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Sample records for growth factor heparin

  1. Synthetic heparin-binding growth factor analogs

    DOEpatents

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  2. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

    2012-04-24

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  3. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

    2009-10-06

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  4. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  5. Heparin-conjugated gelatin as a growth factor immobilization scaffold.

    PubMed

    Nakamura, Shintaro; Kubo, Takafumi; Ijima, Hiroyuki

    2013-05-01

    Tissue engineering requires growth factors, cells and a scaffold to permit effective tissue regeneration. This study aimed to develop a scaffold with a focus on immobilizing growth factors within gelatin. We focused on the extracellular matrix and developed a heparin-conjugated gelatin (Hep-gela). Conjugation was confirmed using the alcian blue assay and X-ray diffraction patterns. The mechanical strength and stability of the Hep-gela gel in protease solution were improved compared with collagen gel. Hep-gela was able to immobilize vascular endothelial growth factor (VEGF) even in the presence of albumin, with an efficiency of 54.2%. Immobilized VEGF promoted proliferation of human umbilical vein endothelial cells. Hep-gela-immobilized VEGF maintained its native biological activity. In summary, Hep-gela has the potential to become an effective material in the field of regenerative medicine.

  6. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

    PubMed

    Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

    2017-02-07

    High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

  7. The activation of fibroblast growth factors by heparin: synthesis, structure, and biological activity of heparin-like oligosaccharides.

    PubMed

    de Paz, J L; Angulo, J; Lassaletta, J M; Nieto, P M; Redondo-Horcajo, M; Lozano, R M; Giménez-Gallego, G; Martín-Lomas, M

    2001-09-03

    An effective strategy has been designed for the synthesis of oligosaccharides of different sizes structurally related to the regular region of heparin; this is illustrated by the preparation of hexasaccharide 1 and octasaccharide 2. This synthetic strategy provides the oligosaccharide sequence containing a D-glucosamine unit at the nonreducing end that is not available either by enzymatic or chemical degradation of heparin. It may permit, after slight modifications, the preparation of oligosaccharide fragments with different charge distribution as well. NMR spectroscopy and molecular dynamics simulations have shown that the overall structure of 1 in solution is a stable right-hand helix with four residues per turn. Hexasaccharide 1 and, most likely, octasaccharide 2 are, therefore, chemically well-defined structural models of naturally occurring heparin-like oligosaccharides for use in binding and biological activity studies. Both compounds 1 and 2 induce the mitogenic activity of acid fibroblast growth factor (FGF1), with the half-maximum activating concentration of 2 being equivalent to that of heparin. Sedimentation equilibrium analysis with compound 2 suggests that heparin-induced FGF1 dimerization is not an absolute requirement for biological activity.

  8. 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

    PubMed Central

    Roberts, Justine J; Farrugia, Brooke L; Green, Rylie A; Rnjak-Kovacina, Jelena; Martens, Penny J

    2016-01-01

    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. PMID:27895888

  9. 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.

    PubMed

    Roberts, Justine J; Farrugia, Brooke L; Green, Rylie A; Rnjak-Kovacina, Jelena; Martens, Penny J

    2016-01-01

    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.

  10. Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    PubMed Central

    Ozen, Evin; Gozukizil, Aysim; Erdal, Esra; Uren, Aykut; Bottaro, Donald P.; Atabey, Nese

    2012-01-01

    The Hepatocyte Growth Factor (HGF)/c-Met signaling pathway regulates hepatocyte proliferation, and pathway aberrations are implicated in the invasive and metastatic behaviors of hepatocellular carcinoma (HCC). In addition to c-Met, heparin acts as a co-receptor to modulate pathway activity. Recently, anti-metastatic and anti-cancer effects of heparin have been reported. However, the role of heparin in the regulation of HGF signaling remains controversial and the effects of heparin on HGF-induced biological responses during hepatocarcinogenesis is not yet defined. In this study we determined the effects of heparin on HGF-induced activities of HCC cells and the underlying molecular mechanisms. Here, we report for the first time that heparin inhibits HGF-induced adhesion, motility and invasion of HCC cells. In addition, heparin reduced HGF-induced activation of c-Met and MAPK in a dose-dependent manner, as well as decreased transcriptional activation and expression of Early growth response factor 1 (Egr1). HGF-induced MMP-2 and MMP-9 activation, and MT1-MMP expression, also were inhibited by heparin. Stable knockdown of Egr1 caused a significant decrease in HGF-induced invasion, as well as the activation and expression of MMPs. Parallel to these findings, the overexpression of Egr1 increased the invasiveness of HCC cells. Our results suggest that Egr1 activates HGF-induced cell invasion through the regulation of MMPs in HCC cells and heparin inhibits HGF-induced cellular invasion via the downregulation of Egr1. Therefore, heparin treatment might be a therapeutic approach to inhibit invasion and metastasis of HCC, especially for patients with active HGF/c-Met signaling. PMID:22912725

  11. Myogenic Growth Factor Present in Skeletal Muscle is Purified by Heparin-Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Kardami, Elissavet; Spector, Dennis; Strohman, Richard C.

    1985-12-01

    A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

  12. Heparin stimulates epidermal growth factor receptor-mediated phosphorylation of tyrosine and threonine residues.

    PubMed

    Revis-Gupta, S; Abdel-Ghany, M; Koland, J; Racker, E

    1991-07-15

    We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1. We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site. To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine. We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF. These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases.

  13. Growth factors-loaded stents modified with hyaluronic acid and heparin for induction of rapid and tight re-endothelialization.

    PubMed

    Choi, Dong Hoon; Kang, Sung Nam; Kim, Seong Min; Gobaa, Samy; Park, Bang Ju; Kim, Ik Hwan; Joung, Yoon Ki; Han, Dong Keun

    2016-05-01

    Rapid re-endothelialization of damaged vessel lining efficiently prevents restenosis and thrombosis and restores original vascular functions. In this study, we designed a novel metallic stent with a heparin-modified surface and used different methods, including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and divinyl sulfone (DVS), to load growth factors. First we loaded heparin into a dopamine-conjugated hyaluronic acid (HA) coating to serve as a growth factor reservoir. In a second step, we took advantage of the heparin-binding domain of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) to gain advanced re-endothelialization capabilities. We demonstrated that DVS technique offered higher amount of growth factor loading. In vitro assessment also showed better capillary-like structure formation and localized gap junctions when DVS coating was employed. This study suggested that growth factor loaded stent modified by HA and heparin provided the advantage to rapid and tight restoration of endothelium.

  14. Heparin-Binding Epidermal Growth Factor-like Growth Factor/Diphtheria Toxin Receptor in Normal and Neoplastic Hematopoiesis

    PubMed Central

    Vinante, Fabrizio; Rigo, Antonella

    2013-01-01

    Heparin-binding EGF-like growth factor (HB-EGF) belongs to the EGF family of growth factors. It is biologically active either as a molecule anchored to the membrane or as a soluble form released by proteolytic cleavage of the extracellular domain. HB-EGF is involved in relevant physiological and pathological processes spanning from proliferation and apoptosis to morphogenesis. We outline here the main activities of HB-EGF in connection with normal or neoplastic differentiative or proliferative events taking place primitively in the hematopoietic microenvironment. PMID:23888518

  15. Heparin decamer bridges a growth factor and an oligolysine by different charge-driven interactions.

    PubMed

    Minsky, Burcu Baykal; Nguyen, Thuy V; Peyton, Shelly R; Kaltashov, Igor A; Dubin, Paul L

    2013-11-11

    Full-length heparin is widely used in tissue engineering applications due its multiple protein-binding sites that allow it to retain growth factor affinity while associating with oligopeptide components of the tissue scaffold. However, the extent to which oligopeptide coupling interferes with cognate protein binding is difficult to predict. To investigate such simultaneous interactions, we examined a well-defined ternary system comprised of acidic fibroblast growth factor (FGF), tetralysine (K4), with a heparin decamer (dp10) acting as a noncovalent coupler. Electrospray ionization mass spectrometry was used to assess binding affinities and complex stoichiometries as a function of ionic strength for dp10·K4 and FGF·dp10. The ionic strength dependence of K4·dp10 formation is qualitatively consistent with binding driven by the release of condensed counterions previously suggested for native heparin with divalent oligopeptides (Mascotti, D. P.; Lohman, T. M. Biochemistry 1995, 34, 2908-2915). On the other hand, FGF binding displays more complex ionic strength dependence, with higher salt resistance. Remarkably, dp10 that can bind two FGF molecules can only bind one tetralysine. The limited binding of K4 to dp10 suggests that the tetralysine might not block growth factor binding, and the 1:1:1 ternary complex is indeed observed. The analysis of mass distribution of the bound dp10 chains in FGF·dp10, FGF2·dp10, and FGF·dp10·K4 complexes indicated that higher degrees of dp10 sulfation promote the formation of FGF2·dp10 and FGF·dp10·K4. Thus, the selectivity of appropriately chosen short heparin chains could be used to modulate growth factor sequestration and release in a way not feasible with heterogeneous native heparin. In support of this, human hepatocellular carcinoma cells (HEP3Bs) treated with FGF·dp10·K4 were found to exhibit biological activity similar to cells treated with FGF.

  16. Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels

    PubMed Central

    Jeon, Oju; Powell, Caitlin; Solorio, Loran D.; Krebs, Melissa D.; Alsberg, Eben

    2013-01-01

    Photocrosslinkable biomaterials are promising for tissue engineering applications due to their capacity to be injected and form hydrogels in situ in a minimally invasive manner. Our group recently reported on the development of photocrosslinked alginate hydrogels with controlled biodegradation rates, mechanical properties, and cell adhesive properties. In this study, we present an affinity-based growth factor delivery system by incorporating heparin into photocrosslinkable alginate hydrogels (HP-ALG), which allows for controlled, prolonged release of therapeutic proteins. Heparin modification had minimal effect on the biodegradation profiles, swelling ratios, and elastic moduli of the hydrogels in media. The release profiles of growth factors from this affinity-based platform were sustained for 3 weeks with no initial burst release, and the released growth factors retained their biological activity. Implantation of bone morphogenetic protein-2 (BMP-2)-loaded photocrosslinked alginate hydrogels induced moderate bone formation around the implant periphery. Importantly, BMP-2-loaded photocrosslinked HP-ALG hydrogels induced significantly more osteogenesis than BMP-2-loaded photocrosslinked unmodified alginate hydrogels, with 1.9-fold greater peripheral bone formation and 1.3-fold greater calcium content in the BMP-2-loaded photocrosslinked HP-ALG hydrogels compared to the BMP-2-loaded photocrosslinked unmodified alginate hydrogels after 8 weeks implantation. This sustained and controllable growth factor delivery system, with independently controllable physical and cell adhesive properties, may provide a powerful modality for a variety of therapeutic applications. PMID:21745508

  17. Synthetic heparin-binding factor analogs

    DOEpatents

    Pena, Louis A [Poquott, NY; Zamora, Paul O [Gaithersburg, MD; Lin, Xinhua [Plainview, NY; Glass, John D [Shoreham, NY

    2010-04-20

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  18. Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow

    PubMed Central

    Forsten-Williams, Kimberly; Zhang, Jun; Fannon, Michael

    2010-01-01

    Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove

  19. Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions

    PubMed Central

    Xu, Ruoyan; Ori, Alessandro; Rudd, Timothy R.; Uniewicz, Katarzyna A.; Ahmed, Yassir A.; Guimond, Scott E.; Skidmore, Mark A.; Siligardi, Giuliano; Yates, Edwin A.; Fernig, David G.

    2012-01-01

    The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit KD values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m−1 s−1 and FGF-9, 130,000 m−1 s−1). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies. PMID:23019343

  20. Heparinized collagen scaffolds with and without growth factors for the repair of diaphragmatic hernia

    PubMed Central

    Brouwer, Katrien M; Wijnen, René M; Reijnen, Daphne; Hafmans, Theo G; Daamen, Willeke F; van Kuppevelt, Toin H

    2013-01-01

    A regenerative medicine approach to restore the morphology and function of the diaphragm in congenital diaphragmatic hernia is especially challenging because of the position and flat nature of this organ, allowing cell ingrowth primarily from the perimeter. Use of porous collagen scaffolds for the closure of surgically created diaphragmatic defects in rats has been shown feasible, but better ingrowth of cells, specifically blood vessels and muscle cells, is warranted. To stimulate this process, heparin, a glycosaminoglycan involved in growth factor binding, was covalently bound to porous collagenous scaffolds (14%), with or without vascular endothelial growth factor (VEGF; 0.4 µg/mg scaffold), hepatocyte growth factor (HGF; 0.5 µg/mg scaffold) or a combination of VEGF + HGF (0.2 + 0.5 µg/mg scaffold). All components were located primarily at the outside of scaffolds. Scaffolds were implanted in the diaphragm of rats and evaluated after 2 and 12 weeks. No herniations or eventrations were observed, and in several cases, growth factor-substituted scaffolds showed macroscopically visible blood vessels at the lung site. The addition of heparin led to an accelerated ingrowth of blood vessels at 2 weeks. In all scaffold types, giant cells and immune cells were present primarily at the liver side of the scaffold, and immune cells and individual macrophages at the lung side; these cell types decreased in number from week 2 to week 12. The addition of growth factors did not influence cellular response to the scaffolds, indicating that further optimization with respect to dosage and release profile is needed. PMID:23867845

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast.

    PubMed

    Hills, Frank A; Mehmet, Huseyin; Sullivan, Mark H

    2012-07-01

    This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Heparin-binding epidermal growth factor-like growth factor regulates fibroblast growth factor-2 expression in aortic smooth muscle cells.

    PubMed

    Peifley, K A; Alberts, G F; Hsu, D K; Feng, S L; Winkles, J A

    1996-08-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a vascular smooth muscle cell (SMC) mitogen and chemotactic factor that is expressed by endothelial cells, SMCs, monocytes/macrophages, and T lymphocytes. Both the membrane-anchored HB-EGF precursor and the secreted mature HB-EGF protein are biologically active; thus, HB-EGF may stimulate SMC growth via autocrine, paracrine, and juxtacrine mechanisms. In the present study, we report that HB-EGF treatment of serum-starved at aortic SMCs can induce fibroblast growth factor (FGF)-2 (basic FGF) gene expression but not FGF-1 (acidic FGF) gene expression. Increased FGF-2 mRNA expression is first detectable at 1 hour after HB-EGF addition, and maximal FGF-2 mRNA levels, corresponding to an approximately 46-fold level of induction, are present at 4 hours. The effect of HB-EGF on FGF-2 mRNA levels appears to be mediated primarily by a transcriptional mechanism and requires de novo synthesized proteins. HB-EGF induction of FGF-2 mRNA levels can be inhibited by treating cells with the anti-inflammatory glucocorticoid dexamethasone or the glycosaminoglycan heparin. Finally, Western blot analyses indicate that HB-EGF-treated SMCs also produce an increased amount of FGF-2 protein. These results indicate that HB-EGF expressed at sites of vascular injury or inflammation in vivo may upregulate FGF-2 production by SMCs.

  4. Heparin-binding epidermal growth factor-like growth factor, a v-Jun target gene, induces oncogenic transformation

    PubMed Central

    Fu, Shu-ling; Bottoli, Ivan; Goller, Martin; Vogt, Peter K.

    1999-01-01

    Jun is a transcription factor belonging to the activator protein 1 family. A mutated version of Jun (v-Jun) transduced by the avian retrovirus ASV17 induces oncogenic transformation in avian cell cultures and sarcomas in young galliform birds. The oncogenicity of Jun probably results from transcriptional deregulation of v-Jun-responsive target genes. Here we describe the identification and characterization of a growth-related v-Jun target, a homolog of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF is strongly expressed in chicken embryo fibroblasts (CEF) transformed by v-Jun. HB-EGF expression is not detectable or is marginal in nontransformed CEF. Using a hormone-inducible Jun-estrogen receptor chimera, we found that HB-EGF expression is correlated with v-Jun activity. In this system, induction of v-Jun is followed within 1 hr by elevated levels of HB-EGF. In CEF infected with various Jun mutants, HB-EGF expression is correlated with the oncogenic potency of the mutant. Constitutive expression of HB-EGF conveys to CEF the ability to grow in soft agar and to form multilayered foci of transformed cells on a solid substrate. These observations suggest that HB-EGF is an effector of Jun-induced oncogenic transformation. PMID:10318950

  5. A generic strategy for co-presentation of heparin-binding growth factors based on CVD polymerization.

    PubMed

    Deng, Xiaopei; Lahann, Joerg

    2012-09-14

    A multifunctional copolymer with both aldehyde and alkyne groups is synthesized by chemical vapor deposition (CVD) for orthogonal co-immobilization of biomolecules. Surface analytical methods including FTIR and XPS are used to confirm the surface modification. Heparin-binding growth factors [basic fibroblast growth factor (bFGF) in this study] can be immobilized through interaction with heparin, which was covalently attached to the CVD surface through an aldehyde-hydrazide reaction. In parallel, an alkyne-azide reaction is used to orthogonally co-immobilize an adhesion peptide as the second biomolecule.

  6. Receptor- and Heparin-Binding Domains of Basic Fibroblast Growth Factor

    NASA Astrophysics Data System (ADS)

    Baird, Andrew; Schubert, David; Ling, Nicholas; Guillemin, Roger

    1988-04-01

    Two functional domains in the primary structure of basic fibroblast growth factor (FGF) have been identified on the basis of their ability to interact with the FGF receptor, bind radiolabeled heparin, and modulate the cellular response to FGF. Peptides derived from these two functional domains can act as partial agonists and antagonists in biological assays of FGF activity. Peptides related to the sequences of FGF-(24-68)-NH2 and FGF-(106-115)-NH2 inhibit thymidine incorporation into 3T3 fibroblasts when they are stimulated by FGF but have no effect when the cells are treated with either platelet-derived growth factor or epidermal growth factor. They also possess partial agonist activity and can stimulate DNA synthesis when tested in the absence of exogenous FGF. The active peptides have no effect on the binding of epidermal growth factor to its receptor on A431 cells and they can modulate the effects of FGF, but not fibronectin, on endothelial cell adhesion. The results suggest the possibility of designing specific analogs of FGF that are capable of inhibiting the biological effects of FGF.

  7. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

    SciTech Connect

    Bashkin, P.; Doctrow, S.; Klagsbrun, M.; Svahn, C.M.; Folkman, J.; Vlodavsky, I. )

    1989-02-21

    Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of {sup 125}I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 x 10{sup 12} binding sites/mm{sup 2} ECM with an apparent k{sub D} of 610 nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 {mu}g/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 {mu}g/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descement's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

  8. Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells.

    PubMed

    Zhuang, Shougang; Kinsey, Gilbert R; Rasbach, Kyle; Schnellmann, Rick G

    2008-03-01

    Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.

  9. Heparin-Binding Epidermal Growth Factor-Like Growth Factor Enhances Aquaporin 3 Expression and Function During Mouse Embryo Implantation.

    PubMed

    Fang, Chuan-Xiang; Nong, Ying-Qi; Liu, Feng-Hua; Fan, Lin; Chen, Ye

    2017-03-01

    Aquaporin 3 (AQP3) is highly expressed in peri-implantation blastocyst trophoblastic cells, indicating its role in cytotrophoblast invasion during embryo implantation. However, the mechanism underlying the regulation of AQP3 expression during embryo implantation remains unclear. In this study, an in vitro co-culture system of blastocysts on a monolayer of uterine endometrial cells was used to mimic in vivo process of embryo attachment and invasion to uterine endometrium and treated with different concentrations of heparin-binding epidermal growth factor-like growth factor (HB-EGF). The results showed that HB-EGF enhanced AQP3 expression in blastocysts in a dose-dependent manner and promoted the attachment and outgrowth of blastocysts on the monolayer of uterine endometrial cells. When the AQP3 activity was inhibited by copper sulfate, both the attachment and outgrowth of blastocysts were inhibited. Furthermore, HB-EGF induced the phosphorylation of EGF receptor (EGFR) and extracellular signal-regulated kinase (ERK). PD153035 (EGFR inhibitor) and U0126 (ERK inhibitor) inhibited AQP3 expression and also the attachment and outgrowth of blastocysts. Collectively, our findings provide the first evidence that HB-EGF stimulates EGFR/ERK signaling to promote AQP3 expression in trophoblastic cells, and AQP3 plays a vital role in HB-EGF-induced embryo implantation.

  10. Heparin Binding–Epidermal Growth Factor-Like Growth Factor for the Regeneration of Chronic Tympanic Membrane Perforations in Mice

    PubMed Central

    Kim, Sungwoo; Varsak, Yasin Kursad; Yang, Yunzhi Peter

    2015-01-01

    We aim to explore the role of epidermal growth factor (EGF) ligand shedding in tympanic membrane wound healing and to investigate the translation of its modulation in tissue engineering of chronic tympanic membrane perforations. Chronic suppurative otitis media (CSOM) is an infected chronic tympanic membrane perforation. Up to 200 million suffer from its associated hearing loss and it is the most common cause of pediatric hearing loss in developing countries. There is a need for nonsurgical treatment due to a worldwide lack of resources. In this study, we show that EGF ligand shedding is essential for tympanic membrane healing as it's inhibition, with KB-R7785, leads to chronic perforation in 87.9% (n=58) compared with 0% (n=20) of controls. We then show that heparin binding–EGF-like growth factor (5 μg/mL), which acts to shed EGF ligands, can regenerate chronic perforations in mouse models with 92% (22 of 24) compared with 38% (10 of 26), also with eustachian tube occlusion with 94% (18 of 19) compared with 9% (2 of 23) and with CSOM 100% (16 of 16) compared with 41% (7 of 17). We also show the nonototoxicity of this treatment and its hydrogel delivery vehicle. This provides preliminary data for a clinical trial where it could be delivered by nonspecialist trained healthcare workers and fulfill the clinical need for a nonsurgical treatment for chronic tympanic membrane perforation and CSOM. PMID:25567607

  11. Structural determinants of heparin-transforming growth factor-β1 interactions and their effects on signaling.

    PubMed

    Lee, Jonathan; Wee, Sheena; Gunaratne, Jayantha; Chua, R J E; Smith, Raymond A A; Ling, Ling; Fernig, David G; Swaminathan, Kunchithapadam; Nurcombe, Victor; Cool, Simon M

    2015-12-01

    Transforming growth factor-β1 (TGF-β1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-β1's activity on hMSCs. Using a proteomics "Protect and Label" approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin (Data are available via ProteomeXchange with identifier PXD002772). Competition assays in an optical biosensor demonstrated that TGF-β1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-β1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14-24 saccharides (dp14-24) in length all increased the phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) after 6 h of stimulation with TGF-β1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-β1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-β1 in hMSCs.

  12. Heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor expression by acute myeloid leukemia cells.

    PubMed

    Vinante, F; Rigo, A; Papini, E; Cassatella, M A; Pizzolo, G

    1999-03-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an EGF family member expressed by numerous cell types that binds to EGF receptor 1 (HER-1) or 4 (HER-4) inducing mitogenic and/or chemotactic activities. Membrane-bound HB-EGF retains growth activity and adhesion capabilities and the unique property of being the receptor for diphtheria toxin (DT). The interest in studying HB-EGF in acute leukemia stems from these mitogenic, chemotactic, and receptor functions. We analyzed the expression of HB-EGF in L428, Raji, Jurkat, Karpas 299, L540, 2C8, HL-60, U937, THP-1, ML-3, and K562 cell lines and in primary blasts from 12 acute myeloid leukemia (AML) cases, by reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blot and by the evaluation of sensitivity to DT. The release of functional HB-EGF was assessed by evaluation of its proliferative effects on the HB-EGF-sensitive Balb/c 3T3 cell line. HB-EGF was expressed by all myeloid and T, but not B (L428, Raji), lymphoid cell lines tested, as well as by the majority (8 of 12) of ex vivo AML blasts. Cell lines (except for the K562 cell line) and AML blasts expressing HB-EGF mRNA underwent apoptotic death following exposure to DT, thus demonstrating the presence of the HB-EGF molecule on their membrane. Leukemic cells also released a fully functional HB-EGF molecule that was mitogenic for the Balb/c 3T3 cell line. Factors relevant to the biology of leukemic growth, such as tumor necrosis factor-alpha (TNF-alpha), 1alpha,25-(OH)2D3, and especially all-trans retinoic acid (ATRA), upregulated HB-EGF mRNA in HL-60 or ML-3 cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced HB-EGF mRNA and acquisition of sensitivity to DT in one previously HB-EGF-negative leukemia case. Moreover, the U937 and Karpas 299 cell lines expressed HER-4 mRNA. This work shows that HB-EGF is a growth factor produced by primary leukemic cells and regulated by ATRA, 1alpha, 25-(OH)2D3, and GM-CSF.

  13. Injectable extracellular matrix derived hydrogel provides a platform for enhanced retention and delivery of a heparin-binding growth factor.

    PubMed

    Seif-Naraghi, Sonya B; Horn, Dinah; Schup-Magoffin, Pamela J; Christman, Karen L

    2012-10-01

    Injectable hydrogels derived from the extracellular matrix (ECM) of decellularized tissues have recently emerged as scaffolds for tissue-engineering applications. Here, we introduce the potential for using a decellularized ECM-derived hydrogel for the improved delivery of heparin-binding growth factors. Immobilization of growth factors on a scaffold has been shown to increase their stability and activity. This can be done via chemical crosslinking, covalent bonding, or by incorporating natural or synthetic growth factor-binding domains similar to those found in vivo in sulfated glycosaminoglycans (GAGs). Many decellularized ECM-derived hydrogels retain native sulfated GAGs, and these materials may therefore provide an excellent delivery platform for heparin-binding growth factors. In this study, the sulfated GAG content of an ECM hydrogel derived from decellularized pericardial ECM was confirmed by Fourier transform infrared spectroscopy and its ability to bind basic fibroblast growth factor (bFGF) was established. Delivery in the pericardial matrix hydrogel increased retention of bFGF both in vitro and in vivo in ischemic myocardium compared to delivery in collagen. In a rodent infarct model, intramyocardial injection of bFGF in pericardial matrix enhanced neovascularization by approximately 112% compared to delivery in collagen. Importantly, the newly formed vasculature was anastomosed with existing vasculature. Thus, the sulfated GAG content of the decellularized ECM hydrogel provides a platform for incorporation of heparin-binding growth factors for prolonged retention and delivery. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  14. Heparinized magnetic mesoporous silica nanoparticles as multifunctional growth factor delivery carriers

    NASA Astrophysics Data System (ADS)

    Wu, Qiang; Liu, Chaoqun; Fan, Luna; Shi, Jiahua; Liu, Zhiqiang; Li, Ruifang; Sun, Liwei

    2012-12-01

    Well-defined magnetic mesoporous silica nanoparticles (MMSNs) with a core/shell structure were prepared via a one pot synthesis. Sphere-like magnetite aggregates were obtained as cores of the final nanoparticles by assembly in the presence of polyvinyl pyrrolidone and cetyltrimethylammonium bromide. The nanoparticles have the property of superparamagnetism with a saturation magnetization value of 20.3 emu g-1. In addition, the combination of heparin and fluorescence-labeled MMSNs endows the resultant particles (denoted as MFMSNs-HP) with magnetism and fluorescence properties, excellent dispersity in the buffer solutions and cell culture media, anticoagulant activity in the blood stream, and the controlled release of basic fibroblast growth factor (bFGF). Furthermore, the bFGF cell viability assays indicate that MFMSNs-HP has nearly no toxicity to human umbilical vein endothelial cells (HUVEC) up to a concentration of 200 μg ml-1, and the proliferation activity of bFGF incorporated into MFMSNs-HP could be retained for at least 6 days. All of these suggest that MFMSNs-HP may serve as a multifunctional carrier for the delivery of growth factors.

  15. Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2

    PubMed Central

    1996-01-01

    Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung

  16. Heparin-binding epidermal growth factor-like growth factor and hepatocyte growth factor inhibit cholestatic liver injury in mice through different mechanisms

    PubMed Central

    Sakamoto, Kouichi; Khai, Ngin Cin; Wang, Yuqing; Irie, Rie; Takamatsu, Hideo; Matsufuji, Hiroshi; Kosai, Ken-Ichiro

    2016-01-01

    In contrast to hepatocyte growth factor (HGF), the therapeutic potential and pathophysiologic roles of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver diseases remain relatively unknown. To address the lack of effective pharmacologic treatments for cholestatic liver injuries, as well as to clarify the biologic features of these growth factors, we explored the effects of HB-EGF and HGF in mice with cholestatic liver injury induced by bile duct ligation (BDL). The mice were assessed 3, 5 and/or 14 days after BDL (acute, subacute and/or chronic phases, respectively) and intravenous injection of adenoviral vector expressing LacZ (control), HB-EGF, HGF, or HB-EGF and HGF. HB-EGF, HGF, or a combination of the growth factors exerted potent antioncotic (antinecrotic), antiapoptotic, anticholestatic, and regenerative effects on hepatocytes in vivo, whereas no robust antiapoptotic or regenerative effects were detected in interlobular bile ducts. Based on serum transaminase levels, the acute protective effects of HB-EGF on hepatocytes were greater than those of HGF. On the other hand, liver fibrosis and cholestasis during the chronic phase were more potently inhibited by HGF compared with HB-EGF. Compared with either growth factor alone, combining HB-EGF and HGF produced greater anticholestatic and regenerative effects during the chronic phase. Taken together, these findings suggest that HB-EGF and HGF inhibited BDL-induced cholestatic liver injury, predominantly by exerting acute cytoprotective and chronic antifibrotic effects, respectively; combining the growth factors enhanced the anticholestatic effects and liver regeneration during the chronic phase. Our results contribute to a better understanding of the pathophysiologic roles of HB-EGF and HGF, as well as to the development of novel effective therapies for cholestatic liver injuries. PMID:27779646

  17. Cooperative heparin-mediated oligomerization of fibroblast growth factor-1 (FGF1) precedes recruitment of FGFR2 to ternary complexes.

    PubMed

    Brown, Alan; Robinson, Christopher J; Gallagher, John T; Blundell, Tom L

    2013-04-16

    Fibroblast growth factors (FGFs) utilize cell surface heparan sulfate as a coreceptor in the assembly of signaling complexes with FGF-receptors on the plasma membrane. Here we undertake a complete thermodynamic characterization of the assembly of the FGF signaling complex using isothermal titration calorimetry. Heparin fragments of defined length are used as chemical analogs of the sulfated domains of heparan sulfate and examined for their ability to oligomerize FGF1. Binding is modeled using the McGhee-von Hippel formalism for the cooperative binding of ligands to a monodimensional lattice. Oligomerization of FGFs on heparin is shown to be mediated by positive cooperativity (α = 6). Heparin octasaccharide is the shortest length capable of dimerizing FGF1 and on longer heparin chains FGF1 binds with a minimal footprint of 4.2 saccharide units. The thermodynamics and stoichiometry of the ternary complex suggest that in solution FGF1 binds to heparin in a trans-dimeric manner before FGFR recruitment.

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

    PubMed Central

    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

  19. Midkine, a heparin-binding growth factor, and its roles in atherogenesis and inflammatory kidney diseases.

    PubMed

    Şalaru, Delia Lidia; Arsenescu-Georgescu, Cătălina; Chatzikyrkou, Christos; Karagiannis, Jaqueline; Fischer, Anja; Mertens, Peter R

    2016-11-01

    The heparin-binding protein midkine is a potent growth factor with emerging roles in numerous inflammatory diseases. Beyond its characterization in embryogenesis and organ development, ample insights into its function have been collected from experimental disease models using knockout animals or knockdown intervention strategies. Here a comprehensive overview on midkine and its functions in atherogenesis and kidney diseases is provided. Molecular clues to key signalling pathways (Akt, ERK, HIF1α) and key events in atherosclerotic vessels link midkine expression with vascular smooth muscle proliferation and (neo)angiogenesis. In acute and chronic kidney diseases, midkine expression is upregulated in tubular as well as endothelial cells. Experimental disease models that mimic diabetic nephropathy and/or immunologic glomerular damage indicate dichotomous midkine activities, with cytoprotective as well as injurious effects. This review also pinpoints the commonalities of the disease models. An understanding of the underlying molecular events will be required in order to design a targeted intervention into cardiovascular or renal diseases as well as inflammatory processes.

  20. Heparin-binding growth factor 1 induces the formation of organoid neovascular structures in vivo.

    PubMed Central

    Thompson, J A; Haudenschild, C C; Anderson, K D; DiPietro, J M; Anderson, W F; Maciag, T

    1989-01-01

    One of the promises of modern molecular biology has been the opportunity to use genetically modified human cells in a patient to permanently restore inborn errors of metabolism. Although it has been possible to introduce genes into mammalian cells and to control their expression, it has proven difficult to introduce mammalian cells as carriers of the modified genetic information into hosts. The successful implantation of selective cells cannot be achieved without adequate vascular support, an essential step toward integration and reconstitution of a new biological function. Although a partial solution to this problem has been found by inducing specific site-directed neovessel formation using heparin-binding growth factor 1 (HBGF-1) adsorbed to a collagen matrix, these implants function for only a short period (weeks). We now report the formation of organoid neovascular structures using polytetrafluoroethylene fibers coated with collagen and HBGF-1 implanted in the peritoneal cavity of the rat. The organoid structures contained readily visible vascular lumina and nonvascular structures that resemble nerve tissue. It was also possible to demonstrate that the vascular system on the implant is continuous with the vascular tree of the host. This feature was used to demonstrate that the organoid structures are capable of sustaining the biological function of implanted normal rat hepatocytes over long periods of time (months) in the homozygous Gunn rat, thereby facilitating future applications involving the delivery of new genetic information. Images PMID:2479012

  1. Production of Heparin-Functionalized Hydrogels for the Development of Responsive and Controlled Growth Factor Delivery Systems

    PubMed Central

    Nie, Ting; Baldwin, Aaron; Yamaguchi, Nori; Kiick, Kristi L.

