A multiwell platform for studying stiffness-dependent cell biology.

Science.gov (United States)

Mih, Justin D; Sharif, Asma S; Liu, Fei; Marinkovic, Aleksandar; Symer, Matthew M; Tschumperlin, Daniel J

2011-01-01

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.

A multiwell platform for studying stiffness-dependent cell biology.

Directory of Open Access Journals (Sweden)

Justin D Mih

Full Text Available Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.

Tumor-produced, active Interleukin-1 β regulates gene expression in carcinoma-associated fibroblasts

International Nuclear Information System (INIS)

Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

2011-01-01

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-β. PDL fibroblasts possess receptor for IL1-β, and its expression is increased 4.56-times in the presence of SCC-25 tumor cells. IL1-β receptor expression in

Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

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Dudas, Jozsef, E-mail: Jozsef.Dudas@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullar, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Bitsche, Mario, E-mail: Mario.Bitsche@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Schartinger, Volker, E-mail: Volker.Schartinger@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Sprinzl, Georg Mathias, E-mail: Georg.Sprinzl@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: Herbert.Riechelmann@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

2011-09-10

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

Matrix compliance and the regulation of cytokinesis

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Savitha Sambandamoorthy

2015-07-01

Full Text Available Integrin-mediated cell adhesion to the ECM regulates many physiological processes in part by controlling cell proliferation. It is well established that many normal cells require integrin-mediated adhesion to enter S phase of the cell cycle. Recent evidence indicates that integrins also regulate cytokinesis. Mechanical properties of the ECM can dictate entry into S phase; however, it is not known whether they also can affect the successful completion of cell division. To address this issue, we modulated substrate compliance using fibronectin-coated acrylamide-based hydrogels. Soft and hard substrates were generated with approximate elastic moduli of 1600 and 34,000 Pascals (Pa respectively. Our results indicate that dermal fibroblasts successfully complete cytokinesis on hard substrates, whereas on soft substrates, a significant number fail and become binucleated. Cytokinesis failure occurs at a step following the formation of the intercellular bridge connecting presumptive daughter cells, suggesting a defect in abscission. Like dermal fibroblasts, mesenchymal stem cells require cell-matrix adhesion for successful cytokinesis. However, in contrast to dermal fibroblasts, they are able to complete cytokinesis on both hard and soft substrates. These results indicate that matrix stiffness regulates the successful completion of cytokinesis, and does so in a cell-type specific manner. To our knowledge, our study is the first to demonstrate that matrix stiffness can affect cytokinesis. Understanding the cell-type specific contribution of matrix compliance to the regulation of cytokinesis will provide new insights important for development, as well as tissue homeostasis and regeneration.

Stiff mutant genes of Phycomyces target turgor pressure and wall mechanical properties to regulate elongation growth rate

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Joseph K. E. Ortega

2012-05-01

Full Text Available Regulation of cell growth is paramount to all living organisms. In plants, algae and fungi, regulation of expansive growth of cells is required for development and morphogenesis. Also, many sensory responses of stage IVb sporangiophores of Phycomyces blakesleeanus are produced by regulating elongation growth rate (growth responses and differential elongation growth rate (tropic responses. Stiff mutant sporangiophores exhibit diminished tropic responses and are found to be defective in at least four genes; madD, madE, madF and madG. Prior experimental research suggests that the defective genes affect growth regulation, but this was not verified. All the growth of the single-celled stalk of the stage IVb sporangiophore occurs in a short region termed the growth zone. Prior experimental and theoretical research indicates that elongation growth rate of the stage IVb sporangiophore can be regulated by controlling the cell wall mechanical properties within the growth zone and the magnitude of the turgor pressure. A quantitative biophysical model for elongation growth rate is required to elucidate the relationship between wall mechanical properties and turgor pressure during growth regulation. In this study, it is hypothesized that the mechanical properties of the wall within the growth zone of stiff mutant sporangiophores are different compared to wild type. A biophysical equation for elongation growth rate is derived for fungal and plant cells with a growth zone. Two strains of stiff mutants are studied, C149 madD120 (- and C216 geo- (-. Experimental results demonstrate that turgor pressure is larger but irreversible deformation rates of the wall within the growth zone and growth zone length are smaller for stiff mutant sporangiophores compared to wild type. These findings explain the diminished tropic responses of the stiff mutant sporangiophores and suggest that the defective genes affect the amount of wall-building material delivered to the inner

Modulation of ROS levels in fibroblasts by altering mitochondria regulates the process of wound healing.

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Janda, Jaroslav; Nfonsam, Valentine; Calienes, Fernanda; Sligh, James E; Jandova, Jana

2016-05-01

Mitochondria are the major source of reactive oxygen species (ROS) in fibroblasts which are thought to be crucial regulators of wound healing with a potential to affect the expression of nuclear genes involved in this process. ROS generated by mitochondria are involved in all stages of tissue repair process but the regulation of ROS-generating system in fibroblasts still remains poorly understood. The purpose of this study was to better understand molecular mechanisms of how the regulation of ROS levels generated by mitochondria may influence the process of wound repair. Cybrid model system of mtDNA variations was used to study the functional consequences of altered ROS levels on wound healing responses in a uniform nuclear background of cultured ρ(0) fibroblasts. Mitochondrial ROS in cybrids were modulated by antioxidants that quench ROS to examine their ability to close the wound. Real-time PCR arrays were used to investigate whether ROS generated by specific mtDNA variants have the ability to alter expression of some key nuclear-encoded genes central to the wound healing response and oxidative stress. Our data suggest levels of mitochondrial ROS affect expression of some nuclear encoded genes central to wound healing response and oxidative stress and modulation of mitochondrial ROS by antioxidants positively affects in vitro process of wound closure. Thus, regulation of mitochondrial ROS-generating system in fibroblasts can be used as effective natural redox-based strategy to help treat non-healing wounds.

Substrate stiffness and oxygen as regulators of stem cell differentiation during skeletal tissue regeneration: a mechanobiological model.

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Darren Paul Burke

Full Text Available Extrinsic mechanical signals have been implicated as key regulators of mesenchymal stem cell (MSC differentiation. It has been possible to test different hypotheses for mechano-regulated MSC differentiation by attempting to simulate regenerative events such as bone fracture repair, where repeatable spatial and temporal patterns of tissue differentiation occur. More recently, in vitro studies have identified other environmental cues such as substrate stiffness and oxygen tension as key regulators of MSC differentiation; however it remains unclear if and how such cues determine stem cell fate in vivo. As part of this study, a computational model was developed to test the hypothesis that substrate stiffness and oxygen tension regulate stem cell differentiation during fracture healing. Rather than assuming mechanical signals act directly on stem cells to determine their differentiation pathway, it is postulated that they act indirectly to regulate angiogenesis and hence partially determine the local oxygen environment within a regenerating tissue. Chondrogenesis of MSCs was hypothesized to occur in low oxygen regions, while in well vascularised regions of the regenerating tissue a soft local substrate was hypothesised to facilitate adipogenesis while a stiff substrate facilitated osteogenesis. Predictions from the model were compared to both experimental data and to predictions of a well established computational mechanobiological model where tissue differentiation is assumed to be regulated directly by the local mechanical environment. The model predicted all the major events of fracture repair, including cartilaginous bridging, endosteal and periosteal bony bridging and bone remodelling. It therefore provides support for the hypothesis that substrate stiffness and oxygen play a key role in regulating MSC fate during regenerative events such as fracture healing.

Matrix stiffness-upregulated LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.

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Wu, Sifan; Zheng, Qiongdan; Xing, Xiaoxia; Dong, Yinying; Wang, Yaohui; You, Yang; Chen, Rongxin; Hu, Chao; Chen, Jie; Gao, Dongmei; Zhao, Yan; Wang, Zhiming; Xue, Tongchun; Ren, Zhenggang; Cui, Jiefeng

2018-05-04

Higher matrix stiffness affects biological behavior of tumor cells, regulates tumor-associated gene/miRNA expression and stemness characteristic, and contributes to tumor invasion and metastasis. However, the linkage between higher matrix stiffness and pre-metastatic niche in hepatocellular carcinoma (HCC) is still largely unknown. We comparatively analyzed the expressions of LOX family members in HCC cells grown on different stiffness substrates, and speculated that the secreted LOXL2 may mediate the linkage between higher matrix stiffness and pre-metastatic niche. Subsequently, we investigated the underlying molecular mechanism by which matrix stiffness induced LOXL2 expression in HCC cells, and explored the effects of LOXL2 on pre-metastatic niche formation, such as BMCs recruitment, fibronectin production, MMPs and CXCL12 expression, cell adhesion, etc. RESULTS: Higher matrix stiffness significantly upregulated LOXL2 expression in HCC cells, and activated JNK/c-JUN signaling pathway. Knockdown of integrin β1 and α5 suppressed LOXL2 expression and reversed the activation of above signaling pathway. Additionally, JNK inhibitor attenuated the expressions of p-JNK, p-c-JUN, c-JUN and LOXL2, and shRNA-c-JUN also decreased LOXL2 expression. CM-LV-LOXL2-OE and rhLOXL2 upregulated MMP9 expression and fibronectin production obviously in lung fibroblasts. Moreover, activation of Akt pathway contributed to LOXL2-induced fibronectin upregulation. LOXL2 in CM as chemoattractant increased motility and invasion of BMCs, implicating a significant role of LOXL2 in BMCs recruitment. Except that, CM-LV-LOXL2-OE as chemoattractant also increased the number of migrated HCC cells, and improved chemokine CXCL12 expression in lung fibroblasts. The number of HCC cells adhered to surface of lung fibroblasts treated with CM-LV-LOXL2-OE was remarkably higher than that of the control cells. These results indicated that the secreted LOXL2 facilitated the motility of HCC cells and

Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

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Chen, Wan-Chun; Lin, Hsi-Hui; Tang, Ming-Jer

2014-09-15

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis. Copyright © 2014 the American Physiological Society.

Regulation of HGF and SDF-1 expression by oral fibroblasts--implications for invasion of oral cancer.

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Daly, Aisling J; McIlreavey, Leanne; Irwin, Chris R

2008-07-01

Invasion and metastasis of oral squamous cell carcinoma (OSCC) is dependent on signals received from stromal fibroblasts present in the surrounding connective tissue. The aim of this study was to investigate the regulation of expression of two important signaling molecules--HGF and SDF-1--by both stromal fibroblasts and their 'activated' form, myofibroblasts, and to determine the role of these two factors in stimulating OSCC cell invasion in vitro. Fibroblasts and myofibroblasts produced similar levels of HGF and SDF-1. IL-1alpha and OSCC cell conditioned medium both stimulated HGF and SDF-1 expression, while TGF-beta(1) inhibited production of each factor. Myofibroblast-derived conditioned medium stimulated OSCC cell invasion through matrigel. Blocking antibodies to both HGF and SDF-1 reduced the level of invasion. In fibroblast-free organotypic raft cultures, addition of HGF and SDF-1 stimulated OSCC cell invasion into the underlying collagen gel, although the pattern of invasion differed from that induced by fibroblasts. Fibroblast-derived HGF and SDF-1 appear to play central roles in the reciprocal interactions between OSCC cells and underlying stromal fibroblasts leading to the local invasion of oral cancer.

SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

Science.gov (United States)

Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

2018-05-01

SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

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Lenartowicz Malgorzata

2016-08-01

Full Text Available Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD or the rare autosomal disorder Wilson disease (WD, respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation.

Past matrix stiffness primes epithelial cells and regulates their future collective migration through a mechanical memory.

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Nasrollahi, Samila; Walter, Christopher; Loza, Andrew J; Schimizzi, Gregory V; Longmore, Gregory D; Pathak, Amit

2017-11-01

During morphogenesis and cancer metastasis, grouped cells migrate through tissues of dissimilar stiffness. Although the influence of matrix stiffness on cellular mechanosensitivity and motility are well-recognized, it remains unknown whether these matrix-dependent cellular features persist after cells move to a new microenvironment. Here, we interrogate whether priming of epithelial cells by a given matrix stiffness influences their future collective migration on a different matrix - a property we refer to as the 'mechanical memory' of migratory cells. To prime cells on a defined matrix and track their collective migration onto an adjoining secondary matrix of dissimilar stiffness, we develop a modular polyacrylamide substrate through step-by-step polymerization of different PA compositions. We report that epithelial cells primed on a stiff matrix migrate faster, display higher actomyosin expression, form larger focal adhesions, and retain nuclear YAP even after arriving onto a soft secondary matrix, as compared to their control behavior on a homogeneously soft matrix. Priming on a soft ECM causes a reverse effect. The depletion of YAP dramatically reduces this memory-dependent migration. Our results present a previously unidentified regulation of mechanosensitive collective cell migration by past matrix stiffness, in which mechanical memory depends on YAP activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular volume regulation and substrate stiffness modulate the detachment dynamics of adherent cells

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Yang, Yuehua; Jiang, Hongyuan

2018-03-01

Quantitative characterizations of cell detachment are vital for understanding the fundamental mechanisms of cell adhesion. Experiments have found that cell detachment shows strong rate dependence, which is mostly attributed to the binding-unbinding kinetics of receptor-ligand bond. However, our recent study showed that the cellular volume regulation can significantly regulate the dynamics of adherent cell and cell detachment. How this cellular volume regulation contributes to the rate dependence of cell detachment remains elusive. Here, we systematically study the role of cellular volume regulation in the rate dependence of cell detachment by investigating the cell detachments of nonspecific adhesion and specific adhesion. We find that the cellular volume regulation and the bond kinetics dominate the rate dependence of cell detachment at different time scales. We further test the validity of the traditional Johnson-Kendall-Roberts (JKR) contact model and the detachment model developed by Wyart and Gennes et al (W-G model). When the cell volume is changeable, the JKR model is not appropriate for both the detachments of convex cells and concave cells. The W-G model is valid for the detachment of convex cells but is no longer applicable for the detachment of concave cells. Finally, we show that the rupture force of adherent cells is also highly sensitive to substrate stiffness, since an increase in substrate stiffness will lead to more associated bonds. These findings can provide insight into the critical role of cell volume in cell detachment and might have profound implications for other adhesion-related physiological processes.

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

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Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

2016-07-01

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

Aging impairs smooth muscle-mediated regulation of aortic stiffness: a defect in shock absorption function?

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Gao, Yuan Z.; Saphirstein, Robert J.; Yamin, Rina; Suki, Bela

2014-01-01

Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of NG-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90–200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aorta's normal shock absorption function in response to stressors. PMID:25128168

Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

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Paola Brun

Full Text Available Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

Energy Technology Data Exchange (ETDEWEB)

Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Büttner, Robert, E-mail: Robert-Buettner@gmx.net [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany); Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Gleinig, Anna, E-mail: annagleinig@yahoo.com [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Richter, Petra, E-mail: P.Richter@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Chen, Yuan, E-mail: Yuan.Chen@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Franz, Marcus, E-mail: Marcus.Franz@med.uni-jena.de [Clinic of Internal Medicine I, Jena University Hospital, 07740 Jena (Germany); Liebmann, Claus, E-mail: Claus.Liebmann@uni-jena.de [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany)

2014-04-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

International Nuclear Information System (INIS)

Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

2014-01-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM TGF , FCM PDGF ) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM B ). FCM TGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM TGF ≫FCM PDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM TGF >FCM PDGF ) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin as sign of EMT. • Results qualify

Macrophages and fibroblasts : key regulators in wound healing, fibrosis and the foreign body reaction

NARCIS (Netherlands)

Ploeger, Diana

2017-01-01

Macrophages and fibroblasts are key regulators in wound healing, fibrosis and foreign body reaction (FBR). After injury macrophages migrate through the extracellular matrix (ECM) towards the wounded area, and adopt a M1 or M2 phenotype. M1 macrophages are associated with tissue injury and

Fibroblast Growth Factor 21 Mediates Glycemic Regulation by Hepatic JNK

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Santiago Vernia

2016-03-01

Full Text Available The cJun NH2-terminal kinase (JNK-signaling pathway is implicated in metabolic syndrome, including dysregulated blood glucose concentration and insulin resistance. Fibroblast growth factor 21 (FGF21 is a target of the hepatic JNK-signaling pathway and may contribute to the regulation of glycemia. To test the role of FGF21, we established mice with selective ablation of the Fgf21 gene in hepatocytes. FGF21 deficiency in the liver caused marked loss of FGF21 protein circulating in the blood. Moreover, the protective effects of hepatic JNK deficiency to suppress metabolic syndrome in high-fat diet-fed mice were not observed in mice with hepatocyte-specific FGF21 deficiency, including reduced blood glucose concentration and reduced intolerance to glucose and insulin. Furthermore, we show that JNK contributes to the regulation of hepatic FGF21 expression during fasting/feeding cycles. These data demonstrate that the hepatokine FGF21 is a key mediator of JNK-regulated metabolic syndrome.

Regulation of Hepatic Stellate Cells and Fibrogenesis by Fibroblast Growth Factors

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Justin D. Schumacher

2016-01-01

Full Text Available Fibroblast growth factors (FGFs are a family of growth factors critically involved in developmental, physiological, and pathological processes, including embryogenesis, angiogenesis, wound healing, and endocrine functions. In the liver, several FGFs are produced basally by hepatocytes and hepatic stellate cells (HSCs. Upon insult to the liver, expression of FGFs in HSCs is greatly upregulated, stimulating hepatocyte regeneration and growth. Various FGF isoforms have also been shown to directly induce HSC proliferation and activation thereby enabling autocrine and paracrine regulation of HSC function. Regulation of HSCs by the endocrine FGFs, namely, FGF15/19 and FGF21, has also recently been identified. With the ability to modulate HSC proliferation and transdifferentiation, targeting FGF signaling pathways constitutes a promising new therapeutic strategy to treat hepatic fibrosis.

Regulation of matrix stiffness on the epithelial-mesenchymal transition of breast cancer cells under hypoxia environment

Science.gov (United States)

Lv, Yonggang; Chen, Can; Zhao, Boyuan; Zhang, Xiaomei

2017-06-01

Substrate stiffness and hypoxia are associated with tumor development and progression, respectively. However, the synergy of them on the biological behavior of human breast cancer cell is still largely unknown. This study explored how substrate stiffness regulates the cell phenotype, viability, and epithelial-mesenchymal transition (EMT) of human breast cancer cells MCF-7 under hypoxia (1% O2). TRITC-phalloidin staining showed that MCF-7 cells transformed from round to irregular polygon with stiffness increase either in normoxia or hypoxia. While being accompanied with the upward tendency from a 0.5- to a 20-kPa substrate, the percentage of cell apoptosis was significantly higher in hypoxia than that in normoxia, especially on the 20-kPa substrate. Additionally, it was hypoxia, but not normoxia, that promoted the EMT of MCF-7 by upregulating hypoxia-inducible factor-1α (HIF-1α), vimentin, Snail 1, and matrix metalloproteinase 2 (MMP 2) and 9 (MMP 9), and downregulating E-cadherin simultaneously regardless of the change of substrate stiffness. In summary, this study discovered that hypoxia and stiffer substrate (20 kPa) could synergistically induce phenotype change, apoptosis, and EMT of MCF-7 cells. Results of this study have an important significance on further exploring the synergistic effect of stiffness and hypoxia on the EMT of breast cancer cells and its molecular mechanism.

The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

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Paweł P. Antończak

2017-07-01

Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

Regulation and inhibition of collagenase expression by long-wavelength ultraviolet radiation in cultured human skin fibroblasts

International Nuclear Information System (INIS)

Petersen, Marta; Hamilton, Tiffani; Haili Li

1995-01-01

The cellular mechanisms responsible for the connective tissue changes produced by chronic exposure to UV light are poorly understood. collagenase, a metalloproteinase, initiates degradation of types I and III collagen and thus plays a key role in the remodeling of dermal collagen. Collagenase synthesis by fibroblasts and keratinocytes involves the protein kinase C (PKC) second messenger system, and corticosteroids have been shown to suppress its synthesis at the level of gene transcription. Long-wavelength UV light (UVA, 320-400 nm) stimulates the synthesis of interstitial collagenase, as well as increasing PKC activity, in human skin fibroblasts in vitro. This study explores the regulation of collagenase expression by UVA in cultured human skin fibroblasts. Specifically, the time course, the effect of actinomycin D, an inhibitor of RNA synthesis, as well as the effect of PKC inhibitors and dexamethansone on expression of collagenase following UVA irradiation were examined. (Author)

Mesenchymal stem cells induce dermal fibroblast responses to injury

International Nuclear Information System (INIS)

Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

2010-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

Cell motility regulation on a stepped micro pillar array device (SMPAD) with a discrete stiffness gradient.

Science.gov (United States)

Lee, Sujin; Hong, Juhee; Lee, Junghoon

2016-02-28

Our tissues consist of individual cells that respond to the elasticity of their environment, which varies between and within tissues. To better understand mechanically driven cell migration, it is necessary to manipulate the stiffness gradient across a substrate. Here, we have demonstrated a new variant of the microfabricated polymeric pillar array platform that can decouple the stiffness gradient from the ECM protein area. This goal is achieved via a "stepped" micro pillar array device (SMPAD) in which the contact area with the cell was kept constant while the diameter of the pillar bodies was altered to attain the proper mechanical stiffness. Using double-step SU-8 mold fabrication, the diameter of the top of every pillar was kept uniform, whereas that of the bottom was changed, to achieve the desired substrate rigidity. Fibronectin was immobilized on the pillar tops, providing a focal adhesion site for cells. C2C12, HeLa and NIH3T3 cells were cultured on the SMPAD, and the motion of the cells was observed by time-lapse microscopy. Using this simple platform, which produces a purely physical stimulus, we observed that various types of cell behavior are affected by the mechanical stimulus of the environment. We also demonstrated directed cell migration guided by a discrete rigidity gradient by varying stiffness. Interestingly, cell velocity was highest at the highest stiffness. Our approach enables the regulation of the mechanical properties of the polymeric pillar array device and eliminates the effects of the size of the contact area. This technique is a unique tool for studying cellular motion and behavior relative to various stiffness gradients in the environment.

LXA4 actions direct fibroblast function and wound closure

International Nuclear Information System (INIS)

Herrera, Bruno S.; Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice; Leung, Kai P.; Van Dyke, Thomas E.

2015-01-01

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A 4 (LXA 4 ), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA 4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA 4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA 4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA 4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA 4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA 4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA 4 receptor (ALX

Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

International Nuclear Information System (INIS)

Hecht, Emelia; Zago, Michela; Sarill, Miles; Rico de Souza, Angela; Gomez, Alvin; Matthews, Jason; Hamid, Qutayba; Eidelman, David H.; Baglole, Carolyn J.

2014-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR −/− ) and wild-type (AhR +/+ ) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR −/− cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR −/− compared to AhR +/+ cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR +/+ fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR +/+ lung fibroblasts in response to serum, corresponding to a decrease in p27 KIP1 protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27 KIP1 in AhR −/− fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR. - Highlights: • The AhR controls proliferation and apoptosis in lung cells. • The AhR regulates the expression of the microRNA miR-196a independent of

Ferrous Iron Up-regulation in Fibroblasts of Patients with Beta Propeller Protein-Associated Neurodegeneration (BPAN).

Science.gov (United States)

Ingrassia, Rosaria; Memo, Maurizio; Garavaglia, Barbara

2017-01-01

Mutations in WDR45 gene, coding for a beta-propeller protein, have been found in patients affected by Neurodegeneration with Brain Iron Accumulation, NBIA5 (also known as BPAN). BPAN is a movement disorder with Non Transferrin Bound Iron (NTBI) accumulation in the basal ganglia as common hallmark between NBIA classes (Hayflick et al., 2013). WDR45 has been predicted to have a role in autophagy, while the impairment of iron metabolism in the different NBIA subclasses has not currently been clarified. We found the up-regulation of the ferrous iron transporter (-)IRE/Divalent Metal Transporter1 and down-regulation of Transferrin receptor in the fibroblasts of two BPAN affected patients with splicing mutations 235+1G>A (BPAN1) and 517_519ΔVal 173 (BPAN2). The BPAN patients showed a concomitant increase of intracellular ferrous iron after starvation. An altered pattern of iron transporters with iron overload is highlighted in BPAN human fibroblasts, supporting for a role of DMT1 in NBIA. We here present a novel element, about iron accumulation, to the existing knowledge in field of NBIA. Attention is focused to a starvation-dependent iron overload, possibly accounting for iron accumulation in the basal ganglia. Further investigation could clarify iron regulation in BPAN.

Possible role of ginsenoside Rb1 in skin wound healing via regulating senescent skin dermal fibroblast.

Science.gov (United States)

Hou, Jingang; Kim, Sunchang

2018-05-05

Cellular senescence suppresses cancer by inducing irreversible cell growth arrest. Nevertheless, senescent cells is proposed as causal link with aging and aging-related pathologies. The physiological beneficial functions of senescent cells are still of paucity. Here we show that senescent human dermal fibroblast accelerates keratinocytes scratch wound healing and stimulates differentiation of fibroblast. Using oxidative stress (100 μM H 2 O 2 exposure for 1 h) induction, we successfully triggered fibroblast senescence and developed senescence associated secretory phenotype (SASP). The induction of SASP was regulated by p38MAPK/MSK2/NF-κB pathway. Interestingly, inhibition of p38MAPK activation only partially suppressed SASP. However, SASP was significantly inhibited by SB747651A, a specific MSK inhibitor. Additionally, we demonstrate that SASP stimulates migration of keratinocytes and myofibroblast transition of fibroblast, through fold-increased secretion of growth factors, platelet-derived growth factor AA (PDGF-AA) and AB (PDGF-AB), transforming growth factor beta 1 (TGF-β1) and beta 2 (TGF-β2), vascular endothelial growth factor A (VEGF-A) and D (VEGF-D), vascular endothelial growth factor receptor 2 (VEGFR2) and 3 (VEGFR3). Importantly, we also confirmed ginsenoside Rb1 promoted SASP-mediated healing process via p38MAPK/MSK2/NF-κB pathway. The results pointed to senescent fibroblast as a potential mechanism of wound healing control in human skin. Further, it provided a candidate targeted for wound therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

Cancer-Associated Fibroblasts Regulate Tumor-Initiating Cell Plasticity in Hepatocellular Carcinoma through c-Met/FRA1/HEY1 Signaling

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Eunice Yuen Ting Lau

2016-05-01

Full Text Available Like normal stem cells, tumor-initiating cells (T-ICs are regulated extrinsically within the tumor microenvironment. Because HCC develops primarily in the context of cirrhosis, in which there is an enrichment of activated fibroblasts, we hypothesized that cancer-associated fibroblasts (CAFs would regulate liver T-ICs. We found that the presence of α-SMA(+ CAFs correlates with poor clinical outcome. CAF-derived HGF regulates liver T-ICs via activation of FRA1 in an Erk1,2-dependent manner. Further functional analysis identifies HEY1 as a direct downstream effector of FRA1. Using the STAM NASH-HCC mouse model, we find that HGF-induced FRA1 activation is associated with the fibrosis-dependent development of HCC. Thus, targeting the CAF-derived, HGF-mediated c-Met/FRA1/HEY1 cascade may be a therapeutic strategy for the treatment of HCC.

Tumor-associated fibrosis as a regulator of tumor immunity and response to immunotherapy.

Science.gov (United States)

Jiang, Hong; Hegde, Samarth; DeNardo, David G

2017-08-01

Tumor-associated fibrosis is characterized by unchecked pro-fibrotic and pro-inflammatory signaling. The components of fibrosis including significant numbers of cancer-associated fibroblasts, dense collagen deposition, and extracellular matrix stiffness, are well appreciated regulators of tumor progression but may also be critical regulators of immune surveillance. While this suggests that the efficacy of immunotherapy may be limited in highly fibrotic cancers like pancreas, it also suggests a therapeutic opportunity to target fibrosis in these tumor types to reawaken anti-tumor immunity. This review discusses the mechanisms by which fibrosis might subvert tumor immunity and how to overcome these mechanisms.

LXA{sub 4} actions direct fibroblast function and wound closure

Energy Technology Data Exchange (ETDEWEB)

Herrera, Bruno S. [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Leung, Kai P., E-mail: kai.p.leung.civ@mail.mil [Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Van Dyke, Thomas E., E-mail: tvandyke@forsyth.org [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States)

2015-09-04

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A{sub 4} (LXA{sub 4}), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA{sub 4} on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA{sub 4} receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA{sub 4} receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA{sub 4} slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA{sub 4} tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA{sub 4} in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF

Fibroblast Growth Factor Signaling in Metabolic Regulation.

Science.gov (United States)

Nies, Vera J M; Sancar, Gencer; Liu, Weilin; van Zutphen, Tim; Struik, Dicky; Yu, Ruth T; Atkins, Annette R; Evans, Ronald M; Jonker, Johan W; Downes, Michael Robert

2015-01-01

The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions.

Fibroblast growth factor signaling in metabolic regulation

Directory of Open Access Journals (Sweden)

Vera eNies

2016-01-01

Full Text Available The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases, and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed.In this review we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease, and to provide starting points for the development of FGF-based therapies against metabolic conditions.

Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

Energy Technology Data Exchange (ETDEWEB)

Hecht, Emelia [Department of Medicine, McGill University, Montreal, Quebec (Canada); Zago, Michela [Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Sarill, Miles [Department of Medicine, McGill University, Montreal, Quebec (Canada); Rico de Souza, Angela [Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Gomez, Alvin; Matthews, Jason [Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON (Canada); Hamid, Qutayba; Eidelman, David H. [Department of Medicine, McGill University, Montreal, Quebec (Canada); Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Baglole, Carolyn J., E-mail: Carolyn.baglole@McGill.ca [Department of Medicine, McGill University, Montreal, Quebec (Canada); Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

2014-11-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR{sup −/−}) and wild-type (AhR{sup +/+}) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR{sup −/−} cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR{sup −/−} compared to AhR{sup +/+} cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR{sup +/+} fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR{sup +/+} lung fibroblasts in response to serum, corresponding to a decrease in p27{sup KIP1} protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27{sup KIP1} in AhR{sup −/−} fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR. - Highlights: • The AhR controls proliferation and apoptosis in lung cells. • The AhR regulates the

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

Directory of Open Access Journals (Sweden)

Hieu T Nim

Full Text Available The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP, an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1 relevant to cardiac literature, and (2 differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10 are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

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Nim, Hieu T; Furtado, Milena B; Costa, Mauro W; Kitano, Hiroaki; Rosenthal, Nadia A; Boyd, Sarah E

2015-01-01

The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

An atomic-force-microscopy study of the structure of surface layers of intact fibroblasts

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Khalisov, M. M.; Ankudinov, A. V.; Penniyaynen, V. A.; Nyapshaev, I. A.; Kipenko, A. V.; Timoshchuk, K. I.; Podzorova, S. A.; Krylov, B. V.

2017-02-01

Intact embryonic fibroblasts on a collagen-treated substrate have been studied by atomic-force microscopy (AFM) using probes of two types: (i) standard probes with tip curvature radii of 2-10 nm and (ii) special probes with a calibrated 325-nm SiO2 ball radius at the tip apex. It is established that, irrespective of probe type, the average maximum fibroblast height is on a level of 1.7 μm and the average stiffness of the probe-cell contact amounts to 16.5 mN/m. The obtained AFM data reveal a peculiarity of the fibroblast structure, whereby its external layers move as a rigid shell relative to the interior and can be pressed inside to a depth dependent on the load only.

N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

LENUS (Irish Health Repository)

Burke, John P

2012-02-01

BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

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Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

2013-01-01

We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

C/EBPα Expression is Partially Regulated by C/EBPβ in Response to DNA Damage and C/EBPα Deficient Fibroblasts Display an Impaired G1 Checkpoint

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Ranjan, Rakesh; Thompson, Elizabeth A.; Yoon, Kyungsil; Smart, Robert C.

2009-01-01

We observed that C/EBPα is highly inducible in primary fibroblasts by DNA damaging agents that induce strand breaks, alkylate and crosslink DNA as well as those that produce bulky DNA lesions. Fibroblasts deficient in C/EBPα (C/EBPα-/-) display an impaired G1 checkpoint as evidenced by inappropriate entry into S-phase in response to DNA damage and these cells also display an enhanced G1 to S transition in response to mitogens. The induction of C/EBPα by DNA damage in fibroblasts does not require p53. EMSA analysis of nuclear extracts prepared from UVB- and MNNG-treated fibroblasts revealed increased binding of C/EBPβ to a C/EBP consensus sequence and ChIP analysis revealed increased C/EBPβ binding to the C/EBPα promoter. To determine whether C/EBPβ has a role in the regulation of C/EBPα we treated C/EBPβ-/- fibroblasts with UVB or MNNG. We observed C/EBPα induction was impaired in both UVB- and MNNG- treated C/EBPβ-/- fibroblasts. Our study reveals a novel role for C/EBPβ in the regulation of C/EBPα in response to DNA damage and provides definitive genetic evidence that C/EBPα has a critical role in the DNA damage G1 checkpoint. PMID:19581927

Regulation by basic fibroblast growth factor of glycosaminoglycan biosynthesis in cultured vascular endothelial cells.

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Kaji, T; Hiraga, S; Ohkawara, S; Inada, M; Yamamoto, C; Kozuka, H; Koizumi, F

1995-05-01

The alteration of glycosaminoglycans (GAGs) in cultured bovine aortic endothelial cells after exposure to basic fibroblast growth factor (bFGF) was investigated. It was found that the incorporation of [3H]glucosamine into GAGs was markedly increased by bFGF in both the cell layer and the conditioned medium; however, that of [35S]sulfate was not changed by the growth factor. These results indicated that bFGF enhanced the sugar-chain formation but did not affect their sulfation in endothelial GAG production. Similar changes were observed in either bovine aortic smooth-muscle cells and human fibroblastic IMR-90 cells to greater and lesser degrees, respectively. Characterization of GAGs in the endothelial cell layer and the conditioned medium revealed that bFGF enhanced both heparan sulfate and the other GAGs to a similar degree. The present data suggest that bFGF may be involved in the regulation of the blood coagulation system via altering GAGs of the vascular tissue when the endothelium was damaged.

Signal Transduction Pathways (MAPKs, NF-κB, and C/EBP) Regulating COX-2 Expression in Nasal Fibroblasts from Asthma Patients with Aspirin Intolerance

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Garcia-Garcia, Francesc Josep; Mullol, Joaquim; Perez-Gonzalez, Maria; Pujols, Laura; Alobid, Isam

2012-01-01

Background Recent studies have revealed that cyclooxygenase-2 (COX-2) expression is down-regulated in aspirin-induced asthma (AIA). Various signal pathways (MAPKs, NF-κB and C/EBP) are involved in COX-2 regulation. Objective To investigate the regulation of COX-2 expression through MAP-kinase pathway activation and nuclear factor translocation in aspirin-induced asthma (AIA). Methods Fibroblasts were isolated from specimens of nasal mucosa (NM, N = 5) and nasal polyps (NP, N = 5). After IL-1β (1 ng/ml) incubation, COX-2 and phosphorylated forms of ERK, JNK and p38 MAPK were measured by Western blot. MAPK’s role in IL-1β-induced COX-2 expression was assessed by treating cells with ERK (PD98059), JNK (SP600125) and p38 MAPK (SB203580) inhibitors (0.1–10 µM) prior to IL-1β exposure. NF-κB and C/EBP nuclear translocation was measured by Western blot and TransAM® after IL-1β (10 ng/ml) exposure. Results No differences were observed in the MAPK phosphorylation time-course between NM and NP-AIA fibroblasts. The p38 MAPK inhibitor at 10 µM significantly reduced IL-1β-induced COX-2 expression in NM fibroblasts (85%). In NP-AIA fibroblasts the COX-2 inhibition (65%) at 1 and 10 µM was not statistically significant compared to non-treated cells. ERK and JNK inhibitors had no significant effect in either the NM or NP-AIA cultures. The effect of IL-1β on NF-κB and C/EBP subunits’ nuclear translocation was similar between NM and NP-AIA fibroblasts. Conclusions These results suggest that p38 MAPK is the only MAPK involved in IL-1β-induced COX-2 expression. NM and NP-AIA fibroblasts have similar MAPK phosphorylation dynamics and nuclear factor translocation (NF-κB and C/EBP). COX-2 downregulation observed in AIA patients appears not to be caused by differences in MAPK dynamics or transcription factor translocation. PMID:23240010

Transcriptome Analysis of Ullrich Congenital Muscular Dystrophy Fibroblasts Reveals a Disease Extracellular Matrix Signature and Key Molecular Regulators.

Directory of Open Access Journals (Sweden)

Sonia Paco

Full Text Available Collagen VI related myopathies encompass a range of phenotypes with involvement of skeletal muscle, skin and other connective tissues. They represent a severe and relatively common form of congenital disease for which there is no treatment. Collagen VI in skeletal muscle and skin is produced by fibroblasts.In order to gain insight into the consequences of collagen VI mutations and identify key disease pathways we performed global gene expression analysis of dermal fibroblasts from patients with Ullrich Congenital Muscular Dystrophy with and without vitamin C treatment. The expression data were integrated using a range of systems biology tools. Results were validated by real-time PCR, western blotting and functional assays.We found significant changes in the expression levels of almost 600 genes between collagen VI deficient and control fibroblasts. Highly regulated genes included extracellular matrix components and surface receptors, including integrins, indicating a shift in the interaction between the cell and its environment. This was accompanied by a significant increase in fibroblasts adhesion to laminin. The observed changes in gene expression profiling may be under the control of two miRNAs, miR-30c and miR-181a, which we found elevated in tissue and serum from patients and which could represent novel biomarkers for muscular dystrophy. Finally, the response to vitamin C of collagen VI mutated fibroblasts significantly differed from healthy fibroblasts. Vitamin C treatment was able to revert the expression of some key genes to levels found in control cells raising the possibility of a beneficial effect of vitamin C as a modulator of some of the pathological aspects of collagen VI related diseases.

Key role of the kidney in the regulation of fibroblast growth factor 23

DEFF Research Database (Denmark)

Mace, Maria L; Gravesen, Eva; Hofman-Bang, Jacob

2015-01-01

was significantly increased in BNX rats. The rapid rise in FGF23 after BNX was independent of parathyroid hormone or FGF receptor signaling. No evidence of early stimulation of FGF23 gene expression in the bone was found. Furthermore, acute severe hyperphosphatemia or hypercalcemia had no impact on intact FGF23......High circulating levels of fibroblast growth factor 23 (FGF23) have been demonstrated in kidney failure, but mechanisms of this are not well understood. Here we examined the impact of the kidney on the early regulation of intact FGF23 in acute uremia as induced by bilateral or unilateral...

Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

International Nuclear Information System (INIS)

Story, M.T.

1989-01-01

To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue

TRX is up-regulated by fibroblast growth factor-2 in lung carcinoma.

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Deng, Zheng-Hao; Cao, Hui-Qiu; Hu, Yong-Bin; Wen, Ji-Fang; Zhou, Jian-Hua

2011-01-01

We have previously shown that exogenous fibroblast growth factor-2 (FGF-2) inhibits apoptosis of the small-cell lung cancer (SCLC) cell line NCI-H446, but the underlying mechanism remains unknown. In this study, the protein profiles of FGF-2-treated and untreated NCI-H446 cells were determined by 2-D gel electrophoresis combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics. Differential expression analysis of the protein profiles after FGF-2 treatment identified a total of 24 protein spots, of which nine were up-regulated and 15 were down-regulated. Four proteins were identified by MALDI-TOF-MS: thioredoxin (TRX), visfatin, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) and Cu/Zn superoxide dismutase (CuZn-SOD). Western blotting revealed that TRX was up-regulated in NCI-H446 and A549 cells treated with FGF-2. Furthermore, immunohistochemical staining confirmed that both FGF-2 and TRX were overexpressed in lung cancer tissues and could be correlated with both lymph node metastasis and clinical stage. These data indicate that TRX may be involved in the FGF-2 signaling pathway. © 2010 The Authors. APMIS © 2010 APMIS.

Substrate stiffness promotes latent TGF-β1 activation in hepatocellular carcinoma

International Nuclear Information System (INIS)

Pang, Mingshu; Teng, Yao; Huang, Jianyong; Yuan, Yuan; Lin, Feng; Xiong, Chunyang

2017-01-01

Hepatocellular carcinoma (HCC) was usually coupled with increased stiffness of the extracellular matrix (ECM) and elevated level of transforming growth factor-β1 (TGF-β1). However, the mechanism by which substrate rigidity modulated TGF-β1 signaling transduction remained unknown. This paper investigated the molecular mechanism of how matrix stiffness regulating TGF-β1 signaling in HCC cells. By means of stiffness tunable collagen I-coated polyacrylamide (PA) gels, we found that the expressions of β1 integrin, p-FAK Y397 and p-Smad2 upregulated on stiffer gels as well as the content of TGF-β1 in culture media of HCC cells, which were inhibited by RGD blocking peptides, Y-27632 (ROCK inhibitor) or Blebbistatin (myosin II inhibitor). Cellular traction force was also significantly higher when plated on stiffer substrates but dramatically decreased after treatment with Y-27632 or Blebbistatin. Furthermore, the upregulation of p-Smad2 in the HCC cells on stiffer PA gels induced by exogenetic latent TGF-β1 was downregulated in the presence of RGD peptides. The nuclear translocation of Smad2 induced by latent TGF-β1 was inhibited by Y-27632 or Blebbistatin. Our results suggested that the extracellular matrix stiffness regulated latent TGF-β1 activation by cytoskeletal tension in HCC cells, showing that matrix stiffness was a key regulator involving the TGF-β1 activity in HCC cells. The current study presented a mechanism of how hepatocirrhosis developed into liver cancer. - Highlights: • TGF-β1 signaling pathway regulated by ECM stiffness was studied in hepatocellular carcinoma. • Matrix stiffness promoted latent TGF-β1 activation via β1 integrin-FAK-Rho GTPase pathway. • A mechanism of how hepatocirrhosis developed into liver cancer was presented.

Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

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Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

2014-10-14

Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

Identification of specific gene expression profiles in fibroblasts derived from middle ear cholesteatoma.

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Yoshikawa, Mamoru; Kojima, Hiromi; Wada, Kota; Tsukidate, Toshiharu; Okada, Naoko; Saito, Hirohisa; Moriyama, Hiroshi

2006-07-01

To investigate the role of fibroblasts in the pathogenesis of cholesteatoma. Tissue specimens were obtained from our patients. Middle ear cholesteatoma-derived fibroblasts (MECFs) and postauricular skin-derived fibroblasts (SFs) as controls were then cultured for a few weeks. These fibroblasts were stimulated with interleukin (IL) 1alpha and/or IL-1beta before gene expression assays. We used the human genome U133A probe array (GeneChip) and real-time polymerase chain reaction to examine and compare the gene expression profiles of the MECFs and SFs. Six patients who had undergone tympanoplasty. The IL-1alpha-regulated genes were classified into 4 distinct clusters on the basis of profiles differentially regulated by SF and MECF using a hierarchical clustering analysis. The messenger RNA expressions of LARC (liver and activation-regulated chemokine), GMCSF (granulocyte-macrophage colony-stimulating factor), epiregulin, ICAM1 (intercellular adhesion molecule 1), and TGFA (transforming growth factor alpha) were more strongly up-regulated by IL-1alpha and/or IL-1beta in MECF than in SF, suggesting that these fibroblasts derived from different tissues retained their typical gene expression profiles. Fibroblasts may play a role in hyperkeratosis of middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth. These fibroblasts may retain tissue-specific characteristics presumably controlled by epigenetic mechanisms.

