increasing initial cell density encouraged cell growth. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well-tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel-implanted in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those containing higher initial cell number yielded better photoreceptor survival. CAC ECT gels offers flexible system design and is a potential treatment option for posterior eye diseases. PMID:27441692

  9. C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration.

    PubMed

    Jóźwiak, Jolanta; Rzhepetskyy, Yuriy; Sobczak, Magdalena; Kocik, Elżbieta; Skórzewski, Radosław; Kłopocka, Wanda; Rędowicz, Maria Jolanta

    2011-02-01

    Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

  10. Dscam1 Forms a Complex with Robo1 and the N-Terminal Fragment of Slit to Promote the Growth of Longitudinal Axons

    PubMed Central

    Alavi, Maryam; Song, Minmin; Gillis, Taylor; Bousum, Adam; Miller, Amanda; Kidd, Thomas

    2016-01-01

    The Slit protein is a major midline repellent for central nervous system (CNS) axons. In vivo, Slit is proteolytically cleaved into N- and C-terminal fragments, but the biological significance of this is unknown. Analysis in the Drosophila ventral nerve cord of a slit allele (slit-UC) that cannot be cleaved revealed that midline repulsion is still present but longitudinal axon guidance is disrupted, particularly across segment boundaries. Double mutants for the Slit receptors Dscam1 and robo1 strongly resemble the slit-UC phenotype, suggesting they cooperate in longitudinal axon guidance, and through biochemical approaches, we found that Dscam1 and Robo1 form a complex dependent on Slit-N. In contrast, Robo1 binding alone shows a preference for full-length Slit, whereas Dscam1 only binds Slit-N. Using a variety of transgenes, we demonstrated that Dscam1 appears to modify the output of Robo/Slit complexes so that signaling is no longer repulsive. Our data suggest that the complex is promoting longitudinal axon growth across the segment boundary. The ability of Dscam1 to modify the output of other receptors in a ligand-dependent fashion may be a general principle for Dscam proteins. PMID:27654876

  11. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  12. The Collagen Family

    PubMed Central

    Ricard-Blum, Sylvie

    2011-01-01

    Collagens are the most abundant proteins in mammals. The collagen family comprises 28 members that contain at least one triple-helical domain. Collagens are deposited in the extracellular matrix where most of them form supramolecular assemblies. Four collagens are type II membrane proteins that also exist in a soluble form released from the cell surface by shedding. Collagens play structural roles and contribute to mechanical properties, organization, and shape of tissues. They interact with cells via several receptor families and regulate their proliferation, migration, and differentiation. Some collagens have a restricted tissue distribution and hence specific biological functions. PMID:21421911

  13. Binding of collagens to an enterotoxigenic strain of Escherichia coli

    SciTech Connect

    Visai, L.; Speziale, P.; Bozzini, S. )

    1990-02-01

    An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity. We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides (alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4) were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure.

  14. The pro alpha 2(V) collagen gene is evolutionarily related to the major fibrillar-forming collagens.

    PubMed Central

    Weil, D; Bernard, M; Gargano, S; Ramirez, F

    1987-01-01

    A number of overlapping cDNA clones, covering 5.2 kb of sequences which code for the human pro alpha 2(V) collagen chain, have been isolated. Analysis of the structural data have indicated a close evolutionary kinship between the pro alpha 2(V) chain and the major fibrillar collagen types. Isolation and analysis of an 8 kb genomic fragment has further supported this notion by revealing a homologous arrangement of nine triple-helical domain exons. These studies have therefore provided conclusive evidence which categorizes the Type V collagen as a member of the Group 1 molecules, or fibrillar-forming collagens. Images PMID:3029669

  15. Oriented collagen nanocoatings for tissue engineering.

    PubMed

    Pastorino, Laura; Dellacasa, Elena; Scaglione, Silvia; Giulianelli, Massimo; Sbrana, Francesca; Vassalli, Massimo; Ruggiero, Carmelina

    2014-02-01

    Collagens are among the most widely present and important proteins composing the human total body, providing strength and structural stability to various tissues, from skin to bone. In this paper, we report an innovative approach to bioactivate planar surfaces with oriented collagen molecules to promote cells proliferation and alignment. The Langmuir-Blodgett technique was used to form a stable collagen film at the air-water interface and the Langmuir-Schaefer deposition was adopted to transfer it to the support surface. The deposition process was monitored by estimating the mass of the protein layers after each deposition step. Collagen films were then structurally characterized by atomic force, scanning electron and fluorescent microscopies. Finally, collagen films were functionally tested in vitro. To this aim, 3T3 cells were seeded onto the silicon supports either modified or not (control) by collagen film deposition. Cells adhesion and proliferation on collagen films were found to be greater than those on control both after 1 (p<0.05) and 7 days culture. Moreover, the functionalization of the substrate surface triggered a parallel orientation of cells when cultured on it. In conclusion, these data demonstrated that the Langmuir-Schaefer technique can be successfully used for the deposition of oriented collagen films for tissue engineering applications.

  16. Development of chitosan-collagen hydrogel incorporated with lysostaphin (CCHL) burn dressing with anti-methicillin-resistant Staphylococcus aureus and promotion wound healing properties.

    PubMed

    Cui, Fuying; Li, Guodong; Huang, Jinjiang; Zhang, Jien; Lu, Min; Lu, Wanying; Huan, Jingning; Huang, Qingshan

    2011-04-01

    Methicillin-resistant Staphylococcus aureus (MRSA) have become increasingly prevalent as nosocomial pathogens, especially in burn patients, which is the leading cause of their death. A drug delivery system of chitosan-collagen hydrogel incorporated with lysostaphin (CCHL) based on the lysostaphin gauze was developed for MRSA infected burn wounds. CCHL scaffold consisted of numerous interconnected sphericles and tubular bodies with an average diameter of 100-200 µm, 20-60-fold swelling, high water retention capacity, and cell proliferation properties. The minimal inhibitory concentration of CCHL was 0.053 U/mL. By the second week after its application on MRSA infected third-degree burn wounds, no bacteria could be detected and the burn wounds had started healing. Therefore, CCHL should be studied further as a promising candidate of burn treatment dressing against MRSA infections for clinics.

  17. Molecular Strategy to Reduce In Vivo Collagen Barrier Promotes Entry of NCX1 Positive Inducible Pluripotent Stem Cells (iPSCNCX1+) into Ischemic (or Injured) Myocardium

    PubMed Central

    Millard, Ronald W.; Yu, Xi-Yong; Luther, Kristin; Xu, Meifeng; Zhao, Ting C.; Yang, Huang-Tian; Qi, Zhihua; LaSance, Kathleen; Ashraf, Muhammad; Wang, Yigang

    2013-01-01

    Objective The purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium. Background We previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium. Methods In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSCNCX1+), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly. Results The number of green fluorescent protein positive (GFP+) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling. Conclusion Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSCNCX1+ penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI. PMID:23990893

  18. Collagen-mediated hemostasis.

    PubMed

    Manon-Jensen, T; Kjeld, N G; Karsdal, M A

    2016-03-01

    Collagens mediate essential hemostasis by maintaining the integrity and stability of the vascular wall. Imbalanced turnover of collagens by uncontrolled formation and/or degradation may result in pathologic conditions such as fibrosis. Thickening of the vessel wall because of accumulation of collagens may lead to arterial occlusion or thrombosis. Thinning of the wall because of collagen degradation or deficiency may lead to rupture of the vessel wall or aneurysm. Preventing excessive hemorrhage or thrombosis relies on collagen-mediated actions. Von Willebrand factor, integrins and glycoprotein VI, as well as clotting factors, can bind collagen to restore normal hemostasis after trauma. This review outlines the essential roles of collagens in mediating hemostasis, with a focus on collagens types I, III, IV, VI, XV, and XVIII.

  19. Biomedical applications of collagens.

    PubMed

    Ramshaw, John A M

    2016-05-01

    Collagen-based biomedical materials have developed into important, clinically effective materials used in a range of devices that have gained wide acceptance. These devices come with collagen in various formats, including those based on stabilized natural tissues, those that are based on extracted and purified collagens, and designed composite, biosynthetic materials. Further knowledge on the structure and function of collagens has led to on-going developments and improvements. Among these developments has been the production of recombinant collagen materials that are well defined and are disease free. Most recently, a group of bacterial, non-animal collagens has emerged that may provide an excellent, novel source of collagen for use in biomaterials and other applications. These newer collagens are discussed in detail. They can be modified to direct their function, and they can be fabricated into various formats, including films and sponges, while solutions can also be adapted for use in surface coating technologies.

  20. Definition of the native and denatured type II collagen binding site for fibronectin using a recombinant collagen system.

    PubMed

    An, Bo; Abbonante, Vittorio; Yigit, Sezin; Balduini, Alessandra; Kaplan, David L; Brodsky, Barbara

    2014-02-21

    Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.

  1. Thermal stability of collagen triple helix.

    PubMed

    Xu, Yujia

    2009-01-01

    Chief among the challenges of characterizing the thermal stability of the collagen triple helix are the lack of the reversibility of the thermal transition and the presence of multiple folding-unfolding steps during the thermal transition which rarely follows the simple two-state, all-or-none mechanism. Despite of the difficulties inherited in the quantitative depiction of the thermal transition of collagen, biophysical studies combined with proteolysis and mutagenesis approaches using full-chain collagens, short synthetic peptides, and recombinant collagen fragments have revealed molecular features of the thermal unfolding of the subdomains of collagen and led to a better understanding of the diverse biological functions of this versatile protein. The subdomain of collagen generally refers to a segment of the long, rope-like triple helical molecule that can unfold cooperatively as an independent unit whose properties (their size, location, and thermal stability) are considered essential for the molecular recognition during the self-assembly of collagen and during the interactions of collagen with other macromolecules. While the unfolding of segments of the triple helix at temperatures below the apparent melting temperature of the molecule has been used to interpret much of the features of the thermal unfolding of full-chain collagens, the thermal studies of short, synthetic peptides have firmly established the molecular basis of the subdomains by clearly demonstrating the close dependence of the thermal stability of a triple helix on the constituent amino acid residues at the X and the Y positions of the characteristic Gly-X-Y repeating sequence patterns of the triple helix. Studies using recombinant collagen fragments further revealed that in the context of the long, linear molecule, the stability of a segment of the triple helix is also modulated by long-range impact of the local interactions such as the interchain salt bridges. Together, the combined approaches

  2. Perlecan antagonizes collagen IV and ADAMTS9/GON-1 in restricting the growth of presynaptic boutons.

    PubMed

    Qin, Jianzhen; Liang, Jingjing; Ding, Mei

    2014-07-30

    In the mature nervous system, a significant fraction of synapses are structurally stable over a long time scale. However, the mechanisms that restrict synaptic growth within a confined region are poorly understood. Here, we identified that in the C. elegans neuromuscular junction, collagens Type IV and XVIII, and the secreted metalloprotease ADAMTS/GON-1 are critical for growth restriction of presynaptic boutons. Without these components, ectopic boutons progressively invade into the nonsynaptic region. Perlecan/UNC-52 promotes the growth of ectopic boutons and functions antagonistically to collagen Type IV and GON-1 but not to collagen XVIII. The growth constraint of presynaptic boutons correlates with the integrity of the extracellular matrix basal lamina or basement membrane (BM), which surrounds chemical synapses. Fragmented BM appears in the region where ectopic boutons emerge. Further removal of UNC-52 improves the BM integrity and the tight association between BM and presynaptic boutons. Together, our results unravel the complex role of the BM in restricting the growth of presynaptic boutons and reveal the antagonistic function of perlecan on Type IV collagen and ADAMTS protein.

  3. Enhancement of flowering and branching phenotype in chrysanthemum by expression of ipt under the control of a 0.821 kb fragment of the LEACO1 gene promoter.

    PubMed

    Khodakovskaya, Mariya; Vanková, Radomira; Malbeck, Jiri; Li, Aizhen; Li, Yi; McAvoy, Richard

    2009-09-01

    The cytokinin biosynthesis gene, isopentenyl transferase (ipt), under the control of an 821 bp fragment of the LEACO1 gene promoter (from Lycopersicon esculentum) was introduced into Dendranthema x grandiflorium 'Iridon' (chrysanthemum). LEACO1(0.821kb)-ipt transgenic lines grown in the vegetative state, exhibited a range of phenotypic changes including increased branching and reduced internode lengths. LEACO1(0.821kb)-ipt transgenic lines grown in the generative state, exhibited increased flower bud count that ranged from 3.8- to 6.7-times the number produced by wild-type plants. Dramatic increases in flower number were associated with a delay of flower bud development and a decrease in flower bud diameter. RT-PCR analysis indicated differences in ipt gene expression between individual transgenic lines that exhibited a range of phenotypes. Within an individual transgenic line, RT-PCR analysis revealed changes in ipt gene expression at different stages of generative shoot development. Expression of ipt in transgenic lines correlated well with high concentrations of the sum total to bioactive cytokinins plus the glucosides and phosphate derivatives of these species, under both vegetative and generative growth conditions. In general, transgenic lines accumulated higher concentrations of both storage-form cytokinins (O-glucosides) and deactivated-form cytokinins (N-glucosides) in generative shoots of than in vegetative shoots. Based on the range of phenotypes observed in various transgenic chrysanthemum lines, we conclude that the LEACO1 (0.821kb) -ipt gene appears to have great potential for use in ornamental crop improvement.

  4. Role of NH2-terminal fragment of Dentin Sialophosphoprotein (DSPP) in dentinogenesis

    PubMed Central

    Gibson, Monica P.; Liu, Qilin; Zhu, Qinglin; Lu, Yongbo; Jani, Priyam; Wang, Xiaofang; Liu, Ying; Paine, Michael L.; Snead, Malcolm L.; Feng, Jian Q.; Qin, Chunlin

    2012-01-01

    DSPP is a large precursor protein that is proteolytically processed into three fragments: a NH2-terminal fragment [dentin sialoprotein (DSP), a proteoglycan form (DSP-PG)] and a COOH-terminal fragment [dentin phosphoprotein (DPP)]. In vitro studies indicate that DPP is a strong initiator and regulator of hydroxyapatite crystal formation and growth, but the role(s) of the NH2-terminal fragment of DSPP (i.e., DSP and DSP-PG) in dentinogenesis remain unclear. This study focuses on the function of the NH2-terminal fragment of DSPP in dentinogenesis. Here, transgenic mouse lines expressing the NH2-terminal fragment of DSPP driven by 3.6-kb type I collagen promoter (Col 1a1) were generated and crossbred with Dspp null mice to obtain mice that express the transgene but lack the endogenous Dspp (Dspp KO/DSP Tg). We found dentin from the Dspp KO/DSP Tg mice was much thinner; more poorly mineralized and remarkably disorganized compared to the Dspp KO mice. The fact that Dspp KO/DSP Tg mice exhibited more severe dentin defects than the Dspp null mice indicates that the NH2-terminal fragment of DSPP may inhibit dentin mineralization or may serve as an antagonist against the accelerating action of DPP and serve to prevent predentin from being mineralized too rapidly during dentionogenesis. PMID:23489896

  5. The Non-phagocytic Route of Collagen Uptake

    PubMed Central

    Madsen, Daniel H.; Ingvarsen, Signe; Jürgensen, Henrik J.; Melander, Maria C.; Kjøller, Lars; Moyer, Amanda; Honoré, Christian; Madsen, Charlotte A.; Garred, Peter; Burgdorf, Sven; Bugge, Thomas H.; Behrendt, Niels; Engelholm, Lars H.

    2011-01-01

    The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen. PMID:21652704

  6. In-situ Damage Assessment of Collagen within Ancient Manuscripts Written on Parchment: A Polarized Raman Spectroscopy Approach

    NASA Astrophysics Data System (ADS)

    Schütz, R.; Rabin, I.; Hahn, O.; Fratzl, P.; Masic, A.

    2010-08-01

    The collection generally known as Qumran scrolls or Dead Sea Scrolls (DSS) comprises some 900 highly fragmented manuscripts (mainly written on parchment) from the Second Temple period. In the years since their manufacture the writing materials have undergone serious deterioration due to a combination of natural ageing and environmental effects. Therefore, understanding quantitatively state of conservation of such manuscripts is a challenging task and a deep knowledge of damage pathways on all hierarchical levels (from molecular up to macroscopic) results of fundamental importance for a correct protection and conservation strategy. However, the degradation of parchments is very complex and not well understood process. Parchment is a final product of processing of animal skin and consist mainly of type I collagen, which is the most abundant constituent of the dermal matrix. Collagen molecule is built by folding of three polypeptide α-chains into a right-handed triple helix. Every α-chain is made by a repetitive sequence of (Gly-X-Y)n, where X and Y are often proline and hydroxyproline. Parallel and staggered collagen triple helices associate into fibrils, which than assemble into fibers. Deterioration of parchment is caused by chemical changes due to gelatinization, oxidation and hydrolysis of the collagen chains, promoted by several factors, summarized as biological and microbiological (bacteria, fungi etc.), heat, light, humidity and pollutants (1, 2). In this work we have focused on studying the collagen within parchments on two different levels of organization (molecular and fibrilar) by applying polarized Raman spectroscopic technique. Beside spectral information related to chemical bonding, polarization anisotropy of some collagen bands (i.e. amide I) has been used to explore organization of collagen on higher levels (three-dimensional arrangement of the triple-helix molecules and their alignment within a fibril of collagen). To this aim we have compared

  7. Direct Visualization of Protease Action on Collagen Triple Helical Structure

    PubMed Central

    Rosenblum, Gabriel; Van den Steen, Philippe E.; Cohen, Sidney R.; Bitler, Arkady; Brand, David D.; Opdenakker, Ghislain; Sagi, Irit

    2010-01-01

    Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease. PMID:20585385

  8. Elevated cysteine-rich protein 61 (CCN1) promotes skin aging via upregulation of IL-1β in chronically sun-exposed human skin.

    PubMed

    Qin, Zhaoping; Okubo, Toru; Voorhees, John J; Fisher, Gary J; Quan, Taihao

    2014-02-01

    Chronic exposure of human skin to solar ultraviolet (UV) irradiation causes premature skin aging, which is characterized by reduced type I collagen production and increased fragmentation of the dermal collagenous extracellular matrix. This imbalance of collagen homeostasis is mediated, in part, by elevated expression of the matricellular protein cysteine-rich protein 61 (CCN1), in dermal fibroblasts, the primary collagen producing cell type in human skin. Here, we report that the actions of CCN1 are mediated by induction of interleukin 1β (IL-1β). CCN1 and IL-1β are strikingly induced by acute UV irradiation, and constitutively elevated in sun-exposed prematurely aged human skin. Elevated CCN1 rapidly induces IL-1β, inhibits type I collagen production, and upregulates matrix metalloproteinase-1, which degrades collagen fibrils. Blockade of IL-1β actions by IL-1 receptor antagonist largely prevents the deleterious effects of CCN1 on collagen homeostasis. Furthermore, knockdown of CCN1 significantly reduces induction of IL-1β by UV irradiation, and thereby partially prevents collagen loss. These data demonstrate that elevated CCN1promotes inflammaging and collagen loss via induction of IL-1β and thereby contributes to the pathophysiology of premature aging in chronically sun-exposed human skin.

  9. 96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen.

    PubMed

    Koshy, P J; Rowan, A D; Life, P F; Cawston, T E

    1999-11-15

    We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added.

  10. Parathyroid hormone linked to a collagen binding domain promotes hair growth in a mouse model of chemotherapy-induced alopecia in a dose-dependent manner.

    PubMed

    Katikaneni, Ranjitha; Ponnapakkam, Tulasi; Seymour, Andrew; Sakon, Joshua; Gensure, Robert

    2014-08-01

    Chemotherapy-induced alopecia is a major source of psychological stress in patients undergoing cancer chemotherapy, and it can influence treatment decisions. Although there is currently no therapy for alopecia, a fusion protein of parathyroid hormone and collagen binding domain (PTH-CBD) has shown promise in animal models. The aim of this study was to determine whether there are dose-dependent effects of PTH-CBD on chemotherapy-induced alopecia in a mouse model. C57BL/6J mice were waxed to synchronize hair follicles; treated on day 7 with vehicle or PTH-CBD (100, 320, and 1000 mcg/kg subcutaneous injection); and treated on day 9 with vehicle or cyclophosphamide (150 mg/kg intraperitoneally). Mice were photographed every 3-4 days and killed on day 63 for histological analysis. Photographs were quantified by gray scale analysis to assess hair content. Mice not receiving chemotherapy showed regrowth of hair 2 weeks after waxing and normal histology after 2 months. Mice receiving chemotherapy alone showed marked hair loss after chemotherapy, which was sustained for 10 days and was followed by rapid regrowth of a normal coat. Histological analysis revealed rapid cycling dystrophic anagen/catagen follicles. Animals receiving chemotherapy and PTH-CBD showed decreased hair loss and more rapid regrowth of hair than that seen with chemotherapy alone (increased hair growth by gray scale analysis, P<0.05), and the effects were dose dependent. Histologically, hair follicles in animals receiving the highest dose of PTH-CBD were in a quiescent phase, similar to that in mice that did not receive chemotherapy. Single-dose subcutaneous administration of PTH-CBD showed dose-dependent effects in minimizing hair loss and speeding up recovery from chemotherapy-induced alopecia.

  11. Chameleon fragmentation

    SciTech Connect

    Brax, Philippe

    2014-02-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ{sup 4} and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  12. Enigmatic insight into collagen

    PubMed Central

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

  13. Collagen and gelatin.

    PubMed

    Liu, Dasong; Nikoo, Mehdi; Boran, Gökhan; Zhou, Peng; Regenstein, Joe M

    2015-01-01

    Collagen and gelatin have been widely used in the food, pharmaceutical, and cosmetic industries due to their excellent biocompatibility, easy biodegradability, and weak antigenicity. Fish collagen and gelatin are of renewed interest, owing to the safety and religious concerns of their mammalian counterparts. The structure of collagen has been studied using various modern technologies, and interpretation of the raw data should be done with caution. The structure of collagen may vary with sources and seasons, which may affect its applications and optimal extraction conditions. Numerous studies have investigated the bioactivities and biological effects of collagen, gelatin, and their hydrolysis peptides, using both in vitro and in vivo assay models. In addition to their established nutritional value as a protein source, collagen and collagen-derived products may exert various potential biological activities on cells in the extracellular matrix through the corresponding food-derived peptides after ingestion, and this might justify their applications in dietary supplements and pharmaceutical preparations. Moreover, an increasing number of novel applications have been found for collagen and gelatin. Therefore, this review covers the current understanding of the structure, bioactivities, and biological effects of collagen, gelatin, and gelatin hydrolysates as well as their most recent applications.

  14. Reactions of lipid-derived malondialdehyde with collagen.

    PubMed

    Slatter, D A; Paul, R G; Murray, M; Bailey, A J

    1999-07-09

    Malondialdehyde is a product of fatty acid oxidation (e.g. from low density lipoprotein) implicated in the damage of proteins such as collagen in the cardiovascular system (Chio, K. J., and Tappel, A. L. (1969) Biochemistry 8, 2821-2827). Its concentration is raised in diabetic subjects probably as a side effect of increased protein glycation. Collagen has enzyme-catalyzed cross-links formed between its individual molecules that are essential for maintaining the structure and flexibility of the collagen fiber. The cross-link dehydro-hydroxylysinonorleucine reacts irreversibly with 10 mM malondialdehyde at least 3 orders of magnitude faster than glucose reactions with lysine or arginine, such that there is little cross-link left after 1 h at 37 degrees C. Other cross-links and glycated elements of collagen are also vulnerable. Several possible products of malondialdehyde with collagen cross-links are proposed, and the potential involvement of collagenous histidine in these reactions is discussed. We have also isolated Ndelta-(2-pyrimidyl)-L-ornithine from collagenous arginine reacted with malondialdehyde. The yields of this product were considerably higher than those from model reactions, being approximately 2 molecules/collagen molecule after 1 day at 37 degrees C in 10 mM malondialdehyde. Collagenous lysine-derived malondialdehyde products may have been present but were not protected from protein acid hydrolysis by standard reduction techniques, thus resulting in a multitude of fragmented products.

  15. Molecular cloning of porcine growth differentiation factor 9 (GDF-9) cDNA and its role in early folliculogenesis: direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis.

    PubMed

    Shimizu, Takashi; Miyahayashi, Yasunori; Yokoo, Masaki; Hoshino, Yumi; Sasada, Hiroshi; Sato, Eimei

    2004-11-01

    Growth differentiation factor-9 (GDF-9) is a growth factor secreted by oocytes in growing ovarian follicles. To investigate the ovarian function of GDF-9 in pigs, we first cloned porcine GDF-9 complementary DNA (cDNA), and then injected its gene fragments into the ovary in gilts. Porcine GDF-9 has open reading frame (ORF) homologies of 81.4%, 84.6%, 84.2%, 72.7% and 72.6% with its human, bovine, ovine, rat and mouse counterparts respectively. Regarding the deduced amino-acid sequence of the mature protein, the corresponding homologies reach 92.1%, 97.8%, 97.0%, 89.6% and 88.1% respectively. To investigate the role of GDF-9 in early folliculogenesis, the ovaries of 2-month-old prepubertal gilts were injected with GDF-9 gene fragments. The injection of porcine GDF-9 gene fragments resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles. These results indicated that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary, and that a technique for direct ovarian injection of GFD-9 gene fragments may contribute to a novel therapy for prevention and treatment of infertility associated with ovarian dysfunction.

