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Sample records for ii-induced collagen type

  1. Type XI Collagen

    DEFF Research Database (Denmark)

    Luo, Yunyun

    2016-01-01

    Type XI collagen is a fibrillary collagen. Type XI collagen is broadly distributed in articular cartilage, testis, trachea, tendons, trabecular bone, skeletal muscle, placenta, lung, and the neoepithelium of the brain. Type XI collagen is able to regulate fibrillogenesis by maintaining the spacing...... and diameter of type II collagen fibrils, and a nucleator for the fibrillogenesis of collagen types I and II. Mutations in type XI collagen are associated with Stickler syndrome, Marshall syndrome, fibrochondrogenesis, otospondylomegaepiphyseal dysplasia deafness, and Weissenbacher–Zweymüller syndrome. Type XI...... collagen binds heparin, heparan sulfate, and dermatan sulfate. Currently there are no biomarkers for type XI collagen....

  2. Suppression of Carrageenan- and Collagen II-Induced Inflammation in Mice by Geranium Oil

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available To obtain experimental evidence on the therapeutic efficacy of essential oils in aromatherapy for inflammatory diseases, we examined the effects of geranium oil on carrageenan-induced and collagen II-induced inflammation in mice, to assess acute and chronic anti-inflammatory activities of the oil. Single intraperitoneal injection of 5μL of geranium oil clearly suppressed the carrageenan-induced footpaw edema and increase in tissue myeloperoxidase activity, and repeated administration of the oil suppressed collagen-induced arthritis. These results revealed that geranium oil suppressed both acute and chronic inflammatory responses in mice

  3. Suppression of Carrageenan- and Collagen II-Induced Inflammation in Mice by Geranium Oil

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    Naho Maruyama

    2006-01-01

    Full Text Available To obtain experimental evidence on the therapeutic efficacy of essential oils in aromatherapy for inflammatory diseases, we examined the effects of geranium oil on carrageenan-induced and collagen II-induced inflammation in mice, to assess acute and chronic anti-inflammatory activities of the oil. Single intraperitoneal injection of 5 μL of geranium oil clearly suppressed the carrageenan-induced footpaw edema and increase in tissue myeloperoxidase activity, and repeated administration of the oil suppressed collagen-induced arthritis. These results revealed that geranium oil suppressed both acute and chronic inflammatory responses in mice.

  4. Changes and significance of IL-25 in chicken collagen II-induced experimental arthritis (CIA).

    Science.gov (United States)

    Kaiwen, Wang; Zhaoliang, Su; Yinxia, Zhao; Siamak, Sandoghchian Shotorbani; Zhijun, Jiao; Yuan, Xue; Heng, Yang; Dong, Zheng; Yanfang, Liu; Pei, Shen; Shengjun, Wang; Qixiang, Shao; Xinxiang, Huang; Liwei, Lu; Huaxi, Xu

    2012-08-01

    Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. It is a systemic inflammatory disease, characterized by chronic, symmetrical, multi-articular synovial arthritis. IL-25 (IL-17E) is a member of the recently emerged cytokine family (IL-17s), which is expressed in Th2 cells and bone marrow-derived mast cells. Unlike the other members of this family, IL-25 is capable of inducing Th2-associated cytokines (IL-4, IL-5, and IL-13) and also promotes the release of some pro-immune factors (IL-6 and IL-8). IL-25 is also a pleiotropic factor, which constitutes a tissue-specific pathological injury and chronic inflammation. In this study, we used chicken collagen II-induced experimental arthritis (CIA) model in DBA/1 mice to investigate the relationship between IL-25 and other inflammatory factors, revealing the possible mechanism in CIA. Our results showed that the expression level of IL-25 was enhanced in the late stage of CIA, and IL-17 was increased in the early stage of the disease. It is well known that IL-17 has a crucial role in the development of RA pathogenesis, and IL-25 plays a significant role in humoral immune. For reasons given above, we suggested that the IL-25 inhibited IL-17 expression to some extent, while enhancing the production of IL-4. It was confirmed that IL-25 not only regulated the cellular immune, but also involved the humoral immune in rheumatoid arthritis.

  5. The Predicted Proteomic Network Associated with the Antiarthritic Action of Qingfu Guanjieshu in Collagen-II-Induced Arthritis in Rats

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    Ting Yu Wang

    2013-01-01

    Full Text Available Qingfu Guanjieshu (QFGJS is an herbal preparation for treating rheumatoid arthritis (RA. Previous studies revealed that QFGJS significantly inhibited experimental arthritis and acute inflammation, accompanied by reduction of proinflammatory cytokines and elevation of anti-inflammatory cytokines. This study aims to identify the targeted proteins and predict the proteomic network associated with the drug action of QFGJS by using 2D gel and MALDI-TOF-MS/MS techniques. Thirty female Wistar rats were evenly grouped as normal and vehicle- and QFGJS-treated CIA rats. The antiarthritic effect of QFGJS was examined with a 19-day treatment course, and the knee synovial tissues of animals from each group were obtained for 2D gel and MALDI-TOF-MS/MS analysis. Results showed that QFGJS significantly ameliorated collagen II-induced arthritis when administrated at 2.8 g/kg body weight for 19 days. 2D gel image analysis revealed 89 differentially expressed proteins in the synovial tissues among the normal and vehicle- and QFGJS-treated CIA rats from over 1000 proteins of which 63 proteins were identified by MALDI-TOF-MS/MS analysis, and 32 proteins were included for classification of functions using Gene Ontology (GO method. Finally, 14 proteins were analyzed using bioinformatics, and a predicted proteomic network related to the anti-arthritic effect of QFGJS was established, and Pgk1 plays a central role.

  6. Substance P ameliorates collagen II-induced arthritis in mice via suppression of the inflammatory response

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    Hong, Hyun Sook [College of Medicine, East-West Medical Research Institute, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul 130-702 (Korea, Republic of); Son, Youngsook, E-mail: ysson@khu.ac.kr [Graduate School of Biotechnology and Department of Genetic Engineering, College of Life Science, Kyung Hee University Global Campus, Seochun-dong, Kiheung-ku, Yong In 441-706 (Korea, Republic of)

    2014-10-10

    Highlights: • SP can increase IL-10 levels and reduce TNF-α and IL-17 levels in RA. • SP causes the increase in T{sub reg}, M2 macrophage, and MSCs in RA. • SP-induced immune suppression leads to the blockade of RA progression. • SP can be used as the therapeutics for autoimmune-related inflammatory diseases. - Abstract: Current rheumatoid arthritis (RA) therapies such as biologics inhibiting pathogenic cytokines substantially delay RA progression. However, patient responses to these agents are not always complete and long lasting. This study explored whether substance P (SP), an 11 amino acids long endogenous neuropeptide with the novel ability to mobilize mesenchymal stem cells (MSC) and modulate injury-mediated inflammation, can inhibit RA progression. SP efficacy was evaluated by paw swelling, clinical arthritis scoring, radiological analysis, histological analysis of cartilage destruction, and blood levels of tumor necrosis factor-alpha (TNF-α) interleukin (IL)-10, and IL-17 in vivo. SP treatment significantly reduced local inflammatory signs, mean arthritis scores, degradation of joint cartilage, and invasion of inflammatory cells into the synovial tissues. Moreover, the SP treatment markedly reduced the size of spleens enlarged by excessive inflammation in CIA, increased IL-10 levels, and decreased TNF-α and IL-17 levels. Mobilization of stem cells and induction of T{sub reg} and M2 type macrophages in the circulation were also increased by the SP treatment. These effect of SP might be associated with the suppression of inflammatory responses in RA and, furthermore, blockade of RA progression. Our results propose SP as a potential therapeutic for autoimmune-related inflammatory diseases.

  7. Inhibitory Effects of Soyeum Pharmacopuncture (SPP on Rheumatoid Arthritis in Collagen II-induced Arthritis (CIA Mice

    Directory of Open Access Journals (Sweden)

    Hwa-Seung Yoo

    2007-06-01

    Full Text Available Objective : The aim is to examine the effect of Soyeum Pharmacopuncture (SPP on collagen-induced arthritis (CIA in DBA/1OlaHsd mice. Methods : To determine the effect of SPP on chronic IFNlammatory joint disease, we induced CIA in DBA/1OlaHsd mice by immunization with bovine type II collagen. Animals were treated with intraperitoneal injection doses of 2 mg/kg of SPP, beginning 3 days before the expected onset of disease symptoms. Inhibitory Effects of SPP were observed by serum levels of TNF-, and IFN-,,, or cell proliferation in the spleen cell culture and histological examination of knee joint. Results : In the CIA Mice, serum levels of TNF-, and IFN-,, production in the spleen cell culture were reduced. At the histopathological examination of knee joint, chondropathy of cartilage in the synovial joint in the SP group was repaired while compared with control group. Conclusion : These results suggest that the SPP may be effective for the prevention and treatment of rheumatoid arthritis disease.

  8. Techniques for Type I Collagen Organization

    Science.gov (United States)

    Anderson-Jackson, LaTecia Diamond

    Tissue Engineering is a process in which cells, engineering, and material methods are used in amalgamation to improve biological functions. The purpose of tissue engineering is to develop alternative solutions to treat or cure tissues and organs that have been severely altered or damaged by diseases, congenital defects, trauma, or cancer. One of the most common and most promising biological materials for tissue engineering to develop scaffolds is Type I collagen. A major challenge in biomedical research is aligning Type I collagen to mimic biological structures, such as ligaments, tendons, bones, and other hierarchal aligned structures within the human body. The intent of this research is to examine possible techniques for organizing Type I collagen and to assess which of the techniques is effective for potential biological applications. The techniques used in this research to organize collagen are soft lithography with solution-assisted sonication embossing, directional freezing, and direct poling. The final concentration used for both soft lithography with solution-assisted sonication embossing and direct poling was 1 mg/ml, whereas for directional freezing the final concentration varied between 4mg/ml, 2mg/ml, and 1 mg/ml. These techniques were characterized using the Atomic Force Microscope (AFM) and Helium Ion Microscope (HIM). In this study, we have found that out of the three techniques, the soft lithography and directional freezing techniques have been successful in organizing collagen in a particular pattern, but not alignment. We concluded alignment may be dependent on the pH of collagen and the amount of acetic acid used in collagen solution. However, experiments are still being conducted to optimize all three techniques to align collagen in a unidirectional arrangement.

  9. BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy

    Science.gov (United States)

    Spagnolli, Ester; Ernande, Laura; Thoonen, Robrecht; Kolodziej, Starsha A.; Leyton, Patricio A.; Cheng, Juan; Tainsh, Robert E. T.; Mayeur, Claire; Rhee, David K.; Wu, Mei. X.; Scherrer-Crosbie, Marielle; Buys, Emmanuel S.; Zapol, Warren M.; Bloch, Kenneth D.; Bloch, Donald B.

    2016-01-01

    Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis. PMID:26873969

  10. BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy.

    Science.gov (United States)

    Shahid, Mohd; Spagnolli, Ester; Ernande, Laura; Thoonen, Robrecht; Kolodziej, Starsha A; Leyton, Patricio A; Cheng, Juan; Tainsh, Robert E T; Mayeur, Claire; Rhee, David K; Wu, Mei X; Scherrer-Crosbie, Marielle; Buys, Emmanuel S; Zapol, Warren M; Bloch, Kenneth D; Bloch, Donald B

    2016-04-15

    Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis.

  11. Ameloblasts express type I collagen during amelogenesis.

    Science.gov (United States)

    Assaraf-Weill, N; Gasse, B; Silvent, J; Bardet, C; Sire, J Y; Davit-Béal, T

    2014-05-01

    Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while post-metamorphic teeth possess enamel only. In first-generation teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.

  12. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

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    Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

    2012-03-15

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

  13. Dermal type I collagen assessment by digital image analysis*

    OpenAIRE

    Brianezi, Gabrielli; Grandi, Fabrizio; Bagatin, Ediléia; Enokihara, Mílvia Maria S. S.; Miot, Hélio Amante [UNESP

    2015-01-01

    Type I collagen is the main dermal component, and its evaluation is relevant to quantitative studies in dermatopathology. However, visual gradation (0 to 4+) has low precision and high subjectivity levels. This study aimed to develop and validate a digital morphometric analysis technique to estimate type I collagen levels in the papillary dermis. Four evaluators visually quantified (0 to 4+) the density of type I collagen in 63 images of forearm skin biopsies marked by immunohistochemistry an...

  14. Type IV collagen-degrading enzyme activity in human serum.

    Directory of Open Access Journals (Sweden)

    Hashimoto,Noriaki

    1988-02-01

    Full Text Available Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful for estimating the degradation of type IV collagen.

  15. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    OpenAIRE

    Song Chen, Toshiyuki Ikoma, Nobuhiro Ogawa, Satoshi Migita, Hisatoshi Kobayashi and Nobutaka Hanagata

    2010-01-01

    Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhib...

  16. Imaging collagen type I fibrillogenesis with high spatiotemporal resolution

    Energy Technology Data Exchange (ETDEWEB)

    Stamov, Dimitar R, E-mail: stamov@jpk.com [JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin (Germany); Stock, Erik [JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin (Germany); Franz, Clemens M [DFG-Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1a, 76131 Karlsruhe (Germany); Jähnke, Torsten; Haschke, Heiko [JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin (Germany)

    2015-02-15

    Fibrillar collagens, such as collagen type I, belong to the most abundant extracellular matrix proteins and they have received much attention over the last five decades due to their large interactome, complex hierarchical structure and high mechanical stability. Nevertheless, the collagen self-assembly process is still incompletely understood. Determining the real-time kinetics of collagen type I formation is therefore pivotal for better understanding of collagen type I structure and function, but visualising the dynamic self-assembly process of collagen I on the molecular scale requires imaging techniques offering high spatiotemporal resolution. Fast and high-speed scanning atomic force microscopes (AFM) provide the means to study such processes on the timescale of seconds under near-physiological conditions. In this study we have applied fast AFM tip scanning to study the assembly kinetics of fibrillar collagen type I nanomatrices with a temporal resolution reaching eight seconds for a frame size of 500 nm. By modifying the buffer composition and pH value, the kinetics of collagen fibrillogenesis can be adjusted for optimal analysis by fast AFM scanning. We furthermore show that amplitude-modulation imaging can be successfully applied to extract additional structural information from collagen samples even at high scan rates. Fast AFM scanning with controlled amplitude modulation therefore provides a versatile platform for studying dynamic collagen self-assembly processes at high resolution. - Highlights: • Continuous non-invasive time-lapse investigation of collagen I fibrillogenesis in situ. • Imaging of collagen I self-assembly with high spatiotemporal resolution. • Application of setpoint modulation to study the hierarchical structure of collagen I. • Observing real-time formation of the D-banding pattern in collagen I.

  17. Imaging collagen type I fibrillogenesis with high spatiotemporal resolution.

    Science.gov (United States)

    Stamov, Dimitar R; Stock, Erik; Franz, Clemens M; Jähnke, Torsten; Haschke, Heiko

    2015-02-01

    Fibrillar collagens, such as collagen type I, belong to the most abundant extracellular matrix proteins and they have received much attention over the last five decades due to their large interactome, complex hierarchical structure and high mechanical stability. Nevertheless, the collagen self-assembly process is still incompletely understood. Determining the real-time kinetics of collagen type I formation is therefore pivotal for better understanding of collagen type I structure and function, but visualising the dynamic self-assembly process of collagen I on the molecular scale requires imaging techniques offering high spatiotemporal resolution. Fast and high-speed scanning atomic force microscopes (AFM) provide the means to study such processes on the timescale of seconds under near-physiological conditions. In this study we have applied fast AFM tip scanning to study the assembly kinetics of fibrillar collagen type I nanomatrices with a temporal resolution reaching eight seconds for a frame size of 500 nm. By modifying the buffer composition and pH value, the kinetics of collagen fibrillogenesis can be adjusted for optimal analysis by fast AFM scanning. We furthermore show that amplitude-modulation imaging can be successfully applied to extract additional structural information from collagen samples even at high scan rates. Fast AFM scanning with controlled amplitude modulation therefore provides a versatile platform for studying dynamic collagen self-assembly processes at high resolution. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    Energy Technology Data Exchange (ETDEWEB)

    Chen Song; Ogawa, Nobuhiro; Migita, Satoshi; Kobayashi, Hisatoshi [Biomaterials Center, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047 (Japan); Ikoma, Toshiyuki [Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, O-okayama 2-12-1, Meguro-ku, Tokyo 152-8550 (Japan); Hanagata, Nobutaka, E-mail: HANAGATA.Nobutaka@nims.go.j [Nanotechnology Innovation Center, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047 (Japan)

    2010-06-15

    Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 {sup 0}C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of {approx}67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

  19. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    Science.gov (United States)

    Chen, Song; Ikoma, Toshiyuki; Ogawa, Nobuhiro; Migita, Satoshi; Kobayashi, Hisatoshi; Hanagata, Nobutaka

    2010-06-01

    Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ~67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

  20. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    Directory of Open Access Journals (Sweden)

    Song Chen, Toshiyuki Ikoma, Nobuhiro Ogawa, Satoshi Migita, Hisatoshi Kobayashi and Nobutaka Hanagata

    2010-01-01

    Full Text Available Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ~67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

  1. [Autoimmune mechanisms toward type I collagen during parodontitis].

    Science.gov (United States)

    2014-04-01

    The article presents original data about autoimmune mechanisms according to the severity of the process during the chronic generalized Parodontitis. The medical examination of 179 patients with different forms of Parodontitis demonstrated that during Parodontitis the synthesis and re-synthesis of type I Collagen is negatively affected and as a result, type I Collagen concentration in the blood serum raises. In addition, the selection of antigen reactive lymphocytes increases toward Type I Collagen, which in turn boosts their quantity in blood and also stimulates the creation of auto-antibodies toward Type I Collagen. This is reflected by the increased quantity of auto-antibodies in the blood serum. The intensity of these processes amplifies as the Parodontitis inflammation becomes more severe. These results demonstrate that the autoimmune process develops toward Type I Collagen during Parodontitis and its intensity reflects the severity of the pathological processes in Parodontitis.

  2. Gap Dependent Rheology in Type I Collagen Gels

    Science.gov (United States)

    Arevalo, Richard; Urbach, Jeffrey; Blair, Daniel

    2010-03-01

    Branched type I collagen fiber networks provide extracellular support in mammalian tissues. The intricate network structure can succumb to partial or complete tearing under sufficient applied strain. Under small shear strains, in vitro collagen gels exhibit strain-stiffening while maintaining overall network integrity. Higher shear strains lead to network failure through discrete yielding events. We perform rheology and confocal-rheology experiments to fully elucidate the strain-stiffening and yielding behavior in these highly nonlinear materials. We apply continuous shear strains to collagen gels confined within the rheometer at fixed gaps. We observe that sheared collagen in the strain-stiffening and yielding regime has an apparent modulus that is strongly dependent on the collagen thickness. Moreover, we demonstrate that network yielding is universally controlled by the ratio of the collagen thickness to the mesh size. These results have broad implications for the interpretation of rheological data of extracellular matrix proteins and for the design of biomimetic scaffolds.

  3. Hyperalgesic priming (type II) induced by repeated opioid exposure: maintenance mechanisms.

    Science.gov (United States)

    Araldi, Dioneia; Ferrari, Luiz F; Levine, Jon D

    2017-07-01

    We previously developed a model of opioid-induced neuroplasticity in the peripheral terminal of the nociceptor that could contribute to opioid-induced hyperalgesia, type II hyperalgesic priming. Repeated administration of mu-opioid receptor (MOR) agonists, such as DAMGO, at the peripheral terminal of the nociceptor, induces long-lasting plasticity expressed, prototypically as opioid-induced hyperalgesia and prolongation of prostaglandin E2-induced hyperalgesia. In this study, we evaluated the mechanisms involved in the maintenance of type II priming. Opioid receptor antagonist, naloxone, induced hyperalgesia in DAMGO-primed paws. When repeatedly injected, naloxone-induced hyperalgesia, and hyperalgesic priming, supporting the suggestion that maintenance of priming involves changes in MOR signaling. However, the knockdown of MOR with oligodeoxynucleotide antisense did not reverse priming. Mitogen-activated protein kinase and focal adhesion kinase, which are involved in the Src signaling pathway, previously implicated in type II priming, also inhibited the expression, but not maintenance of priming. However, when Src and mitogen-activated protein kinase inhibitors were coadministered, type II priming was reversed, in male rats. A second model of priming, latent sensitization, induced by complete Freund's adjuvant was also reversed, in males. In females, the inhibitor combination was only able to inhibit the expression and maintenance of DAMGO-induced priming when knockdown of G-protein-coupled estrogen receptor 30 (GPR30) in the nociceptor was performed. These findings demonstrate that the maintenance of DAMGO-induced type II priming, and latent sensitization is mediated by an interaction between, Src and MAP kinases, which in females is GPR30 dependent.

  4. Enzymatic Breakdown of Type II Collagen in the Human Vitreous

    NARCIS (Netherlands)

    van Deemter, Marielle; Pas, Hendri H.; Kuijer, Roel; van der Worp, Roelofje J.; Hooymans, Johanna M. M.; Los, Leonoor I.

    2009-01-01

    PURPOSE. To investigate whether enzymatic collagen breakdown is an active process in the human vitreous. METHODS. Human donor eyes were used for immunohistochemistry to detect the possible presence of the matrix metalloproteinase (MMP)-induced type II collagen breakdown product col2-3/4C-short in

  5. Enzymatic Breakdown of Type II Collagen in the Human Vitreous

    NARCIS (Netherlands)

    van Deemter, Marielle; Pas, Hendri H.; Kuijer, Roel; van der Worp, Roelofje J.; Hooymans, Johanna M. M.; Los, Leonoor I.

    2009-01-01

    PURPOSE. To investigate whether enzymatic collagen breakdown is an active process in the human vitreous. METHODS. Human donor eyes were used for immunohistochemistry to detect the possible presence of the matrix metalloproteinase (MMP)-induced type II collagen breakdown product col2-3/4C-short in th

  6. [Expression of type VI collagen in arthrofibrosis. An immunohistochemical study].

    Science.gov (United States)

    Zeichen, J; van Griensven, M; Lobenhoffer, P; Bosch, U

    2000-08-01

    Arthrofibrosis is a disabling complication after trauma and surgery due to massive connective tissue proliferation. The etiology and pathogenesis have never been fully understood. A strong immune response may lead to activation and proliferation of fibroblasts with excessive and disordered deposition of matrix proteins. In similar pathological conditions, like lung fibrosis or superficial fibromatoses with fibrotic transformation an increased expression of collagen type VI has been reported. We investigated fibrotic tissue samples taken from 18 patients (average age: 32.7 years), who underwent arthrolysis of the knee joint because of symptomatic arthrofibrosis following ligament injury. The mean interval between trauma and arthrolysis was 13.8 months (range 4-50 months). Tissue samples were taken from the infrapatellar fat pad and intercondylar connective tissue. All samples were stained with HE. The expression of type III and VI collagen was studied immunohistochemically using an immunoperoxidase method for light microscopic visualization. Histologic analysis from patients with arthrofibrosis showed a synovial hyperplasia with cell infiltration and vascular proliferation compared to synovial tissue samples from knee joints without any detectable pathology. Subsynovial an increased deposition of matrix proteins was visible. Type VI collagen was widely distributed as a network subsynovial and around capillary walls. Type III collagen showed a diffuse distribution. Arthrofibrotic tissue is, similar to pathological conditions with fibrotic transformation characterized by an increased expression of collagen type VI. Collagen type VI may play an important role in matrix homeostasis. It serves as an anchoring element between collagen fibers and as a cell binding structure.

  7. Type IV collagen-degrading enzyme activity in human serum.

    OpenAIRE

    Hashimoto, Noriaki; Kobayashi, Michio; Watanabe,Akiharu; Higashi, Toshiro; Tsuji, Takao

    1988-01-01

    Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful...

  8. The decorin sequence SYIRIADTNIT binds collagen type I

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Oldberg, Ake

    2007-01-01

    Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site-directed mut....... These collagen-binding amino acids are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat. This resembles the location of interacting residues in other leucine-rich repeat proteins.......Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site......-directed mutagenesis of this 54-residue-long collagen-binding sequence identifies Arg-207 and Asp-210 in leucine-rich repeat 6 as crucial for the binding to collagen. The synthetic peptide SYIRIADTNIT, which includes Arg-207 and Asp-210, inhibits the binding of full-length recombinant decorin to collagen in vitro...

  9. Cross-reactivity of cell-mediated immunity between interstitial (type I) and basement membrane (type IV) collagens

    OpenAIRE

    1982-01-01

    In the present study, we demonstrate delayed-type hypersensitivity (DTH) to homologous type I collagen that cross-reacts with type IV collagen. Mice immunized with native or denatured type I collagens and challenged with these same antigens or native type IV collagen develop a peak DTH response on day 7. Challenge with denatured type IV collagen or collagenase-treated type IV collagen failed to elicit DTH in type I collagen-sensitized mice. Type I collagen-sensitized spleen cells adoptively t...

  10. Biological role of prolyl 3-hydroxylation in type IV collagen.

    Science.gov (United States)

    Pokidysheva, Elena; Boudko, Sergei; Vranka, Janice; Zientek, Keith; Maddox, Kerry; Moser, Markus; Fässler, Reinhard; Ware, Jerry; Bächinger, Hans Peter

    2014-01-07

    Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.

  11. LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression

    Directory of Open Access Journals (Sweden)

    Yujie Zhang

    2016-03-01

    Full Text Available Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60–70 days. However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6, is specifically involved in type I collagen regulation. In the 5′untranslated region (5’UTR of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5′SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP, 25 kD FK506 binding protein (FKBP25 and RNA helicase A (RHA, contribute to this process.

  12. Size-Dependent Rheology of Type-I Collagen Networks

    OpenAIRE

    Arevalo, Richard C.; Urbach, Jeffrey S.; Blair, Daniel L.

    2010-01-01

    We investigate the system size dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by t...

  13. IL-35 stimulation of CD39+ regulatory T cells confers protection against collagen II-induced arthritis via the production of IL-10.

    Science.gov (United States)

    Kochetkova, Irina; Golden, Sarah; Holderness, Kathryn; Callis, Gayle; Pascual, David W

    2010-06-15

    IL-35 is produced by regulatory T cells, and this novel cytokine can downregulate Th17 cell development and inhibit autoimmune inflammation. In this work, an rIL-35, as a single-chain fusion between murine IL-12p35 and EBV-induced gene 3, was expressed in yeast. This rIL-35 inhibited OVA-specific cellular and Ab responses in OVA-challenged recipients of DO11.10 CD4+ T cells. Likewise, IL-35 inhibited clinical manifestation of collagen-induced arthritis or could cease further disease exacerbation upon initiation of IL-35 treatment. Exogenous IL-35 treatments suppressed Th1 and Th17 cells and promoted CD39 expression by CD4+ T cells. Sorted CD25-CD39+CD4+ T cells from IL-35-treated mice produced IL-10 and, upon adoptive transfer, were sufficiently potent to inhibit subsequent development of inflammation in mice with collagen-induced arthritis, whereas sorted CD25+CD39+CD4+ T cells showed reduced potency. IL-35 treatments of IL-10-/- mice failed to induce protective CD39+CD4+ T cells, demonstrating the effector role of IL-10 by IL-35 immunosuppression.

  14. Telmisartan inhibited angiotensin II-induced collagen metabolic imbalance without directly targeting TGF-β 1/Smad signaling pathway in cardiac fibroblasts.

    Science.gov (United States)

    Zhang, Y; Zhao, N A; Wang, J K; Zhu, S M; Zhu, H L; Liu, B; Cui, Q W; Guan, G C; Tian, G

    2015-12-01

    Cardiac fibrosis is an important pathological process of cardiac remodeling. A large number of studies have shown that telmisartan can attenuate cardiac fibrosis through acting on angiotensin II 1 receptor (AT1R), and TGF-β 1/Smad signaling molecule is an important pathway to achieve this effect. The aim of the study was to clarify whether, with excessive activation of RAAS system, telmisartan could also directly target TGF-β 1/Smad signaling pathway to have the function of anti-cardiac fibrosis. In this study, neonatal rat cardiac fibroblasts were cultured and AngII or TGF-β 1 was administered for treatment or pre-incubation, and then telmisartan was used for 24 hours' incubation. Western blot and enzyme-linked immunosorbent assay (ELISA) tests were performed to detect protein expressions. The results showed that telmisartan could inhibit collagen synthesis and collagen metabolic imbalance under the effect of Ang II, but telmisartan could not have such function in TGF-β 1-induced cardiac fibroblasts. It was further confirmed by western blot method that telmisartan could inhibit TGF-β 1/Smad signaling molecule expression under the effect of Ang II, but telmisartan had no effect on TGF-β 1-induced Smad signaling molecule expression. According to the present study telmisartan played a role of anticardiac fibrosis without directly targeting TGF-β 1/Smad signaling pathway molecule.

  15. Native type II collagen-induced arthritis in the rat. I. Incidence and humoral response to collagen.

    OpenAIRE

    Morgan, K; Clague, R B; Shaw, M J; Holt, P J

    1980-01-01

    An acute inflammatory arthritis has been induced in 76% of rats injected intradermally with native bovine type II collagen emulsified in Freund's complete (CFA) or incomplete (ICFA) adjuvant. The arthritis became chronic in 14 out of 31 rats, and ear and tail lesions were noted in some rats. No arthritis was induced by native type I collagen, denatured type II collagen, rabbit IgG, or buffer alone injected intradermally with adjuvant. Using a solid-phase radioimmunoassay for serum antibodies ...

  16. Localization of type II collagen, long form alpha 1(IX) collagen, and short form alpha 1(IX) collagen transcripts in the developing chick notochord and axial skeleton.

    Science.gov (United States)

    Swiderski, R E; Solursh, M

    1992-06-01

    In this study we compare, by in situ hybridization, the spatial and temporal expression patterns of transcripts of avian type II collagen and the long and short forms of the (alpha 1) chain of type IX collagen during the development of the notochord and axial skeleton. We observed type II collagen and short form type IX collagen transcripts in the developing (stage 25-28) nonchondrogenic notochord. Conversely, long form type IX transcripts were not detectable in the notochord or perinotochordal sheath. Interestingly, all three transcripts colocalized in the developing chondrogenic vertebrae of the axial skeleton as well as in the chondrocranium and Meckel's cartilage. The expression of the short form of type IX collagen in these regions was more restricted than that of the long form. This report provides additional support for a complex regulatory pathway of cartilage marker gene expression in chondrogenic vs. nonchondrogenic tissues during avian embryogenesis.

  17. Size-dependent rheology of type-I collagen networks.

    Science.gov (United States)

    Arevalo, Richard C; Urbach, Jeffrey S; Blair, Daniel L

    2010-10-20

    We investigate the system size-dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by the ratio of the gel thickness to the mesh size. These findings have broad implications for cell-matrix interactions, the interpretation of rheological tissue data, and the engineering of biomimetic scaffolds.

  18. Association of systemic collagen type IV formation with survival among patients undergoing hemodialysis

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Karsdal, Morten A; Rasmussen, Lars M;

    2013-01-01

    The 7S domain of collagen type IV (P4NP_7S) assessed in plasma represents systemic collagen type IV formation. The objective of the study was to investigate the association of systemic collagen type IV formation with survival among patients undergoing hemodialysis.......The 7S domain of collagen type IV (P4NP_7S) assessed in plasma represents systemic collagen type IV formation. The objective of the study was to investigate the association of systemic collagen type IV formation with survival among patients undergoing hemodialysis....

  19. Type V Collagen in Health, Disease, and Fibrosis.

    Science.gov (United States)

    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.

  20. Possible association of elevated serum collagen type IV level with skin sclerosis in systemic sclerosis.

    Science.gov (United States)

    Motegi, Sei-Ichiro; Sekiguchi, Akiko; Fujiwara, Chisako; Toki, Sayaka; Ishikawa, Osamu

    2016-08-29

    Collagen type IV is the primary collagen in the basement membranes around blood vessels and in the dermoepidermal junction in the skin. Perivascular collagen type IV is synthesized by endothelial cells and pericytes, and contributes to the homeostasis and remodeling of blood vessels. It has been well recognized that elevated serum collagen type IV levels are associated with the liver fibrosis. The objective was to examine serum collagen type IV levels and their clinical associations in patients with systemic sclerosis (SSc), and to examine the expression of collagen type IV in the fibrotic skin in SSc. Serum collagen type IV levels in SSc patients and diffuse cutaneous type SSc patients were significantly higher than those in healthy individuals. Serum collagen type IV levels were positively correlated with modified Rodnan total skin score. Serum collagen type IV levels in early stage (disease duration ≤3 years) diffuse cutaneous SSc patients were significantly elevated. Serum collagen type IV levels in SSc patients with digital ulcers (DU) were significantly elevated. In immunohistochemical staining, the expression of collagen type IV around dermal small vessels in the affected skin was reduced compared with those of normal individuals. These results suggest that elevated serum collagen type IV levels may be associated with the skin sclerosis in the early stage of SSc. The measurement of serum collagen type IV levels in SSc patients may be useful as a disease activity marker in skin sclerosis and DU.

  1. Gene expression of collagen types Ⅸ and X in the lumbar disc

    Institute of Scientific and Technical Information of China (English)

    西永明; 胡有谷; 吕振华; 郑红军; 陈岩; 齐宗华

    2004-01-01

    Objective: To study gene expression of collagen types IX and X in human lumbar intervertebral discs during aging and degeneration and to explore the role of collagen types IX and X in disc degeneration.Methods: Fetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene.Results: In fetal intervertebral discs, positive mRNA hybridization signals of type IX collagen were concentrated in the nucleus pulposus and the inner layer of anulns fibrosns. Interstitial matrix of the nucleus pulposns also showed positive type X collagen staining. Positive mRNA hybridization signals of types IX and X were not detected in the middle and outer layers of anulus fibrosus. In adult specimens, expression of type IX collagen mRNA was markedly decreased. No hybridization signals of type X collagen was observed. As for pathological specimens, there was no gene expression of type IX collagen. In severe degenerated discs from adults, there were focal positive expressions of type X collagen. Conclusions: Obvious changes of collagen gene expression occur with aging. Expression of type IX collagen decreases in adult and pathological discs. Results of type X collagen expression suggest that type X collagen is expressed only in older adult and senile discs (I. E., when disc degeneration has already reached a terminal stage ),indicating the terminal stage of degeneration.

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

    Science.gov (United States)

    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

  3. Collagen Type II Enhances Chondrogenesis in Adipose Tissue-Derived Stem Cells by Affecting Cell Shape

    NARCIS (Netherlands)

    Lu, ZuFu; Doulabi, Behrouz Zandieh; Huang, ChunLing; Bank, Ruud A.; Helder, Marco N.

    2010-01-01

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors c

  4. Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape

    NARCIS (Netherlands)

    Lu, Z.; Doulabi, B.Z.; Huang, C.; Bank, R.A.; Helder, M.N.

    2010-01-01

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors c

  5. Collagen Type II Enhances Chondrogenesis in Adipose Tissue-Derived Stem Cells by Affecting Cell Shape

    NARCIS (Netherlands)

    Lu, ZuFu; Doulabi, Behrouz Zandieh; Huang, ChunLing; Bank, Ruud A.; Helder, Marco N.

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors

  6. Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape

    NARCIS (Netherlands)

    Lu, Z.; Doulabi, B.Z.; Huang, C.; Bank, R.A.; Helder, M.N.

    2010-01-01

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors

  7. Water-soluble undenatured type II collagen ameliorates collagen-induced arthritis in mice.

    Science.gov (United States)

    Yoshinari, Orie; Shiojima, Yoshiaki; Moriyama, Hiroyoshi; Shinozaki, Junichi; Nakane, Takahisa; Masuda, Kazuo; Bagchi, Manashi

    2013-11-01

    Earlier studies have reported the efficacy of type II collagen (C II) in treating rheumatoid arthritis (RA). However, a few studies have investigated the ability of the antigenic collagen to induce oral tolerance, which is defined as active nonresponse to an orally administered antigen. We hypothesized that water-soluble undenatured C II had a similar effect as C II in RA. The present study was designed to examine the oral administration of a novel, water-soluble, undenatured C II (commercially known as NEXT-II) on collagen-induced arthritis (CIA) in mice. In addition, the underlying mechanism of NEXT-II was also identified. After a booster dose (collagen-Freund's complete adjuvant), mice were assigned to control CIA group, or NEXT-II treatment group, to which saline and NEXT-II were administered, respectively. The arthritis index in the NEXT-II group was significantly lower compared with the CIA group. Serum IL-6 levels in the NEXT-II group were significantly lower compared with the CIA group, while serum IL-2 level was higher. Furthermore, oral administration of NEXT-II enhanced the proportion of CD4+CD25+T (Treg) cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cells such as forkhead box p3 (Foxp3), transforming growth factor (TGF)-β1, and CD25. These results demonstrated that orally administered water-soluble undenatured C II (NEXT-II) is highly efficacious in the suppression of CIA by inducing CD4+CD25+ Treg cells.

  8. Optically characterizing collagen gels made with different cell types

    Science.gov (United States)

    Levitz, David; Choudhury, Niloy; Vartanian, Keri; Hinds, Monica T.; Hanson, Stephen R.; Jacques, Steven L.

    2009-02-01

    The ability of optical imaging techniques such as optical coherence tomography (OCT) to non-destructively characterize tissue-engineered constructs has generated enormous interest recently. Collagen gels are 3D structures that represent a simple common model of many engineered tissues that contain 2 primary scatterers: collagen and cells. We are testing the ability of OCT data to characterize the remodeling of such collagen-based constructs by 3 different types of cells: vascular smooth muscle cells (SMCs), endothelial cells (ECs), and osteoblasts (OBs). Collagen gels were prepared with SMCs, ECs, and OBs with a seeding density of 1×106 cells/ml; additionally, acellular controls were also prepared. The disk-shaped constructs were allowed to remodel in the incubator for 5 days, with OCT imaging occurring on days 1 and 5. From the OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and imaging conditions to the OCT signal. The degree of gel compaction was determined from the volume of the culture medium that feeds the constructs. We found that gel compaction (relative to the acellular control) occurred in the SMC constructs, but not in the OB or EC constructs. The optical property data showed that at day 5 the SMC constructs had an overall higher reflectivity (lower g) relative to day 1, whereas there was no obvious change in reflectivity of the EC, OB constructs and acellular controls relative to day 1. Moreover, there was a difference in the attenuation of the OB constructs on day 5 relative to day 1, but not in the other constructs. The apparent decrease in anisotropy observed in the SMC constructs, but not in the OB and EC constructs and acellular controls, suggests that OCT is sensitive to the remodeling of the collagen matrix that accompanies gel compaction, and can offer highly localized information on the construct

  9. Second harmonic generation microscopy differentiates collagen type I and type III in COPD

    Science.gov (United States)

    Suzuki, Masaru; Kayra, Damian; Elliott, W. Mark; Hogg, James C.; Abraham, Thomas

    2012-03-01

    The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

  10. Collagen Type I Conduits for the Regeneration of Nerve Defects

    Directory of Open Access Journals (Sweden)

    Silvan Klein

    2016-03-01

    Full Text Available To date, reliable data to support the general use of biodegradable materials for bridging nerve defects are still scarce. We present the outcome of nerve regeneration following type I collagen conduit nerve repair in patients with large-diameter nerve gaps. Ten patients underwent nerve repair using a type I collagen nerve conduit. Patients were re-examined at a minimal follow-up of 14.0 months and a mean follow-up of 19.9 months. Regeneration of nerve tissue within the conduits was assessed by nerve conduction velocity (NCV, a static two-point discrimination (S2PD test, and as disability of arm shoulder and hand (DASH outcome measure scoring. Quality of life measures including patients’ perceived satisfaction and residual pain were evaluated using a visual analog scale (VAS. No implant-related complications were observed. Seven out of 10 patients reported being free of pain, and the mean VAS was 1.1. The mean DASH score was 17.0. The S2PD was below 6 mm in 40%, between 6 and 10 mm in another 40% and above 10 mm in 20% of the patients. Eight out of 10 patients were satisfied with the procedure and would undergo surgery again. Early treatment correlated with lower DASH score levels. The use of type I collagen in large-diameter gaps in young patients and early treatment presented superior functional outcomes.

  11. Age determines the magnitudes of angiotensin II-induced contractions, mRNA, and protein expression of angiotensin type 1 receptors in rat carotid arteries.

    Science.gov (United States)

    Vamos, Zoltan; Cseplo, Peter; Ivic, Ivan; Matics, Robert; Hamar, Janos; Koller, Akos

    2014-05-01

    In this study, we hypothesized that aging alters angiotensin II (Ang II)-induced vasomotor responses and expression of vascular mRNA and protein angiotensin type 1 receptor (AT1R). Thus, carotid arteries were isolated from the following age groups of rats: 8 days, 2-9 months, 12-20 months, and 20-30 months, and their vasomotor responses were measured in a myograph after repeated administrations of Ang II. Vascular relative AT1R mRNA level was determined by quantitative reverse-transcriptase polymerase chain reaction and the AT1R protein density was measured by Western blot. Contractions to the first administration of Ang II increased from 8 days to 6 months and then they decreased to 30 months. In general, second administration of Ang II elicited reduced contractions, but they also increased from 8 days until 2 months and then they decreased to 30 months. Similarly the AT1R mRNA level increased from 8 days to 12 months and then decreased to 30 months. Similarly the AT1R protein density increased from 8 days until 16 months and then they decreased to 30 months. The pattern of these changes correlated with functional vasomotor data. We conclude that aging (newborn to senescence) has substantial effects on Ang II-induced vasomotor responses and AT1R signaling suggesting the importance of genetic programs.

  12. Measurement of Elastic Modulus of Collagen Type I Single Fiber.

    Science.gov (United States)

    Dutov, Pavel; Antipova, Olga; Varma, Sameer; Orgel, Joseph P R O; Schieber, Jay D

    2016-01-01

    Collagen fibers are the main components of the extra cellular matrix and the primary contributors to the mechanical properties of tissues. Here we report a novel approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found in vivo and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. This approach also avoids drying for measurements or visualization, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. The longitudinal component of the single-fiber elastic modulus is between 100 MPa and 360 MPa for samples extracted from different rats and/or different parts of a single tail. Variations are also observed in the fibril-bundle/fibril diameter with an average of 325±40 nm. Since bending forces depend on the diameter to the fourth power, this variation in diameter is important for estimating the range of elastic moduli. The remaining variations in the modulus may be due to differences in composition of the fibril-bundles, or the extent of the proteoglycans constituting fibril-bundles, or that some single fibrils may be of fibril-bundle size.

  13. Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes

    DEFF Research Database (Denmark)

    Sondergaard, B C; Wulf, H; Henriksen, K

    2006-01-01

    chain reaction (RT-PCR). In bovine articular cartilage explants, cartilage degradation was investigated by release of C-terminal telopeptides of collagen type II (CTX-II), induced by tumor necrosis factor-alpha (TNF-alpha) [20 ng/ml] and oncostatin M (OSM) [10 ng/ml], with salmon calcitonin [0.......0001-1 microM]. In vivo, cartilage degradation was investigated in ovariectomized (OVX) rats administered with oral calcitonin [2 mg/kg calcitonin] for 9 weeks. RESULTS: The calcitonin receptor was identified in articular chondrocytes by immunohistochemistry and RT-PCR. Calcitonin concentration......-dependently increased cAMP levels in isolated chondrocytes. Explants cultured with TNF-alpha and OSM showed a 100-fold increase in CTX-II release compared to vehicle-treated controls (Pprotection...

  14. Autoantibodies to Multiple Epitopes on the Non-Collagenous-1 Domain of Type VII Collagen Induce Blisters

    NARCIS (Netherlands)

    Vorobyev, Artem; Ujiie, Hideyuki; Recke, Andreas; Buijsrogge, Jacqueline J. A.; Jonkman, Marcel F.; Pas, Hendrikus; Iwata, Hiroaki; Hashimoto, Takashi; Kim, Soo-Chan; Kim, Jong Hoon; Groves, Richard; Samavedam, Unni; Gupta, Yask; Schmidt, Enno; Zillikens, Detlef; Shimizu, Hiroshi; Ludwig, Ralf J.

    Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease of the skin and mucous membranes, characterized by autoantibodies against type VII collagen (COL7), a major component of anchoring fibrils. Different clinical EBA phenotypes are described, including mechanobullous and

  15. Autoantibodies to Multiple Epitopes on the Non-Collagenous-1 Domain of Type VII Collagen Induce Blisters

    NARCIS (Netherlands)

    Vorobyev, Artem; Ujiie, Hideyuki; Recke, Andreas; Buijsrogge, Jacqueline J. A.; Jonkman, Marcel F.; Pas, Hendrikus; Iwata, Hiroaki; Hashimoto, Takashi; Kim, Soo-Chan; Kim, Jong Hoon; Groves, Richard; Samavedam, Unni; Gupta, Yask; Schmidt, Enno; Zillikens, Detlef; Shimizu, Hiroshi; Ludwig, Ralf J.

    2015-01-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease of the skin and mucous membranes, characterized by autoantibodies against type VII collagen (COL7), a major component of anchoring fibrils. Different clinical EBA phenotypes are described, including mechanobullous and inflamma

  16. Cartilage turnover reflected by metabolic processing of type II collagen

    DEFF Research Database (Denmark)

    Gudmann, Karoline Natasja Stæhr; Wang, Jianxia; Hoielt, Sabine

    2014-01-01

    ). This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab) was raised in mouse, targeting specifically PIIBNP (QDVRQPG) and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP......The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP...

  17. Bio-inspired microstructures in collagen type I hydrogel.

    Science.gov (United States)

    Hosseini, Yahya; Verbridge, Scott S; Agah, Masoud

    2015-06-01

    This article presents a novel technique to fabricate complex type I collagen hydrogel structures, with varying depth and width defined by a single fabrication step. This technique takes advantage of reactive ion etching lag to fabricate three-dimensional (3-D) structures in silicon. Then, a polydimethylsiloxane replica was fabricated utilizing soft lithography and used as a stamp on collagen hydrogel to transfer these patterns. Endothelial cells were seeded on the hydrogel devices to measure their interaction with these more physiologically relevant cell culture surfaces. Confocal imaging was utilized to image the hydrogel devices to demonstrate the robustness of the fabrication technique, and to study the cell-extracellular matrix interaction after cell seeding. In this study, we observed that endothelial cells remodeled the sharp scallops of collagen hydrogel structures and compressed the structures with low degree of slope. Such patterning techniques will enhance the physiological relevance of existing 3-D cell culture platforms by providing a technical bridge between the high resolution yet planar techniques of standard lithography with more complex yet low resolution 3-D printing methods.

  18. The extracellular matrix of Gadus morhua muscle contains types III, V, VI and IV collagens in addition to type I

    DEFF Research Database (Denmark)

    Brüggemann, Dagmar Adeline; Lawson, M.A.

    2005-01-01

    Confocal microscopy and immuno‐histochemistry were used to examine collagens in the extracellular matrix of cod Gadus morhua swimming muscle. In addition to the well known presence of type I fibrous collagen, types III and VI were also found in the myocommata and the endomysium. The beaded collagen...

  19. The discoidin domain receptor DDR2 is a receptor for type X collagen.

    Science.gov (United States)

    Leitinger, Birgit; Kwan, Alvin P L

    2006-08-01

    During endochondral ossification, collagen X is deposited in the hypertrophic zone of the growth plate. Our previous results have shown that collagen X is capable of interacting directly with chondrocytes, primarily via integrin alpha2beta1. In this study, we determined whether collagen X could also interact with the non-integrin collagen receptors, discoidin domain receptors (DDRs), DDR1 or DDR2. The widely expressed DDRs are receptor tyrosine kinases that are activated by a number of different collagen types. Collagen X was found to be a much better ligand for DDR2 than for DDR1. Collagen X bound to the DDR2 extracellular domain with high affinity and stimulated DDR2 autophosphorylation, the first step in transmembrane signalling. Expression of DDR2 in the epiphyseal plate was confirmed by RT-PCR and immunohistochemistry. The spatial expression of DDR2 in the hypertrophic zone of the growth plate is consistent with a physiological interaction of DDR2 with collagen X. Surprisingly, the discoidin domain of DDR2, which fully contains the binding sites for the fibrillar collagens I and II, was not sufficient for collagen X binding. The nature of the DDR2 binding site(s) within collagen X was further analysed. In addition to a collagenous domain, collagen X contains a C-terminal NC1 domain. DDR2 was found to recognise the triple-helical region of collagen X as well as the NC1 domain. Binding to the collagenous region was dependent on the triple-helical conformation. DDR2 autophosphorylation was induced by the collagen X triple-helical region but not the NC1 domain, indicating that the triple-helical region of collagen X contains a specific DDR2 binding site that is capable of receptor activation. Our study is the first to describe a non-fibrillar collagen ligand for DDR2 and will form the basis for further studies into the biological function of collagen X during endochondral ossification.

  20. Modulation of C1-Inhibitor and Plasma Kallikrein Activities by Type IV Collagen

    Directory of Open Access Journals (Sweden)

    Sriram Ravindran

    2012-01-01

    Full Text Available The contact system of coagulation can be activated when in contact with biomaterials. As collagen is being tested in novel biomaterials in this study, we have investigated how type IV collagen affects plasma kallikrein and C1-inhibitor. Firstly, we showed C1-inhibitor binds to type IV collagen with a Kd of 0.86 μM. The effects of type IV collagen on plasma kallikrein, factor XIIa, and β-factor XIIa activity and on C1-inhibitor function were determined. Factor XIIa rapidly lost activity in the presence of type IV collagen, whereas plasma kallikrein and β-factor XIIa were more stable. The rate of inhibition of plasma kallikrein by C1-inhibitor was decreased by type IV collagen in a dose-dependent manner. These studies could be relevant to the properties of biomaterials, which contain collagen, and should be considered in the testing for biocompatibility.

  1. TGF-beta Inhibits Ang II-Induced MAPK p44/42 Signaling in Vascular Smooth Muscle Cells by Ang II Type 1 Receptor Downregulation

    NARCIS (Netherlands)

    Meijering, Bernadet D. M.; van der Wouden, Els A.; Pelgrom, Vincent; Henning, Robert H.; Sharma, Kumar; Deelman, Leo E.

    2009-01-01

    Vascular changes in diabetes are characterized by reduced vasoconstriction and vascular remodeling. Previously, we demonstrated that TGF-beta 1 impairs Ang II-induced contraction through reduced calcium mobilization. However, the effect of TGF-beta 1 on Ang II-induced vascular remodeling is unknown.

  2. Measurement of Elastic Modulus of Collagen Type I Single Fiber.

    Directory of Open Access Journals (Sweden)

    Pavel Dutov

    Full Text Available Collagen fibers are the main components of the extra cellular matrix and the primary contributors to the mechanical properties of tissues. Here we report a novel approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found in vivo and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. This approach also avoids drying for measurements or visualization, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. The longitudinal component of the single-fiber elastic modulus is between 100 MPa and 360 MPa for samples extracted from different rats and/or different parts of a single tail. Variations are also observed in the fibril-bundle/fibril diameter with an average of 325±40 nm. Since bending forces depend on the diameter to the fourth power, this variation in diameter is important for estimating the range of elastic moduli. The remaining variations in the modulus may be due to differences in composition of the fibril-bundles, or the extent of the proteoglycans constituting fibril-bundles, or that some single fibrils may be of fibril-bundle size.

  3. ADHERENCE, PROLIFERATION AND COLLAGEN TURNOVER BY HUMAN FIBROBLASTS SEEDED INTO DIFFERENT TYPES OF COLLAGEN SPONGES

    NARCIS (Netherlands)

    MIDDELKOOP, E; DEVRIES, HJC; RUULS, L; EVERTS, [No Value; WILDEVUUR, CHR; WESTERHOF, W

    We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted

  4. ADHERENCE, PROLIFERATION AND COLLAGEN TURNOVER BY HUMAN FIBROBLASTS SEEDED INTO DIFFERENT TYPES OF COLLAGEN SPONGES

    NARCIS (Netherlands)

    MIDDELKOOP, E; DEVRIES, HJC; RUULS, L; EVERTS, [No Value; WILDEVUUR, CHR; WESTERHOF, W

    1995-01-01

    We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted non-crossl

  5. Different collagen types define two types of idiopathic epiretinal membranes

    OpenAIRE

    Kritzenberger, Michaela; Junglas, Benjamin; Framme, Carsten; Helbig, Horst; Gabel, Veit-Peter; Fuchshofer, Rudolf; Ernst R. Tamm; Hillenkamp, Jost

    2011-01-01

    Abstract Aims: To identify differences in extracellular matrix contents between idiopathic epiretinal membranes (IEM) of cellophane macular reflex (CMRM) or preretinal macular fibrosis (PMFM) type. Methods and results: IEM were analyzed by light and quantitative transmission electron microscopy, immunohistochemistry, and Western blotting. Substantial differences between CMRM and PMFM were observed regarding the nature of extracellular fibrils. In CMRM, the fibrils were thin with...

  6. Synthesis of type III collagen by fibroblasts from the embryonic chick cornea

    OpenAIRE

    1980-01-01

    Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cel...

  7. MMP mediated type V collagen degradation (C5M) is elevated in ankylosing spondylitis

    DEFF Research Database (Denmark)

    Veidal, S S; Larsen, D V; Chen, Xijuan;

    2012-01-01

    Type V collagen has been demonstrated to control fibril formation. The aim of this study was to develop an ELISA capable of detecting a fragment of type V collagen generated by MMP-2/9 and to evaluate the assay as biomarker for ankylosing spondylitis (AS).......Type V collagen has been demonstrated to control fibril formation. The aim of this study was to develop an ELISA capable of detecting a fragment of type V collagen generated by MMP-2/9 and to evaluate the assay as biomarker for ankylosing spondylitis (AS)....

  8. A biomimetic strategy to form calcium phosphate crystals on type I collagen substrate

    Energy Technology Data Exchange (ETDEWEB)

    Xu Zhang [Department of Restorative Dentistry, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road 119074, Singapore (Singapore); Neoh, Koon Gee [Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge 119260, Singapore (Singapore); Kishen, Anil, E-mail: anil.kishen@utoronto.ca [Discipline of Endodontics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON (Canada)

    2010-07-20

    Objective: The aim of this study is to induce mineralization of collagen by introducing phosphate groups onto type I collagen from eggshell membrane (ESM) by treating with sodium trimetaphosphate (STMP). This strategy is based on the hypothesis that phosphate groups introduced on collagen can mimic the nucleating role of phosphorylated non-collagenous proteins bound to collagen for inducing mineralization in natural hard tissue. Method: The collagen membrane was phosphorylated by treating it with a solution of STMP and saturated calcium hydroxide. The phosphorylated collagen was subsequently exposed to a mineralization solution and the pattern of mineralization on the surface of phosphorylated collagen substrate was analyzed. Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), field emission electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and microhardness test were used to characterize the collagen substrate and the pattern of minerals formed on the collagen surface. Results: The FTIR and EDX results indicated that the phosphate groups were incorporated onto the collagen surface by treatment with STMP. During the mineralization process, the plate-like mineral, octacalcium phosphate (OCP), which was initially formed on the surface of ESM, was later transformed into needle-like hydroxyapatite (HAP) as indicated by the SEM, FESEM, EDX and XRD findings. The microhardness test displayed significant increase in the Knoop hardness number of the mineralized collagen. Conclusions: Phosphate groups can be introduced onto type I collagen surface by treating it with STMP and such phosphorylated collagen can induce the mineralization of type I collagen.

  9. Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro.

    Science.gov (United States)

    Nehrer, S; Breinan, H A; Ramappa, A; Shortkroff, S; Young, G; Minas, T; Sledge, C B; Yannas, I V; Spector, M

    1997-01-01

    Synthetic and natural absorbable polymers have been used as vehicles for implantation of cells into cartilage defects to promote regeneration of the articular joint surface. Implants should provide a pore structure that allows cell adhesion and growth, and not provoke inflammation or toxicity when implanted in vivo. The scaffold should be absorbable and the degradation should match the rate of tissue regeneration. To facilitate cartilage repair the chemical structure and pore architecture of the matrix should allow the seeded cells to maintain the chondrocytic phenotype, characterized by synthesis of cartilage-specific proteins. We investigated the behavior of canine chondrocytes in two spongelike matrices in vitro: a collagen-glycosaminoglycan (GAG) copolymer produced from bovine hide consisting of type I collagen and a porous scaffold made of type II collagen by extraction of porcine cartilage. Canine chondrocytes were seeded on both types of matrices and cultured for 3 h, 7 days, and 14 days. The histology of chondrocyte-seeded implants showed a significantly higher percentage of cells with spherical morphology, consistent with chondrocytic morphology, in the type II sponge at each time point. Pericellular matrix stained for proteoglycans and for type II collagen after 14 days. Biochemical analysis of the cell seeded sponges for GAG and DNA content showed increases with time. At day 14 there was a significantly higher amount of DNA and GAG in the type II matrix. This is the first study that directly compares the behavior of chondrocytes in type I and type II collagen matrices. The type II matrix may be of value as a vehicle for chondrocyte implantation on the basis of the higher percentage of chondrocytes retaining spherical morphology and greater biosynthetic activity that was reflected in the greater increase of GAG content.