Relationship between Static Stiffness and Modal Stiffness of Structures

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Tianjian Ji Tianjian Ji

2010-02-01

Full Text Available This paper derives the relationship between the static stiffness and modal stiffness of a structure. The static stiffness and modal stiffness are two important concepts in both structural statics and dynamics. Although both stiffnesses indicate the capacity of the structure to resist deformation, they are obtained using different methods. The former is calculated by solving the equations of equilibrium and the latter can be obtained by solving an eigenvalue problem. A mathematical relationship between the two stiffnesses was derived based on the definitions of two stiffnesses. This relationship was applicable to a linear system and the derivation of relationships does not reveal any other limitations. Verification of the relationship was given by using several examples. The relationship between the two stiffnesses demonstrated that the modal stiffness of the fundamental mode was always larger than the static stiffness of a structure if the critical point and the maximum mode value are at the same node, i.e. for simply supported beam and seven storeys building are 1.5% and 15% respectively. The relationship could be applied into real structures, where the greater the number of modes being considered, the smaller the difference between the modal stiffness and the static stiffness of a structure.

Molecular Cues Guiding Matrix Stiffness in Liver Fibrosis

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Takaoki Saneyasu

2016-01-01

Full Text Available Tissue and matrix stiffness affect cell properties during morphogenesis, cell growth, differentiation, and migration and are altered in the tissue remodeling following injury and the pathological progression. However, detailed molecular mechanisms underlying alterations of stiffness in vivo are still poorly understood. Recent engineering technologies have developed powerful techniques to characterize the mechanical properties of cell and matrix at nanoscale levels. Extracellular matrix (ECM influences mechanical tension and activation of pathogenic signaling during the development of chronic fibrotic diseases. In this short review, we will focus on the present knowledge of the mechanisms of how ECM stiffness is regulated during the development of liver fibrosis and the molecules involved in ECM stiffness as a potential therapeutic target for liver fibrosis.

Cell proliferation in vitro modulates fibroblast collagenase activity

International Nuclear Information System (INIS)

Lindblad, W.J.; Flood, L.

1986-01-01

Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation.

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Wang, Yuanyuan; Chen, Chen; Deng, Ziyu; Bian, Erbao; Huang, Cheng; Lei, Ting; Lv, Xiongwen; Liu, Liping; Li, Jun

2017-03-01

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately. Copyright © 2016. Published by Elsevier B.V.

Fibroblast growth factor 23

African Journals Online (AJOL)

Dr Olaleye

Systemic phosphate homeostasis is maintained through several hormonal mechanisms which involve fibroblast growth factor 23 (FGF-23), α-klotho, vitamin D and parathyroid hormone. FGF-23 is known to be the major regulator of phosphate balance (Mirams et al., 2004). FGF-23 is a phosphaturic hormone, which is.

Association of Gastrocnemius Muscle Stiffness With Passive Ankle Joint Stiffness and Sex-Related Difference in the Joint Stiffness.

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Chino, Kintaro; Takashi, Hideyuki

2017-11-15

Passive ankle joint stiffness is affected by all structures located within and over the joint, and is greater in men than in women. Localized muscle stiffness can be assessed by ultrasound shear wave elastography, and muscle architecture such as fascicle length and pennation angle can be measured by B-mode ultrasonography. Thus, we assessed localized muscle stiffness of the medial gastrocnemius (MG) with consideration of individual variability in the muscle architecture, and examined the association of the muscle stiffness with passive ankle joint stiffness and the sex-related difference in the joint stiffness. Localized muscle stiffness of the MG in 16 men and 17 women was assessed at 10° and 20° plantar flexion, neutral anatomical position, 10° and 20° dorsiflexion. Fascicle length and pennation angle of the MG were measured at these joint positions. Passive ankle joint stiffness was determined by the ankle joint angle-torque relationship. Localized MG muscle stiffness was not significantly correlated with passive ankle joint stiffness, and did not show significant sex-related difference, even when considering the muscle architecture. This finding suggest that muscle stiffness of the MG would not be a prominent factor to determine passive ankle joint stiffness and the sex-related difference in the joint stiffness.

Modeling and simulating the neuromuscular mechanisms regulating ankle and knee joint stiffness during human locomotion.

Science.gov (United States)

Sartori, Massimo; Maculan, Marco; Pizzolato, Claudio; Reggiani, Monica; Farina, Dario

2015-10-01

This work presents an electrophysiologically and dynamically consistent musculoskeletal model to predict stiffness in the human ankle and knee joints as derived from the joints constituent biological tissues (i.e., the spanning musculotendon units). The modeling method we propose uses electromyography (EMG) recordings from 13 muscle groups to drive forward dynamic simulations of the human leg in five healthy subjects during overground walking and running. The EMG-driven musculoskeletal model estimates musculotendon and resulting joint stiffness that is consistent with experimental EMG data as well as with the experimental joint moments. This provides a framework that allows for the first time observing 1) the elastic interplay between the knee and ankle joints, 2) the individual muscle contribution to joint stiffness, and 3) the underlying co-contraction strategies. It provides a theoretical description of how stiffness modulates as a function of muscle activation, fiber contraction, and interacting tendon dynamics. Furthermore, it describes how this differs from currently available stiffness definitions, including quasi-stiffness and short-range stiffness. This work offers a theoretical and computational basis for describing and investigating the neuromuscular mechanisms underlying human locomotion. Copyright © 2015 the American Physiological Society.

The effect of keratinocytes on the biomechanical characteristics and pore microstructure of tissue engineered skin using deep dermal fibroblasts.

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Varkey, Mathew; Ding, Jie; Tredget, Edward E

2014-12-01

Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts: a possible defense against transforming growth factor-ß mediated fibrosis

LENUS (Irish Health Repository)

Watson, Chris J

2012-07-07

AbstractBackgroundMechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts’ response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.Methods and resultsThe effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.ConclusionWe postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

Effects of Mechanical Coupling Between Cardiomyocytes and Cardiac Fibroblasts on Myocardium

Science.gov (United States)

Zorlutuna, Pinar; Nguyen, Trung Dung; Nagarajan, Neerajha

Cardiomyocytes show excitatory responses to stimulation solely by mechanical forces through their stretch-activated ion channels, and can fire action potentials upon mechanical stimulation through a pathway known as mechano-electric feedback. Furthermore, cardiomyocyte (CM) - cardiac fibroblasts (CF) can couple mechanically through cell-cell junctions. Here we investigated the effects of CM and CF mechanical coupling on myocardial physiology and pathology using a bio-nanoindentered coupled with fast calcium imaging and microelectrode arrays. In order to study mechanical signal transmission, we measured the contractile forces generated by CMs, as well as by CFs that were coupled to the CMs. We observed that CFs were beating with the same frequency but at smaller magnitude compared to CMs, and their contractility was dependent on the substrate stiffness. Our results showed that beating CMs actively stretched neighbouring CFs through the deformation of the substrate the cells were seeded on, which promoted the myocardial contractility through mechanical coupling. The results also revealed that CM contractility was propagated greater on soft substrates than stiff ones. Results of this study could help identify the role of the infarcted tissue stiffness and size on heart failure. This study is supported by NSF Grant No: 1530884.

Thyroid hormone increases fibroblast growth factor receptor expression and disrupts cell mechanics in the developing organ of corti

Science.gov (United States)

2013-01-01

Background Thyroid hormones regulate growth and development. However, the molecular mechanisms by which thyroid hormone regulates cell structural development are not fully understood. The mammalian cochlea is an intriguing system to examine these mechanisms, as cellular structure plays a key role in tissue development, and thyroid hormone is required for the maturation of the cochlea in the first postnatal week. Results In hypothyroid conditions, we found disruptions in sensory outer hair cell morphology and fewer microtubules in non-sensory supporting pillar cells. To test the functional consequences of these cytoskeletal defects on cell mechanics, we combined atomic force microscopy with live cell imaging. Hypothyroidism stiffened outer hair cells and supporting pillar cells, but pillar cells ultimately showed reduced cell stiffness, in part from a lack of microtubules. Analyses of changes in transcription and protein phosphorylation suggest that hypothyroidism prolonged expression of fibroblast growth factor receptors, and decreased phosphorylated Cofilin. Conclusions These findings demonstrate that thyroid hormones may be involved in coordinating the processes that regulate cytoskeletal dynamics and suggest that manipulating thyroid hormone sensitivity might provide insight into the relationship between cytoskeletal formation and developing cell mechanical properties. PMID:23394545

MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

Science.gov (United States)

Wang, Daren; Zhang, Hui; Li, Min; Frid, Maria G; Flockton, Amanda R; McKeon, B Alexandre; Yeager, Michael E; Fini, Mehdi A; Morrell, Nicholas W; Pullamsetti, Soni S; Velegala, Sivareddy; Seeger, Werner; McKinsey, Timothy A; Sucharov, Carmen C; Stenmark, Kurt R

2014-01-03

Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti-miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract-binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3' untranslated region of polypyrimidine tract-binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly

Fibroblast growth factor regulates insulin-like growth factor-binding protein production by vascular smooth muscle cells.

Science.gov (United States)

Ververis, J; Ku, L; Delafontaine, P

1994-02-01

Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

International Nuclear Information System (INIS)

Bruzzone, Santina; Battaglia, Florinda; Mannino, Elena; Parodi, Alessia; Fruscione, Floriana; Basile, Giovanna; Salis, Annalisa; Sturla, Laura; Negrini, Simone; Kalli, Francesca; Stringara, Silvia; Filaci, Gilberto

2012-01-01

Highlights: ► ABA is an endogenous hormone in humans, regulating different cell responses. ► ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. ► UV-B irradiation increases ABA content in SSc cultures. ► SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

Effects of dynamic matrix remodelling on en masse migration of fibroblasts on collagen matrices.

Science.gov (United States)

Ozcelikkale, Altug; Dutton, J Craig; Grinnell, Frederick; Han, Bumsoo

2017-10-01

Fibroblast migration plays a key role during various physiological and pathological processes. Although migration of individual fibroblasts has been well studied, migration in vivo often involves simultaneous locomotion of fibroblasts sited in close proximity, so-called ' en masse migration', during which intensive cell-cell interactions occur. This study aims to understand the effects of matrix mechanical environments on the cell-matrix and cell-cell interactions during en masse migration of fibroblasts on collagen matrices. Specifically, we hypothesized that a group of migrating cells can significantly deform the matrix, whose mechanical microenvironment dramatically changes compared with the undeformed state, and the alteration of the matrix microenvironment reciprocally affects cell migration. This hypothesis was tested by time-resolved measurements of cell and extracellular matrix movement during en masse migration on collagen hydrogels with varying concentrations. The results illustrated that a group of cells generates significant spatio-temporal deformation of the matrix before and during the migration. Cells on soft collagen hydrogels migrate along tortuous paths, but, as the matrix stiffness increases, cell migration patterns become aligned with each other and show coordinated migration paths. As cells migrate, the matrix is locally compressed, resulting in a locally stiffened and dense matrix across the collagen concentration range studied. © 2017 The Author(s).

Increased blood pressure and aortic stiffness among abusers of anabolic androgenic steroids

DEFF Research Database (Denmark)

Rasmussen, Jon J; Schou, Morten; Madsen, Per L

2018-01-01

BACKGROUND: Abuse of anabolic androgenic steroids (AAS) is prevalent among recreational athletes and adverse effects on blood pressure (BP) and arterial stiffness could be substantial. Testosterone decreases natriuretic peptides which are key components in BP-regulation and may impair BP-homeosta......BACKGROUND: Abuse of anabolic androgenic steroids (AAS) is prevalent among recreational athletes and adverse effects on blood pressure (BP) and arterial stiffness could be substantial. Testosterone decreases natriuretic peptides which are key components in BP-regulation and may impair BP...

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

Energy Technology Data Exchange (ETDEWEB)

Bruzzone, Santina, E-mail: santina.bruzzone@unige.it [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Battaglia, Florinda [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Mannino, Elena [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Parodi, Alessia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Fruscione, Floriana [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Basile, Giovanna [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Salis, Annalisa; Sturla, Laura [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Negrini, Simone; Kalli, Francesca; Stringara, Silvia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Filaci, Gilberto [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Department of Internal Medicine, Viale Benedetto XV 6, 16132 Genova (Italy); and others

2012-05-25

Highlights: Black-Right-Pointing-Pointer ABA is an endogenous hormone in humans, regulating different cell responses. Black-Right-Pointing-Pointer ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. Black-Right-Pointing-Pointer UV-B irradiation increases ABA content in SSc cultures. Black-Right-Pointing-Pointer SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-{beta} (TGF-{beta}). Conversely, migration toward ABA, but not toward TGF-{beta}, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

Regulation of matrix metalloproteinase-9 expression between gingival fibroblast cells from old and young rats

International Nuclear Information System (INIS)

Kim, Su-Jung; Chung, Yong-Koo; Chung, Tae-Wook; Kim, Jeong-Ran; Moon, Sung-Kwon; Kim, Cheorl-Ho; Park, Young-Guk

2009-01-01

Gingival fibroblast cells (rGF) from aged rats have an age-related decline in proliferative capacity compared with young rats. We investigated G1 phase cell cycle regulation and MMP-9 expression in both young and aged rGF. G1 cell cycle protein levels and activity were significantly reduced in response to interleukin-1β (IL-1β) stimulation with increasing in vitro age. Tumor necrosis factor-α (TNF-α)-induced matrix metalloproteinase-9 (MMP-9) expression was also decreased in aged rGF in comparison with young rGF. Mutational analysis and gel shift assays demonstrated that the lower MMP-9 expression in aged rGF is associated with lower activities of transcription factors NF-κB and AP-1. These results suggest that cell cycle dysregulation and down-regulation of MMP-9 expression in rGF may play a role in gingival remodeling during in vitro aging.

Differential Gene Expression of Fibroblasts: Keloid versus Normal

Directory of Open Access Journals (Sweden)

Michael F. Angel

2002-11-01

Full Text Available Abstract: This study investigated gene regulation and unique gene products in both keloid (KDF and normal (NDF dermal fibroblasts in established cell lines. For gene regulation, NDF versus KDF were compared using Clontech's Atlas™ Human cDNA Expression Array while unique gene products were studied using RNA Fingerprinting Kit. RNA from each sample was converted to cDNA using oligo-dT primers. Down-regulated genes using Atlas Array in KDF were 1 60 S ribosomal protein, 2 Thioredoxin dependent peroxidase, 3 Nuclease sensitive element DNA binding protein, 4 c-myc purine-binding transcription factor, 5 c-AMP dependent protein kinase, and, 6 Heat Shock Protein 90 kDa. Genes that are up regulated in KDF were 1 Tubulin and 2 Heat Shock Protein 27 kDa. With the differential display, we found 17 bands unique to both KDF and NDF. The specific gene and the manner in which they were differentially regulated have direct implications to understanding keloid fibroblast proliferation.

Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts.

LENUS (Irish Health Repository)

Burke, J P

2012-02-01

BACKGROUND: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. METHODS: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and\\/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. RESULTS: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. CONCLUSION: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

Coupling between the Output Force and Stiffness in Different Variable Stiffness Actuators

Directory of Open Access Journals (Sweden)

Amir Jafari

2014-08-01

Full Text Available The fundamental objective in developing variable stiffness actuators is to enable the actuator to deliberately tune its stiffness. This is done through controlling the energy flow extracted from internal power units, i.e., the motors of a variable stiffness actuator (VSA. However, the stiffness may also be unintentionally affected by the external environment, over which, there is no control. This paper analysis the correlation between the external loads, applied to different variable stiffness actuators, and their resultant output stiffness. Different types of variable stiffness actuators have been studied considering springs with different types of nonlinearity. The results provide some insights into how to design the actuator mechanism and nonlinearity of the springs in order to increase the decoupling between the load and stiffness in these actuators. This would significantly widen the application range of a variable stiffness actuator.

RNase-L regulates the stability of mitochondrial DNA-encoded mRNAs in mouse embryo fibroblasts

International Nuclear Information System (INIS)

Chandrasekaran, Krish; Mehrabian, Zara; Li Xiaoling; Hassel, Bret

2004-01-01

Accelerated decrease in the levels of mitochondrial DNA-encoded mRNA (mt-mRNA) occurs in neuronal cells exposed either to the excitatory amino acid, glutamate or to the sodium ionophore, monensin, suggesting a role of mitochondrial RNase(s) on the stability of mt-mRNAs. Here we report that in mouse embryo fibroblasts that are devoid of the interferon-regulated RNase, RNase-L, the monensin-induced decrease in the half-life of mt-mRNA was reduced. In monensin (250 nM)-treated RNase-L +/+ cells the average half-life of mt-mRNA, determined after termination of transcription with actinomycin D, was found to be 3 h, whereas in monensin-treated RNase-L -/- cells the half-life of mt-mRNA was >6 h. In contrast, the stability of nuclear DNA-encoded β-actin mRNA was unaffected. Induction of RNase-L expression in mouse 3T3 fibroblasts further decreased the monensin-induced reduction in mt-mRNA half-life to 1.5 h. The results indicate that the RNase-L-dependent decrease in mtDNA-encoded mRNA transcript levels occurs through a decrease in the half-life of mt-mRNA, and that RNase-L may play a role in the stability of mt-mRNA

Triptonide inhibits the pathological functions of gastric cancer-associated fibroblasts.

Science.gov (United States)

Wang, Zhenfei; Ma, Daguang; Wang, Changshan; Zhu, Zhe; Yang, Yongyan; Zeng, Fenfang; Yuan, Jianlong; Liu, Xia; Gao, Yue; Chen, Yongxia; Jia, Yongfeng

2017-12-01

Direct attacks on tumour cells with chemotherapeutic drugs have the drawbacks of accelerating tumour metastasis and inducing tumour stem cell phenotypes. Inhibition of tumour-associated fibroblasts, which provide nourishment and support to tumour cells, is a novel and promising anti-tumour strategy. However, effective drugs against tumour-associated fibroblasts are currently lacking. In the present study, we explored the possibility of inhibiting the pathological functions of tumour-associated fibroblasts with triptonide. Paired gastric normal fibroblasts (GNFs) and gastric cancer-associated fibroblasts (GCAFs) were obtained from resected tissues. GCAFs showed higher capacities to induce colony formation, migration, and invasion of gastric cancer cells than GNFs. Triptonide treatment strongly inhibited the colony formation-, migration-, and invasion-promoting capacities of GCAFs. The expression of microRNA-301a was higher and that of microRNA-149 was lower in GCAFs than in GNFs. Triptonide treatment significantly down-regulated microRNA-301a expression and up-regulated microRNA-149 expression in GCAFs. Re-establishment of microRNA expression balance increased the production and secretion of tissue inhibitor of metalloproteinase 2, a tumour suppressive factor, and suppressed the production and secretion of IL-6, an oncogenic factor, in GCAFs. Moreover, triptonide treatment abolished the ability of GCAFs to induce epithelial-mesenchymal transition in gastric cancer cells. These results indicate that triptonide inhibits the malignancy-promoting capacity of GCAFs by correcting abnormalities in microRNA expression. Thus, triptonide is a promisingly therapeutic agent for gastric cancer treatment, and traditional herbs may be a valuable source for developing new drugs that can regulate the tumour microenvironment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Cancer-associated fibroblasts regulate keratinocyte cell-cell adhesion via TGF-β-dependent pathways in genotype-specific oral cancer.

Science.gov (United States)

Cirillo, N; Hassona, Y; Celentano, A; Lim, K P; Manchella, S; Parkinson, E K; Prime, S S

2017-01-01

The interrelationship between malignant epithelium and the underlying stroma is of fundamental importance in tumour development and progression. In the present study, we used cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), tumours that are characterized by the loss of genes such as TP53 and p16 INK4A and with extensive loss of heterozygosity, together with CAFs from their more genetically stable (GS) counterparts that have wild-type TP53 and p16 INK4A and minimal loss of heterozygosity (GS-OSCC). Using a systems biology approach to interpret the genome-wide transcriptional profile of the CAFs, we show that transforming growth factor-β (TGF-β) family members not only had biological relevance in silico but also distinguished GU-OSCC-derived CAFs from GS-OSCC CAFs and fibroblasts from normal oral mucosa. In view of the close association between TGF-β family members, we examined the expression of TGF-β1 and TGF-β2 in the different fibroblast subtypes and showed increased levels of active TGF-β1 and TGF-β2 in CAFs from GU-OSCC. CAFs from GU-OSCC, but not GS-OSCC or normal fibroblasts, induced epithelial-mesenchymal transition and down-regulated a broad spectrum of cell adhesion molecules resulting in epithelial dis-cohesion and invasion of target keratinocytes in vitro in a TGF-β-dependent manner. The results demonstrate that the TGF-β family of cytokines secreted by CAFs derived from genotype-specific oral cancer (GU-OSCC) promote, at least in part, the malignant phenotype by weakening intercellular epithelial adhesion. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

OroSTIFF: Face-referenced measurement of perioral stiffness in health and disease.

Science.gov (United States)

Chu, Shin-Ying; Barlow, Steven M; Kieweg, Douglas; Lee, Jaehoon

2010-05-28

A new device and automated measurement technology known as OroSTIFF is described to characterize non-participatory perioral stiffness in healthy adults for eventual application to patients with orofacial movement disorders associated with neuromotor disease, traumatic injury, or congenital clefts of the upper lip. Previous studies of perioral biomechanics required head stabilization for extended periods of time during measurement, which precluded sampling patients with involuntary body/head movements (dyskinesias), or pediatric subjects. The OroSTIFF device is face-referenced and avoids the complications associated with head-restraint. Supporting data of non-participatory perioral tissue stiffness using OroSTIFF are included from 10 male and 10 female healthy subjects. The OroSTIFF device incorporates a pneumatic glass air cylinder actuator instrumented for pressure, and an integrated subminiature displacement sensor to encode lip aperture. Perioral electromyograms were simultaneously sampled to confirm passive muscle state for the superior and inferior divisions of the orbicularis oris muscles. Perioral stiffness, derived as a quotient from resultant force (DeltaF) and interangle span (DeltaX), was modeled with multilevel regression techniques. Real-time calculation of the perioral stiffness function demonstrated a significant quadratic relation between imposed interangle stretch and resultant force. This stiffness growth function also differed significantly between males and females. This study demonstrates the OroSTIFF 'proof-of-concept' for cost-effective non-invasive stimulus generation and derivation of perioral stiffness in a group of healthy unrestrained adults, and a case study to illustrate the dose-dependent effects of Levodopa on perioral stiffness in an individual with advanced Parkinson's disease who exhibited marked dyskinesia and rigidity. Copyright 2010 Elsevier Ltd. All rights reserved.

Generating random walks and polygons with stiffness in confinement

International Nuclear Information System (INIS)

Diao, Y; Ernst, C; Saarinen, S; Ziegler, U

2015-01-01

The purpose of this paper is to explore ways to generate random walks and polygons in confinement with a bias toward stiffness. Here the stiffness refers to the curvature angle between two consecutive edges along the random walk or polygon. The stiffer the walk (polygon), the smaller this angle on average. Thus random walks and polygons with an elevated stiffness have lower than expected curvatures. The authors introduced and studied several generation algorithms with a stiffness parameter s>0 that regulates the expected curvature angle at a given vertex in which the random walks and polygons are generated one edge at a time using conditional probability density functions. Our generating algorithms also allow the generation of unconfined random walks and polygons with any desired mean curvature angle. In the case of random walks and polygons confined in a sphere of fixed radius, we observe that, as expected, stiff random walks or polygons are more likely to be close to the confinement boundary. The methods developed here require that the random walks and random polygons be rooted at the center of the confinement sphere. (paper)

MCPIP1 Regulates Alveolar Macrophage Apoptosis and Pulmonary Fibroblast Activation After in vitro Exposure to Silica.

Science.gov (United States)

Wang, Xingang; Zhang, Yuxia; Zhang, Wei; Liu, Haijun; Zhou, Zewei; Dai, Xiaoniu; Cheng, Yusi; Fang, Shencun; Zhang, Yingming; Yao, Honghong; Chao, Jie

2016-05-01

Silicosis is a fatal and fibrotic pulmonary disease caused by the inhalation of silica. After arriving at the alveoli, silica is ingested by alveolar macrophages (AMOs), in which monocyte chemotactic protein-induced protein 1 (MCPIP1) plays an essential role in controlling macrophage-mediated inflammatory responses. However, the mechanism of action of MCPIP1 in silicosis is poorly understood. Primary rat AMOs were isolated and treated with SiO2 (50 µg/cm(2)). MCPIP1 and AMO activation/apoptosis markers were detected by immunoblotting. MCPIP1 was down-regulated using siRNA in AMOs. The effects of AMOs on fibroblast activation and migration were evaluated using a gel contraction assay, a scratch assay, and a nested collagen matrix migration model. After exposure to SiO2, MCPIP1 was significantly increased in rat AMOs. Activation and apoptosis markers in AMOs were up-regulated after exposure to SiO2 Following siRNA-mediated silencing of MCPIP1 mRNA, the markers of AMO activation and apoptosis were significantly decreased. Rat pulmonary fibroblasts (PFBs) cultured in conditional medium from AMOs treated with MCPIP1 siRNA and SiO2 showed significantly less activation and migration compared with those cultured in conditional medium from AMOs treated with control siRNA and SiO2 CONCLUSION: Our data suggest a vital role for MCPIP1 in AMO apoptosis and PFB activation/migration induced by SiO2. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

FRS2α is Essential for the Fibroblast Growth Factor to Regulate the mTOR Pathway and Autophagy in Mouse Embryonic Fibroblasts

OpenAIRE

Xiang Lin, Yongyou Zhang, Leyuan Liu, Wallace L. McKeehan, Yuemao Shen, Siyang Song, Fen Wang

2011-01-01

Although the fibroblast growth factor (FGF) signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF) in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α)-mediated PI3K/A...

Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

Science.gov (United States)

Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

2018-03-12

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

An Expandable, Inducible Hemangioblast State Regulated by Fibroblast Growth Factor

Directory of Open Access Journals (Sweden)

David T. Vereide

2014-12-01

Full Text Available During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that “trap” murine cells in a proliferative state and endow them with a hemangioblast potential. These “expandable” hemangioblasts (eHBs are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines.

Gene expression profiling following NRF2 and KEAP1 siRNA knockdown in human lung fibroblasts identifies CCL11/Eotaxin-1 as a novel NRF2 regulated gene

Science.gov (United States)

2012-01-01

Background Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system. Methods Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools. Results An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts. Conclusions These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease. PMID:23061798

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

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Janice Kal Van Tam, Koichiro Uto, Mitsuhiro Ebara, Stefania Pagliari, Giancarlo Forte and Takao Aoyagi

2012-01-01

Full Text Available The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.

Heat Shock Protein 90 Inhibitor (17-AAG) Induces Apoptosis and Decreases Cell Migration/Motility of Keloid Fibroblasts.

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Yun, In Sik; Lee, Mi Hee; Rah, Dong Kyun; Lew, Dae Hyun; Park, Jong-Chul; Lee, Won Jai

2015-07-01

The regulation of apoptosis, proliferation, and migration of fibroblasts is altered in keloids. The 90-kDa heat shock protein (heat shock protein 90) is known to play a key role in such regulation. Therefore, the authors investigated whether the inhibition of heat shock protein 90 in keloid fibroblasts could induce apoptosis and attenuate keloid fibroblast proliferation and migration. The authors evaluated heat shock protein 90 expression in keloid tissues with immunohistochemistry. The authors used cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and annexin V/propidium iodide staining for apoptosis, a wound healing model and cell tracking system to assess cell migration, and Akt Western blotting analysis in keloid fibroblasts after inhibition of heat shock protein 90 with 17-allylaminodemethoxygeldanamycin (17-AAG). The expression of heat shock protein 90 in keloid tissues was significantly increased compared with normal tissues. The 17-AAG-treated keloid fibroblasts showed significantly decreased proliferation, promotion of apoptosis, and decreased expression of Akt. Furthermore, a dose-dependent decrease in cell migration was noted after 17-AAG treatment of keloid fibroblasts. The 17-AAG-treated keloid fibroblasts had less directionality to the wound center and migrated a shorter distance. The authors confirmed that the inhibition of heat shock protein 90 in keloid fibroblasts could promote apoptosis and attenuate proliferation and migration of keloid fibroblasts. Therefore, the authors think that the inhibition of heat shock protein 90 is a key factor in the regulation of biological processes in keloids. With further preclinical study, the authors will be able to apply these results clinically for keloid treatment.

Flexural Stiffness of Myosin Va Subdomains as Measured from Tethered Particle Motion

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Michalek, Arthur J.; Kennedy, Guy G.; Warshaw, David M.; Ali, M. Yusuf

2015-01-01

Myosin Va (MyoVa) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton. The motor's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor's structure. Specifically, changes in calcium may regulate motor processivity by altering the motor's lever arm stiffness and thus its interhead communication. In order to measure the flexural stiffness of MyoVa subdomains, we use tethered particle microscopy, which relates the Brownian motion of fluorescent quantum dots, which are attached to various single- and double-headed MyoVa constructs bound to actin in rigor, to the motor's flexural stiffness. Based on these measurements, the MyoVa lever arm and coiled-coil rod domain have comparable flexural stiffness (0.034 pN/nm). Upon addition of calcium, the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm. In addition, the flexural stiffness of the full-length MyoVa construct is an order of magnitude less stiff than both a single lever arm and the coiled-coil rod. This suggests that the MyoVa lever arm-rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network. PMID:26770194

RNA-seq analysis of synovial fibroblasts brings new insights into rheumatoid arthritis

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Heruth Daniel P

2012-12-01

Full Text Available Abstract Background Rheumatoid arthritis (RA is a chronic autoimmune-disease of unknown origin that primarily affects the joints and ultimately leads to their destruction. Growing evidence suggests that synvovial fibroblasts play important roles in the initiation and the perpetuation of RA but underlying molecular mechanisms are not understood fully. In the present study, Illumina RNA sequencing was used to profile two human normal control and two rheumatoid arthritis synvovial fibroblasts (RASFs transcriptomes to gain insights into the roles of synvovial fibroblasts in RA. Results We found that besides known inflammatory and immune responses, other novel dysregulated networks and pathways such as Cell Morphology, Cell-To-Cell Signaling and Interaction, Cellular Movement, Cellular Growth and Proliferation, and Cellular Development, may all contribute to the pathogenesis of RA. Our study identified several new genes and isoforms not previously associated with rheumatoid arthritis. 122 genes were up-regulated and 155 genes were down-regulated by at least two-fold in RASFs compared to controls. Of note, 343 known isoforms and 561 novel isoforms were up-regulated and 262 known isoforms and 520 novel isoforms were down-regulated by at least two-fold. The magnitude of difference and the number of differentially expressed known and novel gene isoforms were not detected previously by DNA microarray. Conclusions Since the activation and proliferation of RASFs has been implicated in the pathogenesis of rheumatoid arthritis, further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying synovial fibroblasts in arthritis and provide new leads of potential therapeutic targets.

Probing the stiffness of isolated nucleoli by atomic force microscopy.

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Louvet, Emilie; Yoshida, Aiko; Kumeta, Masahiro; Takeyasu, Kunio

2014-04-01

In eukaryotic cells, ribosome biogenesis occurs in the nucleolus, a membraneless nuclear compartment. Noticeably, the nucleolus is also involved in several nuclear functions, such as cell cycle regulation, non-ribosomal ribonucleoprotein complex assembly, aggresome formation and some virus assembly. The most intriguing question about the nucleolus is how such dynamics processes can occur in such a compact compartment. We hypothesized that its structure may be rather flexible. To investigate this, we used atomic force microscopy (AFM) on isolated nucleoli. Surface topography imaging revealed the beaded structure of the nucleolar surface. With the AFM's ability to measure forces, we were able to determine the stiffness of isolated nucleoli. We could establish that the nucleolar stiffness varies upon drastic morphological changes induced by transcription and proteasome inhibition. Furthermore, upon ribosomal proteins and LaminB1 knockdowns, the nucleolar stiffness was increased. This led us to propose a model where the nucleolus has steady-state stiffness dependent on ribosome biogenesis activity and requires LaminB1 for its flexibility.

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness.

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Nakagawa, S; Pawelek, P; Grinnell, F

1989-06-01

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

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Stewart-Hutchinson, P.J.; Hale, Christopher M.; Wirtz, Denis; Hodzic, Didier

2008-01-01

The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

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Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

2014-01-01

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

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Malpass, Gloria E., E-mail: gloria.malpass@gmail.com [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu [Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Prasad, G.L., E-mail: prasadg@rjrt.com [R and D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102 (United States); Howlett, Allyn C., E-mail: ahowlett@wakehealth.edu [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States)

2014-09-01

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

Limit cycles and stiffness control with variable stiffness actuators

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Carloni, Raffaella; Marconi, L.

2012-01-01

Variable stiffness actuators realize highly dynamic systems, whose inherent mechanical compliance can be properly exploited to obtain a robust and energy-efficient behavior. The paper presents a control strategy for variable stiffness actuators with the primarily goal of tracking a limit cycle

Effect of phenytoin and age on gingival fibroblast enzymes.

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Surena Vahabi

2014-06-01

Full Text Available The alteration of cytokine balance is stated to exert greater influence on gingival overgrowth compared to the direct effect of the drug on the regulation of extracellular matrix metabolism. The current study evaluated the effect of phenytoin on the regulation of collagen, lysyl oxidase and elastin in gingival fibroblasts.Normal human gingival fibroblasts (HGFs were obtained from 4 healthy children and 4 adults. Samples were cultured with phenytoin. MTT test was used to evaluate the proliferation and ELISA was performed to determine the level of IL1β and PGE2 production by HGFs. Total RNA of gingival fibroblasts was extracted and RT-PCR was performed on samples. Mann-Whitney U test was used to analyze the data with an alpha error level less than 0.05.There was a significant difference in the expression of elastin between the controls and treated samples in both adult and pediatric groups and also in the lysyl oxidase expression of adult controls and treated adults. No significant difference was found between collagen expression in adults.The significant difference in elastin and lysyl oxidase expression between adult and pediatric samples indicates the significant effect of age on their production.

Bone Morphogenetic Protein (BMP-4 and BMP-7 regulate differentially Transforming Growth Factor (TGF-β1 in normal human lung fibroblasts (NHLF

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Lloyd Clare M

2010-06-01

Full Text Available Abstract Background Airway remodelling is thought to be under the control of a complex group of molecules belonging to the Transforming Growth Factor (TGF-superfamily. The Bone Morphogenetic Proteins (BMPs belong to this family and have been shown to regulate fibrosis in kidney and liver diseases. However, the role of BMPs in lung remodelling remains unclear. BMPs may regulate tissue remodelling in asthma by controlling TGF-β-induced profibrotic functions in lung fibroblasts. Methods Cell cultures were exposed to TGF-β1 alone or in the presence of BMP-4 or BMP-7; control cultures were exposed to medium only. Cell proliferation was assessed by quantification of the incorporation of [3H]-thymidine. The expression of the mRNA encoding collagen type I and IV, tenascin C and fibronectin in normal human lung fibroblasts (NHLF was determined by real-time quantitative PCR and the main results were confirmed by ELISA. Cell differentiation was determined by the analysis of the expression of α-smooth muscle actin (α-SMA by western blot and immunohistochemistry. The effect on matrix metalloproteinase (MMP activity was assessed by zymography. Results We have demonstrated TGF-β1 induced upregulation of mRNAs encoding the extracellular matrix proteins, tenascin C, fibronectin and collagen type I and IV when compared to unstimulated NHLF, and confirmed these results at the protein level. BMP-4, but not BMP-7, reduced TGF-β1-induced extracellular matrix protein production. TGF-β1 induced an increase in the activity of the pro-form of MMP-2 which was inhibited by BMP-7 but not BMP-4. Both BMP-4 and BMP-7 downregulated TGF-β1-induced MMP-13 release compared to untreated and TGF-β1-treated cells. TGF-β1 also induced a myofibroblast-like transformation which was partially inhibited by BMP-7 but not BMP-4. Conclusions Our study suggests that some regulatory properties of BMP-7 may be tissue or cell type specific and unveil a potential regulatory role for

Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions.

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He, Xiao-Tao; Wu, Rui-Xin; Xu, Xin-Yue; Wang, Jia; Yin, Yuan; Chen, Fa-Ming

2018-04-15

Accumulating evidence indicates that the physicochemical properties of biomaterials exert profound influences on stem cell fate decisions. However, matrix-based regulation selected through in vitro analyses based on a given cell population do not genuinely reflect the in vivo conditions, in which multiple cell types are involved and interact dynamically. This study constitutes the first investigation of how macrophages (Mφs) in stiffness-tunable transglutaminase cross-linked gelatin (TG-gel) affect the osteogenesis of bone marrow-derived mesenchymal stem cells (BMMSCs). When a single cell type was cultured, low-stiffness TG-gels promoted BMMSC proliferation, whereas high-stiffness TG-gels supported cell osteogenic differentiation. However, Mφs in high-stiffness TG-gels were more likely to polarize toward the pro-inflammatory M1 phenotype. Using either conditioned medium (CM)-based incubation or Transwell-based co-culture, we found that Mφs encapsulated in the low-stiffness matrix exerted a positive effect on the osteogenesis of co-cultured BMMSCs. Conversely, Mφs in high-stiffness TG-gels negatively affected cell osteogenic differentiation. When both cell types were cultured in the same TG-gel type and placed into the Transwell system, the stiffness-related influences of Mφs on BMMSCs were significantly altered; both the low- and high-stiffness matrix induced similar levels of BMMSC osteogenesis. Although the best material parameter for synergistically affecting Mφs and BMMSCs remains unknown, our data suggest that Mφ involvement in the co-culture system alters previously identified material-related influences on BMMSCs, such as matrix stiffness-related effects, which were identified based on a culture system involving a single cell type. Such Mφ-stem cell interactions should be considered when establishing proper matrix parameter-associated cell regulation in the development of biomimetic biomaterials for regenerative applications. The substrate stiffness

Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts.

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Rachel C Williams

Full Text Available Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts.We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts.We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival

Asymmetric migration of human keratinocytes under mechanical stretch and cocultured fibroblasts in a wound repair model.

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Dongyuan Lü

Full Text Available Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

Development of a stiffness-angle law for simplifying the measurement of human hair stiffness.

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Jung, I K; Park, S C; Lee, Y R; Bin, S A; Hong, Y D; Eun, D; Lee, J H; Roh, Y S; Kim, B M

2018-04-01

This research examines the benefits of caffeine absorption on hair stiffness. To test hair stiffness, we have developed an evaluation method that is not only accurate, but also inexpensive. Our evaluation method for measuring hair stiffness culminated in a model, called the Stiffness-Angle Law, which describes the elastic properties of hair and can be widely applied to the development of hair care products. Small molecules (≤500 g mol -1 ) such as caffeine can be absorbed into hair. A common shampoo containing 4% caffeine was formulated and applied to hair 10 times, after which the hair stiffness was measured. The caffeine absorption of the treated hair was observed using Fourier-transform infrared spectroscopy (FTIR) with a focal plane array (FPA) detector. Our evaluation method for measuring hair stiffness consists of a regular camera and a support for single strands of hair. After attaching the hair to the support, the bending angle of the hair was observed with a camera and measured. Then, the hair strand was weighed. The stiffness of the hair was calculated based on our proposed Stiffness-Angle Law using three variables: angle, weight of hair and the distance the hair was pulled across the support. The caffeine absorption was confirmed by FTIR analysis. The concentration of amide bond in the hair certainly increased due to caffeine absorption. After caffeine was absorbed into the hair, the bending angle and weight of the hair changed. Applying these measured changes to the Stiffness-Angle Law, it was confirmed that the hair stiffness increased by 13.2% due to caffeine absorption. The theoretical results using the Stiffness-Angle Law agree with the visual examinations of hair exposed to caffeine and also the known results of hair stiffness from a previous report. Our evaluation method combined with our proposed Stiffness-Angle Law effectively provides an accurate and inexpensive evaluation technique for measuring bending stiffness of human hair. © 2018

Mitochondrial Bioenergetics Is Altered in Fibroblasts from Patients with Sporadic Alzheimer's Disease

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Pérez, María J.; Ponce, Daniela P.; Osorio-Fuentealba, Cesar; Behrens, Maria I.; Quintanilla, Rodrigo A.

2017-01-01

The identification of an early biomarker to diagnose Alzheimer's disease (AD) remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology) in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients. PMID:29056898

Posttraumatic stiff elbow

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Ravi Mittal

2017-01-01

Full Text Available Posttraumatic stiff elbow is a frequent and disabling complication and poses serious challenges for its management. In this review forty studies were included to know about the magnitude of the problem, causes, pathology, prevention, and treatment of posttraumatic stiff elbow. These studies show that simple measures such as internal fixation, immobilization in extension, and early motion of elbow joint are the most important steps that can prevent elbow stiffness. It also supports conservative treatment in selected cases. There are no clear guidelines about the choice between the numerous procedures described in literature. However, this review article disproves two major beliefs-heterotopic ossification is a bad prognostic feature, and passive mobilization of elbow causes elbow stiffness.

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway

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Wang Jun

2012-10-01

Full Text Available Abstract Background Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS to imitate inflammatory conditions. Results In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4 antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB, which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1, compared with transforming growth factor-β1 (TGF-β1. Conclusions Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway.

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Wang, Jun; Si, Yanfang; Wu, Chen; Sun, Lu; Ma, Yudong; Ge, Aili; Li, Baomin

2012-10-17

Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to the pathological process of

Fibroblast growth factor signaling potentiates VE-cadherin stability at adherens junctions by regulating SHP2.

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Kunihiko Hatanaka

Full Text Available The fibroblast growth factor (FGF system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization.In vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity.These results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability.

Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.

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El Ghalbzouri, Abdoelwaheb; Jonkman, Marcel F; Dijkman, Remco; Ponec, Maria

2005-01-01

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.

Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis

International Nuclear Information System (INIS)

Liu, Xueting; Fang, Shencun; Liu, Haijun; Wang, Xingang; Dai, Xiaoniu; Yin, Qing; Yun, Tianwei; Wang, Wei; Zhang, Yingming; Liao, Hong; Zhang, Wei; Yao, Honghong; Chao, Jie

2015-01-01

Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO 2 ). Phagocytosis of SiO 2 in the lung initiates an inflammatory cascade that results in fibroblast proliferation and migration and subsequent fibrosis. Clinical evidence indicates that the activation of alveolar macrophages by SiO 2 produces rapid and sustained inflammation that is characterized by the generation of monocyte chemotactic protein 1 (MCP-1), which induces fibrosis. Pulmonary fibroblast-derived MCP-1 may play a critical role in fibroblast proliferation and migration. Methods and results: Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following results: 1) SiO 2 treatment resulted in the rapid and sustained induction of MCP-1 as well as the elevation of the CC chemokine receptor type 2 (CCR2) protein levels; 2) pretreatment of HPF-a with RS-102895, a specific CCR2 inhibitor, abolished the SiO 2 -induced increase in cell activation and migration in both 2D and 3D culture systems; and 3) RNA interference targeting CCR2 prevented the SiO 2 -induced increase in cell migration. Conclusion: These data demonstrated that the up-regulation of pulmonary fibroblast-derived MCP-1 is involved in pulmonary fibroblast migration induced by SiO 2 . CCR2 was also up-regulated in response to SiO 2 , and this up-regulation facilitated the effect of MCP-1 on fibroblasts. Our study deciphered the link between fibroblast-derived MCP-1 and SiO 2 -induced cell migration. This finding provides novel insight into the potential of MCP-1 in the development of novel therapeutic strategies for silicosis. - Highlights: • Role of pulmonary fibroblast-derived MCP-1 in experimental silicosis was studied. • SiO 2 induced MCP-1 release from cultured human pulmonary fibroblast (HPF-a). • SiO 2 directly activated HPF-a via the MCP-1/CCR2 pathway. • SiO 2 increased HPF-a migration in both 2D and 3D model via the MCP-1/CCR2 pathway. • RNA-i of MCP-1/CCR2

Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis

Energy Technology Data Exchange (ETDEWEB)

Liu, Xueting [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Fang, Shencun [Nine Department of Respiratory Medicine, Nanjing Chest Hospital, Nanjing, Jiangsu 210029 (China); Liu, Haijun [Neurobiology Laboratory, New Drug Screening Centre, China Pharmaceutical University, Nanjing, Jiangsu 210009 (China); Wang, Xingang; Dai, Xiaoniu; Yin, Qing; Yun, Tianwei [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Wang, Wei; Zhang, Yingming [Nine Department of Respiratory Medicine, Nanjing Chest Hospital, Nanjing, Jiangsu 210029 (China); Liao, Hong [Neurobiology Laboratory, New Drug Screening Centre, China Pharmaceutical University, Nanjing, Jiangsu 210009 (China); Zhang, Wei [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Yao, Honghong [Department of Pharmacology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Chao, Jie, E-mail: chaojie@seu.edu.cn [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China)

2015-10-15

Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO{sub 2}). Phagocytosis of SiO{sub 2} in the lung initiates an inflammatory cascade that results in fibroblast proliferation and migration and subsequent fibrosis. Clinical evidence indicates that the activation of alveolar macrophages by SiO{sub 2} produces rapid and sustained inflammation that is characterized by the generation of monocyte chemotactic protein 1 (MCP-1), which induces fibrosis. Pulmonary fibroblast-derived MCP-1 may play a critical role in fibroblast proliferation and migration. Methods and results: Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following results: 1) SiO{sub 2} treatment resulted in the rapid and sustained induction of MCP-1 as well as the elevation of the CC chemokine receptor type 2 (CCR2) protein levels; 2) pretreatment of HPF-a with RS-102895, a specific CCR2 inhibitor, abolished the SiO{sub 2}-induced increase in cell activation and migration in both 2D and 3D culture systems; and 3) RNA interference targeting CCR2 prevented the SiO{sub 2}-induced increase in cell migration. Conclusion: These data demonstrated that the up-regulation of pulmonary fibroblast-derived MCP-1 is involved in pulmonary fibroblast migration induced by SiO{sub 2}. CCR2 was also up-regulated in response to SiO{sub 2}, and this up-regulation facilitated the effect of MCP-1 on fibroblasts. Our study deciphered the link between fibroblast-derived MCP-1 and SiO{sub 2}-induced cell migration. This finding provides novel insight into the potential of MCP-1 in the development of novel therapeutic strategies for silicosis. - Highlights: • Role of pulmonary fibroblast-derived MCP-1 in experimental silicosis was studied. • SiO{sub 2} induced MCP-1 release from cultured human pulmonary fibroblast (HPF-a). • SiO{sub 2} directly activated HPF-a via the MCP-1/CCR2 pathway. • SiO{sub 2} increased HPF-a migration in both 2D and 3D

Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

Directory of Open Access Journals (Sweden)

Rong Yao

Full Text Available Pulmonary fibrosis is one of the most common complications of paraquat (PQ poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR. Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR 1 small-interfering RNA (siRNA group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8 and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05. Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05. APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a

Multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair.

Science.gov (United States)

Chiquet, Matthias; Katsaros, Christos; Kletsas, Dimitris

2015-06-01

Fibroblasts are cells of mesenchymal origin. They are responsible for the production of most extracellular matrix in connective tissues and are essential for wound healing and repair. In recent years, it has become clear that fibroblasts from different tissues have various distinct traits. Moreover, wounds in the oral cavity heal under very special environmental conditions compared with skin wounds. Here, we reviewed the current literature on the various interconnected functions of gingival and mucoperiosteal fibroblasts during the repair of oral wounds. The MEDLINE database was searched with the following terms: (gingival OR mucoperiosteal) AND fibroblast AND (wound healing OR repair). The data gathered were used to compare oral fibroblasts with fibroblasts from other tissues in terms of their regulation and function during wound healing. Specifically, we sought answers to the following questions: (i) what is the role of oral fibroblasts in the inflammatory response in acute wounds; (ii) how do growth factors control the function of oral fibroblasts during wound healing; (iii) how do oral fibroblasts produce, remodel and interact with extracellular matrix in healing wounds; (iv) how do oral fibroblasts respond to mechanical stress; and (v) how does aging affect the fetal-like responses and functions of oral fibroblasts? The current state of research indicates that oral fibroblasts possess unique characteristics and tightly controlled specific functions in wound healing and repair. This information is essential for developing new strategies to control the intraoral wound-healing processes of the individual patient. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Arterial stiffness and cognitive impairment.

Science.gov (United States)

Li, Xiaoxuan; Lyu, Peiyuan; Ren, Yanyan; An, Jin; Dong, Yanhong

2017-09-15

Arterial stiffness is one of the earliest indicators of changes in vascular wall structure and function and may be assessed using various indicators, such as pulse-wave velocity (PWV), the cardio-ankle vascular index (CAVI), the ankle-brachial index (ABI), pulse pressure (PP), the augmentation index (AI), flow-mediated dilation (FMD), carotid intima media thickness (IMT) and arterial stiffness index-β. Arterial stiffness is generally considered an independent predictor of cardiovascular and cerebrovascular diseases. To date, a significant number of studies have focused on the relationship between arterial stiffness and cognitive impairment. To investigate the relationships between specific arterial stiffness parameters and cognitive impairment, elucidate the pathophysiological mechanisms underlying the relationship between arterial stiffness and cognitive impairment and determine how to interfere with arterial stiffness to prevent cognitive impairment, we searched PUBMED for studies regarding the relationship between arterial stiffness and cognitive impairment that were published from 2000 to 2017. We used the following key words in our search: "arterial stiffness and cognitive impairment" and "arterial stiffness and cognitive impairment mechanism". Studies involving human subjects older than 30years were included in the review, while irrelevant studies (i.e., studies involving subjects with comorbid kidney disease, diabetes and cardiac disease) were excluded from the review. We determined that arterial stiffness severity was positively correlated with cognitive impairment. Of the markers used to assess arterial stiffness, a higher PWV, CAVI, AI, IMT and index-β and a lower ABI and FMD were related to cognitive impairment. However, the relationship between PP and cognitive impairment remained controversial. The potential mechanisms linking arterial stiffness and cognitive impairment may be associated with arterial pulsatility, as greater arterial pulsatility

Emmprin, released as a microvesicle in epithelioid sarcoma, interacts with fibroblasts.

Science.gov (United States)

Aoki, Mikiko; Koga, Kaori; Hamasaki, Makoto; Egawa, Nagayasu; Nabeshima, Kazuki

2017-06-01

Emmprin (extracellular matrix metalloproteinase inducer, CD147) is a glycosylated transmembrane protein, consisting of two immunoglobulin domains, that stimulates the production of matrix metalloproteinases (MMPs) by tumor-associated fibroblasts. These effects play important roles in tumor invasion and metastasis. However, the precise mechanisms by which emmprin acts on fibroblasts have not been fully elucidated, especially in sarcoma cells. Previously, we demonstrated that emmprin, expressed in conditioned medium collected from the epithelioid sarcoma cell line (FU-EPS-1), stimulates MMP-2 production via interactions with fibroblasts. In this study, we used microvesicles derived from sarcoma cells, and determined whether emmprin exists in the microvesicles, which enhance the production of MMP-2 via fibroblasts. Microvesicles released from FU-EPS-1 cells were shown to contain full-length emmprin, identified as a 45-kDa protein characterized by polylactosamine glycosylation. Microvesicles collected from FU-EPS-1 cells transfected with emmprin-specific siRNA or transduced with shRNA displayed significantly reduced MMP-2 production by fibroblasts compared with those from control-transfected cells. Our findings show that emmprin is released through microvesicle shedding in sarcoma cells, and emmprin in microvesicles regulates MMP-2 production by influencing the activity of fibroblasts located at sites distant from the tumor cells.

Fuzzy variable impedance control based on stiffness identification for human-robot cooperation

Science.gov (United States)

Mao, Dachao; Yang, Wenlong; Du, Zhijiang

2017-06-01

This paper presents a dynamic fuzzy variable impedance control algorithm for human-robot cooperation. In order to estimate the intention of human for co-manipulation, a fuzzy inference system is set up to adjust the impedance parameter. Aiming at regulating the output fuzzy universe based on the human arm’s stiffness, an online stiffness identification method is developed. A drag interaction task is conducted on a 5-DOF robot with variable impedance control. Experimental results demonstrate that the proposed algorithm is superior.

Identification of pulmonary PDGFRalpha-positive fibroblast specific miRNA and mRNA expression profiles during postnatal lung development

OpenAIRE

Dontireddy, Daria Agnieszka

2015-01-01

BACKGROUND AND AIM The process of alveolarization is tightly regulated and requires the contribution of different subpopulations of fibroblasts such as myofibroblasts, lipofibroblasts and platelet-derived growth factor receptor alpha (PDGFRalpha)-positive fibroblasts. Each of this fibroblasts subset fulfills certain functions during lung development in a time-dependent manner. In particular PDGFRalpha-positive cells are crucial for alveolar septation and myofibroblasts differentiation. PDG...

Estimating Gear Teeth Stiffness

DEFF Research Database (Denmark)

Pedersen, Niels Leergaard

2013-01-01

The estimation of gear stiffness is important for determining the load distribution between the gear teeth when two sets of teeth are in contact. Two factors have a major influence on the stiffness; firstly the boundary condition through the gear rim size included in the stiffness calculation...... and secondly the size of the contact. In the FE calculation the true gear tooth root profile is applied. The meshing stiffness’s of gears are highly non-linear, it is however found that the stiffness of an individual tooth can be expressed in a linear form assuming that the contact length is constant....

Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts.

Science.gov (United States)

Wyman, Anne E; Noor, Zahid; Fishelevich, Rita; Lockatell, Virginia; Shah, Nirav G; Todd, Nevins W; Atamas, Sergei P

2017-06-01

Pulmonary fibrosis is a severe condition with no cure and limited therapeutic options. A better understanding of its pathophysiology is needed. Recent studies have suggested that pulmonary fibrosis may be driven by accelerated aging-related mechanisms. Sirtuins (SIRTs), particularly SIRT1, SIRT3, and SIRT6, are well-known mediators of aging; however, limited data exist on the contribution of sirtuins to lung fibrosis. We assessed the mRNA and protein levels of all seven known sirtuins in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in comparison with lung fibroblasts from healthy controls. These unbiased tests revealed a tendency for all sirtuins to be expressed at lower levels in fibroblasts from patients compared with controls, but the greatest decrease was observed with SIRT7. Similarly, SIRT7 was decreased in lung tissues of bleomycin-challenged mice. Inhibition of SIRT7 with siRNA in cultured lung fibroblasts resulted in an increase in collagen and α-smooth muscle actin (α-SMA). Reciprocally, overexpression of SIRT7 resulted in lower basal and TGF-β-induced levels of COL1A1, COL1A2, COL3A1, and α-SMA mRNAs, as well as collagen and α-SMA proteins. Induced changes in SIRT7 had no effect on endogenous TGF-β mRNA levels or latent TGF-β activation, but overexpression of SIRT7 reduced the levels of Smad3 mRNA and protein. In conclusion, the decline in SIRT7 in lung fibroblasts has a profibrotic effect, which is mediated by changes in Smad3 levels.

Trabecular meshwork stiffness in glaucoma.

Science.gov (United States)

Wang, Ke; Read, A Thomas; Sulchek, Todd; Ethier, C Ross

2017-05-01

Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open-angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho-kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor-β 2 (TGF-β 2 ), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM-based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

Energy Technology Data Exchange (ETDEWEB)

Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

2014-04-04

Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

International Nuclear Information System (INIS)

Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

2014-01-01

Highlights: • LPA 5 inhibits the cell growth and motile activities of 3T3 cells. • LPA 5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA 5 on the cell motile activities inhibited by LPA 1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA 5 in 3T3 cells. • LPA signaling via LPA 5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA 1 –LPA 6 ) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA 1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA 5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA 1 and LPA 5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA 5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA 1

Fibroblastic rheumatism

Directory of Open Access Journals (Sweden)

Jyoti Ranjan Parida

2017-01-01

Full Text Available Fibroblastic rheumatism (FR is a rare dermoarthopathy reported from different parts of the world since 1980. Although the exact cause is unknown, few reports implicate infection may be a triggering event. Patients usually present with multiple skin nodules and polyarthropathy with progressive skin contractures. Laboratory parameters including acute phase reactants are usually normal. The confirmatory diagnosis is based on histopathologic study of skin nodules, which demonstrate fibroblastic proliferation, thickened collagen fibers, dermal fibrosis, and decreased number of elastic fibers. Immunoreactivity for b-catenin, smooth muscle actin, and the monoclonal antibody HHF35 show myofibroblastic differentiation. Treatments with oral prednisolone and other disease-modifying drugs such as methotrexate, infliximab, and interferon have been tried with variable success. In general, skin lesions respond more aptly than joint symptoms indicating that skin fibroblast is more amenable to treatment than synovial fibroblasts. Awareness regarding this orphan disease among clinicians and pathologists will help in more reporting of such cases and finding out optimal treatment regimen.

Artificial muscles with adjustable stiffness

International Nuclear Information System (INIS)

Mutlu, Rahim; Alici, Gursel

2010-01-01

This paper reports on a stiffness enhancement methodology based on using a suitably designed contact surface with which cantilevered-type conducting polymer bending actuators are in contact during operation. The contact surface constrains the bending behaviour of the actuators. Depending on the topology of the contact surface, the resistance of the polymer actuators to deformation, i.e. stiffness, is varied. As opposed to their predecessors, these polymer actuators operate in air. Finite element analysis and modelling are used to quantify the effect of the contact surface on the effective stiffness of a trilayer cantilevered beam, which represents a one-end-free, the-other-end-fixed polypyrrole (PPy) conducting polymer actuator under a uniformly distributed load. After demonstrating the feasibility of the adjustable stiffness concept, experiments were conducted to determine the stiffness of bending-type conducting polymer actuators in contact with a range (20–40 mm in radius) of circular contact surfaces. The numerical and experimental results presented demonstrate that the stiffness of the actuators can be varied using a suitably profiled contact surface. The larger the radius of the contact surface is, the higher is the stiffness of the polymer actuators. The outcomes of this study suggest that, although the stiffness of the artificial muscles considered in this study is constant for a given geometric size, and electrical and chemical operation conditions, it can be changed in a nonlinear fashion to suit the stiffness requirement of a considered application. The stiffness enhancement methodology can be extended to other ionic-type conducting polymer actuators

A variable stiffness joint with electrospun P(VDF-TrFE-CTFE) variable stiffness springs

NARCIS (Netherlands)

Carloni, Raffaella; Lapp, Valerie I.; Cremonese, Andrea; Belcari, Juri; Zucchelli, Andrea

This letter presents a novel rotational variable stiffness joint that relies on one motor and a set of variable stiffness springs. The variable stiffness springs are leaf springs with a layered design, i.e., an electro-active layer of electrospun aligned nanofibers of poly(vinylidene

PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.

Science.gov (United States)

Pincha, Neha; Hajam, Edries Yousaf; Badarinath, Krithika; Batta, Surya Prakash Rao; Masudi, Tafheem; Dey, Rakesh; Andreasen, Peter; Kawakami, Toshiaki; Samuel, Rekha; George, Renu; Danda, Debashish; Jacob, Paul Mazhuvanchary; Jamora, Colin

2018-05-01

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

Probing cellular mechanoadaptation using cell-substrate de-adhesion dynamics: experiments and model.

Science.gov (United States)

S S, Soumya; Sthanam, Lakshmi Kavitha; Padinhateeri, Ranjith; Inamdar, Mandar M; Sen, Shamik

2014-01-01

Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales.

Pharmacological modulation of arterial stiffness.

LENUS (Irish Health Repository)

Boutouyrie, Pierre

2011-09-10

Arterial stiffness has emerged as an important marker of cardiovascular risk in various populations and reflects the cumulative effect of cardiovascular risk factors on large arteries, which in turn is modulated by genetic background. Arterial stiffness is determined by the composition of the arterial wall and the arrangement of these components, and can be studied in humans non-invasively. Age and distending pressure are two major factors influencing large artery stiffness. Change in arterial stiffness with drugs is an important endpoint in clinical trials, although evidence for arterial stiffness as a therapeutic target still needs to be confirmed. Drugs that independently affect arterial stiffness include antihypertensive drugs, mostly blockers of the renin-angiotensin-aldosterone system, hormone replacement therapy and some antidiabetic drugs such as glitazones. While the quest continues for \\'de-stiffening drugs\\

Unidirectional variable stiffness hydraulic actuator for load-carrying knee exoskeleton

Directory of Open Access Journals (Sweden)

Jun Zhu

2017-01-01

Full Text Available This article presents the design and experimental testing of a unidirectional variable stiffness hydraulic actuator for load-carrying knee exoskeleton. The proposed actuator is designed for mimicking the high-efficiency passive behavior of biological knee and providing actively assistance in locomotion. The adjustable passive compliance of exoskeletal knee is achieved through a variable ratio lever mechanism with linear elastic element. A compact customized electrohydraulic system is also designed to accommodate application demands. Preliminary experimental results show the prototype has good performances in terms of stiffness regulation and joint torque control. The actuator is also implemented in an exoskeleton knee joint, resulting in anticipant human-like passive compliance behavior.

TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

Energy Technology Data Exchange (ETDEWEB)

Chen, Xiao-Qing; Zhang, Dao-Liang [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Jiang, Wei-Feng; Zhou, Li [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhao, Liang, E-mail: zhaol_zg@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Wang, Quan-Xing, E-mail: wqxejd@126.com [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Liu, Xu, E-mail: liuxu_xk@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China)

2015-08-14

Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage.

TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

International Nuclear Information System (INIS)

Chen, Xiao-Qing; Zhang, Dao-Liang; Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang; Jiang, Wei-Feng; Zhou, Li; Zhao, Liang; Wang, Quan-Xing; Liu, Xu

2015-01-01

Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage

Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation.

NARCIS (Netherlands)

Scharstuhl, A.; Mutsaers, H.A.M.; Pennings, S.W.C.; Szarek, W.A.; Russel, F.G.M.; Wagener, F.A.D.T.G.

2009-01-01

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and

Measurement and Treatment of Passive Muscle Stiffness

DEFF Research Database (Denmark)

Kirk, Henrik

, which aimed to investigate: 1) The development of a clinical method to evaluate and distinguish neural (reflex mediated stiffness) and non-neural (passive muscle stiffness) components of muscle stiffness in adults with CP by objective and reliable measurements. 2) The association between increased...... and reliability of the method, and argue for the use of the method in the clinical practice. The device is able to distinguish between passive muscle stiffness and reflex-mediated stiffness in subjects with CP. It shows good high intrarater and interrater reliability in evaluation of passive muscle stiffness...... to measure muscle stiffness, and distinguish between passive muscle stiffness and reflex-mediated stiffness. Furthermore, it is a reliable device to measure changes in passive ROM. Treatment of passive muscle stiffness should be directed towards intense training, comprising many repetitions with a functional...

HJURP regulates cellular senescence in human fibroblasts and endothelial cells via a p53-dependent pathway.

Science.gov (United States)

Heo, Jong-Ik; Cho, Jung Hee; Kim, Jae-Ryong

2013-08-01

Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.

Time dependency of morphological remodeling of endothelial cells in response to substrate stiffness

Science.gov (United States)

Goli-Malekabadi, Zahra; Tafazzoli-shadpour, Mohammad; Tamayol, Ali; Seyedjafari, Ehsan

2017-01-01

Introduction: Substrate stiffness regulates cellular behavior as cells experience different stiffness values of tissues in the body. For example, endothelial cells (ECs) covering the inner layer of blood vessels are exposed to different stiffness values due to various pathologic and physiologic conditions. Despite numerous studies, cells by time span sense mechanical properties of the substrate, but the response is not well understood. We hypothesized that time is a major determinant influencing the behavior of cells seeded on substrates of varying stiffness. Methods: We monitored cell spreading, internal structure, 3D topography, and the viability of ECs over 24 hours of culture on polydimethylsiloxane (PDMS) substrates with two different degrees of elastic modulus. Results: Despite significant differences in cell spreading after cell seeding, cells showed a similar shape and internal structure after 24 hours of culture on both soft and stiff substrates. However, 3D topographical images confirmed existence of rich lamellipodia and filopodia around the cells cultured on stiffer PDMS substrates. Conclusion: It was concluded that the response of ECs to the substrate stiffness was time dependent with initial enhanced cellular spreading and viability on stiffer substrates. Results can provide a better comprehension of cell mechanotransduction for tissue engineering applications. PMID:28546952

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

Science.gov (United States)

Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

2018-04-17

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

Alpha8 Integrin (Itga8 Signalling Attenuates Chronic Renal Interstitial Fibrosis by Reducing Fibroblast Activation, Not by Interfering with Regulation of Cell Turnover.

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Ines Marek

Full Text Available The α8 integrin (Itga8 chain contributes to the regulation of cell proliferation and apoptosis in renal glomerular cells. In unilateral ureteral obstruction Itga8 is de novo expressed in the tubulointerstitium and a deficiency of Itga8 results in more severe renal fibrosis after unilateral ureteral obstruction. We hypothesized that the increased tubulointerstitial damage after unilateral ureteral obstruction observed in mice deficient for Itga8 is associated with altered tubulointerstitial cell turnover and apoptotic mechanisms resulting from the lack of Itga8 in cells of the tubulointerstitium. Induction of unilateral ureteral obstruction was achieved by ligation of the right ureter in mice lacking Itga8. Unilateral ureteral obstruction increased proliferation and apoptosis rates of tubuloepithelial and interstitial cells, however, no differences were observed in the tubulointerstitium of mice lacking Itga8 and wild type controls regarding fibroblast or proliferating cell numbers as well as markers of endoplasmic reticulum stress and apoptosis after unilateral ureteral obstruction. In contrast, unilateral ureteral obstruction in mice lacking Itga8 led to more pronounced tubulointerstitial cell activation i.e. to the appearance of more phospho-SMAD2/3-positive cells and more α-smooth muscle actin-positive cells in the tubulointerstitium. Furthermore, a more severe macrophage and T-cell infiltration was observed in these animals compared to controls. Thus, Itga8 seems to attenuate tubulointerstitial fibrosis in unilateral ureteral obstruction not via regulation of cell turnover, but via regulation of TGF-β signalling, fibroblast activation and/or immune cell infiltration.

On gear tooth stiffness evaluation

DEFF Research Database (Denmark)

Pedersen, Niels Leergaard; Jørgensen, Martin Felix

2014-01-01

The estimation of gear stiffness is important for determining the load distribution between the gear teeth when two sets of teeth are in contact. Two factors have a major influence on the stiffness; firstly the boundary condition through the gear rim size included in the stiffness calculation...

EB1 is required for primary cilia assembly in fibroblasts

DEFF Research Database (Denmark)

Schrøder, Jacob M; Schneider, Linda; Christensen, Søren T

2007-01-01

EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and diff...... that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts....

Dynamic stiffness of suction caissons

DEFF Research Database (Denmark)

Ibsen, Lars Bo; Liingaard, Morten; Andersen, Lars

The purpose of this report is to evaluate the dynamic soil-structure interaction of suction caissons for offshore wind turbines. The investigation is limited to a determination of the vertical dynamic stiffness of suction caissons. The soil surrounding the foundation is homogenous with linear...... viscoelastic properties. The dynamic stiffness of the suction caisson is expressed by dimensionless frequency-dependent dynamic stiffness coefficients corresponding to the vertical degree of freedom. The dynamic stiffness coefficients for the foundations are evaluated by means of a dynamic three...

Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture

International Nuclear Information System (INIS)

Marinucci, Lorella; Balloni, Stefania; Bodo, Maria; Carinci, Francesco; Pezzetti, Furio; Stabellini, Giordano; Carmela, Conte; Lumare, Eleonora

2009-01-01

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFβ 3 ) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences

Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

International Nuclear Information System (INIS)

Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

1982-01-01

The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

Influence of Crosslink Density and Stiffness on Mechanical Properties of Type I Collagen Gel

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Shengmao Lin

2015-02-01

Full Text Available The mechanical properties of type I collagen gel vary due to different polymerization parameters. In this work, the role of crosslinks in terms of density and stiffness on the macroscopic behavior of collagen gel were investigated through computational modeling. The collagen fiber network was developed in a representative volume element, which used the inter-fiber spacing to regulate the crosslink density. The obtained tensile behavior of collagen gel was validated against published experimental data. Results suggest that the cross-linked fiber alignment dominated the strain stiffening effect of the collagen gel. In addition, the gel stiffness was enhanced approximately 40 times as the crosslink density doubled. The non-affine deformation was reduced with the increased crosslink density. A positive bilinear correlation between the crosslink density and gel stiffness was obtained. On the other hand, the crosslink stiffness had much less impact on the gel stiffness. This work could enhance our understanding of collagen gel mechanics and shed lights on designing future clinical relevant biomaterials with better control of polymerization parameters.

Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

Science.gov (United States)

Zhou, Dong; Fu, Haiyan; Zhang, Lu; Zhang, Ke; Min, Yali; Xiao, Liangxiang; Lin, Lin; Bastacky, Sheldon I; Liu, Youhua

2017-08-01

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β -catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro , incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication. Copyright © 2017 by the American Society of Nephrology.

Butyrate down regulates BCL-XL and sensitizes human fibroblasts to radiation and chemotherapy induced apoptosis

International Nuclear Information System (INIS)

Chung, Diana H.; Ljungman, Mats; Zhang Fenfen; Chen Feng; McLaughlin, William P.

1997-01-01

Purpose/Objective: Butyrate is a short chain fatty acid that has been implicated in the induction of cell cycle arrest, cell differentiation and apoptosis. The purpose of this study was to determine if butyrate treatment sensitizes cells to radiation or chemotherapy induced apoptosis. Materials and Methods: Normal neonatal human diploid fibroblasts were used throughout this study. Apoptosis was scored and quantified using three different methods. First, cell morphology using propidium iodide and fluorescence microscopy was used to qualitatively determine apoptosis and to quantify the percentage of cells undergoing apoptosis. Second, apoptosis induced DNA degradation was scored by quantifying the amount of cells appearing in a sub-G1 peak using fixed and PI-stained cells and flow cytometry. Third, apoptosis-induced DNA degradation was examined by using an assay involving direct lysis of cells in the wells of agarose gels followed by conventional gel electrophoresis. Western blotting was used to quantify the cellular levels of the apoptosis regulators, Bcl-2, Bcl-XL and Bax. Results: Human diploid fibroblasts, which were resistant to radiation induced apoptosis, were found to undergo massive apoptosis when radiation was combined with butyrate treatment. Sensitization was obtained when butyrate was added before or after radiation although the combination of both pre and post-treatment was the most effective. Butyrate was also found to enhance UV light and cisplatin-induced apoptosis. These findings correlated with a reduction of the apoptosis antagonist Bcl-XL. Bcl-XL levels significantly dropped in a time and dose dependent manner. In addition, butyrate effectively blocked UV-induced accumulation of p53. Conclusion: Our results suggest that butyrate may be an attractive agent to use in combination with radiation or chemotherapy to lower the apoptotic threshold of tumor cells, regardless of the p53 status of the tumor cells

FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis.

Science.gov (United States)

Knüppel, Larissa; Heinzelmann, Katharina; Lindner, Michael; Hatz, Rudolf; Behr, Jürgen; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

2018-04-19

In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.

Method of Measuring the Mismatch of Parasitic Capacitance in MEMS Accelerometer Based on Regulating Electrostatic Stiffness

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Xianshan Dong

2018-03-01

Full Text Available For the MEMS capacitive accelerometer, parasitic capacitance is a serious problem. Its mismatch will deteriorate the performance of accelerometer. Obtaining the mismatch of the parasitic capacitance precisely is helpful for improving the performance of bias and scale. Currently, the method of measuring the mismatch is limited in the direct measuring using the instrument. This traditional method has low accuracy for it would lead in extra parasitic capacitive and have other problems. This paper presents a novel method based on the mechanism of a closed-loop accelerometer. The strongly linear relationship between the output of electric force and the square of pre-load voltage is obtained through theoretical derivation and validated by experiment. Based on this relationship, the mismatch of parasitic capacitance can be obtained precisely through regulating electrostatic stiffness without other equipment. The results can be applied in the design of decreasing the mismatch and electrical adjusting for eliminating the influence of the mismatch.

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

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Hoban PR

2006-06-01

Full Text Available Abstract Background Idiopathic Pulmonary Fibrosis (IPF is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. Methods Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. Results Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p Conclusion These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

International Nuclear Information System (INIS)

Jessup, W.; Dean, R.T.

1983-01-01

Normal rates of pinocytosis of [ 3 H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state were apparently identical in normal and cystic fibrosis cultures. (Auth.)

LSODE, 1. Order Stiff or Non-Stiff Ordinary Differential Equations System Initial Value Problems

International Nuclear Information System (INIS)

Hindmarsh, A.C.; Petzold, L.R.

2005-01-01

1 - Description of program or function: LSODE (Livermore Solver for Ordinary Differential Equations) solves stiff and non-stiff systems of the form dy/dt = f. In the stiff case, it treats the Jacobian matrix df/dy as either a dense (full) or a banded matrix, and as either user-supplied or internally approximated by difference quotients. It uses Adams methods (predictor-corrector) in the non-stiff case, and Backward Differentiation Formula (BDF) methods (the Gear methods) in the stiff case. The linear systems that arise are solved by direct methods (LU factor/solve). The LSODE source is commented extensively to facilitate modification. Both a single-precision version and a double-precision version are available. 2 - Methods: It is assumed that the ODEs are given explicitly, so that the system can be written in the form dy/dt = f(t,y), where y is the vector of dependent variables, and t is the independent variable. LSODE contains two variable-order, variable- step (with interpolatory step-changing) integration methods. The first is the implicit Adams or non-stiff method, of orders one through twelve. The second is the backward differentiation or stiff method (or BDF method, or Gear's method), of orders one through five. 3 - Restrictions on the complexity of the problem: The differential equations must be given in explicit form, i.e., dy/dt = f(y,t). Problems with intermittent high-speed transients may cause inefficient or unstable performance

Potensi Terapeutik Fibroblast Growth Factor 21 terhadap Resistensi Insulin

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Kurniasari Kurniasari

2015-12-01

Full Text Available Fibroblast growth factor 21 (FGF21 merupakan salah satu dari anggota FGF yang berperansebagai faktor endokrin. Hepar dan jaringan adiposa merupakan tempat kerja utama FGF21.Ekspresi FGF21 di hepar diatur oleh peroxisome proliferator activated receptor alpha (PPARαsedangkan di jaringan adiposa diatur oleh peroxisome proliferator activated receptor gamma(PPARγ. Kedua faktor transkripsi tersebut terlibat dalam metabolisme karbohidrat dan lipid. Padaresistensi insulin terdapat hiperglikemia, hiperinsulinemia, dan dislipidemia. Pemberian FGF21pada berbagai studi in vivo dan in vitro telah menunjukan potensi FGF21 dalam mengatasi kelainanakibat resistensi insulin sekaligus meningkatkan sensitivitas jaringan terhadap insulin. Kata kunci: FGF21, PPARγ, PPARα, resistensi insulin Fibroblast Growth Factor 21 (FGF21 Potension in InsulinResistance Treatment Abstract Fibroblast growth factor 21 (FGF21 is a member of FGF family that plays a role as endocrinefactor. Liver and adipose tissue are major target of FGF21. The expression of FGF21 in liveris regulated by peroxisome proliferator activated receptor alpha (PPARα, while peroxisomeproliferator activated receptor gamma (PPARγ regulate FGF21 expression in adipose tissue.Both transcription factors are involved in carbohydrate and lipid metabolism. Hyperglycemia,hyperinsulinemia, and dyslipidemia are observed in insulin resistance. Treatment with FGF21 inin vitro and in vivo study showed that FGF21 have the potential to overcome insulin resistance aswell as increasing tissue’s sensitivity towards insulin. Keywords: FGF21, PPARγ, PPARα, insulin resistance Normal 0 false false false IN X-NONE X-NONE

Pentadecapeptide BPC 157 Enhances the Growth Hormone Receptor Expression in Tendon Fibroblasts

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Chung-Hsun Chang

2014-11-01

Full Text Available BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon.

Properties and determination of the interface stiffness

International Nuclear Information System (INIS)

Du Danxu; Zhang Hao; Srolovitz, David J.

2007-01-01

The chemical potential of a curved interface contains a term that is proportional to the product of the interface curvature and the interface stiffness. In crystalline materials, the interface stiffness is a tensor. This paper examines several basic issues related to the properties of the interface stiffness, especially the determination of the interface stiffness in particular directions (i.e. the commonly used scalar form of the interface stiffness). Of the five parameters that describe an arbitrary grain boundary, only those describing the inclination are crucial for the scalar stiffness. We also examine the influence of crystal symmetry on the stiffness tensor for both free surfaces and grain boundaries. This results in substantial simplifications for cases in which interfaces possess mirror or rotational symmetries. An efficient method for determining the interface stiffness tensor using atomistic simulations is proposed

Overexpression of LncRNA AC067945.2 Down-Regulates Collagen Expression in Skin Fibroblasts and Possibly Correlates with the VEGF and Wnt Signalling Pathways.

Science.gov (United States)

Chen, Ling; Li, Jingyun; Li, Qian; Li, Xue; Gao, Yanli; Hua, Xiangdong; Zhou, Bei; Li, Jun

2018-01-01

Long non-coding RNAs (lncRNAs) are thought to play crucial roles in human diseases. However, the function of lncRNAs in hypertrophic scar formation remains poorly understood. Utilizing qRT-PCR, we explored the expression changes of AC067945.2. Overexpression of AC067945.2 in normal skin fibroblasts was performed by transient plasmid transfection. Western blot was used to check the proteins' expression changes. Cell Counting Kit-8 (CCK-8) assay and Annexin V/7-AAD staining were used to examine cell proliferation and apoptosis, respectively. mRNA-seq was applied to dissect the differentially expressed mRNAs in AC067945.2 overexpressed cells. We also performed ELISA to detect the VEGF secretion. AC067945.2 was down-regulated in hypertrophic scar tissues. Overexpression of AC067945.2 did not affect cell proliferation, but it mildly promoted early apoptosis in normal skin fibroblasts. Furthermore, AC067945.2 overexpression inhibited the expression of COL1A1, COL1A2, COL3A1 and α-SMA proteins. Transforming growth factor-β1 (TGF-β1) could inhibit the expression of AC067945.2. Based on mRNA-seq data, compared with mRNAs in the control group, 138 mRNAs were differentially expressed, including 14 up-regulated and 124 down-regulated transcripts, in the AC067945.2 overexpression group. Gene ontology and pathway analyses revealed that AC067945.2 overexpression was correlated with developmental processes, binding, extracellular region, and the vascular endothelial cell growth factor (VEGF) and Wnt signalling pathways. ELISA confirmed that AC067945.2 overexpression could repress VEGF secretion. Taken together, our data uncovered the functions of a novel lncRNA AC067945.2, which might help us understand the mechanisms regulated by AC067945.2 in the pathogenesis of hypertrophic scar formation. © 2018 The Author(s). Published by S. Karger AG, Basel.

Rac1 and Cdc42 are regulators of HRasV12-transformation and angiogenic factors in human fibroblasts

International Nuclear Information System (INIS)

Appledorn, Daniel M; Dao, Kim-Hien T; O'Reilly, Sandra; Maher, Veronica M; McCormick, J Justin

2010-01-01

The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain. Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays. Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions. The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas transformed human cells, including their ability to form tumors in athymic

Rac1 and Cdc42 are regulators of HRasV12-transformation and angiogenic factors in human fibroblasts

Directory of Open Access Journals (Sweden)

Dao Kim-Hien T

2010-01-01

Full Text Available Abstract Background The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain. Methods Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays. Results Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions. Conclusion(s The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas

Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

Science.gov (United States)

Li, Min; Riddle, Suzette; Zhang, Hui; D'Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J; Hansen, Kirk C; Moldvan, Radu; McKeon, B Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Caánovas, Angela; Medrano, Juan F; Thomas, Milton G; Iloska, Dijana; Plecitá-Hlavatá, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A; El Kasmi, Karim C; Zhang, QingHong; Stenmark, Kurt R

2016-10-11

Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). RNA sequencing, quantitative polymerase chain reaction, 13 C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U- 13 C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD + ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD + ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly

Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

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Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

2015-12-01

Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with

A Novel Variable Stiffness Mechanism Capable of an Infinite Stiffness Range and Unlimited Decoupled Output Motion

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Stefan Groothuis

2014-06-01

Full Text Available In this paper, a novel variable stiffness mechanism is presented, which is capable of achieving an output stiffness with infinite range and an unlimited output motion, i.e., the mechanism output is completely decoupled from the rotor motion, in the zero stiffness configuration. The mechanism makes use of leaf springs, which are engaged at different positions by means of two movable supports, to realize the variable output stiffness. The Euler–Bernoulli leaf spring model is derived and validated through experimental data. By shaping the leaf springs, it is shown that the stiffness characteristic of the mechanism can be changed to fulfill different application requirements. Alternative designs can achieve the same behavior with only one leaf spring and one movable support pin.

Stiffness of desiccating insect wings

International Nuclear Information System (INIS)

Mengesha, T E; Vallance, R R; Mittal, R

2011-01-01

The stiffness of insect wings is typically determined through experimental measurements. Such experiments are performed on wings removed from insects. However, the wings are subject to desiccation which typically leads to an increase in their stiffness. Although this effect of desiccation is well known, a comprehensive study of the rate of change in stiffness of desiccating insect wings would be a significant aid in planning experiments as well as interpreting data from such experiments. This communication presents a comprehensive experimental analysis of the change in mass and stiffness of gradually desiccating forewings of Painted Lady butterflies (Vanessa cardui). Mass and stiffness of the forewings of five butterflies were simultaneously measured every 10 min over a 24 h period. The averaged results show that wing mass declined exponentially by 21.1% over this time period with a time constant of 9.8 h, while wing stiffness increased linearly by 46.2% at a rate of 23.4 μN mm -1 h -1 . For the forewings of a single butterfly, the experiment was performed over a period of 1 week, and the results show that wing mass declined exponentially by 52.2% with a time constant of 30.2 h until it reached a steady-state level of 2.00 mg, while wing stiffness increased exponentially by 90.7% until it reached a steady-state level of 1.70 mN mm -1 . (communication)

Stiffness of desiccating insect wings

Energy Technology Data Exchange (ETDEWEB)

Mengesha, T E; Vallance, R R [Department of Mechanical Engineering, The George Washington University, 738 Phillips Hall, 801 22nd St NW, Washington, DC 20052 (United States); Mittal, R, E-mail: vallance@gwu.edu [Department of Mechanical Engineering, Johns Hopkins University, 126 Latrobe Hall, 3400 N Charles Street, Baltimore, MD 21218 (United States)

2011-03-15

The stiffness of insect wings is typically determined through experimental measurements. Such experiments are performed on wings removed from insects. However, the wings are subject to desiccation which typically leads to an increase in their stiffness. Although this effect of desiccation is well known, a comprehensive study of the rate of change in stiffness of desiccating insect wings would be a significant aid in planning experiments as well as interpreting data from such experiments. This communication presents a comprehensive experimental analysis of the change in mass and stiffness of gradually desiccating forewings of Painted Lady butterflies (Vanessa cardui). Mass and stiffness of the forewings of five butterflies were simultaneously measured every 10 min over a 24 h period. The averaged results show that wing mass declined exponentially by 21.1% over this time period with a time constant of 9.8 h, while wing stiffness increased linearly by 46.2% at a rate of 23.4 {mu}N mm{sup -1} h{sup -1}. For the forewings of a single butterfly, the experiment was performed over a period of 1 week, and the results show that wing mass declined exponentially by 52.2% with a time constant of 30.2 h until it reached a steady-state level of 2.00 mg, while wing stiffness increased exponentially by 90.7% until it reached a steady-state level of 1.70 mN mm{sup -1}. (communication)

The microRNA-132/212 family fine-tunes multiple targets in Angiotensin II signalling in cardiac fibroblasts

DEFF Research Database (Denmark)

Eskildsen, Tilde V; Schneider, Mikael; Sandberg, Maria B

2015-01-01

INTRODUCTION: MicroRNAs (miRNAs) are emerging as key regulators of cardiovascular development and disease; however, the cardiac miRNA target molecules are not well understood. We and others have described the Angiotensin II (AngII)-induced miR-132/212 family as novel regulators of cardiovascular...... in silico and in vitro experiments to identify miR-132/212 molecular targets in primary rat cardiac fibroblasts. RESULTS: MiR-132/212 overexpression increased fibroblast cell size and mRNA arrays detected several hundred genes that were differentially expressed, including a wide panel of receptors...... pathways that fine-tuned by miR-132/212, suggesting a role for this miRNA family as master signalling switches in cardiac fibroblasts. Our data underscore the potential for miRNA tools to manipulate a large array of molecules and thereby control biological function....

Increased biosynthesis and processing of fibronectin in fibroblasts from diabetic mice

International Nuclear Information System (INIS)

Phan-Thanh, L.; Robert, L.; Derouette, J.C.; Labat-Robert, J.

1987-01-01

Diabetic connective tissues exhibit a deranged regulation of extracellular matrix biosynthesis. Fibronectin is shown to be increased in human dermal connective tissue by immunofluorescence, mainly at the dermoepidermal and capillary basement membranes. The rate of fibronectin biosynthesis, excretion, and incorporation in a pericellular polymeric form was investigated using genetically diabetic KK mouse skin and fibroblasts as compared to swiss and C57BL mouse skin and fibroblasts. The rate of incorporation of [ 35 S]methionine into proteins recovered in the culture medium or in deoxycholate and NaDodSO 4 or urea extracts was investigated. The rate of incorporation in the medium and deoxycholate extracts was comparable. However, the relative rate of incorporation of the tracer in the NaDodSO 4 -extractable, pericellular polymeric form was increased in the diabetic KK fibroblasts both for total proteins and for fibronectin. In pulse-chase experiments, the deoxycholate-soluble and NaDodSO 4 -soluble fractions exhibited a precursor-product relationship. The rate of passage of fibronectin from the deoxycholate-soluble (cellular compartment) form to the NaDodSO 4 -soluble (pericellular polymeric) form was strongly accelerated in the diabetic fibroblast cultures. These results confirm the increased rate of synthesis of fibronectin in diabetic fibroblasts as well as its processing from the cellular compartment to the polymeric pericellular form

c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

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Grassilli, E; Bellesia, E; Salomoni, P; Croce, M A; Sikora, E; Radziszewska, E; Tesco, G; Vergelli, M; Latorraca, S; Barbieri, D; Fagiolo, U; Santacaterina, S; Amaducci, L; Tiozzo, R; Sorbi, S; Franceschi, C

1996-09-13

In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

DEFF Research Database (Denmark)

Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

2002-01-01

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

Basic Fibroblast Growth Factor Regulates Persistent ERK Osciliations in Premaligant but not Malignant JB6 Cells

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Weber, Thomas J.; Shankaran, Harish; Wiley, H. S.; Opresko, Lee K.; Chrisler, William B.; Quesenberry, Ryan D.