  16. Isolation of cDNA and genomic DNA clones encoding type II collagen.

    PubMed Central

    Young, M F; Vogeli, G; Nunez, A M; Fernandez, M P; Sullivan, M; Sobel, M E

    1984-01-01

    A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs. Images PMID:6203098

  17. Specific cleavage of human type III and IV collagens by Pseudomonas aeruginosa elastase.

    PubMed Central

    Heck, L W; Morihara, K; McRae, W B; Miller, E J

    1986-01-01

    Purified Pseudomonas aeruginosa elastase cleaved human type III and IV collagens with the formation of specific cleavage products. Furthermore, type I collagen appeared to be slowly cleaved by both P. aeruginosa elastase and alkaline protease. These cleavage fragments from type III and IV collagens were separated from the intact collagen chains by SDS polyacrylamide gradient gel electrophoresis run under reducing conditions, and they were detected by their characteristic Coomassie blue staining pattern. The results of these studies suggest that the pathogenesis of tissue invasion and hemorrhagic tissue necrosis observed in P. aeruginosa infections may be related to the degradation of these collagen types by bacterial extracellular proteases. Images PMID:3079727

  18. Cartilage Fibrils of Mammals are Biochemically Heterogeneous: Differential Distribution of Decorin and Collagen IX

    PubMed Central

    Hagg, Rupert; Bruckner, Peter; Hedbom, Erik

    1998-01-01

    Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents—decorin and collagen IX—in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties. PMID:9660881

  19. Haemorrhoids - a collagen disease?

    PubMed

    Willis, S; Junge, K; Ebrahimi, R; Prescher, A; Schumpelick, V

    2010-12-01

    The cause of haemorrhoidal disease is unknown, epidemiological data and histopathological findings support the hypothesis that reduced connective tissue stability is associated with the incidence of haemorrhoids. Therefore the aim of this study was to analyse the quantity and quality of collagen formation in the corpus cavernosum recti in patients with III°/IV° haemorrhoids in comparison with persons without haemorrhoids. Haemorrhoidectomy specimens of 31 patients with III°/IV° haemorrhoids were examined. The specimens of 20 persons who died a natural death and who had no haemorrhoidal disease served as the controls. The amount of collagen was estimated photometrically by calculating the collagen/protein ratio. The collagen I/III ratio served as parameter for the quality of collagen formation and was calculated using cross polarization spectroscopy. Patients with haemorrhoids had a significantly reduced collagen/protein ratio (42.2 ± 16.2μg/mg vs 72.5±31.0μg/mg; P= 0.02) and a significantly reduced collagen I/III ratio (2.0±0.1 vs 4.6±0.3; P<0.001) compared with persons without haemorrhoidal disease. There was no correlation with patients' age or gender.  There is a fundamental disorder of collagen metabolism in patients with haemorrhoidal disease. It remains unclear whether this is due to exogenous or endogenous influences. © 2010 The Authors. Colorectal Disease © 2010 The Association of Coloproctology of Great Britain and Ireland.

  20. The homeoproteins MAB-18 and CEH-14 insulate the dauer collagen gene col-43 from activation by the adjacent promoter of the Spermatheca gene sth-1 in Caenorhabditis elegans.

    PubMed

    Bando, Tetsuya; Ikeda, Tatsuji; Kagawa, Hiroaki

    2005-04-22

    Genome searches in this study indicate that the nematode Caenorhabditis elegans genome has 2582 bidirectionally oriented genes that account for more than 25% of the total genes. We analyze the transcriptional repression system for one of these predicted bidirectional promoters, which controls the expression of the spermathecal gene sth-1 and collagen gene col-43. These two genes are separated by 1.3 kb and are transcribed bidirectionally. sth-1 is expressed in spermatheca after the L4 stage and col-43 is expressed in the hypodermal cells of the L2d dauer stage. The upstream regions required for the expression of sth-1 and col-43 shared an overlapped control sequence. Two homeoproteins, MAB-18 and CEH-14, were isolated by yeast one-hybrid screening as binding proteins of the overlapped region. MAB-18 bound to two homeodomain-binding sites and interacted with CEH-14 to repress col-43 expression in spermatheca. These results indicate that the two homeoproteins interact with each other to repress col-43 expression in sth-1-expressing tissues. This is the first report of bidirectional gene regulation analysis in the C.elegans genome.

  1. FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Banerjee, Priyam; Guo, Houfu; Liu, Xin; Yu, Jiang; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2017-01-01

    Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities. PMID:28378777

  2. Fragmentation Processes

    NASA Astrophysics Data System (ADS)

    Whelan, Colm T.

    2012-12-01

    Preface; 1. Direct and resonant double-photoionization: from atoms to solids L. Avaldi and G. Stefani; 2. The application of propagation exterior complex scaling to atomic collisions P. L. Bartlett and A. T. Stelbovics; 3. Fragmentation of molecular-ion beams in intense ultra-short laser pulses I. Ben-Itzhak; 4. Atoms with one and two active electrons in strong laser fields I. A. Ivanov and A. S. Kheifets; 5. Experimental aspects of ionization studies by positron and positronium impact G. Laricchia, D. A. Cooke, Á. Kövér and S. J. Brawley; 6. (e,2e) spectroscopy using fragmentation processes J. Lower, M. Yamazaki and M. Takahashi; 7. A coupled pseudostate approach to the calculation of ion-atom fragmentation processes M. McGovern, H. R. J. Walters and C. T. Whelan; 8. Electron Impact Ionization using (e,2e) coincidence techniques from threshold to intermediate energies A. J. Murray; 9. (e,2e) processes on atomic inner shells C. T. Whelan; 10. Spin resolved atomic (e,2e) processes J. Lower and C. T. Whelan; Index.

  3. Effects of endogenous cysteine proteinases on structures of collagen fibres from dermis of sea cucumber (Stichopus japonicus).

    PubMed

    Liu, Yu-Xin; Zhou, Da-Yong; Ma, Dong-Dong; Liu, Zi-Qiang; Liu, Yan-Fei; Song, Liang; Dong, Xiu-Ping; Li, Dong-Mei; Zhu, Bei-Wei; Konno, Kunihiko; Shahidi, Fereidoon

    2017-10-01

    Autolysis of sea cucumber, caused by endogenous enzymes, leads to postharvest quality deterioration of sea cucumber. However, the effects of endogenous proteinases on structures of collagen fibres, the major biologically relevant substrates in the body wall of sea cucumber, are less clear. Collagen fibres were prepared from the dermis of sea cucumber (Stichopus japonicus), and the structural consequences of degradation of the collagen fibres caused by endogenous cysteine proteinases (ECP) from Stichopus japonicus were examined. Scanning electron microscopic images showed that ECP caused partial disaggregation of collagen fibres into collagen fibrils by disrupting interfibrillar proteoglycan bridges. Differential scanning calorimetry and Fourier transform infrared analysis revealed increased structural disorder of fibrillar collagen caused by ECP. SDS-PAGE and chemical analysis indicated that ECP can liberate glycosaminoglycan, hydroxyproline and collagen fragments from collagen fibres. Thus ECP can cause disintegration of collagen fibres by degrading interfibrillar proteoglycan bridges. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Evaluation of nanohydroxyapaptite (nano-HA) coated epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes.

    PubMed

    Chu, Chenyu; Deng, Jia; Man, Yi; Qu, Yili

    2017-09-01

    Collagen is the main component of extracellular matrix (ECM) with desirable biological activities and low antigenicity. Collagen materials have been widely utilized in guided bone regeneration (GBR) surgery due to its abilities to maintain space for hard tissue growth. However, pure collagen lacks optimal mechanical properties. In our previous study, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, with better biological activities and enhanced mechanical properties, may promote osteoblast proliferation, but their effect on osteoblast differentiation is not very significant. Nanohydroxyapatite (nano-HA) is the main component of mineral bone, which possesses exceptional bioactivity properties including good biocompatibility, high osteoconductivity and osteoinductivity, non-immunogenicity and non-inflammatory behavior. Herein, by analyzing the physical and chemical properties as well as the effects on promoting bone regeneration, we have attempted to present a novel EGCG-modified collagen membrane with nano-HA coating, and have found evidence that the novel collagen membrane may promote bone regeneration with a better surface morphology, without destroying collagen backbone. To evaluate the surface morphologies, chemical and mechanical properties of pure collagen membranes, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, nano-HA coated collagen membranes, nano-HA coated EGCG-collagen membranes, (ii) to evaluate the bone regeneration promoted by theses membranes. In the present study, collagen membranes were divided into 4 groups: (1) untreated collagen membranes (2) EGCG cross-linked collagen membranes (3) nano-HA modified collagen membranes (4) nano-HA modified EGCG-collagen membranes. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) were used to evaluate surface morphologies and chemical properties, respectively. Mechanical properties were determined by differential scanning calorimeter (DSC

  5. Short-Fragment DNA Residue from Vaccine Purification Processes Promotes Immune Response to the New Inactivated EV71 Vaccine by Upregulating TLR9 mRNA

    PubMed Central

    Shao, Jie; Gao, Fan; Lin, Hui-Juan; Mao, Qun-Ying; Chen, Pan; Wu, Xing; Yao, Xin; Kong, Wei; Liang, Zheng-Lun

    2016-01-01

    To reduce potential oncogenic long genomic DNA in vaccines, nuclease treatment has been applied in the purification processes. However, this action increased the residue of short-fragment DNA and its effect on vaccine potency was still elusive. In this study, we found residual sf-DNA in an inactivated EV71 vaccine could enhance humoral immune response in mice. Ag stimulation in vitro and vaccine injection in vivo revealed that TLR9 transcription level was elevated, indicating that sf-DNA could activate TLR9. These new findings will help us to understand the molecular mechanism induced by vero-cell culture-derived vaccines. PMID:27082865

  6. Short-Fragment DNA Residue from Vaccine Purification Processes Promotes Immune Response to the New Inactivated EV71 Vaccine by Upregulating TLR9 mRNA.

    PubMed

    Shao, Jie; Gao, Fan; Lin, Hui-Juan; Mao, Qun-Ying; Chen, Pan; Wu, Xing; Yao, Xin; Kong, Wei; Liang, Zheng-Lun

    2016-01-01

    To reduce potential oncogenic long genomic DNA in vaccines, nuclease treatment has been applied in the purification processes. However, this action increased the residue of short-fragment DNA and its effect on vaccine potency was still elusive. In this study, we found residual sf-DNA in an inactivated EV71 vaccine could enhance humoral immune response in mice. Ag stimulation in vitro and vaccine injection in vivo revealed that TLR9 transcription level was elevated, indicating that sf-DNA could activate TLR9. These new findings will help us to understand the molecular mechanism induced by vero-cell culture-derived vaccines.

  7. Nanomechanics of collagen microfibrils

    PubMed Central

    Vesentini, Simone; Redaelli, Alberto; Gautieri, Alfonso

    2013-01-01

    Summary Collagen constitutes one third of the human proteome, providing mechanical stability, elasticity and strength to organisms and is thus the prime construction material in biology. Collagen is also the dominating material in the extracellular matrix where its stiffness controls cell differentiation, growth and pathology. We use atomistic-based hierarchical multiscale modeling to describe this complex biological material from the bottom up. This includes the use and development of large-scale computational modeling tools to investigate several aspects related to collagen-based tissues, including source of visco-elasticity and deformation mechanisms at the nanoscale level. The key innovation of this research is that until now, collagen materials have primarily been described at macroscopic scales, without explicitly understanding the mechanical contributions at the molecular and fibrillar levels. The major impact of this research will be the development of fundamental models of collagenous tissues, important to the design of new scaffolding biomaterials for regenerative medicine as well as for the understanding of collagen-related diseases. PMID:23885342

  8. Collagen V nasal tolerance in experimental model of systemic sclerosis.

    PubMed

    Velosa, Ana Paula Pereira; Teodoro, Walcy Rosolia; de Oliveira, Cristiane Carla; Dos Santos Filho, Antonio; Moutinho, Rodnei Francisco; Santos, Angela Gomes; Vendramini, Margarete Borges Galhardo; Bueno, Cleonice; Parra, Edwin Roger; Capelozzi, Vera Luiza; Yoshinari, Natalino Hajime

    2007-07-01

    Our aim was to study skin remodeling and autoantibody production in an experimental model of scleroderma (SSc), following nasal tolerance with human type V collagen (Col V). Female New Zealand rabbits (n = 12) were immunized with two doses of 1 mg/ml of Col V in complete Freund's adjuvant and additional two boosters in incomplete Freund's adjuvant to induce SSc. After 150 days, half of these immunized rabbits were submitted to type V collagen-induced tolerance receiving a daily nasal administration of 25 mug of Col V. Control animals (n = 6) were only submitted to type V collagen-induced tolerance. Serial skin biopsies were performed on days 0, 150 and 210, and stained with H&E, Masson's trichrome and Picrosirius for morphological and morphometric analysis. Types I, III and V collagen were identified by immunofluorescence. The animals' serum samples were collected to determine anti types I, III, IV and V collagen and antinuclear antibodies (ANA). Skin biopsies from immunized animals confirmed SSc morphology as previously described, such as progressive decrease of papillary dermis, appendages atrophy, increased type I, III and V collagen deposition. Rabbits with Col V-induced nasal tolerance showed reduction of skin involvement, with significant decrease of collagen amount. Humoral immune response did not change with nasal tolerance. Collagen V nasal tolerance promotes regression of skin remodeling process in an experimental model of SSc. We suggest that nasal tolerance with type V collagen can be a promising therapeutic option to treat scleroderma patients.

  9. Augmentation of diabetic wound healing and enhancement of collagen content using nanofibrous glucophage-loaded collagen/PLGA scaffold membranes.

    PubMed

    Lee, Cheng-Hung; Chang, Shang-Hung; Chen, Wei-Jan; Hung, Kuo-Chun; Lin, Yu-Huang; Liu, Shih-Jung; Hsieh, Ming-Jer; Pang, Jong-Hwei S; Juang, Jyuhn-Huarng

    2015-02-01

    This work developed nanofibrous drug-loaded collagen/poly-D-L-lactide-glycolide (PLGA) scaffold membranes that provided the sustained release of glucophage for the wounds associated with diabetes. PLGA, glucophage, and collagen were firstly dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol and were spun into nanofibrous membranes by electrospinning. High-performance liquid chromatography assay was used to characterize the in vivo and in vitro release rates of the pharmaceuticals from the membranes. High concentrations of glucophage were released for over three weeks from the nanofibrous membranes. The nanofibrous glucophage-loaded collagen/PLGA membranes were more hydrophilic than collagen/PLGA membranes and exhibited a greater water-containing capacity. The glucophage-loaded collagen/PLGA membranes markedly promoted the healing of diabetic wounds. Moreover, the collagen content of diabetic rats using drug-eluting membranes was higher than that of the control rats, because of the down-regulation of matrix metalloproteinase 9. The experimental results herein suggest that the nanofibrous glucophage-loaded collagen/PLGA membranes had effect for increasing collagen content in treating diabetic wounds and very effective promoters of the healing of such wounds in the early stages. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes.

    PubMed

    Solursh, M; Jensen, K L; Reiter, R S; Schmid, T M; Linsenmayer, T F

    1986-09-01

    We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences

  11. Glomerular Collagen V Codeposition and Hepatic Perisinusoidal Collagen III Accumulation in Canine Collagen Type III Glomerulopathy.

    PubMed

    Rørtveit, R; Reiten, M R; Lingaas, F; Sveri, S B; Brech, A; Espenes, A; Jansen, J H

    2015-11-01

    Collagen type III glomerulopathy, also known as collagenofibrotic glomerulopathy, is a rare renal disease of unknown pathogenesis. The disease occurs in humans and animals and is characterized by massive glomerular accumulations of collagen type III. In the present study, we describe a Drever dog litter affected by an early onset variant of this glomerular disease, where 4 of 9 puppies developed renal failure within 50 days of age. Necropsy specimens of kidney from the 4 affected cases were studied by light microscopy, electron microscopy, and immunohistochemistry, and characteristic lesions compatible with a diagnosis of collagen type III glomerulopathy were found. In addition, 2 cases showed atypical epithelium in the collecting ducts of the medulla, so-called adenomatoid change. Immunohistochemistry of renal specimens from collagen type III glomerulopathy-affected dogs (n = 10) originating from two different dog strains, the Drever dogs and a mixed-breed strain, demonstrated that the deposited glomerular collagen is composed of a mixture of collagen III and collagen V. The distribution of the collagen V corresponded to the localization of collagen III; however, differences in staining intensity showed that collagen type III is the dominating component. Immunohistochemistry for collagen III (n = 9) and a transmission electron microscopic study (n = 1) showed hepatic perisinusoidal collagen type III deposition in affected cases from both dog strains. This is the first report documenting glomerular accumulations of collagen type V and perisinusoidal liver collagen III deposition in canine collagen type III glomerulopathy. © The Author(s) 2014.

  12. Helicase-like transcription factor (Hltf) regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

    PubMed

    Helmer, Rebecca A; Martínez-Zaguilán, Raul; Dertien, Janet S; Fulford, Candra; Foreman, Oded; Peiris, Vasum; Chilton, Beverly S

    2013-01-01

    HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum) brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf's regulation of the G2/M transition (p=9.726E(-15)) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2) and lysyl (Plod2) collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3) for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus

  13. Development of multifunctional collagen scaffolds directed by collagen mimetic peptides

    NASA Astrophysics Data System (ADS)

    Wang, Yi-Lan (Allen)

    Collagen is widely used for soft tissue replacement and tissue engineering scaffold. Functionalized collagen may offer new and improved applications for collagen-based biomaterials. But passively adsorbed molecules readily diffuse out from collagen matrix, and conventional chemical reactions on collagen are difficult to control and may compromise the biochemical feature of natural collagen. Hence, the aim of this dissertation is to develop a new physical collagen modification method through the non-covalent immobilization of collagen mimetic peptides (CMPs) and CMP derivatives on collagen scaffolds, thereby evading the drawbacks of passive and chemical modifications. Most of the research on CMPs over the past three decades has focused on synthesizing CMPs and understanding the effects of amino acid sequence on the peptide structural stability. Although few attempts have been made to develop biomaterials based on pure CMP, CMP has never used in complex with natural collagen. We demonstrate that CMPs with varying chain lengths have strong propensity to associate with natural 2-D and 3-D collagen substrates. We also show that CMPs can recognize and bind to reconstituted type I collagen fibers as well as collagens of ex vivo human liver tissue. The practical use of CMPs conjugated with linear and multi-arm poly(ethylene glycol)s allows to control cell organization in 2-D collagen substrates. Our cell adhesion studies suggest that under certain conditions (e.g. high incubation temperature, small CMP size), the bound CMP derivatives can be released from the collagen matrix, which may provide new opportunities for manipulating cell behavior especially by dynamically controlling the amount of signaling molecules in the collagen matrix. Polyanionic charged CMP was synthesized to modulate tubulogenesis of endothelial cells by attracting VEGF with 3-D collagen gel and a new PEG hydrogel using bifunctional CMP conjugates was synthesized as physico-chemical crosslinkers for

  14. Collagen Prolyl Hydroxylases are Essential for Breast Cancer Metastasis

    PubMed Central

    Gilkes, Daniele M.; Chaturvedi, Pallavi; Bajpai, Saumendra; Wong, Carmen Chak-Lui; Wei, Hong; Pitcairn, Stephen; Hubbi, Maimon E.; Wirtz, Denis; Semenza, Gregg L.

    2013-01-01

    Metastasis is the leading cause of death among patients with breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies for cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Lastly, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies, and demonstrate that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer. PMID:23539444

  15. Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

    PubMed Central

    1994-01-01

    Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

  16. Collagen structural microheterogeneity and a possible role for glycosylated hydroxylysine in type I collagen

    PubMed Central

    Yamauchi, Mitsuo; Noyes, Claudia; Kuboki, Yoshinori; Mechanic, Gerald L.

    1982-01-01

    A three-chained peptide from type I collagen, crosslinked by hydroxyaldolhistidine, has been isolated from a tryptic digest of 5 M guanidine·HCl-insoluble bovine skin collagen (a small but as yet unknown percentage of the total collagen in whole skin). OsO4/NaIO4 specifically cleaved the crosslink at its double bond into a two-chained crosslink peptide and a single peptide. The sequence of the two-chained peptide containing the bifunctional crosslink was determined after amino acid analysis of the separated peptides. The crosslink consists of an aldehyde derived from hydroxylysine-87 in the aldehyde-containing cyanogen bromide fragment α1CB5ald and an aldehyde derived from the lysine in the COOH-terminal nonhelical region of the α1CB6ald fragment. The α1CB6ald portion of the peptide exhibited structural microheterogeneity, containing the inverted sequence Ala-Lys-His instead of the normal sequence Lys-Ala-His. This indicates that another structural gene exists for α1(I) chain. The original three-chained peptide did not contain any glycosylated hydroxylysine or glycosylated hydroxyaldolhistidine. The lack of glycosylation of hydroxylysine-87 in α1CB5, which is usually glycosylated, allowed formation of the aldehyde, and this, coupled with the sequence inversion, may have allowed formation of the nonreducible crosslink hydroxyaldolhistidine. We suggest that the role of glycosylation, a posttranslational modification, of specific hydroxylysine residues is to prevent their oxidative deamination to aldehydes, thereby precluding formation of complex stable crosslinks. Complex crosslinks would decrease the rate of collagen turnover. The decrease, with time, would increase the population of stable crosslinked collagen molecules, which would eventually accumulate with age. PMID:6961443

  17. Collagen fibrils: nanoscale ropes.

    PubMed

    Bozec, Laurent; van der Heijden, Gert; Horton, Michael

    2007-01-01

    The formation of collagen fibrils from staggered repeats of individual molecules has become "accepted" wisdom. However, for over thirty years now, such a model has failed to resolve several structural and functional questions. In a novel approach, it was found, using atomic force microscopy, that tendon collagen fibrils are composed of subcomponents in a spiral disposition-that is, their structure is similar to that of macroscale ropes. Consequently, this arrangement was modeled and confirmed using elastic rod theory. This work provides new insight into collagen fibril structure and will have wide application-from the design of scaffolds for tissue engineering and a better understanding of pathogenesis of diseases of bone and tendon, to the conservation of irreplaceable parchment-based museum exhibits.

  18. Collagen in organ development

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.

  19. Collagen in organ development

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.

  20. Studies on the molecular significance in the interaction of bilirubin with collagen.

    PubMed

    Nagarajan, Usharani; Gladstone Christopher, Jayakumar; Chandrasekaran, Bangaru; Jonnalagadda, Raghava Rao; Balachandran, Unni Nair; Kohsaku, Kawakami

    2013-10-01

    The present investigation is aimed to understand the physiological significance of bilirubin interaction with collagen. In human skin, collagen absorbs both free bilirubin and serum bound bilirubin from the human system. Interaction between bilirubin and collagen depends on time, temperature and concentration of bilirubin. There is an increase in the aggregation rate of collagen in the presence of biliruibin. At physiological condition, 125 nM of bilirubin is the maximum concentration absorbed by per mg of collagen molecule. Bilirubin accelerates the lateral growth of collagen fibrils by shifting its rate of nucleation. Moreover, collagen-bilirubin complex exhibit a tendency to undergo adsorption onto the surface of the fibroblast cells, showing detrimental effects on fibroblasts proliferations. Based on the collagen binding assays, the binding of bilirubin to collagen is found to be electrostatic in nature, which confirms binding between the amino acid fragment of α1 (I) region of collagen and carboxyl group of bilirubin. The biotinylated bilirubin derivatives show better binding to α1 (I) chain rather than α2 (I) chains which clearly designates that bilirubin shows greater affinity to α1 chains of collagen. This novel approach directs to reduce the occurrence of bilirubin in hyperbilirubinemia patients.

  1. Human Immunodeficiency Virus (HIV)-1 Infects Human Hepatic Stellate Cells and Promotes Collagen I and Monocyte Chemoattractant Protein-1 Expression: Implications for the Pathogenesis of HIV/Hepatitis C Virus–Induced Liver Fibrosis

    PubMed Central

    Tuyama, Ana C.; Hong, Feng; Saiman, Yedidya; Wang, Chuansheng; Ozkok, Derya; Mosoian, Arevik; Chen, Ping; Chen, Benjamin K.; Klotman, Mary E.; Bansal, Meena B.

    2010-01-01

    Patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) develop more rapid fibrosis than those infected with HCV only. In HIV/HCV-coinfected patients, fibrosis progression correlates with HIV RNA levels, suggesting a direct role of HIV in liver fibrogenesis. Chemokine (C-C motif) receptor 5 (CCR5) and cysteine-X-cysteine receptor 4 (CXCR4), the two major coreceptors required for HIV entry into cells, are expressed on activated hepatic stellate cells (HSCs), the principle fibrogenic cell type in the liver. We therefore examined whether HIV can infect HSCs, explored the potential mechanisms of viral entry, and assessed the impact of infection as reflected by the ability of HSCs to transfer virus to T lymphocytes and elicit a proinflammatory and profibrogenic response. We report that the laboratory-adapted viruses HIV-IIIB (CXCR4-tropic or X4) and HIV-BaL (CCR5-tropic or R5) and primary HIV isolates can infect both a human stellate cell line, LX-2, and primary human HSCs. HIV entry and gene expression in HSCs was confirmed using HIV–green fluorescent protein (GFP) expression viral constructs in the presence or absence of the reverse-transcriptase inhibitor azidothymidine. CD4 expression on a subset of primary HSCs was demonstrated using fluorescence-activated cell sorting and immunofluorescence staining. Blocking experiments in the presence of anti-CD4, anti-CXCR4, and anti-CCR5 revealed that HIV entry into HSCs is predominantly CD4/chemokine coreceptor-independent. HIV infection promoted HSC collagen I expression and secretion of the proinflammatory cytokine monocyte chemoattractant protein-1. Furthermore, infected LX-2 cells were capable of transferring GFP-expressing virus to T lymphocytes in a coculture system. Conclusion Taken together, our results suggest a potential role of HIV in liver fibrosis/inflammation mediated through effects on HSCs. The role of early highly active antiretroviral therapy initiation in patients with HIV

  2. Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells.

    PubMed

    Stringa, E; Knäuper, V; Murphy, G; Gavrilovic, J

    2000-06-01

    Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35 degrees C to mimic physiological conditions. PDGF-BB pre-treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by alpha2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on alphavbeta3 integrin. alphavbeta3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-beta tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix.

  3. Potency of Fish Collagen as a Scaffold for Regenerative Medicine

    PubMed Central

    Yamamoto, Kohei; Yanagiguchi, Kajiro

    2014-01-01

    Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine. PMID:24982861

  4. Thermal Memory in Self-Assembled Collagen Fibril Networks

    PubMed Central

    de Wild, Martijn; Pomp, Wim; Koenderink, Gijsje H.