  10. Modulation of C1-Inhibitor and Plasma Kallikrein Activities by Type IV Collagen

    OpenAIRE

    Sriram Ravindran; Marc Schapira; Patston, Philip A.

    2012-01-01

    The contact system of coagulation can be activated when in contact with biomaterials. As collagen is being tested in novel biomaterials in this study, we have investigated how type IV collagen affects plasma kallikrein and C1-inhibitor. Firstly, we showed C1-inhibitor binds to type IV collagen with a Kd of 0.86 μM. The effects of type IV collagen on plasma kallikrein, factor XIIa, and β-factor XIIa activity and on C1-inhibitor function were determined. Factor XIIa rapidly lost activity in the...

  11. Attachment of cells to basement membrane collagen type IV

    OpenAIRE

    1986-01-01

    Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformatio...

  12. Type II collagen C2C epitope in human synovial fluid and serum after knee injury

    DEFF Research Database (Denmark)

    Kumahashi, N; Swärd, P; Larsson, S

    2015-01-01

    PURPOSE: Investigate in a cross-sectional study time-dependent changes of synovial fluid type II collagen epitope C2C concentrations after knee injury and correlate to other joint injury biomarkers. METHODS: Synovial fluid samples were aspirated between 0 days and 7 years after injury (n = 235...... = 0.403, P collagen (r = 0.444, P = 0.003), ARGS-aggrecan (r = 0.337, P ... with an immediate and sustained local degradation of type II collagen....

  13. The spatial organization of Descemet's membrane-associated type IV collagen in the avian cornea

    OpenAIRE

    1990-01-01

    The organization of type IV collagen in the unconventional basement membrane of the corneal endothelium (Descemet's membrane) was investigated in developing chicken embryos using anti-collagen mAbs. Both immunofluorescence histochemistry and immunoelectron microscopy were performed. In mature embryos (greater than 15 d of development), the type IV collagen of Descemet's membrane was present as an array of discrete aggregates of amorphous material at the interface between Descemet's membrane a...

  14. Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes

    NARCIS (Netherlands)

    Rutgers, M.; Saris, Daniël B.F.; Vonk, L.A.; van Rijen, M.H.P.; Akrum, V.; Langeveld, D.; van Boxtel, A.; Dhert, W.J.A.; Creemers, L.B.

    2013-01-01

    Introduction: 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,

  15. Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes

    NARCIS (Netherlands)

    Rutgers, M.; Saris, D.B.F.; Vonk, L.A.; Rijen, van M.H.P.; Akrum, V.; Langeveld, D.; Boxtel, van A.; Dhert, W.J.A.; Creemers, L.B.

    2013-01-01

    Introduction: 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, ho

  16. Type II collagen is transiently expressed during avian cardiac valve morphogenesis.

    Science.gov (United States)

    Swiderski, R E; Daniels, K J; Jensen, K L; Solursh, M

    1994-08-01

    We present new evidence of the temporal and spatial expression of type II collagen in the embryonic chick heart during the very early stages of its development. In particular, we emphasize the distribution of its mRNA and protein during valve formation. Type II collagen as well as several other fibrillar collagens (types I, III, and V) are present in stage 18 endocardial cushion mesenchymal cells. At stage 23, alpha 1 (II) collagen transcripts and the cognate polypeptide colocalize in the atrioventricular valves. As development proceeds, the relative abundance of alpha 1 (II) collagen transcripts decreases during the stages studied (stages 22 to 45; day 3.5 to day 19) as assayed by RNA blotting of extracts of whole hearts. Type II collagen protein was immunologically undetectable in stage 38 (day 12) hearts, although collagens I, III, and V persisted and localize in the valve regions, in the endothelial lining of the heart, and in the epicardium. In keeping with other observations of type II collagen expression in non-chondrogenic regions of a variety of vertebrate embryos, the avian heart also exhibits transient type II collagen expression.

  17. Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy.

    Science.gov (United States)

    McGuire, J D; Gorski, J P; Dusevich, V; Wang, Y; Walker, M P

    2014-10-01

    The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy--teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ

  18. Micromechanical testing of single collagen type I fibrils

    NARCIS (Netherlands)

    Rijt, van der Joost Adrianus Johannes

    2004-01-01

    In this thesis the following subjects will be covered. In chapter 2 the current understanding of the structure and corresponding mechanical properties of collagen and its substructures will be reviewed. In chapter 3 the methodology to enable fixation and subsequent tensile testing of collagen fibril

  19. Pigmentation and melanocyte supply to the epidermis depend on type XVII collagen : Experimental Dermatology

    NARCIS (Netherlands)

    Gostynski, Antoni; Pasmooij, Anna M. G.; Del Rio, Marcela; Diercks, Gilles F.; Pas, Hendrikus; Jonkman, Marcellinus

    Genetic deficiency of type XVII collagen (C17), laminin-332 or type VII collagen causes epidermolysis bullosa (EB). Spontaneous correction of the deficiency, also known as revertant mosaicism, is caused by a second somatic mutation that restores protein expression resulting in clinically healthy

  20. Adjuvant arthritis pretreatment with type II collagen and Mycobacterium butyricum.

    Science.gov (United States)

    Franch, A; Cassany, S; Castellote, C; Castell, M

    1992-11-01

    A treatment previous to adjuvant arthritis induction has been performed with type II collagen (CII) or Mycobacterium butyricum (Mb), which is the inducer of the pathology. Pretreatment was administered in two different ways: a) subcutaneously or intradermally 14 days before arthritis induction, and b) intravenously 3 days before induction. In order to relate the change in inflammation to the corresponding antigen immune response, serum antibodies and delayed type hypersensitivity (DTH) against CII or Mb were studied. Pretreatment with s.c. CII 14 days before induction produced slight protection against arthritis and significantly delayed its onset; systemic inflammation showed good positive correlation with anti-CII antibodies. The CII administered i.v. 3 days before arthritic challenge did not significantly modify the inflammatory process. The use of i.d. subarthritogenic doses of Mb 14 days before induction protected a high percentage of the animals from the posterior arthritic challenge; this protection was accompanied by high anti-Mb antibody titers and DTH reaction. When Mb was given i.v. 3 days before induction, a partial protection of inflammation was observed; arthritis was milder and its onset was delayed. These changes were accompanied by reduced humoral and cellular response to Mb.

  1. Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

    Science.gov (United States)

    Saby, Charles; Buache, Emilie; Brassart-Pasco, Sylvie; El Btaouri, Hassan; Courageot, Marie-Pierre; Van Gulick, Laurence; Garnotel, Roselyne; Jeannesson, Pierre; Morjani, Hamid

    2016-05-03

    Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

  2. Collagen Type III and VI Turnover in Response to Long-Term Immobilization

    DEFF Research Database (Denmark)

    Sun, Shu; Henriksen, Kim; Karsdal, Morten A

    2015-01-01

    BACKGROUND: Muscle mass and function are perturbed by immobilization and remobilization. When muscle mass changes, the quality and quantity of the extracellular matrix protein, particularly the collagens, change with it. In this study, we investigated the temporal profile of three peptide...... biomarkers derived from turnover of collagen type III and type VI in a long-term immobilization and remobilization study. We also compared individual biomarker levels with Lean body Mass (LBM) and changes therein, hypothesizing that these biomarkers would be biomarkers of the remodeling processes associated...... the synthesis of collagen type III; Pro-C6, measured the synthesis of collagen type VI; and C6M measured the degradation of collagen type VI induced by MMP-2 and MMP-9 cleavage. RESULTS: Pro-C3 and Pro-C6 biomarkers are up-regulated with both immobilization and remobilization, whereas C6M is hardly affected...

  3. Fourier transform infrared imaging and infrared fiber optic probe spectroscopy identify collagen type in connective tissues.

    Directory of Open Access Journals (Sweden)

    Arash Hanifi

    Full Text Available Hyaline cartilage and mechanically inferior fibrocartilage consisting of mixed collagen types are frequently found together in repairing articular cartilage. The present study seeks to develop methodology to identify collagen type and other tissue components using Fourier transform infrared (FTIR spectral evaluation of matrix composition in combination with multivariate analyses. FTIR spectra of the primary molecular components of repair cartilage, types I and II collagen, and aggrecan, were used to develop multivariate spectral models for discrimination of the matrix components of the tissues of interest. Infrared imaging data were collected from bovine bone, tendon, normal cartilage, meniscus and human repair cartilage tissues, and composition predicted using partial least squares analyses. Histology and immunohistochemistry results were used as standards for validation. Infrared fiber optic probe spectral data were also obtained from meniscus (a tissue with mixed collagen types to evaluate the potential of this method for identification of collagen type in a minimally-invasive clinical application. Concentration profiles of the tissue components obtained from multivariate analysis were in excellent agreement with histology and immunohistochemistry results. Bone and tendon showed a uniform distribution of predominantly type I collagen through the tissue. Normal cartilage showed a distribution of type II collagen and proteoglycan similar to the known composition, while in repair cartilage, the spectral distribution of both types I and II collagen were similar to that observed via immunohistochemistry. Using the probe, the outer and inner regions of the meniscus were shown to be primarily composed of type I and II collagen, respectively, in accordance with immunohistochemistry data. In summary, multivariate analysis of infrared spectra can indeed be used to differentiate collagen type I and type II, even in the presence of proteoglycan, in

  4. Clinical significance of serum laminin and type-IV collagen levels in cutaneous melanoma patients.

    Science.gov (United States)

    Tas, Faruk; Bilgin, Elif; Karabulut, Senem; Duranyildiz, Derya

    2016-07-01

    Laminin and type-IV collagen constitute a significant portion of the extracellular matrix. The objective of the present study was to evaluate whether the serum concentrations of laminin and type-IV collagen may serve as biomarkers for cutaneous melanoma. Sixty pathologically confirmed melanoma patients were enrolled in the study. Serum laminin and type-IV collagen levels were assessed using an ELISA. Thirty healthy controls were also examined. No significant differences in the baseline serum levels of laminin were identified between melanoma patients and healthy controls (P=0.45). However, the baseline serum levels of type-IV collagen were significantly elevated in melanoma patients compared with those in the control group (PIV collagen (P>0.05). Furthermore, the serum levels of laminin and type-IV collagen had no prognostic value regarding the outcome for melanoma patients (P=0.36 and P=0.26, respectively). While laminin levels showed no diagnostic value, the serum concentrations of type-IV collagen were indicated to serve as a diagnostic marker in patients with cutaneous melanoma. In conclusion, type-IV collagen levels may be used as a diagnostic marker for cutaneous melanoma, while being void of any prognostic value.

  5. The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage.

    Science.gov (United States)

    Shen, G

    2005-02-01

    AUTHOR: Shen G Objective -This review was compiled to explore the role of type X collagen in growth, development and remodeling of articular cartilage by elucidating the linkage between the synthesis of this protein and the phenotypic changes in chondrogenesis and the onset of endochondral ossification. The current studies closely dedicated to elucidating the role of type X collagen incorporating into chondrogenesis and endochondral ossification of articular cartilage were assessed and analyzed to allow for obtaining the mainstream consensus on the bio-molecular mechanism with which type X collagen functions in articular cartilage. There are spatial and temporal correlations between synthesis of type X collagen and occurrence of endochondral ossification. The expression of type X collagen is confined within hypertrophic condrocytes and precedes the embark of endochondral bone formation. Type X collagen facilitates endochondral ossification by regulating matrix mineralization and compartmentalizing matrix components. Type X collagen is a reliable marker for new bone formation in articular cartilage. The future clinical application of this collagen in inducing or mediating endochondral ossification is perceived, e.g. the fracture healing of synovial joints and adaptive remodeling of madibular condyle.

  6. Cells that emerge from embryonic explants produce fibers of type IV collagen.

    Science.gov (United States)

    Chen, J M; Little, C D

    1985-10-01

    Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.

  7. Bending rigidity of type I collagen homotrimer fibrils

    Science.gov (United States)

    Han, Sejin; Leikin, Sergey; Losert, Wolfgang

    2009-03-01

    Normal type I collagen is an α1(I)2α2(I) heterotrimeric triple helix, but α1(I)3 homotrimers are also found in fetal tissues and various pathological conditions, e.g., causing bone fragility and reducing tendon tensile strength. It remains unclear whether homotrimers alter mechanical properties of individual fibrils or affect tissues by altering their organization at a higher level. To address this question, we investigated how homotrimers affect fibril bending rigidity. Homotrimer fibrils have been shown to be more loosely packed so that we expected them to be more susceptible to bending. However, homotrimer fibrils were more rigid despite being thinner and more hydrated. To quantify fibril rigidity, we analyzed their shape by Fourier decomposition, determined the correlation function for the direction along each fibril, and calculated the distribution of local fibril curvature. The estimated persistence length of homotrimer fibrils was 3 ˜ 10 times longer than for heterotrimer fibrils, indicating much higher bending rigidity of homotrimer fibrils.

  8. Thermal denaturation of type I collagen vitrified gels

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Zhiyong, E-mail: zhiyong.xia@jhuapl.edu [The Johns Hopkins University, Applied Physics Laboratory, Laurel, MD 20723 (United States); Calderon-Colon, Xiomara [The Johns Hopkins University, Applied Physics Laboratory, Laurel, MD 20723 (United States); Trexler, Morgana, E-mail: morgana.trexler@jhuapl.edu [The Johns Hopkins University, Applied Physics Laboratory, Laurel, MD 20723 (United States); Elisseeff, Jennifer; Guo, Qiongyu [The Johns Hopkins University, Department of Biomedical Engineering, Baltimore, MD 21231 (United States)

    2012-01-10

    Highlights: Black-Right-Pointing-Pointer We analyzed the denaturation of vitrigels synthesized under different conditions. Black-Right-Pointing-Pointer Overall denaturation kinetics consisted of both reversible and irreversible steps. Black-Right-Pointing-Pointer More stable vitrigels were formed under high level of vitrification. - Abstract: The denaturation kinetics of type I collagen vitrigels synthesized under different vitrification time and temperature were analyzed by the classical Kissinger approach and the advanced model free kinetics (AMFK) using the Vyazovkin algorithm. The AMFK successfully elucidated the overall denaturation into reversible and irreversible processes. Depending on vitrification conditions, the activation energy for the irreversible process ranged from 100 to 200 kJ/mol, and the reversible enthalpy ranged from 250 to 300 kJ/mol. All of these values increased with the vitrification time and temperature, indicating that a more stable and complex structure formed with increased vitrification. The classical Kissinger method predicted the presence of a critical temperate of approximately 60 Degree-Sign C for the transition between reversible and irreversible processes. Scanning electron microscopy revealed the presence of fibril structures in vitrigels both before and after full denaturation; however the fibrils had became thicker and rougher after denaturation.

  9. Collagen Type I as a Ligand for Receptor-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Iris Boraschi-Diaz

    2017-05-01

    Full Text Available Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B, and LAIR-1 of the leukocyte receptor complex (LRC and mannose family receptor uPARAP/Endo180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

  10. Involvement of FOXO transcription factors, TRAIL-FasL/Fas, and sirtuin proteins family in canine coronavirus type II-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Gabriella Marfè

    Full Text Available n our previous study, we have shown that canine coronavirus type II (CCoV-II activates both extrinsic and intrinsic apoptotic pathway in a canine fibrosarcoma cell line (A-72 cells. Herein we investigated the role of Sirtuin and Forkhead box O (FOXO families in this experimental model using Nortern Blot and Western Blot analysis. Our results demonstrated that mitochondrial SIRT3 and SIRT4 protein expression increased from 12 and 24 h post infection (p.i. onwards, respectively, whereas the nuclear SIRT1 expression increased during the first 12 h p.i. followed by a decrease after 36 h p.i., reaching the same level of control at 48 h p.i. Sirtuins interact with/and regulate the activity of FOXO family proteins, and we herein observed that FOXO3A and FOXO1 expression increased significantly and stably from 12 h p.i. onwards. In addition, CCoV-II induces a remarkable increase in the expression of TNF-related apoptosis-inducing ligand (TRAIL, while we observed a slight up-regulation of FasL/Fas at 36 p.i. with a decrease of both proteins at the end of infection. Furthermore, we found that virus infection increased both bax translocation into mitochondria and decreased bcl-2 expression in cytosol in a time-dependent manner.These data suggest that FOXO transcription factors mediate pro-apoptotic effects of CCoV-II, in part due to activation of extrinsic apoptosis pathway, while some Sirtuin family members (such as SIRT3 and SIRT4 may be involved in intrinsic apoptotic pathway. Moreover, these results propose that TRAIL is an important mediator of cell death induced by CCoV-II during in vitro infection.

  11. Skin as marker for collagen type I/III ratio in abdominal wall fascia.

    Science.gov (United States)

    Peeters, E; De Hertogh, G; Junge, K; Klinge, U; Miserez, M

    2014-08-01

    An altered collagen metabolism could play an important role in hernia development. This study compared collagen type I/III ratio and organisation between hernia and control patients, and analysed the correlation in collagen type I/III ratio between skin and abdominal wall fascia. Collagen organisation was analysed in Haematoxylin-Eosin sections of anterior rectus sheath fascia, and collagen type I/III ratio, by crosspolarisation microscopy, in Sirius-Red sections of skin and anterior rectus sheath fascia, of 19 control, 10 primary inguinal, 10 recurrent inguinal, 13 primary incisional and 8 recurrent incisional hernia patients. Compared to control patients [7.2 (IQR = 6.8-7.7) and 7.2 (IQR = 5.8-7.9)], collagen type I/III ratio was significantly lower in skin and anterior rectus sheath fascia of primary inguinal [5.2 (IQR = 3.8-6.3) and 4.2 (IQR = 3.8-4.7)], recurrent inguinal [3.2 (IQR = 3.1-3.6) and 3.3 (IQR = 3-3.7)], primary incisional [3.5 (IQR = 3-3.9) and 3.4 (IQR = 3.3-3.6)] and recurrent incisional hernia [3.2 (IQR = 3.1-3.9) and 3.2 (IQR = 2.9-3.2)] patients; also incisional and recurrent inguinal hernia had lower ratio than primary inguinal hernia patients. Furthermore, collagen type I/III ratio was significantly correlated (r = 0.81; P fascia. Finally, collagen organisation was comparable between hernia and control patients. Furthermore, in both skin and abdominal wall fascia of hernia patients, collagen type I/III ratio was lower compared to control patients, with more pronounced abnormalities in incisional and recurrent inguinal hernia patients. Importantly, collagen type I/III ratio in skin was representative for that in abdominal wall fascia.

  12. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko [China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016 (China); Fujisaki, Hitomi; Hattori, Shunji [Nippi Research Institute of Biomatrix, Toride, Ibaraki 302-0017 (Japan); Tashiro, Shin-ichi [Institute for Clinical and Biomedical Sciences, Kyoto 603-8072 (Japan); Onodera, Satoshi [Department of Clinical and Pharmaceutical Sciences, Showa Pharmaceutical University, Tokyo 194-8543 (Japan); Ikejima, Takashi, E-mail: ikejimat@vip.sina.com [China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016 (China)

    2015-02-20

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.

  13. Effect of papain-based gel on type I collagen--spectroscopy applied for microstructural analysis.

    Science.gov (United States)

    Silva Júnior, Zenildo Santos; Botta, Sergio Brossi; Ana, Patricia Aparecida; França, Cristiane Miranda; Fernandes, Kristianne Porta Santos; Mesquita-Ferrari, Raquel Agnelli; Deana, Alessandro; Bussadori, Sandra Kalil

    2015-06-23

    Considering the improvement of biomaterials that facilitate atraumatic restorative techniques in dentistry, a papain-based gel can be used in the chemomechanical removal of decayed dental tissue. However, there is no information regarding the influence of this gel on the structure of sound collagen. The aim of the present study was to investigate the adsorption of a papain-based gel (Papacarie(TM)) to collagen and determine collagen integrity after treatment. A pilot study was first performed with 10 samples of type I collagen membrane obtained from bovine Achilles deep tendon to compare the influence of hydration (Milli-Q water) on infrared bands of collagen. In a further experiment, 10 samples of type I collagen membrane were used to evaluate the effects of Papacarie(TM) on the collagen microstructure. All analyses were performed using the attenuated total reflectance technique of Fourier transform infrared (ATR-FTIR). The results demonstrated that the application of Papacarie(TM) does not lead to the degradation of collagen and this product can be safely used in minimally invasive dentistry. As the integrity of sound collagen is preserved after the application of the papain-based gel, this product is indicated for the selective removal of infected dentin, leaving the affected dentin intact and capable of re-mineralization.

  14. Interaction of lubricin with type II collagen surfaces: adsorption, friction, and normal forces.

    Science.gov (United States)

    Chang, Debby P; Guilak, Farshid; Jay, Gregory D; Zauscher, Stefan

    2014-02-01

    One of the major constituents of the synovial fluid that is thought to be responsible for chondroprotection and boundary lubrication is the glycoprotein lubricin (PRG4); however, the molecular mechanisms by which lubricin carries out its critical functions still remain largely unknown. We hypothesized that the interaction of lubricin with type II collagen, the main component of the cartilage extracellular matrix, results in enhanced tribological and wear properties. In this study, we examined: (i) the molecular details by which lubricin interacts with type II collagen and how binding is related to boundary lubrication and adhesive interactions; and (ii) whether collagen structure can affect lubricin adsorption and its chondroprotective properties. We found that lubricin adsorbs strongly onto denatured, amorphous, and fibrillar collagen surfaces. Furthermore, we found large repulsive interactions between the collagen surfaces in presence of lubricin, which increased with increasing lubricin concentration. Lubricin attenuated the large friction and also the long-range adhesion between fibrillar collagen surfaces. Interestingly, lubricin adsorbed onto and mediated the frictional response between the denatured and native amorphous collagen surfaces equally and showed no preference on the supramolecular architecture of collagen. However, the coefficient of friction was lowest on fibrillar collagen in the presence of lubricin. We speculate that an important role of lubricin in mediating interactions at the cartilage surface is to attach to the cartilage surface and provide a protective coating that maintains the contacting surfaces in a sterically repulsive state.

  15. Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans

    DEFF Research Database (Denmark)

    Langberg, Henning; Skovgaard, D; Petersen, L J;

    1999-01-01

    1. Physical activity is known to increase type I collagen synthesis measured as the concentration of biomarkers in plasma. By the use of microdialysis catheters with a very high molecular mass cut-off value (3000 kDa) we aimed to determine local type I collagen synthesis and degradation...... in the peritendinous region by measuring interstitial concentrations of a collagen propeptide (PICP; 100 kDa) and a collagen degradation product (ICTP; 9 kDa) as well as an inflammatory mediator (PGE2). 2. Seven trained human runners were studied before and after (2 and 72 h) 3 h of running (36 km). Two microdialysis...... catheters were placed in the peritendinous space ventral to the Achilles' tendon under ultrasound guidance and perfused with a Ringer-acetate solution containing 3H-labelled human type IV collagen and [15-3H(N)]PGE2 for in vivo recovery determination. Relative recovery was 37-59 % (range of the s...

  16. Training-induced changes in peritendinous type I collagen turnover determined by microdialysis in humans

    DEFF Research Database (Denmark)

    Langberg, Henning; Rosendal, L; Kjaer, M

    2001-01-01

    1. Acute exercise is found to increase collagen type I formation locally in peritendinous connective tissue of the Achilles' tendon in humans, as determined from changes in interstitial concentrations of collagen propeptide (PICP) and a collagen degradation product (ICTP) by the use of microdialy...... in local connective tissue of the peritendinous Achilles' region. Early in the process both synthesis and degradation are elevated, whereas later, the anabolic processes are dominating causing a net synthesis of type I collagen in tendon-related tissue in humans.......1. Acute exercise is found to increase collagen type I formation locally in peritendinous connective tissue of the Achilles' tendon in humans, as determined from changes in interstitial concentrations of collagen propeptide (PICP) and a collagen degradation product (ICTP) by the use...... of microdialysis catheters. However, the local collagen type I turnover response to training is unknown. 2. Nineteen young males were studied before and after 4 and 11 weeks of physical training. Microdialysis catheters with a high molecular mass cut-off value (3000 kDa), allowing the determination of PICP...

  17. Determination of markers for collagen type I turnover in peritendinous human tissue by microdialysis

    DEFF Research Database (Denmark)

    Olesen, J L; Langberg, Henning; Heinemeier, K M

    2006-01-01

    Previous results from our group have shown that loading of human tendon elevates tendinous type I collagen production measured by microdialysis. However, exclusion of the observed elevation as a response to trauma from inserting the microdialysis catheters or a possible influence from the collagen...

  18. Propolis Modifies Collagen Types I and III Accumulation in the Matrix of Burnt Tissue

    Directory of Open Access Journals (Sweden)

    Pawel Olczyk

    2013-01-01

    Full Text Available Wound healing represents an interactive process which requires highly organized activity of various cells, synthesizing cytokines, growth factors, and collagen. Collagen types I and III, serving as structural and regulatory molecules, play pivotal roles during wound healing. The aim of this study was to compare the propolis and silver sulfadiazine therapeutic efficacy throughout the quantitative and qualitative assessment of collagen types I and III accumulation in the matrix of burnt tissues. Burn wounds were inflicted on pigs, chosen for the evaluation of wound repair because of many similarities between pig and human skin. Isolated collagen types I and III were estimated by the surface plasmon resonance method with a subsequent collagenous quantification using electrophoretic and densitometric analyses. Propolis burn treatment led to enhanced collagens and its components expression, especially during the initial stage of the study. Less expressed changes were observed after silver sulfadiazine (AgSD application. AgSD and, with a smaller intensity, propolis stimulated accumulation of collagenous degradation products. The assessed propolis therapeutic efficacy, throughout quantitatively and qualitatively analyses of collagen types I and III expression and degradation in wounds matrix, may indicate that apitherapeutic agent can generate favorable biochemical environment supporting reepithelization.

  19. Identification and characterization of the human type II collagen gene (COL2A1).

    NARCIS (Netherlands)

    K.S.E. Cheah (Kathryn); N.G. Stoker; J.R. Griffin; F.G. Grosveld (Frank); E. Solomon

    1985-01-01

    textabstractThe gene contained in the human cosmid clone CosHcol1, previously designated an alpha 1(I) collagen-like gene, has now been identified. CosHcol1 hybridizes strongly to a single 5.9-kilobase mRNA species present only in tissue in which type II collagen is expressed. DNA sequence analysis

  20. Trypsin-mediated enzymatic degradation of type II collagen in the human vitreous

    NARCIS (Netherlands)

    van Deemter, Marielle; Kuijer, Roel; Pas, Hendri Harm; van der Worp, Roelofje Jacoba; Hooymans, Johanna Martina Maria; Los, Leonoor Inge

    2013-01-01

    Purpose: Aging of the vitreous body can result in sight-threatening pathology. One aspect of vitreous aging is liquefaction, which results from the vanishing of collagen fibrils. We investigated the possibility that trypsins are involved in vitreous type II collagen degradation. Methods: Immunohisto

  1. Progress towards discovery of antifibrotic drugs targeting synthesis of type I collagen

    KAUST Repository

    Fritz, Dillon Jeffery

    2011-08-01

    Type I collagen is the most abundant protein in human body. Fibrosis is characterized by excessive synthesis of type I collagen in parenchymal organs. It is a leading cause of morbidity and mortality worldwide, about 45% of all natural deaths are attributable to some fibroproliferative disease. There is no cure for fibrosis. To find specific antifibrotic therapy targeting type I collagen, critical molecular interactions regulating its synthesis must be elucidated. Type I and type III collagen mRNAs have a unique sequence element at the 5\\' end, the 5\\' stem-loop. This stem-loop is not found in any other mRNA. We cloned LARP6 as the protein which binds collagen 5\\' stem-loop with high affinity and specificity. Mutation of the 5\\' stem-loop or knock down of LARP6 greatly diminishes collagen expression. Mice with mutation of the 5\\' stem-loop are resistant to development of liver fibrosis. LARP6 associates collagen mRNAs with filaments composed of nonmuscle myosin; disruption of these filaments abolishes synthesis of type I collagen. Thus, LARP6 dependent collagen synthesis is the specific mechanism of high collagen expression seen in fibrosis. We developed fluorescence polarization (FP) method to screen for drugs that can inhibit binding of LARP6 to 5\\' stem-loop RNA. FP is high when LARP6 is bound, but decreases to low levels when the binding is competed out. Thus, by measuring decrease in FP it is possible to identify chemical compounds that can dissociate LARP6 from the 5\\' stem-loop. The method is simple, fast and suitable for high throughput screening. © 2011 Bentham Science Publishers Ltd.

  2. Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

    Science.gov (United States)

    Wyatt, Karla E-K; Bourne, Jonathan W; Torzilli, Peter A

    2009-05-01

    Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsiloncollagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule.

  3. MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III.

    Science.gov (United States)

    Taddese, Samuel; Jung, Michael C; Ihling, Christian; Heinz, Andrea; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly(775)-Leu(776)- in alpha-2 type I collagen and -Gly(775)-Ile(776)- in alpha-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III. Copyright 2009 Elsevier B.V. All rights reserved.

  4. Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues

    Science.gov (United States)

    Toh, Wei Seong; Gomoll, Andreas H.; Olsen, Bjørn Reino; Spector, Myron

    2014-01-01

    Objective: The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Design: Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti–collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. Results: When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. Conclusions: We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional

  5. 7S Fragment of Type IV Collagen as a Serum Marker of Canine Liver Fibrosis.

    Science.gov (United States)

    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.

  6. Immunohistochemical findings type I and type II collagen in prenatal mouse mandibular condylar cartilage compared with the tibial anlage.

    Science.gov (United States)

    Ishii, M; Suda, N; Tengan, T; Suzuki, S; Kuroda, T

    1998-07-01

    In growing animals the mandibular condylar cartilage serves not only as an articular but also as a growth cartilage, yet, condylar cartilage has some characteristic features that are not found in growth cartilage. For example, some reports suggest that type I collagen, which is not seen in the growth plate cartilage of long bones, is present in the extracellular matrix of condylar cartilage postnatally. Here, the condylar and limb bud cartilage of fetal mice was examined. The distribution of type I and type II collagen in condylar cartilage was already different from that in the limb bud at the first appearance of the cartilage. Type I collagen was demonstrated in the extracellular matrix of the condylar cartilage that first appeared on day 15 of gestation. However, the reaction for type II collagen was much weaker than that for type I collagen. On day 18 of gestation, type I collagen was still found throughout the cell layers but became gradually weaker with depth. Type II collagen was limited exclusively to the deeper layers at this stage. These findings are different from those in the limb bud cartilage, indicating a characteristic feature of the cells in the condylar cartilage present from the prenatal period.

  7. Type I collagen is thermally unstable at body temperature.

    Science.gov (United States)

    Leikina, E; Mertts, M V; Kuznetsova, N; Leikin, S

    2002-02-05

    Measured by ultra-slow scanning calorimetry and isothermal circular dichroism, human lung collagen monomers denature at 37 degrees C within a couple of days. Their unfolding rate decreases exponentially at lower temperature, but complete unfolding is observed even below 36 degrees C. Refolding of full-length, native collagen triple helices does occur, but only below 30 degrees C. Thus, contrary to the widely held belief, the energetically preferred conformation of the main protein of bone and skin in physiological solution is a random coil rather than a triple helix. These observations suggest that once secreted from cells collagen helices would begin to unfold. We argue that initial microunfolding of their least stable domains would trigger self-assembly of fibers where the helices are protected from complete unfolding. Our data support an earlier hypothesis that in fibers collagen helices may melt and refold locally when needed, giving fibers their strength and elasticity. Apparently, Nature adjusts collagen hydroxyproline content to ensure that the melting temperature of triple helical monomers is several degrees below rather than above body temperature.

  8. Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

    Science.gov (United States)

    Yoon, Junghyo; Korkmaz Zirpel, Nuriye; Park, Hyun-Ji; Han, Sewoon; Hwang, Kyung Hoon; Shin, Jisoo; Cho, Seung-Woo; Nam, Chang-Hoon; Chung, Seok

    2016-01-21

    Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix.

  9. Epithelial-to-mesenchymal transition in fibrosis : Collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition

    NARCIS (Netherlands)

    Hosper, Nynke A.; van den Berg, Paul P.; de Rond, Saskia; Popa, Eliane R.; Wilmer, Martijn J.; Masereeuw, Rosalinde; Bank, Ruud A.

    2013-01-01

    The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data

  10. Epithelial-to-mesenchymal transition in fibrosis: collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition.

    NARCIS (Netherlands)

    Hosper, N.A.; Berg, P.P. van den; Rond, S. de; Popa, E.R.; Wilmer, M.J.G.; Masereeuw, R.; Bank, R.A.

    2013-01-01

    The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concernin

  11. Epithelial-to-mesenchymal transition in fibrosis : Collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition

    NARCIS (Netherlands)

    Hosper, Nynke A.; van den Berg, Paul P.; de Rond, Saskia; Popa, Eliane R.; Wilmer, Martijn J.; Masereeuw, Rosalinde; Bank, Ruud A.

    2013-01-01

    The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concernin

  12. Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding.

    Science.gov (United States)

    Mizuno, Kazunori; Boudko, Sergei; Engel, Jürgen; Bächinger, Hans Peter

    2013-06-28

    Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

  13. Degradation of collagen types I, III, IV and V by extracellular proteinases of an oral flagellate Trichomonas tenax.

    Science.gov (United States)

    Bózner, P; Demes, P

    1991-01-01

    Proteinases secreted by an axenic strain of Trichomonas tenax were active against native types I, III, IV and V collagens when evaluated by polyacrylamide gel electrophoresis. Degradation of all four collagen types was temperature dependent. Basement membrane type IV collagen was digested most effectively. An inhibition of all collagenolytic activities by a specific inhibitor of cysteine proteinases, E-64, and activation by a reducing agent, dithiothreitol, indicated the involvement of cysteine proteinases of the oral flagellate in the cleavage of collagen.

  14. DISTRIBUTION OF COLLAGENS TYPE-I, TYPE-III AND TYPE-V IN THE PANCREAS OF RAT, DOG, PIG AND MAN

    NARCIS (Netherlands)

    VANDEIJNEN, JHM; VANSUYLICHEM, PTR; WOLTERS, GHJ; VANSCHILFGAARDE, R

    1994-01-01

    The presence of collagens type I, type III and type V was determined immunohistochemically in pancreatic tissue of rat, pig, dog and man. The reaction to anti-collagen type I is weak (pig, dog) or moderate (rat, man) in the peri-insular region and in the lobar, lobular and acinar septa, whereas the

  15. Elastic and viscoelastic properties of a type I collagen fiber.

    Science.gov (United States)

    Sopakayang, Ratchada; De Vita, Raffaella; Kwansa, Albert; Freeman, Joseph W

    2012-01-21

    A new mathematical model is presented to describe the elastic and viscoelastic properties of a single collagen fiber. The model is formulated by accounting for the mechanical contribution of the collagen fiber's main constituents: the microfibrils, the interfibrillar matrix and crosslinks. The collagen fiber is modeled as a linear elastic spring, which represents the mechanical contribution of the microfibrils, and an arrangement in parallel of elastic springs and viscous dashpots, which represent the mechanical contributions of the crosslinks and interfibrillar matrix, respectively. The linear elastic spring and the arrangement in parallel of elastic springs and viscous dashpots are then connected in series. The crosslinks are assumed to gradually break under strain and, consequently, the interfibrillar is assumed to change its viscous properties. Incremental stress relaxation tests are conducted on dry collagen fibers reconstituted from rat tail tendons to determine their elastic and viscoelastic properties. The elastic and total stress-strain curves and the stress relaxation at different levels of strain collected by performing these tests are then used to estimate the parameters of the model and evaluate its predictive capabilities.

  16. Glycosylation of type II collagen is of major importance for T cell tolerance and pathology in collagen-induced arthritis

    DEFF Research Database (Denmark)

    Bäcklund, Johan; Treschow, Alexandra; Bockermann, Robert;

    2002-01-01

    Type II collagen (CII) is a candidate cartilage-specific autoantigen, which can become post-translationally modified by hydroxylation and glycosylation. T cell recognition of CII is essential for the development of murine collagen-induced arthritis (CIA) and also occurs in rheumatoid arthritis (RA......). The common denominator of murine CIA and human RA is the presentation of an immunodominant CII-derived glycosylated peptide on murine Aq and human DR4 molecules, respectively. To investigate the importance of T cell recognition of glycosylated CII in CIA development after immunization with heterologous CII......, we treated neonatal mice with different heterologous CII-peptides (non-modified, hydroxylated and galactosylated). Treatment with the galactosylated peptide (galactose at position 264) was superior in protecting mice from CIA. Protection was accompanied by a reduced antibody response to CII...

  17. Effects of immobilization and whole-body vibration on rat serum Type I collagen turnover

    Directory of Open Access Journals (Sweden)

    Gürhan Dönmez

    2016-08-01

    Conclusion: Although 1 week of WBV had a positive effect on type I collagen turnover in controls, it is not an efficient method for repairing tissue damage in the early stage following immobilization.

  18. Urinary type IV collagen as a predictor for the incidence of microalbuminuria in young patients with Type 1 diabetes.

    Science.gov (United States)

    Morita, M; Hanai, K; Uchigata, Y

    2014-02-01

    To clarify whether urinary type IV collagen-to-creatinine ratio is a predictor for the incidence of microalbuminuria in patients with Type 1 diabetes. A longitudinal observational cohort study was conducted; the subjects included normoalbuminuric patients diagnosed with Type 1 diabetes before the age of 30 years and who were less than 40 years old at the start of the observation. In total, 225 patients were enrolled (age, mean ± SD: 25 ± 5 years; male: 32.9%). The endpoint was the incidence of microalbuminuria, defined as 30 mg/g Cr ≤ urinary albumin-to-creatinine ratio type IV collagen-to-creatinine ratio levels. During the median follow-up period of 8.8 years (range 1.0-12.8 years), 13 patients with high urinary type IV collagen-to-creatinine ratio progressed to microalbuminuria. Meanwhile, only one patient with low urinary type IV collagen-to-creatinine ratio reached the endpoint. Kaplan-Meier estimates for the time to reach the endpoint were significantly faster for patients with a high ratio than for those with a low ratio (log-rank test, P type IV collagen-to-creatinine ratio was 13.51 (95% CI 1.59-115.02, P = 0.017). When urinary type IV collagen-to-creatinine ratio was treated as a continuous variable, logarithmically transformed urinary type IV collagen-to-creatinine ratio, but not baseline albumin-to-creatinine ratio, was independently associated with reaching the endpoint (hazard ratio 19.23, 95% CI 1.53-242.30, P = 0.022). Urinary type IV collagen may be an important predictor for the incidence of microalbuminuria in young patients with Type 1 diabetes. © 2013 The Authors. Diabetic Medicine © 2013 Diabetes UK.

  19. Goodpasture antigen of the glomerular basement membrane: localization to noncollagenous regions of type IV collagen.

    Science.gov (United States)

    Wieslander, J; Barr, J F; Butkowski, R J; Edwards, S J; Bygren, P; Heinegård, D; Hudson, B G

    1984-01-01

    The glomerular basement membrane antigen in Goodpasture syndrome is a collagenase-resistant molecule with a monomer molecular weight of about 26,000. Type IV collagen isolated from glomerular basement membrane contains collagenase-resistant sequences within its structure. Polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and chemical analysis were used to demonstrate that the collagenase-resistant sequences of type IV collagen contain Goodpasture antigen. Images PMID:6328527

  20. MT1-MMP and type II collagen specify skeletal stem cells and their bone and cartilage progeny

    DEFF Research Database (Denmark)

    Szabova, Ludmila; Yamada, Susan S; Wimer, Helen F

    2009-01-01

    Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic a...

  1. Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis

    National Research Council Canada - National Science Library

    Linsenmayer, T F; Gibney, E; Igoe, F; Gordon, M K; Fitch, J M; Fessler, L I; Birk, D E

    1993-01-01

    Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b...

  2. Expression of collagen type I in unaltered and osteoarthritic menisci of knee joint

    Directory of Open Access Journals (Sweden)

    Sladojević Igor

    2016-01-01

    Full Text Available Introduction. Knee osteoarthritis is a progressive degenerative disease which affects meniscal tissue. The aim of this study was to determine the differences in collagen type I expression in macroscopically unaltered and osteoarthritic menisci, and correlate the expression with the grade of macroscopic damage, age and body mass index of patients, preoperative condition of anterior cruciate ligament, angulation and knee contracture. Material and Methods. The control group consisted of 10 macroscopically unaltered menisci, while the experimental group had 35 osteoarthritic menisci. Besides macroscopic grading of meniscal damage, the analysis of collagen type I expression was determined by immunohistochemical staining with the corresponding antibody using semiquantitative scale scores and quantitative parameters: intensity of expression and stained area size. Results. The results of semiquantitative evaluation showed a statistically significant decrease in collagen type I expression in osteoarthritic menisci, which correlated with an increase in macroscopic damage grade. The results of quantitative evaluation did not show a statistically significant decrease in the expression. In posterior meniscal horns, a more intense collagen type I expression was seen in the women, as well as a positive correlation of quantitatively evaluated expression with body mass index. Collagen type I expression in the anterior horns was significantly lower in varus alignment. Conclusion. In the semiquantitative evaluation, collagen type I expression in osteoarthritic menisci was significantly lower compared to macroscopically unchanged menisci. The decrease in the expression level correlates with the increase in the grade of macroscopic meniscal damage. There was no statistically significant difference in the quantitative evaluation of expression.

  3. Fluoride alters type I collagen expression in the early stages of odontogenesis.

    Science.gov (United States)

    Maciejewska, I; Spodnik, J H; Domaradzka-Pytel, B; Sidor-Kaczmarek, J; Bereznowski, Z

    2006-11-01

    Fluoride alters the expression and post-translational modifications of extracellular matrix proteins in dentin. The aim of our study was to determine the effects of fluoride on type I collagen expression during the early stages of tooth germ development in rats. Pregnant dams were divided into three groups and fed a standard diet. From the fifth day of pregnancy the three groups received tap water with, respectively, trace amounts of fluoride (C), a low fluoride concentration (FL) or and a high fluoride concentration (FH). Changes in type I collagen expression and distribution were evaluated. The expression of type I collagen was restricted to the extracellular spaces of cells of mesenchymal origin. In the youngest animals the most intense immunoreactivity for type I collagen was detected in predentin of the FL group. Although the intensity of immunostaining increased in proportion to the age of the animals, the largest increase in the groups investigated was detected in the FL group. We concluded that a low concentration of fluoride can act as a stimulator of type I collagen deposition in the extracellular matrix of dentin, while high concentrations of fluoride have an opposite effect, acting as an inhibitor of type I collagen formation in dentin.

  4. Effects of collagen types II and X on the kinetics of crystallization of calcium phosphate in biomineralization

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The effects of the components of cartilages matrix on the process of endochondral ossification and the kinetics of crystal growth of calcium phosphate have been studied in the presence of type II or X collagen. During the experiments, type I collagen was added as the seed material. FT-IR analysis shows that calcium phosphate crystallized on the surface of type I collagen was mainly hydroxyapatite. Both type II and X collagens could reduce the growth rate of calcium phosphate crystals, and the effect of type X collagen is more obvious. The reaction was in the fourth order in the presence of type II collagen. The results showed that type II or X collagen had the ability to make Ca2+ accumulate in the process of endochondral ossification, but has little effect on crystal growth and the product of biomineralization.

  5. Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen.

    Science.gov (United States)

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2016-01-01

    3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro(707) and newly identified sites at Pro(716) and Pro(719), at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

  6. MT1-MMP and type II collagen specify skeletal stem cells and their bone and cartilage progeny

    DEFF Research Database (Denmark)

    Szabova, L.; Yamada, S.S.; Wimer, H.;

    2009-01-01

    Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic...... activity associated with the membrane-type 1 matrix metalloproteinase (MT1-MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1-MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1-MMP activity in the type II collagen...

  7. Design of net-charged abc-type collagen heterotrimers

    Science.gov (United States)

    Parmar, Avanish S.; Zahid, Sohail; Belure, Sandeep V.; Young, Robert; Hasan, Nida; Nanda, Vikas

    2013-01-01

    Net-negatively-charged heterospecific A:B:C collagen peptide heterotrimers were designed using an automated computational approach. The design algorithm considers both target stability and the energy gap between the target states and misfolded competing states. Structural characterization indicates the net-negative charge balance on the new designs enhances the specificity of the target state at the expense of its stability. PMID:23603270

  8. Are there autoantibodies reacting against citrullinated peptides derived from type I and type II collagens in patients with rheumatoid arthritis?

    Science.gov (United States)

    Koivula, M; Aman, S; Karjalainen, A; Hakala, M; Risteli, J

    2005-01-01

    Objectives: To assess the possible presence in patients with rheumatoid arthritis (RA) of autoantibodies recognising citrullinated peptides derived from type I and II collagens. Methods: Firstly, the binding of four pairs of synthetic peptides (arginine-containing and artificially citrullinated forms) related to different regions of human type II collagen were tested with sera from 120 patients with RA and 81 controls. Secondly, two similar pairs of peptides related to the carboxy terminal telopeptides of the α1 and α2 chains of human type I collagen were tested. Results: 42–53% of the RA sera showed increased binding of arginine peptides related to type II collagen. However, 12 RA sera bound the citrullinated form of the α1(II) telopeptide more strongly than the corresponding arginine peptide. 20 RA sera bound the citrullinated carboxytelopeptide from the α1 chain of type I collagen (α1(I) telopeptide) more strongly than the respective arginine peptide. The correlation between the autoantibodies to type I and II collagen telopeptides was rs = 0.576, pAnti-cyclic citrullinated peptide (anti-CCP) assay was positive in 71/120 (59%) patients with RA. An anti-CCP assay detects a different subgroup of antibodies than anti-telopeptide assays. However, both anti-telopeptide and anti-CCP antibodies were increased in patients with RA. Conclusion: Some patients with RA were identified whose sera contained antibodies that specifically bound citrullinated peptides related to the carboxy terminal telopeptides of the α1 and α2 chains of type I collagen and the α1 chains of type II collagen (sequences YYXA, FYXA, and YMXA, where X stands for citrulline). PMID:16162901

  9. Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans

    DEFF Research Database (Denmark)

    Langberg, Henning; Skovgaard, D; Petersen, L J;

    1999-01-01

    in the peritendinous region by measuring interstitial concentrations of a collagen propeptide (PICP; 100 kDa) and a collagen degradation product (ICTP; 9 kDa) as well as an inflammatory mediator (PGE2). 2. Seven trained human runners were studied before and after (2 and 72 h) 3 h of running (36 km). Two microdialysis...... increased in blood during running, and returned to baseline in the recovery period, whereas interstitial PGE2 concentration was elevated in the early recovery phase. 4. The findings of the present study indicate that acute exercise induces increased formation of type I collagen in peritendinous tissue...

  10. Biocompatibility of Novel Type I Collagen Purified from Tilapia Fish Scale: An In Vitro Comparative Study

    Directory of Open Access Journals (Sweden)

    Jia Tang

    2015-01-01

    Full Text Available Type I collagen (COL-1 is the prevailing component of the extracellular matrix in a number of tissues including skin, ligament, cartilage, bone, and dentin. It is the most widely used tissue-derived natural polymer. Currently, mammalian animals, including pig, cow, and rat, are the three major sources for purification of COL-1. To reduce the risk of zoonotic infectious diseases transmission, minimize the possibility of immunogenic reaction, and avoid problems related to religious issues, exploration of new sources (other than mammalian animals for the purification of type I collagen is highly desirable. Hence, the purpose of the current study was to investigate the in vitro responses of MDPC-23 to type I collagen isolated from tilapia scale in terms of cellular proliferation, differentiation, and mineralization. The results suggested that tilapia scale collagen exhibited comparable biocompatibility to porcine skin collagen, indicating it might be a potential alternative to type I collagen from mammals in the application for tissue regeneration in oral-maxillofacial area.

  11. Decreased type III collagen expression in human uterine cervix of prolapse uteri

    Science.gov (United States)

    IWAHASHI, MASAAKI; MURAGAKI, YASUTERU

    2011-01-01

    The precise mechanism of prolapse uteri is not fully understood. There is evidence to suggest that abnormalities of collagen, the main component of extracellular matrix, or its repair mechanism, may predispose women to prolapse. To investigate the characteristic structure of human uterine cervix of patients with prolapse uteri, various types of collagen expression in the uterine cervix tissues of the prolapse uteri were compared to those of normal uterine cervix. After informed consent, 36 specimens of uterine cervical tissues were obtained at the time of surgery from 16 postmenopausal women with prolapse uteri (stage III–IV by the Pelvic Organ Prolapse Quantification examination) and 20 postmenopausal women without prolapse uteri (control group). Collagens were extracted from the uterine cervix tissues by salt precipitation methods. The relative levels of various collagens were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The uterine cervix was longer in the patients with prolapse uteri than those of postmenopausal controls without prolapse uteri. The ratios of type III to type I collagen in the uterine cervical tissues were significantly decreased in the prolapse uteri, as compared to those of the postmenopausal uterine cervix without prolapse. These results suggest that decreased type III collagen expression may play an important role in determing the physiology and structure of the uterine cervix tissues of prolapse uteri. PMID:22977496

  12. Determination of three-dimensional molecular orientation of type-I collagen by circularly-polarized second harmonic generation imaging

    Science.gov (United States)

    Zhuo, Guan-Yu; Hung, Wei-Han; Kao, Fu-Jen

    2017-04-01

    The content of collagen is up to 30% existing in mammals. It supports the main component of connective tissues such as skin, ligament, and cartilage. Among various types of collagen, type-I collagen is of the most abundance and has been broadly studied due to the importance in bioscience. Second harmonic generation (SHG) microscopy is an effective tool used to study the collagen organization without labeling. In this study, we used circular polarization instead of linear polarization to retrieve three-dimensional (3D) molecular orientation of type-I collagen with only two cross polarized SHG images without acquiring an image stack of varying polarization.

  13. Stimulation of type I collagen activity in healing of pulp perforation

    Directory of Open Access Journals (Sweden)

    Sri Kunarti

    2008-12-01

    Full Text Available Background: TGF-β1 is a connective tissue stimulant, potential regulator for tissue repair, and promoter in wound healing. The healing of pulp perforation is decided by quantity and quality of new collagen deposition. TGF-β1 upregulates collagen transcription. However, after several weeks production of type I collagen synthesis is stopped and enzymatic degradation of collagen matrix will occur. Purpose: Observe synthesis type I collagen during the process of pulp perforation healing in 7, 14, and 21 days after being treated using TGF-β1. Methods: This research in vivo from orthodontic patients indicated for premolar extraction, between ages 10–15 years. A class V cavity preparation was created in the buccal aspect 1 mm above gingival margin until pulp exposure. Cavity was irrigated slowly with saline solution and dried with a sterile small cotton pellet. The sterile absorbable collagen membrane used, soaked with 5 μl of TGF-β1. It was covered by teflon pledge to separate from glass ionomer cement as restoration. Evaluated on day 7th, 14th, and 21st. All samples were extracted and prepared for immunohystochemical examination. Result: Data were analyzed by t-test. There was significant difference in synthesis type I collagen compared between 7–14 days and 7–21 days after treatment by TGF-β1 also Ca(OH2. There was significant difference compared between TGF-β1 and Ca(OH2 in 14–21 days after treatment (means TGF-β1 > Ca(OH2. Conclusion: Elevation of synthesis type 1 collagen by TGF-β1 in 20 mg/ml.

  14. Distribution of basement membrane type IV collagen alpha chains in ameloblastoma: an immunofluorescence study

    OpenAIRE

    Nakano, K.; Siar, C. H.; Nagai, N.; Naito, I.; Sado, Y.; Nagatsuka, H; Hoh, C; Kurada, K.; Tsujigiwa, H; M. Gunduz

    2002-01-01

    Background: Type IV collagen, a heterotrimeric molecule that exists in six genetically distinct forms, alpha1(IV)-alpha6(IV) is a major structural component of basement membrane (BM) and acts as a scaffold for other BM constituents. Methods: Indirect immunofluorescence using alpha chain-specific monoclonal antibodies was employed to clarify basement membrane (BM) collagen IV distribution in two ameloblastoma, and for comparison, on oral mucosa and tooth germ. Results: Ameloblastoma BM express...

  15. The NC2 domain of type IX collagen determines the chain register of the triple helix.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Type 1 collagen as a potential niche component for CD133-positive glioblastoma cells.

    Science.gov (United States)

    Motegi, Hiroaki; Kamoshima, Yuuta; Terasaka, Shunsuke; Kobayashi, Hiroyuki; Houkin, Kiyohiro

    2014-08-01

    Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133-positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum-containing medium (SCM) or serum-free medium with some growth factors (SFM) and in non-coated (Non-coat) or type 1 collagen-coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non-coat/SCM. Semi-quantitative measurement of CD133-positive cells by immunocytochemistry showed a statistically significant increase of CD133-positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133-positive GSCs and maintain GSCs in adherent culture.

  18. Identification of Type VI Collagen Synthesizing Cells in Human Diabetic Glomerulosclerosis Using Renal Biopsy Sections

    Directory of Open Access Journals (Sweden)

    Mohammed Shawkat Razzaque

    1997-01-01

    Full Text Available Although the role of extracellular matrices in the development of glomerulosclerosis has been discussed widely, the cellular origin of type VI collagen in diabetic nephropathy (DN has remained relatively unexplored. This study reports the distribution and cellular origin of type VI collagen in DN. Type VI collagen‐specific oligonucleotide probes and monoclonal antibody were used to assess the relative expression of mRNA for \\alpha1 (VI chain and its translated protein in paraffin‐embedded renal biopsy sections of DN. By immunohistochemistry, compared to the control, increased deposition of type VI collagen was noted in the diffuse and nodular lesions of diabetic glomeruli. For cellular localization of type VI collagen mRNA, paraffin‐embedded renal sections of the control and DN were hybridized in situ with digoxigenin (Dig‐labeled antisense oligo‐DNA probe complementary to a part of \\alpha1 (VI mRNA. In comparison to the control kidney sections, increased numbers of intraglomerular cells (both mesangial and epithelial cells were positive for α1 (VI mRNA in renal biopsy sections of DN. From the results, we conclude that overexpression of type VI collagen by intraglomerular cells with its increased deposition might significantly contribute to the glomerulosclerosis found in DN.

  19. Methacrylation induces rapid, temperature-dependent, reversible self-assembly of type-I collagen.

    Science.gov (United States)

    Drzewiecki, Kathryn E; Parmar, Avanish S; Gaudet, Ian D; Branch, Jonathan R; Pike, Douglas H; Nanda, Vikas; Shreiber, David I

    2014-09-23

    Type-I collagen self-assembles into a fibrillar gel at physiological temperature and pH to provide a cell-adhesive, supportive, structural network. As such, it is an attractive, popular scaffold for in vitro evaluations of cellular behavior and for tissue engineering applications. In this study, type-I collagen is modified to introduce methacrylate groups on the free amines of the lysine residues to create collagen methacrylamide (CMA). CMA retains the properties of collagen such as self-assembly, biodegradability, and natural bioactivity but is also photoactive and can be rapidly cross-linked or functionalized with acrylated molecules when irradiated with ultraviolet light in the presence of a photoinitiator. CMA also demonstrates unique temperature-dependent behavior. For natural type-I collagen, the overall structure of the fiber network remains largely static over time scales of a few hours upon heating and cooling at temperatures below its denaturation point. CMA, however, is rapidly thermoreversible and will oscillate between a liquid macromer suspension and a semisolid fibrillar hydrogel when the temperature is modulated between 10 and 37 °C. Using a series of mechanical, scattering, and spectroscopic methods, we demonstrate that structural reversibility is manifest across multiple scales from the protein topology of the triple helix up through the rheological properties of the CMA hydrogel. Electron microscopy imaging of CMA after various stages of heating and cooling shows that the canonical collagen-like D-periodic banding ultrastructure of the fibers is preserved. A rapidly thermoreversible collagen-based hydrogel is expected to have wide utility in tissue engineering and drug delivery applications as a biofunctional, biocompatible material. Thermal reversibility also makes CMA a powerful model for studying the complex process of hierarchical collagen self-assembly.

  20. Effects of oral administration of tripeptides derived from type I collagen (collagen tripeptide) on atherosclerosis development in hypercholesterolemic rabbits.

    Science.gov (United States)

    Tang, Lihua; Sakai, Yasuo; Ueda, Yoshimichi; Katsuda, Shogo

    2015-05-01

    Digestion of type I collagen with a collagenase-type protease yields a collagen tripeptide (Ctp) fraction comprising Gly-X-Y sequences that exhibit diverse biological activities. We previously demonstrated that Ctp inhibits the proliferation and migration of cultured aortic smooth muscle cells (SMCs) in vitro. These cells contribute to the pathogenesis of atherosclerosis and other cardiovascular diseases. In order to evaluate the effects of Ctp on atherosclerosis development in vivo, here we used the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit model of familial hypercholesterolemia to determine the effects of oral administration of Ctp for three months. Ctp induced a significant decrease in the area occupied by atherosclerotic plaques in the aorta and in the level of total serum cholesterol. The components of atherosclerotic plaques underwent distinct changes, including reduction in the populations of macrophages and SMCs and a significant decrease in the proportion of macrophages to SMCs. Ctp administration decreased the number of cells in plaques that expressed proliferating cell nuclear antigen and the number of cells with oxidative damage to DNA as indicated by 8-hydroxy-2'-deoxyguanine detection. These findings are the first to define the mechanism underlying the inhibitory effects of Ctp on atherosclerosis development in hypercholesterolemic rabbits, and suggest that Ctp provides an effective therapy for treating atherosclerosis.

