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Sample records for fibroblasts induce heparin

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

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    el Nabout, R.; Martin, M.; Remy, J.; Robert, L.; Lafuma, C. (Laboratoire de Biochimie de Tissu Conjonctif, Creteil (France))

    1989-10-01

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

  2. Angiopoietin-1 prevents severe bleeding complications induced by heparin-like drugs and fibroblast growth factor-2 in mice

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    Jerebtsova, Marina; Das, Jharna R.; Tang, Pingtao; Wong, Edward

    2015-01-01

    Critically ill children can develop bleeding complications when treated with heparin-like drugs. These events are usually attributed to the anticoagulant activity of these drugs. However, previous studies showed that fibroblast growth factor-2 (FGF-2), a heparin-binding growth factor released in the circulation of these patients, could precipitate intestinal hemorrhages in mice treated with the heparin-like drug pentosan polysulfate (PPS). Yet very little is known about how FGF-2 induces bleeding complications in combination with heparin-like drugs. Here, we examined the mechanisms by which circulating FGF-2 induces intestinal hemorrhages in mice treated with PPS. We used a well-characterized mouse model of intestinal hemorrhages induced by FGF-2 plus PPS. Adult FVB/N mice were infected with adenovirus carrying Lac-Z or a secreted form of recombinant human FGF-2, and injected with PPS, at doses that do not induce bleeding complications per se. Mice treated with FGF-2 in combination with PPS developed an intestinal inflammatory reaction that increased the permeability and disrupted the integrity of submucosal intestinal vessels. These changes, together with the anticoagulant activity of PPS, induced lethal hemorrhages. Moreover, a genetically modified form of the endothelial ligand angiopoietin-1 (Ang-1*), which has powerful antipermeability and anti-inflammatory activity, prevented the lethal bleeding complications without correcting the anticoagulant status of these mice. These findings define new mechanisms through which FGF-2 and Ang-1* modulate the outcome of intestinal bleeding complications induced by PPS in mice and may have wider clinical implications for critically ill children treated with heparin-like drugs. PMID:26276817

  3. Heparin modulates the mitogenic activity of fibroblast growth factor by inducing dimerization of its receptor. a 3D view by using NMR.

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    Nieto, Lidia; Canales, Ángeles; Fernández, Israel S; Santillana, Elena; González-Corrochano, Rocío; Redondo-Horcajo, Mariano; Cañada, F Javier; Nieto, Pedro; Martín-Lomas, Manuel; Giménez-Gallego, Guillermo; Jiménez-Barbero, Jesús

    2013-09-23

    In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin-binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin-binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin-binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF-driven mitogenesis.

  4. Heparin-induced thrombocytopenia

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    Shaikh Nissar

    2011-01-01

    Full Text Available In the last 7 decades heparin has remained the most commonly used anticoagulant. Its use is increasing, mainly due to the increase in the number of vascular interventions and aging population. The most feared complication of heparin use is heparin-induced thrombocytopenia (HIT. HIT is a clinicopathologic hypercoagulable, procoagulant prothrombotic condition in patients on heparin therapy, and decrease in platelet count by 50% or to less than 100,000, from 5 to 14 days of therapy. This prothrombotic hypercoagulable state in HIT patient is due to the combined effect of various factors, such as platelet activation, mainly the formation of PF4/heparin/IgG complex, stimulation of the intrinsic factor, and loss of anticoagulant effect of heparin. Diagnosis of HIT is done by clinical condition, heparin use, and timing of thrombocytopenia, and it is confirmed by either serotonin release assay or ELISA assay. Complications of HIT are venous/arterial thrombosis, skin gangrene, and acute platelet activation syndrome. Stopping heparin is the basic initial treatment, and Direct Thrombin Inhibitors (DTI are medication of choice in these patients. A few routine but essential procedures performed by using heparin are hemodialysis, Percutaneous Coronary Intervention, and Cardiopulmonary Bypass; but it cannot be used if a patient develops HIT. HIT patients with unstable angina, thromboembolism, or indwelling devices, such as valve replacement or intraaortic balloon pump, will require alternative anticoagulation therapy. HIT can be prevented significantly by keeping heparin therapy shorter, avoiding bovine heparin, using low-molecular weight heparin, and stopping heparin use for flush and heparin lock.

  5. Heparin-Induced Thrombocytopenia Antibody Test

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    ... Global Sites Search Help? Heparin-induced Thrombocytopenia PF4 Antibody Share this page: Was this page helpful? Also known as: Heparin-PF4 Antibody; HIT Antibody; HIT PF4 Antibody; Heparin Induced Antibody; ...

  6. Exogenous Fibroblast Growth Factor-10 Induces Cystic Lung Development with Altered Target Gene Expression in the Presence of Heparin in Cultures of Embryonic Rat Lung

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    Hashimoto, Shuichi; Nakano, Hiroshi; Suguta, Yuko; Irie, Seiko; Jianhua, Luo; Katyal, Sikardar L.

    2012-01-01

    Objectives Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Methods Embryonic day 14 rat lungs were cultured with FGF-10 (0–250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Results Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh(sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Conclusions Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC

  7. Heparin-induced thrombocytopenia: a general review.

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    Swanson, Joseph M

    2007-01-01

    Unfractionated heparin is widely used for numerous clinical situations. A well-known adverse effect of heparin exposure is thrombocytopenia. Heparin-induced thrombocytopenia is a clinicopathologic syndrome that can be associated with severe complications and significant mortality. The pathophysiology of heparin-induced thrombocytopenia includes an immune-mediated reaction to heparin that activates platelets and results in an acquired hypercoagulability. Diagnosis of heparin-induced thrombocytopenia should incorporate clinical signs and symptoms and laboratory testing for heparin-induced thrombocytopenia antibodies. Therapy should include discontinuation of heparin, initiation of a direct thrombin inhibitor, and eventually therapy with warfarin (only after the platelet count is at least 100 x 10(9)/L).

  8. Current management of heparin-induced thrombocytopenia.

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    Cosmi, Benilde

    2015-12-01

    Heparin-induced thrombocytopenia (HIT) is an immune adverse reaction to heparin (both unfractionated and low-molecular-weight), which is mediated by the formation of IgG antibodies against platelet factor 4-heparin complexes. The IgG/platelet factor 4 immunocomplexes activate platelets with resulting thrombocytopenia, which is not associated with bleeding, but with paradoxical life-threatening thrombotic complications, for coagulation activation. HIT diagnosis requires the assessment of pre-test clinical probability in combination with the measurement of platelet activating antibodies against platelet factor 4-heparin complexes with immunological and functional assays. When HIT is diagnosed, any form of heparin should be stopped and a non-heparin alternative anticoagulant should be started. Argatroban and danaparoid are currently the only drugs licensed for HIT, with different country availability. Bivalirudin is an option in cardiac surgery and procedures in HIT patients.

  9. Short heparin sequences spaced by glycol-split uronate residues are antagonists of fibroblast growth factor 2 and angiogenesis inhibitors.

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    Casu, Benito; Guerrini, Marco; Naggi, Annamaria; Perez, Marta; Torri, Giangiacomo; Ribatti, Domenico; Carminati, Paolo; Giannini, Giuseppe; Penco, Sergio; Pisano, Claudio; Belleri, Mirella; Rusnati, Marco; Presta, Marco

    2002-08-20

    Fibroblast Growth Factor-2 (FGF2) is a major inducer of neovascularization (angiogenesis). Heparin activates FGF2 by favoring formation of ternary complexes with its cellular receptors (FGFRs). Controlled 2-O-desulfation followed by exhaustive periodate oxidation/borohydride reduction has been used to generate sulfation gaps within the prevalent heparin sequences, building-up arrays of pentasulfated trisaccharides (PST, consisting of a 2-O-sulfated iduronic acid flanked by two N,6-disulfated glucosamines) spaced by reduced, glycol-split uronic acid (sU) residues. The structure of the prevalent sequences of the novel heparin derivative has been confirmed by mono- and two-dimensional NMR analysis. NMR spin-lattice relaxation times (T2) and nuclear Overhauser effects suggest that the sU residues act as flexible joints between the PST sequences and cause a marked distortion of the chain conformation of heparin required for formation of ternary complexes. Since the splitting reaction also occurs at the level of the essential glucuronic acid residue of the active site for antithrombin, the heparin derivative has no anticoagulant activity. However, it fully retains the FGF2-binding ability of the original heparin, as shown by its capacity to protect FGF2 from trypsin cleavage and to prevent the formation of heparan sulfate proteoglycan (HSPG)/FGF2/FGFR1 ternary complexes. However, when compared to heparin it showed a reduced capacity to induce FGF2 dimerization and to favor the interaction of [125I]FGF2 with FGFR1 in HSPG-deficient, FGFR1-transfected CHO cells. Accordingly, it was more effective than heparin in inhibiting the mitogenic activity exerted by FGF2 in cultured endothelial cells. Finally, it inhibited angiogenesis in a chick embrio chorioallantoic membrane (CAM) assay in which heparin is inactive.

  10. [Heparin-induced thrombocytopenia. New therapeutical options].

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    Seculini Patiño, Carina E; Tabares, Aldo H

    2016-01-01

    Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse reaction due to antibodies to a multimolecular complex of heparin and platelet factor 4 (PF4) characterized by moderate thrombocytopenia and paradoxical arterial or venous thrombosis. It is a relatively infrequent complication related to the administration of any type of heparin. In patients undergoing percutaneous coronary revascularization or coronary artery by-pass graft the prevalence of HIT is higher than in other clinical settings. Recognizing clinical and laboratory features of HIT allow immediate discontinuation of heparin and the use of alternative anticoagulants to avoid serious thrombotic complications. In this review, we summarize different therapeutic options for the treatment of HIT with special emphasis on direct oral anticoagulants (DOACS) such as dabigatran, rivaroxaban and apixaban. DOACS might represent a therapeutic alternative for HIT treatment.

  11. Delivery of basic fibroblast growth factors from heparinized decellularized adipose tissue stimulates potent de novo adipogenesis.

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    Lu, Qiqi; Li, Mingming; Zou, Yu; Cao, Tong

    2014-01-28

    Scaffolds based on decellularized adipose tissue (DAT) are gaining popularity in adipose tissue engineering due to their high biocompatibility and adipogenic inductive property. However, previous studies involving DAT-derived scaffolds have not fully revealed their potentials for in vivo adipose tissue construction. With the aim of developing a more efficient adipose tissue engineering technique based on DAT, in this study, we investigated the in vivo adipogenic potential of a basic fibroblast growth factor (bFGF) delivery system based on heparinized DAT (Hep-DAT). To generate this system, heparins were cross-linked to mouse DATs by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-Hydroxysuccinimide. The bFGF-binding Hep-DATs were first tested for controlled release ability in vitro and then transplanted subcutaneously. Highly vascularized adipose tissues were formed 6weeks after transplantation. Histology and gene expression analysis revealed that majority of the Hep-DAT scaffolds were infiltrated with host-derived adipose tissues that possessed similar adipogenic and inflammatory gene expression as endogenous adipose tissues. Additionally, strong de novo adipogenesis could also be induced when bFGF-binding Hep-DATs were thoroughly minced and injected subcutaneously. In conclusion, our study demonstrated that bFGF-binding Hep-DAT could be an efficient, biocompatible and injectable adipogenic system for in vivo adipose tissue engineering.

  12. Unfractionated heparin versus low molecular weight heparins for avoiding heparin-induced thrombocytopenia in postoperative patients.

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    Junqueira, Daniela R; Zorzela, Liliane M; Perini, Edson

    2017-04-21

    Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a paradoxical immune reaction resulting in thrombin generation in vivo, which leads to a hypercoagulable state and the potential to initiate venous or arterial thrombosis. A number of factors are thought to influence the incidence of HIT including the type and preparation of heparin (unfractionated heparin (UFH) or low molecular weight heparin (LMWH)) and the heparin-exposed patient population, with the postoperative patient population at higher risk.Although LMWH has largely replaced UFH as a front-line therapy, there is evidence supporting a lack of superiority of LMWH compared with UFH regarding prevention of deep vein thrombosis and pulmonary embolism following surgery, and similar frequencies of bleeding have been described with LMWH and UFH. The decision as to which of these two preparations of heparin to use may thus be influenced by harmful effects such as HIT. We therefore sought to determine the relative impact of UFH and LMWH on HIT in postoperative patients receiving thromboembolism prophylaxis. This is an update of a review first published in 2012. The objective of this review was to compare the incidence of heparin-induced thrombocytopenia (HIT) and HIT complicated by venous thromboembolism in postoperative patients exposed to unfractionated heparin (UFH) versus low molecular weight heparin (LMWH). For this update, the Cochrane Vascular Information Specialist searched the Specialised Register (May 2016), CENTRAL (2016, Issue 4) and trials registries. The authors searched Lilacs (June 2016) and additional trials were sought from reference lists of relevant publications. We included randomised controlled trials (RCTs) in which participants were postoperative patients allocated to receive prophylaxis with UFH or LMWH, in a blinded or unblinded fashion. Studies were excluded if they did not use

  13. Unfractionated heparin versus low molecular weight heparin for avoiding heparin-induced thrombocytopenia in postoperative patients.

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    Junqueira, Daniela R G; Perini, Edson; Penholati, Raphael R M; Carvalho, Maria G

    2012-09-12

    Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a poorly understood paradoxical immune reaction resulting in thrombin generation in vivo, which leads to a hypercoagulable state and the potential to initiate venous or arterial thrombosis. A number of factors are thought to influence the incidence of HIT including the type and preparation of heparin (unfractionated heparin (UFH) or low molecular weight heparin (LMWH)) and the heparin-exposed patient population, with the postoperative patient population presenting a higher risk.Although LMWH has largely replaced UFH as a front-line therapy, there is evidence supporting a lack of superiority of LMWH compared with UFH regarding prevention of deep vein thrombosis and pulmonary embolism following surgery, and similar frequencies of bleeding have been described with LMWH and UFH. The decision as to which of these two preparations of heparin to use may thus be influenced by adverse reactions such as HIT. We therefore sought to determine the relative impact of UFH and LMWH specifically on HIT in postoperative patients receiving thromboembolism prophylaxis. The objective of this review was to compare the incidence of HIT and HIT complicated by thrombosis in patients exposed to UFH versus LMWH in randomised controlled trials (RCTs) of postoperative heparin therapy. The Cochrane Peripheral Vascular Diseases Group searched their Specialised Register (March 2012) and CENTRAL (2012, Issue 2). In addition, the authors searched LILACS (March 2012) and additional trials were sought from reference lists of relevant publications. We were interested in comparing the incidence of HIT occurring during exposure to UFH or LMWH after any surgical intervention. Therefore, we studied RCTs in which participants were postoperative patients allocated to receive UFH or LMWH, in a blinded or unblinded fashion. Eligible studies were

  14. Heparin-Induced Thrombocytopenia: A Comprehensive Clinical Review.

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    Salter, Benjamin S; Weiner, Menachem M; Trinh, Muoi A; Heller, Joshua; Evans, Adam S; Adams, David H; Fischer, Gregory W

    2016-05-31

    Heparin-induced thrombocytopenia is a profoundly dangerous, potentially lethal, immunologically mediated adverse drug reaction to unfractionated heparin or, less commonly, to low-molecular weight heparin. In this comprehensive review, the authors highlight heparin-induced thrombocytopenia's risk factors, clinical presentation, pathophysiology, diagnostic principles, and treatment. The authors place special emphasis on the management of patients requiring procedures using cardiopulmonary bypass or interventions in the catheterization laboratory. Clinical vigilance of this disease process is important to ensure its recognition, diagnosis, and treatment. Misdiagnosis of the syndrome, as well as misunderstanding of the disease process, continues to contribute to its morbidity and mortality.

  15. Reducing the hospital burden of heparin-induced thrombocytopenia: impact of an avoid-heparin program.

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    McGowan, Kelly E; Makari, Joy; Diamantouros, Artemis; Bucci, Claudia; Rempel, Peter; Selby, Rita; Geerts, William

    2016-04-21

    Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction occurring in up to 5% of patients exposed to unfractionated heparin (UFH). We examined the impact of a hospital-wide strategy for avoiding heparin on the incidence of HIT, HIT with thrombosis (HITT), and HIT-related costs. The Avoid-Heparin Initiative, implemented at a tertiary care hospital in Toronto, Ontario, Canada, since 2006, involved replacing UFH with low-molecular-weight heparin (LMWH) for prophylactic and therapeutic indications. Consecutive cases with suspected HIT from 2003 through 2012 were reviewed. Rates of suspected HIT, adjudicated HIT, and HITT, along with HIT-related expenditures were compared in the pre-intervention (2003-2005) and the avoid-heparin (2007-2012) phases. The annual rate of suspected HIT decreased 42%, from 85.5 per 10 000 admissions in the pre-intervention phase to 49.0 per 10 000 admissions in the avoid-heparin phase ( ITALIC! Pheparin phase. To the best of our knowledge, this is the first study demonstrating the success and feasibility of a hospital-wide HIT prevention strategy.

  16. Does ′heparin-induced thrombocytopenia′ hit our minds?

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    Arun R Thangavel

    2016-01-01

    Full Text Available Unfractionated heparin is a widely used drug to prevent deep vein thrombosis and pulmonary emboli in patients at risk. With the advent of newer anticoagulants having lesser side effects, its use has diminished but not out of service. Here, we report a case of deep venous thrombosis, in a patient on prophylactic dose of heparin, which was later found to be a manifestation of heparin-induced thrombocytopenia (HIT. Thrombosis in the presence of heparin prophylaxis should be considered as HIT rather than a failure of anticoagulation.

  17. Trombocitopenia induzida por heparina Heparin-induced thrombocytopenia

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    Fernanda Longhi

    2001-08-01

    Full Text Available O objetivo deste relato é fazer uma revisão a fim de obter informações atualizadas sobre trombocitopenia induzida por heparina, suas manifestações clínicas, seu diagnóstico e seu manejo terapêutico. Após, concluímos que a trombocitopenia induzida por heparina é uma complicação comum em pacientes submetidos a tratamento com heparina, indiferentemente da doença de base. Complicações trombóticas potencialmente fatais têm sido descritas. Por essa razão, se houver suspeita de trombocitopenia induzida por heparina, uma abordagem adequada incluindo suspensão precoce da heparina é mandatória. Produtos que substituem a heparina incluem hirudina e danaparóide sódico. Heparina de baixo peso molecular é contra-indicada.The aim of this paper is to review current information about the clinical manifestations, diagnosis and management of heparin-induced thrombocytopenia. This was achieved by a bibliographic review using Public Medline and consulting Hematology textbooks. From this study we concluded that heparin-induced thrombocytopenia is a common complication of patients exposed to heparin therapy regardless of underlying conditions. Potentially fatal thrombotic complications have been reported. Therefore, if heparin-induced thrombocytopenia is suspected, an adequate approach including early heparin discontinuation is mandatory. Alternative products for heparin include hirudin and danaparoid sodium. Low-molecular-weight heparin is contraindicated.

  18. HEPARIN-INDUCED THROMBOCYTOPAENIA/THROMBOSIS: A ...

    African Journals Online (AJOL)

    2009-12-02

    Dec 2, 2009 ... acid and glucosamine residues. Heparin was ..... which is the amino acid preceding the third cysteine moiety ... In this test visual detection is employed for determination of .... polypeptides extracted from the salivary glands of.

  19. A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor

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    Nguyen, Thi H.; Kim, Sung-Hye; Decker, Caitlin G.; Wong, Darice Y.; Loo, Joseph A.; Maynard, Heather D.

    2013-03-01

    Basic fibroblast growth factor (bFGF) is a protein that plays a crucial role in diverse cellular functions, from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Here, we describe the stabilization of bFGF by covalent conjugation with a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors—such as heat, mild and harsh acidic conditions, storage and proteolytic degradation—unlike native bFGF. Following the application of stress, the conjugate was also significantly more active than the control conjugate system in which the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for the stabilization of heparin-binding growth factors in general.

  20. In situ formation of poly(vinyl alcohol–heparin hydrogels for mild encapsulation and prolonged release of basic fibroblast growth factor and vascular endothelial growth factor

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    Justine J Roberts

    2016-11-01

    Full Text Available Heparin-based hydrogels are attractive for controlled growth factor delivery, due to the native ability of heparin to bind and stabilize growth factors. Basic fibroblast growth factor and vascular endothelial growth factor are heparin-binding growth factors that synergistically enhance angiogenesis. Mild, in situ encapsulation of both basic fibroblast growth factor and vascular endothelial growth factor and subsequent bioactive dual release has not been demonstrated from heparin-crosslinked hydrogels, and the combined long-term delivery of both growth factors from biomaterials is still a major challenge. Both basic fibroblast growth factor and vascular endothelial growth factor were encapsulated in poly(vinyl alcohol-heparin hydrogels and demonstrated controlled release. A model cell line, BaF32, was used to show bioactivity of heparin and basic fibroblast growth factor released from the gels over multiple days. Released basic fibroblast growth factor promoted higher human umbilical vein endothelial cell outgrowth over 24 h and proliferation for 3 days than the poly(vinyl alcohol-heparin hydrogels alone. The release of vascular endothelial growth factor from poly(vinyl alcohol-heparin hydrogels promoted human umbilical vein endothelial cell outgrowth but not significant proliferation. Dual-growth factor release of basic fibroblast growth factor and vascular endothelial growth factor from poly(vinyl alcohol-heparin hydrogels resulted in a synergistic effect with significantly higher human umbilical vein endothelial cell outgrowth compared to basic fibroblast growth factor or vascular endothelial growth factor alone. Poly(vinyl alcohol-heparin hydrogels allowed bioactive growth factor encapsulation and provided controlled release of multiple growth factors which is beneficial toward tissue regeneration applications.

  1. Identification of Receptor and Heparin Binding Sites in Fibroblast Growth Factor 4 by Structure-Based Mutagenesis

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    Bellosta, Paola; Iwahori, Akiyo; Plotnikov, Alexander N.; Eliseenkova, Anna V.; Basilico, Claudio; Mohammadi, Moosa

    2001-01-01

    Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like doma...

  2. Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

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    Jiang, Chao; Wilkinson, Mark C.

    2016-01-01

    The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans. PMID:27030175

  3. Bilateral lower extremity gangrene requiring amputation associated with heparin-induced thrombocytopenia: a case report.

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    Dickinson, Brian P; Lawrence, Peter F

    2007-01-01

    Heparin is a common cause of thrombocytopenia in hospitalized patients. Between 10% and 15% of patients receiving therapeutic doses of heparin develop thrombocytopenia. Heparin-induced thrombocytopenia (HIT) can cause severe bleeding and thrombosis owing to intravascular platelet aggregation. HIT must be distinguished from other causes of thrombocytopenia. Importantly, heparin use is often associated with an early fall in the platelet count that usually occurs within the first 4 days of initiation and recovers without cessation of heparin treatment. This nonimmune heparin-associated thrombocytopenia has not been found to be associated with thrombosis and does not necessitate discontinuation of heparin. The authors present a case report of a 70-year-old man who received heparin therapy following aortic tissue valve replacement and aortic root repair with graft and developed bilateral lower extremity arterial clots 6 days postoperatively in the setting of positive heparin antibody titers. Ultimately the patient required bilateral above-knee amputations.

  4. Delayed-onset heparin-induced thrombocytopenia presenting with multiple arteriovenous thromboses: case report

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    Omran Abbas

    2007-11-01

    Full Text Available Abstract Background Delayed-onset heparin-induced thrombocytopenia with thrombosis, albeit rare, is a severe side effect of heparin exposure. It can occur within one month after coronary artery bypass grafting (CABG with manifestation of different thrombotic events. Case presentation A 59-year-old man presented with weakness, malaise, bilateral lower limb pitting edema and a suspected diagnosis of deep vein thrombosis 18 days after CABG. Heparin infusion was administered as an anticoagulant. Clinical and paraclinical work-up revealed multiple thrombotic events (stroke, renal failure, deep vein thrombosis, large clots in heart chambers and 48 ×103/μl platelet count, whereupon heparin-induced thrombocytopenia was suspected. Heparin was discontinued immediately and an alternative anticoagulant agent was administered, as a result of which platelet count recovered. Heparin-induced thrombocytopenia, which causes thrombosis, is a serious side effect of heparin therapy. It is worthy of note that no case of delayed-onset heparin-induced thrombocytopenia with thrombosis associated with cardiopulmonary bypass surgery has thus far been reported in Iran. Conclusion Delayed-onset heparin-induced thrombocytopenia should be suspected in any patient presenting with arterial or venous thromboembolic disorders after recent heparin therapy, even though the heparin exposure dates back to more than a week prior to presentation; and it should be ruled-out before the initiation of heparin therapy.

  5. Rates of clinically apparent heparin-induced thrombocytopenia for unfractionated heparin vs. low molecular weight heparin in non-surgical patients are low and similar

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    Gerber Jonathan

    2005-04-01

    Full Text Available Abstract With the growing use of low-molecular-weight heparins (LMWH for the treatment and prevention of venous thromboembolism (VTE, it is important to provide an evidence-based comparison with unfractionated heparin (UFH concerning rates of heparin-induced thrombocytopenia (HIT. Such comparisons are essential in clinical decision-making and cost-modeling. In this paper we review data regarding non-surgical (medical patients. We conclude that the lack of uniform evaluation and standardized testing for HIT in the current literature precludes making a reliable estimate of the relative risk of HIT in UFH vs. LMWH in either the treatment or prevention of VTE in non-surgical patients. However, current data suggest that the risk of thrombocytopenia and HIT is low and similar for non-surgical patients who receive either LMWH or UFH.

  6. Intravenous nitroglycerin-induced heparin resistance: a qualitative antithrombin III abnormality.

    Science.gov (United States)

    Becker, R C; Corrao, J M; Bovill, E G; Gore, J M; Baker, S P; Miller, M L; Lucas, F V; Alpert, J A

    1990-06-01

    An ability of intravenous nitroglycerin to interfere with the anticoagulant properties of intravenous heparin would have profound clinical implications. To investigation nitroglycerin-heparin interactions, the following pilot study was performed. Patients (N = 18) admitted to the coronary care unit with a diagnosis of either acute myocardial infarction or unstable angina were divided into four treatment groups: (1) intravenous nitroglycerin and intravenous heparin; (2) intravenous nitroglycerin alone; (3) intravenous heparin alone; or (4) neither intravenous nitroglycerin nor intravenous heparin. Serial determinations of activated partial thromboplastin time (APTT), serum heparin concentration, antithrombin III (ATIII) antigen (ATA), and ATIII activity (ATC) were obtained over a 72-hour period. Overall, patients receiving intravenous nitroglycerin did not differ significantly from other patients in APTT, heparin dose, heparin concentration, ATA, ATC, or ATA/ATC ratio (ATR). However, patients receiving intravenous nitroglycerin at a rate exceeding 350 micrograms per minute had a lower APTT (p less than 0.05), lower ATC (p = 0.02), higher ATR (p = 0.004), and a larger heparin dose requirement than patients receiving lower infusion rates. ATR correlated directly (r = 0.91; p less than 0.05) and ATC inversely (r = -0.78; p less than 0.05) with the intravenous nitroglycerin dose. Serum heparin concentration did not correlate with the intravenous nitroglycerin dose. Intravenous nitroglycerin-induced heparin resistance occurs at a critical nitroglycerin dose. A nitroglycerin-induced qualitative ATIII abnormality may be the underlying mechanism.

  7. Bilateral lower extremity gangrene requiring amputation associated with heparin-induced thrombocytopenia--a case report.

    Science.gov (United States)

    Dickinson, Brian P; De Ugarte, Daniel A; Reil, Todd D; Beseth, Bryce D; Lawrence, Peter F

    2006-01-01

    Heparin use, both prophylactically and therapeutically, is prevalent among hospitalized patients. Patients on heparin may develop a thrombocytopenia that is self-limited. Fewer patients develop a heparin-induced thrombocytopenia that can cause severe bleeding and thrombosis owing to intravascular platelet aggregation. The authors present a case report of heparin-induced thrombocytopenia in a patient who underwent aortic arch and aortic valve replacement that resulted in bilateral above-knee amputations. The patient developed limb ischemia related to heparin-associated thrombosis, but had a delay in antibody seroconversion. Early and accurate diagnosis of heparin-induced thrombocytopenia requires a high clinical suspicion and may be present despite the absence of serum antibodies.

  8. The effect of a heparin-based coacervate of fibroblast growth factor-2 on scarring in the infarcted myocardium.

    Science.gov (United States)

    Chu, Hunghao; Chen, Chien-Wen; Huard, Johnny; Wang, Yadong

    2013-02-01

    Effective delivery of exogenous angiogenic growth factors can provide a new therapy for ischemic diseases. However, clinical translation of growth factor therapies faces multiples challenges; the most significant one is the short half-life of the naked protein. We use heparin and a nontoxic polycation to form an injectable coacervate that protects growth factors and preserves their bioactivities. Here we report the effectiveness of fibroblast growth factor-2 (FGF2) coacervate in reducing scar burden in a mouse myocardial infarction model. The coacervate provides spatial and temporal control of the release of heparin-binding proteins. Coacervate treated animals show lower level of inflammation, fibrosis and cardiomyocyte death in the infarcted myocardium. Histological evaluation indicates that FGF2 coacervate significantly increases the number of endothelial and mural cells and results in stable capillaries and arterioles to at least 6 weeks post injection. Echocardiographic assessment shows that FGF2 coacervate promotes cardiac contractibility and inhibits ventricular dilation, suggesting that the improvement at the tissue level leads to better cardiac functions. On the contrary, identical dosage of free FGF2 shows no statistical difference from saline or vehicle control in histological or functional assessment. Overall, injection of FGF2 coacervate ameliorated the ischemic injury caused by myocardial infarction. The promising data in rodent warrant further examination of the potential of clinical translation of this technology.

  9. Secretion by stimulated murine macrophages of a heparin-binding fibroblast growth activity, distinct from basic FGE and IL-1

    Energy Technology Data Exchange (ETDEWEB)

    Rappolee, D.A.; Banda, M.J.; Werb, Z.

    1986-03-01

    Wound healing requires granulation and formation of neovascularization tissue. These two events require increases in fibroblasts, vascular endothelial, and smooth muscle cells. Macrophages may produce several growth factors which participate in these would healing events. To test this hypothesis they have partially purified a fibroblast growth promoting activity from a murine macrophage cell line (P388 Dl). The activity causes growth in Balb/c and Swiss 3T3 cells as measured by thymidine uptake, nuclear labeling and increase in cell number. PDGF, Basic FGF, and EGF are also mitogenic by thymidine uptake, but purified human IL-1 and recombinant murine IL-1 are not. The activity is pH 2.5-, freeze/thaw-, and dialysis/lyphilyzation-stable. The activity elutes from heparin-Sepharose at 2.0M, but not 0.15m, 0.5M, or 3.0M NaCl. Basic FGF elutes from the same heparin-Sepharose batch at 3.0M, but not at the other three NaCl concentrations. The growth activity is secreted by viable murine macrophage line cells (P388D1, WEHI-3, RAW 264.7) at a 48 hour peak after activating (LPS) or phagocytic stimuli. Unstimulated P388D1 caused growth 1.7 times control whereas stimulation increases the growth 5.1 to 7.1 times control. The optimal activity concentration fails to complement insulin in an assay in which optimal basic FGF concentration complements insulin. These data suggest that the activity may contain a progression factor.

  10. The clinical usefulness of the platelet aggregation test for the diagnosis of heparin-induced thrombocytopenia

    NARCIS (Netherlands)

    Chong, B H; Burgess, J; Ismail, F

    1993-01-01

    The platelet aggregation test is widely used for the diagnosis of heparin-induced thrombocytopenia (HIT), a potentially serious complication of heparin therapy. We have evaluated its sensitivity and specificity in comparison with those of the 14C-serotonin release test. The sensitivity of the platel

  11. Heparin-Induced Cardiac Tamponade and Life-Threatening Hyperkalemia in a Patient with Chronic Hemodialysis

    Directory of Open Access Journals (Sweden)

    Ho-Ming Su

    2005-03-01

    Full Text Available Heparin, a commonly used anticoagulant agent, is frequently used in patients undergoing hemodialysis. As with most medications, heparin has a significant side effect profile. Two of its most important side effects, major bleeding and hyperkalemia, may be devastating without immediate diagnosis and treatment. Major bleeding such as gastrointestinal, genitourinary or intracranial bleeding is occasionally encountered and rarely neglected. However, heparin-induced cardiac tamponade is rarely encountered and may be easily overlooked. Another side effect, heparin-induced hyperkalemia, an unusual but well-described side effect, is frequently forgotten until life-threatening arrhythmia has occurred. We report a case involving a 40-year-old male patient with uremia, who had received heparin for 10 days for deep vein thrombosis in the left lower extremity. Hemopericardium with cardiac tamponade and life-threatening hyperkalemia were both noted in this patient.

  12. Report: Heparin-induced thrombocytopenia associated with cardiopulmonary bypass: Preliminary attempt with recombinant human thrombopoietin therapy.

    Science.gov (United States)

    Yuan, Shi-Min

    2015-09-01

    Recombinant human thrombopoietin (rhTPO) is popularly used for the treatment of chemotherapy-induced thrombocytopenia. However, rhTPO therapy for heparin-induced thrombocytopenia relating to cardiopulmonary bypass has not been previously described. A young patient developed heparin-induced thrombocytopenia during open-heart surgery. Postoperative rhTPO therapy (15000 units injection hypodermatica once daily for consecutive 3 days) made a quick platelet recovery without any side effects. Heparin-induced thrombocytopenia associated with cardiopulmonary bypass is more likely to be benign, and is curable to rhTPO therapy. The preliminary rhTPO administration of heparin-induced thrombocytopenia in association with cardiopulmonary bypass shows satisfactory pharmaceutical effects with lower dose, shorter duration treatment and shorter platelet increase time and recovery time in comparison with those for the treatment of chemotherapy-induced thrombocytopenia. rhTPO therapy does not produce any side effects and it could avoid or minimize necessary blood product infusions.

  13. Arterial and venous complications of heparin-induced thrombocytopenia in burn patients.

    Science.gov (United States)

    Scott, Jeffrey R; Klein, Matthew B; Gernsheimer, Terri; Honari, Shari; Gibbons, Janet; Gibran, Nicole S

    2007-01-01

    Heparin-induced thrombocytopenia (HIT) is an antibody-mediated complication of heparin treatment that can result in a number of devastating thrombotic complications. Given the common use of heparin for deep venous thrombosis prophylaxis in patients with burns, we reviewed the incidence and complications of HIT in our burn center. We performed a retrospective review of all patients treated with heparin at our burn center who underwent testing for HIT from 2001 to 2005. Screening for HIT was performed by platelet factor 4 enzyme-linked immunoassay. Records were reviewed with particular attention to indications for HIT testing, duration of heparin therapy, type of heparin used (fractionated vs unfractionated), indication for heparin use (prophylactic vs therapeutic), treatment of HIT, and complications of HIT. During the 4-year study period, 625 patients received heparin therapy at some point during their hospital course. Of these patients, 43 (6.9%) underwent testing for HIT and 10 of the 43 screened patients (23%) were positive; the incidence among all heparinized burn patients was 1.6%. Thrombotic complications of HIT included arterial thrombosis requiring limb amputation (two patients), deep venous thrombosis (three patients), and pulmonary embolism (two patients). One patient died, presumably secondary to a pulmonary embolism. All patients were anticoagulated after HIT diagnosis, and four patients developed bleeding complications. HIT is a potentially devastating complication of heparin administration. Whereas our overall incidence of HIT was low, HIT+ patients developed significant complications, including arterial and venous thrombosis, pulmonary embolus, limb loss, and death. Treatment for such HIT-related thromboses usually resulted in bleeding complications requiring transfusions. The routine use of heparin for deep venous thrombosis prophylaxis needs to be carefully considered in light of these potential complications.

  14. Emphasis on the Role of PF4 in the Incidence, Pathophysiology and Treatment of Heparin Induced Thrombocytopenia

    OpenAIRE

    Prechel, M Margaret; Walenga, Jeanine M.

    2013-01-01

    Heparin Induced Thrombocytopenia (HIT) is caused by antibodies that recognize platelet factor 4 (PF4) associated with polyanionic glycosaminoglycan drugs or displayed on vascular cell membranes. These antibodies are elicited by multimolecular complexes that can occur when heparin is administered in clinical settings associated with abundant PF4. Heparin binding alters native PF4 and elicits immune recognition and response. While the presence of heparin is integral to immunogenesis, the HIT an...

  15. Heparin inhibits burn-induced spleen cell apoptosis by suppressing interleukin-1 expression

    Institute of Scientific and Technical Information of China (English)

    Zhao Songfeng; Zhang Xiao; Zhang Xiaojian; Shi Xiuqin; Yu Zujiang; Kan Quancheng

    2014-01-01

    Background Epidermal burn injury may trigger significant apoptosis of the spleen cells,which might be caused by a burninduced systemic inflammatory reaction.Heparin has been shown to possess anti-inflammatory properties.Interleukin 1 (IL-1) is centrally important among pro-inflammatory cytokines.We hypothesized that heparin might inhibit burn-induced apoptosis in the spleen via suppression of the IL-1 pathway.Methods Burn injury was performed on IL-1 R+/+ (IL-1 receptor wild-type mouse) and IL-1 R-/-(IL-1 receptor knockout mouse) mice,and they were then treated with heparin,saline or IL-1 receptor antagonist IL-Ra.Apoptosis,IL-1α and IL-1β expression were assessed in the spleens and serum.Survival curve analysis was further applied to elucidate the mechanism of heparin's protective properties.Results Burn induced significant apoptosis (sham:3.6%±2.1% vs.burn:28.8%±5.9%; P <0.001)and remarkable expression o IL-1α and IL-1β in the mouse spleens and serum.Heparin reduced the burn-induced apoptosis in the spleens (heparin treated:8.6%±3.4%,P <0.005),which could be blocked by IL-1Ra.Heparin markedly decreased both IL-1α and IL-1β expression in the spleens and serum of burned mica.IL-1 R-/-mice demonstrated considerably less apoptosis in the spleens and had a higher survival rate after burns.Heparin did not significantly decrease apoptosis in the spleen and the mortality rate in IL-1 R-/-mice after burns.Conclusion Heparin inhibits burn-induced apoptosis of the spleen cells by suppressing IL-1 expression in mice.

  16. Heparin-induced thrombocytopenia: a review of concepts regarding a dangerous adverse drug reaction.

    Science.gov (United States)

    Junqueira, Daniela Rezende Garcia; Carvalho, Maria das Graças; Perini, Edson

    2013-01-01

    Heparin is a natural agent with antithrombotic action, commercially available for therapeutic use as unfractionated heparin and low molecular weight heparin. Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin that promotes antibody-mediated platelet activation. HIT is defined as a relative reduction in platelet count of 50% (even when the platelet count at its lowest level is above>150 x 10(9)/L) occurring within five to 14 days after initiation of the therapy. Thrombocytopenia is the main feature that directs the clinical suspicion of the reaction and the increased risk of thromboembolic complications is the most important and paradoxical consequence. The diagnosis is a delicate issue, and requires a combination of clinical probability and laboratory tests for the detection of platelet activation induced by HIT antibodies. The absolute risk of HIT has been estimated between 1% and 5% under treatment with unfractionated heparin, and less than 1% with low molecular weight heparin. However, high-quality evidence about the risk of HIT from randomized clinical trials is scarce. In addition, information on the frequency of HIT in developing countries is not widely available. This review aims to provide a better understanding of the key features of this reaction and updated information on its frequency to health professionals and other interested parties. Knowledge, familiarity, and access to therapeutic options for the treatment of this adverse reaction are mandatory to minimize the associated risks, improving patient safety.

  17. Polycation-induced benzoperylene probe excimer formation and the ratiometric detection of heparin and heparinase.

    Science.gov (United States)

    Yang, Meiding; Chen, Jian; Zhou, Huipeng; Li, Wenying; Wang, Yan; Li, Juanmin; Zhang, Cuiyun; Zhou, Chuibei; Yu, Cong

    2016-01-15

    A benzoperylene probe excimer emission in an aqueous buffer solution is observed for the first time, and a novel ratiometric fluorescence method based on the probe excimer emission for the sensitive detection of heparin and heparinase is demonstrated. A negatively charged benzoperylene derivative, 6-(benzo[ghi]perylene-1,2-dicarboxylic imide-yl)hexanoic acid (BPDI), was employed. A polycation, poly(diallyldimethylammonium) chloride (poly-DDA), could induce aggregation of BPDI through noncovalent interactions. A decrease of BPDI monomer emission and a simultaneous increase of BPDI excimer emission were observed. Upon the addition of heparin, the strong binding between heparin and poly-DDA caused release of BPDI monomer molecules, and an excimer-monomer emission signal transition was detected. However, after the enzymatic hydrolysis of heparin by heparinase, heparin was hydrolyzed into small fragments, which weakened the competitive binding of heparin to poly-DDA. Poly-DDA induced aggregation of BPDI, and a monomer-excimer emission signal transition was detected. Our assay is simple, rapid, inexpensive, sensitive and selective, which could facilitate the heparin and heparinase related biochemical and biomedical research.

  18. Hemorrhagic bullous dermatosis: a rare heparin-induced cutaneous manifestation.

    Science.gov (United States)

    Govind, Bhuvanesh; Gnass, Esteban; Merli, Geno; Eraso, Luis

    2016-01-01

    Heparin is one of the most widely prescribed medications. Cutaneous reactions distant to the injection site are rare and under-reported in the literature. We present an elderly man with history of CNS lymphoma who underwent treatment of a deep venous thrombosis with enoxaparin and subsequently developed well demarcated bullous lesions within days of heparin initiation. The exact pathophysiology is not well understood. Hemorrhagic bullous dermatosis is a rare cutaneous reaction that is temporally associated with the initiation of heparin products. The handful of cases thus far suggest that regression of these seemingly benign lesions may or may not be associated with dose reduction or discontinuation of heparin products and typically occur within a few weeks. Elderly age appears to be one potential risk factor for development of these rare asymptomatic lesions. Malignancy may have some contributing factor and differentiation between this rare cutaneous manifestation from heparin products and other dermatological findings in patients with malignancy is key. Because of the asymptomatic and self-limiting nature of hemorrhagic bullous dermatoses in the setting of heparin product use, we presume that the reported incidence does not reflect true clinical practice.

  19. [Thrombocytopenia induced by heparin. Diagnosis, treatment, physiopathology: current concepts].

    Science.gov (United States)

    Gruel, Y; Drouet, L

    1986-01-01

    Iatrogenic thrombocytopenia is a rare, but severe complication of treatments with heparin and heparinoids. Mean temporary thrombocytopenia failing to show any complications are usually diagnosed as quite different from acute and delayed thrombocytopenia of which severity depends mainly on thrombotic symptoms demonstrated in 65 p. 100 of cases; the initial evolution of an average thrombocytopenia is not easy to diagnose; it may as well exist a connection between the two diseases, from a physiopathogenic point of view. The diagnosis of severe thrombocytopenia depends:--clinically, on the initial data, delayed as compared with the heparin treatment beginning and existence of arterial and/or venous thrombosis;--biologically, by demonstrating an aggregating activity for platelets in presence of heparin, in the patient plasma. Such an activity requires the suppression of standard heparinotherapy as well as the choice of substitutive anticoagulant treatment in case of evolutive thrombosis. Low molecular weight heparins are prescribed only if in vitro tests of platelet aggregation with the patient's plasma are negative. Antivitamins K are to be used as soon as possible alone or combined with heparin fractions. Antiaggregants are prescribed alone, above all in case of isolated thrombocytopenia and combined with AVK. Treatment of thrombotic complications depends on surgical disobstruction if arterial thrombosis, and use of fibrinolytics if pulmonary embolisms. The acute reaction of some thrombocytopenia to heparin as well as therapeutic difficulties demonstrate the efficiency of an early diagnosis performed thanks to systematic platelet numerations during the first 15 days of a treatment with heparin, as well as to the prevention along with systematic association with aspirin, especially if replaced with AVK.

  20. Heparin-induced thrombocytopenia type II: Innovations in diagnostics and treatment

    Directory of Open Access Journals (Sweden)

    Antonijević Nebojša

    2003-01-01

    Full Text Available Heparin-induced thrombocytopenia (HIT Management of heparin-induced thrombocytopenia (HIT and treatment options have significantly changed recently. Heparin may induce two types of thrombocytopenia. Type I, occurring earlier with a much higher rate of incidence (5-30%, is characterized by mild thrombocytopenia without significant clinical manifestations. Type II is less frequent (0.5-2%, life threatening immune type, develops following a period of minimum 5-7 days upon introduction of heparin therapy (patients earlier treated with heparin are excluded. Type II heparin-induced thrombocytopenia with severely reduced platelet count may be clinically manifested by thrombosis in 20-50% cases within the period of 30 days. HIT is suspected in persons resistant to heparin with relatively reduced platelet count, though HIT is described in person with normal platelet counts, as well. None of available assays used for HIT detection is completely reliable Sensitivity of a highly specific platelet aggregation assay is only 36% sensitivity and specificity of 14C-serotonin release assays amounts to 95% while ELISA using a heparin/platelet factor-4 target has a sensitivity of 85%. Thus, it is sometimes necessary to combine functional and antigen assays. Furthermore, new classes of antigen assays, like antibody detection tests of complexes between heparin and neutrophil-activating peptide-2 as well as those between heparin and interleukin-8, have been used. Current therapy options Current therapy options exclude formerly applied low-molecular-weight heparins due to the existing cross-reactivity of 80–100%. Danaparoid sodium exhibits in vitro cross-reactivity of 10–61%, clinically manifested in less than 5% of patients. Two drugs are drugs of choice in HIT type II treatment: lepirudin, especially in patients without renal failure and argatroban, particularly in patients with renal failure. The following procedures and agents are also efficient: asmapheresis

  1. Heparin-induced multiple electrode aggregometry is a promising and useful functional tool for heparin-induced thrombocytopenia diagnosis: confirmation in a prospective study.

    Science.gov (United States)

    Galea, Vassiliki; Khaterchi, Amir; Robert, Francoise; Gerotziafas, Grigoris; Hatmi, Mohamed; Elalamy, Ismail

    2013-01-01

    Heparin-induced thrombocytopenia (HIT) is a potentially lethal adverse effect of heparin therapy. Accurate and rapid HIT laboratory diagnosis when HIT is suspected is crucial. The combination of an immunological assay with a functional test improves the accuracy of HIT, but functional assays are currently limited to a few laboratories. Multiplate® analyzer (Dynabyte, Munich, Germany) is a practical, semi-automated and easy-to-perform platelet aggregation assay. The aim of this study is to explore whether heparin-induced platelet aggregation in whole blood assessed by Multiplate® (Heparin-induced multiple electrode aggregometry, HIMEA) can replace platelet aggregation test (PAT) in platelet-rich plasma. For this purpose, HIMEA performance in HIT diagnosis was prospectively evaluated. HIMEA and PAT were compared to serotonin-release assay (SRA) in 200 well-characterized consecutive patients suspected for HIT. HIMEA was found to be more sensitive (81% vs. 76%) and more specific (99% vs. 96%) than PAT compared to SRA. Both tests showed a high negative predictive value while HIMEA had a better positive predictive value. HIMEA has overall better performance characteristics than PAT for the detection of HIT platelet-activating antibodies. The combination of an immunological assay with HIMEA could be a feasible option in non-specialized laboratories for HIT diagnosis optimization.

  2. Heparin-Binding Protein (HBP): A Causative Marker and Potential Target for Heparin Treatment of Human Sepsis-Induced Acute Kidney Injury.

    Science.gov (United States)

    Fisher, Jane; Russell, James A; Bentzer, Peter; Parsons, Devyn; Secchia, Stefano; Mörgelin, Matthias; Walley, Keith R; Boyd, John H; Linder, Adam

    2017-09-01

    Sepsis-induced acute kidney injury (AKI) is a common condition with high morbidity and mortality. Neutrophil-derived heparin-binding protein (HBP) induces vascular leakage and is a promising biomarker of sepsis-induced organ dysfunction. It remains unknown if HBP is prognostic of AKI in septic shock and if HBP could play a role in the pathophysiology of sepsis-induced AKI. To determine the association of plasma HBP levels with development of AKI, investigate the role of HBP in the pathophysiology of sepsis-induced AKI, and test the effect of blocking HBP using heparin derivatives. In 296 septic shock patients from the randomized multicenter Vasopressin and Septic Shock Trial (VASST) plasma HBP levels were associated with development of AKI and need for renal replacement therapy (RRT). Human renal tubular cells were exposed to recombinant HBP to evaluate inflammation and heparin derivatives were used to abrogate these effects. Finally, mice were exposed to HBP with and without heparin derivatives and the kidneys examined for signs of inflammation. Plasma HBP levels were significantly higher in patients with AKI and those requiring RRT. HBP levels identified patients with moderate AKI with an area under curve (AUC) of 0.85. HBP increased IL-6 production in renal tubular epithelial cells. Different heparin derivatives abrogated the HBP-induced increased inflammatory response in vitro and in vivo. Elevated plasma HBP is associated with development of sepsis-induced AKI and HBP is involved in its pathophysiology. Our studies suggest that heparin(s) could be tested for efficacy and safety of prevention of sepsis-induced AKI.

  3. Bilateral adrenal haemorrhage associated with heparin-induced thrombocytopaenia during treatment of Fournier gangrene.

    Science.gov (United States)

    Tattersall, Timothy Lee; Thangasamy, Isaac A; Reynolds, Jamie

    2014-10-14

    We present a case of bilateral adrenal haemorrhage (BAH) associated with heparin-induced thrombocytopaenia (HIT) in a 61-year-old man admitted to hospital for the treatment of Fournier's gangrene. He presented to hospital with scrotal swelling and fever, and developed spreading erythaema and a gangrenous scrotum. His scrotum was surgically debrided and intravenous broad-spectrum antibiotics were administered. Unfractionated heparin was given postoperatively for venous thromboembolism prophylaxis. The patient deteriorated clinically 8-11 days postoperatively with delirium, chest pain and severe hypertension followed by hypotension and thrombocytopaenia. Abdominal CT scan revealed bilateral adrenal haemorrhage. Antibodies to the heparin-platelet factor 4 complex were present. HIT-associated BAH was diagnosed and heparin was discontinued. Intravenous bivalirudin and hydrocortisone were started, with rapid improvement in clinical status. BAH is a rare complication of HIT and should be considered in the postoperative patient with unexplained clinical deterioration.

  4. Clinical and laboratory diagnosis of heparin-induced thrombocytopenia: an integrated approach.

    Science.gov (United States)

    Cuker, Adam

    2014-02-01

    Heparin-induced thrombocytopenia (HIT) is a clinicopathologic disorder that predisposes to thrombosis. Diagnosis rests on a compatible clinical picture and laboratory evidence of antiplatelet factor 4 (PF4)/heparin antibodies that activate platelets in a heparin-dependent manner. Rapid and accurate diagnosis is paramount to avoid the perils of misdiagnosis. Clinical evaluation may be guided by scoring systems such as the 4Ts and HIT Expert Probability (HEP) score. Laboratory tests include immunoassays, such as the PF4/heparin enzyme-linked immunosorbent assay (ELISA) and functional tests such as the 14C-serotonin release assay and heparin-induced platelet activation assay. Clinical scoring systems and commercially available immunoassays have high sensitivity but modest specificity. Functional assays are more specific, but they are technically demanding. Novel laboratory assays with faster turnaround times, greater specificity, and lesser technical complexity are in development. A Bayesian approach that combines the 4T score and the PF4/heparin ELISA result may be used to estimate the probability of HIT and guide clinical decision making. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  5. [Diagnosis and treatment of heparin-induced thrombocytopenia (HIT) based on its atypical immunological features].

    Science.gov (United States)

    Miyata, Shigeki; Maeda, Takuma

    2016-03-01

    Heparin-induced thrombocytopenia (HIT) is a prothrombotic side effect of heparin therapy caused by HIT antibodies, i.e., anti-platelet factor 4 (PF4)/heparin IgG with platelet-activating properties. For serological diagnosis, antigen immunoassays are commonly used worldwide. However, such assays do not indicate their platelet-activating properties, leading to low specificity for the HIT diagnosis. Therefore, over-diagnosis is currently the most serious problem associated with HIT. The detection of platelet-activating antibodies using a washed platelet activation assay is crucial for appropriate HIT diagnosis. Recent advances in our understanding of the pathogenesis of HIT include it having several clinical features atypical for an immune-mediated disease. Heparin-naïve patients can develop IgG antibodies as early as day 4, as in a secondary immune response. Evidence for an anamnestic response on heparin re-exposure is lacking. In addition, HIT antibodies are relatively short-lived, unlike those in a secondary immune response. These lines of evidence suggest that the mechanisms underlying HIT antibody formation may be compatible with a non-T cell-dependent immune reaction. These atypical clinical and serological features should be carefully considered while endeavoring to accurately diagnose HIT, which leads to appropriate therapies such as immediate administration of an alternative anticoagulant to prevent thromboembolic events and re-administration of heparin during surgery involving cardiopulmonary bypass when HIT antibodies are no longer detectable.

  6. Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia.

    Science.gov (United States)

    Asada, Reiko; Wanaka, Keiko; Walenga, Jeanine; Prechel, Margaret; Miyashita, Kumiko; Escalante, Vicki; Kaneko, Chieko; Hoshino, Nobuhiro; Oosawa, Mitsuru; Matsuo, Miyako

    2013-01-01

    Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), (14)C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.