    2013-01-01

    Collagen fibrils form extracellular networks that regulate cell functions and provide mechanical strength to tissues. Collagen fibrillogenesis is an entropy-driven process promoted by warming and reversed by cooling. Here, we investigate the influence of noncovalent interactions mediated by the collagen triple helix on fibril stability. We measure the kinetics of cold-induced disassembly of fibrils formed from purified collagen I using turbimetry, probe the fibril morphology by atomic force microscopy, and measure the network connectivity by confocal microscopy and rheometry. We demonstrate that collagen fibrils disassemble by subunit release from their sides as well as their ends, with complex kinetics involving an initial fast release followed by a slow release. Surprisingly, the fibrils are gradually stabilized over time, leading to thermal memory. This dynamic stabilization may reflect structural plasticity of the collagen fibrils arising from their complex structure. In addition, we propose that the polymeric nature of collagen monomers may lead to slow kinetics of subunit desorption from the fibril surface. Dynamic stabilization of fibrils may be relevant in the initial stages of collagen assembly during embryogenesis, fibrosis, and wound healing. Moreover, our results are relevant for tissue repair and drug delivery applications, where it is crucial to control fibril stability. PMID:23823240

  5. Collagen enhances compatibility and strength of glass ionomers.

    PubMed

    Chang, H-J; Wu, C-M; Chang, Y-C; Fanchiang, J-C; Shieh, D-B; Wong, T-Y

    2009-05-01

    Glass ionomers have been used for perforation repair and retrograde filling where biointegration with periodontal tissue is required. Collagen has been demonstrated to promote cellular adhesion and enhance mineral tissue compressive strength. It was hypothesized that an appropriate concentration of collagen integrated into glass ionomer may improve both bio-compatibility and the mechanical properties of the material. By SEM and AFM, we discovered 70-nm granules appearing on the surfaces of glass-ionomer/collagen hybrids. Acid-etching revealed irregularly shaped particles interlinked by membrane-like sheets on the surface of the material with the typical 70-nm granules. WST-1 assay showed that acid-etching significantly enhanced the viability of attached gingival fibroblasts. However, the glass-ionomer/collagen hybrids' combined surface-etching outperformed other groups. The glass-ionomer/collagen hybrids presented enhanced compressive strength when integrated with 0.01% collagen, while higher concentrations of collagen compromised their mechanical property. In summary, collagen improved both the mechanical and biocompatible properties of glass ionomers. Further in vivo study is warranted.

  6. [The genetics of collagen diseases].

    PubMed

    Kaplan, J; Maroteaux, P; Frezal, J

    1986-01-01

    Heritable disorders of collagen include Ehler-Danlos syndromes (11 types are actually known), Larsen syndrome and osteogenesis imperfecta. Their clinical, genetic and biochemical features are reviewed. Marfan syndrome is closely related to heritable disorders of collagen.

  7. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    PubMed

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

  8. A collagen network formation effector from leaves of Premna subscandens.

    PubMed

    Sudo, H; Kijima, K; Otsuka, H; Ide, T; Hirata, E; Takeda, Y; Isaji, M; Kurashina, Y

    1999-09-01

    As a part of the search for biologically active plant products, M cells, which form a collagen fiber network in vitro after a prolonged culture period, were used. The n-BuOH-soluble fraction of a methanol extract of leaves of Premna subscandens exhibited promotion of collagen network formation by M cells. Extensive isolation work guided by a bioassay afforded a phenylethanoid, acteoside, as an active compound.

  9. Technique to accurately quantify collagen content in hyperconfluent cell culture.

    PubMed

    See, Eugene Yong-Shun; Toh, Siew Lok; Goh, James Cho Hong

    2008-12-01

    Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.

  10. Collagen models as a probe in the decay of works of art: synthesis, conformation and immunological studies.

    PubMed

    Zevgiti, Stella; Sakarellos, Constantinos; Sakarellos-Daitsiotis, Maria; Ioakimoglou, Eleni; Panou-Pomonis, Eugenia

    2007-02-01

    Proteinaceous substances such as collagen, casein and albumin have been widely used as binding media in a variety of works of art. Damages of these 'sensitive' materials, mainly caused of the influence of the environment, are responsible for the overall decay of works of art, and their identification is essential to understand ancient technologies, determine the extent of deterioration and help in future restoration and preservation processes. The most commonly used techniques for the identification of proteinaceous binding media are staining techniques, chromatography, spectrometry and immunological methods, although for the latter no systematic studies have been carried out until now. Aiming at contributing to the development of a reliable and reproducible immunoassay for the evaluation of the collagen-based decay of works of art, sequential polypeptides (Pro-X-Gly)n where X represents amino acid residues Val, Lys, Glu and (Hyp-Val-Gly)n were prepared as models of collagen fragments derived from artificially and naturally aged animal collagens. Conformational studies of the polypeptides by CD revealed the occurrence of polyproline II-like structures comparable with those of collagen. Polypeptides and collagen I were administered to animals, and the induced antibodies were used for the immunochemical detection and differentiation of collagen and collagen fragments. The combined application of (i) anti-collagen antibodies, which strongly interact with native collagen, but poorly recognized by artificially aged collagen and (ii) anti-polypeptide antibodies, which do not associate with native collagen, but are strongly recognized by collagen fragments in naturally or artificially aged collagen, is a valuable tool in determining the extent of decay in works of art.

  11. Adipocyte-derived collagen VI affects early mammary tumor progression in vivo, demonstrating a critical interaction in the tumor/stroma microenvironment

    PubMed Central

    Iyengar, Puneeth; Espina, Virginia; Williams, Terence W.; Lin, Ying; Berry, David; Jelicks, Linda A.; Lee, Hyangkyu; Temple, Karla; Graves, Reed; Pollard, Jeffrey; Chopra, Neeru; Russell, Robert G.; Sasisekharan, Ram; Trock, Bruce J.; Lippman, Marc; Calvert, Valerie S.; Petricoin, Emanuel F.; Liotta, Lance; Dadachova, Ekaterina; Pestell, Richard G.; Lisanti, Michael P.; Bonaldo, Paolo; Scherer, Philipp E.

    2005-01-01

    The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen–/– mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and β-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIα3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI. PMID:15841211

  12. Inhibition of Staphylococcus aureus Adherence to Collagen under Dynamic Conditions

    PubMed Central

    Mohamed, Nehal; Teeters, Mark A.; Patti, Joseph M.; Höök, Magnus; Ross, Julia M.

    1999-01-01

    Staphylococcus aureus is the most common etiological agent of bacterial arthritis and acute osteomyelitis and has been shown to bind to type II collagen under static and dynamic conditions. We have previously reported the effect of shear on the adhesion of S. aureus Phillips to collagen and found that this process is shear dependent (Z. Li, M. Höök, J. M. Patti, and J. M. Ross, Ann. Biomed. Eng. 24[Suppl. 1]:S–55). In this study, we used recombinant collagen adhesin fragments as well as polyclonal antibodies generated against adhesin fragments in attempts to inhibit bacterial adhesion. A parallel-plate flow chamber was used in a dynamic adhesion assay, and quantification of adhesion was accomplished by phase contrast video microscopy coupled with digital image processing. We report that both recombinant fragments studied, M19 and M55, and both polyclonal antibodies studied, α-M17 and α-M55, inhibit adhesion to varying degrees and that these processes are shear dependent. The M55 peptide and α-M55 cause much higher levels of inhibition than M19 and α-M17, respectively, at all wall shear rates studied. Our results demonstrate the importance of using a dynamic system in the assessment of inhibitory strategies and suggest the possible use of M55 and α-M55 in clinical applications to prevent infections caused by S. aureus adhesion to collagen. PMID:9916063

  13. Collagen hydrolysate based collagen/hydroxyapatite composite materials

    NASA Astrophysics Data System (ADS)

    Ficai, Anton; Albu, Madalina Georgiana; Birsan, Mihaela; Sonmez, Maria; Ficai, Denisa; Trandafir, Viorica; Andronescu, Ecaterina

    2013-04-01

    The aim of this study was to study the influence of collagen hydrolysate (HAS) on the formation of ternary collagen-hydrolysate/hydroxyapatite composite materials (COLL-HAS/HA). During the precipitation process of HA, a large amount of brushite is resulted at pH = 7 but, practically pure HA is obtained at pH ⩾ 8. The FTIR data reveal the duplication of the most important collagen absorption bands due to the presence of the collagen hydrolysate. The presence of collagen hydrolysate is beneficial for the management of bone and joint disorders such as osteoarthritis and osteoporosis.

  14. Design and synthesis of collagen mimetic peptide derivatives for studying triple helix assembly and collagen mimetic peptide-collagen binding interaction

    NASA Astrophysics Data System (ADS)

    Mo, Xiao

    2008-10-01

    region, one of the thermally unstable domains in the collagen molecule. The binding results at various temperatures were in good agreement with our hypothesis that CMP-collagen binding occurs through strand invasion reaction of the thermally unstable collagen domain. In addition, purple membrane (PM), a protein crystal patch from halobacteria cell membrane, was genetically engineered to display cysteine or histidine groups on the membrane surface with defined nanoscale symmetry. X-ray diffraction results showed that the engineered PM formed a stable 2D PM-like hexagonal crystal lattice (unit cell: 6.2 mm). We utilized the genetically engineered PM as a bio-template for inorganic nanoparticle nucleation and assembly. The specific functional groups (cysteine or histidine) on the surfaces of genetically engineered PMs exhibited designed reactivity for inorganic nanoparticles while promoting the formation of nanoscale 2D particle assembly.

  15. The C-terminal fragment of parathyroid hormone-related peptide promotes bone formation in diabetic mice with low-turnover osteopaenia

    PubMed Central

    Lozano, D; Fernández-de-Castro, L; Portal-Núñez, S; López-Herradón, A; Dapía, S; Gómez-Barrena, E; Esbrit, P

    2011-01-01

    BACKGROUND AND PURPOSE Current data suggest that parathyroid hormone (PTH)-related peptide (PTHrP) domains other than the N-terminal PTH-like domain contribute to its role as an endogenous bone anabolic factor. PTHrP-107-139 inhibits bone resorption, a fact which has precluded an unequivocal demonstration of its possible anabolic action in vivo. We thus sought to characterize the osteogenic effects of this peptide using a mouse model of diabetic low-turnover osteopaenia. EXPERIMENTAL APPROACH PTHrP-107-139 was administered to streptozotocin-induced diabetic mice, with or without bone marrow ablation, for 13 days. Osteopaenia was confirmed by dual-energy X-ray absorptiometry and microcomputed tomography analysis. Histological analysis was performed on paraffin-embedded bone tissue sections by haematoxylin/eosin and Masson's staining, and tartrate-resistent acid phosphatase immunohistochemistry. Mouse bone marrow stromal cells and osteoblastic MC3T3-E1 cells were cultured in normal and/or high glucose (HG) medium. Osteogenic and adipogenic markers were assessed by real-time PCR, and PTHrP and the PTH1 receptor protein expression by Western blot analysis. KEY RESULTS PTHrP-107-139 reversed the alterations in bone structure and osteoblast function, and also promoted bone healing after marrow ablation without affecting the number of osteoclast-like cells in diabetic mice. This peptide also reversed the high-glucose-induced changes in osteogenic differentiation in both bone marrow stromal cells and the more differentiated MC3T3-E1 cells. CONCLUSIONS AND IMPLICATIONS These findings demonstrate that PTHrP-107-139 promotes bone formation in diabetic mice. This mouse model and in vitro cell cultures allowed us to identify various anabolic effects of this peptide in this scenario. PMID:21175568

  16. Collagen remodeling by phagocytosis is determined by collagen substrate topology and calcium-dependent interactions of gelsolin with nonmuscle myosin IIA in cell adhesions

    PubMed Central

    Arora, P. D.; Wang, Y.; Bresnick, A.; Dawson, J.; Janmey, P. A.; McCulloch, C. A.

    2013-01-01

    We examine how collagen substrate topography, free intracellular calcium ion concentration ([Ca2+]i, and the association of gelsolin with nonmuscle myosin IIA (NMMIIA) at collagen adhesions are regulated to enable collagen phagocytosis. Fibroblasts plated on planar, collagen-coated substrates show minimal increase of [Ca2+]i, minimal colocalization of gelsolin and NMMIIA in focal adhesions, and minimal intracellular collagen degradation. In fibroblasts plated on collagen-coated latex beads there are large increases of [Ca2+]i, time- and Ca2+-dependent enrichment of NMMIIA and gelsolin at collagen adhesions, and abundant intracellular collagen degradation. NMMIIA knockdown retards gelsolin recruitment to adhesions and blocks collagen phagocytosis. Gelsolin exhibits tight, Ca2+-dependent binding to full-length NMMIIA. Gelsolin domains G4–G6 selectively require Ca2+ to interact with NMMIIA, which is restricted to residues 1339–1899 of NMMIIA. We conclude that cell adhesion to collagen presented on beads activates Ca2+ entry and promotes the formation of phagosomes enriched with NMMIIA and gelsolin. The Ca2+ -dependent interaction of gelsolin and NMMIIA in turn enables actin remodeling and enhances collagen degradation by phagocytosis. PMID:23325791

  17. The non-phagocytic route of collagen uptake: a distinct degradation pathway.

    PubMed

    Madsen, Daniel H; Ingvarsen, Signe; Jürgensen, Henrik J; Melander, Maria C; Kjøller, Lars; Moyer, Amanda; Honoré, Christian; Madsen, Charlotte A; Garred, Peter; Burgdorf, Sven; Bugge, Thomas H; Behrendt, Niels; Engelholm, Lars H

    2011-07-29

    The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.

  18. Pathogenic prion protein fragment (PrP106-126) promotes human immunodeficiency virus type-1 infection in peripheral blood monocyte-derived macrophages.

    PubMed

    Bacot, Silvia M; Feldman, Gerald M; Yamada, Kenneth M; Dhawan, Subhash

    2015-02-01

    Transfusion of blood and blood products contaminated with the pathogenic form of prion protein Prp(sc), thought to be the causative agent of variant a Creutzfeldt-Jakob disease (vCJD), may result in serious consequences in recipients with a compromised immune system, for example, as seen in HIV-1 infection. In the present study, we demonstrate that treatment of peripheral blood monocyte-derived macrophages (MDM) with PrP106-126, a synthetic domain of PrP(sc) that has intrinsic functional activities related to the full-length protein, markedly increased their susceptibility to HIV-1 infection, induced cytokine secretion, and enhanced their migratory behavior in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Live-cell imaging of MDM cultured in the presence of PrP106-126 showed large cell clusters indicative of cellular activation. Tyrosine kinase inhibitor STI-571, protein kinase C inhibitor K252B, and cyclin-dependent kinase inhibitor olomoucine attenuated PrP106-126-induced altered MDM functions. These findings delineate a previously undefined functional role of PrP106-126-mediated host cell response in promoting HIV-1 pathogenesis.

  19. [Production of recombinant fragments of the Clostridium tetani neurotoxin for the development of new immune-prophylaxis preparations against tetanus].

    PubMed

    Varfolomeeva, N A; Makhotina, O A; Sergeeva, T I; Belyĭ, Iu F

    2003-01-01

    Tetanus belongs to dangerous infection diseases, whose effective prevention can be ensured by vaccines. The acting substance of tetanus vaccines, presently in use, is a partially purified and deprived-of-lethal-action Clostridium tetani neurotoxin. The construction of a subunit preparation on the basis of toxin fragments obtained through gene engineering could be a method aimed at promoting the quality of the used tetanus vaccines. With this goal in mind, we built, within the present case study, the expressing genetic constructions and obtained, in the pure form, an extensive tetanus-vaccine chain with its C-terminal (Hc) fragment, hydride peptides, containing the Hc-fragment and C-terminal fragment of toxin B C. difficile, as well as Hc-fragment and S3 collagen-binding domain of collagenase C. histolyticum. The thus obtained proteins can be used in testing their immunogenic and protective properties, while the conducted study could be a basis for further research of a new-generation vaccine against tetanus and other human infection diseases.

  20. Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

    PubMed Central

    McKleroy, William; Lee, Ting-Hein

    2013-01-01

    Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production. PMID:23564511

  1. Calcium concentration dependent collagen mineralization.

    PubMed

    Niu, Xufeng; Fan, Rui; Tian, Feng; Guo, Xiaolin; Li, Ping; Feng, Qingling; Fan, Yubo

    2017-04-01

    Mineralization of collagen fibrils is a regular combination process of organic and mineral components mainly involving calcium, phosphate and collagen. We report the influence of calcium to the self-assembly of collagen by changing the concentration of calcium ion in the process of mineralization. Low concentration of calcium results in the well collagen self-assembly while poor mineral crystallization. Relatively, high concentration of calcium can hinder collagen self-assembly, whereas it is benefited to mineral crystallization. We also reveal that collagen self-assembly happens in advance of the formation of better mineral crystals. These results interpret the mechanism of collagen mineralization further. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Voorhees, John J; Fisher, Gary J

    2012-09-01

    Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.

  3. Characterization of electrospun nanofiber matrices made of collagen blends as potential skin substitutes.

    PubMed

    Lin, Hsin-Yi; Kuo, Yu-Jen; Chang, Shih-Hsin; Ni, Tsung-Sheng

    2013-04-01

    Collagen and its blends, collagen/polyvinyl alcohol (PVA) and collagen/chitosan/PVA, were made into nanofibers by electrospinning. The nanofibrous matrices were evaluated for their potential as skin substitutes. The addition of PVA to collagen increased the swelling ratio of the nanofibers, their Young's modulus, strain at break and ultimate tensile strength. The addition of chitosan to collagen/PVA reduced its swelling ratio and its strain at break, but increased the Young's modulus and ultimate tensile strength. Both PVA and chitosan stabilized the collagen fibers in an aqueous solution. The addition of PVA, but not chitosan, promoted initial fibroblast cell proliferation on the matrices. Compared to the skin substitute made of pure collagen, the substitutes with PVA and chitosan showed improved structural stability in aqueous solution, better tensile strength and similar or better biocompatibility in vitro.

  4. Liquid Collagen Wound Coverings.

    DTIC Science & Technology

    1992-05-13

    3-compart- metal iodide with a suitable oxidizing agent such as ment sterile blood bag type container which is then heat persulfate, perborate and a...Time oxidizing agent is selected from the group consisting of10 persulfates, perborates , hydrogen peroxide, tertary 2.24 11.25 107 5.0 butyl... perborates , hydrogen peroxide, tertary butyl per- What is claimed is. oxide, alkali metal periodate, hypochlorite salts and free 1. A collagen gel

  5. Characterizing matrix remodeling in collagen gels using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Levitz, David; Hinds, Monica T.; Hanson, Stephen R.; Jacques, Steven L.

    2010-02-01

    Optical coherence tomography (OCT) has shown promise at non-destructively characterizing engineered tissues such as collagen gels. However, as the collagen gels develop, the OCT images lose contrast of structures as the gels develop, making visual assessment difficult. Our group proposed quantitatively characterizing these gels by fitting the optical properties from the OCT signals. In this paper, we imaged collagen gels seeded with smooth muscle cells (SMCs) over a 5-day period and used the data to measure their optical properties. Our results showed that over time, the reflectivity of the samples increased 10-fold, corresponding to a decrease in anisotropy factor g, without much change in the scattering coefficient μs. Overall, the optical properties appeared to be dominated by scattering from the collagen matrix, not the cells. However, SMCs remodeled the collagen matrix, and this collagen remodeling by the cells is what causes the observed changes in optical properties. Moreover, the data showed that the optical properties were sensitive to the activity of matrix metalloproteinases (MMPs), enzymes that break down local collagen fibrils into smaller fragments. Blocking MMPs in the SMC gels greatly impeded both the remodeling process and change in optical properties at day 5. Treating day 1 acellular gels with MMP-8 for 3 hr managed to partially reproduce the remodeling observed in SMC gels at day 5. Altogether, we conclude that matrix remodeling in general, and MMPs specifically, greatly affect the local optical properties of the sample, and OCT is a unique tool that can assess MMP activity in collagen gels both non-destructively and label free.

  6. Osteocalcin/fibronectin-functionalized collagen matrices for bone tissue engineering.

    PubMed

    Kim, S G; Lee, D S; Lee, S; Jang, J-H

    2015-06-01

    Collagen is the most abundant protein found in the extracellular matrix and is widely used to build scaffolds for biomedical applications which are the result of its biocompatibility and biodegradability. In the present study, we constructed a rhOCN/FNIII9-10 fusion protein and rhOCN/FNIII9-10-functionalized collagen matrices and investigated the potential value for bone tissue engineering. In vitro studies carried out with preosteoblastic MC3T3-E1 cells showed that rhOCN/FNIII9-10 fusion protein promoted cell adhesion and the mRNA levels of osteogenic markers including osteocalcin, runt-related transcription factor 2, alkaline phosphatase (ALP), and collagen type I. In addition, rhOCN/FNIII9-10-functionalized collagen matrices showed significant induction of the ALP activity more than rhFNIII9-10-functionalized collagen matrices or collagen matrices alone. These results suggested that rhOCN/FNIII9-10-functionalized collagen matrices have potential for bone tissue engineering.

  7. Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

    PubMed

    Salces-Ortiz, Judit; Ramón, Manuel; González, Carmen; Pérez-Guzmán, M Dolores; Garde, J Julián; García-Álvarez, Olga; Maroto-Morales, Alejandro; Calvo, Jorge H; Serrano, M Magdalena

    2015-01-01

    Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram's fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and

  8. Differences in the Ovine HSP90AA1 Gene Expression Rates Caused by Two Linked Polymorphisms at Its Promoter Affect Rams Sperm DNA Fragmentation under Environmental Heat Stress Conditions

    PubMed Central

    González, Carmen; Pérez-Guzmán, M. Dolores; Garde, J. Julián; García-Álvarez, Olga; Maroto-Morales, Alejandro; Calvo, Jorge H.; Serrano, M. Magdalena

    2015-01-01

    Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram’s fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and

  9. Polarized Raman anisotropic response of collagen in tendon: towards 3D orientation mapping of collagen in tissues.

    PubMed

    Galvis, Leonardo; Dunlop, John W C; Duda, Georg; Fratzl, Peter; Masic, Admir

    2013-01-01

    In this study, polarized Raman spectroscopy (PRS) was used to characterize the anisotropic response of the amide I band of collagen as a basis for evaluating three-dimensional collagen fibril orientation in tissues. Firstly, the response was investigated theoretically by applying classical Raman theory to collagen-like peptide crystal structures. The theoretical methodology was then tested experimentally, by measuring amide I intensity anisotropy in rat tail as a function of the orientation of the incident laser polarization. For the theoretical study, several collagen-like triple-helical peptide crystal structures obtained from the Protein Data Bank were rotated "in plane" and "out of plane" to evaluate the role of molecular orientation on the intensity of the amide I band. Collagen-like peptides exhibit a sinusoidal anisotropic response when rotated "in plane" with respect to the polarized incident laser. Maximal intensity was obtained when the polarization of the incident light is perpendicular to the molecule and minimal when parallel. In the case of "out of plane" rotation of the molecular structure a decreased anisotropic response was observed, becoming completely isotropic when the structure was perpendicular to the plane of observation. The theoretical Raman response of collagen was compared to that of alpha helical protein fragments. In contrast to collagen, alpha helices have a maximal signal when incident light is parallel to the molecule and minimal when perpendicular. For out-of-plane molecular orientations alpha-helix structures display a decreased average intensity. Results obtained from experiments on rat tail tendon are in excellent agreement with the theoretical predictions, thus demonstrating the high potential of PRS for experimental evaluation of the three-dimensional orientation of collagen fibers in biological tissues.

  10. The Biological Function of DMP-1 in Osteocyte Maturation Is Mediated by Its 57-kDa C-terminal Fragment

    PubMed Central

    Lu, Yongbo; Yuan, Baozhi; Qin, Chunlin; Cao, Zhengguo; Xie, Yixia; Dallas, Sarah L; McKee, Marc D; Drezner, Marc K; Bonewald, Lynda F; Feng, Jian Q

    2011-01-01

    Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism. © 2011 American Society for Bone and Mineral Research. PMID:20734454

  11. UV damage of collagen: insights from model collagen peptides.

    PubMed

    Jariashvili, Ketevan; Madhan, Balaraman; Brodsky, Barbara; Kuchava, Ana; Namicheishvili, Louisa; Metreveli, Nunu

    2012-03-01

    Fibrils of Type I collagen in the skin are exposed to ultraviolet (UV) light and there have been claims that collagen photo-degradation leads to wrinkles and may contribute to skin cancers. To understand the effects of UV radiation on collagen, Type I collagen solutions were exposed to the UV-C wavelength of 254 nm for defined lengths of time at 4°C. Circular dichroism (CD) experiments show that irradiation of collagen leads to high loss of triple helical content with a new lower thermal stability peak and SDS-gel electrophoresis indicates breakdown of collagen chains. To better define the effects of UV radiation on the collagen triple-helix, the studies were extended to peptides which model the collagen sequence and conformation. CD studies showed irradiation for days led to lower magnitudes of the triple-helix maximum at 225 nm and lower thermal stabilities for two peptides containing multiple Gly-Pro-Hyp triplets. In contrast, the highest radiation exposure led to little change in the T(m) values of (Gly-Pro-Pro)(10) and (Ala-Hyp-Gly)(10) , although (Gly-Pro-Pro)(10) did show a significant decrease in triple helix intensity. Mass spectroscopy indicated preferential cleavage sites within the peptides, and identification of some of the most susceptible sites of cleavage. The effect of radiation on these well defined peptides gives insight into the sequence and conformational specificity of photo-degradation of collagen.

  12. Heterogeneity of collagens in rabbit cornea: type VI collagen

    SciTech Connect

    Cintron, C.; Hong, B.S.