  1. Angiogenesis and collagen type IV expression in different endothelial cell culture systems.

    Science.gov (United States)

    Bahramsoltani, M; Slosarek, I; De Spiegelaere, W; Plendl, J

    2014-04-01

    In vitro angiogenesis assays constitute an important tool for studying the mechanisms of angiogenesis and for identification of pro- and anti-angiogenic substances. Therefore, endothelial cell and media systems used for in vitro angiogenesis assays are required to mimic the angiogenic process in vivo including endothelial capability to express collagen type IV as a component of the basement membrane. In this study, the expression of collagen type IV and its α chains (α1-6) was investigated in different endothelial cell culture systems in vitro qualitatively and quantitatively. These systems included four different batches of microvascular endothelial cells derived from the human skin, heart and lung, from which only two batches were found to be angiogenic and two batches were classified as non-angiogenic. Distribution of the transcripts of the α chains of collagen type IV was similar in all cell and media systems investigated. However, secretion and deposition of a stable extracellular network of collagen type IV could only be observed in the angiogenic cultures. In conclusion, the consecutive steps of the angiogenic cascade in vivo as well as in vitro depend on an increasing secretion and subsequent extracellular deposition of collagen type IV.

  2. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.

    Science.gov (United States)

    Rajan, Navneeta; Habermehl, Jason; Coté, Marie-France; Doillon, Charles J; Mantovani, Diego

    2006-01-01

    Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at -20 degrees C and lyophilized to obtain a sponge, which could be stored at -80 degrees C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2-3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.

  3. Induction of type XVI collagen expression facilitates proliferation of oral cancer cells.

    Science.gov (United States)

    Ratzinger, Sabine; Grässel, Susanne; Dowejko, Albert; Reichert, Torsten E; Bauer, Richard J

    2011-03-01

    Type XVI collagen belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). Recently, high affinity to integrin alpha1beta1 has been shown allowing cells expressing those integrins to attach and spread on recombinant type XVI collagen. Here, we show that type XVI collagen is overexpressed in dysplastic areas of mucosal epithelium from oral squamous cell carcinoma (OSCC) patients. Induction of its expression in OSCC cell lines (COLXVI cells) leads to an increased expression of Kindlin-1. Moreover, we demonstrate a significantly increased Kindlin-1/beta1-integrin interaction. Additionally, we detected a higher number of activated beta1-integrins in COLXVI cells and found a neo-expression of alpha1 integrin subunit on these cells. FACS analysis revealed a significantly higher amount of COLXVI cells in S-phase and G2/M-phase 6h after synchronisation leading to a markedly higher proliferation activity. Blocking beta1-integrins with a specific antibody resulted in reduced proliferation of COLXVI cells. In summary, we demonstrate that overexpression of type XVI collagen in aberrant oral keratinocytes leads to Kindlin-1 induction, increased Kindlin-1/beta1-integrin interaction, integrin activation and subsequently to a proliferative cellular phenotype.

  4. Multiphoton crosslinking for biocompatible 3D printing of type I collagen.

    Science.gov (United States)

    Bell, Alex; Kofron, Matthew; Nistor, Vasile

    2015-09-03

    Multiphoton fabrication is a powerful technique for three-dimensional (3D) printing of structures at the microscale. Many polymers and proteins have been successfully structured and patterned using this method. Type I collagen comprises a large part of the extracellular matrix for most tissue types and is a widely used cellular scaffold material for tissue engineering. Current methods for creating collagen tissue scaffolds do not allow control of local geometry on a cellular scale. This means the environment experienced by cells may be made up of the native material but unrelated to native cellular-scale structure. In this study, we present a novel method to allow multiphoton crosslinking of type I collagen with flavin mononucleotide photosensitizer. The method detailed allows full 3D printing of crosslinked structures made from unmodified type I collagen and uses only demonstrated biocompatible materials. Resolution of 1 μm for both standing lines and high-aspect ratio gaps between structures is demonstrated and complex 3D structures are fabricated. This study demonstrates a means for 3D printing with one of the most widely used tissue scaffold materials. High-resolution, 3D control of the fabrication of collagen scaffolds will facilitate higher fidelity recreation of the native extracellular environment for engineered tissues.

  5. The Role of Type IV Collagen in Developing Lens in Mouse Fetuses

    Directory of Open Access Journals (Sweden)

    Mehdi Jalali

    2009-09-01

    Full Text Available Objective(sExtracellular matrix (ECM and basement membrane (BM play important roles in many developmental processes during development and after birth. Among the components of the BM, collagen fibers specially type IV are the most important parts. The aim of this study was to determine the time when collagen type IV appears in the BM of lens structure during mouse embryonic development.Materials and MethodsIn this experimental study, 22 female Balb/C mice were randomly selected and were kept under normal condition, finding vaginal plug was assumed as day zero of pregnancy. From embryonic day 10 to 20, all specimens were sacrificed by cervical dislocation and their heads were fixed, serially sectioned and immunohistochemistry study for tracing collagen type IV in lens were carried out.ResultsOur data revealed that collagen type IV appeared at the early stage of gestation day 12 in BM of anterior epithelial lens cells and the amount of this protein gradually increased until days 15-17 in ECM and posterior capsule epithelium. After this period, severe reaction was not observed in any part of the lens.ConclusionThese findings establish the important role of collagen IV in developing optic cup and any changes during critical period of pregnancy may be result in severe visual system defect

  6. [Study on the acid hydrolysis, fiber remodeling and bionics mineralization of rat tail tendon collagen type Ⅰ].

    Science.gov (United States)

    Zhang, Zhan; Zhang, Chun; Guo, Qiaofeng

    2016-05-25

    Objective: To produce bionic bone material that is consistent with human bone in chemical composition and molecular structure using rat tail tendon collagen type Ⅰ. Methods: The typecollagen derived from rat tail was extracted by acetic acid to form collagen fibers. The reconstructed collagen fibers were placed in the mineralized solution to mimic bone mineralization for 2-6 days. Bone mineralization was observed by transmission electron microscopy and electron diffraction.Results: Collagen fibers with characteristic D-Band structure were reconstructed by using rat tail tendon collagen type Ⅰ extracted with acid hydrolysis method. Transmission electron microscopy and electron diffraction showed that calcium hydroxyapatite precursor infiltrated into the collagen fibers, and the collagen fibers were partially mineralized after 2 days of mineralization; the collagen fibers were completely mineralized and bionic bone material of typeⅠ collagen/calcium hydroxyapatite was formed after 6 days of mineralization.Conclusion: The collagen type Ⅰ can be extracted from rat tail tendon by acid hydrolysis method, and can be reformed and mineralized to form the bionic bone material which mimics human bone in chemical composition and the molecular structure.

  7. Autoantibodies to Multiple Epitopes on the Non-Collagenous-1 Domain of Type VII Collagen Induce Blisters.

    Science.gov (United States)

    Vorobyev, Artem; Ujiie, Hideyuki; Recke, Andreas; Buijsrogge, Jacqueline J A; Jonkman, Marcel F; Pas, Hendri H; Iwata, Hiroaki; Hashimoto, Takashi; Kim, Soo-Chan; Hoon Kim, Jong; Groves, Richard; Samavedam, Unni; Gupta, Yask; Schmidt, Enno; Zillikens, Detlef; Shimizu, Hiroshi; Ludwig, Ralf J

    2015-06-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease of the skin and mucous membranes, characterized by autoantibodies against type VII collagen (COL7), a major component of anchoring fibrils. Different clinical EBA phenotypes are described, including mechanobullous and inflammatory variants. Most EBA patients' sera react with epitopes located within the non-collagenous 1 (NC1) domain of human COL7. However, it has remained unclear whether antibody binding to these different epitopes is pathogenically relevant. To address this issue, we generated recombinant proteins covering the entire NC1 domain. IgG reactivity with these proteins was analyzed in sera of 69 EBA patients. Most recognized clusters of epitopes throughout the NC1 domain. No correlation was detected between antibody specificity and clinical phenotype. To study the pathogenicity of antibodies specific to different NC1 subdomains, rabbit antibodies were generated. All these antibodies caused dermal-epidermal separation ex vivo. Antibodies against two of these subdomains were injected into mice carrying null mutations of mouse COL7 and the human COL7 transgene and induced subepidermal blisters. We here document that autoantibodies to COL7, independent of the targeted epitopes, induce blisters both ex vivo and in vivo. In addition, using COL7-humanized mice, we provide in vivo evidence of pathogenicity of autoantibodies binding to human COL7.

  8. Type II Collagen and Gelatin from Silvertip Shark (Carcharhinus albimarginatus) Cartilage: Isolation, Purification, Physicochemical and Antioxidant Properties

    OpenAIRE

    Elango Jeevithan; Bin Bao; Yongshi Bu; Yu Zhou; Qingbo Zhao; Wenhui Wu

    2014-01-01

    Type II acid soluble collagen (CIIA), pepsin soluble collagen (CIIP) and type II gelatin (GII) were isolated from silvertip shark (Carcharhinus albimarginatus) cartilage and examined for their physicochemical and antioxidant properties. GII had a higher hydroxyproline content (173 mg/g) than the collagens and cartilage. CIIA, CIIP and GII were composed of two identical α1 and β chains and were characterized as type II. Amino acid analysis of CIIA, CIIP and GII indicated imino acid contents of...

  9. Surface modification of nanofibrous polycaprolactone/gelatin composite scaffold by collagen type I grafting for skin tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Gautam, Sneh [Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee (India); Chou, Chia-Fu [Institute of Physics, Academia Sinica, Taipei, Taiwan (China); Dinda, Amit K. [Department of Pathology, All India Institute of Medical Science, New Delhi (India); Potdar, Pravin D. [Department of Molecular Medicine and Biology, Jaslok Hospital and Research Centre, Mumbai (India); Mishra, Narayan C., E-mail: mishrawise@gmail.com [Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee (India)

    2014-01-01

    In the present study, a tri-polymer polycaprolactone (PCL)/gelatin/collagen type I composite nanofibrous scaffold has been fabricated by electrospinning for skin tissue engineering and wound healing applications. Firstly, PCL/gelatin nanofibrous scaffold was fabricated by electrospinning using a low cost solvent mixture [chloroform/methanol for PCL and acetic acid (80% v/v) for gelatin], and then the nanofibrous PCL/gelatin scaffold was modified by collagen type I (0.2–1.5 wt.%) grafting. Morphology of the collagen type I-modified PCL/gelatin composite scaffold that was analyzed by field emission scanning electron microscopy (FE-SEM), showed that the fiber diameter was increased and pore size was decreased by increasing the concentration of collagen type I. Fourier transform infrared (FT-IR) spectroscopy and thermogravimetric (TG) analysis indicated the surface modification of PCL/gelatin scaffold by collagen type I immobilization on the surface of the scaffold. MTT assay demonstrated the viability and high proliferation rate of L929 mouse fibroblast cells on the collagen type I-modified composite scaffold. FE-SEM analysis of cell-scaffold construct illustrated the cell adhesion of L929 mouse fibroblasts on the surface of scaffold. Characteristic cell morphology of L929 was also observed on the nanofiber mesh of the collagen type I-modified scaffold. Above results suggest that the collagen type I-modified PCL/gelatin scaffold was successful in maintaining characteristic shape of fibroblasts, besides good cell proliferation. Therefore, the fibroblast seeded PCL/gelatin/collagen type I composite nanofibrous scaffold might be a potential candidate for wound healing and skin tissue engineering applications. - Highlights: • PCL/gelatin/collagen type I scaffold was fabricated for skin tissue engineering. • PCL/gelatin/collagen type I scaffold showed higher fibroblast growth than PCL/gelatin one. • PCL/gelatin/collagen type I might be one of the ideal scaffold for

  10. Role of TGF-beta1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue

    DEFF Research Database (Denmark)

    Heinemeier, Katja; Langberg, Henning; Olesen, Jens L

    2003-01-01

    Mechanical loading of tissue is known to influence local collagen synthesis, and microdialysis studies indicate that mechanical loading of human tendon during exercise elevates tendinous type I collagen production. Transforming growth factor-beta1 (TGF-beta1), a potent stimulator of type I collag...... to exercise, suggest a role for TGF-beta1 in mechanical regulation of local collagen type I synthesis in tendon-related connective tissue in vivo....

  11. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Bai, Jian; Zhang, Na; Hua, Ying; Wang, Bingjian; Ling, Lin; Ferro, Albert; Xu, Biao

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence or absence of metformin (10-200 µM). Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA) and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS) generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  12. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Jian Bai

    Full Text Available Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM in the presence or absence of metformin (10-200 µM. Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  13. The Prognostic and Predictive Value of Soluble Type IV Collagen in Colorectal Cancer

    DEFF Research Database (Denmark)

    Rolff, Hans Christian; Christensen, Ib Jarle; Vainer, Ben

    2016-01-01

    PURPOSE: To investigate the prognostic and predictive biomarker value of type IV collagen in colorectal cancer. EXPERIMENTAL DESIGN: Retrospective evaluation of two independent cohorts of patients with colorectal cancer included prospectively in 2004-2005 (training set) and 2006-2008 (validation...... set). Plasma samples were available from 297 (training set) and 482 (validation set) patients. Type IV collagen determinations were performed using an ELISA. From the training set, 222 tumors were available for IHC. Clinical and follow-up data were retrieved from patient files and national registries....... RESULTS: High levels of type IV collagen showed independent prognostic significance in both cohorts with hazard ratios (HRs; for a one-unit change on the log base 2 scale) of 2.25 [95% confidence intervals (CIs), 1.78-2.84; P

  14. Thyroid follicular adenoma with accumulation of collagen type IV in a common marmoset (Callithrix jacchus).

    Science.gov (United States)

    Kawasako, K; Doi, T; Kanno, T; Wako, Y; Hamamura, M; Tsuchitani, M

    2014-01-01

    A thyroid tumour was identified in a 10-year-old male common marmoset (Callithrix jacchus). The tumour was encapsulated by fibrous connective tissue and compressed the adjacent normal thyroid. The tumour was composed of variably sized and irregularly shaped thyroid follicles lined by a single layer of columnar epithelial cells. Eosinophilic material at the base of the neoplastic cells stained black with periodic acid-methenamine silver and red with periodic acid-Schiff. Immunohistochemistry confirmed that this eosinophilic material was collagen type IV. Ultrastructurally, highly dense and amorphous material was observed at the base of the neoplastic cells. Small vesicles in the basolateral cytoplasm of the neoplastic cells contained similar material to that at the base of the cells. The tumour was diagnosed as a thyroid follicular adenoma with accumulation of collagen type IV. This is the first description of an endocrine tumour with accumulation of collagen type IV in animals.

  15. MT1-MMP and type II collagen specify skeletal stem cells and their bone and cartilage progeny

    DEFF Research Database (Denmark)

    Szabova, L.; Yamada, S.S.; Wimer, H.

    2009-01-01

    Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic...... activity associated with the membrane-type 1 matrix metalloproteinase (MT1-MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1-MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1-MMP activity in the type II collagen......-expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1-MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified...

  16. Smoking is associated with lower amounts of arterial type I collagen and decorin

    DEFF Research Database (Denmark)

    Faarvang, Anne-Sofie; Rørdam Preil, Simone; Switten Nielsen, Patricia

    2016-01-01

    of collagen stainable material was determined. In addition, proteome analysis of matrix molecules and other proteins was performed. RESULTS: The area fraction of collagen stainable material in smokers vs. never-smokers was 29.1% ± 3.8% vs. 43.3% ± 3.6% (mean ± SEM, p = 0.012) in tunica intima, 39.7% ± 5.5% vs....... 56.8% ± 5.6% (mean ± SEM, p = 0.042) in tunica media, and 50.4% ± 3.9% vs. 61.0% ± 3.2% (mean ± SEM, p = 0.046) in tunica adventitia. We discovered significantly lower relative levels of collagen α1(I) (0.68 ± 0.048 vs. 1.02 ± 0.112, mean ± SEM, p = 0.013), collagen α2(I) (0.81 ± 0.046 vs. 1.14 ± 0.......118, mean ± SEM, p = 0.038) and decorin (0.64 ± 0.04 vs. 0.98 ± 0.11, mean ± SEM, p = 0.009) in smokers. CONCLUSIONS: Arterial tissue from active smokers contains decreased amounts of collagen stainable material, as well as type 1 collagen and decorin. These findings may explain some effects of smoking...

  17. Changes in histoanatomical distribution of types I, III and V collagen promote adaptative remodeling in posterior tibial tendon rupture

    Directory of Open Access Journals (Sweden)

    Érika Satomi

    2008-01-01

    Full Text Available INTRODUCTION: Posterior tibial tendon dysfunction is a common cause of adult flat foot deformity, and its etiology is unknown. PURPOSE: In this study, we characterized the morphologic pattern and distribution of types I, III and V collagen in posterior tibial tendon dysfunction. METHOD: Tendon samples from patients with and without posterior tibial tendon dysfunction were stained by immunofluorescence using antibodies against types I, III and V collagen. RESULTS: Control samples showed that type V deposited near the vessels only, while surgically obtained specimens displayed type V collagen surrounding other types of collagen fibers in thicker adventitial layers. Type III collagen levels were also increased in pathological specimens. On the other hand, amounts of collagen type I, which represents 95% of the total collagen amount in normal tendon, were decreased in pathological specimens. CONCLUSION: Fibrillogenesis in posterior tibial tendon dysfunction is altered due to higher expression of types III and V collagen and a decreased amount of collagen type I, which renders the originating fibrils structurally less resistant to mechanical forces.

  18. The alpha 3 chain of type IV collagen induces autoimmune Goodpasture syndrome.

    Science.gov (United States)

    Kalluri, R; Gattone, V H; Noelken, M E; Hudson, B G

    1994-06-21

    Human Goodpasture syndrome is a lethal form of autoimmune disease that is characterized by pulmonary hemorrhage and glomerulonephritis. The tissue injury is mediated by autoantibodies that bind to glomerular and alveolar basement membrane. The target autoantigen is alpha 3(IV) collagen, one of six genetically distinct chains that comprise type IV collagen, and the epitope is sublocalized to the noncollagenous domain (NC1) of the alpha 3 chain. The present study reports the unique capacity of alpha 3(IV)NC1 dimer from bovine kidney to aberrantly engage the immune system of rabbits to respond to self, mimicking the organ-specific form of the human disease, whereas the other chains of type IV collagen are nonpathogenic. However, alpha 3(IV)NC1 hexamer was nonpathogenic, suggesting the exposure of a pathogenic epitope upon dissociation of hexamer into dimers. Exposure of the pathogenic epitope by infection or organic solvents, events which are thought to precede Goodpasture syndrome, may be the principal factor in the etiology of the disease. The pathogenicity of alpha 3(IV) collagen brings full circle a decade of research that has identified four novel chains (alpha 3-alpha 6) of type IV collagen.

  19. Chemical functionalization and stabilization of type I collagen with organic tanning agents

    Energy Technology Data Exchange (ETDEWEB)

    Albu, Madalina Georgiana; Deselnicu, Viorica; Ioannidis, Ioannis; Deselnicu, Dana; Chelaru, Ciprian [Leather and Footwear Research Institute, Bucharest (Romania)

    2015-02-15

    We investigated the interactions between selected organic tanning agents and type I fibrillar collagen as a model fibrillar substrate to enable the fast direct evaluation and validation of interpretations of tanning activity. Type I fibrillar collagen (1%) as gel was used as substrate of tanning and tannic acid, resorcinol- and melamine-formaldehyde and their combination at three concentrations as crosslinking agents (tannins). To evaluate the stability of collagen during tanning, the crosslinked gels at 2.8, 4.5 and 9.0 pHs were freeze-dried as discs which were characterized by FTIR, shrinkage temperature, enzymatic degradation and optical microscopy, and the results were validated by statistical analyses. The best stability was given by combinations between resorcinol- and melamine-formaldehyde at isoelectric pH.

  20. Effects of Oral Administration of Type II Collagen on Rheumatoid Arthritis

    Science.gov (United States)

    Trentham, David E.; Dynesius-Trentham, Roselynn A.; Orav, E. John; Combitchi, Daniel; Lorenzo, Carlos; Sewell, Kathryn Lea; Hafler, David A.; Weiner, Howard L.

    1993-09-01

    Rheumatoid arthritis is an inflammatory synovial disease thought to involve T cells reacting to an antigen within the joint. Type II collagen is the major protein in articular cartilage and is a potential autoantigen in this disease. Oral tolerization to autoantigens suppresses animal models of T cell-mediated autoimmune disease, including two models of rheumatoid arthritis. In this randomized, double-blind trial involving 60 patients with severe, active rheumatoid arthritis, a decrease in the number of swollen joints and tender joints occurred in subjects fed chicken type II collagen for 3 months but not in those that received a placebo. Four patients in the collagen group had complete remission of the disease. No side effects were evident. These data demonstrate clinical efficacy of an oral tolerization approach for rheumatoid arthritis.

  1. Effects of Type I Collagen Degradation on the Durability of Three Adhesive Systems in the Early Phase of Dentin Bonding

    OpenAIRE

    Lin Hu; Yu-hong Xiao; Ming Fang; Yu Gao; Li Huang; An-qi Jia; Ji-hua Chen

    2015-01-01

    Objective This study was designed to evaluate the effects of type I collagen degradation on the durability of three adhesive systems in the early phase of dentin bonding. Methods Bonded dentin specimens were prepared using three different types of adhesive systems. Micro-tensile bond strength and degradation of collagen were tested before, and after 1 month or 4 months of aging in artificial saliva. The relationship between micro-tensile bond strength and collagen degradation was analyzed by ...

  2. Tooth agenesis in osteogenesis imperfecta related to mutations in the collagen type I genes.

    Science.gov (United States)

    Malmgren, B; Andersson, K; Lindahl, K; Kindmark, A; Grigelioniene, G; Zachariadis, V; Dahllöf, G; Åström, E

    2017-01-01

    Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, mainly caused by mutations in the collagen type I genes (COL1A1 and COL1A2). Tooth agenesis is a common feature of OI. We investigated the association between tooth agenesis and collagen type I mutations in individuals with OI. In this cohort study, 128 unrelated individuals with OI were included. Panoramic radiographs were analyzed regarding dentinogenesis imperfecta (DGI) and congenitally missing teeth. The collagen I genes were sequenced in all individuals, and in 25, multiplex ligation-dependent probe amplification was performed. Mutations in the COL1A1 and COL1A2 genes were found in 104 of 128 individuals. Tooth agenesis was diagnosed in 17% (hypodontia 11%, oligodontia 6%) and was more frequent in those with DGI (P = 0.016), and in those with OI type III, 47%, compared to those with OI types I, 12% (P = 0.003), and IV, 13% (P = 0.017). Seventy-five percent of the individuals with oligodontia (≥6 missing teeth) had qualitative mutations, but there was no association with OI type, gender, or presence of DGI. The prevalence of tooth agenesis is high (17%) in individuals with OI, and OI caused by a qualitative collagen I mutation is associated with oligodontia. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Protective Effect of Niacinamide on interleukin-1β-induced Annulus Fibrosus TypeCollagen Degeneration in vitro

    Institute of Scientific and Technical Information of China (English)

    DUAN Deyu; YANG Shuhua; SHAO Zengwu; WANG Hong; XIONG Xiaoqian

    2007-01-01

    The protective effect of niacinamide on interleukin-1β (IL-1β)-induced annulus fibrosus (AF) typecollagen degeneration in vitro and the mechanism were investigated. Chiba's intervertebrai disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, typecollagen degneration group (IL-1β) and treatment group (niacinamide+IL-1β). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and typecollagen immunohistochemical examination, and typecollagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the typecollagen degeneration group (P<0.01). Rate of Caspase-3 positive staining AF cells in the 4 groups was 3.4%, 4.2%, 17.6% and 10.3% respectively. The positive rate in treatment group was lower than in the typecollagen degeneration group (P<0.01). Typecollagen staining demonstrated that lamellar structure and continuity of collagen in treatment group was better reversed than in the degeneration group. RT-PCR revealed that the expression of typecollagen in treatment group was significantly stronger than that in typecollagen degeneration group (P<0.01). It was concluded that niacinamide could effectively inhibit IL-1β stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1β-caused destruction and synthesis inhibition of typecollagen. Niacinamide is of potential for clinical treatment of IVD degeneration.

  4. Subculture of chondrocytes on a collagen type I-coated substrate with suppressed cellular dedifferentiation.

    Science.gov (United States)

    Kino-Oka, Masahiro; Yashiki, Shino; Ota, Yuka; Mushiaki, Yuko; Sugawara, Katsura; Yamamoto, Takeyuki; Takezawa, Toshiaki; Taya, Masahito

    2005-01-01

    To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.

  5. Elevated carboxy terminal cross linked telopeptide of type I collagen in alcoholic cirrhosis

    DEFF Research Database (Denmark)

    Møller, S; Hansen, M; Hillingsø, Jens

    1999-01-01

    BACKGROUND: The carboxy terminal cross linked telopeptide of type I collagen (ICTP) has been put forward as a marker of bone resorption. Patients with alcoholic liver disease may have osteodystrophy. AIMS: To assess circulating and regional concentrations of ICTP in relation to liver dysfunction...

  6. Two major 5 '-untranslated regions for type XVII collagen mRNA

    NARCIS (Netherlands)

    van Zalen, Sebastiaan; Nijenhuis, Albertine; Jonkman, Marcel F.; Pas, Hendri H.

    2006-01-01

    Background: Type XVII collagen is an important structural component of keratinocyte hemidesmosomes and its functional loss in genetic or autoimmune disease results in blistering of the skin. In neoplastic tissue aberrant expression is seen dependent on the stage of the tumor. White the sequence of t

  7. Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering.

    NARCIS (Netherlands)

    Pieper, J.S.; Kraan, P.M. van der; Hafmans, T.G.M.; Kamp, J.; Buma, P.; Susante, J.L.C. van; Berg, W.B. van den; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

    2002-01-01

    The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the genera

  8. Serum type IV collagen level is predictive for esophageal varices in patients with severe alcoholic disease

    Institute of Scientific and Technical Information of China (English)

    Satoshi Mamori; Yasuyuki Searashi; Masato Matsushima; Kenichi Hashimoto; Shinichiro Uetake; Hiroshi Matsudaira; Shuji Ito; Hisato Nakajima; Hisao Tajiri

    2008-01-01

    AIM: To determine factors predictive for esophageal varices in severe alcoholic disease (SAD).METHODS: Abdominal ultrasonography (US) was performed on 444 patients suffering from alcoholism. Forty-four patients found to have splenomegaly and/ or withering of the right liver lobe were defined as those with SAD. SAD patients were examined by upper gastrointestinal (UGI) endoscopy for the presence of esophageal varices. The existence of esophageal varices was then related to clinical variables.RESULTS: Twenty-five patients (56.8%) had esophageal varices. A univariate analysis revealed a significant difference in age and type IV collagen levels between patients with and without esophageal varices. A logistic regression analysis identified type IV collagen as the only independent variable predictive for esophageal varices (P = 0.017). The area under the curve (AUC) for type IV collagen as determined by the receiver operating characteristic (ROC) for predicting esophageal varices was 0.78.CONCLUSION: This study suggests that the level of type IV collagen has a high diagnostic accuracy for the detection of esophageal varices in SAD.

  9. Cartilage collagen type II seromarker patterns in axial spondyloarthritis and psoriatic arthritis

    DEFF Research Database (Denmark)

    Munk, Heidi Lausten; Gudmann, Natasja Staehr; Christensen, Anne Friesgaard

    2016-01-01

    The aim of the study was to assess the possible association between type II collagen turnover seromarkers and disease profile in patients with axial spondyloarthritis (SpA) and psoriatic arthritis (PsA). Outpatients with axial SpA (n = 110) or PsA (n = 101) underwent clinical examination including...

  10. Osteogensis imperfecta type I is commonly due to a COLIAI null allel of type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Willing, M.C.; Pruchno, C.J. (Univ. of Iowa, Iowa City, IA (United States)); Atkinson, M.; Byers, P.H. (Univ. of Washington, Seattle, WA (United States))

    1992-09-01

    Dermal fibroblasts from most individuals with osteogenesis imperfecta (OI) type I produce about half the normal amount of type I procollagen, as a result of decreased synthesis of one of its constituent chains, pro[alpha](I). To test the hypothesis that decreased synthesis of pro[alpha](I) chains results from mutations in the COL1A1 gene, the authors used primer extension with nucleotide-specific chain termination to measure the contribution of individual COL1A1 alleles to the mRNA pool in fibroblasts from affected individuals. A polymorphic Mn/I restriction endonuclease site in the 3'-untranslated region of COL1A1 was used to distinguish the transcripts of the two alleles in heterozygous individuals. Twenty-three individuals from 21 unrelated families were studied. In each case there was marked diminution in steady-state mRNA levels from one COL1A2 allele. Loss of an allele through deletion or rearrangement was not the cause of the diminished COL1A1 mRNA levels. Primer extension with nucleotide-specific chain termination allows identification of the mutant COL1A1 allele in cell strains that are heterozygous for an expressed polymorphism. It is applicable to sporadic cases, to small families, and to large families in whom key individuals are uninformative at the polymorphic sites used in linkage analysis, making it a useful adjunct to the biochemical screening of collagenous proteins for OI. 40 refs., 3 figs., 1 tab.

  11. The structural and optical properties of type III human collagen biosynthetic corneal substitutes

    Science.gov (United States)

    Hayes, Sally; Lewis, Phillip; Islam, M. Mirazul; Doutch, James; Sorensen, Thomas; White, Tomas; Griffith, May; Meek, Keith M.

    2015-01-01

    The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2–9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. PMID:26159106

  12. Physics of soft hyaluronic acid-collagen type II double network gels

    Science.gov (United States)

    Morozova, Svetlana; Muthukumar, Murugappan

    2015-03-01

    Many biological hydrogels are made up of multiple interpenetrating, charged components. We study the swelling, elastic diffusion, mechanical, and optical behaviors of 100 mol% ionizable hyaluronic acid (HA) and collagen type II fiber networks. Dilute, 0.05-0.5 wt% hyaluronic acid networks are extremely sensitive to solution salt concentration, but are stable at pH above 2. When swelled in 0.1M NaCl, single-network hyaluronic acid gels follow scaling laws relevant to high salt semidilute solutions; the elastic shear modulus G' and diffusion constant D scale with the volume fraction ϕ as G' ~ϕ 9 / 4 and D ~ϕ 3 / 4 , respectively. With the addition of a collagen fiber network, we find that the hyaluronic acid network swells to suspend the rigid collagen fibers, providing extra strength to the hydrogel. Results on swelling equilibria, elasticity, and collective diffusion on these double network hydrogels will be presented.

  13. Collagen Type III and VI Turnover in Response to Long-Term Immobilization.

    Directory of Open Access Journals (Sweden)

    Shu Sun

    Full Text Available Muscle mass and function are perturbed by immobilization and remobilization. When muscle mass changes, the quality and quantity of the extracellular matrix protein, particularly the collagens, change with it. In this study, we investigated the temporal profile of three peptide biomarkers derived from turnover of collagen type III and type VI in a long-term immobilization and remobilization study. We also compared individual biomarker levels with Lean body Mass (LBM and changes therein, hypothesizing that these biomarkers would be biomarkers of the remodeling processes associated with immobilization and/or remobilization.In the Berlin bed rest study, 20 young men were recruited and randomly assigned to 8-week's strict bed rest with or without resistive vibration exercise countermeasure. We measured three neo-epitope ELISA kits in the serum samples of this study: Pro-C3, measured the synthesis of collagen type III; Pro-C6, measured the synthesis of collagen type VI; and C6M measured the degradation of collagen type VI induced by MMP-2 and MMP-9 cleavage.Pro-C3 and Pro-C6 biomarkers are up-regulated with both immobilization and remobilization, whereas C6M is hardly affected at all. We found that Pro-C3 and C6M levels are related to LBM at baseline and that high levels of Pro-C6 are associated with smaller changes in muscle mass during both immobilization and remobilization.The Pro-C3 and-C6 biomarkers change likely reflect remodeling changes in response to unloading or reloading, whereas C6M does not appear to respond to unloading. Pro-C3 and C6M levels correlate with LBM at baseline, while Pro-C6 is related to the anabolic and catabolic responses to unloading and reloading.

  14. A metalloprotease secreted by the type II secretion system links Vibrio cholerae with collagen.

    Science.gov (United States)

    Park, Bo R; Zielke, Ryszard A; Wierzbicki, Igor H; Mitchell, Kristie C; Withey, Jeffrey H; Sikora, Aleksandra E

    2015-03-01

    Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.

  15. A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

    Science.gov (United States)

    Park, Bo R.; Zielke, Ryszard A.; Wierzbicki, Igor H.; Mitchell, Kristie C.; Withey, Jeffrey H.

    2015-01-01

    Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program. PMID:25561716

  16. Compositional and in Vitro Evaluation of Nonwoven Type I Collagen/Poly-dl-lactic Acid Scaffolds for Bone Regeneration

    CERN Document Server

    Qiao, Xiangchen; Yang, Xuebin; Tronci, Giuseppe; Wood, David J

    2015-01-01

    Poly-dl-lactic acid (PDLLA) was blended with type I collagen to attempt to overcome the instantaneous gelation of electrospun collagen scaffolds in biological environments. Scaffolds based on blends of type I collagen and PDLLA were investigated for material stability in cell culture conditions (37 {\\deg}C; 5% CO2) in which post-electrospinning glutaraldehyde crosslinking was also applied. The resulting wet-stable webs were cultured with bone marrow stromal cells (HBMSC) for five weeks. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), Fourier transform infra-red spectroscopy (FTIR) and biochemical assays were used to characterise the scaffolds and the consequent cell-scaffold constructs. To investigate any electrospinning-induced denaturation of collagen, identical PDLLA/collagen and PDLLA/gelatine blends were electrospun and their potential to promote osteogenic differentiation investigated. PDLLA/collagen blends with w/w ratios of 40/60, 60/40 and 80/20 resulted in satisfactory...

  17. Transgenic expression of an altered angiotensin type I AT1 receptor resulting in marked modulation of vascular type I collagen.

    Science.gov (United States)

    Yu, Jun; Taylor, Linda; Rich, Celeste; Toselli, Paul; Stone, Philip; Green, Daniel; Warburton, Rod; Hill, Nicholas; Goldstein, Ronald; Polgar, Peter

    2012-05-01

    The angiotensin II (AngII) type I receptor (AT1) was modified by replacing its third intracellular loop and C-terminal tail with the corresponding regions from the bradykinin B2 receptor. Transgenic mice were produced that overexpress this mutated receptor (AB3T). Considerably less collagen content in the intact aorta and in primary aortic smooth muscle cells (aSMCs) cultures was observed in the transgenic mice. On the other hand, elastin content remained unchanged as measured by Western blot, and insoluble amino acid quantitation. The contraction of isolated aortas also remained unaltered. The aSMCs derived from the transgenic mice showed a reduction in AngII responsive type I collagen production. In aSMCs from transgenic mice, the cascade of Akt to the mammalian target rapamycin (mTOR) to p70 S6 kinase (p70S6K) was not AngII activated, while in the aSMCs from wild-type (WT) mice the cascade was AngII activated. Angiotensin activation of Smad2 and Stat3 was also reduced in the AB3T aSMCs. However, no change in the effect of transforming growth factor β (TGFβ) on type I collagen production was observed. Also, the activation of ERK and JNK and G-protein linked signaling remained unaltered in response to AngII. Akt and PI3K activation inhibitors blocked AngII-stimulated type I collagen expression in WT aSMCs, whereas ERK inhibitor had no such effect. Our results point to an Akt/mTOR/p70S6K regulation of collagen production by AngII with participation of Smad2 and Stat3 cascades in this process.

  18. Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin.

    Science.gov (United States)

    Qian, Jun; Okada, Yukari; Ogura, Takayuki; Tanaka, Keisuke; Hattori, Shunji; Ito, Shinji; Satoh, Junko; Takita, Teisuke; Yasukawa, Kiyoshi

    2016-01-01

    Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N- and C-terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N- and in the C-terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N-terminal and in the C-terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS-PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the Km and kcat values increased with increasing temperature (30 to 65 °C), although the kcat /Km values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the Km and kcat values increased with decreasing pH, and the kcat /Km values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.

  19. Laminin and Type IV Collagen Isoform Substitutions Occur in Temporally and Spatially Distinct Patterns in Developing Kidney Glomerular Basement Membranes

    OpenAIRE

    Abrahamson, Dale R.; St. John, Patricia L.; Stroganova, Larysa; Zelenchuk, Adrian; Steenhard, Brooke M.

    2013-01-01

    Kidney glomerular basement membranes (GBMs) undergo laminin and type IV collagen isoform substitutions during glomerular development, which are believed to be required for maturation of the filtration barrier. Specifically, GBMs of earliest glomeruli contain laminin α1β1γ1 and collagen α1α2α1(IV), whereas mature glomeruli contain laminin α5β2γ1 and collagen α3α4α5(IV). Here, we used confocal microscopy to simultaneously evaluate expression of different laminin and collagen IV isoforms in newb...

  20. Influence of oxazolidines and zirconium oxalate crosslinkers on the hydrothermal, enzymatic, and thermo mechanical stability of type 1 collagen fiber

    Energy Technology Data Exchange (ETDEWEB)

    Haroun, Mahdi A. [Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM, Serdang (Malaysia)], E-mail: Mahdiupm@hotmail.com; Khirstova, Palmina K. [People' s Hall 11113, P.O. Box 6272, Khartoum (Sudan); Gasmelseed, Gurashi A. [Juba University, Leather Dept. P.O. Box 12327 Khartoum (Sudan); Covington, Antony D. [Leather Centre, University College Northampton, Northampton (United Kingdom)

    2009-02-20

    Stabilization of type I rat tail tendon (RTT) collagen by crosslink agent oxazolidine and zirconium oxalate was studied to understand the effect on the thermal, enzymatic and mechanical stability of collagen. The results show that both oxazolidine and zirconium oxalate imparts thermal stability to collagen, and oxazolidine exhibits a marked increase in the peak temperature and enthalpy changes when compared to both native and zirconium oxalate tanned RTT. There is a decrease in the peak temperature and the enthalpy changes of oxazolidine tanned RTT fibers after treatment with urea, suggesting the possibility of alterations in the secondary structure of collagen after tanning. Oxazolidine, which forms carbocationic intermediates species in solution, have better crosslinking with collagen as seen from viscometry studies and hence provides better enzymatic stability to collagen than zirconium, which largely forms monomeric species in solution. Zirconium does not seem to change the tensile strength of RTT fibers significantly in wet condition as well as oxazolidine.

  1. Compositional and in Vitro Evaluation of Nonwoven Type I Collagen/Poly-dl-lactic Acid Scaffolds for Bone Regeneration

    Science.gov (United States)

    Qiao, Xiangchen; Russell, Stephen J.; Yang, Xuebin; Tronci, Giuseppe; Wood, David J.

    2015-01-01

    Poly-dl-lactic acid (PDLLA) was blended with type I collagen to attempt to overcome the instantaneous gelation of electrospun collagen scaffolds in biological environments. Scaffolds based on blends of type I collagen and PDLLA were investigated for material stability in cell culture conditions (37 °C; 5% CO2) in which post-electrospinning glutaraldehyde crosslinking was also applied. The resulting wet-stable webs were cultured with bone marrow stromal cells (HBMSC) for five weeks. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), Fourier transform infra-red spectroscopy (FTIR) and biochemical assays were used to characterise the scaffolds and the consequent cell-scaffold constructs. To investigate any electrospinning-induced denaturation of collagen, identical PDLLA/collagen and PDLLA/gelatine blends were electrospun and their potential to promote osteogenic differentiation investigated. PDLLA/collagen blends with w/w ratios of 40/60, 60/40 and 80/20 resulted in satisfactory wet stabilities in a humid environment, although chemical crosslinking was essential to ensure long term material cell culture. Scaffolds of PDLLA/collagen at a 60:40 weight ratio provided the greatest stability over a five-week culture period. The PDLLA/collagen scaffolds promoted greater cell proliferation and osteogenic differentiation compared to HMBSCs seeded on the corresponding PDLLA/gelatine scaffolds, suggesting that any electrospinning-induced collagen denaturation did not affect material biofunctionality within 5 weeks in vitro. PMID:26251924

  2. Compositional and in Vitro Evaluation of Nonwoven Type I Collagen/Poly-dl-lactic Acid Scaffolds for Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Xiangchen Qiao

    2015-08-01

    Full Text Available Poly-dl-lactic acid (PDLLA was blended with type I collagen to attempt to overcome the instantaneous gelation of electrospun collagen scaffolds in biological environments. Scaffolds based on blends of type I collagen and PDLLA were investigated for material stability in cell culture conditions (37 °C; 5% CO2 in which post-electrospinning glutaraldehyde crosslinking was also applied. The resulting wet-stable webs were cultured with bone marrow stromal cells (HBMSC for five weeks. Scanning electron microscopy (SEM, confocal laser scanning microscopy (CLSM, Fourier transform infra-red spectroscopy (FTIR and biochemical assays were used to characterise the scaffolds and the consequent cell-scaffold constructs. To investigate any electrospinning-induced denaturation of collagen, identical PDLLA/collagen and PDLLA/gelatine blends were electrospun and their potential to promote osteogenic differentiation investigated. PDLLA/collagen blends with w/w ratios of 40/60, 60/40 and 80/20 resulted in satisfactory wet stabilities in a humid environment, although chemical crosslinking was essential to ensure long term material cell culture. Scaffolds of PDLLA/collagen at a 60:40 weight ratio provided the greatest stability over a five-week culture period. The PDLLA/collagen scaffolds promoted greater cell proliferation and osteogenic differentiation compared to HMBSCs seeded on the corresponding PDLLA/gelatine scaffolds, suggesting that any electrospinning-induced collagen denaturation did not affect material biofunctionality within 5 weeks in vitro.

  3. Compositional and in Vitro Evaluation of Nonwoven Type I Collagen/Poly-dl-lactic Acid Scaffolds for Bone Regeneration.

    Science.gov (United States)

    Qiao, Xiangchen; Russell, Stephen J; Yang, Xuebin; Tronci, Giuseppe; Wood, David J

    2015-08-05

    Poly-dl-lactic acid (PDLLA) was blended with type I collagen to attempt to overcome the instantaneous gelation of electrospun collagen scaffolds in biological environments. Scaffolds based on blends of type I collagen and PDLLA were investigated for material stability in cell culture conditions (37 °C; 5% CO2) in which post-electrospinning glutaraldehyde crosslinking was also applied. The resulting wet-stable webs were cultured with bone marrow stromal cells (HBMSC) for five weeks. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), Fourier transform infra-red spectroscopy (FTIR) and biochemical assays were used to characterise the scaffolds and the consequent cell-scaffold constructs. To investigate any electrospinning-induced denaturation of collagen, identical PDLLA/collagen and PDLLA/gelatine blends were electrospun and their potential to promote osteogenic differentiation investigated. PDLLA/collagen blends with w/w ratios of 40/60, 60/40 and 80/20 resulted in satisfactory wet stabilities in a humid environment, although chemical crosslinking was essential to ensure long term material cell culture. Scaffolds of PDLLA/collagen at a 60:40 weight ratio provided the greatest stability over a five-week culture period. The PDLLA/collagen scaffolds promoted greater cell proliferation and osteogenic differentiation compared to HMBSCs seeded on the corresponding PDLLA/gelatine scaffolds, suggesting that any electrospinning-induced collagen denaturation did not affect material biofunctionality within 5 weeks in vitro.

  4. Time pattern of exercise-induced changes in type I collagen turnover after prolonged endurance exercise in humans

    DEFF Research Database (Denmark)

    Langberg, Henning; Skovgaard, D; Asp, S;

    2000-01-01

    Type I collagen is known to adapt to physical activity, and biomarkers of collagen turnover indicate that synthesis can be influenced by a single intense exercise bout, but the exact time pattern of these latter changes are largely undescribed. In the present study, 17 healthy young males had...... indicate that type I collagen synthesis is accelerated in response to prolonged strenuous exercise, reaching a peak after 3 days and returning to preexercising levels 5 days after the completion of a marathon run....... their plasma concentrations of the carboxyterminal propeptide of type I procollagen (PICP), a marker of collagen formation, and the immunoactive carboxyterminal cross-linked telopeptide (ICTP), a marker of collagen resorption, measured before and immediately postexercise, as well as 1, 2, 3, 4, 5, and 6 days...

  5. The effect of Nifedipine on the expression of type I collagen in gingival fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Bei Li; Weibin Sun; Yong Ji

    2008-01-01

    Objective:To investigate the effect of nifedipine(calciumchannel blocker) on the expression of collagen in gingival fibroblasts invitro, Methods:Primarily gingival fibroblasts were cultured and incubated with various concentrations of nifedipine(108 μg/L, 360μg/L and 1200 μg/L)for 5 days. Gingival fibroblasts were primarily cultured derived from nifedipine responders and non-responders in the presence of 360 μg/L nifedipine. Enzyme-linked immunosorbent assay was used to evaluate the amount of typecollagen. Cell proliferation was measured by cell counting with evaluating MTT value. Results:The expressions of collagen and cell proliferation were significantly different among the high concentration groups and the others on the fifth day, especially higher in 360 μg/L and 1200 μg/L groups and also different among nifedipine responders and non-responders. Conclusion:The expression of collagen and cell proliferation may be concerned with the biological mechanism for gingival overgrowth.

  6. Cross-linked type I and type II collagenous matrices for the repair of full-thickness articular cartilage defects--a study in rabbits.

    NARCIS (Netherlands)

    Buma, P.; Pieper, J.S.; Tienen, Tony van; Susante, J.L.C. van; Kraan, P.M. van der; Veerkamp, J.H.; Berg, W.B. van den; Veth, R.P.H.; Kuppevelt, A.H.M.S.M. van

    2003-01-01

    The physico-chemical properties of collagenous matrices may determine the tissue response after insertion into full-thickness articular cartilage defects. In this study, cross-linked type I and type II collagen matrices, with and without attached chondroitin sulfate, were implanted into full-thickne

  7. Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I.

    Science.gov (United States)

    Shibayama, Mineko; Serrano-Luna, José de Jesús; Rojas-Hernández, Saúl; Campos-Rodríguez, Rafael; Tsutsumi, Victor

    2003-03-01

    In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.

  8. Is type I alpha 2 collagen gene responsible for intracranial aneurysm in Northeast China?

    Institute of Scientific and Technical Information of China (English)

    Pengfei Wu; Bo Li; Anhua Wu; Yunjie Wang

    2013-01-01

    In this study, we investigated whether a single nucleotide polymorphism (rs42524 G > C) in the type I alpha 2 collagen gene was associated with sporadic ruptured intracranial aneurysm or its clinical characteristics in patients from Northeast China. Genotyping of the rs42524 G > C polymorphism was carried out using a polymerase chain reaction-restriction fragment length polymorphism assay. The data showed that the frequency of the rs42524 GC + CC genotype was significantly higher than the GG genotype among intracranial aneurysm patients whose Hunt and Hess grading scale was > 3. In addition, the rs42524 G > C genotype was found to have a statistically significant association with intracranial aneurysm risk. These findings indicate that the type I alpha 2 collagen gene gene may be involved in a predisposition to intracranial aneurysm in the Northeast Chinese population. Crucially, the rs42524 C allele may be an important risk factor for increased severity of the condition in patients with ruptured intracranial aneurysms.

  9. Interleukin-10 deficiency aggravates angiotensin II-induced cardiac remodeling in mice.

    Science.gov (United States)

    Kwon, Woo-Young; Cha, Hye-Na; Heo, Jung-Yoon; Choi, Jung-Hyun; Jang, Byung Ik; Lee, In-Kye; Park, So-Young

    2016-02-01

    This study examined the role of interleukin (IL)-10 in angiotensin II-induced cardiac remodeling. Angiotensin II was infused subcutaneously (1.1mg/kg/day) for one week in IL-10 knockout and wild-type mice, after which cardiac function and hypertrophy were assessed by echocardiogram. IL-10 gene expression in the heart was increased by angiotensin II infusion. Plasma levels of brain natriuretic peptide (BNP) and gene expression of BNP in the heart were increased by IL-10 deficiency or angiotensin II, and plasma BNP levels were further increased by IL-10 deficiency with angiotensin II. IL-10 deficiency increased the left ventricular dimension, whereas treatment with angiotensin II increased heart weight. Angiotensin II significantly reduced cardiac function in IL-10 knockout mice compared with wild-type mice. Gene expression of tumor necrosis factor-α and interleukin-6 was increased by IL-10 deficiency or angiotensin II infusion, and these increases were further enhanced by IL-10 deficiency with angiotensin II. Gene expression of collagen I/III and collagen III protein levels were increased by angiotensin II but not by IL-10 deficiency. Gene expression of matrix metalloproteinase2/9 was increased by IL-10 deficiency or angiotensin II, and this expression was further increased by IL-10 deficiency with angiotensin II. Akt phosphorylation was increased by IL-10 deficiency or angiotensin II and further increased by IL-10 deficiency with angiotensin II. Phosphorylation of p38 was increased by IL-10 deficiency. These results suggest that IL-10 deficiency causes deterioration in cardiac functions in angiotensin II-infused mice, suggesting that IL-10 plays a protective role against angiotensin II-induced cardiac remodeling. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Irradiation Alters MMP-2/TIMP-2 System and Collagen Type IV Degradation in Brain

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Won Hee [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia (United States); Warrington, Junie P.; Sonntag, William E. [Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (United States); Lee, Yong Woo, E-mail: ywlee@vt.edu [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia (United States); Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia (United States)

    2012-04-01

    Purpose: Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. Methods and Materials: Animals received either whole-brain irradiation (a single dose of 10 Gy {gamma}-rays or a fractionated dose of 40 Gy {gamma}-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR). The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. Results: A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. Conclusions: The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.

  11. Effects of type IV collagen on myogenic characteristics of IGF-I gene-engineered myoblasts.

    Science.gov (United States)

    Ito, Akira; Yamamoto, Masahiro; Ikeda, Kazushi; Sato, Masanori; Kawabe, Yoshinori; Kamihira, Masamichi

    2015-05-01

    Skeletal muscle regeneration requires migration, proliferation and fusion of myoblasts to form multinucleated myotubes. In our previous study, we showed that insulin-like growth factor (IGF)-I gene delivery stimulates the proliferation and differentiation of mouse myoblast C2C12 cells and promotes the contractile force generated by tissue-engineered skeletal muscles. The aim of this study was to investigate the effects of the extracellular matrix on IGF-I gene-engineered C2C12 cells in vitro. Retroviral vectors for doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into C2C12 cells. When cultured on a type IV collagen-coated surface, we observed significant increases in the migration speed and number of IGF-I gene-engineered C2C12 cells with Dox addition, designated as C2C12/IGF (+) cells. Co-culture of C2C12/IGF (+) cells and parental C2C12 cells, which had been cultured in differentiation medium for 3 days, greatly enhanced myotube formation. Moreover, type IV collagen supplementation promoted the fusion of C2C12/IGF (+) cells with differentiated C2C12 cells and increased the number of myotubes with striations. Myotubes formed by C2C12/IGF (+) cells cultured on type IV collagen showed a dynamic contractile activity in response to electrical pulse stimulation. These findings indicate that type IV collagen promotes skeletal muscle regeneration mediated by IGF-I-expressing myoblasts, which may have important clinical implications in the design of myoblast-based therapies.

  12. The Notch pathway mediates the angiotensin II-induced synthesis of extracellular matrix components in podocytes.

    Science.gov (United States)

    Yao, Min; Wang, Xiaomei; Wang, Xiaomeng; Zhang, Tao; Chi, Yanqing; Gao, Feng

    2015-07-01

    The Notch pathway is known to contribute to the development of glomerular disease. Angiotensin II (Ang II), an important member of the renin-angiotensin system, stimulates the accumulation of extracellular matrix components in glomerular disease; however, the exact mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of the Notch pathway on the synthesis of extracellular matrix components in Ang II-stimulated podocytes. Mouse podocytes were stimulated with Ang II (10-6 mol/l). The activation of the Notch pathway was inhibited by a vector carrying short hairpin RNA (shRNA) targeting Notch1 (sh-Notch1) or by γ-secretase inhibitor (GSI). The protein levels of Notch1, Notch intracellular domain 1 (NICD1), hairy and enhancer of split-1 (Hes1), matrix metalloproteinase (MMP)-2, MMP-9, transforming growth factor-β1 (TGF-β1), type IV collagen and laminin were determined by western blot analysis. The Notch1, Hes1, MMP-2, MMP-9, TGF-β1, type IV collagen and laminin mRNA levels were detected by RT-PCR. The MMP-2 and MMP-9 activity was measured using a cell active fluorescence assay kit. The levels of TGF-β1, type IV collagen and laminin were determined in the culture medium of the podocytes by enzyme-linked immunosorbent assay (ELISA). Our results revealed that Ang II upregulated Notch1, NICD1, Hes1, TGF-β1, type IV collagen and laminin expression and downregulated MMP-2 and MMP-9 expression in the cultured podocytes. The inhibition of the Notch pathway by sh-Notch1 or GSI increased MMP-2 and MMP-9 expression, decreased the TGF-β1 level and suppressed type IV collagen and laminin expression. The inhibition of the Notch pathway by sh-Notch1 or GSI also increased MMP-2 and MMP-9 activity, and decreased TGF-β1 levels, type IV collagen levels and laminin secretion. These findings indicate that the Notch pathway potentially mediates the Ang II-induced synthesis of extracellular matrix components in podocytes through the

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2017-03-03

    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.

  15. Biomechanical regulation of type I collagen gene expression in ACLs in organ culture.

    Science.gov (United States)

    Hsieh, Adam H; Sah, Robert L; Paul Sung, K L

    2002-03-01

    In this study, an ex vivo organ culture system that allows the application of controlled loads to the anterior cruciate ligament (ACL) was designed and used to characterize the influence of a step input in mechanical load on gene expression. A procedure for isolating bone-ACL-bone (B-ACL-B) complexes from rat knees was developed. After harvest and 24 hour culture, B-ACL-B complexes exhibited percentages of viability similar to that in intact ACLs (approximately 90%). Application of a physiologically relevant load of 5 N (superimposed on a I N tare load) resulted in changes in levels of mRNA encoding type I collagen. While levels of type I collagen mRNA significantly increased 32+/-13% (mean +/- standard errors of the mean (SEM)) over controls within the first hour of loading, levels decreased significantly to 44+/-9% of control after 2 h. Displacements induced by the 5 N load were measured by video dimensional analysis. Calculated axial strains of 0.141+/-0.034 were achieved rapidly during the first hour and remained essentially unchanged thereafter. These results demonstrate the feasibility of maintaining ligaments in organ culture and illustrate the time course expression of type I collagen following the application of a mechanical load.

  16. Influence of human growth hormone on granulation tissue formation, collagen deposition, and the aminoterminal propeptide of collagen type III in wound chambers in rats.

    Science.gov (United States)

    Rasmussen, L H; Garbarsch, C; Schuppan, D; Moe, D; Hørslev-Pedersen, K; Gottrup, F; Steenfos, H

    1994-01-01

    The influence of growth hormone on granulation tissue formation was investigated in wire mesh cylinders implanted subcutaneously in rats. Two groups of 10 rats (study 1) and 1 group of 12 rats (study 2) were used for the investigation. Growth hormone, 0.02 and 0.2 IU (study 1), 0.05 and 0.2 IU (study 2), or vehicle only, was injected into the cylinders every third day for 16 days. In study 2, wound fluid was aspirated before injection of growth hormone and saved for later analysis of the aminoterminal propeptide of collagen type III. In both studies, growth hormone significantly increased the formation of granulation tissue and of total collagen content dose-dependently, whereas the relative amount of collagen was unaffected by growth hormone treatment. Wound fluid aminopropeptide increased significantly after implantation of the cylinders until day 7, before declining slightly, with no difference between the groups. We conclude that growth hormone stimulated granulation tissue formation and collagen deposition dose-dependently in the wound cylinders when injected every third day. The results suggest that growth hormone treatment does not cause excessive collagen deposition in newly formed granulation tissue.

  17. Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

    Directory of Open Access Journals (Sweden)

    Keith B Neeves

    Full Text Available Microfluidic flow assays (MFA that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T, the rate of platelet accumulation (V(PLT, and platelet surface coverage (SC. A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT and SC at all wall shear rates. A longer Lag(T for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104 and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

  18. Laminin and type IV collagen isoform substitutions occur in temporally and spatially distinct patterns in developing kidney glomerular basement membranes.