    2007-01-01

    Methods to assemble polymeric hydrogels on the basis of noncovalent protein-glycosaminoglycan interactions have been previously demonstrated by us and others and hold promise in the development of receptor-responsive hydrogel materials; improvements in the mechanical properties of such systems would broaden their utility. Thus, in situ crosslinkable and degradable heparin-containing hydrogels were designed for the binding and controlled release of growth factors. Specifically, maleimide-functionalized high molecular weight heparin (HMWH) was synthesized via straightforward chemical methods that permitted facile and controllable modification of carboxylates in HMWH with maleimide groups via control of catalyst and reaction conditions, as assessed via 1H NMR spectroscopy. These modified heparins were crosslinked into hydrogels via reaction with various thiol-functionalized PEGs. The gelation times and elastic moduli of the gels, as assessed through oscillatory rheometry, could be tuned by via control of the functionality of HMWH, the concentration of hydrogel, the identity of the PEG-based crosslinker, as well as the molar ratio between maleimide and thiol groups. The capability of the hydrogels to bind to growth factors was investigated with immunochemical assays. Preliminary studies indicate the controlled release of basic fibroblast growth factor (bFGF) from these materials and suggest their broader use in the design of responsive materials. PMID:17582636

  2. Heparin-binding EGF-like Growth Factor Increases Intestinal Microvascular Blood Flow in Necrotizing Enterocolitis

    PubMed Central

    Yu, Xiaoyi; Radulescu, Andrei; Zorko, Nicholas; Besner, Gail E.

    2009-01-01

    Background & Aims Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates. Although the exact etiology remains unknown, decreased intestinal blood flow is thought to play a critical role. We have shown that heparin-binding EGF-like growth factor (HB-EGF) protects the intestines from injury in a rodent model of NEC. Our current goal was to assess the effect of HB-EGF on intestinal microvascular blood flow and intestinal injury in rat pups subjected to experimental NEC. Methods Newborn rat pups were subjected to stress by exposure to hypoxia, hypothermia, hypertonic feedings and lipopolysaccharide, with some pups receiving HB-EGF (800 μg/kg/dose) added to the feeds. Control animals received breast milk. Intestinal injury was graded using a standard histologic injury scoring system. Microvascular blood flow was assessed by FITC-dextran angiography with fluorescent images subjected to quantification, and by scanning electron microscopy. Results Intestinal microvascular blood flow (defined as the extent of vascular filling with FITC-dextran) was significantly decreased in pups subjected to stress compared to breast fed pups. Stressed pups treated with HB-EGF had significantly increased microvascular blood flow. The changes in villous microvasculature correlated with histologic injury scores, with stressed pups treated with HB-EGF showing decreased histologic injury. Conclusions HB-EGF significantly preserved intestinal microvascular blood flow in pups subjected to experimental NEC, indicating that HB-EGF may play a critical role in the therapy of various diseases manifested by decreased intestinal blood flow, including NEC. PMID:19361505

  3. Characterization of the surface immobilized synthetic heparin binding domain derived from human fibroblast growth factor-2 and its effect on osteoblast differentiation.

    PubMed

    Lee, Jue-Yeon; Choo, Jung-Eun; Choi, Young-Sook; Lee, Kuen-Yong; Min, Do-Sik; Pi, Sung-Hee; Seol, Yang-Jo; Lee, Seung-Jin; Jo, In-Ho; Chung, Chong-Pyoung; Park, Yoon-Jeong

    2007-12-15

    Fibroblast growth factor (FGF)-2 regulates a variety of cellular functions, such as proliferation and differentiation, by binding to cell surface FGF receptors (FGFRs) in the presence of heparin proteoglycans. FGF-2 is known as a heparin-binding growth factor, but the localization of the heparin binding site has not been fully investigated until now. We used two potential heparin binding domains of FGF-2, the residues 105-111 (F105, YKRSRYT) and 119-135 (F119, KRTGQYKLGSKTGPGQK). Peptides could be stably immobilized onto the surface of tissue culture plates. Using solid phase binding assays, we demonstrated that both peptides had higher binding affinity toward heparin compared with nonbinding control sequence. The biological significance of these sites was tested by cell attachment and osteoblast differentiation studies. Cell attachment to the peptides F105 and F119 increased in a dose-dependent manner. Heparin and heparinase treatments decreased cell adhesion to both F105 and F119. This demonstrates that both F105 and F119 interact with cell-surface heparan sulfate proteoglycans, suggesting that FGF-2 has two heparin binding sites. In addition, osteoblast differentiation, confirmed by ALPase activity and mineralization, was increased by surface immobilized peptide F105 and F119. Taken together, these heparin binding peptides could be applied as biological agents enhancing osteoblast differentiation as well as surface modification tools in the tissue regeneration area, especially for bone regeneration. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.

  4. [Expression and significance of heparin binding-epidermal growth factor-like growth factor in paclitaxel-resistant ovarian cancer].

    PubMed

    Tang, Xiaohan; Lu, Meisong; Li, Cuiping; Deng, Suo; Li, Meng

    2014-07-01

    To examine the expression of heparin binding-epidermal growth factor-like growth factor (HB- EGF) in paclitaxel- resistant ovarian cancer and elucidate the relationship between HB-EGF and the resistance of ovarian cancer to paclitaxel. The human ovarian carcinoma cell line A2780 and the paclitaxel- resistant human ovarian carcinoma cell line A2780/Taxol were cultured in vitro. Western blot was used to dectect the expression of HB-EGF protein in A2780 and A2780/Taxol groups. The A2780 cells were treated with cross- reacting material 197 (CRM197 and A2780 + CRM197 group) or dimethyl sulphoxide (DMSO; A2780 group), while the A2780/Taxol cells were treated with CRM197 (A2780/Taxol+CRM197 group) or DMSO (A2780/Taxol group). The effects of CRM197 on growth and proliferation was tested by methyl thiazolyl tetrazolium ( MTT) and the results were showed as absorbance (A). The effects of CRM197 on cell cycles was tested by flow cytometry, while the effects of CRM197 on apoptosis was examined by caspase- 3 activity assay and the results were showed as p- nitroaniline(pNa). In animal experiment, four groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability following CRM197 treatment. Immunohistochemistry was used to determine the expression of HB-EGF protein in A2780 and A2780/Taxol group. The expression level of HB-EGF protein in A2780/Taxol group (2.11 ± 0.41) was significantly higher than that of A2780 group (0.75 ± 0.20; P < 0.01). The inhibition effect of CRM197 on the cell growth of A2780+CRM197 and A2780/Taxol+CRM197 group was accompanied by the acceleration of CRM197 concentration(P < 0.01). When CRM197≥1 µg/ml, the inhibition effect of CRM197 on the cell growth of A2780/Taxol+CRM197 group was significantly higher than that in A2780/Taxol group(P < 0.05). In cell cycle experiment, CRM197 induced the cell-cycle arrest at the G0/G1 phase in A2780+CRM197 cells[(67 ± 4)%] compared with A2780 cells[(54 ± 6)%; P < 0

  5. Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor receptor complex.

    PubMed

    Kan, M; Wang, F; To, B; Gabriel, J L; McKeehan, W L

    1996-10-18

    Polypeptides of the fibroblast growth factor (FGF) family are ubiquitous bioregulators within tissues whose activity is controlled by heparan sulfates within the pericellular matrix. FGF and the ectodomain of their transmembrane tyrosine kinase receptors (FGFR) exhibit heparin-binding domains that when juxtaposed in a FGF middle dotFGFR complex can accommodate a single, potentially bivalent, decameric polysaccharide chain in a ternary complex. Here we show that the interaction of heparin with FGF ligands is not affected by divalent cations. In contrast, the high affinity interaction (apparent Kd = 10 nM) of heparin with FGFR requires Ca2+ or Mg2+ at physiological concentrations. Divalent cations maintain FGFR in a heparan sulfate-dependent state in respect to FGF binding and an FGF- and heparan sulfate-dependent state in respect to autophosphorylation. A model is proposed where divalent cations and heparan sulfate cooperate to maintain FGFR in a conformation that restricts trans-phosphorylation between intracellular kinase domains. The restriction is overcome by FGF or constitutively as a common consequence of diverse mutations in FGFR associated with skeletal and craniofacial abnormalities.

  6. Controlled Delivery of Heparin-Binding EGF-Like Growth Factor Yields Fast and Comprehensive Wound Healing

    PubMed Central

    Johnson, Noah Ray; Wang, Yadong

    2014-01-01

    Wound healing is a dynamic process that relies on coordinated signaling molecules to succeed. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is proven to accelerate healing, however precise control over its application is necessary to reduce side effects and achieve desired therapeutic benefit. To achieve effective growth factor delivery we designed a bioactive heparin-based coacervate. In vitro, HB-EGF released from the coacervate delivery system displayed enhanced bioactivity and promoted human keratinocyte migration while preserving cell proliferative capability. In a mouse excisional full-thickness wound model, controlled release of HB-EGF within the wound significantly accelerated wound closure more effectively than an equal dosage of free HB-EGF. Healing was induced by rapid re-epithelialization, granulation tissue formation, and accompanied by angiogenesis. Consistent with in vitro results, wounds treated with HB-EGF coacervate exhibited enhanced migration of keratinocytes with retained proliferative potential, forming a confluent layer for regained barrier function within 7 days. Collectively, these results suggest that coacervate-based controlled release of HB-EGF may serve as a new therapy to accelerate healing of cutaneous wounds. PMID:23154193

  7. The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    PubMed

    Hospital, Véronique; Nishi, Eiichiro; Klagsbrun, Michael; Cohen, Paul; Seidah, Nabil G; Prat, Annik

    2002-10-01

    Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met(49). Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc-HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca(2+), via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase.

  8. The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    PubMed Central

    Hospital, Véronique; Nishi, Eiichiro; Klagsbrun, Michael; Cohen, Paul; Seidah, Nabil G; Prat, Annik

    2002-01-01

    Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met(49). Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc-HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca(2+), via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase. PMID:12095415

  9. Oligomeric self-association of basic fibroblast growth factor in the absence of heparin-like glycosaminoglycans.

    PubMed Central

    Davis, J C; Venkataraman, G; Shriver, Z; Raj, P A; Sasisekharan, R

    1999-01-01

    Basic fibroblast growth factor (FGF-2) represents a class of heparin-binding growth factors that are stored in the extracellular matrix attached to heparin-like glycosaminoglycans (HLGAGs). It has been proposed that cell surface HLGAGs have a central role in the biological activity of FGF-2, presumably by inducing dimers or oligomers of FGF-2 and leading to the dimerization or oligomerization of FGF receptor and hence signal transduction. We have previously proposed that FGF-2 possesses a natural tendency to self-associate to form FGF-2 dimers and oligomers; HLGAGs would enhance FGF-2 self-association. Here, through a combination of spectroscopic, chemical cross-linking and spectrometric techniques, we provide direct evidence for the self-association of FGF-2 in the absence of HLGAGs, defying the notion that HLGAGs induce FGF-2 oligomerization. Further, the addition of HLGAGs seems to enhance significantly the FGF-2 oligomerization process without affecting the relative percentages of FGF-2 dimers, trimers or oligomers. FGF-2 self-association is consistent with FGF-2's possessing biological activity both in the presence and in the absence of HLGAGs; this leads us to propose that FGF-2 self-association enables FGF-2 to signal both in the presence and in the absence of HLGAGs. PMID:10417324

  10. CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor

    PubMed Central

    1995-01-01

    Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS- modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS- modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS- modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is

  11. 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.

    PubMed

    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.

  12. Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells.

    PubMed

    Hatziapostolou, Maria; Polytarchou, Christos; Katsoris, Panagiotis; Courty, Jose; Papadimitriou, Evangelia

    2006-10-27

    Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.

  13. A Heparin-Mimicking Block Copolymer Both Stabilizes and Increases the Activity of Fibroblast Growth Factor 2 (FGF2)

    PubMed Central

    2016-01-01

    Fibroblast growth factor 2 (FGF2) is a protein involved in cellular functions in applications such as wound healing and tissue regeneration. Stabilization of this protein is important for its use as a therapeutic since the native protein is unstable during storage and delivery. Additionally, the ability to increase the activity of FGF2 is important for its application, particularly in chronic wound healing and the treatment of various ischemic conditions. Here we report a heparin mimicking block copolymer, poly(styrenesulfonate-co-poly(ethylene glycol) methyl ether methacrylate)-b-vinyl sulfonate) (p(SS-co-PEGMA)-b-VS, that contains a segment that enhances the stability of FGF2 and one that binds to the FGF2 receptor. The FGF2 conjugate retained activity after exposure to refrigeration (4 °C) and room temperature (23 °C) for 7 days, while unmodified FGF2 was inactive after these standard storage conditions. A cell study performed with a cell line lacking native heparan sulfate proteoglycans indicated that the conjugated block copolymer facilitated binding of FGF2 to its receptor similar to the addition of heparin to FGF2. A receptor-based enzyme-linked immunosorbant assay (ELISA) confirmed the results. The conjugate also increased the migration of endothelial cells by 80% compared to FGF2 alone. Additionally, the FGF2-p(SS-co-PEGMA)-b-VS stimulated endothelial cell sprouting 250% better than FGF2 at low concentration. These data verify that this rationally designed protein-block copolymer conjugate enhances receptor binding, cellular processes such as migration and tube-like formation, and stability, and suggest that it may be useful for applications in biomaterials, tissue regeneration, and wound healing. PMID:27580376

  14. Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching.

    PubMed

    Takemura, Tsukasa; Hino, Satoshi; Okada, Mituru; Murata, Yuka; Yanagida, Hidehiko; Ikeda, Masaru; Yoshioka, Kazuo; Harris, Raymond C

    2002-06-01

    Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching. The developing metanephros is characterized by growth and differentiation of the ureteric bud and the surrounding mesenchymal tissue. These processes can be influenced by several growth factors, including epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). We examined whether another member of the EGF family of growth factors, heparin-binding epidermal growth factor (HB-EGF), might act as a morphogen in renal epithelial tubulogenesis. Expression of HB-EGF mRNA and immunoreactive protein were examined in fetal, neonatal and adult rat kidneys. For in vitro studies of tubulogenesis, a rat renal epithelial cell line (NRK52E) stably transfected with proHB-EGF (NRKproHB-EGF) was treated with TPA for 30 minutes, washed with 2 mol/L NaCl to remove soluble HB-EGF trapped by cell surface heparan sulfate proteoglycan and replated onto plastic dishes in the absence of fetal calf serum. In further experiments, NRKproHB-EGF were suspended in a type I collagen gel in serum-free media. Northern blot analysis indicated that HB-EGF was strongly expressed in embryonic rat kidney (embryonic days 18-20) and was still increased in the neonatal kidney (day 10), compared to the low basal levels in adult kidney. Immunohistochemical analysis confirmed that immunoreactive HB-EGF expression in the fetal rat kidney was localized predominantly to the ureteric bud. When NRKproHB-EGF were plated onto plastic substrata, they became progressively flattened and enlarged and exhibited filopoidia. By 10 hours after plating, NRKproHB-EGF began to migrate and subsequently developed cell-cell contact and fully established tubular-like structures. Immunoelectron microscopy revealed that the initial recovery of cellular proHB-EGF was localized predominantly to areas of cell-cell attachment. No tubule-like structures were observed in similarly treated NRK

  15. Heparin-conjugated poly(lactic-co-glycolic acid) nanospheres enhance large-wound healing by delivering growth factors in platelet-rich plasma.

    PubMed

    La, Wan-Geun; Yang, Hee Seok

    2015-04-01

    Platelet-rich plasma (PRP) contains many growth factors that are involved in tissue regeneration processes. For successful tissue regeneration, protein growth factors require a delivery vehicle for long-term and sustained release to a defect site in order to maintain their bioactivity. Previously, we showed that heparin-conjugated poly(lactic-co-glycolic acid) nanospheres (HCPNs) can provide long-term delivery of growth factors with affinity for heparin. In this study, we hypothesize that treatment of a skin wound with a mixture of PRP and HCPNs would provide long-term delivery of several growth factors contained in PRP to promote the skin wound healing process with preservation of bioactivity. The release of platelet-derived growth factor-BB (PDGF-BB), contained in PRP, from HCPN with fibrin gel (FG) showed a prolonged release period versus a PRP mixture with FG alone (FG-PRP). Also, growth factors released from PRP with HCPN and FG showed sustained human dermal fibroblast growth for 12 days. Full-thickness skin wound treatment in mice with FG-HCPN-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 9 compared with treatment with FG-HCPN or FG-PRP. The enhanced wound healing using FG-HCPN-PRP may be due to the prolonged release not only of PDGF-BB but also of other growth factors in the PRP. The delivered growth factors accelerated angiogenesis at the wound site.

  16. Studies of molecular docking between fibroblast growth factor and heparin using generalized simulated annealing

    NASA Astrophysics Data System (ADS)

    Pita, Samuel Silva Da Rocha; Fernandes, Tácio Vinício Amorim; Caffarena, Ernesto Raul; Pascutti, Pedro Geraldo

    Since the middle 70s, the main molecular docking problem consists in limitations to treat adequately the degrees of freedom of protein (or a receptor) due to the energy landscape roughness and the high computational cost. Until recently, only few algorithms considering flexible simultaneously both ligand and receptor at low computational cost were developed. As a recent proposed Statistical Mechanics, generalized simulated annealing (GSA) has been employed at diverse works concerning global optimization problems. In this work, we used this method exploring the molecular docking problem taking into account the FGF-2 and heparin complex. Since the requirements of an efficient docking algorithm are accuracy and velocity, we tested the influence of GSA parameters qA (new configuration acceptance index), qV (energy surface visiting index), and qT (temperature decreasing control) on the performance of GSADOCK program. Our simulations showed that as temperature parameter qT increases, qA parameter follows this behavior in the interval ranging from 1.1 to 2.3. We found that the GSA parameters have the best performance for the qA values ranging from 1.1 to 1.3, qV values from 1.3 to 1.5, and qT values from 1.1 to 1.7. Most of good qV values were equal or next the good qT values. Finally, the implemented algorithm is trustworthy and can be employed as a tool of molecular modeling methods. The final version of the program will be free of charge and will be accessible at our home-page or could be requested to the authors for e-mail.

  17. Control of fibroblast growth factor (FGF) 7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs.

    PubMed

    Luo, Yongde; Ye, Sheng; Kan, Mikio; McKeehan, Wallace L

    2006-07-28

    Variation in length, disaccharide composition, and sulfation of heparan sulfate (HS) affects fibroblast growth factor (FGF) signaling. However, it is unclear whether the specific distribution of groups within oligosaccharides or random variations in charge density underlies the effects. Recently we showed that a mixture of undersulfated octasaccharides exhibiting 7 and 8 sulfates (7,8-S-OctaF7) generated from heparin had the highest affinity for FGF7 monitored by salt resistance (>0.60 M salt) of octasaccharide-FGF7 complexes. 7,8-S-OctaF7 also had the highest specific activity for formation of a complex with dimeric FGFR2IIIb competent to bind FGF7. Here we show that when endogenous HS was inhibited by chlorate treatment, 7,8-S-OctaF7 specifically supported FGF7-stimulated DNA synthesis and downstream signaling in FGFR2IIIb-expressing mouse keratinocytes. It failed to support FGF1 signaling in both HS-deficient mouse keratinocytes and 3T3 fibroblasts. In contrast, abundant, more highly sulfated and heterogenous mixtures of octasaccharides with lower affinity (0.30-0.60 M salt) for FGF7 supported FGF1-induced signaling in both cell types. In contrast to the two-component 7,8-S-OctaF7 mixture from FGF7, the high affinity octasaccharide fraction from FGF1 was a heterogeneous mixture with components ranging from 8 to 12 sulfates with 11-S-octasaccharides the most abundant. The high affinity fraction exhibited similar properties to the lower affinity fractions from both FGF1 and FGF7. Octasaccharide mixtures eluting from FGF1 between 0.30 and 0.60 M and above 0.60 M salt were nearly equal in support of FGF1 signaling in fibroblasts and keratinocytes. Both were deficient in support of FGF7-induced signaling in keratinocytes. The results show that both variations in overall charge density and specific distribution of charged groups within HS motifs exhibit FGF-specific control over formation of FGF-HS-FGFR complexes and downstream signaling.

  18. Co-delivery of platelet-derived growth factor (PDGF-BB) and bone morphogenic protein (BMP-2) coated onto heparinized titanium for improving osteoblast function and osteointegration.

    PubMed

    Kim, Sung Eun; Yun, Young-Pil; Lee, Jae Yong; Shim, June-Sung; Park, Kyeongsoon; Huh, Jung-Bo

    2015-12-01

    The aim of this study was to improve osteoblast function by delivering two growth factors, PDGF-BB and BMP-2, incorporated onto heparinized titanium (Hep-Ti) substrate. To achieve co-delivery of PDGF-BB and BMP-2, the surface of anodized Ti was immobilized with heparin, and then the two growth factors were coated onto the Hep-Ti surface. Incorporation of the two growth factors onto Hep-Ti was evaluated by SEM and XPS. Incorporated PDGF-BB and BMP-2 were released from the Hep-Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on PDGF-BB- and BMP-2-immobilized Hep-Ti increased ALP activity, calcium deposition, osteocalcin and osteopontin levels as compared to those grown on PDGF-BB alone- or BMP-2 alone-immobilized Hep-Ti. These results suggested that co-delivery of PDGF-BB and BMP-2 using Hep-Ti substrate will be a promising material for the enhancement of osteoblast function and osteointegration. Copyright © 2013 John Wiley & Sons, Ltd.

  19. A Modular, Plasmin-Sensitive, Clickable Poly(ethylene glycol)-Heparin-Laminin Microsphere System for Establishing Growth Factor Gradients in Nerve Guidance Conduits

    PubMed Central

    Roam, Jacob L.; Yan, Ying; Nguyen, Peter K.; Kinstlinger, Ian S.; Leuchter, Michael K; Hunter, Daniel A.; Wood, Matthew D.; Elbert, Donald L.

    2015-01-01

    Peripheral nerve regeneration is a complex problem that, despite many advancements and innovations, still has sub-optimal outcomes. Compared to biologically derived acelluar nerve grafts and autografts, completely synthetic nerve guidance conduits (NGC), which allow for precise engineering of their properties, are promising but still far from optimal. We have developed an almost entirely synthetic NGC that allows control of soluble growth factor delivery kinetics, cell-initiated degradability and cell attachment. We have focused on the spatial patterning of glial-cell derived human neurotrophic factor (GDNF), which promotes motor axon extension. The base scaffolds consisted of heparin-containing poly(ethylene glycol) (PEG) microspheres. The modular microsphere format greatly simplifies the formation of concentration gradients of reversibly bound GDNF. To facilitate axon extension, we engineered the microspheres with tunable plasmin degradability. ‘Click’ cross-linking chemistries were also added to allow scaffold formation without risk of covalently coupling the growth factor to the scaffold. Cell adhesion was promoted by covalently bound laminin. GDNF that was released from these microspheres was confirmed to retain its activity. Graded scaffolds were formed inside silicone conduits using 3D-printed holders. The fully formed NGC’s contained plasmin-degradable PEG/heparin scaffolds that developed linear gradients in reversibly bound GDNF. The NGC’s were implanted into rats with severed sciatic nerves to confirm in vivo degradability and lack of a major foreign body response. The NGC’s also promoted robust axonal regeneration into the conduit. PMID:26352518

  20. Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

    PubMed Central

    Burgess, W H; Dionne, C A; Kaplow, J; Mudd, R; Friesel, R; Zilberstein, A; Schlessinger, J; Jaye, M

    1990-01-01

    Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma. Images PMID:2167438

  1. [Reverse of the resistance to paclitaxel of the heparin binding-epidermal growth factor-like growth factor inhibitor in ovarian cancer].

    PubMed

    Tang, X H; Lu, M S; Deng, S; Li, M

    2017-02-25

    Objective: To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance of ovarian cancer to paclitaxel. Methods: (1)The effect of CRM197 on the 50% inhibitory concentrations (IC(50)) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF, epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA) vector and saline treatment (MDR1 siRNA group), empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results: (1) In vitro, MTT examination showed that the IC(50) of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3) μmol/L] was significantly lower than the IC(50) in A2780/Taxol group [ (34.1±0.5) μmol/L, P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44±0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72±0.32), respectively (P<0.05). The expression level of EGFR

  2. 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

    PubMed Central

    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

  3. Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity.

    PubMed

    Teran, Madelane; Nugent, Matthew A

    2015-06-26

    Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance and in vitro binding assays. Additionally, we demonstrate that these binding events are relevant to VEGF activity within endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor (VEGFR)-1, NRP-1, and VEGF165 in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF165, VEGFR-2, and monomeric NRP-1 bind weakly to heparin alone yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and ERK1/2 in endothelial cells and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.

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

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

    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.

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

    PubMed

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

    2017-04-01

    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. Graphical Abstract ᅟ.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Modulation of hepatocyte growth factor plasma levels in relation to the dose of exogenous heparin administered: an experimental study in rats.

    PubMed

    Moreno, E; Meneu, J C; Calvo, J; Pérez, B; Sesma, A G; Manrique, A; Vegh, I; Aragón, A M; Grau, M; Gimeno, A; Jiménez, C; Gómez, R; Moreno, A; Abradelo, M; García, I; de la Calle, A

    2005-11-01

    Partial liver transplantation has been consolidated to be a valid treatment option. We sought to understand the factors that modulate and may be harnessed to accelerate hepatocyte regeneration. We sought to determine the impact of heparin on m-hepatocyte growth factor (HGF) plasma concentrations. Sixteen rats were assigned to four groups of four animals each: group A, without heparin; group B, 600 IU/kg; group C, 1000 IU/kg, group D, 1400 IU/kg. Blood samples (0.5 mL) were obtained from each rat at baseline and at 30, 60, 120, and 240 minutes. After the samples were centrifuged to separate supernates from the cell phase they were stored at -20 degrees C in the m-HGF reagent and subsequently tested using enzyme-linked immunosorbent assay. Results were analyzed using SPSS 11.5 statistical software. Among the 16 rats, one died at 110 minutes, just prior to the last extraction. The remaining rats were sacrificed. Mean weight was: 466 +/- 64.24 g with no intergroup differences (P = .149). The comparative results (using Student t test) were: baseline A(1-4) versus A(1-4) 30 minutes: P < .05; baseline A(1-4) versus A(1-4) 60 minutes: P < .05; baseline A(1-4) versus A(1-4) 120 minutes: P = .10 (NS); baseline A(1-4) versus A(1-4) 240 minutes: P = .15 (NS). No significant differences were found among group B: baseline C(1-4) versus C(1-4) 30 minutes and 60 minutes: NS; baseline C(1-4) versus C(1-4) 120 minutes: P < .001; baseline C(1-4) versus C(1-4) 240 minutes: P < .10 (NS). Finally, the results in group D were: baseline D(1-4) versus D(1-4) 30 minutes: NS; baseline D(1-4) versus D(1-4) 60 minutes and 120 minutes: P < .05; baseline D(1-4) versus D(1-4) 240 minutes: P < .0005. When we compared group A to C and D, we detected differences (albeit not when compared to B) with P values = .01. Peak values were obtained at 120 and 240 minutes (225.21 pg/mL and 221.78 pg/mL) among groups C and D. Heparin has a positive effect to increase serum HGF concentrations among rats. The

  8. Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

    PubMed Central

    Eriksson, A. E.; Cousens, L. S.; Matthews, B. W.

    1993-01-01

    The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin. PMID:7691311

  9. Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor

    PubMed Central

    Armant, D. Randall; Kilburn, Brian A.; Petkova, Anelia; Edwin, Samuel S.; Duniec-Dmuchowski, Zophia M.; Edwards, Holly J.; Romero, Roberto; Leach, Richard E.

    2006-01-01

    Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is downregulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O2 (∼2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O2 upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O2, signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O2 and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O2 rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts. PMID:16407398

  10. Delivery system for autologous growth factors fabricated with low-molecular-weight heparin and protamine to attenuate ischemic hind-limb loss in a mouse model.

    PubMed

    Nakamura, Shingo; Takikawa, Megumi; Ishihara, Masayuki; Nakayama, Takefumi; Kishimoto, Satoko; Isoda, Susumu; Ozeki, Yuichi; Sato, Masahiro; Maehara, Tadaaki

    2012-12-01

    Frozen and thawed platelet-rich plasma (PRP) contains high concentrations of various growth factors, such as fibroblast growth factor (FGF)-2, vascular endothelial growth factor, and hepatocyte growth factor. We previously reported that low-molecular-weight heparin/protamine microparticles (LH/P MPs) are useful as biodegradable carriers for the controlled release of FGF-2. In this study, we examined the ability of PRP/LH/P MPs to prevent limb loss in an induced ischemic hind-limb model that used adult BALB/c-nu/nu male mice. One day after inducing ischemia, intramuscular injections of a PRP/LH/P MPs solution were administered into several sites of the ischemic hind limb. Seven days and onward after the injections, the PRP/LH/P MPs-treated and PRP-treated groups recovered from ischemia, as reflected by the improved oxygen saturation. In the PRP-treated group, however, the level of recovery of oxygen saturation after ischemia decreased after 14 days. From the 21st day onward, there was a significant difference between those two groups. In the LH/P MPs-treated group, a partial recovery occurred only in the early period. The saline-treated group (i.e., the control) and the noninjection group (i.e., ischemia only) exhibited no recovery. The limb survival rate at 1 year in the ischemia-induced mice injected with PRP/LH/P MPs was approximately 25 % (two of eight mice) but was absent in the other groups.

  11. Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development.

    PubMed

    Kim, Seonwook; Yang, Lihua; Kim, Seongu; Lee, Richard G; Graham, Mark J; Berliner, Judith A; Lusis, Aldons J; Cai, Lei; Temel, Ryan E; Rateri, Debra L; Lee, Sangderk

    2017-01-01

    The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

  12. Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development

    PubMed Central

    Kim, Seonwook; Yang, Lihua; Kim, Seongu; Lee, Richard G.; Graham, Mark J.; Berliner, Judith A.; Lusis, Aldons J.; Cai, Lei; Temel, Ryan E.; Rateri, Debra L.

    2017-01-01

    Objective The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Approach and results Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. Conclusion This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting. PMID:28792970

  13. Site-directed mutagenesis of heparin-binding EGF-like growth factor (HB-EGF): analysis of O-glycosylation sites and properties.

    PubMed

    Davis-Fleische, K M; Brigstock, D R; Besner, G E

    2001-01-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a 22 kDa, O-glycosylated protein. HeLa cells infected with a recombinant vaccinia virus expressing human HB-EGF produced a secreted, bioactive protein, with Mr 22,000 that was decreased to 14,000 by treatment with O-glycanase. Site-directed mutagenesis of HB-EGF cDNA using oligonucleotide- and PCR-directed techniques was performed to change the potential glycosylation sites, Thr75 and Thr85, to alanine residues to prevent O-glycosylation. Purification and characterization of the mutant proteins demonstrated that: (i) both O-glycosylation sites of HB-EGF are utilized, (ii) HB-EGF secretion does not require O-glycosylation, (iii) removal of O-glycans does not affect proteolytic cleavage of the HB-EGF precursor, nor does it influence HB-EGF intracellular trafficking or subcellular localization, and (iv) HB-EGF produced by HeLa cells is heavily sialylated. Comparisons between glycosylation mutants and wild-type HB-EGF revealed no significant apparent differences in receptor binding activity.

  14. FGF growth factor analogs

    DOEpatents

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY; Takahashi, Kazuyuki [Germantown, MD

    2012-07-24

    The present invention provides a fibroblast growth factor heparin-binding analog of the formula: ##STR00001## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, X, Y and Z are as defined, pharmaceutical compositions, coating compositions and medical devices including the fibroblast growth factor heparin-binding analog of the foregoing formula, and methods and uses thereof.