    1988-05-01

    Normal adult rabbit corneas were digested with 5% pepsin and their collagens extracted with acetic acid. Collagen extracts were fractionated by differential salt precipitation. The 2.5 M NaCl fraction was then redissolved with tris buffer and precipitated with sodium acetate. The precipitate contained a high-molecular-weight disulfide-bonded aggregate which, upon reduction with mercaptoethanol, was converted into three distinct polypeptides having molecular weights between 45 and 66 Kd. These physical characteristics, together with the susceptibility of these polypeptides to collagenase and their amino acid composition, identified the high molecular weight aggregate as type VI collagen. Corneas from neonate rabbits and adult corneas containing 2-week-old scars were organ cultured in the presence of (/sup 14/C) glycine to incorporate radiolabel into collagen. Tissues were digested with 0.02% pepsin and their collagens extracted with formic acid. The total radioactivity of the extracts and tissue residues was determined before the collagens were separated by SDS-polyacrylamide slab gel electrophoresis. Radioactive collagen polypeptides bands were then stained with Coomassie blue, processed for fluorography, and analyzed by densitometry. The results show that: (1) type VI collagen is synthesized by neonate corneas and healing adult corneas; (2) it is not readily solubilized from either corneal tissue by 0.02% pepsin digestion and formic acid extraction; and (3) the proportion of type VI collagen deposited in scar tissue is markedly lower than that found in neonate corneas.

  13. Heterogeneity of collagens in rabbit cornea: type III collagen

    SciTech Connect

    Cintron, C.; Hong, B.S.; Covington, H.I.; Macarak, E.J.

    1988-05-01

    Whole neonate rabbit corneas and adult corneas containing 2-week-old scars were incubated in the presence of (/sup 14/C) glycine. Radiolabeled collagen extracted from the corneas and scar tissue were analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography to determine the types and relative quantity of collagen polypeptides present and synthesized by these tissues. In addition to other collagen types, type III was found in both neonate cornea and scar tissue from adult cornea, albeit in relatively small quantities. Type III collagen in normal cornea was associated with the residue after pepsin digestion and formic acid extraction of the tissue, and the same type of collagen was extracted from scar tissue after similar treatment. Type III collagen-specific monoclonal antibody bound to developing normal corneas and healing adult tissue sections, as determined by immunofluorescence. Antibody binding was localized to the endothelium and growing Descemet's membrane in fetal and neonate corneas, and restricted to the most posterior region of the corneal scar tissue. Although monoclonal antibody to keratan sulfate, used as a marker for stromal fibroblasts, bound to most of the scar tissue, the antibody failed to bind to the posterior scar tissue positive for type III collagen. We conclude that endothelial cells from fetal and neonate rabbit cornea and endothelium-derived fibroblasts from healing wounds of adult cornea synthesize and deposit type III collagen. Moreover, this collagen appears to be incorporated into the growing Descemet's membrane of normal corneas and narrow posterior portion of the scar tissue.

  14. AggLb Is the Largest Cell-Aggregation Factor from Lactobacillus paracasei Subsp. paracasei BGNJ1-64, Functions in Collagen Adhesion, and Pathogen Exclusion In Vitro

    PubMed Central

    Miljkovic, Marija; Strahinic, Ivana; Tolinacki, Maja; Zivkovic, Milica; Kojic, Snezana; Golic, Natasa; Kojic, Milan

    2015-01-01

    Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization. PMID:25955159

  15. Collagen macromolecular drug delivery systems

    SciTech Connect

    Gilbert, D.L.

    1988-01-01

    The objective of this study was to examine collagen for use as a macromolecular drug delivery system by determining the mechanism of release through a matrix. Collagen membranes varying in porosity, crosslinking density, structure and crosslinker were fabricated. Collagen characterized by infrared spectroscopy and solution viscosity was determined to be pure and native. The collagen membranes were determined to possess native vs. non-native quaternary structure and porous vs. dense aggregate membranes by electron microscopy. Collagen monolithic devices containing a model macromolecule (inulin) were fabricated. In vitro release rates were found to be linear with respect to t{sup {1/2}} and were affected by crosslinking density, crosslinker and structure. The biodegradation of the collagen matrix was also examined. In vivo biocompatibility, degradation and {sup 14}C-inulin release rates were evaluated subcutaneously in rats.

  16. A novel combined polyphenol-aldehyde crosslinking of collagen film-Applications in biomedical materials.

    PubMed

    Liu, Ting; Shi, Lu; Gu, Zhipeng; Dan, Weihua; Dan, Nianhua

    2017-03-30

    Despite its crucial role in directing cell fate in healthy and diseased tissues, improvements in physical-chemical properties and biocompatibility of type-I collagen are still needed. In this report, we described combined and facile method to modify collagen. The collagen film was first modified by procyanidins solution, in which, then subjected to further crosslinked by dialdehyde alginate, resulting in collagen-procyanidins-dialdehyde alginate film. The properties of the crosslinked collagen films were investigated and the results were discussed. Results from differential scanning calorimetry and thermo gravimetric analysis suggested that the thermal stabilities of the collagen-procyanidins-dialdehyde alginate film were significantly improved. The mechanical properties of collagen-procyanidins-dialdehyde alginate film in terms of elongation at break and tensile strength increased approximately 2-fold and 3-fold, respectively compare to pure collagen film. In addition, the resistance to collagenase degradation of collagen-procyanidins-dialdehyde alginate film was remarkably promoted. The results from methyltetrazolium assay and confocal laser scanning microscopy showed that no cytotoxicity of collagen film was introduced by the combined crosslinking method. Thus, the novel combined by procyanidins-dialdehyde alginate crosslinking method shown in this study provided a non-toxic and efficient crosslinking method that improved various properties of collagen film, which has great potential applications in biomedical materials.

  17. Impacts of fullerene derivatives on regulating the structure and assembly of collagen molecules.

    PubMed

    Yin, Xiaohui; Zhao, Lina; Kang, Seung-gu; Pan, Jun; Song, Yan; Zhang, Mingyi; Xing, Gengmei; Wang, Fei; Li, Jingyuan; Zhou, Ruhong; Zhao, Yuliang

    2013-08-21

    During cancer development, the fibrous layers surrounding the tumor surface get thin and stiff which facilitates the tumor metastasis. After the treatment of metallofullerene derivatives Gd@C82(OH)22, the fibrous layers become thicker and softer, the metastasis of tumor is then largely suppressed. The effect of Gd@C82(OH)22 was found to be related to their direct interaction with collagen and the resulting impact on the structure of collagen fibrils, the major component of extracellular matrices. In this work we study the interaction of Gd@C82(OH)22 with collagen by molecular dynamics simulations. We find that Gd@C82(OH)22 can enhance the rigidity of the native structure of collagen molecules and promote the formation of an oligomer or a microfibril. The interaction with Gd@C82(OH)22 may regulate further the assembly of collagen fibrils and change the biophysical properties of collagen. The control run with fullerene derivatives C60(OH)24 also indicates that C60(OH)24 can influence the structure and assembly of collagen molecules as well, but to a lesser degree. Both fullerene derivatives can form hydrogen bonds with multiple collagen molecules acting as a "fullerenol-mediated bridge" that enhance the interaction within or among collagen molecules. Compared to C60(OH)24, the interaction of Gd@C82(OH)22 with collagen is stronger, resulting in particular biomedical effects for regulating the biophysical properties of collagen fibrils.

  18. Bacterial collagen-like proteins that form triple-helical structures.

    PubMed

    Yu, Zhuoxin; An, Bo; Ramshaw, John A M; Brodsky, Barbara

    2014-06-01

    A large number of collagen-like proteins have been identified in bacteria during the past 10years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in Escherichia coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35-39°C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.

  19. Bacterial collagen-like proteins that form triple-helical structures

    PubMed Central

    Yu, Zhuoxin; An, Bo; Ramshaw, John A.M.; Brodsky, Barbara

    2014-01-01

    A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions. PMID:24434612

  20. Second harmonic generation in collagen

    NASA Astrophysics Data System (ADS)

    Reiser, Karen M.; Stoller, Patrick; Celliers, Peter; Rubenchik, Alexander; Bratton, Clay; Yankelevich, Diego

    2003-11-01

    Collagen possesses a strong second order nonlinear susceptibility; when it is irradiated with intense laser light, some of the reflected and transmitted light will have twice the frequency of the incident beam, a phenomenon known as second harmonic generation (SHG). Polarization modulation of an ultra-short pulse laser beam can be used to simultaneously measure collagen fiber orientation, SHG intensity, and a parameter related to the second order non-linear susceptibility. This technique has made it possible to discriminate among patterns of fibrillar orientation in many tissues. In the present study the role that organizational complexity plays in the relationship between nonlinear optical properties and collagen structure is investigated. As a component of tissues and organs, collagen"s structure and function is inextricably intertwined with that of the many other matrix components; to what extent do these noncollagenous components affect its nonlinear properties? To answer this, we investigated SHG in two different collagenous tissues, liver and cartilage; in addition we looked at the effect of progressive pathological changes in these tissues on SHG. At the other end of the spectrum, we studied collagen organized at the minimal level of complexity necessary for SHG detection: fibrils generated from solutions containing only a single type of collagen. Data obtained from these studies suggest that collagen"s strong nonlinear susceptibility, a property no other biologically significant macromolecule shares to the same degree, may serve as more than the basis of a novel imaging device for soft tissue. Collagen"s nonlinear optical properties in conjunction with its vast capacity for self-initiated conformational change--through self-assembly, site recognition, post-translational modification, and the like -make it an attractive candidate molecule for any of several demanding engineering applications, such as nanopatterning.

  1. Collagenous colitis: an unrecognised entity.

    PubMed Central

    Bogomoletz, W V; Adnet, J J; Birembaut, P; Feydy, P; Dupont, P

    1980-01-01

    A patient is reported with chronic abdominal pain, diarrhoea, and associated radiological and endoscopic abnormalities of the sigmoid colon. Light and electron microscopic study of colorectal mucosa showed abnormal collagenous thickening of the subepithelial basement membrane. The authors felt that the clinical and morphological features justified a diagnosis of collagenous colitis. Review of the literature suggested that collagenous colitis was still an unrecognised entity. Images Fig. 1 Fig. 2 Fig. 3 PMID:7380341

  2. Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis.

    PubMed

    Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P R O

    2008-02-26

    We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

  3. Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis

    SciTech Connect

    Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P.R.O.

    2008-06-24

    We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or 'collagenolysis.' The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's 'interaction domain,' which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

  4. Effect of microgravity on collagenase deproteinization and EDTA decalcification of bone fragments.

    PubMed

    Simske, S J; Luttges, M W

    1994-09-01

    Undecalcified (n = 140) and decalcified (n = 11) bone fragments were treated with either collagenase (to remove collagen portion; undecalcified n = 64, decalcified n = 11) or EDTA (to remove mineral portion; n = 76) under the reduced gravity environment on US Space Shuttle mission STS-57. The fragments were initially stored in Dulbecco's phosphate buffer solution. After orbit had been established, fragments were exposed to either a neutral buffered collagenase or EDTA solution. Reactions were terminated (neutral buffered formalin for collagenase, 21% CuSO4 5H2O for EDTA) before reentry to earth's atmosphere. Differences in bone samples mass from before flight to after flight were measured. EDTA-treated sample mass was corrected for CuSO4 content. Flight and matched ground (gravitational control) sample showed similar EDTA-induced loss of mineral mass. Collagenase treatments, however, appeared to be more effective in flight samples compared to ground control samples. The flight-exposed, collagenase-treated samples showed significantly more loss of mass than did ground samples. The microgravity environment appeared to promote proteolytic reactions in bone more than the EDTA decalcification reaction.

  5. Effect of microgravity on collagenase deprotoeinization and EDTA decalcification of bone fragments

    NASA Technical Reports Server (NTRS)

    Simske, S. J.; Luttges, M. W.

    1994-01-01

    Undecalcified (n = 140) and decalcified (n = 11) bone fragments were treated with either collagenase (to remove collagen portion; undecalcified n = 64, decalcified n = 11) or EDTA (to remove mineral portion; n= 76) under the reduced gravity environment on US Space Shuttle mission STS-57. The fragments were initially stored in Dulbecco's phosphate buffer solution. After orbit had been established, fragments were exposed to either a neutral buffered collagenase or EDTA solution. Reactions were terminated (neutral buffered formalin for collagenase, 21% CuSO4-5H2O for EDTA) before reentry to earth's atmosphere. Differences in bone samples mass from before flight to after flight were measured. EDTA-treated sample mass was corrected for CuSO4 content. Flight and matched ground (gravitational control) sample showed similar EDTA-induced loss of mineral mass. Collagenase treatments, however, appeared to be more effective in flight samples compared to ground control samples. The flight-exposed, collagenase-treated samples showed significantly more loss than did ground samples. The microgravity environment appeared to promote proteolytic reactions in bone more than the EDTA decalcification reaction.

  6. Effect of collagen type I or type II on chondrogenesis by cultured human articular chondrocytes.

    PubMed

    Rutgers, Marijn; Saris, Daniel B; Vonk, Lucienne A; van Rijen, Mattie H; Akrum, Vanessa; Langeveld, Danielle; van Boxtel, Antonette; Dhert, Wouter J; Creemers, Laura B

    2013-01-01

    Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently, however, fibrillar collagens were shown to promote cartilage degradation. The goal of this study was to evaluate the effects of collagen type I and type II coating on chondrogenic properties of in vitro cultured human chondrocytes, and to investigate if collagen-mediated cartilage degradation occurs. Human chondrocytes of eight healthy cartilage donors were isolated, expanded, and cultured on culture well inserts coated with either collagen type I, type II, or no coating (control). After 28 days of redifferentiation culture, safranin O and immunohistochemical staining for collagen types I, II, X, and Runx2/Cbfa1 were performed and glycosaminoglycan (GAG) and DNA content and release were examined. Further, expression of collagen type I, type II, type X, MMP13, Runx2/Cbfa1, DDR2, α2 and β1 integrin were examined by reverse transcriptase-polymerase chain reaction. The matrix, created by chondrocytes grown on collagen type I- and II-coated membranes, resembled cartilage more than when grown on noncoated membranes as reflected by histological scoring. Immunohistochemical staining did not differ between the conditions. GAG content as well as GAG/DNA were higher for collagen type II-coated cartilage constructs than control. GAG release was also higher on collagen type I- and II-coated constructs. Expression of collagen type X was higher of chondrocytes grown on collagen type II compared to controls, but no collagen X protein could be demonstrated by immunohistochemistry. No effects of collagen coating on DDR2 nor MMP-13 gene expression were found. No differences were observed between collagen types I and II. Chondrocyte culture on collagen type I or II promotes more active matrix production

  7. Porous Collagen Scaffold Reinforced with Surfaced Activated PLLA Nanoparticles

    PubMed Central

    Xu, Cancan; Lu, Wei; Bian, Shaoquan; Liang, Jie; Fan, Yujiang; Zhang, Xingdong

    2012-01-01

    Porous collagen scaffold is integrated with surface activated PLLA nanoparticles fabricated by lyophilizing and crosslinking via EDC treatment. In order to prepare surface-modified PLLA nanoparticles, PLLA was firstly grafted with poly (acrylic acid) (PAA) through surface-initiated polymerization of acrylic acid. Nanoparticles of average diameter 316 nm and zeta potential −39.88 mV were obtained from the such-treated PLLA by dialysis method. Porous collagen scaffold were fabricated by mixing PLLA nanoparticles with collagen solution, freeze drying, and crosslinking with EDC. SEM observation revealed that nanoparticles were homogeneously dispersed in collagen matrix, forming interconnected porous structure with pore size ranging from 150 to 200 μm, irrespective of the amount of nanoparticles. The porosity of the scaffolds kept almost unchanged with the increment of the nanoparticles, whereas the mechanical property was obviously improved, and the degradation was effectively retarded. In vitro L929 mouse fibroblast cells seeding and culture studies revealed that cells infiltrated into the scaffolds and were distributed homogeneously. Compared with the pure collagen sponge, the number of cells in hybrid scaffolds greatly increased with the increment of incorporated nanoparticles. These results manifested that the surface-activated PLLA nanoparticles effectively reinforced the porous collagen scaffold and promoted the cells penetrating into the scaffold, and proliferation. PMID:22448137

  8. Parton fragmentation functions

    NASA Astrophysics Data System (ADS)

    Metz, A.; Vossen, A.

    2016-11-01

    The field of fragmentation functions of light quarks and gluons is reviewed. In addition to integrated fragmentation functions, attention is paid to the dependence of fragmentation functions on transverse momenta and on polarization degrees of freedom. Higher-twist and di-hadron fragmentation functions are considered as well. Moreover, the review covers both theoretical and experimental developments in hadron production in electron-positron annihilation, deep-inelastic lepton-nucleon scattering, and proton-proton collisions.

  9. Fibrillar, fibril-associated and basement membrane collagens of the arterial wall: architecture, elasticity and remodeling under stress.

    PubMed

    Osidak, M S; Osidak, E O; Akhmanova, M A; Domogatsky, S P; Domogatskaya, A S

    2015-01-01

    The ability of a human artery to pass through 150 million liters of blood sustaining 2 billion pulsations of blood pressure with minor deterioration depends on unique construction of the arterial wall. Viscoelastic properties of this construction enable to re-seal the occuring damages apparently without direct immediate participance of the constituent cells. Collagen structures are considered to be the elements that determine the mechanoelastic properties of the wall in parallel with elastin responsible for elasticity and resilience. Collagen scaffold architecture is the function-dependent dynamic arrangement of a dozen different collagen types composing three distinct interacting forms inside the extracellular matrix of the wall. Tightly packed molecules of collagen types I, III, V provide high tensile strength along collagen fibrils but toughness of the collagen scaffold as a whole depends on molecular bonds between distinct fibrils. Apart of other macromolecules in the extracellular matrix (ECM), collagen-specific interlinks involve microfilaments of collagen type VI, meshwork-organized collagen type VIII, and FACIT collagen type XIV. Basement membrane collagen types IV, XV, XVIII and cell-associated collagen XIII enable transmission of mechanical signals between cells and whole artery matrix. Collagen scaffold undergoes continuous remodeling by decomposition promoted with MMPs and reconstitution from newly produced collagen molecules. Pulsatile stress-strain load modulates both collagen synthesis and MMP-dependent collagen degradation. In this way the ECM structure becomes adoptive to mechanical challenges. The mechanoelastic properties of the arterial wall are changed in atherosclerosis concomitantly with collagen turnover both type-specific and dependent on the structure. Improving the feedback could be another approach to restore sufficient blood circulation.

  10. Fragmentation Analysis - Fundamental Processes

    DTIC Science & Technology

    Wausau quartzite and anorthosite of 3.0 to 3.5 inch size were fragmented in this device. An analysis of the fragment distribution results of the drop...disc-shaped specimens of Wausau quartzite, anorthosite , and Felch marble were then fragmented with the impact pendulum device. Computer programs were

  11. Effects of Different Concentrations of Collagenous Peptide from Fish Scales on Osteoblast Proliferation and Osteoclast Resorption.

    PubMed

    Hu, Chung-Hsuan; Yao, Chun-Hsu; Chan, Tzu-Min; Huang, Teng-Le; Sen, Yang; Huang, Chih-Yang; Ho, Chien-Yi

    2016-08-31

    The incidence of osteoporosis has increased among the elderly population. Establishing a model of bone remodeling for screening new drugs is critical to identify safe and effective treatments for osteoporosis. In this study, we established a platform to investigate the therapeutic effects of collagenous peptides extracted from scales of two kinds of fish, namely, sparidae and chanos. These peptides were prepared using seven concentrations of collagenous peptide: 100, 80, 60, 40, 20, 10 and 1 mg/ml. Experimental results indicated that collagenous peptides promoted the proliferation of osteoblasts and inhibited the proliferation of mature osteoclasts; the effective concentration of collagenous peptide-sparidae was 10 mg/ml and that of collagenous peptide-chanos was 40 mg/ml. These findings demonstrate that, to a certain extent, collagenous peptides extracted from fish scales can be used to prevent osteoporosis to assist bone remodeling.

  12. Influence of collagen addition on the thermal and morphological properties of chitosan/xanthan hydrogels.

    PubMed

    Horn, Marilia M; Martins, Virginia C A; Plepis, Ana Maria de Guzzi

    2015-09-01

    This study investigates the collagen influence on thermal and morphological characteristics of chitosan/xanthan hydrogels for potential tissue engineering applications. Anionic collagen was prepared by selective hydrolysis of type I collagen found in bovine tendons. Chitosan was obtained from the partial deacetylation of squid pen β-chitin and xanthan was acquired from Fluka. The hydrogels were obtained in different ratios and were characterized by thermal and morphological analysis. FT-IR suggested only electrostatic interactions between NH3(+) groups of chitosan and COO(-) groups of xanthan and collagen. Thermogravimetric curves showed that hydrogels contain a great amount of water (above 98%) and the presence of collagen does not change this characteristic. Freezing-bound water transition in DSC curves was shifted to higher values due to the increase of water/polymer interaction, mainly when different ratios of chitosan and xanthan were used. SEM images showed sheet-form structures with the presence of collagen promoting an increase in pore size.

  13. Structural study and preliminary biological evaluation on the collagen hydrogel crosslinked by γ-irradiation.

    PubMed

    Zhang, Xiangmei; Xu, Ling; Huang, Xin; Wei, Shicheng; Zhai, Maolin

    2012-11-01

    Under γ-irradiation, concentrated collagen solutions yielded collagen hydrogels and liquid products. The molecular structure of collagen hydrogels and the source of the liquid products were studied. Furthermore, preliminary biological properties of the hydrogels were investigated. The results revealed that crosslinking occurred to form collagen hydrogel and the crosslinking density increased with the increasing of the absorbed dose, and the collagen hydrogels showed enhanced mechanical properties. Meanwhile, collagen underwent radiation degradation and water was squeezed out from hydrogel by contraction of hydrogel, yielding liquid products. Collagen hydrogels induced by γ-irradiation maintained the backbone structure of collagen, and tyrosine partially involved in crosslinking. The irradiated collagen hydrogels have higher denatured temperature, can promote fibroblasts proliferation, and their degradation rate in vivo depended on the absorbed dose. The comprehensive results suggested that the collagen hydrogels prepared by radiation crosslinking preserved the triple helical conformation, possessed improved thermal stability and mechanical properties, excellent biocompatibility, which is expected to favor its application as biomaterials. Copyright © 2012 Wiley Periodicals, Inc.

  14. Fluorescence study on the aggregation of collagen molecules in acid solution influenced by hydroxypropyl methylcellulose.

    PubMed

    Ding, Cuicui; Zhang, Min; Li, Guoying

    2016-01-20

    The effect of hydroxypropyl methylcellulose (HPMC) on the aggregation of collagen molecules with collagen concentrations of 0.25, 0.5 and 1.0mg/mL was studied by fluorescence techniques. On one hand, both the synchronous fluorescence spectra and fluorescence emission spectra showed that there was no change in the fluorescence intensity of collagen intrinsic fluorescence when 30% HPMC was added, while it decreased obviously when HPMC content ≥ 50%. From the two-dimensional fluorescence correlation analysis, it was indicated that collagen molecules in 0.25 and 0.5mg/mL collagen solutions were more sensitive to HPMC than those in 1.0mg/mL collagen solution. On the other hand, the pyrene fluorescence and the fluorescence anisotropy measurements indicated that HPMC inhibited the collagen aggregation for 0.25 and 0.5mg/mL collagen, but promoted it for 1.0mg/mL collagen. The atomic force microscopy images further confirmed the effect of HPMC on collagen with different initial states.

  15. Insulin-like growth factor I enhances collagen synthesis in engineered human tendon tissue.

    PubMed

    Herchenhan, Andreas; Bayer, Monika L; Eliasson, Pernilla; Magnusson, S Peter; Kjaer, Michael

    2015-02-01

    Isolated human tendon cells form 3D tendon constructs that demonstrate collagen fibrillogenesis and feature structural similarities to tendon when cultured under tensile load. The exact role of circulating growth factors for collagen formation in tendon is sparsely examined. We investigated the influence of insulin-like growth factor I (IGF-I) on tendon construct formation in 3D cell culture. Tendon constructs were grown in 0.5 or 10% FBS with or without IGF-I (250 mg/ml) supplementation. Collagen content (fluorometric), mRNA levels (PCR) and fibril diameter (transmission electron microscopy) were determined at 7, 10, 14, 21 and 28 days. IGF-I revealed a stimulating effect on fibril diameter (up to day 21), mRNA for collagen (to day 28), tenomodulin (to day 28) and scleraxis (at days 10 and 14), and on overall collagen content. 10% FBS diminished the development of fibril diameter (day 14), collagen content (at days 21 and 28) and mRNA expression for collagen, tenomodulin and scleraxis. IGF-I supplementation promotes early onset of tensile load induced collagen formation and tendon structural arrangement, whereas the FBS concentration routinely used in cultures diminishes collagen expression, collagen content and fibril formation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Tunability of collagen matrix mechanical properties via multiple modes of mineralization

    PubMed Central

    Smith, Lester J.; Deymier, Alix C.; Boyle, John J.; Li, Zhen; Linderman, Stephen W.; Pasteris, Jill D.; Xia, Younan; Genin, Guy M.; Thomopoulos, Stavros

    2016-01-01

    Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering. PMID:26855755

  17. Effect of collagen sponge and fibrin glue on bone repair

    PubMed Central

    SANTOS, Thiago de Santana; ABUNA, Rodrigo Paolo Flores; de ALMEIDA, Adriana Luisa Gonçalves; BELOTI, Marcio Mateus; ROSA, Adalberto Luiz

    2015-01-01

    ABSTRACT The ability of hemostatic agents to promote bone repair has been investigated using in vitro and in vivo models but, up to now, the results are inconclusive. Objective In this context, the aim of this study was to compare the potential of bone repair of collagen sponge with fibrin glue in a rat calvarial defect model. Material and Methods Defects of 5 mm in diameter were created in rat calvariae and treated with either collagen sponge or fibrin glue; untreated defects were used as control. At 4 and 8 weeks, histological analysis and micro-CT-based histomorphometry were carried out and data were compared by two-way ANOVA followed by Student-Newman-Keuls test when appropriated (p≤0.05). Results Three-dimensional reconstructions showed increased bone formation in defects treated with either collagen sponge or fibrin glue compared with untreated defects, which was confirmed by the histological analysis. Morphometric parameters indicated the progression of bone formation from 4 to 8 weeks. Additionally, fibrin glue displayed slightly higher bone formation rate when compared with collagen sponge. Conclusion Our results have shown the benefits of using collagen sponge and fibrin glue to promote new bone formation in rat calvarial bone defects, the latter being discreetly more advantageous. PMID:26814464

  18. Development of biomimetic tilapia collagen nanofibers for skin regeneration through inducing keratinocytes differentiation and collagen synthesis of dermal fibroblasts.