    Science.gov (United States)

    Abrahamson, Dale R; St John, Patricia L; Stroganova, Larysa; Zelenchuk, Adrian; Steenhard, Brooke M

    2013-10-01

    Kidney glomerular basement membranes (GBMs) undergo laminin and type IV collagen isoform substitutions during glomerular development, which are believed to be required for maturation of the filtration barrier. Specifically, GBMs of earliest glomeruli contain laminin α1β1γ1 and collagen α1α2α1(IV), whereas mature glomeruli contain laminin α5β2γ1 and collagen α3α4α5(IV). Here, we used confocal microscopy to simultaneously evaluate expression of different laminin and collagen IV isoforms in newborn mouse GBMs. Our results show loss of laminin α1 from GBMs in early capillary loop stages and continuous linear deposition of laminin bearing the α5 chain thereafter. In contrast, collagen α1α2α1(IV) persisted in linear patterns into late capillary loop stages, when collagen α3α4α5(IV) first appeared in discontinuous, non-linear patterns. This patchy pattern for collagen α3α4α5(IV) continued into maturing glomeruli where there were lengths of linear, laminin α5-positive GBM entirely lacking either isoform of collagen IV. Relative abundance of laminin and collagen IV mRNAs in newborn and 5-week-old mouse kidneys also differed, with those encoding laminin α1, α5, β1, β2, and γ1, and collagen α1(IV) and α2(IV) chains all significantly declining at 5 weeks, but α3(IV) and α4(IV) were significantly upregulated. We conclude that different biosynthetic mechanisms control laminin and type IV collagen expression in developing glomeruli.

  19. Discoidin Domain Receptor 2 Mediates Collagen-Induced Activation of Membrane-Type 1 Matrix Metalloproteinase in Human Fibroblasts.

    Science.gov (United States)

    Majkowska, Iwona; Shitomi, Yasuyuki; Ito, Noriko; Gray, Nathanael S; Itoh, Yoshifumi

    2017-03-07

    Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity including fibroblasts and invasive cancer cell. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it upregulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation is not clearly understood. In this study we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited collagen-induced activation of proMMP-2 and upregulation of MT1-MMP at the gene and protein level. Interestingly DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signalling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells, as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without non-helical telopeptides region compared to intact collagen fibrils. Those data suggest that DDR2 is a microenvironmental sensor that regulates fibroblasts migration in collagen-rich environment.

  20. Prostaglandins in the perilymph of guinea pig with type II collagen induced ear diseases

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, T.; Chiang, T.; Kitano, H.; Sudo, N.; Kim, S.Y.; Ha, S.; Woo, V.; Wolf, B.; Floyd, R.; Yoo, T.J.

    1986-03-01

    The authors have studied the prostaglandins (PGs) in the perilymph from guinea pig with type II collagen induced autoimmune ear disease. Hartly guinea pigs were immunized with type II collagen in CFA and auditory brain stem responses (ABR) were measured at 2, 3, 4, and 6 months after initial immunization perilymph was obtained and the levels of PGE2 and 6 keto-PGFl..cap alpha.. were measured by radioimmunoassays. Temporal bones were examined for the histopathologic changes. Immunized guinea pigs showed the evidence of hearing loss by ABR. The temporal bones showed the following changes: spiral ganglia degeneration, mild to moderate degree of degeneration in organ of Corti, infrequent very mild endolymphatic hydrops and labrynthitis. The perilymph from immunized animals contained about 5 times more PGE2 and about 3 times more 6 keto-PGFl..cap alpha.. than control animals. However, between these two groups, there was no difference in the CSF and sera levels of PGE2 and 6 keto-PGFl..cap alpha... Thus, this study suggests that these inflammatory mediators might be involved in the pathogenesis of collagen induced autoimmune inner ear disease.

  1. Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium

    National Research Council Canada - National Science Library

    Jabaji, Ziyad; Brinkley, Garrett J; Khalil, Hassan A; Sears, Connie M; Lei, Nan Ye; Lewis, Michael; Stelzner, Matthias; Martín, Martín G; Dunn, James C Y

    2014-01-01

    .... There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells...

  2. Type I Collagen as an Extracellular Matrix for the In Vitro Growth of Human Small Intestinal Epithelium: e107814

    National Research Council Canada - National Science Library

    Ziyad Jabaji; Garrett J Brinkley; Hassan A Khalil; Connie M Sears; Nan Ye Lei; Michael Lewis; Matthias Stelzner; Martín G Martín; James C Y Dunn

    2014-01-01

    .... There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells...

  3. Biochemical markers of type II collagen breakdown and synthesis are positioned at specific sites in human osteoarthritic knee cartilage

    DEFF Research Database (Denmark)

    Jensen, Anne-Christine Bay; Levin Andersen, Thomas; Charni-Ben Tabassi, N;

    2007-01-01

    as for type IIA N propeptide (PIIANP), a biochemical marker reflecting synthesis of type IIA collagen. RESULTS: Helix-II and CTX-II were detected in areas where collagen damage was reported previously, most frequently around chondrocytes, but also frequently in regions not previously investigated......OBJECTIVE: To investigate whether type II collagen turnover markers used for osteoarthritis (OA) activity evaluation in body fluids can be detected at the level of specific histological features of OA cartilage tissue, as well as how they relate with each other at this level. METHODS: Adjacent...... sections were obtained from full-depth cartilage biopsies from 32 OA knees. Immunohistochemistry was performed for Helix-II and CTX-II, which are type II collagen fragments originating from the triple helix and the telopeptide region, respectively, and believed to reflect distinct breakdown events, as well...

  4. [Reactivity of antibodies to collagen types I to IV and antibodies to chondroitin sulfate in the spleen].

    Science.gov (United States)

    Galbavý, S; Ruzicková, M; Surmíková, E; Danihel, L; Porubský, J; Papincák, J; Holesa, S; Trnka, J

    1996-02-01

    Antibodies to collagen type I and III reacted negatively, antibodies to collagen type IV positively with reticulin, trabeculae and circumferent reticulum of lymphatic sheaths, poorly positively with capsula, strongly positively with subcapsular zone. Antibodies to collagen type II reacted positively with capsula, poorly with subcapsular zone, strongly with sinus wall and poorly with trabeculae. They did not react with circumferent reticulum of periarterial lymphoid sheaths. Antibodies to collagen type II and IV reacted positively with central arteries. Antibodies to chondroitinsulphate C reacted poorly and antibodies to chondroitinsulphate B strongly positively with sinus walls and oval cells spread in the white and red pulpa. Antibodies to chondroitin sulphate A reacted similarly as antibodies to chondroitinsulphate B.

  5. Differential co-expression of long and short form type IX collagen transcripts during avian limb chondrogenesis in ovo.

    Science.gov (United States)

    Swiderski, R E; Solursh, M

    1992-05-01

    Using RNA blot analysis of developmentally staged avian limb buds, we demonstrate that transcripts of several cartilage marker genes appear in limb tissue prior to overt chondrogenesis. Type II collagen mRNA, cartilage proteoglycan core protein mRNA, alpha 2(IX) collagen mRNA, and transcripts of the short form alpha 1(IX) collagen chain derived from the downstream promoter are co-expressed in limb tissue approximately 24-36 hours before the appearance of the respective polypeptides in differentiating cartilagenous tissue. Transcripts of the long form alpha 1(IX) collagen chain derived from the upstream promoter appear somewhat later in development; nearly coincident with the immunolocalization of type IX collagen in the cartilage elements of the limb. The spatial distribution of type II and type IX collagen transcripts was analyzed by in situ hybridization. Type II collagen and the long form alpha 1(IX) collagen transcripts co-localized in the chondrogenic elements of the developing forelimb. In contrast, short form alpha 1(IX) collagen transcripts which lack the 5' region encoding the NC4 globular amino-terminal domain were distributed throughout the non-chondrogenic, non-myogenic mesenchymal regions of the limb and were not detectable above background levels in the limb chondrogenic elements. The precocious appearance of several cartilage marker gene transcripts prior to chondrogenesis suggests that multiple levels of gene regulation including alternative promoter use, alternative RNA splicing, alternative polyadenylation, and other post-transcriptional as well as translational mechanisms are active prior to, and during avian limb chondrogenesis.

  6. Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges

    Science.gov (United States)

    Talò, Giuseppe; Lovati, Arianna B.; Pasdeloup, Marielle; Riboldi, Stefania A.; Moretti, Matteo; Mallein-Gerin, Frédéric

    2016-01-01

    Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer in vitro articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated in vivo by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage. PMID:27584727

  7. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    Science.gov (United States)

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  8. Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

    Science.gov (United States)

    2011-01-01

    Background Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N. Methods In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison. Results Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. Conclusion Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving. PMID:21244651

  9. Protective effect of niacinamide on interleukin-1beta-induced annulus fibrosus type II collagen degeneration in vitro.

    Science.gov (United States)

    Duan, Deyu; Yang, Shuhua; Shao, Zengwu; Wang, Hong; Xiong, Xiaoqian

    2007-02-01

    The protective effect of niacinamide on interleukin-1beta (IL-1beta)-induced annulus fibrosus (AF) type II collagen degeneration in vitro and the mechanism were investigated. Chiba's intervertebral disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, type II collagen degneration group (IL-1beta) and treatment group (niacinamide+IL-1beta). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and type II collagen immunohistochemical examination, and type II collagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the type II collagen degeneration group (Pniacinamide could effectively inhibit IL-1beta stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1beta-caused destruction and synthesis inhibition of type II collagen. Niacinamide is of potential for clinical treatment of IVD degeneration.

  10. Influence of Term of Exposure to High-Fat Diet-Induced Obesity on Myocardial Collagen Type I and III

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Danielle Cristina Tomaz da [Departamento de Clínica Médica, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil); Lima-Leopoldo, Ana Paula; Leopoldo, André Soares [Departamento de Esportes, Centro de Educação Física e Desportos da Universidade Federal do Espírito Santo (UFES), Vitória, ES (Brazil); Campos, Dijon Henrique Salomé de; Nascimento, André Ferreira do [Departamento de Clínica Médica, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil); Oliveira, Sílvio Assis Junior de [Escola de Fisioterapia da Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS (Brazil); Padovani, Carlos Roberto [Departamento de Bioestatística do Instituto de Ciências Biológicas da Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil); Cicogna, Antonio Carlos, E-mail: dany.tomaz@gmail.com [Departamento de Clínica Médica, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil)

    2014-02-15

    Obesity is a risk factor for many medical complications; medical research has shown that hemodynamic, morphological and functional abnormalities are correlated with the duration and severity of obesity. Present study determined the influence of term of exposure to high-fat diet-induced obesity on myocardial collagen type I and III. Thirty-day-old male Wistar rats were randomly distributed into two groups: a control (C) group fed a standard rat chow and an obese (Ob) group alternately fed one of four palatable high-fat diets. Each diet was changed daily, and the rats were maintained on their respective diets for 15 (C{sub 15} and Ob{sub 15}) and 30 (C{sub 30} and Ob{sub 30}) consecutive weeks. Obesity was determined by adiposity index. The Ob{sub 15} group was similar to the C{sub 15} group regarding the expression of myocardial collagen type I; however, expression in the Ob{sub 30} group was less than C{sub 30} group. The time of exposure to obesity was associated with a reduction in collagen type I in Ob{sub 30} when compared with Ob{sub 15}. Obesity did not affect collagen type III expression. This study showed that the time of exposure to obesity for 30 weeks induced by unsaturated high-fat diet caused a reduction in myocardial collagen type I expression in the obese rats. However, no effect was seen on myocardial collagen type III expression.

  11. Characteristics of type IV collagen unfolding under various pH conditions as a model of pathological disorder in tissue.

    Science.gov (United States)

    Shimizu, Akio; Kawai, Kenichi; Yanagino, Miki; Wakiyama, Toshiko; Machida, Minoru; Kameyama, Kohji; Naito, Zenya

    2007-07-01

    The overall structure of type IV collagen is the same at neutral and acidic pH, as determined by circular dichroism spectra. The heating rate dependence of denaturation midpoint temperature (T(m)) shows that type IV collagen is unstable at body temperature, similarly to type I collagen. The heating rate dependence of T(m) at neutral pH has two phases, but that at acidic pH apparently has a single phase. The T(m) of the first phase (lower T(m)) at neutral pH is consistent with that at acidic pH, and the activation energy of these phases is consistent, within experimental error. The triple helix region of type IV collagen corresponding to the second phase (higher T(m)) at neutral pH is thermally stable when compared to the triple helical structure at acidic pH. At acidic pH, as the loosely packed and unstable region has spread throughout the whole molecule, the thermal transition is thought to be cooperative and is observed as a single phase. Structural flexibility is related to protein function and assembly; therefore, the unstable structure and increased flexibility of type IV collagen induced at acidic pH may affect diseases accompanied by type IV collagen disorder.

  12. Characterization of type I, II, III, IV, and V collagens by time-resolved laser-induced fluorescence spectroscopy

    Science.gov (United States)

    Marcu, Laura; Cohen, David; Maarek, Jean-Michel I.; Grundfest, Warren S.

    2000-04-01

    The relative proportions of genetically distinct collagen types in connective tissues vary with tissue type and change during disease progression, development, wound healing, aging. This study aims to 1) characterize the spectro- temporal fluorescence emission of fiber different types of collagen and 2) assess the ability of time-resolved laser- induced fluorescence spectroscopy to distinguish between collagen types. Fluorescence emission of commercially available purified samples was induced with nitrogen laser excitation pulses and detected with a MCP-PMT connected to a digital storage oscilloscope. The recorded time-resolved emission spectra displayed distinct fluorescence emission characteristics for each collagen type. The time domain information complemented the spectral domain intensity data for improved discrimination between different collagen types. Our results reveal that analysis of the fluorescence emission can be used to characterize different species of collagen. Also, the results suggest that time-resolved spectroscopy can be used for monitoring of connective tissue matrix composition changes due to various pathological and non-pathological conditions.

  13. Different Organization of Type I Collagen Immobilized on Silanized and Nonsilanized Titanium Surfaces Affects Fibroblast Adhesion and Fibronectin Secretion.

    Science.gov (United States)

    Marín-Pareja, Nathalia; Cantini, Marco; González-García, Cristina; Salvagni, Emiliano; Salmerón-Sánchez, Manuel; Ginebra, Maria-Pau

    2015-09-23

    Silanization has emerged in recent years as a way to obtain a stronger and more stable attachment of biomolecules to metallic substrates. However, its impact on protein conformation, a key aspect that influences cell response, has hardly been studied. In this work, we analyzed by atomic force microscopy (AFM) the distribution and conformation of type I collagen on plasma-treated surfaces before and after silanization. Subsequently, we investigated the effect of the different collagen conformations on fibroblasts adhesion and fibronectin secretion by immunofluorescence analyses. Two different organosilanes were used on plasma-treated titanium surfaces, either 3-chloropropyl-triethoxy-silane (CPTES) or 3-glycidyloxypropyl-triethoxy-silane (GPTES). The properties and amount of the adsorbed collagen were assessed by contact angle, X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and AFM. AFM studies revealed different conformations of type I collagen depending on the silane employed. Collagen was organized in fibrillar networks over very hydrophilic (plasma treated titanium) or hydrophobic (silanized with CPTES) surfaces, the latter forming little globules with a beads-on-a-string appearance, whereas over surfaces presenting an intermediate hydrophobic character (silanized with GPTES), collagen was organized into clusters with a size increasing at higher protein concentration in solution. Cell response was strongly affected by collagen conformation, especially at low collagen density. The samples exhibiting collagen organized in globular clusters (GPTES-functionalized samples) favored a faster and better fibroblast adhesion as well as better cell spreading, focal adhesions formation, and more pronounced fibronectin fibrillogenesis. In contrast, when a certain protein concentration was reached at the material surface, the effect of collagen conformation was masked, and similar fibroblast response was observed in all samples.

  14. Pregnancy Differentially Regulates the Collagens Types I and III in Left Ventricle from Rat Heart

    Science.gov (United States)

    Limon-Miranda, Sarai; Salazar-Enriquez, Diana G.; Muñiz, Jesus; Ramirez-Archila, Mario V.; Sanchez-Pastor, Enrique A.; Andrade, Felipa; Soñanez-Organis, Jose G.; Moran-Palacio, Edgar F.; Virgen-Ortiz, Adolfo

    2014-01-01

    The pathologic cardiac remodeling has been widely documented; however, the physiological cardiac remodeling induced by pregnancy and its reversion in postpartum are poorly understood. In the present study we investigated the changes in collagen I (Col I) and collagen III (Col III) mRNA and protein levels in left ventricle from rat heart during pregnancy and postpartum. Col I and Col III mRNA expression in left ventricle samples during pregnancy and postpartum were analyzed by using quantitative PCR. Data obtained from gene expression show that Col I and Col III in left ventricle are upregulated during pregnancy with reversion in postpartum. In contrast to gene expression, the protein expression evaluated by western blot showed that Col I is downregulated and Col III is upregulated in left ventricle during pregnancy. In conclusion, the pregnancy differentially regulates collagens types I and III in heart; this finding could be an important molecular mechanism that regulates the ventricular stiffness in response to blood volume overload present during pregnancy which is reversed in postpartum. PMID:25147829

  15. Effect of enzymatic hydrolysis on surface activity and surface rheology of type I collagen.

    Science.gov (United States)

    Kezwoń, Aleksandra; Chromińska, Ilona; Frączyk, Tomasz; Wojciechowski, Kamil

    2016-01-01

    We describe the adsorption behaviour and rheological properties of a calf skin type I collagen, and of its hydrolysates obtained using a Clostridium histolyticum collagenase (CHC) under moderate conditions (pH 7, 37°C). The effect of CHC concentration (2×10(-9)-2×10(-6)M) and incubation time (35-85min) was studied and optimised to achieve the highest decrease of surface tension and the highest dilational surface viscoelasticity of the adsorbed layers. SDS-PAGE electrophoresis and reverse-phase high performance liquid chromatography (RP-HPLC) were used to characterise the hydrolysis products. The results show that even simple modifications (heat treatment, pH change, partial hydrolysis) of collagen enhances its surface properties, especially in terms of surface dilational elasticity modulus. The use of low enzyme concentration (CHC-to-collagen molar ratio of 4×10(-3)) and short incubation time (<45min) results in moderately hydrolysed products with the highest ability to lower surface tension (γ=53.9mNm(-1)) forming highly elastic adsorbed layers (surface dilational elasticity, E'=74.5mNm(-1)).

  16. Pregnancy Differentially Regulates the Collagens Types I and III in Left Ventricle from Rat Heart

    Directory of Open Access Journals (Sweden)

    Sarai Limon-Miranda

    2014-01-01

    Full Text Available The pathologic cardiac remodeling has been widely documented; however, the physiological cardiac remodeling induced by pregnancy and its reversion in postpartum are poorly understood. In the present study we investigated the changes in collagen I (Col I and collagen III (Col III mRNA and protein levels in left ventricle from rat heart during pregnancy and postpartum. Col I and Col III mRNA expression in left ventricle samples during pregnancy and postpartum were analyzed by using quantitative PCR. Data obtained from gene expression show that Col I and Col III in left ventricle are upregulated during pregnancy with reversion in postpartum. In contrast to gene expression, the protein expression evaluated by western blot showed that Col I is downregulated and Col III is upregulated in left ventricle during pregnancy. In conclusion, the pregnancy differentially regulates collagens types I and III in heart; this finding could be an important molecular mechanism that regulates the ventricular stiffness in response to blood volume overload present during pregnancy which is reversed in postpartum.

  17. Polymerized-Type I Collagen Induces Upregulation of Foxp3-Expressing CD4 Regulatory T Cells and Downregulation of IL-17-Producing CD4+ T Cells (Th17 Cells in Collagen-Induced Arthritis

    Directory of Open Access Journals (Sweden)

    Janette Furuzawa-Carballeda

    2012-01-01

    Full Text Available Previous studies showed that polymerized-type I collagen (polymerized collagen exhibits potent immunoregulatory properties. This work evaluated the effect of intramuscular administration of polymerized collagen in early and established collagen-induced arthritis (CIA in mice and analyzed changes in Th subsets following therapy. Incidence of CIA was of 100% in mice challenged with type II collagen. Clinimorphometric analysis showed a downregulation of inflammation after administration of all treatments (P<0.05. Histological analysis showed that the CIA-mice group had extensive bone erosion, pannus and severe focal inflammatory infiltrates. In contrast, there was a remarkable reduction in the severity of arthritis in mice under polymerized collagen, methotrexate or methotrexate/polymerized collagen treatment. Polymerized Collagen but not methotrexate induced tissue joint regeneration. Polymerized Collagen and methotrexate/polymerized collagen but not methotrexate alone induces downregulation of CD4+/IL17A+ T cells and upregulation of Tregs and CD4+/IFN-γ+ T cells. Thus, Polymerized Collagen could be an effective therapeutic agent in early and established rheumatoid arthritis by exerting downregulation of autoimmune inflammation.

  18. Collagen type I and decorin expression in tenocytes depend on the cell isolation method

    Directory of Open Access Journals (Sweden)

    Wagenhäuser Markus U

    2012-08-01

    Full Text Available Abstract Backround The treatment of rotator cuff tears is still challenging. Tendon tissue engineering (TTE might be an alternative in future. Tenocytes seem to be the most suitable cell type as they are easy to obtain and no differentiation in vitro is necessary. The aim of this study was to examine, if the long head of the biceps tendon (LHB can deliver viable tenocytes for TTE. In this context, different isolation methods, such as enzymatic digestion (ED and cell migration (CM, are investigated on differences in gene expression and cell morphology. Methods Samples of the LHB were obtained from patients, who underwent surgery for primary shoulder arthroplasty. Using ED as isolation method, 0.2% collagenase I solution was used. Using CM as isolation method, small pieces of minced tendon were put into petri-dishes. After cell cultivation, RT-PCR was performed for collagen type I, collagen type III, decorin, tenascin-C, fibronectin, Scleraxis, tenomodulin, osteopontin and agreccan. Results The total number of isolated cells, in relation to 1 g of native tissue, was 14 times higher using ED. The time interval for cell isolation was about 17 hours using ED and approximately 50 days using CM. Cell morphology in vitro was similar for both isolation techniques. Higher expression of collagen type I could be observed in tenocyte-like cell cultures (TLCC using ED as isolation method (p  Conclusion In summary tenocyte-like cells can be obtained with both isolation methods (ED and CM from the LHB. As no obvious disadvantage could be seen using ED, this method is more suitable for clinical use, as time for cell isolation is shorter and a remarkably higher number of cells can be obtained. However, both isolation methods can further be improved.

  19. Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Shigemi; Ben, T.L.; De Luca, L.M. (National Institutes of Health, Bethesda, MD (USA))

    1988-11-01

    The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10{sup {minus}9} and 10{sup {minus}7} M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

  20. Mechanical implications of the domain structure of fiber-forming collagens: comparison of the molecular and fibrillar flexibilities of the alpha1-chains found in types I-III collagen.

    Science.gov (United States)

    Silver, Frederick H; Horvath, Istvan; Foran, David J

    2002-05-21

    Fibrillar collagens store, transmit and dissipate elastic energy during tensile deformation. Results of previous studies suggest that the collagen molecule is made up of alternating rigid and flexible domains, and extension of the flexible domains is associated with elastic energy storage. In this study, we model the flexibility of the alpha1-chains found in types I-III collagen molecules and microfibrils in order to understand the molecular basis of elastic energy storage in collagen fibers by analysing the areas under conformational plots for dipeptide sequences. Results of stereochemical modeling suggest that the collagen triple helix is made up of rigid and flexible domains that alternate with periods that are multiples of three amino acid residues. The relative flexibility of dipeptide sequences found in the flexible regions is about a factor of five higher than that found for the flexibility of the rigid regions, and the flexibility of types II and III collagen molecules appears to be higher than that found for the type I collagen molecule. The different collagen alpha1-chains were compared by correlating the flexibilities. The results suggest that the flexibilities of the alpha1-chains of types I and III collagen are more closely related than the flexibilities of the alpha1-chains in types I and II and II and III collagen. The flexible domains found in the alpha1-chains of types I-III collagen were found to be conserved in the microfibril and had periods of about 15 amino acid residues and multiples thereof. The flexibility profiles of types I and II collagen microfibrils were found to be more highly correlated than those for types I and III and II and III. These results suggest that the domain structure of the alpha1-chains found in types I-III collagen is an efficient means for storage of elastic energy during stretching while preserving the triple helical structure of the overall molecule. It is proposed that all collagens that form fibers are designed to

  1. Modulation of tumor cell stiffness and migration by type IV collagen through direct activation of integrin signaling pathway.

    Science.gov (United States)

    Chen, Sheng-Yi; Lin, Jo-Shi; Yang, Bei-Chang

    2014-08-01

    Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human β1 integrin or α2β1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of β1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.

  2. Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

    Directory of Open Access Journals (Sweden)

    Barrio Daniel A

    2001-08-01

    Full Text Available Abstract Background The tissue accumulation of protein-bound advanced glycation endproducts (AGE may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS and nitric oxide synthase (NOS expression on these AGE-collagen mediated effects. Results AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP activity. In preosteoblastic MC3T3E1 cells (24-hour culture, proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. Conclusions These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

  3. Development and potential of a biomimetic chitosan/typecollagen scaffold for cartilage tissue engineering

    Institute of Scientific and Technical Information of China (English)

    SHI De-hai; CAI Dao-zhang; ZHOU Chang-ren; RONG Li-min; WANG Kun; XU Yi-chun

    2005-01-01

    Background Damaged articular cartilage has very limited capacity for spontaneous healing. Tissue engineering provides a new hope for functional cartilage repair. Creation of an appropriate cell carrier is one of the critical steps for successful tissue engineering. With the supposition that a biomimetic construct might promise to generate better effects, we developed a novel composite scaffold and investigated its potential for cartilage tissue engineering. Methods Chitosan of 88% deacetylation was prepared via a modified base reaction procedure. A freeze-drying process was employed to fabricate a three-dimensional composite scaffold consisting of chitosan and typecollagen. The scaffold was treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Ultrastructure and tensile strength of the matrix were carried out to assess its physico-chemical properties. After subcutaneous implantation in rabbits, its in vivo biocompatibility and degradability of the scaffold were determined. Its capacity to sustain chondrocyte growth and biosynthesis was evaluated through cell-scaffold co-culture in vitro. Results The fabricated composite matrix was porous and sponge-like with interconnected pores measuring from 100-250 μm in diameter. After cross-linking, the scaffold displayed enhanced tensile strength. Subcutaneous implantation results indicated the composite matrix was biocompatible and biodegradable. In intro cell-scaffold culture showed the scaffold sustained chondrocyte proliferation and differentiation, and maintained the spheric chondrocytic phenotype. As indicated by immunohistochemical staining, the chondrocytes synthesized typecollagen. Conclusions Chitosan and typecollagen can be well blended and developed into a porous 3-D biomimetic matrix. Results of physico-chemical and biological tests suggest the composite matrix satisfies the constraints specified for a tissue-engineered construct and may be used as a chondrocyte

  4. A novel rat tail collagen type-I gel for the cultivation of human articular chondrocytes in low cell density.

    Science.gov (United States)

    Muller-Rath, R; Gavénis, K; Andereya, S; Mumme, T; Schmidt-Rohlfing, B; Schneider, U

    2007-12-01

    Collagen type-I matrix systems have gained growing importance as a cartilage repair device. However, most of the established matrix systems use collagen type-I of bovine origin seeded in high cell densities. Here we present a novel collagen type-I gel system made of rat tail collagen for the cultivation of human chondrocytes in low cell densities. Rat tail collagen type-I gel (CaReS, Arthro Kinetics, Esslingen, Germany) was seeded with human passage 2 chondrocytes in different cell densities to evaluate the optimal cell number. In vitro, the proliferation factor of low density cultures was more than threefold higher compared with high density cultures. After 6 weeks of in vitro cultivation, freshly prepared chondrocytes with an initial cell density of 2x10(5) cells/mL showed a proliferation factor of 33. A cell density of 2x10(5) cells/mL was chosen for in vitro and in vivo cultivation using the common nude mouse model as an in vivo system. Chondrocytes stayed viable as a Live/Dead fluorescence assay and TUNEL staining revealed. During in vitro cultivation, passage 0 cells partly dedifferentiated morphologically. In vivo, passage 0 cells maintained the chondrocyte phenotype and demonstrated an increased synthesis of collagen type-II protein and gene expression compared to passage 2 cells. Passage 2 cells did not redifferentiate in vivo. Cultivating a cell-seeded collagen gel of bovine origin as a control (AtelocollagenTM, Koken, Tokyo, Japan) did not lead to superior results with regard to cell morphology, col-II protein production and col-II gene expression. With the CaReS collagen gel system the best quality of repair tissue was obtained by seeding freshly isolated chondrocytes.

  5. Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthritis

    DEFF Research Database (Denmark)

    Uysal, Hüseyin; Bockermann, Robert; Nandakumar, Kutty S

    2009-01-01

    Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical...... collagen type II (CII) epitope (position 359-369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII...

  6. Type II collagen and gelatin from silvertip shark (Carcharhinus albimarginatus) cartilage: isolation, purification, physicochemical and antioxidant properties.

    Science.gov (United States)

    Jeevithan, Elango; Bao, Bin; Bu, Yongshi; Zhou, Yu; Zhao, Qingbo; Wu, Wenhui

    2014-06-27

    Type II acid soluble collagen (CIIA), pepsin soluble collagen (CIIP) and type II gelatin (GII) were isolated from silvertip shark (Carcharhinus albimarginatus) cartilage and examined for their physicochemical and antioxidant properties. GII had a higher hydroxyproline content (173 mg/g) than the collagens and cartilage. CIIA, CIIP and GII were composed of two identical α1 and β chains and were characterized as type II. Amino acid analysis of CIIA, CIIP and GII indicated imino acid contents of 150, 156 and 153 amino acid residues per 1000 residues, respectively. Differing Fourier transform infrared (FTIR) spectra of CIIA, CIIP and GII were observed, which suggested that the isolation process affected the secondary structure and molecular order of collagen, particularly the triple-helical structure. The denaturation temperature of GII (32.5 °C) was higher than that of CIIA and CIIP. The antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl radicals and the reducing power of CIIP was greater than that of CIIA and GII. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. Accordingly, the physicochemical and antioxidant properties of type II collagens (CIIA, CIIP) and GII isolated from shark cartilage were found to be suitable for biomedical applications.

  7. Type II Collagen and Gelatin from Silvertip Shark (Carcharhinus albimarginatus Cartilage: Isolation, Purification, Physicochemical and Antioxidant Properties

    Directory of Open Access Journals (Sweden)

    Elango Jeevithan

    2014-06-01

    Full Text Available Type II acid soluble collagen (CIIA, pepsin soluble collagen (CIIP and type II gelatin (GII were isolated from silvertip shark (Carcharhinus albimarginatus cartilage and examined for their physicochemical and antioxidant properties. GII had a higher hydroxyproline content (173 mg/g than the collagens and cartilage. CIIA, CIIP and GII were composed of two identical α1 and β chains and were characterized as type II. Amino acid analysis of CIIA, CIIP and GII indicated imino acid contents of 150, 156 and 153 amino acid residues per 1000 residues, respectively. Differing Fourier transform infrared (FTIR spectra of CIIA, CIIP and GII were observed, which suggested that the isolation process affected the secondary structure and molecular order of collagen, particularly the triple-helical structure. The denaturation temperature of GII (32.5 °C was higher than that of CIIA and CIIP. The antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl radicals and the reducing power of CIIP was greater than that of CIIA and GII. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. Accordingly, the physicochemical and antioxidant properties of type II collagens (CIIA, CIIP and GII isolated from shark cartilage were found to be suitable for biomedical applications.

  8. Effects of Endovascular Brachytherapy with 192Ir Afterloading System on Expression of TypeCollagen after Angioplasty

    Institute of Scientific and Technical Information of China (English)

    向定成; 杨传红; 候友贤; 龚志华; 易绍东; 邱建

    2003-01-01

    Objectives To investi-gate the effect and mechanism of endovascularbrachytherapy with 192Ir on expression of typecollagen, metalloproteinases - 1 (MMP - 1) and the tissueinhibitor (TIMP- 1 ) after angioplasty. MethodsRestenotic model of domestic microswine was em-ployed and the iliac arteries were randomized to radi-ation group ( n = 12), which were treated with 20 ~ 25Gy of 192Ir, and non - radiation group ( n = 36) afterangioplasty. The target vessels were harvested in theend of 3 months and 6 months after angioplasty. Im-munohistochemistry and in situ hybridization were usedto detect proteins of typecollagen, MMP-1 andTIMP- 1, and mRNA expression of typecollagen.Results The protein and mRNA of typecollagen,the ratios of TIMP-1/MMP-1 were significantlylower iu radiation group than in non- radiation group( P < 0.05 or 0.01 ). The peak of transcription of typeⅠ collagen mRNA was at 6 months and 3 months in non-radiation group and radiation group respectively.Conclusions Endovascular brachytherapy with192Ir might modify the metabolism of extracellular ma-trix after angioplasty by inhibiting the synthesis of typeⅠ collagen and the activities of MMP - 1 and TIMP - 1.

  9. Exercise-dependent IGF-I, IGFBPs, and type I collagen changes in human peritendinous connective tissue determined by microdialysis

    DEFF Research Database (Denmark)

    Olesen, Jens L; Heinemeier, Katja M; Gemmer, Carsten

    2007-01-01

    -like growth factor I (IGF-I) and its binding proteins (IGFBPs), which are known to stimulate collagen production in animal tendons, may regulate the translation of mechanical loading to collagen synthesis. Systemic and tissue levels of IGF-I, IGFBP, and type I collagen metabolism markers [procollagen I COOH......-terminal propeptide (PICP) and COOH-terminal telopeptide of type I collagen] were measured by microdialysis in peritendinous tissue of the human Achilles tendon in an exercise group (performing a 36-km run, n = 6) and a control group (no intervention, n = 6). An increase in local PICP concentration was seen in both...... and exercise groups after 48 h (P tissue in response to prolonged mechanical loading with part of the increase due to trauma from the sampling...

  10. The non-collagenous domains of the alpha 3 and 4 chains of type IV collagen and their relationship to the Goodpasture antigen.

    Science.gov (United States)

    Savige, J A; Gallicchio, M

    1991-01-01

    The Goodpasture antigen is the target recognized by anti-glomerular basement membrane (GBM) antibodies in anti-GBM disease or Goodpasture's syndrome. This structure is present in all normal GBM, but when serum containing anti-GBM antibodies is used to examine renal tissue from most males with classical Alport's syndrome, the Goodpasture antigen appears to be missing. The nature of the Goodpasture antigen is uncertain although it has been putatively and controversially localized to the non-collagenous domain of a novel type IV collagen chain (alpha 3) by one group, and a short peptide sequence has been published (M2). We have performed several experiments to determine whether M2 represents the Goodpasture antigen and we have also studied the corresponding sequence of the alpha 4 chain of type IV collagen (M3). Firstly, we demonstrated by polymerase chain reaction (PCR) amplification using specific priming oligonucleotides that mRNAs corresponding to M2 and M3 were found within the kidney and that the published sequences were correct. When heterologous antibodies were raised against M2 and M3 these bound specifically to GBM in an ELISA based on collagenase-digested basement membrane and this binding could be inhibited by incubation with collagenase-digested GBM but not with ovalbumin. On further examination of the target molecules using Western blots, the anti-M2 antibody bound to a single high molecular weight band of collagen-digested GBM in contrast to the anti-M3 antibody that bound to the same bands as Goodpasture serum. We then established ELISAs for anti-M2 and anti-M3 activity using the peptides M2 and M3. While rabbit anti-M2 and M3 antibodies bound specifically to their respective peptides in these ELISAs, there was no binding of three high titre Goodpasture's syndrome sera or two sera from Alport's syndrome patients with inhibitable anti-GBM antibody post-renal transplant. We have shown that the sequences of M2 and M3 correspond to proteins present within

  11. Modeling the electromobility of type-I collagen molecules in the electrochemical fabrication of dense and aligned tissue constructs.

    Science.gov (United States)

    Uquillas, Jorge Alfredo; Akkus, Ozan

    2012-08-01

    Isoelectric focusing (IEF) of type-I collagen molecules is a technology with proven efficacy to produce dense and aligned collagen-based biomaterials. The forces and mechanisms during IEF of collagen molecules in carrier ampholyte-free environments remain unknown. This study presents theoretical framework describing the congregation of collagen molecules along the isoelectric point (pI). A single molecule was modeled as a rod-like particle, distributed homogeneously between parallel electrodes. Upon application of electrical current, molecules migrated to the pI. The results showed that self-aggregation of collagen molecules along the pI occurred due to formation of a non-linear pH gradient that rendered the anodic side acidic, and the cathodic side basic. This pH profile and the amphoteric nature of collagen resulted in positively charged molecules at the anode and negatively charged molecules at the cathode. Therefore, repulsive electrostatic forces aided self-aggregation of molecules along the pI. The model could effectively validate the pI of collagen, the pI location, and predict that the instantaneous velocity acting on a molecule at the anode was higher than those velocities at the cathode. This fundamental information represents the baseline theory upon which we can expand our knowledge to the production of biomaterials to engineer soft tissues.

  12. 6β-hydroxytestosterone, a cytochrome P450 1B1 metabolite of testosterone, contributes to angiotensin II-induced hypertension and its pathogenesis in male mice.

    Science.gov (United States)

    Pingili, Ajeeth K; Kara, Mehmet; Khan, Nayaab S; Estes, Anne M; Lin, Zongtao; Li, Wei; Gonzalez, Frank J; Malik, Kafait U

    2015-06-01

    Previously, we showed that Cyp1b1 gene disruption minimizes angiotensin II-induced hypertension and associated pathophysiological changes in male mice. This study was conducted to test the hypothesis that cytochrome P450 1B1-generated metabolites of testosterone, 6β-hydroxytestosterone and 16α-hydroxytestosterone, contribute to angiotensin II-induced hypertension and its pathogenesis. Angiotensin II infusion for 2 weeks increased cardiac cytochrome P450 1B1 activity and plasma levels of 6β-hydroxytestosterone, but not 16α-hydroxytestosterone, in Cyp1b1(+/+) mice without altering Cyp1b1 gene expression; these effects of angiotensin II were not observed in Cyp1b1(-/-) mice. Angiotensin II-induced increase in systolic blood pressure and associated cardiac hypertrophy, and fibrosis, measured by intracardiac accumulation of α-smooth muscle actin, collagen, and transforming growth factor-β, and increased nicotinamide adenine dinucleotide phosphate oxidase activity and production of reactive oxygen species; these changes were minimized in Cyp1b1(-/-) or castrated Cyp1b1(+/+) mice, and restored by treatment with 6β-hydroxytestoterone. In Cyp1b1(+/+) mice, 6β-hydroxytestosterone did not alter the angiotensin II-induced increase in systolic blood pressure; the basal systolic blood pressure was also not affected by this agent in either genotype. Angiotensin II or castration did not alter cardiac, angiotensin II type 1 receptor, angiotensin-converting enzyme, Mas receptor, or androgen receptor mRNA levels in Cyp1b1(+/+) or in Cyp1b1(-/-) mice. These data suggest that the testosterone metabolite, 6β-hydroxytestosterone, contributes to angiotensin II-induced hypertension and associated cardiac pathogenesis in male mice, most probably by acting as a permissive factor. Moreover, cytochrome P450 1B1 could serve as a novel target for developing agents for treating renin-angiotensin and testosterone-dependent hypertension and associated pathogenesis in males.

  13. A novel deletion and two recurrent substitutions on type VII collagen gene in seven Iranian patients with epidermolysis bullosa.

    Science.gov (United States)

    Hamidi, Armita Kakavand; Moghaddam, Mohammad; Hatamnejadian, Nasim; Ebrahimi, Ahmad

    2016-08-01

    Epidermolysis bullosa is one of the most important series of mechano-bullous heritable skin disorders which is categorized into four major types according to the layer that bullae forms within basement membrane zone. In dystrophic form of the disease, blisters are made in the sublamina densa zone, at the level of type VII collagen protein which produce anchoring fibrils. Type VII collagen gene is the only responsible gene for this form. The aim of this study was to survey causative mutations of type VII collagen gene among Iranian patients with epidermolysis bullosa. For this purpose, exons 73-75 were investigated by polymerase chain reaction followed by direct sequencing. In current study, we found three different point mutations in type VII collagen alleles in 7 out of 50 patients. Four patients were homozygous for a new deletion which resulted in frame shift (p.Pro2089fs). Two patients were homozygous for a recurrent glycine substitution (p.G2031S) and one patient was detected with an allele carrying a substitution (p.R2069C). The results emphasized heterogeneity in the type VII collagen gene and will provide a sign for early diagnosis and future study of the disease pathogenesis.

  14. Development of a sensing system to detect C-telopeptide of type-I collagen

    KAUST Repository

    Afsarimanesh, Nasrin

    2016-03-30

    This research work describes a non-invasive and label-free immunosensing technique to detect the C-telopeptide of type-I collagen (CTX-1) by Electrochemical Impedance Spectroscopy (EIS). A planar interdigital capacitive sensor is used to evaluate the properties of the material under test. This sensor was fabricated on the basis of thin film micro-electromechanical system (MEMS) semiconductor device fabrication technology. EIS was used in conjunction with the sensor to detect collagen type-I in blood plasma. At the first stage, the Serum CrossLaps® ELISA was used to measure some known samples in order to obtain a standard curve. Streptavidin agarose was successfully immobilized on the sensing area of the sensor. After that the experiments were done with antibody solution and three known samples of CTX-1, zero concentration which was considered as control, 2.669 ng/ml and 0.798 ng/ml concentration. The results are encouraging for further investigation.

  15. Effects of oral administration of typecollagen on adjuvant arthritis in rat sand its mechanisms

    Institute of Scientific and Technical Information of China (English)

    胡永秀; 赵文明; 钱娴娟; 张力平

    2003-01-01

    Objective To investigate the effects of oral administration of typecollagen (CⅡ) on a djuvant arthritis (AA) in rats and its mechanisms, and to compare the effects of CⅡ with those of the Chinese traditional medicine Tripterygium Polyglycoside a dministered similarly.Methods Arthritis was induced in rats by immunization using Freund's complete adjuvant (FCA). After feeding rats either soluble CⅡ or Tripterygium Polyglycoside, chan ges in degree of articular swelling and articular histological findings were observed in AA rats. Some correlative immunological indexes were measured, includi ng delayed type hypersensitivity (DTH) reaction, anti-collagen and anti-Mycoba cterium tuberculosis (MT) antibody in serum, and levels of IFN-γ and TNF-α i n articular steep in rats.Results Oral administration of CⅡ was able to alleviate both distinctly articular and general symptoms in AA rats, suppress synovium hyperplasia and inflammatory cells infiltration in arthrosis capsule. The effects brought about by CⅡ were stronger than those by Tripterygium Polyglycoside. Oral administration of CⅡ inhibi ted antigen-specific immune response, such as DTH and antibody reaction to CⅡ . In addition, the expression of IFN-γ and TNF-α in joints were locally dow nregulated. Conclusions The therapeutic effect of oral administration of CⅡ is obvious on adjuvant art hritis in rats. Its remedial mechanisms are likely related to the downregulation of both IFN-γ and TNF-α, and the suppression of cell immunity.

  16. Type IV collagen as marker of fibrosis in nonalcoholic liver disease

    Directory of Open Access Journals (Sweden)

    Alvina Alvina

    2016-02-01

    Full Text Available Currently nonalcoholic fatty liver disease (NAFLD and nonalcoholic steatohepatitis (NASH are medical problems associated with the increasing prevalence of diabetes mellitus, obesity, hypertension and hypertriglyceridemia, usually designated as the metabolic syndrome associated with insulin resistance. One study demonstrated an increase in NAFLD prevalence of around 17-33% and in NASH prevalence of 5.7-16.5%. NAFLD comprises a range of mild to severe conditions, from simple steatosis to steatohepatitis, hepatic fibrosis and cirrhosis. The diagnosis of hepatic fibrosis is important for prognosis, stratification for treatment, and monitoring of treatment efficacy. Ultrasonography (USG is a simple method for detecting fatty infiltrates in the liver. USG has a sensitivity of 82-89% and a specificity of 93%, but cannot differentiate between hepatic steatosis and fibrosis. The gold standard for evaluation of hepatic fibrosis is liver biopsy, which however is a painful and invasive procedure. Currently determination of serum type IV collagen has been suggested as an alternative to liver biopsy among the non-invasive methods for evaluation of hepatic fibrosis, as its serum concentration is closely correlated with advanced hepatic fibrosis in NASH. Type IV collagen is one of the components of basement membrane and its serum concentration is indicative of degradation of the extracellular matrix.

  17. Microchimeric fetal cells are recruited to maternal kidney following injury and activate collagen type I transcription.

    Science.gov (United States)

    Bou-Gharios, George; Amin, Farhana; Hill, Peter; Nakamura, Hiroyuki; Maxwell, Patrick; Fisk, Nicholas M

    2011-01-01

    Fetal cells enter the maternal circulation from the early first trimester of pregnancy, where they persist in tissue decades later. We investigated in mice whether fetal microchimeric cells (FMCs) can be detected in maternal kidney, and whether they play a role in kidney homeostasis. FMCs were identified in vivo in two models: one an adaptive model following unilateral nephrectomy, the other an injury via unilateral renal ischaemia reperfusion. Both models were carried out in mothers that had been mated with transgenic mice expressing luciferase transgene under the control of collagen type I, and had given birth to either 1 or 3 litters. FMCs were detected by Y-probe fluorescent in situ hybridization (FISH) and bioluminescence, and the cell number quantified by real-time polymerase chain reaction. In the adaptive model, the remaining kidney showed more cells by all 3 parameters compared with the nephrectomized kidney, while ischaemia reperfusion resulted in higher levels of FMC participation in injured compared to contralateral kidneys. Bioluminescence showed that FMCs switch on collagen type I transcription implicating mesenchymal lineage cells. After injury, Y-probe in situ hydridization was found mainly in the tubular epithelial network. Finally, we compared FMCs with bone marrow cells and found similar dynamics but altered distribution within the kidney. We conclude that FMCs (1) are long-term sequelae of pregnancy and (2) are recruited to the kidney as a result of injury or adaptation, where they activate the transcriptional machinery of matrix proteins.

  18. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

    Science.gov (United States)

    Yeh, Hsi-Yi; Lin, Ting-Yu; Lin, Chen-Huan; Yen, B Linju; Tsai, Ching-Lin; Hsu, Shan-Hui

    2013-01-01

    Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

  19. L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

    Science.gov (United States)

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

    2012-10-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

  20. Type-1 Collagen differentially alters β-catenin accumulation in primary Dupuytren's Disease cord and adjacent palmar fascia cells

    Directory of Open Access Journals (Sweden)

    Gan Bing

    2009-06-01

    Full Text Available Abstract Background Dupuytren's Disease (DD is a debilitating contractile fibrosis of the palmar fascia characterised by excess collagen deposition, contractile myofibroblast development, increased Transforming Growth Factor-β levels and β-catenin accumulation. The aim of this study was to determine if a collagen-enriched environment, similar to in vivo conditions, altered β-catenin accumulation by primary DD cells in the presence or absence of Transforming Growth Factor-β. Methods Primary DD and patient matched, phenotypically normal palmar fascia (PF cells were cultured in the presence or absence of type-1 collagen and Transforming Growth Factor-β1. β-catenin and α-smooth muscle actin levels were assessed by western immunoblotting and immunofluorescence microscopy. Results DD cells display a rapid depletion of cellular β-catenin not evident in patient-matched PF cells. This effect was not evident in either cell type when cultured in the absence of type-1 collagen. Exogenous addition of Transforming Growth Factor-β1 to DD cells in collagen culture negates the loss of β-catenin accumulation. Transforming Growth Factor-β1-induced α-smooth muscle actin, a marker of myofibroblast differentiation, is attenuated by the inclusion of type-1 collagen in cultures of DD and PF cells. Conclusion Our findings implicate type-1 collagen as a previously unrecognized regulator of β-catenin accumulation and a modifier of TGF-β1 signaling specifically in primary DD cells. These data have implications for current treatment modalities as well as the design of in vitro models for research into the molecular mechanisms of DD.

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

    DEFF Research Database (Denmark)

    Veidal, Sanne S.; Karsdal, Morten A.; Nawrocki, Arkadiusz

    2011-01-01

    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......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...... 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....

  2. Role of 17 beta-estradiol on type IV collagen fibers volumetric density in the basement membrane of bladder wall.

    Science.gov (United States)

    de Fraga, Rogerio; Dambros, Miriam; Miyaoka, Ricardo; Riccetto, Cássio Luís Zanettini; Palma, Paulo César Rodrigues

    2007-10-01

    The authors quantified the type IV collagen fibers volumetric density in the basement membrane of bladder wall of ovariectomized rats with and without estradiol replacement. This study was conducted on 40 Wistar rats (3 months old) randomly divided in 4 groups: group 1, remained intact (control); group 2, submitted to bilateral oophorectomy and daily replacement 4 weeks later of 17 beta-estradiol for 12 weeks; group 3, sham operated and daily replacement 4 weeks later of sesame oil for 12 weeks; and group 4, submitted to bilateral oophorectomy and killed after 12 weeks. It was used in immunohistochemistry evaluation using type IV collagen polyclonal antibody to stain the fibers on paraffin rat bladder sections. The M-42 stereological grid system was used to analyze the fibers. Ovariectomy had an increase effect on the volumetric density of the type IV collagen fibers in the basement membrane of rat bladder wall. Estradiol replacement in castrated animals demonstrated a significative difference in the stereological parameters when compared to the castrated group without hormonal replacement. Surgical castration performed on rats induced an increasing volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall and the estradiol treatment had a significant effect in keeping a low volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall.

  3. Anti-type II collagen antibodies detection and avidity in patients with oligoarticular and polyarticular forms of juvenile idiopathic arthritis.

    Science.gov (United States)

    Araujo, Galber R; Fonseca, João E; Fujimura, Patricia T; Cunha-Junior, Jair P; Silva, Carlos H M; Mourão, Ana F; Canhão, Helena; Goulart, Luiz R; Gonçalves, João; Ueira-Vieira, Carlos

    2015-05-01

    Juvenile idiopathic arthritis (JIA) refers to a heterogeneous group of illnesses that have in common the occurrence of chronic joint inflammation in children younger than 16 years of age. The diagnosis is made only on clinical assessment. The identification of antibody markers could improve the early diagnosis, optimizing the clinical management of patients. Type II collagen is one potential autoantigen that has been implicated in the process of arthritis development. The aims of our study were to investigate the occurrence of anti-type II collagen antibodies and also to determine the avidity of the antibody-antigen binding. Ninety-six patients with oligoarticular or polyarticular JIA, 13 patients with ankylosing spondylitis (AS) and 61 healthy controls (HC) were tested for anti-type II collagen antibodies by ELISA and avidity ELISA. Sensitivity and specificity were determined by the receiver operating characteristic (ROC) curve analysis. Forty-two JIA patients (44%) were positive for antibodies against type II collagen. Its detection was significantly higher in JIA patients than in AS patients (p=0.006) and HCs (ppolyarticular JIA, being its presence more prevalent in patients with early disease. It also demonstrates that JIA patients with active disease present antibodies with high avidity against type II collagen.

  4. Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.

    Science.gov (United States)

    Paavola, Kevin J; Sidik, Harwin; Zuchero, J Bradley; Eckart, Michael; Talbot, William S

    2014-08-12

    GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes.

  5. Effect of succinic acid monoethyl ester on hemoglobin glycation and tail tendon collagen properties in type 2 diabetic rats.

    Science.gov (United States)

    Saravanan, Ramalingam; Pari, Leelavinothan

    2008-06-01

    Succinic acid monoethyl ester (EMS) was recently proposed as an insulinotropic agent for the treatment of type 2 diabetes. The aim of the study was to investigate the effect of EMS and metformin administration on tail collagen content and its characteristics in streptozotocin-nicotinamide-induced type 2 diabetic rats. EMS was administered intraperitoneally for 30 days to normal and diabetic rats. In the diabetic rats, a significant increase in the levels of glucose, glycated hemoglobin, hydroxyproline, collagen content, extent glycation, fluorescence, neutral salt, acid and pepsin soluble collagen content was absorbed with a significant decrease in the level of insulin, hemoglobin in streptozotocin-nicotinamide diabetic rats. Moreover, a daily administration of nonglucidic nutrient EMS and metformin significantly decreased the levels of glucose, glycated hemoglobin, hydroxyproline, collagen content, extent glycation, fluorescence, neutral salt, acid and pepsin soluble collagen content, whereas it increased insulin, hemoglobin levels in diabetic rats. The positive influence of nonglucidic nutrient on both collagen content and its properties suggests a potential mechanism for the ability of EMS to delay diabetic complications.

  6. Effects of type I collagen degradation on the durability of three adhesive systems in the early phase of dentin bonding.

    Directory of Open Access Journals (Sweden)

    Lin Hu

    Full Text Available This study was designed to evaluate the effects of type I collagen degradation on the durability of three adhesive systems in the early phase of dentin bonding.Bonded dentin specimens were prepared using three different types of adhesive systems. Micro-tensile bond strength and degradation of collagen were tested before, and after 1 month or 4 months of aging in artificial saliva. The relationship between micro-tensile bond strength and collagen degradation was analyzed by calculating their Pearson's correlation coefficient.Aging induced time-dependent reduction in micro-tensile bond strengths for all the tested adhesive systems, although such reduction for the single-step self-etching adhesive G-Bond (GB was not statistically significant. The bond strength of the two-step self-etching primer adhesive system Clearfil SE Bond (SEB was similar to that of the two-step etch-and-rinse self-priming adhesive system Single Bond 2 (SB, and they were both significantly reduced after one or four months of aging. A negative correlation was found between the degree of collagen degradation and magnitude of micro-tensile bond strength (r = -0.65, p = 0.003. The Pearson's correlation coefficient was 0.426, indicating that 42.6% of the aging-induced reduction in bond strength can be explained by the degradation of collagen.In the early phase of dentin bonding, there was a negative correlation between the degree of collagen degradation and the magnitude of micro-tensile bond strength. The reduction of bond strength was accompanied by the degradation of collagen. These results provide evidence for the causative relationship between the degradation of collagen and the deterioration of dentin-adhesive interface.

  7. Immunohistochemical expression of types I and III collagen antibodies in the temporomandibular joint disc of human foetuses

    Directory of Open Access Journals (Sweden)

    L.O.C. de Moraes

    2011-07-01

    Full Text Available The objective was to study the morphology of the articular disc and analyse the immunohistochemical expression of types I and III collagen markers in the temporomandibular joint (TMJ disc of human foetuses of different gestational ages. Twenty TMJ from human foetuses supplied by Universidade Federal de Uberaba with gestational ages from 17 to 24 weeks were studied. The gestational age of the foetuses was determined by measuring the crown-rump (CR length. Macroscopically, the foetuses were fixed in 10% formalin solution and dissected by removing the skin and subcutaneous tissue and exposing the deep structures. Immunohistochemical markers of type I and III were used to characterize the existence of collagen fibres. Analysis of the immunohistochemical markers of types I and III collagen revealed the presence of heterotypical fibril networks.

  8. Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

    Science.gov (United States)

    Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

    2016-01-01

    The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that

  9. Image analyses of collagen types and thickness in oral sub mucous fibrosis stained with picrosirius red under polarizing microscope

    Directory of Open Access Journals (Sweden)

    Venkatesh V Kamath

    2013-01-01

    Full Text Available Context: Oral submucous fibrosis (OSF is a potentially malignant oral disorder leading to increased fibrosis in the sub-epithelial layer. The collagen in the condition has been a subject of intense scrutiny in an attempt to understand the pathogenesis of the disease. Aim: The present study aims to quantify and qualify the collagen fibers in different histological grades of OSF using picrosirius red stain under the polarizing microscope. The quantification of the fibrosis was carried out using image analysis software and the fibers were graded according to staining hue and intensity into their respective subtypes. Comparison was done with normal mucosa, scar/keloid tissue samples. Materials and Methods: The present study included OSF (n = 50 of differing histological grades, keloid/scar (n = 4 and normal mucosa (n = 6 as control cases. Histological assessment was performed on hematoxylin and eosin stained sections. Picrosirius red stained slides were observed under a polarizing microscope for assessment of collagen subtypes. Quantification of collagen was done under polarizing microscope and image parameters were analyzed using ProReg® Capture Pro 2.8.8 (Lawrence and Mayo India Pvt Ltd, 2011 image analysis software. Results: The epithelial thickness in OSF, scar and keloid is less than that of normal mucosa and progressive decrease in the epithelial thickness is seen in the successive stages of OSF. The fibrosis increases with increasing grades of OSF, was higher in scar and keloid and was highly statistically significant. Type I collagen was more predominant in all stages of OSF, in normal oral mucosa and scar/keloid tissue samples as compared with type III. Though quantitative analysis of the collagen types I and III is possible, with picrosirius red qualitative analysis is an arduous task. The specificity of detection of collagen subtypes was acceptable with the picrosirius red stain, but the sensitivity left a lot to be desired.