  15. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    SciTech Connect

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung; Park, Yoon-Jeong; Chung, Chong-Pyoung

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer HBP sequence identified from HB-EGF has cell penetration activity. Black-Right-Pointing-Pointer HBP inhibits the NF-{kappa}B dependent inflammatory responses. Black-Right-Pointing-Pointer HBP directly blocks phosphorylation and degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer HBP inhibits nuclear translocation of NF-{kappa}B p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-{alpha} and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-{alpha} and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-{kappa}B-dependent inflammatory responses by directly blocking the phosphorylation and degradation of I{kappa}B{alpha} and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-{kappa}B. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  16. Sustained release of hepatocyte growth factor by cationic self-assembling peptide/heparin hybrid hydrogel improves β-cell survival and function through modulating inflammatory response

    PubMed Central

    Liu, Shuyun; Zhang, Lanlan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

    2016-01-01

    Inflammatory response is a major cause of grafts dysfunction in islet transplantation. Hepatocyte growth factor (HGF) had shown anti-inflammatory activity in multiple diseases. In this study, we aim to deliver HGF by self-assembling peptide/heparin (SAP/Hep) hybrid gel to protect β-cell from inflammatory injury. The morphological and slow release properties of SAPs were analyzed. Rat INS-1 β-cell line was treated with tumor necrosis factor α in vitro and transplanted into rat kidney capsule in vivo, and the viability, apoptosis, function, and inflammation of β-cells were evaluated. Cationic KLD1R and KLD2R self-assembled to nanofiber hydrogel, which showed higher binding affinity for Hep and HGF because of electrostatic interaction. Slow release of HGF from cationic SAP/Hep gel is a two-step mechanism involving binding affinity with Hep and molecular diffusion. In vitro and in vivo results showed that HGF-loaded KLD2R/Hep gel promoted β-cell survival and insulin secretion, and inhibited cell apoptosis, cytokine release, T-cell infiltration, and activation of NFκB/p38 MAPK pathways in β-cells. This study suggested that SAP/Hep gel is a promising carrier for local delivery of bioactive proteins in islet transplantation. PMID:27729786

  17. Controlled Dual Delivery of Fibroblast Growth Factor-2 and Interleukin-10 by Heparin-based Coacervate Synergistically Enhances Ischemic Heart Repair

    PubMed Central

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

    2015-01-01

    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. PMID:26370927

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

    PubMed

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and proteolytic processing by a disintegrin and metalloproteinases (ADAM): a regulator of several pathways.

    PubMed

    Taylor, S R; Markesbery, M G; Harding, P A

    2014-04-01

    HB-EGF is a member of the EGF family of ligands that is initially synthesized as a membrane-bound growth factor termed, proHB-EGF. The membrane bound proHB-EGF undergoes extensive proteolytic processing by several metalloproteinases capable of stimulating cellular proliferation. Soluble, mature HB-EGF binds to and activates EGF receptors. HB-EGF is a critical molecular component to a number of normal physiological processes including but not limited to tissue injury and wound healing, reproduction, angiogenesis and recently, adipogenesis. Misexpression of HB-EGF is linked to tumor formation and cancer including hepatocellular, pancreatic, gastric, breast, colon and melanoma, gliomas and glioblastomas. HB-EGF is a likely tool for therapeutic approaches to enhance treatment of injuries as well as a target for prevention of several cancers and obesity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. M2 macrophages induce ovarian cancer cell proliferation via a heparin binding epidermal growth factor/matrix metalloproteinase 9 intercellular feedback loop

    PubMed Central

    Carroll, Molly J.; Kapur, Arvinder; Felder, Mildred; Patankar, Manish S.; Kreeger, Pamela K.

    2016-01-01

    In ovarian cancer, a high ratio of anti-inflammatory M2 to pro-inflammatory M1 macrophages correlates with poor patient prognosis. The mechanisms driving poor tumor outcome as a result of the presence of M2 macrophages in the tumor microenvironment remain unclear and are challenging to study with current techniques. Therefore, in this study we utilized a micro-culture device previously developed by our lab to model concentrated paracrine signaling in order to address our hypothesis that interactions between M2 macrophages and ovarian cancer cells induce tumor cell proliferation. Using the micro-culture device, we determined that co-culture with M2-differentiated primary macrophages or THP-1 increased OVCA433 proliferation by 10–12%. This effect was eliminated with epidermal growth factor receptor (EGFR) or heparin-bound epidermal growth factor (HB-EGF) neutralizing antibodies and HBEGF expression in peripheral blood mononuclear cells from ovarian cancer patients was 9-fold higher than healthy individuals, suggesting a role for HB-EGF in tumor progression. However, addition of HB-EGF at levels secreted by macrophages or macrophage-conditioned media did not induce proliferation to the same extent, indicating a role for other factors in this process. Matrix metalloproteinase-9, MMP-9, which cleaves membrane-bound HB-EGF, was elevated in co-culture and its inhibition decreased proliferation. Utilizing inhibitors and siRNA against MMP9 in each population, we determined that macrophage-secreted MMP-9 released HB-EGF from macrophages, which increased MMP9 in OVCA433, resulting in a positive feedback loop to drive HB-EGF release and increase proliferation in co-culture. Identification of multi-cellular interactions such as this may provide insight into how to most effectively control ovarian cancer progression. PMID:27888810

  1. Heparin localization and fine structure regulate Burkitt's lymphoma growth

    SciTech Connect

    Berry, David; Lynn, David M.; Berry, Eric; Sasisekharan, Ram; Langer, Robert . E-mail: rlanger@mit.edu

    2006-09-29

    Burkitt's lymphoma (BL) is a B-cell malignancy associated with the Epstein-Barr virus (EBV). Mounting evidence has implicated heparan sulfate proteoglycans and heparan sulfate-like glycosaminoglycans (HSGAGs) in the initiation, severity, and progression of the malignancy. The importance of HSGAGs in regulating BL cell growth was therefore examined. Extracellular exogenous heparin inhibited cell growth >30%, while heparin internalized with poly({beta}-amino ester)s promoted proliferation up to 58%. The growth-modulating effects of heparin and internalized heparin were dependent on cell surface HSGAGs, PI3K, and Erk/Mek. Treatment of cells with protamine sulfate or with heparinases potently inhibited proliferation, with the greatest effects induced by heparinase I. Cell surface HSGAGs therefore play an important role in regulating BL proliferation and may offer a potential target for therapeutic intervention.

  2. Heparin-binding epidermal growth factor expression in KATO-III cells after Helicobacter pylori stimulation under the influence of strychnos Nux vomica and Calendula officinalis.

    PubMed

    Hofbauer, Roland; Pasching, Eva; Moser, Doris; Frass, Michael

    2010-07-01

    Previous studies have shown the stimulating effect of Helicobacter pylori on the gene expression of heparin-binding epidermal growth factor (HB-EGF) using the gastric epithelial cell line KATO-III. Strychnos Nux vomica (Nux vomica) and Calendula officinalis are used in highly diluted form in homeopathic medicine to treat patients suffering from gastritis and gastric ulcers. To investigate the influence of Nux vomica and Calendula officinalis on HB-EGF-like growth factor gene expression in KATO-III cells under the stimulation of H. pylori strain N6 using real-time PCR with and without addition of Nux vomica and Calendula officinalis as a 10c or 12c potency. Baseline expression and stimulation were similar to previous experiments, addition of Nux vomica 10c and Calendula officinalis 10c in a 43% ethanolic solution led to a significant reduction of H. pylori induced increase in gene expression of HB-EGF (reduced to 53.12+/-0.95% and 75.32+/-1.16% vs. control; p<0.05), respectively. Nux vomica 12c reduced HB-EGF gene expression even in dilutions beyond Avogadro's number (55.77+/-1.09%; p<0.05). Nux vomica 12c in a 21.5% ethanol showed a smaller effect (71.80+/-3.91%, p<0.05). This effect was only be observed when the drugs were primarily prepared in ethanol, not in aqueous solutions. The data suggest that both drugs prepared in ethanolic solution are potent inhibitors of H. pylori induced gene expression. 2010 Elsevier Ltd. All rights reserved.

  3. Sustained dual release of placental growth factor-2 and bone morphogenic protein-2 from heparin-based nanocomplexes for direct osteogenesis

    PubMed Central

    Liu, Yun; Deng, Li-Zhi; Sun, Hai-Peng; Xu, Jia-Yun; Li, Yi-Ming; Xie, Xin; Zhang, Li-Ming; Deng, Fei-Long

    2016-01-01

    Objective To compare the direct osteogenic effect between placental growth factor-2 (PlGF-2) and bone morphogenic protein-2 (BMP-2). Methods Three groups of PlGF-2/BMP-2-loaded heparin–N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) nanocomplexes were prepared: those with 0.5 μg PlGF-2; with 1.0 μg BMP-2; and with 0.5 μg PlGF-2 combined with 1.0 μg BMP-2. The loading efficiencies and release profiles of these growth factors (GFs) in this nanocomplex system were quantified using enzyme-linked immunosorbent assay, their biological activities were evaluated using cell counting kit-8, cell morphology, and cell number counting assays, and their osteogenic activities were quantified using alkaline phosphatase and Alizarin Red S staining assays. Results The loading efficiencies were more than 99% for the nanocomplexes loaded with just PlGF-2 and for those loaded with both PlGF-2 and BMP-2. For the nanocomplex loaded with just BMP-2, the loading efficiency was more than 97%. About 83%–84% of PlGF-2 and 89%–91% of BMP-2 were stably retained on the nanocomplexes for at least 21 days. In in vitro biological assays, PlGF-2 exhibited osteogenic effects comparable to those of BMP-2 despite its dose in the experiments being lower than that of BMP-2. Moreover, the results implied that heparin-based nanocomplexes encapsulating two GFs have enhanced potential in the enhancement of osteoblast function. Conclusion PlGF-2-loaded heparin–HTCC nanocomplexes may constitute a promising system for bone regeneration. Moreover, the dual delivery of PlGF-2 and BMP-2 appears to have greater potential in bone tissue regeneration than the delivery of either GFs alone. PMID:27042064

  4. Periodate-treated, non-anticoagulant heparin-carrying polystyrene (NAC-HCPS) affects angiogenesis and inhibits subcutaneous induced tumour growth and metastasis to the lung

    PubMed Central

    Ono, K; Ishihara, M; Ishikawa, K; Ozeki, Y; Deguchi, H; Sato, M; Hashimoto, H; Saito, Y; Yura, H; Kurita, A; Maehara, T

    2002-01-01

    Periodate-treated, non-anticoagulant heparin-carrying polystyrene consists of about ten periodate-oxidized, alkaline-degraded low molecular weight-heparin chains linked to a polystyrene core and has a markedly lower anti-coagulant activity than heparin. In this study, we evaluated the effect of non-anticoagulant heparin-carrying polystyrene on tumour growth and metastasis. Non-anticoagulant heparin-carrying polystyrene has a higher activity to inhibit vascular endothelial growth factor-165-, fibroblast growth factor-2- or hepatocyte growth factor-induced human microvascular endothelial cell growth than heparin, ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin, which is probably due to the heparin-clustering effect of non-anticoagulant heparin-carrying polystyrene. Non-anticoagulant heparin-carrying polystyrene inhibited human microvascular endothelial cell, B16 melanoma and Lewis lung cancer cell adhesion to Matrigel-coated plates. Non-anticoagulant heparin-carrying polystyrene also showed strong inhibitory activities in the tubular formation of endothelial cells on Matrigel and B16-melanoma and Lewis lung cancer cell invasion in a Matrigel-coated chamber assay. In vivo studies showed that growth of subcutaneous induced tumours and lung metastasis of B16-melanoma and Lewis lung cancer cells were more effectively inhibited by non-anticoagulant heparin-carrying polystyrene than ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin. Furthermore, non-anticoagulant heparin-carrying polystyrene markedly reduced the number of CD34-positive vessels in subcutaneous Lewis lung cancer tumours, indicating a strong inhibition of angiogenesis. These results suggest that non-anticoagulant heparin-carrying polystyrene has an inhibitory activity on angiogenesis and tumour invasion and may be very useful in cancer therapy. British Journal of Cancer (2002) 86, 1803–1812. doi:10.1038/sj.bjc.6600307 www

  5. Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades.

    PubMed Central

    Ellis, P D; Hadfield, K M; Pascall, J C; Brown, K D

    2001-01-01

    Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1 h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury. PMID:11171084

  6. The anti-tumor effect of cross-reacting material 197, an inhibitor of heparin-binding EGF-like growth factor, in human resistant ovarian cancer

    SciTech Connect

    Tang, Xiao-han; Deng, Suo; Li, Meng; Lu, Mei-song

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer HB-EGF over-expression in A2780/Taxol, A2780/CDDP cells and the matched xenografts. Black-Right-Pointing-Pointer CRM197 induces enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. Black-Right-Pointing-Pointer CRM197 arrests A2780/Taxol and A2780/CDDP cells at G0/G1 phase. Black-Right-Pointing-Pointer CRM197 suppressed the A2780/Taxol and A2780/CDDP growth of xenografts. -- Abstract: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to represent possible chemotherapeutic agent for ovarian cancer. However, the effect of CRM197 on the resistant ovarian carcinoma cells has not been sufficiently elucidated. Here, we found that HB-EGF was over-expressed in a paclitaxel-resistant human ovarian carcinoma cell line (A2780/Taxol) and a cisplatin-resistant cell line (A2780/CDDP), as well as the xenograft mouse tissue samples with these cells. To investigate the possible significance of the HB-EGF over-expression in A2780/Taxol and A2780/CDDP cells, we inhibited HB-EGF expression by CRM197 to investigate the effect of CRM197 treatment on these cells. We observed that CRM197 significantly induced anti-proliferative activity in a dose-dependent manner with the cell-cycle arrest at the G0/G1 phase and enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. The sensitive ovarian carcinoma parental cell line (A2780), A2780/Taxol and A2780/CDDP cells formed tumors in nude mice, and enhanced tumorigenicity was observed in drug-resistant tumors. Furthermore, we observed that CRM197 significantly suppressed the growth of drug-resistant ovarian cancer xenografts in vivo (p < 0.001). These results suggest that CRM197 as an HB-EGF-targeted agent has potent anti-tumor activity in paclitaxel- and cisplatin-resistant ovarian cancer which over-express HB-EGF.

  7. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

    SciTech Connect

    Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi; Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro; Imai, Yasuharu; Higashiyama, Shigeki; Iwamoto, Ryo; Mekada, Eisuke; Takehara, Tetsuo

    2013-07-26

    Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

  8. Lung Cancer-derived Galectin-1 Enhances Tumorigenic Potentiation of Tumor-associated Dendritic Cells by Expressing Heparin-binding EGF-like Growth Factor*

    PubMed Central

    Kuo, Po-Lin; Huang, Ming-Shyan; Cheng, Da-En; Hung, Jen-Yu; Yang, Chih-Jen; Chou, Shah-Hwa

    2012-01-01

    The interaction between cancer cells and their microenvironment is a vicious cycle that enhances the survival and progression of cancer, resulting in metastasis. This study is the first to indicate that lung cancer-derived galectin-1 secretion is responsible for stimulating tumor-associated dendritic cells (TADCs) production of mature heparin-binding EGF-like growth factor (HB-EGF), which, in turn, increases cancer progression. Treatment of galectin-1, present in large amounts in lung cancer conditioned medium and lung cancer patient sera, mimicked the inductive effect of lung cancer conditioned medium on the expression and ectodomain shedding of HB-EGF by TNFα-converting enzyme/a disintegrin and metalloproteinase 9 (ADAM9) and ADAM17. Significant up-regulation of HB-EGF has been seen in tumor-infiltrating CD11c+ dendritic cells in human lung cancer samples. Active cleavage of HB-EGF in TADCs by ADAM9 and ADAM17 is associated with increased protein kinase C δ and Lyn signaling. Enhancement of HB-EGF production in TADCs increased the proliferation, migration, and epithelial-to-mesenchymal transition abilities of lung cancer. In contrast, inhibiting HB-EGF by siRNA suppressed TADC-mediated cancer progression. Moreover, mice injected with galectin-1 knockdown Lewis lung carcinoma showed decreased expression and ectodomain shedding of HB-EGF and reduced incidence of cancer development, resulting in increased survival rates. We demonstrate here for the first time that human and mouse DCs are a source of HB-EGF, an EGFR ligand with tumorigenic properties. Antagonists of the effect of lung cancer-derived galectin-1 on DCs and anti-HB-EGF blocking antibodies could, therefore, have therapeutic potential as antitumor agents. PMID:22291012

  9. Heparin-binding EGF-like growth factor (HB-EGF) overexpression in transgenic mice downregulates insulin-like growth factor binding protein (IGFBP)-3 and -4 mRNA.

    PubMed

    Provenzano, Aaron P; Besner, Gail E; James, Paul F; Harding, Paul A

    2005-03-01

    An in vivo approach was taken to assess the biological significance of heparin-binding EGF-like growth factor (HB-EGF) using transgenic mice. Transgenic mice were generated using the pIRES-EGFP vector expressing a bicistronic mRNA containing both human HB-EGF (hHB-EGF) and enhanced green fluorescent protein (EGFP) coding sequences under the regulation of the cytomegalovirus immediate-early (CMV-IE) promoter. As a marker for transgene expression, EGFP fluorescence in 5 microm tissue sections was evaluated. To confirm HB-EGF expression in EGFP-containing tissues, HB-EGF mRNA was analyzed by RT-PCR and Northern blot analysis. Protein levels of HB-EGF and insulin-like growth factor binding protein-3 (IGFBP-3), a molecule that stabilizes IGFs, which in turn helps to promote growth, were analyzed by Western blot. Also, the weights of transgenic mice were compared with the weights of wild type non-transgenic littermates over a 10-week period. EGFP fluorescence, RT-PCR and Northern analysis of a variety of tissues from hHB-EGF transgenic mice indicate recombinant EGFP/hHB-EGF mRNA expression in kidney, liver, lung and stomach. Western blot analysis confirmed that HB-EGF protein levels were greater in these tissues from hHB-EGF transgenic mice compared to wild type non-transgenic littermates. IGFBP-3 protein was absent in serum of transgenic mice prior to the onset of puberty, but indistinguishable from wild type non-transgenic mice after puberty. Furthermore, IGFBP-3 and IGFBP-4 mRNA were downregulated in the kidney, but not liver or lung of the transgenic mice. In accordance with reduced IGFBP-3 and -4 levels, hHB-EGF transgenic mice exhibited a 20% decrease in weight prior to 6 weeks of age compared to wild type non-transgenic littermates. Our laboratory has generated a biologically functional transgenic mouse model exhibiting increased expression of hHB-EGF in kidney, liver, lung and stomach. Overexpression of hHB-EGF affected the growth rate of these transgenic mice

  10. Role of G protein-coupled receptors (GPCR), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), heparin-binding epidermal growth factor-like growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) in trenbolone acetate-stimulated bovine satellite cell proliferation.

    PubMed

    Thornton, K J; Kamange-Sollo, E; White, M E; Dayton, W R

    2015-09-01

    Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment

  11. Role of hypoxia-related proteins in invasion of ameloblastoma cells: crosstalk between NOTCH1, hypoxia-inducible factor 1α, a disintegrin and metalloproteinase 12, and heparin-binding epidermal growth factor.

    PubMed

    da Costa, Natacha Malu Miranda; Fialho, Amanda Dalla Vechia; Proietti, Carolina Carmine; da Silva Kataoka, Maria Sueli; Jaeger, Ruy Gastaldoni; de Alves-Júnior, Sérgio Melo; de Jesus Viana Pinheiro, João

    2016-07-01

    Ameloblastoma AME is a benign tumour characterized by local invasiveness, high recurrence rates, and diverse histological patterns. The oxygen concentration is reduced in specific areas of the tumour microenvironment, which leads to intratumoral hypoxia. Crosstalk between NOTCH1, a disintegrin and metalloproteinase 12 (ADAM-12), hypoxia-inducible factor 1α (HIF-1α) and heparin-binding epidermal growth factor (HB-EGF) under hypoxic conditions has been implicated in invadopodia formation, tumour invasiveness, and metastasis development. The aim of this study was to analyse the expression of these proteins, in order to further elucidate the mechanisms underlying AME invasiveness. Twenty cases of AME, eight calcifying cystic odontogenic tumours CCOTs and 10 samples of dental follicle were used to investigate the expression of these proteins by immunohistochemistry with the primary antibodies anti-NOTCH1, anti-ADAM-12, anti-HIF-1α, and anti-HB-EGF. Immunostaining results were expressed as the percentage of stained area in images acquired in an AxioScope microscope equipped with an AxioCamHRc camera and a × 40 objective. The results showed that immunoexpression of all proteins was higher in the AME samples than in the CCOT and dental follicle samples (P < 0.05). AME showed an increased presence of proteins associated with tumour invasiveness, which indicates a possible role of these proteins in the biological behaviour of this tumour. © 2015 John Wiley & Sons Ltd.

  12. Distinguishing between anti-platelet factor 4/heparin antibodies that can and cannot cause heparin-induced thrombocytopenia.

    PubMed

    Nazi, I; Arnold, D M; Warkentin, T E; Smith, J W; Staibano, P; Kelton, J G

    2015-10-01

    Many patients exposed to heparin develop antibodies against platelet factor 4 (PF4) and heparin, yet only those antibodies that activate platelets cause heparin-induced thrombocytopenia (HIT). Patients who produce anti-PF4/heparin antibodies without developing HIT either have antibodies that do not cause platelet activation or produce pathogenic antibodies at levels that are insufficient to cause HIT. Understanding the differences between anti-PF4/heparin antibodies with and without HIT will improve test methods and reduce overdiagnosis. To investigate the presence of low levels of platelet-activating antibodies in patients investigated for HIT who had anti-PF4/heparin antibodies but failed to cause platelet activation in the (14) C-serotonin release assay (SRA). We developed a platelet activation assay similar to the SRA using exogenous PF4 without added heparin (PF4-SRA). This assay was able to detect low levels of platelet-activating antibodies. We used this PF4-SRA to test for platelet-activating antibodies in patients investigated for HIT. The PF4-SRA detected platelet-activating antibodies in seven (100%) of seven SRA-positive sera even after the samples were diluted until they were no longer positive in the standard SRA. Platelet-activating antibodies were detected in 14 (36%) of 39 patients who had anti-PF4/heparin antibodies but tested negative in the SRA and did not have clinical HIT. The clinical diagnosis of HIT was confirmed by chart review and concordant with the SRA results. A subset of heparin-treated patients produce subthreshold levels of platelet-activating anti-PF4/heparin antibodies that do not cause HIT. An increase in the titer of these pathogenic antibodies, along with permissive clinical conditions, could lead to HIT. © 2015 International Society on Thrombosis and Haemostasis.

  13. Synthesis of the blood circulating C-terminal fragment of insulin-like growth factor (IGF)-binding protein-4 in its native conformation. Crystallization, heparin and IGF binding, and osteogenic activity.

    PubMed

    Fernández-Tornero, Carlos; Lozano, Rosa M; Rivas, Germán; Jiménez, M Angeles; Ständker, Ludger; Díaz-Gonzalez, Diana; Forssmann, Wolf-Georg; Cuevas, Pedro; Romero, Antonio; Giménez-Gallego, Guillermo

    2005-05-13

    Insulin-like growth factor-binding proteins play a critical role in a wide variety of important physiological processes. It has been demonstrated that both an N-terminal and a C-terminal fragment of insulin-like growth factor-binding protein-4 exist and accumulate in the circulatory system, these fragments accounting for virtually the whole amino acid sequence of the protein. The circulating C-terminal fragment establishes three disulfide bridges, and the binding pattern of these has recently been defined. Here we show that the monodimensional 1H NMR spectrum of the C-terminal fragment is typical of a protein with a relatively close packed tertiary structure. This fragment can be produced in its native conformation in Escherichia coli, without the requirement of further refolding procedures, when synthesis is coupled to its secretion from the cell. The recombinant protein crystallizes with the unit cell parameters of a hexagonal system. Furthermore, it binds strongly to heparin, acquiring a well defined oligomeric structure that interacts with insulin-like growth factors, and promotes bone formation in cultures of murine calvariae.

  14. TGFβ functionalized starPEG-heparin hydrogels modulate human dermal fibroblast growth and differentiation.

    PubMed

    Watarai, Akira; Schirmer, Lucas; Thönes, Stephan; Freudenberg, Uwe; Werner, Carsten; Simon, Jan C; Anderegg, Ulf

    2015-10-01

    Hydrogels are promising biomaterials that can adapt easily to complex tissue entities. Furthermore, chemical modifications enable these hydrogels to become an instructive biomaterial to a variety of cell types. Human dermal fibroblasts play a pivotal role during wound healing, especially for the synthesis of novel dermal tissue replacing the primary fibrin clot. Thus, the control of growth and differentiation of dermal fibroblasts is important to modulate wound healing. In here, we utilized a versatile starPEG-heparin hydrogel platform that can be independently adjusted with respect to mechanical and biochemical properties for cultivating human dermal fibroblasts. Cell-based remodeling of the artificial matrix was ensured by using matrix metalloprotease (MMP) cleavable crosslinker peptides. Attachment and proliferation of fibroblasts on starPEG-heparin hydrogels of differing stiffness, density of pro-adhesive RGD peptides and MMP cleavable peptide linkers were tested. Binding and release of human TGFβ1 as well as biological effect of the pre-adsorbed growth factor on fibroblast gene expression and myofibroblast differentiation were investigated. Hydrogels containing RGD peptides supported fibroblast attachment, spreading, proliferation matrix deposition and remodeling compared to hydrogels without any modifications. Reversibly conjugated TGFβ1 was demonstrated to be constantly released from starPEG-heparin hydrogels for several days and capable of inducing myofibroblast differentiation of fibroblasts as determined by induction of collagen type I, ED-A-Fibronectin expression and incorporation of alpha smooth muscle actin and palladin into F-actin stress fibers. Taken together, customized starPEG-heparin hydrogels could be of value to promote dermal wound healing by stimulating growth and differentiation of human dermal fibroblasts. The increasing number of people of advanced age within the population results in an increasing demand for the treatment of non

  15. Switching of cell growth/detachment on heparin-functionalized thermoresponsive surface for rapid cell sheet fabrication and manipulation.

    PubMed

    Arisaka, Yoshinori; Kobayashi, Jun; Yamato, Masayuki; Akiyama, Yoshikatsu; Okano, Teruo

    2013-06-01

    Heparin-functionalized poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [P(IPAAm-co-CIPAAm)] grafted surface was designed for the switching of cell growth/detachment, achieved by the regulation of affinity binding between basic fibroblast growth factor (bFGF) and immobilized heparin through the temperature-dependent conformational change of grafted P(IPAAm-co-CIPAAm) chains. At 37 °C, bFGF-bound heparin-thermoresponsive surfaces were able to hold the two- to three-fold number of mouse fibroblast (NIH/3T3) cells than both bFGF-physisorbed surface and PIPAAm surface with soluble bFGF after a 3-day cultivation. Bound bFGF via heparin on shrunken grafted P(IPAAm-co-CIPAAm) chains at 37 °C was able to reinforce the formation and stabilization of bFGF-FGF receptor complex, although the activity of physisorbed bFGF on PIPAAm-grafted surfaces was decreased by non-specific and randomly oriented adsorption. At 20 °C, the cultured NIH/3T3 cell sheet with bFGF detached from heparin-functionalized thermoresponsive surface. The release of bFGF from the surfaces was induced by reducing the affinity binding between bFGF and immobilized-heparin due to increasing the mobility of the swollen grafted P(IPAAm-co-CIPAAm) chains. Therefore, heparin-functionalized thermoresponsive surface was able to enhance cell proliferation, and confluent cells detached themselves as a contiguous cell sheet due to switching cell growth by changing temperature. A cell culture system using this surface is useful for rapid cell sheet fabrication and manipulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. [Levels of antiplatelet factor 4-heparin antibodies and 4T score for heparin induced thrombocytopenia].

    PubMed

    Martinuzzo, Marta E; Cerrato, Graciela S; Iglesias Varela, Maria L; Adamczuk, Yolanda P; Pombo, Gonzalo; Forastiero, Ricardo R

    2012-01-01

    Heparin induced thrombocytopenia (HIT) is an immune-mediated disorder due to antibodies anti platelet factor 4-heparin (HPIA). Thrombocytopenia is often moderate but certain patients can develop morbid thrombotic complications. HPIA detection by ELISA has high sensitivity but low specificity, and low titers (without clinical significance) are frequent. A pretest clinical score (4T's) was developed in order to recognize patients that are at high risk of HIT. The aim of this study was to correlate HPIA levels and the 4T's score of consecutive patients derived to our center. We evaluated 84 patients (35 of them developed thrombosis); the clinical questionnaire was sent along with the sample and was analyzed by an investigator who did not know the patients' characteristics, and 4T's scores were calculated before performing the laboratory tests. HPIA were measured by ELISA (Asserachrom HPIA) that detects IgG, IgM and IgA isotypes, (the only reagent available in our country). 4T's score correlated with HPIA levels (rho spearman 0.472, p < 0.001). Patients with 4T's = 6 had higher absorbance percentages than those with = 5 (67 vs. 39%, p < 0.001), and patients with thrombosis also presented higher titers (59 vs. 39%, p = 0.017) than those who did not develop this complication. In conclusion, high titers of HPIA measured by EIA which detects the 3 isotypes, clearly correlate with 4T's score = 6 and are more frequent in patients who develop thrombosis, just as reported when an IgG specific ELISA is used.

  17. Complexes of heparin and platelet factor 4 specifically stimulate T cells from patients with heparin-induced thrombocytopenia/thrombosis.

    PubMed

    Bacsi, S; De Palma, R; Visentin, G P; Gorski, J; Aster, R H

    1999-07-01

    Heparin-induced thrombocytopenia with thrombosis (HITT) is associated with antibodies specific for complexes consisting of heparin and platelet factor 4 (PF4). Studies in individual patients with HITT have demonstrated immunoglobulin (Ig) class switching from IgM to the IgG or IgA isotypes. This transition is thought to require helper T cells, but no studies of the cellular or molecular basis of this process have yet been reported. To characterize T-cell involvement in HITT, peripheral blood mononuclear cells (PBMC) from two patients with classical HITT obtained shortly after the acute episode were restimulated with heparin:PF4 complexes, PF4 alone, heparin alone, and medium alone in the presence of autologous antigen-presenting cells (APC). Responding T cells were then examined using the technique of "spectratyping," in which sequences encoding CDR3 domains of individual V beta (BV) families are amplified and separated by gel electrophoresis. After 14 days in culture with antigen (heparin:PF4 complexes), but not after culture with PF4, heparin, or medium alone, patient cells, but not cells from normal subjects, preferentially expressed T-cell receptor (TCR)-containing beta chains of the BV 5.1 family. Nucleotide sequencing of BV 5.1 TCR CDR3 showed that each patient had a personal repertoire, but also shared a tetrapeptide motif (PGTG). These findings provide evidence that the humoral immune response associated with HITT is driven by helper T cells that presumably recognize peptides derived from PF4. Identification of a common beta-chain CDR3 motif in responding T cells from each of two patients suggests that a limited number of helper TCRs may be used to mount an antibody response to heparin:PF4 complexes. TCR spectratyping appears to offer a new way to examine the molecular basis of pathologic immune responses and may be useful in further studies of HITT and other immune-mediated hematologic disorders.

  18. Preclinical Pharmacokinetics Evaluation of Anti-heparin-binding EGF-like Growth Factor (HB-EGF) Monoclonal Antibody Using Cynomolgus Monkeys via (89)Zr-immuno-PET Study and the Determination of Drug Concentrations in Serum and Cerebrospinal Fluid.

    PubMed

    Kasai, Noriyuki; Adachi, Maiko; Yamano, Kazuya

    2016-02-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KHK2866 is a humanized anti-HB-EGF monoclonal antibody IgG that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. The phase I study of KHK2866 was discontinued because of neuropsychiatric toxicity. In this study, the pharmacokinetics of KHK2866 was evaluated by (89)Zr-immuno-PET study and the determination of drug concentrations in serum and cerebrospinal fluid using cynomolgus monkeys was performed in order to predict neurotoxicity in a reverse-translational manner. KHK2866 was radiolabeled with (89)Zr for preclinical evaluations in normal cynomolgus monkeys and its distribution was analyzed. Furthermore, as a separate study, KHK2866 concentrations in serum and cerebrospinal fluid were determined after administration of a single dose. PET studies with monkeys revealed (89)Zr-KHK2866 accumulation in the liver, spleen and joints of multiple parts, but not in brain. In addition, the pharmacokinetic analyses in serum and CSF demonstrated a low penetration of KHK2866 into the brain. These studies indicate the difficulty of prediction for neuropsychiatric toxicity of monoclonal antibodies in human by means of pharmacokinetic evaluations using cynomolgus monkeys.

  19. Interactions between nattokinase and heparin/GAGs.

    PubMed

    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.

  20. Incorporation of heparin into biomaterials.

    PubMed

    Sakiyama-Elbert, Shelly E

    2014-04-01

    This review provides an overview of the incorporation of heparin into biomaterials with a focus on drug delivery and the use of heparin-based biomaterials for self-assembly of polymer networks. Heparin conjugation to biomaterials was originally explored to reduce the thrombogenicity of materials in contact with blood. Many of the conjugation strategies that were developed for these applications are still popular today for other applications. More recently heparin has been conjugated to biomaterials for drug delivery applications. Many of the delivery approaches have taken advantage of the ability of heparin to bind to a wide variety of growth factors, protecting them from degradation and potentiating interactions with cell surface receptors. More recently, the use of heparin as a base polymer for scaffold fabrication has also been explored, often utilizing non-covalent binding of heparin with peptides or proteins to promote self-assembly of hydrogel networks. This review will highlight recent advances in each of these areas.