    PubMed

    Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

    2015-02-11

    In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides, and its denaturation temperature was 44.99 °C. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4+/CD8+ lymphocytes, and the level of IgG or IgM in Sprague-Dawley rat. The contact angle, tensile strength, and weight loss temperature of collagen nanofibers were 21.2°, 6.72±0.44 MPa, and 300 °C, respectively. The nanofibers could promote the viabilities of human keratinocytes (HaCaTs) and human dermal fibroblasts (HDFs), inducing epidermal differentiation through the gene expression of involucrin, filaggrin, and type I transglutaminase of HaCaTs, and they could also accelerate migration of HaCaTs with the expression of matrix metalloproteinase-9 and transforming growth factor-β1 (TGF-β1). Besides, the nanofibers could upregulate the protien level of Col-I in HDFs both via a direct effect and TGF-β1 secreted from HaCaTs, thus facilitating the formation of collagen fibers. Furthermore, the collagen nanofibers stimulated the skin regeneration rapidly and effectively in vivo. These biological effects could be explained as the contributions from the biomimic extracellular cell matrix structure, hydrophilicity, and the multiple amino acids of the collagen nanofibers.

  19. Selectable fragmentation warhead

    SciTech Connect

    Bryan, C.S.; Paisley, D.L.; Montoya, N.I.; Stahl, D.B.

    1992-12-31

    This report discusses a selectable fragmentation warhead which is capable of producing a predetermined number of fragments from a metal plate, and accelerating the fragments toward a target. A first explosive located adjacent to the plate is detonated at selected number of points by laser-driven slapper detonators. In one embodiment, a smoother-disk and a second explosive, located adjacent to the first explosive, serve to increase acceleration of the fragments toward a target. The ability to produce a selected number of fragments allows for effective destruction of a chosen target.

  20. Selectable fragmentation warhead

    DOEpatents

    Bryan, Courtney S.; Paisley, Dennis L.; Montoya, Nelson I.; Stahl, David B.

    1993-01-01

    A selectable fragmentation warhead capable of producing a predetermined number of fragments from a metal plate, and accelerating the fragments toward a target. A first explosive located adjacent to the plate is detonated at selected number of points by laser-driven slapper detonators. In one embodiment, a smoother-disk and a second explosive, located adjacent to the first explosive, serve to increase acceleration of the fragments toward a target. The ability to produce a selected number of fragments allows for effective destruction of a chosen target.

  1. Epoxy Cross-Linked Collagen and Collagen-Laminin Peptide Hydrogels as Corneal Substitutes

    PubMed Central

    Koh, Li Buay; Islam, Mohammad Mirazul; Mitra, Debbie; Noel, Christopher W.; Merrett, Kimberley; Odorcic, Silvia; Fagerholm, Per; Jackson, William. Bruce; Liedberg, Bo; Phopase, Jaywant; Griffith, May

    2013-01-01

    A bi-functional epoxy-based cross-linker, 1,4-Butanediol diglycidyl ether (BDDGE), was investigated in the fabrication of collagen based corneal substitutes. Two synthetic strategies were explored in the preparation of the cross-linked collagen scaffolds. The lysine residues of Type 1 porcine collagen were directly cross-linked using l,4-Butanediol diglycidyl ether (BDDGE) under basic conditions at pH 11. Alternatively, under conventional methodology, using both BDDGE and 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as cross-linkers, hydrogels were fabricated under acidic conditions. In this latter strategy, Cu(BF4)2·XH2O was used to catalyze the formation of secondary amine bonds. To date, we have demonstrated that both methods of chemical cross-linking improved the elasticity and tensile strength of the collagen implants. Differential scanning calorimetry and biocompatibility studies indicate comparable, and in some cases, enhanced properties compared to that of the EDC/NHS controls. In vitro studies showed that human corneal epithelial cells and neuronal progenitor cell lines proliferated on these hydrogels. In addition, improvement of cell proliferation on the surfaces of the materials was observed when neurite promoting laminin epitope, IKVAV, and adhesion peptide, YIGSR, were incorporated. However, the elasticity decreased with peptide incorporation and will require further optimization. Nevertheless, we have shown that epoxy cross-linkers should be further explored in the fabrication of collagen-based hydrogels, as alternatives to or in conjunction with carbodiimide cross-linkers. PMID:24956085

  2. The NC2 domain of type IX collagen determines the chain register of the triple helix.

    PubMed

    Boudko, Sergei P; Bächinger, Hans Peter

    2012-12-28

    Precise mapping and unraveling the mechanism of interaction or degradation of a certain type of collagen triple helix requires the generation of short and stable collagenous fragments. This is a great challenge especially for hetero-trimeric collagens, where chain composition and register (stagger) are important factors. No system has been reported that can be efficiently used to generate a natural collagenous fragment with exact chain composition and desired chain register. The NC2 domain (only 35-50 residues) of FACIT collagens is a potent trimerization domain. In the case of type IX collagen it provides the efficient selection and hetero-trimerization of three distinct chains. The ability of the NC2 domain to determine the chain register of the triple helix is studied. We generated three possible sequence combinations (α1α1α2, α1α2α1, α2α1α1) of a type I collagen fragment (the binding region for the von Willebrand factor A3 domain) attached to the NC2 domain. In addition, two control combinations were produced that constitute homo-trimers of (α1)(3) or (α2)(3). For the hetero-trimeric constructs, α1α1α2 demonstrated a higher melting temperature than the other two. Binding experiments with the von Willebrand factor A3 domain revealed the homo-trimer of (α1)(3) as the strongest binding construct, whereas the homo-trimer of (α2)(3) showed no binding. For hetero-trimers, α1α1α2 was found to be the strongest binding construct. Differences in thermal stability and binding to the A3 domain unambiguously demonstrate that the NC2 domain of type IX collagen determines not only the chain composition but also the chain register of the adjacent triple helix.

  3. Collagen binding to Staphylococcus aureus

    SciTech Connect

    Holderbaum, D.; Hall, G.S.; Ehrhart, L.A.

    1986-11-01

    Staphylococcus aureus can bind soluble collagen in a specific, saturable manner. We have previously shown that some variability exists in the degree of collagen binding between different strains of heat-killed, formaldehyde-fixed S. aureus which are commercially available as immunologic reagents. The present study demonstrates that live S. aureus of the Cowan 1 strain binds amounts of collagen per organism equivalent to those demonstrated previously in heat-killed, formaldehyde-fixed bacteria but has an affinity over 100 times greater, with Kd values of 9.7 X 10(-11) M and 4.3 X 10(-8) M for live and heat-killed organisms, respectively. Studies were also carried out with S. aureus killed by ionizing radiation, since this method of killing the organism seemed less likely to alter the binding moieties on the surface than did heat killing. Bacteria killed by exposure to gamma radiation bound collagen in a manner essentially indistinguishable from that of live organisms. Binding of collagen to irradiated cells of the Cowan 1 strain was rapid, with equilibrium reached by 30 min at 22 degrees C, and was fully reversible. The binding was not inhibited by fibronectin, fibrinogen, C1q, or immunoglobulin G, suggesting a binding site for collagen distinct from those for these proteins. Collagen binding was virtually eliminated in trypsin-treated organisms, indicating that the binding site has a protein component. Of four strains examined, Cowan 1 and S. aureus ATCC 25923 showed saturable, specific binding, while strains Woods and S4 showed a complete lack of binding. These results suggest that some strains of S. aureus contain high-affinity binding sites for collagen. While the number of binding sites per bacterium varied sixfold in the two collagen-binding strains, the apparent affinity was similar.

  4. Sequence comparison of pepsin-resistant segments of basement-membrane collagen alpha 1(IV) chains from bovine lens capsule and mouse tumour.

    PubMed Central

    Schuppan, D; Glanville, R W; Timpl, R; Dixit, S N; Kang, A H

    1984-01-01

    The C-terminal peptic fragment P1 (about 518 amino acid residues) of bovine lens-capsule collagen alpha 1(IV) chain was cleaved with CNBr and trypsin. The peptides were purified and characterized, allowing their ordering within the P1 fragment by comparison with a corresponding section of mouse collagen alpha 1(IV) chain [Schuppan, Glanville & Timpl (1982) Eur. J. Biochem. 123, 505-512]. About 67% of the sequence of bovine collagen fragment P1 was determined by Edman degradation. Comparison with the sequence of the corresponding mouse collagen fragment P1 showed 76% identity for positions Xaa and Yaa of the triplet structures Gly-Xaa-Yaa. Invariance was found for the positions of two non-triplet interruptions and of 3-hydroxyproline residues, pointing to the functional importance of these structures. PMID:6430279

  5. Electrostatic effects in collagen fibrillization

    NASA Astrophysics Data System (ADS)

    Morozova, Svetlana; Muthukumar, Murugappan

    2014-03-01

    Using light scattering and AFM techniques, we have measured the kinetics of fibrillization of collagen (pertinent to the vitreous of human eye) as a function of pH and ionic strength. At higher and lower pH, collagen triple-peptides remain stable in solution without fibrillization. At neutral pH, the fibrillization occurs and its growth kinetics is slowed upon either an increase in ionic strength or a decrease in temperature. We present a model, based on polymer crystallization theory, to describe the observed electrostatic nature of collagen assembly.

  6. Sterile Keratitis following Collagen Crosslinking.

    PubMed

    Javadi, Mohammad-Ali; Feizi, Sepehr

    2014-01-01

    To report a keratoconic eye that developed severe sterile keratitis and corneal scar after collagen crosslinking necessitating corneal transplantation. A 26-year-old man with progressive keratoconus underwent collagen crosslinking and presented with severe keratitis 72 hours after the procedure. The initial impression was infectious corneal ulcer and a fortified antibiotic regimen was administered. However, the clinical course and confocal microscopy results prompted a diagnosis of sterile keratitis. The eye developed severe corneal scars leading to reduced visual acuity and necessitating corneal transplantation. Sterile keratitis may develop after collagen crosslinking resulting in profound visual loss leading to corneal transplantation.

  7. Golgi fragmentation in Alzheimer's disease

    PubMed Central

    Joshi, Gunjan; Bekier, Michael E.; Wang, Yanzhuang

    2015-01-01

    The Golgi apparatus is an essential cellular organelle for post-translational modifications, sorting, and trafficking of membrane and secretory proteins. Proper functionality of the Golgi requires the formation of its unique cisternal-stacking morphology. The Golgi structure is disrupted in a variety of neurodegenerative diseases, suggesting a common mechanism and contribution of Golgi defects in neurodegenerative disorders. A recent study on Alzheimer's disease (AD) revealed that phosphorylation of the Golgi stacking protein GRASP65 disrupts its function in Golgi structure formation, resulting in Golgi fragmentation. Inhibiting GRASP65 phosphorylation restores the Golgi morphology from Aβ-induced fragmentation and reduces Aβ production. Perturbing Golgi structure and function in neurons may directly impact trafficking, processing, and sorting of a variety of proteins essential for synaptic and dendritic integrity. Therefore, Golgi defects may ultimately promote the development of AD. In the current review, we focus on the cellular impact of impaired Golgi morphology and its potential relationship to AD disease development. PMID:26441511

  8. Bacterial collagen-binding domain targets undertwisted regions of collagen

    PubMed Central

    Philominathan, Sagaya Theresa Leena; Koide, Takaki; Matsushita, Osamu; Sakon, Joshua

    2012-01-01

    Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C-terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly → Ala substitution [(POG)xPOA(POG)y]3, where x + y = 9 and x > 3). 1H–15N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with 15N-labeled CBD demonstrated that the minicollagen binds to a 10 Å wide 25 Å long cleft. Six collagenous peptides each labeled with a nitroxide radical were then titrated with 15N-labeled CBD. CBD binds to either the Gly → Ala substitution site or to the C-terminus of each minicollagen. Small-angle X-ray scattering measurements revealed that CBD prefers to bind the Gly → Ala site to the C-terminus. The HSQC NMR spectra of 15N-labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix. PMID:22898990

  9. Universality of fragment shapes

    PubMed Central

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-01-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism. PMID:25772300

  10. Universality of fragment shapes

    NASA Astrophysics Data System (ADS)

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-03-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism.

  11. Impact of temperature and electrical potentials on the stability and structure of collagen adsorbed on the gold electrode

    NASA Astrophysics Data System (ADS)

    Meiners, Frank; Ahlers, Michael; Brand, Izabella; Wittstock, Gunther

    2015-01-01

    The morphology and structure of collagen type I adsorbed on gold electrodes were studied as a function of electrode potential and temperature by means of capacitance measurements, polarization modulation infrared reflection-absorption spectroscopy and scanning force microscopy at temperatures of 37 °C, 43 °C and 50 °C. The selected temperatures corresponded to the normal body temperature, temperature of denaturation of collagen molecules and denaturation of collagen fibrils, respectively. Independently of the solution temperature, collagen was adsorbed on gold electrodes in the potential range - 0.7 V < E < 0.4 V vs. Ag/AgCl, where the protein film was very stable. Fragments of collagen molecules made a direct contact to the gold surface and water was present in the film. Protein molecules were oriented preferentially with their long axis towards the gold surface. Collagen molecules in the adsorbed state preserved their native triple helical structure even at temperatures corresponding to collagen denaturation in aqueous solutions. Application of E < - 0.75 V vs. Ag/AgCl leads to the swelling of the protein film by water and desorption from the electrode surface. IR spectra provided no evidence of the thermal denaturation of adsorbed collagen molecules. A temperature increase to 50 °C leads to a distortion of the collagen film. The processes of aggregation and fibrilization were preferred over thermal denaturation for collagen adsorbed on the electrode surface and exposed to changing potentials.

  12. Basement Membrane Zone Collagens XV and XVIII/Proteoglycans Mediate Leukocyte Influx in Renal Ischemia/Reperfusion

    PubMed Central

    Zaferani, Azadeh; Talsma, Ditmer T.; Yazdani, Saleh; Celie, Johanna W. A. M.; Aikio, Mari; Heljasvaara, Ritva; Navis, Gerjan J.; Pihlajaniemi, Taina; van den Born, Jacob

    2014-01-01

    Collagen type XV and XVIII are proteoglycans found in the basement membrane zones of endothelial and epithelial cells, and known for their cryptic anti-angiogenic domains named restin and endostatin, respectively. Mutations or deletions of these collagens are associated with eye, muscle and microvessel phenotypes. We now describe a novel role for these collagens, namely a supportive role in leukocyte recruitment. We subjected mice deficient in collagen XV or collagen XVIII, and their compound mutant, as well as the wild-type control mice to bilateral renal ischemia/reperfusion, and evaluated renal function, tubular injury, and neutrophil and macrophage influx at different time points after ischemia/reperfusion. Five days after ischemia/reperfusion, the collagen XV, collagen XVIII and the compound mutant mice showed diminished serum urea levels compared to wild-type mice (all p<0.05). Histology showed reduced tubular damage, and decreased inflammatory cell influx in all mutant mice, which were more pronounced in the compound mutant despite increased expression of MCP-1 and TNF-α in double mutant mice compared to wildtype mice. Both type XV and type XVIII collagen bear glycosaminoglycan side chains and an in vitro approach with recombinant collagen XVIII fragments with variable glycanation indicated a role for these side chains in leukocyte migration. Thus, basement membrane zone collagen/proteoglycan hybrids facilitate leukocyte influx and tubular damage after renal ischemia/reperfusion and might be potential intervention targets for the reduction of inflammation in this condition. PMID:25188209

  13. Fibroblast morphogenesis on 3D collagen matrices: the balance between cell clustering and cell migration.

    PubMed

    da Rocha-Azevedo, Bruno; Grinnell, Frederick

    2013-10-01

    Fibroblast clusters have been observed in tissues under a variety of circumstances: in fibrosis and scar, in the formation of hair follicle dermal papilla, and as part of the general process of mesenchymal condensation that takes place during development. Cell clustering has been shown to depend on features of the extracellular matrix, growth factor environment, and mechanisms to stabilize cell-cell interactions. In vitro studies have shown that increasing the potential for cell-cell adhesion relative to cell-substrate adhesion promotes cell clustering. Experimental models to study fibroblast clustering have utilized centrifugation, hanging drops, and substrata with poorly adhesive, soft and mechanically unstable properties. In this review, we summarize work on a new, highly tractable, cell clustering research model in which human fibroblasts are incubated on the surfaces of collagen matrices. Fibroblast clustering occurs under procontractile growth factor conditions (e.g., serum or the serum lipid agonist lysophosphatidic acid) but not under promigratory growth factor conditions (e.g., platelet-derived growth factor) and can be reversed by switching growth factor environments. Cell contraction plays a dual role in clustering to bring cells closer together and to stimulate cells to organize fibronectin into a fibrillar matrix. Binding of fibroblasts to a shared fibronectin fibrillar matrix stabilizes clusters, and fragmentation of the fibrillar matrix occurs when growth factor conditions are switched to promote cell dispersal.

  14. Fragmentation properties of metals

    SciTech Connect

    Grady, D.E.; Kipp, M.E.

    1996-06-01

    In the present study we are developing an experimental fracture material property test method specific to dynamic fragmentation. Spherical test samples of the metals of interest are subjected to controlled impulsive stress loads by acceleration to high velocities with a light-gas launcher facility and subsequent normal impact on thin plates. Motion, deformation and fragmentation of the test samples are diagnosed with multiple flash radiography methods. The impact plate materials are selected to be transparent to the x-ray method so that only test metal material is imaged. Through a systematic series of such tests, both strain-to-failure and fragmentation resistance properties are determined through this experimental method. Fragmentation property data for several steels, copper, aluminum, tantalum and titanium have been obtained to date. Aspects of the dynamic data have been analyzed with computational methods to achieve a better understanding of the processes leading to failure and fragmentation, and to test an existing computational fragmentation model.

  15. Chitosan and fish collagen as biomaterials for regenerative medicine.

    PubMed

    Hayashi, Yoshihiko; Yamada, Shizuka; Yanagi Guchi, Kajiro; Koyama, Zenya; Ikeda, Takeshi

    2012-01-01

    This chapter focuses and reviews on the characteristics and biomedical application of chitosan and collagen from marine products and advantages and disadvantages of regeneration medicine. The understanding of the production processes of chitosan and collagen and the conformation of these biomaterials are indispensable for promoting the theoretical and practical availability. The initial inflammatory reactions associated with chitosan application to hard and soft tissues need to be controlled before it can be considered for clinical application as scaffold. Further, as chitosan takes too long for biodegradation in vivo, generally it is not suitable for the scaffold for degenerative medicine in especially dental pulp tissue. The collagen extract from the scales of tropical fish has been reported to have a degeneration temperature of 35°C. The properties of biocompatibility and biodegradation of fish atelocollagen are suitable for the scaffold in regenerative medicine.

  16. Human collagen produced in plants

    PubMed Central

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2014-01-01

    Consequential to its essential role as a mechanical support and affinity regulator in extracellular matrices, collagen constitutes a highly sought after scaffolding material for regeneration and healing applications. However, substantiated concerns have been raised with regard to quality and safety of animal tissue-extracted collagen, particularly in relation to its immunogenicity, risk of disease transmission and overall quality and consistency. In parallel, contamination with undesirable cellular factors can significantly impair its bioactivity, vis-a-vis its impact on cell recruitment, proliferation and differentiation. High-scale production of recombinant human collagen Type I (rhCOL1) in the tobacco plant provides a source of an homogenic, heterotrimeric, thermally stable “virgin” collagen which self assembles to fine homogenous fibrils displaying intact binding sites and has been applied to form numerous functional scaffolds for tissue engineering and regenerative medicine. In addition, rhCOL1 can form liquid crystal structures, yielding a well-organized and mechanically strong membrane, two properties indispensable to extracellular matrix (ECM) mimicry. Overall, the shortcomings of animal- and cadaver-derived collagens arising from their source diversity and recycled nature are fully overcome in the plant setting, constituting a collagen source ideal for tissue engineering and regenerative medicine applications. PMID:23941988

  17. Nonlinear microscopy of collagen fibers

    NASA Astrophysics Data System (ADS)

    Strupler, M.; Pena, A.-M.; Hernest, M.; Tharaux, P.-L.; Fabre, A.; Marchal-Somme, J.; Crestani, B.; Débarre, D.; Martin, J.-L.; Beaurepaire, E.; Schanne-Klein, M.-C.

    2007-02-01

    We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in murine models, and that they clearly sort out the fibrotic mice.

  18. Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    PubMed Central

    Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

    2014-01-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

  19. Serpin A1 C-Terminal Peptides as Collagen Turnover Modulators.

    PubMed

    Pascarella, Simona; Tiberi, Caterina; Sabatino, Giuseppina; Nuti, Francesca; Papini, Anna Maria; Giovannelli, Lisa; Rovero, Paolo

    2016-08-19

    The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of type I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpin A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in type I collagen levels in the culture medium of treated cells at micromolar concentrations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Effect of curcumin caged silver nanoparticle on collagen stabilization for biomedical applications.

    PubMed

    Srivatsan, Kunnavakkam Vinjimur; Duraipandy, N; Begum, Shajitha; Lakra, Rachita; Ramamurthy, Usha; Korrapati, Purna Sai; Kiran, Manikantan Syamala

    2015-04-01

    The current study aims at understanding the influence of curcumin caged silver nanoparticle (CCSNP) on stability of collagen. The results indicated that curcumin caged silver nanoparticles efficiently stabilize collagen, indicated by enhanced tensile strength, fibril formation and viscosity. The tensile strength of curcumin caged silver nanoparticle cross-linked collagen and elongation at break was also found to be higher than glutaraldehyde cross-linked collagen. The physicochemical characteristics of curcumin caged nanoparticle cross-linked collagen exhibited enhanced strength. The thermal properties were also good with both thermal degradation temperature and hydrothermal stability higher than native collagen. CD analysis showed no structural disparity in spite of superior physicochemical properties suggesting the significance of curcumin caged nanoparticle mediated cross-linking. The additional enhancement in the stabilization of collagen could be attributed to multiple sites for interaction with collagen molecule provided by curcumin caged silver nanoparticles. The results of cell proliferation and anti-microbial activity assays indicated that curcumin caged silver nanoparticles promoted cell proliferation and inhibited microbial growth making it an excellent biomaterial for wound dressing application. The study opens scope for nano-biotechnological strategies for the development of alternate non-toxic cross-linking agents facilitating multiple site interaction thereby improving therapeutic values to the collagen for biomedical application.

  1. Rheological, biocompatibility and osteogenesis assessment of fish collagen scaffold for bone tissue engineering.

    PubMed

    Elango, Jeevithan; Zhang, Jingyi; Bao, Bin; Palaniyandi, Krishnamoorthy; Wang, Shujun; Wenhui, Wu; Robinson, Jeya Shakila

    2016-10-01

    In the present investigation, an attempt was made to find an alternative to mammalian collagen with better osteogenesis ability. Three types of collagen scaffolds - collagen, collagen-chitosan (CCH), and collagen-hydroxyapatite (CHA) - were prepared from the cartilage of Blue shark and investigated for their physico-functional and mechanical properties in relation to biocompatibility and osteogenesis. CCH scaffold was superior with pH 4.5-4.9 and viscosity 9.7-10.9cP. Notably, addition of chitosan and HA (hydroxyapatite) improved the stiffness (11-23MPa) and degradation rate but lowered the water binding capacity and porosity of the scaffold. Interestingly, CCH scaffolds remained for 3days before complete in-vitro biodegradation. The decreased amount of viable T-cells and higher level of FAS/APO-1 were substantiated the biocompatibility properties of prepared collagen scaffolds. Osteogenesis study revealed that the addition of CH and HA in both fish and mammalian collagen scaffolds could efficiently promote osteoblast cell formation. The ALP activity was significantly high in CHA scaffold-treated osteoblast cells, which suggests an enhanced bone-healing process. Therefore, the present study concludes that the composite scaffolds prepared from fish collagen with higher stiffness, lower biodegradation rate, better biocompatible, and osteogenesis properties were suitable biomaterial for a bone tissue engineering application as an alternative to mammalian collagen scaffolds. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Induction of rat facial nerve regeneration by functional collagen scaffolds.

    PubMed

    Cao, Jiani; Xiao, Zhifeng; Jin, Wei; Chen, Bing; Meng, Danqing; Ding, Wenyong; Han, Sufang; Hou, Xiaoshan; Zhu, Tiansheng; Yuan, Baoyu; Wang, Jing; Liang, Weibang; Dai, Jianwu

    2013-01-01

    Nerve conduit provides a promising strategy for nerve regeneration, and the proper microenvironment in the lumen could improve the regeneration. Our previous work had demonstrated that linear ordered collagen scaffold (LOCS) could effectively guide the oriented growth of axons. Laminin is known as an important nerve growth promoting factor and can facilitate the growth cone formation. In addition, ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) can effectively improve the nerve regeneration after nerve injuries. However, in practice, diffusion caused by the body fluids is the major obstacle in their applications. To retain CNTF or BDNF on the scaffolds, we produced collagen binding CNTF (CBD-CNTF), collagen binding BDNF (CBD-BDNF) and laminin binding CNTF (LBD-CNTF), laminin binding BDNF (LBD-BDNF) respectively. In this work, we developed laminin modified LOCS fibers (L × LOCS) by chemical cross-linking LOCS fibers with laminin. Collagen binding or laminin binding neurotrophic factors were combined with LOCS or L × LOCS, and then filled them into the collagen nerve conduit. They were found to guide the ordered growth of axons, and improve the nerve functional recovery in the rat facial nerve transection model. The combination of CNTF and BDNF greatly enhanced the facial nerve regeneration and functional recovery.

  3. ALCOHOL MODULATION OF MMP AND TIMP EXPRESSION IN THE HEART FAVORS COLLAGEN ACCUMULATION

    PubMed Central

    El Hajj, E.C.; El hajj, M.C.; Voloshenyuk, T.G.; Mouton, A.J.; Khoutorova, E.; Molina, P.E.; Gilpin, N.W.; Gardner, J.D.