  10. Immunohistochemical analysis of type III collagen expression in the lingual mucosa of rats during organogenesis of the tongue.

    Science.gov (United States)

    Iwasaki, Shin-Ichi; Asami, Tomoichiro; Wanichanon, Chaitip; Yoshizawa, Hideki; Aoyagi, Hidekazu

    2008-07-01

    We examined the distribution of immunofluorescence due to immunostaining of type III collagen, differential interference contrast (DIC) images and images obtained in the transmission mode after toluidine blue staining by laser-scanning microscopy of semi-ultrathin sections of epoxy resin-embedded samples, during morphogenesis of the filiform papillae, keratinization of the lingual epithelium, and myogenesis of the rat tongue. Immunoreactivity specific for type III collagen was distributed widely in the mesenchymal connective tissue in fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of one or two layers of cuboidal cells and the lingual muscle was barely recognizable. Immunoreactivity specific for type III collagen was clearly detected on the lamina propria in fetuses on E17 and E19, and it was relatively distinct just beneath the lingual epithelium. Immunoreactivity specific for type III collagen was sparsely distributed on the connective tissue around the developing lingual muscle. In fetuses on E19, the epithelium became clearly stratified and squamous. At postnatal stages from newborn (P0) to postnatal day 14 (P14), keratinization of the lingual epithelium advanced gradually with the development of filiform papillae. On P0, myogenesis of the tongue was almost completed. The intensity of the fluorescence immunoreactivity specific for type III collagen at postnatal stages was almost same as that on E19. The immunoreactivity around the fully mature muscle was relatively distinct between P0 and P14. Thus, type III collagen appeared in conjunction with the morphogenesis of filiform papillae and the keratinization of the lingual epithelium as well as in the connective tissue that surrounded the lingual muscle during myogenesis of the rat tongue.

  11. Serum antibodies to type II collagen in rheumatoid arthritis: comparison of 6 immunological methods and clinical features.

    Science.gov (United States)

    Clague, R B; Firth, S A; Holt, P J; Skingle, J; Greenbury, C L; Webley, M

    1983-10-01

    A collaborative study of 75 selected patients with rheumatoid arthritis (RA) employing 6 different methods for the detection of antibodies to type II collagen showed highly significant correlations between all the assays. The radioimmunoassays showed a greater sensitivity than either the passive haemagglutination or immunofluorescent techniques, and when the native collagen molecule was heat-denatured a higher number of patients showed increased antibody levels. In 33 patients the measurement of serum antibody levels to human, bovine, and rat native type II collagen showed a lack of species specificity, indicating that heterologous collagens can be employed in these assays. A retrospective analysis of the clinical, laboratory, and radiological features in the 41 patients with raised antibody levels and the 34 patients with normal antibody levels showed very few differences, but there was a significantly lower incidence of subcutaneous nodules (24% versus 56%) in patients with raised antibody levels. This study emphasizes the need to standardize assays for the measurement of serum antibody levels to native type II collagen. More extensive studies will be required before the clinical significance of these antibodies can be fully established.

  12. N-Phenethyl caffeamide and photodamage: protecting skin by inhibiting type I procollagen degradation and stimulating collagen synthesis.

    Science.gov (United States)

    Chiang, Hsiu-Mei; Chen, Chien-Wen; Lin, Tzu-Yu; Kuo, Yueh-Hsiung

    2014-10-01

    Skin is mainly damaged by genetic and environmental factors such as ultraviolet (UV) light and pollutants. UV light is a well-known factor that causes various types of skin damage and premature aging. Reactive oxygen species (ROS) are commonly involved in the pathogenesis of skin damage by activating the metalloproteinases that break down type I collagen. This study investigated the antioxidant and antiphotodamage activity and mechanisms of N-phenethyl caffeamide (K36) in human skin fibroblasts. The results indicated that K36 demonstrated strong 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, which dose-dependently reduced the production of UVB-induced intracellular ROS in human dermal fibroblasts. K36 prevented UVB-irradiation-induced type I collagen degradation by inhibiting the expression of matrix metalloproteins-1, -3, and -9 and the phosphorylation of mitogen-activated protein (MAP) kinases. Furthermore, K36 elevated collagen synthesis in skin fibroblasts by inhibiting UVB-induced Smad7 overexpression. K36 downregulated the expression of the transcription factor, activator protein-1 (AP-1). Our results indicated that K36 exhibited antioxidant properties and prevented skin collagen degradation caused by UV exposure and the stimulation of collagen synthesis, which suggests the potential use of K36 in preventing photodamage.

  13. The effects of proteoglycan and type II collagen on T1rho relaxation time of articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Won Seok; Yoo, Hye Jin; Hong, Sung Hwan; Choi, Ja Young [Dept. of Radiology and Institute of Radiation Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul (Korea, Republic of)

    2015-02-15

    To evaluate the effects of proteoglycan and type II collagen within articular cartilage on T1rho relaxation time of articular cartilage. This study was exempted by the institutional and animal review boards, and informed consent was not required. Twelve porcine patellae were assigned to three groups of control, trypsin-treated (proteoglycan-degraded), or collagenase-treated (collagen-degraded). The T1rho images were obtained with a 3 tesla magnetic resonance imaging scanner with a single loop coil. Statistical differences were detected by analysis of variance to evaluate the effects of the enzyme on T1rho relaxation time. Safranin-O was used to stain proteoglycan in the articular cartilage and immunohistochemical staining was performed for type II collagen. Mean T1rho values of the control, trypsin-treated, and collagenase-treated groups were 37.72 +/- 5.82, 57.53 +/- 8.24, and 45.08 +/- 5.31 msec, respectively (p < 0.001). Histology confirmed a loss of proteoglycan and type II collagen in the trypsin- and collagenase-treated groups. Degradation of proteoglycans and collagen fibers in the articular cartilage increased the articular cartilage T1rho value.

  14. Involvement of 90-kuD ribosomal S6 kinase in collagen type Ⅰ expression in rat hepatic fibrosis

    Institute of Scientific and Technical Information of China (English)

    Miao-Fang Yang; Jun Xie; Xiao-Yi Gu; Xiao-Hua Zhang; Andrew K Davey; Shuang-Jie Zhang; Ji-Ping Wang; Ren-Min Zhu

    2009-01-01

    AIM: To investigate the relationship between 90-kuD ribosomal S6 kinase (p90RSK) and collagen type Ⅰ expression during the development of hepatic fibrosis in vivo and in vitro. METHODS: Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type Ⅰ were determined by co-immunofluoresence and confocal microscopy. Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell (HSC) line. The expression of collagen type Ⅰ in HSC-T6 cells was assessed by Western blotting and real-time polymerase chain reaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, then collagen type Ⅰ promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated with or without platelet-derived growth factor (PDGF)-BB at a final concentration of 20 μg/L and the cell growth was determined by MTS conversion. RESULTS: In fibrotic liver tissues, p90RSK was overexpressed in activated HSCs and had a significant positive correlation with collagen type Ⅰ levels. In HSC-T6 cells transfected with RNAi targeted to p90RSK, the expression of collagen type Ⅰ was downregulated (61.8% in mRNA, P < 0.01, 89.1% in protein, P < 0.01). However, collagen type Ⅰ promoter activity was not increased with over-expression of p90RSK and not decreased with low expression either, compared with controls in the same cell line ( P = 0.076). Furthermore, p90RSK siRNA exerted the inhibition of HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation. CONCLUSION: p90RSK is over-expressed in activated HSCs and involved in regulating the abnormal expression of collagen type Ⅰ through initiating the proliferation of HSCs.

  15. Influence of functionalized nanoparticles on conformational stability of type I collagen for possible biomedical applications.

    Science.gov (United States)

    Kandamchira, Aswathy; Selvam, Sangeetha; Marimuthu, Nidhin; Janardhanan, Sreeram Kalarical; Fathima, Nishter Nishad

    2013-12-01

    Collagen-nanoparticle interactions are vital for many biomedical applications including drug delivery and tissue engineering applications. Iron oxide nanoparticles synthesized using starch template according to our earlier reported procedures were functionalized by treating them with Gum Arabic (GA), a biocompatible polysaccharide, so as to enhance the interaction between nanoparticle surfaces and collagen. Viscosity, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) techniques have been used to study the collagen-nanoparticle interactions. The relative viscosity for collagen-nanoparticle conjugate was found to increase with increase in concentration of the nanoparticle within the concentration range investigated, which is due to the aggregation of protein onto the surface of nanoparticle. The CD spectra for the collagen-nanoparticle at different concentration ratios do not have much variation in the Rpn values (ratio of positive peak intensity over negative peak intensity) after functionalization with GA. The variation of molar ellipticity values for collagen-nanoparticle is due to the glycoprotein present in GA. The collagen triple helical structure is maintained after interaction with nanoparticles. The FTIR spectra of native collagen, Coll-Fs (nanoparticle without functionalization) and Coll-FsG (nanoparticle functionalized with GA) show clearly the amide I, II, III bands, with respect to collagen. The ability of polysaccharide stabilized/functionalized nanoparticles to maintain the collagen properties would help in its biomedical applications.

  16. Effects of connective tissue growth factor and collagen type Ⅰ scleroderma

    Institute of Scientific and Technical Information of China (English)

    Xiaoning Yan; Jie Feng; Bingjun Shi

    2007-01-01

    Objective: To investigate the effects of connective tissue growth factor(CTGF) and collagen type Ⅰ(COL-I) on the pathogenesis of scleroderma and explore the relationship between the level of COL-I and CTGF. Methods: 12 mice model of scleroderma was established by the injection of Bleomycin. The level of CTGF and COL-I were detected by immunohistochemical method. The relationship was analyzed between CTGF and COL-I level. As control group, 12 healthy mice were selected. Results: The levels of CTGF and COL-I in sclerotic models were higher than in normal controls (P < 0.05). It was found that there was a correlation between the level of CTGF and COL-I. Conclusion: CTGF and COL-I played an important role in the hardening process of the skin lesions of the mice model, which may be involved in the pathogenesis of scleroderma.

  17. Fast determination of three-dimensional fibril orientation of type-I collagen via macroscopic chirality

    Science.gov (United States)

    Zhuo, Guan-Yu; Chen, Mei-Yu; Yeh, Chao-Yuan; Guo, Chin-Lin; Kao, Fu-Jen

    2017-01-01

    Polarization-resolved second harmonic generation (SHG) microscopy is appealing for studying structural proteins and well-organized biophotonic nanostructures, due to its highly sensitized structural specificity. In recent years, it has been used to investigate the chiroptical effect, particularly SHG circular dichroism (SHG-CD) in biological tissues. Although SHG-CD attributed to macromolecular structures has been demonstrated, the corresponding quantitative analysis and interpretation on how SHG correlates with second-order susceptibility χ(2) under circularly polarized excitations remains unclear. In this study, we demonstrate a method based on macroscopic chirality to elucidate the correlation between SHG-CD and the orientation angle of the molecular structure. By exploiting this approach, three-dimensional (3D) molecular orientation of type-I collagen is revealed with only two cross polarized SHG images (i.e., interactions of left and right circular polarizations) without acquiring an image stack of varying polarization.

  18. Immobilizing collagen type to TiO2film for improvement of biocompatibility

    Institute of Scientific and Technical Information of China (English)

    Xiao-song JIANG; Jun-ying CHEN; Nan HUANG

    2008-01-01

    In this work, using a bio-chemical modification method, collagen type Ⅰ was immobilized on the TiO2 film surface by a silane coupling reagent of aminopropyl-triethoxysilane. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were used to investigate the characteristics of the modified TiO2 film. The thrombus formation ability of the films was studied by in vitro platelet adhesion test. Furthermore, the bio-logical behavior of cultured human umbilical vein endothelial cells (HUVECs) onto different films was investigated by in vitro HUVECs cultured experiment. The results show that the modification can improve the biocompatibility of TiO2 film for applications of biome-dical microcoil hemangioma treatment, etc.

  19. Effects of micropatterned surfaces coated with type I collagen on the proliferation and morphology of tenocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen Xi; Wang Zhi [Institute of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 37 Wainan Guoxue Street, Chengdu 610041, Sichuan (China); Qin Tingwu [Institute of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 37 Wainan Guoxue Street, Chengdu 610041, Sichuan (China)], E-mail: tingwuqin@hotmail.com; Liu Chengjun; Yang Zhiming [Institute of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 37 Wainan Guoxue Street, Chengdu 610041, Sichuan (China)

    2008-11-15

    The effects of micropatterned surfaces coated with type I collagen (CNI) on the proliferation and morphology of rat tail tenocytes were investigated in this study. The micropatterned polydimethylsiloxane substrates were prepared by using the technique of microcontact printing and then coated with different concentrations of CNI by the microfluidic channels technology. After being seeded on the CNI-coated micropatterned substrates, the tenocytes were tested by MTT colorimetric assay at 1-, 3-, 5-, and 7-day time intervals to evaluate the proliferation of tenocytes on the substrates. The alignment and morphology of tenocytes on the CNI-coated substrates after incubation for 1 or 24 h were observed with SEM. The results showed tenocytes proliferated well with increase of CNI concentrations and identically aligned along the grooves of the CNI-coated micropatterned substrates. This could have a potential advantage in construction of engineered tendons in vitro.

  20. Effects of micropatterned surfaces coated with type I collagen on the proliferation and morphology of tenocytes

    Science.gov (United States)

    Chen, Xi; Wang, Zhi; Qin, Ting-Wu; Liu, Cheng-Jun; Yang, Zhi-Ming

    2008-11-01

    The effects of micropatterned surfaces coated with type I collagen (CNI) on the proliferation and morphology of rat tail tenocytes were investigated in this study. The micropatterned polydimethylsiloxane substrates were prepared by using the technique of microcontact printing and then coated with different concentrations of CNI by the microfluidic channels technology. After being seeded on the CNI-coated micropatterned substrates, the tenocytes were tested by MTT colorimetric assay at 1-, 3-, 5-, and 7-day time intervals to evaluate the proliferation of tenocytes on the substrates. The alignment and morphology of tenocytes on the CNI-coated substrates after incubation for 1 or 24 h were observed with SEM. The results showed tenocytes proliferated well with increase of CNI concentrations and identically aligned along the grooves of the CNI-coated micropatterned substrates. This could have a potential advantage in construction of engineered tendons in vitro.

  1. Type XV collagen in human colonic adenocarcinomas has a different distribution than other basement membrane zone proteins.

    Science.gov (United States)

    Amenta, P S; Briggs, K; Xu, K; Gamboa, E; Jukkola, A F; Li, D; Myers, J C

    2000-03-01

    In situ carcinomas must penetrate their own basement membrane to be classified as invasive, and subsequently infiltrate surrounding connective tissue and cross vascular basement membranes to metastasize hematogenously. Accordingly, in many studies, integral basement membrane components, including type IV collagen, laminin, and heparan sulfate proteoglycan, have been localized in a spectrum of tumors to gain insight into their role in neoplasia. A number of recently identified extracellular matrix molecules and isoforms of the aforementioned proteins have been localized to the basement membrane zone, illustrating another level of biochemical heterogeneity in these structures. As the complexity of these matrices becomes more apparent, their roles in maintaining homeostasis and in tumor biology falls into question. Of the new group of collagens localized to the basement membrane zone, type XV was the first to be characterized (Cell Tissue Res, 286:493-505, 1996). This nonfibrillar collagen has a nearly ubiquitous distribution in normal human tissues via a strong association with basement membrane zones, suggesting that it functions to adhere basement membrane to the underlying stroma. To begin investigation of this protein in malignant tumors, we have localized type XV in human colonic adenocarcinomas and compared its distribution with that of type IV collagen and laminin. Collagens XV and IV and laminin were found in all normal and colonic epithelial, muscle, fat, neural, and vascular basement membrane zones, as shown previously. In moderately differentiated, invasive adenocarcinomas, laminin and type IV collagen were sometimes observed as continuous, linear deposits around some of the malignant glands, but more often they were seen in either discontinuous deposits or were completely absent. In contrast, type XV collagen was characterized as virtually absent from the basement membrane zones of malignant glandular elements in moderately differentiated tumors

  2. Matrix metalloproteinase-9-mediated type III collagen degradation as a novel serological biochemical marker for liver fibrogenesis

    DEFF Research Database (Denmark)

    Veidal, Sanne S; Vassiliadis, Efstathios; Barascuk, Natasha

    2010-01-01

    During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via their acti......During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via...... their activity in the proteolytic degradation of extracellular macromolecules such as collagens, resulting in the generation of specific cleavage fragments. These neo-epitopes may be used as markers of fibrosis....

  3. Electrodeposition of platinum nanoclusters on type I collagen modified electrode and its electrocatalytic activity for methanol oxidation

    Science.gov (United States)

    Sun, Yujing; Sun, Lanlan; Xu, Fugang; Guo, Cunlan; Liu, Zhelin; Zhang, Yue; Yang, Tao; Li, Zhuang

    2009-05-01

    We firstly reported a novel polymer matrix fabricated by type I collagen and polymers, and this matrix can be used as nanoreactors for electrodepositing platinum nanoclusters (PNCs). The type I collagen film has a significant effect on the growth of PNCs. The size of the platinum nanoparticles could be readily tuned by adjusting deposition time, potential and the concentration of electrolyte, which have been verified by field-emitted scanning electron microscopy (FE-SEM). Furthermore, cyclic voltammetry (CV) has demonstrated that the as-prepared PNCs can catalyze methanol directly with higher activity than that prepared on PSS/PDDA film, and with better tolerance to poisoning than the commercial E-TEK catalyst. The collagen-polymer matrix can be used as a general reactor to electrodeposit other metal nanostructures.

  4. Therapeutic effects of LLT, a novel immunosuppressant, on typecollagen- induced arthritis in DBA/1 mice

    Institute of Scientific and Technical Information of China (English)

    RuZHOU; WeiTANG; Pei-lanHE; Xiao-yuLI; Yi-fuYANG; Jian-pingZUO

    2005-01-01

    AIM To evaluate the therapeutic effects of LLT on the type II collagen-induced arthritis in DBA/1 mice. LLT is a new compound derived from triptolide. LLT had been demonstrated in vitro and in vivo for its immunosuppressive activities, and our results showed LLT with low toxicity and high efficiency. LLTis a more drug-like compound comparing with the parent compound. METHODS Male DBA/1 mice were immunized at the tail base with 125 ( g of Bovine type II collagen emulsified in CFA containing Mycobacterium tuberculosis and boosted with collagen emulsified in IFA 3 weeks later. The severity of arthritis was graded visually and expressed using the arthritic index. The arthritic score of each mouse was obtained by scoring each limb as severity 0 to 4 and taken the sum of the scores of the four limbs.

  5. Suppression of collagen-induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of type II collagen.

    Science.gov (United States)

    Iizuka, Mana; Wakasa, Yuhya; Tsuboi, Hiroto; Asashima, Hiromitsu; Hirota, Tomoya; Kondo, Yuya; Matsumoto, Isao; Takaiwa, Fumio; Sumida, Takayuki

    2014-10-01

    Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We previously reported that altered peptide ligands (APLs) of type II collagen (CII256-271) suppress the development of collagen-induced arthritis (CIA). In this study, we generated transgenic rice expressing CII256-271 and APL6 contained in fusion proteins with the rice storage protein glutelin in the seed endosperm. These transgene products successfully and stably accumulated at high levels (7-24 mg/g seeds) in protein storage vacuoles (PB-II) of mature seeds. We examined the efficacy of these transgenic rice seeds by performing oral administration of the seeds to CIA model mice that had been immunized with CII. Treatment with APL6 transgenic rice for 14 days significantly inhibited the development of arthritis (based on clinical score) and delayed disease onset during the early phase of arthritis. These effects were mediated by the induction of IL-10 from CD4(+ ) CD25(-) T cells against CII antigen in splenocytes and inguinal lymph nodes (iLNs), and treatment of APL had no effect on the production of IFN-γ, IL-17, IL-2 or Foxp3(+) Treg cells. These findings suggest that abnormal immune suppressive mechanisms are involved in the therapeutic effect of rice-based oral vaccine expressing high levels of APLs of type II collagen on the autoimmune disease CIA, suggesting that the seed-based mucosal vaccine against CIA functions via a unique mechanism.

  6. Osteogenesis imperfecta type I: Molecular heterogeneity for COL1A1 null alleles of type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Willing, M.C.; Deschenes, S.P.; Pitts, S.H.; Arikat, H.; Roberts, E.J.; Scott, D.A.; Slayton, R.L. [Univ. of Iowa, Iowa City, IA (United States); Byers, P.H. [Univ. of Washington, Seattle, WA (United States)

    1994-10-01

    Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 {open_quotes}null{close_quotes} allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5{prime} donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon, that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype. 58 refs., 4 figs., 1 tab.

  7. Arrangement of type IV collagen on NH₂ and COOH functionalized surfaces.

    Science.gov (United States)

    Coelho, Nuno Miranda; González-García, Cristina; Salmerón-Sánchez, Manuel; Altankov, George

    2011-12-01

    Apart from the paradigm that cell-biomaterials interaction depends on the adsorption of soluble adhesive proteins we anticipate that upon distinct conditions also other, less soluble ECM proteins such as collagens, associate with the biomaterials interface with consequences for cellular response that might be of significant bioengineering interest. Using atomic force microscopy (AFM) we seek to follow the nanoscale behavior of adsorbed type IV collagen (Col IV)--a unique multifunctional matrix protein involved in the organization of basement membranes (BMs) including vascular ones. We have previously shown that substratum wettability significantly affects Col IV adsorption pattern, and in turn alters endothelial cells interaction. Here we introduce two new model surfaces based on self-assembled monolayers (SAMs), a positively charged -NH(2) , and negatively charged -COOH surface, to learn more about their particular effect on Col IV behavior. AFM studies revealed distinct pattern of Col IV assembly onto the two SAMs resembling different aspects of network-like structure or aggregates (suggesting altered protein conformation). Moreover, the amount of adsorbed FITC-labeled Col IV was quantified and showed about twice more protein on NH(2) substrata. Human umbilical vein endothelial cells attached less efficiently to Col IV adsorbed on negatively charged COOH surface judged by altered cell spreading, focal adhesions formation, and actin cytoskeleton development. Immunofluorescence studies also revealed better Col IV recognition by both α(1) and α(2) integrins on positively charged NH(2) substrata resulting in higher phosphorylated focal adhesion kinase recruitment in the focal adhesion complexes. On COOH surface, no integrin clustering was observed. Taken altogether these results, point to the possibility that combined NH(2) and Col IV functionalization may support endothelization of cardiovascular implants.

  8. Phenotypic characterization of type II collagen-induced arthritis in Wistar rats.

    Science.gov (United States)

    Song, Hou-Pan; Li, Xin; Yu, Rong; Zeng, Guang; Yuan, Zhen-Yi; Wang, Wei; Huang, Hui-Yong; Cai, Xiong

    2015-10-01

    The aim of the present study was to determine a more specific, efficient and simple method for the induction of collagen-induced arthritis (CIA) in rats. Different strains of rats were injected at the base of the tail with bovine type II collagen (CII) emulsified in incomplete Freund's adjuvant (IFA). The onset and severity of arthritis were evaluated by clinical assessment. The established CIA model was analyzed using a comprehensive examination of clinical, hematological, histological and radiological parameters. The results demonstrated that Wistar rats were the most susceptible strain to CIA followed by Wistar Furth rats, with Sprague Dawley rats being the least susceptible. Following primary and booster immunization, female Wistar rats developed severe arthritis, with an incidence of >83% and low variability in clinical signs. The development of arthritis was accompanied by a significantly elevated erythrocyte sedimentation rate compared with that in the control rats. The radiographic examination revealed bone matrix resorption, considerable soft tissue swelling, periosteal new bone formation and bone erosion in the arthritic joints of the CIA rats. Histopathologically, the synovial joints of CIA rats were characterized by synovial hyperplasia, pannus formation, marked cellular infiltration, bone and cartilage erosion and narrowing of the joint space. The administration of an intradermal injection of only 200 µg bovine CII emulsified in IFA at the base of the tail therefore leads to the successful development of a CIA rat model. This well-characterized CIA rat model could be specifically used to study the pathophysiology of human rheumatoid arthritis as well as to test and develop anti-arthritic agents for humans.

  9. Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.

    Directory of Open Access Journals (Sweden)

    Ziyad Jabaji

    Full Text Available We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet

  10. Matrix metalloproteinase-9-mediated type III collagen degradation as a novel serological biochemical marker for liver fibrogenesis

    DEFF Research Database (Denmark)

    Veidal, Sanne S; Vassiliadis, Efstathios; Barascuk, Natasha

    2010-01-01

    During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via their acti...

  11. Development and utilization of a bovine type I collagen microfibril model

    Science.gov (United States)

    The structure of fibrous collagen, a long triple helix that self-associates in a staggered array to form a matrix of fibrils, fibers and fiber bundles, makes it uniquely suitable as a scaffold for biomaterial engineering. A major challenge for this application is to stabilize collagen structure by m...

  12. Data on cell spread area and directional contraction in human umbilical vein endothelial cells on fibronectin and on collagen type I-coated micro-posts

    Directory of Open Access Journals (Sweden)

    Jing-Jing Han

    2016-03-01

    Full Text Available Fibronectin and collagen type I are abundant extracellular matrix proteins that modulate cell mechanics and they regulate angiogenic sprouting. In this data article, fibronectin- or collagen type I-coated micro-posts were used to examine the traction force, cell spread area and directional contraction of human umbilical vein endothelial cells (HUVECs.

  13. Effect of ionic liquids on the different hierarchical order of type I collagen.

    Science.gov (United States)

    Mehta, Ami; Rao, J Raghava; Fathima, Nishter Nishad

    2014-05-01

    The effect of ionic liquids (ILs) on proteins has been gaining huge interest due to easy tunability of cation and anion for generating the desired effect. This study explores the effect of alkyl imidazolium chloride ILs on collagen at molecular, inter-fibrillar and skin matrix level. Circular dichroic studies reveal that at the molecular level, the secondary structure of collagen was not affected by imidazolium ILs and there was no change in thermal stability as well. However, collagen at the inter-fibrillar level behaved differently. With increase in concentration of ILs, remarkable decrease in thermal stability of rat tail tendon (RTT) collagen fibers with marginal swelling effect was seen. SEM micrographs of skin matrix treated with IL show opening up of pores. This kind of exquisite behavior of ILs at different hierarchal order of collagen indicates that ILs are endowed with potential lyotropic action, which can be judiciously employed for biomedical applications.

  14. Transforming growth factor β1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Xiaodong Pan

    Full Text Available Transforming growth factor-beta (TGF-β, a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1 expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

  15. Millicurrent stimulation of human articular chondrocytes cultivated in a collagen type-I gel and of human osteochondral explants

    Directory of Open Access Journals (Sweden)

    Silny Jiri

    2010-08-01

    Full Text Available Abstract Background Here we investigate the effect of millicurrent treatment on human chondrocytes cultivated in a collagen gel matrix and on human osteochondral explants. Methods Human chondrocytes from osteoarthritic knee joints were enzymatically released and transferred into a collagen type-I gel. Osteochondral explants and cell-seeded gel samples were cultivated in-vitro for three weeks. Samples of the verum groups were stimulated every two days by millicurrent treatment (3 mA, sinusoidal signal of 312 Hz amplitude modulated by two super-imposed signals of 0.28 Hz, while control samples remained unaffected. After recovery, collagen type-I, type-II, aggrecan, interleukin-1β, IL-6, TNFα and MMP13 were examined by immunohistochemistry and by real time PCR. Results With regard to the immunostainings 3 D gel samples and osteochondral explants did not show any differences between treatment and control group. The expression of all investigated genes of the 3 D gel samples was elevated following millicurrent treatment. While osteochondral explant gene expression of col-I, col-II and Il-1β was nearly unaffected, aggrecan gene expression was elevated. Following millicurrent treatment, IL-6, TNFα, and MMP13 gene expression decreased. In general, the standard deviations of the gene expression data were high, resulting in rarely significant results. Conclusions We conclude that millicurrent stimulation of human osteoarthritic chondrocytes cultivated in a 3 D collagen gel and of osteochondral explants directly influences cell metabolism.

  16. Clinical utility of C-terminal telopeptide of type 1 collagen in multiple myeloma.

    Science.gov (United States)

    Ting, Kay R; Brady, Jennifer J; Hameed, Abdul; Le, Giao; Meiller, Justine; Verburgh, Estelle; Bayers, Christopher; Benjamin, Dalia; Anderson, Kenneth C; Richardson, Paul G; Dowling, Paul; Clynes, Martin; Fitzgibbon, Maria C; O'Gorman, Peter

    2016-04-01

    Myeloma bone disease (MBD) is a major cause of morbidity in multiple myeloma (MM). We investigated bone turnover markers (BTM) as relapse predictors and biomarkers for monitoring MBD. We measured C-terminal telopeptide of type I collagen (CTX-1), and Procollagen type 1 N Propeptide (P1NP) in 86 MM patients and 26 controls. CTX-1 was higher in newly diagnosed patients compared to control, remission and relapse (P < 0·05), and decreased following treatment. In the setting of relapse, a CTX-1 rise greater than the calculated least significant change (LSC) was observed in 26% of patients 3-6 months prior to relapse (P = 0·007), and in 60·8% up to 3 months before relapse (P = 0·015). Statistically significant changes in CTX-1 levels were also observed in patients who were with and without bisphosphonate therapy at the time of relapse. In patients with normal renal function, mean CTX-1 level was highest in the newly diagnosed group (0·771 ± 0·400 μg/l), and lowest in the remission group (0·099 ± 0·070 μg/l) (P < 0·0001). P1NP levels were not statistically different across the patient groups. We conclude that CTX-1, measured on an automated hospital laboratory platform, has a role in routine treatment monitoring and predicting relapse of MBD, even in patients on bisphosphonates.

  17. Altered stress fibers and integrin expression in the Malpighian epithelium of Drosophila type IV collagen mutants

    Directory of Open Access Journals (Sweden)

    András A. Kiss

    2016-06-01

    Full Text Available Basement membranes (BMs are highly specialized extracellular matrices (ECMs that provide support and polarization cues for epithelial cells. Proper adhesion to the BM is pivotal in epithelial cell function and survival. Type IV collagens are the predominant components of all types of BMs, that form an irregular, polygonal lattice and serve as a scaffold for numerous other BM components and BM-associated cells. Mutations in the ubiquitous human BM components COL4A1 and COL4A2 cause a multisystem disorder involving nephropathy. Affected patients develop renal dysfunction and chronic kidney failure with or without hematuria. Mouse Col4a1 and Col4a2 mutants recapitulate the human symptoms. In vertebrates, excretion is accomplished by the kidneys and by the Malpighian tubules in insects, including the fruit fly Drosophila. Our present results with dominant, temperature-sensitive mutation of the Drosophila col4a1 gene demonstrate altered integrin expression and amplified effects of mechanical stress on the Malpighian epithelial cytoskeleton.

  18. A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

    DEFF Research Database (Denmark)

    Faassen, A E; Schrager, J A; Klein, D J

    1992-01-01

    motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG...... with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I...... collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG...

  19. MMP Mediated Degradation of Type VI Collagen Is Highly Associated with Liver Fibrosis - Identification and Validation of a Novel Biochemical Marker Assay

    DEFF Research Database (Denmark)

    Veidal, Sanne Skovgard; Karsdal, Morten Asser; Vassiliadis, Efstathios

    2011-01-01

    Background and Aims: During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small...... fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis. Methods: Mass spectrometric...... analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon...

  20. Inhibition on the production of collagen type Ⅰ, Ⅲ of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid

    Institute of Scientific and Technical Information of China (English)

    Wen-Bin Liu; Chang-Qing Yang; Wei Jiang; Yi-Qing Wang; Jing-Sheng Guo; Bo-Ming He; Ji-Yao Wang

    2003-01-01

    AIM: To investigate the inhibition effects on the productionof collagen type I, Ⅲ secreted by activated rat hepatic stellatecells (rHSCs) by antisense tissue inhibitors of metalloproteinase1 (TIMP-1) recombinant plasmid through elevating interstitialcollagenase activity.METHODS: rHSCs were extracted from normal rat liverby pronase and collagenase digestion and purified bycentrifugal elutriation, and were cultured on plastic dishesuntil they were activated to a myofibroblastic phenotypeafter 7-10 days. RT-Nest-PCR and gene recombinanttechniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryoticcells. The recombinant plasmid and the pcDNA3 emptyplasmid were transfected in rHSCs by Effectene (QIAGEN)separately. Cells were selected after growing in DMEMcontaining 400 μg/ml G418 for 2-3 weeks. Expression ofexogenous gene was assessed by Northern blot, andexpression oflIMP-1 in rHSCs was determined by Northernblot and Western blot. We tested the interstitial collagenaseactivity with FITC-labled type I collagen as substrate.Ultimately, we quantified the type Ⅰ, Ⅲ collagen byWestern blot.RESULTS: The exogenous antisense TIMP-1 recombinantplasmid could be expressed in rHSCs well, which couldblock the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNAlevel (P<0.05); the ratio of TIMP-1/β-actin was 0.31, 0.98and 1.32 separately at protein level (P<0.05); It mightelevate active and latent interstitial collagenase activity,the collagenase activity was 0.3049, 0.1411 and 0.1196respectively. (P<0.05), which led to promotion thedegradation of type Ⅰ, Ⅲ collagen, the ratio of collagen I/β-actin was 0.63, 1.78 and 1.92 separately (P<0.05); andthe ratio of collagen Ⅲ/β-actin was 0.59, 1.81 and 1.98separately (P<0.05).CONCLUSION: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on theproduction of type Ⅰ, Ⅲ collagens

  1. Hip Dysplasia in Children With Osteogenesis Imperfecta: Association With Collagen Type I C-Propeptide Mutations.

    Science.gov (United States)

    Kishta, Waleed; Abduljabbar, Fahad H; Gdalevitch, Marie; Rauch, Frank; Hamdy, Reggie; Fassier, François

    Osteogenesis imperfecta (OI) is a heritable skeletal disorder characterized by bone fragility and short stature that is usually due to mutations in 1 of the 2 genes that code for collagen type I α-chains. The association between hip dysplasia and OI has not been systematically investigated. In this single-center study, we retrospectively reviewed all cases of OI associated with hip dysplasia to describe clinical characteristics and the effect of therapy. We reviewed the charts of 687 patients with OI who were seen at the Shriners Hospital for Children in Montreal between 1999 and 2013 to identify patients with a diagnosis of hip dysplasia. Clinical characteristics and the course after therapeutic interventions were extracted from the charts. Hip dysplasia was diagnosed in 8 hips of 5 patients (4 boys, 1 girl; age at diagnosis ranged between 3 wk and 27 mo old). The prevalence of hip dysplasia and OI was therefore 0.87% (per patient). In 4 of the 5 patients (80%), OI was caused by mutations affecting the C-propeptide of collagen type I, which is otherwise rare in OI. Among the 26 patients with C-propeptide mutations followed at our institution, 4 (15%) had hip dysplasia. Pavlik harness treatment was attempted in 2 patients (3 hips) but was not effective in either case and resulted in avascular necrosis of 1 hip. Femoral varus derotational shortening osteotomies using a telescopic rod were performed in all 8 hips along with a closed reduction in 4 hips and an open reduction in 4 hips. Concomitant pelvic osteotomies were performed in 2 hips (1 patient). Surgery resulted in redislocation of 1 hip; all other surgically treated hips remained reduced. Clinical screening for hip dysplasia is difficult in OI owing to the bowing of the proximal femur and the risk of causing fractures. OI patients with positive C-propeptide mutation should therefore be screened for hip dysplasia by use of ultrasound. Presence of a C-propeptide mutation appears to be a risk factor for hip

  2. A comparative evaluation of bovine-derived xenograft (Bio-Oss Collagen) and type I collagen membrane (Bio-Gide) with bovine-derived xenograft (Bio-Oss Collagen) and fibrin fibronectin sealing system (TISSEEL) in the treatment of intrabony defects: A clinico-radiographic study

    OpenAIRE

    Palachur, Deepthi; K V Prabhakara Rao; Murthy, K. Raja V.; Kishore, D. Trinath; Reddy, M. Narendra; Bhupathi, Anitha

    2014-01-01

    Background and Objectives: The purpose of this study was to compare the efficacy of bovine-derived xenograft (Bio-Oss Collagen) and Type I collagen membrane (Bio-Gide) with bovine-derived xenograft (Bio-Oss Collagen) and fibrin fibronectin sealing system (TISSEEL) in the treatment of periodontal infrabony defects. Materials and Methods: Fourteen healthy patients in the age range of 20 to 60 years, showing bilateral or contralateral infrabony defects were selected. The defects were assigned ra...

  3. Role of α1 and α2 chains of type IV collagen in early fibrotic lesions of idiopathic interstitial pneumonias and migration of lung fibroblasts.

    Science.gov (United States)

    Urushiyama, Hirokazu; Terasaki, Yasuhiro; Nagasaka, Shinya; Terasaki, Mika; Kunugi, Shinobu; Nagase, Takahide; Fukuda, Yuh; Shimizu, Akira

    2015-08-01

    Early fibrotic lesions are thought to be the initial findings of fibrogenesis in idiopathic interstitial pneumonias, but little is known about their properties. Type IV collagen comprises six gene products, α1-α6, and although it is known as a major basement membrane component, its abnormal deposition is seen in fibrotic lesions of certain organs. We studied the expression of type I and III collagen and all α chains of type IV collagen in lung specimens from patients with usual interstitial pneumonia (UIP) or organizing pneumonia (OP) via immunohistochemistry. With cultured lung fibroblasts, we analyzed the expression and function of all α chains of type IV collagen via immunohistochemistry, western blotting, real-time quantitative PCR, and a Boyden chamber migration assay after the knockdown of α1 and α2 chains. Although we observed type I and III collagens in early fibrotic lesions of both UIP and OP, we found type IV collagen, especially α1 and α2 chains, in early fibrotic lesions of UIP but not OP. Fibroblasts enhanced the expression of α1 and α2 chains of type IV collagen after transforming growth factor-β1 stimulation. Small interfering RNA against α1 and α2 chains increased fibroblast migration, with upregulated phosphorylation of focal adhesion kinase (FAK), and adding medium containing fibroblast-produced α1 and α2 chains reduced the increased levels of fibroblast migration and phosphorylation of FAK. Fibroblasts in OP were positive for phosphorylated FAK but fibroblasts in UIP were not. These results suggest that fibroblasts in UIP with type IV collagen deposition, especially α1 and α2 chains, have less ability to migrate from early fibrotic lesions than fibroblasts in OP without type IV collagen deposition. Thus, type IV collagen deposition in early fibrotic lesions of UIP may be implicated in refractory pathophysiology including migration of lesion fibroblasts via a FAK pathway.

  4. Structural Basis for Platelet Collagen Responses by the Immune-type Receptor Glycoprotein VI

    Energy Technology Data Exchange (ETDEWEB)

    Horii,K.; Kahn, M.; Herr, A.

    2006-01-01

    Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of heart attack and stroke, and drugs to block platelet activation have successfully reduced cardiovascular morbidity and mortality. In humans and mice, collagen activation of platelets is mediated by glycoprotein VI (GPVI), a receptor that is homologous to immune receptors but bears little sequence similarity to known matrix protein adhesion receptors. Here we present the crystal structure of the collagen-binding domain of human GPVI and characterize its interaction with a collagen-related peptide. Like related immune receptors, GPVI contains 2 immunoglobulin-like domains arranged in a perpendicular orientation. Significantly, GPVI forms a back-to-back dimer in the crystal, an arrangement that could explain data previously obtained from cell-surface GPVI inhibition studies. Docking algorithms identify 2 parallel grooves on the GPVI dimer surface as collagen-binding sites, and the orientation and spacing of these grooves precisely match the dimensions of an intact collagen fiber. These findings provide a structural basis for the ability of an immunetype receptor to generate signaling responses to collagen and for the development of GPVI inhibitors as new therapies for human cardiovascular disease.

  5. Type VII Collagen is Enriched in the Enamel Organic Matrix Associated with the Dentin-Enamel Junction of Mature Human Teeth

    Science.gov (United States)

    McGuire, Jacob D.; Walker, Mary P.; Mousa, Ahmad; Wang, Yong; Gorski, Jeff P.

    2014-01-01

    The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin. PMID:24594343

  6. Changes in Cytokines and Aggrecan ARGS Neoepitope in Synovial Fluid and Serum and in C-Terminal Crosslinking Telopeptide of Type II Collagen and N-Terminal Crosslinking Telopeptide of Type I Collagen in Urine Over Five Years After Anterior Cruciate Ligament Rupture

    DEFF Research Database (Denmark)

    Struglics, André; Larsson, Staffan; Kumahashi, Nobuyuki

    2015-01-01

    OBJECTIVE: To prospectively monitor levels of proinflammatory cytokines and aggrecan ARGS neoepitope in synovial fluid and serum as well as levels of C-terminal crosslinking telopeptide of type II collagen (CTX-II) and N-terminal crosslinking telopeptide of type I collagen (NTX-I) in urine after...... of aggrecan and type II collagen. Cytokine levels remained increased up to 5 years after injury, indicative of extended local inflammation in the joint....

  7. Glycosylation modulates melanoma cell α2β1 and α3β1 integrin interactions with type IV collagen.

    Science.gov (United States)

    Stawikowski, Maciej J; Aukszi, Beatrix; Stawikowska, Roma; Cudic, Mare; Fields, Gregg B

    2014-08-01

    Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382-393 but right in the middle of α3β1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.

  8. Altered spatiotemporal expression of collagen types I, III, IV, and VI in Lpar3-deficient peri-implantation mouse uterus.

    Science.gov (United States)

    Diao, Honglu; Aplin, John D; Xiao, Shuo; Chun, Jerold; Li, Zuguo; Chen, Shiyou; Ye, Xiaoqin

    2011-02-01

    Lpar3 is upregulated in the preimplantation uterus, and deletion of Lpar3 leads to delayed uterine receptivity in mice. Microarray analysis revealed that there was higher expression of Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3(-/-) uterus compared to Day 3.5 wild-type (WT) uterus. Since extracellular matrix (ECM) remodeling is indispensable during embryo implantation, and dynamic spatiotemporal alteration of specific collagen types is part of this process, this study aimed to characterize the expression of four main uterine collagen types: fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3(-/-) uterus. An observed delay of COL III and COL VI clearance in the Lpar3(-/-) uterus may be associated with higher preimplantation expression of Col3a1 and Col6a3. There was also delayed clearance of COL I and delayed deposition of COL IV in the decidual zone in the Lpar3(-/-) uterus. These changes were different from the effects of 17beta-estradiol and progesterone on uterine collagen expression in ovariectomized WT uterus, indicating that the altered collagen expression in Lpar3(-/-) uterus is unlikely to be a result of alterations in ovarian hormones. Decreased expression of several genes encoding matrix-degrading metallo- and serine proteinases was observed in the Lpar3(-/-) uterus. These results demonstrate that pathways downstream of LPA3 are involved in the dynamic remodeling of ECM in the peri-implantation uterus.

  9. Effects of bosentan on collagen type I synthesis on in vitro culture of scleroderma skin fibroblasts

    Directory of Open Access Journals (Sweden)

    S. Soldano

    2011-01-01

    Full Text Available The present study evaluated the effects of a non-selective endothelin (ETA/B receptors antagonist, on collagen type I (COLI synthesis on in vitro culture of scleroderma (SSc skin fibroblasts (Fb. Fb were obtained from skin biopsies of 6 female SSc patients (mean age 64. 1±6 years, after informed consent and Ethical Committee Approval. Cells were treated with endothelin-I [ET-I, 100nM] for 24 and 48 hrs, pre-treated for I hr with ETA/B receptors antagonist [10nM] alone or followed by ET-I for 24 and 48 hrs. Untreated Fb were used as controls. Immunocytochemistry and western blot analysis were performed to evaluate COLI synthesis. ET-I increased COLI synthesis both at 24 and 48 hrs when compared to controls. ETA/B receptor antagonost blocks the increased COLI synthesis ET-I-mediated both at 24 and 48 hrs vs. ET-I. Results showed that ET-I receptors blockage by ETA/B receptors antagonist might prevent the excessive synthesis of COLI, supporting its positive action in the management of skin fibrosis.

  10. Asiaticoside induces type I collagen synthesis and osteogenic differentiation in human periodontal ligament cells.

    Science.gov (United States)

    Nowwarote, Nunthawan; Osathanon, Thanaphum; Jitjaturunt, Peachaya; Manopattanasoontorn, Sukuman; Pavasant, Prasit

    2013-03-01

    Asiaticoside, an active ingredient extracted from Centella asiatica, has been widely used to promote wound healing. In this study, the effects of asiaticoside on proliferation, protein synthesis, and osteogenic differentiation in human periodontal ligament cells (HPDLs) were investigated. HPDLs were treated with asiaticoside at concentrations of 25, 50, and 100 µg/mL. Cell number was determined by MTT assay. The mRNA expression was analyzed by reverse transcription-polymerase chain reaction. Western blot analysis and immunocytochemistry were used to confirm protein synthesis. Osteogenic differentiation was determined by alkaline phosphatase activity, osteoblast marker gene expression, and in vitro mineralization. The results showed that asiaticoside treatment, ranging from 25 to 100 mg/mL, had no effect on cytotoxicity or cell proliferation. When HPDLs were treated with asiaticoside in serum-free medium, dose-dependent increases in the levels of fibronectin and collagen type I mRNA and protein were observed at 72 h. Moreover, asiaticoside attenuated matrix metalloproteinase-1 but enhanced tissue inhibitor of metalloproteinase-1 mRNA expression. The addition of asiaticoside to osteogenic medium resulted in an increase in alkaline phosphatase enzymatic activity, up-regulation of osteoblast marker gene mRNA expression, and enhancement of mineralization by HPDLs. These results suggest the potential application of asiaticoside for enhancing periodontal tissue healing.

  11. Detection of sequence variability of the collagen type IIalpha 1 3' variable number of tandem repeat.

    Science.gov (United States)

    van Meurs, J B; Arp, P P; Fang, Y; Slagboom, P E; Meulenbelt, I; van Leeuwen, J P; Pols, H A; Uitterlinden, A G

    2000-11-01

    The variable number of tandem repeat (VNTR) 3' of the collagen type II (COL2A1) gene has been shown to be highly variable with a complex molecular structure. In a previous pilot experiment we observed discordance between methods to genotype this informative marker. To further investigate the extent and molecular nature of this discordance, we genotyped a random sample of 207 Caucasian individuals with two genotyping methods and sequenced new alleles. We compared single-strand (SS) analysis, which is based on detection of size differences between the different alleles, and heteroduplex analysis (HA), which is sensitive to both size and sequence differences. Overall, 26% of discordance between the two methods was detected. Approximately two thirds of this discordance was caused by subdivision of SS-alleles 13R1 and 14R2 into HA-alleles 4A + 4B and 3B + 3C, respectively. Sequence analysis of the COL2A1 VNTR alleles 4B and 3C showed that these alleles differed in sequence, but not in size, from already described SS-alleles, which explains why they escape detection by SS. The 4B allele is a frequent allele in the population (14%) and is, therefore, important to distinguish in association studies. We conclude that HA is a reliable method when the described optimized electrophoretic conditions are used. HA is a sensitive genotyping method to document allelic diversity at this locus, which can distinguish more alleles compared to the SS method.

  12. Collagen type I alpha 1 gene polymorphism in premature ovarian failure

    Directory of Open Access Journals (Sweden)

    Vujović Svetlana

    2013-01-01

    Full Text Available Introduction. Premature ovarian failure (POF is characterized by amenorrhea, hypergonadotropism and hypoestrogenism in women bellow 40 years. Osteoporosis is one of the late complications of POF. Objective. To correlate collagen type I alpha1 (COLIA1 gene polymorphism with bone mineral density (BMD in women with POF. Methods. We determined the COLIA1 genotypes SS, Ss, ss in 66 women with POF. Single nucleotide polymorphism (G to T substitution within the Sp 1-binding site in the first intron of the COLIA1 gene was assessed by polymerase chain reaction (PCR followed by single-stranded conformation polymorphism (SSCP analysis. Bone mineral density (BMD was measured at the lumbar spine region by dual X-ray absorptiometry. Statistics: Kruskal-Wallis ANOVA, Chisquare test, Spearman correlation test. Results. The relative distribution of COLIA1 genotype alleles was SS - 54.4%, Ss - 41.0% and ss - 4.5%. No significant differences were found between genotype groups in body mass index, age, duration of amenorrhea or BMD. A significant positive correlation was observed between BMI and parity. Conclusion. The COLIA1 gene is just one of many genes influencing bone characteristics. It may act as a marker for differences in bone quantity and quality, bone fragility and accelerated bone loss in older women. However, in young women with POF, COLIA1 cannot identify those at higher risk for osteoporosis. [Projekat Ministarstva nauke Republike Srbije, br. ON 173056

  13. 3D collagen type I matrix inhibits the antimigratory effect of doxorubicin

    Directory of Open Access Journals (Sweden)

    Millerot-Serrurot Emilie

    2010-08-01

    Full Text Available Abstract Background The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D context which simulates a natural microenvironment. Methods To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation. Results We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected β1 integrin expression nor the state of phosphorylation of FAK and RhoA. Conclusion This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.

  14. RB1CC1 Protein Suppresses Type II Collagen Synthesis in Chondrocytes and Causes Dwarfism*

    Science.gov (United States)

    Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

    2011-01-01

    RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

  15. Fibrochondrogenesis results from mutations in the COL11A1 type XI collagen gene.

    Science.gov (United States)

    Tompson, Stuart W; Bacino, Carlos A; Safina, Nicole P; Bober, Michael B; Proud, Virginia K; Funari, Tara; Wangler, Michael F; Nevarez, Lisette; Ala-Kokko, Leena; Wilcox, William R; Eyre, David R; Krakow, Deborah; Cohn, Daniel H

    2010-11-12

    Fibrochondrogenesis is a severe, autosomal-recessive, short-limbed skeletal dysplasia. In a single case of fibrochondrogenesis, whole-genome SNP genotyping identified unknown ancestral consanguinity by detecting three autozygous regions. Because of the predominantly skeletal nature of the phenotype, the 389 genes localized to the autozygous intervals were prioritized for mutation analysis by correlation of their expression with known cartilage-selective genes via the UCLA Gene Expression Tool, UGET. The gene encoding the α1 chain of type XI collagen (COL11A1) was the only cartilage-selective gene among the three candidate intervals. Sequence analysis of COL11A1 in two genetically independent fibrochondrogenesis cases demonstrated that each was a compound heterozygote for a loss-of-function mutation on one allele and a mutation predicting substitution for a conserved triple-helical glycine residue on the other. The parents who were carriers of missense mutations had myopia. Early-onset hearing loss was noted in both parents who carried a loss-of-function allele, suggesting COL11A1 as a locus for mild, dominantly inherited hearing loss. These findings identify COL11A1 as a locus for fibrochondrogenesis and indicate that there might be phenotypic manifestations among carriers.

  16. Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction.

    Science.gov (United States)

    Coelho, N Miranda; González-García, C; Planell, J A; Salmerón-Sánchez, M; Altankov, G

    2010-06-09

    Considering the structural role of type IV collagen (Col IV) in the assembly of the basement membrane (BM) and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass) and hydrophobic trichloro(octadecyl)silane (ODS) surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50microg/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine - nearly single molecular size - network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC) attach less efficiently to the aggregated Col IV (on ODS), as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both alpha1 and alpha2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.

  17. Type IV Collagens and Basement Membrane Diseases: Cell Biology and Pathogenic Mechanisms.

    Science.gov (United States)

    Mao, Mao; Alavi, Marcel V; Labelle-Dumais, Cassandre; Gould, Douglas B

    2015-01-01

    Basement membranes are highly specialized extracellular matrices. Once considered inert scaffolds, basement membranes are now viewed as dynamic and versatile environments that modulate cellular behaviors to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to neighboring cells, basement membranes serve as reservoirs of growth factors that direct and fine-tune cellular functions. Type IV collagens are a major component of all basement membranes. They evolved along with the earliest multicellular organisms and have been integrated into diverse fundamental biological processes as time and evolution shaped the animal kingdom. The roles of basement membranes in humans are as complex and diverse as their distributions and molecular composition. As a result, basement membrane defects result in multisystem disorders with ambiguous and overlapping boundaries that likely reflect the simultaneous interplay and integration of multiple cellular pathways and processes. Consequently, there will be no single treatment for basement membrane disorders, and therapies are likely to be as varied as the phenotypes. Understanding tissue-specific pathology and the underlying molecular mechanism is the present challenge; personalized medicine will rely upon understanding how a given mutation impacts diverse cellular functions.

  18. Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction

    Directory of Open Access Journals (Sweden)

    NM Coelho

    2010-06-01

    Full Text Available Considering the structural role of type IV collagen (Col IV in the assembly of the basement membrane (BM and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass and hydrophobic trichloro(octadecylsilane (ODS surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50μg/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine – nearly single molecular size – network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC attach less efficiently to the aggregated Col IV (on ODS, as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both α1 and α2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.

  19. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Møller, Jonas Bech

    2015-01-01

    producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human...... embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed...... by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid...

  20. Evaluation of anti-citrullinated type II collagen and anti-citrullinated vimentin antibodies in patients with juvenile idiopathic arthritis

    Science.gov (United States)

    2013-01-01

    Background To determine the prevalence and significance of anti-citrullinated vimentin and anti-citrullinated type II collagen antibodies and elucidate their role in the disease process of juvenile idiopathic arthritis (JIA). Methods Sera were obtained from 95 patients with various subtypes of JIA, 19 systemic lupus erythematosus (SLE) patients, and 10 healthy children. Antibodies were measured in the sera against citrullinated and native type II collagen and vimentin (vim1-16 and vim 59-74) by enzyme-linked immunosorbent assay. Samples were compared to anti-cyclic citrullinated peptide (anti-CCP) antibody and rheumatoid factor (RF) isotypes, and our previously measured anti-citrullinated fibrinogen and α-enolase antibodies on the same patient population, in addition to erythrocyte sedimentation rate and C-reactive protein. The relationship between the anti-citrullinated antibody profile and disease activity and joint damage were also investigated. Results Twenty-three JIA patients (24%) demonstrated reactivity to anti-citrullinated type II collagen. Ten JIA patients (10.5%) demonstrated reactivity to anti-citrullinated vimentin 1–16 antibodies and 7 (7.4%) to anti-citrullinated vimentin 59–74 antibodies. One IgM RF-positive polyarticular patient was positive for all 5 of the citrullinated autoantibodies tested. Thirty-seven different subsets of patients were identified based on their anti-citrullinated autoantibody and RF isotype profile. No significant associations were noted with anti-citrullinated type II collagen and anti-citrullinated vimentin antibodies with joint damage or disease activity. Anti-citrullinated vimentin 59–74 antibodies demonstrated the highest overall specificity at 89.7%, with anti-citrullinated vimentin 1–16 and anti-citrullinated type II collagen antibodies at 86.2%. Conclusion This study demonstrates that antibodies to multiple citrullinated epitopes are present in the sera of patients with various subtypes of JIA. It also

  1. Glucocorticoids inhibit the synthesis rate of type III collagen, but do not affect the hepatic clearance of its aminoterminal propeptide (PIIINP)

    DEFF Research Database (Denmark)

    Hansen, M; Stoltenberg, M; Høst, N B;

    1995-01-01

    The aminoterminal propeptide of type III procollagen (PIIINP) is a marker of type III collagen metabolism. The serum concentration of PIIINP is increased during inflammation, probably reflecting stimulated biosynthesis of type III collagen. Serum PIIINP decreases during glucocorticoid treatment....... This has been interpreted as an inhibited biosynthesis of type III collagen. However, circulating PIIINP is extracted by the liver, and the decrease in serum PIIINP may also be caused by an increased hepatic elimination. In the present study we investigated the influence of intravenous methylprednisolone...

  2. Tissue elasticity displayed by elastography and its correlation with the characteristics of collagen type I and type III in prostatic stroma

    Directory of Open Access Journals (Sweden)

    Jie Tang

    2014-04-01

    Full Text Available We investigated the prostate elasticity displayed by elastography and its correlation with the content and distribution of collagen type I (Col1 and type III (Col3. A total of 62 patients underwent transrectal real-time tissue elastography (TRTE examinations. Targeted biopsies were performed after 12-core systematic biopsy. The tissues corresponding to the elastograms were stained with picric acid-sirius red. The distribution of Col1 and type Col3 was observed, and the collagen volume fraction (CVF of these two types of collagen fibers was calculated. The CVFs of Col1 in the stiff and soft groups were 0.05 ± 0.02 and 0.02 ± 0.01 (P = 0.002, respectively. The CVFs of Col3 in the stiff and soft groups were 0.05 ± 0.04 and 0.07 ± 0.03 (P = 0.13, respectively. The circular analysis results showed that collagen fibers were disorganized both in the soft and stiff groups. Col1 and Col3 were mainly cross-linked, and some parallelization was observed in the sections. The distributions of Col1 and Col3 were different between the stiff and soft groups (P = 0.03. In conclusion, the texture of the prostate is due to the content of Col1 and its relative correlation with Col3.