  7. Fibrogenic Lung Injury Induces Non-Cell-Autonomous Fibroblast Invasion.

    Science.gov (United States)

    Ahluwalia, Neil; Grasberger, Paula E; Mugo, Brian M; Feghali-Bostwick, Carol; Pardo, Annie; Selman, Moisés; Lagares, David; Tager, Andrew M

    2016-06-01

    Pathologic accumulation of fibroblasts in pulmonary fibrosis appears to depend on their invasion through basement membranes and extracellular matrices. Fibroblasts from the fibrotic lungs of patients with idiopathic pulmonary fibrosis (IPF) have been demonstrated to acquire a phenotype characterized by increased cell-autonomous invasion. Here, we investigated whether fibroblast invasion is further stimulated by soluble mediators induced by lung injury. We found that bronchoalveolar lavage fluids from bleomycin-challenged mice or patients with IPF contain mediators that dramatically increase the matrix invasion of primary lung fibroblasts. Further characterization of this non-cell-autonomous fibroblast invasion suggested that the mediators driving this process are produced locally after lung injury and are preferentially produced by fibrogenic (e.g., bleomycin-induced) rather than nonfibrogenic (e.g., LPS-induced) lung injury. Comparison of invasion and migration induced by a series of fibroblast-active mediators indicated that these two forms of fibroblast movement are directed by distinct sets of stimuli. Finally, knockdown of multiple different membrane receptors, including platelet-derived growth factor receptor-β, lysophosphatidic acid 1, epidermal growth factor receptor, and fibroblast growth factor receptor 2, mitigated the non-cell-autonomous fibroblast invasion induced by bronchoalveolar lavage from bleomycin-injured mice, suggesting that multiple different mediators drive fibroblast invasion in pulmonary fibrosis. The magnitude of this mediator-driven fibroblast invasion suggests that its inhibition could be a novel therapeutic strategy for pulmonary fibrosis. Further elaboration of the molecular mechanisms that drive non-cell-autonomous fibroblast invasion consequently may provide a rich set of novel drug targets for the treatment of IPF and other fibrotic lung diseases.

  8. Beryllium induces premature senescence in human fibroblasts.

    Science.gov (United States)

    Coates, Shannon S A; Lehnert, Bruce E; Sharma, Sunil; Kindell, Susan M; Gary, Ronald K

    2007-07-01

    After cells have completed a sufficient number of cell divisions, they exit the cell cycle and enter replicative senescence. Here, we report that beryllium causes proliferation arrest with premature expression of the principal markers of senescence. After young presenescent human fibroblasts were treated with 3 microM BeSO(4) for 24 h, p21 cyclin-dependent kinase inhibitor mRNA increased by >200%. Longer periods of exposure caused mRNA and protein levels to increase for both p21 and p16(Ink4a), a senescence regulator that prevents pRb-mediated cell cycle progression. BeSO(4) also caused dose-dependent induction of senescence-associated beta-galactosidase activity (SA-beta-gal). Untreated cells had 48 relative fluorescence units (RFU)/microg/h of SA-beta-gal, whereas 3 microM BeSO(4) caused activity to increase to 84 RFU/microg/h. In chromatin immunoprecipitation experiments, BeSO(4) caused p53 protein to associate with its DNA binding site in the promoter region of the p21 gene, indicating that p53 transcriptional activity is responsible for the large increase in p21 mRNA elicited by beryllium. Forced expression of human telomerase reverse transcriptase (hTERT) rendered HFL-1 cells incapable of normal replicative senescence. However, there was no difference in the responsiveness of normal HFL-1 fibroblasts (IC(50) = 1.9 microM) and hTERT-immortalized cells (IC(50) = 1.7 microM) to BeSO(4) in a 9-day proliferation assay. The effects of beryllium resemble those of histone deacetylase-inhibiting drugs, which also cause large increases in p21. However, beryllium produced no changes in histone acetylation, suggesting that Be(2+) acts as a novel and potent pharmacological inducer of premature senescence.

  9. Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.

    Science.gov (United States)

    Farwell, Sara Lynn N; Kanyi, Daniela; Hamel, Marianne; Slee, Joshua B; Miller, Elizabeth A; Cipolle, Mark D; Lowe-Krentz, Linda J

    2016-03-01

    Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.

  10. Natural language processor as a tool to assess heparin induced thrombocytopenia awareness.

    Science.gov (United States)

    Tafur, Alfonso J; McBane, Robert D; Wysokinski, Waldemar E; Gregg, Melissa S; Daniels, Paul R; Mohr, David N

    2012-01-01

    The life-threatening consequences of heparin induced thrombocytopenia (HIT) may be prevented with early recognition, prompt heparin withdrawal and direct thrombin inhibitor use. To determine the level of HIT awareness, electronic term recognition software can be used to query the electronic medical record (EMR) to assess the thought process and test ordering behavior of health care providers confronted with falling platelet counts. We sought to assess the awareness of HIT in a large teaching institution using these tools. Mayo Clinic databases were queried to identify a cohort of hospitalized adults receiving heparin (06/1/08-06/1/09). Serial platelet counts for each patient were scrutinized for a 50% decrement from baseline. "Clinician awareness" was defined by mention of HIT (determined by electronic term recognition software) within the hospital record by any member of the healthcare team or requisition of platelet factor 4/heparin antibody testing. During this time period, 34,694 adults were hospitalized and 24,956 received heparin. Only 3,239 (13%) patients had more than 1 platelet count during the hospital stay. Of 199 patients (6.1%) with ≥50% platelet count drop, clinician awareness was 36%. The absolute platelet count was the only independent variable associated with HIT awareness (P awareness are low at this large teaching institution. Software tools for monitoring awareness and providing realtime alerts of significant platelet count decrements may be useful.

  11. Heparin-Induced Thrombocytopenia in a Patient with Essential Thrombocythemia: A Case Based Update

    Directory of Open Access Journals (Sweden)

    Edva Noel

    2015-01-01

    Full Text Available Vascular thrombosis is a common clinical feature of both essential thrombocythemia (ET and heparin-induced thrombocytopenia (HIT. The development of HIT in a patient with ET is rare and underrecognized. We report the case of a 77-year-old woman with preexisting ET, who was admitted with acute coronary syndrome, and IV heparin was started. She was exposed to unfractionated heparin (UFH 5 days prior to this admission. Decrease in platelet count was noted, and HIT panel was sent. Heparin was discontinued. Patient developed atrial fibrillation, and Dabigatran was started. On day three, patient also developed multiple tiny cerebral infarctions and acute right popliteal DVT. On day ten of admission, HIT panel was positive, and Dabigatran was changed to Lepirudin. Two days later, Lepirudin was also discontinued because patient developed pseudoaneurysm on the right common femoral artery at the site of cardiac catheterization access. A progressive increase in the platelet count was noted after discontinuing heparin. Physicians should be aware of the coexistence of HIT and ET, accompanied challenges of the prompt diagnosis, and initiation of appropriate treatment.

  12. Atomic features of an autoantigen in heparin-induced thrombocytopenia (HIT).

    Science.gov (United States)

    Cai, Zheng; Zhu, Zhiqiang; Greene, Mark I; Cines, Douglas B

    2016-07-01

    Autoantigen development is poorly understood at the atomic level. Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). In solution, PF4 exists as an equilibrium among monomers, dimers and tetramers. Structural studies of these interacting components helped delineate a multi-step process involved in the pathogenesis of HIT. First, heparin binds to the 'closed' end of the PF4 tetramer and stabilizes its conformation; exposing the 'open' end. Second, PF4 arrays along heparin/GAG chains, which approximate tetramers, form large antigenic complexes that enhance antibody avidity. Third, pathogenic HIT antibodies bind to the 'open' end of stabilized PF4 tetramers to form an IgG/PF4/heparin ternary immune complex and also to propagate the formation of 'ultralarge immune complexes' (ULCs) that contain multiple IgG antibodies. Fourth, ULCs signal through FcγRIIA receptors, activating platelets and monocytes directly and generating thrombin, which transactivates hematopoietic and endothelial cells. A non-pathogenic anti-PF4 antibody prevents tetramer formation, binding of pathogenic antibody, platelet activation and thrombosis, providing a new approach to manage HIT. An improved understanding of the pathogenesis of HIT may lead to novel diagnostics and therapeutics for this autoimmune disease.

  13. Heparin suppresses cyclosporine-induced endothelin-1 synthesis in rat endothelial cells.

    Science.gov (United States)

    Yokokawa, K; Kohno, M; Minami, M; Yasunari, K; Mandal, A K; Yoshikawa, J

    1998-01-01

    Cyclosporine stimulates vasoconstrictor endothelin-1 (ET-1) synthesis. This study examined the effect of heparin on cyclosporine-induced ET-1 synthesis in Wistar rat aortic endothelial cells in culture. Cyclosporine (0.01-5 mumol/L) stimulated ET-1 mRNA expression in a dose-dependent manner. A nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA) (10(-5) mol/L), did not affect cyclosporine-induced ET-1 mRNA expression. Heparin (1-20 U/ml) suppressed cyclosporine-induced ET-1 mRNA expression in a dose-dependent manner. The inhibitory effect of heparin was blunted in the presence of either L-NMMA (10(-5) mol/L) or calmodulin inhibitors such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (5 x 10(-5) mol/L) or calmidazolium (5 x 10(-5) mol/L) in the presence of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (0.1 mmol/L). These results suggest that heparin suppresses cyclosporine-induced ET-1 mRNA expression via both NO- and calmodulin-dependent pathways.

  14. Heparin improves organ microcirculatory disturbances in caerulein-induced acute pancreatitis in rats

    Institute of Scientific and Technical Information of China (English)

    Marek Dobosz; Lucjanna Mionskowska; Stanislaw Ha(a); Sebastian Dobrowolski; Dariusz Dymecki; Zdzislaw Wajda

    2004-01-01

    AIM: Microcirculatory disturbances are important early pathophysiological events in various organs during acute pancreatitis. The aim of the study was to evaluate changes in microperfusion of the pancreas, liver, kidney, stomach,colon, skeletal muscle, and to investigate the influence of heparin on the organ microcirculation in caerulein-induced experimental acute pancreatitis.METHODS: Acute pancreatitis was induced by 4 intraperitoneal injections of caerulein (Cn) (15 μg/kg). The organ microcirculation was measured by laser Doppler flowmetry. Serum interleukin 6 and hematocrit levels were analysed.RESULTS: Acute pancreatitis resulted in a significant drop of microperfusion in all examined organs. Heparin administration (2×2.5 mg/kg) improved the microcirculation in pancreas (36.9±4% vs 75.9±10%), liver (56.6±6% vs 75.2±16%), kidney (45.1±6% vs 79.3±5%), stomach (65.2±8% vs78.1±19%), colon (69.8±6% vs102.5±19%),and skeletal muscle (59.2±6% vs 77.9±13%). Heparin treatment lowered IL-6 (359.0±66 U/mL vs288.5±58 U/mL)and hematocrit level (53±4% vs 46±3%).CONCLUSION: Heparin administration has a positive influence on organ microcirculatory disturbances accompanying experimental Ch-induced acute pancreatitis.

  15. Metabolites of Hypoxic Cardiomyocytes Induce the Migration of Cardiac Fibroblasts.

    Science.gov (United States)

    Shi, Huairui; Zhang, Xuehong; He, Zekun; Wu, Zhiyong; Rao, Liya; Li, Yushu

    2017-01-01

    The migration of cardiac fibroblasts to the infarct region plays a major role in the repair process after myocardial necrosis or damage. However, few studies investigated whether early hypoxia in cardiomyocytes induces the migration of cardiac fibroblasts. The purpose of this study was to assess the role of metabolites of early hypoxic cardiomyocytes in the induction of cardiac fibroblast migration. Neonatal rat heart tissue was digested with a mixture of trypsin and collagenase at an appropriate ratio. Cardiomyocytes and cardiac fibroblasts were cultured via differential adhesion. The cardiomyocyte cultures were subjected to hypoxia for 2, 4, 6, 8, 10, and 12 h. The supernatants of the cardiomyocyte cultures were collected to determine the differences in cardiac fibroblast migration induced by hypoxic cardiomyocyte metabolites at various time points using a Transwell apparatus. Meanwhile, ELISA was performed to measure TNF-α, IL-1β and TGF-β expression levels in the cardiomyocyte metabolites at various time points. The metabolites of hypoxic cardiomyocytes significantly induced the migration of cardiac fibroblasts. The induction of cardiac fibroblast migration was significantly enhanced by cardiomyocyte metabolites in comparison to the control after 2, 4, and 6 h of hypoxia, and the effect was most significant after 2 h. The expression levels of TNF-α, IL-1β, IL-6, and TGF-β were substantially increased in the metabolites of cardiomyocytes, and neutralization with anti-TNF-α and anti-IL-1β antibodies markedly reduced the induction of cardiac fibroblast migration by the metabolites of hypoxic cardiomyocytes. The metabolites of early hypoxic cardiomyocytes can induce the migration of cardiac fibroblasts, and TNF-α and IL-1β may act as the initial chemotactic inducers. © 2017 The Author(s) Published by S. Karger AG, Basel.

  16. Apoptosis of wound fibroblasts induced by oxidative stress.

    Science.gov (United States)

    Takahashi, Atsushi; Aoshiba, Kazutetsu; Nagai, Atsushi

    2002-06-01

    Irreversible lung parenchymal injury is usually healed by fibrosis, which depends on the abilities of fibroblasts to proliferate, migrate into the wound, and survive. Because the lung is frequently exposed to increased oxidative stress, which is thought to mediate apoptosis, we examined whether oxidative stress induces apoptosis in fibroblasts during wound healing. We performed an in vitro scratch wound assay where cultured fibroblast monolayers were exposed to H2O2 (10-500 microM) after artificial wounding. Apoptosis was evaluated by nuclear staining with Hoechst33342 or terminal deoxynucleotidyl transferase (TdT)-mediated nucleotide nick end-labeling (TUNEL). Intracellular oxidants were assessed with the peroxide-sensitive fluorochrome carboxydichlorodihydrofluorescein (CDCF). We found that repopulating fibroblasts at the wound margin, but not quiescent fibroblasts at the intact site, selectively underwent oxidant accumulation and apoptosis in response to H2O2 exposure. Some of the apoptotic cells had incorporated bromodeoxyuridine (BrdU), an indicator of proliferating cells. These results suggest that oxidative stress selectively induces apoptosis in fibroblasts that are stimulated to proliferate and/or migrate into the wound. Fibroblast apoptosis induced by oxidative stress during wound repopulation may be relevant to intractable wound healing.

  17. Analysis of the fibroblast growth factor receptor (FGFR) signalling network with heparin as coreceptor: evidence for the expansion of the core FGFR signalling network.

    Science.gov (United States)

    Xu, Ruoyan; Rudd, Timothy R; Hughes, Ashley J; Siligardi, Giuliano; Fernig, David G; Yates, Edwin A

    2013-05-01

    The evolution of the fibroblast growth factor (FGF)-FGF receptor (FGFR) signalling system has closely followed that of multicellular organisms. The abilities of nine FGFs (FGF-1 to FGF-9; examples of FGF subfamilies 1, 4, 7, 8, and 9) and seven FGFRs or isoforms (FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, and FGFR4) to support signalling in the presence of heparin, a proxy for the cellular heparan sulfate coreceptor, were assembled into a network. A connection between two FGFRs was defined as their mutual ability to signal with a particular FGF. The network contained a core of four receptors (FGFR1c, FGFR2c, FGFR3c, and FGFR4) with complete connectivity and high redundancy. Analysis of the wider network indicated that neither FGF-3 nor FGF-7 was well connected to this core of four receptors, and that divergence of a precursor of FGF subgroups 1, 4 and 9 from FGF subgroup 8 may have allowed expansion from a three-member FGFR core signalling system to the four-member core network. This increases by four-fold the number of possible signalling combinations. Synchrotron radiation CD spectra of the FGFs with heparin revealed no overall common structural change, suggesting the existence of distinct heparin-binding sites throughout the FGFs. The approach provides a potential method of identifying agents capable of influencing particular FGF-FGFR combinations, or areas of the signalling network, for experimental or therapeutic purposes.

  18. Acidosis induced by carbon dioxide insufflation decreases heparin potency: a risk factor for thrombus formation.

    Science.gov (United States)

    Gorter, Karin A M; Stehouwer, Marco C; Van Putte, Bart P; Vlot, Eline A; Urbanus, Rolf T

    2017-04-01

    Since the introduction of CO2 insufflation during open heart surgery in our hospital, we incidentally observed thrombus formation in the dissected heart, in the pericardium and in the cardiotomy reservoir of the cardiopulmonary bypass system. Furthermore, we measured very high levels of pCO2, causing severe acidosis, in stagnant blood in the pericardium and cardiotomy reservoir. In this in vitro study, we assessed the influence of acidosis and hypothermia on heparin potency and thrombin formation. We assessed heparin potency in function of pH (pH 5.0-7.4) and temperature (24-37°C) by comparing the activated partial thromboplastin time in platelet-poor plasma between samples with and without unfractionated heparin. We measured thrombin formation in platelet-poor plasma by means of fluorescent, calibrated, automated thrombography in function of pH (pH 5.0-7.4) and temperature (24-37°C). The parameters of interest were the endogenous thrombin potential and the peak amount of thrombin generation. The major finding of this study is the significant decrease in the efficiency of unfractionated heparin in delaying thrombus formation at acidotic (pH 5.0-7.0) conditions (p=0.034-0.05). Furthermore, we found that thrombin formation is significantly increased at hypothermic (24-34°C) conditions (p=acidosis may lead to a decreased heparin potency. Acidosis, as induced by CO2 insufflation, may predispose patients to incidental thrombus formation in stagnant blood in the open thorax and in the cardiotomy reservoir. Hypothermia might further increase this risk. Therefore, we recommend reconsidering the potential advantages and disadvantages of using CO2 insufflation during cardiopulmonary bypass.

  19. Heparin-binding secretory transforming gene (hst) facilitates rat lactotrope cell tumorigenesis and induces prolactin gene transcription.

    OpenAIRE

    Shimon, I; Hüttner, A; Said, J; Spirina, O M; Melmed, S

    1996-01-01

    We have shown previously that human prolactinomas express transforming sequences of the heparin-binding secretory transforming gene (hst) which encodes fibroblast growth factor-4 (FGF-4). To elucidate the role of hst in pituitary tumorigenesis we treated primary rat pituitary and pituitary tumor cell cultures with recombinant FGF-4 and also stably transfected pituitary cell lines with full-length human hst cDNA. Transfectants were screened for hst mRNA expression and FGF-4 production. FGF-4 (...

  20. Electrostatic Forces as Dominant Interactions Between Proteins and Polyanions: an ESI MS Study of Fibroblast Growth Factor Binding to Heparin Oligomers

    Science.gov (United States)

    Minsky, Burcu Baykal; Dubin, Paul L.; Kaltashov, Igor A.

    2017-02-01

    The interactions between fibroblast growth factors (FGFs) and their receptors (FGFRs) are facilitated by heparan sulfate (HS) and heparin (Hp), highly sulfated biological polyelectrolytes. The molecular basis of FGF interactions with these polyelectrolytes is highly complex due to the structural heterogeneity of HS/Hp, and many details still remain elusive, especially the significance of charge density and minimal chain length of HS/Hp in growth factor recognition and multimerization. In this work, we use electrospray ionization mass spectrometry (ESI MS) to investigate the association of relatively homogeneous oligoheparins (octamer, dp8, and decamer, dp10) with acidic fibroblast growth factor (FGF-1). This growth factor forms 1:1, 2:1, and 3:1 protein/heparinoid complexes with both dp8 and dp10, and the fraction of bound protein is highly dependent on protein/heparinoid molar ratio. Multimeric complexes are preferentially formed on the highly sulfated Hp oligomers. Although a variety of oligomers appear to be binding-competent, there is a strong correlation between the affinity and the overall level of sulfation (the highest charge density polyanions binding FGF most strongly via multivalent interactions). These results show that the interactions between FGF-1 and Hp oligomers are primarily directed by electrostatics, and also demonstrate the power of ESI MS as a tool to study multiple binding equilibria between proteins and structurally heterogeneous polyanions.

  1. Inhaled unfractionated heparin improves abnormalities of alveolar coagulation, fibrinolysis and inflammation in endotoxemia-induced lung injury rats

    Institute of Scientific and Technical Information of China (English)

    WANG Zong-yu; WU Sheng-nan; ZHU Zhao-zhong; YANG Ba-xian; ZHU Xi

    2013-01-01

    Background Acute lung injury/acute respiratory distress syndrome presents with not only local inflammation,but also pulmonary coagulopathy which is characterized by an alveolar procoagulant response,anticoagulant inhibition,fibrinolytic supression and fibrin deposition.We thus had hypothesized that if aerosolized unfractionated heparin was inhaled into alveolar spaces,it could block the procoagulant tendency,lessen depletion of coagulation factors,and even influence the inflammatory response.We also assessed the effects of different administration regimens of heparin.Methods Male Wistar rats were given inhaled heparin starting 30 minutes before (prophylactic heparin) or 2 hours after (therapeutic heparin) intravenous lipopolysaccharide (LPS) was administered at 6-hour intervals; control groups received inhaled normal saline with or without being exposed to LPS.Thrombin-antithrombin complexes,activated protein C,tissue type and urokinase type plasminogen activators (t-PNu-PA),plasminogen activator inhibitor-1 (PAl-1),tumor necrosis factor-α,interleukin-6 in bronchoalveolar lavage,and lung tissue myeloperoxidase activity,and histology score were measured at three time-points.PAI-1/(t-PA + u-PA) was calculated based on the before-mentioned parameters.Statistical analysis was made using one-way analysis of variance (ANOVA) with post hoc test or Student's t test in the case of heterogeneity of variance.Results An alveolar procoagulant reaction,depressed fibrinolysis,and inflammatory response occurred in endotoxemia-induced lung injury.Local prophylactic application of heparin attenuated coagulation and early inflammation,promoted fibrinolysis,and reduced the histology score.Therapeutic application of heparin had similar,but weaker effects.Conclusions Intrapulmonary application of unfractionated heparin by inhalation might inhibit alveolar procoagulant reaction and the early inflammatory response,promote flbrinolysis,and alleviate pulmonary pathology in endotoxemia-induced

  2. [Fondaparinux as an alternative anticoagulant in heparin-induced thrombocytopenia in the patient with a ventricular assist device].

    Science.gov (United States)

    Cegarra-Sanmartín, V; Paniagua, P; Galán, J; Muñoz, C; Moral, M V

    2013-11-01

    Heparin-induced thrombocytopenia is a reaction associated with the use of this drug. It occurs in up to 3% of patients treated for at least 5 days. Its treatment is to stop the heparin, and according to patient needs, replace it with another anticoagulant. We present a patient who, after a heart transplant, and the need for a ventricular assist device, required anticoagulation. The patient developed heparin-induced thrombocytopenia. Heparin was stopped and anticoagulation was replaced by fondaparinux. The peri-operative complications and the management of the coagulation are described. Copyright © 2012 Sociedad Española de Anestesiología, Reanimación y Terapéutica del Dolor. Published by Elsevier España. All rights reserved.

  3. Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring.

    Science.gov (United States)

    Crawford, Justin; Nygard, Karen; Gan, Bing Siang; O'Gorman, David Brian

    2015-02-01

    Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix-associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type-I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three-dimensional type-I collagen lattices. Inhibition of Rho-associated protein kinase activity significantly attenuated the effects of matrix-associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix-associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho-associated protein kinase-dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.

  4. Cerebral venous thrombosis and heparin-induced thrombocytopenia in an 18-year old male with severe ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    Gudrun Scheving Thorsteinsson; Maria Magnussson; Lena M Hallberg; Nils Gunnar Wahlgren; Fredrik Lindgren; Petter Malmborg; Thomas H Casswall

    2008-01-01

    The risk of thromboembolism is increased in inflammatory bowel disease and its symptoms may be overlooked. Furthermore, its treatment can be complex and is not without complications. We describe a case of an adolescent boy who developed a cerebral sinus venous thrombosis during a relapse of his ulcerative colitis and who, while on treatment with heparin,developed heparin-induced thrombocytopenia (HIT).The treatment was then switched to fondaparinux, a synthetic and selective inhibitor of activated factor x.

  5. 肝素诱导血小板减少症%Heparin-induced thrombocytopenia

    Institute of Scientific and Technical Information of China (English)

    贾海; 褚燕琦; 李炜; 袁继勇

    2011-01-01

    1例57岁男性患者因行溶栓术,给予尿激酶200000U,1次/6h泵入;肝素钠12 500 U入0.9%氯化钠注射液50ml进行24h微量泵泵入.溶栓治疗当日血小板计数为126×109/L,第3天为99×109/L,第4天降至65×109/L.考虑为肝素诱导血小板减少症,停用肝素钠,改为阿加曲班抗凝,并继续应用尿激酶行第2次溶栓术.停用肝素钠后血小板计数即回升,第2、4天分别为93×109/L、113×109/L,第5天恢复到术前水平.%A 57-year-old male patient received urokinase 200 000 U via pump every six hours and heparin sodium 12 500 U in 0. 9% chloride sodium 50 ml via minipump for 24 hours for thrombolytic therapy. In same day, the platelet level was 126 × 109/L and, on day 3 and 4, the platelet count respectively decreased to 99 × 109/L and 65 × 109/L. Heparin-induced thrombocytopenia was considered. Heparin sodium was stopped and switched to argatroban. The patient then underwent the second thrombolytic therapy with urokinase. The platelet count increased at once after heparin sodium discontinuation and, on days 2 and 4, his platelet level was 93 × 109/L and 113 × 109/L, respectively. His platelet count returned to a baseline level on day 5.

  6. Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor

    NARCIS (Netherlands)

    Bos, Gert W.; Scharenborg, Nicole M.; Poot, André A.; Engbers, Gerard H.M.; Beugeling, Tom; Aken, van Willem G.; Feijen, Jan

    1999-01-01

    Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a conflu

  7. Ceramide-induced apoptosis in rabbit corneal fibroblasts.

    Science.gov (United States)

    Kim, Tae-im; Pak, Jhang Ho; Tchah, Hungwon; Lee, Seung-ah; Kook, Michael S

    2005-01-01

    To evaluate the effect of various ceramides on the apoptosis of corneal fibroblasts and to determine the pathway on which they act. Corneal fibroblasts isolated and cultured from New Zealand white rabbits were exposed to various concentrations of ceramide types II and VI and phytoceramide types II and VI, and their apoptotic response was evaluated using an LDH assay and Hoechst and Annexin V staining. Corneal fibroblasts were preincubated with various concentrations of the CPP32-like protease inhibitor Z-VAD-FMK, the caspase-8 inhibitor IETD-CHO, and the caspase-9 inhibitor Z-LEHD-FMK before treatment with ceramide, and apoptotic response was assayed by LDH assay. In addition, cells treated with ceramide or phytoceramide were stained with an antibody to cytochrome c. At concentrations of 20 microM and higher, all 4 ceramides increased fibroblast apoptotic response significantly after 12 hours. Hoechst staining showed shrinkage of the cytoplasm, formation of apoptotic bodies, and nuclear fragmentation after ceramide exposure, and Annexin V staining showed small vesicles around the cell membrane. The CPP32-like protease inhibitor reduced the apoptotic response to all 4 ceramides. The specific caspase-8 inhibitor reduced the apoptotic response to ceramide type VI and phytoceramide types II and VI, whereas the specific caspase-9 inhibitor significantly reduced the apoptotic response to phytoceramide types II and VI. Following exposure to ceramides, corneal fibroblasts stained positively with antibody to cytochrome c. Ceramide induced apoptosis in cultured corneal fibroblasts. This apoptosis involved the caspase cascade and the mitochondrial pathway.

  8. Successful Management of Heparin-Induced Thrombocytopenia Using Argatroban in a Very Old Woman: A Case Report

    Directory of Open Access Journals (Sweden)

    A. Putot

    2013-01-01

    Full Text Available Thrombosis due to heparin-induced thrombocytopenia (HIT is rare but has a severe prognosis. Its management is not always easy, particularly in old patients with renal insufficiency. A 95-year-old woman was hospitalized for dyspnea. Curative treatment with unfractionated heparin was started because pulmonary embolism was suspected. Disseminated intravascular coagulation was then suspected because of thrombocytopenia, hypoprothrombinemia, hypofibrinogenemia, and a positive ethanol gelation test. The first immunoassay for HIT was negative. On the 12th day of hospitalization, bilateral cyanosis of the toes occurred associated with recent deep bilateral venous and arterial thrombosis at duplex ultrasound. New biological tests confirmed HIT and led us to stop heparin and to start argatroban with a positive clinical and biological evolution. Venous and arterial thrombosis associated with thrombocytopenia during heparin treatment must be considered HIT whatever the biological test results are. Argatroban is a good alternative treatment in the elderly.

  9. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Keiko Miyoshi

    2015-01-01

    Full Text Available Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs, human dermal fibroblasts (hDFs, and hOF-derived induced pluripotent stem cells (hOF-iPSCs, linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

  10. Reprogramming fibroblasts into induced pluripotent stem cells with Bmi

    Institute of Scientific and Technical Information of China (English)

    Jai-Hee Moon; June Seok Heo; Jun Sung Kim; Eun Kyoung Jun; Jung Han Lee; Aeree Kim; Jonggun Kim

    2011-01-01

    Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4,Sox2,and Klf4 in combination with c-Myc.Recently,Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells.Here,we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells,and,in combination with Oct4,can replace Sox2,Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells.Furthermore,activation of sonic hedgehog signaling (by Shh,purmorphamine,or oxysterol) compensates for the effects of Bmil,and,in combination with Oct4,reprograms mouse embryonic and adult fibroblasts into iPS cells.One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile,epigenetic status,and in vitro and in vivo differentiation into all three germ layers,as well as teratoma formation and germline transmission in vivo.These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2,KIf4,and N-Myc allows iPS generation via the addition of Oct4.

  11. Controlled dual delivery of fibroblast growth factor-2 and Interleukin-10 by heparin-based coacervate synergistically enhances ischemic heart repair.

    Science.gov (United States)

    Chen, William C W; Lee, Brandon G; Park, Dae Woo; Kim, Kyobum; Chu, Hunghao; Kim, Kang; Huard, Johnny; Wang, Yadong

    2015-12-01

    Myocardial infarction (MI) causes myocardial necrosis, triggers chronic inflammatory responses, and leads to pathological remodeling. Controlled delivery of a combination of angiogenic and immunoregulatory proteins may be a promising therapeutic approach for MI. We investigated the bioactivity and therapeutic potential of an injectable, heparin-based coacervate co-delivering an angiogenic factor, fibroblast growth factor-2 (FGF2), and an anti-inflammatory cytokine, Interleukin-10 (IL-10) in a spatially and temporally controlled manner. Coacervate delivery of FGF2 and IL-10 preserved their bioactivities on cardiac stromal cell proliferation in vitro. Upon intramyocardial injection into a mouse MI model, echocardiography revealed that FGF2/IL-10 coacervate treated groups showed significantly improved long-term LV contractile function and ameliorated LV dilatation. FGF2/IL-10 coacervate substantially augmented LV myocardial elasticity. Additionally, FGF2/IL-10 coacervate notably enhanced long-term revascularization, especially at the infarct area. In addition, coacervate loaded with 500 ng FGF2 and 500 ng IL-10 significantly reduced LV fibrosis, considerably preserved infarct wall thickness, and markedly inhibited chronic inflammation at the infarct area. These results indicate that FGF2/IL-10 coacervate has notably greater therapeutic potential than coacervate containing only FGF2. Overall, our data suggest therapeutically synergistic effects of FGF-2/IL-10 coacervate, particularly coacervate with FGF2 and 500 ng IL-10, for the treatment of ischemic heart disease.

  12. Effect Of Heparin Sodium In Protection Of The Lens Against Cataract Induced With Intravitreal Injection Of Sodium Selenite- In Rabbits

    Directory of Open Access Journals (Sweden)

    Bahaa A. Abdul-Hussein

    2015-08-01

    Full Text Available Abstract Objective To evaluate the possible protective role of heparin sodium eye drops against sodium selenite induced cataract in rabbits.Materials and Methods A group of 18 adult rabbits Oryctologus cuniculus were divided into 3 groups each one of 6 rabbits normal group without treatment and induction control group received DW pre and post induction of cataract and heparin sodium group received heparin sodium eye drops pre and post induction of cataract. The cataract had been induced by intravitreal injection of 0.1ml sodium selenite 0.01 wv in the right eye. Results Heparin sodium was effective in prevention of cataract and the mean score of opacity was 0.170.01 at the end of trial period in stead of the expected score 4 0.00 which observed in DW group and there was non significant difference comparing to pre induction p0.05. Conclusions Heparin sodium eye drops exerted a detectable preventive effect against sodium selenite - induced cataract in rabbits also it was found to be apparently safe and tolerable along the trial period.

  13. Radiation-Induced Differentiation in Human Lung Fibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sa-Rah; Ahn, Ji-Yeon; Han, Young-Soo; Shim, Jie-Young; Yun, Yeon-Sook; Song, Jie-Young [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-10-15

    One of the most common tumors in many countries is lung cancer and patients with lung cancer may take radiotherapy. Although radiotherapy may have its own advantages, it can also induce serious problems such as acute radiation pneumonitis and pulmonary fibrosis. Pulmonary fibrosis is characterized by excessive production of {alpha}-SMA and accumulation of extracellular matrix (ECM) such as collagen and fibronectin. There has been a great amount of research about fibrosis but the exact mechanism causing the reaction is not elucidated especially in radiation-induced fibrosis. Until now it has been known that several factors such as transforming growth factor (TGF-{beta}), tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are related to fibrosis. Among them TGF-{beta} with Smad signaling is known to be the main stream and other signaling molecules such as MAPK, ERK and JNK (3) also participates in the process. In addition to those above factors, it is thought that more diverse and complicate mechanisms may involve in the radiationinduced fibrosis. Therefore, to investigate the underlying mechanisms in radiation induced fibrosis, first of all, we confirmed whether radiation induces trans differentiation in human normal lung fibroblasts. Here, we suggest that not only TGF-{beta} but also radiation can induce trans differentiation in human lung fibroblast WI-38 and IMR-90.

  14. Interactions between nattokinase and heparin/GAGs.

    Science.gov (United States)

    Zhang, Fuming; Zhang, Jianhua; Linhardt, Robert J

    2015-12-01

    Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK's potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6-O-sulfo as well as the N-sulfo groups but not the 2-O-sulfo groups within heparin, are essential for heparin's interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin's interaction with antithrombin and fibroblast growth factors.

  15. Generation of human induced pluripotent stem cells from dermal fibroblasts

    OpenAIRE

    2008-01-01

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ...

  16. Bioengineered heparin

    Science.gov (United States)

    Lord, Megan S; Whitelock, John M

    2014-01-01

    Heparin is a widely used drug for the control of blood coagulation. The majority of heparin that is produced commercially is derived from animal sources, is poly-disperse in nature and therefore ill-defined in structure. This makes regulation of heparin challenging with respect to identifying its absolute structural identity, purity, and efficacy. This raises the question as to whether there might be alternative methods of producing commercial grade heparin. The commentary highlights ways that we might manufacture heparin using bioengineering approaches to yield a successful therapeutic replacement for animal-derived heparin in the future. PMID:24902029

  17. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  18. An Anti-Coagulation Conundrum: Implantation of Total Artificial Heart in a Patient with Heparin-Induced Thrombocytopenia Type II

    Science.gov (United States)

    Cios, Theodore J.; Salamanca-Padilla, Yuliana; Guvakov, Dmitri

    2017-01-01

    Patient: Male, 44 Final Diagnosis: Heparin-induced thrombocytopenia Type II Symptoms: Congestive heart failure • short of breath Medication: — Clinical Procedure: LVAD explantation • TAH insertion Specialty: Anesthesiology Objective: Rare co-existance of disease or pathology Background: Heparin-induced thrombocytopenia (HIT) is a rare but life-threatening complication of heparin administration. It can present a major clinical dilemma for physicians caring for patients requiring life-saving urgent or emergent cardiac surgery. Studies have been published examining the use of alternative anticoagulants for patients undergoing cardiopulmonary bypass (CPB), however, evidence does not clearly support any particular approach. Presently, there are no large-scale, prospective randomized studies examining the impact of alternative anticoagulants on clinical outcomes for HIT-positive patients requiring cardiac surgery. Case Report: We present the case of a patient who underwent SynCardia Total Artificial Heart (TAH) implantation following a recent left ventricular assist device (LVAD) placement. The patient was receiving argatroban for type II HIT with anuric renal failure, and developed a thrombus which occluded the inflow cannula of the LVAD. Based on a published study and after establishing consensus with the surgical, anesthesiology, perfusion, and hematology teams, we decided to use tirofiban as an antiplatelet agent to inhibit the platelet aggregation induced by heparin, and ultimately used heparin as the anticoagulant for cardiopulmonary bypass. Conclusions: When selecting anticoagulation for a HIT-positive patient requiring CPB, so that benefits outweigh risks, it is of paramount importance that the decision be based on a multitude of factors. The team caring for the patient should have a shared mental model and be familiar with the pharmacology, devices used, and local practices. These three elements should be integrated with patient-specific comorbidities

  19. [Contaminated heparins].

    Science.gov (United States)

    Monneret, C

    2008-08-01

    In January 2008, following the detection of severe allergic reaction, several batches of heparins were removed from the United-States market. Although less dramatic, comparable side effects were also reported in Germany but not in France despite the fact that low-weight heparins, obtained from contaminated batches of unfractionated heparins, were used to limit shortage. So far, tainted injectable heparin has been linked to over 80 deaths in the USA. Analyses demonstrated that such tainted heparins were contaminated by high levels of chondroïtin persulfate (5-20%), a cheaper hemi-synthetic product. All batches were furnished by several Chinese chemical industries, China representing 50% of all heparins produced worldwide. Thus, contamination of the heparin supply is a worldwide problem. Following this event, the efficiency of the quality insurance, particularly analytical controls before proceeding, remains questionable. The strict respect of the pharmaceutical chain is urgently required to avoid any kind of quality problem in the future.

  20. Extracellular matrix heparin induces alteration of the cell adhesion during brain development

    NARCIS (Netherlands)

    Ushakova, GA; Nikonenko, IR; Nikonenko, AG; Skibo, GG

    2002-01-01

    The studies of neuronal cell-glycosaminoglycan interactions indicate an increasing interest in the question of how heparin can mediate adhesion properties of the cell. We have found that high levels of both N-CAM concentration and heparin-binding activity were noticed in the early stages of brain fo

  1. Inhibition of surface induced coagulation by preadsorption of albumin-heparin conjugates

    NARCIS (Netherlands)

    Hennink, W.E.; Kim, S.W.; Feijen, J.

    1984-01-01

    Surface coatings of the albumin-heparin conjugates were developed to improve the blood compatibility of polymeric materials. Glass, PVC, Biomer and cellulose acetate were coated with albumin-heparin conjugate and its adsorption and desorption behavior on glass in particular was studied using 3H and

  2. Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

    Science.gov (United States)

    Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

    2014-05-01

    To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and

  3. The role of calcineurin in the lung fibroblasts proliferation and collagen synthesis induced by basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    陈亚红; 赵鸣武; 符民桂; 姚婉贞; 唐朝枢

    2003-01-01

    Objective To investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).Methods We used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using 32P-labelled substrate. In the primary culture of lung fibroblasts, 3H-thymidine (3H-TdR) and 3H-proline incorporation methods were used to study the effect of cyclosporin A(CsA), an inhibitor of calcineurin, on the lung fibroblast DNA and collagen synthesis stimulated by bFGF. Results We found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1±2.0 pmol Pi/mg pr/min). CsA(10-8-10-6mol/L) inhibited lung fibroblast,3H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by20%, 46% and 66%(P<0.01). CsA(10-7-10-6mol/L) inhibited 3H-proline incorporation in lung fibroblasts stimulated by bFGF, with the inhibitory rates by 21% and 37%(P<0.01). In a culture medium, CsA (10-8-10-6mol/L) inhibited 3H-proline secretion induced by bFGF in a dose-dependent manner, with the inhibitory rates by 19%,29%(P<0.05) and 56% (P<0.01). CsA (10-7mol/L) could inhibit calcineurin activity by 44% in lung fibroblasts(P<0.01). Conclusions Calcineurin is expressed in lung tissue and has phosphatase activity. It is involved in the bFGF stimulated lung fibroblast DNA and collagen synthesis.

  4. 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

  5. Physiological loading of tendons induces scleraxis expression in epitenon fibroblasts.

    Science.gov (United States)

    Mendias, Christopher L; Gumucio, Jonathan P; Bakhurin, Konstantin I; Lynch, Evan B; Brooks, Susan V

    2012-04-01

    Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting fibroblast proliferation and matrix synthesis during the embryonic development of tendons. Mice with a targeted inactivation of scleraxis (Scx(-/-)) fail to properly form limb tendons, but the role that scleraxis has in regulating the growth and adaptation of tendons of adult organisms is unknown. To determine if scleraxis expression changes in response to a physiological growth stimulus to tendons, we subjected adult mice that express green fluorescent protein (GFP) under the control of the scleraxis promoter (ScxGFP) to a 6-week-treadmill training program designed to induce adaptive growth in Achilles tendons. Age matched sedentary ScxGFP mice were used as controls. Scleraxis expression was sparsely observed in the epitenon region of sedentary mice, but in response to treadmill training, scleraxis was robustly expressed in fibroblasts that appeared to be emerging from the epitenon and migrating into the superficial regions of tendon fascicles. Treadmill training also led to an increase in scleraxis, tenomodulin, and type I collagen gene expression as measured by qPCR. These results suggest that in addition to regulating the embryonic formation of limb tendons, scleraxis also appears to play an important role in the adaptation of adult tendons to physiological loading.

  6. [The long-term therapy of familial hypercholesterolemia with heparin-induced extracorporeal LDL precipitation].

    Science.gov (United States)

    Roth, R; Köster, W; Wanner, C; Andre, M; Orth, M; Wieland, H; Schollmeyer, P

    1992-07-24

    The long-term tolerance to and effectiveness of heparin-induced extracorporeal LDL precipitation (HELP) in combination with lipid reducing drugs and diet was tested in six patient (5 males, 1 female; mean age 48 +/- 4 years). Follow-up period was over 50 months, in one patient over 24 months, while one man had a sudden cardiac death 57 weeks after starting treatment. The study was divided into three phases. In phase I (24 months) treatment consisted of HELP and conventional lipid-reducing drugs; in phase II (12 months) of lovastatin (80 mg daily) and cholestyramine (12-24 g daily); and phase III (14 months) of HELP, lovastatin and cholestyramine. In phase I it was possible to lower the pre-treatment level of LDL-cholesterol from 306 +/- 18 mg/dl to 173 +/- 13 mg/dl (43.5%). A similar effect (from 307 +/- 21 mg/dl to 155 +/- 17 mg/dl [-49.5%]) was obtained in phase II. The resumption of HELP reduced the pre-treatment LDL concentration to 136 +/- 9 mg/dl (-55.7%). The various treatment regimens were well tolerated. Biochemical data remained unchanged except for iron loss requiring substitution. Thus combined HELP, lovastatin and ion exchange offer for the first time an effective and reliable means in familial hypercholesterolaemia of clearly reducing long-term the mean LDL cholesterol level below the atherosclerosis threshold of 120 mg/dl.

  7. Microchannel-patterned and heparin micro-contact-printed biodegradable composite membranes for tissue-engineering applications.

    Science.gov (United States)

    Baran, Erkan T; Tuzlakoğlu, Kadriye; Salgado, António; Reis, Rui L

    2011-06-01

    Microchannel-patterned starch-poly(capro-lactone)/hydydroxyapatite (SPCL-HA) and starch-poly(lactic acid) (SPLA) composite membranes were produced for use as a laminated tissue-engineering scaffold that incorporates both physical and biochemical patterns. For this purpose, SPCL (30% starch) blended with inorganic hydroxyl apatite (50%) and SPLA (50% starch) membranes were made with compressive moulding. Consequently, the microchannel structures (width 102 µm, 174 µm intervals) were developed on the composite membranes by means of micro-patterned metal mould(s) and hydraulic pressing. An elastomer poly(dimetylsiloxane) stamp was used to transfer heparin as a biochemical cue over the microchannel surfaces by micro-contact printing (µCP). Toluidine blue staining of developed capillaries and heparin µCP-coated membranes showed that heparin was transferred predominantly over the microchannel surfaces. Fibroblast cell culture over the microchannel-formed and heparin µCP-modified SPCL-HA and SPLA membranes showed distinct growth patterns. In contrast to the uniform cell layer formed on unmodified microchannels, the cells were bridging across the grooves of heparin-printed microchannels. At extended culture periods, the heparin-printed microchannels were covered with a layer of fibroblast cells without cellular ingrowths inside. This study indicated that the topographical pattern could induce an organization of fibroblasts only with the biochemical cue and the cells' functions can be controlled spatially over the microchannels by using both cues.

  8. Catastrophic heparin-induced thrombocytopenia/thrombosis syndrome related to the use of a Port-A-Cath in a breast cancer patient receiving chemotherapy.

    Science.gov (United States)

    Dai, Ming-Shen; Hsieh, An-Tie; Chao, Tsu-Yi

    2004-07-01

    Heparin-induced thrombocytopenia and thrombosis (HIT/T) syndrome is usually triggered by an immune response after repeated administration of heparin. The syndrome is strongly associated with limb deep vein thrombosis and is potentially life-threatening if unrecognized. We describe the case of a patient with compartment syndrome of the left forearm complicated by HIT/T that developed after Port-A-Cath implantation through the left subclavian vein. Prompt recognition of HIT/T, immediate withdrawal of heparin, and timely institution of thrombolytic therapy successfully prevented limb loss.

  9. Bovine trophectoderm cell lines induced from bovine fibroblasts with reprogramming factors

    Science.gov (United States)

    Bovine trophectoderm (TE) cells were induced [induced bovine trophectoderm-like (iBT)] from bovine fetal liver-derived fibroblasts, and other bovine fetal fibroblasts, after viral-vector transduction with either four or six reprogramming factors (RF), including POU5F1, KLF4, SOX2, C-MYC, SV40 large ...

  10. Refractory heparin induced thrombocytopenia with thrombosis (HITT) treated with therapeutic plasma exchange and rituximab as adjuvant therapy.

    Science.gov (United States)

    Schell, Amy M; Petras, Melissa; Szczepiorkowski, Zbigniew M; Ornstein, Deborah L

    2013-10-01

    We report a case of refractory heparin-induced thrombocytopenia with thrombosis (HITT) with prolonged thrombocytopenia and multiple thrombotic complications that failed to improve despite aggressive treatment. A 60 year old female with a prior history of venous thromboembolism was admitted with an acute pulmonary embolism, and developed HITT after several days on heparin therapy. She suffered multiple complications including bilateral venous limb gangrene, acute renal failure, and refractory thrombocytopenia, leading us to use multimodality therapy including therapeutic plasma exchange (TPE) and rituximab immunosuppression. The patient had transient improvements in her thrombocytopenia with TPE, and rituximab was added in an attempt to reduce antibody production. She eventually required bilateral limb amputation, and only after removal of the gangrenous limbs did her platelet count show sustained improvement. We discuss the possible contribution of infection to her prolonged course.

  11. Heparin-induced platelet aggregation (H-IPA): dose/response relationship for two low molecular weight (LMW) heparin preparations (CY 216 and CY 222)

    Energy Technology Data Exchange (ETDEWEB)

    Brace, L.D.; Fareed, J.

    1986-03-01

    The authors have previously demonstrated that heparin and a LMW heparin derivative (PK 10169) causes platelet aggregation in a dose-dependent manner that can be inhibited by antagonists of the thromboxane pathway. Using fractions of these agents separated on the basis of molecular weight (MW) by gel permeation chromatography, the authors showed that H-IPA was directly dependent upon the MW of the agents tested. In order to further examine this MW dependence, the authors tested two other LMW heparin preparations, CY 216 and CY 222 and subfractions of these agents separated on the basis of MW. Citrate anticoagulated whole blood was drawn from drug-free normal healthy donors whose platelets aggregated when heparin was added to their platelet-rich plasma (PRP). PRP was prepared, various concentrations of the agents or their subfractions were added and aggregation was monitored for 40 minutes at 37/sup 0/C. The results demonstrate that like heparin and PK 10169, CY 216 and CY 222 caused platelet aggregation in a dose and MW dependent manner. Fractions with MW less than 2500 daltons caused aggregation only at concentrations exceeding the therapeutic range of the agents. The authors conclude that the ability to cause H-IPA is an inherent property of heparin and its fractions.

  12. Dioxin induces genomic instability in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Merja Korkalainen

    Full Text Available Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI, i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD. Mouse embryonic fibroblasts (C3H10T1/2 were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days. For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay, was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

  13. Mercury-induced micronuclei in skin fibroblasts of beluga whales

    Energy Technology Data Exchange (ETDEWEB)

    Gauthier, J.M.; Dubeau, H.; Rassart, E. [Univ. du Quebec, Montreal, Quebec (Canada). Dept. des Sciences Biologiques

    1998-12-01

    Beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high incidence of cancer observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei assay was used to test the genotoxic potential of mercury compounds in skin fibroblasts of an Arctic beluga whale. Both mercuric chloride (Hg) and methylmercury (MeHg) induced a highly significant dose-response increase of micronucleated cells. Statistically significant increases in micronucleated cells were observed for 0.5, 5, and 20 {micro}g/ml Hg and 0.05, 0.5, and 2 {micro}g/ml MeHg when compared to control cultures. Concentrations of 0.5, 5, and 20 {micro}g/ml Hg induced a two-, three- and fourfold increase of micronucleated cells, respectively. Treatment with MeHg was one order of magnitude more potent in inducing micronuclei and in inhibiting cell proliferation than Hg. Although results of this in vitro study do not imply that mercury compounds are involved in the etiology of cancer in St. Lawrence beluga whales, significant increases in micronuclei frequency were found at low concentrations of MeHg that are believed to be comparable to concentrations present in certain whales of this population.

  14. Metformin Inhibits Angiotensin II-Induced Differentiation of Cardiac Fibroblasts into Myofibroblasts

    OpenAIRE

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

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibr...

  15. Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts.

    Science.gov (United States)

    Shaukat, Irfan; Barré, Lydia; Venkatesan, Narayanan; Li, Dong; Jaquinet, Jean-Claude; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2016-01-01

    Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-β-D-xylopyranoside, a competitive inhibitor of β4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-β1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-β1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of β4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of β4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis.

  16. Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts

    Science.gov (United States)

    Shaukat, Irfan; Barré, Lydia; Venkatesan, Narayanan; Li, Dong; Jaquinet, Jean-Claude; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2016-01-01

    Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-β-D-xylopyranoside, a competitive inhibitor of β4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-β1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-β1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of β4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of β4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis. PMID:26751072

  17. 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.

  18. Structural Stability of Human Fibroblast Growth Factor-1 Is Essential for Protective Effects Against Radiation-Induced Intestinal Damage

    Energy Technology Data Exchange (ETDEWEB)

    Nakayama, Fumiaki, E-mail: f_naka@nirs.go.jp [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Umeda, Sachiko [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Yasuda, Takeshi [Department of Radiation Emergency Medicine, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba (Japan); Asada, Masahiro; Motomura, Kaori; Suzuki, Masashi [Signaling Molecules Research Laboratory, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki (Japan); Zakrzewska, Malgorzata [Faculty of Biotechnology, University of Wroclaw (Poland); Imamura, Toru [Signaling Molecules Research Laboratory, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki (Japan); Imai, Takashi [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan)

    2013-02-01

    Purpose: Human fibroblast growth factor-1 (FGF1) has radioprotective effects on the intestine, although its structural instability limits its potential for practical use. Several stable FGF1 mutants were created increasing stability in the order, wild-type FGF1, single mutants (Q40P, S47I, and H93G), Q40P/S47I, and Q40P/S47I/H93G. This study evaluated the contribution of the structural stability of FGF1 to its radioprotective effect. Methods and Materials: Each FGF1 mutant was administered intraperitoneally to BALB/c mice in the absence of heparin 24 h before or after total body irradiation (TBI) with {gamma}-rays at 8-12 Gy. Several radioprotective effects were examined in the jejunum. Results: Q40P/S47I/H93G could activate all subtypes of FGF receptors in vitro much more strongly than the wild-type without endogenous or exogenous heparin. Preirradiation treatment with Q40P/S47I/H93G significantly increased crypt survival more than wild-type FGF1 after TBI at 10 or 12 Gy, and postirradiation treatment with Q40P/S47I/H93G was effective in promoting crypt survival after TBI at 10, 11, or 12 Gy. In addition, crypt cell proliferation, crypt depth, and epithelial differentiation were significantly promoted by postirradiation treatment with Q40P/S47I/H93G. The level of stability of FGF1 mutants correlated with their mitogenic activities in vitro in the absence of heparin; however, preirradiation treatment with the mutants increased the crypt number to almost the same level as Q40P/S47I/H93G. When given 24 h after TBI at 10 Gy, all FGF1 mutants increased crypt survival more than wild-type FGF1, and Q40P/S47I/H93G had the strongest mitogenic effects in intestinal epithelial cells after radiation damage. Moreover, Q40P/S47I/H93G prolonged mouse survival after TBI because of the repair of intestinal damage. Conclusion: These findings suggest that the structural stability of FGF1 can contribute to the enhancement of protective effects against radiation-induced intestinal

  19. Modified Heparin

    Institute of Scientific and Technical Information of China (English)

    XIONG; Jian

    2001-01-01

    Heparin, the naturally occurring blood anticoagulating agent, is a sulphated polysaccharide. In addition to famous anticoagulation, It also has other important pharmacological actions, such as preventing thrombosis, immunological accommodation, smooth muscle hyperplasia, antivirus etc.  ……

  20. Modified Heparin

    Institute of Scientific and Technical Information of China (English)

    XIONG Jian; YE Jun; GUAN Jialun; LI Lin

    2001-01-01

    @@ Heparin, the naturally occurring blood anticoagulating agent, is a sulphated polysaccharide. In addition to famous anticoagulation, It also has other important pharmacological actions, such as preventing thrombosis, immunological accommodation, smooth muscle hyperplasia, antivirus etc.