    2013-01-01

    Background Chronic alcohol consumption has been shown in human and animal studies to result in collagen accumulation, myocardial fibrosis, and heart failure. Cardiac fibroblasts produce collagen and regulate extracellular matrix (ECM) homeostasis through the synthesis and activity of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP), with the balance of MMPs/TIMPs determining the rate of collagen turnover. Dynamic changes of MMP and TIMP expression were reported in alcohol induced hepatic fibrosis; however, the effect of alcohol on MMP/TIMP balance in the heart and cardiac fibroblasts is unknown. We hypothesized that alcohol exposure alters cardiac fibroblast MMP and TIMP expression to promote collagen accumulation in the heart. Methods Cardiac fibroblasts isolated from adult rats were cultured in the presence of alcohol (12.5–200 mM) for 48 hrs. MMP, TIMP, and collagen type I and III expression were assayed by Western blot analysis. Hydroxyproline (HPro) was used as a marker of collagen production. The in vivo cardiac effects of ethanol were determined using rats exposed to ethanol vapor for two weeks, resulting in blood alcohol levels of 150–200 mg/dl. Cardiac collagen volume fraction (CVF), as well as MMP, TIMP and collagen expression, was assessed. Results Ethanol exposed rats exhibited upregulation of TIMP-1, -3 and -4 in the heart, with no significant increases in MMPs. Cardiac fibroblasts exhibited transformation to a profibrotic phenotype following exposure to alcohol. These changes were reflected by increased α-smooth muscle actin and collagen I and III expression, as well as increased collagen secretion. In vivo ethanol exposure also produced fibrosis, indicated by increased CVF and expression of collagens. Conclusion Alcohol exposure modulates cardiac fibroblast MMP/TIMP expression favoring a profile associated with collagen accumulation. Our data suggest that this disrupted MMP/TIMP profile may contribute to the development of

  4. Health Promotion

    PubMed Central

    Karmali-Rawji, Shameela; Kassim-Lakha, Shaheen; Taylor, Karmel

    1992-01-01

    Perceived lack or loss of control, stress, a rapidly again population and rising costs of health care necessitate effective health promotion and disease prevention in the elderly. In a collaborative health promotion effort, the private sector, public sector, and community partners have joined to increase the South Asian elders' sense of control over the decisions and circumstances that affect their everyday lives. The project was designed to help elders come to terms with the fragmentation of their extended families, cultural alienation, decreased autonomy, need for information, and greater risk of cardiovascular disease. Imagesp622-a

  5. An extracellular proteasome releases endostatin from human collagen XVIII.

    PubMed

    Reiss-Pistilli, Maria L V; Schuppan, Detlef; Barroso, Madalena M S; Assunção-Miranda, Iranaia; Farias, Shirley; Lery, Letícia; Bauer, Michael; Juliano, Luiz; Juliano, Maria A; Coelho-Sampaio, Tatiana

    2017-02-01

    Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

  6. Cigarette smoke-induced collagen destruction; key to chronic neutrophilic airway inflammation?

    PubMed

    Overbeek, Saskia A; Braber, Saskia; Koelink, Pim J; Henricks, Paul A J; Mortaz, Esmaeil; LoTam Loi, Adele T; Jackson, Patricia L; Garssen, Johan; Wagenaar, Gerry T M; Timens, Wim; Koenderman, Leo; Blalock, J Edwin; Kraneveld, Aletta D; Folkerts, Gert

    2013-01-01

    Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP. Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE. This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.

  7. Linear Ordered Collagen Scaffolds Loaded with Collagen-Binding Basic Fibroblast Growth Factor Facilitate Recovery of Sciatic Nerve Injury in Rats

    PubMed Central

    Ma, Fukai; Xiao, Zhifeng; Chen, Bing; Hou, Xianglin

    2014-01-01

    Natural biological functional scaffolds, consisting of biological materials filled with promoting elements, provide a promising strategy for the regeneration of peripheral nerve defects. Collagen conduits have been used widely due to their excellent biological properties. Linear ordered collagen scaffold (LOCS) fibers are good lumen fillers that can guide nerve regeneration in an ordered direction. In addition, basic fibroblast growth factor (bFGF) is important in the recovery of nerve injury. However, the traditional method for delivering bFGF to the lesion site has no long-term effect because of its short half-life and rapid diffusion. Therefore, we fused a specific collagen-binding domain (CBD) peptide to the N-terminal of native basic fibroblast growth factor (NAT-bFGF) to retain bFGF on the collagen scaffolds. In this study, a natural biological functional scaffold was constructed using collagen tubes filled with collagen-binding bFGF (CBD-bFGF)-loaded LOCS to promote regeneration in a 5-mm rat sciatic nerve transection model. Functional evaluation, histological investigation, and morphometric analysis indicated that the natural biological functional scaffold retained more bFGF at the injury site, guided axon growth, and promoted nerve regeneration as well as functional restoration. PMID:24188561

  8. Collagen Hydrogel Scaffold and Fibroblast Growth Factor-2 Accelerate Periodontal Healing of Class II Furcation Defects in Dog

    PubMed Central

    Momose, Takehito; Miyaji, Hirofumi; Kato, Akihito; Ogawa, Kosuke; Yoshida, Takashi; Nishida, Erika; Murakami, Syusuke; Kosen, Yuta; Sugaya, Tsutomu; Kawanami, Masamitsu

    2016-01-01

    Objective: Collagen hydrogel scaffold exhibits bio-safe properties and facilitates periodontal wound healing. However, regenerated tissue volume is insufficient. Fibroblast growth factor-2 (FGF2) up-regulates cell behaviors and subsequent wound healing. We evaluated whether periodontal wound healing is promoted by application of collagen hydrogel scaffold in combination with FGF2 in furcation defects in beagle dogs. Methods: Collagen hydrogel was fabricated from bovine type I collagen with an ascorbate-copper ion cross-linking system. Collagen hydrogel was mingled with FGF2 and injected into sponge-form collagen. Subsequently, FGF2 (50 µg)/collagen hydrogel scaffold and collagen hydrogel scaffold alone were implanted into class II furcation defects in dogs. In addition, no implantation was performed as a control. Histometric parameters were assessed at 10 days and 4 weeks after surgery. Result: FGF2 application to scaffold promoted considerable cell and tissue ingrowth containing numerous cells and blood vessel-like structure at day 10. At 4 weeks, reconstruction of alveolar bone was stimulated by implantation of scaffold loaded with FGF2. Furthermore, periodontal attachment, consisting of cementum-like tissue, periodontal ligament-like tissue and Sharpey’s fibers, was also repaired, indicating that FGF2-loaded scaffold guided self-assembly and then re-established the function of periodontal organs. Aberrant healing, such as ankylosis and root resorption, was not observed. Conclusion: FGF2-loaded collagen hydrogel scaffold possessed excellent biocompatibility and strongly promoted periodontal tissue engineering, including periodontal attachment re-organization. PMID:27583044

  9. Nanomechanics of Type I Collagen.

    PubMed

    Varma, Sameer; Orgel, Joseph P R O; Schieber, Jay D

    2016-07-12

    Type I collagen is the predominant collagen in mature tendons and ligaments, where it gives them their load-bearing mechanical properties. Fibrils of type I collagen are formed by the packing of polypeptide triple helices. Higher-order structures like fibril bundles and fibers are assembled from fibrils in the presence of other collagenous molecules and noncollagenous molecules. Curiously, however, experiments show that fibrils/fibril bundles are less resistant to axial stress compared to their constituent triple helices-the Young's moduli of fibrils/fibril bundles are an order-of-magnitude smaller than the Young's moduli of triple helices. Given the sensitivity of the Young's moduli of triple helices to solvation environment, a plausible explanation is that the packing of triple helices into fibrils perhaps reduces the Young's modulus of an individual triple helix, which results in fibrils having smaller Young's moduli. We find, however, from molecular dynamics and accelerated conformational sampling simulations that the Young's modulus of the buried core of the fibril is of the same order as that of a triple helix in aqueous phase. These simulations, therefore, suggest that the lower Young's moduli of fibrils/fibril bundles cannot be attributed to the specific packing of triple helices in the fibril core. It is not the fibril core that yields initially to axial stress. Rather, it must be the portion of the fibril exposed to the solvent and/or the fibril-fibril interface that bears the initial strain. Overall, this work provides estimates of Young's moduli and persistence lengths at two levels of collagen's structural assembly, which are necessary to quantitatively investigate the response of various biological factors on collagen mechanics, including congenital mutations, posttranslational modifications and ligand binding, and also engineer new collagen-based materials.

  10. Fragment Hazard Investigation Program

    DTIC Science & Technology

    1978-10-01

    53 Ballistic Density (k) ............................................. 53 Ejection A ngle (a...54 Ejection Velocity (V) ................................................. 54 DEVELOPMENT OF EMPIRICAL RELATION...5S 54 Fragment Weight Versus Gamma for Test QD-155-08 ......................... 56 55 Fragment Range Versus Ejection Angle as a Function of

  11. Fragments and Coherence

    ERIC Educational Resources Information Center

    Watson, Anne

    2008-01-01

    Can teachers contact the inner coherence of mathematics while working in a context fragmented by always-new objectives, criteria, and initiatives? How, more importantly, can learners experience the inner coherence of mathematics while working in a context fragmented by testing, modular curricular, short-term learning objectives, and lessons that…

  12. Fragmentation of fullerenes

    NASA Astrophysics Data System (ADS)

    Chancey, Ryan T.; Oddershede, Lene; Harris, Frank E.; Sabin, John R.

    2003-04-01

    We have performed classical molecular-dynamics simulations of the fragmentation collisions of neutral fullerenes (C24, C60, C100, and C240) with a hard wall. The interactions between the carbon atoms are modeled by a Tersoff potential and the position of each carbon atom at each time step is calculated using a sixth-order predictor-corrector method. The statistical distribution of the fragments depends on impact energy. At low energies, the fragment distribution appears symmetric, with both the large and small fragment distributions well fitted by an exponential function of the same exponent, the value of which decreases with impact energy. At intermediate energies, the distribution of the smallest fragments can be fitted equally well by a power law or an exponential function. At high impact energies, the entire fragmentation pattern is well described by a single exponential function, the exponent increasing with energy. The observed tendencies in fragment distributions as well as the obtained exponents are in agreement with experimental observations. The fragmentation behavior of the four investigated fullerenes is very similar, and it is noted that C60 appears to be the most stable.

  13. Effect of ultrasonication on the fibril-formation and gel properties of collagen from grass carp skin.

    PubMed

    Jiang, Ying; Wang, Haibo; Deng, Mingxia; Wang, Zhongwen; Zhang, Juntao; Wang, Haiyin; Zhang, Hanjun

    2016-02-01

    Controlling the fibril-formation process of collagen in vitro to fabricate novel biomaterials is a new area in the field of collagen research. This study aimed to determine the effect of ultrasonication on collagen fibril formation and the properties of the resulting collagen gels. Native collagen, extracted from the skin of grass carp, self-assembled under ultrasonic conditions (at different ultrasonic power and duration). The self-assembly kinetics, fibrillar morphology, and physical and cell growth-promoting properties of the collagen gels were analyzed and compared. The results showed that the self-assembly rate of collagen was increased by ultrasonication at the nucleation stage. The resulting fibrils exhibited smaller diameters and D-periodicity lengths than that of the untreated collagen samples (p<0.05). The viscoelasticity and textural properties of collagen gels also changed after ultrasonication at the nucleation stage. Texture profile analysis and cell proliferation assays showed that ultrasonication produced softer collagen gel colloids, which were more suitable for cell proliferation than the untreated collagen gels.

  14. Parathyroid hormone linked to a collagen binding domain (PTH-CBD) promotes hair growth in a mouse model of chemotherapy-induced alopecia in a dose-dependent manner

    PubMed Central

    Katikaneni, Ranjitha; Ponnapakkam, Tulasi; Seymour, Andrew; Sakon, Joshua; Gensure, Robert

    2014-01-01

    Chemotherapy-induced alopecia is a major source of psychological stress in patients undergoing cancer chemotherapy, and can influence treatment decisions. While there is currently no therapy, PTH-CBD, a fusion protein of parathyroid hormone and collagen binding domain, has shown promise in animal models. Objective To determine if there are dose-dependent effects of PTH-CBD on chemotherapy-induced alopecia in a mouse model. Methods C57BL/6J mice were waxed to synchronize hair follicles; treated on day 7 with vehicle or PTH-CBD (100, 320 and 1000 mcg/kg subcutaneous injection); treated on day 9 with vehicle or cyclophosphamide (150 mg/kg i.p.). Mice were photographed every 3–4 days and sacrificed on day 63 for histological analysis. Photographs were quantified by grey scale analysis to assess hair content. Results Mice not receiving chemotherapy showed regrowth of hair 2 weeks following waxing, and normal histology after 2 months. Mice receiving chemotherapy alone showed marked hair loss after chemotherapy, which was sustained for 10 days and was followed by rapid regrowth of a normal coat. Histology revealed rapid cycling dystrophic anagen/catagen follicles. Animals receiving chemotherapy and PTH-CBD showed decreased hair loss and more rapid regrowth of hair than that seen with chemotherapy alone (increased hair growth by grey scale analysis, p<0.05), and the effects were dose dependent. Histologically, hair follicles in animals receiving the highest dose of PTH-CBD were in a quiescent phase, similar to mice which did not receive chemotherapy. Conclusions Single dose subcutaneous administration of PTH-CBD showed dose-dependent effects in minimizing hair loss and speeding recovery from chemotherapy-induced alopecia. PMID:24710191

  15. The genes COL4A5 and COL4A6, coding for basement membrane collagen chains alpha 5(IV) and alpha 6(IV), are located head-to-head in close proximity on human chromosome Xq22 and COL4A6 is transcribed from two alternative promoters.

    PubMed Central

    Sugimoto, M; Oohashi, T; Ninomiya, Y

    1994-01-01

    The genes for the alpha 5(IV) and alpha 6(IV) chains of human basement membrane collagen type IV have been found together on chromosome X at segment q22 and have been reported to be arranged in a head-to-head fashion. Here we report the 5' flanking sequences of COL4A5 and COL4A6 and that COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion. Analysis of the sequence immediately upstream of the transcription start sites revealed some features of housekeeping genes--i.e., the lack of a TATA motif and the presence of CCAAT and CTC boxes. Further analysis revealed that COL4A6 contains two alternative promoters that control the generation of two different transcripts. One transcription start site (from exon 1') is 442 bp away from the transcription start site of COL4A5, while an alternative transcription start site (from exon 1) is located 1050 bp from the first one and drives the expression of a second transcript that encodes an alpha 6(IV) chain with a different signal peptide. Reverse transcription-PCR experiments revealed that the transcript from exon 1' is abundant in placenta, whereas the transcript from exon 1 is more frequently found in kidney and lung. These results provide additional clues to answering the general question of what mechanisms are used to generate unique basement membrane structures in different tissues. Images PMID:7972123

  16. Fragmentation of Shells

    NASA Astrophysics Data System (ADS)

    Wittel, F.; Kun, F.; Herrmann, H. J.; Kröplin, B. H.

    2004-07-01

    We present a theoretical and experimental study of the fragmentation of closed thin shells made of a disordered brittle material. Experiments were performed on brown and white hen egg shells under two different loading conditions: impact with a hard wall and explosion by a combustible mixture. Both give rise to power law fragment size distributions. A three-dimensional discrete element model of shells is worked out. Based on simulations of the model, we give evidence that power law fragment mass distributions arise due to an underlying phase transition which proved to be abrupt for explosion and continuous for impact. We demonstrate that the fragmentation of closed shells defines a new universality class of fragmentation phenomena.

  17. Hydroperoxide formation in model collagens and collagen type I.

    PubMed

    Madison, S A; McCallum, J E B; Rojas Wahl, R U

    2002-02-01

    Protein hydroperoxides represent a relatively new concept in understanding biological oxidation chemistry. Here, we show with post-column-chemiluminescence that this sometimes remarkably stable and yet reactive species can be formed in collagen models and collagen type I when submitted to oxidative stress as exemplified by the Fenton reaction. These findings are supported by mass spectrometry and iodometry. Using (Proline-hydroxyproline-glycine)(10) (POG)(10), those hydroperoxides are stable for hours at room temperature and can give rise to free radicals in the presence of ferrous sulphate, as evidenced by EPR spin trapping with DMPO. Possible implications for biological systems are discussed with emphasis on collagen in the extracellular matrix in skin as a major type of connective tissue.

  18. Enhanced stabilization of collagen by furfural.

    PubMed

    Lakra, Rachita; Kiran, Manikantan Syamala; Usha, Ramamoorthy; Mohan, Ranganathan; Sundaresan, Raja; Korrapati, Purna Sai

    2014-04-01

    Furfural (2-furancarboxaldehyde), a product derived from plant pentosans, has been investigated for its interaction with collagen. Introduction of furfural during fibril formation enhanced the thermal and mechanical stability of collagen. Collagen films treated with furfural exhibited higher denaturation temperature (Td) (p<0.04) and showed a 3-fold increase in Young's modulus (p<0.04) at higher concentration. Furfural and furfural treated collagen films did not have any cytotoxic effect. Rheological characterization showed an increase in shear stress and shear viscosity with increasing shear rate for treated collagen. Circular dichroism (CD) studies indicated that the furfural did not have any impact on triple helical structure of collagen. Scanning electron microscopy (SEM) of furfural treated collagen exhibited small sized porous structure in comparison with untreated collagen. Thus this study provides an alternate ecologically safe crosslinking agent for improving the stability of collagen for biomedical and industrial applications.

  19. Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen.

    PubMed

    Milan, Anna M; Sugars, Rachael V; Embery, Graham; Waddington, Rachel J

    2006-06-01

    Dentine phosphoprotein (DPP) has been proposed to both promote and inhibit mineral deposition during dentinogenesis. The present study aimed to investigate the molecular interactions of DPP and dephosphorylated DPP (DPP-p) with hydroxyapatite (HAP). Bovine DPP was purified and dephosphorylated by alkaline phosphatase to obtain DPP-p. DPP and DPP-p adsorption to HAP was determined along with their ability, when free in solution or bound to collagen, to influence HAP-induced crystal growth. Absorption isotherms suggested that lower DPP concentrations (1.5-6.25 microg ml(-1)) demonstrated a reduced affinity for HAP compared with higher protein concentrations (12.5-50.0 microg ml(-1)). Dephosphorylated DPP had a much reduced affinity for HAP compared with DPP. Dentine phosphoprotein inhibited seeded HAP crystal growth, in a dose-dependent manner, whilst removal of the phosphate groups reduced this inhibition. When bound to collagen fibrils, DPP significantly promoted the rate of HAP crystal growth over 0-8 min. Conversely, DPP-p and collagen significantly decreased the rate of crystal growth over 0-18 min. These results indicate a major role for the phosphate groups present on DPP in HAP crystal growth. In addition, concentration-dependent conformational changes to DPP, and the interaction with other matrix components, such as collagen, are important in predicting its dual role in the mineralization of dentine.

  20. The pathway of collagen secretion.

    PubMed

    Malhotra, Vivek; Erlmann, Patrik

    2015-01-01

    COPII vesicles mediate export of secretory cargo from the endoplasmic reticulum (ER). However, a standard COPII vesicle with a diameter of 60-90 nm is too small to export collagens that are composed of rigid triple helices of up to 400 nm in length. How do cells pack and secrete such bulky molecules? This issue is fundamentally important, as collagens constitute approximately 25% of our dry body weight and are essential for almost all cell-cell interactions. Recently, a potential mechanism for the biogenesis of mega-transport carriers was identified, involving packing collagens and increasing the size of COPII coats. Packing is mediated by TANGO1, which binds procollagen VII in the lumen and interacts with the COPII proteins Sec23/Sec24 on the cytoplasmic side of the ER. Cullin3, an E3 ligase, and its specific adaptor protein, KLHL12, ubiquitinate Sec31, which could increase the size of COPII coats. Recruitment of these proteins and their specific interactors into COPII-mediated vesicle biogenesis may be all that is needed for the export of bulky collagens from the ER. Nonetheless, we present an alternative pathway in which TANGO1 and COPII cooperate to export collagens without generating a mega-transport carrier.

  1. CMG2/ANTXR2 regulates extracellular collagen VI which accumulates in hyaline fibromatosis syndrome

    PubMed Central

    Bürgi, Jérôme; Kunz, Béatrice; Abrami, Laurence; Deuquet, Julie; Piersigilli, Alessandra; Scholl-Bürgi, Sabine; Lausch, Ekkehart; Unger, Sheila; Superti-Furga, Andrea; Bonaldo, Paolo; van der Goot, F. Gisou

    2017-01-01

    Loss-of-function mutations in capillary morphogenesis gene 2 (CMG2/ANTXR2), a transmembrane surface protein, cause hyaline fibromatosis syndrome (HFS), a severe genetic disorder that is characterized by large subcutaneous nodules, gingival hypertrophy and severe painful joint contracture. Here we show that CMG2 is an important regulator of collagen VI homoeostasis. CMG2 loss of function promotes accumulation of collagen VI in patients, leading in particular to nodule formation. Similarly, collagen VI accumulates massively in uteri of Antxr2−/− mice, which do not display changes in collagen gene expression, and leads to progressive fibrosis and sterility. Crossing Antxr2−/− with Col6a1−/− mice leads to restoration of uterine structure and reversion of female infertility. We also demonstrate that CMG2 may act as a signalling receptor for collagen VI and mediates its intracellular degradation. PMID:28604699

  2. CMG2/ANTXR2 regulates extracellular collagen VI which accumulates in hyaline fibromatosis syndrome.

    PubMed

    Bürgi, Jérôme; Kunz, Béatrice; Abrami, Laurence; Deuquet, Julie; Piersigilli, Alessandra; Scholl-Bürgi, Sabine; Lausch, Ekkehart; Unger, Sheila; Superti-Furga, Andrea; Bonaldo, Paolo; van der Goot, F Gisou

    2017-06-12

    Loss-of-function mutations in capillary morphogenesis gene 2 (CMG2/ANTXR2), a transmembrane surface protein, cause hyaline fibromatosis syndrome (HFS), a severe genetic disorder that is characterized by large subcutaneous nodules, gingival hypertrophy and severe painful joint contracture. Here we show that CMG2 is an important regulator of collagen VI homoeostasis. CMG2 loss of function promotes accumulation of collagen VI in patients, leading in particular to nodule formation. Similarly, collagen VI accumulates massively in uteri of Antxr2(-/-) mice, which do not display changes in collagen gene expression, and leads to progressive fibrosis and sterility. Crossing Antxr2(-/-) with Col6a1(-/-) mice leads to restoration of uterine structure and reversion of female infertility. We also demonstrate that CMG2 may act as a signalling receptor for collagen VI and mediates its intracellular degradation.

  3. Covalent Immobilization of Collagen on Titanium through Polydopamine Coating to Improve Cellular Performances of MC3T3-E1 Cells

    PubMed Central

    Yu, Xiaohua; Walsh, John; Wei, Mei

    2014-01-01

    Surface modification of orthopedic implants is critical for improving the clinical performance of these medical devices. Herein, collagen was covalently immobilized onto a titanium implant surface via a novel adherent polydopamine coating inspired by mussel adhesive proteins. The formation and composition of the collagen coating was characterized using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Fluorescent labeled collagen was also used to examine the formation and uniformity of the collagen coating. The resultant collagen coating with a polydopamine supporting substrate demonstrated better uniformity and distribution on the titanium surface compared to a physical adsorption of collagen. The covalent immobilized collagen coating is biologically active, as evidenced by its ability to enhance MC3T3-E1 cell adhesion, support cell proliferation and promote early stage osteogenic differentiation of pre-osteoblasts. Our study suggests covalent immobilization of collagen through the polydopamine coating might be an efficient way to improve the cellular performance of implant surfaces. PMID:24932406

  4. Covalent Immobilization of Collagen on Titanium through Polydopamine Coating to Improve Cellular Performances of MC3T3-E1 Cells.

    PubMed

    Yu, Xiaohua; Walsh, John; Wei, Mei

    2013-11-21

    Surface modification of orthopedic implants is critical for improving the clinical performance of these medical devices. Herein, collagen was covalently immobilized onto a titanium implant surface via a novel adherent polydopamine coating inspired by mussel adhesive proteins. The formation and composition of the collagen coating was characterized using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Fluorescent labeled collagen was also used to examine the formation and uniformity of the collagen coating. The resultant collagen coating with a polydopamine supporting substrate demonstrated better uniformity and distribution on the titanium surface compared to a physical adsorption of collagen. The covalent immobilized collagen coating is biologically active, as evidenced by its ability to enhance MC3T3-E1 cell adhesion, support cell proliferation and promote early stage osteogenic differentiation of pre-osteoblasts. Our study suggests covalent immobilization of collagen through the polydopamine coating might be an efficient way to improve the cellular performance of implant surfaces.

  5. The Role of Collagen Quaternary Structure in the Platelet:Collagen Interaction

    PubMed Central

    Brass, Lawrence F.; Bensusan, Howard B.

    1974-01-01

    We have investigated whether collagen queternary structure is required for the platelet: collagen interaction. Quaternary structure refers to the assembly of collagen monomers (tropocollagen) into polymers (native-type fibrils). Purified monomeric collagen was prepared from acetic acid extracts of fetal calfskin. Polymeric collagen was prepared by dispersion of bovine Achilles tendon collagen and by incubation of monomeric collagen at 37°C and pH 7.4. The state of polymerization was confirmed by electron microscopy. Release of platelet serotonin in the absence of platelet aggregation was used to determine the effectiveness of the platelet: collagen interaction. All forms of collagen produced serotonin release only after a lag period, but polymeric collagen gave a shorter lag period than did monomeric collagen. Monomeric collagen was also quanidinated selectively to convert collagen lysine groups to homoarginine, while leaving the arrangement of polar groups intact. Guanidination of monomeric collagen increased the rate of polymerization and reduced the lag time in serotonin release. Glucosamine (17 mM) retarded polymerization and inhibited the release of platelet serotonin by monomeric collagen but had little effect on release produced by thrombin or polymeric collagen. At the same concentration, glucosamine did not reduce the sensitivity of platelets to stimulation by collagen or block the platelet: collagen interaction. The only effect of glucosamine was on the collagen: collagen interaction. Galactosamine had a similar effect, but glucose, galactose, and N-acetylglycosamine had no effect. We conclude from this data that collagen monomers cannot effectively interact with platelets and that, therefore, collagen quaternary structure has a role in the recognition of collagen by platelets. PMID:4215825

  6. Opaque rock fragments

    SciTech Connect

    Abhijit, B.; Molinaroli, E.; Olsen, J.