  3. Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

    Science.gov (United States)

    Bockman, R S; Guidon, P T; Pan, L C; Salvatori, R; Kawaguchi, A

    1993-08-01

    Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

  4. Collagen type I Density on dental pulp inflamation of sprague-dawley rats following the application of trigona sp propolis from south sulawesi province,Indonesia

    OpenAIRE

    Ardo Sabir, Dr.drg. M.Kes

    2015-01-01

    The Result show That there is no significants difference of the collagen fibers density among 4 time periods of each group and among 5 groups of each time periods. The aim was to analyse the collagen type I density as the result of trigona sp propolis application in the dental pulp inflamation of sprague-dawley rats.

  5. Both Hyaluronan and Collagen Type II Keep Proteoglycan 4 (Lubricin) at the Cartilage Surface in a Condition That Provides Low Friction during Boundary Lubrication

    NARCIS (Netherlands)

    Majd, Sara Ehsani; Kuijer, Roel; Koewitsch, Alexander; Groth, Thomas; Schmidt, Tannin A.; Sharma, Prashant K.

    2014-01-01

    Wear resistant and ultralow friction in synovial joints is the outcome of a sophisticated synergy between the major macromolecules of the synovial fluid, e.g., hyaluronan (HA) and proteoglycan 4 (PRG4), with collagen type II fibrils and other non-collagenous macromolecules of the cartilage superfici

  6. [Experimental study on effects of acupoint application with Leima type II plaster on collagen-induced arthritis in rats].

    Science.gov (United States)

    Li, Peng; Fang, Jian-Qiao; Zhou, Ya-Feng

    2011-09-01

    To observe the therapeutic effect of acupoint application with Leima type II plaster on collagen-induced arthritis (CIA) in rats and probe its mechanism. Bovine type II collagen was injected intradermally into the middle line of the back to induce CIA model with 48 Wistar rats. Then the rats were randomly divided into a model control group (group A), a matrix control group (group B), acupoint application group with plaster of low concentration (group C) and high concentration plaster group (group D), 12 rats in each group. Group C and group D were treated with low and high concentration of Leima type II plaster, and "Shenzhu" (GV 12), "Zhiyang" (GV 9) and "Mingmen" (GV 4) were selected, each application for about 15 hours, once each day for 30 days. Group B was used the same method of acupoint application except using non-drug matrix plaster, and group A was not given any treatment. The morphous and the histopathological changes of affection joint were observed. The paw edema volume after 30 days treatment in group C was significantly lower than that in group B (P plaster has a good therapeutic effect on CIA rats and the protective mechanism is related to the reduction of anti-type II collagen antibody level so as to carry out anti-inflammatory effect and immunosuppression.

  7. Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

    NARCIS (Netherlands)

    Blumbach, K.; Bastiaansen-Jenniskens, Y.M.; Groot, J. de; Paulsson, M.; Osch, G.J.V.M. van; Zaucke, F.

    2009-01-01

    Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins

  8. Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

    NARCIS (Netherlands)

    Blumbach, K.; Bastiaansen-Jenniskens, Y.M.; Groot, J. de; Paulsson, M.; Osch, G.J.V.M. van; Zaucke, F.

    2009-01-01

    Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins

  9. Evaluation of chondrocyte growth in the highly porous scaffolds made by fused deposition manufacturing (FDM) filled with type II collagen.

    Science.gov (United States)

    Yen, Hung-Jen; Tseng, Ching-Shiow; Hsu, Shan-Hui; Tsai, Ching-Lin

    2009-06-01

    Highly porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds for cartilage tissue engineering were fabricated in this study using the fused deposition manufacturing (FDM) process and were further modified by type II collagen. The average molecular weight of PLGA decreased to about 60% of the original value after the melt-extrusion process. Type II collagen exhibited sponge-like structure and filled the macroporous FDM scaffolds. An increase of the fiber spacing resulted in an increase of the porosity. The storage modulus of FDM scaffolds with a large fiber spacing was comparable to that of the native porcine articular cartilage. Although the FDM hybrid scaffolds were swollen in various extents after 28 days of in vitro culture, the seeded chondrocytes were well distributed in the interior of the scaffolds with a large fiber spacing and neocartilage was formed around the scaffolds. The study also suggested that a low processing temperature may be required to produce PLGA precision scaffolds using FDM.

  10. Nitrated type III collagen as a biological marker of nitric oxide-mediated synovial tissue metabolism in osteoarthritis

    DEFF Research Database (Denmark)

    Richardot, P; Charni-Ben Tabassi, N; Toh, L

    2009-01-01

    OBJECTIVES: Nitric oxide (NO) is a major mediator of joint tissue inflammation and damage in osteoarthritis (OA) and mediates the nitration of tyrosine (Y*) residues in proteins. We investigated the nitration of type III collagen, a major constituent of synovial membrane, in knee OA. METHODS...... in healthy controls and significantly correlated with C-reactive protein values (r=0.40, Pcollagen N-telopeptide is increased in the synovial tissue of patients with knee OA. Measurements of serum IIINys level may be useful for the clinical......: A polyclonal antibody directed against the nitrated QY*DSY*DVKSG sequence from type III collagen N-telopeptide was generated. Synovial tissues from patients with knee OA (n=4) and rheumatoid arthritis (RA, n=4) were analyzed by immunohistochemistry for IIINys. Serum IIINys levels were measured by enzyme...

  11. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Miki Takeuchi

    2015-10-01

    Full Text Available Granule cells (GCs are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs. Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.

  12. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.

    Science.gov (United States)

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-10-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.

  13. Regional alterations of type I collagen in rat tibia induced by skeletal unloading

    Science.gov (United States)

    Shiiba, Masashi; Arnaud, Sara B.; Tanzawa, Hideki; Kitamura, Eiji; Yamauchi, Mitsuo

    2002-01-01

    Skeletal unloading induces loss of mineral density in weight-bearing bones that leads to inferior bone mechanical strength. This appears to be caused by a failure of bone formation; however, its mechanisms still are not well understood. The objective of this study was to characterize collagen, the predominant matrix protein in bone, in various regions of tibia of rats that were subjected to skeletal unloading by 4 weeks tail suspension. Sixteen male Sprague-Dawley rats (4 months old) were divided into tail suspension and ambulatory controls (eight rats each). After the tail suspension, tibias from each animal were collected and divided into five regions and collagen was analyzed. The collagen cross-linking and the extent of lysine (Lys) hydroxylation in unloaded bones were significantly altered in proximal epiphysis, diaphysis, and, in particular, proximal metaphysis but not in distal regions. The pool of immature/nonmineralized collagen measured by its extractability with a chaotropic solvent was significantly increased in proximal metaphysis. These results suggest that skeletal unloading induced an accumulation of post-translationally altered nonmineralized collagen and that these changes are bone region specific. These alterations might be caused by impaired osteoblastic function/differentiation resulting in a mineralization defect.

  14. Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

    Institute of Scientific and Technical Information of China (English)

    Juxue Li; Enkui Duan; Chenglin Miao; Weixiang Guo; Liwei Jia; Jiaxi Zhou; Baohua Ma; Sha Peng; Shuang Liu; Yujing Cao

    2008-01-01

    The enrichment and identification of human epidermal stem cells (EpSCs) are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rapidly adherent keratinocytes into three populations that range in size from 5-7 μm (population A), to 7-9 μm (population B), to >9 μm (population C) in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of βl-integrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency (CFE). When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

  15. Use of synovium-derived stromal cells and chitosan/collagen type I scaffolds for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Gong Zhongcheng; Lin Zhaoquan [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830054 (China); Xiong Hui; Long Xing; Wei Lili; Li Jian; Wu Yang, E-mail: xinglong1957@yahoo.com.c [State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079 (China)

    2010-10-01

    The objective was to investigate synovium-derived stromal cells (SDSCs) coupled with chitosan/collagen type I (CS/COL-I) scaffolds for cartilage engineering. CS/COL-I scaffolds were fabricated through freeze-drying and cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. SDSCs were isolated from synovium and cultured onto CS/COL-I scaffolds, constructs of which were incubated in serum-free chondrogenic medium with sequential application of TGF-{beta}1 and bFGF for up to 21 days and then implanted into nude mice. The physical characteristics of the scaffolds were examined. The quality of the in vitro constructs was assessed in terms of DNA content by PicoGreen assay and cartilaginous matrix by histological examination. The implants of the constructs were evaluated by histological and immunohistochemical examinations and reverse transcription PCR. Results indicated that the CS/COL-I scaffold showed porous structures, and the DNA content of SDSCs in CS/COL-I scaffolds increased at 1 week culture time. Both of the constructs in vitro and the implants were examined with positive stained GAGs histologically and the implants with positive collagen type II immunohistochemically. RT-PCR of the implants indicated that aggrecan and collagen type II expressed. It suggested that SDSCs coupled with CS/COL-I scaffolds treated sequentially with TGF-{beta}1 and bFGF in vitro were highly competent for engineered cartilage formation in vitro and in vivo.

  16. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

    Science.gov (United States)

    Guilbert, Marie; Roig, Blandine; Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-02-23

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

  17. Bioinspired coupled helical coils for soft tissue engineering of tubular structures - Improved mechanical behavior of tubular collagen type I templates.

    Science.gov (United States)

    Janke, H P; Bohlin, J; Lomme, R M L M; Mihaila, S M; Hilborn, J; Feitz, W F J; Oosterwijk, E

    2017-09-01

    The design of constructs for tubular tissue engineering is challenging. Most biomaterials need to be reinforced with supporting structures such as knittings, meshes or electrospun material to comply with the mechanical demands of native tissues. In this study, coupled helical coils (CHCs) were manufactured to mimic collagen fiber orientation as found in nature. Monofilaments of different commercially available biodegradable polymers were wound and subsequently fused, resulting in right-handed and left-handed polymer helices fused together in joints where the filaments cross. CHCs of different polymer composition were tested to determine the tensile strength, strain recovery, hysteresis, compressive strength and degradation of CHCs of different composition. Subsequently, seamless and stable hybrid constructs consisting of PDSII® USP 2-0 CHCs embedded in porous collagen type I were produced. Compared to collagen alone, this hybrid showed superior strain recovery (93.5±0.9% vs 71.1±12.6% in longitudinal direction; 87.1±6.6% vs 57.2±4.6% in circumferential direction) and hysteresis (18.9±2.7% vs 51.1±12.0% in longitudinal direction; 11.5±4.6% vs 46.3±6.3% in circumferential direction). Furthermore, this hybrid construct showed an improved Young's modulus in both longitudinal (0.5±0.1MPavs 0.2±0.1MPa; 2.5-fold) and circumferential (1.65±0.07MPavs (2.9±0.3)×10(-2)MPa; 57-fold) direction, respectively, compared to templates created from collagen alone. Moreover, hybrid template characteristics could be modified by changing the CHC composition and CHCs were produced showing a mechanical behavior similar to the native ureter. CHC-enforced templates, which are easily tunable to meet different demands may be promising for tubular tissue engineering. Most tubular constructs lack sufficient strength and tunability to comply with the mechanical demands of native tissues. Therefore, we embedded coupled helical coils (CHCs) produced from biodegradable polymers - to

  18. ISOLATION AND DETERMINATION OF TYPE I COLLAGEN FROM TILAPIA (Oreochromis niloticus WASTE

    Directory of Open Access Journals (Sweden)

    S.Sujithra

    2013-06-01

    Full Text Available Tilapia are one of the most widely introduced fish globally that has clearly emerged as a promising group in aquaculture. Oreochromis niloticus was the first Tilapia species to be taken up for large Scaleaquaculture. It is consumed widely due to its deliciousness and rich source of protein. During its processing, the scales, Fins, Skins etc are expelled out as waste Acid solubilized collagen (ASC and Pepsin Solubilizedcollagen (PSC were extracted from these processing wastes. Initial extraction by acid yielded 22% of collagen and subsequent digestion with pepsin yielded 56% on dry weight basis. The total protein of ASC and PSC was determined by Bradford method which contains 68.34mg/ml,23.24 mg/ml respectively. The FT-IR Spectrumshowed that ASC and PSC are helpful in prediction and confirmation of Secondary structure of proteins. The denaturation temperature of ASC was 32⁰C while for PSC it is 29⁰C.SEM micrograph showed the fibrousnature of Collagen. This report indicates that Tilapia waste might be useful as a new source of collagen apart from usual bovine and pig skin.

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

    Science.gov (United States)

    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.

  20. Multilocus sequence typing analysis of Streptococcus mutans strains with the cnm gene encoding collagen-binding adhesin.

    Science.gov (United States)

    Lapirattanakul, Jinthana; Nakano, Kazuhiko; Nomura, Ryota; Leelataweewud, Pattarawadee; Chalermsarp, Narumon; Klaophimai, Arthit; Srisatjaluk, Ratchapin; Hamada, Shigeyuki; Ooshima, Takashi

    2011-11-01

    Streptococcus mutans is one of the oral pathogens associated with infective endocarditis (IE). With respect to bacterial binding ability to the extracellular matrix, the Cnm protein, a cell surface collagen-binding adhesin of S. mutans, is known as one of the possible virulence factors with regard to IE. In this study, we aimed to determine the distribution of the cnm gene, which encodes Cnm, in a large number of clinical isolates of S. mutans from Thai subjects. Then, the cnm-positive strains were classified using a multilocus sequence typing (MLST) scheme, which we constructed previously. In addition, the data were analysed together with our previous MLST data of cnm-positive strains from Japan and Finland in order to evaluate the clonal relationship among S. mutans strains harbouring the cnm gene. The cnm gene was detected in 12.4 % of all 750 Thai isolates, and serotype f showed the highest rate of detection (54.5 %). According to the MLST data, two clonal complex groups were revealed as the important clones related to cnm-positive S. mutans from various origins of isolation. Moreover, the collagen-binding properties of S. mutans strains with the cnm gene were significantly greater than those of strains without the gene, although four cnm-negative strains classified into two sequence types (STs), ST110 and ST136, showed extremely high collagen-binding rates suggesting the presence of additional genes involved with collagen binding in these STs. Taken together, these results provided information on both epidemiological as well as evolutional aspects of S. mutans possessing the cnm gene.

  1. Comparative analysis of the noncollagenous NC1 domain of type IV collagen: identification of structural features important for assembly, function, and pathogenesis.

    OpenAIRE

    Netzer, K. O.; Suzuki, K; Itoh, Y.; Hudson, B.G.; Khalifah, R. G.

    1998-01-01

    Type IV collagen alpha1-alpha6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, alpha1-like and alpha2-like, and suggested tha...

  2. Physical characterization of succinylated type I collagen by Raman spectra and MALDI-TOF/MS and in vitro evaluation for biomedical applications

    Science.gov (United States)

    Kumar, Ramadhar; Sripriya, R.; Balaji, S.; Senthil Kumar, M.; Sehgal, P. K.

    2011-05-01

    In this study, we report on physical and in vitro biological characterization of succinylated collagen (SC). SC was prepared by succinylation of type I bovine tendon collagen. SC swells and dissolves in physiological pH buffers (pH 7.4) Biocompatibility of SC to collagen for fibroblasts was comparable but L6 myoblasts showed pronounced proliferation and differentiation with SC. Using the MALDI-TOF/MS technique, SC was found with increased molecular mass by 16,359 Da per molecule which corresponds to about 54 succinyl groups covalently linked to the collagen strand. Raman spectroscopy revealed the retention of triple helical structure conformation in the presence of linked succinyl groups. New peaks near 1737, 1675 and 1420 cm -1 and decreased intensities near 2440 and 488 cm -1 provides the most convenient marker bands for succinylation of collagen. The intense band regions near 2856-2934, 2724, and 1445 cm -1 also confirms the existence of succinyl groups.

  3. Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods.

    Science.gov (United States)

    Buckley, Mike

    2016-03-24

    Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of

  4. Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods

    Directory of Open Access Journals (Sweden)

    Mike Buckley

    2016-03-01

    Full Text Available Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF as well as in-depth liquid chromatography (LC-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I chain sequence variation, in comparison to the alpha 1(I chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the

  5. Basement membrane zone remodeling during appendageal development in human fetal skin. The absence of type VII collagen is associated with gelatinase-A (MMP2) activity.

    Science.gov (United States)

    Karelina, T V; Bannikov, G A; Eisen, A Z

    2000-02-01

    Epithelial cell adhesion, migration, and differentiation are controlled by interactions at the basement membrane zone (BMZ). Type VII collagen is the major collagenous component of anchoring fibrils that are essential for the attachment of the epidermis to the dermis. Gelatinase A (MMP-2) is believed to be necessary for the degradation of type VII collagen. In this study we have examined the in vivo distribution of type VII collagen and gelatinase A (Gel A) in the developing human epidermis and its appendages. At 13-15 wk of gestation a marked decrease in type VII collagen immunoreactivity was seen in the BMZ surrounding invading appendageal buds; however, type VII collagen mRNA was strongly expressed in the budding epidermal keratinocytes adjacent to the BMZ. At these stages, Gel A-positive mesenchymal-like cells were found scattered throughout the stroma with numerous Gel A-containing cells in direct contact with the developing appendageal buds. In situ zymography was used to show Gel A-activity in vivo. Gel A-mediated lysis was present at the interface between the appendageal buds and the underlying BMZ. By 20-25 wk of gestational age, immunostaining for type VII collagen protein was absent from the BMZ surrounding the distal portion of invading appendageal epithelial cords of both hair follicles and sweat glands. In contrast, type VII collagen mRNA was present in the basal keratinocytes adjacent to the BMZ surrounding the distal portion of these invading appendageal epithelial cords. At these stages Gel A-positive cells were present in the stroma directly adjacent to the distal portion of developing appendageal cords that lacked type VII collagen. In situ zymography showed zones of Gel A-mediated stromal lysis at the distal portion of developing appendageal cords. Interestingly, no differences were seen in the distribution of type IV collagen in the BMZ of both budding and resting fetal epidermis. These observations suggest that the absence of type VII collagen

  6. 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

    DEFF Research Database (Denmark)

    Barascuk, Natasha; Vassiliadis, Efstathios; Larsen, Lise

    2011-01-01

    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....

  7. Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

    Directory of Open Access Journals (Sweden)

    Hisatomi Keiko

    2012-06-01

    Full Text Available Abstract Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF. We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

  8. Reversal of tolerance induced by transplantation of skin expressing the immunodominant T cell epitope of rat type II collagen entitles development of collagen-induced arthritis but not graft rejection

    DEFF Research Database (Denmark)

    Bäcklund, Johan; Treschow, Alexandra; Firan, Mihail

    2002-01-01

    collagen (CI), e.g. in skin, are tolerized against rat CII and resistant to CIA. In this study we transplanted skin from TSC transgenic mice onto non-transgenic CIA-susceptible littermates to investigate whether introduction of this epitope to a naïve immune system would lead to T cell priming and graft......Collagen-induced arthritis (CIA) is induced in H-2(q) mice after immunization with rat type II collagen (CII). The immunodominant T cell epitope on heterologous CII has been located to CII256-270. We have previously shown that TSC transgenic mice, which express the heterologous epitope in type I...... rejection or instead to tolerance and arthritis protection. Interestingly, TSC grafts were accepted and not even immunization of recipient mice with CII in adjuvant induced graft rejection. Instead, TSC skin recipients displayed a reduced T and B cell response to CII and were also protected from arthritis...

  9. Effect of surfactants on surface activity and rheological properties of type I collagen at air/water interface.

    Science.gov (United States)

    Kezwoń, Aleksandra; Góral, Ilona; Frączyk, Tomasz; Wojciechowski, Kamil

    2016-12-01

    We describe the effect of three synthetic surfactants (anionic - sodium dodecyl sulfate (SDS), cationic - cetyltrimethylammonium bromide (CTAB) and nonionic - Triton X-100 (TX-100)) on surface properties of the type I calf skin collagen at the air/water interface in acidic solutions (pH 1.8). The protein concentration was fixed at 5×10(-6)molL(-1) and the surfactant concentration was varied in the range 5×10(-6)molL(-1)-1×10(-4)molL(-1), producing the protein/surfactant mixtures with molar ratios of 1:1, 1:2, 1:5, 1:10 and 1:20. An Axisymmetric Drop Shape Analysis (ADSA) method was used to determine the dynamic surface tension and surface dilatational moduli of the mixed adsorption layers. Two spectroscopic techniques: UV-vis spectroscopy and fluorimetry allowed us to determine the effect of the surfactants on the protein structure. The thermodynamic characteristic of the mixtures was studied using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Modification of the collagen structure by SDS at low surfactant/protein ratios has a positive effect on the mixture's surface activity with only minor deterioration of the rheological properties of the adsorbed layers. The collagen/CTAB mixtures do not show that pronounced improvement in surface activity, while rheological properties are significantly deteriorated. The mixtures with non-ionic TX-100 do not show any synergistic effects in surface activity.

  10. Nitrosonifedipine ameliorates angiotensin II-induced vascular remodeling via antioxidative effects.

    Science.gov (United States)

    Sakurada, Takumi; Ishizawa, Keisuke; Imanishi, Masaki; Izawa-Ishizawa, Yuki; Fujii, Shoko; Tominaga, Erika; Tsuneishi, Teppei; Horinouchi, Yuya; Kihira, Yoshitaka; Ikeda, Yasumasa; Tomita, Shuhei; Aihara, Ken-ichi; Minakuchi, Kazuo; Tsuchiya, Koichiro; Tamaki, Toshiaki

    2013-01-01

    Nifedipine is unstable under light and decomposes to a stable nitroso analog, nitrosonifedipine (NO-NIF). The ability of NO-NIF to block calcium channels is quite weak compared with that of nifedipine. Recently, we have demonstrated that NO-NIF reacts with unsaturated fatty acid leading to generate NO-NIF radical, which acquires radical scavenging activity. However, the effects of NO-NIF on the pathogenesis related with oxidative stress, such as atherosclerosis and hypertension, are unclear. In this study, we investigated the effects of NO-NIF on angiotensin II (Ang II)-induced vascular remodeling. Ang II-induced thickening and fibrosis of aorta were inhibited by NO-NIF in mice. NO-NIF decreased reactive oxygen species (ROS) in the aorta and urinary 8-hydroxy-20-deoxyguanosine. Ang II-stimulated mRNA expressions of p22(phox), CD68, F4/80, monocyte chemoattractant protein-1, and collagen I in the aorta were inhibited by NO-NIF. Moreover, NO-NIF inhibited Ang II-induced cell migration and proliferation of vascular smooth muscle cells (VSMCs). NO-NIF reduced Ang II-induced ROS to the control level detected by dihydroethidium staining and lucigenin chemiluminescence assay in VSMCs. NO-NIF suppressed phosphorylations of Akt and epidermal growth factor receptor induced by Ang II. However, NO-NIF had no effects on intracellular Ca(2+) increase and protein kinase C-δ phosphorylation induced by Ang II in VSMCs. The electron paramagnetic resonance spectra indicated the continuous generation of NO-NIF radical of reaction with cultured VSMCs. These findings suggest that NO-NIF improves Ang II-induced vascular remodeling via the attenuation of oxidative stress.

  11. Mechanical properties of collagen fibrils

    OpenAIRE

    Wenger, M. P. E.; Bozec, L.; Horton, M. A.; Mesquida, P

    2007-01-01

    The formation of collagen fibers from staggered subfibrils still lacks a universally accepted model. Determining the mechanical properties of single collagen fibrils ( diameter 50 - 200 nm) provides new insights into collagen structure. In this work, the reduced modulus of collagen was measured by nanoindentation using atomic force microscopy. For individual type 1 collagen fibrils from rat tail, the modulus was found to be in the range from 5 GPa to 11.5 GPa ( in air and at room temperature)...

  12. Hepatocyte Growth Factor Suppresses Transforming Growth Factor-Beta-1 and Type III Collagen in Human Primary Renal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Shan Mou

    2009-11-01

    Full Text Available Tubulointerstitial changes in the diabetic kidney correlate closely with renal fibrosis, and transforming growth factor-beta-1 (TGF-β1 is thought to play a key role in this process. In contrast, hepatocyte growth factor (HGF has shown therapeutic effects on injured renal tubules in animal models. This study was undertaken to test the hypothesis that the preventive effects of HGF may result from interventions in TGF-β1-mediated signaling and collagen III secretion. We examined the expression of HGF/HGF receptor (c-Met and TGF-β1 in renal fibroblasts at multiple time points. The effects of recombinant human HGF on TGF-β1 expression were studied by RT-PCR and Western blotting, and the levels of collagen III were measured by ELISA. In the high-glucose condition, the expression of HGF and c-Met in renal fibroblasts was detected as early as 6 hours following cell culture while the level of TGF-β1 peaked at 96 hours. The addition of recombinant human HGF to the culture media dose-dependently inhibited TGF-β1 mRNA expression and reduced collagen III secretion by 34%. These results indicate that, during hyperglycemia, HGF inhibits TGF-β1 signaling and type III collagen activation in interstitial fibroblasts. Furthermore, we should recognize that changes in the balance between HGF and TGF-β1 might be decisive in the pathogenesis of chronic renal fibrosis. Therefore, administration of HGF to restore this balance may offer a novel therapeutic intervention in managing renal fibrogenesis in diabetic nephropathy.

  13. Motor physical therapy affects muscle collagen type I and decreases gait speed in dystrophin-deficient dogs.

    Directory of Open Access Journals (Sweden)

    Thaís P Gaiad

    Full Text Available Golden Retriever Muscular Dystrophy (GRMD is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD maintained their routine of activities of daily living. At t0 (pre and t1 (post-physical therapy, collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy, mediolateral (Fz and craniocaudal (Fx ground reaction forces (GRF were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000. The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.

  14. Eccentric rehabilitation exercise increases peritendinous type I collagen synthesis in humans with Achilles tendinosis

    DEFF Research Database (Denmark)

    Langberg, Henning; Ellingsgaard, H; Madsen, T

    2007-01-01

    It has been shown that 12 weeks of eccentric heavy resistance training can reduce pain in runners suffering from chronic Achilles tendinosis, but the mechanism behind the effectiveness of this treatment is unknown. The present study investigates the local effect of an eccentric training regime...... of heavy-resistance eccentric training apart from their regular training and soccer activity. Before and after the training period the tissue concentration of indicators of collagen turnover was measured by the use of the microdialysis technique. After training, collagen synthesis was increased...... in the healthy tendons. The clinical effect of the 12 weeks of eccentric training was determined by using a standardized loading procedure of the Achilles tendons showing a decrease in pain in all the chronic injured tendons (VAS before 44+/-9, after 13+/-9; P...

  15. Both hyaluronan and collagen type II keep proteoglycan 4 (lubricin) at the cartilage surface in a condition that provides low friction during boundary lubrication.

    Science.gov (United States)

    Majd, Sara Ehsani; Kuijer, Roel; Köwitsch, Alexander; Groth, Thomas; Schmidt, Tannin A; Sharma, Prashant K

    2014-12-09

    Wear resistant and ultralow friction in synovial joints is the outcome of a sophisticated synergy between the major macromolecules of the synovial fluid, e.g., hyaluronan (HA) and proteoglycan 4 (PRG4), with collagen type II fibrils and other non-collagenous macromolecules of the cartilage superficial zone (SZ). This study aimed at better understanding the mechanism of PRG4 localization at the cartilage surface. We show direct interactions between surface bound HA and freely floating PRG4 using the quartz crystal microbalance with dissipation (QCM-D). Freely floating PRG4 was also shown to bind with surface bound collagen type II fibrils. Albumin, the most abundant protein of the synovial fluid, effectively blocked the adsorption of PRG4 with HA, through interaction with C and N terminals on PRG4, but not that of PRG4 with collagen type II fibrils. The above results indicate that collagen type II fibrils strongly contribute in keeping PRG4 in the SZ during cartilage articulation in situ. Furthermore, PRG4 molecules adsorbed very well on mimicked SZ of absorbed HA molecules with entangled collagen type II fibrils and albumin was not able to block this interaction. In this last condition PRG4 adsorption resulted in a coefficient of friction (COF) of the same order of magnitude as the COF of natural cartilage, measured with an atomic force microscope in lateral mode.

  16. Collagen turnover after tibial fractures

    DEFF Research Database (Denmark)

    Joerring, S; Krogsgaard, M; Wilbek, H

    1994-01-01

    Collagen turnover after tibial fractures was examined in 16 patients with fracture of the tibial diaphysis and in 8 patients with fracture in the tibial condyle area by measuring sequential changes in serological markers of turnover of types I and III collagen for up to 26 weeks after fracture...... collagen. A group comparison showed characteristic sequential changes in the turnover of types I and III collagen in fractures of the tibial diaphysis and tibial condyles. The turnover of type III collagen reached a maximum after 2 weeks in both groups. The synthesis of type I collagen reached a maximum...... after 2 weeks in the diaphyseal fractures and after 6 weeks in the condylar fractures. The degradation of type I collagen increased after 4 days and reached a maximum at 2 weeks in both groups. The interindividual variation was wide. On a group basis, the turnover of types I and III collagen had...

  17. Four patients with Sillence type I osteogenesis imperfecta and mild bone fragility, complicated by left ventricular cardiac valvular disease and cardiac tissue fragility caused by type I collagen mutations

    DEFF Research Database (Denmark)

    Vandersteen, Anthony M; Lund, Allan M; Ferguson, David J P

    2014-01-01

    Osteogenesis imperfecta (OI) type I is a hereditary disorder of connective tissue (HDCT) characterized by blue or gray sclerae, variable short stature, dentinogenesis imperfecta, hearing loss, and recurrent fractures from infancy. We present four examples of OI type I complicated by valvular heart...... disease and associated with tissue fragility. The diagnosis of a type I collagen disorder was confirmed by abnormal COL1A1 or COL1A2 gene sequencing. One patient was investigated with electrophoresis of collagens from cultured skin fibroblasts, showing structurally abnormal collagen type I, skin biopsy...

  18. De Novo and Inherited Mutations in COL4A2, Encoding the Type IV Collagen α2 Chain Cause Porencephaly

    Science.gov (United States)

    Yoneda, Yuriko; Haginoya, Kazuhiro; Arai, Hiroshi; Yamaoka, Shigeo; Tsurusaki, Yoshinori; Doi, Hiroshi; Miyake, Noriko; Yokochi, Kenji; Osaka, Hitoshi; Kato, Mitsuhiro; Matsumoto, Naomichi; Saitsu, Hirotomo

    2012-01-01

    Porencephaly is a neurological disorder characterized by fluid-filled cysts or cavities in the brain that often cause hemiplegia. It has been suggested that porencephalic cavities result from focal cerebral degeneration involving hemorrhages. De novo or inherited heterozygous mutations in COL4A1, which encodes the type IV α1 collagen chain that is essential for structural integrity for vascular basement membranes, have been reported in individuals with porencephaly. Most mutations occurred at conserved Gly residues in the Gly-Xaa-Yaa repeats of the triple-helical domain, leading to alterations of the α1α1α2 heterotrimers. Here we report on two individuals with porencephaly caused by a heterozygous missense mutation in COL4A2, which encodes the type IV α2 collagen chain. Mutations c.3455G>A and c.3110G>A, one in each of the individuals, cause Gly residues in the Gly-Xaa-Yaa repeat to be substituted as p.Gly1152Asp and p.Gly1037Glu, respectively, probably resulting in alterations of the α1α1α2 heterotrimers. The c.3455G>A mutation was found in the proband's mother, who showed very mild monoparesis of the left upper extremity, and the maternal elder uncle, who had congenital hemiplegia. The maternal grandfather harboring the mutation is asymptomatic. The c.3110G>A mutation occurred de novo. Our study confirmed that abnormalities of the α1α1α2 heterotrimers of type IV collagen cause porencephaly and stresses the importance of screening for COL4A2 as well as for COL4A1. PMID:22209246

  19. Influence of Biodentine® - A Dentine Substitute - On Collagen Type I Synthesis in Pulp Fibroblasts In Vitro.

    Science.gov (United States)

    Nikfarjam, Frangis; Beyer, Kim; König, Anke; Hofmann, Matthias; Butting, Manuel; Valesky, Eva; Kippenberger, Stefan; Kaufmann, Roland; Heidemann, Detlef; Bernd, August; Zöller, Nadja Nicole

    2016-01-01

    Preserving a patient's own teeth-even in a difficult situation-is nowadays preferable to surgical intervention and therefore promotes development of suitable dental repair materials. Biodentine®, a mineral trioxide aggregate substitute, has been used to replace dentine in a bioactive and biocompatible manner in both the dental crown and the root. The aim of our study was to evaluate the influence of Biodentine® on pulp fibroblasts in vitro. For this study, one to five Biodentine® discs with a diameter of 5.1mm were incubated in DMEM. To obtain Biodentine® suspensions the media were collected and replaced with fresh medium every 24h for 4 days. Primary pulp cells were isolated from freshly extracted wisdom teeth of 20-23 year old patients and incubated with the Biodentine® suspensions. Proliferation, cell morphology, cell integrity and cell viability were monitored. To evaluate the effect of Biodentine® on collagen type I synthesis, the secretion of the N-terminal domain of pro-collagen type I (P1NP) and the release of transforming growth factor-β1 (TGF-β1) were quantified. None of the Biodentine® suspensions tested influenced cell morphology, proliferation or cell integrity. The cell viability varied slightly depending on the suspension used. However, the concentrations of P1NP of all pulp fibroblast cultures treated for 24h with the moderate to high Biodentine® concentration containing suspensions of day 1 were reduced to 5% of the control. Furthermore, a significant TGF-β1 reduction was observed after treatment with these suspensions. It could be shown that Biodentine® is biocompatible. However, dissolved particles of the moderate to high concentrated Biodentine® suspensions 24h after mixing induce a significant reduction of TGF-β1 release and reduce the secretion of collagen type I of primary pulp fibroblasts.

  20. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-02-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  1. EFFECT OF LOW SELENIUM ON CHONDROCYTE DIFFERENTIATION AND DIFFERENTIAL EXPRESSION OF COLLAGEN TYPES Ⅰ , Ⅱ AND X IN ARTICULAR CARTILAGE FROM MINI-PIGS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective According to the distribution of low selenium areas, low nutrition state of the residents and the affecting cartilage growth and articular cartilage of Kashin-Beck Disease(KBD),the chondrocyte differentia- tion and differential expression of collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage from Chinese mini-pigs treated with low selenium were investigated in order to gain insight into the effects of these conditions on chondrocyte differ- entiation in KBD cartilage. Mothods Eleven male juvenile mini-pigs, aged from 4 weeks to 6 weeks after birth, were divided into 3 groups. The Se content in the diet of the “low Se” group was 0. 035mg/kg diet, and 0. 175 mg/kg diet in the control. For Se-supplemented group 0. 390mg /kg diet was added. The content of Se in blood was assayed at the beginning and at the end of each experiment. Samples of articular cartilage were taken from the right femur condylus, and collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage were analyzed by immunohistochemistry and in situ hybridization. Results ①All cartilage samples from juvenile mini-pigs fed with low selenium diet revealed a re- duction in typecollagen mRNA expression in the hypertrophic chondrocytes as shown by in situ hybridization, and reduced typecollagen deposition in the lower hypertrophic zone as shown by immunohistochemistry. ②Addition of selenium to the diet restored the typecollagen to normal level. ③Typecollagen was evenly distributed over the entire articular cartilage in all experimental and control groups. Typecollagen mRNA signals were most prominent in the upper articular layer as well as in the hypertrophic zone in all groups. Typecollagen expression was restrict- ed to the zone of endochondral ossification in all experimental groups and the control. Conclusion Low selenium has an down-regulatory role on the synthesis and deposition of collagen type Ⅹ in hypertrophic chondrocytes in articular cartilage of

  2. Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo

    DEFF Research Database (Denmark)

    Ågren, Magnus S; Schnabel, Reinhild; Christensen, Lise H

    2015-01-01

    /ml) in the absence or presence of the nonselective MMP inhibitor GM6001 for 8 days. The basal culture conditions promoted type I collagen catabolism that was accelerated by TNF-α (p...Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10ng...... were associated with increased collagen degradation. TNF-α increased secretion of MMP-1 (p

  3. Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional therapy of type 1 diabetes - Relevance of glycated collagen products versus HbA(1c) as markers of diabetic complications

    NARCIS (Netherlands)

    Monnier, VM; Bautista, O; Kenny, D; Sell, DR; Fogarty, J; Dahms, W; Cleary, PA; Lachin, J; Genuth, S

    1999-01-01

    The relationships between long-term intensive control of glycemia and indicators of skin collagen glycation (furosine), glycoxidation (pentosidine and N-epsilon-[carboxymethyl]-lysine [CML]), and crosslinking (acid and pepsin solubility) were examined in 216 patients with type 1 diabetes from the pr

  4. Transforming growth factor-β1/Smad3-independent epithelial–mesenchymal transition in type I collagen glomerulopathy

    Directory of Open Access Journals (Sweden)

    Brodeur AC

    2017-08-01

    Full Text Available Amanda C Brodeur,1–3 Anna M Roberts-Pilgrim,3 Kimberlee L Thompson,1 Craig L Franklin,4 Charlotte L Phillips2,3 1Department of Biomedical Sciences, Missouri State University, Springfield, MO, USA; 2Department of Child Health, University of Missouri, Columbia, MO, USA; 3Department of Biochemistry, University of Missouri, Columbia, MO, USA; 4Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA Abstract: The glomerulofibrotic Col1a2-deficient mouse model demonstrates glomerular homotrimeric type I collagen deposition in mesangial and subendothelial spaces. In this report, we investigate the role of transforming growth factor β1 (TGF-β1 in myofibroblast activation and epithelial–mesenchymal transition (EMT in this glomerulopathy. Immunohistochemical analyses of glomerular α-sma, desmin, vimentin, and proliferating cell nuclear antigen demonstrated parietal epithelial cell proliferation and EMT in late stages of the glomerulopathy in the Col1a2-deficient mice. Glomerular TGF-β1 RNA and protein were not elevated in 1- and 3-month-old mice as determined by quantitative reverse transcriptase-polymerase chain reaction and protein immunoassay analyses. To investigate further whether TGF-β1 plays a role in the glomerulopathy outside of the 1- and 3-month time periods, the Col1a2-deficient mice were bred with Smad3 knockout mice. If the glomerular fibrosis in the Col1a2-deficient mice is mediated by the TGF-β1/Smad3 transcription pathway, it was hypothesized that the resultant Col1a2-deficient/Smad3-deficient mice would exhibit attenuated glomerular homotrimer deposition. However, the Col1a2-deficient/Smad3-deficient kidneys were similarly affected as compared to age-matched Col1a2-deficient kidneys, suggesting that homotrimeric type I collagen deposition in the Col1a2-deficient mouse is independent of TGF-β1/Smad3 signaling. Deposition of homotrimeric type I collagen appears to be the initiating event in this

  5. Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

    Science.gov (United States)

    Parreno, Justin; Raju, Sneha; Niaki, Mortah Nabavi; Andrejevic, Katarina; Jiang, Amy; Delve, Elizabeth; Kandel, Rita

    2014-10-16

    This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.

  6. Induction of rat yolk sac carcinomas with consistent pattern of laminin, entactin, and type IV collagen biosynthesis

    DEFF Research Database (Denmark)

    Wewer, U

    1984-01-01

    Twenty-four yolk sac carcinomas in Lewis rats were experimentally induced by puncturing the pregnant uterine wall with a hypodermic needle at day 9-13 of gestation. Morphologically, the tumours were composed of parietal- and visceral yolk sac carcinoma and to a less degree of trophoblastic giant ...... to their biosynthesis of the basement membrane components laminin, entactin, and type IV collagen. This model system offers a simple approach to inducing rat yolk sac carcinomas for further morphological and biochemical characterization of the basement membrane....

  7. Hepatitis B virus infects hepatic stellate cells and affects their proliferation and expression of collagen type

    Institute of Scientific and Technical Information of China (English)

    LIU Xuan; ZHU Sheng-tao; YOU Hong; CONG Min; LIU Tian-hui; WANG Bao-en; JIA Ji-dong

    2009-01-01

    Background Hepatitis B is at particularly high risk of fibrosis progression. Unfortunately, the mechanism of hepatic fibrogenesis induced by hepatitis B virus (HBV) has not been fully understood to date. The aim of this study was to observe whether HBV can infect hepatic stellate cells (HSCs), and to examine the effects of HBV or HBV S protein (HBs) on the proliferation and collagen type Ⅰ expression of HSCs.Methods The supernatants of HepG2.2.15 cells which contained HBV-DNA or HBs were added to LX-2 cells for 72 hours. Cell survival was determined by MTT assay. HBV particles in LX-2 cells were detected by transmission electron microscopy. The expression of HBs and HBV C protein (HBc) was determined by confocal fluorescence microscopy. The expression levels of HBV-DNA were measured by real-time PCR. The cellular collagen type Ⅰ mRNA and protein levels were quantified by reverse transcription-PCR and ELISA, respectively.Results High concentrations of HBV (1.2x105-5.0x105 copies/ml) or HBs (1.25-20 μg/ml) inhibited the proliferation of LX-2 cells, while low concentrations of HBV (1.0x103-6.2x104 copies/ml) or HBs (0.04-0.62 μg/ml) promoted the proliferation. After treating LX-2 cells with HBV for 72 hours, about 42 nm HBV-sized particles and strong expression of HBs and HBc were found in the cytoplasm of LX-2 cells. HBV-DNA in the culture medium of LX-2 cells decreased at 24 hours, rose at 48 hours and thereafter, decreased again at 72 hours. The mRNA and protein expression of cellular collagen type Ⅰ in LX-2 cells were significantly increased by HBV infection but not by recombinant HBs. Conclusions HBV and HBs affect the proliferation of HSCs; HBV can transiently infect and replicate in cultured HSCs and express HBs and HBc in vitro. Furthermore, HBV can significantly increase the expression of collagen type Ⅰ mRNA and protein in HSCs.

  8. Testosterone delays vascular smooth muscle cell senescence and inhibits collagen synthesis via the Gas6/Axl signaling pathway.

    Science.gov (United States)

    Chen, Yan-qing; Zhao, Jing; Jin, Cheng-wei; Li, Yi-hui; Tang, Meng-xiong; Wang, Zhi-hao; Zhang, Wei; Zhang, Yun; Li, Li; Zhong, Ming

    2016-06-01

    Testosterone deficiency is associated with a higher incidence of cardiovascular diseases in men. However, its effect on cell senescence, which plays a causal role in vascular aging, remains unclear. Here, we tested the hypothesis that testosterone alleviated vascular smooth muscle cell (VSMC) senescence and collagen synthesis via growth arrest-specific protein 6 (Gas6)/Axl- and Akt/FoxO1a-dependent pathways. Testosterone significantly ameliorated angiotensin II-induced VSMC senescence and collagen overexpression. In addition, testosterone inhibited angiotensin II-induced matrix metalloproteinase-2 (MMP-2) activity, which played a pivotal role in facilitating age-related collagen deposition. Testosterone increased the expression of tissue inhibitor of metalloproteinase-2 but decreased the expression of MMP-2 and membrane type-1 metalloproteinase which contributed to increase MMP-2 activity. The effects on VSMCs senescence and collagen synthesis were mediated by restoration of angiotensin II-induced downregulation of Gas6 and Axl expression and a subsequent reduction of Akt and FoxO1a phosphorylation. The effects of testosterone were reversed by a Gas6 blocker, Axl-Fc, and a specific inhibitor of Axl, R428. Treatment of VSMCs with PI3K inhibitor LY294002 abrogated the downregulating effect of testosterone on MMP-2 activity. Furthermore, when FoxO1a expression was silenced by using a specific siRNA, the inhibitory effect of testosterone on MMP-2 activity was revered as well, that indicated this process was Akt/FoxO1a dependence. Taken together, Gas6/Axl and Akt/FoxO1a were involved in protective effects of testosterone on VSMCs senescence and collagen synthesis. Our results provide a novel mechanism underlying the protective effect of testosterone on vascular aging and may serve as a theoretical basis for testosterone replacement therapy.

  9. ROLE OF GLYCAEMIA LEVEL IN THE DEVELOPMENT OF INTERSTITIAL COLLAGEN IN PATIENTS WITH CORONARY HEART DISEASE AND TYPE 2 DIABETES

    Directory of Open Access Journals (Sweden)

    T. Rudenko

    2015-12-01

    Full Text Available A role of blood glucose levels in the development of interstitial collagen has been studied in 84 patients (53 women and 31 men, average age 60 ± 2.4 years with coronary heart disease (CHD. All patients were divided into twocomparable groups: a study group including patients with coronary heart disease andtype 2 diabetes mellitus (DM and a control group consisting of patients with coronary heart disease without DM. All patients received standard medical therapy as recommended by the European Society of Cardiology. The level of blood glucose in both groups was assessed by the standard technique, a degree of interstitial collagen volume fraction (ICVF was measured using the formula of J. Shirani et al. The data were processed by parametric and nonparametric statistical methods. It has been proved that hyperglycemia in type 2 diabetes contributes to the development of ICVF, the degree of which increases with the rise of blood glucose level. A high level ofICVF in patients with coronary heart disease and diabetes type 2 can be a predictor of myocardial dyssynchrony development and heart failure progression, therefore, a close monitoring and timely correction of changes of blood glucose levelsare recommended to prevent the complication development. ICVF evaluation should become a routine diagnostic method in all patients with type 2 diabetes.

  10. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the [alpha]1 (XVIII) collagen gene to mouse chromosome 10 and human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Oh, S.P.; Warman, M.L.; Timmons, S.; Olsen, B.R.; Knoll, J.H.M. (Harvard Medical School, Boston, MA (United States)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Cheng, Sou-De (Children' s Hospital/Harvard Medical School, Boston, MA (United States))

    1994-02-01

    Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which the authors have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, they report the isolation of human cDNAs and genomic DNAs encoding the [alpha]1 (XVIII) collagen chain. Utilizing a genomic clone as probe, they have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, they have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2. 16 refs., 5 figs.

  11. Multi-Hierarchical Self-Assembly of Collagen Mimetic Peptides into AAB Type Heterotrimers, Nanofibers and Hydrogels Driven by Charged Pair Interactions

    Science.gov (United States)

    O'Leary, Lesley Russell

    2011-12-01

    Replicating the multi-hierarchical self-assembly of collagen (peptide chain to triple helix to nanofiber and, finally, to a hydrogel) has long attracted scientists, both from the fundamental science perspective of supramolecular chemistry and for the potential biomedical applications perceived in tissue engineering. In terms of triple helical formation, collagen is the most abundant protein in the human body with at least 28 types, yet research involving collagen mimetic systems has only recently began to consider the innate ability of collagen to control helix composition and register. Collagen triple helices can be homotrimeric or heterotrimeric and while some types of natural collagen form only one specific composition of helix, others can form multiple. It is critical to fully understand and, if possible, reproduce the control that native collagen has on helix composition and register. In terms of nanofiber formation, many approaches to drive the self-assembly of synthetic systems through the same steps as natural collagen have been partially successful, but none have simultaneously demonstrated all levels of structural assembly. In this work, advancements in the ability to control helix composition and replicate the multi-hierarchical assembly of collagen are described. Both positive and negative design for the assembly of AAB type collagen heterotrimers were utilized by promoting heterotrimer formation though the use of charged amino acids to form intra-helix electrostatic interactions, while simultaneously discouraging homotrimers, resulting in the identification of multiple peptide systems with full control over the composition of the resulting triple helix. Similar salt-bridged hydrogen bonds between charged residues were incorporated into nanofiber forming peptides, one of which successfully assembled into sticky-ended triple helices, nanofibers with characteristic triple helical packing visible in the solution state, and strong hydrogels that are

  12. Biomimetic Intrafibrillar Mineralization of Type I Collagen with Intermediate Precursors-loaded Mesoporous Carriers

    Science.gov (United States)

    Zhang, Wei; Luo, Xiao-juan; Niu, Li-na; Yang, Hong-ye; Yiu, Cynthia K.Y.; Wang, Tian-da; Zhou, Li-qun; Mao, Jing; Huang, Cui; Pashley, David H.; Tay, Franklin R.

    2015-01-01

    Limited continuous replenishment of the mineralization medium is a restriction for in-situ solution-based remineralization of hypomineralized body tissues. Here, we report a process that generated amine-functionalized mesoporous silica nanoparticles for sustained release of biomimetic analog-stabilized amorphous calcium phosphate precursors. Both two-dimensional and three-dimensional collagen models can be intrafibrillarly mineralized with these released fluidic intermediate precursors. This represents an important advance in the translation of biomineralization concepts into regimes for in-situ remineralization of bone and teeth. PMID:26053330

  13. Sera from patients with anti-GBM nephritis including goodpasture syndrome show heterogenous reactivity to recombinant NC1 domain of type IV collagen alpha chains.

    Science.gov (United States)

    Dehan, P; Weber, M; Zhang, X; Reeders, S T; Foidart, J M; Tryggvason, K

    1996-11-01

    Goodpasture (GP) syndrome is defined by the clinical association of pulmonary haemorrhage with rapidly progressive glomerulonephritis. The disease is caused by pathogenic autoantibodies directed against type IV collagen, which is a major structural component of glomerular basement membranes (GBM). The non-collagenous domains (NC1) of all six human type IV collagen alpha chains was produced in E. coli as recombinant fusion proteins with glutathione-S transferase. Sera from 10 patients with different types of anti-GBM nephritis, including GP syndrome, were tested for reactivity with the six proteins using immunoblotting of denatured and reduced proteins and ELISA without reduction. All 10 sera reacted with the alpha 3 (IV) collagen chain by immunoblotting and ELISA. One serum also recognized the alpha 2(IV), alpha 4(IV), alpha 5(IV) and alpha 6(IV) chains by immunoblotting. ELISA measurements revealed reactivity of several other sera with alpha 2(IV), alpha 4(IV) or alpha 6(IV) but not with alpha 5(IV) collagen chains. No reactivity was observed with the alpha 1(IV) chain. Autoantibodies in anti-GBM nephritis may not be directed only against the alpha 3(IV) collagen chain and they frequently recognize conformational epitopes.

  14. Enigmatic insight into collagen

    Directory of Open Access Journals (Sweden)

    Shrutal Narendra Deshmukh

    2016-01-01

    Full Text Available 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.

  15. [The genetics of collagen diseases].

    Science.gov (United States)

    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.

  16. Enhancement of chondrogenic differentiation of rabbit mesenchymal stem cells by oriented nanofiber yarn-collagen type I/hyaluronate hybrid

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Xianyou; Wang, Wei; Liu, Shen [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China); Wu, Jinglei [Biomaterials and Tissue Engineering Laboratory, College of Chemistry and Chemical Engineering and Biological Engineering, Donghua University, Shanghai 201620 (China); Li, Fengfeng [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China); Cao, Lei [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011 (China); Liu, Xu-dong [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China); Mo, Xiumei, E-mail: xmm@dhu.edu.cn [Biomaterials and Tissue Engineering Laboratory, College of Chemistry and Chemical Engineering and Biological Engineering, Donghua University, Shanghai 201620 (China); Fan, Cunyi, E-mail: fancunyish@126.com [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China)

    2016-01-01

    Cartilage defects cause joint pain and loss of mobility. It is crucial to induce the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by both biological and structural signals in cartilage tissue engineering. Sponge-like scaffolds fabricated using native cartilage extracellular matrix components can induce the BMSC differentiation by biological signals and limited structural signals. In this study, an oriented poly(L-lactic acid)-co-poly(ε-caprolactone) P(LLA-CL)/collagen type I (Col-I) nanofiber yarn mesh, fabricated by dynamic liquid electrospinning served as a skeleton for a freeze-dried Col-I/hyaluronate (HA) chondral phase (SPONGE) containing both structural and biological signals to guide BMSC chondrogenic differentiation. In vitro results show that the Yarn Col-I/HA hybrid scaffold (Yarn-CH) promotes orientation, adhesion and proliferation of BMSCs better than SPONGE. Furthermore, BMSCs seeded on the Yarn-CH scaffold demonstrated a large increase in the glycosaminoglycan content and expression of collagen type II following a 21-day culture. - Highlights: • An oriented yarn was used as the skeleton of the sponge-like scaffold. • Both structural and biological signals were given for BMSC chondrogenic differentiation. • Yarn-CH promotes orientation and chondrogenesis differentiation of BMSCs. • Yarn-CH reproduces the superficial zone of the cartilage.

  17. Type II collagen-hyaluronan hydrogel – a step towards a scaffold for intervertebral disc tissue engineering

    Directory of Open Access Journals (Sweden)

    L Calderon

    2010-09-01

    Full Text Available Intervertebral disc regeneration strategies based on stem cell differentiation in combination with the design of functional scaffolds is an attractive approach towards repairing/regenerating the nucleus pulposus. The specific aim of this study was to optimise a composite hydrogel composed of type II collagen and hyaluronic acid (HA as a carrier for mesenchymal stem cells. Hydrogel stabilisation was achieved by means of 1-ethyl-3(3-dimethyl aminopropyl carbodiimide (EDC and N-hydroxysuccinimide (NHS cross-linking. Optimal hydrogel properties were determined by investigating different concentrations of EDC (8mM, 24mM and 48mM. Stable hydrogels were obtained independent of the concentration of carbodiimide used. The hydrogels cross-linked by the lowest concentration of EDC (8mM demonstrated high swelling properties. Additionally, improved proliferation of seeded rat mesenchymal stem cells (rMSCs and hydrogel stability levels in culture were observed with this 8mM cross-linked hydrogel. Results from this study indicate that EDC/NHS (8mM cross-linked type II collagen/HA hydrogel was capable of supporting viability of rMSCs, and furthermore their differentiation into a chondrogenic lineage. Further investigations should be conducted to determine its potential as scaffold for nucleus pulposus regeneration/repair.

  18. Mechanical properties and solubility in water of corn starch-collagen composite films: Effect of starch type and concentrations.

    Science.gov (United States)

    Wang, Kun; Wang, Wenhang; Ye, Ran; Liu, Anjun; Xiao, Jingdong; Liu, Yaowei; Zhao, Yana

    2017-02-01

    This study investigated the possibility of enhancing the properties of collagen with three different maize starches: waxy maize starch, normal starch, and high amylose starch. Scanning electron microscopy images revealed that starch-collagen films had a rougher surface compared to pure collagen films which became smoother upon heating. Amylose starch and normal starch increased the tensile strength of unheated collagen films in both dry and wet states, while all starches increased tensile strength of collagen film by heating. Depending upon the amylose content and starch concentrations, film solubility in water decreased with the addition of starch. DSC thermograms demonstrated that addition of all starches improved the thermal stability of the collagen film. Moreover, X-ray diffraction results indicated that except for high amylose starch, the crystallinity of both starch and collagen was significantly decreased when subject to heating. FTIR spectra indicated that intermolecular interactions between starch and collagen were enhanced upon heating.

  19. Enzyme-linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S)

    DEFF Research Database (Denmark)

    Leeming, Diana J; Nielsen, Mette J; Dai, Yueqin

    2012-01-01

    Aim:  The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100...... were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P collagen...... expression in BDL rats (r = 0.49, P collagen in CCL4 treated livers (P 

  20. Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Londei, M.; Savill, C.M.; Verhoef, A.; Brennan, F.; Leech, Z.A.; Feldmann, M. (Charing Cross Sunley Research Centre, London (England)); Duance, V. (Bristol Laboratory (England)); Maini, R.N. (Kennedy Institute of Rheumatology, London (England))

    1989-01-01

    Rheumatoid arthritis is an autoimmune disease characterized by T-cell infiltration of the synovium of joints. Analysis of the phenotype and antigen specificity of the infiltrating cells may thus provide insight into the pathogenesis of rheumatoid arthritis. T cells were cloned with interleukin 2, a procedure that selects for in vivo-activated cells. All clones had the CD4 CDW29 phenotype. Their antigen specificity was tested by using a panel of candidate joint autoantigens. Four of 17 reacted against autologous blood mononuclear cells. Two clones proliferated in response to collagen type II. After 21 months, another set of clones was derived from synovial tissue of the same joint. One of eight clones tested showed a strong proliferative response against collagen type II. The uncloned synovial T cells of a third operation from another joint also responded to collagen type II. The persistence of collagen type II-specific T cells in active rheumatoid joints over a period of 3 years suggests that collagen type II could be one of the autoantigens involved in perpetuating the inflammatory process in rheumatoid arthritis.

  1. Chondrocyte-seeded type I/III collagen membrane for autologous chondrocyte transplantation

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Lenz, Philipp; Kreuz, Peter C;

    2010-01-01

    PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT-CS...... Repair Society (ICRS) rating, the percentage of patients rated A (normal) and B (nearly normal) increased from 33.9% preoperatively to 92.5% at 24 months after ACT-CS. IKDC and Lysholm scores increased from 50.1 points (SD, 13.4) and 60.5 points (SD, 9.4), respectively, to 76.1 points (SD, 15.2) (P...... CS as a salvage procedure. The rate of failures in patients with isolated cartilage defects was 5.9%. CONCLUSIONS: ACT-CS...