  1. K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes

    SciTech Connect

    Hattori, Yutaka; Odagiri, Hiroki; Nakatani, Hiroshi; Miyagawa, Kiyoshi; Naito, Kenichiro; Sakamoto, Hiromi; Katoh, Osamu; Yoshida, Teruhiko; Sugimura, Takashi; Terada, Masaaki )

    1990-08-01

    DNA fragments amplified in a stomach cancer-derived cell line, KATO-III, were previously identified by the in-gel DNA renaturation method, and a 0.2-kilobase-pair fragment of the amplified sequence was subsequently cloned. By genomic walking, a portion of the exon of the gene flanking this 0.2-kilobase-pair fragment was cloned, and the gene was designated as K-sam ({und K}ATO-III cell-derived {und s}tomach cancer {und am}plified gene). The K-sam cDNAs, corresponding to the 3.5-kilobase K-sam mRNA, were cloned from the KATO-III cells. Sequence analysis revealed that this gene coded for 682 amino acid residues that satisfied the characteristics of the receptor tyrosine kinase. The K-sam gene had significant homologies with bek, FLG, and chicken basic fibroblast growth factor receptor gene. The K-sam gene was amplified in KATO-III cells with the major transcript of 3.5-kilobases in size. This gene was also expressed in some other stomach cancer cells, a small cell lung cancer, and germ cell tumors.

  2. K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes.

    PubMed Central

    Hattori, Y; Odagiri, H; Nakatani, H; Miyagawa, K; Naito, K; Sakamoto, H; Katoh, O; Yoshida, T; Sugimura, T; Terada, M

    1990-01-01

    DNA fragments amplified in a stomach cancer-derived cell line, KATO-III, were previously identified by the in-gel DNA renaturation method, and a 0.2-kilobase-pair fragment of the amplified sequence was subsequently cloned. By genomic walking, a portion of the exon of the gene flanking this 0.2-kilobase-pair fragment was cloned, and the gene was designated as K-sam (KATO-III cell-derived stomach cancer amplified gene). The K-sam cDNAs, corresponding to the 3.5-kilobase K-sam mRNA, were cloned from the KATO-III cells. Sequence analysis revealed that this gene coded for 682 amino acid residues that satisfied the characteristics of the receptor tyrosine kinase. The K-sam gene had significant homologies with bek, FLG, and chicken basic fibroblast growth factor receptor gene. The K-sam gene was amplified in KATO-III cells with the major transcript of 3.5-kilobases in size. This gene was also expressed in some other stomach cancer cells, a small cell lung cancer, and germ cell tumors. Images PMID:2377625

  3. Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

    PubMed

    Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

    2007-12-01

    Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

  4. Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

    PubMed Central

    Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

    2007-01-01

    Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

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

    NASA Astrophysics Data System (ADS)

    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) and three binding pathways in the PF4/long 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

  6. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  7. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  8. Identification of heparin-binding EGF-like growth factor (HB-EGF) as a biomarker for lysophosphatidic acid receptor type 1 (LPA1) activation in human breast and prostate cancers.

    PubMed

    David, Marion; Sahay, Debashish; Mege, Florence; Descotes, Françoise; Leblanc, Raphaël; Ribeiro, Johnny; Clézardin, Philippe; Peyruchaud, Olivier

    2014-01-01

    Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA₁₋₆). LPA receptor type 1 (LPA₁) signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA₁ is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA₁ is known to induce IL-6 and IL-8 secretion, as also do LPA₂ and LPA₃. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA₁,₂,₆; MDA-MB-231: LPA1,2; MCF-7: LPA₂,₆). Among the set of genes upregulated by LPA only in LPA₁-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF) was inhibited by LPA₁-₃ antagonists (Ki16425, Debio0719). Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA₁ (MDA-B02/LPA₁) and downregulated for LPA₁ (MDA-B02/shLPA1), respectively. At a clinical level, we quantified the expression of LPA₁ and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA₁. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in quantifying LPA

  9. Interactions between nattokinase and heparin/GAGs

    PubMed Central

    Zhang, Fuming

    2015-01-01

    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 a 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. PMID:26412225

  10. Efficacy of unfractionated heparin, low molecular weight heparin and both combined for releasing total and free tissue factor pathway inhibitor.

    PubMed

    Altman, R; Scazziota, A; Rouvier, J

    1998-01-01

    Unfractionated heparin (UFH) exerts its anticoagulant properties by increasing the inactivation of thrombin and activated factor X by antithrombin III. Apart from this main action release of tissue factor pathway inhibitor (TFPI) from endothelial cells could also be important for the antithrombotic activity of heparins. Four different heparin preparations were injected subcutaneously into 5 healthy volunteers 1 week apart: (1) UFH 2,500 IU fix dose (FixUFH), (2) 1 mg/kg body weight of low molecular weight heparin (LMWH), (3) the combined LMWH-adjusted dose plus UFH 2,500 IU fix dose (ComHep) and (4) UFH 2,500 IU/10 kg body weight (UFHvar). Plasma samples were drawn before and 1, 2, 4, 6, 12 and 24 h afterwards. FixUFH did not affect the concentration of total and free TFPI. Total TFPI increased in the 1st hour after LMWH injection from 74 to 124 ng/ml (p < 0.01), after ComHep from 82 to 144 ng/ml (p < 0.01), and after UFHvar from 91 to 113 ng/ml (p < 0.05). All observed elevations were significant at the peak value (+/- 2 h, p < 0.01 compared with baselines). The increase of free TFPI produced by UFHvar (74.5 and 70.5 ng/ml) was significantly higher than with LMWH (42.8 and 38.0 ng/ml) at 2 and 4 h (p < 0.001 and p < 0.01, respectively). UFHvar and ComHep but not LMWH produced a statistically significant increase of free TFPI compared with FixUFH at 2, 4 and 6 h (p < 0. 01). We concluded that at comparable therapeutic doses, subcutaneous UFHvar released more free TFPI than LMWH and ComHep. A synergism between LMWH and low dose of UFH was found in 4-, 6- and 12-hour blood samples.

  11. Heparin chain-length dependence of factor Xa inhibition by antithrombin in plasma.

    PubMed

    Rezaie, Alireza R

    2007-01-01

    Heparin anticoagulants function by enhancing the inhibition of coagulation proteases by the serpin antithrombin (AT). A direct evaluation of the specific anti-factor Xa (fXa) activity of therapeutic heparins in the physiologically relevant plasma-based clotting assays has not been feasible since thrombin, the final protease of the cascade, is the primary target for inhibition by AT in the presence of heparin. To circumvent this problem, we developed an assay in which the native AT in plasma was replaced with an AT mutant which exhibits identical affinity for heparin and near normal reactivity for fXa, but does not react with thrombin and other coagulation proteases in either the absence or presence of heparin. This assay was used to distinguish the anti-fXa activity of different molecular weight heparins from their anti-thrombin activity in clotting assays which were initiated by the triggers of either the extrinsic or intrinsic coagulation pathway. The results suggest that the acceleration of fXa inhibition by AT exhibits a marked heparin chain-length dependence, with fondaparinux (a pentasaccharide) having the lowest and unfractionated heparin having the highest effect. Interestingly, comparative studies revealed that the fondaparinux-catalyzed acceleration of thrombin inhibition by AT also contributes to the prolongation of the clotting time, possibly suggesting that the anticoagulant function of the therapeutic pentasaccharide is mediated though the inhibition of both fXa and thrombin.

  12. Chemical Conjugate of Low Molecular Weight Heparin and Suramin Fragment Inhibits Tumor Growth Possibly by Blocking VEGF165.

    PubMed

    Park, Jooho; Kim, Ji-young; Hwang, Seung Rim; Mahmud, Foyez; Byun, Youngro

    2015-11-02

    Low molecular weight heparin (LMWH) and its derivatives have been reported to possess antiangiogenic effect via electrostatic interaction with various angiogenic growth factors such as VEGF165. However, clinical applications of LMWH for anticancer therapy have been restricted due to its anticoagulant effect and insufficient therapeutic efficacy. To overcome these limitations and enhance the antiangiogenic efficacy, LMWH was conjugated with suramin fragments that have a binding affinity to the heparin-binding domain (HBD) of proteins. The conjugation of suramin fragments to LMWH enhanced the antiangiogenic effect of LMWH by increasing the binding affinity to VEGF165, while decreasing its anticoagulant activity. The chemical conjugate of LMWH and suramin fragments (LHsura) showed a substantial inhibitory effect on VEGF165-mediated cell proliferation, migration, and tube formation of HUVECs without significant cytotoxicity in vitro. Finally, we confirmed the anticancer effect of LHsura (61.4% vs control) in a SCC7-bearing mouse model.

  13. [Fibroblast growth factor-2].

    PubMed

    Faitová, J

    2004-01-01

    Fibroblast growth factor-2 is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 occurs in several isoforms resulting from alternative initiations of traslation: an 18 kDa cytoplasmic isoform and four larger molecular weight nuclear isoforms (22, 22.5, 24 and 34 kDa). It acts mainly through a paracrine/autocrine mechanism involving high affinity transmembrane receptors and heparan sulfate proteoglycan low affinity receptors. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and plays an important role in mesoderm induction, stimulates the growth and development of the new blood vessels (angiogenesis), normal wound healing and tissue development. FGF-2 positively regulates hematopoiesis by acting on various cellular targets: stromal cells, early and committed hematopoietic progenitors and possibly some mature blood cells. FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.

  14. Anti-platelet factor 4/heparin antibodies from patients with heparin-induced thrombocytopenia provoke direct activation of microvascular endothelial cells.

    PubMed

    Blank, Miri; Shoenfeld, Yehuda; Tavor, Sigal; Praprotnik, Sonja; Boffa, Marie Claire; Weksler, Babette; Walenga, M Jeanine; Amiral, Jean; Eldor, Amiram

    2002-02-01

    Heparin-induced thrombocytopenia (HIT) is a serious complication that occurs in approximately 1-5% of patients treated with heparin and may be associated with severe thrombotic events. HIT is mediated by antibodies directed mostly to epitope(s) formed by complexes between heparin or other anionic mucopolysaccharides and platelet factor 4 (PF4). Anti-PF4/heparin IgG antibodies from six patients with HIT were affinity purified and assessed for interaction with human microvascular and macrovascular endothelial cells (EC). The antibodies directly activated primary cultures of human bone marrow microvascular EC (HBMEC) and SV40 immortalized HBMEC (TrHBMEC) only in the presence of PF4, but did not activate macrovascular human umbilical vein EC (HUVEC) under the same conditions. These antibodies were found to bind to TrHBMEC through the F(ab)(2) portion of the anti-PF4/heparin IgG. TrHBMEC activation was characterized by an augmented release of IL-6, von Willebrand factor, soluble thrombomodulin, and by an elevated expression of the adhesion molecules P-selectin, E-selectin and vascular cellular endothelial molecule-I to different degrees. Enhanced monocyte adhesion to PF4/heparin antibody-treated TrHBMEC (33-72% adhesion) was also observed. None of these effects occurred with unstimulated HUVEC. However, pre-treatment of HUVEC with tumor necrosis factor-alpha resulted in the same changes observed with microvascular EC exposed to the HIT antibodies. Our findings indicate that anti-PF4/heparin antibodies directly activate microvascular EC while interaction with macrovascular EC requires pre-activation. These results may explain some of the specific clinical manifestations in HIT.

  15. The relative molecular mass dependence of the anti-factor Xa properties of heparin.

    PubMed Central

    Ellis, V; Scully, M F; Kakkar, V V

    1986-01-01

    The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 X 10(7) M-1 X min-1, compared with 6.3 X 10(4) M-1 X min-1 in the absence of heparin, and this rose progressively to 4.2 X 10(8) M-1 X min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III. PMID:3800942

  16. Structural and functional characterization of full-length heparin-binding growth associated molecule.

    PubMed Central

    Hampton, B S; Marshak, D R; Burgess, W H

    1992-01-01

    Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor. Images PMID:1550956

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

    SciTech Connect

    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.

  18. Modulation of factors involved in placental haemostasis and angiogenesis by low-molecular-weight-heparins.

    PubMed

    Grandone, Elvira; Chinni, Elena; Villani, Michela; Sciannamè, Natale; Tiscia, Giovanni L; Favuzzi, Giovanni; Cappucci, Filomena; Petruzzelli, Francesco; Margaglione, Maurizio

    2016-11-01

    In placentae from uneventful pregnancies a direct relationship between expression of tissue factor (TF) and tissue-factor pathway inhibitor type 2 (TFPI2) was found, as well as between TF and vascular endothelial growth factor (VEGF). Furthermore, placentae from gestational vascular complications (GVCs) lack these correlations. Aims of the present study are (1) to evaluate a possible role of low-molecular-weight-heparins (LMWHs) in the modulation of the expression of TF, TFPI, TFPI2 and VEGF in placentae from thrombophilic women and (2) to study the possible role of endothelium in the placental expression of markers involved in haemostasis and angiogenesis. Fourteen pregnancies in thrombophilic women and 11 uneventful pregnancies in non-thrombophilic women were studied and placentae collected. From each placenta total RNA was obtained. Expression of TF, TFPI, TFPI2 and VEGF was evaluated. Human Vein Endothelial Cells were incubated with increasing doses of LMWH and expression of TF, TFPI and VEGF was measured. Expression of all the markers analyzed in placentae from treated pregnancies was similar to that observed in placentae from uneventful ones. A significant direct relationship between TF and TFPI2, as well as TF and VEGF, was observed in cases treated with LMWHs and controls. Furthermore, the expression of TF and its inhibitors and VEGF in endothelial cells was modulated by LMWH. Present data suggest that LMWH during pregnancy in thrombophilic women restores the relationship between markers of haemostasis and angiogenesis. Furthermore, the endothelium is likely to play an important role in this phenomenon.

  19. Contact factor deficiencies and cardiopulmonary bypass surgery: detection of the defect and monitoring of heparin.

    PubMed

    van Veen, Joost Jair; Laidlaw, Stuart; Swanevelder, Justin; Harvey, Nicholas; Watson, Chris; Kitchen, Steve; Makris, Mike

    2009-03-01

    Contact factor pathway deficiencies do not cause surgical bleeding but make heparin monitoring by the activated partial thromboplastin time (APTT) and activated clotting time (ACT) unreliable. Heparin monitoring during cardiopulmonary bypass (CPB) surgery in these patients is particularly challenging. Here we describe heparin monitoring during CPB using the chromogenic anti Xa assay in two patients with severe factor XII deficiency (FXII < 0.01 U/mL) and one patient with severe prekallikrein (PK) deficiency (PK < 0.01 U/mL). Anti Xa levels of the three patients during CPB varied between 3.8 and 4.8 U/mL in keeping with a control group (mean anti Xa 4.5 U/mL and ACT > 480 s). There were no bleeding or thrombotic complications. We also found that detection of severe PK deficiency by the APTT in the PK deficient patient was dependent on the reagent used and discuss the sensitivity of different APTT reagents for contact factor deficiencies. We conclude that the sensitivity of APTT methods for contact pathway deficiencies is highly variable and although insensitivity is not a clinical problem in terms of bleeding, it can be a cause of discrepancy between different APTT reagents and the ACT. This can lead to confusion about a possible haemorrhagic tendency and delays in surgery. If these patients need to undergo cardiac surgery requiring high dose heparin treatment, monitoring by chromogenic anti Xa assay is a good alternative.

  20. Sustaining neovascularization of a scaffold through staged release of vascular endothelial growth factor-A and platelet-derived growth factor-BB.

    PubMed

    Davies, Neil H; Schmidt, Christian; Bezuidenhout, Deon; Zilla, Peter

    2012-01-01

    Tissue regeneration into a three-dimensional scaffold requires the stimulation of blood vessel ingrowth. We have employed a freely interconnecting porous scaffold developed by us to determine the utility of a covalently bound heparin surface coating for the delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) in vivo. The heparin surface was shown to release VEGF far more rapidly than PDGF-BB in vitro (VEGF: 75 ng/h for 24 h; PDGF-BB: 86 pg/h for >7 days). In rat subcutaneous implants, at 10 days the heparin surface alone increased vessel ingrowth substantially (p<0.05 vs. unmodified scaffold), release of VEGF resulted in a further increase (p<0.05 vs. heparinized scaffold), whereas PDGF-BB had no additional effect. The increase induced by the combination of growth factors was similar to VEGF alone. After 2 months, PDGF-BB, but not VEGF delivery, resulted in a substantial increase in vascularization above that induced by heparin (p<0.05). At the longer time point the combination of growth factors was similar to PDGF-BB. However, only the combination of growth factors significantly elevated the number of ingrowing arterioles (p<0.05 vs. heparinized scaffold). Thus, the covalent modification of a porous scaffold with heparin allows for the differential release of VEGF and PDGF-BB that results in both a rapid and sustained increase in scaffold vascularization.

  1. Structural mechanism for heparin-binding of the third Kunitz domain of human tissue factor pathway inhibitor.

    PubMed

    Mine, Shouhei; Yamazaki, Toshio; Miyata, Toshiyuki; Hara, Saburo; Kato, Hisao

    2002-01-08

    Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factor VIIa and Xa through its K1 and K2 domain, respectively, and the inhibitory activity is enhanced by heparin. The function of the K3 domain of TFPI has not been established, but the domain probably harbors a heparin binding site (HBS-2). We determined the three-dimensional solution structure of the TFPI K3 domain (Glu182-Gly242) by heteronuclear multidimensional NMR. The results showed that the molecule is composed of one antiparallel beta-sheet and one alpha-helix, and in overall structure is very similar to the K2 domain, with the rms deviation of 1.55 A for the 58 defined C(alpha) positions. However, the surface electrostatic properties of both domains are different each other. The lack of inhibitory activity of the K3 domain is explained by the absence of electrostatic interaction with factor Xa over a large surface area. A titration experiment with size-fractionated heparin showed that a heparin binding site was located in the vicinity of the alpha-helix. In this region, a positively charged cluster is formed by Lys213, Lys232, and Lys240, and the negatively charged sulfate groups of heparin bind there. The enhancement of inhibitory activity by heparin probably was not due to a conformational change to TFPI itself. It is likely that heparin simply increases the local concentration of TFPI on the cell surface and stabilizes the initial complex that forms.

  2. Pharmacokinetic and pharmacodynamic interactions between palifermin and heparin.

    PubMed

    Yang, Bing-Bing; Gillespie, Brad; Smith, Brian; Smith, William; Lissmats, Agneta; Rudebeck, Mattias; Kullenberg, Torbjörn; Olsson, Birgitta

    2015-10-01

    Oral mucositis, a severe complication during chemo- and/or radiotherapy, is prevented with palifermin treatment, a recombinant human keratinocyte growth factor (KGF/FGF-7). The FGF family belongs to the larger family of heparin-binding growth factors. Because it has been shown that heparin modulates binding of KGF to the KGF receptor and subsequently affects cellular proliferation induced by the KGF mitogenic signal, it is critical to understand the drug-drug interactions between palifermin and heparin, particularly because of heparin's narrow therapeutic margin. Two studies were performed in healthy subjects to characterize the effect of palifermin on the pharmacodynamics of heparin (activated partial thromboplastin time) and evaluate the impact of heparin on the pharmacokinetics and pharmacodynamics (Ki67 staining of buccal mucosal tissue) of palifermin. Results demonstrated a pronounced pharmacokinetic interaction; heparin coadministration increased the palifermin AUC 4- to 5-fold and decreased its half-life by 40%-45%, suggesting an approximate 70%-80% decrease in palifermin clearance and volume of distribution. These changes in the pharmacokinetics of palifermin during coadministration of heparin, however, did not affect the pharmacodynamic effect of palifermin, or the anticoagulant activity of heparin, and did not lead to increased safety findings. Therefore, these results suggest that dose adjustments for heparin and palifermin are not warranted when administered concurrently.

  3. Heparin-chitosan nanoparticle functionalization of porous poly(ethylene glycol) hydrogels for localized lentivirus delivery of angiogenic factors

    PubMed Central

    Thomas, Aline M.; Gomez, Andrew J.; Palma, Jaime L.; Yap, Woon Teck

    2014-01-01

    Hydrogels have been extensively used for regenerative medicine strategies given their tailorable mechanical and chemical properties. Gene delivery represents a promising strategy by which to enhance the bioactivity of the hydrogels, though the efficiency and localization of gene transfer have been challenging. Here, we functionalized porous poly(ethylene glycol) hydrogels with heparin-chitosan nanoparticles to retain the vectors locally and enhance lentivirus delivery while minimizing changes to hydrogel architecture and mechanical properties. The immobilization of nanoparticles, as compared to homogeneous heparin and/or chitosan, is essential to lentivirus immobilization and retention of activity. Using this gene-delivering platform, we over-expressed the angiogenic factors sonic hedgehog (Shh) and vascular endothelial growth factor (Vegf) to promote blood vessel recruitment to the implant site. Shh enhanced endothelial recruitment and blood vessel formation around the hydrogel compared to both Vegf-delivering and control hydrogels. The nanoparticle-modified porous hydrogels for delivering gene therapy vectors can provide a platform for numerous regenerative medicine applications. PMID:25023395

  4. The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides.

    PubMed Central

    Barrowcliffe, T W; Havercroft, S J; Kemball-Cook, G; Lindahl, U

    1987-01-01

    The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results. PMID:3606581

  5. Heparin interacts with elongation factor 1α of Cryptosporidium parvum and inhibits invasion

    PubMed Central

    Inomata, Atsuko; Murakoshi, Fumi; Ishiwa, Akiko; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Cagayat Recuenco, Frances; Horimoto, Taisuke; Kato, Kentaro

    2015-01-01

    Cryptosporidium parvum is an apicomplexan parasite that can cause serious watery diarrhea, cryptosporidiosis, in human and other mammals. C. parvum invades gastrointestinal epithelial cells, which have abundant glycosaminoglycans on their cell surface. However, little is known about the interaction between C. parvum and glycosaminoglycans. In this study, we assessed the inhibitory effect of sulfated polysaccharides on C. parvum invasion of host cells and identified the parasite ligands that interact with sulfated polysaccharides. Among five sulfated polysaccharides tested, heparin had the highest, dose-dependent inhibitory effect on parasite invasion. Heparan sulfate-deficient cells were less susceptible to C. parvum infection. We further identified 31 parasite proteins that potentially interact with heparin. Of these, we confirmed that C. parvum elongation factor 1α (CpEF1α), which plays a role in C. parvum invasion, binds to heparin and to the surface of HCT-8 cells. Our results further our understanding of the molecular basis of C. parvum infection and will facilitate the development of anti-cryptosporidial agents. PMID:26129968

  6. Heparin-induced thrombocytopenia.

    PubMed

    Shaikh, Nissar

    2011-01-01

    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.

  7. Collection of neural inducing factors from PA6 cells using heparin solution and their immobilization on plastic culture dishes for the induction of neurons from embryonic stem cells.

    PubMed

    Yamazoe, Hironori; Murakami, Yoshinobu; Mizuseki, Kenji; Sasai, Yoshiki; Iwata, Hiroo

    2005-10-01

    Embryonic stem (ES) cells have the ability to replicate themselves and differentiate into various mature cells. Recently, dopaminergic neurons were efficiently induced from ES cells using mouse stromal cells (PA6 cells) as a feeder cell layer. This simple procedure seems to be very efficient to obtain dopamine-releasing cells for future clinical cell transplantation treatment of Parkinson's disease. In this study, we prepared stock solutions containing neural inducing factors (NIFs) by washing PA6 cells with phosphate-buffered saline containing heparin. ES cells grew successfully in culture media supplemented with 33 v/v% NIFs stock solution, and the rate of neural differentiation of ES cell progeny increased with increasing heparin concentration in the culture media. In addition, NIFs-immobilized surfaces were prepared by exposing polyethyleneimine-modified surfaces to NIFs stock solutions. The NIFs-immobilized culture dish effectively supported cell growth as the culture medium supplemented with NIFs stock did, but its induction effect to dopaminergic neurons from ES cells was much smaller than free NIFs. NIFs stock solutions have two different activities. One can stimulate cell growth and the other induces differentiation of ES cells to the neural fate when heparin existed. The former factors were effectively immobilized on the culture dish, but those that induce differentiation may not be. Further optimization is required.

  8. The clinical significance and risk factors of anti-platelet factor 4/heparin antibody on maintenance hemodialysis patients: a two-year prospective follow-up.

    PubMed

    Zhao, Delong; Sun, Xuefeng; Yao, Li; Lin, Hongli; Li, Jijun; Zhao, Jiuyang; Zhang, Zhimin; Lun, Lide; Zhang, Jianrong; Li, Mingxu; Huang, Qi; Yang, Yang; Jiang, Shimin; Wang, Yong; Zhu, Hanyu; Chen, Xiangmei

    2013-01-01

    Heparin-induced thrombocytopenia is an immune response mediated by anti-PF4/heparin antibody, which is clinically characterized by thrombocytopenia and thromboembolic events. In this study, a prospective and multi-center clinical investigation 1) determined the positive rate of anti-PF4/heparin antibody in maintenance hemodialysis patients in China, 2) identified the related risk factors, and 3) further explored the effect of the anti-PF4/heparin antibody on bleeding, thromboembolic events, and risk of death in the patients. The serum anti-PF4/heparin antibody was measured in 661 patients from nine hemodialysis centers, detected by IgG-specific ELISA and followed by confirmation with excess heparin. Risk factors of these patients were analyzed. Based on a two-year follow-up, the association between the anti-PF4/heparin antibody and bleeding, thromboembolic events, and risk of death in the patients was investigated. 1) The positivity rate of the anti-PF4/heparin antibody in maintenance hemodialysis patients was 5.6%. With diabetes as an independent risk factor, the positivity rate of the anti-PF4/heparin antibody decreased in the patients undergoing weekly dialyses ≥3 times. 2) The positivity rate of the anti-PF4/heparin antibody was not related to the occurrence of clinical thromboembolic events and was not a risk factor for death within two years in maintenance hemodialysis patients. 3) Negativity for the anti-PF4/heparin antibody combined with a reduction of the platelet count or combined with the administration of antiplatelet drugs yielded a significant increase in bleeding events. However, the composite determination of the anti-PF4/heparin antibody and thrombocytopenia, as well as the administration of antiplatelet drugs, was not predictive for the risk of thromboembolic events in the maintenance hemodialysis patients. A single detection of the anti-PF4/heparin antibody did not predict the occurrence of clinical bleeding, thromboembolic events, or risk of

  9. Polycistronic expression of human platelet factor 4 with heparin-neutralizing activity in Escherichia coli.

    PubMed

    Duan, Yitao; Wang, Zhe; Wu, Wei; Fang, Zhenjiang; Huang, He

    2012-01-01

    Human platelet factor 4 (hPF4) was evaluated as a clinical alternative to protamine for heparin neutralization, a protector against radiation injury and an anti-neoplastic. To achieve high-level expression of hPF4, expression vectors pET-28a(+)-nf PF4 (n=4, 5, 6) containing n tandem repeats of PF4 were constructed and transformed into the Escherichia coli BL21(DE3) strain. A higher expression level, about 45% of the total proteins (TP), was obtained for E. coli BL21(DE3)/pET28a(+)-nf PF4 (n=4, 5, 6). The purified His-PF4 protein was further identified by cleavage with enterokinase and MS, and its heparin-neutralizing activity was determined by colony formation assay. This study represents a novel approach to large-scale production of PF4 in E. coli, one that might be applied to large-scale production of PF4 protein for possible clinical application. It also provides theoretical points for the expression and purification of other small-molecule peptides.

  10. Factors affecting bone growth.

    PubMed

    Gkiatas, Ioannis; Lykissas, Marios; Kostas-Agnantis, Ioannis; Korompilias, Anastasios; Batistatou, Anna; Beris, Alexandros

    2015-02-01

    Bone growth and development are products of the complex interactions of genetic and environmental factors. Longitudinal bone growth depends on the growth plate. The growth plate has 5 different zones-each with a different functional role-and is the final target organ for longitudinal growth. Bone length is affected by several systemic, local, and mechanical factors. All these regulation systems control the final length of bones in a complicated way. Despite its significance to bone stability, bone growth in width has not been studied as extensively as longitudinal bone growth. Bone growth in width is also controlled by genetic factors, but mechanical loading regulates periosteal apposition. In this article, we review the most recent data regarding bone growth from the embryonic age and analyze the factors that control bone growth. An understanding of this complex system is important in identifying metabolic and developmental bone diseases and fracture risk.

  11. Subcellular localization of the heparin-neutralizing factor in blood platelets.

    PubMed Central

    Da Prada, M; Jakábová, M; Lüscher, E F; Pletscher, A; Richards, J G

    1976-01-01

    1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc. Images Plate 1 Plate 2 PMID:950602

  12. Bioengineered heparin

    PubMed Central

    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

  13. Single-step synthesis of heparin-doped polypyrrole nanoparticles for delivery of angiogenic factor.

    PubMed

    Xiong, Gordon M; Yap, Yi Zhen; Choong, Cleo

    2016-04-01

    To perform one-pot synthesis of heparin-immobilized polypyrrole (PPy) nanoparticles and evaluate the use of these nanoparticles for the delivery of VEGF. Heparin-stabilized synthesis of PPy nanoparticles was performed via oxidative polymerization. VEGF-bound PPy-heparin nanoparticles were delivered to endothelial cells and bioactivity of VEGF was assessed by Matrigel tube formation. Size-controllable synthesis of heparin-doped PPy nanoparticles was achieved, and heparin promoted the conjugation of VEGF. Angiogenic activity of the VEGF-conjugated PPy nanoparticles was verified. Heparin-doped PPy nanoparticles can be synthesized using one-pot reaction and provide a delivery platform by which VEGF can be conjugated onto.

  14. Proteolytic Processing Regulates Placental Growth Factor Activities*

    PubMed Central

    Hoffmann, Daniel C.; Willenborg, Sebastian; Koch, Manuel; Zwolanek, Daniela; Müller, Stefan; Becker, Ann-Kathrin A.; Metzger, Stephanie; Ehrbar, Martin; Kurschat, Peter; Hellmich, Martin; Hubbell, Jeffrey A.; Eming, Sabine A.

    2013-01-01

    Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth. PMID:23645683

  15. Heparin inhibits human coronary artery smooth muscle cell migration.

    PubMed

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

    1998-09-01

    Heparin, an anticoagulant, has been shown to reduce neointimal proliferation and restenosis following vascular injury in experimental studies, but the clinical trials of heparin in coronary balloon angioplasty have been negative. The current study, therefore, examined the effect of heparin on basal or stimulated migration by serum and platelet-derived growth factor (PDGF)-BB in cultured human coronary artery smooth muscle cells (SMCs) by Boyden's chamber method. In addition, the reversibility of the heparin effect on human coronary artery SMC migration was examined. Fetal calf serum (FCS) and PDGF-BB stimulated SMC migration in a concentration-dependent manner. Heparin in moderate to high concentration (10 to 100 U/mL) exhibited concentration-related inhibition of FCS- and PDGF-BB-stimulated SMC migration; however, a low concentration (1 U/mL) of heparin had no inhibitory effects. Heparin also had weak inhibitory effects on nonstimulated SMC migration. The SMCs that were exposed to a high concentration (100 U/mL) of heparin for 6 hours were capable of migrating after a short lag period of removal of heparin from the culture medium. These SMCs also showed recovery of responses to FCS and PDGF-BB by migrating significantly greater than the nonstimulated level. Furthermore, heparin-containing medium did not contain detached cells. These results indicate that heparin inhibits human coronary artery SMC migration, especially when stimulated by FCS or PDGF-BB, and that this inhibitory effect of heparin is reversible and not simply a function of killing cells.

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

    SciTech Connect

    Story, M.T. )

    1989-05-01

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

  17. Antibodies to platelet factor 4-heparin complex and outcome in hemodialysis patients with diabetes.

    PubMed

    Krane, Vera; Berger, Mario; Lilienthal, Jürgen; Winkler, Karl; Schambeck, Christian; Wanner, Christoph

    2010-05-01

    Hemodialysis patients with type 2 diabetes exhibit an excessive cardiovascular risk and regularly receive heparin. We tested whether antibodies to the platelet factor 4-heparin complex (PF4-H-AB) contribute to outcome. In 1255 hemodialysis patients with type 2 diabetes, the German Diabetes Dialysis Study evaluated the effect of atorvastatin (20 mg/d) versus placebo. In a post hoc analysis, the association among PF4-H-ABs, biochemistry, and prespecified, centrally adjudicated end points (combined cardiovascular end point [CVE], all-cause mortality, sudden death, myocardial infarction, stroke) was investigated. During 4 years, 460 patients reached the CVE; 605 died, 159 of sudden death. Myocardial infarction and stroke occurred in 199 and 97 patients, respectively. Positive PF4-H-AB status was found in 231 (18.7%) of 1236 tested patients and was associated with lower albumin, higher C-reactive protein, and arrhythmia. In a multivariate model adjusted for demographics, comorbidities, and biochemistry, PF4-H-ABs were associated with sudden death. No significant association between PF4-H-ABs and all-cause mortality, myocardial infarction, stroke, or the CVE was observed. Detecting an interaction between acetylsalicylic acid and PF4-H-ABs regarding sudden death and mortality, we found that the association between PF4-H-ABs and outcomes was restricted to patients with acetylsalicylic acid use, most likely because of indication bias. In hemodialysis patients who have type 2 diabetes and are treated with acetylsalicylic acid, PF4-H-ABs are associated with sudden and all-cause death. Further studies are needed to elucidate this association.