  1. Generation of human induced pluripotent stem cells from dermal fibroblasts.

    Science.gov (United States)

    Lowry, W E; Richter, L; Yachechko, R; Pyle, A D; Tchieu, J; Sridharan, R; Clark, A T; Plath, K

    2008-02-26

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ectopic expression of the defined transcription factors KLF4, OCT4, SOX2, and C-MYC. The resultant cell lines are morphologically indistinguishable from human embryonic stem cells (HESC) generated from the inner cell mass of a human preimplantation embryo. Consistent with these observations, human iPS cells share a nearly identical gene-expression profile with two established HESC lines. Importantly, DNA fingerprinting indicates that the human iPS cells were derived from the donor material and are not a result of contamination. Karyotypic analyses demonstrate that reprogramming of human cells by defined factors does not induce, or require, chromosomal abnormalities. Finally, we provide evidence that human iPS cells can be induced to differentiate along lineages representative of the three embryonic germ layers indicating the pluripotency of these cells. Our findings are an important step toward manipulating somatic human cells to generate an unlimited supply of patient-specific pluripotent stem cells. In the future, the use of defined factors to change cell fate may be the key to routine nuclear reprogramming of human somatic cells.

  2. Lithium Attenuates TGF-β1-Induced Fibroblasts to Myofibroblasts Transition in Bronchial Fibroblasts Derived from Asthmatic Patients

    Directory of Open Access Journals (Sweden)

    Marta Michalik

    2012-01-01

    Full Text Available Bronchial asthma is a chronic disorder accompanied by phenotypic transitions of bronchial epithelial cells, smooth muscle cells, and fibroblasts. Human bronchial fibroblasts (HBFs derived from patients with diagnosed asthma display predestination towards TGF-β-induced phenotypic switches. Since the interference between TGF-β and GSK-3β signaling contributes to pathophysiology of chronic lung diseases, we investigated the effect of lithium, a nonspecific GSK-3β inhibitor, on TGF-β1-induced fibroblast to myofibroblast transition (FMT in HBF and found that the inhibition of GSK-3β attenuates TGF-β1-induced FMT in HBF populations derived from asthmatic but not healthy donors. Cytoplasmically sequestrated β-catenin, abundant in TGF-β1/LiCl-stimulated asthmatic HBFs, most likely interacts with and inhibits the nuclear accumulation and signal transduction of Smad proteins. These data indicate that the specific cellular context determines FMT-related responses of HBFs to factors interfering with the TGF-β signaling pathway. They may also provide a mechanistic explanation for epidemiological data revealing coincidental remission of asthmatic syndromes and their recurrence upon the discontinuation of lithium therapy in certain psychiatric diseases.

  3. Fibroblast growth factor 10 protects neuron against oxygen-glucose deprivation injury through inducing heme oxygenase-1.

    Science.gov (United States)

    Li, Yong-Hua; Yang, Li-Ye; Chen, Wei; Li, Ying-Ke; Yuan, Hong-Bin

    2015-01-02

    Fibroblast growth factors (FGFs) are a family of structurally related heparin-binding proteins with diverse biological functions. FGFs participate in mitogenesis, angiogenesis, cell proliferation, development, differentiation and cell migration. Here, we investigated the potential effect of FGF10, a member of FGFs, on neuron survival in oxygen-glucose deprivation (OGD) model. In primary cultured mouse cortical neurons upon OGD, FGF10 treatment (100 and 1000 ng/ml) attenuated the decrease of cell viability and rescued the LDH release. Tuj-1 immunocytochemistry assay showed that FGF10 promoted neuronal survival. Apoptosis assay with Annexin V+PI by flow cytometry demonstrated that FGF10 treatment reduced apoptotic cell proportion. Moreover, immunoblotting showed that FGF10 alleviated the cleaved caspase-3 upregulation caused by OGD. FGF10 treatment also depressed the OGD-induced increase of caspase-3, -8 and -9 activities. At last, we found FGF10 triggered heme oxygenase-1 (HO-1) protein expression rather than hypoxia-inducible factor-1 (HIF-1), AMP-activated protein kinase (AMPK) signaling and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling. Knockdown of HO-1 by siRNA partly abolished the neuroprotection of FGF10 in OGD model. In summary, our observations provide the first evidence for the neuroprotective function of FGF10 against ischemic neuronal injury and suggest that FGF10 may be a promising agent for treatment of ischemic stroke.

  4. Fibroblast-like synoviocytes induce calcium mineral formation and deposition.

    Science.gov (United States)

    Sun, Yubo; Mauerhan, David R; Franklin, Atiya M; Zinchenko, Natalia; Norton, Harry James; Hanley, Edward N; Gruber, Helen E

    2014-01-01

    Calcium crystals are present in the synovial fluid of 65%-100% patients with osteoarthritis (OA) and 20%-39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  5. Fibroblast-Like Synoviocytes Induce Calcium Mineral Formation and Deposition

    Directory of Open Access Journals (Sweden)

    Yubo Sun

    2014-01-01

    Full Text Available Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA and 20%–39% patients with rheumatoid arthritis (RA. This study sought to investigate the role of fibroblast-like synoviocytes (FLSs in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s. The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  6. Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism: Implications for breast cancer prevention

    National Research Council Canada - National Science Library

    Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Lamb, Rebecca; Hulit, James; Howell, Anthony; Sotgia, Federica; Rubin, Emanuel; Lisanti, Michael P

    2013-01-01

    ...) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation...

  7. Interleukin-19 Downregulates Interleukin-4-Induced Eotaxin Production in Human Nasal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Masaaki Higashino

    2011-01-01

    Conclusions: These results suggest that IL-19 down-regulates IL-4-induced eotaxin production via SOCS-1 in human nasal fibroblasts. In non-hematopoietic cells in AR, IL-19 might be an immunosuppressive factor.

  8. Radiation-induced gene expression in human subcutaneous fibroblasts is predictive of radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Rødningen, Olaug Kristin; Børresen-Dale, Anne-Lise; Alsner, Jan

    2008-01-01

    with variable risk of RIF (grouped into five classes from low to high risk) were irradiated with two different schemes: 1x3.5Gy with RNA isolated 2 and 24h after irradiation, and a fractionated scheme with 3x3.5Gy in intervals of 24h with RNA isolated 2h after the last dose. RNA was also isolated from non......BACKGROUND AND PURPOSE: Breast cancer patients show a large variation in normal tissue reactions after ionizing radiation (IR) therapy. One of the most common long-term adverse effects of ionizing radiotherapy is radiation-induced fibrosis (RIF), and several attempts have been made over the last...... years to develop predictive assays for RIF. Our aim was to identify basal and radiation-induced transcriptional profiles in fibroblasts from breast cancer patients that might be related to the individual risk of RIF in these patients. MATERIALS AND METHODS: Fibroblast cell lines from 31 individuals...

  9. Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jon M Carthy

    Full Text Available Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

  10. Fibroblast nemosis induces angiogenic responses of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Enzerink, Anna, E-mail: anna.enzerink@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland); Rantanen, Ville, E-mail: ville.rantanen@helsinki.fi [Computational Systems Biology Laboratory, Institute of Biomedicine and Genome-Scale Biology Research Program, University of Helsinki, P.O. BOX 63, 00014 Helsinki (Finland); Vaheri, Antti, E-mail: antti.vaheri@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland)

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  11. Heparin-Induced Thrombocytopenia Associated with Massive Intracardiac Thrombosis: A Case Report

    Directory of Open Access Journals (Sweden)

    Atheer Ahmed

    2012-01-01

    Full Text Available A 60-years old patient was admitted to a community hospital with septic arthritis. He was treated with antibiotics and subcutaneous unfractionated heparin (UH was used for venous thromboprophylaxis. After three days, he developed leg deep venous thrombosis and was treated with IV heparin. One day later, the patient developed pulmonary emboli, which was found using ventilation/perfusion scan. He was transferred to the University Hospital for further management. Upon arrival, antibiotic and intravenous UH were continued. Trans-Esophageal Echocardiogram showed a thrombus in the right atrium, a small portion of which extended to the left atrium through a patent foramen ovale. Another large thrombus was noted in the right ventricle, which extended to the pulmonary artery. Review of the patient’s medical records revealed a halving of his platelet count three days following the heparin administration. Therefore, HIT seemed very likely. Intravenous UH was stopped and an emergency thrombectomy was performed. ELISA testing of HIT antibodies came negative. This made HIT diagnosis unlikely and the patient received dalteparin. A week later, as the platelet count declined again, HIT antibodies’ testing using ELISA and C-14 serotonin release was repeated, and both assays were positive. Argatroban was restarted and the platelet count normalized.

  12. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    Science.gov (United States)

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  13. The level of heparin-induced antibodies in correlation with the result of the flow cytometric functional assay in the patients with suspected HIT.

    Science.gov (United States)

    Maličev, Elvira; Maček Kvanka, Marjeta; Klemenc, Polona; Rožman, Primož

    2017-09-13

    Heparin can induce the formation of antibodies against a heparin complex with a platelet factor 4 (PF4), leading to platelet activation and the development of heparin-induced thrombocytopaenia (HIT). Because screening ELISA does not discriminate between platelet activating and non-activating anti-heparin/PF4 antibodies, each positive result is confirmed by an additional functional assay. We analysed 1004 sera of patients with suspected HIT. Optical density (OD) values of ELISA-positive results were correlated with the risk for a positive result with our functional flow cytometric assay. Only 10.7% were ELISA positive and 59.8% of those were positive with the functional assay. The positive functional assay was found in 23.4% of patients with OD2.0. Although our results showed that higher ELISA OD values increasethe possibility of the presence of platelet-activating anti-heparin/PF4 antibodies - , there is no need for improving ELISA cut-off value for positive result. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  14. Intravenous lipid and heparin infusion-induced elevation in free fatty acids and triglycerides modifies circulating androgen levels in women: a randomized, controlled trial.

    Science.gov (United States)

    Mai, K; Bobbert, T; Reinecke, F; Andres, J; Maser-Gluth, C; Wudy, S A; Möhlig, M; Weickert, M O; Hartmann, M F; Schulte, H M; Diederich, S; Pfeiffer, A F H; Spranger, J

    2008-10-01

    The polycystic ovarian syndrome (PCOS) is characterized by hyperandrogenism and associated with obesity and impaired glucose metabolism. Despite the high prevalence of PCOS and the considerable clinical impact, the precise interplay between metabolism and hyperandrogenemia is not entirely clear. The objective of the study was to analyze the effects of iv lipid and heparin infusion on circulating androgen levels in healthy women. This was a randomized, controlled, crossover trial. The study was conducted at an endocrinology center. Patients included 12 healthy young women during the early follicular phase of two subsequent cycles. After an overnight fast, a 20% lipid/heparin or a saline/heparin infusion was administered in random order for 330 min. A detailed characterization of androgen metabolism was performed. Elevations in free fatty acids and triglycerides, induced by lipid/heparin infusion, elevates the levels of androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), testosterone, 5alpha-dihydrotestosterone, estrone, and 17beta-estradiol. Urinary excretion of DHEA, DHEAS, 5-androstene-3beta,17beta-diol, and the sum of urinary excreted DHEA and its 16-hydroxylated downstream metabolites, 16alpha-hydroxy-DHEA and 5-androstene-3beta,16alpha,17beta-triol, were reduced. The mechanism of iv lipid and heparin infusion-induced elevation of circulating androgens described here might contribute to the development of hyperandrogenism in women with PCOS and suggests that lowering of hyperlipidemia might be a potential therapeutic target in patients with PCOS to treat hyperandrogenemia.

  15. Inhibition by medroxyprogesterone acetate of interleukin-1β-induced collagen degradation by corneal fibroblasts.

    Science.gov (United States)

    Zhou, Hongyan; Kimura, Kazuhiro; Orita, Tomoko; Nishida, Teruo; Sonoda, Koh-Hei

    2012-06-28

    To examine the effect of medroxyprogesterone 17-acetate (MPA) on interleukin-1β (IL-1β)-induced collagen degradation by corneal fibroblasts. Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without MPA. Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression or activity of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) was evaluated by immunoblot analysis or gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was examined by immunoblot analysis. Cell proliferation and viability were evaluated by measurement of bromodeoxyuridine incorporation and the release of lactate dehydrogenase, respectively. MPA inhibited IL-1β-induced collagen degradation by corneal fibroblasts in a concentration- and time-dependent manner. MMP expression and activation as well as TIMP expression in corneal fibroblasts exposed to IL-1β were also inhibited by MPA. MPA had no effect on cell proliferation or viability. MPA inhibited the IL-1β-induced phosphorylation of p38 MAPK without affecting that of the MAPKs ERK or JNK. IL-1β-induced MMP expression and activation as well as collagen degradation were also blocked by the p38 MAPK inhibitor SB203580. MPA inhibited MMP expression and thereby suppressed collagen degradation by corneal fibroblasts induced by IL-1β. Furthermore, inhibition of p38 MAPK phosphorylation by MPA may contribute to its inhibition of collagen degradation.

  16. Release of glomerular heparan-/sup 35/SO/sub 4/ proteoglycan by heparin from glomeruli of streptozocin-induced diabetic rats

    Energy Technology Data Exchange (ETDEWEB)

    Klein, D.J.; Oegema, T.R. Jr.; Brown, D.M.

    1989-01-01

    Abnormalities in the incorporation of heparan sulfate proteoglycan into the glomerular basement membrane have been implicated in the pathogenesis of various proteinuric states, including diabetes mellitus. To understand further the interactions between proteoglycans and glomerular extracellular matrices, glomeruli were isolated from normal and streptozocin-induced diabetic rats after in vivo exposure to 35S-labeled sulfate and were treated with heparin in vitro. Heparin treatment released a unique heparan sulfate proteoglycan from glomerular cell surface or extracellular matrix proteoglycan receptors. Another, smaller heparan sulfate proteoglycan was the most abundant proteoglycan released into medium and was released constitutively in medium with or without added heparin. While the two heparin-extracted proteoglycans copurified on anion-exchange and gel-filtration chromatographic columns, they were resolved by composite 0.6% agarose--1.8% polyacrylamide gel electrophoresis. Glomeruli from diabetic rats contained decreased proportions of the heparin-releasable heparan sulfate proteoglycan and more constitutively released heparan sulfate proteoglycan. The apparent molecular weight and intrinsic charge of the heparin-released proteoglycan mixture and the apparent molecular weight and sulfation pattern of their 35S-labeled glycosaminoglycan chains after nitrous acid deaminative cleavage were similar in the two groups. A brief trypsin digestion of heparin-treated glomeruli released proportionately less integral membrane and extracellular matrix 35S-labeled proteoglycans and 35S-labeled glycopeptides from diabetic glomeruli than form control glomeruli. Elution of these 35S-labeled macromolecules from anion-exchange columns and migration in agarose-polyacrylamide gels were similar in the two groups.

  17. Budd-Chiari syndrome and heparin-induced thrombocytopenia in polycythemia vera: Successful treatment with repeated TIPS and interferon alpha

    Directory of Open Access Journals (Sweden)

    Akoum Riad

    2009-01-01

    Full Text Available Polycythemia vera (PV is a common cause of Budd-Chiari syndrome (BCS and portal vein thrombosis (PVT. The postpartum period is a precipitating cofactor. An additional heparin-induced thrombocytopenia/thrombosis (HIT/T leads to a life-threatening condition in which transjugular intrahepatic portosystemic shunting (TIPS seems to be the only life-saving procedure. We describe the case of a subacute BCS and PVT in the late postpartum period. The diagnosis was established using CT scan, MRI, and Doppler ultrasonography of abdominal vessels and the laboratory findings were compatible with PV. After a successful creation of TIPS, a HIT/T worsened the hemorrhagic and thrombotic picture. TIPS procedure was successfully repeated and heparin was replaced with Fondaparinux and then vitamin K antagonist. The treatment with interferon alpha-2A, started after the normalization of liver functions, resulted in a complete remission within 6 months. The JAK2 V617F mutation clone remained undetectable after 2 years′ follow-up.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jian Bai

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

  20. Fibroblast growth factor 10 protects neuron against oxygen–glucose deprivation injury through inducing heme oxygenase-1

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yong-Hua; Yang, Li-Ye; Chen, Wei; Li, Ying-Ke, E-mail: liyingke6f@126.com; Yuan, Hong-Bin, E-mail: yuanhongbin6f@126.com

    2015-01-02

    Highlights: • FGF10 attenuates OGD induced injury in cortical neuron. • FGF10 reduces OGD triggered ROS level in cortical neuron. • FGF10 induces HO-1 expression upon OGD stimuli in cortical neuron. • Knockdown of HO-1 impairs the neuroprotection of FGF10 in OGD model. - Abstract: Fibroblast growth factors (FGFs) are a family of structurally related heparin-binding proteins with diverse biological functions. FGFs participate in mitogenesis, angiogenesis, cell proliferation, development, differentiation and cell migration. Here, we investigated the potential effect of FGF10, a member of FGFs, on neuron survival in oxygen–glucose deprivation (OGD) model. In primary cultured mouse cortical neurons upon OGD, FGF10 treatment (100 and 1000 ng/ml) attenuated the decrease of cell viability and rescued the LDH release. Tuj-1 immunocytochemistry assay showed that FGF10 promoted neuronal survival. Apoptosis assay with Annexin V + PI by flow cytometry demonstrated that FGF10 treatment reduced apoptotic cell proportion. Moreover, immunoblotting showed that FGF10 alleviated the cleaved caspase-3 upregulation caused by OGD. FGF10 treatment also depressed the OGD-induced increase of caspase-3, -8 and -9 activities. At last, we found FGF10 triggered heme oxygenase-1 (HO-1) protein expression rather than hypoxia-inducible factor-1 (HIF-1), AMP-activated protein kinase (AMPK) signaling and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling. Knockdown of HO-1 by siRNA partly abolished the neuroprotection of FGF10 in OGD model. In summary, our observations provide the first evidence for the neuroprotective function of FGF10 against ischemic neuronal injury and suggest that FGF10 may be a promising agent for treatment of ischemic stroke.

  1. Successful Use of Alternative Anticoagulants in the Management of Heparin-induced Thrombocytopenia with Thrombotic Complications: Report of 5 cases and review of literature.

    LENUS (Irish Health Repository)

    Alkindi, Salam

    2011-08-01

    Heparin is one of the most frequently used anticoagulants. It is easy to use, but can be associated with life-threatening side effects. One of these is heparin-induced thrombocytopenia syndrome (HITS), which develops in about 3-5% of patients exposed to heparin and is associated with thrombosis in 1% of cases. We report here the successful treatment of five patients with HITS who were treated with alternative anticoagulants namely danaparoid or hirudin. The median time between their exposure to heparin and onset of symptoms and or signs was 10.2 days (range 7-14 days). Platelet counts decreased to a mean of 38.4 x 10(9) \\/l (12-82 x 10(9)\\/l). All five patients had evidence of thrombosis; four patients had clinical and radiological evidence of pulmonary emboli, one patient had confirmed deep vein thrombosis (DVT) and one patient had extensive skin necrosis of the thighs and abdomen. Platelet aggregation test were positive in two patients, inconclusive in one patient and negative in two patients. Two patients were anticoagulated with danaparoid and three with hirudin until their platelet counts returned to normal between 4 and 14 days (average 6 days) following the recognition of the syndrome. Our patients had significant morbidity, but no mortality. Immediate withdrawal of heparin is of paramount importance and introduction of alternative anticoagulant is necessary in the presence of thrombosis.

  2. Heparin-induced thrombocytopenia (HIT) causing portosplenic, superior mesenteric, and splenic vein thrombosis resulting in splenic rupture and pulmonary emboli formation.

    Science.gov (United States)

    Lammering, Jeanne C; Wang, David S; Shin, Lewis K

    2012-01-01

    Heparin-induced thrombocytopenia (HIT) is a life-threatening complication of heparin administration. Of the few reported cases of HIT-associated intra-abdominal thrombosis, none to our knowledge provide multidetector-row computed tomography (MDCT) imaging findings or emphasize its utility in diagnosis. We describe a case of HIT with MDCT images demonstrating extensive intra-abdominal thrombosis and end-organ complications including splenic rupture and pulmonary emboli. This case emphasizes the potential role of MDCT in the rapid detection of HIT-related thromboembolic complications in patients with nonspecific abdominal pain.

  3. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

  4. HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    Science.gov (United States)

    We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cel...

  5. Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts.

    Science.gov (United States)

    Lu, Jiamei; Zhu, Yanting; Feng, Wei; Pan, Yilin; Li, Shaojun; Han, Dong; Liu, Lu; Xie, Xinming; Wang, Guizuo; Li, Manxiang

    2014-09-01

    Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13- induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.

  6. Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts

    Indian Academy of Sciences (India)

    Jiamei Lu; Yanting Zhu; Wei Feng; Yilin Pan; Shaojun Li; Dong Han; Lu Liu; Xinming Xie; Guizuo Wang; Manxiang Li

    2014-09-01

    Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13-induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.

  7. Effects of icariin on cytokine-induced ankylosing spondylitis with fibroblastic osteogenesis and its molecular mechanism.

    Science.gov (United States)

    Jia, Chunrong; Liu, Hongxiao; Li, Min; Wu, Zhikui; Feng, Xinghua

    2014-01-01

    The aim of this study is to explore the effects of icariin on cytokine induced ankylosing spondylitis fibroblast osteogenesis type expression and its molecular mechanism. The normal fibroblasts were collected as normal control group, and the fibroblasts of hip joint capsule of AS patients were collected, which were respectively added in fetal bovine serum (group AS), fetal bovine serum and cytokines (BMP-2+TGF-beta 1) (group AS), and cell factor solution (icariin group), and observed of the osteogenic expression of fibroblast, to evaluate the impact of Icariin on it. The ALP activity, the content of collagen, osteocalcin content and cbfa1mRNA and OCmRNA of fibroblast of AS group increased compared to the normal control group and AS control group (P < 0.01), indicating that icariin can significantly inhibit the above changes (P < 0.01). Icariin can inhibit fibroblast further osteogenic differentiation through inhibiting the effect of cytokines on the fibroblast osteogenesis type markers and osteogenic gene expression and osteogenic differentiation.

  8. 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

  9. Lipoprotein glomerulopathy treated with LDL-apheresis (Heparin-induced Extracorporeal Lipoprotein Precipitation system: a case report

    Directory of Open Access Journals (Sweden)

    Rivasi Paolo

    2009-12-01

    Full Text Available Abstract Introduction Lipoprotein glomerulopathy is a glomerulonephritis which was described for the first time by Saito in 1989 and is currently acknowledged as a separate nosological entity. It is histologically characterized by a marked dilatation of the glomerular capillaries and the presence of lipoprotein thrombi in the glomerular lumens. The dyslipidemic profile is similar to that of type III dyslipoproteinemia with Apolipoprotein E values that are often high; proteinuria and renal dysfunction are present. Proteinuria often does not respond to steroid and cytostatic treatments. The phenotypic expression of lipoprotein glomerulopathy is most probably correlated to a genetic alteration of the lipoprotein metabolism (mutation of the Apolipoprotein E coding gene. In literature, lipoprotein glomerulopathies have mainly been reported in Japanese and Chinese subjects, except for three cases in the Caucasian race, reported in France and the USA. Case presentation We describe the case of a 60-year-old female, Caucasian patient suffering from lipoprotein glomerulopathy, carrier of a new mutation on the Apolipoprotein E gene (Apolipoprotein EMODENA, and treated successfully with low density lipoprotein-apheresis with the Heparin induced extracorporeal lipoprotein precipitation system. After a first phase of therapeutic protocol with statins, the patient was admitted for nephrotic syndrome, renal failure and hypertension. Since conventional treatment alone was not able to control dyslipidemia, aphaeretic treatment with heparin-induced Extracorporeal Lipoprotein Precipitation - apheresis (HELP-apheresis was started to maintain angiotensin converting enzyme inhibitor therapy for the treatment of hypertension. Treatment with HELP-apheresis led to a complete remission of the proteinuria in a very short time (four months, as well as control of hypercholesterolemia and renal function recovery. Conclusion According to this case of lipoprotein glomerulopathy

  10. IL-13 induces YY1 through the AKT pathway in lung fibroblasts.

    Science.gov (United States)

    Guo, Jia; Yao, Hongwei; Lin, Xin; Xu, Haodong; Dean, David; Zhu, Zhou; Liu, Gang; Sime, Patricia

    2015-01-01

    A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13's downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 in vitro. In vivo YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation.

  11. A genome-wide association study of heparin-induced thrombocytopenia using an electronic medical record

    DEFF Research Database (Denmark)

    Karnes, Jason H; Cronin, Robert M; Rollin, Jerome

    2015-01-01

    . Here, we performed a genome-wide association study (GWAS) and candidate gene study using HIT cases and controls identified using electronic medical records (EMRs) coupled to a DNA biobank and attempted to replicate GWAS associations in an independent cohort. We subsequently investigated influences......-heparin treated patients (OR 3.09; 1.14-8.13; p=0.02). In the candidate gene study, SNPs at HLA-DRA were nominally associated with HIT (OR 0.25; 0.15-0.44; p=2.06×10(-6)). Further study of TDAG8 and HLA-DRA SNPs is warranted to assess their influence on the risk of developing HIT....

  12. Centrifugal force induces human ligamentum flavum fibroblasts inflammation through activation of JNK and p38 pathways.

    Science.gov (United States)

    Chao, Yuan-Hung; Tsuang, Yang-Hwei; Sun, Jui-Sheng; Sun, Man-Ger; Chen, Ming-Hong

    2012-01-01

    Inflammation has been proposed to be an important causative factor in ligamentum flavum hypertrophy. However, the mechanisms of mechanical load on inflammation of ligamentum flavum remain unclear. In this study, we used an in vitro model of human ligamentum flavum fibroblasts subjected to centrifugal force to elucidate the effects of mechanical load on cultured human ligamentum flavum fibroblasts; we further studied its molecular and biochemical mechanisms. Human ligamentum flavum fibroblasts were obtained from six patients undergoing lumbar spine surgery. Monolayer cultures of human ligamentum flavum fibroblasts were subjected to different magnitudes of centrifugal forces. Cell viability, cell death, biochemical response, and molecular response to centrifugal forces were analyzed. It was found that centrifugal stress significantly suppressed cell viability without inducing cell death. Centrifugal force at 67.1 g/cm(2) for 60 min significantly increases the production of prostaglandin E2 and nitric oxide as well as gene expression of proinflammatory cytokines, including interleukin (IL)-1α, IL-1β and IL-6, showed that centrifugal force-dependent induction of cyclooxygense-2 and inducible NO synthase required JNK and p38 mitogen-activated protein kinase, but not ERK 1/2 activities. This study suggested that centrifugal force does induce inflammatory responses in human ligamentum flavum fibroblasts. The activation of both JNK and p38 mitogen-activated protein kinase mechanotransduction cascades is a crucial intracellular mechanism that mediates cyclooxygense-2/prostaglandin E2 and inducible NO synthase/nitric oxide production.

  13. Matrine inhibits proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB

    Institute of Scientific and Technical Information of China (English)

    WU Yan-an; GAO Chun-fang; WANG Hao; HUANG Chao; KONG Xian-tao

    2001-01-01

    To study the effect of matrine on proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB (PDGF-BB). Methods: Mouse skin fibroblasts were obtained from newborn ⅠCR mice and propagated in vitro. Proliferation of cell was analyzed by mitochondrial reduction of tetrazolium salt MTT and actual cell count. Results: Matrine (50 to 500 μg/ml) caused dose-dependent reduction of serum-stimulated cell growth. Growth inhibition was totally reversed after removal of the drug. Matrine also inhibited PDGF-BB induced cell growth dose-dependently. Conclusion: Matrine exhibits potent anti-proliferation effect on mouse skin fibroblast. This effect appears to be mediated by decrease of PDGF-induced growth. These results suggest that matrine might have preventive and therapeutic implication in skin fibrosis.

  14. Fondaparinux for intra and perioperative anticoagulation in patients with heparin-induced thrombocytopenia candidates for peripheral vascular surgery: Report of 4 cases.

    Science.gov (United States)

    Illuminati, Giulio; Calio', Francesco G; Pizzardi, Giulia; Amatucci, Chiara; Masci, Federica; Palumbo, Piergaspare

    2016-01-01

    Intra and perioperative anticoagulation in patients with heparin induced thrombocytopenia (HIT), candidates for peripheral vascular surgery remains a challenge, as the best alternative to heparin has not yet been established. We evaluated the off-label use of fondaparinux in four patients with HIT, undergoing peripheral vascular surgery procedures. Four patients of whom 3 men of a mean age of 66 years, with proven heparin induced thrombocytopenia (HIT) underwent two axillo-femoral bypasses, one femoro-popliteal bypass and one resection of a splenic artery aneurysm under fondaparinux. No intra or perioperative bleeding or thrombosis of new onset was observed. In the absence of a valid alternative to heparin for intra and perioperative anticoagulation in HIT, several other anticoagulants can be used in an off-label setting. However, no general consensus exist on which should be the one of choice. In this small series fondaparinux appeared to be both safe and effective. These preliminary results seem to justify the off-label use of fondaparinux for intra and perioperative anticoagulation in patients with HIT, candidates for peripheral vascular surgery interventions. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts.

    Science.gov (United States)

    Liu, Haijun; Dai, Xiaoniu; Cheng, Yusi; Fang, Shencun; Zhang, Yingming; Wang, Xingang; Zhang, Wei; Liao, Hong; Yao, Honghong; Chao, Jie

    2016-01-15

    Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

  16. Midkine, heparin-binding growth factor, blocks kainic acid-induced seizure and neuronal cell death in mouse hippocampus

    Directory of Open Access Journals (Sweden)

    Lim In J

    2010-03-01

    Full Text Available Abstract Background Midkine (MK, a member of the heparin-binding growth factor family, which includes MK and pleiotrophin, is known to possess neurotrophic and neuroprotective properties in the central nervous system. Previous studies have shown that MK is an effective neuroprotective agent in reducing retinal degeneration caused by excessive light and decreasing hippocampal neuronal death in ischemic gerbil brain. The present study was undertaken to investigate whether MK acts as an anticonvulsant in kainic acid (KA-induced seizure in mouse and blocks KA-mediated neuronal cell death in hippocampus. Results Increased expression of MK was found in hippocampus of mouse following seizures induced by intracerebroventricular injection of KA, and MK expression was found in glial fibrillary acidic protein (GFAP-positive astrocytes. Concurrent injection of MK and KA attenuated KA-induced seizure activity and cell death of hippocampal neurons including pyramidal cells and glutamic acid decarboxylase 67 (GAD67-positive GABAergic interneurons in the CA3 and hilar area. Conclusion The results of the present study indicate that MK functions as an anticonvulsant and neuroprotective agent in hippocampus during KA-induced seizures.

  17. Heparin-induced thrombocytopenia: reducing misdiagnosis via collaboration between an inpatient anticoagulation pharmacy service and hospital reference laboratory.

    Science.gov (United States)

    Burnett, Allison E; Bowles, Harmony; Borrego, Matthew E; Montoya, Tiffany N; Garcia, David A; Mahan, Charles

    2016-11-01

    Misdiagnosis of heparin-induced thrombocytopenia (HIT) is common and exposes patients to high-risk therapies and potentially serious adverse events. The primary objective of this study was to evaluate the impact of collaboration between an inpatient pharmacy-driven anticoagulation management service (AMS) and hospital reference laboratory to reduce inappropriate HIT antibody testing via pharmacist intervention and use of the 4T pre-test probability score. Secondary objectives included clinical outcomes and cost-savings realized through reduced laboratory testing and decreased unnecessary treatment of HIT. This was a single center, pre-post, observational study. The hospital reference laboratory contacted the AMS when they received a blood sample for an enzyme-linked immunosorbent HIT antibody (HIT Ab). Trained pharmacists prospectively scored each HIT Ab ordered by using the 4T score with subsequent communication to physicians recommending for or against processing and reporting of lab results. Utilizing retrospective chart review and a database for all patients with a HIT Ab ordered during the study period, we compared the incidence of HIT Ab testing before and after implementation of the pharmacy-driven 4T score intervention. Our intervention significantly reduced the number of inappropriate HIT Ab tests processed (176 vs. 63, p < 0.0001), with no increase in thrombotic or hemorrhagic events. Overall incidence of suspected and confirmed HIT was <3 and <0.005 %, respectively. Overall cost savings were $75,754 (US) or 62 % per patient exposed to heparin between the pre and post intervention groups. Collaboration between inpatient pharmacy AMS and hospital reference laboratories can result in reduction of misdiagnosis of HIT and significant cost savings with similar safety.

  18. Accuracy of a prediction model for heparin-induced thrombocytopenia (HIT): an analysis based on individual patient data.

    Science.gov (United States)

    Junqueira, Daniela Rezende Garcia; Viana, Thércia Guedes; Carvalho, Maria das Graças; Perini, Edson

    2011-08-17

    Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction associated with thrombosis, and its paradoxical nature is a challenging issue for the diagnosis. The '4Ts' scoring system represents a simple and efficient way to improve clinical diagnoses of the syndrome. This system classifies patients as having high, intermediate, and low clinical probability for HIT. However, uncertainty remains concerning its clinical meaning, thus weakening the diagnostic value of this screening instrument. We analyzed the diagnostic test accuracy based on individual patient data extracted from published primary scientific studies. This study focused on 186 cases of treatment with heparin, which later evolved into a clinical suspicion of HIT. Upon choosing the most appropriate reference laboratory, the accuracy of the 4Ts was analyzed using the receiver operator characteristic curve analysis. Half of the positive cases (57.1%) were classified as having a high score, while 25.5% of the negative cases were classified as a having low score for HIT. Slightly more than half of all patients (53.2%) were classified as having an intermediate score. As such, the pre-test instrument would most likely fail to distinguish between diseased and nondiseased patients in a relevant number of cases. The calculated accuracy of the summary indicates that the 4Ts can be considered a good, but not a defining, test. Further studies are warranted regarding clinical score systems, either alone or in combination with laboratory tests, in an attempt to improve the early diagnosis of this adverse drug reaction and to provide better care for at-risk patients. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. The role of interleukin-8 produced by tumor induced fibroblasts in the development of cutaneous melanoma

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To determine the role of interleukin-8(IL-8)produced by tumor induced fibroblasts in the development of cutaneous melanoma.Methods B16 melanoma cells induced L929 fibroblasts phenotype was transdifferentiated to myofibroblasts(MF)by co-culture in vitro.MF was monitored by morphology and immunophenotype for a-SMA.The level of IL-8 was detected by ELISA.The effect on B16 cell proliferation rate was estimated using MTT method in vitro.Melanoma implanting model was constructed in C57 mice.Results L929...

  20. Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Wei ZHAO; Zhong Xiang LIN; Zhi Qian ZHANG

    2004-01-01

    To examine the role of gap junctions in cell senescence,the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore,cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis. p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin (10 mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis,elevation of p53 expression,loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.

  1. Differential effects of planktonic and biofilm MRSA on human fibroblasts.

    Science.gov (United States)

    Kirker, Kelly R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

  2. 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.

  3. Comparison of the therapeutic effects of sildenafil citrate, heparin and neuropeptides in a rat model of acetic acid-induced gastric ulcer.

    Science.gov (United States)

    Kalayci, Mehmet; Kocdor, Mehmet Ali; Kuloglu, Tuncay; Sahin, İbrahim; Sarac, Mehmet; Aksoy, Aziz; Yardim, Meltem; Dalkilic, Semih; Gursu, Onur; Aydin, Suna; Akkoc, Ramazan Fazil; Ugras, Meltem; Artas, Gokhan; Ozercan, İbrahim Hanifi; Ugur, Kader; Aydin, Suleyman

    2017-10-01

    The purpose of our investigative work has been to determine whether there can be therapeutic roles in the administration of sildenafil citrate, heparin and several neuropeptides on an animal model where gastric ulcers were induced with acetic acid, and to compare their efficacy. The animals were divided into 13 groups, with 4 animals in each. Gastric ulcers was induced in the animals of 12 groups with one untreated group being left as the control (Group I - control; given normal saline (NS)). The other groups were: Group II (ulcer+NS); Group III (5mg/kg sildenafil citrate, low dose); Group IV (10mg/kg sildenafil citrate, high dose); Group V (0.6mg/kg heparin, low dose); Group VI (6mg/kg heparin, high dose); Group VII (20nmol/kg des-acyl ghrelin); Group VIII (40nmol/kg des-acyl ghrelin); Group IX (4nmol/kg acyl ghrelin); Group X (8nmol/kg acly ghrelin); Group XI (20pmol/kg Nesfatin-1); Group XII (15nmol/kg Obestatin) and Group XIII (5nmol/kg Neuropeptide Y). Gastric neuropeptide expression was measured using an immunohistochemical method, and the amount in circulation was detected using ELISA. To compare with no treatment, the controls and other treatment groups, we recorded loss of the surface epithelium of the stomach, erosion, bleeding and inflammatory cell infiltration in the upper halves of the gastric glands. The muscularis and the layers beneath it were, however, apparently normal. The gastric mucosa healed with little or no inflammation when sildenafil citrate, low dose heparin, ghrelin, NUCB2/Nesfatin-1, obestatin, Neuropeptide Y were administered. Overall the data indicate that low dose heparin, and especially sildenafil citrate and neuropeptides, can be used clinically as an alternative approach in the treatment of the gastric ulcer. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

    Directory of Open Access Journals (Sweden)

    Yinglong Sa

    2015-04-01

    Full Text Available Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1 has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1, Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

  5. Heparin-induced thrombocytopenia and endovascular procedures: report of two cases Trombocitopenia induzida por heparina e tratamento endovascular: relato de dois casos

    Directory of Open Access Journals (Sweden)

    Christiane Campolina Furquim Werneck

    2009-09-01

    Full Text Available Heparin-induced thrombocytopenia (HIT is a transient disorder caused by platelet-activating antibodies against platelet factor 4 (PF4-heparin complexes. Clinically it translates into arterial or venous thrombosis and carries high morbidity and mortality. The use of large doses of heparin during endovascular repair of abdominal aortic aneurysm could increase the incidence of HIT. We report two cases associating the use of heparin during endovascular repair of abdominal aortic aneurysm with the development of HIT.Trombocitopenia induzida por heparina (TIH é um distúrbio transitório causado pela ativação de anticorpos anti-plaquetários contra o fator plaquetário 4 (FP4 combinado com a molécula de heparina, formando complexos que clinicamente se traduzem em tromboses arteriais ou venosas com alta morbimortalidade. O uso de altas doses de heparina durante procedimentos endovasculares para o tratamento de aneurisma de aorta abdominal pode aumentar a incidência de TIH. Relatamos dois casos em que o uso de heparina durante tratamento endovascular de aneurisma de aorta abdominal estava associado ao aparecimento de TIH.

  6. Deep vein thrombosis, ecythyma gangrenosum and heparin-induced thrombocytopenia occurring in a man with a heterozygous Factor V Leiden mutation

    Directory of Open Access Journals (Sweden)

    Mariya Apostolova

    2012-11-01

    Full Text Available Skin necrosis and limb gangrene are occasional thrombotic manifestations of anticoagulation therapy. We report a man heterozygous for the Factor V Leiden (FVL mutation, and with a history of recurrent deep venous thrombosis, who initially presented with a necrotic skin lesion of the right flank while on warfarin therapy with a therapeutic international normalized ratio. Warfarin was discontinued and he received intravenous heparin. Thereafter he developed thrombocytopenia and pedal erythema and was diagnosed with heparin-induced thrombocytopenia (HIT. Heparin was replaced with argatroban. He ultimately underwent bilateral below-knee amputations for the thrombotic complications of the HIT. The initial necrotic lesion healed with antibiotics and wound care. Pathologic examination of multiple biopsy specimens revealed two separate lesions. One was necrotic tissue infiltrated with methicillin resistant Staphylococcus aureus having features of ecthyma gangrenosum. The second showed thrombotic changes consistent with HIT. The case illustrates the differential diagnosis of skin necrosis and limb gangrene in patients on warfarin and heparin, and also the clinical complexities that can occur in a FVL heterozygote.

  7. Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide

    Institute of Scientific and Technical Information of China (English)

    Bing FENG; Lai-ji MA; Jin-jing YAO; Yun FANG; Yan-ai MEI; Shao-min WEI

    2013-01-01

    Oat contains different components that possess antioxidant properties;no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level.Therefore,the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide(H2O2).Kjeldahl determination,phenol-sulfuric acid method,and high-performance liquid chromatography(HPLC)analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%,of which 97.43% are peptides with a molecular range from 438.56 to 1301.01 Da.Assays for 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling(TUNEL)assay for apoptosis showed that administration of H2O2 in human dermal fibroblasts caused cell damage and apoptosis.Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H2O2,but application oat peptides with H2O2 at same time did not.Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H2O2-induced decrease of superoxide dismutase(SOD)and the inhibition of malondialdehyde(MDA).The results demonstrate that oat peptides possess antioxidant activity and are effective against H2O2-induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level.Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury.

  8. Advanced glycation end products (AGEs) and their receptor (RAGE) induce apoptosis of periodontal ligament fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li, D.X.; Deng, T.Z.; Lv, J.; Ke, J. [Department of Stomatology, Air Force General Hospital PLA, Haidian District, Beijing (China)

    2014-09-19

    Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01) and increased apoptosis (11.31±1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

  9. Protective effect of kaempferol on LPS plus ATP-induced inflammatory response in cardiac fibroblasts.

    Science.gov (United States)

    Tang, Xi-Lan; Liu, Jian-Xun; Dong, Wei; Li, Peng; Li, Lei; Hou, Jin-Cai; Zheng, Yong-Qiu; Lin, Cheng-Ren; Ren, Jun-Guo

    2015-02-01

    Inflammatory response is an important mechanism in the pathogenesis of cardiovascular diseases. Cardiac fibroblasts play a crucial role in cardiac inflammation and might become a potential therapeutic target in cardiovascular diseases. Kaempferol, a flavonoid commonly existing in many edible fruits, vegetables, and Chinese herbs, is well known to possess anti-inflammatory property and thus has a therapeutic potential for the treatment of inflammatory diseases. To date, the effect of kaempferol on cardiac fibroblasts inflammation is unknown. In this study, we investigated the anti-inflammatory effect of kaempferol on lipopolysaccharide (LPS) plus ATP-induced cardiac fibroblasts and explored the underlying mechanisms. Our results showed that kaempferol at concentrations of 12.5 and 25 μg/mL significantly suppressed the release of TNF-α, IL-1β, IL-6, and IL-18 and inhibited activation of NF-κB and Akt in LPS plus ATP-induced cardiac fibroblasts. These findings suggest that kaempferol attenuates cardiac fibroblast inflammation through suppression of activation of NF-κB and Akt.

  10. Advanced glycation end products (AGEs and their receptor (RAGE induce apoptosis of periodontal ligament fibroblasts

    Directory of Open Access Journals (Sweden)

    D.X. Li

    2014-12-01

    Full Text Available Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL fibroblasts induced by advanced glycation end products (AGEs and their receptor (RAGE. We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA, bovine serum albumin (BSA alone, or given no treatment (control. Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01 and increased apoptosis (11.31±1.73%, P<0.05. Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

  11. Advanced glycation end products (AGEs) and their receptor (RAGE) induce apoptosis of periodontal ligament fibroblasts.

    Science.gov (United States)

    Li, D X; Deng, T Z; Lv, J; Ke, J

    2014-12-01

    Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80 ± 5.50%, PAGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

  12. S100A4 amplifies TGF-β-induced fibroblast activation in systemic sclerosis

    DEFF Research Database (Denmark)

    Tomcik, Michal; Palumbo-Zerr, Katrin; Zerr, Pawel

    2015-01-01

    the expression of S100A4 to stimulate the release of collagen in SSc fibroblasts and induce fibrosis. Since S100A4 is essentially required for the pro-fibrotic effects of TGF-β and neutralising antibodies against S100A4 are currently evaluated, S100A4 might be a candidate for novel antifibrotic therapies....

  13. Sensitivity of fibroblast growth factor 23 measurements in tumor-induced osteomalacia

    DEFF Research Database (Denmark)

    Imel, Erik A; Peacock, Munro; Pitukcheewanont, Pisit

    2006-01-01

    Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome of hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum 1,25-dihydryxyvitamin-D concentration, myopathy, and osteomalacia. Fibroblast growth factor 23 (FGF23) is a phosphaturic protein overexpressed in tumors...

  14. Sensitivity of fibroblast growth factor 23 measurements in tumor-induced osteomalacia.

    NARCIS (Netherlands)

    Imel, E.A.; Peacock, M.; Pitukcheewanont, P.; Heller, H.J.; Ward, L.M.; Shulman, D.; Kassem, M.; Rackoff, P.; Zimering, M.; Dalkin, A.; Drobny, E.; Colussi, G.; Shaker, J.L.; Hoogendoorn, E.H.; Hui, S.L.; Econs, M.J.

    2006-01-01

    CONTEXT: Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome of hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum 1,25-dihydryxyvitamin-D concentration, myopathy, and osteomalacia. Fibroblast growth factor 23 (FGF23) is a phosphaturic protein overexpressed in tu

  15. Utilization of 4T score to determine the pretest probability of heparin-induced thrombocytopenia in a community hospital in upstate New York

    Directory of Open Access Journals (Sweden)

    Yazan Samhouri

    2016-09-01

    Full Text Available Background: Thrombocytopenia is common in hospitalized patients. Heparin-induced thrombocytopenia (HIT is a life-threatening condition which can lead to extensive thrombosis. Diagnosis of HIT relies on clinical suspicion determined by 4T score and immunoassays through testing for anti-PF4/heparin antibodies. Clinical practice guidelines published by the American Society of Hematology in 2013 recommended use of the 4T score before ordering the immunoassays as a measure of pretest probability. The purpose of this study was to evaluate the utilization of 4T score before ordering anti-PF4/heparin antibodies at Unity Hospital. Methods: We did a retrospective chart review for patients who are 18 years or older, admitted to Unity Hospital between July 1, 2013, and December 31, 2014, and had anti-PF4/heparin antibodies ordered. Subjects who had prior history of HIT or had end-stage renal disease on hemodialysis were excluded. After calculating 4T score retrospectively, we calculated the proportion of patients who had 4T score documented prior to ELISA testing and proportion of ELISA tests, which were not indicated due to a 4T score less than or equal to 3 using Minitab 16. Results: Review of 123 patients, with an average age of 69.4 years, showed that testing was indicated in 18 patients. Six subjects had positive results, and testing was indicated in all of them. 4T score was documented in three patients. This quality improvement study showed that 4T score documentation rate at Unity Hospital is 2.4%. Anti-PF4/heparin antibody testing was indicated in 14.6%. This test is being overused in thrombocytopenia work up at Unity Hospital, costing $9,345. The topic was reviewed for residents. A prompt and calculator for 4T score were added to electronic medical records before ordering the test as a step to improve high value care.

  16. Alpha-Tocopherol Counteracts the Cytotoxicity Induced by Ochratoxin A in Primary Porcine Fibroblasts

    DEFF Research Database (Denmark)

    Fusi, Elenora; Rebucci, Raffaella; Pecorini, Chiara

    2010-01-01

    The aims of the current study were to determine the half-lethal concentration of ochratoxin A (OTA) as well as the levels of lactate dehydrogenase release and DNA fragmentation induced by OTA in primary porcine fibroblasts, and to examine the role of α-tocopherol in counteracting its toxicity....... Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P lactate dehydrogenase release and DNA damage in both fibroblast cultures. These findings indicate that α...

  17. Morphological change of skin fibroblasts induced by UV Irradiation is involved in photoaging.

    Science.gov (United States)

    Yamaba, Hiroyuki; Haba, Manami; Kunita, Mayumi; Sakaida, Tsutomu; Tanaka, Hiroshi; Yashiro, Youichi; Nakata, Satoru

    2016-08-01

    Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line ɑ1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.

  18. Heparin Binding Epidermal Growth Factor-Like Growth Factor Heals Chronic Tympanic Membrane Perforations With Advantage Over Fibroblast Growth Factor 2 and Epidermal Growth Factor in an Animal Model.

    Science.gov (United States)

    Santa Maria, Peter Luke; Weierich, Kendall; Kim, Sungwoo; Yang, Yunzhi Peter

    2015-08-01

    That heparin binding epidermal growth factor-like growth factor (HB-EGF) heals chronic tympanic membrane (TM) perforations at higher rates than fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) in an animal model. A nonsurgical treatment for chronic TM perforation would benefit those unable to access surgery or those unable to have surgery, as well as reducing the cost of tympanoplasty. Growth factor (GF) treatments have been reported in the literature with variable success with the lack of a suitable animal providing a major obstacle. The GFs were tested in a validated mouse model of chronic TM perforation. A bioabsorbable hydrogel polymer was used to deliver the GF at a steady concentration as it dissolved over 4 weeks. A control (polymer only, n = 18) was compared to polymer loaded with HB-EGF (5 μg/ml, n = 18), FGF2 (100 μg/ml, n = 19), and EGF (250 μg/ml, n = 19). Perforations were inspected at 4 weeks. The healing rates, as defined as 100% perforation closure, were control (5/18, 27.8%), HB-EGF (15/18, 83.3%), FGF2 (6/19, 31.6%), and EGF (3/19, 15.8%). There were no differences between FGF2 (p = 0.80) and EGF (p = 0.31) with control healing rates. HB-EGF (p = 0.000001) showed a significant difference for healing. The HB-EGF healed TMs showed layers similar to a normal TM, whereas the other groups showed a lack of epithelial migration. This study confirms the advantage of HB-EGF over two other commonly used growth factors and is a promising nonsurgical treatment of chronic TM perforations.

  19. Effect of heparin on high glucose induced proliferation and expression of matrix metalloproteinases in normal human mesangial cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qiao-ling; Arima Terukatsu; Yasumoto Yuichiro; Tsukamoto Masatoshi; Nozaki Tsuyoshi; Sogabe Atsushi; Harada Kouji; ZHANG Yi-xiang; LIN Xiao-yan; ZHANG Yang-de

    2005-01-01

    Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process.Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar matrix (ECM) components may increase, and that degradation of ECM may decrease in DN. It was reported heparin could inhibit mesangial cells proliferation in vitro. The main aim of this study is to explore whether heparin inhibits proliferation of mesangial cells grown in high glucose concentration and to measure the effect of heparin on matrix metalloproteinases (MMPs) expression in mesangial cells. Methods The medium contained either low glucose (5 mmol/L) or high glucose (25 mmol/L). The concentrations of heparin in the culture medium were 0, 25, 50,100, 200 or 400 μg/mL. A metabolic (WST-1) assay was used to measure mesangial cell proliferation and Western blot analysis was used to measure MMPs expression of mesangial cells. Results Normal human mesangial cell (NHMC) proliferation was higher in high glucose (HG) medium than in low glucose (LG) medium. They showed a 1.93 fold expansion after 72 h in high glucose in contrast to a 1.63 fold expansion in low glucose. In the presence of heparin, mesangial cells proliferation was inhibited, which was more obvious at high glucose concentrations than at low glucose concentrations. In high glucose, with heparin concentration of 50, 100, 200 and 400 μg/mL, the mesangial cells showed a 0. 61 fold, 0.52 fold, 0.52 fold and 0.41 fold reductions in cell number compared to cells grown without heparin. In low glucose, only concentrations of 200 μg/mL and 400 μg/mL showed reduction in cell number, namely 0.54 fold and 0.45 fold, when compared to cells grown without heparin. In Western blot analysis,MMP1, MMP2, MMP3 and MMP9 was expressed by mesangial cells expressed in both high and low glucose concentrations, which was more prominent in high glucose medium. Incubation of heparin further increased expression of

  20. Effects of Baicalin on Expression of Inducible Nitric Oxide Synthase in Cultured Fibroblasts Stimulated by Cytokines

    Institute of Scientific and Technical Information of China (English)

    毕新岭; 顾军; 聂本勇; 李泉; 刘辉; 米庆胜

    2004-01-01

    Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α(TNF-α), interferon-γ (IFN-γ), interleukin-8 (IL-S) in different groups. iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results: Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1. 0× 106 U/L TNF-α, 0.2× 106U/L IFN-γ, 0.2× 106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50 μg/ mi of baicalin. Conclusion: Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.

  1. Effects of Balcalin on Expression of Inducible Nitric Oxide Synthase In Cultured Fibroblasts Stimulated by Cytokines

    Institute of Scientific and Technical Information of China (English)

    毕新岭; 顾军; 聂本勇; 李泉; 刘辉; 米庆胜

    2004-01-01

    Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α((TNF-α), interferon-γ(IFN-γ), interleukin-8 (IL-8) in different groups, iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results. Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1.0×106 U/L TNF-α, 0.2×106U/L IFN-γ, 0.2×106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50μg/ ml of baicalin. Conclusion. Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.