  2. Induction of tolerance against the arthritogenic antigen with type-II collagen peptide-linked soluble MHC class II molecules

    Science.gov (United States)

    Park, Yoon-Kyung; Jung, Sundo; Park, Se-Ho

    2016-01-01

    In murine collagen-induced arthritis (CIA), self-reactive T cells can recognize peptide antigens derived from type-II collagen (CII). Activation of T cells is an important mediator of autoimmune diseases. Thus, T cells have become a focal point of study to treat autoimmune diseases. In this study, we evaluated the efficacy of recombinant MHC class II molecules in the regulation of antigen-specific T cells by using a self peptide derived from CII (CII260-274; IAGFKGEQGPKGEPG) linked to mouseI-Aq in a murine CIA model. We found that recombinant I-Aq/CII260-274 molecules could be recognized by CII-specific T cells and inhibit the same T cells in vitro. Furthermore, the development of CIA in mice was successfully prevented by in vivo injection of recombinant I-Aq/CII260-274 molecules. Thus, treatment with recombinant soluble MHC class II molecules in complex with an immunodominant self-peptide might offer a potential therapeutic for chronic inflammation in autoimmune disease such as rheumatoid arthritis. [BMB Reports 2016; 49(6): 331-336 PMID:26779996

  3. Immunological detection of the type V collagen propeptide fragment, PVCP-1230, in connective tissue remodeling associated with liver fibrosis

    DEFF Research Database (Denmark)

    Vassiliadis, Efstathios; Veidal, Sanne Skovgård; Simonsen, Henrik

    2011-01-01

    AIM: Liver fibrosis involves excessive remodeling and deposition of fibrillar extracellular matrix (ECM) components, which leads to malfunction of the organ, causing significant morbidity and mortality. The aim of this study was to assess whether levels of a type V collagen fragment, the propeptide...... CO5-1230, indicate the amount of collagen deposited during liver fibrosis. METHODS: A specific competitive enzyme-linked immunosorbent assay (ELISA) was developed to measure CO5-1230 levels. The sequence TAALGDIMGH located at the start of the C-terminal propeptide between amino acid position 1230......, for up to 20 weeks. RESULTS: The ELISA was capable of measuring CO5-1230 in serum specifically, with an intra-assay variation of 3.46% and inter-assay variation of 5.09%. Mean CO5-1230 levels were significantly elevated in CCl(4) rats compared with controls [8 weeks: 57.4 ng/mL, controls 45.5 ng/mL (P...

  4. Collagenous gastroduodenitis.

    Science.gov (United States)

    Rustagi, Tarun; Rai, Mridula; Scholes, John V

    2011-10-01

    Collagenous gastroduodenitis is a rare histopathologic entity characterized by marked subepithelial collagen deposition with associated mucosal inflammatory infiltrate. Only 4 cases have been reported, of which 3 had associated collagenous colitis. Collagenous gastroduodenitis without colonic involvement is exceptionally rare with only 1 case reported so far in the literature. We present a case of a 68-year-old woman with dyspepsia and mild anemia, who was found to have nodular gastric and duodenal mucosa on endoscopic examination. Histopathology showed collagenous gastroduodenitis. To the best of our knowledge, this is the second (and first in English literature) reported case of isolated collagenous gastroduodenitis.

  5. Long-term changes in collagen formation expressed by serum carboxyterminal propeptide of type-I procollagen and relation to left ventricular function after acute myocardial infarction

    DEFF Research Database (Denmark)

    Poulsen, S H; Høst, N B; Egstrup, K

    2001-01-01

    The purpose of this study was to investigate the long-term sequential changes in serum levels of the carboxyterminal propeptide of type-I procollagen (s-PICP), which is a marker of type-I collagen synthesis, and to assess its clinical value in relation to left ventricular (LV) function...

  6. The Staphylococcus aureus lineage-specific markers collagen adhesin and toxic shock syndrome toxin 1 distinguish multilocus sequence typing clonal complexes within spa clonal complexes

    NARCIS (Netherlands)

    Deurenberg, Ruud H; Rijnders, Michelle I A; Sebastian, Silvie; Welling, Maaike A; Beisser, Patrick S; Stobberingh, Ellen E

    2009-01-01

    Spa typing/based upon repeat pattern (BURP) sometimes cannot differentiate multilocus sequence typing (MLST) clonal complexes (CCs) within spa-CCs. It has been observed previously that virulence factors, such as collagen adhesin (CNA) and toxic shock syndrome toxin 1 (TSST-1), are associated with

  7. The neo-epitope specific PRO-C3 ELISA measures true formation of type III collagen associated with liver and muscle parameters

    DEFF Research Database (Denmark)

    Nielsen, Mette J; Nedergaard, Anders F; Sun, Shu;

    2013-01-01

    AIM: The present study describes the assessment of true formation of type III collagen in different pathologies using a neo-epitope specific competitive Enzyme-linked immunosorbent assay (ELISA) towards the N-terminal propeptide of type III collagen (PRO-C3). METHODS: The monoclonal antibody...... was raised against the N-protease mediated cleavage site of the N-terminal propeptide of type III collagen and a competitive ELISA was developed using the selected antibody. The assay was evaluated in relation to neo-epitope specificity, technical performance, and as a marker for liver fibrosis and muscle...... mass using the rat carbon tetrachloride (CCl4) model and a study of immobilization induced muscle loss in humans, respectively. RESULTS: The ELISA was neo-epitope specific, technically stable and can be assessed in serum and plasma samples. In the CCl4 liver fibrosis model it was observed that serum...

  8. Development and application of a new Silent reporter system to quantitate the activity of enhancer elements in the type II Collagen Gene.

    Science.gov (United States)

    Ito, Kazuo; Shinomura, Tamayuki

    2016-07-01

    Type II collagen is a major component of cartilage, which provide structural stiffness to the tissue. As a sufficient amount of type II collagen is critical for maintaining the biomechanical properties of cartilage, its expression is tightly regulated in chondrocytes. Therefore, it is essential to elucidate in detail the transcriptional mechanism that controls expression of type II collagen, in particular by two enhancer elements we recently discovered. To systematically analyze and compare enhancer activities, we developed a novel reporter assay system that exploits site-specific integration of promoter and enhancer elements to activate a transcriptionally silent reporter gene. Using this system, we found that the enhancer elements have distinct characteristics, with one exhibiting additive effects and the other exhibiting synergistic effects when repeated in tandem.

  9. Myocardial repair with long-term and low-dose administration of a nitric oxide synthesis inhibitor. Myofibroblasts, type III collagen and fibronectin

    Directory of Open Access Journals (Sweden)

    Pessanha Mônica Gomes

    1999-01-01

    Full Text Available OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.

  10. An unusual case of pulmonary-renal syndrome associated with defects in type IV collagen composition and anti-glomerular basement membrane autoantibodies.

    Science.gov (United States)

    Charytan, David; MacDonald, Brian; Sugimoto, Hikaru; Pastan, Stephen; Staton, Gerald; Hennigar, Randy; Kalluri, Raghu

    2005-04-01

    Commercial serological assays for the presence of anti-glomerular basement membrane (GBM) antibodies are thought to be indicative of Goodpasture's syndrome. We report a case in which commercial tests inaccurately suggested that a patient with a pulmonary-renal syndrome had Goodpasture's disease. Additional laboratory testing using recombinant type IV collagen NC1 domain proteins showed that the autoantibodies in question were not directed against the Goodpasture antigen (the alpha3NC1 domain), but against the alpha2NC1 domain of type IV collagen. Our findings represent the first known case of human autoantibodies to the alpha2NC1 domain. Further investigation showed that this patient has decreased alpha3 and alpha5 chain expression in the GBM and defects in type IV collagen, resembling abnormalities in patients with Alport's syndrome.

  11. Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes

    Directory of Open Access Journals (Sweden)

    Natasja Stæhr Gudmann

    2014-10-01

    Full Text Available The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP. This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab was raised in mouse, targeting specifically PIIBNP (QDVRQPG and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6–2.2 nM, human amniotic fluid (163–188 nM and sera from different animal species, e.g., fetal bovine serum (851–901 nM with general good linearity (100% (SD 7.6 recovery and good intra- and inter-assay variation (CV% < 10. Dose (0.1 to 100 ng/mL and time (7, 14 and 21 days dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX and human cartilage explants (HEX upon stimulation with insulin-like growth factor (IGF-1, transforming growth factor (TGF-β1 and fibroblastic growth factor-2 (FGF-2. TGF-β1 and IGF-1 in concentrations of 10–100 ng/mL significantly (p < 0.05 induced release of PIIBNP in BEX compared to conditions without treatment (WO. In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.

  12. NMR studies demonstrate a unique AAB composition and chain register for a heterotrimeric type IV collagen model peptide containing a natural interruption site.

    Science.gov (United States)

    Xiao, Jianxi; Sun, Xiuxia; Madhan, Balaraman; Brodsky, Barbara; Baum, Jean

    2015-10-02

    All non-fibrillar collagens contain interruptions in the (Gly-X-Y)n repeating sequence, such as the more than 20 interruptions found in chains of basement membrane type IV collagen. Two selectively doubly labeled peptides are designed to model a site in type IV collagen with a GVG interruption in the α1(IV) and a corresponding GISLK sequence within the α2(IV) chain. CD and NMR studies on a 2:1 mixture of these two peptides support the formation of a single-component heterotrimer that maintains the one-residue staggering in the triple-helix, has a unique chain register, and contains hydrogen bonds at the interruption site. Formation of hydrogen bonds at interruption sites may provide a driving force for self-assembly and chain register in type IV and other non-fibrillar collagens. This study illustrates the potential role of interruptions in the structure, dynamics, and folding of natural collagen heterotrimers and forms a basis for understanding their biological role.

  13. MMP Mediated Degradation of Type IV Collagen Alpha 1 and Alpha 3 Chains Reflects Basement Membrane Remodeling in Experimental and Clinical Fibrosis - Validation of Two Novel Biomarker Assays

    DEFF Research Database (Denmark)

    Sand, Jannie Marie; Larsen, Lise Skakkebæk; Hogaboam, Cory;

    2013-01-01

    Fibrosis is characterized by excessive tissue remodeling resulting from altered expression of various growth factors, cytokines and proteases. We hypothesized that matrix metalloproteinase (MMP) mediated degradation of type IV collagen, a main component of the basement membrane, will release...... peptide fragments (neo-epitopes) into the circulation. Here we present the development of two competitive enzyme-linked immunosorbent assays (ELISAs) for assessing the levels of specific fragments of type IV collagen α1 (C4M12a1) and α3 (C4M12a3) chains in serum as indicators of fibrosis....

  14. Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    刘刚; 胡蕴玉; 赵建宁; 吴苏稼; 熊卓; 吕荣

    2004-01-01

    Objective: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). Methods: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm×1.2 cm×2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [3H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). Results: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm±141cpm vs. 1797 cpm±118 cpm, P=0.017; 8 h, 2311 cpm±113 cpm vs. 1891 cpm±103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7th day (1021 cpm±159 cpm vs. 451 cpm±67 cpm, P=0.002), the 14th day (1472 cpm±82 cpm vs. 583 cpm±67 cpm, P<0.001) and 21th day (1728 cpm±78 cpm vs. 632 cpm±55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls. Conclusions: Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.

  15. Plasma Pro-C3 (N-terminal type III collagen propeptide) predicts fibrosis progression in patients with chronic hepatitis C

    DEFF Research Database (Denmark)

    Nielsen, Mette J.; Veidal, Sanne S.; Karsdal, Morten A.

    2015-01-01

    BACKGROUND & AIMS: Fibrogenesis results in release of certain extracellular matrix protein fragments into the circulation. We evaluated the diagnostic and prognostic performance of two novel serological markers, the precisely cleaved N-terminal propeptide of type III collagen (Pro-C3) and a peptide...... of helical collagen type III degradation (C3M), in chronic hepatitis C (CHC) patients. METHOD: Pro-C3 and C3M were measured by ELISA in plasma from CHC patients (n = 194) from a prior phase II antifibrotic trial (NCT00244751). Plasma samples and paired liver biopsies were obtained at baseline and after 1...

  16. Collagenous sprue

    DEFF Research Database (Denmark)

    Soendergaard, Christoffer; Riis, Lene Buhl; Nielsen, Ole Haagen

    2014-01-01

    Collagenous sprue is a rare clinicopathological condition of the small bowel. It is characterised by abnormal subepithelial collagen deposition and is typically associated with malabsorption, diarrhoea and weight loss. The clinical features of collagenous sprue often resemble those of coeliac dis...... disease and together with frequent histological findings like mucosal thinning and intraepithelial lymphocytosis the diagnosis may be hard to reach without awareness of this condition. While coeliac disease is treated using gluten restriction, collagenous sprue is, however, not improved...

  17. Computational Characterization of Type I collagen-based Extra-cellular Matrix

    Science.gov (United States)

    Liang, Long; Jones, Christopher Allen Rucksack; Lin, Daniel; Jiao, Yang; Sun, Bo

    2015-03-01

    A model of extracellular matrix (ECM) of collagen fibers has been built, in which cells could communicate with distant partners via fiber-mediated long-range-transmitted stress states. The ECM is modeled as a spring-like fiber network derived from skeletonized confocal microscopy data. Different local and global perturbations have been performed on the network, each followed by an optimized global Monte-Carlo (MC) energy minimization leading to the deformed network in response to the perturbations. In the optimization, a highly efficient local energy update procedure is employed and force-directed MC moves are used, which results in a convergence to the energy minimum state 20 times faster than the commonly used random displacement trial moves in MC. Further analysis and visualization of the distribution and correlation of the resulting force network reveal that local perturbations can give rise to global impacts: the force chains formed with a linear extent much further than the characteristic length scale associated with the perturbation sites and average fiber length. This behavior provides a strong evidence for our hypothesis of fiber-mediated long-range force transmission in ECM networks and the resulting long-range cell-cell mechanical signaling. ASU Seed Grant.

  18. Kaempferol inhibits fibroblast collagen synthesis, proliferation and activation in hypertrophic scar via targeting TGF-β receptor type I.

    Science.gov (United States)

    Li, Hongwei; Yang, Liu; Zhang, Yuebing; Gao, Zhigang

    2016-10-01

    Hypertrophic scar (HPS) formation is a debilitating condition that results in pain, esthetic symptom and loss of tissue function. So far, no satisfactory therapeutic approach has been available for HPS treatment. In this study, we discovered that a natural small molecule, kaempferol, could significantly inhibit HPS formation in a mechanical load-induced mouse model. Our results also demonstrated that kaempferol remarkably attenuated collagen synthesis, proliferation and activation of fibroblasts in vitro and in vivo. Western blot analysis further revealed that kaempferol significantly down-regulated Smad2 and Smad3 phosphorylation in a dose-dependent manner. At last, we found that such bioactivity of kaempferol which resulted from the inhibition of TGF-β1/Smads signaling was induced by the selective binding of kaempferol to TGF-β receptor type I (TGFβRI). These findings suggest that kaempferol could be developed into a promising agent for the treatment of HPS or other fibroproliferative disorders.

  19. Safety and immunogenicity of a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis in normal rats.

    Science.gov (United States)

    Juan, Long; Xiao, Zhao; Song, Yun; Zhijian, Zhang; Jing, Jin; Kun, Yu; Yuna, Hao; Dongfa, Dai; Lili, Ding; Liuxin, Tan; Fei, Liang; Nan, Liu; Fang, Yuan; Yuying, Sun; Yongzhi, Xi

    2015-01-01

    Current clinically available treatments for rheumatoid arthritis (RA) fail to cure the disease or unsatisfactorily halt disease progression. To overcome these limitations, the development of therapeutic DNA vaccines and boosters may offer new promising strategies. Because type II collagen (CII) as a critical autoantigen in RA and native chicken type II collagen (nCCII) has been used to effectively treat RA, we previously developed a novel therapeutic DNA vaccine encoding CCII (pcDNA-CCOL2A1) with efficacy comparable to that of the current "gold standard", methotrexate(MTX). Here, we systemically evaluated the safety and immunogenicity of the pcDNA-CCOL2A1 vaccine in normal Wistar rats. Group 1 received only a single intramuscular injection into the hind leg with pcDNA-CCOL2A1 at the maximum dosage of 3 mg/kg on day 0; Group 2 was injected with normal saline (NS) as a negative control. All rats were monitored daily for any systemic adverse events, reactions at the injection site, and changes in body weights. Plasma and tissues from all experimental rats were collected on day 14 for routine examinations of hematology and biochemistry parameters, anti-CII IgG antibody reactivity, and histopathology. Our results indicated clearly that at the maximum dosage of 3 mg/kg, the pcDNA-CCOL2A1 vaccine was safe and well-tolerated. No abnormal clinical signs or deaths occurred in the pcDNA-CCOL2A1 group compared with the NS group. Furthermore, no major alterations were observed in hematology, biochemistry, and histopathology, even at the maximum dose. In particularly, no anti-CII IgG antibodies were detected in vaccinated normal rats at 14 d after vaccination; this was relevant because we previously demonstrated that the pcDNA-CCOL2A1 vaccine, when administered at the therapeutic dosage of 300 μg/kg alone, did not induce anti-CII IgG antibody production and significantly reduced levels of anti-CII IgG antibodies in the plasma of rats with established collagen-induced arthritis

  20. NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion.

    Science.gov (United States)

    Cattaruzza, Sabrina; Nicolosi, Pier Andrea; Braghetta, Paola; Pazzaglia, Laura; Benassi, Maria Serena; Picci, Piero; Lacrima, Katia; Zanocco, Daniela; Rizzo, Erika; Stallcup, William B; Colombatti, Alfonso; Perris, Roberto

    2013-06-01

    In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb to the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells closely correlate with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild-type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.

  1. Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

    Science.gov (United States)

    Muhonen, Virpi; Salonius, Eve; Haaparanta, Anne-Marie; Järvinen, Elina; Paatela, Teemu; Meller, Anna; Hannula, Markus; Björkman, Mimmi; Pyhältö, Tuomo; Ellä, Ville; Vasara, Anna; Töyräs, Juha; Kellomäki, Minna; Kiviranta, Ilkka

    2016-05-01

    The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  2. Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

    Science.gov (United States)

    Liu, Xiaoling; Xu, Qian; Liu, Weiwei; Yao, Guodong; Zhao, Yeli; Xu, Fanxing; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Yamato, Masayuki; Ikejima, Takashi

    2017-09-20

    Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

  3. Elastic energy storage in human articular cartilage: estimation of the elastic modulus for type II collagen and changes associated with osteoarthritis.

    Science.gov (United States)

    Silver, Frederick H; Bradica, Gino; Tria, Alfred

    2002-03-01

    The viscoelastic mechanical properties of normal and osteoarthritic articular were analyzed based on data reported by Kempson [in: Adult Articular Cartilage (1973)] and Silver et al. (Connect. Tissue Res., 2001b). Results of the analysis of tensile elastic stress-strain curves suggest that the elastic modulus of cartilage from the superficial zone is approximately 7.0 GPa parallel and 2.21 GPa perpendicular to the cleavage line pattern. Collagen fibril lengths in the superficial zone were found to be approximately 1265 microm parallel and 668 microm perpendicular to the cleavage line direction. The values for the elastic modulus and fibril lengths decreased with increased extent of osteoarthritis. The elastic modulus of type II collagen parallel to the cleavage line pattern in the superficial zone approaches that of type I collagen in tendon, suggesting that elastic energy storage occurs in the superficial zone due to the tensile pre-tension that exists in this region. Decreases in the elastic modulus associated with osteoarthritis reflect decreased ability of cartilage to store elastic energy, which leads to cartilage fibrillation and fissure formation. We hypothesize that under normal physiological conditions, collagen fibrils in cartilage function to store elastic energy associated with weight bearing and locomotion. Enzymatic cleavage of cartilage proteoglycans and collagen observed in osteoarthritis may lead to fibrillation and fissure formation as a result of impaired energy storage capability of cartilage.

  4. Phenotypic expressions of a Gly154Arg mutation in type II collagen in two unrelated patients with spondyloepimetaphyseal dysplasia (SEMD)

    Energy Technology Data Exchange (ETDEWEB)

    Kaitila, I.; Marttinen, E. [Helsinki Univ. Hospital (Finland); Koerkkoe, J.; Ala-Kokko, L. [Thomas Jefferson Univ., Philadelphia, PA (United States)

    1996-05-03

    Type II collagenopathies consist of chondrodysplasia ranging from lethal to mild in severity. A large number of mutations has been found in the COL2A1 gene. Glycine substitutions have been the most common types of mutation. Genotype-phenotype correlations in type II collagenopathies have not been established, partly because of insufficient clinical and radiographic description of the patients. We found a glycine-to-arginine substitution at position 154 in type II collagen in two unrelated isolated propositi with spondyloepimetaphyseal dysplasia and provide a comparative clinical and radiographic analysis from birth to young adulthood for this condition. The clinical phenotype was disproportionate short stature with varus/valgus deformities of the lower limbs requiring corrective osteotomies, and lumbar lordosis. The skeletal radiographs showed an evolution from short tubular bones, delayed epiphyseal development, and mild vertebral involvement to severe metaphyseal dysplasia with dappling irregularities, and hip {open_quotes}dysplasia.{close_quotes} The metaphyseal abnormalities disappeared by adulthood. 27 refs., 11 figs., 1 tab.

  5. Type II collagen peptide is able to accelerate embryonic chondrocyte differentiation: an association with articular cartilage matrix resorption in osteoarthrosis

    Directory of Open Access Journals (Sweden)

    Elena Vasil'evna Chetina

    2010-01-01

    Conclusion. The effect of CP on gene expression and collagen decomposition activity depends on the morphotype of embryonic chondrocytes. Lack of effect of CP on collagen decomposition activity in both the embryonic hypertrophic chondrocytes and the cartilage explants from OA patients supports the hypothesis that the hypertrophic morphotype is a dominant morphotype of articular chondrocytes in OA. Moreover, collagen decomposition products can be involved in the resorption of matrix in OA and in the maintenance of chronic nature of the pathology.

  6. Two novel COL1A1 mutations in patients with osteogenesis imperfecta (OI) affect the stability of the collagen type I triple-helix.

    Science.gov (United States)

    Witecka, Joanna; Auguściak-Duma, Aleksandra M; Kruczek, Anna; Szydło, Anna; Lesiak, Marta; Krzak, Maria; Pietrzyk, Jacek J; Männikkö, Minna; Sieroń, Aleksander L

    2008-01-01

    Osteogenesis imperfecta (OI) is a bone dysplasia caused by mutations in the COL1A1 and COL1A2 genes. Although the condition has been intensely studied for over 25 years and recently over 800 novel mutations have been published, the relation between the location of mutations and clinical manifestation is poorly understood. Here we report missense mutations in COL1A1 of several OI patients. Two novel mutations were found in the D1 period. One caused a substitution of glycine 200 by valine at the N-terminus of D1 in OI type I/IV, lowering collagen stability by 50% at 34 degrees C. The other one was a substitution of valine 349 by phenylalanine at the C-terminus of D1 in OI type I, lowering collagen stability at 37.5 degrees C. Two other mutations, reported before, changed amino residues in D4. One was a lethal substitution changing glycine 866 to serine in genetically identical twins with OI type II. That mutated amino acid was near the border of D3 and D4. The second mutation changed glycine 1040 to serine located at the border of D4 and D0.4, in a proband manifesting OI type III, and lowered collagen stability at 39 degrees C (2 degrees C lower than normal). Our results confirm the hypothesis on a critical role of the D1 and D4 regions in stabilization of the collagen triple-helix. The defect in D1 seemed to produce a milder clinical type of OI, whereas the defect in the C-terminal end of collagen type caused the more severe or lethal types of OI.

  7. Expression and characterization of common carp (Cyprinus carpio) matrix metalloproteinase-2 and its activity against type I collagen.

    Science.gov (United States)

    Wang, Ci; Zhan, Chun-Lan; Cai, Qiu-Feng; Du, Cui-Hong; Liu, Guang-Ming; Su, Wen-Jin; Cao, Min-Jie

    2014-05-10

    Matrix metalloproteinases (MMPs) play essential roles in the metabolism of animal collagen while few reports are available for MMPs in aquatic animals. In this study, we report the complete sequence of matrix metalloproteinase-2 (MMP-2) gene from common carp (Cyprinus carpio) skeletal muscle. The full-length cDNA of MMP-2 was 2792bp which contains an open reading frame of 1974bp, corresponding to a protein of 657 amino acid residues. Based on the structural feature of MMP-2, the gene of the catalytic domain containing 351 amino acid residues was cloned and expressed in Escherichia coli. SDS-PAGE showed that the truncated recombinant MMP-2 (trMMP-2) with molecular mass of approximately 38kDa was in the form of inclusion body. The trMMP-2 was further purified by immobilized metal ion affinity chromatography. After renaturation, similar to native MMP-2, the trMMP-2 exhibited high hydrolyzing activity toward gelatin as appeared on gelatin zymography and optimal activity was at pH 8.0 and 40°C. The activity of the trMMP-2 was completely suppressed by metalloproteinase inhibitors, including EDTA, EGTA and 1,10-phenanthroline while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca(2+) was necessary for the gelatinolytic activity, suggesting it is a calcium-dependent metalloproteinase. Moreover, the trMMP-2 effectively hydrolyzed native type I collagen at 37°C and even at 4°C, implying its potential application value as a collagenase for preparation of biologically active oligopeptides.

  8. Serum cross-linked n-telopeptides of type 1 collagen (NTx in patients with solid tumors

    Directory of Open Access Journals (Sweden)

    Fernando Jablonka

    Full Text Available CONTEXT AND OBJECTIVE: Cross-linked N-telopeptides of type I collagen (NTx increase in concentration in situations in which bone resorption is increased, such as osteoporosis and bone metastasis (BM. We aimed to evaluate the serum concentrations of NTx in a sample of patients with several types of solid tumors. DESIGN AND SETTING: Cross-sectional analytical study with a control group in a tertiary public hospital. METHODS: We performed the quantitative enzyme-linked immunosorbent assay (ELISA on serum NTx levels in 19 subjects without a history of cancer and 62 patients with various solid tumors who had been referred for a bone scan. Three experienced analysts read all bone scans. RESULTS: The serum NTx levels in patients with cancer and BM, with cancer but without BM and without cancer were 46.77 ± 2.58, 32.85 ± 2.05 and 22.32 ± 2.90 respectively (P < 0.0001. We did not find any significant correlations of serum NTx with age, gender, history of bone pain, tumor type and bone alkaline phosphatase levels. We found a significant correlation between serum NTx and alkaline phosphatase levels (R² = 0.08; P = 0.022. CONCLUSIONS: Serum NTx levels are significantly higher in patients with solid tumors and bone metastases than they are in patients without bone metastases and in normal controls.

  9. A novel urinary biomarker of type VI collagen formation and endotrophin is associated with loss of kidney function in patients with diabetic nephropathy

    DEFF Research Database (Denmark)

    Genovese, Federica; Rasmussen, Daniel; Nielsen, Signe Holm

    2017-01-01

    -stage renal disease. Fibrosis is characterized by a dysregulated remodeling of the extracellular matrix (ECM). Collagen type VI (COL VI) is a crucial ECM molecule for the control of tissue organization. It is present at the interface of the glomerular basement membrane and interstitial matrix and its levels...

  10. Elevated carboxy terminal cross linked telopeptide of type I collagen in alcoholic cirrhosis: relation to liver and kidney function and bone metabolism

    DEFF Research Database (Denmark)

    Møller, S; Hansen, M; Hillingso, J

    1999-01-01

    BACKGROUND: The carboxy terminal cross linked telopeptide of type I collagen (ICTP) has been put forward as a marker of bone resorption. Patients with alcoholic liver disease may have osteodystrophy. AIMS: To assess circulating and regional concentrations of ICTP in relation to liver dysfunction...

  11. Low sensitivity of type VII collagen enzyme-linked immunosorbent assay in epidermolysis bullosa acquisita : serration pattern analysis on skin biopsy is required for diagnosis

    NARCIS (Netherlands)

    Terra, J. B.; Jonkman, M. F.; Diercks, G. F. H.; Pas, H. H.

    BackgroundThe type VII collagen (coll VII) enzyme-linked immunosorbent assay (ELISA) has been reported to have high sensitivity (>93%) and specificity (>96%) for diagnosing epidermolysis bullosa acquisita (EBA) in patients who are seropositive on indirect immunofluorescence on salt-split skin (SSS).

  12. Low sensitivity of type VII collagen enzyme-linked immunosorbent assay in epidermolysis bullosa acquisita : serration pattern analysis on skin biopsy is required for diagnosis

    NARCIS (Netherlands)

    Terra, J. B.; Jonkman, M. F.; Diercks, G. F. H.; Pas, H. H.

    2013-01-01

    BackgroundThe type VII collagen (coll VII) enzyme-linked immunosorbent assay (ELISA) has been reported to have high sensitivity (>93%) and specificity (>96%) for diagnosing epidermolysis bullosa acquisita (EBA) in patients who are seropositive on indirect immunofluorescence on salt-split skin (SSS).

  13. Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP) - Assessment of corresponding epitopes

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Larsen, D.V.; Zhang, C.

    2010-01-01

    Objectives: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. Methods: Monoclonal antibodies were raised against...

  14. Combined expression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type Ⅱ in rhesus monkey lumbar intervertebral disc cells in vitro

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; KONG Jie; CHEN Bo-hua; HU You-gu

    2010-01-01

    Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor (CTGF) and tissue inhibitor of metalloprotease-1(TIMP-1) expression mediated by adeno-associated virus (AAV) on collagen type Ⅱ and proteoglycan levels.The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.Methods Rhesus monkey lumbar intervertebral disc nucleus pulposus cells (NPCs) were isolated by enzyme digestion,cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection (MOl) of 106.The expression of collagen type Ⅱ and proteoglycan was measured using RT-PCR and Western blotting.The synthetic rate of proteoglycan was measured using 35S incorporation.Results Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1,rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected.Compared to the control,CTGF promoted the synthesis of collagen type Ⅱ and proteoglycan.TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type Ⅱ.Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type Ⅱ.Conclusions Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan.CTGF expression can also enhance collagen type Ⅱ protein synthesis.Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type Ⅱ to levels greater than transduction of a single gene alone.Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.

  15. Four patients with Sillence type I osteogenesis imperfecta and mild bone fragility, complicated by left ventricular cardiac valvular disease and cardiac tissue fragility caused by type I collagen mutations.

    Science.gov (United States)

    Vandersteen, Anthony M; Lund, Allan M; Ferguson, David J P; Sawle, Philip; Pollitt, Rebecca C; Holder, Susan E; Wakeling, Emma; Moat, Neil; Pope, F Michael

    2014-02-01

    Osteogenesis imperfecta (OI) type I is a hereditary disorder of connective tissue (HDCT) characterized by blue or gray sclerae, variable short stature, dentinogenesis imperfecta, hearing loss, and recurrent fractures from infancy. We present four examples of OI type I complicated by valvular heart disease and associated with tissue fragility. The diagnosis of a type I collagen disorder was confirmed by abnormal COL1A1 or COL1A2 gene sequencing. One patient was investigated with electrophoresis of collagens from cultured skin fibroblasts, showing structurally abnormal collagen type I, skin biopsy showed unusual histology and abnormal collagen fibril ultra-structure at electron microscopy. The combined clinical, surgical, histological, ultra-structural, and molecular genetic data suggest the type I collagen defect as contributory to cardiac valvular disease. The degree of tissue fragility experienced at cardiac surgery in these individuals, also reported in a small number of similar case reports, suggests that patients with OI type I need careful pre-operative assessment and consideration of the risks and benefits of cardiac surgery.

  16. Preparation and characterization of injectable fibrillar type I collagen and evaluation for pseudoaneurysm treatment in a pig model.

    NARCIS (Netherlands)

    Geutjes, P.J.; Vliet, J.A. van der; Faraj, K.A.; Vries, N. de; Moerkerk, H.T.B. van; Wismans, P.G.P.; Hendriks, T.; Daamen, W.F.; Kuppevelt, A.H.M.S.M. van

    2010-01-01

    OBJECTIVE: Despite the efficacy of collagen in femoral artery pseudoaneurysm treatment, as reported in one patient study, its use has not yet gained wide acceptance in clinical practice. In this particular study, the collagen was not described in detail. To further investigate the potential of

  17. Oral Administration of Shark Type II Collagen Suppresses Complete Freund’s Adjuvant-Induced Rheumatoid Arthritis in Rats

    Science.gov (United States)

    Chen, Lijuan; Bao, Bin; Wang, Nanping; Xie, Jing; Wu, Wenhui

    2012-01-01

    Objective: Shark type II collagen (SCII) is extracted as a glycoprotein from the cartilage of blue shark (Prionace glauca). We aim to confirm the effects of oral tolerance of SCII on inflammatory and immune responses to the ankle joint of rheumatoid-arthritis rats induced by Complete Freund’s Adjuvant (CFA). Materials and Methods: The onset of rheumatoid arthritis (RA) was observed 14 ± x days after injection of CFA. Rats in the control group were treated with acetic acid by oral administration (0.05 mmol kg−1d−1, days 14–28), while rats in experimental groups were treated by oral administration with SCII (1 or 3 mg kg−1d−1, days 14–28), Tripterygium wilfordii polyglycosidium (TWP) (10 mg kg−1d−1, days 14–28), and bovine type II collagen from US (US-CII) (1 mg kg−1d−1, days 14–28), respectively. The severity of arthritis was evaluated by the articular swelling. The immunological indexes observed included delayed type hypersensitivity (DTH) reaction, the level of interleukins 10 (IL-10) in rat blood serum and morphological characterization. Mixed lymphocyte culture (MLC) was performed to investigate the relationship between T cell apoptosis and specific immune tolerance induced by SCII. Results: Treatment with SCII for 2 weeks significantly attenuated the acute inflammation. The rats orally administrated with SCII at the level of 3 mg kg−1d−1 (SCII 3) and US-CII had decreased DTH reaction compared with rats in control group. Rats treated with SCII 3 had the highest level of IL-10 with 102 pg/mL. SCII with concentration of 10 μg/L could help to significantly enhance level of Fas/Apo-1 in T cell in vitro. The result of histological staining indicated that the recovery of the articular membranes of ankle joint in SCII 3 group was greatly enhanced. Conclusions: Our results suggest that appropriate dose of SCII can not only ameliorate symptoms but also modify the disease process of Complete-Freunds-Adjuvant-induced arthritis. Oral

  18. Oral Administration of Shark Type II Collagen Suppresses Complete Freund's Adjuvant-Induced Rheumatoid Arthritis in Rats.

    Science.gov (United States)

    Chen, Lijuan; Bao, Bin; Wang, Nanping; Xie, Jing; Wu, Wenhui

    2012-03-28

    Shark type II collagen (SCII) is extracted as a glycoprotein from the cartilage of blue shark (Prionace glauca). We aim to confirm the effects of oral tolerance of SCII on inflammatory and immune responses to the ankle joint of rheumatoid-arthritis rats induced by Complete Freund's Adjuvant (CFA). The onset of rheumatoid arthritis (RA) was observed 14 ± x days after injection of CFA. Rats in the control group were treated with acetic acid by oral administration (0.05 mmol kg-1d-1, days 14-28), while rats in experimental groups were treated by oral administration with SCII (1 or 3 mg kg-1d-1, days 14-28), Tripterygium wilfordii polyglycosidium (TWP) (10 mg kg-1d-1, days 14-28), and bovine type II collagen from US (US-CII) (1 mg kg-1d-1, days 14-28), respectively. The severity of arthritis was evaluated by the articular swelling. The immunological indexes observed included delayed type hypersensitivity (DTH) reaction, the level of interleukins 10 (IL-10) in rat blood serum and morphological characterization. Mixed lymphocyte culture (MLC) was performed to investigate the relationship between T cell apoptosis and specific immune tolerance induced by SCII. Treatment with SCII for 2 weeks significantly attenuated the acute inflammation. The rats orally administrated with SCII at the level of 3 mg kg-1d-1 (SCII 3) and US-CII had decreased DTH reaction compared with rats in control group. Rats treated with SCII 3 had the highest level of IL-10 with 102 pg/mL. SCII with concentration of 10 μg/L could help to significantly enhance level of Fas/Apo-1 in T cell in vitro. The result of histological staining indicated that the recovery of the articular membranes of ankle joint in SCII 3 group was greatly enhanced. Our results suggest that appropriate dose of SCII can not only ameliorate symptoms but also modify the disease process of Complete-Freunds-Adjuvant-induced arthritis. Oral administration of SCII might be a potential candidate as a novel drug for further

  19. Oral Administration of Shark Type II Collagen Suppresses Complete Freund’s Adjuvant-Induced Rheumatoid Arthritis in Rats

    Directory of Open Access Journals (Sweden)

    Wenhui Wu

    2012-03-01

    Full Text Available Objective: Shark type II collagen (SCII is extracted as a glycoprotein from the cartilage of blue shark (Prionace glauca. We aim to confirm the effects of oral tolerance of SCII on inflammatory and immune responses to the ankle joint of rheumatoid-arthritis rats induced by Complete Freund’s Adjuvant (CFA. Materials and Methods: The onset of rheumatoid arthritis (RA was observed 14 ± x days after injection of CFA. Rats in the control group were treated with acetic acid by oral administration (0.05 mmol kg−1d−1, days 14–28, while rats in experimental groups were treated by oral administration with SCII (1 or 3 mg kg−1d−1, days 14–28, Tripterygium wilfordii polyglycosidium (TWP (10 mg kg−1d−1, days 14–28, and bovine type II collagen from US (US-CII (1 mg kg−1d−1, days 14–28, respectively. The severity of arthritis was evaluated by the articular swelling. The immunological indexes observed included delayed type hypersensitivity (DTH reaction, the level of interleukins 10 (IL-10 in rat blood serum and morphological characterization. Mixed lymphocyte culture (MLC was performed to investigate the relationship between T cell apoptosis and specific immune tolerance induced by SCII. Results: Treatment with SCII for 2 weeks significantly attenuated the acute inflammation. The rats orally administrated with SCII at the level of 3 mg kg−1d−1 (SCII 3 and US-CII had decreased DTH reaction compared with rats in control group. Rats treated with SCII 3 had the highest level of IL-10 with 102 pg/mL. SCII with concentration of 10 μg/L could help to significantly enhance level of Fas/Apo-1 in T cell in vitro. The result of histological staining indicated that the recovery of the articular membranes of ankle joint in SCII 3 group was greatly enhanced. Conclusions: Our results suggest that appropriate dose of SCII can not only ameliorate symptoms but also modify the disease process of Complete-Freunds-Adjuvant-induced arthritis. Oral

  20. Low-level light-emitting diode therapy increases mRNA expressions of IL-10 and type I and III collagens on Achilles tendinitis in rats.

    Science.gov (United States)

    Xavier, Murilo; de Souza, Renato Aparecido; Pires, Viviane Araújo; Santos, Ana Paula; Aimbire, Flávio; Silva, José Antônio; Albertini, Regiane; Villaverde, Antonio Balbin

    2014-01-01

    The present study investigated the effects of low-level light-emitting diode (LED) therapy (880 ± 10 nm) on interleukin (IL)-10 and type I and III collagen in an experimental model of Achilles tendinitis. Thirty male Wistar rats were separated into six groups (n = 5), three groups in the experimental period of 7 days, control group, tendinitis-induced group, and LED therapy group, and three groups in the experimental period of 14 days, tendinitis group, LED therapy group, and LED group with the therapy starting at the 7th day after tendinitis induction (LEDT delay). Tendinitis was induced in the right Achilles tendon using an intratendinous injection of 100 μL of collagenase. The LED parameters were: optical power of 22 mW, spot area size of 0.5 cm(2), and irradiation time of 170 s, corresponding to 7.5 J/cm(2) of energy density. The therapy was initiated 12 h after the tendinitis induction, with a 48-h interval between irradiations. The IL-10 and type I and III collagen mRNA expression were evaluated by real-time polymerase chain reaction at the 7th and 14th days after tendinitis induction. The results showed that LED irradiation increased IL-10 (p < 0.001) in treated group on 7-day experimental period and increased type I and III collagen mRNA expression in both treated groups of 7- and 14-day experimental periods (p < 0.05), except by type I collagen mRNA expression in LEDT delay group. LED (880 nm) was effective in increasing mRNA expression of IL-10 and type I and III collagen. Therefore, LED therapy may have potentially therapeutic effects on Achilles tendon injuries.

  1. Interleukin-1 inhibits Sox9 and collagen type Ⅱ expression via nuclear factor-κB in the cultured human intervertebral disc cells

    Institute of Scientific and Technical Information of China (English)

    YU Zhan-ge; XU Ning; WANG Wen-bo; PAN Shang-ha; LI Ke-shen; LIU Jia-kun

    2009-01-01

    Background The most significant biological change in intervertebral disc degeneration is the decrease of chondrocyte specific gene and protein expression of Sox9 and collagen type Ⅱ. Interleukin-1 (IL-1) is not expressed in the normal intervertebral disc tissue but increases in the degenerated intervertebral disc tissue. This suggests that IL-1 may play a role in regulation of the expression of Sox9 and collagen type Ⅱ.Methods Human intervertebral disc cells were isolated and cultured. Sox9 and collagen type Ⅱ expression during treatment with IL-1, with or without the nuclear factor-κB (NF-κB) activity inhibitor curcumin, were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the activity of the NF-κB signaling pathway was detected by the electrophoretic mobility shift assay (EMSA).Results IL-1 lowered the mRNA level and protein expression of Sox9 and collagen type Ⅱ in the cultured intervertebral disc cells in a dose dependent manner (P 0.05). IL-1 at concentrations of 0.1 ng/ml, 1 ng/ml and 10 ng/ml could stimulate the activity of NF-κB in the intervertebral disc cells in a dose dependent manner (P <0.05) that was inhibited by curcumin.Conclusions We demonstrated the previously unknown function of IL-1 in inhibiting Sox9 and collagen type II via NF-κB in the intervertebral disc cells. This inhibition can be attenuated by curcumin, which is an effective NF-κB activity inhibitor.

  2. Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

    Directory of Open Access Journals (Sweden)

    L.R. Ferreira

    2003-01-01

    Full Text Available Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

  3. The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen.

    Directory of Open Access Journals (Sweden)

    Marta Bober

    Full Text Available Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid. The streptococcal leucine rich (Slr protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1 and emm1 mutant strain (MC25 had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.

  4. Discoidin domain receptor 2-microRNA 196a-mediated negative feedback against excess type I collagen expression is impaired in scleroderma dermal fibroblasts.

    Science.gov (United States)

    Makino, Katsunari; Jinnin, Masatoshi; Aoi, Jun; Hirano, Ayaka; Kajihara, Ikko; Makino, Takamitsu; Sakai, Keisuke; Fukushima, Satoshi; Inoue, Yuji; Ihn, Hironobu

    2013-01-01

    Systemic sclerosis (SSc) is characterized by excess collagen deposition in the skin, due to intrinsic transforming growth factor-β (TGF-β) activation. We tried to determine the expression and the role of discoidin domain receptor 2 (DDR2) in SSc. The expression of DDR2 mRNA and protein was significantly decreased in SSc dermal fibroblasts, which was recovered by knocking down TGF-β. The knockdown of DDR2 in normal fibroblasts induced microRNA-196a expression, which led to type I collagen downregulation, indicating that DDR2 itself has a negative effect on microRNA-196a expression and inducible effect on collagen expression. In SSc fibroblasts, however, the DDR2 knockdown did not affect TGF-β signaling and microRNA-196a expression. The microRNA-196a levels were significantly decreased in normal fibroblasts treated with TGF-β and in SSc fibroblasts. Taken together our data indicate that, in SSc fibroblasts, intrinsic TGF-β stimulation induces type I collagen expression, and also downregulates DDR2 expression. This probably acts as a negative feedback mechanism against excess collagen expression, as a decreased DDR2 expression is supposed to stimulate the microRNA-196a expression and further change the collagen expression. However, in SSc fibroblasts the microRNA-196a expression was downregulated by TGF-β signaling. DDR2-microRNA-196a pathway may be a previously unreported negative feedback system, and its impairment may be involved in the pathogenesis of SSc.

  5. Simvastatin inhibits transforming growth factor-β1-induced expression of type I collagen, CTGF, and α-SMA in keloid fibroblasts.

    Science.gov (United States)

    Mun, Je-Ho; Kim, Young-Mi; Kim, Byung-Soo; Kim, Jae-Ho; Kim, Moon-Bum; Ko, Hyun-Chang

    2014-01-01

    Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor, is used to reduce cholesterol levels. Accumulating evidence has revealed the immunomodulatory and anti-inflammatory effects of simvastatin that prevent cardiovascular diseases. In addition, the beneficial effects of statins on fibrosis of various organs have been reported. However, the functional effect of statins on dermal fibrosis of keloids has not yet been explored. The objective of this study was to determine whether simvastatin could affect dermal fibrosis associated with keloids. We examined the effect of simvastatin on transforming growth factor (TGF)-β1-induced production of type I collagen, connective tissue growth factor (CTGF or CCN2), and α-smooth muscle actin (α-SMA). Keloid fibroblasts were cultured and exposed to different concentrations of simvastatin in the presence of TGF-β1, and the effects of simvastatin on TGF-β1-induced collagen and CTGF production in keloid fibroblasts were determined. The type I collagen, CTGF, and α-SMA expression levels and the Smad2 and Smad3 phosphorylation levels were assessed by Western blotting. The effect of simvastatin on cell viability was evaluated by assessing the colorimetric conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Simvastatin suppressed TGF-β1-induced type I collagen, CTGF, and α-SMA production in a concentration-dependent manner. The TGF-β1-induced Smad2 and Smad3 phosphorylation levels were abrogated by simvastatin pretreatment. The inhibition of type I collagen, CTGF, and α-SMA expression by simvastatin was reversed by geranylgeranyl pyrophosphate, suggesting that the simvastatin-induced cellular responses were due to inhibition of small GTPase Rho involvement. A RhoA activation assay showed that preincubation with simvastatin significantly blocked TGF-β1-induced RhoA activation. The Rho-associated coiled kinase inhibitor Y27632 abrogated TGF-β1-induced production of type I collagen

  6. Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein-47 and collagen type I in rat hepatic stellate cells in vitro.

    Science.gov (United States)

    Xiang, Xian-Hong; Jiang, Tian-Peng; Zhang, Shuai; Song, Jie; Li, Xing; Yang, Jian-Yong; Zhou, Shi

    2015-07-01

    Pirfenidone (esbiret) is an established anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis. In the present study, the dose-dependent effects of pirfenidone on the cell cycle, proliferation and expression of heat shock protein (HSP)-47 and collagen type I in a cultured rat hepatic stellate cell line (HSC-T6) were investigated. Following pirfenidone treatment, cell proliferation was determined using the cell counting kit-8 assay and the cell cycle was measured using flow cytometry. HSP-47 expression was estimated using western blot analysis and collagen type I mRNA was assessed using reverse transcription quantitative polymerase chain reaction. Pirfenidone induced significant dose-dependent inhibition of proliferation in HSC-T6 cells. Cell viability was unaffected by treatment with pirfenidone (0, 10 or 100 µM) for 24 and 72 h. However, after 24 h, HSC-T6 cells exhibited dose-dependent decreases in HSP-47 protein and collagen I mRNA levels. In conclusion, pirfenidone inhibited HSC-T6 cell proliferation, arrested the cell cycle and reduced the expression of HSP-47 and collagen type I, indicating that pirfenidone may be a promising drug in the treatment of liver fibrosis.

  7. A comparative study on collagen type I and hyaluronic acid dependent cell behavior for osteochondral tissue bioprinting.

    Science.gov (United States)

    Park, Ju Young; Choi, Jong-Cheol; Shim, Jin-Hyung; Lee, Jung-Seob; Park, Hyoungjun; Kim, Sung Won; Doh, Junsang; Cho, Dong-Woo

    2014-09-01

    Bioprinting is a promising technique for engineering composite tissues, such as osteochondral tissues. In this study, as a first step toward bioprinting-based osteochondral tissue regeneration, we systematically examined the behavior of chondrocytes and osteoblasts to hyaluronic acid (HA) and type I collagen (Col-1) hydrogels. First, we demonstrated that cells on hydrogels that were comprised of major native tissue extracellular matrix (ECM) components (i.e. chondrocytes on HA hydrogels and osteoblasts on Col-1 hydrogels) exhibited better proliferation and cell function than cells on non-native ECM hydrogels (i.e., chondrocytes on Col-1 hydrogels and osteoblasts on HA hydrogels). In addition, cells located near their native ECM hydrogels migrated towards them. Finally, we bioprinted three-dimensional (3D) osteochondral tissue-mimetic structures composed of two compartments, osteoblast-encapsulated Col-1 hydrogels and chondrocyte-encapsulated HA hydrogels, and found viability and functions of each cell type were well maintained within the 3D structures up to 14 days in vitro. These results suggest that with proper choice of hydrogel materials, bioprinting-based approaches can be successfully applied for osteochondral tissue regeneration.

  8. Rheological Properties of GuHCl/TypeCollagen Dispersions%盐酸胍/Ⅰ型胶原分散体系的流变性

    Institute of Scientific and Technical Information of China (English)

    步红红; 焦燕妮; 兰文军; 庄晓丽; 戴肖南; 李天铎

    2016-01-01

    采用胃蛋白酶降解法从猪皮中提取了胶原蛋白,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳( SDS-PAGE)确定为Ⅰ型胶原;红外及紫外光谱表明胶原分子中存在三螺旋结构.分别采用小幅振荡剪切法、恒定剪切速率法及滞后环法研究了盐酸胍浓度及作用时间对Ⅰ型胶原蛋白体系流变性的影响.研究表明,随盐酸胍浓度的增大及作用时间的延长,盐酸胍/胶原分散体系由假塑性流体逐渐接近于牛顿流体.在所研究的盐酸胍浓度范围(0~6.0 mol/L)内,盐酸胍/胶原分散体系的触变性类型随盐酸胍浓度的增大发生正触变性-复合触变性-负触变性的转变;随盐酸胍作用时间的延长(6~48 h),盐酸胍浓度为1.0 mol/L的胶原分散体系的触变性类型发生复合触变性-负触变性的转变.本文的研究结果有助于加深对触变性产生机理的认识.%Collagen was extracted from the outer skin of pig using a pepsin digestion method. The isolated collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis( SDS-PAGE) , Fourier transform infrared spectroscopy(FTIR) and ultraviolet(UV). According to the electrophoretic pattern, the isolated collagen consisted of two different α-chains(α1 , α2 ) and one β-chain, indicating typecollagen. FTIR and UV results confirmed the presence of triple helical structure in the collagen molecule. The rheologi-cal properties of GuHCl/typeⅠcollagen dispersions were investigated as a function of guanidine hydrochloride ( GuHCl) concentration and action time. The results of flow curves indicated that the pseudoplastic flow behavior of GuHCl/collagen dispersions gradually close to the Newtonian fluid with the increase of GuHCl concentration in the range of 0—6. 0 mol/L and action time in the range of 6—48 h. The linear oscillatory shear, steady shear and thixotropic loop experiments were performed to investigate thixotropic behavior of

  9. Linkage of the gene that encodes the alpha 1 chain of type V collagen (COL5A1) to type II Ehlers-Danlos syndrome (EDS II).

    Science.gov (United States)

    Loughlin, J; Irven, C; Hardwick, L J; Butcher, S; Walsh, S; Wordsworth, P; Sykes, B

    1995-09-01

    Ehlers-Danlos syndrome (EDS) is a group of heritable disorders of connective tissue with skin, ligaments and blood vessels being the main sites affected. The commonest variant (EDS II) exhibits an autosomal dominant mode of inheritance and is characterized by joint hypermobility, cigarette paper scars, lax skin and excessive bruising. As yet no gene has been linked to EDS II, nor has linkage been established to a specific region of the genome. However, several candidate genes encoding proteins of the extracellular matrix have been excluded. Using an intragenic simple sequence repeat polymorphism, we report linkage of the COL5A1 gene, which encodes the alpha 1(V) chain of type V collagen, to EDS II. A maximum LOD score (Zmax) for linkage of 8.3 at theta = 0.00 was generated for a single large pedigree.

  10. Collagen type XII and versican are present in the early stages of cartilage tissue formation by both redifferentating passaged and primary chondrocytes.

    Science.gov (United States)

    Taylor, Drew W; Ahmed, Nazish; Parreno, Justin; Lunstrum, Gregory P; Gross, Allan E; Diamandis, Eleftherios P; Kandel, Rita A

    2015-02-01

    Current approaches to cartilage tissue engineering require a large number of chondrocytes. Although chondrocyte numbers can be expanded in monolayer culture, the cells dedifferentiate and unless they can be redifferentiated are not optimal to use for cartilage repair. We took advantage of the differential effect of culture conditions on the ability of passaged and primary chondrocytes to form cartilage tissue to dissect out the extracellular matrix (ECM) molecules produced and accumulated in the early stages of passaged cell cartilage tissue formation as we hypothesized that passaged bovine cells that form cartilage accumulate a pericellular matrix that differs from cells that do not form cartilage. Twice passaged bovine chondrocytes (P2) (cartilage forming), or as a control primary chondrocytes (P0) (which do not generate cartilage), were cultured on three-dimensional membrane inserts in serum-free media. P2 redifferentiation was occurring during the first 8 days as indicated by increased expression of the chondrogenic genes Sox9, collagen type II, aggrecan, and COMP, suggesting that this is an appropriate time period to examine the ECM. Mass spectrometry showed that the P2 secretome (molecules released into the media) at 1 week had higher levels of collagen types I, III, and XII, and versican while type II collagen and COMP were found at higher levels in the P0 secretome. There was increased collagen synthesis and retention by P2 cells compared to P0 cells as early as 3 days of culture. Confocal microscopy showed that types XII, III, and II collagen, aggrecan, versican, and decorin were present in the ECM of P2 cells. In contrast, collagen types I, II, and III, aggrecan, and decorin were present in the ECM of P0 cells. As primary chondrocytes grown in serum-containing media, a condition that allows for the generation of cartilage tissue in vitro, also accumulate versican and collagen XII, this study suggests that these molecules may be necessary to provide a

  11. The Goodpasture autoantigen. Structural delineation of two immunologically privileged epitopes on alpha3(IV) chain of type IV collagen.

    Science.gov (United States)

    Kalluri, R; Sun, M J; Hudson, B G; Neilson, E G

    1996-04-12

    The family of type IV collagen comprises six chains numbered alpha1 through alpha6. The alpha3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the alpha3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co-inhibited Goodpasture autoantibodies binding to denatured human alpha3(IV) NC1 monomer by nearly 80%, with the helix-junction region of the alpha3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated alpha3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant alpha3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the alpha3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C-terminal epitope of the alpha3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in

  12. Different clinical and biochemical phenotypes resulting from the same substitution at the same glycine residue in different chains of the type I collagen molecule

    Energy Technology Data Exchange (ETDEWEB)

    Paepe, A. De; Nuytinck, L. [Univ. of Gent, Brussels (Belgium); Spotila, L. [Jefferson Institute of Molecular Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    Mutations in type I collagen produce osteogenesis imperfecta (OI; brittle bone disease), ranging in severity from lethal to very mild. This phenotypic variation is largely determined by the position and nature of the mutation. Because type I collagen consists of two {alpha}1 chains and one {alpha}2 chain, {alpha}1(I) mutations are generally regarded to have more serious consequences than {alpha}2(I) mutations. We have characterized a point mutation causing substitution of serine for glycine at position 661 of the {alpha}1(I) chain in a child with severe OI. This is precisely the same substitution that had been detected in the {alpha}2(I) chain in a woman with post-menopausal osteoporosis. She and two of her sons were heterozygous and the third son was homozygous as a result of uniparental disomy. Biochemical collagen profiles were studied in each of the patients and compared with a control. Medium and cell-layer collagens were overmodified in all patients. Overmodification was pronounced in the patient with the {alpha}1(I) mutation, but mild in the patients with the {alpha}2(I) mutation, being slightly less evident in the heterozygotes than in the homozygote. Thermal stability assays showed that the melting temperature of the mutant {alpha}1(I) chains was reduced by 3{degrees}C, whereas the melting curves in the patients with the {alpha}2(I) mutation were not significantly different from the control. These results show that the type of {alpha}-chain harboring the mutation influences the fundamental biochemical behavior of type I collagen molecules and strikingly emphasizes the predominant role of {alpha}1(I) chains compared with {alpha}2(I) in this respect.

  13. Cloning of the human type XVII collagen gene (COL17A1), and detection of novel mutations in generalized atrophic benign epidermolysis bullosa

    Energy Technology Data Exchange (ETDEWEB)

    Gatalica, B.; Pulkkinen, L.; Li, K. [Thomas Jefferson Univ., Philadelphia, PA (United States)] [and others

    1997-02-01

    Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal variant of junctional epidermolysis bullosa (JEB). Previous findings have suggested that type XVII collagen is the candidate gene for mutations in this disease. We now have cloned the entire human type XVII collagen gene (COL17A1) and have elucidated its intron-exon organization. The gene comprises 56 distinct exons, which span {approximately}52 kb of the genome, on the long arm of chromosome 10. It encodes a polypeptide, the {alpha}1(XVII) chain, consisting of an intracellular globular domain, a transmembrane segment, and an extracellular domain that contains 15 separate collagenous subdomains, the largest consisting of 242 amino acids. We also have developed a strategy to identify mutations in COL17A1 by use of PCR amplification of genomic DNA, using primers placed on the flanking introns. The PCR products are scanned for sequence variants by heteroduplex analysis using conformation-sensitive gel electrophoresis and then are subjected to direct automated sequencing. We have identified several intragenic polymorphisms in COL17A1, as well as mutations, in both alleles, in two Finnish families with GABEB. The probands in both families showed negative immunofluorescence staining with an anti-type XVII collagen antibody. In one family, the proband was homozygous for a 5-bp deletion, 2944del5, which resulted in frameshift and a premature termination codon of translation. The proband in the other family was a compound heterozygote, with one allele containing the 2944del5 mutation and the other containing a nonsense mutation, Q1023X. These results expand the mutation database in different variants of JEB, and they attest to the functional importance of type XVII collagen as a transmembrane component of the hemidesmosomes at the dermal/epidermal junction. 48 refs., 9 figs., 3 tabs.