  18. Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor

    SciTech Connect

    Tsai, M.; Takeishi, Takashi; Geissler, E.N. ); Thompson, H.; Metcalfe, D.D. ); Langley, K.E.; Zsebo, K.M.; Galli, S.J. )

    1991-07-15

    The authors investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF{sup 164} (rrSCF{sup 164}) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of connective tissue-type mast cells (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF{sup 164} induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mast cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC> BMCMC maintained in rrSCF{sup 164} not only proliferated but also matured. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.

  19. The modulation of biodistribution of stem cells by anchoring lipid-conjugated heparin on the cell surface.

    PubMed

    Kim, Jong Chul; Tae, Giyoong

    2015-11-10

    Heparin is a bioactive glycosaminoglycan that can interact with various extracellular matrix (ECM) proteins and growth factors. Lipid-conjugated heparin was synthesized, and was used to coat adipose-derived stem cells (ADSCs) by physical insertion on the cell membrane. Coating of lipid-conjugated heparin with two lipid moieties on ADSCs was stable for 24h in vitro. Biodistribution of heparin-coated ADSCs upon intravenous injection in mice was analyzed by In-Vivo Imaging System (IVIS), and showed enhanced accumulation in the liver and spleen while reduced entrapment in the lung. Thus, the coating of ADSCs with lipid-conjugated heparin could significantly modulate the biodistribution of cells.

  20. Attachment of Flexible Heparin Chains to Gelatin Scaffolds Improves Endothelial Cell Infiltration

    PubMed Central

    Leijon, Jonas; Carlsson, Fredrik; Brännström, Johan; Sanchez, Javier; Larsson, Rolf; Nilsson, Bo; Rosenquist, Magnus

    2013-01-01

    Long-term survival of implanted cells requires oxygen and nutrients, the need for which is met by vascularization of the implant. The use of scaffolds with surface-attached heparin as anchoring points for angiogenic growth factors has been reported to improve this process. We examined the potential role of surface modification of gelatin scaffolds in promoting endothelial cell infiltration by using a unique macromolecular conjugate of heparin as a coating. Compared to other heparin coatings, this surface modification provides flexible heparin chains, representing a new concept in heparin conjugation. In vitro cell infiltration of scaffolds was assessed using a three-dimensional model in which the novel heparin surface, without growth factors, showed a 2.5-fold increase in the number of infiltrating endothelial cells when compared to control scaffolds. No additional improvement was achieved by adding growth factors (vascular endothelial growth factor and/or fibroblast growth factor-2) to the scaffold. In vivo experiments confirmed these results and also showed that the addition of angiogenic growth factors did not significantly increase the endothelial cell infiltration but increased the number of inflammatory cells in the implanted scaffolds. The endothelial cell-stimulating ability of the heparin surface alone, combined with its growth factor-binding capacity, renders it an interesting candidate surface treatment to create a prevascularized site prepared for implantation of cells and tissues, in particular those sensitive to inflammation but in need of supportive revascularization, such as pancreatic islets of Langerhans. PMID:23327585

  1. Synthesis and structural study of two new heparin-like hexasaccharides.

    PubMed

    Lucas, Ricardo; Angulo, Jesús; Nieto, Pedro M; Martín-Lomas, Manuel

    2003-07-07

    Two new heparin-like hexasaccharides, 5 and 6, have been synthesised using a convergent block strategy and their solution conformations have been determined by NMR spectroscopy and molecular modelling. Both hexasaccharides contain the basic structural motif of the regular region of heparin but with negative charge distributions which have been designed to get insight into the mechanism of fibroblast growth factors (FGFs) activation.

  2. Growth factors for nanobacteria

    NASA Astrophysics Data System (ADS)

    Ciftcioglu, Neva; Kajander, E. Olavi

    1999-12-01

    Nanobacteria are novel microorganisms recently isolated from fetal bovine serum and blood of cows and humans. These coccoid, gram negative bacteria in alpha-2 subgroup of Proteobacteria grow slowly under mammalian cell culture conditions but not in common media for microbes. Now we have found two different kinds of culture supplement preparations that improve their growth and make them culturable in the classical sense. These are supernatant fractions of conditioned media obtained from 1 - 3 months old nanobacteria cultures and from about a 2 weeks old Bacillus species culture. Both improved multiplication and particle yields and the latter increased their resistance to gentamicin. Nanobacteria cultured with any of the methods shared similar immunological property, structure and protein pattern. The growth supporting factors were heat-stabile and nondialyzable, and dialysis improved the growth promoting action. Nanobacteria formed stony colonies in a bacteriological medium supplemented with the growth factors. This is an implication that nanobacterial growth is influenced by pre-existing bacterial flora.

  3. Peptide growth factors, part A

    SciTech Connect

    Barnes, D.; Sirbasku, D.A.

    1987-01-01

    This book contains information on the following topics: Epidermal Growth Factor;Transforming Growth Factors;Bone and Cartilage Growth Factors;Somatomedin/Insulin-Like Growth Factors;Techniques for the Study of Growth Factor Activity;Assays, Phosphorylation, and Surface Membrane Effects.

  4. New microbial growth factor.

    PubMed Central

    Bok, S H; Casida, L E

    1977-01-01

    A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a new microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight, and it has high specific activity. When added to the diets for a meadow vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain. PMID:327929

  5. New microbial growth factor

    NASA Technical Reports Server (NTRS)

    Bok, S. H.; Casida, L. E., Jr.

    1977-01-01

    A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a previously unknown microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight and has high specific activity. When added to the diets for a meadow-vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain.

  6. New microbial growth factor

    NASA Technical Reports Server (NTRS)

    Bok, S. H.; Casida, L. E., Jr.

    1977-01-01

    A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a previously unknown microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight and has high specific activity. When added to the diets for a meadow-vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain.

  7. Effect of basic fibroblast growth factor on angiogenesis in the infarcted porcine heart.

    PubMed

    Watanabe, E; Smith, D M; Sun, J; Smart, F W; Delcarpio, J B; Roberts, T B; Van Meter, C H; Claycomb, W C

    1998-02-01

    Administration of growth factors is emerging as a new therapeutic approach for the enhancement of collateral vessel formation in the ischemic heart. We have investigated the effects of intramyocardial delivery of FGF-2 in the presence and absence of heparin on angiogenesis in a porcine model of myocardial infarction. Yorkshire pigs were subjected to myocardial infarction by the placement of an embolization coil in the left anterior descending artery (n = 5). Four to five weeks after creation of an infarct, FGF-2 (10 micrograms) alone or in complex with heparin, heparan sulfate, or heparin agarose beads was injected either into the normal myocardium or along the infarct border area. Histologic evaluation of each injection site was performed 4 to 5 weeks post-injection. The effect of FGF-2 on angiogenesis was evaluated by determining the number of capillaries (diameter < 20 microns (and arterioles (> 20 microns with tunica media) in each area observed. The number of capillaries were not affected by the treatment of FGF-2 both in normal myocardium and infarct border area. However, in the normal myocardium, the number of arterioles were increased with the treatment of FGF-2 alone (85 +/- 59%, P < 0.04), FGF-2 plus heparin (281 +/- 193%, P < 0.004) and FGF-2-coated heparin beads (241 +/- 141%, P < 0.01), as compared to control. Delivery of FGF-2 into the infarct border area, also increased the number of arterioles when FGF-2 was given with heparin (736 +/- 154%, P < 0.001) or heparin beads (700 +/- 109%, P < 0.001), as compared to control. FGF-2 administered with heparin was the most effective method of enhancing angiogenesis as compared to FGF-2 alone, FGF-2 plus heparan sulfate, or FGF-2 coated heparin agarose beads.

  8. Nitric oxide degradation of heparin and heparan sulphate.

    PubMed Central

    Vilar, R E; Ghael, D; Li, M; Bhagat, D D; Arrigo, L M; Cowman, M K; Dweck, H S; Rosenfeld, L

    1997-01-01

    NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines. PMID:9182706

  9. Heparin treatment increases thioredoxin interacting protein expression in hepatocellular carcinoma cells.

    PubMed

    Gunes, Aysim; Iscan, Evin; Topel, Hande; Avci, Sanem Tercan; Gumustekin, Mukaddes; Erdal, Esra; Atabey, Nese

    2015-08-01

    Heparins play an important role in cell growth, differentiation, migration and invasion. However, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. To determine the effect of heparin on gene expression, we performed a cDNA microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. In this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. We determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. We showed the importance of heparin mediated histone modifications and down-regulation of Enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxin-interacting protein. When we tested biological significance of these data; we observed that cells overexpressing thioredoxin-interacting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. Interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. In conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. This study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis.

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

    PubMed

    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=<0.001-0.01). Based on the results of our in-vitro study, we conclude that 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.

  11. Sulfation patterns determine cellular internalization of heparin-like polysaccharides

    PubMed Central

    Raman, Karthik; Mencio, Caitlin; Desai, Umesh R.; Kuberan, Balagurunathan

    2013-01-01

    Heparin is a highly sulfated polysaccharide which serves biologically relevant roles as an anticoagulant and anti-cancer agent. While it is well known that modification of heparin’s sulfation pattern can drastically influence its ability to bind growth factors and other extracellular molecules, very little is known about the cellular uptake of heparin and the role sulfation patterns serve in affecting its internalization. In this study, we chemically synthesized several fluorescently-labeled heparins consisting of a variety of sulfation patterns. These polysaccharides were thoroughly characterized using anion exchange chromatography and size exclusion chromatography. Subsequently, we utilized flow cytometry and confocal imaging to show that sulfation patterns differentially affect the amount of heparin uptake in multiple cell types. This study provides the first comprehensive analysis of the effect of sulfation pattern on the cellular internalization of heparin or heparan sulfate like polysaccharides. The results of this study expand current knowledge regarding heparin internalization and provide insights into developing more effective heparin-based drug conjugates for applications in intracellular drug delivery. PMID:23398560

  12. The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells

    PubMed Central

    2011-01-01

    Background Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit in vitro and in vivo the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop. Methods A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. In vitro, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. In vivo, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. In vivo anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay. Results Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. In vitro, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an in vivo Matrigel™ plug assay in mice Conclusions Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on in vitro and in vivo growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTP

  13. Inhibition of factor IXa by the pegnivacogin system during cardiopulmonary bypass: a potential substitute for heparin. A study in baboons.

    PubMed

    Bel, Alain; Borik, Wasseem; Davidson, Simon; Helies, Jean-Marie; Stimmer, Lev; Fremes, Stephen; Zelenkofske, Steven; Rusconi, Christopher; Alexander, John; Alexander, David; Menasché, Philippe; Pepper, John

    2016-02-01

    Heparin and protamine are standard for anticoagulation and reversal for cardiopulmonary bypass (CPB). The REGADO biosciences protocol 1 (REG1) anticoagulant system, consisting of the Factor IXa (FIXa)-inhibitor pegnivacogin and its reversal agent (anivamersen), has been studied in patients undergoing coronary catheterization and in CPB in sheep and pigs. Prior to first human use in CPB, we wanted to test the safety and efficacy of REG1 in a primate model. Fourteen baboons undergoing 2 h of CPB followed by 1 h of reperfusion were studied. Three received heparin/protamine and 11 received 1 of 2 doses of pegnivacogin followed by anivamersen. Thrombin-generating capacity was tested in additional in vitro experiments. Targeted drug levels and near-complete FIXa inhibition were achieved. Bypass was run uneventfully in all animals without any clotting in the circuit and bleeding was minimal in the two groups. However, in contrast to heparin-treated baboons, those receiving pegnivacogin/anivamersen displayed thrombi in the bypass cannulae upon cannulation and kidney cortical infarcts. Inter-species comparisons revealed that in the presence of high levels of FIXa inhibition, tissue factor-mediated thrombin generation in baboons was much higher than that in other species. These data highlight the limitations of the baboon model for assessing factor-specific coagulation inhibitors during CPB. The justification for Phase 1 human studies using REG1 for CPB is unclear. © The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

  14. Growth factors and cytokines in acute renal failure.

    PubMed

    Harris, R C

    1997-04-01

    The mammalian kidney is susceptible to injury by ischemia/reperfusion and toxins, and regeneration after injury is characterized by hyperplasia and recovery of the damaged epithelial cells that line the tubules. Locally produced growth factors may serve as mediators of nephrogenesis and differentiation during renal development and of renal regeneration after acute injury. In cultured cells, administration of one or a mixture of growth factors to quiescent cells will initiate progression through the cell cycle and cell division. In the adult kidney, cell division normally is very low, but will increase up to 10-fold after acute injury. In addition to proliferation after lethal injury, there also is cellular repair in cells that have undergone sublethal injury. Recent studies indicate that growth factors inhibit programmed cell death in response to acute injury. Growth factors also may initiate or promote protein and lipid biosynthesis and provide an intracellular milieu that promotes cellular repair. In addition to cellular repair, growth factors also may be involved in the re-establishment of cell-extracellular matrix and cell-cell integrity. Finally, growth factors may limit injury by decreasing the factors that induce damage. Increased local renal expression of growth factors in response to acute injury include heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I), transforming growth factor-beta, parathyroid hormone-related peptide, and acidic fibroblast growth factor. In a number of experimental models of acute renal injury, administration of exogenous growth factors has been shown to accelerate both structural and functional recovery. Specifically, EGF, IGF-1, and HGF all have been shown to be effective in this regard. These studies are reviewed and potential therapeutic uses of growth factors and cytokines will be discussed.

  15. Pharmacokinetics of danaparoid sodium, dalteparin sodium and heparin determined by inhibitory effect on the activated coagulation factor X activity after single intravenous administration in rabbits.

    PubMed

    Ishida, M; Nakada, Y; Horiuchi, M; Sakamoto, F

    1998-08-01

    The inhibitory effect on the activated coagulation factor X activity (anti-Xa activity) in plasma and urine of danaparoid sodium (DAS, CAS 9005-49-6) was compared with that of dalteparin sodium (DLS, CAS 9041-08-1) and heparin (CAS 9005-49-6) after single intravenous administration at a dose of 640 anti-Xa U/kg to male rabbits. The elimination of half-life of DAS was 9.90 h and was 6.0 times longer than that of DLS and 16.5 times longer than that of heparin. The area under the plasma concentration-time curve (AUC) of DAS was 47.13 +/- 14.55 anti-Xa U.h/ml and was 2.4 times larger than that of DLS and 2.9 times larger than that of heparin. The urinary cumulative excretion of anti-Xa activity of DAS and DLS was 42.6 +/- 6.4% and 16.4 +/- 0.8% of dose, respectively, in 24 h after dosing, respectively. But the anti-Xa activity in urine was not detected at any sampling points after administration of heparin. DAS has a longer elimination half-life and a higher renal excretion of anti-Xa activity than that of DLS and heparin. Therefore, in comparison to DLS and heparin, it seems that the anticoagulant activity of DAS has a long duration.

  16. Sulodexide induces hepatocyte growth factor release in humans.

    PubMed

    Borawski, Jacek; Dubowski, Miroslaw; Pawlak, Krystyna; Mysliwiec, Michal

    2007-03-08

    Heparin influences numerous pleiotropic growth factors, including hepatocyte growth factor (HGF), partially by their release from endothelial and extracellular matrix stores. The effects of sulodexide, a heparin-like glycosaminoglycan medication of growing importance in medicine, on HGF liberation are not known. We performed a 2-week open-label sulodexide trial in healthy male volunteers. The drug was initially administered intravenously (i.v.) in a single dose of 1200 Lipoprotein Lipase Releasing Units (LRU), then -- orally for 12 days (500 LRU twice a day), and -- again by i.v. route (1200 LRU) on day 14. Intravenous sulodexide injections were repeatedly found to induce marked and reproducible increases in immunoreactive plasma HGF levels (more than 3500% vs baseline after 10 min, and more than 1200% after 120 min), and remained unchanged when measured 120 min following oral sulodexide administration. The percentage increments in plasma HGF evoked by i.v. sulodexide at both time points and on both days inversely correlated with baseline levels of the growth factor. On day 14, the HGF levels after 120 min and their percentage increase vs baseline were strongly and directly dependent on i.v. sulodexide dose per kg of body weight. This study shows that sulodexide has a novel, remarkable and plausibly biologically important stimulating effect on the release of pleiotropic hepatocyte growth factor in humans.

  17. Supramolecular polymeric peptide amphiphile vesicles for the encapsulation of basic fibroblast growth factor.

    PubMed

    Loh, Xian Jun; del Barrio, Jesús; Lee, Tung-Chun; Scherman, Oren A

    2014-03-21

    The synthesis of a supramolecular double hydrophilic peptide-conjugated polymer held together by cucurbit[8]uril (CB[8]) ternary complexation and its subsequent temperature triggered self-assembly into vesicles are described. Basic fibroblast growth factor can be easily loaded into the vesicles under benign conditions and their bioactivities can be preserved without the need for excipients such as heparin.

  18. Assessment of recombinant factor VIIa as an antidote for bleeding induced in the rabbit by low molecular weight heparin.

    PubMed

    Chan, S; Kong, M; Minning, D M; Hedner, U; Marder, V J

    2003-04-01

    While protamine sulfate reverses the anticoagulant effect of standard heparin, there currently is no effective antidote for low molecular weight heparin (LMWH)-induced bleeding. Recently, recombinant activated factor VII (rFVIIa) was approved by the FDA for use in hemophilia patients with factor (F)VIII or FIX inhibitors. However, this new pro-hemostatic agent has potential utility in other clinical scenarios. In this study, we utilized a well-characterized rabbit ear puncture model to test the efficacy of rFVIIa to reverse LMWH-induced prolonged bleeding. Animals were first treated with bolus intravenous LMWH (1800 anti-FXa U kg(-1)) which increased the primary bleeding time approximately fourfold and raised the plasma anti-FXa activity immediately and continuously throughout the 90-min experiment. In a randomized and blinded fashion, animals then received either rFVIIa (400 microg kg(-1)) or placebo by bolus intravenous injection, following which the ear puncture bleeding times were measured, along with blood levels of heparin (anti-FXa activity) and FVII. FVII activity increased 5.3-fold over baseline in treated animals, decreasing by only 24% over the full observation period. The rFVIIa-treated animals showed a slight decrease in bleeding time immediately after injection, but there was no statistically significant difference in bleeding after rFVIIa or placebo administration. In this study using a rabbit ear bleeding model, rFVIIa was not an effective antidote to LMWH-induced bleeding. However, the bolus injection of LMWH produced a very high blood anti-FXa level, which may have precluded rFVIIa effectiveness.

  19. Heparin-dependent regulation of fibronectin matrix conformation

    PubMed Central

    Hubbard, Brant; Buczek-Thomas, Jo Ann; Nugent, Matthew A.; Smith, Michael L.

    2013-01-01

    Extracellular matrix (ECM) conformation is regulated by a variety of stimuli in vivo, including mechanical forces and allosteric binding partners, and these conformational changes contribute to the regulation of cell behavior. Heparin and heparan sulfate, for example, have been shown to regulate the sequestration and presentation of numerous growth factors, including vascular endothelial growth factor, on the heparin 2 binding domain in fibronectin (Fn). However, mechanical force also alters Fn conformation, indicating that the growth factor binding region may be co-regulated by both heparin and mechanical force. Herein, we describe a simple antibody-based method for evaluating the conformation of the heparin 2 binding domain in Fn, and use it to determine the relative contributions of heparin and mechanical strain to the regulation of Fn conformation. We achieved specificity in quantifying conformational changes in this region of Fn by measuring the ratio of two fluorescent monoclonal antibodies, one that is insensitive to Fn conformational changes and a second whose binding is reduced or enhanced by non-equilibrium conformational changes. Importantly, this technique is shown to work on Fn adsorbed on surfaces, single Fn fibers, and Fn matrix fibers in cell culture. Using our dual antibody approach, we show that heparin and mechanical strain co-regulate Fn conformation in matrix fibrils, which is the first demonstration of heparin-dependent regulation of Fn in its physiologically-relevant fibrillar state. Furthermore, the dual antibody approach utilizes commercially available antibodies and simple immunohistochemistry, thus making it accessible to a wide range of scientists interested in Fn mechanobiology. PMID:24148804

  20. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells [published erratum appears in J Cell Biol 1990 Mar;110(3):863

    PubMed Central

    1989-01-01

    The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect. PMID:2592420

  1. Pigment epithelium-derived factor (PEDF) shares binding sites in collagen with heparin/heparan sulfate proteoglycans.

    PubMed

    Sekiya, Atsushi; Okano-Kosugi, Hitomi; Yamazaki, Chisato M; Koide, Takaki

    2011-07-29

    Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.

  2. Adeno-Associated Viral Vectors Based on Serotype 3b Use Components of the Fibroblast Growth Factor Receptor Signaling Complex for Efficient Transduction

    PubMed Central

    Messina, Emily L.; Nienaber, Jeffrey; Daneshmand, Mani; Villamizar, Nestor; Samulski, Jude; Milano, Carmelo

    2012-01-01

    Abstract Adeno-associated virus type 3b (AAV3b) has been largely ignored by gene therapists because of the inability of vectors based on this serotype to transduce target tissues efficiently. Here we describe a phenomenon unique to AAV3b in that vectors based on this serotype mediate enhanced transduction in the presence of heparin. Among the many biological functions attributed to heparin, its interaction with, and ability to regulate, several growth factors (GFs) and growth factor receptors (GFRs) has been well characterized. Using GFR-overexpressing cell lines, soluble GFs and heparins, as well as specific GFR inhibitors, we have demonstrated a requirement for fibroblast growth factor receptor-2 (FGFR2) and FGF1 in the heparin-mediated augmentation of AAV3b vector transduction. In contrast to AAV2, we establish that heparin can be used as an adjunct with AAV3b to further increase transduction in a variety of cells and target tissues, additionally suggesting that AAV3b may be an attractive viral vector for clinical use during procedures in which heparin is used. In summary, AAV3b exhibits FGFR2-dependent, markedly enhanced transduction efficiency in the presence of heparin and FGFs, which could make it a useful vector for gene therapy in a variety of human diseases. PMID:22680698

  3. Adeno-associated viral vectors based on serotype 3b use components of the fibroblast growth factor receptor signaling complex for efficient transduction.

    PubMed

    Messina, Emily L; Nienaber, Jeffrey; Daneshmand, Mani; Villamizar, Nestor; Samulski, Jude; Milano, Carmelo; Bowles, Dawn E

    2012-10-01

    Adeno-associated virus type 3b (AAV3b) has been largely ignored by gene therapists because of the inability of vectors based on this serotype to transduce target tissues efficiently. Here we describe a phenomenon unique to AAV3b in that vectors based on this serotype mediate enhanced transduction in the presence of heparin. Among the many biological functions attributed to heparin, its interaction with, and ability to regulate, several growth factors (GFs) and growth factor receptors (GFRs) has been well characterized. Using GFR-overexpressing cell lines, soluble GFs and heparins, as well as specific GFR inhibitors, we have demonstrated a requirement for fibroblast growth factor receptor-2 (FGFR2) and FGF1 in the heparin-mediated augmentation of AAV3b vector transduction. In contrast to AAV2, we establish that heparin can be used as an adjunct with AAV3b to further increase transduction in a variety of cells and target tissues, additionally suggesting that AAV3b may be an attractive viral vector for clinical use during procedures in which heparin is used. In summary, AAV3b exhibits FGFR2-dependent, markedly enhanced transduction efficiency in the presence of heparin and FGFs, which could make it a useful vector for gene therapy in a variety of human diseases.

  4. Factors Influencing ACT After Intravenous Bolus Administration of 100 IU/kg of Unfractionated Heparin During Cardiac Catheterization in Children.

    PubMed

    Muster, Ileana; Haas, Thorsten; Quandt, Daniel; Kretschmar, Oliver; Knirsch, Walter

    2016-01-01

    Anticoagulation using intravenous bolus administration of unfractionated heparin (UFH) aims to prevent thromboembolic complications in children undergoing cardiac catheterization (CC). Optimal UFH dosage is needed to reduce bleeding complications. We analyzed the effect of bolus UFH on activated clotting time (ACT) in children undergoing CC focusing on age-dependent, anesthesia-related, or disease-related influencing factors. This retrospective single-center study of 183 pediatric patients receiving UFH during CC analyzed ACT measured at the end of CC. After bolus administration of 100 IU UFH/kg body weight, ACT values between 105 and 488 seconds were reached. Seventy-two percent were within target level of 160 to 240 seconds. Age-dependent differences were not obtained ( P = .407). The ACT values were lower due to hemodilution (total fluid and crystalloid administration during CC, both P < .001), with premedication of acetylsalicylic acid ( P = .014) and low-molecular-weight heparin ( P = .049). Arterial thrombosis (3.85%), venous thrombosis (0.55%), and bleeding (1.65%) following CC did not correlate with ACT values but occurred more frequently in children between 1 month and 1 year of age (91%). In conclusion, with a bolus of 100 IU UFH/kg, an ACT target level of 160 to 240 seconds can be achieved during CC in children in 72%, which is influenced by hemodilution and anticoagulant and antiplatelet premedication but not by age.

  5. Discovery of Allosteric Modulators of Factor XIa by Targeting Hydrophobic Domains Adjacent to its Heparin-Binding Site

    PubMed Central

    Karuturi, Rajesh; Al-Horani, Rami A.; Mehta, Shrenik C.; Gailani, David; Desai, Umesh R.

    2013-01-01

    To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the VMAX of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively-charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggest the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-element strategy may be widely applicable for discovering SAMs of other GBPs. PMID:23451707

  6. Heparin-functionalized polymeric biomaterials in tissue engineering and drug delivery applications

    PubMed Central

    Liang, Yingkai; Kiick, Kristi L.

    2014-01-01

    Heparin plays an important role in many biological processes, via its interaction with various proteins, and hydrogels and nanoparticles comprising heparin exhibit attractive properties such as anticoagulant activity, growth factor binding, as well as antiangiogenic and apoptotic effects, making them great candidates for emerging applications. Accordingly, this review summarizes recent efforts in the preparation of heparin-based hydrogels and formation of nanoparticles, as well as the characterization of their properties and applications. The challenges and future perspectives for heparin-based materials are also discussed. Prospects are promising for heparin-containing polymeric biomaterials in diverse applications ranging from cell carriers for promoting cell differentiation to nanoparticle therapeutics for cancer treatment. PMID:23911941

  7. Pituitary follicular cells produce basic fibroblast growth factor

    SciTech Connect

    Ferrara, N.; Schweigerer, L.; Neufeld, G.; Mitchell, R.; Gospodarowicz, D.

    1987-08-01

    Cultured monolayers of bovine pituitary follicular cells, which transport ions, contain high amounts of mitogenic activity for endothelial cells which, on the basis of gene expression analysis, heparin-Sepharose elution profile, bioassay, immunoblotting, radioimmunoassay, and radioreceptor assay, has been identified as basic fibroblast growth factor (bFGF). These data indicate that follicular cells may be a major source of bFGF in the pituitary gland. Considering that bFGF has been proposed to play a role in paracrine regulation of pituitary hormone secretion, the data also suggest that these cells may exert important local regulatory functions.

  8. Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light.

    PubMed Central

    LaVail, M M; Unoki, K; Yasumura, D; Matthes, M T; Yancopoulos, G D; Steinberg, R H

    1992-01-01

    Recent demonstrations of survival-promoting activity by neurotrophic agents in diverse neuronal systems have raised the possibility of pharmacological therapy for inherited and degenerative disorders of the central nervous system. We have shown previously that, in the retina, basic fibroblast growth factor delays photoreceptor degeneration in Royal College of Surgeons rats with inherited retinal dystrophy and that the growth factor reduces or prevents the rapid photoreceptor degeneration produced by constant light in the rat. This light-damage model now provides an efficient way to assess quantitatively the survival-promoting activity in vivo of a number of growth factors and other molecules. We report here that photoreceptors can be significantly protected from the damaging effects of light by intravitreal injection of eight different growth factors, cytokines, and neurotrophins that typically act through several distinct receptor families. In addition to basic fibroblast growth factor, those factors providing a high degree of photoreceptor rescue include brain-derived neurotrophic factor, ciliary neurotrophic factor, interleukin 1 beta, and acidic fibroblast growth factor; those with less activity include neurotrophin 3, insulin-like growth factor II, and tumor necrosis factor alpha; those showing little or no protective effect are nerve growth factor, epidermal growth factor, platelet-derived growth factor, insulin, insulin-like growth factor I, heparin, and laminin. Although we used at least one relatively high concentration of each agent (the highest available), it is still possible that other concentrations or factor combinations might be more protective. Injecting heparin along with acidic fibroblast growth factor or basic fibroblast growth factor further enhanced the degree of photoreceptor survival and also suppressed the increased incidence of macrophages produced by either factor, especially basic fibroblast growth factor. These results now provide the

  9. The effect of heparinized decellularized scaffolds on angiogenic capability.

    PubMed

    Wu, Qiong; Li, Yi; Wang, Yujia; Li, Li; Jiang, Xin; Tang, Jing; Yang, Hao; Zhang, Jie; Bao, Ji; Bu, Hong

    2016-12-01

    The immobilization of heparin, a new and versatile approach to the surface modification of decellularized tissues, has the potential to greatly improve the hemocompatibility of engineered tissue constructs derived from decellularized organs. We report on porcine decellularized liver scaffolds (DLSs) heparinized by the end-point attachment (EPA) technique. The heparinized DLSs (HEP-DLSs) have the ability to bind and slowly release heparin-binding growth factors. We hypothesized that DLS-immobilized heparin acts as an antithrombotic coating reagent and binds vascular endothelial growth factor (VEGF) to induce angiogenesis in the DLSs. Human umbilical vein endothelial cells (HUVECs) seeded on HEP-VEGF-DLSs attached and remained bioactive. Using the chicken chorioallantoic membrane (CAM) assay, we found that the HEP-VEGF-DLSs induced a significant and rapid enhancement of angiogenesis compared with native DLSs. Scaffolds were implanted in the greater omentum of rats and evaluated after 7, 14, 21, and 28 days. There were significant increases in the numbers of blood vessels in the HEP-VEGF-DLSs compared with native DLSs at all time-points. The modified method introduced in this article could overcome obstacles faced by conventional matrices that lack the ability to induce rapid and sufficient vascularization. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3021-3030, 2016.

  10. Growth hormone, growth factors, and acromegaly

    SciTech Connect

    Ludecke, D.K.; Tolis, G.T.

    1987-01-01

    This book contains five sections, each consisting of several papers. The section headings are: Biochemistry and Physiology of GH and Growth Factors, Pathology of Acromegaly, Clinical Endocrinology of Acromegaly, Nonsurgical Therapy of Acromegaly, and Surgical Therapy of Acromegaly.

  11. Stabilization of growth factors relevant to wound healing by a plant cell wall biomaterial.

    PubMed

    Ni, Yawei; Turner, Debra; Yates, Kenneth; Tizard, Ian

    2007-10-01

    Stabilization of growth factors in a wound environment is critical to the wound healing process. Here we report on the stabilization of key growth factors by a unique plant cell wall biomaterial. MicroSheets are clear cell wall fragments isolated from the non-living water storage cells in the pulp or inner gel of Aloe vera L., which has widely been used for wound healing. It was found that MicroSheets bind to a subset of heparin binding growth factors including basic fibroblast growth factor (bFGF) and keratinocyte growth factor (KGF), two key growth factors in the wound healing process. The binding of bFGF and KGF to MicroSheets was inhibited by heparin and also by a pectic substance isolated from the MicroSheets, indicating that the binding was mediated by this pectic component of the MicroSheet. The binding protected the growth factors against protease digestion. Furthermore, the protective effect was also demonstrated with KGF against digestion by wound fluids and by measuring the biological activity. Thus, these results showed that MicroSheets can stabilize certain critical growth factors in wounds and thereby promote the healing process. Incorporation of a material like MicroSheets provides an important functional element in wound dressings, i. e., growth factor stabilization.

  12. Nanofibrous heparin and heparin-mimicking multilayers as highly effective endothelialization and antithrombogenic coatings.

    PubMed

    Nie, Chuanxiong; Ma, Lang; Cheng, Chong; Deng, Jie; Zhao, Changsheng

    2015-03-09

    Combining the advantages of the fibrous nanostructure of carbon nanotubes (CNTs) and the bioactivities of heparin/heparin-mimicking polyanions, functional nanofibrous heparin or heparin-mimicking multilayers were constructed on PVDF membrane with highly promoted endothelialization and antithrombogenic activities. Oxidized CNT (oCNT) was first functionalized with water-soluble chitosan (polycation), then enwrapped with heparin or a typical sulfonated heparin-mimicking polymers (poly(sodium 4-styrenesulfonate-co-sodium methacrylate)) to construct the multilayers. Then, the surface-deposited multilayers were constructed via electrostatic layer-by-layer assembly of the functionalized oCNTs. The scanning electron microscope and atom force microscope images confirmed that the coated multilayers exhibited nanofibrous and porous structure. The live/dead cell staining and cell viability assay results indicated that the coated nanofibrous multilayers had excellent compatibility with endothelial cells. The cell morphology observation further confirmed the promotion ability of surface endothelialization due to the coated heparin/heparin-mimicking multilayers. Further systematical evaluation on blood compatibility revealed that the surface heparin/heparin-mimicking multilayer-coated membranes also had significantly improved blood compatibility including restrained platelet adhesion and activation, prolonged blood clotting times, and inhibited activation of coagulation and complement factors. In summary, the proposed nanofibrous multilayers integrated endothelialization and antithrombogenic properties; meanwhile, the heparin-mimicking coating validated comparable performances as heparin coating. Herein, it is expected that the surface coating of nanofibrous multilayers, especially the facilely constructed heparin-mimicking coating, may have great application potential in biomedical fields.