2010-05-02

basic fibroblast growth factor (bFGF or FGF2) plays an important role in epidermal wound healing in vivo and is associated with a persistent increased in the extracellular signal-regulated kinase (ERK) pathway in vitro. Here we have examined whether bFGF induces the closure of an experimental scratch wound in JB6 mouse epidermal cells and have explored the regulation of the ERK pathway by bFGF in the context of kinase oscillations. bFGF stimulation is associated with increases in cellular phospho-ERK and phospho-c-Jun levels. In addition, bFGF increases cell proliferation and a change in cell morphology (stellate appearance) in a dose-dependent fashion (0.1 – 100 ng/ml). bFGF treatment also promoted the closure of an experimental scratch wound in vitro. JB6 cells were stably transfected with an ERK1-GFP chimera to follow temporal ERK subcellular distribution patterns. We observe a persistent upregulation of the ERK pathway, as evidenced by a significant increase in nuclear ERK1-GFP levels at time points up to 24 hr after bFGF treatment. Interestingly, at the single cell level, ERK is observed to oscillate between nuclear and cytosolic compartments in response to bFGF treatment. Because this oscillatory behavior is asynchronous in the cell population, it is only clearly resolved at the single cell level. Collectively, data presented here are consistent with an important role for bFGF in wound healing and suggest a more complex regulation of the ERK pathway by bFGF than has previously been appreciated.

Shoulder Stiffness : Current Concepts and Concerns

NARCIS (Netherlands)

Itoi, Eiji; Arce, Guillermo; Bain, Gregory I.; Diercks, Ronald L.; Guttmann, Dan; Imhoff, Andreas B.; Mazzocca, Augustus D.; Sugaya, Hiroyuki; Yoo, Yon-Sik

Shoulder stiffness can be caused by various etiologies such as immobilization, trauma, or surgical interventions. The Upper Extremity Committee of ISAKOS defined the term "frozen shoulder" as idiopathic stiff shoulder, that is, without a known cause. Secondary stiff shoulder is a term that should be

Expression of tenascin-C in a rat vocal fold injury model and its regulation of fibroblasts.

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Li, Juan; Liu, Yiqiong; Wang, Yiming; Xu, Wen

2018-03-23

Tenascin-C (Tnc) is an extracellular matrix (ECM) glycoprotein that plays a vital role in wound healing and fibrotic disease. Tnc is highly upregulated soon after vocal fold injury, but its function in the vocal fold has not yet been defined. In this study, we investigated Tnc expression in a rat vocal fold injury model in vivo and its roles in fibroblasts in vitro. In vivo and in vitro. Tnc mRNA and protein expression levels were quantified on days 3, 7, 14, 28, and 56 after vocal fold injury in Sprague-Dawley rats. In vitro, immunocytochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed in primary rat vocal fold fibroblasts following Tnc or transforming growth factor (TGF)-β1 stimulation to investigate the phenotypic effects. Tnc mRNA and protein expression was upregulated dramatically on days 3 and 7 after injury, and significant differences were observed by qRT-PCR (P vocal fold fibroblasts. Following incubation with Tnc for 72 hours, α-smooth muscle actin, collagen I, and fibronectin expression was significantly upregulated (P vocal fold fibroblast migration, transdifferentiation, and ECM protein synthesis in vitro. Tnc was induced by TGF-β1 in a SMAD3-dependent manner. Transient expression of Tnc is likely to promote regeneration, but its potential role in fibrosis requires further study. NA Laryngoscope, 2018. © 2018 The American Laryngological, Rhinological and Otological Society, Inc.

The link between exercise and titin passive stiffness.

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Lalande, Sophie; Mueller, Patrick J; Chung, Charles S

2017-09-01

What is the topic of this review? This review focuses on how in vivo and molecular measurements of cardiac passive stiffness can predict exercise tolerance and how exercise training can reduce cardiac passive stiffness. What advances does it highlight? This review highlights advances in understanding the relationship between molecular (titin-based) and in vivo (left ventricular) passive stiffness, how passive stiffness modifies exercise tolerance, and how exercise training may be therapeutic for cardiac diseases with increased passive stiffness. Exercise can help alleviate the negative effects of cardiovascular disease and cardiovascular co-morbidities associated with sedentary behaviour; this may be especially true in diseases that are associated with increased left ventricular passive stiffness. In this review, we discuss the inverse relationship between exercise tolerance and cardiac passive stiffness. Passive stiffness is the physical property of cardiac muscle to produce a resistive force when stretched, which, in vivo, is measured using the left ventricular end diastolic pressure-volume relationship or is estimated using echocardiography. The giant elastic protein titin is the major contributor to passive stiffness at physiological muscle (sarcomere) lengths. Passive stiffness can be modified by altering titin isoform size or by post-translational modifications. In both human and animal models, increased left ventricular passive stiffness is associated with reduced exercise tolerance due to impaired diastolic filling, suggesting that increased passive stiffness predicts reduced exercise tolerance. At the same time, exercise training itself may induce both short- and long-term changes in titin-based passive stiffness, suggesting that exercise may be a treatment for diseases associated with increased passive stiffness. Direct modification of passive stiffness to improve exercise tolerance is a potential therapeutic approach. Titin passive stiffness itself may

Mechanosensation across borders: fibroblasts inside a macroporous scaffold sense and respond to the mechanical environment beyond the scaffold walls.

Science.gov (United States)

Könnig, D; Herrera, A; Duda, G N; Petersen, A

2018-01-01

In tissue defects, cells face distinct mechanical boundary conditions, but how this influences early stages of tissue regeneration remains largely unknown. Biomaterials are used to fill defects but also to provide specific mechanical or geometrical signals. However, they might at the same time shield mechanical information from surrounding tissues that is relevant for tissue functionalisation. This study investigated how fibroblasts in a soft macroporous biomaterial scaffold respond to distinct mechanical environments while they form microtissues. Different boundary stiffnesses counteracting scaffold contraction were provided via a newly developed in vitro setup. Online monitoring over 14 days revealed 3.0 times lower microtissue contraction but 1.6 times higher contraction force for high vs. low stiffness. This difference was significant already after 48 h, a very early stage of microtissue growth. The microtissue's mechanical and geometrical adaptation indicated a collective cellular behaviour and mechanical communication across scaffold pore walls. Surprisingly, the stiffness of the environment influenced cell behaviour even inside macroporous scaffolds where direct cell-cell contacts are hindered. Mechanical communication between cells via traction forces is essential for tissue adaptation to the environment and should not be blocked by rigid biomaterials. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Efficient Method for Calculating the Composite Stiffness of Parabolic Leaf Springs with Variable Stiffness for Vehicle Rear Suspension

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Wen-ku Shi

2016-01-01

Full Text Available The composite stiffness of parabolic leaf springs with variable stiffness is difficult to calculate using traditional integral equations. Numerical integration or FEA may be used but will require computer-aided software and long calculation times. An efficient method for calculating the composite stiffness of parabolic leaf springs with variable stiffness is developed and evaluated to reduce the complexity of calculation and shorten the calculation time. A simplified model for double-leaf springs with variable stiffness is built, and a composite stiffness calculation method for the model is derived using displacement superposition and material deformation continuity. The proposed method can be applied on triple-leaf and multileaf springs. The accuracy of the calculation method is verified by the rig test and FEA analysis. Finally, several parameters that should be considered during the design process of springs are discussed. The rig test and FEA analytical results indicate that the calculated results are acceptable. The proposed method can provide guidance for the design and production of parabolic leaf springs with variable stiffness. The composite stiffness of the leaf spring can be calculated quickly and accurately when the basic parameters of the leaf spring are known.

Comparison of the transcriptomes of mouse skin derived precursors (SKPs and SKP-derived fibroblasts (SFBs by RNA-Seq.

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Yujie Mao

Full Text Available Skin-derived precursors (SKPs from dermis possess the capacities of self-renewal and multipotency. In vitro and in vivo studies demonstrated that they can differentiate into fibroblasts. However, little is known about the molecular mechanism of the differentiation of SKPs into fibroblasts. Here we compare the transcriptomes of mouse SKPs and SKP-derived fibroblasts (SFBs by RNA-Seq analysis, trying to find differences in gene expression between the two kinds of cells and then elucidate the candidate genes that may play important roles in the differentiation of SKPs into fibroblasts. A total of 1971 differentially expressed genes (DEGs were identified by RNA-Seq, which provided abundant data for further analysis. Gene Ontology enrichment analysis revealed that genes related to cell differentiation, cell proliferation, protein binding, transporter activity and membrane were significantly enriched. The most significantly up-regulated genes Wnt4, Wisp2 and Tsp-1 and down-regulated genes Slitrk1, Klk6, Agtr2, Ivl, Msx1, IL15, Atp6v0d2, Kcne1l and Thbs4 may play important roles in the differentiation of SKPs into fibroblasts. KEGG analysis showed that DEGs were significantly enriched in the TGF-β signaling pathway, Wnt signaling pathway and Notch signaling pathway, which have been previously proven to regulate the differentiation and self-renewal of various stem cells. These identified DEGs and pathways could facilitate further investigations of the detailed molecular mechanisms, making it possible to take advantage of the potential therapeutic applications of SKPs in skin regeneration in the future.

Cyclic stress at mHz frequencies aligns fibroblasts in direction of zero strain.

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Uta Faust

Full Text Available Recognition of external mechanical signals is vital for mammalian cells. Cyclic stretch, e.g. around blood vessels, is one such signal that induces cell reorientation from parallel to almost perpendicular to the direction of stretch. Here, we present quantitative analyses of both, cell and cytoskeletal reorientation of umbilical cord fibroblasts. Cyclic strain of preset amplitudes was applied at mHz frequencies. Elastomeric chambers were specifically designed and characterized to distinguish between zero strain and minimal stress directions and to allow accurate theoretical modeling. Reorientation was only induced when the applied stretch exceeded a specific amplitude, suggesting a non-linear response. However, on very soft substrates no mechanoresponse occurs even for high strain. For all stretch amplitudes, the angular distributions of reoriented cells are in very good agreement with a theory modeling stretched cells as active force dipoles. Cyclic stretch increases the number of stress fibers and the coupling to adhesions. We show that changes in cell shape follow cytoskeletal reorientation with a significant temporal delay. Our data identify the importance of environmental stiffness for cell reorientation, here in direction of zero strain. These in vitro experiments on cultured cells argue for the necessity of rather stiff environmental conditions to induce cellular reorientation in mammalian tissues.

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

NARCIS (Netherlands)

Honing, van der H.S.; Ruijter, de N.C.A.; Emons, A.M.C.; Ketelaar, T.

2010-01-01

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm.

The hallmarks of fibroblast ageing.

Science.gov (United States)

Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

2014-06-01

Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Hydration Status Is Associated with Aortic Stiffness, but Not with Peripheral Arterial Stiffness, in Chronically Hemodialysed Patients

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Daniel Bia

2015-01-01

Full Text Available Background. Adequate fluid management could be essential to minimize high arterial stiffness observed in chronically hemodialyzed patients (CHP. Aim. To determine the association between body fluid status and central and peripheral arterial stiffness levels. Methods. Arterial stiffness was assessed in 65 CHP by measuring the pulse wave velocity (PWV in a central arterial pathway (carotid-femoral and in a peripheral pathway (carotid-brachial. A blood pressure-independent regional arterial stiffness index was calculated using PWV. Volume status was assessed by whole-body multiple-frequency bioimpedance. Patients were first observed as an entire group and then divided into three different fluid status-related groups: normal, overhydration, and dehydration groups. Results. Only carotid-femoral stiffness was positively associated (P<0.05 with the hydration status evaluated through extracellular/intracellular fluid, extracellular/Total Body Fluid, and absolute and relative overhydration. Conclusion. Volume status and overload are associated with central, but not peripheral, arterial stiffness levels with independence of the blood pressure level, in CHP.

Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

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Ioana Alesutan

2013-09-01

Full Text Available Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc, is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM and cell morphology analysed by scanning ion conductance microscopy (SICM. Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

Fibroblast growth factor receptors in breast cancer.

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Wang, Shuwei; Ding, Zhongyang

2017-05-01

Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

International Nuclear Information System (INIS)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

2005-01-01

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

Lack of co-ordinate expression of the alpha1(I) and alpha1(III) procollagen genes in fibroblast clonal cultures.

Science.gov (United States)

Yamaguchi, Y; Crane, S; Zhou, L; Ochoa, S M; Falanga, V

2000-12-01

Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.

Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Co-Repressor C-terminal Binding Protein-1

Science.gov (United States)

Li, Min; Riddle, Suzette; Zhang, Hui; D’Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J.; Hansen, Kirk C.; Moldvan, Radu; McKeon, B. Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Cánovas, Angela; Medrano, Juan F.; Thomas, Milton G.; Iloska, Dijana; Plecita-Hlavata, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A.; El Kasmi, Karim C.; Zhang, Qinghong; Stenmark, Kurt R.

2016-01-01

Background Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells including fibroblasts in pulmonary hypertension (PH). Herein, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and pro-inflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional co-repressor C-terminal binding protein 1 (CtBP1). Methods RNA-Sequencing, qPCR, 13C-NMR, fluorescence-lifetime imaging, mass spectrometry-based metabolomics and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure NADH/NAD+ ratio in bovine and human adventitial fibroblasts, and mouse lung tissues. Immunohistochemistry was utilized to assess CtBP1 expression in the whole lung tissues. CtBP1 siRNA and the pharmacologic inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were utilized to abrogate CtBP1 activity in cells and hypoxic mice. Results We found adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with IPAH (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with IPAH and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB, or genetically blocking CtBP1 using siRNA, upregulated the cyclin-dependent genes (p15 and p21) and pro-apoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. ChIP analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated

VCODE, Ordinary Differential Equation Solver for Stiff and Non-Stiff Problems

International Nuclear Information System (INIS)

Cohen, Scott D.; Hindmarsh, Alan C.

2001-01-01

1 - Description of program or function: CVODE is a package written in ANSI standard C for solving initial value problems for ordinary differential equations. It solves both stiff and non stiff systems. In the stiff case, it includes a variety of options for treating the Jacobian of the system, including dense and band matrix solvers, and a preconditioned Krylov (iterative) solver. 2 - Method of solution: Integration is by Adams or BDF (Backward Differentiation Formula) methods, at user option. Corrector iteration is by functional iteration or Newton iteration. For the solution of linear systems within Newton iteration, users can select a dense solver, a band solver, a diagonal approximation, or a preconditioned Generalized Minimal Residual (GMRES) solver. In the dense and band cases, the user can supply a Jacobian approximation or let CVODE generate it internally. In the GMRES case, the pre-conditioner is user-supplied

The mechanical memory of lung myofibroblasts.

Science.gov (United States)

Balestrini, Jenna L; Chaudhry, Sidharth; Sarrazy, Vincent; Koehler, Anne; Hinz, Boris

2012-04-01

Fibroblasts differentiate into the highly synthetic and contractile myofibroblast phenotype when exposed to substrates with an elastic modulus corresponding to pathologically stiff fibrotic tissue. Cellular responses to changes in substrate stiffness are typically analyzed after hours or days, which does not enable the monitoring of myofibroblast persistence, a hallmark of fibrosis. To determine long-lasting effects on the fibrotic behavior of lung fibroblasts, we followed a novel approach of explanting and repeatedly passaging fibroblasts on silicone substrates with stiffness representing various states of lung health. Fibrotic activity was determined by assaying for myofibroblast proliferation, cell contractility, expression of α-smooth muscle actin, extracellular matrix and active TGFβ1. As predicted, myofibroblast activity was low on healthy soft substrates and increased with increasing substrate stiffness. However, explanting and mechanically priming lung fibroblasts for 3 weeks on pathologically stiff substrates resulted in sustained myofibroblast activity even after the cells were returned to healthy soft cultures for 2 weeks. Such primed cells retained higher fibrotic activity than cells that had been exclusively cultured on soft substrates, and were not statistically different from cells continuously passaged on stiff surfaces. Inversely, priming lung fibroblasts for 3 weeks on soft substrates partially protected from myofibroblast activation after the shift to stiff substrates. Hence, mechano-sensed information relating to physical conditions of the local cellular environment could permanently induce fibrotic behavior of lung fibroblasts. This priming effect has important implications for the progression and persistence of aggressive fibrotic diseases such as idiopathic pulmonary fibrosis. This journal is © The Royal Society of Chemistry 2012

The effects of acoustic vibration on fibroblast cell migration.

Science.gov (United States)

Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

2016-12-01

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

An acoustic startle alters knee joint stiffness and neuromuscular control.

Science.gov (United States)

DeAngelis, A I; Needle, A R; Kaminski, T W; Royer, T R; Knight, C A; Swanik, C B

2015-08-01

Growing evidence suggests that the nervous system contributes to non-contact knee ligament injury, but limited evidence has measured the effect of extrinsic events on joint stability. Following unanticipated events, the startle reflex leads to universal stiffening of the limbs, but no studies have investigated how an acoustic startle influences knee stiffness and muscle activation during a dynamic knee perturbation. Thirty-six individuals were tested for knee stiffness and muscle activation of the quadriceps and hamstrings. Subjects were seated and instructed to resist a 40-degree knee flexion perturbation from a relaxed state. During some trials, an acoustic startle (50 ms, 1000 Hz, 100 dB) was applied 100 ms prior to the perturbation. Knee stiffness, muscle amplitude, and timing were quantified across time, muscle, and startle conditions. The acoustic startle increased short-range (no startle: 0.044 ± 0.011 N·m/deg/kg; average startle: 0.047 ± 0.01 N·m/deg/kg) and total knee stiffness (no startle: 0.036 ± 0.01 N·m/deg/kg; first startle 0.027 ± 0.02 N·m/deg/kg). Additionally, the startle contributed to decreased [vastus medialis (VM): 13.76 ± 33.6%; vastus lateralis (VL): 6.72 ± 37.4%] but earlier (VM: 0.133 ± 0.17 s; VL: 0.124 ± 0.17 s) activation of the quadriceps muscles. The results of this study indicate that the startle response can significantly disrupt knee stiffness regulation required to maintain joint stability. Further studies should explore the role of unanticipated events on unintentional injury. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Soft Robotic Haptic Interface with Variable Stiffness for Rehabilitation of Neurologically Impaired Hand Function

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Frederick Sebastian

2017-12-01

Full Text Available The human hand comprises complex sensorimotor functions that can be impaired by neurological diseases and traumatic injuries. Effective rehabilitation can bring the impaired hand back to a functional state because of the plasticity of the central nervous system to relearn and remodel the lost synapses in the brain. Current rehabilitation therapies focus on strengthening motor skills, such as grasping, employ multiple objects of varying stiffness so that affected persons can experience a wide range of strength training. These devices have limited range of stiffness due to the rigid mechanisms employed in their variable stiffness actuators. This paper presents a novel soft robotic haptic device for neuromuscular rehabilitation of the hand, which is designed to offer adjustable stiffness and can be utilized in both clinical and home settings. The device eliminates the need for multiple objects by employing a pneumatic soft structure made with highly compliant materials that act as the actuator of the haptic interface. It is made with interchangeable sleeves that can be customized to include materials of varying stiffness to increase the upper limit of the stiffness range. The device is fabricated using existing 3D printing technologies, and polymer molding and casting techniques, thus keeping the cost low and throughput high. The haptic interface is linked to either an open-loop system that allows for an increased pressure during usage or closed-loop system that provides pressure regulation in accordance to the stiffness the user specifies. Preliminary evaluation is performed to characterize the effective controllable region of variance in stiffness. It was found that the region of controllable stiffness was between points 3 and 7, where the stiffness appeared to plateau with each increase in pressure. The two control systems are tested to derive relationships between internal pressure, grasping force exertion on the surface, and displacement using

Preparation and biological evaluation of a fibroblast growth factor-2-apatite composite layer on polymeric material

Energy Technology Data Exchange (ETDEWEB)

Sasaki, Kenkichi; Kamitakahara, Masanobu; Ioku, Koji [Graduate School of Environmental Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Oyane, Ayako [Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Hyodo, Koji [Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-2-1 Namiki, Tsukuba, Ibaraki 305-8564 (Japan); Ito, Atsuo; Sogo, Yu, E-mail: a-oyane@aist.go.j [Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566 (Japan)

2010-12-15

A polymeric percutaneous device with good biocompatibility and resistance to bacterial infection is required clinically. In this study, a fibroblast growth factor-2 (FGF-2)-hydroxyapatite (HAp) composite layer (FHAp layer) was formed on the surfaces of ethylene-vinyl alcohol copolymer (EVOH) specimens using a coating process in a supersaturated calcium phosphate solution supplemented with FGF-2. FGF-2 in the FHAp layer retained its biological activity to promote proliferation of fibroblasts. The EVOH specimens coated with HAp and FHAp layers were percutaneously implanted in the scalp of rats. Not only the HAp layer but also the FHAp layer showed good biocompatibility, and FGF-2 showed no harmful effects on the skin tissue responses to the implanted specimen as long as 14 d. No significantly higher infection resistance was verified for the FHAp layer over the HAp layer, although an FHAp layer coated on a metallic percutaneous device for bone fixation demonstrated higher resistance to bacterial infection over an HAp layer in the previous study. The efficacy of FHAp layers coated on percutaneous implants in resistance to bacterial infection depends on physical factors including fixation condition, stiffness and movement of implants.

Krüppel-Like Factor 4 Is a Regulator of Proinflammatory Signaling in Fibroblast-Like Synoviocytes through Increased IL-6 Expression

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Xinjing Luo

2016-01-01

Full Text Available Human fibroblast-like synoviocytes play a vital role in joint synovial inflammation in rheumatoid arthritis (RA. Proinflammatory cytokines induce fibroblast-like synoviocyte activation and dysfunction. The inflammatory mediator Krüppel-like factor 4 is upregulated during inflammation and plays an important role in endothelial and macrophage activation during inflammation. However, the role of Krüppel-like factor 4 in fibroblast-like synoviocyte activation and RA inflammation remains to be defined. In this study, we identify the notion that Krüppel-like factor 4 is higher expressed in synovial tissues and fibroblast-like synoviocytes from RA patients than those from osteoarthritis patients. In vitro, the expression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes is induced by proinflammatory cytokine tumor necrosis factor-α. Overexpression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes robustly induced interleukin-6 production in the presence or absence of tumor necrosis factor-α. Conversely, knockdown of Krüppel-like factor 4 markedly attenuated interleukin-6 production in the presence or absence of tumor necrosis factor-α. Krüppel-like factor 4 not only can bind to and activate the interleukin-6 promoter, but also may interact directly with nuclear factor-kappa B. These results suggest that Krüppel-like factor 4 may act as a transcription factor mediating the activation of fibroblast-like synoviocytes in RA by inducing interleukin-6 expression in response to tumor necrosis factor-α.

Real-Time Vision-Based Stiffness Mapping †.

Science.gov (United States)

Faragasso, Angela; Bimbo, João; Stilli, Agostino; Wurdemann, Helge Arne; Althoefer, Kaspar; Asama, Hajime

2018-04-26

This paper presents new findings concerning a hand-held stiffness probe for the medical diagnosis of abnormalities during palpation of soft-tissue. Palpation is recognized by the medical community as an essential and low-cost method to detect and diagnose disease in soft-tissue. However, differences are often subtle and clinicians need to train for many years before they can conduct a reliable diagnosis. The probe presented here fills this gap providing a means to easily obtain stiffness values of soft tissue during a palpation procedure. Our stiffness sensor is equipped with a multi degree of freedom (DoF) Aurora magnetic tracker, allowing us to track and record the 3D position of the probe whilst examining a tissue area, and generate a 3D stiffness map in real-time. The stiffness probe was integrated in a robotic arm and tested in an artificial environment representing a good model of soft tissue organs; the results show that the sensor can accurately measure and map the stiffness of a silicon phantom embedded with areas of varying stiffness.

Real-Time Vision-Based Stiffness Mapping †

Directory of Open Access Journals (Sweden)

Angela Faragasso

2018-04-01

Full Text Available This paper presents new findings concerning a hand-held stiffness probe for the medical diagnosis of abnormalities during palpation of soft-tissue. Palpation is recognized by the medical community as an essential and low-cost method to detect and diagnose disease in soft-tissue. However, differences are often subtle and clinicians need to train for many years before they can conduct a reliable diagnosis. The probe presented here fills this gap providing a means to easily obtain stiffness values of soft tissue during a palpation procedure. Our stiffness sensor is equipped with a multi degree of freedom (DoF Aurora magnetic tracker, allowing us to track and record the 3D position of the probe whilst examining a tissue area, and generate a 3D stiffness map in real-time. The stiffness probe was integrated in a robotic arm and tested in an artificial environment representing a good model of soft tissue organs; the results show that the sensor can accurately measure and map the stiffness of a silicon phantom embedded with areas of varying stiffness.

Dynamic stiffness of suction caissons - vertical vibrations

Energy Technology Data Exchange (ETDEWEB)

Ibsen, Lars Bo; Liingaard, M.; Andersen, Lars

2006-12-15

The dynamic response of offshore wind turbines are affected by the properties of the foundation and the subsoil. The purpose of this report is to evaluate the dynamic soil-structure interaction of suction caissons for offshore wind turbines. The investigation is limited to a determination of the vertical dynamic stiffness of suction caissons. The soil surrounding the foundation is homogenous with linear viscoelastic properties. The dynamic stiffness of the suction caisson is expressed by dimensionless frequency-dependent dynamic stiffness coefficients corresponding to the vertical degree of freedom. The dynamic stiffness coefficients for the foundations are evaluated by means of a dynamic three-dimensional coupled Boundary Element/Finite Element model. Comparisons are made with known analytical and numerical solutions in order to evaluate the static and dynamic behaviour of the Boundary Element/Finite Element model. The vertical frequency dependent stiffness has been determined for different combinations of the skirt length, Poisson's ratio and the ratio between soil stiffness and skirt stiffness. Finally the dynamic behaviour at high frequencies is investigated. (au)

IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells?

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Jennifer R Bailey

Full Text Available BACKGROUND: Fibrosis is a serious consequence of Crohn's disease (CD, often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. METHODS: Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f CD and compared with cancer control (C, ulcerative colitis (UC and uninvolved (u CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants. RESULTS: In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R α1 was expressed by intestinal muscle smooth muscle, nerve and KIR(+ cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR(+CD45(+CD56(+/-CD3(- were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13. CONCLUSIONS: The data indicate that in fibrotic intestinal muscle of Crohn's patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1(+, KIR(+ innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Structure of rat acidic fibroblast growth factor at 1.4 A resolution

DEFF Research Database (Denmark)

Kulahin, Nikolaj; Kiselyov, Vladislav; Kochoyan, Artur

2007-01-01

Fibroblast growth factors (FGFs) constitute a family of 22 structurally related heparin-binding polypeptides that are involved in the regulation of cell growth, survival, differentiation and migration. Here, a 1.4 A resolution X-ray structure of rat FGF1 is presented. Two molecules are present...

Observer-Based Human Knee Stiffness Estimation.

Science.gov (United States)

Misgeld, Berno J E; Luken, Markus; Riener, Robert; Leonhardt, Steffen

2017-05-01

We consider the problem of stiffness estimation for the human knee joint during motion in the sagittal plane. The new stiffness estimator uses a nonlinear reduced-order biomechanical model and a body sensor network (BSN). The developed model is based on a two-dimensional knee kinematics approach to calculate the angle-dependent lever arms and the torques of the muscle-tendon-complex. To minimize errors in the knee stiffness estimation procedure that result from model uncertainties, a nonlinear observer is developed. The observer uses the electromyogram (EMG) of involved muscles as input signals and the segmental orientation as the output signal to correct the observer-internal states. Because of dominating model nonlinearities and nonsmoothness of the corresponding nonlinear functions, an unscented Kalman filter is designed to compute and update the observer feedback (Kalman) gain matrix. The observer-based stiffness estimation algorithm is subsequently evaluated in simulations and in a test bench, specifically designed to provide robotic movement support for the human knee joint. In silico and experimental validation underline the good performance of the knee stiffness estimation even in the cases of a knee stiffening due to antagonistic coactivation. We have shown the principle function of an observer-based approach to knee stiffness estimation that employs EMG signals and segmental orientation provided by our own IPANEMA BSN. The presented approach makes realtime, model-based estimation of knee stiffness with minimal instrumentation possible.

Inhibition of fibroblast growth by Notch1 signaling is mediated by induction of Wnt11-dependent WISP-1.

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Zhao-Jun Liu

Full Text Available Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM. They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox embryonic fibroblasts (MEFs. Notch1-deficient (Notch1(-/- MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1 in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441, which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1. Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4 in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

Science.gov (United States)

McCoy, Sara S; Reed, Tamra J; Berthier, Celine C; Tsou, Pei-Suen; Liu, Jianhua; Gudjonsson, Johann E; Khanna, Dinesh; Kahlenberg, J Michelle

2017-11-01

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Icariin Regulates Cellular Functions and Gene Expression of Osteoarthritis Patient-Derived Human Fibroblast-Like Synoviocytes

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Lianhong Pan

2017-12-01

Full Text Available Synovial inflammation plays an important role in the pathogenesis and progress of osteoarthritis (OA. There is an urgent need to find safe and effective drugs that can reduce the inflammation and regulate the pathogenesis of cytokines of the OA disease. Here, we investigated the effect of icariin, the major pharmacological active component of herb Epimedium on human osteoarthritis fibroblast-like synoviocytes (OA–FLSs. The OA–FLSs were isolated from patients with osteoarthritis and cultured in vitro with different concentrations of icariin. Then, cell viability, proliferation, and migration were investigated; MMP14, GRP78, and IL-1β gene expression levels were detected via qRT-PCR. Icariin showed low cytotoxicity to OA–FLSs at a concentration of under 10 μM and decreased the proliferation of the cells at concentrations of 1 and 10 μM. Icariin inhibited cell migration with concentrations ranging from 0.1 to 1 μM. Also, the expression of three cytokines for the pathogenesis of OA which include IL-1β, MMP14 and GRP78 was decreased by the various concentrations of icariin. These preliminary results imply that icariin might be an effective compound for the treatment of OA disease.

Directional cell migration and chemotaxis in wound healing response to PDGF-AA are coordinated by the primary cilium in fibroblasts

DEFF Research Database (Denmark)

Schneider, Linda; Cammer, Michael; Lehman, Jonathan

2010-01-01

Cell motility and migration play pivotal roles in numerous physiological and pathophysiological processes including development and tissue repair. Cell migration is regulated through external stimuli such as platelet-derived growth factor-AA (PDGF-AA), a key regulator in directional cell migration....... Here we used micropipette analysis to show that a normal chemosensory response to PDGF-AA in fibroblasts requires the primary cilium. In vitro and in vivo wound healing assays revealed that in ORPK mouse (IFT88(Tg737Rpw)) fibroblasts, where ciliary assembly is defective, chemotaxis towards PDGF-AA...

Curcumin induces apoptosis and inhibits prostaglandin E(2) production in synovial fibroblasts of patients with rheumatoid arthritis.

Science.gov (United States)

Park, Cheol; Moon, Dong-Oh; Choi, Il-Whan; Choi, Byung Tae; Nam, Taek-Jeong; Rhu, Chung-Ho; Kwon, Taeg Kyu; Lee, Won Ho; Kim, Gi-Young; Choi, Yung Hyun

2007-09-01

Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by hyperplasia of the synovial fibroblasts, which is partly the result of decreased apoptosis. This study investigated the mechanisms through which curcumin, a polyphenolic compound from the rhizome of Curcuma longa, exerts its anti-proliferative action in the synovial fibroblasts obtained from patients with RA. Exposure of the synovial fibroblasts to curcumin resulted in growth inhibition and the induction of apoptosis, as measured by MTT assay, fluorescent microscopy and Annexin-V-based assay. RT-PCR and immunoblotting showed that treating the cells with curcumin resulted in the down-regulation of anti-apoptotic Bcl-2 and the X-linked inhibitor of the apoptosis protein as well as the up-regulation of pro-apoptotic Bax expression in a concentration-dependent manner. Curcumin-induced apoptosis was also associated with the proteolytic activation of caspase-3 and caspase-9, and the concomitant degradation of poly(ADP-ribose) polymerase protein. Furthermore, curcumin decreased the expression levels of the cyclooxygenase (COX)-2 mRNA and protein without causing significant changes in the COX-1 levels, which was correlated with the inhibition of prostaglandin E(2) synthesis. These results show that curcumin might help identify a new therapeutic pathway against hyperplasia of the synovial fibroblasts in RA.

Optimization of a quasi-zero-stiffness isolator

International Nuclear Information System (INIS)

Carrella, A.; Brennan, M. J.; Waters, T. P.

2007-01-01

The frequency range over which a mount can isolate a mass from a vibrating base (or vice versa) is often limited by the mount stiffness required to support the weight of the mass. This compromise can be made more favourable by employing non-linear mounts with a softening spring characteristic such that small excursions about the static equilibrium position result in small dynamic spring forces and a correspondingly low natural frequency. This paper concerns the force-displacement characteristic of a so-called quasi-zero-stiffness (QZS) mechanism which is characterised by an appreciable static stiffness but very small (theoretically zero) dynamic stiffness. The mechanism studied comprises a vertical spring acting in parallel with two further springs which, when inclined at an appropriate angle to the vertical, produce a cancelling negative stiffness effect. Analysis of the system shows that a QZS characteristic can be obtained if the systems parameters (angle of inclination and ratio of spring stiffness) are opportunely chosen. By introducing the additional criterion that the displacement of the system be largest without exceeding a desired (low) value of stiffness an optimal set of parameter values is derived. Under sufficiently large displacements the stiffness of the QZS mechanism can eventually exceed that of the simple mass-spring system and criteria for this detrimental scenario to arise are presented

Progranulin Overproduction Due to Fli-1 Deficiency Contributes to the Resistance of Dermal Fibroblasts to Tumor Necrosis Factor in Systemic Sclerosis.

Science.gov (United States)

Ichimura, Yohei; Asano, Yoshihide; Akamata, Kaname; Noda, Shinji; Taniguchi, Takashi; Takahashi, Takehiro; Toyama, Tetsuo; Tada, Yayoi; Sugaya, Makoto; Sato, Shinichi; Kadono, Takafumi

2015-12-01

Progranulin is a growth factor that is active in wound repair and is an antagonist of tumor necrosis factor (TNF) receptors, regulating fibroblast activation, angiogenesis, and inflammation. Because long-standing activation of gene programs related to wound healing is a hallmark of systemic sclerosis (SSc), we sought to investigate the role of progranulin in SSc. Progranulin expression levels in human and murine skin samples were determined by immunohistochemical analysis and quantitative reverse transcription-polymerase chain reaction. The role of progranulin in fibroblast activation was examined using a gene-silencing technique. Progranulin levels in serum obtained from 60 patients with SSc and 16 healthy control subjects were determined by enzyme-linked immunosorbent assay. Progranulin expression was increased in SSc dermal fibroblasts compared with normal dermal fibroblasts, both in vivo and in vitro. Transcription factor Fli-1, a deficiency of which is involved in the activation of SSc dermal fibroblasts, served as a potent repressor of the progranulin gene, and Fli-1(+/-) mice and bleomycin-treated wild-type mice exhibited up-regulated expression of progranulin in dermal fibroblasts. SSc dermal fibroblasts were resistant to the antifibrotic effect of TNF, but this resistance was reversed by gene silencing of progranulin. Serum progranulin levels were elevated in patients with early diffuse cutaneous SSc (dcSSc), especially in those with inflammatory skin symptoms, and were positively correlated with the C-reactive protein level. Progranulin overproduction due to Fli-1 deficiency may contribute to the constitutive activation of SSc dermal fibroblasts by antagonizing the antifibrotic effect of TNF. Progranulin may also be involved in the inflammatory process associated with progressive skin sclerosis in early dcSSc. © 2015, American College of Rheumatology.

The dependence of MG63 osteoblast responses to (meth)acrylate-based networks on chemical structure and stiffness.

Science.gov (United States)

Smith, Kathryn E; Hyzy, Sharon L; Sunwoo, Moonhae; Gall, Ken A; Schwartz, Zvi; Boyan, Barbara D

2010-08-01

The cell response to an implant is regulated by the implant's surface properties including topography and chemistry, but less is known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene glycol) dimethacrylate (PEGDMA; Mn approximately 750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60 MPa. To further decouple chemistry and stiffness, three networks comprised of 2-hydroxyethyl methacrylate (2HEMA) and PEGDMA or DEGDMA were also designed that exhibited a range of moduli similar to the PEGDMA-DEGDMA networks. MG63 cells were cultured on each surface and tissue culture polystyrene (TCPS), and the effect of copolymer composition on cell number, osteogenic markers (alkaline phosphatase specific activity and osteocalcin), and local growth factor production (OPG, TGF-beta1, and VEGF-A) were assessed. Cells exhibited a more differentiated phenotype on the PEGDMA-DEGDMA copolymers compared to the 2HEMA-PEGDMA copolymers. On the PEGDMA-DEGDMA system, cells exhibited a more differentiated phenotype on the stiffest surface indicated by elevated osteocalcin compared with TCPS. Conversely, cells on 2HEMA-PEGDMA copolymers became more differentiated on the less stiff 2HEMA surface. Growth factors were regulated in a differential manner. These results indicate that copolymer chemistry is the primary regulator of osteoblast differentiation, and the effect of stiffness is secondary to the surface chemistry. 2010 Elsevier Ltd. All rights reserved.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

Science.gov (United States)

Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

2011-08-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

Science.gov (United States)

Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

2011-01-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

Science.gov (United States)

Fujiwara, Toshihiro; Dohi, Teruyuki; Maan, Zeshaan N; Rustad, Kristine C; Kwon, Sun Hyung; Padmanabhan, Jagannath; Whittam, Alexander J; Suga, Hirotaka; Duscher, Dominik; Rodrigues, Melanie; Gurtner, Geoffrey C

2017-07-04

Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-β1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Circular RNA profiling reveals that circCOL3A1-859267 regulate type I collagen expression in photoaged human dermal fibroblasts

International Nuclear Information System (INIS)

Peng, Yating; Song, Xiaojing; Zheng, Yue; Wang, Xinyi; Lai, Wei

2017-01-01

Production of type I collagen declines is a main characteristic during photoaging, but the mechanism is still not fully understood. Circular RNAs (circRNAs) are a class of newly identified non-coding RNAs with regulatory potency by sequestering miRNAs like a sponge. It's more stable than linear RNAs, and would be a useful tool for regulation of gene expression. However, the role of circRNAs in collagen expression during photoaging is still unclear. Here we performed deep sequencing of RNA generated from UVA irradiated and no irradiated human dermal fibroblasts (HDFs) and identified 29 significantly differentially expressed circRNAs (fold change ≥ 1.5, P < 0.05), 12 circRNAs were up-regulated and 17 circRNAs were down-regulated.3 most differentially expressed circRNAs were verified by qRT-PCR and the down-regulated circCOL3A1-859267 exhibited the most significantly altered in photoaged HDFs. Overexpression of circCOL3A1-859267 inhibited UVA-induced decrease of type I collagen expression and silencing of it reduced type I collagen intensity. Via a bioinformatic method, 44 miRNAs were predicted to binding with circCOL3A1-859267, 5 of them have been confirmed or predicted to interact with type I collagen. This study show that circCOL3A1-859267 regulate type I collagen expression in photoaged HDFs, suggesting it may be a novel target for interfering photoaging.

Constitutive Modelling of Resins in the Stiffness Domain

Science.gov (United States)

Klasztorny, M.

2004-09-01

An analytic method for inverting the constitutive compliance equations of viscoelasticity for resins is developed. These equations describe the HWKK/H rheological model, which makes it possible to simulate, with a good accuracy, short-, medium- and long-term viscoelastic processes in epoxy and polyester resins. These processes are of first-rank reversible isothermal type. The time histories of deviatoric stresses are simulated with three independent strain history functions of fractional and normal exponential types. The stiffness equations are described by two elastic and six viscoelastic constants having a clear physic meaning (three long-term relaxation coefficients and three relaxation times). The time histories of axiatoric stresses are simulated as perfectly elastic. The inversion method utilizes approximate constitutive stiffness equations of viscoelasticity for the HWKK/H model. The constitutive compliance equations for the model are a basis for determining the exact complex shear stiffness, whereas the approximate constitutive stiffness equations are used for determining the approximate complex shear stiffness. The viscoelastic constants in the stiffness domain are derived by equating the exact and approximate complex shear stiffnesses. The viscoelastic constants are obtained for Epidian 53 epoxy and Polimal 109 polyester resins. The accuracy of the approximate constitutive stiffness equations are assessed by comparing the approximate and exact complex shear stiffnesses. The constitutive stiffness equations for the HWKK/H model are presented in uncoupled (shear/bulk) and coupled forms. Formulae for converting the constants of shear viscoelasticity into the constants of coupled viscoelasticity are given as well.

Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

Science.gov (United States)

Butt, Hira; Mehmood, Azra; Ali, Muhammad; Tasneem, Saba; Anjum, Muhammad Sohail; Tarar, Moazzam N; Khan, Shaheen N; Riazuddin, Sheikh

2017-09-01

Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. Fibroblasts were treated with 100μM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNβ and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

Upregulation of NOXA by 10-Hydroxycamptothecin plays a key role in inducing fibroblasts apoptosis and reducing epidural fibrosis

Directory of Open Access Journals (Sweden)

Jihang Dai

2017-01-01

Full Text Available The fibrosis that develops following laminectomy or discectomy often causes serious complications, and the proliferation of fibroblasts is thought to be the major cause of epidural fibrosis. 10-Hydroxycamptothecin (HCPT has been proven to be efficient in preventing epidural fibrosis, but the exact mechanism is still unclear. NOXA is a significant regulator of cell apoptosis, which has been reported to be beneficial in the treatment of fibrosis. We performed a series of experiments, both in vitro and in vivo, to explore the intrinsic mechanism of HCPT that underlies the induction of apoptosis in fibroblasts, and also to investigate whether HCPT has positive effects on epidural fibrosis following laminectomy in rats. Fibroblasts were cultured in vitro and stimulated by varying concentrations of HCPT (0, 1, 2, 4 µg/ml for various durations (0, 24, 48, 72 h; the effect of HCPT in inducing the apoptosis of fibroblasts was investigated via Western blots and TUNEL assay. Our results showed that HCPT could induce apoptosis in fibroblasts and up-regulate the expression of NOXA. Following the knockdown of NOXA in fibroblasts, the results of Western blot analysis showed that the level of apoptotic markers, such as cleaved-PARP and Bax, was decreased. The results from the TUNEL assay also showed a decreased rate of apoptosis in NOXA-knocked down fibroblasts. For the in vivo studies, we performed a laminectomy at the L1-L2 levels in rats and applied HCPT of different concentrations (0.2, 0.1, 0.05 mg/ml and saline locally; the macroscopic histological assessment, hydroxyproline content analysis and histological staining were performed to evaluate the effect of HCPT on reducing epidural fibrosis. The TUNEL assay in epidural tissues showed that HCPT could obviously induce apoptosis in fibroblasts in a dose-dependent manner. Also, immunohistochemical staining showed that the expression of NOXA increased as the concentrations of HCPT increased. Our findings are

The stiffness change and the increase in the ultimate capacity for a stiff pile resulting from a cyclic loading

DEFF Research Database (Denmark)

Lada, Aleksandra; Ibsen, Lars Bo; Nicolai, Giulio

In the paper the experimental results of small-scale tests on a stiff monopile are presented to outline the change in stiffness during the cyclic loading and the change in the ultimate pile capacity. The results confirm the increase of stiffness and the increase in bearing capacity resulting from...

Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

DEFF Research Database (Denmark)

Berzina, Zane; Solanko, Lukasz M; Mehadi, Ahmed S

2018-01-01

/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous...... but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM...

Analytical study of a quasi-zero stiffness coupling using a torsion magnetic spring with negative stiffness

Science.gov (United States)

Zheng, Yisheng; Zhang, Xinong; Luo, Yajun; Zhang, Yahong; Xie, Shilin

2018-02-01

By now, many translation quasi-zero stiffness (QZS) mechanisms have been proposed to overcome the restriction between the isolation frequency range and the load bearing capacity of linear isolators. The couplings of rotor systems undertake the functions of transmitting static driving torque and isolating disturbing torque simultaneously, which creates the demand of torsion QZS mechanisms. Hence a QZS coupling is presented in this paper, where a torsion magnetic spring (TMS) composed of two coaxial ring magnet arrangements in repulsive configuration is employed to produce negative torsion stiffness to counteract the positive stiffness of a rubber spring. In this paper, the expressions of magnetic torque and stiffness are given firstly and verified by finite element simulations; and the effect of geometric parameters of the TMS on its stiffness characteristic is analyzed in detail, which contributes to the optimal design of the TMS. Then dynamic analysis of the QZS coupling is performed and the analytical expression of the torque transmissibility is achieved based on the Harmonic Balance Method. Finally, simulation of the torque transmissibility is carried out to reveal how geometric parameters of the TMS affect the isolation performance.

Structure of rat acidic fibroblast growth factor at 1.4 Å resolution

International Nuclear Information System (INIS)

Kulahin, Nikolaj; Kiselyov, Vladislav; Kochoyan, Arthur; Kristensen, Ole; Kastrup, Jette Sandholm; Berezin, Vladimir; Bock, Elisabeth; Gajhede, Michael

2007-01-01

The structure of rat acidic fibroblast growth factor was determined and compared with those of human, bovine and newt origin. The rat and human structures were found to be very similar. Fibroblast growth factors (FGFs) constitute a family of 22 structurally related heparin-binding polypeptides that are involved in the regulation of cell growth, survival, differentiation and migration. Here, a 1.4 Å resolution X-ray structure of rat FGF1 is presented. Two molecules are present in the asymmetric unit of the crystal and they coordinate a total of five sulfate ions. The structures of human, bovine and newt FGF1 have been published previously. Human and rat FGF1 are found to have very similar structures

Stiffness of Railway Soil-Steel Structures

Directory of Open Access Journals (Sweden)

Machelski Czesław

2015-12-01

Full Text Available The considerable influence of the soil backfill properties and that of the method of compacting it on the stiffness of soil-steel structures is characteristic of the latter. The above factors (exhibiting randomness become apparent in shell deformation measurements conducted during construction and proof test loading. A definition of soil-shell structure stiffness, calculated on the basis of shell deflection under the service load, is proposed in the paper. It is demonstrated that the stiffness is the inverse of the deflection influence function used in structural mechanics. The moving load methodology is shown to be useful for testing, since it makes it possible to map the shell deflection influence line also in the case of group loads (concentrated forces, as in bridges. The analyzed cases show that the shell’s span, geometry (static scheme and the height of earth fill influence the stiffness of the structure. The soil-steel structure’s characteristic parameter in the form of stiffness k is more suitable for assessing the quality of construction works than the proposed in code geometric index ω applied to beam structures. As shown in the given examples, parameter k is more effective than stiffness parameter λ used to estimate the deformation of soil-steel structures under construction. Although the examples concern railway structures, the methodology proposed in the paper is suitable also for road bridges.

Stiffness of Railway Soil-Steel Structures

Science.gov (United States)

Machelski, Czesław

2015-12-01

The considerable influence of the soil backfill properties and that of the method of compacting it on the stiffness of soil-steel structures is characteristic of the latter. The above factors (exhibiting randomness) become apparent in shell deformation measurements conducted during construction and proof test loading. A definition of soil-shell structure stiffness, calculated on the basis of shell deflection under the service load, is proposed in the paper. It is demonstrated that the stiffness is the inverse of the deflection influence function used in structural mechanics. The moving load methodology is shown to be useful for testing, since it makes it possible to map the shell deflection influence line also in the case of group loads (concentrated forces), as in bridges. The analyzed cases show that the shell's span, geometry (static scheme) and the height of earth fill influence the stiffness of the structure. The soil-steel structure's characteristic parameter in the form of stiffness k is more suitable for assessing the quality of construction works than the proposed in code geometric index ω applied to beam structures. As shown in the given examples, parameter k is more effective than stiffness parameter λ used to estimate the deformation of soil-steel structures under construction. Although the examples concern railway structures, the methodology proposed in the paper is suitable also for road bridges.

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts.

Science.gov (United States)

Wang, Zhen-Fei; Ma, Da-Guang; Zhu, Zhe; Mu, Yong-Ping; Yang, Yong-Yan; Feng, Li; Yang, Hao; Liang, Jun-Qing; Liu, Yong-Yan; Liu, Li; Lu, Hai-Wen

2017-12-28

To investigate the inhibitory effect of astragaloside IV on the pathological functions of cancer-associated fibroblasts, and to explore the underlying mechanism. Paired gastric normal fibroblast (GNF) and gastric cancer-associated fibroblast (GCAF) cultures were established from resected tissues. GCAFs were treated with vehicle control or different concentrations of astragaloside IV. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs, and astragaloside IV-treated GCAFs, and used to culture BGC-823 human gastric cancer cells. Proliferation, migration and invasion capacities of BGC-823 cells were determined by MTT, wound healing, and Transwell invasion assays, respectively. The action mechanism of astragaloside IV was investigated by detecting the expression of microRNAs and the expression and secretion of the oncogenic factor, macrophage colony-stimulating factor (M-CSF), and the tumor suppressive factor, tissue inhibitor of metalloproteinase 2 (TIMP2), in different groups of GCAFs. The expression of the oncogenic pluripotency factors SOX2 and NANOG in BGC-823 cells cultured with different conditioned media was also examined. GCAFs displayed higher capacities to induce BGC-823 cell proliferation, migration, and invasion than GNFs ( P GNFs, GCAFs expressed a lower level of microRNA-214 ( P < 0.01) and a higher level of microRNA-301a ( P < 0.01). Astragaloside IV treatment significantly up-regulated microRNA-214 expression ( P < 0.01) and down-regulated microRNA-301a expression ( P < 0.01) in GCAFs. Reestablishing the microRNA expression balance subsequently suppressed M-CSF production ( P < 0.01) and secretion ( P < 0.05), and elevated TIMP2 production ( P < 0.01) and secretion ( P < 0.05). Consequently, the ability of GCAFs to increase SOX2 and NANOG expression in BGC-823 cells was abolished by astragaloside IV. Astragaloside IV can inhibit the pathological functions of GCAFs by correcting their dysregulation of microRNA expression, and it is

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

International Nuclear Information System (INIS)

Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

2011-01-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

Energy Technology Data Exchange (ETDEWEB)

Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

2011-07-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

Colorectal cancer cell-derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway.

Science.gov (United States)

Dai, Guangyao; Yao, Xiaoguang; Zhang, Yubin; Gu, Jianbin; Geng, Yunfeng; Xue, Fei; Zhang, Jingcheng

2018-04-01

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Single motor–variable stiffness actuator using bistable switching mechanisms for independent motion and stiffness control

NARCIS (Netherlands)

Groothuis, Stefan; Carloni, Raffaella; Stramigioli, Stefano

This paper presents a proof of concept of a variable stiffness actuator (VSA) that uses only one (high power) input motor. In general, VSAs use two (high power) motors to be able to control both the output position and the output stiffness, which possibly results in a heavy, and bulky system. In

Adventitial fibroblasts induce a distinct proinflammatory/profibrotic macrophage phenotype in pulmonary hypertension.

Science.gov (United States)

El Kasmi, Karim C; Pugliese, Steven C; Riddle, Suzette R; Poth, Jens M; Anderson, Aimee L; Frid, Maria G; Li, Min; Pullamsetti, Soni S; Savai, Rajkumar; Nagel, Maria A; Fini, Mehdi A; Graham, Brian B; Tuder, Rubin M; Friedman, Jacob E; Eltzschig, Holger K; Sokol, Ronald J; Stenmark, Kurt R

2014-07-15

Macrophage accumulation is not only a characteristic hallmark but is also a critical component of pulmonary artery remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Using multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, and primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive pulmonary arteries (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL-6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL-4/IL-13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation, complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, and deficiency in C/EBPβ or HIF1 attenuated fibroblast-driven macrophage activation. These findings challenge the current paradigm of IL-4/IL-13-STAT6-mediated alternative macrophage activation as the sole driver of vascular remodeling in PH, and uncover a cross-talk between adventitial fibroblasts and macrophages in which paracrine IL-6-activated STAT3, HIF1α, and C/EBPβ signaling are critical for macrophage activation and polarization. Thus, targeting IL-6 signaling in macrophages by completely inhibiting C/EBPβ or HIF1α or by partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL-6 and absent IL-4/IL-13 signaling. Copyright © 2014 by The American Association of Immunologists

Big bang nucleosynthesis with a stiff fluid

International Nuclear Information System (INIS)

Dutta, Sourish; Scherrer, Robert J.

2010-01-01

Models that lead to a cosmological stiff fluid component, with a density ρ S that scales as a -6 , where a is the scale factor, have been proposed recently in a variety of contexts. We calculate numerically the effect of such a stiff fluid on the primordial element abundances. Because the stiff fluid energy density decreases with the scale factor more rapidly than radiation, it produces a relatively larger change in the primordial helium-4 abundance than in the other element abundances, relative to the changes produced by an additional radiation component. We show that the helium-4 abundance varies linearly with the density of the stiff fluid at a fixed fiducial temperature. Taking ρ S10 and ρ R10 to be the stiff fluid energy density and the standard density in relativistic particles, respectively, at T=10 MeV, we find that the change in the primordial helium abundance is well-fit by ΔY p =0.00024(ρ S10 /ρ R10 ). The changes in the helium-4 abundance produced by additional radiation or by a stiff fluid are identical when these two components have equal density at a 'pivot temperature', T * , where we find T * =0.55 MeV. Current estimates of the primordial 4 He abundance give the constraint on a stiff fluid energy density of ρ S10 /ρ R10 <30.

Synovial DKK1 expression is regulated by local glucocorticoid metabolism in inflammatory arthritis.

Science.gov (United States)

Hardy, Rowan; Juarez, Maria; Naylor, Amy; Tu, Jinwen; Rabbitt, Elizabeth H; Filer, Andrew; Stewart, Paul M; Buckley, Christopher D; Raza, Karim; Cooper, Mark S

2012-10-18

Inflammatory arthritis is associated with increased bone resorption and suppressed bone formation. The Wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Local glucocorticoid production is also significantly increased during joint inflammation. Therefore, we investigated how locally derived glucocorticoids and inflammatory cytokines regulate DKK1 synthesis in synovial fibroblasts during inflammatory arthritis. We examined expression and regulation of DKK1 in primary cultures of human synovial fibroblasts isolated from patients with inflammatory arthritis. The effect of TNFα, IL-1β and glucocorticoids on DKK1 mRNA and protein expression was examined by real-time PCR and ELISA. The ability of inflammatory cytokine-induced expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to sensitise fibroblasts to endogenous glucocorticoids was explored. Global expression of Wnt signalling and target genes in response to TNFα and glucocorticoids was assessed using a custom array. DKK1 expression in human synovial fibroblasts was directly regulated by glucocorticoids but not proinflammatory cytokines. Glucocorticoids, but not TNFα, regulated expression of multiple Wnt agonists and antagonists in favour of inhibition of Wnt signalling. However, TNFα and IL-1β indirectly stimulated DKK1 production through increased expression of 11β-HSD1. These results demonstrate that in rheumatoid arthritis synovial fibroblasts, DKK1 expression is directly regulated by glucocorticoids rather than TNFα. Consequently, the links between synovial inflammation, altered Wnt signalling and bone remodelling are not direct but are dependent on local activation of endogenous glucocorticoids.

Direct measurement of the intrinsic ankle stiffness during standing.

Science.gov (United States)

Vlutters, M; Boonstra, T A; Schouten, A C; van der Kooij, H

2015-05-01

Ankle stiffness contributes to standing balance, counteracting the destabilizing effect of gravity. The ankle stiffness together with the compliance between the foot and the support surface make up the ankle-foot stiffness, which is relevant to quiet standing. The contribution of the intrinsic ankle-foot stiffness to balance, and the ankle-foot stiffness amplitude dependency remain a topic of debate in the literature. We therefore developed an experimental protocol to directly measure the bilateral intrinsic ankle-foot stiffness during standing balance, and determine its amplitude dependency. By applying fast (40 ms) ramp-and-hold support surface rotations (0.005-0.08 rad) during standing, reflexive contributions could be excluded, and the amplitude dependency of the intrinsic ankle-foot stiffness was investigated. Results showed that reflexive activity could not have biased the torque used for estimating the intrinsic stiffness. Furthermore, subjects required less recovery action to restore balance after bilateral rotations in opposite directions compared to rotations in the same direction. The intrinsic ankle-foot stiffness appears insufficient to ensure balance, ranging from 0.93±0.09 to 0.44±0.06 (normalized to critical stiffness 'mgh'). This implies that changes in muscle activation are required to maintain balance. The non-linear stiffness decrease with increasing rotation amplitude supports the previous published research. With the proposed method reflexive effects can be ruled out from the measured torque without any model assumptions, allowing direct estimation of intrinsic stiffness during standing. Copyright © 2015 Elsevier Ltd. All rights reserved.

CDCP1 identifies a CD146 negative subset of marrow fibroblasts involved with cytokine production.

Directory of Open Access Journals (Sweden)

Mineo Iwata

Full Text Available In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs Protein Kinase C delta (PKC-δ. When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression.

Insulin-like growth factor binding protein-6 delays replicative senescence of human fibroblasts

DEFF Research Database (Denmark)

Micutkova, Lucia; Diener, Thomas; Li, Chen

2011-01-01

Cellular senescence can be induced by a variety of mechanisms, and recent data suggest a key role for cytokine networks to maintain the senescent state. Here, we have used a proteomic LC-MS/MS approach to identify new extracellular regulators of senescence in human fibroblasts. We identified 26 e...

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

International Nuclear Information System (INIS)

Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

2014-01-01

Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

Energy Technology Data Exchange (ETDEWEB)

Fuchigami, Takao [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kibe, Toshiro [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Nishizawa, Yoshiaki [Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Hijioka, Hiroshi; Fujii, Tomomi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ueda, Masahiro [Natural Science Centre for Research and Education, Kagoshima University, 1-21-24 Koorimoto, Kagoshima 890-8580 (Japan); Nakamura, Norifumi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kiyono, Tohru [Department of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuouku, Tokyo 104-0045 (Japan); Kishida, Michiko, E-mail: kmichiko@m2.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

2014-09-05

Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

Observed variations of monopile foundation stiffness

DEFF Research Database (Denmark)

Kallehave, Dan; Thilsted, C.L.; Diaz, Alberto Troya

2015-01-01

full-scale measurements obtained from one offshore wind turbine structure located within Horns Reef II offshore wind farm. Data are presented for a 2.5 years period and covers normal operating conditions and one larger storm event. A reduction of the pile-soil stiffness was observed during the storm...... events, followed by a complete regain to a pre-storm level when the storm subsided. In additional, no long term variations of the pile-soil stiffness was observed. The wind turbine is located in dense to very dense sand deposits.......The soil-structure stiffness of monopile foundations for offshore wind turbines has a high impact on the fatigue loading during normal operating conditions. Thus, a robust design must consider the evolution of pile-soil stiffness over the lifetime of the wind farm. This paper present and discuss...

Stiff Hands

Science.gov (United States)

... All Topics A-Z Videos Infographics Symptom Picker Anatomy Bones Joints Muscles Nerves Vessels Tendons About Hand Surgery What is a Hand Surgeon? What is a Hand Therapist? Media Find a Hand Surgeon Home Anatomy Stiff Hands Email to a friend * required fields ...

A Stewart isolator with high-static-low-dynamic stiffness struts based on negative stiffness magnetic springs

Science.gov (United States)

Zheng, Yisheng; Li, Qingpin; Yan, Bo; Luo, Yajun; Zhang, Xinong

2018-05-01

In order to improve the isolation performance of passive Stewart platforms, the negative stiffness magnetic spring (NSMS) is employed to construct high static low dynamic stiffness (HSLDS) struts. With the NSMS, the resonance frequencies of the platform can be reduced effectively without deteriorating its load bearing capacity. The model of the Stewart isolation platform with HSLDS struts is presented and the stiffness characteristic of its struts is studied firstly. Then the nonlinear dynamic model of the platform including both geometry nonlinearity and stiffness nonlinearity is established; and its simplified dynamic model is derived under the condition of small vibration. The effect of nonlinearity on the isolation performance is also evaluated. Finally, a prototype is built and the isolation performance is tested. Both simulated and experimental results demonstrate that, by using the NSMS, the resonance frequencies of the Stewart isolator are reduced and the isolation performance in all six directions is improved: the isolation frequency band is increased and extended to a lower-frequency level.

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

Science.gov (United States)

Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

2018-01-01

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Cyclooxygenase-2 and hypoxia-regulated proteins are modulated by basic fibroblast growth factor in acute renal failure

Directory of Open Access Journals (Sweden)

Sandra Villanueva

2012-01-01

Full Text Available Acute renal failure (ARF can be caused by injuries that induce tissue hypoxia, which in turn can trigger adaptive or inflammatory responses. We previously showed the participation of basic fibroblast growth factor (FGF-2 in renal repair. Based on this, the aim of this study was to analyze the effect of FGF-2 signaling pathway manipulation at hypoxia-induced protein levels, as well as in key proteins from the vasoactive systems of the kidney. We injected rat kidneys with FGF-2 recombinant protein (r-FGF or FGF-2 receptor antisense oligonucleotide (FGFR2-ASO after bilateral ischemia, and evaluated the presence of iNOS, EPO and HO-1, in representation of hypoxia-induced proteins, as well as COX-2, renin, kallikrein, and B2KR, in representation of the vasoactive systems of the kidney. A reduction in iNOS, HO-1, EPO, renin, kallikrein, B2KR, and in renal damage was observed in animals treated with r-FGF. The opposite effect was found with FGF-2 receptor down-regulation. In contrast, COX-2 protein levels were higher in kidneys treated with r-FGF and lower in those that received FGFR2-ASO, as compared to saline treated kidneys. These results suggest that the protective role of FGF-2 in the pathogenesis of ARF induced by I/R is a complex process, through which a differential regulation of metabolic pathways takes place.

Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

DEFF Research Database (Denmark)

Su, J; Muranjan, M; Sap, J

1999-01-01

of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...... these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect...

Independent control of matrix adhesiveness and stiffness within a 3D self-assembling peptide hydrogel.

Science.gov (United States)

Hogrebe, Nathaniel J; Reinhardt, James W; Tram, Nguyen K; Debski, Anna C; Agarwal, Gunjan; Reilly, Matthew A; Gooch, Keith J

2018-04-01

A cell's insoluble microenvironment has increasingly been shown to exert influence on its function. In particular, matrix stiffness and adhesiveness strongly impact behaviors such as cell spreading and differentiation, but materials that allow for independent control of these parameters within a fibrous, stromal-like microenvironment are very limited. In the current work, we devise a self-assembling peptide (SAP) system that facilitates user-friendly control of matrix stiffness and RGD (Arg-Gly-Asp) concentration within a hydrogel possessing a microarchitecture similar to stromal extracellular matrix. In this system, the RGD-modified SAP sequence KFE-RGD and the scrambled sequence KFE-RDG can be directly swapped for one another to change RGD concentration at a given matrix stiffness and total peptide concentration. Stiffness is controlled by altering total peptide concentration, and the unmodified base peptide KFE-8 can be included to further increase this stiffness range due to its higher modulus. With this tunable system, we demonstrate that human mesenchymal stem cell morphology and differentiation are influenced by both gel stiffness and the presence of functional cell binding sites in 3D culture. Specifically, cells 24 hours after encapsulation were only able to spread out in stiffer matrices containing KFE-RGD. Upon addition of soluble adipogenic factors, soft gels facilitated the greatest adipogenesis as determined by the presence of lipid vacuoles and PPARγ-2 expression, while increasing KFE-RGD concentration at a given stiffness had a negative effect on adipogenesis. This three-component hydrogel system thus allows for systematic investigation of matrix stiffness and RGD concentration on cell behavior within a fibrous, three-dimensional matrix. Physical cues from a cell's surrounding environment-such as the density of cell binding sites and the stiffness of the surrounding material-are increasingly being recognized as key regulators of cell function

Fibroblast cultures in duchenne muscular dystrophy

International Nuclear Information System (INIS)

Ionasescu, V.; Lara-Braud, C.; Zellweger, H.; Ionasescu, R.; Burmeister, L.

1977-01-01

Primary skin fibroblast cultures were grown from forearm pinch skin biopsies obtained from 24 patients with Duchenne muscular dystrophy (DMD) and ten normal controls matched for sex and age. The first subcultures were grown for 7 days and incubated with L-( 3 H)-proline for 24 hours. Intracellular collagen incoption was significantly decreased (2.2 X) and extracellular collagen incorporation significantly increased (1.8 X) in fibroblast cultures from patients with DMD by both collagenase assay and polyacrylamide gel electrophoresis. The synthesis of noncollagen proteins showed low values from the DMD fibroblast cultures. The alterations in synthesis and secretion of collagen and noncollagen proteins were characteristic only for the log phase of DMD fibroblasts. (author)

A novel energy-efficient rotational variable stiffness actuator

NARCIS (Netherlands)

Rao, S.; Carloni, Raffaella; Stramigioli, Stefano

This paper presents the working principle, the design and realization of a novel rotational variable stiffness actuator, whose stiffness can be varied independently of its output angular position. This actuator is energy-efficient, meaning that the stiffness of the actuator can be varied by keeping

Stiffness and the automatic selection of ODE codes

International Nuclear Information System (INIS)

Shampine, L.F.

1984-01-01

The author describes the basic ideas behind the most popular methods for the numerical solution of ordinary differential equations (ODEs). He takes up the qualitative behavior of solutions of ODEs and its relation ot the propagation of numerical error. Codes for ODEs are intended either for stiff problems or for non-stiff problems. The difference is explained. Users of codes do not have the information needed to recognize stiffness. A code, DEASY, which automatically recognizes stiffness and selects a suitable method is described

Multi-fingered haptic palpation utilizing granular jamming stiffness feedback actuators

International Nuclear Information System (INIS)

Li, Min; Sareh, Sina; Seneviratne, Lakmal D; Wurdemann, Helge A; Althoefer, Kaspar; Ranzani, Tommaso; Dasgupta, Prokar

2014-01-01

This paper describes a multi-fingered haptic palpation method using stiffness feedback actuators for simulating tissue palpation procedures in traditional and in robot-assisted minimally invasive surgery. Soft tissue stiffness is simulated by changing the stiffness property of the actuator during palpation. For the first time, granular jamming and pneumatic air actuation are combined to realize stiffness modulation. The stiffness feedback actuator is validated by stiffness measurements in indentation tests and through stiffness discrimination based on a user study. According to the indentation test results, the introduction of a pneumatic chamber to granular jamming can amplify the stiffness variation range and reduce hysteresis of the actuator. The advantage of multi-fingered palpation using the proposed actuators is proven by the comparison of the results of the stiffness discrimination performance using two-fingered (sensitivity: 82.2%, specificity: 88.9%, positive predicative value: 80.0%, accuracy: 85.4%, time: 4.84 s) and single-fingered (sensitivity: 76.4%, specificity: 85.7%, positive predicative value: 75.3%, accuracy: 81.8%, time: 7.48 s) stiffness feedback. (paper)

The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

Science.gov (United States)

Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

2017-04-06

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

A novel variable stiffness mechanism for dielectric elastomer actuators

Science.gov (United States)

Li, Wen-Bo; Zhang, Wen-Ming; Zou, Hong-Xiang; Peng, Zhi-Ke; Meng, Guang

2017-08-01

In this paper, a novel variable stiffness mechanism is proposed for the design of a variable stiffness dielectric elastomer actuator (VSDEA) which combines a flexible strip with a DEA in a dielectric elastomer minimum energy structure. The DEA induces an analog tuning of the transverse curvature of the strip, thus conveniently providing a voltage-controllable flexural rigidity. The VSDEA tends to be a fully flexible and compact structure with the advantages of simplicity and fast response. Both experimental and theoretical investigations are carried out to reveal the variable stiffness performances of the VSDEA. The effect of the clamped location on the bending stiffness of the VSDEA is analyzed, and then effects of the lengths, the loading points and the applied voltages on the bending stiffness are experimentally investigated. An analytical model is developed to verify the availability of this variable stiffness mechanism, and the theoretical results demonstrate that the bending stiffness of the VSDEA decreases as the applied voltage increases, which agree well with the experimental data. Moreover, the experimental results show that the maximum change of the relative stiffness can reach about 88.80%. It can be useful for the design and optimization of active variable stiffness structures and DEAs for soft robots, vibration control, and morphing applications.

A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

DEFF Research Database (Denmark)

Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

1992-01-01

major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

Can a Soft Robotic Probe Use Stiffness Control Like a Human Finger to Improve Efficacy of Haptic Perception?

Science.gov (United States)

Sornkarn, Nantachai; Nanayakkara, Thrishantha

2017-01-01

When humans are asked to palpate a soft tissue to locate a hard nodule, they regulate the stiffness, speed, and force of the finger during examination. If we understand the relationship between these behavioral variables and haptic information gain (transfer entropy) during manual probing, we can improve the efficacy of soft robotic probes for soft tissue palpation, such as in tumor localization in minimally invasive surgery. Here, we recorded the muscle co-contraction activity of the finger using EMG sensors to address the question as to whether joint stiffness control during manual palpation plays an important role in the haptic information gain. To address this question, we used a soft robotic probe with a controllable stiffness joint and a force sensor mounted at the base to represent the function of the tendon in a biological finger. Then, we trained a Markov chain using muscle co-contraction patterns of human subjects, and used it to control the stiffness of the soft robotic probe in the same soft tissue palpation task. The soft robotic experiments showed that haptic information gain about the depth of the hard nodule can be maximized by varying the internal stiffness of the soft probe.

Lower Body Stiffness Modulation Strategies in Well Trained Female Athletes.

Science.gov (United States)

Millett, Emma L; Moresi, Mark P; Watsford, Mark L; Taylor, Paul G; Greene, David A

2016-10-01

Millett, EL, Moresi, MP, Watsford, ML, Taylor, PG, and Greene, DA. Lower body stiffness modulation strategies in well trained female athletes. J Strength Cond Res 30(10): 2845-2856, 2016-Lower extremity stiffness quantifies the relationship between the amount of leg compression and the external load to which the limb are subjected. This study aimed to assess differences in leg and joint stiffness and the subsequent kinematic and kinetic control mechanisms between athletes from various training backgrounds. Forty-seven female participants (20 nationally identified netballers, 13 high level endurance athletes and 14 age and gender matched controls) completed a maximal unilateral countermovement jump, drop jump and horizontal jump to assess stiffness. Leg stiffness, joint stiffness and associated mechanical parameters were assessed with a 10 camera motion analysis system and force plate. No significant differences were evident for leg stiffness measures between athletic groups for any of the tasks (p = 0.321-0.849). However, differences in joint stiffness and its contribution to leg stiffness, jump performance outcome measures and stiffness control mechanisms were evident between all groups. Practitioners should consider the appropriateness of the task utilised in leg stiffness screening. Inclusion of mechanistic and/or more sports specific tasks may be more appropriate for athletic groups.

Fibroblast growth factor receptor 4 regulates tumor invasion by coupling fibroblast growth factor signaling to extracellular matrix degradation

DEFF Research Database (Denmark)

Sugiyama, Nami; Varjosalo, Markku; Meller, Pipsa

2010-01-01

/stroma border and tumor invasion front. The strongest overall coexpression was found in prostate carcinoma. Studies with cultured prostate carcinoma cell lines showed that the FGFR4-R388 variant, which has previously been associated with poor cancer prognosis, increased MT1-MMP-dependent collagen invasion......Aberrant expression and polymorphism of fibroblast growth factor receptor 4 (FGFR4) has been linked to tumor progression and anticancer drug resistance. We describe here a novel mechanism of tumor progression by matrix degradation involving epithelial-to-mesenchymal transition in response...... to membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) induction at the edge of tumors expressing the FGFR4-R388 risk variant. Both FGFR4 and MT1-MMP were upregulated in tissue biopsies from several human cancer types including breast adenocarcinomas, where they were partially coexpressed at the tumor...

Stiffness Evolution in Frozen Sands Subjected to Stress Changes

KAUST Repository

Dai, Sheng; Santamarina, Carlos

2017-01-01

Sampling affects all soils, including frozen soils and hydrate-bearing sediments. The authors monitor the stiffness evolution of frozen sands subjected to various temperature and stress conditions using an oedometer cell instrumented with P-wave transducers. Experimental results show the stress-dependent stiffness of freshly remolded sands, the dominant stiffening effect of ice, creep after unloading, and the associated exponential decrease in stiffness with time. The characteristic time for stiffness loss during creep is of the order of tens of minutes; therefore it is inevitable that frozen soils experience sampling disturbances attributable to unloading. Slow unloading minimizes stiffness loss; conversely, fast unloading causes a pronounced reduction in stiffness probably attributable to the brittle failure of ice or ice-mineral bonding.

Stiffness Evolution in Frozen Sands Subjected to Stress Changes

KAUST Repository

Dai, Sheng

2017-04-21

Sampling affects all soils, including frozen soils and hydrate-bearing sediments. The authors monitor the stiffness evolution of frozen sands subjected to various temperature and stress conditions using an oedometer cell instrumented with P-wave transducers. Experimental results show the stress-dependent stiffness of freshly remolded sands, the dominant stiffening effect of ice, creep after unloading, and the associated exponential decrease in stiffness with time. The characteristic time for stiffness loss during creep is of the order of tens of minutes; therefore it is inevitable that frozen soils experience sampling disturbances attributable to unloading. Slow unloading minimizes stiffness loss; conversely, fast unloading causes a pronounced reduction in stiffness probably attributable to the brittle failure of ice or ice-mineral bonding.

Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.

Science.gov (United States)

Ara, Toshiaki; Kurata, Kazuyuki; Hirai, Kaname; Uchihashi, Takayuki; Uematsu, Takashi; Imamura, Yasuhiro; Furusawa, Kiyohumi; Kurihara, Saburo; Wang, Pao-Li

2009-02-01

A major factor in the pathogenesis of periodontal disease, which is one of the biofilm infectious diseases, is thought to be lipopolysaccharide (LPS), owing to its ability to cause inflammation and promote tissue destruction. Moreover, the elimination of pathogens and their component LPSs is essential for the successful treatment of periodontal disease. Lipopolysaccharide tolerance is a mechanism that prevents excessive and prolonged responses of monocytes and macrophages to LPS. Since persistence of inflammation is necessary for inflammatory cytokine production, cells other than monocytes and macrophages are thought to maintain the production of cytokines in the presence of LPS. In this study, we investigated whether human gingival fibroblasts (HGFs), the most abundant structural cell in periodontal tissue, might be able to maintain inflammatory cytokine production in the presence of LPS bynot displaying LPS tolerance. Human gingival fibroblasts were pretreated with LPS (from Porphyromonas gingivalis and Escherichia coli) and then treated with LPS, and the amounts of interleukin (IL)-6 and IL-8 in the cell culture supernatants were measured. The expression of negative regulators of LPS signalling (suppressor of cytokine signalling-1, interleukin-1 receptor-associated-kinase M and SH2 domain-containing inositol-5-phosphatase-1) was also examined in LPS-treated HGFs. Human gingival fibroblasts did not display LPS tolerance but maintained production of IL-6 and IL-8 when pretreated with LPS, followed by secondary LPS treatment. Lipopolysaccharide-treated HGFs did not express negative regulators. These results demonstrate that HGFs do not show LPS tolerance and suggest that this characteristic of HGFs sustains the inflammatory response in the presence of virulence factors.

Triceps surae short latency stretch reflexes contribute to ankle stiffness regulation during human running.

Directory of Open Access Journals (Sweden)

Neil J Cronin

Full Text Available During human running, short latency stretch reflexes (SLRs are elicited in the triceps surae muscles, but the function of these responses is still a matter of controversy. As the SLR is primarily mediated by Ia afferent nerve fibres, various methods have been used to examine SLR function by selectively blocking the Ia pathway in seated, standing and walking paradigms, but stretch reflex function has not been examined in detail during running. The purpose of this study was to examine triceps surae SLR function at different running speeds using Achilles tendon vibration to modify SLR size. Ten healthy participants ran on an instrumented treadmill at speeds between 7 and 15 km/h under 2 Achilles tendon vibration conditions: no vibration and 90 Hz vibration. Surface EMG from the triceps surae and tibialis anterior muscles, and 3D lower limb kinematics and ground reaction forces were simultaneously collected. In response to vibration, the SLR was depressed in the triceps surae muscles at all speeds. This coincided with short-lasting yielding at the ankle joint at speeds between 7 and 12 km/h, suggesting that the SLR contributes to muscle stiffness regulation by minimising ankle yielding during the early contact phase of running. Furthermore, at the fastest speed of 15 km/h, the SLR was still depressed by vibration in all muscles but yielding was no longer evident. This finding suggests that the SLR has greater functional importance at slow to intermediate running speeds than at faster speeds.

Experimental Challenges to Stiffness as a Transport Paradigm

Science.gov (United States)

Luce, T. C.

2017-10-01

Transport in plasmas is treated experimentally as a relationship between gradients and fluxes in analogy to the random-walk problem. Gyrokinetic models often predict strong increases in local flux for small increases in local gradient when above a threshold, holding all other parameters fixed. This has been named `stiffness'. The radial scalelength is then expected to vary little with source strength as a result of high stiffness. To probe the role of ExB shearing on stiffness in the DIII-D tokamak, two neutral beam injection power scans in H-mode plasmas were specially crafted-one with constant, low torque and one with increasing torque. The ion heat, electron heat, and ion toroidal momentum transport do not show expected signatures of stiffness, while the ion particle transport does. The ion heat transport shows the clearest discrepancy; the normalized heat flux drops with increasing inverse ion temperature scalelength. ExB shearing affects the transport magnitude, but not the scalelength dependence. Linear gyrofluid (TGLF) and nonlinear gyrokinetic (GYRO) predictions show stiff ion heat transport around the experimental profiles. The ion temperature gradient required to match the ion heat flux with increasing auxiliary power is not correctly described by TGLF, even when parameters are varied within the experimental uncertainties. TGLF also underpredicts transport at smaller radii, but overpredicts transport at larger radii. Independent of the theory/experiment comparison, it is not clear that the theoretical definition of stiffness yields any prediction about parameter scans such as the power scans here, because the quantities that must be held fixed to quantify stiffness are varied. A survey of recent literature indicated that profile resilience is routinely attributed to stiffness, but simple model calculations show profile resilience does not imply stiffness. Taken together, these observations challenge the use of local stiffness as a paradigm for explaining

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

2015-02-27

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

International Nuclear Information System (INIS)

Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

2015-01-01

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β 1 (TGF-β 1 )-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β 1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β 1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β 1

Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

International Nuclear Information System (INIS)

White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

2010-01-01

Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-β, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten -/- fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten -/- cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten -/- cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

Stiffness Evaluation of High Temperature Superconductor Bearing Stiffness for 10 kWh Superconductor Flywheel Energy Storage System

International Nuclear Information System (INIS)

Park, B. J.; Jung, S. Y.; Lee, J. P.; Park, B. C.; Kim, C. H.; Han, S. C.; Du, S. G.; Han, Y. H.; Sung, T. H.

2009-01-01

A superconductor flywheel energy storage(SFES) system is mainly act an electro-mechanical battery which transfers mechanical energy into electrical form and vice versa. SFES system consists of a pair of non-contacting High Temperature Superconductor (HTS) bearings with a very low frictional loss. But it is essential to design an efficient HTS bearing considering with rotor dynamic properties through correct calculation of stiffness in order to support a huge composite flywheel rotor with high energy storage density. Static properties of HTS bearings provide data to solve problems which may occur easily in a running system. Since stiffness to counter vibration is the main parameter in designing an HTS bearing system, we investigate HTS bearing magnetic force through static properties between the Permanent Magnet(PM) and HTS. We measured axial / radial stiffness and found bearing stiffness can be easily changed by activated vibration direction between PM and HTS bulk. These results are used to determine the optimal design for a 10 kWh SFES.

Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens

Energy Technology Data Exchange (ETDEWEB)

Montani, Claudia [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Steimberg, Nathalie; Boniotti, Jennifer [Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia (Italy); Biasiotto, Giorgio; Zanella, Isabella [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Department of Molecular and Translational Medicine, University of Brescia, Brescia (Italy); Diafera, Giuseppe [Integrated Systems Engineering (ISE), Milan (Italy); Biunno, Ida [IRGB-CNR, Milan (Italy); IRCCS-Multimedica, Milan (Italy); Caimi, Luigi [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Department of Molecular and Translational Medicine, University of Brescia, Brescia (Italy); Mazzoleni, Giovanna [Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia (Italy); Di Lorenzo, Diego, E-mail: diego.dilorenzo@yahoo.it [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy)

2014-11-01

Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of β-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential. - Highlights: • We here characterized the first cell line derived from an estrogen reporter mouse. • In the RCCS cells express an immortalized behavior but not a transformed phenotype. • The RCCS provides a system for

Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts

International Nuclear Information System (INIS)

Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

2014-01-01

Highlights: • Uniaxial stretching activates Ca 2+ signaling in human lung fibroblasts. • Stretch-induced intracellular Ca 2+ elevation is mainly via Ca 2+ influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca 2+ influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca 2+ concentration ([Ca 2+ ] i ) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca 2+ ] i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca 2+ ] i . The stretch-induced [Ca 2+ ] i elevation was attenuated in Ca 2+ -free solution. In contrast, the increase of [Ca 2+ ] i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd 3+ , ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca 2+ ] i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca 2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP

Intracellular Calreticulin Regulates Multiple Steps in Fibrillar Collagen Expression, Trafficking, and Processing into the Extracellular Matrix*

OpenAIRE

Van Duyn Graham, Lauren; Sweetwyne, Mariya T.; Pallero, Manuel A.; Murphy-Ullrich, Joanne E.

2009-01-01

Calreticulin (CRT), a chaperone and Ca2+ regulator, enhances wound healing, and its expression correlates with fibrosis in animal models, suggesting that CRT regulates production of the extracellular matrix. However, direct regulation of collagen matrix by CRT has not been previously demonstrated. We investigated the role of CRT in the regulation of fibrillar collagen expression, secretion, processing, and deposition in the extracellular matrix by fibroblasts. Mouse embryonic fibroblasts defi...

Stiffness and damping in mechanical design

National Research Council Canada - National Science Library

Rivin, Eugene I

1999-01-01

... important conceptual issues are stiffness of mechanical structures and their components and damping in mechanical systems sensitive to and/or generating vibrations. Stiffness and strength are the most important criteria for many mechanical designs. However, although there are hundreds of books on various aspects of strength, and strength issues ar...

In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1

NARCIS (Netherlands)

de Haan, G; Weersing, E; Dontje, B; van Os, R; Bystrykh, LV; Vellenga, E; Miller, G

The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed

Platelets stimulate fibroblast-mediated contraction of collagen gels

Directory of Open Access Journals (Sweden)

Lundahl Joachim

2003-10-01

Full Text Available Abstract Background Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF and transforming growth factor-β (TGF-β contribute to this effect. Methods Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing, were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β1 concentrations were measured in culture supernatants by ELISA. Results Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM of initial area vs. 48.0% ± 0.4 at 48 hours; P 1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction. Conclusion We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.

The Effect of Shoe Insole Stiffness on Leg Stiffness during Stance Phase of Running in Two Different Speeds ‎among Active Men

Directory of Open Access Journals (Sweden)

Zeinab Tazike-Lemeski

2016-08-01

Full Text Available Introduction: The effect of shoe insoles with different characteristics and in different running speeds on lower-limb stiffness is still ‎controversial. The aim of this study was to investigate the effect of two types of insoles (soft and semi-rigid in two ‎different running speeds on leg stiffness during stance phase of running among active men.‎ Materials and Methods: ‎15 male students without any background of lower extremity injury were selected. Subjects were asked to run with ‎two controlled velocities of 3.0 ± 0.2 and 5.0 ± 0.1 m/s in control and insole conditions (soft and semi-rigid on a ‎force plate, placed on the middle of 15-meter runway. The cinematics and cinetics of motion were measured and ‎calculated using 5 video cameras and one force plate. The leg stiffness was achieved via dividing the vertical ‎ground reaction force by leg compression. Two-factor repeated measures ANOVA was used to test the hypothesis at ‎the significance level of P £ 0.050.‎ Results: There was a significant difference between the two types of insoles on leg stiffness. In fact, semi-rigid insole significantly increased leg stiffness (P < 0.001. However, this discrepancy was not related to the running speed (P = 0.999. In addition, there was no significant difference between the two different speeds on leg stiffness (P = 0.632. Conclusion: It seems that the increase in shoe insole stiffness may increase the leg stiffness. Furthermore, the effect of insole ‎stiffness is not related to the running speed, and leg stiffness will remains constant in low to medium running speeds.‎

Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

Directory of Open Access Journals (Sweden)

Xuemei Zhang

2016-10-01

Full Text Available Myostatin (MSTN can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

Rac inhibition reverses the phenotype of fibrotic fibroblasts.

Directory of Open Access Journals (Sweden)

Shi-wen Xu

Full Text Available BACKGROUND: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA, type I collagen and CCN2 (connective tissue growth factor, CTGF. The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies. METHODS AND FINDINGS: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766. CONCLUSION: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.

RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells.

Directory of Open Access Journals (Sweden)

Andreea Iren Serban

Full Text Available AGEs accumulation in the skin affects extracellular matrix (ECM turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE's proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.

Heterogeneity in Fibroblast Proliferation and Survival in Idiopathic Pulmonary Fibrosis

Directory of Open Access Journals (Sweden)

David Michael Habiel

2014-01-01

Full Text Available Idiopathic pulmonary fibrosis (IPF is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. Myofibroblasts have been shown to arise from interstitial fibroblasts, epithelial to mesenchymal transition of type II alveolar epithelial cells, and the differentiation of recruited fibrocytes. There are many mechanisms that are utilized by these cells for survival, proliferation and persistent activation including up-regulation of cytokines (i.e. Interlukin 6 (IL-6, cytokine receptors (i.e. Interlukin 6 Receptor 1 (IL-6R1, Glycoprotein 130 (gp130 and C-C Chemokine Receptor type 7 (CCR7 and innate pattern recognition receptors (PRRs; i.e. Toll Like Receptor 9 (TLR9. In this review, we will discuss the role of the cytokines IL-6 and CCL21, their receptors and the pattern recognition receptor (PRR, TLR9, in fibroblast recruitment, activation, survival and differentiation into myofibroblasts in IPF.

[Regulation of airway stem cell proliferation in idiopathic pulmonary fibrosis].

Science.gov (United States)

Yang, S X; Wu, Q; Sun, X; Li, X; Li, K; Xu, L; Li, Y; Zhang, Q Y; Zhang, Y C; Chen, H Y

2016-09-01

To investigate the effect of fibroblasts on regulating airway stem cell proliferation in idiopathic pulmonary fibrosis. Lung cell suspension was prepared from β-actin-GFP mice. Airway stem cells were obtained by fluorescence activated cell sorting and co-cultured with lung fibroblasts. The fibroblasts were treated with TGF-β inhibitor SB43142. The expression of growth factors FGF1/2 and the effect of FGF1/2 on stem cell proliferation were observed. The cloning efficiency of airway stem cells, when co-cultured with normal lung fibroblast cells for 8 days, was (3.5±1.1)%, while the cloning efficiency was reduced to (0.04±0.04)% when co-cultured with lung fibroblasts from idiopathic pulmonary fibrosis patients. The difference between the 2 groups was statistically significant(P=0.002 5). TGF-β receptor inhibitor SB431542 increased lung fibroblast growth factors FGF1/2 expression.FGF1 mRNA expression was increased to the experimental group 0.005 5 from 0.000 2 in the control group.FGF2 mRNA expression of the amount raised to the experimental group 0.000 15 from 0.000 8 in the control group.FGF1/2 promoted the growth of airway stem cells. After FGF1/2 was co-cultured with normal lung fibroblast cells for 8 days, the cloning efficiency of airway stem cells was (0.3±0.1)%. During the development of idiopathic pulmonary fibrosis, fibroblast secreted FGF1/2 regulate airway stem cell proliferation.

The stable stiffness triangle - drained sand during deformation cycles

DEFF Research Database (Denmark)

Sabaliauskas, Tomas; Ibsen, Lars Bo

2017-01-01

Cyclic, drained sand stiffness was observed using the Danish triaxial appa- ratus. New, deformation dependant soil property (the stable stiffness triangle) was detected. Using the the stable stiffness triangle, secant stiffness of drained sand was plausible to predict (and control) even during ir...... findings can find application in off-shore, seismic and other engi- neering practice, or inspire new branches of research and modelling wherever dynamic, cyclic or transient loaded sand is encountered....

Pipe elbow stiffness coefficients including shear and bend flexibility factors for use in direct stiffness codes

International Nuclear Information System (INIS)

Perry, R.F.

1977-01-01

Historically, developments of computer codes used for piping analysis were based upon the flexibility method of structural analysis. Because of the specialized techniques employed in this method, the codes handled systems composed of only piping elements. Over the past ten years, the direct stiffness method has gained great popularity because of its systematic solution procedure regardless of the type of structural elements composing the system. A great advantage is realized with a direct stiffness code that combines piping elements along with other structural elements such as beams, plates, and shells, in a single model. One common problem, however, has been the lack of an accurate pipe elbow element that would adequately represent the effects of transverse shear and bend flexibility factors. The purpose of the present paper is to present a systematic derivation of the required 12x12 stiffness matrix and load vectors for a three dimensional pipe elbow element which includes the effects of transverse shear and pipe bend flexibility according to the ASME Boiler and Pressure Vessel Code, Section III. The results are presented analytically and as FORTRAN subroutines to be directly incorporated into existing direct stiffness codes. (Auth.)

Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression

Science.gov (United States)

Liu, Fei; Mih, Justin D.; Shea, Barry S.; Kho, Alvin T.; Sharif, Asma S.; Tager, Andrew M.

2010-01-01

Tissue stiffening is a hallmark of fibrotic disorders but has traditionally been regarded as an outcome of fibrosis, not a contributing factor to pathogenesis. In this study, we show that fibrosis induced by bleomycin injury in the murine lung locally increases median tissue stiffness sixfold relative to normal lung parenchyma. Across this pathophysiological stiffness range, cultured lung fibroblasts transition from a surprisingly quiescent state to progressive increases in proliferation and matrix synthesis, accompanied by coordinated decreases in matrix proteolytic gene expression. Increasing matrix stiffness strongly suppresses fibroblast expression of COX-2 (cyclooxygenase-2) and synthesis of prostaglandin E2 (PGE2), an autocrine inhibitor of fibrogenesis. Exogenous PGE2 or an agonist of the prostanoid EP2 receptor completely counteracts the proliferative and matrix synthetic effects caused by increased stiffness. Together, these results demonstrate a dominant role for normal tissue compliance, acting in part through autocrine PGE2, in maintaining fibroblast quiescence and reveal a feedback relationship between matrix stiffening, COX-2 suppression, and fibroblast activation that promotes and amplifies progressive fibrosis. PMID:20733059

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

Science.gov (United States)

Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

MiR-25 regulates Wwp2 and Fbxw7 and promotes reprogramming of mouse fibroblast cells to iPSCs.

Directory of Open Access Journals (Sweden)

Dong Lu

Full Text Available miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs.We screened 52 miRNAs cloned in a piggybac (PB vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation.We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.

Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum).

Science.gov (United States)

Satoh, Akira; Cummings, Gillian M C; Bryant, Susan V; Gardiner, David M

2010-01-15

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a "critical-size defect" (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment. Copyright 2009 Elsevier Inc. All rights reserved.

Direct measurement of the intrinsic ankle stiffness during standing

NARCIS (Netherlands)

Vlutters, Mark; Vlutters, M.; Boonstra, Tjitske; Schouten, Alfred Christiaan; van der Kooij, Herman

2015-01-01

Ankle stiffness contributes to standing balance, counteracting the destabilizing effect of gravity. The ankle stiffness together with the compliance between the foot and the support surface make up the ankle-foot stiffness, which is relevant to quiet standing. The contribution of the intrinsic

Operator-Based Preconditioning of Stiff Hyperbolic Systems

International Nuclear Information System (INIS)

Reynolds, Daniel R.; Samtaney, Ravi; Woodward, Carol S.

2009-01-01

We introduce an operator-based scheme for preconditioning stiff components encountered in implicit methods for hyperbolic systems of partial differential equations posed on regular grids. The method is based on a directional splitting of the implicit operator, followed by a characteristic decomposition of the resulting directional parts. This approach allows for solution to any number of characteristic components, from the entire system to only the fastest, stiffness-inducing waves. We apply the preconditioning method to stiff hyperbolic systems arising in magnetohydro- dynamics and gas dynamics. We then present numerical results showing that this preconditioning scheme works well on problems where the underlying stiffness results from the interaction of fast transient waves with slowly-evolving dynamics, scales well to large problem sizes and numbers of processors, and allows for additional customization based on the specific problems under study

Egr-1 Upregulates Siva-1 Expression and Induces Cardiac Fibroblast Apoptosis

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Karin Zins

2014-01-01

Full Text Available The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

Wound Healing Activity of Extracts and Formulations of Aloe vera, Henna, Adiantum capillus-veneris, and Myrrh on Mouse Dermal Fibroblast Cells.

Science.gov (United States)

Negahdari, Samira; Galehdari, Hamid; Kesmati, Mahnaz; Rezaie, Anahita; Shariati, Gholamreza

2017-01-01

Among the most important factors in wound healing pathways are transforming growth factor beta1 and vascular endothelial growth factor. Fibroblasts are the main cell in all phases wound closure. In this study, the extracts of plant materials such as Adiantum capillus-veneris , Commiphora molmol , Aloe vera , and henna and one mixture of them were used to treatment of normal mouse skin fibroblasts. Cytotoxic effects of each extract and their mixture were assessed on mouse skin fibroblasts cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We performed migration assays to assess migration properties of mouse skin fibroblasts cells in response to the extracts. Changes in the gene expression of the Tgf β1 and Vegf-A genes were monitored by real-time polymerase chain reaction. A. capillus-veneris , C. molmol and henna extract improved the expression of Tgfβ1 gene. All used extracts upregulated the expression of Vegf-A gene and promoted the migration of mouse fibroblast cells in vitro . The present study demonstrated that the mentioned herbal extracts might be effective in wound healing, through the improvement in the migration of fibroblast cells and regulating the gene expression of Tgfβ1 and Vegf-A genes in fibroblast cells treated with extracts.

The study of stiffness modulus values for AC-WC pavement

Science.gov (United States)

Lubis, AS; Muis, Z. A.; Iskandar, T. D.

2018-02-01

One of the parameters of the asphalt mixture in order for the strength and durability to be achieved as required is the stress-and-strain showing the stiffness of a material. Stiffness modulus is a very necessary factor that will affect the performance of asphalt pavements. If the stiffness modulus value decreases there will be a cause of aging asphalt pavement crack easily when receiving a heavy load. The high stiffness modulus asphalt concrete causes more stiff and resistant to bending. The stiffness modulus value of an asphalt mixture material can be obtained from the theoretical (indirect methods) and laboratory test results (direct methods). For the indirect methods used Brown & Brunton method, and Shell Bitumen method; while for the direct methods used the UMATTA tool. This study aims to determine stiffness modulus values for AC-WC pavement. The tests were conducted in laboratory that used 3 methods, i.e. Brown & Brunton Method, Shell Bitumen Method and Marshall Test as a substitute tool for the UMATTA tool. Hotmix asphalt made from type AC-WC with pen 60/70 using a mixture of optimum bitumen content was 5.84% with a standard temperature variation was 60°C and several variations of temperature that were 30, 40, 50, 70 and 80°C. The stiffness modulus value results obtained from Brown & Brunton Method, Shell Bitumen Method and Marshall Test which were 1374,93 Mpa, 235,45 Mpa dan 254,96 Mpa. The stiffness modulus value decreases with increasing temperature of the concrete asphalt. The stiffness modulus value from the Bitumen Shell method and the Marshall Test has a relatively similar value.The stiffness modulus value from the Brown & Brunton method is greater than the Bitumen Shell method and the Marshall Test, but can not measure the stiffness modulus value at temperature above 80°C.

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

DEFF Research Database (Denmark)

Dabelsteen, S; Wandall, H H; Grøn, B

1997-01-01

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expres......Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF m......RNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa, from gingiva, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal...

Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts.

Science.gov (United States)

Cieslik, Katarzyna A; Trial, JoAnn; Crawford, Jeffrey R; Taffet, George E; Entman, Mark L

2014-05-01

Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-β), due to reduced expression of TGF-β receptor I (TβRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-β promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-β signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TβRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ". © 2013. Published by Elsevier Ltd. All rights reserved.

Simvastatin Ameliorates Matrix Stiffness-Mediated Endothelial Monolayer Disruption.

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Marsha C Lampi

Full Text Available Arterial stiffening accompanies both aging and atherosclerosis, and age-related stiffening of the arterial intima increases RhoA activity and cell contractility contributing to increased endothelium permeability. Notably, statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase inhibitors whose pleiotropic effects include disrupting small GTPase activity; therefore, we hypothesized the statin simvastatin could be used to attenuate RhoA activity and inhibit the deleterious effects of increased age-related matrix stiffness on endothelial barrier function. Using polyacrylamide gels with stiffnesses of 2.5, 5, and 10 kPa to mimic the physiological stiffness of young and aged arteries, endothelial cells were grown to confluence and treated with simvastatin. Our data indicate that RhoA and phosphorylated myosin light chain activity increase with matrix stiffness but are attenuated when treated with the statin. Increases in cell contractility, cell-cell junction size, and indirect measurements of intercellular tension that increase with matrix stiffness, and are correlated with matrix stiffness-dependent increases in monolayer permeability, also decrease with statin treatment. Furthermore, we report that simvastatin increases activated Rac1 levels that contribute to endothelial barrier enhancing cytoskeletal reorganization. Simvastatin, which is prescribed clinically due to its ability to lower cholesterol, alters the endothelial cell response to increased matrix stiffness to restore endothelial monolayer barrier function, and therefore, presents a possible therapeutic intervention to prevent atherogenesis initiated by age-related arterial stiffening.

Adventitial Fibroblasts induce a distinct Pro-inflammatory/Pro-fibrotic Macrophage Phenotype in Pulmonary Hypertension

Science.gov (United States)

El Kasmi, Karim C.; Pugliese, Steven C.; Riddle, Suzette R.; Poth, Jens M.; Anderson, Aimee L.; Frid, Maria G.; Li, Min; Pullamsetti, Soni S.; Savai, Rajkumar; Nagel, Maria A.; Fini, Mehdi A.; Graham, Brian B.; Tuder, Rubin M.; Friedman, Jacob E.; Eltzschig, Holger K.; Sokol, Ronald J.; Stenmark, Kurt R.

2014-01-01

Macrophage accumulation is not only a characteristic hallmark but also a critical component of pulmonary artery (PA) remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Utilizing multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, as well as primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive Pas (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL4/IL13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation while complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, while deficiency in C/EBPβ or HIF1 attenuated fibroblast driven macrophage activation. These findings challenge the current paradigm of IL4/IL13-STAT6 mediated alternative macrophage activation as the sole driver of vascular remodeling in PH and uncover a crosstalk between adventitial fibroblasts and macrophages in which paracrine IL6 activated STAT3, HIF1, and C/EBPβ signaling is critical for macrophage activation and polarization. Thus, targeting IL6 signaling in macrophages by completely inhibiting C/EBPβ, HIF1a or partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL6 and absent IL4/IL13 signaling. PMID:24928992

Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

Energy Technology Data Exchange (ETDEWEB)

Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin, E-mail: salah.ahmed@wsu.edu

2015-09-15

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. - Highlights: • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA). • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS. • ASK1 may be used as a potential therapeutic target in RA. • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS. • Thymoquinone might serve as a potential small

Analysis of Dynamic Stiffness of Bridge Cap-Pile System

Directory of Open Access Journals (Sweden)

Jinhui Chu

2018-01-01

Full Text Available In order to investigate the applicability of dynamic stiffness for bridge cap-pile system, a laboratory test was performed. A numerical model was also built for this type of system. The impact load was applied on the cap top and the dynamic stiffness was analysed. Then, the effect of the effective friction area between pile and soil was also considered. Finally, the dynamic stiffness relationship between the single pile and the cap-pile system was also compared. The results show that the dynamic stiffness is a sensitive index and can well reflect the static characteristics of the pile at the elastic stage. There is a significant positive correlation between the vertical dynamic stiffness index and bearing capacity of the cap-pile system in the similar formation environment. For the cap-pile system with four piles, the dynamic stiffness is about four times as large as the single pile between 10 and 20 Hz.

Stiff quantum polymers

OpenAIRE

Kleinert, H.

2009-01-01

At ultralow temperatures, polymers exhibit quantum behavior, which is calculated here for the second and fourth moments of the end-to-end distribution in the large-stiffness regime. The result should be measurable for polymers in wide optical traps.

Arterial Stiffness in Children: Pediatric Measurement and Considerations

Science.gov (United States)

Savant, Jonathan D.; Furth, Susan L.; Meyers, Kevin E.C.

2014-01-01

Background Arterial stiffness is a natural consequence of aging, accelerated in certain chronic conditions, and predictive of cardiovascular events in adults. Emerging research suggests the importance of arterial stiffness in pediatric populations. Methods There are different indices of arterial stiffness. The present manuscript focuses on carotid-femoral pulse wave velocity and pulse wave analysis, although other methodologies are discussed. Also reviewed are specific measurement considerations for pediatric populations and the literature describing arterial stiffness in children with certain chronic conditions (primary hypertension, obesity, diabetes, chronic kidney disease, hypercholesterolemia, genetic syndromes involving vasculopathy, and solid organ transplant recipients). Conclusions The measurement of arterial stiffness in children is feasible and, under controlled conditions, can give accurate information about the underlying state of the arteries. This potentially adds valuable information about the functionality of the cardiovascular system in children with a variety of chronic diseases well beyond that of the brachial artery blood pressure. PMID:26587447

Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

Directory of Open Access Journals (Sweden)

Bin Shan

Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

The NF-κB family member RelB regulates microRNA miR-146a to suppress cigarette smoke-induced COX-2 protein expression in lung fibroblasts.

Science.gov (United States)

Zago, Michela; Rico de Souza, Angela; Hecht, Emelia; Rousseau, Simon; Hamid, Qutayba; Eidelman, David H; Baglole, Carolyn J

2014-04-21

Diseases due to cigarette smoke exposure, including chronic obstructive pulmonary disease (COPD) and lung cancer, are associated with chronic inflammation typified by the increased expression of cyclooxygenase-2 (COX-2) protein. RelB is an NF-κB family member that suppresses cigarette smoke induction of COX-2 through an unknown mechanism. The ability of RelB to regulate COX-2 expression may be via miR-146a, a miRNA that attenuates COX-2 in lung fibroblasts. In this study we tested whether RelB attenuation of cigarette smoke-induced COX-2 protein is due to miR-146a. Utilizing pulmonary fibroblasts deficient in RelB expression, together with siRNA knock-down of RelB, we show the essential role of RelB in diminishing smoke-induced COX-2 protein expression despite robust activation of the canonical NF-κB pathway and subsequent induction of Cox-2 mRNA. RelB did not regulate COX-2 protein expression at the level of mRNA stability. Basal levels of miR-146a were significantly lower in Relb-deficient cells and cigarette smoke increased miR-146a expression only in Relb-expressing cells. Inhibition of miR-146a had no effects on Relb expression or induction of Cox-2 mRNA by cigarette smoke but significantly increased COX-2 protein. These data highlight the potential of a RelB-miR-146a axis as a novel regulatory pathway that attenuates inflammation in response to respiratory toxicants. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Analysis of primary cilia in directional cell migration in fibroblasts

DEFF Research Database (Denmark)

Christensen, Søren Tvorup; Veland, Iben; Schwab, Albrecht

2013-01-01

summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by micropipette addition of PDGFRα ligands to cultures of fibroblasts. These methods should be useful not only in studying cell migration....... In particular, platelet-derived growth factor receptor alpha (PDGFRα) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We...

Magnitude and duration of stretch modulate fibroblast remodeling.

Science.gov (United States)

Balestrini, Jenna L; Billiar, Kristen L

2009-05-01

Mechanical cues modulate fibroblast tractional forces and remodeling of extracellular matrix in healthy tissue, healing wounds, and engineered matrices. The goal of the present study is to establish dose-response relationships between stretch parameters (magnitude and duration per day) and matrix remodeling metrics (compaction, strength, extensibility, collagen content, contraction, and cellularity). Cyclic equibiaxial stretch of 2-16% was applied to fibroblast-populated fibrin gels for either 6 h or 24 h/day for 8 days. Trends in matrix remodeling metrics as a function of stretch magnitude and duration were analyzed using regression analysis. The compaction and ultimate tensile strength of the tissues increased in a dose-dependent manner with increasing stretch magnitude, yet remained unaffected by the duration in which they were cycled (6 h/day versus 24 h/day). Collagen density increased exponentially as a function of both the magnitude and duration of stretch, with samples stretched for the reduced duration per day having the highest levels of collagen accumulation. Cell number and failure tension were also dependent on both the magnitude and duration of stretch, although stretch-induced increases in these metrics were only present in the samples loaded for 6 h/day. Our results indicate that both the magnitude and the duration per day of stretch are critical parameters in modulating fibroblast remodeling of the extracellular matrix, and that these two factors regulate different aspects of this remodeling. These findings move us one step closer to fully characterizing culture conditions for tissue equivalents, developing improved wound healing treatments and understanding tissue responses to changes in mechanical environments during growth, repair, and disease states.

Oxidized DNA induces an adaptive response in human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Kostyuk, Svetlana V., E-mail: svet.kostyuk@gmail.com [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Tabakov, Viacheslav J.; Chestkov, Valerij V.; Konkova, Marina S.; Glebova, Kristina V.; Baydakova, Galina V.; Ershova, Elizaveta S.; Izhevskaya, Vera L. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Baranova, Ancha, E-mail: abaranov@gmu.edu [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Center for the Study of Chronic Metabolic Diseases, School of System Biology, George Mason University, Fairfax, VA 22030 (United States); Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

2013-07-15

Highlights: • We describe the effects of gDNAOX on human fibroblasts cultivated in serum withdrawal conditions. • gDNAOX evokes an adaptive response in human fibroblasts. • gDNAOX increases the survival rates in serum starving cell populations. • gDNAOX enhances the survival rates in cell populations irradiated at 1.2 Gy dose. • gDNAOX up-regulates NRF2 and inhibits NF-kappaB-signaling. - Abstract: Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA{sup OX}. The levels of cfDNA{sup OX} are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by H{sub 2}O{sub 2}in vitro (gDNA{sup OX}) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA{sup OX} on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA{sup OX} evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2 Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA{sup OX} leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of PSNA, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA{sup OX} and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA{sup OX} inhibits NF-κB signaling. gDNA{sup OX} is a model for oxidized cfDNA{sup OX} that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA

Fibroblast growth factor 21 has no direct role in regulating fertility in female mice

Directory of Open Access Journals (Sweden)

Garima Singhal

2016-08-01

Full Text Available Objective: Reproduction is an energetically expensive process. Insufficient calorie reserves, signaled to the brain through peripheral signals such as leptin, suppress fertility. Recently, fibroblast growth factor 21 (FGF21 was implicated as a signal from the liver to the hypothalamus that directly inhibits the hypothalamic–gonadotropin axis during fasting and starvation. However, FGF21 itself increases metabolic rate and can induce weight loss, which suggests that the effects of FGF21 on fertility may not be direct and may reflect changes in energy balance. Methods: To address this important question, we evaluated fertility in several mouse models with elevated FGF21 levels including ketogenic diet fed mice, fasted mice, mice treated with exogenous FGF21 and transgenic mice over-expressing FGF21. Results: We find that ketogenic diet fed mice remain fertile despite significant elevation in serum FGF21 levels. Absence of FGF21 does not alter transient infertility induced by fasting. Centrally infused FGF21 does not suppress fertility despite its efficacy in inducing browning of inguinal white adipose tissue. Furthermore, a high fat diet (HFD can restore fertility of female FGF21-overexpressing mice, a model of growth restriction, even in the presence of supraphysiological serum FGF21 levels. Conclusions: We conclude that FGF21 is not a direct physiological regulator of fertility in mice. The infertility observed in FGF21 overexpressing mice is likely driven by the increased energy expenditure and consequent excess calorie requirements resulting from high FGF21 levels. Keywords: FGF21, Fertility, Leptin, Hypothalamic action

Toll-like receptor 9 mediated responses in cardiac fibroblasts.

Directory of Open Access Journals (Sweden)

Ingrid Kristine Ohm

Full Text Available Altered cardiac Toll-like receptor 9 (TLR9 signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β. Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088 or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

Ball Bearing Stiffnesses- A New Approach Offering Analytical Expressions

Science.gov (United States)

Guay, Pascal; Frikha, Ahmed

2015-09-01

Space mechanisms use preloaded ball bearings in order to withstand the severe vibrations during launch.The launch strength requires the calculation of the bearing stiffness, but this calculation is complex. Nowadays, there is no analytical expression that gives the stiffness of a bearing. Stiffness is computed using an iterative algorithm such as Newton-Raphson, to solve the nonlinear system of equations.This paper aims at offering a simplified analytical approach, based on the assumption that the contact angle is constant. This approach gives analytical formulas of the stiffness of preloaded ball bearing.

Matrix metalloproteinase inhibition reduces contraction by dupuytren fibroblasts.

Science.gov (United States)

Townley, William A; Cambrey, Alison D; Khaw, Peng T; Grobbelaar, Adriaan O

2008-11-01

Dupuytren's disease is a common fibroproliferative condition of the hand characterized by fibrotic lesions (nodules and cords), leading to disability through progressive digital contracture. Although the etiology of the disease is poorly understood, recent evidence suggests that abnormal matrix metalloproteinase (MMP) activity may play a role in cell-mediated collagen contraction and tissue scarring. The aim of this study was to investigate the efficacy of ilomastat, a broad-spectrum MMP inhibitor, in an in vitro model of Dupuytren fibroblast-mediated contraction. Nodule-derived and cord-derived fibroblasts were isolated from Dupuytren patients; carpal ligament-derived fibroblasts acted as control. Stress-release fibroblast-populated collagen lattices (FPCLs) were used as a model of contraction. FPCLs were allowed to develop mechanical stress (48 hours) during treatment with ilomastat (0-100 micromol/L), released, and allowed to contract over a 48-hour period. Contraction was estimated by measuring lattice area compared with untreated cells or treatment with a control peptide. MMP-1, MMP-2, and MT1-MMP levels were assessed by zymography, Western blotting, and enzyme-linked immunosorbent assay. Nodule-derived fibroblasts contracted lattices (69% +/- 2) to a greater extent than did cord-derived (55% +/- 3) or carpal ligament-derived (55% +/- 1) fibroblasts. Exposure to ilomastat led to significant inhibition of lattice contraction by all fibroblasts, although a reduction in lattice contraction by nodule-derived fibroblasts was most prominent (84% +/- 8). In addition, treatment with ilomastat led to a concomitant suppression of MMP-1 and MMP-2 activity, whereas MT1-MMP activity was found to be upregulated. Our results demonstrate that inhibition of MMP activity results in a reduction in extracellular matrix contraction by Dupuytren fibroblasts and suggest that MMP activity may be a critical target in preventing recurrent contracture caused by this disease.

Evaluation of feedforward and feedback contributions to hand stiffness and variability in multijoint arm control.

Science.gov (United States)

He, Xin; Du, Yu-Fan; Lan, Ning

2013-07-01

The purpose of this study is to validate a neuromechanical model of the virtual arm (VA) by comparing emerging behaviors of the model to those of experimental observations. Hand stiffness of the VA model was obtained by either theoretical computation or simulated perturbations. Variability in hand position of the VA was generated by adding signal dependent noise (SDN) to the motoneuron pools of muscles. Reflex circuits of Ia, Ib and Renshaw cells were included to regulate the motoneuron pool outputs. Evaluation of hand stiffness and variability was conducted in simulations with and without afferent feedback under different patterns of muscle activations during postural maintenance. The simulated hand stiffness and variability ellipses captured the experimentally observed features in shape, magnitude and orientation. Steady state afferent feedback contributed significantly to the increase in hand stiffness by 35.75±16.99% in area, 18.37±7.80% and 16.15±7.15% in major and minor axes; and to the reduction of hand variability by 49.41±21.19% in area, 36.89±12.78% and 18.87±23.32% in major and minor axes. The VA model reproduced the neuromechanical behaviors that were consistent with experimental data, and it could be a useful tool for study of neural control of posture and movement, as well as for application to rehabilitation.

Serum 25-hydroxyvitamin D is associated with both arterial and ventricular stiffness in healthy subjects.

Science.gov (United States)

Şeker, Taner; Gür, Mustafa; Kuloğlu, Osman; Kalkan, Gülhan Yüksel; Şahin, Durmuş Yıldıray; Türkoğlu, Caner; Elbasan, Zafer; Baykan, Ahmet Oytun; Gözübüyük, Gökhan; Çaylı, Murat

2013-12-01

Vitamin D regulates the renin-angiotensin system, suppresses proliferation of vascular smooth muscle and improves endothelial cell dependent vasodilatation. These mechanisms may play a role on pathogenesis of arterial and left ventricular stiffness. We aimed to investigate the association between serum 25-hydroxyvitamin D with arterial and left ventricular stiffness in healthy subjects. We studied 125 healthy subjects without known cardiovascular risk factors or overt heart disease (mean age: 60.2 ± 11.9 years). Serum 25-hydroxyvitamin D was measured using a direct competitive chemiluminescent immunoassay. The subjects were divided into two groups according to the serum vitamin D level; vitamin D sufficient (≥ 20 ng/ml, n = 56) and vitamin D deficient (stiffness such as E/A and E/E' were measured. Pulse wave velocity (PWV), which reflects arterial stiffness, was calculated using the single-point method via the Mobil-O-Graph(®) ARC solver algorithm. Systolic blood pressure, level of serum calcium, PWV and E/E' values were higher and E/A values were lower in vitamin D deficient group compared with vitamin D sufficient group. Multiple linear regression analysis showed that vitamin D level was independently associated with E/E' (β = -0.364, pstiffness as well as systolic blood pressure in healthy subjects. Copyright © 2013 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Plant fibre composites - porosity and stiffness

DEFF Research Database (Denmark)

Madsen, Bo; Thygesen, Anders; Lilholt, Hans

2009-01-01

Plant fibre composites contain typically a relatively large amount of porosity which influences their performance. A model, based on a modified rule of mixtures, is presented to include the influence of porosity on the composite stiffness. The model integrates the volumetric composition...... of the composites with their mechanical properties. The fibre weight fraction is used as an independent parameter to calculate the complete volumetric composition. A maximum obtainable stiffness of the composites is calculated at a certain transition fibre weight fraction, which is characterised by a best possible...... combination of high fibre volume fraction and low porosity. The model is validated with experimental data from the literature on several types of composites. A stiffness diagram is presented to demonstrate that the calculations can be used for tailoring and design of composites with a given profile...

Fibroblast adhesion and activation onto micro-machined titanium surfaces.

Science.gov (United States)

Guillem-Marti, J; Delgado, L; Godoy-Gallardo, M; Pegueroles, M; Herrero, M; Gil, F J

2013-07-01

Surface modifications performed at the neck of dental implants, in the manner of micro-grooved surfaces, can reduce fibrous tissue encapsulation and prevent bacterial colonization, thereby improving fibrointegration and the formation of a biological seal. However, the applied procedures are technically complex and/or time consuming methods. The aim of this study was to analyse the fibroblast behaviour on modified titanium surfaces obtained, applying a simple and low-cost method. An array of titanium surfaces was obtained using a commercial computerized numerical control lathe, modifying the feed rate and the cutting depth. To elucidate the potential ability of the generated surfaces to activate connective tissue cells, a thorough gene (by real time - qPCR) and protein (by western blot or zymography) expression and cellular response characterization (cell morphology, cell adhesion and cell activation by secreting extracellular matrix (ECM) components and their enzyme regulators) was performed. Micro-grooved surfaces have statistically significant differences in the groove's width (approximately 10, 50 and 100 μm) depending on the applied advancing fixed speed. Field emission scanning electron microscopy images showed that fibroblasts oriented along the generated grooves, but they were only entirely accommodated on the wider grooves (≥50 μm). Micro-grooved surfaces exhibited an earlier cell attachment and activation, as seen by collagen Iα1 and fibronectin deposition and activation of ECM remodelling enzymes, compared with the other surfaces. However, fibroblasts could remain in an activated state on narrower surfaces (fibrotic response. © 2012 John Wiley & Sons A/S.

Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts.

Science.gov (United States)

Anitua, Eduardo; de la Fuente, María; Ferrando, Marcos; Quintana, Fernando; Larreategui, Zaloa; Matorras, Roberto; Orive, Gorka

2016-11-01

To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann-Whitney U-test). We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in "in vivo" endometrial development and especially in human embryo implantation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fibroblasts in fibrosis: novel roles and mediators

Directory of Open Access Journals (Sweden)

Ryan Thomas Kendall

2014-05-01

Full Text Available Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types—most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of noncancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve nonspecific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

Science.gov (United States)

Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

Science.gov (United States)

Vitt, Anton; Slizen, Veronica; Boström, Elisabeth A; Yucel-Lindberg, Tülay; Kats, Anna; Sugars, Rachael V; Gustafsson, Anders; Buhlin, Kåre

2017-10-01

Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E 2 , interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE 2 , IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE 2 , IL-6, IL-8 or MMP-1 by gingival fibroblasts. Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

Is chronic obstructive pulmonary disease associated with increased arterial stiffness?

DEFF Research Database (Denmark)

Janner, Julie H; McAllister, David A; Godtfredsen, Nina S

2012-01-01

We hypothesize that airflow limitation is associated with increasing arterial stiffness and that having COPD increases a non-invasive measure of arterial stiffness - the aortic augmentation index (AIx) - independently of other CVD risk factors.......We hypothesize that airflow limitation is associated with increasing arterial stiffness and that having COPD increases a non-invasive measure of arterial stiffness - the aortic augmentation index (AIx) - independently of other CVD risk factors....

A Rapid Aeroelasticity Optimization Method Based on the Stiffness characteristics

OpenAIRE

Yuan, Zhe; Huo, Shihui; Ren, Jianting

2018-01-01

A rapid aeroelasticity optimization method based on the stiffness characteristics was proposed in the present study. Large time expense in static aeroelasticity analysis based on traditional time domain aeroelasticity method is solved. Elastic axis location and torsional stiffness are discussed firstly. Both torsional stiffness and the distance between stiffness center and aerodynamic center have a direct impact on divergent velocity. The divergent velocity can be adjusted by changing the cor...

LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

Directory of Open Access Journals (Sweden)

Jean Albrengues

2014-06-01

Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

Damper modules with adapted stiffness ratio

Energy Technology Data Exchange (ETDEWEB)

Sonnenburg, R.; Stretz, A. [ZF Sachs AG, Entwicklungszentrum, Schweinfurt (Germany)

2011-07-15

A mechanism for the excitation of piston rod vibrations in automotive damper modules is discussed by a simple model. An improved nonlinear model based on elasticity effects leads to good simulation results. It is shown theoretically and experimentally that the adaptation of the stiffness of the piston rod bushing to the ''stiffness'' of the damper force characteristic can eliminate the piston rod oscillations completely. (orig.)

Rotational and peak torque stiffness of rugby shoes.

Science.gov (United States)

Ballal, Moez S; Usuelli, Federico Giuseppe; Montrasio, Umberto Alfieri; Molloy, Andy; La Barbera, Luigi; Villa, Tomaso; Banfi, Giuseppe

2014-09-01

Sports people always strive to avoid injury. Sports shoe designs in many sports have been shown to affect traction and injury rates. The aim of this study is to demonstrate the differing stiffness and torque in rugby boots that are designed for the same effect. Five different types of rugby shoes commonly worn by scrum forwards were laboratory tested for rotational stiffness and peak torque on a natural playing surface generating force patterns that would be consistent with a rugby scrum. The overall internal rotation peak torque was 57.75±6.26 Nm while that of external rotation was 56.55±4.36 Nm. The Peak internal and external rotational stiffness were 0.696±0.1 and 0.708±0.06 Nm/deg respectively. Our results, when compared to rotational stiffness and peak torques of football shoes published in the literature, show that shoes worn by rugby players exert higher rotational and peak torque stiffness compared to football shoes when tested on the same natural surfaces. There was significant difference between the tested rugby shoes brands. In our opinion, to maximize potential performance and lower the potential of non-contact injury, care should be taken in choosing boots with stiffness appropriate to the players main playing role. Copyright © 2014 Elsevier Ltd. All rights reserved.

Static stiffness modeling of a novel hybrid redundant robot machine

International Nuclear Information System (INIS)

Li Ming; Wu Huapeng; Handroos, Heikki

2011-01-01

This paper presents a modeling method to study the stiffness of a hybrid serial-parallel robot IWR (Intersector Welding Robot) for the assembly of ITER vacuum vessel. The stiffness matrix of the basic element in the robot is evaluated using matrix structural analysis (MSA); the stiffness of the parallel mechanism is investigated by taking account of the deformations of both hydraulic limbs and joints; the stiffness of the whole integrated robot is evaluated by employing the virtual joint method and the principle of virtual work. The obtained stiffness model of the hybrid robot is analytical and the deformation results of the robot workspace under certain external load are presented.

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

International Nuclear Information System (INIS)

Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.; Varga, John

2008-01-01

Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2 -/- ) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2 -/- MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2 -/- MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2 -/- MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts

Stiffness requirement of flexible skin for variable trailing-edge camber wing

Institute of Scientific and Technical Information of China (English)

无

2010-01-01

The method for analyzing the deformation of flexible skin under the air loads was developed based on the panel method and finite element method.The deformation of flexible skin under air pressures and effects of the local deformation on the aerodynamic characteristics were discussed.Numerical results show that the flexible skin on the upper surface of trailing-edge will bubble under the air loads and the bubble has a powerful effect on the aerodynamic pressure near the surface of local deforma-tion.Then the stiffness requirements for flexible skin of variable trailing-edge were given by using the Jacobs rule,i.e.,the maximum displacement of skin is not greater than 0.1% of wing chord.Results show that the in-plane stiffness can be reduced by increasing the ratio of bending stiffness to in-plane stiffness.Although the deformation of flexible skin increases with the in-plane stiffness decreasing,it depends on the bending stiffness.When the bending stiffness exceeds critical value,the deformation of flexible skin only depends on the bending stiffness and has nothing to do with the in-plane stiffness.The conclusions can be used for the structural design of flexible skin.

Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

2014-10-10

Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

Subspace methods for identification of human ankle joint stiffness.

Science.gov (United States)

Zhao, Y; Westwick, D T; Kearney, R E

2011-11-01

Joint stiffness, the dynamic relationship between the angular position of a joint and the torque acting about it, describes the dynamic, mechanical behavior of a joint during posture and movement. Joint stiffness arises from both intrinsic and reflex mechanisms, but the torques due to these mechanisms cannot be measured separately experimentally, since they appear and change together. Therefore, the direct estimation of the intrinsic and reflex stiffnesses is difficult. In this paper, we present a new, two-step procedure to estimate the intrinsic and reflex components of ankle stiffness. In the first step, a discrete-time, subspace-based method is used to estimate a state-space model for overall stiffness from the measured overall torque and then predict the intrinsic and reflex torques. In the second step, continuous-time models for the intrinsic and reflex stiffnesses are estimated from the predicted intrinsic and reflex torques. Simulations and experimental results demonstrate that the algorithm estimates the intrinsic and reflex stiffnesses accurately. The new subspace-based algorithm has three advantages over previous algorithms: 1) It does not require iteration, and therefore, will always converge to an optimal solution; 2) it provides better estimates for data with high noise or short sample lengths; and 3) it provides much more accurate results for data acquired under the closed-loop conditions, that prevail when subjects interact with compliant loads.

MiR-124 down-regulation is critical for cancer associated fibroblasts-enhanced tumor growth of oral carcinoma

Energy Technology Data Exchange (ETDEWEB)

Li, Xia, E-mail: dentistlx@163.com [Department of Stomatology, School of Stomatology and medicine, Foshan Stomatology Hospital, Foshan University, Foshan 528000 (China); Fan, Qinqiao [Department of Hepatobiliary Surgery, Cancer Center, Chenzhou No.1 People' s Hospital, Chenzhou 423000 (China); Li, Jinyun [Department of Head and Neck Surgery, The Affiliated Cancer Hospital of Xiangya School of Medicine, Center South University, Changsha 410013 (China); Song, Jing; Gu, Yangcong [Department of Stomatology, School of Stomatology and medicine, Foshan Stomatology Hospital, Foshan University, Foshan 528000 (China)

2017-02-01

Cancer associated fibroblasts (CAFs) are known to be involved in initiation, progression and metastasis of various cancers. However, the molecular mechanism of how CAFs affects the biological function of oral cancer (OC) has not been fully-addressed. In this study, we demonstrated that miR-124 was downregulated in oral CAFs and oral cancer cells (OCCs) when compared with matched normal fibroblasts (NFs). Hypermethylation in the promoter region of miR-124 genes was accounted for its downregulation. Interestingly, CAFs but not NFs exerted promotion effect on OCCs cell proliferation, migration and tumor growth in CAFs/NFs-OCCs co-culture. Furthermore, we identified Chemokine (C-C motif) ligand 2 (CCL2) and Interleukin 8 (IL-8) as two direct targets of miR-124. Over-expression of miR-124 in CAFs-OCCs co-culture abrogated CAFs-promoted OCCs cell growth and migration, and this inhibitory effect can be rescued by addition of CCL2 and IL-8. Finally, we showed that restoration of miR-124 expression by lentiviral infection or formulated miR-124 injection inhibited oral tumor growth in vivo suggesting miR-124 rescue could be a potential rationale for therapeutic applications in oral cancer in the future. - Highlights: • miR-124 was downregulated in oral cancer cells and cancer associated fibroblasts. • Hypermethylation in the promoter region was accounted for miR-124 downregulation. • CCL2 and IL-8 are two direct targets of miR-124. • miR-124 rescue could be a potential rationale for oral cancer therapy.

MiR-124 down-regulation is critical for cancer associated fibroblasts-enhanced tumor growth of oral carcinoma

International Nuclear Information System (INIS)

Li, Xia; Fan, Qinqiao; Li, Jinyun; Song, Jing; Gu, Yangcong

2017-01-01

Cancer associated fibroblasts (CAFs) are known to be involved in initiation, progression and metastasis of various cancers. However, the molecular mechanism of how CAFs affects the biological function of oral cancer (OC) has not been fully-addressed. In this study, we demonstrated that miR-124 was downregulated in oral CAFs and oral cancer cells (OCCs) when compared with matched normal fibroblasts (NFs). Hypermethylation in the promoter region of miR-124 genes was accounted for its downregulation. Interestingly, CAFs but not NFs exerted promotion effect on OCCs cell proliferation, migration and tumor growth in CAFs/NFs-OCCs co-culture. Furthermore, we identified Chemokine (C-C motif) ligand 2 (CCL2) and Interleukin 8 (IL-8) as two direct targets of miR-124. Over-expression of miR-124 in CAFs-OCCs co-culture abrogated CAFs-promoted OCCs cell growth and migration, and this inhibitory effect can be rescued by addition of CCL2 and IL-8. Finally, we showed that restoration of miR-124 expression by lentiviral infection or formulated miR-124 injection inhibited oral tumor growth in vivo suggesting miR-124 rescue could be a potential rationale for therapeutic applications in oral cancer in the future. - Highlights: • miR-124 was downregulated in oral cancer cells and cancer associated fibroblasts. • Hypermethylation in the promoter region was accounted for miR-124 downregulation. • CCL2 and IL-8 are two direct targets of miR-124. • miR-124 rescue could be a potential rationale for oral cancer therapy.

Intracellular pH in increased after transformation of Chinese hamster embryo fibroblasts

International Nuclear Information System (INIS)

Ober, S.S.; Pardee, A.B.