  2. Dexamethasone inhibition of IL-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.

    Science.gov (United States)

    Lu, Ying; Fukuda, Ken; Liu, Yang; Kumagai, Naoki; Nishida, Teruo

    2004-09-01

    Corticosteroids regulate the functions of inflammatory cells. The purpose of the present study was to investigate the effect of dexamethasone on collagen degradation by corneal fibroblasts, an underlying cause of corneal ulceration. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen and in the absence or presence of IL-1beta or dexamethasone. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was evaluated by immunoblot analysis, gelatin zymography, and reverse transcription and real-time polymerase chain reaction. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was assessed by immunoblot analysis. Dexamethasone inhibited IL-1beta-induced collagen degradation by corneal fibroblasts in a dose-dependent manner. Both the synthesis and activation of MMPs and the expression of TIMPs were inhibited by dexamethasone, as was the activity of plasmin in culture supernatants. Dexamethasone also inhibited the IL-1beta-induced phosphorylation of the MAPKs extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not that of p38. Dexamethasone exerted multiple effects on the MMP-TIMP system in corneal fibroblasts and thereby inhibited IL-1beta-induced collagen degradation by these cells. Inhibition of the IL-1beta-induced activation of ERK and JNK may contribute to these effects of dexamethasone. Copyright Association for Research in Vision and Ophthalmology

  3. Hemocompatibility and oxygenation performance of polysulfone membranes grafted with polyethylene glycol and heparin by plasma-induced surface modification.

    Science.gov (United States)

    Wang, Weiping; Zheng, Zhi; Huang, Xin; Fan, Wenling; Yu, Wenkui; Zhang, Zhibing; Li, Lei; Mao, Chun

    2016-05-14

    Polyethylene glycol (PEG) and heparin (Hep) were grafted onto polysulfone (PSF) membrane by plasma-induced surface modification to prepare PSF-PEG-Hep membranes used for artificial lung. The effects of plasma treatment parameters, including power, gas type, gas flow rate, and treatment time, were investigated, and different PEG chains were bonded covalently onto the surface in the postplasma grafting process. Membrane surfaces were characterized by water contact angle, PEG grafting degree, attenuated total reflectance-Fourier transform infrared spectroscopy, ultraviolet-visible spectrophotometry, X-ray photoelectron spectroscopy, critical water permeability pressure, and scanning electron microscopy. Protein adsorption, platelet adhesion, and coagulation tests showed significant improvement in the hemocompatibility of PSF-PEG-Hep membranes compared to pristine PSF membrane. Gas exchange tests through PSF-PEG6000-Hep membrane showed that when the flow rate of porcine blood reached 5.0 L/min, the permeation fluxes of O2 and CO2 reached 192.6 and 166.9 mL/min, respectively, which were close to the gas exchange capacity of a commercial membrane oxygenator. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

  4. Antithrombin-Ⅲ without concomitant heparin improves endotoxin-induced acute lung injury rats by inhibiting the activation of mitogen-activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    SUN Hui-ming; HONG Ling-zhi; SHEN Xiao-kun; LIN Xin-qing; SONG Yong; SHI Yi

    2009-01-01

    Background Antithrombin-Ⅲ (AT-Ⅲ), the major inhibitor of thrombin in plasma, also has anti-inflammation property and might have positive effect on sepsis. The present study aimed to investigate the effects of AT-Ⅲ on inflammatory reaction and pulmonary protection in endotoxin-induced acute lung injury (ALI) rat.Methods Sixty male Sprague-Dawley rats were randomly assigned equally to normal control group, ALl group, AT-Ⅲ treatment group, AT-Ⅲ+heparin treatment group, and heparin treatment group. The pulmonary vascular permeability index (PVPI) was measured by single nuclide tracer technique. The activity of AT-Ⅲ in plasma was determined by the method of synthetic chromogenic substrata. Tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) levels in serum were determined by enzyme-linked immunosorbent assay. The expressions of lung tissue mitogen-activated protein kinases (ERK1/2, P38 and JNK MAPK) were determined by Western blotting.Results Rats had significantly improved lung histopathology in the AT-Ⅲ treatment group and heparin treatment group compared with the ALI group. The PVPI of the ALI group was 0.38±0.04, significantly higher than that of the normal control group (0.20±0.02, P <0.01), AT-Ⅲ treatment group (0.30±0.04, P <0.01) and heparin treatment group (0.28±0.04,P <0.01) respectively. There were no significant differences of PVPI in the ALl group and AT-Ⅲ+heparin treatment group.The activity of AT-Ⅲ in plasma in the ALl group was (76±8)%, significantly lower than that of the normal control group ((96±11)%, P <0.05) and AT-Ill treatment group ((105±17)%, P <0.05) respectively. The serum levels of TNF-α and IL-6 of the ALI group were (2.770±0.373) pg/L and (1.615±0.128) ng/ml respectively, significantly higher than those of the normal control group ((0.506±0.093) pg/L and (0.233±0.047) ng/ml respectively, all P <0.01), AT-Ⅲ treatment group ((1.774±0.218) μg/L and (1.140±0145) ng/ml respectively, all P <0.01) and

  5. Successful liver transplantation in a patient with splanchnic vein thrombosis and pulmonary embolism due to polycythemia vera with Jak2v617f mutation and heparin-induced thrombocytopenia.

    Science.gov (United States)

    Biagioni, Emanuela; Pedrazzi, Paola; Marietta, Marco; Di Benedetto, Fabrizio; Villa, Erica; Luppi, Mario; Girardis, Massimo

    2013-10-01

    Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin treatment resulting in a severe acquired thrombophilic condition with an associated mortality of about 10 %. We report the first case of successful urgent liver transplantation (LT) in a patient with end-stage liver disease due to a Budd-Chiari syndrome, portal vein thrombosis and pulmonary embolism due to acquired thrombophilia associated to polycythemia vera carrying JAK2V617F gene mutation and HIT in the acute phase. Lepirudin was used to provide anticoagulation in the LT perioperative period that was performed without haemorrhagic and thrombotic complications despite the donor received heparin during liver explantation.

  6. 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

  7. QUANTITATIVE STUDY ON APOPTOSIS INDUCED BY ELECTROMAGNETIC PULSES IN NIH- 3T3 FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    ZHAO Mei - lan; CAO Xiao - zhe; WANG De - wen; GU Qing- yang; LIU Jie

    2002-01-01

    Aim: To observe the apoptotic changes following exposure to EMP and to explore the possible injury mechanism in NIH - 3T3 fibroblasts. Methods: Following NIH - 3T3 cells were exposed to EMP,the proliferation and viability of NIH - 3T3 fibroblasts were estimated by hemacytometer and MTT Measurements. Apoptotic rate was measured by flow cytometer. The imnmohistochemical SP method was used to determine bcl- 2, p53. The positively stained cells were analyzed with CMIAS- Ⅱ image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. Results: The proliferation and viability of NIH- 3T3 cells were markedly inhibited and significant apoptosis was induced following exposure to EMP.EMP could increase the number of non- adherent cells, the highest ratio (33.9%) of non- adherent cells also occurred at 6h. The As70 value of MTT decreased immediately at 1 h, 6h following the cells were exposed as compared with the control (P < 0.05). The highest ratio of apoptosis was 15.07% at 6h, then decreased gradually. Down - regulation of bcl - 2 and up - regulation of p53 were induced by EMP ( P < 0.05). Conclusions: EMP could promote apoptosis of NIH - 3T3 fibroblasts. EMP could also down - regulate bcl - 2 level and up - regulate p53 level in NIH - 3T3 fibroblasts. Bcl - 2 and p53 gene may take part in the process of apoptosis.

  8. Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts.

    Science.gov (United States)

    Turner, Bradley J; Lopes, Elizabeth C; Cheema, Surindar S

    2004-04-01

    High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.

  9. Ets-1 targeted by microrna-221 regulates angiotensin II-induced renal fibroblast activation and fibrosis.

    Science.gov (United States)

    Di, Jia; Jiang, Lei; Zhou, Yang; Cao, Hongdi; Fang, Li; Wen, Ping; Li, Xiurong; Dai, Chunsun; Yang, Junwei

    2014-01-01

    Fibroblast activation is one of the most important mechanisms for Angiotensin II (Ang II) in promoting renal fibrosis. Transcription factor Ets-1 is recognized to play a key role in kidney diseases. However, the role and mechanisms of Ets-1 in Ang-II induced fibroblast activation and kidney fibrosis are not fully understood. Mice were treated with Ang II via osmotic mini-pumps or Ang II expression plasmid (pAng II). Cultured normal rat kidney interstitial fibroblast (NRK-49F) cells were incubated with Ang II. Role of Ets-1 in renal fibrosis and fibroblast activation were assessed by Western blot, Immunohistochemical staining'MTT, Boyden chamber and Immunofluorescence staining. Effects of miR-221 on Ets-1 and fibroblast activation were investigated by MTT, Boyden chamber, Western blot and Q-PCR. We found that Ets-1 was up-regulated in fibrotic kidneys. Similarly, Ang II could activate NRK-49F cells as demonstrated by up-regulated α-SMA and fibronectin(FN) expression and enhanced cell proliferation and migration. Ang II also induced Ets-1 expression in NRK-49F cells in a dose and time dependent manner. Knock-down of Ets-1 by RNA interference attenuated Ang II-induced activation of NRK-49F cells. Ets-1 was previously reported as a target of microRNA-221 (miR-221). In Ang II-induced fibrotic kidney, miR-221 was down-regulated. Similar results were observed in Ang II treated NRK-49F cells. Ectopic expression of miR-221 mimic attenuated the up-regulation of Ets-1 by Ang II in NRK-49F cells, which further prevented the activation of NRK-49F cells. However, the inhibitor of miR-221 aggravated Ang II induced Ets-1 expression and NRK-49F cells activation. Our study suggests that miR-221/Ets-1 axis takes an important role in mediating AngII induced interstitial fibroblast activation and renal fibrosis. © 2014 S. Karger AG, Basel.

  10. Adipose Tissue-Derived Stromal Cells Inhibit TGF-beta 1-Induced Differentiation of Human Dermal Fibroblasts and Keloid Scar-Derived Fibroblasts in a Paracrine Fashion

    NARCIS (Netherlands)

    Spiekman, Maroesjka; Przybyt, Ewa; Plantinga, Josee A.; Gibbs, Susan; van der Lei, Berend; Harmsen, Martin C.

    2014-01-01

    Background: Adipose tissue-derived stromal cells augment wound healing and skin regeneration. It is unknown whether and how they can also influence dermal scarring. The authors hypothesized that adipose tissue-derived stromal cells inhibit adverse differentiation of dermal fibroblasts induced by the

  11. Fibroblast growth factor-1 attenuates TGF-β1-induced lung fibrosis.

    Science.gov (United States)

    Shimbori, Chiko; Bellaye, Pierre-Simon; Xia, Jiaji; Gauldie, Jack; Ask, Kjetil; Ramos, Carlos; Becerril, Carina; Pardo, Annie; Selman, Moises; Kolb, Martin

    2016-10-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibroblast and myofibroblast proliferation, and extensive deposition of extracellular matrix (ECM). Fibroblast growth factor-1 (FGF-1) belongs to the FGF family and has been shown to inhibit fibroblast collagen production and differentiation into myofibroblasts, and revert epithelial-mesenchymal transition by inhibiting TGF-β1 signalling pathways. However, the precise role of FGF-1 in pulmonary fibrosis has not yet been elucidated. In this study, we explore the mechanisms underlying the anti-fibrogenic effect of FGF-1 in pulmonary fibrosis in vitro and in vivo by prolonged transient overexpression of FGF-1 (AdFGF-1) and TGF-β1 (AdTGF-β1) using adenoviral vectors. In vivo, FGF-1 overexpression markedly attenuated TGF-β1-induced pulmonary fibrosis in rat lungs when given both concomitantly, or delayed, by enhancing proliferation and hyperplasia of alveolar epithelial cells (AECs). AdFGF-1 also attenuated the TGF-β1 signalling pathway and induced FGFR1 expression in AECs. In vitro, AdFGF-1 prevented the increase in α-SMA and the decrease in E-cadherin induced by AdTGF-β1 in normal human lung fibroblasts, primary human pulmonary AECs, and A549 cells. Concomitantly, AdTGF-β1-induced Smad2 phosphorylation was significantly reduced by AdFGF-1 in both cell types. AdFGF-1 also attenuated the increase in TGFβR1 protein and mRNA levels in fibroblasts. In AECs, AdFGF-1 decreased TGFβR1 protein by favouring TGFβR1 degradation through the caveolin-1/proteasome pathway. Furthermore, FGFR1 expression was increased in AECs, whereas it was decreased in fibroblasts. In serum of IPF patients, FGF-1 levels were increased compared to controls. Interestingly, FGF-1 expression was restricted to areas of AEC hyperplasia, but not α-SMA-positive areas in IPF lung tissue. Our results demonstrate that FGF-1 may have preventative and therapeutic effects on TGF-β1-driven pulmonary fibrosis via inhibiting

  12. PPAR-γ inhibits IL-13-induced collagen production in mouse airway fibroblasts.

    Science.gov (United States)

    Lu, Jiamei; Liu, Lu; Zhu, Yanting; Zhang, Yonghong; Wu, Yuanyuan; Wang, Guizuo; Zhang, Dexin; Xu, Jing; Xie, Xinming; Ke, Rui; Han, Dong; Li, Shaojun; Feng, Wei; Xie, Mei; Liu, Yun; Fang, Ping; Shi, Hongyang; He, Ping; Liu, Yuan; Sun, Xiuzhen; Li, Manxiang

    2014-08-15

    Interleukin-13 (IL-13) plays an important role in extracellular matrix production of airway remodeling in asthma. Activation of PPAR-γ has been shown to inhibit the occurrence of airway fibrosis in asthma, yet it remains unknown whether the effect of PPAR-γ on suppression of airway fibrosis is associated with the inhibition of IL-13 signaling. In the present study, primary cultured airway fibroblasts were stimulated with IL-13, and JAK inhibitor, PDGF receptor blocker and MEK inhibitor were applied to investigate the involvement of these pathways in IL-13-induced collagen production. Our results demonstrate that IL-13 dose- and time-dependently induced collagen production in primary cultured mouse airway fibroblasts; this effect was blocked by inhibition of JAK/STAT6 signal pathway. IL-13 also stimulated JAK/STAT6-dependent PDGF production, elevation of PDGF in turn activated ERK1/2 MAPK and caused collagen production. Activation of PPAR-γ by rosiglitazone reduced IL-13-induced collagen expression by suppression of STAT6-driven PDGF production. Our results indicate that activation of JAK/STAT6 signal and subsequent PDGF generation and ERK1/2 MAPK activation mediate IL-13-induced collagen production in airway fibroblasts. This study suggests that activation of PPAR-γ might be a novel strategy for the treatment of asthma partially by inhibition of airway fibrosis.

  13. Cutaneous reactions to heparin therapy: when are they caused by heparin allergy?

    Directory of Open Access Journals (Sweden)

    Giuliana Zisa

    2013-03-01

    Full Text Available Introduction: Little is known about the incidence and causes of heparin-induced skin lesions. The most commonly reported causes are delayed-type hypersensitivity reactions. We describe 3 patients who were referred to our staff between March and October 2009 for suspected heparin allergies. All were scheduled to undergo major surgery (cardiovascular or orthopedic. Materials and methods: All 3 patients reported the development of itchy, erythematous rashes a few days after the subcutaneous administration of heparin (nadroparin calcium in cases 1 and 2, unspecified in case 3. Each of them underwent a diagnostic work-up for heparin allergy, which included prick and intradermal tests with commonly used heparins and patch testing with undiluted heparins and disinfectants. Results: Patch tests with disinfectants were negative in all 3 cases. In case 2, all allergological tests were negative. In cases 1 and 3, delayed positivity emerged for nadroparin calcium and at least one other heparin tested. Intravenous and/or subcutaneous provocation testing was done with an alternative heparin which produced negative results in skin tests (heparin sodium in case 1, pentasaccharide fondaparinux in case 3. In both cases the alternative drug was tolerated. After our evaluation, all 3 patients underwent surgery with no heparin-related complications. Discussion: The presenting clinical features in these 3 cases provided no information on which reactions were likely to be allergic: all 3 patients presented with similar local delayed reaction. The allergic reactions were identified only after cutaneous testing.

  14. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Zhang, YouEn; Wang, JiaNing; Li, Hua; Yuan, LiangJun; Wang, Lei; Wu, Bing; Ge, JunBo

    2015-11-01

    In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

  15. HEMA but not TEGDMA induces autophagy in human gingival fibroblasts

    Science.gov (United States)

    Teti, Gabriella; Orsini, Giovanna; Salvatore, Viviana; Focaroli, Stefano; Mazzotti, Maria C.; Ruggeri, Alessandra; Mattioli-Belmonte, Monica; Falconi, Mirella

    2015-01-01

    Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3 mmol/L of HEMA or 3 mmol/L of TEGDMA for 24, 48, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase – 3 and PARP) and autophagy (beclin – 1 and LC3B I/II) were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis. PMID:26483703

  16. HEMA but not TEGDMA Induces Autophagy in Human Gingival Fibroblasts.

    Directory of Open Access Journals (Sweden)

    gabriella eteti

    2015-10-01

    Full Text Available Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA and triethylene glycol dimethacrylate (TEGDMA to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3mmol/L of HEMA or 3mmol/L of TEGDMA for 24 h, 48h, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase – 3 and PARP and autophagy (beclin – 1 and LC3B I/II were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis.

  17. Heparin-induced amyloid fibrillation of β2 -microglobulin explained by solubility and a supersaturation-dependent conformational phase diagram.

    Science.gov (United States)

    So, Masatomo; Hata, Yasuko; Naiki, Hironobu; Goto, Yuji

    2017-05-01

    Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative and classical view of amyloid fibrillation: amyloid fibrils are crystal-like precipitates of denatured proteins formed above solubility upon breaking supersaturation. Various additives accelerate and then inhibit amyloid fibrillation in a concentration-dependent manner, suggesting that the combined effects of stabilizing and destabilizing forces affect fibrillation. Heparin, a glycosaminoglycan and anticoagulant, is an accelerator of fibrillation for various amyloidogenic proteins. By using β2 -microglobulin, a protein responsible for dialysis-related amyloidosis, we herein examined the effects of various concentrations of heparin on fibrillation at pH 2. In contrast to previous studies that focused on accelerating effects, higher concentrations of heparin inhibited fibrillation, and this was accompanied by amorphous aggregation. The two-step effects of acceleration and inhibition were similar to those observed for various salts. The results indicate that the anion effects caused by sulfate groups are one of the dominant factors influencing heparin-dependent fibrillation, although the exact structures of fibrils and amorphous aggregates might differ between those formed by simple salts and matrix-forming heparin. We propose that a conformational phase diagram, accommodating crystal-like amyloid fibrils and glass-like amorphous aggregates, is important for understanding the effects of various additives. © 2017 The Protein Society.

  18. Artocarpin-enriched (Artocarpus altilis) Heartwood Extract Provides Protection Against UVB-induced Mechanical Damage in Dermal Fibroblasts.

    Science.gov (United States)

    Tiraravesit, Narisara; Humbert, Philippe; Robin, Sophie; Tissot, Marion; Viennet, Céline; Viyoch, Jarupa

    2017-10-01

    This study aimed to evaluate the protective effect of artocarpin-enriched (Artocarpus altilis) heartwood extract on the mechanical properties of UVB-irradiated fibroblasts. Human skin fibroblasts were pretreated with 50 μg/mL(-1) extract and later irradiated with UVB (200 mJ/cm(-2) ). They were then cultured within three-dimensional of free-floating and tense collagen lattices. The pretreatment of fibroblasts with the extract prior to UVB radiation showed cells protection against UVB-induced suppression of α-SMA expression, fibroblast migration and contraction. These results reveal that the extract prevents mechanical damages induced by UVB irradiation in fibroblast-embedded collagen lattices, and therefore, has a potential as a natural photo-protectant. © 2017 The American Society of Photobiology.

  19. Schisandrin B protects against solar irradiation-induced oxidative injury in BJ human fibroblasts.

    Science.gov (United States)

    Chiu, Po Yee; Lam, Philip Y; Yan, Chung Wai; Ko, Kam Ming

    2011-06-01

    The effects of schisandrin B (Sch B) and its analogs on solar irradiation-induced oxidative injury were examined in BJ human fibroblasts. Sch B and schisandrin C (Sch C) increased cellular reduced glutathione (GSH) level and protected against solar irradiation-induced oxidative injury. The photoprotection was paralleled by decreases in the elastases-type protease activity and matrix-metalloproteinases-1 expression in solar-irradiated fibroblasts. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production. The results suggest that by virtue of its pro-oxidant action and the subsequent glutathione antioxidant response, Sch B or Sch C may offer the prospect of preventing skin photo-aging.

  20. Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

    Science.gov (United States)

    Korpela, J; Kulomaa, M; Tuohimaa, P; Vaheri, A

    1983-01-01

    Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process. Images Fig. 2. Fig. 3. PMID:6315397

  1. Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

    Science.gov (United States)

    Korpela, J; Kulomaa, M; Tuohimaa, P; Vaheri, A

    1983-01-01

    Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process.

  2. Proteome oxidative carbonylation during oxidative stress-induced premature senescence of WI-38 human fibroblasts

    DEFF Research Database (Denmark)

    Le Boulch, Marine; Ahmed, Emad K; Rogowska-Wrzesinska, Adelina

    2017-01-01

    Accumulation of oxidatively damaged proteins is a hallmark of cellular and organismal ageing, and is also a phenotypic feature shared by both replicative senescence and stress-induced premature senescence of human fibroblasts. Moreover, proteins that are building up as oxidized (i.e. the "Oxi......-proteome") during ageing and age-related diseases represent a restricted set of cellular proteins, indicating that certain proteins are more prone to oxidative carbonylation and subsequent intracellular accumulation. The occurrence of specific carbonylated proteins upon oxidative stress induced premature senescence...... of WI-38 human fibroblasts and their follow-up identification have been addressed in this study. Indeed, it was expected that the identification of these proteins would give insights into the mechanisms by which oxidatively damaged proteins could affect cellular function. Among these proteins, some...

  3. Proliferation of chicken fibroblasts induced by light-emitting diodes: a comparative trial for different wavelengths

    Science.gov (United States)

    Vinck, Elke; Cagnie, B.; Declercq, H.; Cornelissen, R.; Cambier, D.

    2003-12-01

    The effectiveness and applicability of a variety of light sources, in the treatment of wounds has thoroughly been investigated, in vitro as well as in vivo. The current commercial availability of Light Emitting Diode (LED) sources therefore also invites research to explore the effect of low power infrared, red and green light on wound healing, e.g. by means of fibroblast proliferation. Therefore a controlled and randomized study on cultured embryonic chicken fibroblasts was conducted. The fibroblasts were irradiated during three consecutive days, at several wavelengths (950 nm, 660 nm and 570 nm) and a respective power output of 160 mW, 80 mW or 10 mW. Treatment duration varied from 1 minute to 3 minutes to obtain a surface energy density of 0.9 J/cm2 (infrared and red light) or 0.2 J/cm2 (green light). Statistical analysis revealed that LED irradiation for all three wavelengths induced a higher rate of proliferation in comparison of the control group. This difference was statistically significant (p cells, than the red (p < .001) an the infrared probe (p < .001). Furthermore, the red probe provided a higher increase (p < .001) than the IR probe. LED irradiation results in an increased fibroblast proliferation in vitro. This outcome postulates beneficial stimulatory effects of LED at the applied wavelength, energy density and power output on wound healing in vivo. Further investigation is necessary to examine this hypothesis.

  4. Perioperative care in an adolescent patient with heparin-induced thrombocytopenia for placement of a cardiac assist device and heart transplantation: case report and literature review

    Directory of Open Access Journals (Sweden)

    Kamata M

    2017-02-01

    Full Text Available Mineto Kamata,1 Roby Sebastian,1,2 Patrick I McConnell,3 Daniel Gomez,4 Aymen Naguib,1,2 Joseph D Tobias1,2,5 1Department of Anesthesiology and Pain Medicine, Nationwide Children’s Hospital, 2Department of Anesthesiology and Pain Medicine, The Ohio State University College of Medicine, 3Department of Cardiothoracic Surgery, Nationwide Children’s Hospital, 4Cardiovascular Perfusion Services and Heart Center, Nationwide Children’s Hospital and The Ohio State University, 5Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, USA Abstract: Heparin-induced thrombocytopenia (HIT can cause life-threatening complications following the administration of heparin. Discontinuation of all sources of heparin exposure and the use of alternative agents for anticoagulation are necessary when HIT is suspected or diagnosed. We present the successful use of bivalirudin anticoagulation in an adolescent patient during cardiopulmonary bypass who underwent both placement of a left ventricular assist device and subsequent heart transplantation within a 36-hour period. The pathophysiology and diagnosis of HIT are reviewed, previous reports of the use of direct thrombin inhibitors for cardiac surgery are presented, and potential dosing regimens for bivalirudin are discussed. Keywords: bivalirudin, anticoagulation, cardiopulmonary bypass, heart transplant

  5. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    OpenAIRE

    Shan-Shan Liu; Hao-Yan Wang; Jun-Ming Tang; Xiu-Mei Zhou

    2013-01-01

    The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II) on collagen synthesis in hypoxic human lung fibroblast (HLF) cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression levels in human ...

  6. Asbestos-Induced Mesothelial to Fibroblastic Transition Is Modulated by the Inflammasome.

    Science.gov (United States)

    Thompson, Joyce K; MacPherson, Maximilian B; Beuschel, Stacie L; Shukla, Arti

    2017-03-01

    Despite the causal relationship established between malignant mesothelioma (MM) and asbestos exposure, the exact mechanism by which asbestos induces this neoplasm and other asbestos-related diseases is still not well understood. MM is characterized by chronic inflammation, which is believed to play an intrinsic role in the origin of this disease. We recently found that asbestos activates the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in a protracted manner, leading to an up-regulation of IL-1β and IL-18 production in human mesothelial cells. Combined with biopersistence of asbestos fibers, we hypothesize that this creates an environment of chronic IL-1β signaling in human mesothelial cells, which may promote mesothelial to fibroblastic transition (MFT) in an NLRP3-dependent manner. Using a series of experiments, we found that asbestos induces a fibroblastic transition of mesothelial cells with a gain of mesenchymal markers (vimentin and N-cadherin), whereas epithelial markers, such as E-cadherin, are down-regulated. Use of siRNA against NLRP3, recombinant IL-1β, and IL-1 receptor antagonist confirmed the role of NLRP3 inflammasome-dependent IL-1β in the process. In vivo studies using wild-type and various inflammasome component knockout mice also revealed the process of asbestos-induced mesothelial to fibroblastic transition and its amelioration in caspase-1 knockout mice. Taken together, our data are the first to suggest that asbestos induces mesothelial to fibroblastic transition in an inflammasome-dependent manner. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  7. Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

    OpenAIRE

    Korpela, J; Kulomaa, M; Tuohimaa, P; Vaheri, A.

    1983-01-01

    Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metab...

  8. Role of nuclear factor-kappaB in interleukin-1-induced collagen degradation by corneal fibroblasts.

    Science.gov (United States)

    Lu, Ying; Fukuda, Ken; Li, Qin; Kumagai, Naoki; Nishida, Teruo

    2006-09-01

    The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration. The role of nuclear factor (NF)-kappaB in the IL-1-induced degradation of collagen by corneal fibroblasts that underlies corneal ulceration was investigated. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen with or without IL-1 and sulfasalazine, an inhibitor of NF-kappaB activation. Collagen degradation was assessed from the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The release of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) into culture supernatants was examined by immunoblot analysis and gelatin zymography, and the cellular abundance of MMP and TIMP mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis. The phosphorylation and degradation of the NF-kappaB-inhibitory protein IkappaB-alpha were examined by immunoblot analysis. The subcellular localization and DNA binding activity of the p65 subunit of NF-kappaB were evaluated by immunofluorescence analysis and with a colorimetric assay, respectively. The transactivation activity of NF-kappaB was assessed with a reporter gene assay. Sulfasalazine inhibited IL-1-induced collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also inhibited the stimulatory effects of IL-1 on the synthesis or activation of various MMPs in a concentration-dependent manner. IL-1 induced the phosphorylation and degradation of IkappaB-alpha, the nuclear translocation and up-regulation of the DNA binding activity of the p65 subunit of NF-kappaB, and the activation of NF-kappaB in a manner sensitive to sulfasalazine. These results suggest that NF-kappaB contributes to the IL-1-induced degradation of collagen by corneal fibroblasts and is therefore a potential therapeutic target for treatment of corneal ulcers.

  9. Endothelial glycocalyx degradation induces endogenous heparinization in patients with severe injury and early traumatic coagulopathy

    DEFF Research Database (Denmark)

    Ostrowski, Sisse R; Johansson, Pär I

    2012-01-01

    There is emerging evidence that early trauma-induced coagulopathy (TIC) is mechanistically linked to disruption of the vascular endothelium and its glycocalyx, assessed by thrombomodulin and syndecan 1, respectively. This study evaluated if degradation of the endothelial glycocalyx and ensuing...

  10. Heparin release from thermosensitive hydrogels

    NARCIS (Netherlands)

    Gutowska, Anna; Bae, You Han; Feijen, Jan; Kim, Sung Wan

    1992-01-01

    Thermosensitive hydrogels (TSH) were synthesized and investigated as heparin releasing polymers for the prevention of surface induced thrombosis. TSH were synthesized with N-isopropyl acrylamide (NiPAAm) copolymerized with butyl methacrylate (BMA) (hydrophobic) or acrylic acid (AAc) (hydrophilic) co

  11. Bivalirudin dosing adjustments for reduced renal function with or without hemodialysis in the management of heparin-induced thrombocytopenia.

    Science.gov (United States)

    Tsu, Laura V; Dager, William E

    2011-10-01

    While not approved by the Food and Drug Administration for treatment of heparin-induced thrombocytopenia (HIT), except in patients undergoing percutaneous interventions, the direct thrombin inhibitor bivalirudin is a treatment option that is gaining use. An initial dose of bivalirudin 0.15-0.2 mg/kg/h, adjusted to an activated partial thromboplastin time (aPTT) of 1.5-2.5 times the baseline value, has been suggested. Initial dosing in patients with renal dysfunction, including those on hemodialysis, is unclear. To evaluate initial bivalirudin dosing requirements in patients with and without renal dysfunction, including patients on different forms of dialysis. A retrospective analysis of 135 patients treated with bivalirudin for HIT between June 2004 and October 2009 was conducted at a tertiary care medical center. The patients were divided into groups, based on renal function. Patients receiving dialysis were divided into 3 subgroups based on the mode of hemodialysis: intermittent hemodialysis (IHD, n = 24), sustained low-efficiency daily diafiltration (SLEDD, n = 12), or continuous renal replacement therapy (CRRT, n = 5). Patients not receiving dialysis were separated into 3 subgroups based on calculated creatinine clearance (CrCl): CrCl >60 mL/min (n = 52), CrCl 30-60 mL/min (n = 26), and CrCl 60 mL/min), patients with differing degrees of renal dysfunction (CrCl 30-60 and doses of bivalirudin to achieve aPTT goal (0.13 vs 0.08 vs 0.05 mg/kg/h, respectively; p CRRT) also required dose reductions (0.07, 0.09, and 0.07 mg/kg/h) compared with patients with normal renal function, but higher dosing requirements than patients not receiving dialysis with CrCl dose of bivalirudin to reach a therapeutic aPTT goal. Slightly higher doses may be observed in patients receiving hemodialysis.

  12. The role of interleukin-8 produced by tumor induced fibroblasts in the development of cutaneous melanoma

    Institute of Scientific and Technical Information of China (English)

    Cui Rong; Feng Jie; Cao Haozhe; Zhou Xi; Zhang Xin; Yan Xiaoning

    2008-01-01

    Objective To determine the role of interleukin-8 (IL-8) produced by tumor induced fibroblasts in the development of cutaneous melanoma. Methods B16 melanoma cells induced L929 fibroblasts phenotype was transdifferentiated to myofibroblasts (MF) by co-culture in vitro. MF was monitored by morphology and immunophenotype for a-SMA. The level of IL-8 was detected by ELISA. The effect on B16 cell proliferation rate was estimated using MIT method in vitro. Melanoma implanting model was constructed in C57 mice. Results L929 MF phenotype could be modulated by B16 melanoma cells-derived transforming growth factor-β1 (TGF-β1) and elevated the levels of IL-8. L929 MF did not influence the B16 melanoma cells viability in vitro, but shortened the time of tumor formation and increased the incidence rates of tumors in C57 implanting model mice. Conclusion Fibroblasts can be activated by tumor cells and produce IL-8, which acts as an inflammatory cytokine promoting the development of cutaneous melanoma.

  13. Low-Dose UVA Radiation-Induced Adaptive Response in Cultured Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhongrong Liu

    2012-01-01

    Full Text Available Objective. To investigate the mechanism of the adaptive response induced by low-dose ultraviolet A (UVA radiation. Methods. Cultured dermal fibroblasts were irradiated by a lethal dose of UVA (86.4 J/cm2 with preirradiation of single or repetitive low dose of UVA (7.2 J/cm2. Alterations of cellular morphology were observed by light microscope and electron microscope. Cell cycle and cellular apoptosis were assayed by flow cytometer. The extent of DNA damage was determined by single-cell gel electrophoresis (SCGE. Results. The cultured dermal fibroblasts, with pretreatment of single or repetitive irradiation of 7.2 J/cm2 UVA relieved toxic reaction of cellular morphology and arrest of cell cycle, decreased apoptosis ratio, reduced DNA chain breakage, and accelerated DNA repair caused by subsequent 86.4 J/cm2 UVA irradiation. Compared with nonpretreatment groups, all those differences were significant (P<0.01 or P<0.05. Conclusions. The adaptation reaction might depend on the accumulated dose of low-dose UVA irradiation. Low-dose UVA radiation might induce adaptive response that may protect cultured dermal fibroblasts from the subsequent challenged dose of UVA damage. The duration and protective capability of the adaptive reaction might be related to the accumulated dose of low-dose UVA Irradiation.

  14. A new feature of Mpl receptor: ligand-induced transforming activity in FRE rat fibroblasts.

    Science.gov (United States)

    Challier, C; Cocault, L; Flon, M; Pauchard, M; Porteu, F; Gisselbrecht, S; Souyri, M

    2000-04-13

    Mpl is the receptor for thrombopoietin, the primary regulator of platelet production by megakaryocytes. Upon stimulation by its ligand, Mpl receptor induces proliferation and differentiation of hematopoietic cell lines of various origins. In this paper, we show that Mpl is also able to transform FRE rat fibroblasts in the presence of MGDF (pegylated Megakaryocyte Growth and Development Factor), a modified form of its ligand. We also demonstrate that upon MGDF stimulation Mpl receptor activates the classical transduction pathways described for hematopoietic cell lines in FRE cells. Introduction of Mpl deletion mutants in FRE cells allowed us to demonstrate that the C-terminal region of the Mpl intracytoplasmic domain, which is involved in hematopoietic differentiation, is necessary for the transformation process. Within that region, site-directed mutagenesis showed that the Y112 residue, which is required for Shc phosphorylation, is essential for rat fibroblast transformation by Mpl/MGDF, suggesting the involvement of Shc in Mpl-mediated transformation. Interestingly, we showed that transformation correlated with strong and sustained MAPK activation. Neither Jak2, Stat3 nor Stat5 phosphorylation was sufficient to induce the transformation process. Taken altogether, our results suggest the oncogenicity of Mpl in fibroblastic cells in the presence of its ligand.

  15. Heparin-induced extracorporeal LDL-precipitation(HELP):Mode of action,safety and clinical experience(3)

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ Safety and practicability of the HELP procedure Compared to standard plasma exchange, HELP-apheresis is much better tolerated, primarily be-cause heparin rather than citrate is used for anti-coagulation and therefore problems due to hypercalce-mia do not play a role.

  16. Heparin increases the infectivity of Human Papillomavirus type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

    NARCIS (Netherlands)

    Cerqueira, C.; Liu, Y.; Kuhling, L.; Chai, W.; Hafezi, W.; Kuppevelt, T.H. van; Kuhn, J.E.; Feizi, T.; Schelhaas, M.

    2013-01-01

    Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin resu

  17. Protective Effect of Strawberry Extract against Inflammatory Stress Induced in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Massimiliano Gasparrini

    2017-01-01

    Full Text Available A protracted pro-inflammatory state is a major contributing factor in the development, progression and complication of the most common chronic pathologies. Fruit and vegetables represent the main sources of dietary antioxidants and their consumption can be considered an efficient tool to counteract inflammatory states. In this context an evaluation of the protective effects of strawberry extracts on inflammatory stress induced by E. coli LPS on human dermal fibroblast cells was performed in terms of viability assays, ROS and nitrite production and biomarkers of oxidative damage of the main biological macromolecules. The results demonstrated that strawberry extracts exerted an anti-inflammatory effect on LPS-treated cells, through an increase in cell viability, and the reduction of ROS and nitrite levels, and lipid, protein and DNA damage. This work showed for the first time the potential health benefits of strawberry extract against inflammatory and oxidative stress in LPS-treated human dermal fibroblast cells.

  18. SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells.

    Science.gov (United States)

    Pan, Chuanying; Hicks, Amy; Guan, Xuan; Chen, Hong; Bishop, Colin E

    2010-04-01

    Induced pluripotent stem (iPS) cells can be derived from human somatic cells by cellular reprogramming. This technology provides a potential source of non-controversial therapeutic cells for tissue repair, drug discovery, and opportunities for studying the molecular basis of human disease. Normally, mouse embryonic fibroblasts (MEFs) are used as feeder layers in the initial derivation of iPS lines. The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cells reprogrammed from somatic cells using lentiviral expressed reprogramming factors. In our study, iPS cells expressed common pluripotency markers, displayed human embryonic stem cells (hESCs) morphology and unmethylated promoters of NANOG and OCT4. These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.

  19. Heparin induces an accumulation of atherogenic lipoproteins during hemodialysis in normolipidemic end-stage renal disease patients.

    Science.gov (United States)

    Barbagallo, Carlo M; Noto, Davide; Cefalù, Angelo B; Ganci, Antonia; Giammarresi, Carlo; Panno, Donata; Cusumano, Gaspare; Greco, Massimiliano; Di Gaudio, Francesca; Averna, Maurizio R

    2015-07-01

    Dyslipidemias may account for the excess of cardiovascular mortality in end-stage renal disease (ESRD). Lipoprotein studies in ESRD patients are usually relative to prehemodialysis samples even if significative changes may occur after dialysis. In this study, we aimed to investigate the effects of ESRD on triglyceride-rich lipoproteins (TRL) subpopulations distribution and acute change following hemodialytic procedures, including the relative contribution of heparin administration. We selected a group of normolipidemic male middle-aged ESRD patients free of any concomitant disease affecting lipoprotein remnant metabolism compared with controls. We separated TRL subfractions according to density and apoE content and evaluated the changes of these particles after hemodialytic procedures with or without heparin. ESRD subjects had higher TRL subfractions, with the exception of apoE-rich particles, lower high-density lipoprotein (HDL) largest subclasses, and a smaller low-density lipoprotein peak particle size than controls. After a hemodialytic standard procedure with heparin, we demonstrated a significant reduction of triglyceride, an increase of HDL-cholesterol levels, and a raise of small very-low-density lipoprotein, intermediate-density lipoproteins (IDL), apoE-rich particles, and non-HDL-cholesterol levels. When hemodialysis was performed without heparin, no significant changes were observed. In the absence of concomitant hyperlipidemic triggers, ESRD patients show significant lipoprotein abnormalities before dialysis, but without any increased remnant particles concentrations. We speculate that hemodialysis, in particular heparin administration during this procedure, leads to a massive atherogenic TRLs production because of the acute stimulation of the dysfunctional lipolytic system not followed by an efficient removal, determining a recurrent lipoprotein remnant accumulation. © 2014 International Society for Hemodialysis.

  20. The role of side stream dark field microvasculature imaging in a rare case of vancomycin-resistant enterococcal endocarditis complicated by heparin-induced thrombocytopenia

    Directory of Open Access Journals (Sweden)

    Janak Bechar

    2016-01-01

    Full Text Available Sidestream dark field (SDF imaging allows direct visualization of microvascular architecture and function. We examine the role of an SDF imaging device in visualizing the sub-lingual microvasculature as a surrogate for splanchnic microperfusion. We demonstrate good correlation between current monitoring techniques and the SDF imaging device in a rare case of vancomycin-resistant enterococcal (VRE sepsis along with heparin-induced thrombocytopenia (HIT. To the best of our knowledge, VRE endocarditis with concurrent HIT has not been described in literature. The role of SDF imaging may predict the earlier need for escalation of care, improving morbidity and mortality.

  1. Unfractionated and low-molecular-weight heparin and the phosphodiesterase inhibitors, IBMX and cilostazol, block ex vivo Equid Herpesvirus type-1-induced platelet activation

    Directory of Open Access Journals (Sweden)

    Tracy Stokol

    2016-11-01

    Full Text Available Equid herpes virus type-1 (EHV-1 is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins or platelet inhibitors that impede post-receptor thrombin signaling (phosphodiesterase [PDE] antagonists would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque forming unit/cell in the presence or absence of unfractionated heparin (UFH, low-molecular-weight (LMWH heparin or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1 to 0.2 U/mL anti-Factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX or isoenzyme-3 selective (cilostazol PDE antagonists inhibit ex vivo EHV-1-induced platelet activation

  2. Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice

    Directory of Open Access Journals (Sweden)

    Yinghua Song

    2013-10-01

    Full Text Available OBJECTIVE: To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice. METHODS: Skin fibroblasts that were isolated from three systemic scleroderma (SSc patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM. Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I procollagen (COL1A1, alpha 1 (III procollagen (COL3A1, matrix metalloproteinase (MMP-1 and tissue inhibitor of matrix metalloproteinase (TIMP-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA. Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR. RESULTS: Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3. CONCLUSION: Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a

  3. Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

    Science.gov (United States)

    Meyer-Ter-Vehn, Tobias; Katzenberger, Barbara; Han, Hong; Grehn, Franz; Schlunck, Günther

    2008-09-01

    The transdifferentiation of Tenon fibroblasts to myofibroblasts is a pivotal step in filtering bleb scarring. It is mediated by the cytokine TGF-beta, Rho-dependent contractility, and cell-matrix interactions in an interdependent fashion. HMG-CoA-reductase inhibitors (statins) have been shown to inhibit Rho-GTPase signaling; therefore, the authors studied the influence of lovastatin on TGF-beta-mediated myofibroblast transdifferentiation to assess the potential use of statins in wound healing modulation. Human Tenon fibroblasts were grown in culture, pretreated with lovastatin, lovastatin and mevalonate, or specific inhibitors of farnesyl transferase or geranylgeranyl transferase and were stimulated with TGF-beta1. alpha-Smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) transcription were assessed by real-time PCR. alpha-SMA protein expression and localization were studied by Western blot and confocal immunofluorescence microscopy. Cell contractility was determined in collagen gel contraction assays. Phosphorylation of the signaling proteins Smad-2/3 and p38 were detected by Western blot, and Smad-2/3 localization was determined by confocal immunofluorescence microscopy. Lovastatin inhibited TGF-beta-induced CTGF transcription, alpha-SMA expression and incorporation into actin stress fibers, and subsequent collagen gel contraction. These effects were reversed by mevalonate. The inhibition of geranylgeranyl transferase but not farnesyl transferase blocked TGF-beta-induced alpha-SMA expression. Lovastatin decreased TGF-beta-induced p38 activation, whereas Smad-2/3 phosphorylation and nuclear translocation were preserved. Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human Tenon fibroblasts, most likely by interfering with Rho-signaling. Statins may, therefore, serve to inhibit scarring after filtering glaucoma surgery.

  4. Data on cytochrome c oxidase assembly in mice and human fibroblasts or tissues induced by SURF1 defect.

    Science.gov (United States)

    Kovářová, Nikola; Pecina, Petr; Nůsková, Hana; Vrbacký, Marek; Zeviani, Massimo; Mráček, Tomáš; Viscomi, Carlo; Houštěk, Josef

    2016-06-01

    This paper describes data related to a research article entitled "Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects" [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1 (-/-) ) and control (SURF1 (+/+) ) mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX), to reversible inhibition of mitochondrial translation in SURF1 (-/-) mouse and SURF1 patient fibroblast cell lines.

  5. Ionizing radiation-induced adaptive response in fibroblasts under both monolayer and 3-dimensional conditions.

    Science.gov (United States)

    Zhao, Yinlong; Zhong, Rui; Sun, Liguang; Jia, Jie; Ma, Shumei; Liu, Xiaodong

    2015-01-01

    To observe the adaptive response (AR) induced by ionizing radiation in human fibroblasts under monolayer and 3-dimensional (3-D) condition. Three kinds of fibroblasts were cultured under both monolayer and 3-D condition. Immunofluorescent staining was used to detect the γ-H2AX foci and the morphological texture. Trypan blue staining was used to detect the cell death. Western blot was used to detect the expressions of γ-H2AX, p53 and CDKN1A/p21 (p21). We found that DNA damage increased in a dose-dependent and time-dependent manner after high doses of radiation. When cells were pretreated with a priming low dose of radiation followed by high dose radiation, DNA damage was attenuated under both monolayer and 3-D condition, and the adaptive response (AR) was induced. Additionally, the morphology of cells under monolayer and 3-D conditions were different, and radiation also induced AR according to morphological texture analysis. Priming low dose radiation induced AR both under monolayer and 3-D condition. Interestingly, 3-D microenvironment made cells more sensitive to radiation. The expression of p53 and p21 was changed and indicated that they might participate in the regulation of AR.

  6. Inhibition by rebamipide of cytokine-induced or lipopolysaccharide-induced chemokine synthesis in human corneal fibroblasts.

    Science.gov (United States)

    Fukuda, Ken; Ishida, Waka; Tanaka, Hiroshi; Harada, Yosuke; Fukushima, Atsuki

    2014-12-01

    The dry-eye drug rebamipide has mucin secretagogue activity in and anti-inflammatory effects on corneal epithelial cells. Corneal stromal fibroblasts (transdifferentiated keratocytes) function as immune modulators in the pathogenesis of chronic ocular allergic inflammation and in innate immune responses at the ocular surface. The possible anti-inflammatory effects of rebamipide on human corneal stromal fibroblasts were examined. Serum-deprived cells were incubated for 1 h with rebamipide and then for various times in the additional absence or presence of cytokines or bacterial lipopolysaccharide (LPS). The release of chemokines into culture supernatants was determined with ELISAs. The intracellular abundance of chemokine mRNAs was quantitated by reverse transcription and real-time PCR analysis. Degradation of the nuclear factor κB (NFκB) inhibitor IκBα was detected by immunoblot analysis. Rebamipide suppressed the release of interleukin (IL)-8 and the upregulation of IL-8 mRNA induced by tumour necrosis factor α (TNF-α) or LPS in corneal fibroblasts. It also inhibited eotaxin-1 (CCL-11) expression at the protein and mRNA levels induced by the combination of TNF-α and IL-4. In addition, rebamipide attenuated the degradation of IκBα induced by TNF-α or LPS. Rebamipide inhibited the synthesis of chemokines by corneal fibroblasts in association with suppression of NFκB signalling. Rebamipide may therefore prove effective for the treatment of corneal stromal inflammation associated with allergy or bacterial infection. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  7. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: taehoon@novacelltech.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer 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 {beta}1 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

  8. Resonance Light Scattering Imaging Determination of Heparin

    Institute of Scientific and Technical Information of China (English)

    Hong Ping GUO; Cheng Zhi HUANG; Jian LING

    2006-01-01

    A laser-induced resonance light scattering (RLS) imaging method to determine heparin is described based on the high light scattering emission power of the aggregation species of heparin with α, β, γ, δtetra(4-trimethylaminoniumphenyl)prophyrin (TAPP) in solution. By imaging the light scattering signals of the aggregation species, we proposed the method to determine the heparin with a detection range of 0.02 - 0.6 μg/mL and the detection limit (3 σ) of 1.3 ng/mL.

  9. Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by {sup 15}N NMR relaxation methods

    Energy Technology Data Exchange (ETDEWEB)

    Canales-Mayordomo, Angeles; Fayos, Rosa [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain); Angulo, Jesus; Ojeda, Rafael [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Martin-Pastor, Manuel [Unidad de RM y Unidad de RMN de Biomoleculas Asociada al CSIC, Laboratorio de Estructura e Estructura de Biomoleculas Jose Carracido (Spain); Nieto, Pedro M.; Martin-Lomas, Manuel [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain)], E-mail: jjbarbero@cib.csic.es

    2006-08-15

    The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR)

  10. Control of IBMIR Induced by Fresh and Cryopreserved Hepatocytes by Low Molecular Weight Dextran Sulfate Versus Heparin.

    Science.gov (United States)

    Gustafson, Elisabet; Asif, Sana; Kozarcanin, Huda; Elgue, Graciela; Meurling, Staffan; Ekdahl, Kristina N; Nilsson, Bo

    2017-01-24

    Rapid destruction of hepatocytes after hepatocyte transplantation has hampered the application of this procedure clinically. The instant blood-mediated inflammatory reaction (IBMIR) is a plausible underlying cause for this cell loss. The present study was designed to evaluate the capacity of low molecular weight dextran sulfate (LMW-DS) to control these initial reactions from the innate immune system. Fresh and cryopreserved hepatocytes were tested in an in vitro whole-blood model using ABO-compatible blood. The ability to elicit IBMIR and the capacity of LMW-DS (100 μg/ml) to attenuate the degree of activation of the cascade systems were monitored. The effect was also compared to conventional anticoagulant therapy using unfractionated heparin (1 IU/ml). Both fresh and freeze-thawed hepatocytes elicited IBMIR to the same extent. LMW-DS reduced the platelet loss and maintained the cell counts at the same degree as unfractionated heparin, but controlled the coagulation and complement systems significantly more efficiently than heparin. LMW-DS also attenuated the IBMIR elicited by freeze-thawed cells. Therefore, LMW-DS inhibits the cascade systems and maintains the cell counts in blood triggered by both fresh and cryopreserved hepatocytes in direct contact with ABO-matched blood. LMW-DS at a previously used and clinically applicable concentration (100 μg/ml) inhibits IBMIR in vitro and is therefore a potential IBMIR inhibitor in hepatocyte transplantation.

  11. Perioperative use of iloprost in cardiac surgery patients diagnosed with heparin-induced thrombocytopenia-reactive antibodies or with true HIT (HIT-reactive antibodies plus thrombocytopenia): An 11-year experience.

    Science.gov (United States)

    Palatianos, George; Michalis, Alkiviadis; Alivizatos, Petros; Lacoumenda, Stavroula; Geroulanos, Stefanos; Karabinis, Andreas; Iliopoulou, Eugenia; Soufla, Giannoula; Kanthou, Chryso; Khoury, Mazen; Sfyrakis, Petros; Stavridis, George; Astras, George; Vassili, Maria; Antzaka, Christina; Marathias, Katerina; Kriaras, Ioannis; Tasouli, Androniki; Papadopoulos, Kyrillos; Katafygioti, Marina; Matoula, Nikoletta; Angelidis, Antonios; Melissari, Euthemia

    2015-07-01

    Thrombocytopenia and thromboembolism(s) may develop in heparin immune-mediated thrombocytopenia (HIT) patients after reexposure to heparin. At the Onassis Cardiac Surgery Center, 530 out of 17,000 patients requiring heart surgery over an 11-year period underwent preoperative HIT assessment by ELISA and a three-point heparin-induced platelet aggregation assay (HIPAG). The screening identified 110 patients with HIT-reactive antibodies, out of which 46 were also thrombocytopenic (true HIT). Cardiac surgery was performed in HIT-positive patients under heparin anticoagulation and iloprost infusion. A control group of 118 HIT-negative patients received heparin but no iloprost during surgery. For the first 20 patients, the dose of iloprost diminishing the HIPAG test to ≤5% was determined prior to surgery by in vitro titration using the patients' own plasma and donor platelets. In parallel, the iloprost "target dose" was also established for each patient intraoperatively, but before heparin administration. Iloprost was infused initially at 3 ng/kg/mL and further adjusted intraoperatively, until ex vivo aggregation reached ≤5%. As a close correlation was observed between the "target dose" identified before surgery and that established intraoperatively, the remaining 90 patients were administered iloprost starting at the presurgery identified "target dose." This process significantly reduced the number of intraoperative HIPAG reassessments needed to determine the iloprost target dose, and reduced surgical time, while maintaining similar primary clinical outcomes to controls. Therefore, infusion of iloprost throughout surgery, under continuous titration, allows cardiac surgery to be undertaken safely using heparin, while avoiding life-threatening iloprost-induced hypotension in patients diagnosed with HIT-reactive antibodies or true HIT. © 2015 Wiley Periodicals, Inc.

  12. Heparin-Induced Thrombocytopenia

    Science.gov (United States)

    ... My alerts Sign In Join Give up seat Facebook Twitter Home About this Journal Editorial Board General Statistics Circulation Doodle → Blip the Doodle Information for Advertisers Author Reprints Commercial Reprints Customer Service and Ordering ...

  13. Regulation of hypoxia-inducible factor-1α in human buccal mucosal fibroblasts stimulated with arecoline

    Directory of Open Access Journals (Sweden)

    Yung-Chuan Ho

    2017-06-01

    Full Text Available Hypoxia-inducible factor (HIF-1α is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare HIF-1α expression from fibroblasts derived from human normal buccal mucosa and oral submucous fibrosis (OSF specimens and further to explore the potential mechanisms that may lead to induce HIF-1α expression. OSF buccal mucosal fibroblasts (BMFs demonstrated significantly higher HIF-1α mRNA expression than normal BMFs (p<0.005. Arecoline, the major areca nut alkaloid, was also found to elevate HIF-1α mRNA expression in a dose-dependent manner (p<0.05. Moreover, arecoline-induced HIF-1α expression was downregulated by mitogen-activated protein kinase inhibitor U0126, phosphatidylinositol 3-kinase inhibitor LY294002, p38 inhibitor SB203580, cyclooxygenase-2 inhibitor NS-398, and glutathione precursor N-acetyl-L-cysteine (p<0.05. Taken together, hypoxia plays an important role in the pathogenesis of areca quid chewing-associated OSF. These pharmacological agents may be further used as chemoprevention agents for OSF.