  13. Mesoglycan and sulodexide act as stabilizers and protectors of fibroblast growth factors (FGFs).

    PubMed

    Tardieu, M; Bourin, M C; Desgranges, P; Barbier, P; Barritault, D; Caruelle, J P

    1994-01-01

    Heparin and heparan sulfate proteoglycans (HSPGs) stabilize FGFs which belong to heparin-binding growth factors (HBGFs) on active conformation. They also strongly potentiate their mitogenic activity on many cell types, and protect them against thermal denaturation and enzymatic degradation. In the present work we have tested two heparin-like substances named mesoglycan and sulodexide obtained from bovine intestinal mucosal extracts. These products are used as heparin, in various of therapeutic fields such as atherosclerosis or antithrombotic therapy. The compositions of mesoglycan and sulodexide are partially known and include chondroitin, dermatan and heparan sulfate. We have shown that mesoglycan and sulodexide potentiated the mitogenic activity of FGF1 and FGF2. The magnitude of this effect was identical with that of heparin used as a control substance but at double concentration. Mesoglycan and sulodexide also exerted stabilizing and protective effects on FGFs for heat denaturation and enzymatic degradation. The suppression of the protective properties after heparinase treatment of mesoglycan and sulodexide indirectly demonstrated the presence of heparan sulfate which was shown to represent about 60% of the commercial products.

  14. Binding Potency of Heparin Immobilized on Activated Charcoal for DNA Antibodies.

    PubMed

    Snezhkova, E A; Tridon, A; Evrard, B; Nikolaev, V G; Uvarov, V Yu; Tsimbalyuk, R S; Ivanuk, A A; Komov, V V; Sakhno, L A

    2016-02-01

    In vitro experiments showed that heparin adsorbed on activated charcoal can bind antibodies raised against native and single-stranded DNA in a diluted sera pool with a high level of these DNA. Thus, heparin used as anticoagulant during hemosorption procedure can demonstrate supplementary therapeutic activity resulting from its interaction with various agents involved in acute and chronic inflammatory reactions such as DNA- and RNA-binding substances, proinflammatory cytokines, complement components, growth factors, etc. Research and development of heparin-containing carbonic adsorbents for the therapy of numerous inflammatory and autoimmune diseases seems to be a promising avenue in hematology.

  15. Anti-Platelet Factor 4/Heparin Antibody Plays a Significant Role in Progression of Arterial Stiffness among Hemodialysis Patients

    PubMed Central

    Kuo, Chieh; Tsai, Chiang-Chin; Chen, Chien-An; Tsai, Yueh-Feng; Chen, Yen-Hsun

    2017-01-01

    Background Arterial stiffness is a determinant of cardiovascular disease in end stage renal disease. Hemodialysis patients may develop anti-platelet factor 4/heparin antibody (PF4-H Ab) because of heparin treatment in dialysis. We tested whether PF4-H Ab was associated with progression of arterial stiffness in a 3-year follow-up. Methods We enrolled 74 hemodialysis patients and studied their clinical, biochemical and arterial stiffness measurement with brachial-ankle pulse wave velocity (baPWV) over 3 years. Baseline and changes in baPWV after 3 years (ΔbaPWV) were collected and compared with related clinical and biochemical parameters. PF4-H Ab was evaluated by the enzyme-linked immunosorbent assay and titer ≥ 0.4 was defined to have PF4-H Ab. Results We found a positive PF4-H Ab status in 25 of 74 patients. Mean baPWV was 16.1 ± 3.8 (m/s) at baseline and 17.6 ± 4.0 (m/s) after 3 years. Mean ΔbaPWV was 3.4 ± 2.2 (m/s) in the PF4-H Ab positive group, and 0.6 ± 1.2 (m/s) in the PF4-H Ab negative group. Baseline baPWV was only significantly associated with age (β = 0.49, p < 0.01). ΔbaPWV was significantly different between the PF4-H Ab positive and negative groups (p < 0.01). In multivariate regression analysis, only PF4-H Ab was positively associated with ΔbaPWV (β = 0.71, p < 0.01). Conclusions Our study concluded that PF4-H Ab was associated with progression of arterial stiffness in hemodialysis patients. PMID:28344423

  16. Heparin dopant increases the electrical stability, cell adhesion, and growth of conducting polypyrrole/poly(L,L-lactide) composites.

    PubMed

    Meng, Shiyun; Rouabhia, Mahmoud; Shi, Guixin; Zhang, Ze

    2008-11-01

    Polypyrrole (PPy) is a promising conductive polymer for tissue engineering and bioelectrical applications. However, its electrical conductivity deteriorates easily in aqueous conditions. Cell adhesion to PPy is also relatively poor. The goal of this study was to simultaneously improve the electrical stability of and cell adhesion to PPy by using heparin (HE) as dopant, for HE is both a polyanion and an important glycosaminoglycan in cell membranes and extracellular matrix. PPy particles doped with HE were synthesized through emulsion polymerization using Fenton's reagent as an oxidant. X-ray photoelectron spectroscopy (XPS), infrared and scanning electron microscopy (SEM) were used to investigate the PPy particles. Conductive biodegradable membranes of 10(2) to 10(3) Omega/square were prepared from 5% (w) PPy with various amounts of HE and 95% (w) poly(L,L-lactide) (PPy/PLLA). Azure A staining was employed to quantify the HE exposed on the surface of the PPy particles and PPy/PLLA membranes. The distribution of HE on membranes was demonstrated by DAPI staining. Results showed that HE was incorporated into the PPy particles as counterions and presented on particle surface. A unique "filament"-like morphology of the PPy preparation was observed at high-HE content. The electrical stability of the PPy/PLLA membranes was tested in saline at 37 degrees C for 500 h. Human skin fibroblasts were used to test the cell adhesion capacity. The conductive membranes containing HE-doped PPy particles recorded significantly increased electrical stability, cell adhesion, and growth. The electrically more stable and cell adhesive conductive biodegradable membrane may act as a platform for various biomedical applications.

  17. Current management of heparin-induced thrombocytopenia.

    PubMed

    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.

  18. Factors Associated with Continuous Low Dose Heparin Infusion for Central Venous Catheter Patency in Critically Ill Children Worldwide

    PubMed Central

    Onyeama, Sara-Jane N; Hanson, Sheila J; Dasgupta, Mahua; Hoffmann, Raymond G; Faustino, Edward Vincent S

    2016-01-01

    Objective To identify patient, hospital and central venous catheter (CVC) factors that may influence the use of low dose heparin infusion (LDHI) for CVC patency in critically ill-children. Design Secondary analysis of an international multicenter observational study. Setting 59 Pediatric Intensive Care Units (PICUs) over four study dates in 2012, involving 7 countries. Patients Children less than 18 years of age with a CVC, admitted to a participating unit and enrolled in the completed PROTRACT study were included. All overflow patients were excluded. Interventions None. Measurements and Main Results Of the 2,484 patients in the PROTRACT study, 1,312 patients had a CVC. 507 of those patients used LDHI. The frequency of LDHI was compared across various patient, hospital and CVC factors using chi-squared, Mann-Whitney and Fisher's exact tests. In the multivariate analysis, age was not a significant factor for LDHI use. Patients with pulmonary hypertension had decreased LDHI use while those with active surgical or trauma diagnoses had increased LDHI use. All central CVC insertion sites were more likely to use LDHI when compared to peripherally inserted CVCs. The Asia-Pacific region showed increased LDHI use, along with community hospitals and smaller ICUs (<10 beds). Conclusion Patient, CVC, and hospital factors are associated with the use of LDHI in critically ill children. Further study is needed to evaluate the efficacy and persistence of LDHI use. PMID:27362853

  19. Molecular Insights into the Klotho-Dependent, Endocrine Mode of Action of Fibroblast Growth Factor 19 Subfamily Members

    SciTech Connect

    Goetz,R.; Beenken, A.; Ibrahimi, O.; Kalinina, J.; Olsen, S.; Eliseenkova, A.; Xu, C.; Neubert, T.; Zhang, F.; et al.

    2007-01-01

    Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/{beta}Klotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between {beta} strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the {beta}1-{beta}2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/{beta}Klotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.

  20. Effect of gentamicin on heparin activity.

    PubMed

    Tyler, L S; Rehder, T L; Davis, R B

    1981-04-01

    The importance of the effect of gentamicin on heparin activity was investigated. Heparin activity was assessed using the activated partial thromboplastin time (APTT), thrombin clotting time (TCT), and Factor X heparin assay. Drug concentrations used were 0.1, 0.2, 0.3, and 0.4 units/ml heparin with 40, 80, and 400 microgram/ml gentamicin, in vitro in human plasma. The drugs were precipitated at concentrations of 10, 20, 30, and 40 units/ml heparin with 4 and 40 mg/ml gentamicin. After centrifuging, the supernate was diluted to 0.1-0.4 units/ml heparin for assay. There was no change in heparin activity in the presence of gentamicin (in the unprecipitated solutions) as measured by the TCT, Factor X assay, and the APTT; however, APTT was prolonged by gentamicin. In the precipitated samples, heparin activity was lost in the precipitate. No significant clinical interaction that would affect the therapeutic efficacy of heparin was demonstrated except for a pharmaceutical incompatibility between gentamicin and heparin. The APTT, performed with ellagic acid as an activator, is prolonged by gentamicin.

  1. Growth factors and myometrium: biological effects in uterine fibroid and possible clinical implications

    PubMed Central

    Ciarmela, Pasquapina; Islam, Md. Soriful; Reis, Fernando M.; Gray, Peter C.; Bloise, Enrrico; Petraglia, Felice; Vale, Wylie; Castellucci, Mario

    2011-01-01

    BACKGROUND Growth factors are proteins secreted by a number of cell types that are capable of modulating cellular growth, proliferation and cellular differentiation. It is well accepted that uterine cellular events such as proliferation and differentiation are regulated by sex steroids and their actions in target tissues are mediated by local production of growth factors acting through paracrine and/or autocrine mechanisms. Myometrial mass is ultimately modified in pregnancy as well as in tumour conditions such as leiomyoma and leiomyosarcoma. Leiomyomas, also known as fibroids, are benign tumours of the uterus, considered to be one of the most frequent causes of infertility in reproductive years in women. METHODS For this review, we searched the database MEDLINE and Google Scholar for articles with content related to growth factors acting on myometrium; the findings are hereby reviewed and discussed. RESULTS Different growth factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and TGF-β perform actions in myometrium and in leiomyomas. In addition to these growth factors, activin and myostatin have been recently identified in myometrium and leiomyoma. CONCLUSIONS Growth factors play an important role in the mechanisms involved in myometrial patho-physiology. PMID:21788281

  2. Factors predicting recurrence of chronic subdural haematoma: the influence of intraoperative irrigation and low-molecular-weight heparin thromboprophylaxis.

    PubMed

    Tahsim-Oglou, Yasemin; Beseoglu, Kerim; Hänggi, Daniel; Stummer, Walter; Steiger, Hans-Jakob

    2012-06-01

    Burr-hole drainage has become the accepted treatment of choice for chronic subdural haematoma (cSDH), although still burdened with a major recurrence rate. The current analysis was initiated to determine management-related risk factors for recurrence, i.e. postoperative low-molecular-weight heparin thromboprophylaxis, and the importance of rinsing the subdural space. Two-hundred and forty-seven patients with computerised tomography (CT) defined symptomatic cSDH were managed by two burr-hole trepanations and drainage between January 2005 and November 2008. Postoperative thromboprophylaxis with 40 mg enoxaparine daily was given only during the first half of the study period. For the current analysis the amount of rinsing fluid, postoperative low-dose thromboprophylaxis, as well as age and gender, bilaterality, preoperative and postoperative blood coagulation studies, platelet counts and decrease of subdural fluid on early postoperative CT, were recorded and correlated with recurrence. Statistical calculation was done by univariate and multivariate analysis. A total of 62 of 247 patients needed revision surgery for recurrence (25.1 %). Recurrence rates were significantly lower in the patients treated without postoperative enoxaparine (18.84 %) than in the group with postoperative low-dose enoxaparine thromboprophylaxis (32.11 %) and enoxaparine was administered in a higher proportion of the patients suffering recurrence (P = 0.013). A median intraoperative irrigation volume of 863 ml saline was used in the patients suffering recurrence and 1,500 ml in patients without recurrence (P < 0.001). The median age was slightly higher in the patients suffering from recurrence. Male gender predominated in both groups but was slightly more pronounced in the recurrence group. Preoperative and postoperative platelet counts and plasmatic coagulation indices did not differ significantly between the groups. Relative residual subdural fluid collection on early postoperative CT

  3. ON THE ORIGIN OF HEPARIN

    PubMed Central

    Oliver, Jean; Bloom, Frank; Mangieri, Carmen

    1947-01-01

    1. The spontaneous mast cell tumor of the dog contains heparin. 2. The cytoplasmic particulate content of the tumor mast cells varies with their anaplasia. This conclusion is based on the following findings: (a) in the immature cell of the more malignant tumor the particulate matter appeared in the living cells by phase microscopy to be composed of greyish illdefined particles or as a fine, weakly metachromatic granulation in the fixed and stained preparation; (b) in the mature cells of a relatively benign mast cell tumor, both in the living cell and in stained preparations, the particulate matter occurred in the form of discrete, dense, and strongly metachromatic granules, resembling those of the normal mast cell. 3. The heparin content was large (fifty times that of dog liver) in the growth with mature cells and only moderate (1.7 times) in that with immature cells. 4. Since there may be a great amount of greyish particulate matter (or fine stained granules) in a tumor of relatively low heparin content, it is suggested that this material represents an early or precursor phase in the development of heparin. 5. This possibility and the fact that the blood stream may be invaded by mature tumor mast cells of large heparin content without evident disturbance in the coagulability of the blood suggest the value of a comprehensive biochemical study of the heparin of mast cell tumors. PMID:19871658

  4. Heparin fragments inhibit human vascular smooth muscle cell proliferation in vitro

    SciTech Connect

    Selden, S.C.; Johnson, W.V.; Maciag, T.

    1986-03-01

    The authors have examined the effect of heparin on human abdominal aortic smooth muscle cell growth. Cell proliferation was inhibited by more than 90% at a concentration of 20 ..mu..g/ml in a 12 day growth assay using heparin from Sigma, Upjohn or Calbiochem. Additionally, 200 ..mu..g/ml Upjohn heparin inhibits /sup 3/H-thymidine incorporation by 50% in short term assays using serum or purified platelet-derived growth factor (25-100ng/ml) to initiate the cell cycle. Homogeneous size classes of heparin fragments were prepared by nitrous acid cleavage and BioGel P-10 filtration chromatography. Deca-, octa-, hexa-, tetra-, and di-saccharides inhibited proliferation by 90% at concentrations of 280, 320, 260, 180 and 100 ..mu..g/ml, respectively, in a 12 day growth assay. These data confirm the work of Castellot et.al. and extend the range of inhibitory fragments down to the tetra- and di-saccharide size. These data suggest, therefore, that di-saccharide subunit of heparin is sufficient to inhibit vascular smooth muscle cell proliferation. The authors are now examining the role of the anhydromannose moiety on the reducing end of the nitrous acid generated fragments as a possible mediator of heparin-induced inhibition of vascular smooth muscle cell proliferation.

  5. Aggrecan-mimetic, glycosaminoglycan-containing nanoparticles for growth factor stabilization and delivery.

    PubMed

    Place, Laura W; Sekyi, Maria; Kipper, Matt J

    2014-02-10

    The direct delivery of growth factors to sites of tissue healing is complicated by their relative instability. In many tissues, the glycosaminoglycan (GAG) side chains of proteoglycans like aggrecan stabilize growth factors in the pericellular and extracellular space, creating a local reservoir that can be accessed during a wound healing response. GAGs also regulate growth factor-receptor interactions at the cell surface. Here we report the development of nanoparticles for growth factor delivery that mimic the size, GAG composition, and growth factor binding and stabilization of aggrecan. The aggrecan-mimetic nanoparticles are easy to assemble, and their structure and composition can be readily tuned to alter their physical and biological properties. We use basic fibroblast growth factor (FGF-2) as a model heparin-binding growth factor, demonstrating that aggrecan-mimetic nanoparticles can preserve its activity for more than three weeks. We evaluate FGF-2 activity by measuring both the proliferation and metabolic activity of bone marrow stromal cells to demonstrate that chondroitin sulfate-based aggrecan mimics are as effective as aggrecan, and heparin-based aggrecan mimics are superior to aggrecan as delivery vehicles for FGF-2.

  6. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... coagulation factor X (Stuart factor) or procedures based on the neutralization of heparin by protamine sulfate...

  7. The binding of chondroitin sulfate to pleiotrophin/heparin-binding growth-associated molecule is regulated by chain length and oversulfated structures.

    PubMed

    Maeda, Nobuaki; Fukazawa, Nobuna; Hata, Toshihiro

    2006-02-24

    Pleiotrophin is an 18-kDa heparin-binding growth factor, which uses chondroitin sulfate (CS) proteoglycan, PTPzeta as a receptor. It has been suggested that the D-type structure (GlcA(2S)beta1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTPzeta and pleiotrophin. Here, we analyzed the interaction of shark cartilage CS-D with pleiotrophin using a surface plasmon resonance biosensor to reveal the importance of D-type structure. CS-D was partially digested with chondroitinase ABC, and fractionated using a Superdex 75pg column. The > or =18-mer CS fractions showed significant binding to pleiotrophin, and the longer fractions had stronger affinity for pleiotrophin than the shorter ones. The approximately 46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (K(D) = approximately 30 nM), and the binding reactions fitted the bivalent analyte model. However, when the density of the immobilized pleiotrophin was lowered, the strength of affinity remarkably decreased (K(D) = approximately 2.5 microM), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20 approximately 24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (K(D) = 3 approximately 20 microM), which seemed to be monovalent. When approximately 22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (K(D) = 0.36 approximately >10 microM), and the affinity correlated with the amounts of D- and E- (GlcAbeta1-3GalNAc(4S,6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS approximately 20 mer as a unit and by the amounts of oversulfated structures.

  8. Injectable gelatin derivative hydrogels with sustained vascular endothelial growth factor release for induced angiogenesis

    PubMed Central

    Li, Zhe; Qu, Tiejun; Ding, Chen; Ma, Chi; Sun, Hongchen; Li, Shirong; Liu, Xiaohua

    2014-01-01

    Injectable biomaterials are attractive for soft tissue regeneration because they are handled in a minimally invasive manner and can easily adapt to complex defects. However, inadequate vascularization of the injectable constructs has long been a barrier, leading to necrosis or volume reduction after implantation. In this work, we developed a three-step process to synthesize injectable gelatin-derived hydrogels that are capable of controlling growth factor delivery to induce angiogenesis. In our approach, tyramine was first introduced into gelatin chains to provide enzymatical crosslinking points for gel formation after injection. Next, heparin, a polysaccharide with binding domains to many growth factors, was covalently linked to the tyramine-modified gelatin. Finally, vascular endothelial growth factor (VEGF) was incorporated into the gelatin derivative by binding with the heparin in the gelatin derivative, and an injectable gel with controlled VEGF release was formed by an enzymatic catalytic reaction with hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The gelation time, mechanical properties and degradation of the gel was readily tailored by the gelatin concentration and the ratio of H2O2/HRP. Binding VEGF to heparin stabilizes this growth factor, protects it from denaturation and proteolytic degradation, and subsequently prolongs the sustained release. An in vitro release study and bioactivity assay indicated that the VEGF was released in a sustained manner with high bioactivity for over 3 weeks. Furthermore, a chicken chorioallantoic membrane (CAM) assay and animal experiments were performed to evaluate in vivo bioactivity of the VEGF released from the hydrogels. After 5 days of incubation on CAM, the number of blood vessels surrounding the heparin-modified hydrogels was 2.4-fold increase than that of the control group. Deeper and denser cell infiltration and angiogenesis in the heparin-modified gelatin/VEGF gels were observed than in the controls

  9. Injectable gelatin derivative hydrogels with sustained vascular endothelial growth factor release for induced angiogenesis.

    PubMed

    Li, Zhe; Qu, Tiejun; Ding, Chen; Ma, Chi; Sun, Hongchen; Li, Shirong; Liu, Xiaohua

    2015-02-01

    Injectable biomaterials are attractive for soft tissue regeneration because they are handled in a minimally invasive manner and can easily adapt to complex defects. However, inadequate vascularization of the injectable constructs has long been a barrier, leading to necrosis or volume reduction after implantation. In this work, we developed a three-step process to synthesize injectable gelatin-derived hydrogels that are capable of controlling growth factor delivery to induce angiogenesis. In our approach, tyramine was first introduced into gelatin chains to provide enzymatic crosslinking points for gel formation after injection. Next, heparin, a polysaccharide with binding domains to many growth factors, was covalently linked to the tyramine-modified gelatin. Finally, vascular endothelial growth factor (VEGF) was incorporated into the gelatin derivative by binding with the heparin in the gelatin derivative, and an injectable gel with controlled VEGF release was formed by an enzymatic catalytic reaction with hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The gelation time, mechanical properties and degradation of the gel was readily tailored by the gelatin concentration and the ratio of H2O2/HRP. Binding VEGF to heparin stabilizes this growth factor, protects it from denaturation and proteolytic degradation and subsequently prolongs the sustained release. An in vitro release study and bioactivity assay indicated that the VEGF was released in a sustained manner with high bioactivity for over 3 weeks. Furthermore, a chicken chorioallantoic membrane (CAM) assay and animal experiments were performed to evaluate in vivo bioactivity of the VEGF released from the hydrogels. After 5 days of incubation on CAM, the number of blood vessels surrounding the heparin-modified hydrogels was increased by 2.4-fold compared with that of the control group. Deeper and denser cell infiltration and angiogenesis in the heparin-modified gelatin/VEGF gels were observed compared to

  10. A case of a severe factor XI deficiency in patient undergoing hemodialysis without the use of heparin.

    PubMed

    Takamizawa, Yasuyuki; Araki, Makoto; Yoshida, Noriko; Yoshioka, Teruaki; Miura, Kohei

    2014-12-01

    Factor XI (FXI) deficiency is a rare hematologic disease, and shows a less severe bleeding tendency compared with what is generally observed in patients with hemophilia A and B. FXI has received a lot of attention in recent years as a new therapeutic target. Here, we present a case of 59-year-old male patient with chronic renal failure. The patient was found to have a markedly prolonged activated partial thromboplastin time (aPTT) during routine preoperative blood test before an arteriovenous fistula surgery. Finally, he was diagnosed with FXI deficiency. More than 6 months after the start of hemodialysis, no sign of blood clotting in the extracorporeal circuit has been observed. Of note, the patient did not receive any anticoagulant during hemodialysis, and he did not show any bleeding tendency even with aPTT more than 120 s and FXI activity below 3% of normal in a patient with renal failure. To our knowledge, this is the first case report to demonstrate FXI deficiency exhibiting anticoagulant effect equivalent to heparin in dialysis.

  11. The binding of pentapeptides to biological and synthetic high affinity heparin.

    PubMed

    Flengsrud, Ragnar; Antonsen, Simen Gjelseth

    2015-11-01

    Pentapeptides have been shown to bind the synthetic heparin fondaparinux (Arixtra) as well the biological heparins dalteparin (Fragmin) and salmon heparin. In contrast to heparin binding consensus sequences, the pentapeptides are acidic or neutral, with no arginine or histidine residue. The peptides showed an effect on in vitro heparin anti-factor X activity with a reduction of fondaparinux activity by 65-95%. Heparin binding was further studied by using peptide solid phase chromatography and NMR analysis.

  12. A composite fibrin-based scaffold for controlled delivery of bioactive pro-angiogenetic growth factors.

    PubMed

    Briganti, Enrica; Spiller, Dario; Mirtelli, Chiara; Kull, Silvia; Counoupas, Claudio; Losi, Paola; Senesi, Sonia; Di Stefano, Rossella; Soldani, Giorgio

    2010-02-25

    The aim of this study was to fabricate and characterize in vitro a novel composite scaffold that, combining good mechanical properties with a controlled and sustained release of bioactive pro-angiogenetic growth factors, should be useful for angiogenesis induction in organs/tissues in which is also necessary to give resistance and mechanical strength. Composite scaffolds, constituted by a synthetic biocompatible material, a poly(ether)urethane-polydimethylsiloxane blend, and a biological polymer, the fibrin, were manufactured by spray, phase-inversion technique. During the manufacturing process heparin and heparin-binding growth factors, such as VEGF(165) and bFGF, were incorporated into the fibrin layer. Microscopical examinations showed a homogeneous fibrin layer firmly adherent on top of the synthetic material. Tensile tests highlighted the high elasticity of the composite scaffold and its capability to maintain integrity up to high deformation. VEGF(165) and bFGF release were controlled by fibrinogen concentration, whereas it was not affected by heparin concentration, as revealed by ELISA assay. The biological activity of the released growth factors was maintained as demonstrated by HUVEC proliferation. Finally, scaffolds induced a low monocyte mRNA expression of inflammatory markers (IL-8, L-SEL, LFA-1 and iNOS). In conclusion, the new composite scaffolds, once implanted, providing a co-localization and temporal distribution of bioactive VEGF and bFGF in addition to good mechanical properties, may be useful to stimulate new vessels formation in ischemic tissues.

  13. Assessment of anti-factor Xa activity of heparin in binary parenteral nutrition admixtures for premature neonates.

    PubMed

    Foinard, A; Perez, M; Barthélémy, C; Lannoy, D; Flamein, F; Storme, L; Tournoys, A; Décaudin, B; Odou, P

    2015-07-01

    An in vitro study was carried out to determine the anti-Xa activity of heparin in binary parenteral nutrition (BPN) admixtures for premature neonates in our neonatal intensive care unit (NICU) after a 24-hour infusion, as well as to assess drug interaction with a 50% glucose solution. Two types of bags were prepared: (1) BPN admixtures (composition defined in the NICU) including sodium heparin at 77 UI/mL and (2) bags containing only G50% with sodium heparin at 193 UI/mL. The anti-Xa activity of heparin was measured in bags at T0, after the 24-hour infusion and in eluates at the outlet of the infusion line after 24hours, using a validated chromogenic anti-Xa method. Comparisons of the mean concentration observed with the theoretical value for anti-Xa activity were performed with the Student t-test. Mean values of anti-Xa activity do not differ significantly from the values expected for all conditions. We found a slight variation in anti-Xa activity when infused over 24hours for both types of bags, with and without in-line filtration, showing that heparin remains stable during this infusion period in both BPN admixtures and G50%.

  14. Interstitial fibrosis and growth factors.

    PubMed Central

    Lasky, J A; Brody, A R

    2000-01-01

    Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused by a variety of inhaled agents including mineral particles, organic dusts, and oxidant gases. The disease afflicts millions of individuals worldwide, and there are no effective therapeutic approaches. A major reason for this lack of useful treatments is that few of the molecular mechanisms of disease have been defined sufficiently to design appropriate targets for therapy. Our laboratory has focused on the molecular mechanisms through which three selected peptide growth factors could play a role in the development of IPF. Hundreds of growth factors and cytokines could be involved in the complex disease process. We are studying platelet-derived growth factor because it is the most potent mesenchymal cell mitogen yet described, transforming growth factor beta because it is a powerful inducer of extracellular matrix (scar tissue) components by mesenchymal cells, and tumor necrosis factor alpha because it is a pleiotropic cytokine that we and others have shown is essential for the development of IPF in animal models. This review describes some of the evidence from studies in humans, in animal models, and in vitro, that supports the growth factor hypothesis. The use of modern molecular and transgenic technologies could elucidate those targets that will allow effective therapeutic approaches. Images Figure 1 Figure 2 PMID:10931794

  15. Growth factors in synaptic function

    PubMed Central

    Poon, Vivian Y.; Choi, Sojoong; Park, Mikyoung

    2013-01-01

    Synapses are increasingly recognized as key structures that malfunction in disorders like schizophrenia, mental retardation, and neurodegenerative diseases. The importance and complexity of the synapse has fuelled research into the molecular mechanisms underlying synaptogenesis, synaptic transmission, and plasticity. In this regard, neurotrophic factors such as netrin, Wnt, transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), and others have gained prominence for their ability to regulate synaptic function. Several of these factors were first implicated in neuroprotection, neuronal growth, and axon guidance. However, their roles in synaptic development and function have become increasingly clear, and the downstream signaling pathways employed by these factors have begun to be elucidated. In this review, we will address the role of these factors and their downstream effectors in synaptic function in vivo and in cultured neurons. PMID:24065916

  16. Heparins that block VEGF-A-mediated von Willebrand factor fiber generation are potent inhibitors of hematogenous but not lymphatic metastasis

    PubMed Central

    Desch, Anna; Mayer, Frank Thomas; Koett, Julian; Nowak, Kai; Karampinis, Ioannis; Bohlmann, Michael K.; Umansky, Viktor; Bauer, Alexander Thomas

    2016-01-01

    Von Willebrand factor (VWF) serves as a nidus for platelet aggregation and thrombosis. We hypothesize that VWF fibers contribute to the development of venous thromboembolism (VTE) and to metastasis formation. Here, we show that vascular and lymphatic endothelial cells (ECs) express VWF in vitro and release VWF fibers after activation by tumor cell supernatants. In contrast, an ex vivo analysis of primary mouse tumors revealed the presence of VWF fibers in the blood microvasculature but not in lymphatic vessels. Unlike the anticoagulant Fondaparinux, an inhibitor of thrombin generation, the low-molecular-weight heparin (LMWH) Tinzaparin inhibited VWF fiber formation and vessel occlusion in tumor vessels by blocking thrombin-induced EC activation and vascular endothelial growth factor-A (VEGF-A)-mediated VWF release. Intradermal tumor cell inoculation in VWF- and ADAMTS13-deficient mice did not alter lymph node metastases compared with wild type animals. Interestingly, multiple tumor-free distal organs exhibited hallmarks of malignancy-related VTE, including luminal VWF fibers, platelet-rich thrombi and vessel occlusions. Furthermore, ADAMTS13 deficiency, characterized by prolonged intraluminal VWF network lifetimes, resulted in a severely increased number of metastatic foci in an experimental model of hematogenous lung seeding. Treatment with Tinzaparin inhibited tumor-induced release of VWF multimers, impeded platelet aggregation and decreased lung metastasis. Thus, our data strongly suggest a critical role of luminal VWF fibers in determining the occurrence of thrombosis and cancer metastasis. Moreover, the findings highlight LMWHs as therapeutic strategy to treat thrombotic complications while executing anti-metastatic activities. PMID:27602496

  17. Structural and Functional Basis of CXCL12 (stromal cell-derived factor-1 alpha) Binding to Heparin

    SciTech Connect

    Murphy,J.; Cho, Y.; Sachpatzidis, A.; Fan, C.; Hodsdon, M.; Lolis, E.

    2007-01-01

    CXCL12 (SDF-1a) and CXCR4 are critical for embryonic development and cellular migration in adults. These proteins are involved in HIV-1 infection, cancer metastasis, and WHIM disease. Sequestration and presentation of CXCL12 to CXCR4 by glycosaminoglycans (GAGs) is proposed to be important for receptor activation. Mutagenesis has identified CXCL12 residues that bind to heparin. However, the molecular details of this interaction have not yet been determined. Here we demonstrate that soluble heparin and heparan sulfate negatively affect CXCL12-mediated in vitro chemotaxis. We also show that a cluster of basic residues in the dimer interface is required for chemotaxis and is a target for inhibition by heparin. We present structural evidence for binding of an unsaturated heparin disaccharide to CXCL12 attained through solution NMR spectroscopy and x-ray crystallography. Increasing concentrations of the disaccharide altered the two-dimensional 1H-15N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to {beta}-strands in the dimer interface. The second includes the amino-terminal loop and the a-helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His25, Lys27, and Arg41 of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala20, Arg21, Asn30, and Lys64. This is the first x-ray structure of a CXC class chemokine in complex with glycosaminoglycans. Based on the observation of two heparin binding sites, we propose a mechanism in which GAGs bind around CXCL12 dimers as they sequester and present CXCL12 to CXCR4.

  18. Destabilization, oligomerization and inhibition of the mitogenic activity of acidic fibroblast-growth factor by aurintricarboxylic acid.

    PubMed

    Lozano, R M; Rivas, G; Giménez-Gallego, G

    1997-08-15

    The triphenylmethane derivative aurintricarboxylic acid has been used to inhibit angiogenesis, vascular smooth muscle cell proliferation and cell transformation, an effect that has been attributed to its relatively nonspecific inhibitory activity of protein-nucleic acid interactions. Here, we show that this compound binds to acidic fibroblast growth factor, a prototypic member of a family of protein mitogens activated by heparin, altering its physicochemical properties and decreasing its mitogenic activity. Counteraction of the effects of aurintricarboxylic acid by heparin shows that the two compounds have opposite and reversible effects on acidic fibroblast growth factor structure and biological activity. The studies reported here may contribute to a deeper understanding of the inhibition of fibroblast-growth-factor-dependent mitogenesis of relevance to future pharmacologic developments.