  14. Effect of angiotensin Ⅱ and angiotensin Ⅱ type 1 receptor antagonist on the proliferation,contraction and collagen synthesis in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    LIU Jun; GONG Hao; ZHANG Zhong-tao; WANG Yu

    2008-01-01

    Background Angiotensin Ⅱ(Ang Ⅱ)is a very important vasoactive peptide that acts upon hepatic stellate cells(HSCs),which are major effector cells in hepatic cirrhosis and portal hypertension.The present study was aimed to investigate the effects of Ang Ⅱ and angiotensin Ⅱ type 1 receptor antagonist(AT1RA)on the proliferation,contraction and collagen synthesis in HSCs.Methods HSC-T6 rat hepatic stellate cell Iine was studied.The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by3 H-thymidine incorporation.The effects of angiotensin Ⅱ and AT1 RA on HSCs contraction were studied by analVSIs of the contraction of the collagen Iattice.CelI culture media were analyzed by RT-PCR to detect secretion of collagen Ⅰ(Col Ⅰ),collagen Ⅲ(Col Ⅲ)and transforming growth factor β1 (TGF-β1)by enzyme Iinked Immunosorbent assay.HSC was harvested to measure collagen Ⅰ,collagen Ⅲ and tissue inhibitor of metalloproteinase-1(TIMP-1)mRNA expression.Results Ang Ⅱ((1 x10-10-1×10-4)mol/L)stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells.AT1 RA inhibited angiotensin Ⅱ induced proliferation of HSCs.A Iinear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin Ⅱ(1×10-9-1×10-5mol/L)and with time over 48 hours.ATlRA blocks angiotensin Ⅱ induced contraction of collagen Iattice.Coll,Col Ⅲ and TGF-β1 levels of the Ang Ⅱ group were higher than those of control group and this increase was downregulated by AT1RA.The mRNA expressions of ColⅠ,CoI Ⅲ and TIMP-1 were higher in HSCs from the Ang Ⅱ group than the control group and downregulated by AT1RA.Conclusions Angiotensin Ⅱ increased DNA synthesis and proliferation of HSCs in a dose-dependent manner,stimulated the contraction of HSCs dose-and time-dependently.Angiotensin also promoted excretion of Col Ⅰ,ColⅢand TGF-β1 Ievels and stimulated Col Ⅰ,Col Ⅲ and

  15. Increased Levels of Type I and III Collagen and Hyaluronan in Scleroderma Skin

    DEFF Research Database (Denmark)

    Søndergaard, Klaus; Heickendorff, Lene; L, Risteli

    1997-01-01

    The aminoterminal propeptide of type III procollagen (PIIINP) and the carboxyterminal propeptide of type I procollagen (PICP) and hyaluronan (HA) were measured in plasma and suction blister fluid from 13 systemic sclerosis patients and 11 healthy volunteers. Suction blisters and skin biopsies were...... from the transition zone between normal skin and scleroderma, and uninvolved abdominal skin of patients. The median value of suction blister PIIINP from the transition zone was 38% higher than suction blister PIIINP from uninvolved skin. PIIINP was localized to the dermis by immunohistochemical...... techniques. PICP and HA levels in blisters from the transition zone were 87% and 53%, respectively, above the levels measured in uninvolved skin. Furthermore, PICP and HA blister levels from the transition zone were 67% and 63%, respectively, higher than the levels measured in healthy volunteers. In plasma...

  16. Increased Levels of Type I and III Collagen and Hyaluronan in Scleroderma Skin

    DEFF Research Database (Denmark)

    Søndergaard, Klaus; Heickendorff, Lene; L, Risteli

    1997-01-01

    The aminoterminal propeptide of type III procollagen (PIIINP) and the carboxyterminal propeptide of type I procollagen (PICP) and hyaluronan (HA) were measured in plasma and suction blister fluid from 13 systemic sclerosis patients and 11 healthy volunteers. Suction blisters and skin biopsies were...... from the transition zone between normal skin and scleroderma, and uninvolved abdominal skin of patients. The median value of suction blister PIIINP from the transition zone was 38% higher than suction blister PIIINP from uninvolved skin. PIIINP was localized to the dermis by immunohistochemical...... techniques. PICP and HA levels in blisters from the transition zone were 87% and 53%, respectively, above the levels measured in uninvolved skin. Furthermore, PICP and HA blister levels from the transition zone were 67% and 63%, respectively, higher than the levels measured in healthy volunteers. In plasma...

  17. Temperature-Responsive Gelation of Type I Collagen Solutions Involving Fibril Formation and Genipin Crosslinking as a Potential Injectable Hydrogel

    OpenAIRE

    Shunji Yunoki; Yoshimi Ohyabu; Hirosuke Hatayama

    2013-01-01

    We investigated the temperature-responsive gelation of collagen/genipin solutions using pepsin-solubilized collagen (PSC) and acid-solubilized collagen (ASC) as substrates. Gelation occurred in the PSC/genipin solutions at genipin concentrations 0–2 mM under moderate change in temperature from 25 to 37°C. The PSC/genipin solutions exhibited fluidity at room temperature for at least 30 min, whereas the ASC/genipin solutions rapidly reached gel points. In specific cases PSC would be preferred o...

  18. Matrix Metalloproteinase 2 (MMP-2) Plays a Critical Role in the Softening of Common Carp Muscle during Chilled Storage by Degradation of Type I and V Collagens.

    Science.gov (United States)

    Xu, Chao; Wang, Cheng; Cai, Qiu-Feng; Zhang, Qian; Weng, Ling; Liu, Guang-Ming; Su, Wen-Jin; Cao, Min-Jie

    2015-12-30

    Matrix metalloproteinases (MMPs) are proposed to play important roles in the degradation of collagens, thus causing the post-mortem softening of fish muscle, although the specific mechanism remains largely unresolved. Previously, we reported the existence of gelatinase-like proteinases in common carp (Cyprinus carpio) muscle. The primary structures of these proteinases, however, have never been investigated. In the present study, two MMPs with molecular masses of 66 and 65 kDa were purified to homogeneity from common carp muscle by ammonium sulfate fractionation and a series of column chromatographies. Matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) analysis indicated that they are completely identical to MMP-2 from common carp. During chilled storage of common carp at 4 °C, the enzymatic activity of MMP-2 increased to 212% in 12 h while the texture profile increased over the first 2 h and gradually decreased. On the other hand, type V collagen was purified to homogeneity and a specific polyclonal antibody against this protein was prepared. Both type I and V collagens were effectively hydrolyzed by MMP-2 at 30 °C and even at 4 °C. Furthermore, injection of metalloproteinase proteinase inhibitor EDTA into the blood vessel of live common carp suppressed post-mortem tenderization significantly. All of these results confirmed that MMP-2 is a major proteinase responsible for the degradation of collagens, resulting in the softening of fish muscle during chilled storage.

  19. Computational Study of a Heterostructural Model of Type I Collagen and Implementation of an Amino Acid Potential Method Applicable to Large Proteins

    Directory of Open Access Journals (Sweden)

    Jay Eifler

    2014-02-01

    Full Text Available Collagen molecules are the primary structural proteins of many biological systems. Much progress has been made in the study of the structure and function of collagen, but fundamental understanding of its electronic structures at the atomic level is still lacking. We present the results of electronic structure and bonding calculations of a specific model of type I collagen using the density functional theory-based method. Information on density of states (DOS, partial DOS, effective charges, bond order values, and intra- and inter-molecular H-bonding are obtained and discussed. We further devised an amino-acid-based potential method (AAPM to circumvent the full self-consistent field (SCF calculation that can be applied to large proteins. The AAPM is validated by comparing the results with the full SCF calculation of the whole type I collagen model with three strands. The calculated effective charges on each atom in the model retained at least 95% accuracy. This technique provides a viable and efficient way to study the electronic structure of large complex biomaterials at the ab initio level.

  20. ABNORMAL TYPE-III COLLAGEN PRODUCED BY AN EXON-17-SKIPPING MUTATION OF THE COL3A1 GENE IN EHLERS-DANLOS SYNDROME TYPE-IV IS NOT INCORPORATED INTO THE EXTRACELLULAR-MATRIX

    NARCIS (Netherlands)

    CHIODO, AA; SILLENCE, DO; COLE, WG; BATEMAN, JF

    1995-01-01

    A novel heterozygous mutation of the COL3Al gene that encodes the alpha 1(III) chains of type III collagen was identified in a family with the: acrogeric form of Ehlers-Danlos syndrome type IV (EDS-IV). Cultured dermal fibroblasts produced normal and shortened alpha 1(III) chains. The triple helix o

  1. Plexin D1 determines body fat distribution by regulating the type V collagen microenvironment in visceral adipose tissue.

    Science.gov (United States)

    Minchin, James E N; Dahlman, Ingrid; Harvey, Christopher J; Mejhert, Niklas; Singh, Manvendra K; Epstein, Jonathan A; Arner, Peter; Torres-Vázquez, Jesús; Rawls, John F

    2015-04-07

    Genome-wide association studies have implicated PLEXIN D1 (PLXND1) in body fat distribution and type 2 diabetes. However, a role for PLXND1 in regional adiposity and insulin resistance is unknown. Here we use in vivo imaging and genetic analysis in zebrafish to show that Plxnd1 regulates body fat distribution and insulin sensitivity. Plxnd1 deficiency in zebrafish induced hyperplastic morphology in visceral adipose tissue (VAT) and reduced lipid storage. In contrast, subcutaneous adipose tissue (SAT) growth and morphology were unaffected, resulting in altered body fat distribution and a reduced VAT:SAT ratio in zebrafish. A VAT-specific role for Plxnd1 appeared conserved in humans, as PLXND1 mRNA was positively associated with hypertrophic morphology in VAT, but not SAT. In zebrafish plxnd1 mutants, the effect on VAT morphology and body fat distribution was dependent on induction of the extracellular matrix protein collagen type V alpha 1 (col5a1). Furthermore, after high-fat feeding, zebrafish plxnd1 mutant VAT was resistant to expansion, and excess lipid was disproportionately deposited in SAT, leading to an even greater exacerbation of altered body fat distribution. Plxnd1-deficient zebrafish were protected from high-fat-diet-induced insulin resistance, and human VAT PLXND1 mRNA was positively associated with type 2 diabetes, suggesting a conserved role for PLXND1 in insulin sensitivity. Together, our findings identify Plxnd1 as a novel regulator of VAT growth, body fat distribution, and insulin sensitivity in both zebrafish and humans.

  2. Angiotensin II Induced Cardiac Dysfunction on a Chip.

    Directory of Open Access Journals (Sweden)

    Renita E Horton

    Full Text Available In vitro disease models offer the ability to study specific systemic features in isolation to better understand underlying mechanisms that lead to dysfunction. Here, we present a cardiac dysfunction model using angiotensin II (ANG II to elicit pathological responses in a heart-on-a-chip platform that recapitulates native laminar cardiac tissue structure. Our platform, composed of arrays of muscular thin films (MTF, allows for functional comparisons of healthy and diseased tissues by tracking film deflections resulting from contracting tissues. To test our model, we measured gene expression profiles, morphological remodeling, calcium transients, and contractile stress generation in response to ANG II exposure and compared against previous experimental and clinical results. We found that ANG II induced pathological gene expression profiles including over-expression of natriuretic peptide B, Rho GTPase 1, and T-type calcium channels. ANG II exposure also increased proarrhythmic early after depolarization events and significantly reduced peak systolic stresses. Although ANG II has been shown to induce structural remodeling, we control tissue architecture via microcontact printing, and show pathological genetic profiles and functional impairment precede significant morphological changes. We assert that our in vitro model is a useful tool for evaluating tissue health and can serve as a platform for studying disease mechanisms and identifying novel therapeutics.

  3. Angiotensin II Induced Cardiac Dysfunction on a Chip.

    Science.gov (United States)

    Horton, Renita E; Yadid, Moran; McCain, Megan L; Sheehy, Sean P; Pasqualini, Francesco S; Park, Sung-Jin; Cho, Alexander; Campbell, Patrick; Parker, Kevin Kit

    2016-01-01

    In vitro disease models offer the ability to study specific systemic features in isolation to better understand underlying mechanisms that lead to dysfunction. Here, we present a cardiac dysfunction model using angiotensin II (ANG II) to elicit pathological responses in a heart-on-a-chip platform that recapitulates native laminar cardiac tissue structure. Our platform, composed of arrays of muscular thin films (MTF), allows for functional comparisons of healthy and diseased tissues by tracking film deflections resulting from contracting tissues. To test our model, we measured gene expression profiles, morphological remodeling, calcium transients, and contractile stress generation in response to ANG II exposure and compared against previous experimental and clinical results. We found that ANG II induced pathological gene expression profiles including over-expression of natriuretic peptide B, Rho GTPase 1, and T-type calcium channels. ANG II exposure also increased proarrhythmic early after depolarization events and significantly reduced peak systolic stresses. Although ANG II has been shown to induce structural remodeling, we control tissue architecture via microcontact printing, and show pathological genetic profiles and functional impairment precede significant morphological changes. We assert that our in vitro model is a useful tool for evaluating tissue health and can serve as a platform for studying disease mechanisms and identifying novel therapeutics.

  4. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Yoshino A

    2016-08-01

    Full Text Available Atsushi Yoshino,1 Natalia Polouliakh,1–3 Akira Meguro,1 Masaki Takeuchi,1,4 Tatsukata Kawagoe,1 Nobuhisa Mizuki1 1Department of Ophthalmology and Visual Science, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 2Sony Computer Science Laboratories Inc., Fundamental Research Laboratories, 3Systems Biology Institute, Tokyo, Japan; 4Inflammatory Disease Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Abstract: Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. Keywords: fish egg, antiaging, gene expression analysis, antioxidative gene, phylogenetic footprinting analysis

  5. The inversa type of recessive dystrophic epidermolysis bullosa is caused by specific arginine and glycine substitutions in type VII collagen

    NARCIS (Netherlands)

    van den Akker, Peter C.; Mellerio, Jemima E.; Martinez, Anna E.; Liu, Lu; Meijer, Rowdy; Dopping-Hepenstal, Patricia J. C.; van Essen, Anthonie J.; Scheffer, Hans; Hofstra, Robert M. W.; McGrath, John A.; Jonkman, Marcel F.

    2011-01-01

    Background The inversa type of recessive dystrophic epidermolysis bullosa (RDEB-I) is a rare variant of dystrophic epidermolysis bullosa, characterised by blistering in the body flexures, trunk, and mucosa. The cause of this specific distribution is unknown. So far, 20 COL7A1 genotypes have been des

  6. The inversa type of recessive dystrophic epidermolysis bullosa is caused by specific arginine and glycine substitutions in type VII collagen

    NARCIS (Netherlands)

    Akker, P.C. van den; Mellerio, J.E.; Martinez, A.E.; Liu, L.; Meijer, R.; Dopping-Hepenstal, P.J.; Essen, A.J. van; Scheffer, H.; Hofstra, R.M.; McGrath, J.A.; Jonkman, M.F.

    2011-01-01

    BACKGROUND: The inversa type of recessive dystrophic epidermolysis bullosa (RDEB-I) is a rare variant of dystrophic epidermolysis bullosa, characterised by blistering in the body flexures, trunk, and mucosa. The cause of this specific distribution is unknown. So far, 20 COL7A1 genotypes have been de

  7. Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types

    DEFF Research Database (Denmark)

    Heinemeier, K M; Olesen, J L; Haddad, F

    2007-01-01

    Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle-tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-beta-1 (TGF......-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-beta-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX...... induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly...

  8. 骨髓间充质干细胞移植对胶原诱导性关节炎滑膜增殖的影响及机制%Effect of mesenchymal stem cells transplantation on the proliferation of synovinm in rats with collagen II induced arthritis

    Institute of Scientific and Technical Information of China (English)

    尹纪伟; 赵福涛; 王艳玲; 徐素粉

    2011-01-01

    目的 探讨骨髓间充质干细胞(MSCs)移植对胶原诱导性大鼠(CIA)滑膜增殖的影响及可能的作用机制.方法 分离培养SD大鼠MSCs.将40只SD大鼠,随机分为5组,空白对照组、早期MSCs治疗组(初次诱导的同时注射MSCs)、晚期MSCs治疗组(再次诱导的同时注射MSCs)及其相应对照组早期CIA对照组(初次诱导的同时注射生理盐水)和晚期CIA对照组(再次诱导的同时注射生理盐水).MSCs和生理盐水均从尾静脉注入.疗效观察包括关节炎指数(AI)、关节病理评分及ELISA检测血清血管内皮细胞生长因子(VEGF)、肿瘤坏死因子(TNF)-α和IL-17等.结果 (1)早期治疗组和晚期治疗组的AI、关节病理评分较相应对照组明显下降(P<0.05);(2)早期治疗组与晚期治疗组比较,AI与关节病理评分较低,差异有统计学意义(P<0.05);(3)早期治疗组、晚期治疗组血清VEGF、TNF-α、IL-17水平降低(P<0.05),与AI和关节病理评分呈显著正相关(P<0.05).结论 MSCs移植可减轻CIA的滑膜增殖,其机制有可能是通过下调VEGF、TNF-α和IL-17的表达来实现的.%Objective To investigate the influence and the probably mechanism of the mesenchymal stem cells(MSCs)transplantation in the collagen Ⅱ induced arthritis's synovial proliferation.Methods MSCs from SD rats bone marrow were isolated and expanded.40 rats were randomly divided into 5 groups, including:normal control group,early MSCs treatment group(MSCs were transplanted by the time the rats were induced for the first time),later MSCs treatment group(MSCs were transplanted by the time the rats were induced for the second time),early CIA control group(Normal saline were injected by the time the rats were induced for the first time)and later CIA control group(Normal saline were injected by the time the rats were induced for the second time).MSCs and normal saline were injected through tail venus.The observed parameters included arthritis index

  9. Bioengineered collagens

    Science.gov (United States)

    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

  10. Quantitative and qualitative analysis of collagen types in the fascia transversalis of inguinal hernia patients A fascia transversalis na etiopatogenia da hérnia inguinal

    Directory of Open Access Journals (Sweden)

    Alberto Luiz Monteiro Meyer

    2007-09-01

    Full Text Available BACKGROUND: Inguinal hernia is the second most common surgical case in our field. The anatomical factors alone are not enough to explain the inguinal hernia. Studies show changes in the proportion and quantity of collagen fibers in the developing of inguinal hernia. The greater production of collagen type III compared to the type I could justify the thinning of the fascia transversalis and its weakness. AIM: To determine the quantitative and qualitative changes of collagen in the fascia transversalis in inguinal hernia patients and compare them to findings from corpses without inguinal hernia. METHOD: Prospective case-control study based on the biopsy of fascia transversalis of 27 patients and 24 corpses. The technique used was hematoxylin-eosin and picrosirius colorimetry. RESULTS: The medium percent area of collagen (types I + III and collagen type I, in both groups, show no statistic difference. The quantity of collagen type III was greater in the patients. Patients classified with Nyhus IIIa presented greater quantity of collagen type III. CONCLUSION: There is no significant difference in the quantity of collagen in the fascia transversalis of patients compared to the controls. An increase in the quantity of collagen type III was found in patients with inguinal hernia and a greater quantity in those patients classified with Nyhus IIIa.RACIONAL: A hérnia inguinal é a segunda afecção cirúrgica mais comum em nosso meio. Os fatores anatômicos, isoladamente, não são suficientes para explicar a ocorrência das hérnias inguinais. Estudos apontam alterações na proporção e quantidade de fibras colágenas no desenvolvimento da hérnia inguinal. A maior produção de colágeno tipo III em relação ao tipo I poderia justificar o adelgaçamento da fascia transversalis e sua fraqueza. OBJETIVO: Determinar as alterações quantitativas e qualitativas de colágeno na fascia transversalis de doentes com hérnia inguinal e compará-las com

  11. Polymerized-Type I Collagen Downregulates Inflammation and Improves Clinical Outcomes in Patients with Symptomatic Knee Osteoarthritis Following Arthroscopic Lavage: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial

    Directory of Open Access Journals (Sweden)

    Janette Furuzawa-Carballeda

    2012-01-01

    Full Text Available Objectives. Polymerized-type I collagen (polymerized collagen is a downmodulator of inflammation and cartilage regenerator biodrug. Aim. To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA. Methods. Patients (n=19 were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n=10 or 2 mL of placebo (n=9 during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS, WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII and erythrocyte sedimentation rate (ESR were determined. Results. Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P<0.05 from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. Conclusion. Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA.

  12. Effects of valsartan on angiotensin II-induced migration of human coronary artery smooth muscle cells.

    Science.gov (United States)

    Kohno, M; Ohmori, K; Nozaki, S; Mizushige, K; Yasunari, K; Kano, H; Minami, M; Yoshikawa, J

    2000-11-01

    The migration as well as proliferation of coronary artery medial smooth muscle cells (SMC) into the intima is proposed to be an important process of intimal thickening in coronary atherosclerosis. In the current study, we examined the effects of the angiotensin type 1 receptor antagonist valsartan on angiotensin II (Ang II)-induced migration of cultured human coronary artery SMC using Boyden's chamber methods. Ang II significantly stimulated human coronary artery SMC migration in a concentration-dependent manner between 10(-6) and 10(-8) mol/l when cells of passage 4 to 6 were used. However, the migration response to Ang II was moderately decreased in cells of passage 10 to 12, and was markedly decreased in cells of passage 15 to 17, compared to that of passage 4 to 6. Ang II-induced migration was blocked by the Ang II type 1 (AT1) receptor antagonist valsartan in a concentration-dependent manner. By contrast, the Ang II type 2 (AT2) receptor antagonist PD 123319 did not affect Ang II-induced migration. Ang II modestly increased the cell number of human coronary artery SMC after a 24-h incubation. This increase in cell numbers was also clearly blocked by valsartan, but not by PD 123319. These results indicate that Ang II stimulates migration as well as proliferation via AT1 receptors in human coronary artery SMC when cells of passage 4 to 6 are used. Valsartan may prevent the progression of coronary atherosclerosis through an inhibition of Ang II-induced migration and proliferation in these cells, although in vivo evidence is lacking.

  13. Contraction-induced Mmp13 and-14 expression by goat articular chondrocytes in collagen type I but not type II gels

    NARCIS (Netherlands)

    Berendsen, Agnes D.; Vonk, Lucienne A.; Zandieh-Doulabi, Behrouz; Everts, Vincent; Bank, Ruud A.

    2012-01-01

    Collagen gels are promising scaffolds to prepare an implant for cartilage repair but several parameters, such as collagen concentration and composition as well as cell density, should be carefully considered, as they are reported to affect phenotypic aspects of chondrocytes. In this study we

  14. Contraction-induced Mmp13 and-14 expression by goat articular chondrocytes in collagen type I but not type II gels

    NARCIS (Netherlands)

    Berendsen, Agnes D.; Vonk, Lucienne A.; Zandieh-Doulabi, Behrouz; Everts, Vincent; Bank, Ruud A.

    2012-01-01

    Collagen gels are promising scaffolds to prepare an implant for cartilage repair but several parameters, such as collagen concentration and composition as well as cell density, should be carefully considered, as they are reported to affect phenotypic aspects of chondrocytes. In this study we investi

  15. Reconstruction of a hard connective tissue utilizing a pressed silk sheet and type-I collagen as the scaffold for fibroblasts.

    Science.gov (United States)

    Takezawa, Toshiaki; Ozaki, Katsuyuki; Takabayashi, Chiyuki

    2007-06-01

    A pressed silk sheet is a new biomaterial composed of a network of numerous cocoon filaments and having excellent mechanical strength and shape stability compared to a cotton-gauze sheet. To reconstruct a hard connective tissue using the silk sheet and type-I collagen as the scaffold for fibroblasts, three different three-dimensional floating culture systems were designed. "On sheet" system: fibroblasts were seeded on the silk sheet coated with collagen and the cell-attached sheet was cultured. "In gel" system: fibroblasts and the silk sheet were co-embedded in a collagen gel and the gel was cultured. "On vitrigel" system: fibroblasts were seeded on both sides of a collagen vitrigel involving the silk sheet and the vitrigel was cultured. The fibroblasts in all culture systems grew and formed disk-shaped connective tissue models involving the silk sheet by 14 days of culture. The "on sheet" and "on vitrigel" models retained a maximum elastic load of about 23 kgf and an ultimate tensile load of about 3.6 kgf, which were almost the same as for the individual silk sheet. However, the "in gel" system showed a low value for the tensile load. Cell damage following application of mechanical stress was lowest in the "on vitrigel" system. These data demonstrated the advantage of the "on vitrigel" system in reconstructing hard connective tissues. Such a novel culture method would contribute to a regenerative medicine for the failure of ligaments, tendons, and other connective tissues.

  16. Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

    Science.gov (United States)

    Rothdiener, Miriam; Hegemann, Miriam; Uynuk-Ool, Tatiana; Walters, Brandan; Papugy, Piruntha; Nguyen, Phong; Claus, Valentin; Seeger, Tanja; Stoeckle, Ulrich; Boehme, Karen A.; Aicher, Wilhelm K.; Stegemann, Jan P.; Hart, Melanie L.; Kurz, Bodo; Klein, Gerd; Rolauffs, Bernd

    2016-01-01

    Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype. PMID:27775041

  17. Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

    Science.gov (United States)

    Rothdiener, Miriam; Hegemann, Miriam; Uynuk-Ool, Tatiana; Walters, Brandan; Papugy, Piruntha; Nguyen, Phong; Claus, Valentin; Seeger, Tanja; Stoeckle, Ulrich; Boehme, Karen A.; Aicher, Wilhelm K.; Stegemann, Jan P.; Hart, Melanie L.; Kurz, Bodo; Klein, Gerd; Rolauffs, Bernd

    2016-10-01

    Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

  18. New lethal disease involving type I and III collagen defect resembling geroderma osteodysplastica, De Barsy syndrome, and Ehlers-Danlos syndrome IV.

    Science.gov (United States)

    Jukkola, A; Kauppila, S; Risteli, L; Vuopala, K; Risteli, J; Leisti, J; Pajunen, L

    1998-06-01

    We describe the clinical findings and biochemical features of a male child suffering from a so far undescribed lethal connective tissue disorder characterised by extreme hypermobility of the joints, lax skin, cataracts, severe growth retardation, and insufficient production of type I and type III procollagens. His features are compared with Ehlers-Danlos type IV, De Barsy syndrome, and geroderma osteodysplastica, as these disorders show some symptoms and signs shared with our patient. The child died because of failure of the connective tissue structures joining the skull and the spine, leading to progressive spinal stenosis. The aortic valve was translucent and insufficient. The clinical symptoms and signs, together with histological findings, suggested a collagen defect. Studies on both skin fibroblast cultures and the patient's serum showed reduced synthesis of collagen types I and III at the protein and RNA levels. The sizes of the mRNAs and newly synthesised proteins were normal, excluding gross structural abnormalities. These findings are not in accordance with any other collagen defect characterised so far.

  19. Preclinical evaluation of collagen type I scaffolds, including gelatin-collagen microparticles and loaded with a hydroglycolic Calendula officinalis extract in a lagomorph model of full-thickness skin wound.

    Science.gov (United States)

    Millán, D; Jiménez, R A; Nieto, L E; Linero, I; Laverde, M; Fontanilla, M R

    2016-02-01

    Previously, we have developed collagen type I scaffolds including microparticles of gelatin-collagen type I (SGC) that are able to control the release of a hydroglycolic extract of the Calendula officinalis flower. The main goal of the present work was to carry out the preclinical evaluation of SGC alone or loaded with the C. officinalis extract (SGC-E) in a lagomorph model of full-thickness skin wound. A total of 39 rabbits were distributed in three groups, of 13 animals each. The first group was used to compare wound healing by secondary intention (control) with wound healing observed when wounds were grafted with SGC alone. Comparison of control wounds with wounds grafted with SGC-E was performed in the second group, and comparison of wounds grafted with SGC with wounds grafted with SGC-E was performed in the third group. Clinical follow-ups were carried in all animals after surgery, and histological and histomorphometric analyses were performed on tissues taken from the healed area and healthy surrounding tissue. Histological and histomorphometric results indicate that grafting of SGC alone favors wound healing and brings a better clinical outcome than grafting SGC-E. In vitro collagenase digestion data suggested that the association of the C. officinalis extract to SGC increased the SGC-E cross-linking, making it difficult to degrade and affecting its biocompatibility.

  20. Comparative analysis of the noncollagenous NC1 domain of type IV collagen: identification of structural features important for assembly, function, and pathogenesis.

    Science.gov (United States)

    Netzer, K O; Suzuki, K; Itoh, Y; Hudson, B G; Khalifah, R G

    1998-06-01

    Type IV collagen alpha1-alpha6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, alpha1-like and alpha2-like, and suggested that vertebrate alpha3/alpha4 sequences evolved before alpha1/alpha2 and alpha5/alpha6. About one fifth of NC1 residues were identified to confer either the alpha1 or alpha2 group-specificity. These residues accumulate opposite charge in subdomain B of alpha1 (positive) and alpha2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic beta-strands and one short alpha-helix with a significant hydrophobic moment. The existence of opposite charges in the alpha-helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) toward a peptide substrate, though not to [14C]-gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying proteinase inhibitor function.

  1. Photobiomodulation therapy on collagen type I and III, vascular endothelial growth factor, and metalloproteinase in experimentally induced tendinopathy in aged rats.

    Science.gov (United States)

    Marques, Anna Cristina de Farias; Albertini, Regiane; Serra, Andrey Jorge; da Silva, Evela Aparecida Pereira; de Oliveira, Vanessa Lima Cavalcante; Silva, Luciana Miatto; Leal-Junior, Ernesto Cesar Pinto; de Carvalho, Paulo de Tarso Camillo

    2016-12-01

    This study investigates the effect of photobiomodulation therapy (PBMT) on collagen type I and III, matrix metalloproteinase (MMP), and vascular endothelial growth factor (VEGF) in experimentally induced tendinopathy in female aged rats. Tendinopathy was induced by the Achilles tendoncollagenase peritendinous. Forty-two Wistar rats (Norvegicus albinus) were used; groups consisted of 36 aged animals (18 months old; mean body weight, 517.7 ± 27.54 g) and 6 adult animals (12 weeks old; mean body weight, 266± 19.30 g). The animals were divided into three groups: control, aged tendinopathy, and aged tendinopathy PBMT; the aged groups were subdivided based on time to euthanasia: 7, 14, and 21 days. PBMT involved a gallium-arsenide-aluminum laser (Theralaser, DMC®) with active medium operating at wavelength 830 ± 10 nm, 50 mW power, 0.028 cm(2) laser beam, 107 J/cm(2) energy density, 1.8 W/cm(2) power density, and an energy of 3 J per point. The laser was applied by direct contact with the left Achilles tendon during 60 s per point at a frequency of three times per week, until the euthanasia date (7, 14, and 21 days). VEGF, MMP-3, and MMP-9 were analyzed by immunohistochemistry, and collagen type I and III by Sirius red. PBMT increased the deposition of collagen type I and III in a gradual manner, with significant differences relative to the group aged tendonitis (p tendinopathy (p < 0.001). PBMT, therefore, increased the production of collagen type I and III, downregulated the expression of MMP-3 and MMP-9, and upregulated that of VEGF, with age and age-induced hormonal deficiency.

  2. Physical and mechanical properties of cross-linked type I collagen scaffolds derived from bovine, porcine, and ovine tendons.

    Science.gov (United States)

    Ghodbane, Salim A; Dunn, Michael G

    2016-11-01

    Collagen scaffolds are often utilized in tissue engineering applications where their performance depends on physical and mechanical properties. This study investigated the effects of collagen source (bovine, porcine, and ovine tendon) on properties of collagen sponge scaffolds cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS). Scaffolds were tested for tensile and compressive properties, stability (resistance to enzymatic degradation), pore size, and swelling ratio. No significant differences in tensile modulus were observed, but ovine scaffolds had significantly greater ultimate strain, stress, and toughness relative to bovine and porcine scaffolds. No significant differences in compressive properties, pore size, or swelling ratio were observed as a function of collagen source. Ovine scaffolds were more resistant to collagenase degradation compared to bovine samples, which were more resistant than porcine scaffolds. In comparison to bovine scaffolds, ovine scaffolds performed equivalently or superiorly in all evaluations, and porcine scaffolds were equivalent in all properties except enzymatic stability. These results suggest that collagen sponges derived from bovine, porcine, and ovine tendon have similar physical and mechanical properties, and are all potentially suitable materials for various tissue engineering applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2685-2692, 2016.

  3. Framework of collagen type I - vasoactive vessels structuring invariant geometric attractor in cancer tissues: insight into biological magnetic field.

    Directory of Open Access Journals (Sweden)

    Jairo A Díaz

    Full Text Available In a previous research, we have described and documented self-assembly of geometric triangular chiral hexagon crystal-like complex organizations (GTCHC in human pathological tissues. This article documents and gathers insights into the magnetic field in cancer tissues and also how it generates an invariant functional geometric attractor constituted for collider partners in their entangled environment. The need to identify this hierarquic attractor was born out of the concern to understand how the vascular net of these complexes are organized, and to determine if the spiral vascular subpatterns observed adjacent to GTCHC complexes and their assembly are interrelational. The study focuses on cancer tissues and all the macroscopic and microscopic material in which GTCHC complexes are identified, which have been overlooked so far, and are rigorously revised. This revision follows the same parameters that were established in the initial phase of the investigation, but with a new item: the visualization and documentation of external dorsal serous vascular bed areas in spatial correlation with the localization of GTCHC complexes inside the tumors. Following the standard of the electro-optical collision model, we were able to reproduce and replicate collider patterns, that is, pairs of left and right hand spin-spiraled subpatterns, associated with the orientation of the spinning process that can be an expansion or contraction disposition of light particles. Agreement between this model and tumor data is surprisingly close; electromagnetic spiral patterns generated were identical at the spiral vascular arrangement in connection with GTCHC complexes in malignant tumors. These findings suggest that the framework of collagen type 1 - vasoactive vessels that structure geometric attractors in cancer tissues with invariant morphology sets generate collider partners in their magnetic domain with opposite biological behavior. If these principles are incorporated

  4. Collagen type-I leads to in vivo matrix mineralization and secondary stabilization of Mg-Zr-Ca alloy implants.

    Science.gov (United States)

    Mushahary, Dolly; Wen, Cuie; Kumar, Jerald Mahesh; Lin, Jixing; Harishankar, Nemani; Hodgson, Peter; Pande, Gopal; Li, Yuncang

    2014-10-01

    Biodegradable magnesium-zirconia-calcium (Mg-Zr-Ca) alloy implants were coated with Collagen type-I (Coll-I) and assessed for their rate and efficacy of bone mineralization and implant stabilization. The phases, microstructure and mechanical properties of these alloys were analyzed using X-ray diffraction (XRD), optical microscopy and compression test, respectively, and the corrosion behavior was established by their hydrogen production rate in simulated body fluid (SBF). Coll-I extracted from rat tail, and characterized using fourier transform infrared (FT-IR) spectroscopy, was used for dip-coating the Mg-based alloys. The coated alloys were implanted into the femur bones of male New Zealand white rabbits. In vivo bone formation around the implants was quantified by measuring the bone mineral content/density (BMC/BMD) using dual-energy X-ray absorptiometry (DXA). Osseointegration of the implant and new bone mineralization was visualized by histological and immunohistochemical analysis. Upon surface coating with Coll-I, these alloys demonstrated high surface energy showing enhanced performance as an implant material that is suitable for rapid and efficient new bone tissue induction with optimal mineral content and cellular properties. The results demonstrate that Coll-I coated Mg-Zr-Ca alloys have a tendency to form superior trabecular bone structure with better osteoinduction around the implants and higher implant secondary stabilization, through the phenomenon of contact osteogenesis, compared to the control and uncoated ones in shorter periods of implantation. Hence, Coll-I surface coating of Mg-Zr-Ca alloys is a promising method for expediting new bone formation in vivo and enhancing osseointegration in load bearing implant applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Ethyl pyruvate therapy attenuates experimental severe arthritis caused by type II collagen (CII) in the mouse (CIA).

    Science.gov (United States)

    Di Paola, R; Mazzon, E; Galuppo, M; Esposito, E; Bramanti, P; Fink, M P; Cuzzocrea, S

    2010-01-01

    This study tested the hypothesis that ethyl pyruvate (EP), a simple aliphatic ester with anti-inflammatory effects, can reduce type II collagen-induced mouse arthritis (CIA). DBA/1J mice were used for the study, developing erosive hind paw arthritis when immunized with CII in an emulsion in complete Freund?s adjuvant (CFA). The incidence of CIA was 100 percent by day 28 in the CII-challenged mice, and the severity of CIA progressed over a 35-day period with radiographic evaluation revealing focal resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint margins. EP-treatment (40 mg/kg/day i.p.) starting at the onset of arthritis (day 25) ameliorated the clinical signs at days 26-35 and improved histological status in the joint and paw. Immunohistochemical analysis for nitrotyrosine, poly (ADP-ribose) (PAR), inducible nitric oxide synthase (iNOS) revealed a positive staining in inflamed joints from mice subjected to CIA, while no staining was observed for HO-1 and Nrf-2 in the same group. The degree of staining for nitrotyrosine, PAR, iNOS, was significantly reduced in CII-challenged mice treated with the EP. Immuno-positive-staining for HO-1 and Nrf-2 was observed instead, in joints obtained from the EP-treated group. Plasma levels of TNF-α, IL-6 and the joint tissue levels of macrophage inflammatory protein (MIP)-1α and MIP-2 were also significantly reduced by EP treatment. Thirty-five days after immunization, EP-treatment significantly increased plasma levels of IL-10. These data demonstrate that EP treatment exerts an anti-inflammatory effect during chronic inflammation and is able to ameliorate the tissue damage associated with CIA.

  6. A Novel Regulatory Mechanism of Type II Collagen Expression via a SOX9-dependent Enhancer in Intron 6.

    Science.gov (United States)

    Yasuda, Hideyo; Oh, Chun-do; Chen, Di; de Crombrugghe, Benoit; Kim, Jin-Hoi

    2017-01-13

    Type II collagen α1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1. However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5-10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. Furthermore, SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, although both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing.

    Science.gov (United States)

    Jettanacheawchankit, Suwimon; Sasithanasate, Siriruk; Sangvanich, Polkit; Banlunara, Wijit; Thunyakitpisal, Pasutha

    2009-04-01

    Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival fibroblasts (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; keratinocyte growth factor-1 (KGF-1), vascular endothelial growth factor (VEGF), and type I collagen production; and oral wound healing in rats. [(3)H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol; and Carbopol containing 0.5%, 1%, and 2% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of 2, 4, 8, and 16 mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between 2 - 16 mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of fibroblast proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.

  8. Evaluation of Magnetic Nanoparticle-Labeled Chondrocytes Cultivated on a Type II Collagen-Chitosan/Poly(Lactic-co-Glycolic) Acid Biphasic Scaffold.

    Science.gov (United States)

    Su, Juin-Yih; Chen, Shi-Hui; Chen, Yu-Pin; Chen, Wei-Chuan

    2017-01-04

    Chondral or osteochondral defects are still controversial problems in orthopedics. Here, chondrocytes labeled with magnetic nanoparticles were cultivated on a biphasic, type II collagen-chitosan/poly(lactic-co-glycolic acid) scaffold in an attempt to develop cultures with trackable cells exhibiting growth, differentiation, and regeneration. Rabbit chondrocytes were labeled with magnetic nanoparticles and characterized by scanning electron microscopy (SEM), transmission electron (TEM) microscopy, and gene and protein expression analyses. The experimental results showed that the magnetic nanoparticles did not affect the phenotype of chondrocytes after cell labeling, nor were protein and gene expression affected. The biphasic type II collagen-chitosan/poly(lactic-co-glycolic) acid scaffold was characterized by SEM, and labeled chondrocytes showed a homogeneous distribution throughout the scaffold after cultivation onto the polymer. Cellular phenotype remained unaltered but with increased gene expression of type II collagen and aggrecan, as indicated by cell staining, indicating chondrogenesis. Decreased SRY-related high mobility group-box gene (Sox-9) levels of cultured chondrocytes indicated that differentiation was associated with osteogenesis. These results are encouraging for the development of techniques for trackable cartilage regeneration and osteochondral defect repair which may be applied in vivo and, eventually, in clinical trials.

  9. Novel feline autoimmune blistering disease resembling bullous pemphigoid in humans: IgG autoantibodies target the NC16A ectodomain of type XVII collagen (BP180/BPAG2).

    Science.gov (United States)

    Olivry, T; Chan, L S; Xu, L; Chace, P; Dunston, S M; Fahey, M; Marinkovich, M P

    1999-07-01

    In humans and dogs, bullous pemphigoid (BP) is an autoimmune blistering disease associated with the production of basement membrane autoantibodies that target the 180-kd type XVII collagen (BP180, BPAG2) and/or the 230-kd plakin epidermal isoform BPAG1e (BP230). In two adult cats, an acquired dermatosis and stomatitis was diagnosed as BP subsequent to the fulfillment of the following criteria: 1) presence of cutaneous vesicles, erosions, and ulcers; 2) histologic demonstration of subepidermal vesiculation with inflammatory cells, including eosinophils; 3) in vivo deposition of IgG autoantibodies at the epidermal basement membrane zone; and 4) serum IgG autoantibodies targeting a 180-kd epidermal protein identified as type XVII collagen. In both cats, the antigenic epitopes targeted by IgG autoantibodies were shown to be situated in the NC16A ectodomain of type XVII collagen, a situation similar to that of humans and dogs with BP. Feline BP therefore can be considered a clinical, histopathologic, and immunologic homologue of BP in humans and dogs.

  10. The severity of Osteogenesis imperfecta and type I collagen pattern in human skin as determined by nonlinear microscopy: proof of principle of a diagnostic method.

    Directory of Open Access Journals (Sweden)

    Javier Adur

    Full Text Available BACKGROUND: The confirmatory diagnosis of Osteogenesis Imperfecta (OI requires invasive, commonly bone biopsy, time consuming and destructive methods. This paper proposes an alternative method using a combination of two-photon excitation fluorescence (TPEF and second-harmonic generation (SHG microscopies from easily obtained human skin biopsies. We show that this method can distinguish subtypes of human OI. METHODOLOGY/PRINCIPAL FINDINGS: Different aspects of collagen microstructure of skin fresh biopsies and standard H&E-stained sections of normal and OI patients (mild and severe forms were distinguished by TPEF and SHG images. Moreover, important differences between subtypes of OI were identified using different methods of quantification such as collagen density, ratio between collagen and elastic tissue, and gray-level co-occurrence matrix (GLCM image-pattern analysis. Collagen density was lower in OI dermis, while the SHG/autofluorescence index of the dermis was significantly higher in OI as compared to that of the normal skin. We also showed that the energy value of GLCM texture analysis is useful to discriminate mild from severe OI and from normal skin. CONCLUSIONS/SIGNIFICANCE: This work demonstrated that nonlinear microscopy techniques in combination with image-analysis approaches represent a powerful tool to investigate the collagen organization in skin dermis in patients with OI and has the potential to distinguish the different types of OI. The procedure outlined in this paper requires a skin biopsy, which is almost painless as compared to the bone biopsy commonly used in conventional methods. The data presented here complement existing clinical diagnostic techniques and can be used as a diagnostic procedure to confirm the disease, evaluate its severity and treatment efficacy.

  11. [Collagenous colitis].

    Science.gov (United States)

    Lindström, C G

    1991-05-01

    Collagenous colitis is now regarded by an overwhelming majority of authors as a clinicopathological entity and has been taken up as a such in many text-books and diagnostic atlases (Morson & Dawson, 1990, Fenoglio-Preiser et al., 1989, Whitehead 1985, Whitehead 1989). A good, detailed review of cases of collagenous colitis published up to 1988 was performed by Perri et al. Collagenous colitis was also presented to a wider medical public through a clinicopathological conference case at Massachusetts General Hospital (Case 29-1988). Finally it may be added that collagenous colitis has been included in the new fourth edition of Robbins Pathologic Basis of Disease (Cotran, Kumar, Robbins, 1989), where the possibility of an autoimmune disease is stressed.

  12. Potential in two types of collagen scaffolds for urological tissue engineering applications - Are there differences in growth behaviour of juvenile and adult vesical cells?

    Science.gov (United States)

    Leonhäuser, D; Vogt, M; Tolba, R H; Grosse, J O

    2016-02-01

    The aging society has a deep impact on patient care in urology. The number of patients in need of partial or whole bladder wall replacement is increasing simultaneously with the number of cancer incidents. Therefore, urological research requires a model of bladder wall replacement in adult and elderly people. Two types of porcine collagen I/III scaffolds were used in vitro for comparison of cell growth of two different pig breeds at different growth stages. Scaffolds were characterised with scanning electron and laser scanning microscopy. Urothelial and detrusor smooth muscle cells were isolated from 15 adult Göttingen minipigs and 15 juvenile German Landrace pigs. Growth behaviour was examined in cell culture and seeded onto the collagen scaffolds via immunohistochemistry, two-photon laser scanning microscopy and a viability assay. The collagen scaffolds showed different structured surfaces which are appropriate for seeding of the two different cell types. Moisturisation of the scaffolds resulted in a change of the structure. Cell growth of German Landrace urothelial cells and smooth muscle cells was significantly higher than cell growth of the Göttingen minipig cells. Seeding of scaffolds with both cell types from both pig races was possible which could be shown by immunohistochemistry and two-photon laser scanning microscopy. Growth behaviour on the scaffolds was significantly increased for the German Landrace compared to Göttingen minipig. Nevertheless, seeding with the adult Göttingen minipig cells resulted in a closed layer on the surface and urothelial cells and smooth muscle cells showed increasing growth until day 14. The results show that these collagen scaffolds are adequate for the seeding with vesical cells. Moreover, they seem appropriate for the use as an in vitro model for the adult or elderly as the cells of the adult Göttingen minipig too, show good growth behaviour. © The Author(s) 2015.

  13. Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the [alpha]I(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid

    Energy Technology Data Exchange (ETDEWEB)

    Wallis, G.A.; Rash, B.; Sweetman, W.A.; Thomas, J.T.; Grant, M.E.; Boot-Handford, R.P. (Univ. of Manchester (United Kingdom)); Super, M. (Royal Manchester Children' s Hospital, Manchester (United Kingdom)); Evans, G. (Robert Jones Orthopaedic Hospital, Oswestry (United Kingdom))

    1994-02-01

    Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the site of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. The authors used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, they identified two individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitutions at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development. 36 refs., 5 figs.

  14. UV damage of collagen: insights from model collagen peptides.

    Science.gov (United States)

    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.

  15. Identification of the alpha 3 chain of type IV collagen as the common autoantigen in antibasement membrane disease and Goodpasture syndrome.

    Science.gov (United States)

    Kalluri, R; Wilson, C B; Weber, M; Gunwar, S; Chonko, A M; Neilson, E G; Hudson, B G

    1995-10-01

    Antiglomerular basement membrane (GBM) antibodies can cause glomerulonephritis or pulmonary hemorrhage by themselves or Goodpasture syndrome when they occur together. It is unknown if variations in antibody reactivity contribute to the different patterns of organ involvement seen in this disease. This study examines the reactivity of the alpha 1-alpha 6 NC1 domains of Type IV collagen, the putative autoantigen, in sera from patients with anti-GBM antibodies after various clinical presentations of lung hemorrhage and renal injury. Serum or plasma containing anti-GBM antibodies from 35 patients with combined glomerulonephritis and pulmonary hemorrhage, 19 with glomerulonephritis alone, and 4 with pulmonary hemorrhage alone were compared with samples from 19 normal controls and 32 patients with other kidney diseases. Four different immunologic assays were performed with bovine alpha 1-alpha 6(IV) and recombinant human type alpha 1-alpha 5(IV) collagen NC1 domains. The study found that the anti-GBM antibodies from all patients reacted with the alpha 3(IV) NC1 (85% exclusively). Additional limited reactivity with the alpha 1(IV) NC1 and alpha 4(IV) NC1 was found in 15 and 3%, respectively. This non-alpha 3(IV) NC1 reactivity was most frequent in the patients with anti-GBM antibodies and glomerulonephritis alone. None of the patients had reactivity to other basement membrane components like laminin, fibronectin, heparan sulfate proteoglycan, entactin, or the 7S and triple helical fragments of Type IV collagen. The observed alpha-chain NC1 reactivity was confined to patients with anti-GBM antibodies with no additional reactivities detected among a large number of other kidney diseases controls. The correlation of alpha 1-alpha 6(IV) NC1 reactivity in a large number of patients with anti-GBM antibodies defined by classic assays definitively establishes that reactivity to alpha 3(IV) NC1 domains is both sufficient and necessary for the expression of autoimmune disease

  16. Interleukin-13 induces collagen type-1 expression through matrix metalloproteinase-2 and transforming growth factor-β1 in airway fibroblasts in asthma.

    Science.gov (United States)

    Firszt, Rafael; Francisco, Dave; Church, Tony D; Thomas, Joseph M; Ingram, Jennifer L; Kraft, Monica

    2014-02-01

    Airway remodelling is a feature of asthma that contributes to loss of lung function. One of the central components of airway remodelling is subepithelial fibrosis. Interleukin (IL)-13 is a key T-helper 2 cytokine and is believed to be the central mediator of allergic asthma including remodelling, but the mechanism driving the latter has not been elucidated in human asthma. We hypothesised that IL-13 stimulates collagen type-1 production by the airway fibroblast in a matrix metalloproteinase (MMP)- and transforming growth factor (TGF)-β1-dependent manner in human asthma as compared to healthy controls. Fibroblasts were cultured from endobronchial biopsies in 14 subjects with mild asthma and 13 normal controls that underwent bronchoscopy. Airway fibroblasts were treated with various mediators including IL-13 and specific MMP-inhibitors. IL-13 significantly stimulated collagen type-1 production in asthma compared to normal controls. Inhibitors of MMP-2 significantly attenuated collagen production in asthma but had no effect in normal controls. IL-13 significantly increased total and active forms of TGF-β1, and this activation was blocked using an MMP-2 inhibitor. IL-13 activated endogenous MMP-2 in asthma patients as compared to normal controls. In an ex vivo model, IL-13 potentiates airway remodelling through a mechanism involving TGF-β1 and MMP-2. These effects provide insights into the mechanism involved in IL-13-directed airway remodelling in asthma.

  17. Time course of antibodies against IgG and type II collagen in adjuvant arthritis. Role of mycobacteria administration in antibody production.

    Science.gov (United States)

    Franch, A; Cassany, S; Castellote, C; Castell, M

    1994-02-01

    The aim of this study was to elucidate, during the time course of adjuvant arthritis, the existence of antibodies directed to IgG (rheumatoid factor-like) and antibodies against type II collagen. In a second study, we also studied the relation between antibody production, arthritic process and mycobacteria administration. We have demonstrated the presence of antibodies to IgG and type II collagen by means of ELISA techniques. This reactivity appeared on day 7 post-induction, decreased later, and increased progressively from day 21 until last day studied (day 56 post-induction). We have also quantified antibodies against a soluble fraction of Mycobacterium butyricum, the inductor of the disease. Anti-mycobacteria antibodies appeared during the first seven days after induction, but from day 14, when systemic inflammation began, their levels suddenly increased. There is a positive correlation between anti-mycobacteria antibody levels and articular swelling. Anti-IgG and anti-collagen antibody production was not directly linked to arthritic process since these antibodies were synthesized when M. butyricum was administered intraperitoneally, which does not induce arthritis. Anti-mycobacteria antibody concentration was higher when arthritis induction by mycobacterial was successful than when it was unsuccessful.

  18. Ehlers-Danlos syndrome type IV: unusual congenital anomalies in a mother and son with a COL3A1 mutation and a normal collagen III protein profile.

    Science.gov (United States)

    Kroes, H Y; Pals, G; van Essen, A J

    2003-03-01

    A mother and son with Ehlers-Danlos syndrome (EDS) type IV and unusual congenital anomalies are described. The congenital anomalies include, in the mother, amniotic band-like constrictions on one hand, a unilateral clubfoot, and macrocephaly owing to normal-pressure hydrocephaly and, in the son, an esophageal atresia and hydrocephaly. Protein analysis of collagen III in cultured fibroblasts of the mother showed no abnormalities. However, DNA analysis of the COL3A1 gene revealed a pathogenic mutation (388G-->T) in both the mother and the son. The possible relationship between the observed congenital anomalies and EDS IV are discussed. We stress that DNA analysis of COL3A1 should be performed in all patients when there is a strong suspicion of EDS IV, despite negative findings in a collagen protein analysis. Copyright Blackwell Munksgaard, 2003

  19. Misbalance in type III collagen formation/degradation as a novel serological biomarker for penetrating (Montreal B3) Crohn's disease

    NARCIS (Netherlands)

    van Haaften, W. T.; Mortensen, J. H.; Karsdal, M. A.; Bay-Jensen, A. C.; Dijkstra, G.; Olinga, P.

    Background: Misbalances in extracellular matrix turnover are key factors in the development of stricturing (Montreal B2) and penetrating (Montreal B3) Crohn's disease. Aim: To determine whether serological markers for collagen formation and degradation could serve as biomarkers for complications of

  20. Temperature-Responsive Gelation of Type I Collagen Solutions Involving Fibril Formation and Genipin Crosslinking as a Potential Injectable Hydrogel

    Directory of Open Access Journals (Sweden)

    Shunji Yunoki

    2013-01-01

    Full Text Available We investigated the temperature-responsive gelation of collagen/genipin solutions using pepsin-solubilized collagen (PSC and acid-solubilized collagen (ASC as substrates. Gelation occurred in the PSC/genipin solutions at genipin concentrations 0–2 mM under moderate change in temperature from 25 to 37°C. The PSC/genipin solutions exhibited fluidity at room temperature for at least 30 min, whereas the ASC/genipin solutions rapidly reached gel points. In specific cases PSC would be preferred over ASC as an injectable gel system. The temperature-responsive gelation of PSC/genipin solutions was due to temperature responses to genipin crosslinking and collagen fibril formation. The elastic modulus of the 0.5% PSC/genipin gel system could be adjusted in a range of 2.5 to 50 kPa by the PSC and genipin concentrations, suggesting that a PSC/genipin solution is a potential injectable gel system for drug and cell carriers, with mechanical properties matching those of living tissues.

  1. Changes of MMP-1 and collagen type Ialpha1 by UVA, UVB and IRA are differentially regulated by Trx-1.

    Science.gov (United States)

    Buechner, Nicole; Schroeder, Peter; Jakob, Sascha; Kunze, Kerstin; Maresch, Tanja; Calles, Christian; Krutmann, Jean; Haendeler, Judith

    2008-07-01

    Exposure of human skin to solar radiation, which includes ultraviolet (UV) radiation (UVA and UVB) visible light and infrared radiation, induces skin aging. The effects of light have been attributed to irradiation-induced reactive oxygen species (ROS) formation, but the specific signaling pathways are not well understood. Detrimental effects of solar radiation are dermal diseases and photoaging. Exposure of cultured human dermal fibroblasts to UVA, UVB or IRA increased ROS formation in vitro. One important redox regulator is the oxidoreductase thioredoxin-1 (Trx). Trx is ubiquitously expressed and has anti-oxidative and anti-apoptotic properties. Besides its function to reduce H(2)O(2), Trx binds to and regulates transcription factors. The aim of this study was to investigate whether Trx influences the regulation of MMP-1 and collagen Ialpha1 by UVA, UVB and IRA. We irradiated human dermal fibroblasts with UVA, UVB and IRA. UVA, UVB and IRA upregulated MMP-1 expression. Trx inhibited UVA-induced MMP-1 upregulation in a NFkappaB dependent manner. UVA, UVB and IRA reduced collagen Ialpha1 expression. Incubation with Trx inhibited the effects of UVB and IRA on collagen Ialpha1 expression. In conclusion, MMP-1 and collagen Ialpha1, which play important roles in aging processes, seems to be regulated by different transcriptional mechanisms and Trx can only influence distinct signaling pathways induced by UVA, UVB and probably IRA. Thus, Trx may serve as an important contributor to an "anti-aging therapeutic cocktail".

  2. Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ.

    Science.gov (United States)

    Canty-Laird, Elizabeth G; Lu, Yinhui; Kadler, Karl E

    2012-01-15

    Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late emb