    1987-05-01

    The authors describe a new, rare, but petrogenetically significant variety of rock fragments from Holocene detrital sediments. Approximately 50% of the opaque heavy mineral concentrates from Holocene siliciclastic sands are polymineralic-Fe-Ti oxide particles, i.e., they are opaque rock fragments. About 40% to 70% of these rock fragments show intergrowth of hm + il, mt + il, and mt + hm +/- il. Modal analysis of 23,282 opaque particles in 117 polished thin sections of granitic and metamorphic parent rocks and their daughter sands from semi-arid and humid climates show the following relative abundances. The data show that opaque rock fragments are more common in sands from igneous source rocks and that hm + il fragments are more durable. They assume that equilibrium conditions existed in parent rocks during the growth of these paired minerals, and that the Ti/Fe ratio did not change during oxidation of mt to hm. Geothermometric determinations using electron probe microanalysis of opaque rock fragments in sand samples from Lake Erie and the Adriatic Sea suggest that these rock fragments may have equilibrated at approximately 900/sup 0/ and 525/sup 0/C, respectively.

  7. Fragmentation trees reloaded.

    PubMed

    Böcker, Sebastian; Dührkop, Kai

    2016-01-01

    Untargeted metabolomics commonly uses liquid chromatography mass spectrometry to measure abundances of metabolites; subsequent tandem mass spectrometry is used to derive information about individual compounds. One of the bottlenecks in this experimental setup is the interpretation of fragmentation spectra to accurately and efficiently identify compounds. Fragmentation trees have become a powerful tool for the interpretation of tandem mass spectrometry data of small molecules. These trees are determined from the data using combinatorial optimization, and aim at explaining the experimental data via fragmentation cascades. Fragmentation tree computation does not require spectral or structural databases. To obtain biochemically meaningful trees, one needs an elaborate optimization function (scoring). We present a new scoring for computing fragmentation trees, transforming the combinatorial optimization into a Maximum A Posteriori estimator. We demonstrate the superiority of the new scoring for two tasks: both for the de novo identification of molecular formulas of unknown compounds, and for searching a database for structurally similar compounds, our method SIRIUS 3, performs significantly better than the previous version of our method, as well as other methods for this task. SIRIUS 3 can be a part of an untargeted metabolomics workflow, allowing researchers to investigate unknowns using automated computational methods.Graphical abstractWe present a new scoring for computing fragmentation trees from tandem mass spectrometry data based on Bayesian statistics. The best scoring fragmentation tree most likely explains the molecular formula of the measured parent ion.

  8. Impacts of fullerene derivatives on regulating the structure and assembly of collagen molecules

    NASA Astrophysics Data System (ADS)

    Yin, Xiaohui; Zhao, Lina; Kang, Seung-Gu; Pan, Jun; Song, Yan; Zhang, Mingyi; Xing, Gengmei; Wang, Fei; Li, Jingyuan; Zhou, Ruhong; Zhao, Yuliang

    2013-07-01

    During cancer development, the fibrous layers surrounding the tumor surface get thin and stiff which facilitates the tumor metastasis. After the treatment of metallofullerene derivatives Gd@C82(OH)22, the fibrous layers become thicker and softer, the metastasis of tumor is then largely suppressed. The effect of Gd@C82(OH)22 was found to be related to their direct interaction with collagen and the resulting impact on the structure of collagen fibrils, the major component of extracellular matrices. In this work we study the interaction of Gd@C82(OH)22 with collagen by molecular dynamics simulations. We find that Gd@C82(OH)22 can enhance the rigidity of the native structure of collagen molecules and promote the formation of an oligomer or a microfibril. The interaction with Gd@C82(OH)22 may regulate further the assembly of collagen fibrils and change the biophysical properties of collagen. The control run with fullerene derivatives C60(OH)24 also indicates that C60(OH)24 can influence the structure and assembly of collagen molecules as well, but to a lesser degree. Both fullerene derivatives can form hydrogen bonds with multiple collagen molecules acting as a ``fullerenol-mediated bridge'' that enhance the interaction within or among collagen molecules. Compared to C60(OH)24, the interaction of Gd@C82(OH)22 with collagen is stronger, resulting in particular biomedical effects for regulating the biophysical properties of collagen fibrils.During cancer development, the fibrous layers surrounding the tumor surface get thin and stiff which facilitates the tumor metastasis. After the treatment of metallofullerene derivatives Gd@C82(OH)22, the fibrous layers become thicker and softer, the metastasis of tumor is then largely suppressed. The effect of Gd@C82(OH)22 was found to be related to their direct interaction with collagen and the resulting impact on the structure of collagen fibrils, the major component of extracellular matrices. In this work we study the interaction

  9. Fragmentation of monoclonal antibodies

    PubMed Central

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  10. Severe disruption and disorganization of dermal collagen fibrils in early striae gravidarum.

    PubMed

    Wang, F; Calderone, K; Do, T T; Smith, N R; Helfrich, Y R; Johnson, T R B; Kang, S; Voorhees, J J; Fisher, G J

    2017-08-17

    Striae gravidarum (SG), or stretch marks of pregnancy, begin as erythematous streaks, and mature into hypopigmented atrophic bands. To investigate molecular alterations that may promote atrophy of SG, we investigated dermal type I collagen fibrils, which provide human skin with support. We obtained skin samples of recently developed, erythematous abdominal SG from pregnant women. To examine the organization of collagen fibrils, second-harmonic generation imaging was performed using multiphoton microscopy. Immunostaining was used to determine protein expression and localization of type I procollagen, the precursor of type I collagen fibrils. Real-time polymerase chain reaction was used to determine gene expression levels. In control (hip) and stretched, normal-appearing perilesional abdominal skin, dermal collagen fibrils were organized as tightly packed, interwoven bundles. In SG, collagen bundles appeared markedly separated, especially in the mid-to-deep dermis. In the spaces separating bundles, loosely packed wavy collagen fibrils lacking organization as bundles were present. These disorganized fibrils persisted into the postpartum period and failed to form densely packed bundles. Numerous large fibroblasts displaying type I procollagen expression were in close proximity to the disorganized fibrils, suggesting that the fibrils are newly synthesized. Supporting this possibility, immunostaining and gene expression of type I procollagen were increased throughout the dermis of SG. Early SG display marked separation of collagen bundles and emergence of disorganized collagen fibrils that fail to form bundles. These alterations may reflect ineffective repair of collagen bundles disrupted by intense skin stretching. Persistent disruption of the collagenous extracellular matrix likely promotes formation and atrophy of SG. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  11. A conceptual framework to describe the ecology of fragmented landscapes and implications for conservation and management.

    PubMed

    del Castillo, Rafael F

    2015-09-01

    The study of the ecology of fragmented landscapes has been dominated by two assumptions: the unique unidirectional path from larger to smaller fragments and the negligible role of fragment species on fragment properties. An accurate conceptualization of fragmented landscapes requires consideration of the age and origin of the fragments, i.e., direct fragmentation or reverse fragmentation (generation or increase of vegetated fragments by colonization), and the habitat modifications of fragment species (autogenic processes). Colonization and autogenic processes alter the fragments' composition and function. Fragment metrics affect colonization. Autogenic processes are antagonized by disturbances and modulated by abiotic inputs. Fragment alterations by autogenic processes may explain the continuous species substitution detected in some fragments or the species persistence in others. Reverse fragmentation, a natural process in commonly disturbed landscapes, challenges the avoidance-of-habitat disturbance as the ultimate strategy for biodiversity conservation and stresses the importance of pioneer species that promote succession as resilience elements in fragmented landscapes. Among-fragment diversity, generated by local disturbances, can be essential for the resilience of fragmented landscapes, suggesting that conservation and habitat utilization can be complementary processes. Traditional agroforestry systems that depend on disturbance, fragmentation, colonization, and autogenic processes may provide important insights into fragmentation ecology.

  12. Excessive collagen turnover products are released during colorectal cancer progression and elevated in serum from metastatic colorectal cancer patients

    PubMed Central

    Kehlet, S. N.; Sanz-Pamplona, R.; Brix, S.; Leeming, D. J.; Karsdal, M. A.; Moreno, V.

    2016-01-01

    During cancer progression, the homeostasis of the extracellular matrix becomes imbalanced with an excessive collagen remodeling by matrix metalloproteinases. As a consequence, small protein fragments of degraded collagens are released into the circulation. We have investigated the potential of protein fragments of collagen type I, III and IV as novel biomarkers for colorectal cancer. Specific fragments of degraded type I, III and IV collagen (C1M, C3M, C4M) and type III collagen formation (Pro-C3) were assessed in serum from colorectal cancer patients, subjects with adenomas and matched healthy controls using well-characterized and validated ELISAs. Serum levels of the biomarkers were significantly elevated in colorectal cancer patients compared to subjects with adenomas (C1M, Pro-C3, C3M) and controls (C1M, Pro-C3). When patients were stratified according to their tumour stage, all four biomarkers were able to differentiate stage IV metastatic patients from all other stages. Combination of all markers with age and gender in a logistic regression model discriminated between metastatic and non-metastatic patients with an AUROC of 0.80. The data suggest that the levels of these collagen remodeling biomarkers may be a measure of tumour activity and invasiveness and may provide new clinical tools for monitoring of patients with advanced stage colorectal cancer. PMID:27465284

  13. Universality in Fragmentation

    NASA Astrophysics Data System (ADS)

    Åström, J. A.; Holian, B. L.; Timonen, J.

    2000-04-01

    Fragmentation of a two-dimensional brittle solid by impact and ``explosion,'' and a fluid by ``explosion'' are all shown to become critical. The critical points appear at a nonzero impact velocity, and at infinite explosion duration, respectively. Within the critical regimes, the fragment-size distributions satisfy a scaling form qualitatively similar to that of the cluster-size distribution of percolation, but they belong to another universality class. Energy balance arguments give a correlation length exponent that is exactly one-half of its percolation value. A single crack dominates fragmentation in the slow-fracture limit, as expected.

  14. Recovery of Uranium Fragments

    NASA Astrophysics Data System (ADS)

    James, H. R.; McElrue, D. H.; Winter, R. E.

    2002-07-01

    We describe a theory for calculating the penetration of fragments into foam. Comparisons with regular projectiles show that the drag term is similar in value to the analogous term in aerodynamics. This, plus the simple model used to describe porosity, enables the theory to be used in predicting the levels of stress present when uranium fragments are arrested in foam catchers. Consequently the theory can be used to assist in the design of catchers which will not distort uranium fragments travelling at 1-3 km/s. The theory is tested against experiments using some current designs.

  15. Mandibular Cartilage Collagen Network Nanostructure

    PubMed Central

    Vanden Berg-Foels, Wendy S.

    2015-01-01

    Background Mandibular condyle cartilage (MCC) has a unique structure among articular cartilages; however, little is known about its nanoscale collagen network architecture, hampering design of regeneration therapies and rigorous evaluation of regeneration experiment outcomes in preclinical research. Helium ion microscopy is a novel technology with a long depth of field that is uniquely suited to imaging open 3D collagen networks at multiple scales without obscuring conductive coatings. Objective The objective of this research was to image, at the micro- and nanoscales, the depth-dependent MCC collagen network architecture. Design MCC was collected from New Zealand white rabbits. Images of MCC zones were acquired using helium ion, transmission electron, and light microscopy. Network fibril and canal diameters were measured. Results For the first time, the MCC was visualized as a 3D collagen fibril structure at the nanoscale, the length scale of network assembly. Fibril diameters ranged from 7 to 110 nm and varied by zone. The articular surface was composed of a fine mesh that was woven through thin layers of larger fibrils. The fibrous zone was composed of approximately orthogonal lamellae of aligned fibrils. Fibrocyte processes surrounded collagen bundles forming extracellular compartments. The proliferative, mature, and hypertrophic zones were composed of a branched network that was progressively remodeled to accommodate chondrocyte hypertrophy. Osteoid fibrils were woven around osteoblast cytoplasmic processes to create numerous canals similar in size to canaliculi of mature bone. Conclusion This multiscale investigation advances our foundational understanding of the complex, layered 3D architecture of the MCC collagen network. PMID:27375843

  16. Structural hierarchy controls deformation behavior of collagen.

    PubMed

    Pradhan, Shashindra M; Katti, Kalpana S; Katti, Dinesh R

    2012-08-13

    The structure of collagen, the most abundant protein in mammals, consists of a triple helix composed of three helical polypeptide chains. The deformation behavior of collagen is governed by molecular mechanisms that involve the interaction between different helical hierarchies found in collagen. Here, we report results of Steered Molecular Dynamics study of the full-length collagen molecule (~290 nm). The collagen molecule is extended at various pulling rates ranging from 0.00003/ps to 0.012/ps. These simulations reveal a new level of hierarchy exhibited by collagen: helicity of the triple chain. This level of hierarchy is apparent at the 290 nm length and cannot be observed in the 7-9 nm models often described to evaluate collagen mechanics. The deformation mechanisms in collagen are governed by all three levels of hierarchy, helicity of single chain (level-1), helical triple helix (level-2), and hereby described helicity of the triple chain (level-3). The mechanics resulting from the three levels is described by an interlocking gear analogy. In addition, remarkably, the full-length collagen does not show much unwinding of triple helix unlike that exhibited by short collagen models. Further, the full-length collagen does not show significant unwinding of the triple helix, unlike that exhibited by short collagen. Also reported is that the interchain hydrogen bond energy in the full-length collagen is significantly smaller than the overall interchain nonbonded interaction energies, suggesting that the nonbonded interactions have far more important role than hydrogen bonds in the mechanics of collagen. However, hydrogen bonding is essential for the triple helical conformation of the collagen. Hence, although mechanics of collagen is controlled by nonbonded interchain interaction energies, the confirmation of collagen is attributed to the interchain hydrogen bonding.

  17. Fragmentation in Biaxial Tension

    SciTech Connect

    Campbell, G H; Archbold, G C; Hurricane, O A; Miller, P L

    2006-06-13

    We have carried out an experiment that places a ductile stainless steel in a state of biaxial tension at a high rate of strain. The loading of the ductile metal spherical cap is performed by the detonation of a high explosive layer with a conforming geometry to expand the metal radially outwards. Simulations of the loading and expansion of the metal predict strain rates that compare well with experimental observations. A high percentage of the HE loaded material was recovered through a soft capture process and characterization of the recovered fragments provided high quality data, including uniform strain prior to failure and fragment size. These data were used with a modified fragmentation model to determine a fragmentation energy.

  18. Type V Collagen in Health, Disease, and Fibrosis.

    PubMed

    Mak, Ki M; Png, Chien Yi M; Lee, Danielle J

    2016-05-01

    Type V collagen (COLV) is a regulatory fibril-forming collagen. It has at least three different molecular isoforms-α1(V)2 α2(V), α1(V)3, and α1(V)α2(V)α3(V)-formed by combinations of three different polypeptide α chains-α1(V), α2(V), and α3(V). COL V is a relatively minor collagen of the extracellular matrix (ECM). Morphologically, COLV occurs as heterotypic fibrils with type I collagen (COLI), microfilaments, or 12-nm-thick fibrils. COLV is synthesized in various mesenchymal cells and its gene expression is modulated by TGF-β and growth factors. While resistant to digestion by interstitial collagenases, native and denatured COLV are degraded by metalloproteinases and gelatinases, thereby promoting ECM remodeling. COLV interacts with matrix collagens and structural proteins, conferring structural integrity to tissue scaffolds. It binds matrix macromolecules, modulating cellular behavior, and functions. COLV co-assembles with COLI into heterotypic fibrils in the cornea and skin dermis, acting as a dominant regulator of collagen fibrillogenesis. COLV deficiency is associated with loss of corneal transparency and classic Ehlers-Danlos syndrome, while COLV overexpression is found in cancer, granulation tissue, inflammation, atherosclerosis, and fibrosis of lungs, skin, kidneys, adipose tissue, and liver. COLV isoform containing the α3(V) chain is involved in mediating pancreatic islet cell functions. In the liver, COLV is a minor but regular component of the ECM. Increases in COLV are associated with both early and advanced hepatic fibrosis. The neoepitopes of COLV have been shown to be a useful noninvasive serum biomarker for assessing fibrotic progression and resolution in experimental hepatic fibrosis. COLV is multifunctional in health, disease, and fibrosis.

  19. Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization.

    PubMed

    Burton, Brianne; Gaspar, Anne; Josey, David; Tupy, Jindra; Grynpas, Marc D; Willett, Thomas L

    2014-04-01

    Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness. Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture. Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For

  20. Analysis of mineral deposition in turkey tendons and self-assembled collagen fibers using mechanical techniques.

    PubMed

    Freeman, Joseph W; Silver, Frederick H

    2004-01-01

    During limb movement and locomotion, animals store elastic energy in the tendons of the feet, legs, and other limbs. In the turkey, much of the force generated by the gastrocnemius muscle during locomotion is stored as elastic energy through deformation of the tendon. During growth and development, the leg tendons in some avians, including turkeys, mineralize and result in an increase in tensile strength and modulus. The purpose of our study was to evaluate the effects of mineralization on elastic energy storage and transmission in turkey tendons. Elastic and viscous stress-strain curves and elastic energy storage behavior were used to compare the behavior of mineralized turkey gastrocnemius tendons and mineralized self-assembled type I collagen fibers. Based on analysis of these two systems, we concluded that a simple mineralized fibrillar collagenous substrate can mimic the behavior of a more complex fibrillar collagenous substrate such as mineralized turkey tendon; however, the exact mechanism of mineralization may be different between the two substrates. Changes in mechanical properties of turkey tendon were consistent with a model in which mineralization appears to increase the effective collagen fibril length by efficiently transferring stress between neighboring collagen fibrils. Mineralization in self-assembled collagen fibers increased elastic energy storage less efficiently as compared with turkey tendon suggesting that the noncollagenous components of mineralizing tissue may act to promote collagen fibril to collagen fibril interactions.

  1. The evolution of fibrillar collagens: a sea-pen collagen shares common features with vertebrate type V collagen.

    PubMed

    Tillet, E; Franc, J M; Franc, S; Garrone, R

    1996-02-01

    The extracellular matrix of marine primitive invertebrates (sponges, polyps and jellyfishes) contains collagen fibrils with narrow diameters. From various data, it has been hypothesized that these primitive collagens could represent ancestral forms of the vertebrate minor collagens, i.e., types V or XI. Recently we have isolated a primitive collagen from the soft tissues of the sea-pen Veretillum cynomorium. This report examines whether the sea-pen collagen shares some features with vertebrate type V collagen. Rotary shadowed images of acid-soluble collagen molecules extracted from beta-APN treated animals, positive staining of segment-long-spacing crystallites precipitated from pepsinized collagen, Western blots of the pepsinized alpha1 and alpha2 chains with antibodies to vertebrate types I, III and V collagens, and in situ gold immunolabeling of ECM collagen fibrils were examined. Our results showed that the tissue form of the sea-pen collagen is a 340-nm threadlike molecule, which is close to the vertebrate type V collagen with its voluminous terminal globular domain, the distribution of most of its polar amino-acid residues, and its antigenic properties.

  2. Endothelial Cell Growth and Differentiation on Collagen-Immobilized Polycaprolactone Nanowire Surfaces.

    PubMed

    Leszczak, Victoria; Baskett, Dominique A; Popat, Ketul C

    2015-06-01

    The success of cardiovascular implants is associated with the development of an endothelium on material surface, critical to the prevention of intimal hyperplasia, calcification and thrombosis. A thorough understanding of the interaction between vascular endothelial cells and the biomaterial involved is essential in order to have a successful application which promotes healing and regeneration through integration with native tissue. In this study, we have developed collagen immobilized nanostructured surfaces with controlled arrays of high aspect ratio nanowires for the growth and maintenance of human microvascular endothelial cells (HMVECs). The nanowire surfaces were fabricated from polycaprolactone using a novel nanotemplating technique, and were immobilized with collagen utilizing an aminolysis method. The collagen immobilized nanowire surfaces were characterized using contact angle measurements, scanning electron microscopy and X-ray photoelectron spectroscopy. Human microvascular endothelial cells were used to evaluate the efficacy of the collagen immobilized nanowire surfaces to promote cell adhesion, proliferation, viability and differentiation. The results presented here indicate significantly higher cellular adhesion, proliferation and viability on nanowire and collagen immobilized surfaces as compared to the control surface. Further, HMVECs have a more elongated body and low shape factor on nanostructured surfaces. The differentiation potential of collagen immobilized nanowire surfaces was also evaluated by immunostaining and western blotting for key endothelial cell markers that are expressed when human microvascular endothelial cells are differentiated. Results indicate that expression of VE-cadherin is increased on collagen immobilized surfaces while the expression of von Willebrand factor is statistically similar on all surfaces.

  3. A biomaterial composed of collagen and solubilized elastin enhances angiogenesis and elastic fiber formation without calcification.

    PubMed

    Daamen, Willeke F; Nillesen, Suzan T M; Wismans, Ronnie G; Reinhardt, Dieter P; Hafmans, Theo; Veerkamp, Jacques H; van Kuppevelt, Toin H

    2008-03-01

    Elastin is the prime protein in elastic tissues that contributes to elasticity of, for example, lung, aorta, and skin. Upon injury, elastic fibers are not readily replaced, which hampers tissue regeneration. Incorporation of solubilized elastin (hydrolyzed insoluble elastin fibers or elastin peptides) in biomaterials may improve regeneration, because solubilized elastin is able to promote proliferation as well as elastin synthesis. Porous biomaterials composed of highly purified collagen without and without elastin fibers or solubilized elastin were prepared by freezing and lyophilization. Solubilized elastin formed spherical structures that were incorporated in the collagenous part of the scaffolds and that persisted after chemical crosslinking of the scaffolds. Crosslinked scaffolds were subcutaneously implanted in young Sprague Dawley rats. Collagen-solubilized elastin and collagen scaffolds showed no calcification in this sensitive calcification model, in contrast to scaffolds containing elastin fibers. Collagen-solubilized elastin scaffolds also induced angiogenesis, as revealed by type IV collagen staining, and promoted elastic fiber synthesis, as shown with antibodies against rat elastin and fibrillin-1. It is concluded that scaffolds produced from collagen and solubilized elastin present a non-calcifying biomaterial with a capacity for soft-tissue regeneration, especially in relation to elastic fiber synthesis.

  4. [Disc electrophoresis of collagen protein (author's transl)].

    PubMed

    Reitmayr, P; Verzár, F

    1975-01-01

    The composition of proteins extracted from tendon collagen is investigated by disc electrophoresis. No qualitative differences can be demonstrated between young and old collagen. The action of formaldehyde and methionine on the tendons has no effect on the electrophoretic picture.

  5. Collagen crosslinks in chondromalacia of the patella.

    PubMed

    Väätäinen, U; Kiviranta, I; Jaroma, H; Arokosi, J; Tammi, M; Kovanen, V

    1998-02-01

    The aim of the study was to determine collagen concentration and collagen crosslinks in cartilage samples from chondromalacia of the patella. To study the extracellular matrix alterations associated to chondromalacia, we determined the concentration of collagen (hydroxyproline) and its hydroxylysylpyridinoline and lysylpyridinoline crosslinks from chondromalacia foci of the patellae in 12 patients and 7 controls from apparently normal cadavers. The structure of the collagen network in 8 samples of grades II-IV chondromalacia was examined under polarized light microscopy. The full-thickness cartilage samples taken with a surgical knife from chondromalacia lesions did not show changes in collagen, hydroxylysylpyridinoline and lysylpyridinoline concentration as compared with the controls. Polarized light microscopy showed decreased birefringence in the superficial cartilage of chondromalacia lesions, indicating disorganization or disappearance of collagen fibers in this zone. It is concluded that the collagen network shows gradual disorganization with the severity of chondromalacia lesion of the patella without changes in the concentration or crosslinks of collagen.

  6. Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.

    PubMed Central

    Chiang, T M; Rinaldy, A; Kang, A H

    1997-01-01

    A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen. PMID:9239397

  7. Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.

    PubMed

    Chiang, T M; Rinaldy, A; Kang, A H

    1997-08-01

    A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.

  8. Degradation of the COL1 domain of type XIV collagen by 92-kDa gelatinase.

    PubMed

    Sires, U I; Dublet, B; Aubert-Foucher, E; van der Rest, M; Welgus, H G

    1995-01-20

    Type XIV collagen is a newly described member of the fibril-associated collagens with interrupted triple helices (FACITs). Expression of this collagen has been localized to various embryonic tissues, suggesting that it has a functional role in development. All FACITs thus far described (types IX, XII, XIV, and XVI) contain a highly homologous carboxyl-terminal triple helical domain designated COL1. We have studied the capacity of various matrix metalloproteinases (interstitial collagenase, stromelysin, matrilysin, and 92-kDa gelatinase) to degrade the COL1 domain of collagen XIV. We found that only 92-kDa gelatinase cleaves COL1. Furthermore, digestion of whole native collagen XIV by the 92-kDa gelatinase indicates that this enzyme specifically attacks the carboxyl-terminal triple helix-containing region of the molecule. COL1 is cleaved by 92-kDa gelatinase at 30 degrees C, a full 5-6 degrees C below the melting temperature (Tm) of this domain; native collagen XIV is also degraded at 30 degrees C. In comparison to interstitial collagenase degradation of its physiologic native type I collagen substrate, the 92-kDa enzyme cleaved COL1 (XIV) with comparable catalytic efficacy. Interestingly, following thermal denaturation of the COL1 fragment, its susceptibility to 92-kDa gelatinase increases, but only to a degree that leaves it several orders of magnitude less sensitive to degradation than denatured collagens I and III. These data indicate that native COL1 and collagen XIV are readily and specifically cleaved by 92-kDa gelatinase. They also suggest a role for 92-kDa gelatinase activity in the structural tissue remodeling of the developing embryo.