  19. Low-density lipoprotein apheresis reduces platelet factor 4 on the surface of platelets: a possible protective mechanism against heparin-induced thrombocytopenia and thrombosis.

    PubMed

    Tanhehco, Yvette C; Rux, Ann H; Sachais, Bruce S

    2011-05-01

    Heparin-induced thrombocytopenia and thrombosis (HITT) is characterized by thrombocytopenia due to the formation of antibodies against heparin : platelet factor 4 (PF4) complexes. Despite the exposure to heparin during treatment and predisposition of patients with atherosclerosis to HITT, HITT in patients undergoing low-density lipoprotein (LDL) apheresis is rare. We investigated the possibility that LDL apheresis decreases PF4 on platelet (PLT) surfaces and/or plasma HITT antibody levels, either of which would disfavor HITT. We enrolled 25 patients undergoing LDL apheresis at the Hospital of the University of Pennsylvania. Blood samples were drawn before and after treatment. Plasma samples were drawn proximal and distal to the LA-15 treatment column. PF4, HITT antibodies, heparin levels, and P-selectin were measured. No patient had clinical symptoms of HITT. The LA-15 column was found to efficiently remove PF4. PF4 levels in peripheral blood plasma did not change significantly after LDL apheresis. However, PLT surface PF4 significantly decreased after treatment. HITT antibodies were found in only two patients and were nonfunctional. PLT surface P-selectin did not change during treatment. We have demonstrated that LDL apheresis via dextran sulfate absorption removes plasma PF4 and reduces the amount of PF4 on the surface of circulating PLTs. Reduced surface PF4 may decrease antibody formation and/or recognition by HITT antibodies. These data provide a potential explanation for the near lack of HITT in hypercholesterolemic patients undergoing LDL apheresis. They also suggest the possibility that LDL apheresis using dextran sulfate adsorption may have therapeutic value in the treatment of HITT. © 2010 American Association of Blood Banks.

  20. Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

    PubMed Central

    Hartmann, G.; Krug, A.; Waller-Fontaine, K.; Endres, S.

    1996-01-01

    BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for

  1. Dynamic properties of biologically active synthetic heparin-like hexasaccharides.

    PubMed

    Angulo, Jesús; Hricovíni, Milos; Gairi, Margarida; Guerrini, Marco; de Paz, José Luis; Ojeda, Rafael; Martín-Lomas, Manuel; Nieto, Pedro M

    2005-10-01

    A complete study of the dynamics of two synthetic heparin-like hexasaccharides, D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (1) and -->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHAc-6-SO4-alpha-(1-->4)-L-IdoA-alpha-(1-->4)-D-GlcNHSO3-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (2), has been performed using 13C-nuclear magnetic resonance (NMR) relaxation parameters, T1, T2, and heteronuclear nuclear Overhauser effect (NOEs). Compound 1 is constituted from sequences corresponding to the major polysaccharide heparin region, while compound 2 contains a sequence never found in natural heparin. They differ from each other only in sulphation patterns, and are capable of stimulating fibroblast growth factors (FGFs)-1 induced mitogenesis. Both oligosaccharides exhibit a remarkable anisotropic overall motion in solution as revealed by their anisotropic ratios (tau /tau||), 4.0 and 3.0 respectively. This is a characteristic behaviour of natural glycosaminoglycans (GAG) which has also been observed for the antithrombin (AT) binding pentasaccharide D-GlcNHSO3-6-SO4-alpha-(1-->4)-D-GlcA-beta-(1-->4)-D-GlcNHSO3-(3,6-SO4)-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-1-->Me (3) (Hricovíni, M., Guerrini, M., Torri, G., Piani, S., and Ungarelli, F. (1995) Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr. Res., 277, 11-23). The motional properties observed for 1 and 2 provide additional support to the suitability of these compounds as heparin models in agreement with previous structural (de Paz, J.L., Angulo, J., Lassaletta, J.M., Nieto, P.M., Redondo-Horcajo, M., Lozano, R.M., Jiménez-Gallego, G., and Martín-Lomas, M. (2001) The activation of fibroblast growth factors by heparin: synthesis, structure and biological activity of heparin-like oligosaccharides. Chembiochem, 2, 673-685; Ojeda, R

  2. Heparin-induced thrombocytopaenia.

    PubMed

    Gounden, Ronald; Blockman, Marc

    2008-01-01

    Heparin-induced thrombocytopaenia (HIT) is an acquired, transient prothrombotic disorder caused by heparin. The predominant problem is the creation of a prothrombotic milieu, accompanied by a fall in the platelet count. This explains the apparent paradox of thrombosis in the face of thrombocytopaenia and why non-heparin antithrombotic agents are integral to its management.

  3. Heparin functionalized polyaspartamide/polyester scaffold for potential blood vessel regeneration.

    PubMed

    Pitarresi, Giovanna; Fiorica, Calogero; Palumbo, Fabio Salvatore; Rigogliuso, Salvatrice; Ghersi, Giulio; Giammona, Gaetano

    2014-05-01

    An interesting issue in tissue engineering is the development of a biodegradable vascular graft able to substitute a blood vessel and to allow its complete regeneration. Here, we report a new scaffold potentially useful as a synthetic vascular graft, produced through the electrospinning of α,β-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-D,L-aspartamide-graft-polylactic acid (PHEA-EDA-g-PLA) in the presence of polycaprolactone (PCL). The scaffold degradation profile has been evaluated as well as the possibility to bind heparin to electrospun fibers, being it a known anticoagulant molecule able to bind growth factors. In vitro cell compatibility has been investigated using human vascular endothelial cells (ECV 304) and the ability of heparinized PHEA-EDA-g-PLA/PCL scaffold to retain basic fibroblast growth factor has been evaluated in comparison with not heparinized sample.

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

    PubMed

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

    2016-03-04

    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.

  5. Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

    PubMed Central

    Balestreire, Elena M.

    2007-01-01

    The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling. PMID:17287395

  6. Effect of sulodexide on plasma transforming growth factor-beta1 in healthy volunteers.

    PubMed

    Borawski, Jacek; Dubowski, Miroslaw; Pawlak, Krystyna; Mysliwiec, Michal

    2010-02-01

    It is unknown whether the glycosaminoglycan drug sulodexide interferes with transforming growth factor-beta1--a member of heparin-binding family and a potent regulator of human biology and diseases. Hence, a 2-week pilot study was performed in 11 healthy men. Sulodexide was initially administered intravenously in a single dose, then--orally for 12 days and--again intravenously on study completion. Initial injection had no effect on activated form of the growth factor measured in plasma after 10 and 120 min; no change was also observed after 120 min from drug ingestion on day 7. On final intravenous administration, the growth factor levels increased by almost 60% after 10 min and remained elevated; the 120-min levels directly correlated with sulodexide dosage. Baseline cytokine levels decreased during the 2-week trial by more than 50%. In conclusion, transforming growth factor-beta1 release and likely downregulation of its expression may constitute novel pharmacological effects of sulodexide.

  7. The autoactivation of factor XII (Hageman factor) induced by low-Mr heparin and dextran sulphate. The effect of the Mr of the activating polyanion.

    PubMed Central

    Silverberg, M; Diehl, S V

    1987-01-01

    Human Factor XII is known to undergo autoactivation in the presence of dextran sulphate of Mr 500,000. We have now studied the dependence of this reaction on the Mr of the dextran sulphate by using fractions resolved by gel filtration. We have found that autoactivation can be induced by dextran sulphate fractions with Mr as low as 3000, and there is a marked dependence of the rate constant of autoactivation on the Mr value. Fractions with Mr below 8000 gave very low rates of autoactivation; there was a sharp increase in the rate obtained when the Mr of the dextran sulphate was greater than 10,000. Various preparations of heparin were also able to support the autoactivation of Factor XII and gave a very similar relationship between molecular size and reaction rate. The data provide support for the hypothesis that the mechanism by which the 'surface' acts in contact activation involves the presence, on the same particle, of multiple binding sites for the proteins. PMID:2449171

  8. Epidermal Growth Factor-Like Growth Factors Prevent Apoptosis of Alcohol-Exposed Human Placental Cytotrophoblast Cells1

    PubMed Central

    Wolff, Garen S.; Chiang, Po Jen; Smith, Susan M.; Romero, Roberto; Armant, D. Randall

    2007-01-01

    Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0–100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1–2 h of exposure to 50 mM alcohol. Exposure to 25–50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism. PMID:17392498

  9. Heparinized polyurethanes: in vitro and in vivo studies.

    PubMed

    Heyman, P W; Cho, C S; McRea, J C; Olsen, D B; Kim, S W

    1985-04-01

    Heparin immobilization chemistry using alkyl spacer arms was adapted to optimize yield on polyurethane (PU) surfaces. The resultant biological activity of immobilized heparin (HI) was examined in vitro and in vivo, and compared with a heparin releasing (HR) system. Immobilized heparin retained its ability to bind and inactivate thrombin and Factor Xa; nonspecific coagulation factor binding was insignificant. Such activity cannot be attributed to the leakage of improperly bound heparin. Immobilized heparin-polyurethane catheters implanted in canine femoral and jugular veins for 1 h periods exhibited significant reduction in thrombus formation compared with untreated PU contralateral controls. Polyurethane catheters coated with a 9% heparin dispersion in PU (HR) system provided even greater improvement in antithrombogenicity.

  10. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  11. Recurrent heparin-induced thrombocytopenia due to heparin rinsing before priming the machine in a hemodialysis patient: A case report.

    PubMed

    Lim, Jin Han; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Kim, Won

    2016-10-13

    Heparin has remained the most commonly used anticoagulant for patients undergoing hemodialysis. It is usually safe to use but can have severe adverse effects in some cases. Heparin-induced thrombocytopenia (HIT) is a life-threatening complication of exposure to heparin. It results from an autoantibody directed against endogenous platelet factor 4 (PF4) in complex with heparin, which activates platelets and can cause catastrophic arterial and venous thromboses. Here, we present the case of an 80-year-old woman with a recent diagnosis of chronic renal failure who developed acute HIT (platelet count nadir, 15 × 10(9) /L) on day 7 of hemodialysis performed with routine heparin anticoagulation, who despite subsequent heparin-free hemodialysis (with argatroban and warfarin) developed recurrent HIT (complicated by acute cerebral infarction) on day 11 that we attributed to "rinsing" of the circuit with heparin-containing saline (3,000 units of unfractionated heparin, with subsequent saline washing) performed pre-dialysis as per routine. After stopping heparin rinsing, the platelet count recovered completely, without further thrombotic or other sequelae. Our experience indicates that for patients with acute HIT, besides the well-known practice of using non-heparin anticoagulation during dialysis and avoiding heparin "locking" of dialysis catheters, it is also important to avoid inadvertent rinsing of the circuit with heparin during preparation for hemodialysis.

  12. Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis.

    PubMed

    Relf, M; LeJeune, S; Scott, P A; Fox, S; Smith, K; Leek, R; Moghaddam, A; Whitehouse, R; Bicknell, R; Harris, A L

    1997-03-01

    Angiogenesis is a significant prognostic factor in breast cancer, but the factors that control angiogenesis in vivo are not well defined. Multiple angiogenic polypeptides are known, and we have determined the expression of seven of these in primary human breast cancers; the relationship of expression to estrogen receptor and vascular density was also examined. Vascular endothelial growth factor (VEGF) and its four isoforms (121, 165, 189, and 206 amino acids), transforming growth factor (TGF)-beta1, pleiotrophin, acidic and basic fibroblast growth factor (FGF), placental growth factor, and thymidine phosphorylase (platelet-derived endothelial cell growth factor) were quantitated by RNase protection analysis. beta-FGF was also measured by ELISA. The estrogen receptor (ER), epidermal growth factor receptor, and vascular density were analyzed in 64 primary breast cancers. All tumors expressed at least six different vascular growth factors. VEGF was most abundant, and the transcript for the 121-amino acid form predominated. Other angiogenic factors expressed at high levels were thymidine phosphorylase and TGF-beta1. Expression of most of the angiogenic factors did not correlate with that of ER or vascular density. However, thymidine phosphorylase did, with a correlation coefficient of 0.3 (P = 0.03). There were significant associations of pleiotrophin with acidic FGF expression (P = 0.001) and TGF-beta with platelet-derived endothelial cell growth factor expression (P = 0.001). Thus, angiogenesis may involve a coordinate regulation of some vascular growth factors. High VEGF expression correlated with poor prognosis in univariate analysis (P = 0.03), as did ER and epidermal growth factor receptor expression. Basic FGF was also assessed by ELISA and was more highly expressed in tumors than normal breast tissues (median, 346 microg/ml cytosol; range, 54-1323 versus median, 149; range, 32-509; P = 0.01). Implications for therapy are that broad spectrum agents that block

  13. Physiological factors influencing capillary growth.

    PubMed

    Egginton, S

    2011-07-01

    (1) Angiogenesis (growth of new capillaries from an existing capillary bed) may result from a mismatch in microvascular supply and metabolic demand (metabolic error signal). Krogh examined the distribution and number of capillaries to explore the correlation between O(2) delivery and O(2) consumption. Subsequently, the heterogeneity in angiogenic response within a muscle has been shown to reflect either differences in fibre type composition or mechanical load. However, local control leads to targetted angiogenesis in the vicinity of glycolytic fibre types following muscle stimulation, or oxidative fibres following endurance training, while heterogeneity of capillary spacing is maintained during ontogenetic growth. (2) Despite limited microscopy resolution and lack of specific markers, Krogh's interest in the structure of the capillary wall paved the way for understanding the mechanisms of capillary growth. Angiogenesis may be influenced by the response of perivascular or stromal cells (fibroblasts, macrophages and pericytes) to altered activity, likely acting as a source for chemical signals modulating capillary growth such as vascular endothelial growth factor. In addition, haemodynamic factors such as shear stress and muscle stretch play a significant role in adaptive remodelling of the microcirculation. (3) Most indices of capillarity are highly dependent on fibre size, resulting in possible bias because of scaling. To examine the consequences of capillary distribution, it is therefore helpful to quantify the area of tissue supplied by individual capillaries. This allows the spatial limitations inherent in most models of tissue oxygenation to be overcome generating an alternative approach to Krogh's tissue cylinder, the capillary domain, to improve descriptions of intracellular oxygen diffusion. © 2010 The Author. Acta Physiologica © 2010 Scandinavian Physiological Society.

  14. Heparin pharmacokinetics and pharmacodynamics.

    PubMed

    Kandrotas, R J

    1992-05-01

    Heparin was discovered approximately 75 years ago and has been used extensively for the last 50 years to treat thromboembolic disorders. An endogenous glycosaminoglycan, heparin is found largely in the liver, lung and intestine. It is available for exogenous administration both as unfractionated and low molecular weight heparin. Unfractionated heparin is a heterogenous mixture of polysaccharide chains of varying length resulting in a range of molecular weights from 3000 to 30,000D while low molecular weight heparin ranges from 3000 to 6000D. Heparin produces its antithrombotic effect by binding to antithrombin III and this complex then binds to thrombin. In order to accomplish this a total of 18 to 22 monosaccharide units is necessary including a specific pentasaccharide binding site for antithrombin III. After either subcutaneous or intravenous injection heparin is distributed primarily within the intravascular space. A short distribution phase is seen which is thought to correspond to endothelial cell binding and internalisation. The disposition curve for unfractionated heparin has a unique concave-convex shape which is the result of combined saturable and nonsaturable elimination mechanisms. The nonsaturable elimination mechanism is renal and is the primary route of elimination for low molecular weight heparins. For this reason, the concave-convex pattern is not seen with low molecular weight preparations. Both forms of heparin are useful antithrombotic agents; however, the correlation between the antithrombotic effect and an in vitro laboratory test for either type still needs further clarification.

  15. Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

    PubMed

    Bittoun, P; Bagheri-Yarmand, R; Chaubet, F; Crépin, M; Jozefonvicz, J; Fermandjian, S

    1999-06-15

    CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

  16. Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

    PubMed

    Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

    2015-01-01

    An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

  17. Adipose Tissue and Extracellular Matrix Development by Injectable Decellularized Adipose Matrix Loaded with Basic Fibroblast Growth Factor.

    PubMed

    Zhang, Shipin; Lu, Qiqi; Cao, Tong; Toh, Wei Seong

    2016-04-01

    There is a significant need for soft-tissue replacements in the field of reconstructive surgery. Decellularized adipose tissues were heparin crosslinked and loaded with basic fibroblast growth factor (bFGF). This injectable system was evaluated for its adipogenic and angiogenic capabilities for in vivo adipose tissue regeneration. Decellularized adipose tissues were harvested from the inguinal fat pads of C57BL/6J mice, minced, and heparinized before being loaded with bFGF. Decellularized adipose tissues without bFGF served as a control. In vivo adipose neotissue formation, neovascularization, and volume stability were evaluated over a period of 12 weeks. After 6 or 12 weeks, mice were killed and the newly formed adipose tissues, together with the contralateral endogenous adipose tissues, were harvested for gross, volumetric, histologic, and immunohistochemical analysis. Decellularized adipose tissues that were heparinized and loaded with bFGF induced significant de novo adipose neotissue formation, with progressive tissue growth and neovascularization from 6 to 12 weeks. The adipose neotissues exhibited mature adipose morphology and extracellular matrix that closely resembled that of the endogenous adipose tissue. In contrast, decellularized adipose tissues without bFGF induced limited adipose neotissue formation and were completely resorbed by the end of 12 weeks. This study demonstrates the high efficiency of heparinized decellularized adipose tissue matrix loaded with bFGF in promoting adipose neotissue formation and neovascularization with long-term volume stability.

  18. Enhanced Survival and Engraftment of Transplanted Stem Cells using Growth Factor Sequestering Hydrogels

    PubMed Central

    Jha, Amit K.; Tharp, Kevin M.; Ye, Jianqin; Santiago-Ortiz, Jorge L.; Jackson, Wesley M.; Stahl, Andreas; Schaffer, David V.; Yeghiazarians, Yerem; Healy, Kevin E.

    2015-01-01

    We have generated a bioinspired tunable system of hyaluronic acid (HyA)-based hydrogels for Matrix-Assisted Cell Transplantation (MACT). With this material, we have independently evaluated matrix parameters such as adhesion peptide density, mechanical properties, and growth factor sequestering capacity, to engineer an environment that imbues donor cells with a milieu that promotes survival and engraftment with host tissues after transplantation. Using a versatile population of Sca-1+/CD45− cardiac progenitor cells (CPCs), we demonstrated that the addition of heparin in the HyA hydrogels was necessary to coordinate the presentation of TGFβ1 and to support the trophic functions of the CPCs via endothelial cell differentiation and vascular like tubular network formation. Presentation of exogenous TGFβ1 by binding with heparin improved differentiated CPC function by sequestering additional endogenously-produced angiogenic factors. Finally, we demonstrated that TGFβ1 and heparin-containing HyA hydrogels can promote CPC survival when implanted subcutaneously into murine hind-limbs and encouraged their participation in the ensuing neovascular response, including blood vessels that had anastomosed with the host’s blood vessels. PMID:25682155

  19. Heparin-induced thrombocytopenia in pediatrics.

    PubMed

    Severin, T; Sutor, A H

    2001-06-01

    As in adult patients, heparin is used for prophylaxis and treatment of thromboembolism in newborns, children, and adolescents. Patients receiving heparin are potentially at risk to develop heparin-induced thrombocytopenia (HIT). HIT type II has been extensively described in the adult population; only a few reports address HIT type II in pediatric patients (total of 15 neonates, 4 young children, 12 older children and adolescents). The available data are discussed, and the case of a patient with recurrent thrombosis and HIT type II without thrombocytopenia is presented. The review of the literature reveals that HIT type II occurs especially in neonates and adolescents, corresponding to the two age peaks of thrombosis in pediatric patients. Risk factors for thrombosis include hereditary factors, immobilization, and surgery. HIT complications are severe and partly lead to life-threatening thromboembolism. In three patients, an increasing heparin demand was found. In five cases, thrombocytopenia was absent. Heparin was replaced mostly by danaparoid sodium; in three patients hirudin was used as an alternative anticoagulant. HIT type II represents a potentially dangerous complication of heparin therapy in pediatric patients and should be taken into consideration whenever heparin is given for prophylactic or therapeutic use in newborns, children, or adolescents.

  20. [Neuronal growth factors--neurotrophins].

    PubMed

    Meyer, M; Rasmussen, J Z

    1999-04-05

    Neurotrophic factors are polypeptides primarily known to regulate the survival and differentiation of nerve cells during the development of the peripheral and central nervous systems. The neurotrophic factors act via specific receptors after retrograde axonal transport from the nerve fibre target areas back to the cell bodies, and locally through autocrine and paracrine mechanisms linked to nerve cell activity. In the mature nervous system, neurotrophic factors maintain morphological and neurochemical characteristics of nerve cells and promote activity-dependent dynamic/plastic changes in the synaptic contacts between nerve cells by strengthening functionally active synaptic connections. Induction and increased production of neurotrophic factors in relation to neural injuries are thought to serve protective and reparative purposes. Specific neurotrophic factors have thus been shown to protect nerve cells in a number of experimental models for neurodegenerative diseases, such as Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis, just as specific neurotrophic factors have been shown to stimulate regenerative growth of both peripheral and central nerve fibres. Today, problems with continuous and localized delivery of specific neurotrophins or combinations thereof into the nervous system appear to be the most important obstacle for more widespread clinical application.

  1. Comparative pharmacodynamic assessment of the antiangiogenesis activity of heparin and low-molecular-weight heparin fractions: structure-function relationship.

    PubMed

    Mousa, Shaker A

    2013-01-01

    Effects of unfractionated heparin and low-molecular-weight heparins (LMWHs) on human microvascular endothelial cell sprouting (tube formation assay) in vitro were determined. Antiangiogenesis efficacy of commercially available LMWHs tinzaparin and enoxaparin in the chick chorioallantoic membrane (CAM) model of growth factor-induced angiogenesis was compared. The LMWH tinzaparin was fractionated into different molecular weight (MW) pools by size exclusion chromatography; they inhibited CAM angiogenesis depending on their MW distribution, with optimal inhibition at 8 to 12 kDa and no inhibition at <2 kDa. Tinzaparin demonstrated greater antiangiogenesis efficacy than enoxaparin (P < .001); these CAM results correlated with the endothelial tube formation assay results (P < .001, tinzaparin vs enoxaparin). These data point to the variable antiangiogenesis efficacy of different LMWHs as a function of MW and perhaps other structural differences. Our hypothesis confirmed a relationship between lower release of tissue factor pathway inhibitor by lower MW fractions of tinzaparin or enoxaparin leading to reduced antiangiogenesis efficacy.

  2. Injectable fibroblast growth factor-2 coacervate for persistent angiogenesis

    PubMed Central

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

    2011-01-01

    Enhancing the maturity of the newly formed blood vessels is critical for the success of therapeutic angiogenesis. The maturation of vasculature relies on active participation of mural cells to stabilize endothelium and a basal level of relevant growth factors. We set out to design and successfully achieved robust angiogenesis using an injectable polyvalent coacervate of a polycation, heparin, and fibroblast growth factor-2 (FGF2). FGF2 was loaded into the coacervate at nearly 100% efficiency. In vitro assays demonstrated that the matrix protected FGF2 from proteolytic degradations. FGF2 released from the coacervate was more effective in the differentiation of endothelial cells and chemotaxis of pericytes than free FGF2. One injection of 500 ng of FGF2 in the coacervate elicited comprehensive angiogenesis in vivo. The number of endothelial and mural cells increased significantly, and the local tissue contained more and larger blood vessels with increased circulation. Mural cells actively participated during the whole angiogenic process: Within 7 d of the injection, pericytes were recruited to close proximity of the endothelial cells. Mature vasculature stabilized by vascular smooth muscle cells persisted till at least 4 wk. On the other hand, bolus injection of an identical amount of free FGF2 induced weak angiogenic responses. These results demonstrate the potential of polyvalent coacervate as a new controlled delivery platform. PMID:21808045

  3. Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential

    SciTech Connect

    Delli-Bovi, P.; Curatola, A.M.; Newman, K.M.; Sato, Y.; Moscatelli, D.; Hewick, R.M.; Rifkin, D.B.; Basilico, C.

    1988-07-01

    The authors recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, the authors have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unkike basic and acidic FGFs, the K-FGF protein is cleaved after a single peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. They have used the conditioned medium from transfected COS-1 cells to test the K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium consistent with an autorcrine mechanism of growth. The authors have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

  4. Antithrombin and heparin.

    PubMed

    Carrell, R; Skinner, R; Warden, M; Whisstock, J

    1995-08-01

    Antithrombin, the main inhibitor of thrombosis in blood, is bound and activated by the heparin-like side-chains that line the small vasculature. We now have good depictions of the heparin-binding site on antithrombin, and of the way in which mutations at this site cause thrombotic disease. The interaction of heparin with antithrombin is, however, a kinetic one, with binding being followed by formation of a complex with thrombin and then release from the heparin. Our understanding of the processes involved is currently based on crystallographic models but, for a mobile mechanism, these merely provide snapshots - what is needed is a movie.

  5. Growth and growth factors in diabetes mellitus.

    PubMed Central

    Salardi, S; Tonioli, S; Tassoni, P; Tellarini, M; Mazzanti, L; Cacciari, E

    1987-01-01

    Growth of 79 children with diabetes was analysed at diagnosis and again after one to 10.7 years of treatment with insulin. Both sexes were tall at onset, whereas at the last observation boys alone showed significant growth retardation. Height standard deviation score (SDS), however, showed no significant fall either in 32 subjects reassessed after five years of disease or in 18 subjects examined at full stature. Skeletal maturity was not significantly impaired after treatment. Pubertal growth spurt was reduced, especially in girls and in subjects with onset of disease at or around puberty. We found no significant correlation between height and height velocity SDS and glycosylated haemoglobin values or secretion of growth hormone during the arginine test. Somatomedin C values were correlated with height velocity SDS in prepubertal boys. The results of this study suggest that there are interferences in the growth of children with diabetes but that they do not seem to have a significant influence on adult height. PMID:3813637

  6. Heparin-binding peptide amphiphile supramolecular architectures as platforms for angiogenesis and drug delivery

    NASA Astrophysics Data System (ADS)

    Chow, Lesleyann W.

    A fascinating phenomenon in nature is the self-assembly of molecules into a functional, hierarchical structure. In the past decade, the Stupp Laboratory has developed several classes of self-assembling biomaterials, one of which is the synthetic peptide amphiphile (PA). Self-assembling PAs are attractive and versatile biomolecules that can be customized for specific applications in regenerative medicine. In particular, a heparin-binding peptide amphiphile (HBPA) containing a specific heparin-binding peptide sequence was used here to induce angiogenesis and serve as a delivery vehicle for growth factors and small hydrophobic molecules. Throughout this dissertation, the HBPA/heparin system is used in different architectures for a variety of regenerative medicine applications. In one aspect of this work, hybrid scaffolds made from HBPA/heparin gelled on a poly(L-lactic acid) (PLLA) fiber mesh were used to promote angiogenesis to facilitate pancreatic islet transplantation for the treatment of type 1 diabetes. Delivery of growth factors with HBPA/PLLA scafflolds increased vessel density in vivo and correlated with improved transplant outcomes in a streptozotocin-induced diabetic mouse model. Soluble HBPA nanofiber architectures were also useful for islet transplantation applications. These nanofibers were used at concentrations below gelation to deliver growth factors into the dense islet cell aggregate, promoting cell survival and angiogenesis in vitro. The nanostructures infiltrated the islets and promoted the retention of heparin and growth factors within the islet. Another interesting growth factor release system discussed here is the HBPA membrane structure. HBPA was found to self-assemble with hyaluronic acid, a large biopolymer found in the body, into macroscopic, hierarchically-ordered membranes. Heparin was incorporated into these membranes and affected the membrane's mechanical properties and growth factor release. Human mesenchymal stem cells were also shown

  7. Enhanced skin wound healing by a sustained release of growth factors contained in platelet-rich plasma.

    PubMed

    Yang, Hee Seok; Shin, Jaehoon; Bhang, Suk Ho; Shin, Jung Youn; Park, Jooyeon; Im, Gun Il; Kim, Chang Sung; Kim, Byung Soo

    2011-11-30

    Platelet-rich plasma (PRP) contains growth factors that promote tissue regeneration. Previously, we showed that heparin-conjugated fibrin (HCF) exerts the sustained release of growth factors with affinity for heparin. Here, we hypothesize that treatment of skin wound with a mixture of PRP and HCF exerts sustained release of several growth factors contained in PRP and promotes skin wound healing. The release of fibroblast growth factor 2, platelet-derived growth factor-BB, and vascular endothelial growth factor contained in PRP from HCF was sustained for a longer period than those from PRP, calcium-activated PRP (C-PRP), or a mixture of fibrin and PRP (F-PRP). Treatment of full-thickness skin wounds in mice with HCF-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 12 compared to treatment with either C-PRP or F-PRP. Enhanced skin regeneration observed in HCF-PRP group may have been at least partially due to enhanced angiogenesis in the wound beds. Therefore, this method could be useful for skin wound treatment.

  8. Effect of Heparin Oligomer Chain Length on the Activation of Valvular Interstitial Cells

    PubMed Central

    Pedron, Sara; Kasko, Andrea M.; Peinado, Carmen; Anseth, Kristi S.

    2010-01-01

    A key event in connective tissue remodeling involves the transformation of fibroblasts to myofibroblasts, also revealed by expression of α-smooth muscle actin (α-SMA). However, misregulation of this transition can lead to fibrosis, an overgrowth and hardening of tissue due to excess extracellular matrix deposition, a process that is linked to heart valve disease and many others. Both disease treatment and regenerative strategies would benefit from strategies for the controlled delivery and presentation of bioactive factors that can promote or suppress this transformation. In this regard, the ability of heparin to complex a plethora of growth factors offers a broad range of possibilities for this purpose. Here, the effects of heparin chain length and structure on valvular interstitial cell (VIC) phenotypic expression were explored. Heparin from porcine intestinal mucosa was depolymerized with heparinase and fractionated to obtain oligosaccharides of different sizes. VICs cultured with octasaccharides and decasaccharides exhibited higher expression of a-SMA when compared to other saccharides and full-length heparin. No activation of VICs was observed in response to full-length heparin presence in media. PMID:20446725

  9. Cardiopulmonary Bypass Without Heparin.

    PubMed

    Rehfeldt, Kent H; Barbara, David W

    2016-03-01

    Due to familiarity, short half-life, ease of monitoring, and the availability of a reversal agent, heparin remains the anticoagulant of choice for cardiac operations requiring cardiopulmonary bypass (CPB). However, occasionally patients require CPB but should not receive heparin, most often because of acute or subacute heparin-induced thrombocytopenia (HIT). In these cases, if it is not feasible to wait for the disappearance of HIT antibodies, an alternative anticoagulant must be selected. A number of non-heparin anticoagulant options have been explored. However, current recommendations suggest the use of a direct thrombin inhibitor such as bivalirudin. This review describes the use of heparin alternatives for the conduct of CPB with a focus on the direct thrombin inhibitors.

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

    PubMed

    Seculini Patiño, Carina E; Tabares, Aldo H

    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. Heparin depolymerization by immobilized heparinase: A review.

    PubMed

    Bhushan, Indu; Alabbas, Alhumaidi; Sistla, Jyothi C; Saraswat, Rashmi; Desai, Umesh R; Gupta, Ram B

    2017-06-01

    Heparin is a member of the glycosaminoglycan (GAG) family composed of glucosamine and uronic acid units containing O-sulfo, N-acetyl and N-sulfo groups, which are alternating in the chain and linked by 1→4 manner. It is a naturally occurring anticoagulant that prevents the formation of clots and their growth within blood. Certain low molecular weight heparins (LMWHs) are considered as better therapeutic agents than natural heparin because of the reduced side effects and smaller risk of bleeding. LMWHs can be produced from heparin by chemical or enzymatic depolymerizations. Heparinases catalyze the cleavage of glycosidic linkage between amino sugars and uronic acids in heparin. There are three kinds of heparinases which are frequently used for depolymerization of heparin. Despite wide range of applications of heparinases in health care, their use still has been hampered due to poor stability and high cost. To overcome this problem heparinases are recommended for immobilization to reduce the cost of product and enhance stability. Heparinases have been successfully immobilized using various methods and supports, mostly for deheparinization of blood through extracorporeal devices. The focus of this review is to present the current status of heparinase immobilization including various supports and methods used, stability and applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Structure of rat acidic fibroblast growth factor at 1.4 Å resolution

    SciTech Connect

    Kulahin, Nikolaj; Kochoyan, Arthur; Berezin, Vladimir; Bock, Elisabeth; Gajhede, Michael

    2007-02-01

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

  13. Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients.