  14. High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shao-Yun, E-mail: jiangshaoyun@yahoo.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Wei, Cong-Cong; Shang, Ting-Ting; Lian, Qi; Wu, Chen-Xuan [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Deng, Jia-Yin, E-mail: yazhou2991@126.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer High glucose significantly induced TLR2 expression in gingival fibroblasts. Black-Right-Pointing-Pointer High glucose increased NF-{kappa}B p65 nuclear activity, IL-1{beta} and TNF-{alpha} levels. Black-Right-Pointing-Pointer PKC-{alpha}/{delta}-TLR2 pathway is involved in periodontal inflammation under high glucose. -- Abstract: Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p < 0.05). Also, high glucose increased nuclear factor kappa B (NF-{kappa}B) p65 nuclear activity, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-l{beta} (IL-1{beta}) levels. Protein kinase C (PKC)-{alpha} and {delta} knockdown with siRNA significantly decreased TLR2 and NF-{kappa}B p65 expression (p < 0.05), whereas inhibition of PKC-{beta} had no effect on TLR2 and NF-{kappa}B p65 under high glucose (p < 0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-{kappa}B expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-{alpha} and IL-1{beta} secretion via inducing TLR2 through PKC-{alpha} and PKC-{delta} in human gingival fibroblasts.

  15. THE EFFECTS OF HSP27 ON THE CYTOTOXICITY OF RAT EMBRYO FIBROBLAST INDUCED BY CISPLATIN

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the protective effects of heat shock protein 2700(Hsp27)on cis- platin inducing cytotoxicity in a temperature mutant SV40 large T antigen transformed rat embryo fibroblast (P1-Hsp27). Methods The cytotoxical effects of cisplatin on the proliferation status of P1-Hsp27 cells in the presence or absence of Hsp27 were measured by MTT assay. Results Cisplatin possessed dose-dependent cyto- toxicity on P1-Hsp27 cells. 48h after treatment, about 50% cells were dead in those cells exposed to 200μmol cisplatin. However, no obvious protective effects of Hsp27 on cisplatin induced cytotoxicity could be observed (P>0.05),except in those cells exposed to 500μmol cisplatin 12h after treatment. Conclusion Hsp27 has no obvious protective effects on cisplatin inducing cytotoxicity.

  16. Knockdown of CDK2AP1 in primary human fibroblasts induces p53 dependent senescence.

    Directory of Open Access Journals (Sweden)

    Khaled N Alsayegh

    knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.

  17. Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

    Science.gov (United States)

    Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

    2013-01-01

    Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID

  18. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    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.

  19. Late infusion of cloned marrow fibroblasts stimulates endogenous recovery from radiation-induced lung injury.

    Directory of Open Access Journals (Sweden)

    Mineo Iwata

    Full Text Available In the current study, we used a canine model of radiation-induced lung injury to test the effect of a single i.v. infusion of 10×10(6/kg of marrow fibroblasts on the progression of damage following 15 Gy exposure to the right lung. The fibroblasts, designated DS1 cells, are a cloned population of immortalized cells isolated from a primary culture of marrow stromal cells. DS1 cells were infused at week 5 post-irradiation when lung damage was evident by imaging with high-resolution computed tomography (CT. At 13 weeks post-irradiation we found that 4 out of 5 dogs receiving DS1 cells had significantly improved pulmonary function compared to 0 out of 5 control dogs (p = 0.047, Fisher's Exact. Pulmonary function was measured as the single breath diffusion capacity-hematocrit (DLCO-Hct, the total inspiratory capacity (IC, and the total lung capacity (TLC, which differed significantly between control and DS1-treated dogs; p = 0.002, p = 0.005, and p = 0.004, respectively. The DS1-treated dogs also had less pneumonitis detected by CT imaging and an increased number of TTF-1 (thyroid transcription factor 1, NKX2-1 positive cells in the bronchioli and alveoli compared to control dogs. Endothelial-like progenitor cells (ELC of host origin, detected by colony assays, were found in peripheral blood after DS1 cell infusion. ELC numbers peaked one day after infusion, and were not detectable by 7 days. These data suggest that infusion of marrow fibroblasts stimulates mobilization of ELC, which is associated with a reduction in otherwise progressive radiation-induced lung injury. We hypothesize that these two observations are related, specifically that circulating ELC contribute to increased angiogenesis, which facilitates endogenous lung repair.

  20. Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds

    Science.gov (United States)

    Nguyen, Thi-Huong; Greinacher, Andreas; Delcea, Mihaela

    2015-05-01

    Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers). Analysis of the force-distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (=8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought close to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand-receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8- and 12-mers. These observations are particularly important to understand processes in which PF4-heparin interactions are involved and to develop new heparin-derived drugs.Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly

  1. Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation

    Directory of Open Access Journals (Sweden)

    Y Boza

    2010-12-01

    Full Text Available The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM, primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001. HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05. The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.

  2. Interleukin-21 induces migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

    Science.gov (United States)

    Xing, R; Jin, Y; Sun, L; Yang, L; Li, C; Li, Z; Liu, X; Zhao, J

    2016-05-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone erosion. Fibroblast-like synoviocytes (FLS) play a pivotal role in RA pathogenesis through aggressive migration and matrix invasion, and certain proinflammatory cytokines may affect synoviocyte invasion. Whether interleukin (IL)-21 influences this process remains controversial. Here, we evaluated the potential regulatory effect of IL-21 on the migration, invasion and matrix metalloproteinase (MMP) expression in RA-FLS. We found that IL-21 promoted the migration, invasion and MMP (MMP-2, MMP-3, MMP-9, MMP-13) production in RA-FLS. Moreover, IL-21 induced activation of the phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription-3 (STAT-3) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, and blockage of these pathways [PI3K/protein kinase B (AKT) inhibitor LY294002, STAT-3 inhibitor STA-21 and ERK1/2 inhibitor PD98059] attenuated IL-21-induced migration and secretion of MMP-3 and MMP-9. In conclusion, our results suggest that IL-21 promotes migration and invasion of RA-FLS. Therefore, therapeutic strategies targeting IL-21 might be effective for the treatment of RA.

  3. Recombinant N-Terminal Slit2 Inhibits TGF-β-Induced Fibroblast Activation and Renal Fibrosis.

    Science.gov (United States)

    Yuen, Darren A; Huang, Yi-Wei; Liu, Guang-Ying; Patel, Sajedabanu; Fang, Fei; Zhou, Joyce; Thai, Kerri; Sidiqi, Ahmad; Szeto, Stephen G; Chan, Lauren; Lu, Mingliang; He, Xiaolin; John, Rohan; Gilbert, Richard E; Scholey, James W; Robinson, Lisa A

    2016-09-01

    Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-β Here, we examined whether Slit2 also controls TGF-β-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-β-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis.

  4. Adiponectin induces CXCL1 secretion from cancer cells and promotes tumor angiogenesis by inducing stromal fibroblast senescence.

    Science.gov (United States)

    Cai, Lun; Xu, Shengyuan; Piao, Chunmei; Qiu, Shulan; Li, Huihua; Du, Jie

    2016-11-01

    Adiponectin is an adipocyte-specific adipocytokine with proliferative and pro-angiogenic effects that regulates many biological processes, including immunity, insulin resistance, and inflammation. The oncogenic role of adiponectin has been implicated in several cancer types. Stromal cells within tumor contribute tumor growth and angiogenesis; however, it is not clear that how adiponectin regulates stromal cell-mediated tumorigenesis. In this study, using the tumor xenograft models, we demonstrated that tumor development was severely impaired in mouse subcutaneous cancer tissue and metastasis tumor tissue in adiponectin knockout mice. Our results indicated adiponectin deficiency resulted in decrease of blood vessel and stromal senescent fibroblasts in subcutaneous and metastasis tumor tissue. These observations were confirmed in vitro, in which co-cultured tumor cells and fibroblasts treated with adiponectin promoted ECs tube formation. A secretion of CXCL1 by adiponectin-treated tumor cells was observed during the process of inducing stromal fibroblast senescence. Furthermore, stromal cells senescence was through p53 and p16 pathways. Taken together, our results indicate that adiponectin promotes stromal cell senescence within invasive colon cancer contributing to angiogenesis and tumor growth in part through the production of CXCL1 and may serve as a therapeutic target for tumor patients. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  5. Cordyceps militaris Extract Protects Human Dermal Fibroblasts against Oxidative Stress-Induced Apoptosis and Premature Senescence

    Directory of Open Access Journals (Sweden)

    Jun Myoung Park

    2014-09-01

    Full Text Available Oxidative stress induced by reactive oxygen species (ROS is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs. Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS.

  6. Cordyceps militaris Extract Protects Human Dermal Fibroblasts against Oxidative Stress-Induced Apoptosis and Premature Senescence

    Science.gov (United States)

    Park, Jun Myoung; Lee, Jong Seok; Lee, Ki Rim; Ha, Suk-Jin; Hong, Eock Kee

    2014-01-01

    Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS. PMID:25230212

  7. Expression of fibroblast growth factor-2 and fibroblast growth factor receptor-1 protein in the hippocampus in rats exhibiting chronic stress-induced depression

    Institute of Scientific and Technical Information of China (English)

    Gonglin Hou; Mingming Tang

    2011-01-01

    There is evidence that the expression of members of the fibroblast growth factor (FGF) protein family is altered in post-mortem brains of humans suffering from major depressive disorder. The present study examined whether the expression of fibroblast growth factor-2 (FGF2) and fibroblast growth factor receptor-1 (FGFR1) protein is altered following chronic stress in an animal model. Rats were exposed to 35 days of chronic unpredictable mild stress, and then tested using open-field and sucrose consumption tests. Compared with the control group, rats in the chronic stress group exhibited obvious depressive-like behaviors, including anhedonia, anxiety and decreased mobility. The results of western blot analysis and immunohistochemical analysis revealed a downregulation of the expression of FGF2 and FGFR1 in the hippocampus of rats, particularly in the CA1, CA3 and dentate gyrus. This decreased expression is in accord with the results of post-mortem studies in humans with major depressive disorder. These findings suggest that FGF2 and FGFR1 proteins participate in the pathophysiology of depressive-like behavior, and may play an important role in the mechanism of chronic stress-induced depression.

  8. Variability in heparin sensitivity of appt reagents in heparinized plasma in vitro and patients receiving heparin

    National Research Council Canada - National Science Library

    Fernanda Daniela Serralvo; Jacinta Ludovico Zamboti; Maria Emilia Favero; José Wander Bregano

    2015-01-01

    ... used for its determination. Objective: To evaluate the sensibility to heparin in the different reagents used to determine APPT in samples of heparinized plasma in vitro and in patients using non-fractioned heparin (NFH...

  9. A spectroscopic study of interaction of cationic dyes with heparin

    Directory of Open Access Journals (Sweden)

    R. Nandini

    2010-01-01

    Full Text Available The interaction of two cationic dyes namely, acridine orange and pinacyanol chloride with an anionic polyelectrolyte, heparin, has been investigated by spectrophotometric method.The polymer induced metachromasy in the dyes resulting in the shift of the absorption maxima of the dyes towards shorter wavelengths. The stability of the complexes formed between acridine orange and heparin was found to be lesser than that formed between pinacyanol chloride and heparin. This fact was further confirmed by reversal studies using alcohols, urea and surfactants. The interaction of acridine orange with heparin has also been investigated fluorimetrically.The interaction parameters revealed that binding between acridine orange and heparin arises due to electrostatic interaction while that between pinacyanol chloride and heparin is found to involve both electrostatic and hydrophobic forces. The effect of the structure of the dye in inducing metachromasy has also been discussed.

  10. P190B RhoGAP overexpression in the developing mammary epithelium induces TGFβ-dependent fibroblast activation.

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    Melissa Gillette

    Full Text Available Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC morphogenesis. Increased extracellular matrix (ECM deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The

  11. The molecular mechanism of leptin secretion and expression induced by aristolochic acid in kidney fibroblast.

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    Tsung-Chieh Lin

    Full Text Available BACKGROUND: Leptin is a peptide hormone playing pivotal role in regulating food intake and energy expenditure. Growing evidence has suggested the pro-inflammatory and fibrogenic properties of leptin. In addition, patients with renal fibrosis have higher level of plasma leptin, which was due to the increased leptin production. Aristolochic acid (AA is a botanical toxin characterized to associate with the development of renal fibrosis including tubulointerstitial fibrosis. However, whether leptin is upregulated to participate in AA-induced kidney fibrosis remain completely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, leptin expression was increased by sublethal dose of AA in kidney fibroblast NRK49f determined by enzyme-linked immunosorbent assay and Western blot. Data from real-time reverse transcriptase-polymerase chain reaction revealed that leptin was upregulated by AA at transcriptional level. DNA binding activity of CCAAT enhancer binding protein α (C/EBP α, one of the transcription factors for leptin gene, was enhanced in DNA affinity precipitation assay and chromatin immunoprecipitation experiments. Knockdown of C/EBP α expression by small interfering RNA markedly reduced AA-induced leptin expression. Moreover, AA promoted Akt interaction with p-PDK1, and increased phosphorylated activation of Akt. Akt knockdown, and inhibition of Akt signaling by LY294002 and mTOR inhibitor rapamycin reduced leptin expression. Furthermore, treatment of LY294002 or rapamycin significantly suppressed AA-induced C/EBP α DNA-binding activity. These results suggest that Akt and C/EBP α activation were involved in AA-regulated leptin expression. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the first that AA could induce secretion and expression of fibrogenic leptin in kidney fibroblasts, which reveal potential involvement of leptin in the progression of kidney fibrosis in aristolochic acid nephropathy.

  12. Disruption of Calcium Signaling in Fibroblasts and Attenuation of Bleomycin-Induced Fibrosis by Nifedipine.

    Science.gov (United States)

    Mukherjee, Subhendu; Ayaub, Ehab A; Murphy, James; Lu, Chao; Kolb, Martin; Ask, Kjetil; Janssen, Luke J

    2015-10-01

    Fibrotic lung disease afflicts millions of people; the central problem is progressive lung destruction and remodeling. We have shown that external growth factors regulate fibroblast function not only through canonical signaling pathways but also through propagation of periodic oscillations in Ca(2+). In this study, we characterized the pharmacological sensitivity of the Ca(2+)oscillations and determined whether a blocker of those oscillations can prevent the progression of fibrosis in vivo. We found Ca(2+) oscillations evoked by exogenously applied transforming growth factor β in normal human fibroblasts were substantially reduced by 1 μM nifedipine or 1 μM verapamil (both L-type blockers), by 2.7 μM mibefradil (a mixed L-/T-type blocker), by 40 μM NiCl2 (selective at this concentration against T-type current), by 30 mM KCl (which partially depolarizes the membrane and thereby fully inactivates T-type current but leaves L-type current intact), or by 1 mM NiCl2 (blocks both L- and T-type currents). In our in vivo study in mice, nifedipine prevented bleomycin-induced fibrotic changes (increased lung stiffness, overexpression of smooth muscle actin, increased extracellular matrix deposition, and increased soluble collagen and hydroxyproline content). Nifedipine had little or no effect on lung inflammation, suggesting its protective effect on lung fibrosis was not due to an antiinflammatory effect but rather was due to altering the profibrotic response to bleomycin. Collectively, these data show that nifedipine disrupts Ca(2+) oscillations in fibroblasts and prevents the impairment of lung function in the bleomycin model of pulmonary fibrosis. Our results provide compelling proof-of-principle that interfering with Ca(2+) signaling may be beneficial against pulmonary fibrosis.

  13. TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment.

    Science.gov (United States)

    Degryse, Amber L; Tanjore, Harikrishna; Xu, Xiaochuan C; Polosukhin, Vasiliy V; Jones, Brittany R; Boomershine, Chad S; Ortiz, Camila; Sherrill, Taylor P; McMahon, Frank B; Gleaves, Linda A; Blackwell, Timothy S; Lawson, William E

    2011-06-01

    The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.

  14. The chromene sargachromanol E inhibits ultraviolet A-induced ageing of skin in human dermal fibroblasts.

    Science.gov (United States)

    Kim, J-A; Ahn, B-N; Kong, C-S; Kim, S-K

    2013-05-01

    Skin ageing is influenced by environmental factors such as ultraviolet (UV) radiation. The effects of UV radiation on skin functions should be investigated using human in vitro models to understand the mechanisms of skin ageing. Additionally, marine algae provide a valuable source for identifying and extracting biologically active substances. In this study, sargachromanol E was isolated from a marine brown alga, Sargassum horneri, and its inhibitory effect on skin ageing was investigated using UVA-irradiated dermal fibroblasts. Formation of intracellular reactive oxygen species (ROS), lipid peroxidation and protein oxidation induced by UVA irradiation were investigated in UVA-irradiated human dermal fibroblasts. The levels of matrix metalloproteinases (MMPs) were determined by reverse-transcriptase polymerase chain reaction and Western blot analysis. Sargachromanol E did not exhibit any significant cytotoxicity or phototoxicity in UVA-exposed dermal fibroblasts. Additionally, sargachromanol E suppressed intracellular formation of ROS, membrane protein oxidation, lipid peroxidation and expression of collagenases such as MMP-1, MMP-2 and MMP-9, all of which are caused by UVA exposure. It was further found that these inhibitions were related to an increase in the expression of the tissue inhibitor of metalloproteinase (TIMP) genes, TIMP1 and TIMP2. Moreover, we have shown that the transcriptional activation of activator protein 1 (AP-1) signalling caused by UVA irradiation was inhibited by treatment with sargachromanol E. This study suggests that UVA irradiation modulates MMP expression via the transcriptional activation of AP-1 signalling, whereas treatment with sargachromanol E protected cell damage caused by UVA irradiation. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

  15. Apigenin prevents ultraviolet-B radiation induced cyclobutane pyrimidine dimers formation in human dermal fibroblasts.

    Science.gov (United States)

    Britto, S Mary; Shanthakumari, D; Agilan, B; Radhiga, T; Kanimozhi, G; Prasad, N Rajendra

    2017-09-01

    Exposure to solar ultraviolet-B (UVB) radiation leads to the formation of cyclobutane pyrimidine dimers (CPDs). We investigated the protective effect of apigenin against UVB-induced CPDs formation in human dermal fibroblasts cells (HDFa). For this purpose, HDFa cells were treated with apigenin (15μM) prior to UVB irradiation (20mJ/cm(2)); DNA damage and subsequent molecular end points were observed. Exposure to UVB radiation increased significant CPDs formation in HDFa cells and the frequencies of CPDs were reduced by treatment with apigenin (15μM). UVB-induced CPDs downregulates the expression of nucleotide excision repair (NER) genes such as xeroderma pigmentosum complementation group C, B, G and F (XPC, XPB, XPG and XPF), transcription factor II human (TFIIH) and excision repair cross-complementation group 1 (ERCC1) in HDFa cells. Conversely, apigenin treatment restored UVB-induced loss of NER proteins in HDFa cells, which indicates its preventive effect against CPDs formation. Besides, single low dose UVB-exposure induced nuclear fragmentation, apoptotic frequency and apoptotic proteins expression (Bax and Caspase-3) have been prevented by the apigenin pretreatment. Furthermore, apigenin exhibits strong UV absorbance property and showed 10.08 SPF value. Thus, apigenin can protect skin cells against UVB-induced CPDs formation probably through its sunscreen effect. Hence, apigenin can be considered as an effective protective agent against UV induced skin damages. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

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    Qiao Lu

    Full Text Available Human induced pluripotent stem cells (hiPSCs have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.

  17. Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

    Science.gov (United States)

    Lu, Qiao; Yu, Meixing; Shen, Chongyang; Chen, Xiaoping; Feng, Ting; Yao, Yongchao; Li, Jinrong; Li, Hong; Tu, Wenwei

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.

  18. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

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    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  19. Epigallocatechin-3-gallate suppresses TNF-alpha -induced production of MMP-1 and -3 in rheumatoid arthritis synovial fibroblasts.

    Science.gov (United States)

    Yun, Hee-Jin; Yoo, Wan-Hee; Han, Myung-Kwan; Lee, Young-Rae; Kim, Jong-Suk; Lee, Sang-Il

    2008-11-01

    Rheumatoid arthritis (RA) synovial fibroblasts produce matrix metaloproteinases (MMPs), which destroy cartilage and bone in RA joint. Tumor necrosis factor-alpha (TNF-alpha) is one of the most important mediator leading to MMP production in RA synovial fibroblasts. Here we show that epigallocatechin-3-Gallate (EGCG) suppresses TNF-alpha-induced production of MMP-1 and MMP-3 in RA synovial fibroblasts, which was accompanied by inhibition of mitogen activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways. EGCG treatment resulted in dose-dependent inhibition of TNF-alpha-induced production of MMP-1 and MMP-3 at the protein and mRNA levels in RA synovial fibroblast. EGCG treatment also inhibited TNF-alpha-induced phosphorylation of MAPKs, such as ERK1/2, p38, JNK. Electrophoretic mobility shift assay revealed that EGCG inhibits binding of AP-1 proteins to its response elements in synovial fibroblast treated. Thus, EGCG may play a role in regulating inflammation and bone destruction in RA patients.

  20. Mitochondrial iron and energetic dysfunction distinguish fibroblasts and induced neurons from pantothenate kinase-associated neurodegeneration patients.

    Science.gov (United States)

    Santambrogio, Paolo; Dusi, Sabrina; Guaraldo, Michela; Rotundo, Luisa Ida; Broccoli, Vania; Garavaglia, Barbara; Tiranti, Valeria; Levi, Sonia

    2015-09-01

    Pantothenate kinase-associated neurodegeneration is an early onset autosomal recessive movement disorder caused by mutation of the pantothenate kinase-2 gene, which encodes a mitochondrial enzyme involved in coenzyme A synthesis. The disorder is characterised by high iron levels in the brain, although the pathological mechanism leading to this accumulation is unknown. To address this question, we tested primary skin fibroblasts from three patients and three healthy subjects, as well as neurons induced by direct fibroblast reprogramming, for oxidative status, mitochondrial functionality and iron parameters. The patients' fibroblasts showed altered oxidative status, reduced antioxidant defence, and impaired cytosolic and mitochondrial aconitase activities compared to control cells. Mitochondrial iron homeostasis and functionality analysis of patient fibroblasts indicated increased labile iron pool content and reactive oxygen species development, altered mitochondrial shape, decreased membrane potential and reduced ATP levels. Furthermore, analysis of induced neurons, performed at a single cell level, confirmed some of the results obtained in fibroblasts, indicating an altered oxidative status and signs of mitochondrial dysfunction, possibly due to iron mishandling. Thus, for the first time, altered biological processes have been identified in vitro in live diseased neurons. Moreover, the obtained induced neurons can be considered a suitable human neuronal model for the identification of candidate therapeutic compounds for this disease.

  1. A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion.

    Science.gov (United States)

    Coppe, Jean-Philippe; Boysen, Megan; Sun, Chung Ho; Wong, Brian J F; Kang, Mo K; Park, No-Hee; Desprez, Pierre-Yves; Campisi, Judith; Krtolica, Ana

    2008-07-01

    Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.

  2. Lidocaine-induced apoptosis of gingival fibroblasts: participation of cAMP and PKC activity.

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    Villarruel, Emmanuel Quinteros; Borda, Enri; Sterin-Borda, Leonor; Orman, Betina

    2011-08-01

    Local anaesthetics are drugs that prevent or relieve pain by interrupting nervous conduction and are the most commonly used drugs in dentistry. Their main targets of action are voltage-dependent Na+ channels. The Na+ channel is modulated by phosphorylation of two enzymes: PKA (protein kinase A) and PKC (protein kinase C). We studied the ability of lidocaine to modulate programmed cell death of human gingival fibroblasts and the mechanisms involved in this process. Lidocaine (10-5 to 10-7 M) stimulated apoptosis in primary cultures and the caspase-3 activity in a concentration-dependent manner. The stimulatory effect of lidocaine on apoptosis was attenuated in the presence of HA 1004 (PKA inhibitor) and stimulated by staurosporine and Go 6976 (PKC inhibitors). Lidocaine-induced apoptotic nuclei correlated positively with cAMP accumulation and negatively with PKC activity. These results show that lidocaine promotes apoptosis in human gingival fibroblasts at concentrations used for local anaesthesia. The mechanism involves PKA stimulation and PKC inhibition, which in turn stimulates caspase-3 and leads to programmed cell death.

  3. Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih [Institute of Biomedical Sciences, Taiwan (China)] [and others

    1995-08-01

    Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.

  4. Oral heparin: status review

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    Gomez-Orellana Isabel

    2006-05-01

    Full Text Available Abstract Unfractionated heparin and low molecular weight heparin are the most commonly used antithrombotic and thromboprophylactic agents in hospital practice. Extended out-of-hospital treatment is inconvenient in that these agents must be administered parenterally. Current research is directed at development of a safe and effective oral antithrombotic agent as an alternative for the effective, yet difficult to use vitamin K antagonists. A novel drug delivery technology that facilitates transport of drugs across the gastrointestinal epithelium has been harnessed to develop an oral dosage form of unfractionated heparin. Combining unfractionated heparin with the carrier molecule, sodium N-(8 [2-hydroxybenzoyl]amino caprylate, or SNAC has markedly increased the gastrointestinal absorption of this drug. Preclinical and clinical studies to-date suggests that oral heparin-SNAC can confer a clinical efficacious effect; further confirmation is sought in planned clinical trials.

  5. Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells.

    Science.gov (United States)

    Ogawa, Shin-ichiro; Tokumoto, Yasuhito; Miyake, Jun; Nagamune, Teruyuki

    2011-08-01

    Induced pluripotent stem cells (iPSCs) prepared from somatic cells might become a novel therapeutic tool in regenerative medicine, especially for the central nervous system (CNS). In this study, we attempted to induce O4-positive (O4(+)) oligodendrocytes from adult human fibroblast-derived iPSCs in vitro. We used two adult human iPSC cell lines, 201B7 and 253G1. 201B7 was induced by four-gene transduction (oct4, sox2, klf4, c-myc), and 253G1 was induced by three-gene transduction (oct4, sox2, klf4). We treated these cells with two in vitro oligodendrocyte-directed differentiation protocols that were optimized for human embryonic stem cells. One protocol used platelet-derived growth factor as the major mitogen for oligodendrocyte lineage cells, and the other protocol used epidermal growth factor (EGF) as the mitogen. Although the differentiation efficiency was low (less than 0.01%), we could induce O4(+) oligodendrocytes from 253G1 cells using the EGF-dependent differentiation protocol. This is the first report of the in vitro induction of oligodendrocytes differentiation from human iPSCs.

  6. Arecoline-induced myofibroblast transdifferentiation from human buccal mucosal fibroblasts is mediated by ZEB1.

    Science.gov (United States)

    Chang, Yu-Chao; Tsai, Chung-Hung; Lai, You-Liang; Yu, Cheng-Chia; Chi, Wan-Yu; Li, Jung Jung; Chang, Wen-Wei

    2014-04-01

    Oral submucous fibrosis (OSF) is considered as a pre-cancerous condition of the oral mucosa and is highly associated with habitual areca quid chewing. Arecoline is the major alkaloid in areca quid and is thought to be involved in the pathogenesis of OSF. Our previous studies have demonstrated that arecoline could induce epithelial-mesenchymal transition (EMT)-related factors in primary human buccal mucosal fibroblasts (BMFs). Therefore, we investigated the expression of zinc finger E-box binding homeobox 1 (ZEB1), which is a well-known transcriptional factor in EMT, in OSF tissues and its role in arecoline-induced myofibroblast transdifferentiation from BMFs. The expression of ZEB1, as well as the myofibroblast marker α-smooth muscle actin (α-SMA), was significantly increased in OSF tissues, respectively. With immunofluorescence analysis, arecoline induced the formation of α-SMA-positive stress fibres in BMFs expressing nuclear ZEB1. Arecoline also induced collagen contraction of BMFs in vitro. By chromatin immunoprecipitation, the binding of ZEB1 to the α-SMA promoter in BMFs was increased by arecoline. The promoter activity of α-SMA in BMFs was also induced by arecoline, while knockdown of ZEB1 abolished arecoline-induced α-SMA promoter activity and collagen contraction of BMFs. Long-term exposure of BMFs to arecoline induced the expression of fibrogenic genes and ZEB1. Silencing of ZEB1 in fibrotic BMFs from an OSF patient also suppressed the expression of α-SMA and myofibroblast activity. Inhibition of insulin-like growth factor receptor-1 could suppress arecoline-induced ZEB1 activation in BMFs. Our data suggest that ZEB1 may participate in the pathogenesis of areca quid-associated OSF by activating the α-SMA promoter and inducing myofibroblast transdifferentiation from BMFs. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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

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    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

  8. Transforming growth factor-β1 involved in urotensin Ⅱ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yong-gang; HU Yan-chao; MAO Yan-yan; WEI Rui-hong; BAO Shi-lin; WU Li-biao; KUANG Ze-jian

    2010-01-01

    Background Urotensin Ⅱ (UⅡ) is a new vasoconstrictive peptide that may activate the adventitial fibroblasts.Transforming growth factor-β1 (TGF-β1) is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-β1 is involved in UⅡ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.Methods Adventitial fibroblasts were prepared by the explant culture method. TGF-β1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression of α-smooth nuscle actin (α-SM-actin), the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR (real-time RT-PCR) and Western blotting, respectively.Results UⅡ stimulated the secretion of TGF-β1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% (P <0.01), than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10-8 mol/L of UⅡ (P <0.01), which was increased by 59.9%,compared with in the control group (P <0.01). The secretion of TGF-β1 induced by UⅡ was significantly blocked by SB-710411 (10-7 mol/L), a specific antagonist of UⅡ receptor. In addition, both UⅡ (10-8 mol/L) and TGF-β1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UⅡ was inhibited by the specific neutralizing antibody (20 μg/ml) of TGF-β1, while the α-SM-actin expression stimulated by TGF-β1 (20 ng/ml)was inhibited by SB-710411 (10-7 mol/L), the UⅡ receptor antagonist.Conclusion This study suggests that UⅡ could induce TGF-β1 secretion in adventitial fibroblasts via UT activation, and TGF-β1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UⅡ, and TGF-β1

  9. Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction

    Science.gov (United States)

    Pena, Ana-Maria; Fagot, Dominique; Olive, Christian; Michelet, Jean-François; Galey, Jean-Baptiste; Leroy, Frédéric; Beaurepaire, Emmanuel; Martin, Jean-Louis; Colonna, Anne; Schanne-Klein, Marie-Claire

    2010-09-01

    Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

  10. Connective tissue growthfactor induces transforma-tion of renal fibroblasts into myofibroblasts

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Connective tissue growth factor (CTGF) is thought to be a specific mediator of TGF-β profibrogenic effect, but the role of CTGF in the transformation of renal fibroblasts to myofibroblasts, which are the most important host cells in the renal chronic fibrosis, is still unknown.Aimed at observing the biological effect of CTGF, we set up a cell line overexpressing CTGF, and analyzed the amount of myofibroblast and the level of extracellular matrix Collagen Ⅲ mRNA in cultured cells. Our results show that CTGF can directly induce myofibroblasts formation, and increase the level of Collagen Ⅲ mRNA. This suggests that CTGF may be a novel pharmacotherapeutical target protein for the interference with renal fibrosis, thus providing useful theoretic possibility for clinical prevention of chronic progression of kidney disease.``

  11. Dynamic changes of apoptosis in duck embryo fibroblasts induced by new type Gosling viral enteritis virus

    Institute of Scientific and Technical Information of China (English)

    Shun Chen; Anchun Cheng; Mingshu Wang; Xiaoyue Chen

    2008-01-01

    The monolayer duck embryo fibroblast (DEF) cells were experimentally infected with new type Gosling viral enteritis virus (NGVEV) and the dynamic changes of apoptosis were detected at different time points after NGVEV infection by transmission electron microscopy (TEM), DNA agarose gel electrophoresis and Annexin V-FITC/PI stained fluorescence-activated cell sorter (FACS). The result shows that NGVEV can induce infected cells undergoing apoptosis and changing regularly. A series of characteristic apoptotic morphological changes including shrinkage of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies, wereobserved by TEM. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. And using flow cytometry analysis of Annexin V-FITC/PI staining, the dead, viable, apoptotic and necrotic cells could be analyzed quantitatively.

  12. Inactivation of Heparin by Cationically Modified Chitosan

    Directory of Open Access Journals (Sweden)

    Barbara Lorkowska-Zawicka

    2014-06-01

    Full Text Available This study was performed to evaluate the ability of N-(2-hydroxypropyl-3-tri methylammonium chitosan chloride (HTCC, the cationically modified chitosan, to form biologically inactive complexes with unfractionated heparin and thereby blocking its anticoagulant activity. Experiments were carried out in rats in vivo and in vitro using the activated partial thromboplastin time (APTT and prothrombin time (PT tests for evaluation of heparin anticoagulant activity. For the first time we have found that HTCC effectively neutralizes anticoagulant action of heparin in rat blood in vitro as well as in rats in vivo. The effect of HTCC on suppression of heparin activity is dose-dependent and its efficacy can be comparable to that of protamine-the only agent used in clinic for heparin neutralization. HTCC administered i.v. alone had no direct effect on any of the coagulation tests used. The potential adverse effects of HTCC were further explored using rat experimental model of acute toxicity. When administered i.p. at high doses (250 and 500 mg/kg body weight, HTCC induced some significant dose-dependent structural abnormalities in the liver. However, when HTCC was administered at low doses, comparable to those used for neutralization of anticoagulant effect of heparin, no histopathological abnormalities in liver were observed.

  13. High-LET Radiation Induced Chromosome Aberrations in Normal and Ataxia Telangiectasia Fibroblast Cells

    Science.gov (United States)

    Kawata, Tetsuya; George, Ms Kerry; Cucinotta, Francis A.; Shigematsu, Naoyuki; Ito, Hisao; Furusawa, Yoshiya; Uno, Takashi

    We investigated the effects of heavy ions beams on chromosomal aberrations in normal and AT cells. Normal and AT fibroblast cells arrested at G0/G1 phase were irradiated with 2 Gy of X-rays, 490 MeV/u Silicon (LET 55 keV/micron), 500 MeV/u Iron (LET 185 keV/micron) and 200 MeV/u Iron (LET 440 keV/micron) particles, and then cells were allowed to repair for 24 hours at 37 degrees before subculture. Calyculin-A induced PCC method was employed to collect G2/M chromosomes and whole DNA probes 1 and 3 were used to analyze chromosomal aberrations such as color-junctions, deletions, simple exchanges (incomplete and reciprocal exchanges) and complex-type exchanges. The percentages of aberrant cells were higher when normal and AT cells were exposed to heavy ions compared to X-rays, and had a tendency to increase with increasing LET up to 185 keV/micron and then decreased at 440 keV/micron. When the frequency of color-junctions per cell was compared after X-ray exposure, AT cells had around three times higher frequency of color-junctions (mis-rejoining) than normal cells. However, at 185 keV/micron there was no difference in the frequency of color-junctions between two cell lines. It was also found that the frequency of simple exchanges per cell was almost constant in AT cells regardless LET levels, but it was LET dependent for normal cells. Interestingly, the frequency of simple exchanges was higher for normal fibroblast cells when it was compared at 185 keV/micron, but AT cells had more complex-type exchanges at the same LET levels. Heavy ions are more efficient in inducing chromosome aberrations in normal and AT cells compared to X-rays, and the aberration types between normal and AT fibroblast appeared different probably due to difference in the ATM gene function.

  14. Plasmid-based generation of induced neural stem cells from adult human fibroblasts

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    Philipp Capetian

    2016-10-01

    Full Text Available Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72% and glial cells (9% astrocytes, 6% oligodendrocytes. Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts. Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside

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

    Energy Technology Data Exchange (ETDEWEB)

    Lerner, Chad A.; Rutagarama, Pierrot; Ahmad, Tanveer; Sundar, Isaac K.; Elder, Alison; Rahman, Irfan, E-mail: irfan_rahman@urmc.rochester.edu

    2016-09-02

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

  16. Heparin pharmacovigilance in Brazil.

    Science.gov (United States)

    Junqueira, Daniela Rezende Garcia; Viana, Thércia Guedes; Peixoto, Eliane R de M; Barros, Fabiana C R de; Carvalho, Maria das Graças; Perini, Edson

    2011-01-01

    To investigate the biological origin of injectable unfractioned heparin available in Brazilian market by discussing the impact of the profile of commercial products and the changes in heparin monograph on the drug safety. The Anvisa data base for the Registered Products of Pharmaceutical Companies and the Dictionary of Pharmaceutical Specialties (DEF 2008/2009) were searched. A survey with industries having an active permission for marketing the drug in Brazil was conducted. Five companies were granted a permission to market unfractioned heparin in Brazil. Three of them are porcine in origin and two of them are bovine in origin, with only one explicitly showing this information in the package insert. The effectiveness and safety of heparin studied in non-Brazilian populations may not represent the Brazilian reality, since most countries no longer produce bovine heparin. The currently marketed heparin has approximately 10% less anticoagulant activity than that previously produced and this change may have clinical implications. Evidence about the lack of dose interchangeability between bovine and porcine heparins and the unique safety profile of these drugs indicates the need to follow the treatment and the patients' response. Events threatening the patient's safety must be reported to the pharmacovigilance system in each particular country.

  17. IGF-I induced genes in stromal fibroblasts predict the clinical outcome of breast and lung cancer patients

    Directory of Open Access Journals (Sweden)

    Herrmann Richard

    2010-01-01

    Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-I signalling is important for cancer initiation and progression. Given the emerging evidence for the role of the stroma in these processes, we aimed to characterize the effects of IGF-I on cancer cells and stromal cells separately. Methods We used an ex vivo culture model and measured gene expression changes after IGF-I stimulation with cDNA microarrays. In vitro data were correlated with in vivo findings by comparing the results with published expression datasets on human cancer biopsies. Results Upon stimulation with IGF-I, breast cancer cells and stromal fibroblasts show some common and other distinct response patterns. Among the up-regulated genes in the stromal fibroblasts we observed a significant enrichment in proliferation associated genes. The expression of the IGF-I induced genes was coherent and it provided a basis for the segregation of the patients into two groups. Patients with tumours with highly expressed IGF-I induced genes had a significantly lower survival rate than patients whose tumours showed lower levels of IGF-I induced gene expression (P = 0.029 - Norway/Stanford and P = 7.96e-09 - NKI dataset. Furthermore, based on an IGF-I induced gene expression signature derived from primary lung fibroblasts, a separation of prognostically different lung cancers was possible (P = 0.007 - Bhattacharjee and P = 0.008 - Garber dataset. Conclusion Expression patterns of genes induced by IGF-I in primary breast and lung fibroblasts accurately predict outcomes in breast and lung cancer patients. Furthermore, these IGF-I induced gene signatures derived from stromal fibroblasts might be promising predictors for the response to IGF-I targeted therapies. See the related commentary by Werner and Bruchim: http://www.biomedcentral.com/1741-7015/8/2

  18. Neuroligin-1 induces neurite outgrowth through interaction with neurexin-1ß and activation of fibroblast growth factor receptor-1

    DEFF Research Database (Denmark)

    Gjørlund, Michelle D; Nielsen, Janne; Pankratova, Stanislava;

    2012-01-01

    the neuritogenic effect of NLGN1 in cultures of hippocampal neurons. Our results show that NLGN1, both in soluble and membrane-bound forms, induces neurite outgrowth that depends on the interaction with NRXN1ß and on activation of fibroblast growth factor receptor-1. In addition, we demonstrate that a synthetic...

  19. Bacillus Calmette Guerin induces fibroblast activation both directly and through macrophages in a mouse bladder cancer model.

    Directory of Open Access Journals (Sweden)

    Catalina Lodillinsky

    Full Text Available BACKGROUND: Bacillus Calmette-Guerin (BCG is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.

  20. Novel Experimental and Clinical Therapeutic Uses of Low-Molecular-Weight Heparin/Protamine Microparticles

    Directory of Open Access Journals (Sweden)

    Shingo Nakamura

    2012-01-01

    Full Text Available Low-molecular-weight heparin/protamine microparticles (LMW-H/P MPs were produced as a carrier for heparin-binding growth factors (GFs and for various adhesive cells. A mixture of low-molecular-weight heparin (MW: approximately 5000 Da, 6.4 mg/mL and protamine (MW: approximately 3000 Da, 10 mg/mL at a ratio of 7:3 (vol:vol yields a dispersion of microparticles (0.5–3 µm in diameter. LMW-H/P MPs immobilize, control the release and protect the activity of GFs. LMW-H/P MPs can also bind to cell surfaces, causing these cells to interact with the LMW-H/P MPs, inducing cells/MPs-aggregate formation and substantially promoting cellular viability. Furthermore, LMW-H/P MPs can efficiently bind to tissue culture plates and retain the binding of important GFs, such as fibroblast growth factor (FGF-2. The LMW-H/P MPs-coated matrix with various GFs or cytokines may provide novel biomaterials that can control cellular activity such as growth and differentiation. Thus, LMW-H/P MPs are an excellent carrier for GFs and various cells and are an efficient coating matrix for cell cultures.

  1. Fibroblast Growth Factor 21 Deficiency Attenuates Experimental Colitis-Induced Adipose Tissue Lipolysis

    Directory of Open Access Journals (Sweden)

    Liming Liu

    2017-01-01

    Full Text Available Aims. Nutrient deficiencies are common in patients with inflammatory bowel disease (IBD. Adipose tissue plays a critical role in regulating energy balance. Fibroblast growth factor 21 (FGF21 is an important endocrine metabolic regulator with emerging beneficial roles in lipid homeostasis. We investigated the impact of FGF21 in experimental colitis-induced epididymal white adipose tissue (eWAT lipolysis. Methods. Mice were given 2.5% dextran sulfate sodium (DSS ad libitum for 7 days to induce colitis. The role of FGF21 was investigated using antibody neutralization or knockout (KO mice. Lipolysis index and adipose lipolytic enzymes were determined. In addition, 3T3-L1 cells were pretreated with IL-6, followed by recombinant human FGF21 (rhFGF21 treatment; lipolysis was assessed. Results. DSS markedly decreased eWAT/body weight ratio and increased serum concentrations of free fatty acid (FFA and glycerol, indicating increased adipose tissue lipolysis. eWAT intracellular lipolytic enzyme expression/activation was significantly increased. These alterations were significantly attenuated in FGF21 KO mice and by circulating FGF21 neutralization. Moreover, DSS treatment markedly increased serum IL-6 and FGF21 levels. IL-6 pretreatment was necessary for the stimulatory effect of FGF21 on adipose lipolysis in 3T3-L1 cells. Conclusions. Our results demonstrate that experimental colitis induces eWAT lipolysis via an IL-6/FGF21-mediated signaling pathway.

  2. Tumor-associated fibroblast-conditioned medium induces CDDP resistance in HNSCC cells.

    Science.gov (United States)

    Steinbichler, Teresa Bernadette; Metzler, Veronika; Pritz, Christian; Riechelmann, Herbert; Dudas, Jozsef

    2016-01-19

    EMT (epithelial to mesenchymal transition) contributes to tumor progression and metastasis. We aimed to investigate the effects of EMT on CDDP resistance in HNSCC (head and neck squamous cell carcinoma)-cells. EMT was induced using conditioned medium from a tumor cell/fibroblast co-culture. HNSCC cells were alternatively treated with TGF-β1. The response to CDDP was evaluated with viability and clonogenic assays. Treatment of SCC-25/ Detroit 562 cells with conditioned medium increased viability of the tumor cells. Moreover, it doubled the IC50 of CDDP of SCC-25 cells from 6.2 μM to 13.1 μM (p cells was increased following treatment with conditioned medium from 13.1 μM to 26.8 μM (p cells treated with co-culture conditioned medium than in controls (p 0.1). Cell free medium from a co-culture was able to induce EMT in HNSCC cells. Co-culture treated HNSCC cells revealed increased viability and were less sensitive to CDDP treatment. TGF-β1 also induced a mesenchymal phenotype, but did not alter resistance to CDDP in HNSCC cells.

  3. Activation of Sirt1 by resveratrol inhibits TNF-α induced inflammation in fibroblasts.

    Directory of Open Access Journals (Sweden)

    Xiaoxia Zhu

    Full Text Available Inflammation is one of main mechanisms of autoimmune disorders and a common feature of most diseases. Appropriate suppression of inflammation is a key resolution to treat the diseases. Sirtuin1 (Sirt1 has been shown to play a role in regulation of inflammation. Resveratrol, a potent Sirt1 activator, has anti-inflammation property. However, the detailed mechanism is not fully understood. In this study, we investigated the anti-inflammation role of Sirt1 in NIH/3T3 fibroblast cell line. Upregulation of matrix metalloproteinases 9 (MMP-9, interleukin-1beta (IL-1β, IL-6 and inducible nitric oxide synthase (iNOS were induced by tumor necrosis factor alpha (TNF-α in 3T3 cells and resveratrol suppressed overexpression of these pro-inflammatory molecules in a dose-dependent manner. Knockdown of Sirt1 by RNA interference caused 3T3 cells susceptible to TNF-α stimulation and diminished anti-inflammatory effect of resveratrol. We also explored potential anti-inflammatory mechanisms of resveratrol. Resveratrol reduced NF-κB subunit RelA/p65 acetylation, which is notably Sirt1 dependent. Resveratrol also attenuated phosphorylation of mammalian target of rapamycin (mTOR and S6 ribosomal protein (S6RP while ameliorating inflammation. Our data demonstrate that resveratrol inhibits TNF-α-induced inflammation via Sirt1. It suggests that Sirt1 is an efficient target for regulation of inflammation. This study provides insight on treatment of inflammation-related diseases.

  4. Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells

    Directory of Open Access Journals (Sweden)

    Phillips Stacia L

    2006-04-01

    Full Text Available Abstract Background We have demonstrated that in some human cancer cells both chronic mild heat and ionizing radiation exposures induce a transient block in S and G2 phases of the cell cycle. During this delay, cyclin B1 protein accumulates to supranormal levels, cyclin B1-dependent kinase is activated, and abrogation of the G2/M checkpoint control occurs resulting in mitotic catastrophe (MC. Results Using syngenic mouse embryonic fibroblasts (MEF with wild-type or mutant p53, we now show that, while both cell lines exhibit delays in S/G2 phase post-irradiation, the mutant p53 cells show elevated levels of cyclin B1 followed by MC, while the wild-type p53 cells present both a lower accumulation of cyclin B1 and a lower frequency of MC. Conclusion These results are in line with studies reporting the role of p53 as a post-transcriptional regulator of cyclin B1 protein and confirm that dysregulation of cyclin B1 promote radiation-induced MC. These findings might be exploited to design strategies to augment the yield of MC in tumor cells that are resistant to radiation-induced apoptosis.

  5. Ras-inducible immortalized fibroblasts: focus formation without cell cycle deregulation.

    Science.gov (United States)

    Jacobsen, Kivin; Groth, Anja; Willumsen, Berthe M

    2002-05-02

    The Ras oncogene transforms cultured murine fibroblasts into malignant, focus-forming cells, whose lack of contact inhibition is evidenced by high saturation densities. In order to investigate the reversibility of Ras transformation, as well as the kinetics of Ras-induced changes, cell lines that conditionally express oncogenic Ras were constructed. Both focus formation and increased saturation density were inducible and fully reversible. In exponentially growing cells, oncogenic Ras-expression had no effect on proliferation rates, Erk phosphorylation, or the level of cyclin D1, and Ras-induction did not confer serum-independent growth. As expected, growth to high density in uninduced cells led to quiescence with a low level of cyclin D1 and no active Erk; in this setting, Ras induction prevented full downregulation of cyclin D1 and inactivation of Erk. Our results show that Ras expression to a level sufficient for transformation leads to relatively subtle effects on known downstream targets, and that the focus formation and increased saturation density growth induced by Ras is not a result of growth factor independence.

  6. STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

    Directory of Open Access Journals (Sweden)

    Stefan Kippenberger

    Full Text Available Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

  7. STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

    Science.gov (United States)

    Kippenberger, Stefan; Zöller, Nadja; Kleemann, Johannes; Müller, Jutta; Kaufmann, Roland; Hofmann, Matthias; Bernd, August; Meissner, Markus; Valesky, Eva

    2015-01-01

    Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk) without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk) and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

  8. Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.

    Science.gov (United States)

    Conte, Enrico; Gili, Elisa; Fagone, Evelina; Fruciano, Mary; Iemmolo, Maria; Vancheri, Carlo

    2014-07-16

    Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways.

  9. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    Science.gov (United States)

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated.

  10. Gingival fibroblasts as a promising source of induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hiroshi Egusa

    Full Text Available BACKGROUND: Induced pluripotent stem (iPS cells efficiently generated from accessible tissues have the potential for clinical applications. Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort. METHODOLOGY/PRINCIPAL FINDINGS: We herein demonstrate iPS cell generation from adult wild-type mouse gingival fibroblasts (GFs via introduction of four factors (Oct3/4, Sox2, Klf4 and c-Myc; GF-iPS-4F cells or three factors (the same as GF-iPS-4F cells, but without the c-Myc oncogene; GF-iPS-3F cells without drug selection. iPS cells were also generated from primary human gingival fibroblasts via four-factor transduction. These cells exhibited the morphology and growth properties of embryonic stem (ES cells and expressed ES cell marker genes, with a decreased CpG methylation ratio in promoter regions of Nanog and Oct3/4. Additionally, teratoma formation assays showed ES cell-like derivation of cells and tissues representative of all three germ layers. In comparison to mouse GF-iPS-4F cells, GF-iPS-3F cells showed consistently more ES cell-like characteristics in terms of DNA methylation status and gene expression, although the reprogramming process was substantially delayed and the overall efficiency was also reduced. When transplanted into blastocysts, GF-iPS-3F cells gave rise to chimeras and contributed to the development of the germline. Notably, the four-factor reprogramming efficiency of mouse GFs was more than 7-fold higher than that of fibroblasts from tail-tips, possibly because of their high proliferative capacity. CONCLUSIONS/SIGNIFICANCE: These results suggest that GFs from the easily obtainable gingival tissues can be readily reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular reprogramming and pluripotency for future clinical applications

  11. Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

    Science.gov (United States)

    Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

    2015-07-01

    It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

  12. Biodentine and mineral trioxide aggregate induce similar cellular responses in a fibroblast cell line.

    Science.gov (United States)

    Corral Nuñez, Camila M; Bosomworth, Helen J; Field, Claire; Whitworth, John M; Valentine, Ruth A

    2014-03-01

    The aim of this study was to assess the cell viability and messenger RNA expression of interleukin (IL)-1α and IL-6 in 3T3 fibroblast cells when in direct contact with Biodentine (Septodont, Saint Maur de Fossés, France) and mineral trioxide aggregate (MTA). Biodentine and MTA were coated onto coverslips and allowed to set. An uncoated coverslip and one coated with GC Fuji IX (GC Corporation, Tokyo, Japan) were used as controls. Coverslips were cultured with 3T3 fibroblast cells. Cell viability was assessed quantitatively using AlamarBlue dye (Serotec, Oxford, UK) after 3, 6, 24, and 72 hours. Morphologic cell changes of 3T3 cells in contact with BD and MTA were observed by scanning electron microscopy, and cytokine expression was assessed at the messenger RNA level by semiquantitative reverse-transcription polymerase chain reaction after 3 and 24 hours of direct contact with the materials. Cells in contact with Biodentine and MTA showed similar viability to untreated control cells at all time points, with the exception of 6 hours when viability was decreased with both treatments. Examination by scanning electron microscopy revealed cells adhering to most of the Biodentine surface after 24 hours. However, for MTA samples, significantly fewer cells were observed. The messenger RNA expression of IL-1α and IL-6 by cells in contact with Biodentine was similar to cells in contact with MTA. Biodentine and MTA showed similar cytotoxicity and induced a similar pattern of cytokine expression. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  13. Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G2/M accumulation in cardiac fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Xu Dong LIAO; Xiao Hui WANG; Hai Jing JIN; Lan Ying CHEN; Quan CHEN

    2004-01-01

    Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study,a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes,but not cardiac fibroblasts,underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψm) reduction,indicative of mitochondrial permeability transition pore (PTP)opening. Cyclosporin A,an inhibitor of PTP,inhibited stretch-induced cyto c release,△ψm reduction and apoptosis,suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins,including Bax and Bad,in cardiomyocytes,but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψm reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery,probably by targeting to PTP. Interestingly,the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis,suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore,we showed that p21 was upregulated and cyclin B l was downregulated only in cardiac fibroblasts,which may be associated with G2/M accumulation in response to mechanical stretch.

  14. Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts.