  9. The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern.

    PubMed Central

    Elima, K; Eerola, I; Rosati, R; Metsäranta, M; Garofalo, S; Perälä, M; De Crombrugghe, B; Vuorio, E

    1993-01-01

    Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3'-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3'-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice. Images Figure 3 Figure 5 Figure 6 PMID:8424763

  10. Wavelength-Dependent Conformational Changes of Collagen in Mid-IR Ablation

    NASA Astrophysics Data System (ADS)

    Hutson, M. Shane; Xiao, Yaowu; Guo, Mingsheng

    2006-03-01

    Single pulses of the Mark-III free electron laser have been used to ablate porcine corneas at a fluence of 240 J/cm^2 and wavelengths of 2.77 and 6.45 μm. As previously characterized, the non-volatile ablation debris shows evidence of wavelength-dependent collagen fragmentation. We have measured micro-Raman spectra of the debris and the ablation crater to determine if any wavelength-dependent conformational changes have taken place. Comparison of the spectra from two different wavelengths shows that a 938 cm-1 Raman band -- assignable to the peptide CC=O-Cα stretch of collagen -- loses substantial intensity during 6.45-μm ablation, but not in 2.77-μm ablation. This intensity decrease may be associated with a reduction of collagen triple-helix structure. Other spectral techniques yield mixed results; signatures for the loss of triple-helix structure are evident in UV-CD and some aspects of ^13C-NMR spectra, but not in FTIR spectra. Raman measurements on thermally-treated corneal slices display similar changes at high temperatures, suggesting that higher protein temperatures are reached during ablation at 6.45 μm when compared to 2.77 μm. These observations suggest that any pre-vaporization loss of protein structural integrity includes not only collagen fragmentation, but also a loss of collagen triple-helix structure.

  11. Biology, chemistry and pathology of collagen

    SciTech Connect

    Fleischmajer, R.; Olsen, B.R.; Kuhn, K.

    1985-01-01

    This book consists of five parts and a section of poster papers. Some of the articles are: Structure of the Type II Collagen Gene; Structural and Functional Analysis of the Genes for ..cap alpha..2(1) and ..cap alpha..1(III) collagens; Structure and Expression of the Collagen Genes of C. Elegans; Molecular Basis of Clinical Heterogeneity in the Ehlers-Danlos Syndrome; and Normal and Mutant Human Collagen Genes.

  12. Valproic acid suppresses collagen by selective regulation of Smads in conjunctival fibrosis.

    PubMed

    Seet, Li-Fong; Toh, Li Zhen; Finger, Sharon N; Chu, Stephanie W L; Stefanovic, Branko; Wong, Tina T

    2016-03-01

    Overproduction of type I collagen is associated with a wide range of fibrotic diseases as well as surgical failure such as in glaucoma filtration surgery (GFS). Its modulation is therefore of clinical importance. Valproic acid (VPA) is known to reduce collagen in a variety of tissues with unclear mechanism of action. In this report, we demonstrate that VPA inhibited collagen production in both conjunctival fibroblasts and the mouse model of GFS. In fibroblasts, VPA decreased type I collagen expression which intensified with longer drug exposure and suppressed steady-state type I collagen promoter activity. Moreover, VPA decreased Smad2, Smad3 and Smad4 but increased Smad6 expression with a similar intensity-exposure profile. Reduction of Smad3 using small hairpin RNA and/or overexpression of Smad6 resulted in decreased collagen expression which was exacerbated when VPA was simultaneously present. Furthermore, fibrogenic TGF-β2 failed to induce collagen when VPA was present, as opposed to the myofibroblast markers, beta-actin, alpha-smooth muscle actin and tenascin-C, which were elevated by TGF-β2. VPA suppressed p3TP-Lux luciferase activity and selectively rescued Smad6 expression from suppression by TGF-β2. Notably, SMAD6 overexpression reduced the effectiveness of TGF-β2 in inducing collagen expression. In corroboration, VPA inhibited type I collagen but increased Smad6 expression in the late phase of wound healing in the mouse model of GFS. Taken together, our data indicate that VPA has the capacity to effectively suppress both steady-state and fibrogenic activation of type I collagen expression by modulating Smad expression. Hence, VPA is potentially applicable as an anti-fibrotic therapeutic by targeting collagen. Key message: • VPA modulates type I collagen expression via members of the Smad family. • VPA suppresses Smad2, Smad3 and Smad4 but upregulates Smad6. • Smad3 and Smad6 are involved in VPA regulation of steady-state collagen expression.

  13. The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior

    PubMed Central

    An, Bo; DesRochers, Teresa M.; Qin, Guokui; Xia, Xiaoxia; Thiagarajan, Geetha; Brodsky, Barbara; Kaplan, David

    2012-01-01

    Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in E. coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyrogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications. PMID:23088839

  14. The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior.

    PubMed

    An, Bo; DesRochers, Teresa M; Qin, Guokui; Xia, Xiaoxia; Thiagarajan, Geetha; Brodsky, Barbara; Kaplan, David L

    2013-01-01

    Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications.

  15. Biomimetic Analogs for Collagen Biomineralization

    PubMed Central

    Gu, L.; Kim, Y.K.; Liu, Y.; Ryou, H.; Wimmer, C.E.; Dai, L.; Arola, D.D.; Looney, S.W.; Pashley, D.H.; Tay, F.R.

    2011-01-01

    Inability of chemical phosphorylation of sodium trimetaphosphate to induce intrafibrillar mineralization of type I collagen may be due to the failure to incorporate a biomimetic analog to stabilize amorphous calcium phosphates (ACP) as nanoprecursors. This study investigated adsorption/desorption characteristics of hydrolyzed and pH-adjusted sodium trimetaphosphate (HPA-Na3P3O9) to collagen. Based on those results, a 5-minute treatment time with 2.8 wt% HPA-Na3P3O9 was used in a single-layer reconstituted collagen model to confirm that both the ACP-stabilization analog and matrix phosphoprotein analog must be present for intrafibrillar mineralization. The results of that model were further validated by complete remineralization of phosphoric-acid-etched dentin treated with the matrix phosphoprotein analog and lined with a remineralizing lining composite, and with the ACP-stabilization analog supplied in simulated body fluid. An understanding of the basic processes involved in intrafibrillar mineralization of reconstituted collagen fibrils facilitates the design of novel tissue engineering materials for hard tissue repair and regeneration. PMID:20940362

  16. The materials science of collagen.

    PubMed

    Sherman, Vincent R; Yang, Wen; Meyers, Marc A

    2015-12-01

    Collagen is the principal biopolymer in the extracellular matrix of both vertebrates and invertebrates. It is produced in specialized cells (fibroblasts) and extracted into the body by a series of intra and extracellular steps. It is prevalent in connective tissues, and the arrangement of collagen determines the mechanical response. In biomineralized materials, its fraction and spatial distribution provide the necessary toughness and anisotropy. We review the structure of collagen, with emphasis on its hierarchical arrangement, and present constitutive equations that describe its mechanical response, classified into three groups: hyperelastic macroscopic models based on strain energy in which strain energy functions are developed; macroscopic mathematical fits with a nonlinear constitutive response; structurally and physically based models where a constitutive equation of a linear elastic material is modified by geometric characteristics. Viscoelasticity is incorporated into the existing constitutive models and the effect of hydration is discussed. We illustrate the importance of collagen with descriptions of its organization and properties in skin, fish scales, and bone, focusing on the findings of our group.

  17. Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy.

    PubMed

    Cui, Jason Z; Tehrani, Arash Y; Jett, Kimberly A; Bernatchez, Pascal; van Breemen, Cornelis; Esfandiarei, Mitra

    2014-09-01

    In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.

  18. Echinoid skeleton: absence of a collagenous matrix.

    PubMed

    Klein, L; Currey, J D

    1970-09-18

    Lack of hydroxyproline and proline in the calcified distal spines and Aristotle's lantern of the echinoderm Strongylocentrotus indicated the absence of a collagenous matrix. The fact that the small amount of collagen present in the base of the spines and in the test with sutures was removed by bacterial collagenase indicates that this collagen was not calcified.

  19. The binding capacity of α1β1-, α2β1- and α10β1-integrins depends on non-collagenous surface macromolecules rather than the collagens in cartilage fibrils.

    PubMed

    Woltersdorf, Christian; Bonk, Melanie; Leitinger, Birgit; Huhtala, Mikko; Käpylä, Jarmo; Heino, Jyrki; Gil Girol, Christian; Niland, Stephan; Eble, Johannes A; Bruckner, Peter; Dreier, Rita; Hansen, Uwe

    2017-02-10

    Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of β1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1β1-, α2β1- and α10β1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.

  20. Soybean-fragmented proteoglycans against skin aging.

    PubMed

    Barba, Clara; Alonso, Cristina; Sánchez, Isabel; Suñer, Elisa; Sáez-Martín, L C; Coderch, Luisa

    2017-08-01

    The majority of age-dependent skin changes happen in the dermis layer inducing changes in skin collagen and in the proteoglycans. The main aim of this work is to study the efficacy of a Proteum serum, containing soybean-fragmented proteoglycans, against skin aging. In vitro tests were performed to evaluate the Proteum serum ability on activating the production of collagen and proteoglycans. An in vivo long-term study was performed to determine the efficacy of the Proteum serum when applied on skin. Protection of healthy skin against detergent-induced dermatitis and the antioxidant properties of the applied Proteum serum were also studied. The in vitro tests demonstrated that the Proteum serum was able to elevate the production of molecules which are essential for supporting the dermal extracellular matrix organization. These results were correlated by the in vivo measurements where a clear trend on improving the measured skin parameters due to the Proteum serum application was found. A beneficial effect of the Proteum serum was demonstrated with an improvement in the skin roughness and a reinforcement of the skin barrier function. Moreover, a significant protector effect on human stratum corneum against lipids peroxides (LPO) was demonstrated.

  1. The collagenous gastroenteritides: similarities and differences.

    PubMed

    Gopal, Purva; McKenna, Barbara J

    2010-10-01

    Collagenous gastritis, collagenous sprue, and collagenous colitis share striking histologic similarities and occur together in some patients. They also share some drug and disease associations. Pediatric cases of collagenous gastritis, however, lack most of these associations. The etiologies of the collagenous gastroenteritides are not known, so it is not clear whether they are similar because they share pathogeneses, or because they indicate a common histologic response to varying injuries. The features, disease and drug associations, and the inquiries into the pathogenesis of these disorders are reviewed.

  2. Collagen: a network for regenerative medicine

    PubMed Central

    Pawelec, K. M.; Best, S. M.

    2016-01-01

    The basic building block of the extra-cellular matrix in native tissue is collagen. As a structural protein, collagen has an inherent biocompatibility making it an ideal material for regenerative medicine. Cellular response, mediated by integrins, is dictated by the structure and chemistry of the collagen fibers. Fiber formation, via fibrillogenesis, can be controlled in vitro by several factors: pH, ionic strength, and collagen structure. After formation, fibers are stabilized via cross-linking. The final bioactivity of collagen scaffolds is a result of both processes. By considering each step of fabrication, scaffolds can be tailored for the specific needs of each tissue, improving their therapeutic potential. PMID:27928505

  3. The linker-free covalent attachment of collagen to plasma immersion ion implantation treated polytetrafluoroethylene and subsequent cell-binding activity.

    PubMed

    Bax, Daniel V; McKenzie, David R; Weiss, Anthony S; Bilek, Marcela M M

    2010-03-01

    It is desirable that polymers used for the fabrication of prosthetic implants promote biological functions such as cellular adhesion, differentiation and viability. In this study, we have used plasma immersion ion implantation (PIII) to modify the surface of polytetrafluoroethylene (PTFE), thereby modulating the binding mechanism of collagen. The amount of collagen bound to the polymer surface following PIII-treatment was similar to that bound by non-covalent physisorption. In a manner consistent with previous enzyme and tropoelastin binding data, the collagen bound to the PIII-treated PTFE surface was resistant to sodium dodecyl sulfate (SDS) elution whilst collagen bound to the untreated surface was fully removed. This demonstrates the capability of PIII-treated surfaces to covalently attach collagen without employing chemical linking molecules. Only the collagen bound to the PIII-treated PTFE surface supported human dermal fibroblast attachment and spreading. This indicates that collagen on the PIII-treated surface possesses increased adhesive activity as compared to that on the untreated surface. Cell adhesion was inhibited by EDTA when the collagen was bound to PIII-treated PTFE, as expected for integrin involvement. Additionally this adhesion was sensitive to the conformation of the bound collagen. Increased actin cytoskeletal assembly was observed on cells spreading onto collagen-coated PIII-treated PTFE compared to the collagen-coated untreated PTFE. These data demonstrate the retention of collagen's biological properties following its attachment to PIII-treated PTFE, suggesting advantages for tissue engineering and prosthetic design.

  4. The collagen receptor uPARAP/Endo180 in tissue degradation and cancer (Review).

    PubMed

    Melander, Maria C; Jürgensen, Henrik J; Madsen, Daniel H; Engelholm, Lars H; Behrendt, Niels

    2015-10-01

    The collagen receptor uPARAP/Endo180, the product of the MRC2 gene, is a central component in the collagen turnover process governed by various mesenchymal cells. Through the endocytosis of collagen or large collagen fragments, this recycling receptor serves to direct basement membrane collagen as well as interstitial collagen to lysosomal degradation. This capacity, shared only with the mannose receptor from the same protein family, endows uPARAP/Endo180 with a critical role in development and homeostasis, as well as in pathological disruptions of the extracellular matrix structure. Important pathological functions of uPARAP/Endo180 have been identified in various cancers and in several fibrotic conditions. With a particular focus on matrix turnover in cancer, this review presents the necessary background for understanding the function of uPARAP/Endo180 at the molecular and cellular level, followed by an in-depth survey of the available knowledge of the expression and role of this receptor in various types of cancer and other degenerative diseases.

  5. The collagen receptor uPARAP/Endo180 in tissue degradation and cancer (Review)

    PubMed Central

    MELANDER, MARIA C.; JÜRGENSEN, HENRIK J.; MADSEN, DANIEL H.; ENGELHOLM, LARS H.; BEHRENDT, NIELS

    2015-01-01

    The collagen receptor uPARAP/Endo180, the product of the MRC2 gene, is a central component in the collagen turnover process governed by various mesenchymal cells. Through the endocytosis of collagen or large collagen fragments, this recycling receptor serves to direct basement membrane collagen as well as interstitial collagen to lysosomal degradation. This capacity, shared only with the mannose receptor from the same protein family, endows uPARAP/Endo180 with a critical role in development and homeostasis, as well as in pathological disruptions of the extracellular matrix structure. Important pathological functions of uPARAP/Endo180 have been identified in various cancers and in several fibrotic conditions. With a particular focus on matrix turnover in cancer, this review presents the necessary background for understanding the function of uPARAP/Endo180 at the molecular and cellular level, followed by an in-depth survey of the available knowledge of the expression and role of this receptor in various types of cancer and other degenerative diseases. PMID:26316068

  6. Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

    PubMed Central

    1987-01-01

    Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross- hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth. PMID:3558480

  7. Collagen interactions: Drug design and delivery.

    PubMed

    An, Bo; Lin, Yu-Shan; Brodsky, Barbara

    2016-02-01

    Collagen is a major component in a wide range of drug delivery systems and biomaterial applications. Its basic physical and structural properties, together with its low immunogenicity and natural turnover, are keys to its biocompatibility and effectiveness. In addition to its material properties, the collagen triple-helix interacts with a large number of molecules that trigger biological events. Collagen interactions with cell surface receptors regulate many cellular processes, while interactions with other ECM components are critical for matrix structure and remodeling. Collagen also interacts with enzymes involved in its biosynthesis and degradation, including matrix metalloproteinases. Over the past decade, much information has been gained about the nature and specificity of collagen interactions with its partners. These studies have defined collagen sequences responsible for binding and the high-resolution structures of triple-helical peptides bound to its natural binding partners. Strategies to target collagen interactions are already being developed, including the use of monoclonal antibodies to interfere with collagen fibril formation and the use of triple-helical peptides to direct liposomes to melanoma cells. The molecular information about collagen interactions will further serve as a foundation for computational studies to design small molecules that can interfere with specific interactions or target tumor cells. Intelligent control of collagen biological interactions within a material context will expand the effectiveness of collagen-based drug delivery.

  8. Collagen gene expression during limb cartilage differentiation

    PubMed Central

    1986-01-01

    As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen mRNA occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II collagen mRNA occurs which parallels the progressive accumulation of cartilage matrix by cells. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen mRNA are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen. PMID:3754261

  9. Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines

    PubMed Central

    Koshy, P; Henderson, N; Logan, C; Life, P; Cawston, T; Rowan, A

    2002-01-01

    Objective: To investigate whether interleukin 17 (IL17), derived specifically from T cells, can promote type II collagen release from cartilage. The ability of IL17 to synergise with other proinflammatory mediators to induce collagen release from cartilage, and what effect anti-inflammatory agents had on this process, was also assessed. Methods: IL17 alone, or in combination with IL1, IL6, oncostatin M (OSM), or tumour necrosis factor α (TNFα), was added to bovine nasal cartilage explant cultures. Proteoglycan and collagen release were determined. Collagenolytic activity was determined by bioassay. Chondroprotective effects of IL4, IL13, transforming growth factor ß1 (TGFß1) and insulin-like growth factor-1 (IGF1) were assessed by inclusion in the explant cultures. Results: IL17 alone stimulated a dose dependent release of proteoglycan and type II collagen from bovine nasal cartilage explants. Suboptimal doses of IL17 synergised potently with TNFα, IL1, OSM, and IL6 to promote collagen degradation. This collagen release was completely inhibited by tissue inhibitor of metalloproteinase-1 and BB-94 (a synthetic metalloproteinase inhibitor), and was significantly reduced by IL4, IL13, TGFß1, and IGF1. In IL17 treated chondrocytes, mRNA expression for matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 was detected. Moreover, a synergistic induction of these MMPs was seen when IL17 was combined with other proinflammatory cytokines. Conclusions: IL17 can, alone and synergistically in combination with other proinflammatory cytokines, promote chondrocyte mediated MMP dependent type II collagen release from cartilage. Because levels of all these proinflammatory cytokines are raised in rheumatoid synovial fluids, this study suggests that IL17 may act as a potent upstream mediator of cartilage collagen breakdown in inflammatory joint diseases. PMID:12117676

  10. Control of dense collagen gel scaffolds for tissue engineering through measurement and modelling of hydraulic permeability

    NASA Astrophysics Data System (ADS)

    Serpooshan, Vahid

    Among various natural biopolymers, type I collagen gels have demonstrated the highest potential as biomimetic scaffolds for tissue engineering (TE). However, the successful application of collagen gels requires a greater understanding of the relationship between their microstructure and physical-mechanical properties. Therefore, a precise method to modulate collagen gel microstructure in order to attain optimal scaffold properties for diverse biomedical applications is necessary. This dissertation describes a new approach to produce collagen gels with defined microstructures, quantified by hydraulic permeability ( k), in order to optimize scaffold properties for TE applications. It was hypothesized that the measurement of k can be used to study the role of microstructure in collagen gel properties, as well as cell function and cell-scaffold interactions. Applying increasing levels of plastic compression (PC) to the highly hydrated collagen gels resulted in an increase in collagen fibrillar density, reduced Happel model derived k values, increased gel stiffness, promoted MSC metabolic activity, osteogenic differentiation, and mineral deposition, while cell-induced gel contraction diminished. Thus, collagen gels with lower k and higher stiffness values exhibited greater potential for bone tissue engineering. Correlating between collagen gel microstructure, k, and fibroblast function within collagen gels indicated that increasing the level of PC yielded a reduction in pore size and an increase in fibril bundle diameter. Decrease in k values resulted in a decrease in gel contraction and an increase in cell metabolic activity. An increase in cell density accelerated contraction. Therefore, fibroblast function within collagen gels can be optimised by a balance between the microstructure, k, and cell seeding density. Developing a micromechanical model to measure experimental k of collagen gels during confined compression revealed the formation of a dense collagen lamella

  11. Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.

    PubMed

    McConnell, James C; O'Connell, Oliver V; Brennan, Keith; Weiping, Lisa; Howe, Miles; Joseph, Leena; Knight, David; O'Cualain, Ronan; Lim, Yit; Leek, Angela; Waddington, Rachael; Rogan, Jane; Astley, Susan M; Gandhi, Ashu; Kirwan, Cliona C; Sherratt, Michael J; Streuli, Charles H

    2016-01-08

    additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness. These preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population.

  12. IMPACT fragmentation model developments

    NASA Astrophysics Data System (ADS)

    Sorge, Marlon E.; Mains, Deanna L.

    2016-09-01

    The IMPACT fragmentation model has been used by The Aerospace Corporation for more than 25 years to analyze orbital altitude explosions and hypervelocity collisions. The model is semi-empirical, combining mass, energy and momentum conservation laws with empirically derived relationships for fragment characteristics such as number, mass, area-to-mass ratio, and spreading velocity as well as event energy distribution. Model results are used for several types of analysis including assessment of short-term risks to satellites from orbital altitude fragmentations, prediction of the long-term evolution of the orbital debris environment and forensic assessments of breakup events. A new version of IMPACT, version 6, has been completed and incorporates a number of advancements enabled by a multi-year long effort to characterize more than 11,000 debris fragments from more than three dozen historical on-orbit breakup events. These events involved a wide range of causes, energies, and fragmenting objects. Special focus was placed on the explosion model, as the majority of events examined were explosions. Revisions were made to the mass distribution used for explosion events, increasing the number of smaller fragments generated. The algorithm for modeling upper stage large fragment generation was updated. A momentum conserving asymmetric spreading velocity distribution algorithm was implemented to better represent sub-catastrophic events. An approach was developed for modeling sub-catastrophic explosions, those where the majority of the parent object remains intact, based on estimated event energy. Finally, significant modifications were made to the area-to-mass ratio distribution to incorporate the tendencies of different materials to fragment into different shapes. This ability enabled better matches between the observed area-to-mass ratios and those generated by the model. It also opened up additional possibilities for post-event analysis of breakups. The paper will discuss

  13. Aging decreases collagen IV expression in vivo in the dermo-epidermal junction and in vitro in dermal fibroblasts: possible involvement of TGF-β1.

    PubMed

    Feru, Jezabel; Delobbe, Etienne; Ramont, Laurent; Brassart, Bertrand; Terryn, Christine; Dupont-Deshorgue, Aurelie; Garbar, Christian; Monboisse, Jean-Claude; Maquart, Francois-Xavier; Brassart-Pasco, Sylvie

    2016-08-01

    Collagen IV is a major component of the dermo-epidermal junction (DEJ). To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV. Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H2O2 concentrations. Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-β1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-β1-neutralizing antibody to fibroblast cultures decreased collagen IV expression. Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-β1 signalling.

  14. MicroRNA-21 Promotes Proliferation of Fibroblast-Like Synoviocytes through Mediation of NF-κB Nuclear Translocation in a Rat Model of Collagen-Induced Rheumatoid Arthritis

    PubMed Central

    Xian, Pei-Feng; Yang, Lu; Wang, Sheng-Xu

    2016-01-01

    MicroRNA-21 (miR-21) is overexpressed in patients with rheumatoid arthritis (RA). This study was designed to investigate the effect and mechanism of miR-21 on cell proliferation in fibroblast-like synoviocytes (FLS) of RA. FLS were primary-cultured from a rat RA model. RA-FLS and normal FLS were infected with lentivirus (anti-miR-21 or pro-miR-21) for overexpression or downregulation of miR-21, respectively. The effects of miR-21 overexpression or inhibition on nucleoprotein NF-κB levels and FLS cell proliferation were evaluated by western blotting and MTT assays. The effects of an inhibitor of NF-κB nuclear translocation (BAY 11-7082) were also evaluated. The results showed that the levels of miR-21 and nucleoprotein NF-κB were increased in FLS of RA model rats compared to the control group. Downregulation of miR-21 in RA FLS led to a significant decrease in nucleoprotein NF-κB levels and cell proliferation rates compared to the antinegative control (NC) group. However, miR-21 overexpression in normal FLS resulted in a significant increase of nucleoprotein NF-κB levels and cell proliferation rates compared to the pro-NC group. The effects of miR-21 overexpression were reversed by BAY 11-7082. We concluded that upregulated miR-21 in FLS in RA model rats may promote cell proliferation by facilitating NF-κB nuclear translocation, thus affecting the NF-κB pathway. PMID:27429986

  15. MicroRNA-21 Promotes Proliferation of Fibroblast-Like Synoviocytes through Mediation of NF-κB Nuclear Translocation in a Rat Model of Collagen-Induced Rheumatoid Arthritis.

    PubMed

    Chen, Ying; Xian, Pei-Feng; Yang, Lu; Wang, Sheng-Xu

    2016-01-01

    MicroRNA-21 (miR-21) is overexpressed in patients with rheumatoid arthritis (RA). This study was designed to investigate the effect and mechanism of miR-21 on cell proliferation in fibroblast-like synoviocytes (FLS) of RA. FLS were primary-cultured from a rat RA model. RA-FLS and normal FLS were infected with lentivirus (anti-miR-21 or pro-miR-21) for overexpression or downregulation of miR-21, respectively. The effects of miR-21 overexpression or inhibition on nucleoprotein NF-κB levels and FLS cell proliferation were evaluated by western blotting and MTT assays. The effects of an inhibitor of NF-κB nuclear translocation (BAY 11-7082) were also evaluated. The results showed that the levels of miR-21 and nucleoprotein NF-κB were increased in FLS of RA model rats compared to the control group. Downregulation of miR-21 in RA FLS led to a significant decrease in nucleoprotein NF-κB levels and cell proliferation rates compared to the antinegative control (NC) group. However, miR-21 overexpression in normal FLS resulted in a significant increase of nucleoprotein NF-κB levels and cell proliferation rates compared to the pro-NC group. The effects of miR-21 overexpression were reversed by BAY 11-7082. We concluded that upregulated miR-21 in FLS in RA model rats may promote cell proliferation by facilitating NF-κB nuclear translocation, thus affecting the NF-κB pathway.

  16. WOUND HEALING AND COLLAGEN FORMATION

    PubMed Central

    Ross, Russell; Benditt, Earl P.

    1961-01-01

    The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed. PMID:14494202

  17. Immunostimulation effect of jellyfish collagen.

    PubMed

    Sugahara, Takuya; Ueno, Masashi; Goto, Yoko; Shiraishi, Ryusuke; Doi, Mikiharu; Akiyama, Koichi; Yamauchi, Satoshi

    2006-09-01

    Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 degrees C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by human PBL was stimulated 100- and 17-fold respectively. Co