1987-01-01

These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pH/sub i/) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. pH measurements were made with [ 14 C]-benzoic acid. This increase of pH/sub i/ in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na + /H + -antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pH/sub i/ regulation in the tumorigenic CHEF/18 derivatives lies in the Na + /H + antiporter itself. Further studies to determine the biological significance of an increased pH/sub i/ show that the external pH (pH 0 )-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pH/sub i/ in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pH/sub i/ can contribute to the acquisition of altered growth properties

Proteomic Analysis Shows Constitutive Secretion of MIF and p53-associated Activity of COX-2−/− Lung Fibroblasts

Directory of Open Access Journals (Sweden)

Mandar Dave

2017-12-01

Full Text Available The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF in lung fibroblasts derived from COX-2−/− but not wild-type (WT or COX-1−/− mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2−/− fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2−/− was “prostaglandin-independent.” GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2−/− cells. Furthermore, COX-2−/− fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

Science.gov (United States)

Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

2014-01-01

Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

Pulling a polymer with anisotropic stiffness near a sticky wall

International Nuclear Information System (INIS)

Tabbara, R; Owczarek, A L

2012-01-01

We solve exactly a two-dimensional partially directed walk model of a semi-flexible polymer that has one end tethered to a sticky wall, while a pulling force away from the adsorbing surface acts on the free end of the walk. This model generalizes a number of previously considered adsorption models by incorporating individual horizontal and vertical stiffness effects, in competition with a variable pulling angle. A solution to the corresponding generating function is found by means of the kernel method. While the phases and related phase transitions are similar in nature to those found previously the analysis of the model in terms of its physical variables highlights various novel structures in the shapes of the phase diagrams and related behaviour of the polymer. We review the results of previously considered sub-cases, augmenting these findings to include analysis with respect to the model’s physical variables—namely, temperature, pulling force, pulling angle away from the surface, stiffness strength and the ratio of vertical to horizontal stiffness potentials, with our subsequent analysis for the general model focusing on the effect that stiffness has on this pulling angle range. In analysing the model with stiffness we also pay special attention to the case where only vertical stiffness is included. The physical analysis of this case reveals behaviour more closely resembling that of an upward pulling force acting on a polymer than it does of a model where horizontal stiffness acts. The stiffness–temperature phase diagram exhibits re-entrance for low temperatures, previously only seen for three-dimensional or co-polymer models. For the most general model we delineate the shift in the physical behaviour as we change the ratio of vertical to horizontal stiffness between the horizontal-only and the vertical-only stiffness regimes. We find that a number of distinct physical characteristics will only be observed for a model where the vertical stiffness dominates

Chromosome aberration induction in human diploid fibroblast and epithelial cells

International Nuclear Information System (INIS)

Scott, D.

1986-01-01

The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

Melatonin suppresses acrolein-induced IL-8 production in human pulmonary fibroblasts.

Science.gov (United States)

Kim, Gun-Dong; Lee, Seung Eun; Kim, Tae-Ho; Jin, Young-Ho; Park, Yong Seek; Park, Cheung-Seog

2012-04-01

Cigarette smoke (CS) causes harmful alterations in the lungs and airway structures and functions that characterize chronic obstructive pulmonary disease (COPD). In addition to COPD, active cigarette smoking causes other respiratory diseases and diminishes health status. Furthermore, recent studies show that, α, β-unsaturated aldehyde acrolein in CS induces the production of interleukin (IL)-8, which is known to be related to bronchitis, rhinitis, pulmonary fibrosis, and asthma. In addition, lung and pulmonary fibroblasts secrete IL-8, which has a chemotactic effect on leukocytes, and which in turn, play a critical role in lung inflammation. On the other hand, melatonin regulates circadian rhythm homeostasis in humans and has many other effects, which include antioxidant and anti-inflammatory effects, as demonstrated by the reduced expressions of iNOS, IL-1β, and IL-6 and increased glutathione (GSH) and superoxide dismutase activities. In this study, we investigated whether melatonin suppresses acrolein-induced IL-8 secretion in human pulmonary fibroblasts (HPFs). It was found that acrolein-induced IL-8 production was accompanied by increased levels of phosphorylation of Akt and extracellular signal-regulated kinases (ERK1/2) in HPFs, and that melatonin suppressed IL-8 production in HPFs. These results suggest that melatonin suppresses acrolein-induced IL-8 production via ERK1/2 and phosphatidylinositol 3-kinase (PI3K)/Akt signal inhibition in HPFs. © 2011 John Wiley & Sons A/S.

MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

International Nuclear Information System (INIS)

Li, Xiaoou; Liu, Lian; Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan; Wen, Fuqiang

2014-01-01

Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis

MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Li, Xiaoou [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Liu, Lian [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Wen, Fuqiang, E-mail: wenfuqiang.scu@gmail.com [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China)

2014-11-28

Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.

Influence of stiffness on CHF for horizontal tubes under LPLF conditions

Energy Technology Data Exchange (ETDEWEB)

Baburajan, P.K. [Nuclear Safety Analysis Division, AERB, Niyamak Bhavan, 400094 (India); Bisht, Govind Singh [Department of Mechanical Engineering, IIT Bombay, 400076 (India); Gaikwad, Avinash J. [Nuclear Safety Analysis Division, AERB, Niyamak Bhavan, 400094 (India); Prabhu, S.V., E-mail: svprabhu@iitb.ac.in [Department of Mechanical Engineering, IIT Bombay, 400076 (India)

2014-10-01

Highlights: • Effect of stiffness on the CHF in horizontal tube under LPLF conditions is studied. • CHF increases with the increase in stiffness. • Correlation for the prediction of CHF as a function of stiffness is developed. • Correlation for mass flux at CHF in terms of stiffness and initial mass flux is given. • RELAP5 is capable of predicting the effect of stiffness on CHF. - Abstract: Studies reported in the past on critical heat flux (CHF) are mostly limited to vertical flow, large channel diameter, high pressure and high mass flux. Since horizontal flow is commonly encountered in boiler tubes, refrigerating equipments and nuclear reactor fuel channels (PHWR), there is a need to understand horizontal flow CHF, generate sufficient experimental database and to develop reliable predictive method. Few studies are reported on the effect of upstream flow restrictions on flow instabilities and CHF. The present work investigates the effect of upstream flow restriction on CHF in horizontal flow at near atmospheric pressure conditions. In the present study, stiffness is defined as the ratio of upstream flow restriction pressure drop to the test section pressure drop. The classification of a flow boiling system as soft or stiff on the basis of quantification of the stiffness is attempted. Experimental data shows an increase in the CHF with the increase in the stiffness for a given initial mass flux. A correlation for the prediction of CHF under various stiffness conditions is developed. A correlation is suggested to predict the mass flux at CHF as a function of stiffness and initial mass flux. Modeling and transient analysis of the stiffness effect on CHF is carried out using the thermal hydraulic system code RELAP5. The predicted phenomena are in agreement with the experimental observations.

Fatigue crack paths under the influence of changes in stiffness

Directory of Open Access Journals (Sweden)

G. Kullmer

2016-02-01

Full Text Available An important topic of the Collaborative Research Centre TRR 30 of the Deutsche Forschungsgemeinschaft (DFG is the crack growth behaviour in graded materials. In addition, the growth of cracks in the neighbourhood of regions and through regions with different material properties belongs under this topic. Due to the different material properties, regions with differing stiffness compared to the base material may arise. Regions with differing stiffness also arise from ribs, grooves or boreholes. Since secure findings on the propagation behaviour of fatigue cracks are essential for the evaluation of the safety of components and structures, the growth of cracks near changes in stiffness has to be considered, too. Depending on the way a crack penetrates the zone of influence of such a change in stiffness and depending on whether this region is more compliant or stiffer than the surrounding area the crack may grow towards or away from this region. Both cases result in curved crack paths that cannot be explained only by the global loading situation. To evaluate the influence of regions with differing stiffness on the path of fatigue cracks the paths and the stress intensity factors of cracks growing near and through regions with differing stiffness are numerically determined with the program system ADAPCRACK3D. Therefore, arrangements of changes in stiffness modelled as material inclusions with stiffness properties different from the base material or modelled as ribs and grooves are systematically varied to develop basic conclusions about the crack growth behaviour near and through changes in stiffness.

On prestress stiffness analysis of bolt-plate contact assemblies

DEFF Research Database (Denmark)

Pedersen, Niels Leergaard; Pedersen, Pauli

2008-01-01

, but with finite element (FE) and contact analysis, it is possible to find the stiffness of the member. In the case of many connections and for practical applications, it is not suitable to make a full FE analysis. The purpose of the present paper is to find simplified expressions for the stiffness of the member......, including the case when the width of the member is limited. The calculation of the stiffness is based on the FE, including the solution to the contact problem, and we express the stiffness as a function of the elastic energy in the structure, whereby the definition of the displacements related...

Low frequency noise reduction using stiff light composite panels

Institute of Scientific and Technical Information of China (English)

DENG Yongchang; LIN Weizheng

2003-01-01

The experiment presented in this paper is to investigate and analyze the noise reduction at low frequency using stiff light composite panels. Since these composite panels are made of lightweight and stiff materials, this actuation strategy will enable the creation of composite panels for duct noise control without using traditional heavy structural mass. The results suggest that the mass-spring resonance absorption in the case of a comparatively stiff thick panel with a thin flexible plate is more efficient with minimum weight, when subjected to low-frequency (<500 Hz). The efficiency of the panel absorber depends on the mass of the thin flexible plate and the stiffness of the panel.

Elastin in large artery stiffness and hypertension

Science.gov (United States)

Wagenseil, Jessica E.; Mecham, Robert P.

2012-01-01

Large artery stiffness, as measured by pulse wave velocity (PWV), is correlated with high blood pressure and may be a causative factor in essential hypertension. The extracellular matrix components, specifically the mix of elastin and collagen in the vessel wall, determine the passive mechanical properties of the large arteries. Elastin is organized into elastic fibers in the wall during arterial development in a complex process that requires spatial and temporal coordination of numerous proteins. The elastic fibers last the lifetime of the organism, but are subject to proteolytic degradation and chemical alterations that change their mechanical properties. This review discusses how alterations in the amount, assembly, organization or chemical properties of the elastic fibers affect arterial stiffness and blood pressure. Strategies for encouraging or reversing alterations to the elastic fibers are addressed. Methods for determining the efficacy of these strategies, by measuring elastin amounts and arterial stiffness, are summarized. Therapies that have a direct effect on arterial stiffness through alterations to the elastic fibers in the wall may be an effective treatment for essential hypertension. PMID:22290157

Advanced damper with negative structural stiffness elements

International Nuclear Information System (INIS)

Dong, Liang; Lakes, Roderic S

2012-01-01

Negative stiffness is understood as the occurrence of a force in the same direction as the imposed deformation. Structures and composites with negative stiffness elements enable a large amplification in damping. It is shown in this work, using an experimental approach, that when a flexible flat-ends column is aligned in a post-buckled condition, a negative structural stiffness and large hysteresis (i.e., high damping) can be achieved provided the ends of the column undergo tilting from flat to edge contact. Stable axial dampers with initial modulus equivalent to that of the parent material and with enhanced damping were designed and built using constrained negative stiffness effects entailed by post-buckled press-fit flat-ends columns. Effective damping of approximately 1 and an effective stiffness–damping product of approximately 1.3 GPa were achieved in such stable axial dampers consisting of PMMA columns. This is a considerable improvement for this figure of merit (i.e., the stiffness–damping product), which generally cannot exceed 0.6 GPa for currently used damping layers. (paper)

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain.

Science.gov (United States)

Iwashita, Misato; Kataoka, Noriyuki; Toida, Kazunori; Kosodo, Yoichi

2014-10-01

Accumulating evidence implicates the significance of the physical properties of the niche in influencing the behavior, growth and differentiation of stem cells. Among the physical properties, extracellular stiffness has been shown to have direct effects on fate determination in several cell types in vitro. However, little evidence exists concerning whether shifts in stiffness occur in vivo during tissue development. To address this question, we present a systematic strategy to evaluate the shift in stiffness in a developing tissue using the mouse embryonic cerebral cortex as an experimental model. We combined atomic force microscopy measurements of tissue and cellular stiffness with immunostaining of specific markers of neural differentiation to correlate the value of stiffness with the characteristic features of tissues and cells in the developing brain. We found that the stiffness of the ventricular and subventricular zones increases gradually during development. Furthermore, a peak in tissue stiffness appeared in the intermediate zone at E16.5. The stiffness of the cortical plate showed an initial increase but decreased at E18.5, although the cellular stiffness of neurons monotonically increased in association with the maturation of the microtubule cytoskeleton. These results indicate that tissue stiffness cannot be solely determined by the stiffness of the cells that constitute the tissue. Taken together, our method profiles the stiffness of living tissue and cells with defined characteristics and can therefore be utilized to further understand the role of stiffness as a physical factor that determines cell fate during the formation of the cerebral cortex and other tissues. © 2014. Published by The Company of Biologists Ltd.

Evaluating pulp stiffness from fibre bundles by ultrasound

Science.gov (United States)

Karppinen, Timo; Montonen, Risto; Määttänen, Marjo; Ekman, Axel; Myllys, Markko; Timonen, Jussi; Hæggström, Edward

2012-06-01

A non-destructive ultrasonic tester was developed to measure the stiffness of pulp bundles. The mechanical properties of pulp are important when estimating the behaviour of paper under stress. Currently available pulp tests are tedious and alter the fibres structurally and mechanically. The developed tester employs (933 ± 15) kHz tweezer-like ultrasonic transducers and time-of-flight measurement through (9.0 ± 2.5) mm long and (0.8 ± 0.1) mm thick fibre bundles kept at (19.1 ± 0.4) °C and (62 ± 1)% RH. We determined the stiffness of soft wood pulps produced by three kraft pulping modifications: standard kraft pulp, (5.2 ± 0.4) GPa, prehydrolysis kraft pulp, (4.3 ± 0.4) GPa, and alkali extracted prehydrolysis kraft pulp, (3.3 ± 0.4) GPa. Prehydrolysis and alkali extraction processes mainly lowered the hemicellulose content of the pulps, which essentially decreased the fibre-wall stiffness hence impairing the stiffness of the fibre networks. Our results indicate that the method allows ranking of pulps according to their stiffness determined from bundle-like samples taken at an early phase of the papermaking process.

A new variable stiffness suspension system: passive case

Directory of Open Access Journals (Sweden)

O. M. Anubi

2013-02-01

Full Text Available This paper presents the design, analysis, and experimental validation of the passive case of a variable stiffness suspension system. The central concept is based on a recently designed variable stiffness mechanism. It consists of a horizontal control strut and a vertical strut. The main idea is to vary the load transfer ratio by moving the location of the point of attachment of the vertical strut to the car body. This movement is controlled passively using the horizontal strut. The system is analyzed using an L2-gain analysis based on the concept of energy dissipation. The analyses, simulation, and experimental results show that the variable stiffness suspension achieves better performance than the constant stiffness counterpart. The performance criteria used are; ride comfort, characterized by the car body acceleration, suspension deflection, and road holding, characterized by tire deflection.

Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque

Directory of Open Access Journals (Sweden)

Min Ji Choi

2015-06-01

Full Text Available Transforming growth factor-β1 (TGF-β1 has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD. The mothers against decapentaplegic homolog 7 (SMAD7 is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator and induced the expression of poly (ADP-ribose polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.

Association of Parental Hypertension With Arterial Stiffness in Nonhypertensive Offspring

DEFF Research Database (Denmark)

Andersson, Charlotte; Quiroz, Rene; Enserro, Danielle

2016-01-01

High arterial stiffness seems to be causally involved in the pathogenesis of hypertension. We tested the hypothesis that offspring of parents with hypertension may display higher arterial stiffness before clinically manifest hypertension, given that hypertension is a heritable condition. We compa......, in this community-based sample of young, nonhypertensive adults, we observed greater arterial stiffness in offspring of parents with hypertension. These observations are consistent with higher vascular stiffness at an early stage in the pathogenesis of hypertension.......High arterial stiffness seems to be causally involved in the pathogenesis of hypertension. We tested the hypothesis that offspring of parents with hypertension may display higher arterial stiffness before clinically manifest hypertension, given that hypertension is a heritable condition. We...... compared arterial tonometry measures in a sample of 1564 nonhypertensive Framingham Heart Study third-generation cohort participants (mean age: 38 years; 55% women) whose parents were enrolled in the Framingham Offspring Study. A total of 468, 715, and 381 participants had 0 (referent), 1, and 2 parents...

Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts

DEFF Research Database (Denmark)

Hansen, Daniel Bloch; Friis, Martin Barfred; Hoffmann, Else Kay

2012-01-01

The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine...... are significantly increased following hyperosmotic exposure. Swelling-induced ROS production in NIH3T3 fibroblasts is generated by NOX4 and by increasing total ROS, by either exogenous application of H(2)O(2) or overexpressing NOX4, we demonstrate that TonEBP activity and taurine influx are regulated negatively...... by ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low...

VARIABLE STIFFNESS HAND PROSTHESIS: A SYSTEMATIC REVIEW

Directory of Open Access Journals (Sweden)

S. Cecilia Tapia-Siles

2017-06-01

Full Text Available Prosthetics is an important field in engineering due to the large number of amputees worldwide and the associated problems such as limited functionality of the state of the art. An important functionality of the human hand is its capability of adjusting the stiffness of the joints depending on the currently performed task. For the development of new technology it is important to understand the limitations of existing resources. As part of our efforts to develop a variable stiffness grasper for developing countries a systematic review was performed covering technology of body powered and myoelectric hand prosthesis. Focus of the review is readiness of prosthetic hands regarding their capability of controlling the stiffness of the end effector. Publications sourced through three different digital libraries were systematically reviewed on the basis of the PRISMA standard. We present a search strategy as well as the PRISMA assessment of the resulting records which covered 321 publications. The records were assessed and the results are presented for the ability of devices to control their joint stiffness. The review indicates that body powered prosthesis are preferred to myoelectric hands due to the reduced cost, the simplicity of use and because of their inherent ability to provide feedback to the user. Stiffness control was identified but has not been fully covered in the current state of the art. In addition we summarise the identified requirements on prosthetic hands as well as related information which can support the development of new prosthetics.

Vascular endothelial growth factor and basic fibroblast growth factor expression positively correlates with angiogenesis and peritumoural brain oedema in astrocytoma

International Nuclear Information System (INIS)

Jang, F.F.; Wei, W.

2008-01-01

Astrocytoma is the most malignant intracranial neoplasm and is characterized by high neovascularization and peritumoural brain oedema. Angiogenesis is a complicated process in oncogenesis regulated by the balance between angiogenic and antiangiogenic factors. The expression of two angiogenic growth factors, vascular endothelial growth factor and basic fibroblast growth factor were investigated using immunohistochemistry for astrocytoma from 82 patients and 11 normal human tissues. The expression of vascular endothelial growth factor and basic fibroblast growth factor positively correlate with the pathological grade of astrocytoma, microvessel density numbers and brain oedema, which may be responsible for the increased tumour neovascularization and peritumoural brain oedema. The results support the idea that inhibiting vascular endothelial growth factor and basic fibroblast growth factor are useful for the treatment of human astrocytoma and to improve patient's clinical outcomes and prognosis. (author)

Effect of long-term isometric training on core/torso stiffness.

Science.gov (United States)

Lee, Benjamin C Y; McGill, Stuart M

2015-06-01

Although core stiffness enhances athletic performance traits, controversy exists regarding the effectiveness of isometric vs. dynamic core training methods. This study aimed to determine whether long-term changes in stiffness can be trained, and if so, what is the most effective method. Twenty-four healthy male subjects (23 ± 3 years; 1.8 ± 0.06 m; 77.5 ± 10.8 kg) were recruited for passive and active stiffness measurements before and after a 6-week core training intervention. Twelve subjects (22 ± 2 years; 1.8 ± 0.08 m; 78.3 ± 12.3 kg) were considered naive to physical and core exercise. The other 12 subjects (24 ± 3 years; 1.8 ± 0.05 m; 76.8 ± 9.7 kg) were Muay Thai athletes (savvy). A repeated-measures design compared core training methods (isometric vs. dynamic, with a control group) and subject training experience (naive vs. savvy) before and after a 6-week training period. Passive stiffness was assessed on a "frictionless" bending apparatus and active stiffness assessed through a quick release mechanism. Passive stiffness increased after the isometric training protocol. Dynamic training produced a smaller effect, and as expected, there was no change in the control group. Active stiffness did not change in any group. Comparisons between subject and training groups did not reveal any interactions. Thus, an isometric training approach was superior in terms of enhancing core stiffness. This is important since increased core stiffness enhances load bearing ability, arrests painful vertebral micromovements, and enhances ballistic distal limb movement. This may explain the efficacy reported for back and knee injury reduction.

PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors.

Science.gov (United States)

Gaber, Zachary B; Butler, Samantha J; Novitch, Bennett G

2013-10-01

Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment.

Doubling potential of fibroblasts from different species after ionising radiation

International Nuclear Information System (INIS)

Macieira-Coelho, A.; Diatloff, C.; Malaise, E.

1976-01-01

It is stated that whereas chicken fibroblasts invariably die after a certain number of doublings in vitro, and this fact is never altered by chemical or physical agents, mouse fibroblasts invariably acquire spontaneously an infinite growth potential. In the human species fibroblasts never acquire spontaneously the capacity to divide for ever, although they can become permanent cell lines after treatment with certain viruses. This behaviour of fibroblasts in vitro has been attributed to different nutritional requirements. Experiments are described with human and mouse fibroblasts in which it was found that the response to ionising radiation matches the relative tendencies of the fibroblasts to yield permanent cell lines. Irradiation was commenced during the phase of active proliferation. Human fibroblast cultures irradiated with 100 R stopped dividing earlier than the controls, whereas cultures irradiated with 200, 300 and 500 R had the same lifespan as the control cultures. Cultures irradiated with 400 R showed the longest survival. With mouse fibroblasts the growth curves of the irradiated cells were of the same type as in the controls, but recovery occurred earlier. The results indicated that ionising radiation accelerates a natural phenomenon; in cells with a limited growth potential (chicken) it shortens the lifespan, whereas in cells that can acquire an unlimited growth potential (mouse) it accelerates acquisition of this potential; human fibroblasts showed an intermediate response, since ionising radiation neither established the cultures as with mouse cells nor reduced the number of cells produced as with chicken fibroblasts. Possible explanations for the different behaviour of the species are offered. (U.K.)

Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

DEFF Research Database (Denmark)

Barker, Holly E; Bird, Demelza; Lang, Georgina

2013-01-01

models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α...

[Metabolic syndrome and aortic stiffness].

Science.gov (United States)

Simková, A; Bulas, J; Murín, J; Kozlíková, K; Janiga, I

2010-09-01

The metabolic syndrome (MS) is a cluster of risk factors that move the patient into higher level of risk category of cardiovascular disease and the probability of type 2 diabetes mellitus manifestation. Definition of MS is s based on the presence of selected risk factors as: abdominal obesity (lager waist circumpherence), atherogenic dyslipidemia (low value of HDL-cholesterol and increased level of triglycerides), increased fasting blood glucose (or type 2 DM diagnosis), higher blood pressure or antihypertensive therapy. In 2009 there were created harmonizing criteria for MS definition; the condition for assignment of MS is the presence of any 3 criteria of 5 mentioned above. The underlying disorder of MS is an insulin resistance or prediabetes. The patients with MS more frequently have subclinical (preclinical) target organ disease (TOD) which is the early sings of atherosclerosis. Increased aortic stiffness is one of the preclinical diseases and is defined by pathologically increased carotidofemoral pulse wave velocity in aorta (PWV Ao). With the aim to assess the influence of MS on aortic stiffness we examined the group of women with arterial hypertension and MS and compare them with the group of women without MS. The aortic stiffness was examined by Arteriograph--Tensiomed, the equipment working on the oscillometric principle in detection of pulsations of brachial artery. This method determines the global aortic stiffness based on the analysis of the shape of pulse curve of brachial artery. From the cohort of 49 pts 31 had MS, the subgroups did not differ in age or blood pressure level. The mean number of risk factors per person in MS was 3.7 comparing with 1.7 in those without MS. In the MS group there was more frequently abdominal obesity present (87% vs 44%), increased fasting blood glucose (81% vs 22%) and low HDL-cholesterol level. The pulse wave velocity in aorta, PWV Ao, was significantly higher in patients with MS (mean value 10,19 m/s vs 8,96 m

Carcinoma associated fibroblasts (CAFs) promote breast cancer motility by suppressing mammalian Diaphanous-related formin-2 (mDia2).

Science.gov (United States)

Dvorak, Kaitlyn M; Pettee, Krista M; Rubinic-Minotti, Kaitlin; Su, Robin; Nestor-Kalinoski, Andrea; Eisenmann, Kathryn M

2018-01-01

The tumor microenvironment (TME) promotes tumor cell invasion and metastasis. An important step in the shift to a pro-cancerous microenvironment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are present in a majority of solid tumors and can directly promote tumor cell motility via cytokine, chemokine and growth factor secretion into the TME. The exact effects that the TME has upon cytoskeletal regulation in motile tumor cells remain enigmatic. The conserved formin family of cytoskeleton regulating proteins plays an essential role in the assembly and/or bundling of unbranched actin filaments. Mammalian Diaphanous-related formin 2 (mDia2/DIAPH3/Drf3/Dia) assembles a dynamic F-actin cytoskeleton that underlies tumor cell migration and invasion. We therefore sought to understand whether CAF-derived chemokines impact breast tumor cell motility through modification of the formin-assembled F-actin cytoskeleton. In MDA-MB-231 cells, conditioned media (CM) from WS19T CAFs, a human breast tumor-adjacent CAF line, significantly and robustly increased wound closure and invasion relative to normal human mammary fibroblast (HMF)-CM. WS19T-CM also promoted proteasome-mediated mDia2 degradation in MDA-MB-231 cells relative to control HMF-CM and WS21T CAF-CM, a breast CAF cell line that failed to promote robust MDA-MB-231 migration. Cytokine array analysis of CM identified up-regulated secreted factors in WS19T relative to control WS21T CM. We identified CXCL12 as a CM factor influencing loss of mDia2 protein while increasing MDA-MB-231 cell migration. Our data suggest a mechanism whereby CAFs promote tumor cell migration and invasion through CXCL12 secretion to regulate the mDia2-directed cytoskeleton in breast tumor cells.

Phenotype change and migration of adventitial fibroblasts during postangioplasty

International Nuclear Information System (INIS)

Wang Yongli; Zhang Jiaxing; He Nengshu; Si Tongguo; Fan Hailun; Ge Xihong; Xu Rui

2006-01-01

Objective: To verify fibroblasts translocation from adventitia into neointima by labeling adventitia cells with bromodeoxyuridine (BrDU) after angioplasty, and to explore the relationship of adventitial fibroblast with restenosis. Methods: Vascular restenosis model was created by injured intima of common carotid artery (CCA) of mouse with guide wire, adventitial fibroblasts were labeled with BrDU, and dynamic distribution of myofibroblasts in adventitia, media and neoitima was observed at different times (3 d, 7 d, 14 d and 28 d) by means of single/double-label immunohistochemistry, light microscope, electronic microscope and image analysis system. Results: 1.Immunohistochemistry: More adventitial fibroblasts combined with BrDU could be found in adventitia on the 3rd day of postangioplasty, and the number of this kind of cells reached the peak on 7th day, and at the same time fibroblasts changed their phenotypes and became myofibroblasts, which produced α-actin and extracellular matrix (ECM). On 14th day, the number of the positive cells decreased in adventitia, increased in media and neointima associated with intima thickening; on 28th day, while the number of fibroblasts labeled by BrDU returned to the basic-line in adventitia, media and intima, nevertheless, intima thickening and vascular stenosis and intimal ELM precipitation were still present. There were significant differences in the number of fibroblasts labeled with BrDU located in three layers of artery (P<0.05). 2. Electronic microscope: After angioplasty, the plasm of fibroblasts became rich, mitochondrious and increase of Golgi apparatus; and the amount of rough endoplasmic reticulums rose with more secretory granules, together with a great amount of collagen synthesized forming the microfilaments; on days of 7th and 14th, the wide pseudopodia of myofibroblasts could be found extending into the windows on the external elastic lamina (ELL) and the internal elastic lamina (ILL); and showing the tendency

Epidermal growth factor receptor-induced activato protein 1 activity controls density-dependent growht inhibition in normal rat kidney fibroblasts.

NARCIS (Netherlands)

Hornberg, J.J.; Dekker, H.; Peters, P.H.J.; Langerak, P.; Westerhoff, H.V.; Lankelma, J.; Zoelen, E.J.J.

2006-01-01

Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these

Sway‐dependent changes in standing ankle stiffness caused by muscle thixotropy

Science.gov (United States)

Sakanaka, Tania E.; Lakie, Martin

2016-01-01

Key points The passive stiffness of the calf muscles contributes to standing balance, although the properties of muscle tissue are highly labile.We investigated the effect of sway history upon intrinsic ankle stiffness and demonstrated reductions in stiffness of up to 43% during conditions of increased baseline sway.This sway dependence was most apparent when using low amplitude stiffness‐measuring perturbations, and the short‐range stiffness component was smaller during periods of high sway.These characteristics are consistent with the thixotropic properties of the calf muscles causing the observed changes in ankle stiffness.Periods of increased sway impair the passive stabilization of standing, demanding more active neural control of balance. Abstract Quiet standing is achieved through a combination of active and passive mechanisms, consisting of neural control and intrinsic mechanical stiffness of the ankle joint, respectively. The mechanical stiffness is partly determined by the calf muscles. However, the viscoelastic properties of muscle are highly labile, exhibiting a strong dependence on movement history. By measuring the effect of sway history upon ankle stiffness, the present study determines whether this lability has consequences for the passive stabilization of human standing. Ten subjects stood quietly on a rotating platform whose axis was collinear with the ankle joint. Ankle sway was increased by slowly tilting this platform in a random fashion, or decreased by fixing the body to a board. Ankle stiffness was measured by using the same platform to simultaneously apply small, brief perturbations (ankle stiffness by up to 43% compared to the body‐fixed condition. Normal quiet stance was associated with intermediate values. The effect was most apparent when using smaller perturbation amplitudes to measure stiffness (0.1 vs. 0.6 deg). Furthermore, torque responses exhibited a biphasic pattern, consisting of an initial steep rise followed by a

Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

Science.gov (United States)

Reisner, P D; Brandt, P C; Vanaman, T C

1997-01-01

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

Determination of Ankle and Metatarsophalangeal Stiffness During Walking and Jogging.

Science.gov (United States)

Mager, Fabian; Richards, Jim; Hennies, Malika; Dötzel, Eugen; Chohan, Ambreen; Mbuli, Alex; Capanni, Felix

2018-05-29

Forefoot stiffness has been shown to influence joint biomechanics. However, little or no data exists on metatarsophalangeal stiffness. Twenty-four healthy rearfoot strike runners were recruited from a staff and student population at the University of Central Lancashire. Five repetitions of shod, self-selected speed level walking and jogging were performed. Kinetic and kinematic data were collected using retro-reflective markers placed on the lower limb and foot, to create a three-segment foot model using the Calibrated Anatomical System Technique. Ankle and metatarsophalangeal moments and angles were calculated. Stiffness values were calculated using a linear best fit line of moment versus of angle plots. Paired t-tests were used to compare values between walking and jogging conditions. Significant differences were seen in ankle range of motion (ROM), but not in metatarsophalangeal ROM. Maximum moments were significantly greater in the ankle during jogging, but these were not significantly different at the metatarsophalangeal joint. Average ankle joint stiffness exhibited significantly lower stiffness when walking compared to jogging. However, the metatarsophalangeal joint exhibited significantly greater stiffness when walking compared to jogging. A greater understanding of forefoot stiffness may inform the development of footwear, prosthetic feet and orthotic devices, such as ankle-foot orthoses for walking and sporting activities.

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

Energy Technology Data Exchange (ETDEWEB)

Tashiro, Kanae [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Shishido, Mayumi [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Fujimoto, Keiko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan); Hirota, Yuko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Yo, Kazuyuki; Gomi, Takamasa [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Tanaka, Yoshitaka, E-mail: tanakay@bioc.phar.kyushu-u.ac.jp [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan)

2014-01-03

Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

International Nuclear Information System (INIS)

Tashiro, Kanae; Shishido, Mayumi; Fujimoto, Keiko; Hirota, Yuko; Yo, Kazuyuki; Gomi, Takamasa; Tanaka, Yoshitaka

2014-01-01

Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility

Dynamically tuned magnetostrictive spring with electrically controlled stiffness

Science.gov (United States)

Scheidler, Justin J.; Asnani, Vivake M.; Dapino, Marcelo J.

2016-03-01

This paper presents the design and testing of an electrically controllable magnetostrictive spring that has a dynamically tunable stiffness (i.e., a magnetostrictive Varispring). The device enables in situ stiffness tuning or stiffness switching for vibration control applications. Using a nonlinear electromechanical transducer model and an analytical solution of linear, mechanically induced magnetic diffusion, Terfenol-D is shown to have a faster rise time to stepped voltage inputs and a significantly higher magnetic diffusion cut-off frequency relative to Galfenol. A Varispring is manufactured using a laminated Terfenol-D rod. Further rise time reductions are achieved by minimizing the rod’s diameter and winding the electromagnet with larger wire. Dynamic tuning of the Varispring’s stiffness is investigated by measuring the Terfenol-D rod’s strain response to dynamic, compressive, axial forces in the presence of sinusoidal or square wave control currents. The Varispring’s rise time is \\lt 1 ms for 1 A current switches. Continuous modulus changes up to 21.9 GPa and 500 Hz and square wave modulus changes (dynamic {{Δ }}E effect) up to 12.3 GPa and 100 Hz are observed. Stiffness tunability and tuning bandwidth can be considerably increased by operating about a more optimal bias stress and improving the control of the electrical input.

The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs as a co-culture in vitro

Directory of Open Access Journals (Sweden)

Król Magdalena

2012-03-01

Full Text Available Abstract Background It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs in vitro. Results A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p Conclusion The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.

The intriguing role of fibroblasts and c-Jun in the chemopreventive and therapeutic effect of finasteride on xenograft models of prostate cancer

Directory of Open Access Journals (Sweden)

Yi-Nong Niu

2016-01-01

Full Text Available In a large clinical trial, finasteride reduced the rate of low-grade prostate cancer (PCa while increasing the incidence of high-grade cancer. Whether finasteride promotes the development of high-grade tumors remains controversial. We demonstrated the role of fibroblasts and c-Jun in chemopreventive and therapeutic effect of finasteride on xenograft models of PCa. LNCaP (PC3 cells or recombinants of cancer cells and fibroblasts were implanted in male athymic nude mice treated with finasteride. Tumor growth, cell proliferation, apoptosis, p-Akt, and p-ERK1/2 were evaluated. In LNCaP (PC3 mono-grafted models, finasteride did not change the tumor growth. In recombinant-grafted models, fibroblasts and c-Jun promoted tumor growth; finasteride induced proliferation of LNCaP cells and repressed PC3 cell apoptosis. When c-Jun was knocked out, fibroblasts and/or finasteride did not promote the tumor growth. Finasteride inhibited p-Akt and p-ERK1/2 in mono-culture cancer cells while stimulating the same signaling molecules in the presence of fibroblasts. Reduced p-Akt and p-ERK1/2 were noted in the presence of c-Jun−/− fibroblasts. Fibroblasts and c-Jun promote PCa growth; finasteride further stimulates tumor growth with promoted proliferation, repressed apoptosis, and up-regulated pro-proliferative molecular pathway in the presence of fibroblasts and c-Jun. Stromal-epithelial interactions play critical roles in finasteride′s therapeutic effects on PCa. Our findings have preliminary implications in using finasteride as a chemopreventive or therapeutic agent for PCa patients.

Effects of mechanical properties and geometric conditions on stiffness of Hyperboloid Shallow Shell

Directory of Open Access Journals (Sweden)

Zhao Lihong

2015-01-01

Full Text Available The experiment models based on the hyperboloid shallow shells that represent automobile panel's surface features are established. The effects of material properties and geometric conditions condition on the stiffness of hyperboloid shallow shell are investigated experimentally. The influences of panel thickness and geometric conditions on stiffness are very obvious. Stiffness increases with increasing of the panel thickness, and stiffness doubled as increasing in thickness with 0.1 mm. The effect of thickness on stiffness is far greater than that of blank holding force. The greater the arc height of punch, the greater the stiffness. And stiffness increases nearly by five times with arc height of punch is from 3mm to 9mm. The effect of arc height of punch on stiffness is far greater than that of materials mechanical properties. The stiffness is varied with different panel material properties by the same forming and stiffness test conditions. The decrease of yield strength is beneficial to the panel stiffness. The appropriate choice of materials and forming process condition is important in meeting necessary requirements for the energy-saving, lightweight and reducing wind resistance design in automotive industry.

Intraventricular filling under increasing left ventricular wall stiffness and heart rates

Science.gov (United States)

Samaee, Milad; Lai, Hong Kuan; Schovanec, Joseph; Santhanakrishnan, Arvind; Nagueh, Sherif

2015-11-01

Heart failure with normal ejection fraction (HFNEF) is a clinical syndrome that is prevalent in over 50% of heart failure patients. HFNEF patients show increased left ventricle (LV) wall stiffness and clinical diagnosis is difficult using ejection fraction (EF) measurements. We hypothesized that filling vortex circulation strength would decrease with increasing LV stiffness irrespective of heart rate (HR). 2D PIV and hemodynamic measurements were acquired on LV physical models of varying wall stiffness under resting and exercise HRs. The LV models were comparatively tested in an in vitro flow circuit consisting of a two-element Windkessel model driven by a piston pump. The stiffer LV models were tested in comparison with the least stiff baseline model without changing pump amplitude, circuit compliance and resistance. Increasing stiffness at resting HR resulted in diminishing cardiac output without lowering EF below 50% as in HFNEF. Increasing HR to 110 bpm in addition to stiffness resulted in lowering EF to less than 50%. The circulation strength of the intraventricular filling vortex diminished with increasing stiffness and HR. The results suggest that filling vortex circulation strength could be potentially used as a surrogate measure of LV stiffness. This research was supported by the Oklahoma Center for Advancement of Science and Technology (HR14-022).

The effect of short-term isometric training on core/torso stiffness.

Science.gov (United States)

Lee, Benjamin; McGill, Stuart

2017-09-01

"Core" exercise is a basic part of many physical training regimens with goals ranging from rehabilitation of spine and knee injuries to improving athletic performance. Core stiffness has been proposed to perform several functions including reducing pain by minimising joint micro-movements, and enhancing strength and speed performance. This study probes the links between a training approach and immediate but temporary changes in stiffness. Passive and active stiffness was measured on 24 participants; 12 having little to no experience in core training (inexperienced), and the other 12 being athletes experienced to core training methods; before and after a 15 min bout of isometric core exercises. Passive stiffness was assessed on a "frictionless" bending apparatus and active stiffness assessed via a quick release mechanism. Short-term isometric core training increased passive and active stiffness in most directions for both inexperienced and experienced participants, passive left lateral bend among experienced participants being the exception (P core stiffness, in this case following a single session. This may influence performance and injury resilience for a brief period.

Dynamic Stiffness Transfer Function of an Electromechanical Actuator Using System Identification

Science.gov (United States)

Kim, Sang Hwa; Tahk, Min-Jea

2018-04-01

In the aeroelastic analysis of flight vehicles with electromechanical actuators (EMAs), an accurate prediction of flutter requires dynamic stiffness characteristics of the EMA. The dynamic stiffness transfer function of the EMA with brushless direct current (BLDC) motor can be obtained by conducting complicated mathematical calculations of control algorithms and mechanical/electrical nonlinearities using linearization techniques. Thus, system identification approaches using experimental data, as an alternative, have considerable advantages. However, the test setup for system identification is expensive and complex, and experimental procedures for data collection are time-consuming tasks. To obtain the dynamic stiffness transfer function, this paper proposes a linear system identification method that uses information obtained from a reliable dynamic stiffness model with a control algorithm and nonlinearities. The results of this study show that the system identification procedure is compact, and the transfer function is able to describe the dynamic stiffness characteristics of the EMA. In addition, to verify the validity of the system identification method, the simulation results of the dynamic stiffness transfer function and the dynamic stiffness model were compared with the experimental data for various external loads.

Monophasic Pulsed 200-μA Current Promotes Galvanotaxis With Polarization of Actin Filament and Integrin α2β1 in Human Dermal Fibroblasts.

Science.gov (United States)

Uemura, Mikiko; Maeshige, Noriaki; Koga, Yuka; Ishikawa-Aoyama, Michiko; Miyoshi, Makoto; Sugimoto, Masaharu; Terashi, Hiroto; Usami, Makoto

2016-01-01

The monophasic pulsed microcurrent is used to promote wound healing, and galvanotaxis regulation has been reported as one of the active mechanisms in the promotion of tissue repair with monophasic pulsed microcurrent. However, the optimum monophasic pulsed microcurrent parameters and intracellular changes caused by the monophasic pulsed microcurrent have not been elucidated in human dermal fibroblasts. The purpose of this study was to investigate the optimum intensity for promoting galvanotaxis and the effects of electrical stimulation on integrin α2β1 and actin filaments in human dermal fibroblasts. Human dermal fibroblasts were treated with the monophasic pulsed microcurrent of 0, 100, 200, or 300 μA for 8 hours, and cell migration and cell viability were measured 24 hours after starting monophasic pulsed microcurrent stimulation. Polarization of integrin α2β1 and lamellipodia formation were detected by immunofluorescent staining 10 minutes after starting monophasic pulsed microcurrent stimulation. The migration toward the cathode was significantly higher in the cells treated with the 200-μA monophasic pulsed microcurrent than in the controls (P microcurrent did not alter the migration ratio. The electrostimulus of 200 μA also promoted integrin α2β1 polarization and lamellipodia formation at the cathode edge (P microcurrent intensity to promote migration toward the cathode, and this intensity could regulate polarization of migration-related intracellular factors in human dermal fibroblasts.

Martial arts training attenuates arterial stiffness in middle aged adults.

Science.gov (United States)

Douris, Peter C; Ingenito, Teresa; Piccirillo, Barbara; Herbst, Meredith; Petrizzo, John; Cherian, Vincen; McCutchan, Christopher; Burke, Caitlin; Stamatinos, George; Jung, Min-Kyung

2013-09-01

Arterial stiffness increases with age and is related to an increased risk of coronary artery disease. Poor trunk flexibility has been shown to be associated with arterial stiffness in middle-aged subjects. The purpose of our research study was to measure arterial stiffness and flexibility in healthy middle-aged martial artists compared to age and gender matched healthy sedentary controls. Ten martial artists (54.0 ± 2.0 years), who practice Soo Bahk Do (SBD), a Korean martial art, and ten sedentary subjects (54.7 ± 1.8 years) for a total of twenty subjects took part in this cross-sectional study. Arterial stiffness was assessed in all subjects using pulse wave velocity (PWV), a recognized index of arterial stiffness. Flexibility of the trunk and hamstring were also measured. The independent variables were the martial artists and matched sedentary controls. The dependent variables were PWV and flexibility. There were significant differences, between the SBD practitioners and sedentary controls, in PWV (P = 0.004), in trunk flexibility (P= 0.002), and in hamstring length (P= 0.003). The middle-aged martial artists were more flexible in their trunk and hamstrings and had less arterial stiffness compared to the healthy sedentary controls. The flexibility component of martial art training or flexibility exercises in general may be considered as a possible intervention to reduce the effects of aging on arterial stiffness.

Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski

OpenAIRE

Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

2012-01-01

Ski is a transcriptional regulator that has been considered an oncoprotein, given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski−/− mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chroma...

Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts

DEFF Research Database (Denmark)

Hansen, Daniel Bloch; Guerra, Barbara; Jacobsen, Jack Hummeland

2011-01-01

Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume...... is a critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na(+) dependent transporter TauT and taurine release are increased...... and reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA(2) activity, exposure...

NOD2 and TLR2 ligands trigger the activation of basophils and eosinophils by interacting with dermal fibroblasts in atopic dermatitis-like skin inflammation

Science.gov (United States)

Jiao, Delong; Wong, Chun-Kwok; Qiu, Huai-Na; Dong, Jie; Cai, Zhe; Chu, Man; Hon, Kam-Lun; Tsang, Miranda Sin-Man; Lam, Christopher Wai-Kei

2016-01-01

The skin of patients with atopic dermatitis (AD) has a unique predisposition for colonization by Staphylococcus aureus (S. aureus), which contributes to the inflammation and grim prognosis of AD. Although the mechanism underlying the S. aureus-induced exacerbation of AD remains unclear, recent studies have found a pivotal role for pattern recognition receptors in regulating the inflammatory responses in S. aureus infection. In the present study, we used a typical mouse model of AD-like skin inflammation and found that S. aureus-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and toll-like receptor 2 (TLR2) ligands exacerbated AD-like symptoms, which were further deteriorated by the in vivo expansion of basophils and eosinophils. Subsequent histological analyses revealed that dermal fibroblasts were pervasive in the AD-like skin lesions. Co-culture of human dermal fibroblasts with basophils and eosinophils resulted in a vigorous cytokine/chemokine response to the NOD2/TLR2 ligands and the enhanced expression of intercellular adhesion molecule-1 on the dermal fibroblasts. Basophils and eosinophils were primarily responsible for the AD-related cytokine/chemokine expression in the co-cultures. Direct intercellular contact was necessary for the crosstalk between basophils and dermal fibroblasts, while soluble mediators were sufficient to mediate the eosinophil–fibroblast interactions. Moreover, the intracellular p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and nuclear factor-kappa B signaling pathways were essential for NOD2/TLR2 ligand-mediated activation of basophils, eosinophils, and dermal fibroblasts in AD-related inflammation. This study provides the evidence of NOD2/TLR2-mediated exacerbation of AD through activation of innate immune cells and therefore sheds light on a novel mechanistic pathway by which S. aureus contributes to the pathophysiology of AD. PMID:26388234

Viability test of fish scale collagen (Oshpronemus gouramy on baby hamster kidney fibroblasts-21 fibroblast cell culture

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Chiquita Prahasanti

2018-04-01

Full Text Available Aim: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy by evaluating its viability against baby hamster kidney fibroblasts-21. Materials and Methods: Collagen was extracted from gouramy fish scales (O. gouramy with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. Results: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed value of 0.754 (p>00025. Conclusion: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

International Nuclear Information System (INIS)

Klemm, Anna H.; Diez, Gerold; Alonso, Jose-Luis; Goldmann, Wolfgang H.

2009-01-01

Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

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Jaroslaw Suchanski

Full Text Available In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first

Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

Science.gov (United States)

Suchanski, Jaroslaw; Tejchman, Anna; Zacharski, Maciej; Piotrowska, Aleksandra; Grzegrzolka, Jedrzej; Chodaczek, Grzegorz; Nowinska, Katarzyna; Rys, Janusz; Dziegiel, Piotr; Kieda, Claudine; Ugorski, Maciej

2017-01-01

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such

Stiffness Coefficients of Mortise and Tenon Joints used on Wooden Window Profiles

Directory of Open Access Journals (Sweden)

Milan Podlena

2016-04-01

Full Text Available Samples of corner joints of wooden rectangular windows, with widths of 78 and 92 mm, were used to determine the stiffness of tenon and mortise joints. Two series of samples were loaded statically in the angular plane of compression and tension, so that the bending moment could be derived. The objective of the experiment was to determine the existing correlations between the stiffness in maximum strength and the stiffness in the elastic area for both types of tests. After strength tests were carried out, the annual ring width of the samples was measured to determine whether this factor affects the stiffness of the joints. The results showed that there was a relatively strong correlation between the stiffness in the elastic area and the maximum load. A two-factor analysis of variance confirmed that the type of load did not affect the stiffness of the joint, but the type of joint (width does significantly affect the stiffness. Therefore, the width of annual rings was positively correlated with the stiffness of the joints.

Variable stiffness and damping MR isolator

Energy Technology Data Exchange (ETDEWEB)

Zhang, X Z; Wang, X Y; Li, W H; Kostidis, K [University of Wollongong, School of Mechanical, Materials and Mechatronic Engineering, NSW 2522 (Australia)], E-mail: weihuali@uow.edu.au

2009-02-01

This paper presents the development of a magnetorheological (MR) fluid-based variable stiffness and damping isolator for vibration suppressions. The MR fluid isolator used a sole MR control unit to achieve the variable stiffness and damping in stepless and relative large scope. A mathematical model of the isolator was derived, and a prototype of the MR fluid isolator was fabricated and its dynamic behavior was measured in vibration under various applied magnetic fields. The parameters of the model under various magnetic fields were identified and the dynamic performances of isolator were evaluated.

Effect of lumped mass and support stiffness on pipe seismic response

International Nuclear Information System (INIS)

Chang, P.S.Y.

1986-01-01

In performing pipe stress analysis, generic support stiffness values are typically used to predict the response of the piping systems. Consistent design of every support to match the generic stiffness value is difficult. The difference between the actual and generic stiffness may affect the results of pipe stresses and support reactions. The objective of this study is to develop an acceptance criteria for the actual support stiffness and to avoid unnecessary reanalysis. The support mass in the restraint direction and mass within the pipe span can also affect the piping system behavior and this study will discuss this mass effect as well. Added mass and change in support stiffness will cause the piping system to shift frequency

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

International Nuclear Information System (INIS)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

2012-01-01

Highlights: ► We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. ► YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. ► There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. ► The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. ► The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts

International Nuclear Information System (INIS)

Xinbo Cheng; Nge Cheong; Ya Wang; Iliakis, George

1996-01-01

Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or γ-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21 cip1/waf1/sdi1 remains inducible. The results demonstrate a growth-stage and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells

Optimization of a variable-stiffness skin for morphing high-lift devices

International Nuclear Information System (INIS)

Thuwis, G A A; Abdalla, M M; Gürdal, Z

2010-01-01

One of the possibilities for the next generation of smart high-lift devices is to use a seamless morphing structure. A passive composite variable-stiffness skin as a solution to the dilemma of designing the structure to have high enough stiffness to withstand aerodynamic loading and low stiffness to enable morphing is proposed. The variable-stiffness skin is achieved by allowing for a spatial fibre angle and skin thickness variation on a morphing high-lift system. The stiffness distribution is tailored to influence the deformation of the structure beneficially. To design a realistic stiffness distribution, it is important to take aerodynamic and actuation loads into account during the optimization. A two-dimensional aero-servo-elastic framework is created for this purpose. Skin optimization is performed using a gradient-based optimizer, where sensitivity information is found through application of the adjoint method. The implementation of the aero-servo-elastic environment is addressed and initial optimization results presented. The results indicate that a variable-stiffness skin increases the design space. Moreover, the importance of taking the change in aerodynamic loads due to morphing skin deformation into account during optimization is demonstrated

circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica.

Science.gov (United States)

Cao, Zhouli; Xiao, Qingling; Dai, Xiaoniu; Zhou, Zewei; Jiang, Rong; Cheng, Yusi; Yang, Xiyue; Guo, Huifang; Wang, Jing; Xi, Zhaoqing; Yao, Honghong; Chao, Jie

2017-12-13

Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO 2 -induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO 2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO 2 . SiO 2 induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO 2 , inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO 2 . circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO 2 . Our study elucidated a link between SiO 2 -induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

Fibroblast-derived CXCL12 promotes breast cancer metastasis by facilitating tumor cell intravasation.

Science.gov (United States)

Ahirwar, Dinesh K; Nasser, Mohd W; Ouseph, Madhu M; Elbaz, Mohamad; Cuitiño, Maria C; Kladney, Raleigh D; Varikuti, Sanjay; Kaul, Kirti; Satoskar, Abhay R; Ramaswamy, Bhuvaneswari; Zhang, Xiaoli; Ostrowski, Michael C; Leone, Gustavo; Ganju, Ramesh K

2018-05-03

The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.

Protective role of microRNA-29a in denatured dermis and skin fibroblast cells after thermal injury

Directory of Open Access Journals (Sweden)

Jie Zhou

2016-03-01

Full Text Available Our previous study has suggested that downregulated microRNA (miR-29a in denatured dermis might be involved in burn wound healing. However, the exact role of miR-29a in healing of burn injury still remains unclear. Here, we found that expression of miR-29a was notably upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury, and thereafter gradually downregulated compared with control group. By contrast, the expression of collagen, type I, alpha 2 (COL1A2 and vascular endothelial growth factor (VEGF-A were first reduced and subsequently upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury. We further identified COL1A2 as a novel target of miR-29a, which is involved in type I collagen synthesis, and showed that miR-29a negatively regulated the expression level of COL1A2 in skin fibroblast cells. In addition, VEGF-A, another target gene of miR-29a, was also negatively mediated by miR-29a in skin fibroblast cells. Inhibition of miR-29a expression significantly promoted the proliferation and migration of skin fibroblast cells after thermal injury, and knockdown of COL1A2 and VEGF-A reversed the effects of miR-29a on the proliferation and migration of skin fibroblast cells. Furthermore, we found that Notch2/Jagged2 signaling was involved in miR-29a response to burn wound healing. Our findings suggest that downregulated miR-29a in denatured dermis may help burn wound healing in the later phase, probably via upregulation of COL1A2 and VEGF-A expression, which can further enhance type I collagen synthesis and angiogenesis.

Accurate spring constant calibration for very stiff atomic force microscopy cantilevers

Energy Technology Data Exchange (ETDEWEB)

Grutzik, Scott J.; Zehnder, Alan T. [Field of Theoretical and Applied Mechanics, Cornell University, Ithaca, New York 14853 (United States); Gates, Richard S.; Gerbig, Yvonne B.; Smith, Douglas T.; Cook, Robert F. [Nanomechanical Properties Group, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 (United States)

2013-11-15

There are many atomic force microscopy (AFM) applications that rely on quantifying the force between the AFM cantilever tip and the sample. The AFM does not explicitly measure force, however, so in such cases knowledge of the cantilever stiffness is required. In most cases, the forces of interest are very small, thus compliant cantilevers are used. A number of methods have been developed that are well suited to measuring low stiffness values. However, in some cases a cantilever with much greater stiffness is required. Thus, a direct, traceable method for calibrating very stiff (approximately 200 N/m) cantilevers is presented here. The method uses an instrumented and calibrated nanoindenter to determine the stiffness of a reference cantilever. This reference cantilever is then used to measure the stiffness of a number of AFM test cantilevers. This method is shown to have much smaller uncertainty than previously proposed methods. An example application to fracture testing of nanoscale silicon beam specimens is included.

Accurate spring constant calibration for very stiff atomic force microscopy cantilevers

International Nuclear Information System (INIS)

Grutzik, Scott J.; Zehnder, Alan T.; Gates, Richard S.; Gerbig, Yvonne B.; Smith, Douglas T.; Cook, Robert F.

2013-01-01

There are many atomic force microscopy (AFM) applications that rely on quantifying the force between the AFM cantilever tip and the sample. The AFM does not explicitly measure force, however, so in such cases knowledge of the cantilever stiffness is required. In most cases, the forces of interest are very small, thus compliant cantilevers are used. A number of methods have been developed that are well suited to measuring low stiffness values. However, in some cases a cantilever with much greater stiffness is required. Thus, a direct, traceable method for calibrating very stiff (approximately 200 N/m) cantilevers is presented here. The method uses an instrumented and calibrated nanoindenter to determine the stiffness of a reference cantilever. This reference cantilever is then used to measure the stiffness of a number of AFM test cantilevers. This method is shown to have much smaller uncertainty than previously proposed methods. An example application to fracture testing of nanoscale silicon beam specimens is included

Exchange stiffness of Ca-doped YIG

Science.gov (United States)

Avgin, I.; Huber, D. L.

1994-05-01

An effective medium theory for the zero-temperature exchange stiffness of uncompensated Ca-doped YIG is presented. The theory is based on the assumption that the effect of the Ca impurities is to produce strong, random ferromagnetic interactions between spins on the a and d sublattices. In the simplest version of the theory, a fraction, x, of the ad exchange integrals are large and positive, x being related to the Ca concentration. The stiffness is calculated as function of x for arbitrary perturbed ad exchange integral, Jxad. For Jxad≳(1/5)‖8Jaa+3Jdd‖, with Jaa and Jdd denoting the aa and dd exchange integrals, respectively, there is a critical concentration, Xc, such that when x≳Xc, the stiffness is complex. It is suggested that Xc delineates the region where there are significant departures from colinearity in the ground state of the Fe spins. Extension of the theory to a model where the Ca doping is assumed to generate Fe4+ ions on the tetrahedral sites is discussed. Possible experimental tests of the theory are mentioned.

Bone mineralization is regulated by signaling cross talk between molecular factors of local and systemic origin: the role of fibroblast growth factor 23.

Science.gov (United States)

Sapir-Koren, Rony; Livshits, Gregory

2014-01-01

Body phosphate homeostasis is regulated by a hormonal counter-balanced intestine-bone-kidney axis. The major systemic hormones involved in this axis are parathyroid hormone (PTH), 1,25-dihydroxyvitamin-D, and fibroblast growth factor-23 (FGF23). FGF23, produced almost exclusively by the osteocytes, is a phosphaturic hormone that plays a major role in regulation of the bone remodeling process. Remodeling composite components, bone mineralization and resorption cycles create a continuous influx-efflux loop of the inorganic phosphate (Pi) through the skeleton. This "bone Pi loop," which is formed, is controlled by local and systemic factors according to phosphate homeostasis demands. Although FGF23 systemic actions in the kidney, and for the production of PTH and 1,25-dihydroxyvitamin-D are well established, its direct involvement in bone metabolism is currently poorly understood. This review presents the latest available evidence suggesting two aspects of FGF23 bone local activity: (a) Regulation of FGF23 production by both local and systemic factors. The suggested local factors include extracellular levels of Pi and pyrophosphate (PPi), (the Pi/PPi ratio), and another osteocyte-derived protein, sclerostin. In addition, 1,25-dihydroxyvitamin-D, synthesized locally by bone cells, may contribute to regulation of FGF23 production. The systemic control is achieved via PTH and 1,25-dihydroxyvitamin-D endocrine functions. (b) FGF23 acts as a local agent, directly affecting bone mineralization. We support the assumption that under balanced physiological conditions, sclerostin, by para- autocrine signaling, upregulates FGF23 production by the osteocyte. FGF23, in turn, acts as a mineralization inhibitor, by stimulating the generation of the major mineralization antagonist-PPi. © 2014 International Union of Biochemistry and Molecular Biology.

Wide-range stiffness gradient PVA/HA hydrogel to investigate stem cell differentiation behavior.

Science.gov (United States)

Oh, Se Heang; An, Dan Bi; Kim, Tae Ho; Lee, Jin Ho

2016-04-15

Although stiffness-controllable substrates have been developed to investigate the effect of stiffness on cell behavior and function, the use of separate substrates with different degrees of stiffness, substrates with a narrow range stiffness gradient, toxicity of residues, different surface composition, complex fabrication procedures/devices, and low cell adhesion are still considered as hurdles of conventional techniques. In this study, a cylindrical polyvinyl alcohol (PVA)/hyaluronic acid (HA) hydrogel with a wide-range stiffness gradient (between ∼20kPa and ∼200kPa) and cell adhesiveness was prepared by a liquid nitrogen (LN2)-contacting gradual freezing-thawing method that does not use any additives or specific devices to produce the stiffness gradient hydrogel. From an in vitro cell culture using the stiffness gradient PVA/HA hydrogel, it was observed that human bone marrow mesenchymal stem cells have favorable stiffness ranges for induction of differentiation into specific cell types (∼20kPa for nerve cell, ∼40kPa for muscle cell, ∼80kPa for chondrocyte, and ∼190kPa for osteoblast). The PVA/HA hydrogel with a wide range of stiffness spectrum can be a useful tool for basic studies related with the stem cell differentiation, cell reprogramming, cell migration, and tissue regeneration in terms of substrate stiffness. It is postulated that the stiffness of the extracellular matrix influences cell behavior. To prove this concept, various techniques to prepare substrates with a stiffness gradient have been developed. However, the narrow ranges of stiffness gradient and complex fabrication procedures/devices are still remained as limitations. Herein, we develop a substrate (hydrogel) with a wide-range stiffness gradient using a gradual freezing-thawing method which does not need specific devices to produce a stiffness gradient hydrogel. From cell culture experiments using the hydrogel, it is observed that human bone marrow mesenchymal stem cells have

Collagen expression in fibroblasts with a novel LMNA mutation

International Nuclear Information System (INIS)

Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

2007-01-01

Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy

Optimization of a variable-stiffness skin for morphing high-lift devices

NARCIS (Netherlands)

Thuwis, G.A.A.; Abdalla, M.M.; Gürdal, Z.

2010-01-01

One of the possibilities for the next generation of smart high-lift devices is to use a seamless morphing structure. A passive composite variable-stiffness skin as a solution to the dilemma of designing the structure to have high enough stiffness to withstand aerodynamic loading and low stiffness to

Design of a Variable Stiffness Soft Dexterous Gripper

Science.gov (United States)

Nefti-Meziani, Samia; Davis, Steve

2017-01-01

Abstract This article presents the design of a variable stiffness, soft, three-fingered dexterous gripper. The gripper uses two designs of McKibben muscles. Extensor muscles that increase in length when pressurized are used to form the fingers of the gripper. Contractor muscles that decrease in length when pressurized are then used to apply forces to the fingers through tendons, which cause flexion and extension of the fingers. The two types of muscles are arranged to act antagonistically and this means that by raising the pressure in all of the pneumatic muscles, the stiffness of the system can be increased without a resulting change in finger position. The article presents the design of the gripper, some basic kinematics to describe its function, and then experimental results demonstrating the ability to adjust the bending stiffness of the gripper's fingers. It has been demonstrated that the fingers' bending stiffness can be increased by more than 150%. The article concludes by demonstrating that the fingers can be closed loop position controlled and are able to track step and sinusoidal inputs. PMID:29062630

Bone metabolism and arterial stiffness after renal transplantation.

Science.gov (United States)

Cseprekál, Orsolya; Kis, Eva; Dégi, Arianna A; Kerti, Andrea; Szabó, Attila J; Reusz, György S

2014-01-01

To assess the relationship between bone and vascular disease and its changes over time after renal transplantation. Metabolic bone disease (MBD) is common in chronic kidney disease (CKD) and is associated with cardiovascular (CV) disease. Following transplantation (Tx), improvement in CV disease has been reported; however, data regarding changes in bone disease remain controversial. Bone turnover and arterial stiffness (pulse wave velocity (PWV)) were assessed in 47 Tx patients (38 (3-191) months after Tx). Bone alkaline phosphatase (BALP), osteocalcin (OC) and beta-crosslaps were significantly higher in Tx patients, and decreased significantly after one year. There was a negative correlation between BALP, OC and steroid administered (r = -0.35; r = -0.36 respectively). PWV increased in the Tx group (1.15 SD). In patients with a follow up of bone turnover and arterial stiffness are present following kidney transplantation. While bone turnover decreases with time, arterial stiffness correlates initially with bone turnover, after which the influence of cholesterol becomes significant. Non-invasive estimation of bone metabolism and arterial stiffness may help to assess CKD-MBD following renal transplantation.

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

Science.gov (United States)

Demirci, Selami; Doğan, Ayşegül; Apdik, Hüseyin; Tuysuz, Emre Can; Gulluoglu, Sukru; Bayrak, Omer Faruk; Şahin, Fikrettin

2018-01-01

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fıbroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Graphene oxide improves the biocompatibility of collagen membranes in an in vitro model of human primary gingival fibroblasts.

Science.gov (United States)

De Marco, Patrizia; Zara, Susi; De Colli, Marianna; Radunovic, Milena; Lazović, Vladimir; Ettorre, Valeria; Di Crescenzo, Antonello; Piattelli, Adriano; Cataldi, Amelia; Fontana, Antonella

2017-09-13

Commercial collagen membranes are used in oral surgical procedures as scaffolds for bone deposition in guided bone regeneration. Here, we have enriched them with graphene oxide (GO) via a simple non-covalent functionalization, exploiting the capacity of oxygenated carbon functional moieties of GO to interact through hydrogen bonding with collagen. In the present paper, the GO-coated membranes have been characterized in terms of stability, nano-roughness, biocompatibility and induction of inflammatory response in human primary gingival fibroblast cells. The obtained coated membranes are demonstrated not to leak GO in the bulk solution, and to change some features of the membrane, such as stiffness and adhesion between the membrane and the atomic force microscopy (AFM) tip. Moreover, the presence of GO increases the roughness and the total surface exposed to the cells, as demonstrated by AFM analyses. The obtained material is biocompatible, and does not induce inflammation in the tested cells.

Identification of a parametric, discrete-time model of ankle stiffness.

Science.gov (United States)

Guarin, Diego L; Jalaleddini, Kian; Kearney, Robert E

2013-01-01

Dynamic ankle joint stiffness defines the relationship between the position of the ankle and the torque acting about it and can be separated into intrinsic and reflex components. Under stationary conditions, intrinsic stiffness can described by a linear second order system while reflex stiffness is described by Hammerstein system whose input is delayed velocity. Given that reflex and intrinsic torque cannot be measured separately, there has been much interest in the development of system identification techniques to separate them analytically. To date, most methods have been nonparametric and as a result there is no direct link between the estimated parameters and those of the stiffness model. This paper presents a novel algorithm for identification of a discrete-time model of ankle stiffness. Through simulations we show that the algorithm gives unbiased results even in the presence of large, non-white noise. Application of the method to experimental data demonstrates that it produces results consistent with previous findings.

Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts.

Science.gov (United States)

Liu, Wei; Krump, Nathan A; MacDonald, Margo; You, Jianxin

2018-02-15

Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse ( Mus musculus ), rabbit ( Oryctolagus cuniculus ), rat ( Rattus norvegicus ), chimpanzee ( Pan troglodytes ), rhesus macaque ( Macaca mulatta ), patas monkey ( Erythrocebus patas ), common woolly monkey ( Lagothrix lagotricha ), red-chested mustached tamarin ( Saguinus labiatus ), and tree shrew ( Tupaia belangeri ). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression. IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro.

Science.gov (United States)

Zhang, Chengliang; Zhang, Yanfeng; Zhu, Hong; Hu, Jiajia; Xie, Zhongshang

2018-06-01

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Extension of elastic stiffness formula for leaf type holddown spring assembly

Energy Technology Data Exchange (ETDEWEB)

Song, Kee Nam; Kang, Heung Seok; Yoon, Kyung Ho; Kim, Hyung Kyu [Korea Atomic Energy Research Institute, Taejon (Korea)

1998-09-01

Based on the Euler beam theory and the strain energy method, an elastic stiffness formula of the holddown spring assembly consisting of several leaves was previously derived. The formula was known to be useful to estimate the elastic stiffness of the holddown spring assembly only with the geometric data and the material properties of the leaf. Recently, it was reported that the elastic stiffness from the formula deviated much from the test results as the number of leaves was increased. In this study, in order to resolve such an increasing deviation as the increasing number of leaves, the formula has been extended to be able to consider normal forces and friction forces acting on interfaces between the leaves. The elastic stiffness analysis on specimens of leaf type holddown springs has been carried out using the extended formula and the analysis results are compared with the test results. As a result of comparisons, it is found that the extended formula is able to evaluate the elastic stiffness of the holddown spring assembly within an error range of 10%, irrespective of the number of leaves. In addition, it is found that the effect of shear forces and axial forces on the elastic stiffness of the holddown spring assembly is only below 0.2% of the elastic stiffness, and therefore the greatest portion of the elastic stiffness of the holddown spring assembly is attributed to the bending moment. (author). 13 refs., 10 figs., 12 tabs.

Dynamic Functional Stiffness Index of the Ankle Joint During Daily Living.

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Argunsah Bayram, Hande; Bayram, Mehmed B

2018-03-30

Exploring ankle joint physiologic functional stiffness is crucial for improving the design of prosthetic feet that aim to mimic normal gait. We hypothesized that ankle joint stiffness would vary among the different activities of daily living and that the magnitude of the stiffness would indicate the degree of energy storage element sufficiency in terms of harvesting and returning energy. We examined sagittal plane ankle moment versus flexion angle curves from 12 healthy subjects during the daily activities. The slopes of these curves were assessed to find the calculated stiffness during the peak energy return and harvest phases. For the energy return and harvest phases, stiffness varied from 0.016 to 0.283 Nm/kg° and 0.025 and 0.858 Nm/kg°, respectively. The optimum stiffness during the energy return phase was 0.111 ± 0.117 Nm/kg° and during the energy harvest phase was 0.234 ± 0.327 Nm/kg°. Ankle joint stiffness varied significantly during the activities of daily living, indicating that an energy storage unit with a constant stiffness would not be sufficient in providing energy regenerative gait during all activities. The present study was directed toward the development of a complete data set to determine the torque-angle properties of the ankle joint to facilitate a better design process. Copyright © 2017 The American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

Measuring the correlation between cell mechanics and myofibroblastic differentiation during maturation of 3D microtissues

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Zhao, Ruogang; Wang, Weigang; Boudou, Thomas; Chen, Christopher; Reich, Daniel

2013-03-01

Tissue stiffness and cellular contractility are two of the most important biomechanical factors regulating pathological transitions of encapsulated cells, such as the differentiation of fibroblasts into myofibroblasts - a key event contributing to tissue fibrosis. However, a quantitative correlation between tissue stiffness and cellular contraction and myofibroblast differentiation has not yet been established in 3D environments, mainly due to the lack of suitable 3D tissue culture models that allow both tissue remodeling and simultaneous measurement of the cell/tissue mechanics. To address this, we have developed a magnetic microtissue tester system that allows the remodeling of arrays of cell-laden 3D collagen microtissues and the measurement of cell and tissue mechanics using magnetically actuated elastomeric microcantilevers. By measuring the development of cell/tissue mechanical properties and the expression level of α-smooth muscle actin (α-SMA, a marker for myofibroblast differentiation) during a 6 day culture period, we found microtissue stiffness increased by 45% and α-SMA expression increased by 38%, but tissue contraction forces only increased by 10%, indicating that tissue stiffness may be the predominant mechanical factor for regulation of myofibroblast differentiation. This study provides new quantitative insight into the regulatory effect of cell and tissue mechanics on cellular function. Supported in part by NIH grant HL090747

Effects of fibroblast growth factor-2 on the expression and regulation of chemokines in human dental pulp cells.

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Kim, Young-Suk; Min, Kyung-San; Jeong, Dong-Ho; Jang, Jun-Hyeog; Kim, Hae-Won; Kim, Eun-Cheol

2010-11-01

Fibroblast growth factor-2 (FGF-2) participates in both hematopoiesis and osteogenesis; however, the effects of FGF-2 on chemokines during odontoblastic differentiation have not been reported. This study investigated whether human dental pulp cells (HDPCs) treated with FGF-2 could express chemokines during differentiation into odontoblastic cells and sought to identify its underlying mechanism of action. To analyze differentiation, we measured alkaline phosphatase (ALP) activity, calcified nodule formation by alizarin red staining, and marker RNA (mRNA) expression by reverse-transcriptase polymerase chain reaction (RT-PCR). Expression of chemokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and MIP-3α, were evaluated by RT-PCR. ALP activity, the mineralization, and mRNA expression for odontoblastic markers were enhanced by FGF-2 in HDPCs. FGF-2 also up-regulated the expression of IL-6, IL-8, MCP-1, MIP-1α, and MIP-3α mRNAs, which were attenuated by inhibitors of p38, ERK1/2 and p38 MAP kinases, protein kinase C, phosphoinositide-3 kinase, and NF-κB. Taken together, these data suggest that FGF-2 plays a role not only as a differentiation inducing factor in the injury repair processes of pulpal tissue but also as a positive regulator of chemokine expression, which may help in tissue engineering and pulp regeneration using HDPCs. However, the fate of odontoblastic or osteoblastic differentiation, effective local delivery for FGF-2, interaction of chemotatic and odontogenic factors, and other limitations will need to be overcome before a major modality for the treatment of pulp disease. Copyright © 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Vitamin D3 as a Novel Regulator of Basic Fibroblast Growth Factor in Chronic Rhinosinusitis with Nasal Polyposis

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Sansoni, E. Ritter; Sautter, Nathan B.; Mace, Jess C.; Smith, Timothy L.; Yawn, James R.; Lawrence, Lauren A.; Schlosser, Rodney J.; Soler, Zachary M.; Mulligan, Jennifer K.

2015-01-01

Background The immunopathogenesis of chronic rhinosinusitis (CRS) is largely unknown, but it is thought that different inflammatory profiles are responsible for the different CRS subtypes. 25-hydroxyvitamin-D (25-VD3) has been shown to alter inflammatory mediators in other disease processes and 25VD3 deficiency is associated with CRS with nasal polyps (CRSwNP), but it is unknown if 25VD3 levels impacts local inflammation in CRS. This study investigated the correlation between plasma 25-VD3 and sinonasal mucus MCP-1, RANTES and bFGF levels in patients with CRS. Methods Study subjects undergoing endoscopic sinus surgery (ESS) for CRS were prospectively enrolled from January 2012-August 2014. Control subjects included patients undergoing ESS for non-inflammatory pathology. Blood and sinonasal mucus were collected at the time of ESS. Plasma 25-VD3 was measured by ELISA and mucus levels of MCP-1, RANTES, and bFGF by cytometric bead array (CBA). Results A total of 57 patients were enrolled and categorized as CRS without nasal polyps (CRSsNP) (n=31), CRSwNP (n=14) and controls (n=12). No significant correlation was found between MCP-1 and 25-VD3. There was a significant negative correlation between 25-VD3 and RANTES (r= −0.612; p=0.026) and bFGF (r= −0.578; p=0.039) in CRSwNP patients; however, there was no significant correlation in CRSsNP patients. Conclusion This data suggests that 25-VD3 may play a role in regulation of RANTES and bFGF expression in CRSwNP. This may occur through regulation of nasal polyp fibroblasts or other immune cells. Further investigation is warranted to better elucidate the role of RANTES, bFGF and 25-VD3 in CRSwNP. PMID:25561293

Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease

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Gourdie, Robert G.; Dimmeler, Stefanie; Kohl, Peter

2016-01-01

Our understanding of cardiac fibroblast functions has moved beyond their roles in heart structure and extracellular matrix generation, and now includes contributions to paracrine, mechanical and electrical signalling during ontogenesis and normal cardiac activity. Fibroblasts have central roles in pathogenic remodelling during myocardial ischaemia, hypertension and heart failure. As key contributors to scar formation, they are crucial for tissue repair after interventions including surgery and ablation. Novel experimental approaches targeting cardiac fibroblasts are promising potential therapies for heart disease. Indeed, several existing drugs act, at least partially, through effects on cardiac connective tissue. This Review outlines the origins and roles of fibroblasts in cardiac development, homeostasis and disease; illustrates the involvement of fibroblasts in current and emerging clinical interventions; and identifies future targets for research and development. PMID:27339799

Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

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Restu Syamsul Hadi

2014-08-01

Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

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Linda Yulianti

2015-08-01

Full Text Available Asiatiocoside, a saponin component isolated from Centella asiatica can improve wound healing by promoting the proliferation of human dermal fibroblasts (HDF and synthesis of collagen. The skin-renewing cells and type I and III collagen synthesis decrease with aging, resulting in the reduction of skin elasticity and delayed wound healing. Usage of natural active compounds from plants in wound healing should be evaluated and compared to retinoic acid as an active agent that regulates wound healing. The aim of this study was to compare and evaluate the effect of asiaticoside and retinoic acid to induce greater cell proliferation and type I and III collagen synthesis in human dermal fibroblast. Methods Laboratory experiments were conducted using human dermal fibroblasts (HDF isolated from human foreskin explants. Seven passages of HDF were treated with asiaticoside and retinoic acid at several doses and incubated for 24 and 48 hours. Cell viability in all groups was tested with the MTT assay to assess HDF proliferation. Type I and III collagen synthesis was examined using the respective ELISA kits. Analysis of variance was performed to compare the treatment groups. Results Asiaticoside had significantly stronger effects on HDF proliferation than retinoic acid (p<0.05. The type III collagen production was significantly greater induction with asiaticoside compared to retinoic acid (p<0.05. Conclusion Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

IL-34 Expression in Gingival Fibroblasts, Gingival Crevicular Fluid and Gingival Tissue

OpenAIRE

Kreidly, Mariam

2014-01-01

IL-34 is a protein associated with bone degenerative diseases but the role in periodontal disease is unknown. The aim of this study was to assess the expression of IL-34 in primary human gingival fibroblasts (GF) and investigate if the expression is regulated by the pro-inflammatory cytokines interleukin-1 (IL-1β) and tumor necrosis factor α(TNF-α). We also investigated if IL-34 is detectible in gingival crevicular fluid (GCF) in healthy, gingivitis and periodontitis sites. Furthermore, we e...

Effects of ascorbate and B-aminopropionitrile on tendon fibroblast migration in vitro

International Nuclear Information System (INIS)

Nelson, J.M.; Cohen, I.K.; Diegelmann, R.F.

1986-01-01

Ascorbate (Asc) stimulates collagen synthesis and hydroxylation whereas beta-aminopropionitrile (BAPN) inhibits collagen cross-link formation. In this study, an in vitro model employing isolated chicken tendon biopsies (2 mm in dia.) in a fibrin clot has been used to examine the effect of Asc and BAPN on tendon fibroblast migration and proliferation. After 5 days in culture with either Asc (0.1 mM), or BAPN (0.5, 1.0, 2.0 mM), cell migration was measured using an automatic planimeter and cell proliferation was quantitated by 125 IUDR incorporation into DNA. In cultures treated with Asc alone, cell migration was enhanced by approximately 33% compared to controls without ascorbate (5.9 mm 2 vs. 4.4 mm 2 ; p < 0.05) with no significant effect on cell proliferation. In contrast, cultures incubated with increasing concentrations of BAPN (0.5 - 2.0 mM) displayed a dose-dependent decrease (up to 9.6-fold at 2 mM) in fibroblast migration into the clot. The inhibitory effect of BAPN on cell migration was not due to a corresponding inhibition of fibroblast proliferation. These observations suggest that Asc enhanced collagen formation and thus allowed greater cell migration into the fibrin matrix. In contrast, in the absence of a mature, cross-linked collagen matrix following exposure to BAPN, cell migration was sub-optimal. These in vitro studies support the hypothesis that modulation of the collagen matrix may be a useful means of regulating tissue repair in vivo

Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

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Julie A Wallace

Full Text Available Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

High-Mobility Group Box 1 Mediates Fibroblast Activity via RAGE-MAPK and NF-κB Signaling in Keloid Scar Formation

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Jihee Kim

2017-12-01

Full Text Available Emerging studies have revealed the involvement of high-mobility group box 1 (HMGB1 in systemic fibrotic diseases, yet its role in the cutaneous scarring process has not yet been investigated. We hypothesized that HMGB1 may promote fibroblast activity to cause abnormal cutaneous scarring. In vitro wound healing assay with normal and keloid fibroblasts demonstrated that HMGB1 administration promoted the migration of both fibroblasts with increased speed and a greater traveling distance. Treatment of the HMGB1 inhibitor glycyrrhizic acid (GA showed an opposing effect on both activities. To analyze the downstream mechanism, the protein levels of extracellular signal-regulated kinase (ERK 1/2, protein kinase B (AKT, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB were measured by western blot analysis. HMGB1 increased the expression levels of ERK1/2, AKT, and NF-κB compared to the control, which was suppressed by GA. HMGB1 promoted both normal and keloid fibroblasts migration to a degree equivalent to that achieved with TGF-β. We concluded that HMGB1 activates fibroblasts via the receptor for advanced glycation end product (RAGE—mitogen-activated protein kinases (MAPK and NF-κB interaction signaling pathways. Further knowledge of the relationship of HMGB1 with skin fibrosis may lead to a promising clinical approach to manage abnormal scarring.

High-Mobility Group Box 1 Mediates Fibroblast Activity via RAGE-MAPK and NF-κB Signaling in Keloid Scar Formation.

Science.gov (United States)

Kim, Jihee; Park, Jong-Chul; Lee, Mi Hee; Yang, Chae Eun; Lee, Ju Hee; Lee, Won Jai

2017-12-28

Emerging studies have revealed the involvement of high-mobility group box 1 (HMGB1) in systemic fibrotic diseases, yet its role in the cutaneous scarring process has not yet been investigated. We hypothesized that HMGB1 may promote fibroblast activity to cause abnormal cutaneous scarring. In vitro wound healing assay with normal and keloid fibroblasts demonstrated that HMGB1 administration promoted the migration of both fibroblasts with increased speed and a greater traveling distance. Treatment of the HMGB1 inhibitor glycyrrhizic acid (GA) showed an opposing effect on both activities. To analyze the downstream mechanism, the protein levels of extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (AKT), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were measured by western blot analysis. HMGB1 increased the expression levels of ERK1/2, AKT, and NF-κB compared to the control, which was suppressed by GA. HMGB1 promoted both normal and keloid fibroblasts migration to a degree equivalent to that achieved with TGF-β. We concluded that HMGB1 activates fibroblasts via the receptor for advanced glycation end product (RAGE)-mitogen-activated protein kinases (MAPK) and NF-κB interaction signaling pathways. Further knowledge of the relationship of HMGB1 with skin fibrosis may lead to a promising clinical approach to manage abnormal scarring.

Parametric study of roof diaphragm stiffness requirements

International Nuclear Information System (INIS)

Jones, W.D.; Tenbus, M.A.

1991-01-01

A common assumption made in performing a dynamic seismic analysis for a building is that the roof/floor system is open-quotes rigidclose quotes. This assumption would appear to be reasonable for many of the structures found in nuclear power plants, since many of these structures are constructed of heavily reinforced concrete having floor/roof slabs at least two feet in thickness, and meet the code requirements for structural detailing for seismic design. The roofs of many Department of Energy (DOE) buildings at the Oak Ridge Y-12 Plant in Oak Ridge, Tennessee, have roofs constructed of either metal, precast concrete or gypsum plank deck overlaid with rigid insulation, tar and gravel. In performing natural phenomena hazard assessments for one such facility, it was assumed that the existing roof performed first as a flexible diaphragm (zero stiffness) and then, rigid (infinitely stiff). For the flexible diaphragm model it was determined that the building began to experience significant damage around 0.09 g's. For the rigid diaphragm model it was determined that no significant damage was observed below 0.20 g's. A Conceptual Design Report has been prepared for upgrading/replacing the roof of this building. The question that needed to be answered here was, open-quotes How stiff should the new roof diaphragm be in order to satisfy the rigid diaphragm assumption and, yet, be cost effective?close quotes. This paper presents a parametric study of a very simple structural system to show that the design of roof diaphragms needs to consider both strength and stiffness (frequency) requirements. This paper shows how the stiffness of a roof system affects the seismically induced loads in the lateral, vertical load resisting elements of a building and provides guidance in determining how open-quotes rigidclose quotes a roof system should be in order to accomplish a cost effective design

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

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Khamaisi, Mogher; Katagiri, Sayaka; Keenan, Hillary; Park, Kyoungmin; Maeda, Yasutaka; Li, Qian; Qi, Weier; Thomou, Thomas; Eschuk, Danielle; Tellechea, Ana; Veves, Aris; Huang, Chenyu; Orgill, Dennis Paul; Wagers, Amy; King, George L

2016-03-01

Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

Biochemical mechanisms of skin radiation burns inhibition and healing by the volumetric autotransplantation of fibroblasts and of keratinocytes with fibroblasts composition

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L. V. Altukhova

2015-09-01

Full Text Available Mechanisms of influence of volumetric autotransplantation of fibroblasts and of the mixture of fibroblasts and keratinocytes on the development of the local 3rd degree X-ray burn and the radiation skin ulcer in guinea pigs were investigated. We used deepadministration into the irradiation zone on its perimeter of 6 doses, which contained (150–160×103 fibroblasts and (130–140×103 keratinocytes in 100 µl. It is shown that this autotransplantation carried out 1 hour after the irradiation, and then every 24 hours, reduces the area of burn on the 35th day, compared to the control by 63%. Radiation ulcer appears on the 10th day after irradiation and is completely healed on the 25th day. With the same regimen of administration of only fibroblasts containing (200–210×103 cells in 100 µl, these parameters of treatment were equal to 31% on 4th and 35th day, respectively. It is shown that as a result of radiation in the area of burn the level of gene expression of collagen types I and III, elastin, fibronectin, vinculin, decorin, hyaluronansynthases 1, 2, 3, matrix metalloproteinases 1, 2, 3, 7, 9 and hyaluronidase is reduced. Besides, in the burn area the level of gene expression of transforming growth factor α, fibroblast growth factors 1, 2, 8 and anti-inflammatory cytokines – interleukin 10 and transforming growth factor-β1 – is reduced, while the level of gene expression of proinflammatory cytokine (interleykin1β increases. Both types of autotransplantation cause the growth of the expression level of all the structural genes and regulatory proteins of biopolymers and decrease in the expression level of interleukin 1β, which leads to activation of tissue regeneration and healing of the burn wound. Reasonsfor the higher efficiency of autotransplantation using the mixture of fibroblasts and keratinocytes compared to autotransplantation by fibroblasts only are both the larger total number of live cells regularly replacing dead cells in

Comparative study of diastolic filling under varying left ventricular wall stiffness

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Mekala, Pritam; Santhanakrishnan, Arvind

2014-11-01

Pathological remodeling of the human cardiac left ventricle (LV) is observed in hypertensive heart failure as a result of pressure overload. Myocardial stiffening occurs in these patients prior to chronic maladaptive changes, resulting in increased LV wall stiffness. The goal of this study was to investigate the change in intraventricular filling fluid dynamics inside a physical model of the LV as a function of wall stiffness. Three LV models of varying wall stiffness were incorporated into an in vitro flow circuit driven by a programmable piston pump. Windkessel elements were used to tune the inflow and systemic pressure in the model with least stiffness to match healthy conditions. Models with stiffer walls were comparatively tested maintaining circuit compliance, resistance and pump amplitude constant. 2D phase-locked PIV measurements along the central plane showed that with increase in wall stiffness, the peak velocity and cardiac output inside the LV decreased. Further, inflow vortex ring propagation toward the LV apex was reduced with increasing stiffness. The above findings indicate the importance of considering LV wall relaxation characteristics in pathological studies of filling fluid dynamics.