    Science.gov (United States)

    Audo, Rachel; Deschamps, Véronique; Hahne, Michael; Combe, Bernard; Morel, Jacques

    2007-01-01

    Synovial hyperplasia in rheumatoid arthritis (RA) has been associated with apoptosis deficiency of RA fibroblast-like synoviocytes (FLSs). Celecoxib is a non-steroidal anti-inflammatory drug that has been demonstrated to induce apoptosis in some cellular systems. We have therefore examined the dose- and time-dependent effects of celecoxib on RA FLS viability. Treatment of RA FLSs with celecoxib for 24 hours reduced their viability in a dose-dependent manner. Analysis of celecoxib-treated RA FLSs for their content of apoptotic and necrotic cells by Annexin V staining and TO-PRO-3 uptake displayed only few apoptotic cells. Caspase 3, a key mediator of apoptosis, was not activated in celecoxib-treated RA FLSs, and the presence of specific caspase 3 or pan-caspase inhibitors did not affect celecoxib-induced cell death. Moreover, we could not detect other signs of apoptosis, such as cleavage of poly(ADP-ribose) polymerase, caspase 8 or 9, or DNA fragmentation. We therefore conclude that apoptosis is not the major death pathway in celecoxib-treated RA FLSs.

  15. Phosphatidylinositol 3-kinase inhibitor, LY294002, induced senescence-like changes in human diploid fibroblasts

    Institute of Scientific and Technical Information of China (English)

    李淑萍; 张宗玉; 童坦君

    2003-01-01

    Objective To reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.Methods Using a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association β-galactosidase staining as well as senescence association CKIs, p16 INK4 and p21 Cip1 protein expressions were all measured in the low passages of 2BS cells.Results Both 25 μmol/L and 50 μmol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G 1 phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association β-galactosidase (P<0.05). In addition, LY294002 could induce time-course expressions of p16 INK4 and p21 Cip1 in 2BS cell lines.Conclusions PI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two enescence associated CKIs,p16 INK4 and p21 Cip1, might be involved in this senescence phenotype proceeding in 2BS cell lines.

  16. Dietary moderately oxidized oil induces expression of fibroblast growth factor 21 in the liver of pigs

    Directory of Open Access Journals (Sweden)

    Varady Juliane

    2012-03-01

    Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21, whose expression is induced by peroxisome proliferator-activated receptor α (PPARα, has been recently identified as a novel metabolic regulator which plays a crucial role in glucose homeostasis, lipid metabolism, insulin sensitivity and obesity. Previous studies have shown that administration of oxidized fats leads to an activation of PPARα in the liver. Therefore, the present study investigated the hypothesis that feeding of oxidized fats causes an induction of FGF21 in the liver. Methods Twenty four crossbred pigs were allocated to two groups of 12 pigs each and fed nutritionally adequate diets with either fresh rapeseed oil or oxidized rapeseed oil prepared by heating at a temperature of 175°C for 72 h. Results In pigs fed the oxidized fat mRNA abundance and protein concentrations of FGF21 in liver were significantly increased (P P P Conclusion The present study shows for the first time that administration of an oxidized fat induces the expression of FGF21 in the liver, probably mediated by activation of PPARα. Induction of FGF21 could be involved in several effects observed in animals administered an oxidized fat.

  17. The profile of lysosomal exoglycosidases in replicative and stress-induced senescence in early passage human fibroblasts

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    Małgorzata Knaś

    2012-07-01

    Full Text Available The aim of the present study was to assess the profiles of the exoglycosidases: N-acetyl-β-hexosoaminidase, β glucuronidase and β galactosidase, α mannosidase and α fucosidase in fibroblast culture undergoing replicative and stress-induced senescence. Half of the cell culture was grown in normal conditions, without the stressor, and the other half of the cell was treated with 0.15 mM tert-butylhydroperoxide. The activities of total N-acetyl-β-hexosoaminidase as well as β glucuronidase in the cell lysate were determined in duplicates using the method of Marciniak et al. The activities of β galactosidase, α mannosidase and α fucosidase in the cell lysate were determined in duplicates using the method of Chatteriee et al. with the modification by Zwierz et al. The activities of the exoglycosidases examined, with the exception of β glucuronidase, showed a significant increase between individual days of the experiment in both non-stressed and stressed fibroblast cell culture. On each day of the experiment, in the cell lysate of stressed fibroblasts, the activities of exoglycosidases were significantly higher compared to the non-stressed cells. There were very strong correlations between SA-β-GAL staining and b galactosidase activity on individual days of the experiment in both non-stressed and stressed fibroblast cell culture. Replicative and stress-induced senescence results in significant changes to the level of lysosomal exoglycosidases, and results in enhanced lysosomal degradative capacity.

  18. Aloin Protects Skin Fibroblasts from Heat Stress-Induced Oxidative Stress Damage by Regulating the Oxidative Defense System.

    Directory of Open Access Journals (Sweden)

    Fu-Wei Liu

    Full Text Available Oxidative stress is commonly involved in the pathogenesis of skin damage induced by environmental factors, such as heat stress. Skin fibroblasts are responsible for the connective tissue regeneration and the skin recovery from injury. Aloin, a bioactive compound in Aloe vera, has been reported to have various pharmacological activities, such as anti-inflammatory effects. The aim of this study was to investigate the protective effect of aloin against heat stress-mediated oxidative stress in human skin fibroblast Hs68 cells. Hs68 cells were first incubated at 43°C for 30 min to mimic heat stress. The study was further examined if aloin has any effect on heat stress-induced oxidative stress. We found that aloin protected Hs68 cells against heat stress-induced damage, as assessed by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay. Aloin protected Hs68 cells by regulating reactive oxygen species production and increasing the levels of glutathione, cytosolic and mitochondrial superoxide dismutase. Aloin also prevented the elevation of thiobarbituric acid reactive substances and the reduction of 8-OH-dG induced by heat stress. These results indicated that aloin protected human skin fibroblasts from heat stress-induced oxidative stress damage by regulating the oxidative defense system.

  19. Endoglin negatively regulates transforming growth factor beta1-induced profibrotic responses in intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts isolated from strictures in Crohn\\'s disease (CD) exhibit reduced responsiveness to stimulation with transforming growth factor (TGF) beta1. TGF-beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. The membrane glycoprotein, endoglin, is a negative regulator of TGF-beta1. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies of patients undergoing intestinal resection for CD strictures or from control patients. Endoglin expression was assessed using confocal microscopy, flow cytometry and western blot. The effect of small interfering (si) RNA-mediated knockdown and plasmid-mediated overexpression of endoglin on fibroblast responsiveness to TGF-beta1 was assessed by examining smad phosphorylation, smad binding element (SBE) promoter activity, connective tissue growth factor (CTGF) expression and ability to contract collagen. RESULTS: Crohn\\'s stricture fibroblasts expressed increased constitutive cell-surface and whole-cell endoglin relative to control cells. Endoglin co-localized with filamentous actin. Fibroblasts treated with siRNA directed against endoglin exhibited enhanced TGF-beta1-mediated smad-3 phosphorylation, and collagen contraction. Cells transfected with an endoglin plasmid did not respond to TGF-beta1 by exhibiting SBE promoter activity or producing CTGF. CONCLUSION: Fibroblasts from strictures in CD express increased constitutive endoglin. Endoglin is a negative regulator of TGF-beta1 signalling in the intestinal fibroblast, modulating smad-3 phosphorylation, SBE promoter activity, CTGF production and collagen contraction.

  20. Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts

    NARCIS (Netherlands)

    Lundvig, D.M.S.; Pennings, S.W.C.; Brouwer, K.M.; Mtaya-Mlangwa, M.; Mugonzibwa, E.A.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den; Wagener, F.A.D.T.G.

    2015-01-01

    Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-beta1 play a key role in these processes. The persistence of (myo)fibroblasts and their exces

  1. Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts

    NARCIS (Netherlands)

    Lundvig, D.M.S.; Pennings, S.W.C.; Brouwer, K.M.; Mtaya-Mlangwa, M.; Mugonzibwa, E.A.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den; Wagener, F.A.D.T.G.

    2015-01-01

    Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-beta1 play a key role in these processes. The persistence of (myo)fibroblasts and their

  2. Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.

    Directory of Open Access Journals (Sweden)

    Pavel Rossner

    Full Text Available The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells and tested their response to treatment with benzo[a]pyrene (B[a]P and extractable organic matter (EOM from ambient air particles <2.5 µm (PM2.5 collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs including the levels of bulky DNA adducts and the nucleotide excision repair (NER response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9 and absence (-S9 of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (-S9; the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.

  3. Polyguluronate sulfate and its oligosaccharides but not heparin promotes FGF19/FGFR1c signaling

    Science.gov (United States)

    Lan, Ying; Zeng, Xuan; Guo, Zhihua; Zeng, Pengjiao; Hao, Cui; Zhao, Xia; Yu, Guangli; Zhang, Lijuan

    2017-06-01

    Fibroblast growth factor 19(FGF19) functions as a hormone by affecting glucose metabolism. FGF19 improves glucose tolerance when overexpressed in mice with impaired glucose tolerance or diabetes. A functional cellular FGF19 receptor consists of FGF receptor (FGFR) and glycosaminoglycan complexed with either α Klotho or β Klotho. Interestingly, in mice with diet-induced diabetes, a single injection of FGF1 is enough to restore blood sugar levels to a healthy range. FGF1 binds heparin with high affinity whereas FGF19 does not, indicating that polysaccharides other than heparin might enhance FGF19/FGFR signaling. Using a FGFs/FGFR1c signaling-dependent BaF3 cell proliferation assay, we discovered that polyguluronate sulfate (PGS) and its oligosaccharides, PGS12 and PGS25, but not polyguluronate (PG), a natural marine polysaccharide, enhanced FGF19/FGFR1c signaling better than that of heparin based on 3H-thymidine incorporation. Interestingly, PGS6, PGS8, PGS10, PGS12, PGS25, and PGS, but not PG, had comparable FGF1/FGFR1c signal-stimulating activity compared to that of heparin. These results indicated that PGS and its oligosaccharides were excellent FGF1/FGFR1c and FGF19/FGFR1c signaling enhancers at cellular level. Since the inexpensive PGS and PGS oligosaccharides can be absorbed through oral route, these seaweed-derived compounds merit further investigation as novel agents for the treatment of type 2 diabetes through enhancing FGF1/FGFR1c and FGF19/FGFR1c signaling in future.

  4. Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium.

    Science.gov (United States)

    Zhang, Xi; Shen, Man-Ru; Xu, Zhen-Dong; Hu, Zhe; Chen, Chao; Chi, Ya-Li; Kong, Zhen-Dong; Li, Zi-Fu; Li, Xiao-Tong; Guo, Shi-Lei; Xiong, Shao-Hu; Zhang, Chuan-Sen

    2014-05-01

    Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the

  5. Distinct cell stress responses induced by ATP restriction in quiescent human fibroblasts

    Directory of Open Access Journals (Sweden)

    Nirupama Yalamanchili

    2016-10-01

    Full Text Available Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated transcription factors and altered transcription factor subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.

  6. Magnetite nanoparticles induced adaptive mechanisms counteract cell death in human pulmonary fibroblasts.

    Science.gov (United States)

    Radu, Mihaela; Dinu, Diana; Sima, Cornelia; Burlacu, Radu; Hermenean, Anca; Ardelean, Aurel; Dinischiotu, Anca

    2015-10-01

    Magnetite nanoparticles (MNP) have attracted great interest for biomedical applications due to their unique chemical and physical properties, but the MNP impact on human health is not fully known. Consequently, our study proposes to highlight the biochemical mechanisms that underline the toxic effects of MNP on a human lung fibroblast cell line (MRC-5). The cytotoxicity generated by MNP in MRC-5 cells was dose and time-dependent. MNP-treated MRC-5 cells accumulated large amount of iron and reactive oxygen species (ROS) and exhibited elevated antioxidant scavenger enzymes. Reduced glutathione (GSH) depletion and enhanced lipid peroxidation (LPO) processes were also observed. The cellular capacity to counteract the oxidative damage was sustained by high levels of heat shock protein 60 (Hsp60), a protein that confers resistance against ROS attack and inhibition of cell death. While significant augmentations in nitric oxide (NO) and prostaglandine E2 (PGE2) levels were detected after 72 h of MNP-exposure only, caspase-1 was activated earlier starting with 24h post-treatment. Taken together, our results suggest that MRC-5 cells have the capacity to develop cell protection mechanisms against MNP. Detailed knowledge of the mechanisms induced by MNP in cell culture could be essential for their prospective use in various in vivo biochemical applications.

  7. High-LET radiation-induced aberrations in prematurely condensed G2 chromosomes of human fibroblasts

    Science.gov (United States)

    Kawata, T.; Gotoh, E.; Durante, M.; Wu, H.; George, K.; Furusawa, Y.; Cucinotta, F. A.; Dicello, J. F. (Principal Investigator)

    2000-01-01

    PURPOSE: To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIAL AND METHODS: Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290MeV/u), silicon (490MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. RESULTS: The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. CONCLUSIONS: Compared with gamma-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.

  8. Passiflora tarminiana fruits reduce UVB-induced photoaging in human skin fibroblasts.

    Science.gov (United States)

    Bravo, Karent; Duque, Luisa; Ferreres, Federico; Moreno, Diego A; Osorio, Edison

    2017-03-01

    Skin aging is a complex process that is strongly affected by UV radiation, which stimulates the production of reactive oxygen species (ROS) in the epidermis and dermis and subsequently causes skin damage. Among the major consequences are increased collagen degradation and reduced collagen synthesis. Previous reports have demonstrated the beneficial effects of polyphenols for healthy skin. Passiflora tarminiana Coppens & V.E. Barney, a species of the Passifloraceae family, is widely distributed in South America and is rich in flavonoids. We show that UVB radiation increases metalloproteinase 1 (MMP-1) and reduces procollagen production in human dermal fibroblast (HDF) cells in a dose- and time-dependent manner. We examined the antioxidant and antiaging effects of the extract and fractions of P. tarminiana fruits. The fractions showed high polyphenol content (620mg EAG/g) and antioxidant activity, as measured by ORAC (4097μmol ET/g) and ABTS (2992μmol ET/g) assays. The aqueous fraction drastically inhibited the collagenase enzyme (IC50 0.43μg/mL). The extract and fractions presented photoprotective effects by reducing UVB-induced MMP-1 production, increasing UVB-inhibited procollagen production, and decreasing ROS production after UVB irradiation in HDF. Finally, the polyphenol contents of the extracts and fractions from P. tarminiana were analyzed by HPLC-DAD-ESI-MS(n), and procyanidins and glycosylated flavonoids were identified. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. The Effects of Amphiregulin Induced MMP-13 Production in Human Osteoarthritis Synovial Fibroblast

    Directory of Open Access Journals (Sweden)

    Yi-Te Chen

    2014-01-01

    Full Text Available Osteoarthritis (OA belongs to a group of degenerative diseases. Synovial inflammation, cartilage abrasion, and subchondral sclerosis are characteristics of OA. Researchers do not fully understand the exact etiology of OA. However, matrix metalloproteinases (MMPs, which are responsible for cartilage matrix degradation, play a pivotal role in the progression of OA. Amphiregulin (AREG binds to the EGF receptor (EGFR and activates downstream proteins. AREG is involved in a variety of pathological processes, such as the development of tumors, inflammatory diseases, and rheumatoid arthritis. However, the relationship between AREG and MMP-13 in OA synovial fibroblasts (SFs remains unclear. We investigated the signaling pathway involved in AREG-induced MMP-13 production in SFs. AREG caused MMP-13 production in a concentration- and time-dependent manner. The results of using pharmacological inhibitors and EGFR siRNA to block EGFR revealed that the EGFR receptor was involved in the AREG-mediated upregulation of MMP-13. AREG-mediated MMP-13 production was attenuated by PI3K and Akt inhibitors. The stimulation of cells by using AREG activated p65 phosphorylation and p65 translocation from the cytosol to the nucleus. Our results provide evidence that AREG acts through the EGFR and activates PI3K, Akt, and finally NF-kappaB on the MMP-13 promoter, thus contributing to cartilage destruction during osteoarthritis.

  10. Phenotypes induced by NM causing α-skeletal muscle actin mutants in fibroblasts, Sol 8 myoblasts and myotubes

    Directory of Open Access Journals (Sweden)

    Vandamme Drieke

    2009-03-01

    Full Text Available Abstract Background Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with α-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects. Findings We conducted the same biochemical experiments for twelve new actin mutants associated with nemaline myopathy. We observed folding and polymerization defects. Immunostainings of these and eight other mutants in transfected cells revealed typical cellular defects such as nemaline rods or aggregates, decreased incorporation in F-actin structures, membrane blebbing, the formation of thickened actin fibres and cell membrane blebbing in myotubes. Conclusion Our results confirm that NM associated α-actin mutations induce a range of defects at the biochemical level as well as in cultured fibroblasts and muscle cells.

  11. Substrate stiffness influences TGF-β1-induced differentiation of bronchial fibroblasts into myofibroblasts in airway remodeling.

    Science.gov (United States)

    Shi, Yanling; Dong, Yuhui; Duan, Yiyuan; Jiang, Xuemei; Chen, Cheng; Deng, Linhong

    2013-02-01

    Chronic inflammation and remodeling of the bronchial wall are basic hallmarks of asthma. During the process of bronchial wall remodeling, inflammatory factors, such as transforming growth factor-β1 (TGF-β1), are known to induce the differentiation of fibroblasts into myofibroblasts, which leads to excessive synthesis and secretion of extracellular matrix (ECM) proteins, thus thickening and stiffening the basement membrane. However, it has not been thoroughly studied whether or not substrate stiffening affects the TGF-β1‑induced myofibroblast differentiation. In the present study, the influence of substrate stiffness on the process of bronchial fibroblast differentiation into myofibroblasts in the presence of TGF-β1 was investigated. To address this question, we synthesized polydimethylsiloxane (PDMS) substrates with varying degrees of stiffness (Young's modulus of 1, 10 and 50 kPa, respectively). We cultured bronchial fibroblasts on the substrates of varying stiffness in media containing TGF-β1 (10 ng/ml) to stimulate the differentiation of fibroblasts into myofibroblasts. Myofibroblast differentiation was examined using semi-quantitative RT-PCR for the expression of α-smooth muscle actin (α-SMA) mRNA and collagen I mRNA, the enzyme-linked immunosorbent assay method was used to assess the expression of collagen I protein and western blotting to assess the expression of α-SMA protein. The optical magnetic twisting cytometry (OMTC) method was used for the changing of cell mechanical properties. Our findings suggest that when fibroblasts were incubated with TGF-β1 (10 ng/ml) on substrate of varying stiffness, the differentiation of fibroblasts into myofibroblasts was enhanced by increasing substrate stiffness. Compared with those cultured on substrate with Young's modulus of 1 kPa, the mRNA and protein expression of collagen I and α-SMA of fibroblasts cultured on substrates with Young's modulus of 10 and 50 kPa were increased. Furthermore, with the

  12. Severe heparin osteoporosis in pregnancy.

    OpenAIRE

    Griffiths, H. T.; Liu, D T

    1984-01-01

    A case of severe osteoporosis following administration of low dose subcutaneous heparin in pregnancy is reported. Possible reasons for the condition are suggested which caution against the indiscriminate use of subcutaneous heparin in pregnancy.

  13. Angiogenic efficacy of Heparin on chick chorioallantoic membrane

    Directory of Open Access Journals (Sweden)

    Rema Reji

    2012-04-01

    Full Text Available Abstract Heparin is an anticoagulant agent known to have diverse effects on angiogenesis with some reports suggesting that it can induce angiogenesis while a few have indicated of its inhibitory property. Cancer patients treated for venous thromboembolism with low molecular heparin had a better survival than the unfractionated heparin (UFH. Heparin is known to interact with various angiogenic growth factors based on its sulfation modifications within the glycosaminoglycan chains. Therefore it is important to study the mechanism of action of heparin of different molecular weight to understand its angiogenic property. In this concern, we examined the angiogenic response of higher molecular weight Heparin (15 kDa of different concentrations using late CAM assay. Growth of blood vessels in terms of their length and size was measured and thickness of the CAM was calculated morphometrically. The observed increase in the thickness of the CAM is suggestive of the formation of capillary like structures at the treated region. Analysis of the diffusion pattern showed internalized action of heparin that could affect gene expression leading to proliferation of endothelial cells. Angiogenesis refers to formation of new blood vessels from the existing ones and occurrence of new blood vessels at the treated area strongly confirms that heparin of 15 kDa molecular weight has the ability to induce angiogenesis on CAM vascular bed in a dose dependent manner. The results demonstrate the affinity of heparin to induce angiogenesis and provide a novel mechanism by which heparin could be used in therapeutics such as in wound healing process.

  14. Interleukin-8 induces motile behavior and loss of focal adhesions in primary fibroblasts

    DEFF Research Database (Denmark)

    Dunlevy, J R; Couchman, J R

    1995-01-01

    Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutrophil migration. Although fibroblasts are known to secrete IL-8, the actions of this cytokine on fibroblasts have not been previously reported. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8....... Additionally, video microscopy assays using heart conditioned medium depleted with the IL-8 antibody show an increase in the percentage of stationary cells, a consequent decrease in the percentage of migrating cells, and a twofold increase in the mitotic rate. Interleukin-1 alpha and tumor necrosis factor...

  15. CD11c+ CD8+ T Cells Reduce Renal Fibrosis Following Ureteric Obstruction by Inducing Fibroblast Apoptosis

    Directory of Open Access Journals (Sweden)

    Haidong Wang

    2016-12-01

    Full Text Available Tubulointerstitial fibrosis is a common consequence of various kidney diseases that lead to end-stage renal failure, and lymphocyte infiltration plays an important role in renal fibrosis. We previously found that depletion of cluster of differentiation 8+ (CD8+ T cells increases renal fibrosis following ureteric obstruction, and interferon-γ (IFN-γ-expressing CD8+ T cells contribute to this process. CD8+ T cells are cytotoxic T cells; however, whether their cytotoxic effect reduces fibrosis remains unknown. This study showed that CD8+ T cells isolated from obstructed kidney showed mRNA expression of the cytotoxicity-related genes perforin 1, granzyme A, granzyme B, and FAS ligand; additionally, CD8 knockout significantly reduced the expression levels of these genes in obstructed kidney. Infiltrated CD8+ T cells were distributed around fibroblasts, and they are associated with fibroblast apoptosis in obstructed kidney. Moreover, CD11c+ CD8+ T cells expressed higher levels of the cytotoxicity-related genes than CD11c− CD8+ T cells, and infiltrated CD11c+ CD8+ T cells in obstructed kidney could induce fibroblast death in vitro. Results indicated that induction of fibroblast apoptosis partly contributed to the effect of CD8+ T cells on reduction of renal fibrosis. Given that inflammatory cells are involved in fibrosis, our results suggest that kidney fibrosis is a multifactorial process involving different arms of the immune system.

  16. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

    OpenAIRE

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-01-01

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single c...

  17. Salidroside protects against premature senescence induced by ultraviolet B irradiation in human dermal fibroblasts.

    Science.gov (United States)

    Mao, G-X; Xing, W-M; Wen, X-L; Jia, B-B; Yang, Z-X; Wang, Y-Z; Jin, X-Q; Wang, G-F; Yan, J

    2015-06-01

    Salidroside, the predominant component of a Chinese herbal medicine, Rhodiola rosea L., becomes an attractive bio-agent due to its multifunction. Although it is well proposed that this herbal medicine may have photoprotective effect according to the folk hearsay, the direct supportive experimental evidences linking the drug with skin ageing have rarely been reported so far. The study was conducted to investigate the photoprotective role of salidrosdie and its related mechanisms in vitro. First, a premature senescence model induced by UVB irradiation (250 mJ cm(-2)) in human dermal fibroblasts (HDFs) was established, and senescent phenotypes were evaluated by cell morphology, cell proliferation, senescence-associated beta-galactosidase (SA-β-gal) activity and cell cycle distribution. Then the photoprotective effect of salidroside was investigated. Cells were pre-treated with various doses of salidroside (1, 5 and 10 μM) followed by the sublethal dosage of UVB exposure and then were harvested for various detections, including senescence-associated phenotypes and molecules, alteration of oxidative stress, matrix metalloproteinase-1 (MMP-1) secretion and inflammatory response. Pre-treatment of salidroside dose dependently reversed the senescent state of HDFs induced by UVB as evidenced by elevated cell viability, decreased SA-β-gal activity and relieving of G1/G0 cell cycle arrest. UVB-induced increased protein expression of cyclin-dependent kinase (CDK) inhibitors p21(WAF) (1) and p16(INK) (4) was also repressed by salidrosdie treatment in a dose-dependent manner. Meanwhile, the increment of malondialdehyde (MDA) level in UVB-irradiated HDFs was inhibited upon salidroside treatment. Additionally, salidroside significantly attenuated UVB-induced synthesis of MMP-1 as well as the production of IL-6 and TNF-α in HDFs. Our data provided the evidences for the protective role of salidroside against UVB-induced premature senescence in HDFs probably via its anti

  18. Roles of cyclooxygenase-2 in microvascular endothelial cell proliferation induced by basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background The level of basic fibroblast growth factor (bFGF) increases rapidly after cerebral ischemia. However, the molecular mechanisms for the effects of bFGF on cerebral microvascular endothelial cells (cMVECs) have not yet been fully elucidated. In this study, a murine cMVEC line, bend.3, was employed to study the effects of bFGF on cyclooxygenase (COX) expression and its downstream effects in cMVECs. Methods After treatment with bFGF, RT-PCR and Western blotting analyses were carried out to evaluate the changes in COX-2 mRNA and protein expression, respectively. Ml-r assays were performed to measure cell proliferation. The prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) concentrations in the culture medium were measured by enzyme-linked immunosorbent assay (ELISA). Results COX-2 mRNA and protein expressions in bEnd.3 cells were induced by bFGF in time- and dose-dependent manners. The bFGF-induced COX-2 upregulation led to enhanced PGE2 production by bEnd.3 cells, and this effect was abolished by the selective COX-2 inhibitor NS-398. bFGF also increased VEGF production by bend.3 cells, and this effect was blocked by NS-398 and the EP1/2 (PGE2 receptors) antagonist AH6809. Furthermore, exogenous PGE2 increased VEGF production in bend.3 cells, and AH6809 blocked this effect. Conclusion bFGF increases VEGF production in an autocrine manner by increasing COX-2-generated PGE2 in cMVECs and subsequently stimulates MVEC proliferation and angiogenesis.

  19. Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells.

    Science.gov (United States)

    Wei, Qinzhi; Huang, Haiyan; Yang, Linqing; Yuan, Jianhui; Yang, Xiaohua; Liu, Yungang; Zhuang, Zhixiong

    2014-04-01

    Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 μM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type. Copyright © 2012 Wiley Periodicals, Inc.

  20. Heparin platelet factor 4 antibody positivity in pseudothrombocytopenia.

    Science.gov (United States)

    Balcik, Ozlem Sahin; Akdeniz, Derya; Cipil, Handan; Uysal, Sema; Isik, Ayse; Kosar, Ali

    2012-01-01

    Pseudothrombocytopenia (PTCP) is a laboratory event of platelet clustering related to drugs used for anticoagulation. This condition is engendered by autoantibodies against platelets in usually EDTA-anticoagulated blood. Pseudothrombocytopenia has no clinical significance but when evaluated as true thrombocytopenia, this misconception may lead to unnecessary diagnostic procedures. Heparin-induced thrombocytopenia with thrombosis (HITT) is a complication of heparin treatment caused by heparin platelet factor 4 (HPF-4) antibodies, leading to platelet activation and hypercoagulability. In our study, 48 patients with PTCP and 36 healthy volunteers were included. Heparin platelet factor 4 antibody positivity was detected in 12 patients from PTCP group; nobody from control group had. Citrated serum samples and peripheral blood smears showed normal platelet count. Of the 4 patients using heparin derivative, 1 (2.1%) had antibody positivity but without any bleeding symptoms. In conclusion, HPF-4 antibody positivity might be a risk factor for PTCP. Clinicians should be aware of this kind of condition.

  1. The hypoxia-inducible factor-1α activates ectopic production of fibroblast growth factor 23 in tumor-induced osteomalacia

    Science.gov (United States)

    Zhang, Qian; Doucet, Michele; Tomlinson, Ryan E; Han, Xiaobin; Quarles, L Darryl; Collins, Michael T; Clemens, Thomas L

    2016-01-01

    Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome in which ectopic production of fibroblast growth factor 23 (FGF23) by non-malignant mesenchymal tumors causes phosphate wasting and bone fractures. Recent studies have implicated the hypoxia-inducible factor-1α (HIF-1α) in other phosphate wasting disorders caused by elevated FGF23, including X-linked hypophosphatemic rickets and autosomal dominant hypophosphatemia. Here we provide evidence that HIF-1α mediates aberrant FGF23 in TIO by transcriptionally activating its promoter. Immunohistochemical studies in phosphaturic mesenchymal tumors resected from patients with documented TIO showed that HIF-1α and FGF23 were co-localized in spindle-shaped cells adjacent to blood vessels. Cultured tumor tissue produced high levels of intact FGF23 and demonstrated increased expression of HIF-1α protein. Transfection of MC3T3-E1 and Saos-2 cells with a HIF-1α expression construct induced the activity of a FGF23 reporter construct. Prior treatment of tumor organ cultures with HIF-1α inhibitors decreased HIF-1α and FGF23 protein accumulation and inhibited HIF-1α-induced luciferase reporter activity in transfected cells. Chromatin immunoprecipitation assays confirmed binding to a HIF-1α consensus sequence within the proximal FGF23 promoter, which was eliminated by treatment with a HIF-1α inhibitor. These results show for the first time that HIF-1α is a direct transcriptional activator of FGF23 and suggest that upregulation of HIF-1α activity in TIO contributes to the aberrant FGF23 production in these patients. PMID:27468359

  2. Continual Cell Deformation Induced via Attachment to Oriented Fibers Enhances Fibroblast Cell Migration

    OpenAIRE

    Sisi Qin; Vincent Ricotta; Marcia Simon; Clark, Richard A.F.; Rafailovich, Miriam H.

    2015-01-01

    Fibroblast migration is critical to the wound healing process. In vivo, migration occurs on fibrillar substrates, and previous observations have shown that a significant time lag exists before the onset of granulation tissue. We therefore conducted a series of experiments to understand the impact of both fibrillar morphology and migration time. Substrate topography was first shown to have a profound influence. Fibroblasts preferentially attach to fibrillar surfaces, and orient their cytoplasm...

  3. Heparin Leakage in Central Venous Catheters by Hemodynamic Transport

    Science.gov (United States)

    Barbour, Michael; McGah, Patrick; Gow, Kenneth; Aliseda, Alberto

    2014-11-01

    Central venous catheters (CVCs), placed in the superior vena cava for hemodialysis, are routinely filled with heparin, an anticoagulant, while not in use to maintain patency and prevent thrombus formation at the catheter tip. However, the heparin-lock procedure places the patient at risk for systemic bleeding incidences, as heparin is known to leak into the blood stream. We propose that the driving mechanism behind heparin leakage is advective-diffusive transport due to the pulsatile blood flow surrounding the catheter tip. This novel hypothesis is based on Planar Laser Induced Fluorescence (PLIF) measurements of heparin transport from a CVC placed inside an in vitro pulsatile flow loop and validated with CFD simulations. The results show an initial, fast (catheter lumen, where concentration is still high, that is insufficient at replenishing the lost heparin at the tip. These results, which estimate leakage rates consistent with published in vivo data, predict that the concentration of heparin at the catheter tip is effectively zero for the majority of the interdialytic phase, rendering the heparin lock ineffective.

  4. Mitochondrial vulnerability and increased susceptibility to nutrient-induced cytotoxicity in fibroblasts from leigh syndrome French canadian patients.

    Directory of Open Access Journals (Sweden)

    Yan Burelle

    Full Text Available Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC, a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol, or modulate fatty acid (L-carnitine or Krebs cycle metabolism (propionate are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise

  5. Mitochondrial Vulnerability and Increased Susceptibility to Nutrient-Induced Cytotoxicity in Fibroblasts from Leigh Syndrome French Canadian Patients

    Science.gov (United States)

    Burelle, Yan; Thompson Legault, Julie; Boucher, Gabrielle; Morin, Charles; Coderre, Lise; Des Rosiers, Christine

    2015-01-01

    Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the

  6. Effects of low molecular weight heparin on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid-induced colitis

    Institute of Scientific and Technical Information of China (English)

    Bing Xia; Hong Han; Ke-Jian Zhang; Jin Li; Guang-Song Guo; Ling-Ling Gong; Xian-Chang Zeng; Jun-Yan Liu

    2004-01-01

    AIM: To observe the effects of Iow molecular weight heparin (LMWH) on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis.METHODS: Colitis was induced in female Sprauge-Dawley rats by colonic administration of 2, 4, 6-TNBS. LMWH, a dalteparin (150 U/kg, 300 U/kg) was subcutaneously administrated one hour before induction of colitis and went on once a day for 6 days. Then a half dose was given for the normal saline once a day for 14 days after treated by TNBS.Animals were sacrificed at 24 h, days 7 and 14 after induction of colitis. The colon was excised for the evaluation of macroscopic and histological findings and TNF-a immunohistochemical assay. Platelet surface P-selectin expression was determined by radioimmunoassay and serum IL-8 production was assayed by ELISA method.RESULTS: LMWH treatment in a dose of 300 U/kg for 14 days significantly improved colonic inflammation by histological examination. Serum IL-8 production in the 300 U/kg treatment group was more significantly decreased at day 14 than that at 24 h (P<0.05). However, platelet surface P-selectin expression and TNF-a staining in colonic tissue were not significantly different among the three groups.CONCLUSION: LMWH has an anti-inflammatory effect on TNBS induced colitis in rats. The effect is possibly related to inhibition of proinfiammatory cytokine IL-8, but not involved platelet surface P-selectin expression.

  7. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

    Science.gov (United States)

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-07-14

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts.

  8. Improved wound healing in pressure-induced decubitus ulcer with controlled release of basic fibroblast growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Wei [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Wang Hailun [Department of Dermatology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Jin Faguang [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)], E-mail: nidewenzhang@163.com; Yu Chunyan [Department of Dermatology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Chu Dongling [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Wang Lin [Department of Internal Medicine, 316 Hospital of PLA, Beijing 100093 (China); Lu Xian [93942 Unit Hospital of PLA, Xianyang 710012 (China)

    2008-07-14

    The purpose was to evaluate the efficacy of the wound dressing containing basic fibroblast growth factor (bFGF)-loaded microspheres on promoting healing in pressure-induced decubitus ulcer. In this study, the pressure-induced ulcer in swine was used as a model to demonstrate the hypothesis that controlled release of bFGF has the potential to provide optimal healing milieu for chronic wounds in the repair process. Average size of the microspheres was 14.36 {+-} 3.56 {mu}m and the network gelatin sponges were characterized with an average pore size of 80-160 {mu}m. Both the in vitro release efficiency and the protein bioactivity revealed that bFGF was released from the microspheres in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of fibroblasts. Pressure-induced ulcer was created at 500 g/cm{sup 2} pressure loaded on swine dorsal skin 12 h daily for 2 consecutive days. After removal of the pressure load, the gelatin sponge containing bFGF gelatin microspheres or bFGF in solution was implanted into the wound. Swine were sacrificed at 7, 14, and 21 days after implantation, and a full-thickness biopsy was taken and stained for histological analysis. It was observed that controlled release of bFGF provided an accelerated recovery in the wound areas. Histological investigations showed that the dressings were biocompatible and had capability of proliferating fibroblasts and inducing neovascularisation. The present study implied the clinical potential of gelatin sponge with bFGF microspheres to promote the healing in pressure-induced decubitus ulcer.

  9. Cellular uptake and activity of heparin functionalised cerium oxide nanoparticles in monocytes.

    Science.gov (United States)

    Ting, S R Simon; Whitelock, John M; Tomic, Romana; Gunawan, Cindy; Teoh, Wey Yang; Amal, Rose; Lord, Megan S

    2013-06-01

    Cerium oxide nanoparticles (nanoceria) are effective in scavenging intracellular reactive oxygen species (ROS). In this study nanoceria synthesized by flame spray pyrolysis (dXRD = 12 nm) were functionalised with heparin via an organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria were functionalised with approximately 130 heparin molecules per nanoparticle as determined by thermo gravimetric analysis. Heparin functionalised nanoceria were more effectively internalised by the human monocyte cell line, U937, and U937 cells that had been activated with phorbol 12 myristate 13-acetate (PMA) than bare nanoceria. The heparin functionalised nanoceria were also more effective in scavenging ROS than nanoceria in both activated and unactivated U937 cells. Heparin coupled nanoceria were found to be biologically active due to their ability to bind fibroblast growth factor 2 and signal through FGF receptor 1. Additionally, the heparin-coupled nanoceria, once internalised by the cells, were found to be degraded by 48 h. Together these data demonstrated that heparin enhanced the biological properties of nanoceria in terms of cellular uptake and ROS scavenging, while the nanoceria themselves were more effective at delivering heparin intracellularly than exposing cells to heparin in solution.

  10. Heparinized poly(vinyl alcohol)-small intestinal submucosa composite membrane for coronary covered stents

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Tao; Wang Guixue; Qiu Juhui; Luo Lailong [Bioengineering College and ' 111 Project' Laboratory of Biomechanics and Tissue Repair, Chongqing University, Chongqing (China); Zhang Guoquan, E-mail: wjjt72@live.c [Histology and Embryology Department, Medical College of China People Armed Police Forces, Tianjin (China)

    2009-04-15

    To develop a novel coating material for coronary covered stents, we prepared a kind of composite membrane which contains polyvinyl alcohol (PVA) and porcine small intestinal submucosa (SIS) powders crosslinked and heparinized by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The amount of immobilized heparin increased with increasing ratios of EDC:heparin, and the maximum amount was approximately 60 {mu}g heparin per milligram SIS powder at a weight ratio of EDC:heparin of 2. Uniaxial tensile and balloon inflation testing suggested that the composite membrane crosslinked by lower EDC concentration is more flexible and elastic. The clotting time (APTT and PT) of the heparinized PVA-SIS membrane was longer than that of the unheparinized membrane. The number of adherent platelets on the heparinized PVA-SIS composite membrane was about 25% of the unheparininzed, and there was no sign of accumulation and almost no pseudopodium was observed. The endothelial cells were amicable with the heparinized and unheparinized PVA-SIS composite membranes. In in vivo implantation tests, we observed a thin capsule formed by several layers of fibroblasts surrounding the implants. These results showed that the heparinized PVA-SIS composite membrane has potential biomechanical and biological properties as a coating material for coronary covered stent.

  11. Proteome alteration induced by hTERT transfection of human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Riou Jean-François

    2008-04-01

    Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

  12. Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

    Institute of Scientific and Technical Information of China (English)

    LiGU; Yuan-jueZHU; Zi-jianGUO; Xing-xiangXU; Wen-bingXU

    2004-01-01

    AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-β1 and the role of interferon (IFN)-γ, dexamethasone (DEX) in the fibroblast-myofibroblast differentiation. METHODS: α-Smooth muscle actin (α-SMA), Smad2/3, and Smad7 protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline, α-SMA and collagen III mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-β1, IFN-γ 200 μg/L markedly blocked TGF-β1-induced α-SMA protein expression (PO.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-β1-induced α-SMA expression (P<0.01). IFN-γ 200 μg/L did not block TGF-β1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-131 induced fibroblastmyofibroblast differentiation in a Smad proteins-dependent manner. IFN-γ could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism withTGF-β1. Differentiated myofibroblasts, however, were resistant to both IFN-γ and DEX.

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

    Directory of Open Access Journals (Sweden)

    Xiaodong Pan

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

  14. Alternative splicing of fibroblast growth factor receptor 3 produces a secreted isoform that inhibits fibroblast growth factor-induced proliferation and is repressed in urothelial carcinoma cell lines.

    Science.gov (United States)

    Tomlinson, Darren C; L'Hôte, Corine G; Kennedy, Wendy; Pitt, Eva; Knowles, Margaret A

    2005-11-15

    Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that play key roles in proliferation, differentiation, and tumorigenesis. FGFR3 was identified as the major family member expressed in both normal human urothelium and cultured normal human urothelial (NHU) cells and was expressed as the IIIb isoform. We also identified a splice variant, FGFR3 Delta8-10, lacking exons encoding the COOH-terminal half of immunoglobulin-like domain III and the transmembrane domain. Previous reports have assumed that this is a cancer-specific splice variant. We showed that FGFR3 Delta8-10 is a normal transcript in NHU cells and is translated, N-glycosylated, and secreted. Primary urothelium expressed high levels of FGFR3 transcripts. In culture, levels were reduced in actively proliferating cells but increased at confluence and as cells approached senescence. Cells overexpressing FGFR3 IIIb showed FGF1-induced proliferation, which was inhibited by the addition of FGFR3 Delta8-10. In bladder tumor cell lines derived from aggressive carcinomas, there were significant alterations in the relative expression of isoforms including an overall decrease in the proportion of FGFR3 Delta8-10 and predominant expression of FGFR3 IIIc in some cases. In summary, alternative splicing of FGFR3 IIIb in NHU cells represents a normal mechanism to generate a transcript that regulates proliferation and in bladder cancer, the ratio of FGFR3 isoforms is significantly altered.

  15. Characterization of PF4-Heparin Complexes by Photon Correlation Spectroscopy and Zeta Potential.

    Science.gov (United States)

    Bertini, Sabrina; Fareed, Jawed; Madaschi, Laura; Risi, Giulia; Torri, Giangiacomo; Naggi, Annamaria

    2017-01-01

    Heparin-induced thrombocytopenia (HIT) is associated with antibodies to complexes between heparin and platelet factor 4 (PF4), a basic protein usually found in platelet alpha granules. Heparin-induced thrombocytopenia antibodies preferentially recognize macromolecular complexes formed between positively charged PF4 and polyanionic heparins over a narrow range of molar ratios. The aim of this work was to study the complexes that human PF4 forms with heparins from various species, such as porcine, bovine, and ovine; heparins from various organs, such as mucosa and lung; and different low-molecular-weight heparins (LMWHs) at several stoichiometric ratios to evaluate their sizes and charges by photo correlation spectroscopy and zeta potential measurements. The resulting data of the PF4 complexes with unfractionated heparins (UFHs), LMWHs and their fractions, and oligosaccharide components suggest that the size of aggregates is not only a simple function of average molecular weight but also of the molecular weight distribution of the sample. Moreover, it was found that lower concentrations of the tested ovine-derived mucosal heparin are required to form the large PF4/heparin complexes as compared to mucosal porcine and bovine heparin.

  16. Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors.

    Science.gov (United States)

    Deng, Yanfei; Liu, Qingyou; Luo, Chan; Chen, Shibei; Li, Xiangping; Wang, Caizhu; Liu, Zhenzhen; Lei, Xiaocan; Zhang, Huina; Sun, Hongliang; Lu, Fenghua; Jiang, Jianrong; Shi, Deshun

    2012-09-01

    Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.

  17. Generation of colonies of induced trophoblast cells during standard reprogramming of porcine fibroblasts to induced pluripotent stem cells.

    Science.gov (United States)

    Ezashi, Toshihiko; Matsuyama, Haruyo; Telugu, Bhanu Prakash V L; Roberts, R Michael

    2011-10-01

    During reprogramming of porcine mesenchymal cells with a four-factor (POU5F1/SOX2/KLF4/MYC) mixture of vectors, a fraction of the colonies had an atypical phenotype and arose earlier than the recognizable porcine induced pluripotent stem (iPS) cell colonies. Within days after each passage, patches of cells with an epithelial phenotype formed raised domes, particularly under 20% O(2) conditions. Relative to gene expression of the iPS cells, there was up-regulation of genes for transcription factors associated with trophoblast (TR) lineage emergence, e.g., GATA2, PPARG, MSX2, DLX3, HAND1, GCM1, CDX2, ID2, ELF5, TCFAP2C, and TEAD4 and for genes required for synthesis of products more typical of differentiated TR, such as steroids (HSD17B1, CYP11A1, and STAR), pregnancy-associated glycoproteins (PAG6), and select cytokines (IFND, IFNG, and IL1B). Although POU5F1 was down-regulated relative to that in iPS cells, it was not silenced in the induced TR (iTR) cells over continued passage. Like iPS cells, iTR cells did not senesce on extended passage and displayed high telomerase activity. Upon xenografting into immunodeficient mice, iTR cells formed nonhemorrhagic teratomas composed largely of layers of epithelium expressing TR markers. When cultured under conditions that promoted embryoid body formation, iTR cells formed floating spheres consisting of a single epithelial sheet whose cells were tethered laterally by desmosome-like structures. In conclusion, reprogramming of porcine fibroblasts to iPS cells generates, as a by-product, colonies composed of self-renewing populations of TR cells, possibly containing TR stem cells.

  18. Intestinal ellagitannin metabolites ameliorate cytokine-induced inflammation and associated molecular markers in human colon fibroblasts.

    Science.gov (United States)

    Giménez-Bastida, Juan A; Larrosa, Mar; González-Sarrías, Antonio; Tomás-Barberán, Francisco; Espín, Juan C; García-Conesa, María-Teresa

    2012-09-12

    Pomegranate ellagitannins (ETs) are transformed in the gut to ellagic acid (EA) and its microbiota metabolites, urolithin A (Uro-A) and urolithin B (Uro-B). These compounds exert anti-inflammatory effects in vitro and in vivo. The aim of this study was to investigate the effects of Uro-A, Uro-B, and EA on colon fibroblasts, cells that play a key role in intestinal inflammation. CCD18-Co colon fibroblasts were exposed to a mixture of Uro-A, Uro-B, and EA, at concentrations comparable to those found in the colon (40 μM Uro-A, 5 μM Uro-B, 1 μM EA), both in the presence or in the absence of IL-1β (1 ng/mL) or TNF-α (50 ng/mL), and the effects on fibroblast migration and monocyte adhesion were determined. The levels of several growth factors and adhesion cytokines were also measured. The mixture of metabolites significantly inhibited colon fibroblast migration (∼70%) and monocyte adhesion to fibroblasts (∼50%). These effects were concomitant with a significant down-regulation of the levels of PGE(2), PAI-1, and IL-8, as well as other key regulators of cell migration and adhesion. Of the three metabolites tested, Uro-A exhibited the most significant anti-inflammatory effects. The results show that a combination of the ET metabolites found in colon, urolithins and EA, at concentrations achievable in the intestine after the consumption of pomegranate, was able to moderately improve the inflammatory response of colon fibroblasts and suggest that consumption of ET-containing foods has potential beneficial effects on gut inflammatory diseases.

  19. 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.

  20. Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

    OpenAIRE

    Lin, Audrey; Hokugo, Akishige; Choi, Jae; Nishimura, Ichiro

    2010-01-01

    Wounds created in the oral cavity heal rapidly and leave minimal scarring. We have examined a role of a previously isolated cDNA from oral wounds encoding wound inducible transcript-3.0 (wit3.0), also known as fibroblast growth factor receptor 1 oncogene partner 2 (FGFR1OP2). FGFR1OP2/wit3.0 was highly expressed in oral wound fibroblasts without noticeable up-regulation of α-smooth muscle actin. In silico analyses, denaturing and nondenaturing gel Western blot, and immunocytology together dem...

  1. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    Science.gov (United States)

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

  2. Protective effect of chromene isolated from Sargassum horneri against UV-A-induced damage in skin dermal fibroblasts.

    Science.gov (United States)

    Kim, Jung-Ae; Ahn, Byul-Nim; Kong, Chang-Suk; Kim, Se-Kwon

    2012-08-01

    Skin homoeostasis is interrupted during UV-A irradiation. How the UV-A-altered skin components influences photoageing of skin should be investigated using human in vitro models that are important for understanding skin ageing. In this study, chromene compound, sargachromenol, was isolated from Sargassum horneri, and its potency on inhibition of photoageing was investigated in UV-A-irradiated dermal fibroblasts. Effects of sargachromenol on the prevention of photoageing were evaluated by measuring ROS production, membrane protein oxidation, lipid peroxidation and ageing-related gene expression in UV-A-irradiated human skin dermal fibroblasts. The results indicated that treatment with sargachromenol suppressed the collagenase matrix metalloproteinases (MMPs), MMP-1, MMP-2 and MMP-9 expression without any cytotoxicity and phototoxicity. It was further found that these inhibitions were because of increase in the expression of TIMP-1 and TIMP-2 genes. Furthermore, we confirmed that the UV-A-induced transcriptions of AP-1 signalling pathway were regulated by sargachromenol treatment in UV-A-irradiated dermal fibroblasts. © 2012 John Wiley & Sons A/S.

  3. Chemerin induces CCL2 and TLR4 in synovial fibroblasts of patients with rheumatoid arthritis and osteoarthritis.

    Science.gov (United States)

    Eisinger, Kristina; Bauer, Sabrina; Schäffler, Andreas; Walter, Roland; Neumann, Elena; Buechler, Christa; Müller-Ladner, Ulf; Frommer, Klaus W

    2012-02-01

    Chemerin stimulates migration of leukocytes to sites of inflammation and also increases inflammatory signaling in chondrocytes suggesting a function of chemerin in joint inflammation. Synovial fibroblasts (SF) are critically involved in synovitis and subsequent cartilage destruction. Here, we analyzed whether synovial fibroblasts express chemerin and its receptor CMKLR1. Further, the role of chemerin in synovial fibroblast chemotaxis, proliferation, insulin response and release of inflammatory proteins was studied. Synovial tissue sections were labeled with chemerin antibody and chemerin was measured in synovial fluid by ELISA. Chemerin mRNA and protein as well as CMKLR1 expression were determined in SFs from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Effects of chemerin on cytokines, chemokines and matrix metalloproteinases (MMP), and on proliferation, migration and insulin signaling were analyzed appropriately. SFs expressed CMKLR1 and chemerin mRNA, and chemerin protein was found in cell supernatants of synovial fibroblasts. Immunohistochemistry detected chemerin in synovial tissue predominantly localized within the lining layer. Chemerin was present in synovial fluids of RA, OA and psoriatic arthritis patients in similar concentrations. Chemerin neither increased IL-6 levels nor MMP-2 or -9 activity in SFs. Also, it did not act as a chemoattractant for these cells. With respect to intracellular signaling, neither basal nor insulin-mediated phosphorylation of Akt was affected. However, chemerin significantly increased TLR4 mRNA and synthesis of CCL2 in SFs while CCL4 and -5 were not altered. Cell proliferation of SFs, however, was modestly reduced by chemerin. These data show that human SFs express both chemerin and its receptor. As chemerin enhanced expression of TLR4 and induced release of CCL2 in SFs, a role of this protein in innate immune system-associated joint inflammation is proposed. Copyright © 2011 Elsevier Inc. All rights

  4. RAGE activation induces invasiveness of RA fibroblast-like synoviocytes in vitro

    NARCIS (Netherlands)

    Steenvoorden, M.M.C.; Toes, R.E.M.; Ronday, H.K.; Huizinga, T.W.J.; Groot, J. de

    2007-01-01

    Ligands for the receptor for advanced glycation endproducts (RAGE) are increased in RA synovial fluid (SF), serum and synovium. Since RAGE is present on fibroblast-like synoviocytes (FLS), the present study investigates whether the RAGE ligands HMGB-1 and AGEs are able to stimulate the characteristi

  5. RAGE activation induces invasiveness of RA fibroblast-like synoviocytes in vitro

    NARCIS (Netherlands)

    Steenvoorden, M.M.C.; Toes, R.E.M.; Ronday, H.K.; Huizinga, T.W.J.; Groot, J. de

    2007-01-01

    Ligands for the receptor for advanced glycation endproducts (RAGE) are increased in RA synovial fluid (SF), serum and synovium. Since RAGE is present on fibroblast-like synoviocytes (FLS), the present study investigates whether the RAGE ligands HMGB-1 and AGEs are able to stimulate the

  6. RAGE activation induces invasiveness of RA fibroblast-like synoviocytes in vitro

    NARCIS (Netherlands)

    Steenvoorden, M.M.C.; Toes, R.E.M.; Ronday, H.K.; Huizinga, T.W.J.; Groot, J. de

    2007-01-01

    Ligands for the receptor for advanced glycation endproducts (RAGE) are increased in RA synovial fluid (SF), serum and synovium. Since RAGE is present on fibroblast-like synoviocytes (FLS), the present study investigates whether the RAGE ligands HMGB-1 and AGEs are able to stimulate the characteristi

  7. Nanoscale topography-induced modulation of fundamental cell behaviors of rabbit corneal keratocytes, fibroblasts, and myofibroblasts.

    Science.gov (United States)

    Pot, Simon A; Liliensiek, Sara J; Myrna, Kathern E; Bentley, Ellison; Jester, James V; Nealey, Paul F; Murphy, Christopher J

    2010-03-01

    Keratocyte-to-myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast, and myofibroblast cell behavior. Primary rabbit corneal keratocytes, fibroblasts, and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400-, 800-, 1200-, 1600-, 2000-, and 4000-nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged, and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours, and individual cell trajectories and migration rates were calculated. Keratocytes, fibroblasts, and myofibroblasts aligned and elongated to pitch sizes larger than 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes, with a transition zone around the 800- to 1200-nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges larger than 1000 nm but lacked directional guidance on submicron and nanoscale topographic features and on planar surfaces. Keratocytes remained essentially immobile. Corneal stromal cells elongated, aligned, and migrated, differentially guided by substratum topographic features. All cell types failed to respond to topographic features approximating the dimensions of individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.

  8. Evaluating the potential of poly(beta-amino ester nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Bhise NS

    2013-12-01

    Full Text Available Nupura S Bhise,1,* Karl J Wahlin,2,* Donald J Zack,2–4 Jordan J Green1,21Department of Biomedical Engineering, Translational Tissue Engineering Center, and Institute for Nanobiotechnology, 2Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, 3Solomon H Snyder Department of Neuroscience, Department of Molecular Biology and Genetics, and Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Institut de la Vision, Paris, France*These authors contributed equally to this workBackground: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs from human fibroblasts.Methods: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.Results: 1-(3-aminopropyl-4-methylpiperazine end-terminated poly(1,4-butanediol diacrylate-co-4-amino-1-butanol polymer (B4S4E7 self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available

  9. Mitomycin C induces fibroblasts apoptosis and reduces epidural fibrosis by regulating miR-200b and its targeting of RhoE.

    Science.gov (United States)

    Sun, Yu; Ge, Yingbin; Fu, Yuxuan; Yan, Lianqi; Cai, Jun; Shi, Kun; Cao, Xiaojian; Lu, Chun

    2015-10-15

    Mitomycin C (MMC) is known to reduce epidural fibrosis, but the underlying mechanisms have not yet been elucidated. Aberrant miR-200b expressions have been reported in multiple types of fibrotic tissues from many diseases. The aim of this study was to clarify the mechanism by which MMC induces fibroblasts apoptosis and reduces epidural fibrosis. The expression of miR-200b in human fibroblasts was determined after MMC treatment, and the targeted association between miR-200b and RhoE was determined using the luciferase activity assay. The effects of MMC and miR-200b on human fibroblasts apoptosis were evaluated using flow cytometry and western blot analysis. The effects of MMC and miR-200b on epidural fibrosis were evaluated using the Rydell classification, hydroxyproline content, apoptotic cell count and histological analysis. The study revealed that MMC could significantly downregulate miR-200b expression and induce human fibroblasts apoptosis. The direct downregulation of miR-200b could induce human fibroblasts apoptosis. Furthermore, we identified the binding sequence for miR-200b within the 3' untranslated region of RhoE. RhoE was confirmed to be a direct target of miR-200b, and RhoE itself acted as a promoter of fibroblasts apoptosis. The inhibition of miR-200b increased fibroblasts apoptosis and reduced epidural fibrosis in rats, which was in accordance with the effect of MMC. This study suggests that MMC induces fibroblasts apoptosis and reduces epidural fibrosis by regulating miR-200b expression and its targeting of RhoE.

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

    Science.gov (United States)

    Yao, Rong; Cao, Yu; He, Ya-rong; Lau, Wayne Bond; Zeng, Zhi; Liang, Zong-an

    2015-01-01

    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 (ppulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.

  11. Mechanisms of Multi-walled Carbon Nanotubes-Induced Oxidative Stress and Genotoxicity in Mouse Fibroblast Cells.

    Science.gov (United States)

    Alarifi, Saud; Ali, Daoud

    2015-01-01

    The extensive production and wide application of carbon nanotubes have made investigations of its toxic potentials necessary. In the present study, we explored the underlying mechanism through which multi-walled carbon nanotubes (MWCNTs) induce toxicity in mouse fibroblast cells (L929). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and neutral red uptake viability assays were used to examine mechanisms of cytotoxicity. Dose and time-dependent cytotoxicity was observed in L929 cells. The MWCNTs significantly increased the generation of reactive oxygen species, lipid peroxidation, superoxide dismutase, and decreased glutathione. It was observed that the MWCNTs induced caspase 3 activity. The highest DNA strand breakage was detected by comet assay at 300 µg/mL of MWCNTs. Thus, the data indicate that MWCNTs induced cytotoxicity and apoptosis in L929 cells via oxidative stress.

  12. Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.

    Science.gov (United States)

    Cheng, Fang; Bourseau-Guilmain, Erika; Belting, Mattias; Fransson, Lars-Åke; Mani, Katrin

    2016-06-01

    There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein (APP) of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the APP is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576, there is increased processing of the APP to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced

  13. Autophagy is involved in TGF-β1-induced protective mechanisms and formation of cancer-associated fibroblasts phenotype in tumor microenvironment.

    Science.gov (United States)

    Liu, Fang-Lan; Mo, En-Pan; Yang, Liu; Du, Jun; Wang, Hong-Sheng; Zhang, Huan; Kurihara, Hiroshi; Xu, Jun; Cai, Shao-Hui

    2016-01-26

    Transforming growth factor-β1 (TGF-β1) present in tumor microenvironment acts in a coordinated fashion to either suppress or promote tumor development. However, the molecular mechanisms underlying the effects of TGF-β1 on tumor microenvironment are not well understood. Our clinical data showed a positive association between TGF-β1 expression and cancer-associated fibroblasts (CAFs) in tumor microenvironment of breast cancer patients. Thus we employed starved NIH3T3 fibroblasts in vitro and 4T1 cells mixed with NIH3T3 fibroblasts xenograft model in vivo to simulate nutritional deprivation of tumor microenvironment to explore the effects of TGF-β1. We demonstrated that TGF-β1 protected NIH3T3 fibroblasts from Star-induced growth inhibition, mitochondrial damage and cell apoptosis. Interestingly, TGF-β1 induced the formation of CAFs phenotype in starvation (Star)-treated NIH3T3 fibroblasts and xenografted Balb/c mice, which promoted breast cancer tumor growth. In both models, autophagy agonist rapamycin increased TGF-β1-induced protective effects and formation of CAFs phenotypes, while autophagy inhibitor 3-methyladenine, Atg5 knockdown or TGF-β type I receptor kinase inhibitor LY-2157299 blocked TGF-β1 induced these effects. Taken together, our results indicated that TGF-β/Smad autophagy was involved in TGF-β1-induced protective effects and formation of CAFs phenotype in tumor microenvironment, which may be used as therapy targets in breast cancer.

  14. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    Energy Technology Data Exchange (ETDEWEB)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Li, Xiao-Dong [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Hong, Mo-Na [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Chen, Qi-Zhi [Shanghai Institute of Hypertension, Shanghai (China); Han, Wei-Qing, E-mail: whan020@gmail.com [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Gao, Ping-Jin, E-mail: gaopingjin@sibs.ac.cn [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China)

    2016-04-29

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

  15. Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

    Science.gov (United States)

    Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-01-04

    Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  16. Fibroblast-Specific Deletion of Hypoxia Inducible Factor-1 Critically Impairs Murine Cutaneous Neovascularization and Wound Healing.

    Science.gov (United States)

    Duscher, Dominik; Maan, Zeshaan N; Whittam, Alexander J; Sorkin, Michael; Hu, Michael S; Walmsley, Graham G; Baker, Hutton; Fischer, Lauren H; Januszyk, Michael; Wong, Victor W; Gurtner, Geoffrey C

    2015-11-01

    Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1α, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1α knockout mouse model. The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1α knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1α knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds. Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p wound closure and vascularity (p fibroblasts results in delayed wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.

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

    Science.gov (United States)

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

    2017-03-07

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

  18. A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent.

    Science.gov (United States)

    Bett, Cyrus; Grgac, Ksenija; Long, Dianna; Karfunkle, Michael; Keire, David A; Asher, David M; Gregori, Luisa

    2017-01-23

    In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans. Thus, US heparin is currently sourced only from pig intestines. Availability of alternative sources of crude heparin, a life-saving drug, would benefit public health. Bovine heparin is an obvious option, but BSE clearance by the bovine heparin manufacturing process should be evaluated. To this end, using hamster 263K scrapie as a surrogate for BSE agent, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked scrapie agent. We removed aliquots from each step and analyzed them for residual abnormal prion protein (PrP(TSE)) using a sensitive in vitro method, real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity by 3.6 log10 and removed PrP(TSE), measured as seeding activity, by 3.4 log10. NaOH treatment was the most effective removal step tested. We also investigated NaOH at different concentrations and pH: the results showed that as much as 5.2 log10 of PrP(TSE) seeding activity was removed at pH 12.5. Thus, changes to the concentration, treatment time, and temperature of alkaline extraction might further improve removal. Our results, using a basic heparin manufacturing process, inform efforts to reintroduce safe bovine heparin in the USA.

  19. CALCITONIN GENE-RELATED PEPTIDE INDUCES FIBROBLASTS FROM HIP PSEUDOCAPSULE TO PRODUCE CYTOKINES

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate calcitonin gene-related peptide (CGRP) involvement in the process of aseptic loosening. Methods We isolated fibroblasts from periprosthetic pseudocapsular tissue at the time of revision hip arthroplasty performed due to aseptic loosening. Fibroblasts were incubated for 24 h in the presence of CGRP, and the levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the media were determined using enzyme-linked immunosorbent assay kit. Results We found that the levels of IL-1β, IL-6, and TNF-α increased in a time- and concentration-dependent manner. ConclusionThese inflammatory cytokines play an important role in the development of periprosthetic osteolysis and implant loosening. Our results suggest that CGRP may be involved in the pathogenesis of aseptic loosening.

  20. Preventive effects of tamarind seed coat extract on UVA-induced alterations in human skin fibroblasts.

    Science.gov (United States)

    Phetdee, Khemjira; Rakchai, Racharat; Rattanamanee, Kwanchai; Teaktong, Thanasak; Viyoch, Jarupa

    2014-01-01

    One of the most damaging actions on skin is from solar radiation, particularly from its ultraviolet (UV) component, through the formation of oxidative species. Thus, an antioxidant strategy that prevents the formation of these oxidants could form the basis of an efficacious cutaneous protectant. Many herbal materials contain antioxidant polyphenols, and this study assessed the possibility that tamarind seed coat extract could fulfill this role. An alcoholic extract of the tamarind (Tamarindus indica L.) seed coat showed stronger antioxidant activity (2,2-diphenyl-1-picrylhydrazyl inhibition, EC(50) = 12.9 μg/ml) than L-ascorbic acid (EC(50) = 22.9 μg/ml) and α-tocopherol (EC(50) = 29.3 μg/ml). In cultured fibroblasts taken from human skin, hydrogen peroxide (100-1000 μM) damaged 62-92% of the cells compared to only 35-47% when the cells were preincubated in extract (200 μg/ml) for 24 h. UVA (40 J/cm2) irradiation of human fibroblasts damaged 25% of the cells but the death rate was reduced to 10% with extract. UV irradiation increased the proportion of cells arrest in G(0)/G(1) phase (from 59% to 78%) but this was largely prevented by the extract (64%), according to flow cytometry. Intracellular total glutathione of UVA-irradiated cells pretreated with the extract increased to 10-25% compared to the non-pretreated group at 24-72 h after irradiation. Fibroblasts typically increased matrix metalloproteinase-1 secretion after photodamage, and this is prevented by the extract. This is the first report showing that tamarind seed coat extract is an antioxidant and can protect human skin fibroblasts from cellular damage produced by UVA and thus may form the foundation for an antiaging cosmetic.

  1. Continual Cell Deformation Induced via Attachment to Oriented Fibers Enhances Fibroblast Cell Migration

    Science.gov (United States)

    Qin, Sisi; Ricotta, Vincent; Simon, Marcia; Clark, Richard A. F.; Rafailovich, Miriam H.

    2015-01-01

    Fibroblast migration is critical to the wound healing process. In vivo, migration occurs on fibrillar substrates, and previous observations have shown that a significant time lag exists before the onset of granulation tissue. We therefore conducted a series of experiments to understand the impact of both fibrillar morphology and migration time. Substrate topography was first shown to have a profound influence. Fibroblasts preferentially attach to fibrillar surfaces, and orient their cytoplasm for maximal contact with the fiber edge. In the case of en-mass cell migration out of an agarose droplet, fibroblasts on flat surfaces emerged with an enhanced velocity, v = 52μm/h, that decreases to the single cell value, v = 28μm/h within 24 hours and remained constant for at least four days. Fibroblasts emerging on fibrillar surfaces emerged with the single cell velocity, which remained constant for the first 24 hours and then increased reaching a plateau with more than twice the initial velocity within the next three days. The focal adhesions were distributed uniformly in cells on flat surfaces, while on the fibrillar surface they were clustered along the cell periphery. Furthermore, the number of focal adhesions for the cells on the flat surfaces remained constant, while it decreased on the fibrillar surface during the next three days. The deformation of the cell nuclei was found to be 50% larger on the fiber surfaces for the first 24 hours. While the mean deformation remained constant on the flat surface, it increased for the next three days by 24% in cells on fibers. On the fourth day, large actin/myosin fibers formed in cells on fibrillar surfaces only and coincided with a change from the standard migration mechanism involving extension of lamellipodia, and retraction of the rear, to one involving strong contractions oriented along the fibers and centered about the nucleus. PMID:25774792

  2. Continual cell deformation induced via attachment to oriented fibers enhances fibroblast cell migration.

    Directory of Open Access Journals (Sweden)

    Sisi Qin

    Full Text Available Fibroblast migration is critical to the wound healing process. In vivo, migration occurs on fibrillar substrates, and previous observations have shown that a significant time lag exists before the onset of granulation tissue. We therefore conducted a series of experiments to understand the impact of both fibrillar morphology and migration time. Substrate topography was first shown to have a profound influence. Fibroblasts preferentially attach to fibrillar surfaces, and orient their cytoplasm for maximal contact with the fiber edge. In the case of en-mass cell migration out of an agarose droplet, fibroblasts on flat surfaces emerged with an enhanced velocity, v = 52μm/h, that decreases to the single cell value, v = 28μm/h within 24 hours and remained constant for at least four days. Fibroblasts emerging on fibrillar surfaces emerged with the single cell velocity, which remained constant for the first 24 hours and then increased reaching a plateau with more than twice the initial velocity within the next three days. The focal adhesions were distributed uniformly in cells on flat surfaces, while on the fibrillar surface they were clustered along the cell periphery. Furthermore, the number of focal adhesions for the cells on the flat surfaces remained constant, while it decreased on the fibrillar surface during the next three days. The deformation of the cell nuclei was found to be 50% larger on the fiber surfaces for the first 24 hours. While the mean deformation remained constant on the flat surface, it increased for the next three days by 24% in cells on fibers. On the fourth day, large actin/myosin fibers formed in cells on fibrillar surfaces only and coincided with a change from the standard migration mechanism involving extension of lamellipodia, and retraction of the rear, to one involving strong contractions oriented along the fibers and centered about the nucleus.

  3. Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

    Institute of Scientific and Technical Information of China (English)

    Li GU; Yuan-jue ZHU; Zi-jian GUO; Xing-xiang XU; Wen-bing XU

    2004-01-01

    AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-β1 and the role of interferon (IFN)-γ, dexamethasone (DEX)in the fibroblast-myofibroblast differentiation. METHODS: α-Smooth muscle actin (α-SMA), Smad2/3, and Smad7protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline. α-SMA and collagen Ⅲ mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-β1, IFN-γ 200 μg/L markedly blocked TGF-β1-induced α-SMA protein expression (P<0.01),collagen protein (P<0.01) and mRNA (P<0.05) expression, and myofibroblasts morphological transformation, but DEX 10 μmol/L augmented TGF-β1-induced α-SMA expression (P<0.01). For myofibroblasts, both IFN-γ 200 μg/L and DEX 10 μmol/L did not inhibit α-SMA expression (P>0.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-β1-induced α-SMA expression (P<0.01). IFN-γ 200 μg/L did not block TGF-β1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-β1 induced fibroblastmyofibroblast differentiation in a Smad proteins-dependent manner. IFN-γ could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism with TGF-β1. Differentiated myofibroblasts, however, were resistant to both IFN-γ and DEX.

  4. Hypoxia induces a phenotypic switch of fibroblasts to myofibroblasts through a MMP-2/TIMP mediated pathway: Implications for venous neointimal hyperplasia in hemodialysis access

    Science.gov (United States)

    Misra, Sanjay; Fu, Alex A.; Misra, Khamal D.; Shergill, Uday M.; Leof, Edward B; Mukhopadhyay, Debabrata

    2010-01-01

    Purpose Hemodialysis grafts fail because of venous neointimal hyperplasia formation caused by adventitial fibroblasts which have become myofibroblasts (α-smooth muscle actin positive cells) and migrate to the neointima. There is increased expression of hypoxia inducible factor-1 alpha (HIF-1α in venous neointimal hyperplasia formation in experimental animal model and clinical samples. We hypothesized that under hypoxic stimulus (HIF-1α fibroblasts will convert to myofibroblasts through a matrix metalloproteinase-2 (MMP-2) mediated pathway. Materials and methods Murine AKR-2B fibroblasts were made hypoxic or normoxic for 24, 48, and 72 hours. Protein expression for HIF-1α, α-smooth muscle actin, MMP-2, MMP-9, TIMP-1, and TIMP-2 was performed to determine the kinetic changes of these proteins. Immunostaining for α-smooth muscle actin, collagen, and fibronectin was performed. Results At all time points, there was significantly increased expression of HIF-1α in the hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was significantly increased expression α-smooth muscle actin at all time points which peaked by 48 hours in hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was a significant increase in the expression of active MMP-2 by 48-72 hours and a significant increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) by 48-72 hours by hypoxic fibroblasts (P<0.05). By 72 hours, there was significant increase in TIMP-2 expression (P<0.05). Immunohistochemical analysis demonstrated increased expression for α-smooth muscle actin, collagen, and fibronectin as the length of hypoxia increased. Conclusions Under hypoxia, fibroblasts will convert to myofibroblasts through a MMP-2 mediated pathway which may provide insight into the mechanism of venous neointimal hyperplasia. PMID:20434368

  5. Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hypoxic Pulmonary Vascular Remodeling

    Institute of Scientific and Technical Information of China (English)

    Yong-Liang JIANG; Ai-Guo DAI; Qi-Fang LI; Rui-Cheng HU

    2006-01-01

    The muscularization of non-muscular pulmonary arterioles is animportant pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-β 1 (TGF-β 1) can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of α-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-β1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-β1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling.

  6. Genome-wide microarray analysis of human fibroblasts in response to γ radiation and the radiation-induced bystander effect.

    Science.gov (United States)

    Kalanxhi, Erta; Dahle, Jostein

    2012-01-01

    Radiation-induced bystander effects have been studied extensively due to their potential implications for cancer therapy and radiation protection; however, a complete understanding of the molecular mechanisms remains to be elucidated. In this study, we monitored transcriptional responses to γ radiation in irradiated and bystander fibroblasts simultaneously employing a genome-wide microarray approach to determine factors that may be modulated in the generation or propagation of the bystander effect. For the microarray data we employed analysis at both the single-gene and gene-set level to place the findings in a biological context. Unirradiated bystander fibroblasts that were recipients of growth medium harvested from irradiated cultures 2 h after exposure to 2 Gy displayed transient enrichment in gene sets belonging to ribosome, oxidative phosphorylation and neurodegenerative disease pathways associated with mitochondrial dysfunctions. The response to direct irradiation was characterized by induction of signaling and apoptosis genes and the gradual formation of a cellular immune response. A set of 14 genes, many of which were regulated by p53, were found to be induced early after irradiation (prior to medium transfer) and may be important in the generation or propagation of the bystander effect.

  7. UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Tatiana Grohmann Ortolan

    Full Text Available DNA damage induced by ultraviolet (UV radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR and the other specific for transcribed DNA (TCR, and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient and XP-C (GGR-deficient primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high, defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.

  8. Titanium dioxide nanoparticles induce matrix metalloprotease 1 in human pulmonary fibroblasts partly via an interleukin-1β-dependent mechanism.

    Science.gov (United States)

    Armand, Lucie; Dagouassat, Maylis; Belade, Esther; Simon-Deckers, Angélique; Le Gouvello, Sabine; Tharabat, Chantal; Duprez, Corinne; Andujar, Pascal; Pairon, Jean-Claude; Boczkowski, Jorge; Lanone, Sophie

    2013-03-01

    Exposure to titanium dioxide (TiO2) nanoparticles (NPs) is associated with lung remodeling, but the underlying mechanisms are unknown. Matrix metalloprotease (MMP)-1 is an important actor in matrix homeostasis and could therefore participate in TiO2 NP effects. Our aim was to evaluate the effects of TiO2 NPs on MMP-1 expression and activity in lung pulmonary fibroblasts and to understand the underlying mechanisms and assess the importance of the physicochemical characteristics of the particles in these effects. Human pulmonary fibroblasts (MRC-5 cell line and primary cells) were exposed to 10 or 100 μg/cm(2) TiO2 (two anatases, two anatase/rutile mix, one rutile NP, and one micrometric) and carbon black (CB) NPs for 6 to 48 hours. We examined cell viability, MMP-1 expression and activity, and the implication of oxidative stress, transforming growth factor (TGF)-β, extracellular MMP inducer, and IL-1β in MMP-1 expression. All TiO2 NPs induced MMP-1 (mRNA and protein expression), repression of procollagen-1, and α-actin expression, but only the two anatase/rutile mix induced MMP-1 activity. Micrometric TiO2 had smaller effects than TiO2 NPs, and CB NPs did not induce MMP-1. MMP-1 induction by TiO2 NPs was not related to TGF-β, oxidative stress, or EMPRIN expression but was related to IL-1β expression, which partly drives MMP-1 induction by two TiO2 NPs (one anatase/rutile mix and the rutile one). Taken together, our results show that TiO2 NPs are potent inducers and regulators of MMP-1 expression and activity, partly via an IL-1β-dependent mechanism. This may explain TiO2 lung remodeling effects.

  9. Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

    Directory of Open Access Journals (Sweden)

    Yukihiro Nishikawa

    Full Text Available Withaferin A (WA, a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD. WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L. Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

  10. The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

    Directory of Open Access Journals (Sweden)

    Hiromi Murase

    2013-01-01

    Full Text Available Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB. Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8 cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS- sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation.

  11. Role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in rheumatic diseases

    Institute of Scientific and Technical Information of China (English)

    ZHU Li-xiu; ZHANG Hai-hong; MEI Yi-fang; ZHAO Yan-ping; ZHANG Zhi-yi

    2012-01-01

    Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily of structurally related cytokines and is known to induce proliferation,migration,differentiation,apoptotic cell death,inflammation,and angiogenesis.These physiological processes are induced by the binding of TWEAK to fibroblast growth factor-inducible 14 (Fn14),a highly inducible cell-surface receptor that is linked to several intracellular signaling pathways,including the nuclear factor-κB (NF-κB) pathway.This review discusses the role of the TWEAK-Fn14 axis in several rheumatic diseases and the potential therapeutic benefits of modulation of the TWEAK-Fn14 pathway.

  12. Differential gene expression before and after ionizing radiation of subcutaneous fibroblasts identifies breast cancer patients resistant to radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Alsner, Jan; Rødningen, Olaug K.; Overgaard, Jens

    2007-01-01

    -induced changes in gene expression in fibroblasts, whether differential expression is more pronounced when looking at the fold induction levels, taking into account the differences in background expression levels between patients, and whether there is a linear correlation between individual risk of RIF...... and changes in radiation-induced gene expression in fibroblasts. MATERIAL AND METHODS: Gene expression was analysed by quantitative real-time PCR before and after a fractionated scheme with 3x3.5Gy/3 days in fibroblasts derived from 26 patients with breast cancer treated with post-mastectomy radiotherapy....... RESULTS: Robust radiation-induced changes in gene expression were observed, with differential gene expression between low and high risk patients being most pronounced for the fold induction level ('after' value divided by 'before' value for each patient). When including patients with intermediate risk...

  13. Differential gene expression before and after ionizing radiation of subcutaneous fibroblasts identifies breast cancer patients resistant to radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Alsner, Jan; Rødningen, Olaug K.; Overgaard, Jens

    2007-01-01

    -induced changes in gene expression in fibroblasts, whether differential expression is more pronounced when looking at the fold induction levels, taking into account the differences in background expression levels between patients, and whether there is a linear correlation between individual risk of RIF...... and changes in radiation-induced gene expression in fibroblasts. MATERIAL AND METHODS: Gene expression was analysed by quantitative real-time PCR before and after a fractionated scheme with 3x3.5Gy/3 days in fibroblasts derived from 26 patients with breast cancer treated with post-mastectomy radiotherapy....... RESULTS: Robust radiation-induced changes in gene expression were observed, with differential gene expression between low and high risk patients being most pronounced for the fold induction level ('after' value divided by 'before' value for each patient). When including patients with intermediate risk...

  14. Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Salahuddin, E-mail: Salah.Ahmed@utoledo.edu [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V. [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Bhansali, Pravin; Tillekeratne, L.M. Viranga [Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States)

    2013-07-15

    In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

  15. Antifibrotic effect of heparin on liver fibrosis model in rats

    Institute of Scientific and Technical Information of China (English)

    Binita; Shah; Gaurang; Shah

    2012-01-01

    AIM: To evaluate the effect of chronic thrombin inhibition by heparin on experimentally induced chronic liver injury (liver fibrosis) in rats. METHODS: Chronic liver injury (liver fibrosis) was induced in Wistar rats by oral administration of carbon tetrachloride (CCl 4 ) for 7 wk, an animal model with persistent severe hepatic fibrosis. Intravenous administration of the thrombin antagonist (heparin) started 1 wk after the start of CCl 4 intoxication for 6 wk. After completion of treatment (7 wk), markers of hepatic dysfunction were measured and changes evaluated histopathologically. RESULTS: Higher serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total, direct and indirect bilirubin levels, as well as lower fibrinogen levels, were found in CCl 4 intoxicated rats. Heparin, silymarin and combination of drug (heparin and silymarin) treatment for 6 wk prevented a rise in SGOT, SGPT, ALP, total, direct and indirect bilirubin levels and improved fibrinogen levels. Deterioration in hepatic function determined by the fibrosis area was retarded, as evident from hepatic histopathology. Total protein levels were not changed in all groups.CONCLUSION: Heparin, a thrombin antagonist, preserved hepatic function and reduced severity of hepatic dysfunction/fibrogenesis. Combination of heparin and silymarin produced additional benefits on liver fibrosis.

  16. Bmx regulates LPS-induced IL-6 and VEGF production via mRNA stability in rheumatoid synovial fibroblasts.

    Science.gov (United States)

    Palmer, Christine D; Mutch, Brenda E; Page, Theresa H; Horwood, Nicole J; Foxwell, Brian M J

    2008-06-13

    Discordant cytokine production is characteristic of chronic inflammatory conditions like rheumatoid arthritis (RA), and anti-cytokine therapeutics are becoming routinely used to treat RA in the clinic. Fibroblasts from rheumatoid synovium have been shown to contribute to cytokine production in inflamed joints; likewise these cells also produce cytokines in response to inflammatory mediators signalling through Toll like receptors (TLRs). Tyrosine kinase activity is essential to LPS-induced cytokine production, and we have previously implicated a role for the Tec kinase, Bmx, in inflammatory cytokine production. Here we show that Bmx kinase activity in RASF is increased following LPS stimulation and that Bmx is involved in the regulation of LPS-induced IL-6 and VEGF production via mRNA stabilisation. This is an important insight into the regulation of VEGF, which is involved in a wide range of different pathologies, and may lead to more effective design of novel anti-inflammatory/angiogenic therapeutics for conditions such as RA.

  17. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2015-11-27

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  18. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts.

    Science.gov (United States)

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-06-14

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model.

  19. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    Science.gov (United States)

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  20. Celiac anti-type 2 transglutaminase antibodies induce differential effects in fibroblasts from celiac disease patients and from healthy subjects.

    Science.gov (United States)

    Paolella, Gaetana; Lepretti, Marilena; Barone, Maria Vittoria; Nanayakkara, Merlin; Di Zenzo, Marina; Sblattero, Daniele; Auricchio, Salvatore; Esposito, Carla; Caputo, Ivana

    2017-03-01

    Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

  1. The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts.

    Science.gov (United States)

    Opländer, Christian; Cortese, Miriam M; Korth, Hans-Gert; Kirsch, Michael; Mahotka, Csaba; Wetzel, Wiebke; Pallua, Norbert; Suschek, Christoph V

    2007-09-01

    Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.

  2. STAT6-dependent collagen synthesis in human fibroblasts is induced by bovine milk

    OpenAIRE

    Stefan Kippenberger; Nadja Zöller; Johannes Kleemann; Jutta Müller; Roland Kaufmann; Matthias Hofmann; August Bernd; Markus Meissner; Eva Valesky

    2015-01-01

    Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold,...

  3. Imaging Features that Discriminate between Foci Induced by High-and Low-LET Radiation in Human Fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Costes, Sylvain V.; Boissiere, Arnaud; Ravani, Shraddha; Romano,Raquel; Parvin, Bahram; Barcellos-Hoff, Mary Helen

    2006-10-08

    In this study, we investigated the formation ofradiation-induced foci in normal human fibroblasts exposed to X rays or130 keV/mum nitrogen ions using antibodies to phosphorylated proteinkinase ataxia telangiectasia mutated (ATMp) and histone H2AX(gamma-H2AX). High-content automatic image analysis was used to quantifythe immunofluorescence of radiation-induced foci. The size ofradiation-induced foci increased for both proteins over a 2-h periodafter nitrogen-ion irradiation, while the size of radiation-induced focidid not change after exposure to low-LET radiation. The number ofradiation-induced ATMp foci showed a more rapid rise and greaterfrequency after X-ray exposure and was resolved more rapidly such thatthe frequency of radiation-induced foci decreased by 90 percent comparedto 60 percent after exposure to high-LET radiation 2 h after 30 cGy. Incontrast, the kinetics of radiation-induced gamma-H2AX focus formationwas similar for high- and low-LET radiation in that it reached a plateauearly and remained constant for up to 2 h. High-resolution 3D images ofradiation-induced gamma-H2AX foci and dosimetry computation suggest thatmultiple double-strand breaks from nitrogen ions are encompassed withinlarge nuclear domains of 4.4 Mbp. Our work shows that the size andfrequency of radiation-induced foci vary as a function of radiationquality, dose, time and protein target. Thus, even though double-strandbreaks and radiation-induced foci are correlated, the dynamic nature ofboth contradicts their accepted equivalence for low doses of differentradiation qualities.

  4. Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.

    Science.gov (United States)

    Pugh, Raymond J; Slee, Joshua B; Farwell, Sara Lynn N; Li, Yaqiu; Barthol, Trista; Patton, Walter A; Lowe-Krentz, Linda J

    2016-03-01

    Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.

  5. High Sulfation and a High Molecular Weight Are Important for Anti-hepcidin Activity of Heparin

    Science.gov (United States)

    Asperti, Michela; Naggi, Annamaria; Esposito, Emiliano; Ruzzenenti, Paola; Di Somma, Margherita; Gryzik, Magdalena; Arosio, Paolo; Poli, Maura

    2016-01-01

    Heparins are efficient inhibitors of hepcidin expression even in vivo, where they induce an increase of systemic iron availability. Heparins seem to act by interfering with BMP6 signaling pathways that control the expression of liver hepcidin, causing the suppression of SMAD1/5/8 phosphorylation. The anti-hepcidin activity persists also when the heparin anticoagulant property is abolished or reduced by chemical reactions of oxidation/reduction (glycol-split, Gs-Heparins) or by high sulfation (SS-Heparins), but the structural characteristics needed to optimize this inhibitory activity have not been studied in detail. To this aim we analyzed three different heparins (Mucosal Heparin, the Glycol split RO-82, the partially desulfated glycol-split RO-68 and the oversulfated SSLMWH) and separated them in fractions of molecular weight in the range 4–16 kD. Since the distribution of the negative charges in heparins contributes to the activity, we produced 2-O- and 6-O-desulfated heparins. These derivatives were analyzed for the capacity to inhibit hepcidin expression in hepatic HepG2 cells and in mice. The two approaches produced consistent results and showed that the anti-hepcidin activity strongly decreases with molecular weight below 7 kD, with high N-acetylation and after 2-O and 6-O desulfation. The high sulfation and high molecular weight properties for efficient anti-hepcidin activity suggest that heparin is involved in multiple binding sites. PMID:26955355

  6. Deep vein thrombosis, ecythyma gangrenosum and heparin-induced thrombocytopenia occurring in a man with a heterozygous Factor V Leiden mutation

    OpenAIRE

    Mariya Apostolova; Baoying Weng; Pote, Harry H.; Harold Ashcraft; Curtis Goldblatt; Paul V. Woolley

    2012-01-01

    Skin necrosis and limb gangrene are occasional thrombotic manifestations of anticoagulation therapy. We report a man heterozygous for the Factor V Leiden (FVL) mutation, and with a history of recurrent deep venous thrombosis, who initially presented with a necrotic skin lesion of the right flank while on warfarin therapy with a therapeutic international normalized ratio. Warfarin was discontinued and he received intravenous heparin. Thereafter he developed thrombocytopenia and pedal erythema ...

  7. Epigenetic Characterization of the FMR1 Promoter in Induced Pluripotent Stem Cells from Human Fibroblasts Carrying an Unmethylated Full Mutation

    Science.gov (United States)

    de Esch, Celine E.F.; Ghazvini, Mehrnaz; Loos, Friedemann; Schelling-Kazaryan, Nune; Widagdo, W.; Munshi, Shashini T.; van der Wal, Erik; Douben, Hannie; Gunhanlar, Nilhan; Kushner, Steven A.; Pijnappel, W.W.M. Pim; de Vrij, Femke M.S.; Geijsen, Niels; Gribnau, Joost; Willemsen, Rob

    2014-01-01

    Summary Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene. PMID:25358783

  8. Epigenetic Characterization of the FMR1 Promoter in Induced Pluripotent Stem Cells from Human Fibroblasts Carrying an Unmethylated Full Mutation

    Directory of Open Access Journals (Sweden)

    Celine E.F. de Esch

    2014-10-01

    Full Text Available Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs of an unmethylated full mutation (uFM individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene.

  9. Overexpression of N-acetylgalactosamine-4-sulphatase induces a multiple sulphatase deficiency in mucopolysaccharidosis-type-VI fibroblasts.

    Science.gov (United States)

    Anson, D S; Muller, V; Bielicki, J; Harper, G S; Hopwood, J J

    1993-09-15

    High-titre stocks of an amphotropic retrovirus, constructed so as to express a full-length cDNA encoding the human lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4-sulphatase) from the cytomegalovirus immediate early promoter, were used to infect skin fibroblasts from a clinically severe mucopolysaccharidosis type VI (MPS VI) patient. The infected MPS VI cells showed correction of the enzymic defect with the enzyme being expressed at high levels and in the correct subcellular compartment. Surprisingly this did not result in correction of glycosaminoglycan turnover as measured by accumulation of 35S in metabolically labelled cells. We demonstrate that this is apparently caused by an induced reduction of the activities of other lysosomal sulphatases, presumably due to competition for a sulphatase-specific processing mechanism by the over-expressed 4-sulphatase. The level of steroid sulphatase, which is a microsomal sulphatase, was also reduced. Infection of skin fibroblasts from a second, clinically mildly affected, MPS VI patient with the same virus also resulted in no significant change in the level of glycosaminoglycan storage. However, in this case the cause of the observed phenomenon was less clear. These results are of obvious practical importance when considering gene therapy for a sulphatase deficiency such as MPS VI and also provide possible new avenues for exploration of the processes involved in sulphatase synthesis and genetically determined multiple sulphatase deficiency.

  10. Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts.

    Science.gov (United States)

    Paggetti, Jerome; Haderk, Franziska; Seiffert, Martina; Janji, Bassam; Distler, Ute; Ammerlaan, Wim; Kim, Yeoun Jin; Adam, Julien; Lichter, Peter; Solary, Eric; Berchem, Guy; Moussay, Etienne

    2015-08-27

    Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

  11. PGE2 induces IL-6 in orbital fibroblasts through EP2 receptors and increased gene promoter activity: implications to thyroid-associated ophthalmopathy.

    Directory of Open Access Journals (Sweden)

    Nupur Raychaudhuri

    Full Text Available BACKGROUND: IL-6 plays an important role in the pathogenesis of Graves' disease and its orbital component, thyroid-associated ophthalmopathy (TAO. Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE(2, underlying the inflammatory phenotype of these cells. METHODOLOGY/PRINCIPAL FINDINGS: Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE(2 on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP(2 receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE(2-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE(2 provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE(2 promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts. CONCLUSION/SIGNIFICANCE: These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO.

  12. 肝素诱导的血小板减少症患者体外循环抗凝管理%Anticoagulation Management in Patients with Heparin-induced Thrombocytopenia Undergoing Extracorporeal Circulation

    Institute of Scientific and Technical Information of China (English)

    周伯颐

    2012-01-01

    Patients with heparin-induced thrombocytopenia have a poor prognosis and high mortality, thus surgical risk under extracorporeal circulation increased. Early diagnosis, safe and effective alternative anticoagulation strategy are crucial for these patients to receive extracorporeal circulation surgery. This review focuses on the pathophysiology, laboratory examination, diagnosis and alternative anticoagulation strategy of extracorporeal circulation for patients with heparin-induced thrombocytopenia.%肝素诱导的血小板减少症患者预后差,病死率高,大大增加了体外循环手术的风险.对其早期诊断和选择一种安全有效的药物替代肝素抗凝是这类患者接受体外循环手术的关键.我们对肝素诱导的血小板减少症发病机制、实验室检查、诊断以及体外循环抗凝管理进行综述.

  13. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  14. Exposure to titanium dioxide and other metallic oxide nanoparticles induces cytotoxicity on human neural cells and fibroblasts

    Directory of Open Access Journals (Sweden)

    James C K Lai

    2008-12-01

    Full Text Available James C K Lai1, Maria B Lai1, Sirisha Jandhyam1, Vikas V Dukhande1, Alok Bhushan1, Christopher K Daniels1, Solomon W Leung21Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, and Biomedical Research Institute; 2Department of Civil and Environmental Engineering, College of Engineering and Biomedical Research Institute, Idaho State University, Pocatello, ID, USAAbstract: The use of titanium dioxide (TiO2 in various industrial applications (eg, production of paper, plastics, cosmetics, and paints has been expanding thereby increasing the occupational and other environmental exposure of these nanoparticles to humans and other species. However, the health effects of exposure to TiO2 nanoparticles have not been systematically assessed even though recent studies suggest that such exposure induces inflammatory responses in lung tissue and cells. Because the effects of such nanoparticles on human neural cells are unknown, we have determined the putative cytotoxic effects of these nanoparticles on human astrocytes-like astrocytoma U87 cells and compared their effects on normal human fibroblasts. We found that TiO2 micro- and nanoparticles induced cell death on both human cell types in a concentration-related manner. We further noted that zinc oxide (ZnO nanoparticles were the most effective, TiO2 nanoparticles the second most effective, and magnesium oxide (MgO nanoparticles the least effective in inducing cell death in U87 cells. The cell death mechanisms underlying the effects of TiO2 micro- and nanoparticles on U87 cells include apoptosis, necrosis, and possibly apoptosis-like and necrosis-like cell death types. Thus, our findings may have toxicological and other pathophysiological implications on exposure of humans and other mammalian species to metallic oxide nanoparticles.Keywords: cytotoxicity of titanium dioxide micro- and nanoparticles, cytotoxicity of zinc oxide and magnesium oxide nanoparticles, human neural cells

  15. Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan.

    Science.gov (United States)

    Pfefferkorn, E R

    1984-01-01

    Treatment of human fibroblasts with human recombinant gamma interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth of the parasite was measured by a plaque assay 7 days after infection or by the incorporation of [3H]uracil 1 or 2 days after infection. The antitoxoplasma activity induced in the host cells by gamma interferon was strongly dependent upon the tryptophan concentration of the medium. Progressively higher minimal inhibitory concentrations of gamma interferon were observed as the tryptophan concentration in the culture medium was increased. Treatment with gamma interferon did not make the cells impermeable to tryptophan. The kinetics of [3H]tryptophan uptake into the acid-soluble pools of control and gamma interferon-treated cultures were identical during the first 48 sec. Thereafter uptake of [3H]tryptophan into the acid-soluble pool of control fibroblasts reached the expected plateau after 96 sec. In contrast, uptake of [3H]tryptophan continued for at least 12 min in the gamma interferon-treated cultures. At that time, the acid-soluble pool of the gamma interferon-treated cultures contained 8 times the radioactivity of the control cultures. This continued accumulation was the result of rapid intracellular degradation of [3H]tryptophan into kynurenine and N-formylkynurenine that leaked slowly from the cells. These two metabolites were also recovered from the medium of cultures treated for 1 or 2 days with gamma interferon. Human recombinant alpha and beta interferons, which have no antitoxoplasma activity, did not induce any detectable degradation of tryptophan. Several hypotheses are presented to explain how the intracellular degradation of tryptophan induced by gamma interferon could restrict the growth of an obligate intracellular parasite. Images PMID:6422465

  16. [Influence of ASODN to the human tenon's fibroblasts in expressing CTGF induced by transforming growth factor beta2].

    Science.gov (United States)

    Hu, Yi-Zhen; Wang, Yu-Hong; Cao, Yang; Zhang, Ming-Chang

    2008-02-01

    To investigate the effect of connective tissue growth factor's antisense oligonucleotides (ASODN) on the growth of human tenon' s capsule fibroblasts (HTF) induced by transforming growth factor beta2 (TGF-beta2) in vitro. It was a experimental study. HTF was collected from glaucoma patients and cultured. The 5-6 passage was used for experiments. The HTF induced by TGF-beta2 was divided into the following groups: N group: normal HTF; T group: HTF induced by TGF-beta2; A group: CTGF ASODN antisense:5'-TACTGGCGGCGGTCAT-3' encapsulated with liposome; S group: sense 5'-ATGACCGCCGCCAGTA-3' encapsulated with liposome; D group: HTF encapsulated with liposome only. The activity of HTF treated by different concentrations of liposome was detected using methylthianolyldiphenyl tetrazolium bromide (MT) colorimetry. The expression of CTGF was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry assays. The expression of fibronectin (Fn) was examined by Western blot and immunocytochemistry assays. Liposome-ASODN (A group) significantly (F=15.25, 204.88, 19.73, 90.00; P HTF induced by TGF-beta2 compared with S and D group. However, Liposome alone (T group) has no significant impact in HTF growth compared with T group (t = 0.90, 2.32, 0.75, 2.11; P > 0.05). CTGF-ASODN inhibits the CTGF and Fn expression of HTF induced by TGF-beta2, which may delay the formation of scar in glaucoma filtering surgery.

  17. Characterization of arecoline-induced effects on cytotoxicity in normal human gingival fibroblasts by global gene expression profiling.

    Science.gov (United States)

    Chiang, Shang-Lun; Jiang, Shih-Sheng; Wang, Yi-Jou; Chiang, Horn-Che; Chen, Ping-Ho; Tu, Hung-Pin; Ho, Kun-Yen; Tsai, Yu-Shan; Chang, I-Shou; Ko, Ying-Chin

    2007-11-01

    Areca nut is the most widely used psychoactive substance and an important environmental risk factor for development of oral premalignant lesions and cancer. Arecoline, the major alkaloid of areca nut, has been known to cause cytotoxicity and genotoxicity in mammalian cells in vivo and in vitro and even contributes to carcinogenicity. However, the susceptible genes accounting for arecoline-induced damage in normal human oral cells are still lacking, which possibly involves in initial molecular damage via alternation of gene expression level on biological pathways. The present study was undertaken to characterize the toxic effects of arecoline in gene expression profiling on normal human gingival fibroblasts (HGF) using cDNA microarray and quantitative real-time reverse transcription PCR. The cytotoxicity of arecoline on HGF-1 cell line was elevated in a dose-dependent manner (p arecoline determined from dose-response curve of the cytotoxicity, a large number of genes were significantly repressed than induced by arecoline in global gene expression profiling. Five induced- and seven repressed genes including glutathione synthetase were further validated, and their gene expression changes were increased in a dose-dependent manner in a concentration range of 50-150 microg/ml. In conclusion, we proposed a tentative model to explain arecoline-induced effects on contribution of oral pathogenesis. The findings identified that 12 susceptible genes can potentially serve as biomarkers of arecoline-induced damage in betel chewers.

  18. Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts.

    Science.gov (United States)

    Park, Jihwan; Kwon, Yoo-Wook; Ham, Seokjin; Hong, Chang-Pyo; Seo, Seonghye; Choe, Moon Kyung; Shin, So-I; Lee, Choon-Soo; Kim, Hyo-Soo; Roh, Tae-Young

    2017-01-01

    The generation of induced pluripotent stem cell (iPSC), a substitute for embryonic stem cell (ESC), requires the proper orchestration of a transcription program at the chromatin level. Our recent approach for the induction of pluripotent stem cells from fibroblasts using protein extracts from mouse ESCs could overcome the potential tumorigenicity risks associated with random retroviral integration. Here, we examine the epigenetic modifications and the transcriptome of two types of iPSC and of partially reprogrammed iPSCs (iPSCp) generated independently from adult cardiac and skin fibroblasts to assess any perturbations of the transcription program during reprogramming. The comparative dissection of the transcription profiles and histone modification patterns at lysines 4 and 27 of histone H3 of the iPSC, iPSCp, ESC, and somatic cells revealed that the iPSC was almost completely comparable to the ESC, regardless of their origins, whereas the genes of the iPSCp were dysregulated to a larger extent. Regardless of the origins of the somatic cells, the fibroblasts induced using the ESC protein extracts appear to be completely reprogrammed into pluripotent cells, although they show unshared marginal differences in their gene expression programs, which may not affect the maintenance of stemness. A comparative investigation of the iPSCp generated by unwanted reprogramming showed that the two groups of genes on the pathway from somatic cells to iPSC might function as sequential reprogramming-competent early and late responders to the induction stimulus. Moreover, some of the divergent genes expressed only in the iPSCp were associated with many tumor-related pathways. Faithful transcriptional reprogramming should follow epigenetic alterations to generate induced pluripotent stem cells from somatic cells. This genome-wide comparison enabled us to define the early and late responder genes during the cell reprogramming process to iPSC. Our results indicate that the cellular

  19. In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

    DEFF Research Database (Denmark)

    Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens

    2008-01-01

    BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins....... MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively...... in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity. Udgivelsesdato: 2008-Mar...

  20. Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

    Directory of Open Access Journals (Sweden)

    Ha Na Kim

    2017-05-01

    Full Text Available Heparin and heparan sulfate are structurally-related carbohydrates with therapeutic applications in anticoagulation, drug delivery, and regenerative medicine. This study explored the effect of different bioreactor conditions on the production of heparin/heparan sulfate chains via the recombinant expression of serglycin in mammalian cells. Tissue culture flasks and continuously-stirred tank reactors promoted the production of serglycin decorated with heparin/heparan sulfate, as well as chondroitin sulfate, while the serglycin secreted by cells in the tissue culture flasks produced more highly-sulfated heparin/heparan sulfate chains. The serglycin produced in tissue culture flasks was effective in binding and signaling fibroblast growth factor 2, indicating the utility of this molecule in drug delivery and regenerative medicine applications in addition to its well-known anticoagulant activity.

  1. A hyaluronic acid-based compound inhibits fibroblast senescence induced by oxidative stress in vitro and prevents oral mucositis in vivo.

    Science.gov (United States)

    Cirillo, Nicola; Vicidomini, Antonio; McCullough, Michael; Gambardella, Antonio; Hassona, Yazan; Prime, Stephen S; Colella, Giuseppe

    2015-07-01

    Virtually all patients receiving radio- and chemotherapy for cancer develop oral mucositis, a severe and highly debilitating condition. The onset of mucositis is thought to involve the production of reactive oxygen species (ROS) in the submucosa. Here we investigated a possible protective effect of a commercial formulation of hyaluronic acid (HA) enriched with amino acids (Mucosamin(®)) against the damage induced by oxidative stress both in vitro and in vivo. Transient exposure of normal human oral fibroblasts to hydrogen peroxide (H(2)O(2)) led to irreversible senescence, as demonstrated by sustained increase in the levels of p16(INK4A) and SA-βGal. Conditioned media from senescent fibroblasts induced detrimental effects on keratinocytes, as shown by reduced metabolic activity and migration capability. Pre-treatment with Mucosamin(®) prevented H(2)O(2) -induced, but not TGF-β-induced, fibroblast senescence with a concomitant reduction of fibroblast-induced loss of keratinocyte vitality and functional activity. Finally, data from a case-series of patients undergoing radio/chemotherapy strongly suggested that prophylactic use of the hyaluronic acid-based compound in the form of a spray may be effective in preventing the onset of oral mucositis.

  2. 21 CFR 864.7525 - Heparin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and... HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A heparin assay is a device used to determine the level of the anticoagulant heparin in the...

  3. Stromal Fibroblasts from the Interface Zone of Triple Negative Breast Carcinomas Induced Epithelial-Mesenchymal Transition and its Inhibition by Emodin

    Science.gov (United States)

    Wang, Hao-Yu; Hung, Chao-Ming; Lin, Ying-Chao; Ho, Chi-Tang; Way, Tzong-Der

    2017-01-01

    “Triple negative breast cancer” (TNBC) is associated with a higher rate and earlier time of recurrence and worse prognosis after recurrence. In this study, we aimed to examine the crosstalk between fibroblasts and TNBC cells. The fibroblasts were isolated from TNBC patients’ tissue in tumor burden zones, distal normal zones and interface zones. The fibroblasts were indicated as cancer-associated fibroblasts (CAFs), normal zone fibroblasts (NFs) and interface zone fibroblasts (INFs). Our study found that INFs grew significantly faster than NFs and CAFs in vitro. The epithelial BT20 cells cultured with the conditioned medium of INFs (INFs-CM) and CAFs (CAFs-CM) showed more spindle-like shape and cell scattering than cultured with the conditioned medium of NFs (NFs-CM). These results indicated that factors secreted by INFs-CM or CAFs-CM could induce the epithelial-mesenchymal transition (EMT) phenotype in BT20 cells. Using an in vitro co-culture model, INFs or CAFs induced EMT and promoted cancer cell migration in BT20 cells. Interestingly, we found that emodin inhibited INFs-CM or CAFs-CM-induced EMT programming and phenotype in BT20 cells. Previous studies reported that CAFs and INFs-secreted TGF-β promoted human breast cancer cell proliferation, here; our results indicated that TGF-β initiated EMT in BT20 cells. Pretreatment with emodin significantly suppressed the TGF-β-induced EMT and cell migration in BT20 cells. These results suggest that emodin may be used as a novel agent for the treatment of TNBC. PMID:28060811

  4. [Heparin immune thrombocytopenia in a hemodialysis patient. A case report. Literature review].

    Science.gov (United States)

    Benítez, M; González Gómez, I; González Carmelo, I; Palma, A; Cruz, S; Rodríguez, E; Amián, A; Fernández M, F; Merino, M J; Fernández G, F; Suárez, C; Onaindía, J M; González, J

    2007-01-01

    Heparin-induced thrombocytopenia (HIT) is an important side effect of heparin therapy associated with significant morbidity and mortality if unrecognized. The platelet count typically falls below 150,000/ll 5-14 days after heparin is started. Thrombosis is the major clinical complication. We present the case gives a patient that develops a deep vein thrombosis ilio-femoral left, with trombocytopenia, one week after beginning treatment with haemodialysis in which Ac anti heparin is detected by test PaGIA (Particle Gel Inmuno Assay.

  5. Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action.

    Science.gov (United States)

    Jones, M; Tussey, L; Athanasou, N; Jackson, D G

    2000-03-17

    The CD44 glycoprotein is expressed in multiple isoforms on a variety of cell types where it functions as a receptor for hyaluronan-mediated motility. Recently, interest has centered on CD44 heparan sulfate proteoglycan (HSPG) isoforms because of their potential to sequester heparin-binding growth factors and chemokines. Expression of these isoforms on ectodermal cells has recently been shown to regulate limb morphogenesis via presentation of fibroblast growth factor (FGF) 4/FGF 8 while expression on tumor cells was shown to sequester hepatocyte growth factor and promote tumor dissemination. To date, however, CD44 HSPG expression in tissue macrophages and lymphocytes has not been adequately investigated, despite the fact these cells actively synthesize growth factors and chemokines and indirect evidence that monocyte CD44 sequesters macrophage inflammatory protein-1beta. Here we show primary human monocytes rather than lymphocytes express CD44 HSPGs, but only following in vitro differentiation to macrophages or activation with the proinflammatory cytokine interleukin-1alpha or bacterial lipopolysaccharide. Furthermore, we show these isoforms are preferentially modified with heparan rather than chondroitin sulfate, bind the macrophage-derived growth factors FGF-2, vascular endothelial growth factor, and heparin-binding epidermal growth factor with varying affinities (K(d) 25-330 nM) and in the case of FGF-2, can stimulate productive binding to the high affinity tyrosine kinase FGF receptor 1 (FGFR1). In contrast, we find no evidence for significant binding to C-C chemokines. Last, we confirm by immunofluorescent antibody staining that inflamed synovial membrane macrophages express CD44 HSPGs and that expression is greatest in cells containing high FGF-2 levels. These results suggest a paracrine role for macrophage CD44 HSPG isoforms in the regulation of growth factor action during inflammation.

  6. Fibroblast Growth Factor Receptor-2 Contributes to the Basic Fibroblast Growth Factor-Induced Neuronal Differentiation in Canine Bone Marrow Stromal Cells via Phosphoinositide 3-Kinase/Akt Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Rei Nakano

    Full Text Available Bone marrow stromal cells (BMSCs are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2 and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR, phosphatidylinositol 3-kinase (PI3K and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs.

  7. Dimerization capacities of FGF2 purified with or without heparin-affinity chromatography.

    Directory of Open Access Journals (Sweden)

    Natalia Platonova

    Full Text Available Fibroblast growth factor-2 (FGF2 is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.

  8. Fibroblast-specific expression of AC6 enhances beta-adrenergic and prostacyclin signaling and blunts bleomycin-induced pulmonary fibrosis.

    Science.gov (United States)

    Liu, Xiaoqiu; Li, Fengying; Sun, Shu Qiang; Thangavel, Muthusamy; Kaminsky, Joseph; Balazs, Louisa; Ostrom, Rennolds S

    2010-06-01

    Pulmonary fibroblasts regulate extracellular matrix production and degradation and are critical in maintenance of lung structure, function, and repair, but they also play a central role in lung fibrosis. cAMP-elevating agents inhibit cytokine- and growth factor-stimulated myofibroblast differentiation and collagen synthesis in pulmonary fibroblasts. In the present study, we overexpressed adenylyl cyclase 6 (AC6) in pulmonary fibroblasts and measured cAMP production and collagen synthesis. AC6 overexpression enhanced cAMP production and the inhibition of collagen synthesis mediated by isoproterenol and beraprost, but not the responses to butaprost or PGE(2). To examine if increased AC6 expression would impact the development of fibrosis in an animal model, we generated transgenic mice that overexpress AC6 under a fibroblast-specific promoter, FTS1. Lung fibrosis was induced in FTS1-AC6(+/-) mice and littermate controls by intratracheal instillation of saline or bleomycin. Wild-type mice treated with bleomycin showed extensive peribronchial and interstitial fibrosis and collagen deposition. By contrast, FTS1-AC6(+/-) mice displayed decreased fibrotic development, lymphocyte infiltration (as determined by pathological scoring), and lung collagen content. Thus, AC6 overexpression inhibits fibrogenesis in the lung by reducing pulmonary fibroblast-mediated collagen synthesis and myofibroblast differentiation. Because AC6 overexpression does not lead to enhanced basal or PGE(2)-stimulated levels of cAMP, we conclude that endogenous catecholamines or prostacyclin is produced during bleomycin-induced lung fibrosis and that these signals have antifibrotic potential.

  9. Engineered heparins as new anticoagulant drugs.

    Science.gov (United States)

    Vaidyanathan, Deepika; Williams, Asher; Dordick, Jonathan S; Koffas, Mattheos A G; Linhardt, Robert J

    2017-03-01

    Heparin is an anionic polysaccharide that is widely used as a clinical anticoagulant. This glycosaminoglycan is prepared from animal tissues in metric ton quantities. Animal-sourced heparin is also widely used in the preparation of low molecular weight heparins that are gaining in popularity as a result of their improved pharmacological properties. The recent contamination of pharmaceutical heparin together with concerns about increasing demand for this life saving drug and the fragility of the heparin supply chain has led the scientific community to consider other potential sources for heparin. This review examines progress toward the preparation of engineered heparins through chemical synthesis, chemoenzymatic synthesis, and metabolic engineering.

  10. Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

    DEFF Research Database (Denmark)

    Rønn, L C; Doherty, P; Holm, A;

    2000-01-01

    The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently...... identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons...... from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1...

  11. Induced pluripotent stem cells (iPSCs derived from cerebrotendinous xanthomatosis (CTX patient's fibroblasts carrying a R395S mutation

    Directory of Open Access Journals (Sweden)

    Philip Höflinger

    2016-09-01

    Full Text Available Induced pluripotent stem cells (iPSCs were generated from dermal fibroblasts from a 60-year-old cerebrotendinous xanthomatosis (CTX patient, carrying a homozygous mutation c. [1183C>A]; p. R395S in CYP27A1. Episomal plasmids encoding the pluripotency genes OCT4, SOX2, KLF4, L-MYC and LIN28 were introduced via electroporation. The generated line iPS-CTX-R395S has no sign of plasmid integration or chromosomal aberration and retained the mutation site in CYP27A1. Furthermore, iPSCs express pluripotency markers and are able to differentiate in all germ layers in vitro. The generated line may be a useful tool for disease modelling of CTX.

  12. Fibroblast growth factor-21 is induced in human skeletal muscles by hyperinsulinemia

    DEFF Research Database (Denmark)

    Hojman, Pernille; Pedersen, Maria; Nielsen, Anders Rinnov

    2009-01-01

    OBJECTIVE: Fibroblast growth factor-21 (FGF-21) is a potent metabolic regulator, which in animal models has been shown to improve glucose metabolism and insulin sensitivity. Recently, FGF-21 was shown to be expressed and secreted from murine muscle cells in response to insulin stimulation. RESEARCH...... DESIGN AND METHODS: We studied muscular FGF-21 expression and plasma FGF-21 after acute insulin stimulation in young healthy men during a hyperinsulinemic-euglycemic clamp. Furthermore, we investigated systemic levels and muscle FGF-21 expression in humans with or without insulin resistance and chronic...... elevated insulin. RESULTS: FGF-21 was barely detectable in young healthy men before insulin infusion. After 3 or 4 h of insulin infusion during a hyperinsulinemic-euglycemic clamp, muscular FGF-21 expression increased significantly. Plasma FGF-21 followed the same pattern. In individuals with chronic...

  13. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells.

    Science.gov (United States)

    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells.

  14. Bisdemethoxycurcumin Increases Sirt1 to Antagonize t-BHP-Induced Premature Senescence in WI38 Fibroblast Cells

    Directory of Open Access Journals (Sweden)

    Ying-Bo Li

    2013-01-01

    Full Text Available Curcuminoids are well known for their capabilities to combat risk factors that are associated with ageing and cellular senescence. Recent reports have demonstrated that curcuminoids can extend the lifespan of model organisms. However, the underlying mechanisms by which these polyphenic compounds exert these beneficial effects remain unknown. In this study, t-BHP-induced premature senescence model in human fibroblasts was chosen to explore the protective effects of a curcuminoid, bisdemethoxycurcumin (BDMC, on cellular senescence. The results demonstrated that BDMC attenuated oxidative stress-induced senescence-like features which include the induction of an enlarged cellular appearance, higher frequency of senescence-associated β-galactosidase staining activity, appearance of senescence-associated heterochromatic foci in nuclei, decrease in proliferation capability, and alteration in related molecules such as p16 and retinoblastoma protein. Notably, we found that BDMC treatment activated Sirt1/AMPK signaling pathway. Moreover, downregulating Sirt1 by the pharmacological inhibitor nicotianamine or small interfering RNA blocked BDMC-mediated protection against t-BHP-mediated decrease in proliferation. These results suggested that BDMC prevented t-BHP-induced cellular senescence, and BDMC-induced Sirt1 may be a mechanism mediating its beneficial effects.

  15. Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Yingying; Chen, Xi; Yu, Dehai [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Tao [Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Cui, Jiuwei; Wang, Guanjun [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Hu, Ji-Fan, E-mail: jifan@stanford.edu [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Wei, E-mail: jdyylw@163.com [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China)

    2015-09-10

    Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

  16. Arctiin induces an UVB protective effect in human dermal fibroblast cells through microRNA expression changes.

    Science.gov (United States)

    Lee, Ghang Tai; Cha, Hwa Jun; Lee, Kwang Sik; Lee, Kun Kook; Hong, Jin Tae; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan; Bae, Seunghee

    2014-03-01

    Ultraviolet (UV) radiation induces severe alterations in the molecular and cellular components of normal human dermal fibroblast (NHDF) cells by disrupting many intracellular transduction cascades. Although UV responses have been well documented at the genome and proteome levels, UV protective effects have not been elucidated at these levels. The aim of the present study was to demonstrate that arctiin, a phytochemical isolated from the plant Arctium lappa, induced a protective effect against UVB radiation by changing microRNA (miRNA) expression profiles. Using flow cytometry, and water-soluble tetrazolium salt (WST-1)-based cell viability, wound healing, and DNA repair assays we showed that pretreatment with arctiin prior to UVB irradiation reduced UVB-induced apoptosis, cell migration defects, and DNA damage in NHDF cells. It was also found that arctiin‑induced UVB protection is associated with altered miRNA expression profiles. Bioinformatic analysis revealed that the deregulated miRNAs were functionally involved in mitogen-activated protein kinase (MAPK) signaling and cancer signaling pathways. The results suggest that arctiin acts as a UVB protective agent by altering specific miRNA expression in NHDF cells.

  17. Heparin versus low molecular weight heparin K 2165 in chronic hemodialysis patients: a randomized cross-over study.

    Science.gov (United States)

    Borm, J J; Krediet, R; Sturk, A; ten Cate, J W

    1986-01-01

    Ten patients on chronic intermittent hemodialysis treatment received either unfractionated heparin or low molecular weight (LMW) heparin K 2165 in a single-blinded randomized cross-over study to assess: effects on hemostasis and ex vivo platelet functions, and effectiveness, i.e. prevention of fibrin formation in the extracorporeal circuit. The 20 dialysis treatments were without untoward side effects, for both drugs used. The variation in the plasma anti-Xa activities was significantly less during K 2165 treatment than during heparinization. No differences between the drugs were observed regarding the Ivy bleeding time, platelet count and platelet aggregation (spontaneous, and induced by ADP and collagen). Plasma platelet factor 4 levels did not increase under K 2165 to such an extent as under heparin. Both drugs did not influence the plasma levels of beta-thromboglobulin, thromboxane B2 and platelet serotonin content. K 2165 did not affect platelet adhesion to collagen, in contrast to heparin which substantially inhibited platelet adhesion. Under both treatments, 4 minor clots were observed in 4 artificial kidneys, despite plasma anti-Xa levels in between 0.19 and 0.46 U/ml. K 2165 may therefore be considered as effective an anticoagulant as heparin, with less effects on ex vivo platelet functions.

  18. Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

    Energy Technology Data Exchange (ETDEWEB)

    Han, Young-Hyun; Garron, Marie-Line; Kim, Hye-Yeon; Kim, Wan-Seok; Zhang, Zhenqing; Ryu, Kyeong-Seok; Shaya, David; Xiao, Zhongping; Cheong, Chaejoon; Kim, Yeong Shik; Linhardt, Robert J.; Jeon, Young Ho; Cygler, Miroslaw; (SNU); (Korea BSI); (McGill); (UST-Korea); (Rensselaer)

    2010-01-12

    Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a {beta}-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an ({alpha}/{alpha}){sub 6} fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.

  19. Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Secher, Jan Ole Bertelsen; Ceylan, Ahmet; Mazzoni, Gianluca;

    2017-01-01

    Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed...

  20. Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Secher, Jan O; Ceylan, Ahmet; Mazzoni, Gianluca

    2017-01-01

    Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to sh...

  1. Protective effects of basic fibroblast growth factor in the development of emphysema induced by interferon-γ.

    Science.gov (United States)

    Lee, Byung-Jae; Moon, Hyung-Geun; Shin, Tae-Seop; Jeon, Seong Gyu; Lee, Eun-Young; Gho, Yong Song; Lee, Chun Geun; Zhu, Zhou; Elias, Jack A; Kim, Yoon-Keun

    2011-04-30

    Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-γ in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-β, is important in wound healing. We investigated the role of FGF2 in IFN-γ-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-γ-induced emphysema, lung targeted IFN-γ transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 μg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-γ in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-γ but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-γ-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.

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

    Science.gov (United States)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K; Ahmed, Salahuddin

    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 (1-5μM) 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 (pinhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (pTNF-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 (pTNF-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.

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

    Science.gov (United States)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin

    2015-01-01

    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. PMID:26134265

  4. Low molecular weight heparins prevent thrombin-induced thrombo-embolism in mice despite low anti-thrombin activity. Evidence that the inhibition of feed-back activation of thrombin generation confers safety advantages over direct thrombin inhibition.

    Science.gov (United States)

    Momi, S; Nasimi, M; Colucci, M; Nenci, G G; Gresele, P

    2001-03-01

    Thrombin-induced thromboembolism in mice is a model in which the feed-back clotting activation produced by the injected enzyme greatly contributes to fibrin accumulation in lungs and to mortality. Using this model we have previously shown that activated human protein C (aPC), by interrupting endogenous clotting activation at a high level (factors Va and VIIIa), prevents mortality inducing only a minor hemostatic impairment. With the same model we have now compared the antithrombotic and prohemorrhagic effects of two low molecular weight heparins (LMWHs), reviparin and tinzaparin, which are expected to inhibit preferentially the positive feed-back triggered by thrombin (anti Xa activity), with those of unfractionated heparin (UFH) and PEG-hirudin, which inhibit mainly or exclusively thrombin activity (anti IIa activity). Pulmonary thromboembolism was induced in mice by i.v. injection of bovine thrombin (1,000U/kg). Drugs (from 0.12 to 1.2 mg/kg) were given as bolus injection 2 min prior to thrombin challenge and mortality was assessed within 15 min. The bleeding time was assessed by a tail tip transection model. Activated partial thromboplastin time (aPTT), thrombin clotting time (TcT), fibrinogen assay and anti Xa activity determination were performed in citrated plasma from saline- or drug-treated animals. All drugs protected mice from thrombin-induced mortality in a dose-dependent way. At comparable antithrombotic dosages, the anti IIa activity generated in plasma (assessed by TcT) was highest with UFH, intermediate with tinzaparin and very low with reviparin. Accordingly, the fibrinogen drop, which is caused mainly by the injected thrombin, was prevented by the heparins to an extent that was fairly well related to their anti IIa activity. aPTT and bleeding time, used as measures of hemorrhagic risk, were markedly more prolonged by UFH than by reviparin. Tinzaparin, instead, had an intermediate effect. Interestingly, PEG-hirudin, at equipotent antithrombotic

  5. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells

    Energy Technology Data Exchange (ETDEWEB)

    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito, E-mail: y-ihara@wakayama-med.ac.jp

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells. - Highlights: • Hsc70 siRNA treatment suppressed the expression of Hsc70 but induced the expression of Hsp70 in NRK-49F cells. • Hsc70 siRNA treatment suppressed the activation of Smad2/3 in the cells treated with TGF-β. • Hsc70 interacted with Smad2/3 on stimulation with TGF-β in the cells. • Hsp70 did not influence the TGF-β-induced activation of Smad2/3 in the cells overexpressing Hsp70.

  6. Inhibition by a selective IkappaB kinase-2 inhibitor of interleukin-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.

    Science.gov (United States)

    Kondo, Yukiko; Fukuda, Ken; Adachi, Tadafumi; Nishida, Teruo

    2008-11-01

    Corneal ulcer results from excessive collagen degradation in the corneal stroma. Interleukin (IL)-1 promotes this process by activating signaling molecules that include nuclear factor (NF)-kappaB and stimulating the synthesis of matrix metalloproteinases (MMPs) in corneal fibroblasts. NF-kappaB activation is mediated by phosphorylation of the inhibitor IkappaB by IkappaB kinase (IKK)-2 and consequent IkappaB degradation. The authors investigated the effects of the IKK-2 inhibitor [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) on collagen degradation by corneal fibroblasts. Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels. Collagen degradation was evaluated by spectrophotometric quantitation of hydroxyproline in culture supernatants subjected to acid-heat hydrolysis. Expression of MMPs was evaluated by immunoblot analysis, gelatin zymography, and real-time reverse transcription polymerase chain reaction analysis. The phosphorylation and degradation of IkappaBalpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively. IL-1beta-induced collagen degradation by corneal fibroblasts was inhibited by TPCA-1 in a concentration- and time-dependent manner. TPCA-1 inhibited the IL-1beta-induced expression of MMP-1, -3, and -9 in these cells at both the mRNA and protein levels and the IL-1beta-induced activation of pro-MMP-2. In contrast to dexamethasone, TPCA-1 inhibited the phosphorylation and degradation of IkappaBalpha and the nuclear translocation of NF-kappaB induced by IL-1beta. An IKK-2 inhibitor blocked IL-1beta-induced collagen degradation by corneal fibroblasts by inhibiting the activation of the NF-kappaB signaling pathway and the upregulation of MMPs. IKK-2 inhibitors are thus potential alternatives to dexamethasone for the treatment of corneal ulcer.

  7. Heparin Characterization: Challenges and Solutions

    Science.gov (United States)

    Jones, Christopher J.; Beni, Szabolcs; Limtiaco, John F. K.; Langeslay, Derek J.; Larive, Cynthia K.

    2011-07-01

    Although heparin is an important and widely prescribed pharmaceutical anticoagulant, its high degree of sequence microheterogeneity and size polydispersity make molecular-level characterization challenging. Unlike nucleic acids and proteins that are biosynthesized through template-driven assembly processes, heparin and the related glycosaminoglycan heparan sulfate are actively remodeled during biosynthesis through a series of enzymatic reactions that lead to variable levels of O- and N-sulfonation and uronic acid epimers. As summarized in this review, heparin sequence information is determined through a bottom-up approach that relies on depolymerization reactions, size- and charge-based separations, and sensitive mass spectrometric and nuclear magnetic resonance experiments to determine the structural identity of component oligosaccharides. The structure-elucidation process, along with its challenges and opportunities for future analytical improvements, is reviewed and illustrated for a heparin-derived hexasaccharide.

  8. Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakahara, Takehiro; Sato, Hiroko; Shimizu, Takehisa; Tanaka, Toru; Matsui, Hiroki; Kawai-Kowase, Keiko; Sato, Mahito; Iso, Tatsuya; Arai, Masashi [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Kurabayashi, Masahiko, E-mail: mkuraba@med.gunma-u.ac.jp [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2010-04-02

    Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling upstream of the Runx2-mediated activation of osteoblastic program in vascular smooth muscle cells (VSMC) is poorly understood. We examined the effects of fibroblast growth factor-2 (FGF-2), an important regulator of bone formation, on osteoblastic differentiation of VSMC. Stimulation of cultured rat aortic SMC (RASMC) with FGF-2 induced the expression of the osteoblastic markers osteopontin (OPN) and osteocalcin. Luciferase assays showed that FGF-2 induced osteocyte-specific element (OSE)-dependent transcription. Downregulation of Runx2 by siRNA repressed the basal and FGF-2-stimulated expression of the OPN gene in RASMC. FGF-2 produced hydrogen peroxide in RASMC, as evaluated by fluorescent probe. Induction of OPN expression by FGF-2 was inhibited not only by PD98059 (MEK1 inhibitor) and PP1 (c-Src inhibitor), but also by an antioxidant, N-acetyl cysteine. Nuclear extracts from FGF-2-treated RASMC exhibited increased DNA-binding of Runx2 to its target sequence. Immunohistochemistry of human coronary atherectomy specimens and calcified aortic tissues showed that expression of FGF receptor-1 and Runx2 was colocalized. In conclusion, these results suggest that FGF-2 plays a role in inducing osteoblastic differentiation of VSMC by activating Runx2 through mitogen-activated protein kinase (MAPK)-dependent- and oxidative stress-sensitive-signaling pathways.

  9. Extracellular matrix elasticity modulates TGF-β-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.

    Science.gov (United States)

    Meyer-ter-Vehn, Tobias; Han, Hong; Grehn, Franz; Schlunck, Günther

    2011-11-25

    Extracellular matrix and the cytokine TGF-β influence scar formation in an interdependent fashion. In this study, the impact of extracellular matrix elasticity on TGF-β-induced signal transduction and myofibroblast transdifferentiation was examined. Primary human tenon fibroblasts were seeded on collagen-coated glass coverslips (rigid environment) or collagen or polyacrylamide gels (elastic environment) of different compliance and stimulated with TGF-β. Myofibroblast transdifferentiation was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis for the marker gene α-smooth muscle actin (SMA), and SMA incorporation into stress fibers was determined by confocal immunofluorescence microscopy. CTGF transcription was assessed by RT-qPCR. Signaling pathways were examined by Western blot using phosphospecific antibodies and by immunofluorescence microscopy. TGF-β-dependent myofibroblast transdifferentiation was enhanced in a stiff environment. Increasing matrix elasticity attenuated TGF-β-induced myofibroblast transdifferentiation and the associated CTGF expression. TGF-β-induced p38 activation was reduced on elastic substrates. The results suggest that matrix elasticity influences TGF-β-dependent activation of p38 signaling and subsequent myofibroblast transdifferentiation. Biomechanical cues represent an important determinant of scarring processes. Therefore, cellular signals elicited by mechanotransduction deserve consideration in the design of novel antifibrotic strategies.

  10. Fibre-induced lipid peroxidation leads to DNA adduct formation in Salmonella typhimurium TA104 and rat lung fibroblasts.

    Science.gov (United States)

    Howden, P J; Faux, S P

    1996-03-01

    Certain end-products of lipid peroxidation bind to DNA forming a fluorescent chromophore. Incubation of both Salmonella typhimurium TA104 and a rat lung fibroblast cell line, RFL-6, with various types of mineral fibre resulted in a time- and dose-dependent increase in DNA fluorescence. The increase in DNA fluorescence was shown to be directly related to the amount of iron that could be mobilized from the fibre surface using in vitro studies in the absence of cells or bacteria. Crocidolite and man-made vitreous fibre-21 (MMVF-21) mobilized significant quantities of iron and were significantly more active than chrysotile and refactory ceramic fibre-1 (RCF-1). Fibre-induced malondialdehyde-DNA adduct formation, the fluorescent product, was increased by incubating cells with buthionine sulfoximine and ameliorated by co-treatment with N-acetylcysteine, indicating a protective role for glutathione. Similarly, vitamin E was also shown to inhibit DNA adduct formation. These results suggest that mineral fibre-induced lipid peroxidation produced genotoxic products which can diffuse into nucleus and interact with cellular DNA. In conclusion, fibre-induced lipid peroxidation may be a possible mechanism in the genotoxic action of fibrous materials.

  11. Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jian Chen

    Full Text Available Induced pluripotent stem cell (iPSC technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs and fibroblasts (F-iPSCs. This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (p<0.05, of which 63 remained significant after correcting for genome-wide analysis (FDR <0.05. The most striking difference was the lower level of expression detected for numerous members of the all four HOX gene families in neurons derived from T-iPSCs. In addition, an increased level of expression was seen for several transcription factors expressed in the developing forebrain (FOXP2, OTX1, and LHX2, for example. Overall, pathway analysis revealed that differentially expressed genes that showed higher levels of expression in neurons derived from T-iPSCs were enriched for genes implicated in schizophrenia (SZ. The findings suggest that neurons derived from T-iPSCs are suitable for disease

  12. S100A8 and S100A9 Are Induced by Decreased Hydration in the Epidermis and Promote Fibroblast Activation and Fibrosis in the Dermis.

    Science.gov (United States)

    Zhong, Aimei; Xu, Wei; Zhao, Jingling; Xie, Ping; Jia, Shengxian; Sun, Jiaming; Galiano, Robert D; Mustoe, Thomas A; Hong, Seok J

    2016-01-01

    The most critical function of the epidermis is to prevent water loss and maintain skin homeostasis. Disruption of the functional skin barrier causes delayed wound healing, hypertrophic scarring, and many skin diseases. Herein, we show that reduced hydration increases the expression of S100 protein family members, S100A8/S100A9, in stratified keratinocyte culture and human ex vivo skin culture. Immunohistological analyses show that S100A8/A9 are highly expressed in the epidermis of human hypertrophic scar and keloid tissues. Reduced hydration demonstrates activation of fibroblasts in the keratinocyte-fibroblast co-culture. In contrast, knockdown of S100A8 or S100A9 by RNA interference in keratinocytes failed to activate fibroblasts. Pretreatment with pharmacological blockers of S100A8/A9 receptors, Toll-like receptor 4 and receptor for advanced glycation end products, inhibits fibroblast activation induced by recombinant S100A8/A9 proteins. Moreover, we observe that local delivery of S100A8 protein results in a marked increase in hypertrophic scarring in the in vivo rabbit ear scar model. Our results indicate that hydration status promotes fibroblast activation and fibrosis by directly affecting the expression of inflammatory signaling in keratinocytes, thereby strongly suggesting S100A8/A9 to be novel targets in preventing scarring.

  13. Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound.

    Science.gov (United States)

    Tsukamoto, Akira; Higashiyama, Satoru; Yoshida, Kenji; Watanabe, Yoshiaki; Furukawa, Katsuko S; Ushida, Takashi

    2011-12-01

    An increase in cytoplasmic calcium (Ca(2+) increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca(2+) in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca(2+) in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca(2+) was observed; however, no increase in Ca(2+) was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca(2+) in cells subjected to pulsed ultrasound.

  14. Reducing X-Ray Induced Oxidative Damages in Fibroblasts with Graphene Oxide

    Directory of Open Access Journals (Sweden)

    Yong Qiao

    2014-06-01

    Full Text Available A major issue of X-ray radiation therapy is that normal cells can be damaged, limiting the amount of X-rays that can be safely delivered to a tumor. This paper describes a new method based on graphene oxide (GO to protect normal cells from oxidative damage by removing free radicals generated by X-ray radiation using grapheme oxide (GO. A variety of techniques such as cytotoxicity, genotoxicity, oxidative assay, apoptosis, γ-H2AX expression, and micro-nucleus assay have been used to assess the protective effect of GO in cultured fibroblast cells. It is found that although GO at higher concentration (100 and 500 µg/mL can cause cell death and DNA damage, it can effectively remove oxygen free radicals at a lower concentration of 10 µg/mL. The level of DNA damage and cell death is reduced by 48%, and 39%, respectively. Thus, low concentration GO can be used as an effective radio-protective agent in occupational and therapeutic settings.

  15. Bioengineered heparins and heparan sulfates.

    Science.gov (United States)

    Fu, Li; Suflita, Matthew; Linhardt, Robert J

    2016-02-01

    Heparin and heparan sulfates are closely related linear anionic polysaccharides, called glycosaminoglycans, which exhibit a number of important biological and pharmacological activities. These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of animal cells. While heparan sulfate is a widely distributed membrane and extracellular glycosaminoglycan, heparin is found primarily intracellularly in the granules of mast cells. While heparin has historically received most of the scientific attention for its anticoagulant activity, interest has steadily grown in the multi-faceted role heparan sulfate plays in normal and pathophysiology. The chemical synthesis of these glycosaminoglycans is largely precluded by their structural complexity. Today, we depend on livestock animal tissues for the isolation and the annual commercial production of hundred ton quantities of heparin used in the manufacture of anticoagulant drugs and medical device coatings. The variability of animal-sourced heparin and heparan sulfates, their inherent impurities, the limited availability of source tissues, the poor control of these source materials and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans, driven by both therapeutic applications and as probes to study their natural functions. This review focuses on the complex biology of these glycosaminoglycans in human health and disease, and the use of recombinant technology in the chemoenzymatic synthesis and metabolic engineering of heparin and heparan sulfates. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Facile Conjugation of Heparin onto Titanium Surfaces via Dopamine Inspired Coatings for Improving Blood Compatibility

    Institute of Scientific and Technical Information of China (English)

    LI Guicai; XIE Bing; PAN Changjiang; YANG Ping; DING Hongyan; HUANG Nan

    2014-01-01

    Immobilization of heparin on biomaterials surface has been proven to be an effective strategy for preventing thrombus formation. However, the procedures of most immobilization methods (physical adsorption, covalent linkage, electrostatic interaction) are complicated and time-consuming. In the present study, heparin with various concentrations immobilized on a titanium (Ti) substrate via polydopamine layer for improving blood compatibility was investigated. Water contact angle measurement showed that the immobilization of heparin resulted in an increase of the hydrophilicity. X-ray photoelectron spectroscopy (XPS) and Toluidine Blue O (TBO) tests displayed that the heparin molecules were successfully immobilized on Ti surface. The evaluations of blood compatibility (hemolysis rate, APTT, platelet adhesion and activation, fibrinogen conformational change) showed that the immobilization of heparin decreased hemolysis rate, prolonged blood coagulation time, reduced platelets adhesion and activation, and induced less fibrinogen conformational change. Moreover, a significant inhibition of blood coagulation and platelet adhesion was obtained when the heparin concentration was higher than 5 mg/mL, indicating that only with a certain surface densities could heparin perform its anticoagulant properties well. The results suggest that the immobilization of heparin via polydopamine layer can confer excellent antithrombotic properties, and the heparin immobilization method via polydopamine layer provides an alternative approach for other biomolecules immobilization on biomaterials surface. Thus it is envisaged that this method will be potentially useful for the surface modification of blood-contacting biomaterials.

  17. Unfractionated heparin suppresses lipopolysaccharide-induced monocyte chemoattractant protein-1 expression in human microvascular endothelial cells by blocking Krüppel-like factor 5 and nuclear factor-κB pathway.

    Science.gov (United States)

    Li, Xu; Li, Xin; Zheng, Zhen; Liu, Yina; Ma, Xiaochun

    2014-10-01

    Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), apart from anticoagulant activities, contain a variety of biological properties such as anti-inflammatory actions possibly affecting sepsis. Chemokines are vital for promoting the movement of circulating leukocytes to the site of infection and are involved in the pathogenesis of sepsis. The purpose of this study was to investigate the effects and potential mechanisms of UFH on lipopolysaccharide (LPS)-induced chemokine production in human pulmonary microvascular endothelial cells (HPMECs). HPMECs were pretreated with UFH (0.1 U/ml and 1 U/ml), 15 min prior to stimulation with LPS (10 μg/ml). Cells were cultured under various experimental conditions for 2 h and 6 h for analysis. UFH markedly decreased LPS-induced interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression in HPMECs. UFH also attenuated the secretion of these chemokines in culture supernatants. In addition, UFH blocked the chemotactic activities of LPS-stimulated HPMECs supernatants on monocytes migration as expected. UFH inhibited LPS-induced Krüppel-like factor 5 (KLF-5) mRNA and protein levels. Concurrently, UFH reduced nuclear factor (NF)-κB nuclear translocation. Importantly, transfection with siRNA targeting KLF-5 reduced NF-κB activation and chemokines expression. These results demonstrate that interfering with KLF-5 mediated NF-κB activation might contribute to the inhibitory effects of chemokines and monocytes migration by UFH in LPS-stimulated HPMECs.

  18. Application of a Persistent Heparin Treatment Inhibits the Malignant Potential of Oral Squamous Carcinoma Cells Induced by Tumor Cell-Derived Exosomes.

    Science.gov (United States)

    Sento, Shinya; Sasabe, Eri; Yamamoto, Tetsuya

    2016-01-01

    Exosomes are 30-100 nm-sized membranous vesicles, secreted from a variety of cell types into their surrounding extracellular space. Various exosome components including lipids, proteins, and nucleic acids are transferred to recipient cells and affect their function and activity. Numerous studies have showed that tumor cell-derived exosomes play important roles in tumor growth and progression. However, the effect of exosomes released from oral squamous cell carcinoma (OSCC) into the tumor microenvironment remains unclear. In the present study, we isolated exosomes from OSCC cells and investigated the influence of OSCC cell-derived exosomes on the tumor cell behavior associated with tumor development. We demonstrated that OSCC cell-derived exosomes were taken up by OSCC cells themselves and significantly promoted proliferation, migration, and invasion through the activation of the PI3K/Akt, MAPK/ERK, and JNK-1/2 pathways in vitro. These effects of OSCC cell-derived exosomes were obviously attenuated by treatment with PI3K, ERK-1/2, and JNK-1/2 pharmacological inhibitors. Furthermore, the growth rate of tumor xenografts implanted into nude mice was promoted by treatment with OSCC cell-derived exosomes. The uptake of exosomes by OSCC cells and subsequent tumor progression was abrogated in the presence of heparin. Taken together, these data suggest that OSCC cell-derived exosomes might be a novel therapeutic target and the use of heparin to inhibit the uptake of OSCC-derived exosomes by OSCC cells may be useful for treatment.

  19. EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-β signaling in lung fibroblasts

    Directory of Open Access Journals (Sweden)

    Inagaki Hirofumi

    2006-01-01

    Full Text Available Abstract Background Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice. Methods Seven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL fluid and for induction of messenger RNA (mRNA of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT-polymerase chainreaction (PCR on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3 protein assay by western blotting in MLg2908. Results Bleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-β. The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-β, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF

  20. Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

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    Che, Xiajing; Wang, Qin; Xie, Yuanyuan; Xu, Weijia; Shao, Xinghua; Mou, Shan, E-mail: shan_mou@126.com; Ni, Zhaohui, E-mail: doctor_nzh@126.com

    2015-09-04

    Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production in a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.

  1. The Effect of (--Epigallocatechin-3-Gallate on IL-1β Induced IL-8 Expression in Orbital Fibroblast from Patients with Thyroid-Associated Ophthalmopathy.

    Directory of Open Access Journals (Sweden)

    Ji-Young Lee

    Full Text Available Orbital fibroblasts have been reported to be an important effector cells for the development of thyroid-associated ophthalmopathy (TAO. Orbital fibroblasts secrete various inflammatory cytokines in response to an inflammatory stimulation, leading to TAO-related tissue swelling. It has also been reported that (--epigallocatechin-3-gallate (EGCG, a major polyphenolic constituent of green tea, has antioxidant and anti-inflammatory properties. In the current study, we investigated the issue of whether or how EGCG affects the interleukin (IL-1β-induced secretion of IL-8 in human orbital fibroblasts from TAO patients. Treatment with EGCG significantly reduced the level of IL-1β-induced secretion of IL-8 and the expression of IL-8 mRNA. IL-1β-induced the degradation of IκBα, and the phosphorylation of p38 and ERK, and the IL-1β-induced expression of IL-8 mRNA was inhibited by specific inhibitors, such as BAY-117085 for NF-kB, SB203580 for p38, and PD98059 for ERK. In addition, treatment with EGCG inhibited the IL-1β-induced degradation of IκBα, and the phosphorylation of p38 and ERK. However, pre-treatment with antioxidants, NVN and NAC, which suppressed ROS generation, did not reduce IL-8 expression in IL-1β-treated orbital fibroblasts, suggesting that the IL-1β-induced IL-8 expression is not mediated by the generation of ROS. These results show that EGCG suppresses the IL-1β-induced expression of IL-8 through inhibition of the NF-κB, p38, and ERK pathways. These findings could contribute to the development of new types of EGCG-containing pharmacological agents for use in the treatment of TAO.

  2. Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts.

    Science.gov (United States)

    Chang, M-C; Tsai, Y-L; Chen, Y-W; Chan, C-P; Huang, C-F; Lan, W-C; Lin, C-C; Lan, W-H; Jeng, J-H

    2013-02-01

    Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases. © 2012 John Wiley & Sons A/S.

  3. HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1

    Directory of Open Access Journals (Sweden)

    Gallagher John

    2006-01-01

    Full Text Available Abstract Background Heparin/heparan sulfate (HS proteoglycans are found in the extracellular matrix (ECM and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF, cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin. Results Our results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities. Conclusion Our results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.

  4. Reprogramming of sheep fibroblasts into pluripotency under a drug-inducible expression of mouse-derived defined factors.

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    Yang Li

    Full Text Available Animal embryonic stem cells (ESCs provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs, have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes.

  5. Antiaging Effect of Pine Pollen in Human Diploid Fibroblasts and in a Mouse Model Induced by D-Galactose

    Directory of Open Access Journals (Sweden)

    Gen-Xiang Mao

    2012-01-01

    Full Text Available The present paper was designed to investigate the effect of pine pollen against aging in human diploid fibroblast 2BS cells and in an accelerated aging model, which was established by subcutaneous injections with D-galactose daily for 8 weeks in C57BL/6J mice. Pine pollen (1 mg/mL and 2 mg/mL is proved to delay the replicative senescence of 2BS cells as evidenced by enhanced cell proliferation, decreased SA-β-Gal activity, and reversed expression of senescence-associated molecular markers, such as p53, p21Waf1, p16INK4a, PTEN, and p27Kip1 in late PD cells. Besides, pine pollen reversed D-galactose-induced aging effects in neural activity and inflammatory cytokine levels, as indicated by improved memory latency time and reduced error rate in step-down test and decreased concentrations of IL-6 and TNF-α in model mice. Similar to the role of AGEs (advanced glycation endproducts formation inhibitor aminoguanidine (AG, pine pollen inhibited D-galactose-induced increment of AGEs levels thus reversed the aging phenotypes in model mice. Furthermore, the declined antioxidant activity was obviously reversed upon pine pollen treatment, which may account for its inhibitory effect on nonenzymatic glycation (NEG in vivo. Our finding presents pine pollen as an attractive agent with potential to retard aging and attenuate age-related diseases in humans.

  6. The role of hexon in egg drop syndrome virus (EDSV) inducing apoptosis in duck embryo fibroblast cells.

    Science.gov (United States)

    Qi, Xuefeng; Xu, Jiamin; Wang, Zugui; Wang, Xueping; Wang, Jingyu

    2017-07-17

    Although extensive efforts have been made to understand adenovirus infection in human cells, little is known for egg drop syndrome virus (EDSV) infection in the avian-derived cells. In this study, the effects of EDSV infection as well as the possible role hexon protein, the main building block of the EDSV capsid, on apoptosis induction in duck embryo fibroblast (DEF) cells was examined. Flow cytometry analysis and TUNEL assay revealed that EDSV infection induced significant apoptosis in DEF cells compared with mock infected cells. Interestingly, the increase of the apoptosis rate detected in EDSV infected DEF cells were accompanied by an increased virus load in cells in a time-dependent manner. Furthermore, a time-dependent decrease in hexon protein expression levels in hexon transfected DEF cells in parallel with a gradual decrease in TUNEL-labeling cells was also observed in the current study. In addition, caspase activity detection and western blot analysis indicates that either EDSV infection or EDSV hexon transfection both induced apoptosis of DEF cells via activating both the exogenous and the mitochondrial pathway. Copyright © 2017. Published by Elsevier Ltd.

  7. Modulation of fibroblast growth factor receptor expression and signalling during retinoic acid-induced differentiation of Tera-2 teratocarcinoma cells.

    Science.gov (United States)

    Pertovaara, L; Tienari, J; Vainikka, S; Partanen, J; Saksela, O; Lehtonen, E; Alitalo, K

    1993-02-26

    We have analyzed the regulation of fibroblast growth factor receptors (FGFRs) during retinoic acid (RA) induced differentiation of Tera-2 human embryonal carcinoma cells. Undifferentiated Tera-2 cells expressed mRNAs for all four known FGFRs. Their differentiation led to loss of FGFR-4 mRNA expression and mRNA levels for FGFR-2 and FGFR-3 were considerably downregulated, whereas the mRNA levels for FGFR-1 remained unaltered. A substantial decrease in binding of K-FGF was found to occur upon RA-induced differentiation of the cells. In undifferentiated Tera-2 cells FGF stimulation caused an increase of c-fos mRNA, and c-jun mRNAs, but no increase of junB mRNA, whereas in the differentiated cells, FGFs strongly stimulated the expression of all three genes. Thus differentiation of the Tera-2 cells leads to marked changes in FGFR gene expression as well as to complex alterations in their responses to exogenous FGFs.

  8. Notch Signaling Mediates TNF-α-Induced IL-6 Production in Cultured Fibroblast-Like Synoviocytes from Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Zhijun Jiao

    2012-01-01

    Full Text Available It has been reported that Notch family proteins are expressed in synovium tissue and involved in the proliferation of synoviocyte from rheumatoid arthritis (RA. The aim of this paper was to investigate whether Notch signaling mediated TNF-α-induced cytokine production of cultured fibroblast-like synoviocytes (FLSs from RA. Exposure of RA FLSs to TNF-α (10 ng/ml led to increase of Hes-1, a target gene of Notch signaling, and a marked upregulation of Notch 2, Delta-like 1, and Delta-like 3 mRNA levels. Blockage of Notch signaling by a γ-secretase inhibitor (DAPT inhibited IL-6 secretion of RA FLSs in response to TNF-α while treatment with recombinant fusion protein of Notch ligand Delta-like 1 promoted such response. TNF-α stimulation also induced IL-6 secretion in OA FLSs; however, the Hes-1 level remained unaffected. Our data confirm the functional involvement of Notch pathway in the pathophysiology of RA FLSs which may provide a new target for RA therapy.

  9. A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

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    Pan Hui; Kantharia, Sarah [Department of Biomedical Engineering, State University of New York-Stony Brook, Stony Brook, NY 11794-2580 (United States); Jiang Hongliang [Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027 (China); Chen Weiliam, E-mail: weiliam.chen@nyumc.org [Division of Wound Healing and Regenerative Medicine, Department of Surgery, New York University School of Medicine, New York, NY 10016 (United States)

    2011-12-15

    Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

  10. 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

  11. [Malignant transformation of human fibroblasts by neutrons and by gamma radiation: Relationship to mutations induced

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1993-12-31

    A brief overview if provided of selected reports presented at the International Symposium on Molecular Mechanisms of Radiation- and Chemical Carcinogen-Induced Cell Transformation held at Mackinac Island, Michigan on September 19-23, 1993.

  12. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Linda Yuliati

    2015-12-01

    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.

  13. Metformin inhibits aldosterone-induced cardiac fibroblast activation, migration and proliferation in vitro, and reverses aldosterone+salt-induced cardiac fibrosis in vivo.

    Science.gov (United States)

    Mummidi, Srinivas; Das, Nitin A; Carpenter, Andrea J; Kandikattu, Hemanthkumar; Krenz, Maike; Siebenlist, Ulrich; Valente, Anthony J; Chandrasekar, Bysani

    2016-09-01

    The overall goals of this study were to investigate whether metformin exerts anti-fibrotic effects in aldosterone (Aldo)+salt-treated wild type mouse hearts, and determine the underlying molecular mechanisms in isolated adult cardiac fibroblasts (CF). In vitro, Aldo induced CF activation, migration, and proliferation, and these effects were inhibited by metformin. Further, Aldo induced PPM1A (Protein Phosphatase Magnesium Dependent 1A) activation and inhibited AMPK phosphorylation. At a pharmacologically relevant concentration, metformin restored AMPK activation, and inhibited Aldo-induced Nox4/H2O2-dependent TRAF3IP2 induction, pro-inflammatory cytokine expression, and CF migration and proliferation. Further, metformin potentiated the inhibitory effects of spironolactone, a mineralocorticoid receptor antagonist, on Aldo-induced collagen expression, and CF migration and proliferation. These results were recapitulated in vivo, where metformin reversed Aldo+salt-induced oxidative stress, suppression of AMPK activation, TRAF3IP2 induction, pro-inflammatory cytokine expression, and cardiac fibrosis, without significantly modulating systolic blood pressure. These in vitro and in vivo data indicate that metformin has the potential to reduce adverse cardiac remodeling in hypertensive heart disease.

  14. Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Claudia V.; Oliveira, Paulo J. [CNC—Center for Neuroscience and Cell Biology, University of Coimbra (Portugal); Will, Yvonne [Compound Safety Prediction, Pfizer Global Research and Development, Groton, CT (United States); Nadanaciva, Sashi, E-mail: sashi.nadanaciva@pfizer.com [Compound Safety Prediction, Pfizer Global Research and Development, Groton, CT (United States)

    2012-10-15

    Mitochondrial DNA (mtDNA) variations including single nucleotide polymorphisms (SNPs) have been proposed to be involved in idiosyncratic drug reactions. However, current in vitro and in vivo models lack the genetic diversity seen in the human population. Our hypothesis is that different cell strains with distinct mtDNA SNPs may have different mitochondrial bioenergetic profiles and may therefore vary in their response to drug-induced toxicity. Therefore, we used an in vitro system composed of four strains of mouse embryonic fibroblasts (MEFs) with mtDNA polymorphisms. We sequenced mtDNA from embryonic fibroblasts isolated from four mouse strains, C57BL/6J, MOLF/EiJ, CZECHII/EiJ and PERA/EiJ, with the latter two being sequenced for the first time. The bioenergetic profile of the four strains of MEFs was investigated at both passages 3 and 10. Our results showed that there were clear differences among the four strains of MEFs at both passages, with CZECHII/EiJ having a lower mitochondrial robustness when compared to C57BL/6J, followed by MOLF/EiJ and PERA/EiJ. Seven drugs known to impair mitochondrial function were tested for their effect on the ATP content of the four strains of MEFs in both glucose- and galactose-containing media. Our results showed that there were strain-dependent differences in the response to some of the drugs. We propose that this model is a useful starting point to study compounds that may cause mitochondrial off-target toxicity in early stages of drug development, thus decreasing the number of experimental animals used. -- Highlights: ► mtDNA SNPs may be linked to individual predisposition to drug-induced toxicity. ► CZECHII/EiJ and PERA/EiJ mtDNA was sequenced for the first time in this study. ► Strain-dependent mitochondrial capacity differences were measured. ► Strain-dependent differences in response to mitochondrial toxicants were observed.

  15. Gene Expression Profile Changes and Cellular Responses to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Kidane, Yared; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Rohde, Larry; Wu, Honglu

    2016-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. In addition, DNA in space can be damaged by toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage affects the accuracy of the radiation risk assessment for astronauts and the mutation rate in microorganisms. Although possible synergistic effects of space radiation and microgravity have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on cellular responses to DNA damage, confluent human fibroblast cells (AG1522) flown on the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB). Damages in the DNA were quantified by immunofluorescence staining for ?-H2AX, which showed similar percentages of different types of stained cells between flight and ground. However, there was a slight shift in the distribution of the ?-H2AX foci number in the flown cells with countable foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. A microarray analysis of gene expressions in response to bleomycin treatment was also performed. Comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. Similar responses at the RNA level between different gravity conditions were also observed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly

  16. Unfractionated heparin dosing for venous thromboembolism in morbidly obese patients: case report and review of the literature.

    Science.gov (United States)

    Myzienski, April E; Lutz, Mark F; Smythe, Maureen A

    2010-03-01

    Unfractionated heparin infusion therapy is often administered using a weight-based dosing strategy for the treatment of venous thromboembolism. In the last several decades, the prevalence of obesity in the United States has increased significantly. The applicability of weight-based heparin dosing recommendations in the obese and morbidly obese population is uncertain, as limited data are available. We describe a 388-kg man who was started on an intravenous infusion of heparin according to hospital protocol for suspected pulmonary embolism. The patient was given a 5000-unit heparin bolus followed by an initial heparin infusion rate of 1500 units/hour, the maximum initial rate specified in the protocol. After additional infusion rate adjustments, a therapeutic activated partial thromboplastin time (aPTT) was reached 55 hours later with a heparin infusion rate of 3650 units/hour. Due to concerns of heparin-induced thrombocytopenia, heparin therapy was discontinued, and fondaparinux 5 mg/day was started. However, heparin therapy was restarted 4 days later for persistent, refractory hypoxemia and recurrent concerns of possible pulmonary embolism. During this second course, a therapeutic aPTT was achieved with a heparin infusion rate of 3550 units/hour. The patient developed bloody pulmonary secretions (with a therapeutic aPTT), necessitating the discontinuation of the heparin infusion. The patient later died after developing pulseless electrical activity. Standard weight-based heparin dosing protocols that specify maximum doses for initial bolus and infusion rates can result in significant delays in time to achieve therapeutic anticoagulation in the obese and morbidly obese patient. Despite limited data on heparin dosing in obesity, we recommend the use of a dosing weight to determine initial heparin dosing when treating venous thromboembolism in morbidly obese patients. It is reasonable to consider one of the following formulas: dosing weight = ideal body weight (IBW

  17. Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts

    Directory of Open Access Journals (Sweden)

    Ara Toshiaki

    2012-03-01

    Full Text Available Abstract Objective Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL-6, IL-8, and the chemical mediator prostaglandin E2 (PGE2 are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA inhibitor H-89 suppresses lipopolysaccharide (LPS-induced IL-8 production by human gingival fibroblasts (HGFs. In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8 and PGE2 by HGFs were examined. Methods HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP, aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE2 levels were evaluated by ELISA. Results H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE2 production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE2 production. Up to 10 μg/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at 100 μg/ml. Similarly, 0.01 μg/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at concentrations of 0.1 and 1 μg/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE2 production. Conclusion These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE2 production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 μg/ml in serum. These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.

  18. Heparin-induced thrombocytopenia: a review of concepts regarding a dangerous adverse drug reaction Trombocitopenia induzida por heparina: revisão de conceitos de uma importante reação adversa a medicamentos

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    Daniela Rezende Garcia Junqueira

    2013-04-01

    Full Text Available Heparin is a natural agent with antithrombotic action, commercially available for therapeutic use as unfractionated heparin and low molecular weight heparin. Heparin-induced thrombocytopenia (HIT is a serious adverse reaction to heparin that promotes antibodymediated platelet activation. HIT is defined as a relative reduction in platelet count of 50% (even when the platelet count at its lowest level is above > 150 x 10(9/L occurring within five to 14 days after initiation of the therapy. Thrombocytopenia is the main feature that directs the clinical suspicion of the reaction and the increased risk of thromboembolic complications is the most important and paradoxical consequence. The diagnosis is a delicate issue, and requires a combination of clinical probability and laboratory tests for the detection of platelet activation induced by HIT antibodies. The absolute risk of HIT has been estimated between 1% and 5% under treatment with unfractionated heparin, and less than 1% with low molecular weight heparin. However, high-quality evidence about the risk of HIT from randomized clinical trials is scarce. In addition, information on the frequency of HIT in developing countries is not widely available. This review aims to provide a better understanding of the key features of this reaction and updated information on its frequency to health professionals and other interested parties. Knowledge, familiarity, and access to therapeutic options for the treatment of this adverse reaction are mandatory to minimize the associated risks, improving patient safety.A heparina é um agente natural com ação antitrombótica, sendo disponibilizadas para uso terapêutico a heparina não fracionadaeaheparina de baixo peso molecular. A trombocitopenia induzida por heparina (TIH é uma reação adversa grave às heparinas mediada por anticorpos que promovem ativação de plaquetas. A TIH é definida como uma redução rela- tiva na contagem de plaquetas de 50% (mesmo se

  19. Interleukin-21 Induces Proliferation and Proinflammatory Cytokine Profile of Fibroblast-like Synoviocytes of Patients with Rheumatoid Arthritis.

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    Xing, R; Yang, L; Jin, Y; Sun, L; Li, C; Li, Z; Zhao, J; Liu, X

    2016-01-01

    Fibroblast-like synoviocytes (FLS) play a pivotal role in the pathogenesis of rheumatoid arthritis (RA) through aggressive proliferation and invasion, and certain proinflammatory cytokines may affect synoviocyte proliferation. To evaluate whether interleukin-21 (IL-21) could promote proliferation and proinflammatory cytokine production by RA-FLS, immunohistochemistry and immunoblotting were performed to observe the expression of IL-21 receptor (IL-21R) in synovial tissues and FLS from RA and osteoarthritis (OA) patients. The MTS assay was used to analyse RA-FLS proliferation. The concentrations of IL-6 and tumour necrosis factor-α (TNF-α) in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). The signalling pathways triggered by IL-21 were characterized by immunoblotting. IL-21R was upregulated in the synovial tissues and FLS of RA patients as compared with OA patients. IL-21 stimulated RA-FLS proliferation and promoted the production of TNF-α and IL-6 and blockade of IL-21/IL-21R pathway with IL-21R.Fc attenuated IL-21-induced proliferation and secretion of TNF-α and IL-6. Moreover, IL-21 induced activation of the ERK1/2, PI3K/AKT and STAT3 pathways, and blockade of these pathways attenuated IL-21-induced proliferation and secretion of TNF-α and IL-6. These results suggest that IL-21 could promote RA-FLS proliferation and production of proinflammatory cytokines. Therefore, therapeutic strategies targeting IL-21 might be effective for the treatment of RA.

  20. TNFR1 and TNFR2 differentially mediate TNF-α-induced inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes.

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    Zhang, Hongfeng; Xiao, Weiguo

    2017-04-01

    TNF-α has long been implicated in the progression of rheumatoid arthritis (RA). However, how the receptors of TNF-α, namely TNFR1 and TNFR2, mediate TNF-α-induced inflammatory responses in fibroblast-like synoviocytes (FLS) in RA has not been elucidated. In the present study, primary FLS cells were isolated from RA patients and treated with TNF-α in vitro. The exogenous TNF-α induced the expression and release of endogenous TNF-α in FLS. In addition, TNF-α led to gradual downregulation of TNFR1 following 1 h treatment. By contrast, the expression of TNFR2 was markedly upregulated after 12 h treatment with TNF-α. Moreover, following TNF-α treatment, the expression of interleukin (IL)-2, IL-6, and IL-8 was gradually increased with time, but their mRNA levels dropped significantly at 48 h. We further investigated the differential functions of TNFR1 and TNFR2 in FLS by conducting siRNA-mediated knockdown. The TNF-α autocrine was inhibited to a greater extent in TNFR1-silenced FLS compared with TNFR2-silenced FLS. Silencing of TNFR1, not TNFR2, activated intrinsic apoptosis and inhibited TNF-α-induced cytokine production in FLS. These results suggest that TNFR1 is the major pro-inflammatory mediator of TNF-α in FLS, whereas TNFR2, which is upregulated in response to prolonged TNF-α stimulation, may act as an immunosuppressor in FLS for the prevention of overwhelming inflammatory reactions.

  1. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

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    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  2. Cyr61 induces IL-6 production by fibroblast-like synoviocytes promoting Th17 differentiation in rheumatoid arthritis.

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    Lin, Jinpiao; Zhou, Zhou; Huo, Rongfen; Xiao, Lianbo; Ouyang, Guilin; Wang, Li; Sun, Yue; Shen, Baihua; Li, Dangsheng; Li, Ningli

    2012-06-01

    Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17-dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvβ5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4(+) T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.

  3. Interleukin-35 upregulates OPG and inhibits RANKL in mice with collagen-induced arthritis and fibroblast-like synoviocytes.

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    Li, Y; Li, D; Li, Y; Wu, S; Jiang, S; Lin, T; Xia, L; Shen, H; Lu, J

    2016-04-01

    IL-35 is a novel anti-inflammatory cytokine, but the exact role of IL-35 in the progression of RA remains unclear, especially associated with osteoporosis and bone erosion. The present research has not been reported. Our purpose is to study how IL-35 affects RA bone destruction. This study investigated the effect of interleukin-35 (IL-35) on OPG and RANKL expression in collagen-induced arthritis (CIA) in rats and in cultured fibroblast-like synoviocytes (FLS). Thirty DBA/1J mice were randomly assigned to three groups (n = 10 per group): the control group, the CIA group, and the CIA + IL-35 group. Collagen-induced arthritis was induced by immunization with collagen. IL-35 was intraperitoneally injected daily for 10 days, starting from the 24(th) day after immunization. FL