    PubMed

    Leonardi, A; Sathe, S; Bortolotti, M; Beaton, A; Sack, R

    2009-05-01

    To detect the presence of multiple mediators and growth factors in tears of vernal keratoconjunctivitis (VKC) patients with active disease using stationary phase antibody arrays. Tears were collected from 12 normal subjects (CT) and 24 active VKC patients. Tears were centrifuged and successively probed using three microwell plate arrays specific for: (i) cytokines: interleukin (IL)-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-13, interferon-gamma and tumour necrosis factor-alpha; (ii) growth factors: basic fibroblast growth factor (bFGF), platelet-derived growth factor, thrombopoietin, angiopoietin-2, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), keratocyte growth factor, tissue inhibitor of metalloprotease (TIMP)-1 and heparin-binding epithelial growth factor (HB-EGF) and (iii) matrix metalloprotease (MMP)-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1 and TIMP-2. Interleukin-8 signals were detected in all CT and highly detected in all VKC samples. The Th2-type cytokines, IL-4, IL-5 and IL-10 were detected only in tears of VKC patients. Signals for bFGF, HB-EGF, VEGF and HGF were detected in 41-87% of VKC samples and in few CT samples. Only TIMP-1 and TIMP-2 were found in all normal and patient tear samples, whereas MMP-1, MMP-2, MMP-3, MMP-9 and MMP-10 were highly present in all VKC samples. Stationary phase antibody array methodology was useful for the screening of various cytokines, growth factors and MMPs in tears. These analyses identified in tears of VKC patients previously unreported factors including MMP-3 and MMP-10 and multiple proteases, growth factors and cytokines, which may all play an important role in the pathogenesis of conjunctival inflammation.

  14. Heparin management in a patient with thyroid storm.

    PubMed

    Belchikov, Yuly G; Marotta, Sandra E

    2010-04-01

    Management of atrial fibrillation during thyroid storm includes anticoagulation for risk of clot propagation. Physiologic changes that occur in patients with thyroid storm may lead to heparin resistance and inappropriate anticoagulation. Factors contributing to heparin resistance include antithrombin deficiency, increased heparin clearance, and increased levels of factor VIII. We describe a 30-year-old woman who was hospitalized with thyroid storm. She subsequently developed atrial fibrillation, and unfractionated heparin was started. Over the next 4 days, the heparin infusion rate was titrated to an exaggerated dose of 2100 units/hour (33 units/kg/hr) in order to attain a therapeutic response. By hospital day 7, her atrial fibrillation had resolved; the heparin infusion was discontinued, and the patient remained clinically stable with no sequelae. The exact mechanism of heparin resistance in thyroid storm is unknown; however, data have shown a positive correlation between factor VIII and thyroxine levels. This patient had an elevated thyroxine level of 32.5 microg/dl, which suggested that an increased factor VIII level was the probable mechanism of heparin resistance. Recognition of possible heparin resistance in patients with thyroid storm will allow clinicians to promptly identify appropriate interventions to ensure adequate anticoagulation in this high-risk patient population.

  15. Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

    NASA Astrophysics Data System (ADS)

    Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

    1983-11-01

    Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

  16. Mussel-inspired one-step adherent coating rich in amine groups for covalent immobilization of heparin: hemocompatibility, growth behaviors of vascular cells, and tissue response.

    PubMed

    Yang, Ying; Qi, Pengkai; Wen, Feng; Li, Xiangyang; Xia, Qin; Maitz, Manfred F; Yang, Zhilu; Shen, Ru; Tu, Qiufen; Huang, Nan

    2014-08-27

    Heparin, an important polysaccharide, has been widely used for coatings of cardiovascular devices because of its multiple biological functions including anticoagulation and inhibition of intimal hyperplasia. In this study, surface heparinization of a commonly used 316L stainless steel (SS) was explored for preparation of a multifunctional vascular stent. Dip-coating of the stents in an aqueous solution of dopamine and hexamethylendiamine (HD) (PDAM/HD) was presented as a facile method to form an adhesive coating rich in primary amine groups, which was used for covalent heparin immobilization via active ester chemistry. A heparin grafting density of about 900 ng/cm(2) was achieved with this method. The retained bioactivity of the immobilized heparin was confirmed by a remarkable prolongation of the activated partial thromboplastin time (APTT) for about 15 s, suppression of platelet adhesion, and prevention of the denaturation of adsorbed fibrinogen. The Hep-PDAM/HD also presented a favorable microenvironment for selectively enhancing endothelial cell (EC) adhesion, proliferation, migration and release of nitric oxide (NO), and at the same time inhibiting smooth muscle cell (SMC) adhesion and proliferation. Upon subcutaneous implantation, the Hep-PDAM/HD exhibited mitigated tissue response, with thinner fibrous capsule and less granulation formation compared to the control 316L SS. This number of unique functions qualifies the heparinized coating as an attractive alternative for the design of a new generation of stents.

  17. The Role of Heparin-Binding EGF-Like Growth Factor in Breast Cancer

    DTIC Science & Technology

    1996-10-01

    analysis: Human breast cancer cells were lysed using lysis buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 1% Nonidet - P40 , 0.5% deoxycholate, 5 mM...protein (MBP) assay: MCF-10A nontransformed and transformed cells were lysed in a buffer containing 1% Nonidet - P40 , 0.5 % deoxycholate, 20 mM Tris, pH 7.5

  18. Transforming growth factor alpha, Shope fibroma growth factor, and vaccinia growth factor can replace myxoma growth factor in the induction of myxomatosis in rabbits.

    PubMed

    Opgenorth, A; Nation, N; Graham, K; McFadden, G

    1993-02-01

    The epidermal growth factor (EGF) homologues encoded by vaccinia virus, myxoma virus, and malignant rabbit fibroma virus have been shown to contribute to the pathogenicity of virus infection upon inoculation of susceptible hosts. However, since the primary structures of these growth factors and the disease profiles induced by different poxvirus genera vary substantially, the degree to which the various EGF homologues perform similar roles in viral pathogenesis remains unclear. In order to determine whether different EGF-like growth factors can perform qualitatively similar functions in the induction of myxomatosis in rabbits, we created recombinant myxoma virus variants in which the native growth factor, myxoma growth factor (MGF), was disrupted and replaced with either vaccinia virus growth factor, Shope fibroma growth factor, or rat transforming growth factor alpha. Unlike the control virus containing an inactivated MGF gene, which caused marked attenuation of the disease syndrome and substantially less proliferation of the epithelial cell layers in the conjunctiva and respiratory tract, the recombinant myxoma virus strains expressing heterologous growth factors produced infections which were both clinically and histopathologically indistinguishable from wild-type myxomatosis. We conclude that these poxviral and cellular EGF-like growth factors, which are diverse with respect to primary structure and origin, have similar biological functions in the context of myxoma virus pathogenesis and are mitogenic for the same target cells.

  19. Growth Factors in Proliferative Diabetic Retinopathy

    PubMed Central

    Khan, Zia Ali

    2003-01-01

    Many growth factors are implicated in the pathogenesis of proliferative diabetic retinopathy. Alteration of growth factors and their receptors in diabetes has been shown in both experimental and clinical studies. Sustained hyperglycemia resulting from long-standing diabetes leads to several biochemical abnormalities that consequently result in retinal hypoxia. Retinal oxygenation state regulates various growth factors that promote angiogenesis in order to meet the oxygen demands of the tissue. However, unregulated expression of these growth factors and induction of complex cascades leading to augmentation of other proangiogenic factors, which may not be regulated by tissue oxygenation, leads to uncontrolled retinal neovascularization and blindness in diabetic patients. PMID:14668050

  20. Loading of VEGF to the heparin cross-linked demineralized bone matrix improves vascularization of the scaffold.

    PubMed

    Chen, Lei; He, Zhengquan; Chen, Bing; Yang, Maojin; Zhao, Yannan; Sun, Wenjie; Xiao, Zhifeng; Zhang, Jing; Dai, Jianwu

    2010-01-01

    Deficient vascularization is one of the prominent shortcomings of porous tissue-engineering scaffolds, which results in insufficient oxygen and nutrients transportation. Here, heparin cross-linked demineralized bone matrices (HC-DBM) pre-loaded with vascular endothelial growth factor (VEGF) were designed to promote cells and new microvessels invasion into the matrices. After being chemical crosslinked with heparin by N-hydroxysuccinimide and N-(3-di-methylaminopropyl)-N'-ethylcarbodiimide, the scaffold could bind more VEGF than the non-crosslinked one and achieve localized and sustained delivery. The biological activity of VEGF binding on heparinized collagen was demonstrated by promoting endothelial cells proliferation. Evaluation of the angiogenic potential of heparinized DBM loaded with VEGF was further investigated by subcutaneous implantation. Improved angiogenesis of heparinized DBM loaded with VEGF was observed from haematoxylin-eosin staining and immunohistochemistry examination. The results demonstrated that heparin cross-linked DBM binding VEGF could be a useful strategy to stimulate cells and blood vessels invasion into the scaffolds.

  1. Differential Mitogenic Effects of Single Chain Hepatocyte Growth Factor (HGF)/Scatter Factor and HGF/NK1 following Cleavage by Factor Xa*

    PubMed Central

    Pediaditakis, Peter; Monga, Satdarshan P. S.; Mars, Wendy M.; Michalopoulos, George K.

    2007-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF. PMID:11832492

  2. Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells.

    PubMed

    Das, Jharna R; Gutkind, J Silvio; Ray, Patricio E

    2016-01-01

    Renal endothelial cells (REc) are the first target of HIV-1 in the kidney. The integrity of REc is maintained at least partially by heparin binding growth factors that bind to heparan sulfate proteoglycans located on their cell surface. However, previous studies showed that the accumulation of two heparin-binding growth factors, Vascular Endothelial Cell Growth Factor-A (VEGF-A) and Fibroblast Growth Factor-2 (FGF-2), in combination with the viral protein Tat, can precipitate the progression of HIV-renal diseases. Nonetheless, very little is known about how these factors affect the behavior of REc in HIV+ children. We carried out this study to determine how VEGF-A, FGF-2, and HIV-Tat, modulate the cytoskeletal structure and permeability of cultured REc, identify key signaling pathways involved in this process, and develop a functional REc assay to detect HIV+ children affected by these changes. We found that VEGF-A and FGF-2, acting in synergy with HIV-Tat and heparin, affected the cytoskeletal structure and permeability of REc through changes in Rho-A, Src, and Rac-1 activity. Furthermore, urine samples from HIV+ children with renal diseases, showed high levels of VEGF-A and FGF-2, and induced similar changes in cultured REc and podocytes. These findings suggest that FGF-2, VEGF-A, and HIV-Tat, may affect the glomerular filtration barrier in HIV+ children through the induction of synergistic changes in Rho-A and Src activity. Further studies are needed to define the clinical value of the REc assay described in this study to identify HIV+ children exposed to circulating factors that may induce glomerular injury through similar mechanisms.

  3. Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells

    PubMed Central

    Das, Jharna R; Gutkind, J. Silvio; Ray, Patricio E

    2016-01-01

    Renal endothelial cells (REc) are the first target of HIV-1 in the kidney. The integrity of REc is maintained at least partially by heparin binding growth factors that bind to heparan sulfate proteoglycans located on their cell surface. However, previous studies showed that the accumulation of two heparin-binding growth factors, Vascular Endothelial Cell Growth Factor-A (VEGF-A) and Fibroblast Growth Factor-2 (FGF-2), in combination with the viral protein Tat, can precipitate the progression of HIV-renal diseases. Nonetheless, very little is known about how these factors affect the behavior of REc in HIV+ children. We carried out this study to determine how VEGF-A, FGF-2, and HIV-Tat, modulate the cytoskeletal structure and permeability of cultured REc, identify key signaling pathways involved in this process, and develop a functional REc assay to detect HIV+ children affected by these changes. We found that VEGF-A and FGF-2, acting in synergy with HIV-Tat and heparin, affected the cytoskeletal structure and permeability of REc through changes in Rho-A, Src, and Rac-1 activity. Furthermore, urine samples from HIV+ children with renal diseases, showed high levels of VEGF-A and FGF-2, and induced similar changes in cultured REc and podocytes. These findings suggest that FGF-2, VEGF-A, and HIV-Tat, may affect the glomerular filtration barrier in HIV+ children through the induction of synergistic changes in Rho-A and Src activity. Further studies are needed to define the clinical value of the REc assay described in this study to identify HIV+ children exposed to circulating factors that may induce glomerular injury through similar mechanisms. PMID:27097314

  4. Growth factors and acute renal failure.

    PubMed

    Hirschberg, R; Ding, H

    1998-03-01

    During acute renal injury, there are alterations in the expression of several growth factors and their receptors in the kidney. The increased expression of several growth factors and/or their receptors at sites of nephron injury suggests important contributions to repair. Exogenous administration of some growth factors, such as IGF-I, EGF and HGF, accelerates recovery of renal function in experimental acute renal failure (ARF). In ARF growth factors act through several mechanisms, which may include altered cell cycle regulation and mitogenesis, differentiation of recovered cells, regulation of apoptosis, improved renal hemodynamics, and others. There is evidence for interactions of growth factors with other growth factors as well as with other genes resulting in complex orchestration of biologic events contributing to recovery from ARF.

  5. Heparin-induced thrombocytopenia with thrombotic sequelae: a review.

    PubMed

    Goor, Yoav; Goor, Odelia; Eldor, Amiram

    2002-08-01

    Heparin-induced thrombocytopenia (HIT) occurs in 1-5% of patients treated with heparin. The pathogenesis involves the formation of antibodies to heparin-platelet factor 4 complexes, and the major clinical sequelae are thrombotic. Diagnosis is based on a combination of clinical and laboratory data. Treatment consists of stopping heparin, but, insofar as the risk of thrombosis remains high, treatment by alternative antithrombotic agents is indicated. Most clinical experience has been with danaparoid sodium and hirudin. The use of low-molecular-weight heparins (LMWH) in subsequent HIT episodes has been described, but is not recommended, especially with the introduction of new agents, such as oral thrombin inhibitors and pentasaccharides, which are hoped to reduce the use of heparins and the occurrence of HIT.

  6. Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells

    SciTech Connect

    Kobayashi, M.; Shimada, K.; Ozawa, T. )

    1990-09-01

    Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of (35S)sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and (3H)leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation.

  7. Outbreak of Adverse Reactions Associated with Contaminated Heparin

    PubMed Central

    Blossom, David B.; Kallen, Alexander J.; Patel, Priti R.; Elward, Alexis; Robinson, Luke; Gao, Ganpan; Langer, Robert; Perkins, Kiran M.; Jaeger, Jennifer L.; Kurkjian, Katie M.; Jones, Marilyn; Schillie, Sarah F.; Shehab, Nadine; Ketterer, Daniel; Venkataraman, Ganesh; Kishimoto, Takashi Kei; Shriver, Zachary; McMahon, Ann W.; Austen, K. Frank; Kozlowski, Steven; Srinivasan, Arjun; Turabelidze, George; Gould, Carolyn V.; Arduino, Matthew J.; Sasisekharan, Ram

    2013-01-01

    BACKGROUND In January 2008, the Centers for Disease Control and Prevention began a nationwide investigation of severe adverse reactions that were first detected in a single hemodialysis facility. Preliminary findings suggested that heparin was a possible cause of the reactions. METHODS Information on clinical manifestations and on exposure was collected for patients who had signs and symptoms that were consistent with an allergic-type reaction after November 1, 2007. Twenty-one dialysis facilities that reported reactions and 23 facilities that reported no reactions were included in a case–control study to identify facility-level risk factors. Unopened heparin vials from facilities that reported reactions were tested for contaminants. RESULTS A total of 152 adverse reactions associated with heparin were identified in 113 patients from 13 states from November 19, 2007, through January 31, 2008. The use of heparin manufactured by Baxter Healthcare was the factor most strongly associated with reactions (present in 100.0% of case facilities vs. 4.3% of control facilities, P<0.001). Vials of heparin manufactured by Baxter from facilities that reported reactions contained a contaminant identified as oversulfated chondroitin sulfate (OSCS). Adverse reactions to the OSCS-contaminated heparin were often characterized by hypotension, nausea, and shortness of breath occurring within 30 minutes after administration. Of 130 reactions for which information on the heparin lot was available, 128 (98.5%) occurred in a facility that had OSCS-contaminated heparin on the premises. Of 54 reactions for which the lot number of administered heparin was known, 52 (96.3%) occurred after the administration of OSCS-contaminated heparin. CONCLUSIONS Heparin contaminated with OSCS was epidemiologically linked to adverse reactions in this nationwide outbreak. The reported clinical features of many of the cases further support the conclusion that contamination of heparin with OSCS was the cause

  8. Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor.

    PubMed

    Huang, Min; Vitharana, Samadhi N; Peek, Laura J; Coop, Tina; Berkland, Cory

    2007-05-01

    Angiogenesis has long been a desired therapeutic approach to improve clinical outcomes of conditions typified by ischemia. Vascular endothelial growth factor (VEGF) has demonstrated the ability to generate new blood vessels in vivo, but trials using intravenous delivery have not yet produced clinical success. Localized, sustained delivery of VEGF has been proven necessary to generate blood vessels as demonstrated by implantable, controlled release devices. Ultimately, nanoparticles delivered by intravenous injection may be designed to accumulate in target tissues and sustain the local VEGF concentration; however, injectable nanosuspensions that control the release of stabilized VEGF must first be developed. In this study, we utilize the heparin binding domain of VEGF to bind the polyanion dextran sulfate, resulting in an enhanced thermal stability of VEGF. Coacervation of the VEGF-bound dextran sulfate with selected polycations (chitosan, polyethylenimine, or poly-L-lysine) produced nanoparticles approximately 250 nm in diameter with high VEGF encapsulation efficiency (50-85%). Release of VEGF from these formulations persisted for >10 days and maintained high VEGF activity as determined by ELISA and a mitogenic bioassay. Chitosan-dextran sulfate complexes were preferred because of their biodegradability, desirable particle size ( approximately 250 nm), entrapment efficiency ( approximately 85%), controlled release (near linear for 10 days), and mitogenic activity.

  9. Intranasal epidermal growth factor treatment rescues neonatal brain injury

    NASA Astrophysics Data System (ADS)

    Scafidi, Joseph; Hammond, Timothy R.; Scafidi, Susanna; Ritter, Jonathan; Jablonska, Beata; Roncal, Maria; Szigeti-Buck, Klara; Coman, Daniel; Huang, Yuegao; McCarter, Robert J.; Hyder, Fahmeed; Horvath, Tamas L.; Gallo, Vittorio

    2014-02-01

    There are no clinically relevant treatments available that improve function in the growing population of very preterm infants (less than 32 weeks' gestation) with neonatal brain injury. Diffuse white matter injury (DWMI) is a common finding in these children and results in chronic neurodevelopmental impairments. As shown recently, failure in oligodendrocyte progenitor cell maturation contributes to DWMI. We demonstrated previously that the epidermal growth factor receptor (EGFR) has an important role in oligodendrocyte development. Here we examine whether enhanced EGFR signalling stimulates the endogenous response of EGFR-expressing progenitor cells during a critical period after brain injury, and promotes cellular and behavioural recovery in the developing brain. Using an established mouse model of very preterm brain injury, we demonstrate that selective overexpression of human EGFR in oligodendrocyte lineage cells or the administration of intranasal heparin-binding EGF immediately after injury decreases oligodendroglia death, enhances generation of new oligodendrocytes from progenitor cells and promotes functional recovery. Furthermore, these interventions diminish ultrastructural abnormalities and alleviate behavioural deficits on white-matter-specific paradigms. Inhibition of EGFR signalling with a molecularly targeted agent used for cancer therapy demonstrates that EGFR activation is an important contributor to oligodendrocyte regeneration and functional recovery after DWMI. Thus, our study provides direct evidence that targeting EGFR in oligodendrocyte progenitor cells at a specific time after injury is clinically feasible and potentially applicable to the treatment of premature children with white matter injury.

  10. Growth factors, nutrient signaling, and cardiovascular aging

    PubMed Central

    Fontana, Luigi; Vinciguerra, Manlio; Longo, Valter D.

    2012-01-01

    Growth factors regulated by specific macronutrients have been shown to promote aging and accelerate mortality in the great majority of the organisms studied. In particular, the enzymes activated by growth hormone (GH), insulin and insulin-like growth factor 1 (IGF-I) in mammals and their orthologs in simple model organisms represent perhaps the best-understood proteins involved in the aging process. Dietary restriction (DR), which reduces the level of IGF-I and of other growth factors, has been associated with protection from diabetes, cancer, and cardiovascular diseases and deficiencies in GH signaling and IGF-I are strongly associated with protection from cancer and diabetes in both mice and humans, but their role in cardiac function and cardiovascular diseases is controversial. Here we review the link between growth factors, cardiac function and heart disease with focus on the cardioprotective and sensitizing effect of growth factors in both model organisms and humans. PMID:22499903

  11. Stability and biological activity evaluations of PEGylated human basic fibroblast growth factor

    PubMed Central

    Hadadian, Shahin; Shamassebi, Dariush Norouzian; Mirzahoseini, Hasan; Shokrgozar, Mohamad Ali; Bouzari, Saeid; Sepahi, Mina

    2015-01-01

    Background: Human basic fibroblast growth factor (hBFGF) is a heparin-binding growth factor and stimulates the proliferation of a wide variety of cells and tissues causing survival properties and its stability and biological activity improvements have received much attention. Materials and Methods: In the present work, hBFGF produced by engineered Escherichia coli and purified by cation exchange and heparin affinity chromatography, was PEGylated under appropriate condition employing 10 kD polyethylene glycol. The PEGylated form was separated by size exclusion chromatography. Structural, biological activity, and stability evaluations were performed using Fourier transform infrared (FITR) spectroscopy, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and effect denaturing agent, respectively. Results: FITR spectroscopy revealed that both PEGylated and native forms had the same structures. MTT assay showed that PEGyalated form had a 30% reduced biological activity. Fluorescence spectrophotometry indicated that the PEGylated form denatured at higher concentrations of guanidine HCl (1.2 M) compared with native, which denatured at 0.8 M guanidine HCl. Conclusions: PEGylation of hBFGF makes it more stable against denaturing agent but reduces its bioactivity up to 30%. PMID:26605215

  12. Shape changes induced by biologically active peptides and nerve growth factor in blood platelets of rabbits.

    PubMed

    Gudat, F; Laubscher, A; Otten, U; Pletscher, A

    1981-11-01

    1 Nerve growth factor (NGF), substance P (SP) and thymopoietin all caused shape change reactions of rapid onset in rabbit platelets. NGF had the highest maximal effect, and SP the lowest EC50 (concentration causing half maximal shape change). The action of SP was reversible within 5 min, whereas that of NGF lasted for at least 1 h. A series of other peptides were inactive. 2 After preincubation of platelets with SP, a second application of SP no longer caused a shape change reaction, whereas the effect of NGF was not influenced. 3 An oxidized NGF-derivative without biological activity did not cause a shape change reaction, neither did epidermal growth factor. 4 Prostaglandin E1 (PGE1) and pretreatment of the platelets with 3% butanol, which counteract the shape changes caused by 5-hydroxytryptamine (5-HT) and adenosine 3',5'-diphosphate, also antagonized those induced by NGF and SP. Neither heparin nor methysergide, an antagonist of 5-HT-receptors, influenced the shape change induced by NGF or SP. The action of NGF was also antagonized by a specific antibody to NGF. 5 Thymopoietin, like the basic polypeptide polyornithine (mol. wt. 40,000) was not antagonized by PGE1 and butanol. Heparin, which counteracted the effect of polyornithine, did not influence that of thymopoietin. 6 In conclusion, different modes of action are involved in the shape change of blood platelets induced by polypeptides and proteins. SP and NGF may act by stimulating specific membrane receptors.

  13. Autocrine growth factors and solid tumor malignancy.

    PubMed Central

    Walsh, J. H.; Karnes, W. E.; Cuttitta, F.; Walker, A.

    1991-01-01

    The ability of malignant cells to escape the constraint that normally regulate cell growth and differentiation has been a primary focus of attention for investigators of cancer cell biology. An outcome of this attention has been the discovery that the protein products of oncogenes play a role in the activation of growth signal pathways. A second outcome, possibly related to abnormal oncogene expression, has been the discovery that malignant cells frequently show an ability to regulate their own growth by the release of autocrine growth modulatory substances. Most important, the growth of certain malignant cell types has been shown to depend on autocrine growth circuits. A malignant tumor whose continued growth depends on the release of an autocrine growth factor may be vulnerable to treatment with specific receptor antagonists or immunoneutralizing antibodies designed to break the autocrine circuit. Information is rapidly emerging concerning autocrine growth factors in selected human solid tissue malignancy. Images PMID:1926844

  14. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.

    1993-01-01

    Long-term manned space travel will require a better understanding of skeletal muscle atrophy which results from microgravity. Astronaut strength and dexterity must be maintained for normal mission operations and for emergency situations. Although exercise in space slows the rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity/tension/exercise help to maintain muscle size by altering protein synthesis and/or degradation rate should ultimately allow pharmacological intervention to prevent muscle atrophy in microgravity. The overall objective is to examine some of the basic biochemical processes involved in tension-induced muscle growth. With an experimental in vitro system, the role of exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth are examined. Differentiated avian skeletal myofibers can be 'exercised' in tissue culture using a newly developed dynamic mechanical cell stimulator device which simulates different muscle activity patterns. Patterns of mechanical activity which significantly affect muscle growth and metabolic characteristics were found. Both exogenous and endogenous growth factors are essential for tension-induced muscle growth. Exogenous growth factors found in serum, such as insulin, insulin-like growth factors, and steroids, are important regulators of muscle protein turnover rates and mechanically-induced muscle growth. Endogenous growth factors are synthesized and released into the culture medium when muscle cells are mechanically stimulated. At least one family of mechanically induced endogenous factors, the prostaglandins, help to regulate the rates of protein turnover in muscle cells. Endogenously synthesized IGF-1 is another. The interaction of muscle mechanical activity and these growth factors in the regulation of muscle protein turnover rates with our in vitro model system is studied.

  15. Enoxaparin for unstable angina and ancrod for cardiac surgery following heparin allergy.

    PubMed

    Smith, R E; Townsend, G E; Berry, B R; Bowen, T

    1996-05-01

    To describe a patient who presented with heparin allergy and required alternate anticoagulation for unstable angina and coronary artery bypass surgery. To review therapeutic alternatives to porcine heparin for patients with hypersensitivity or intolerance to standard heparin anticoagulation. A 74-year-old man with a 15-year-old coronary artery bypass graft presented to the emergency room with unstable angina and was scheduled for urgent coronary artery revascularization. A bolus dose of porcine heparin was administered followed by a continuous infusion. Shortly afterward the patient developed a type I allergic reaction to the porcine heparin that was confirmed by rechallenge. Three alternatives to porcine heparin were tried, including bovine lung heparin, low-molecular-weight heparin (enoxaparin), and ancrod. The patient was found to be cross-sensitive to bovine lung heparin, but tolerated enoxaparin for unstable angina without cross-sensitivity. Anticoagulation for cardiopulmonary bypass was achieved with an infusion of ancrod that was later reversed with cryoprecipitate. The patient was discharged postoperatively on day 5 without the complication of excessive bleeding. Type I allergic reaction to unfractionated heparin is a rare occurrence and could be the result of a variety of factors. Possible causes for the reaction include a porcine protein, a preservative contained in the heparin solution, or a hapten formed between heparin and a plasma protein. We considered four alternatives to heparin anticoagulation: rush desensitization, bovine lung heparin, low-molecular-weight heparin, and ancrod. The patient was cross-sensitive to bovine lung heparin, but was able to tolerate low-molecular-weight heparin (enoxaparin). This was unexpected because enoxaparin is derived from unfractionated porcine heparin. Testing for cross-sensitivity had no value in this case, as two negative subcutaneous test doses were followed by dramatic reactions when the drugs were given

  16. Roles for Growth Factors in Cancer Progression

    PubMed Central

    Witsch, Esther; Sela, Michael; Yarden, Yosef

    2011-01-01

    Under physiological conditions, cells receive fate-determining signals from their tissue surroundings, primarily in the form of polypeptide growth factors. Integration of these extracellular signals underlies tissue homeostasis. Although departure from homeostasis and tumor initiation are instigated by oncogenic mutations rather than by growth factors, the latter are the major regulators of all subsequent steps of tumor progression, namely clonal expansion, invasion across tissue barriers, angiogenesis, and colonization of distant niches. Here, we discuss the relevant growth factor families, their roles in tumor biology, as well as the respective downstream signaling pathways. Importantly, cancer-associated activating mutations that impinge on these pathways often relieve, in part, the reliance of tumors on growth factors. On the other hand, growth factors are frequently involved in evolvement of resistance to therapeutic regimens, which extends the roles for polypeptide factors to very late phases of tumor progression and offers opportunities for cancer therapy. PMID:20430953

  17. Low risk of symptomatic venous thromboembolic events during growth factor administration for PBSC mobilization.

    PubMed

    Naina, H V; Pruthi, R K; Inwards, D J; Dingli, D; Litzow, M R; Ansell, S M; William, H J; Dispenzieri, A; Buadi, F K; Elliott, M A; Gastineau, D A; Gertz, M A; Hayman, S R; Johnston, P B; Lacy, M Q; Micallef, I N; Porrata, L F; Kumar, S

    2011-02-01

    The use of erythropoietic agents has been associated with an increased risk of venous thromboembolic events (VTEs), especially in patients with underlying malignancies. However, it is not known whether there is an increased risk of VTE associated with granulocyte growth factors. We reviewed 621 patients undergoing PBSC mobilization using granulocyte growth factors, alone or in combination with CY. Patients with a diagnosis of AL amyloidosis (AL: 114; 18%), multiple myeloma (MM: 278; 44%) Hodgkin lymphoma (HL: 20; 3%) or non-Hodgkin lymphoma (NHL: 209; 33%) were included. Symptomatic VTE occurred in six (0.97%) patients: two AL, two MM and two NHL. Of the six patients, two had pulmonary embolism, one developed deep vein thrombosis and three developed symptomatic catheter related thrombosis. Two patients with AL had heparin-induced thrombocytopenia and thrombosis. We found a low incidence of VTE among patients undergoing PBSC mobilization.

  18. (*) Central Growth Factor Loaded Depots in Bone Tissue Engineering Scaffolds for Enhanced Cell Attraction.

    PubMed

    Quade, Mandy; Knaack, Sven; Akkineni, Ashwini Rahul; Gabrielyan, Anastasia; Lode, Anja; Rösen-Wolff, Angela; Gelinsky, Michael

    2017-08-01

    Tissue engineering, the application of stem and progenitor cells in combination with an engineered extracellular matrix, is a promising strategy for bone regeneration. However, its success is limited by the lack of vascularization after implantation. The concept of in situ tissue engineering envisages the recruitment of cells necessary for tissue regeneration from the host environment foregoing ex vivo cell seeding of the scaffold. In this study, we developed a novel scaffold system for enhanced cell attraction, which is based on biomimetic mineralized collagen scaffolds equipped with a central biopolymer depot loaded with chemotactic agents. In humid milieu, as after implantation, the signaling factors are expected to slowly diffuse out of the central depot forming a gradient that stimulates directed cell migration toward the scaffold center. Heparin, hyaluronic acid, and alginate have been shown to be capable of depot formation. By using vascular endothelial growth factor (VEGF) as model factor, it was demonstrated that the release kinetics can be adjusted by varying the depot composition. While alginate and hyaluronic acid are able to reduce the initial burst and prolong the release of VEGF, the addition of heparin led to a much stronger retention that resulted in an almost linear release over 28 days. The biological activity of released VEGF was proven for all variants using an endothelial cell proliferation assay. Furthermore, migration experiments with endothelial cells revealed a relationship between the degree of VEGF retention and migration distance: cells invaded deepest in scaffolds containing a heparin-based depot indicating that the formation of a steep gradient is crucial for cell attraction. In conclusion, this novel in situ tissue engineering approach, specifically designed to recruit and accommodate endogenous cells upon implantation, appeared highly promising to stimulate cell invasion, which in turn would promote vascularization and finally new

  19. Involvement of Receptor-like Protein Tyrosine Phosphatase ζ/RPTPβ and Its Ligand Pleiotrophin/Heparin-binding Growth-associated Molecule (HB-GAM) in Neuronal Migration

    PubMed Central

    Maeda, Nobuaki; Noda, Masaharu

    1998-01-01

    Pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) is a specific ligand of protein tyrosine phosphatase ζ (PTPζ)/receptor-like protein tyrosine phosphatase β (RPTPβ) expressed in the brain as a chondroitin sulfate proteoglycan. Pleiotrophin and PTPζ isoforms are localized along the radial glial fibers, a scaffold for neuronal migration, suggesting that these molecules are involved in migratory processes of neurons during brain development. In this study, we examined the roles of pleiotrophin-PTPζ interaction in the neuronal migration using cell migration assay systems with glass fibers and Boyden chambers. Pleiotrophin and poly-l-lysine coated on the substratums stimulated cell migration of cortical neurons, while laminin, fibronectin, and tenascin exerted almost no effect. Pleiotrophin-induced and poly-l-lysine–induced neuronal migrations showed significant differences in sensitivity to various molecules and reagents. Polyclonal antibodies against the extracellular domain of PTPζ, PTPζ-S, an extracellular secreted form of PTPζ, and sodium vanadate, a protein tyrosine phosphatase inhibitor, added into the culture medium strongly suppressed specifically the pleiotrophin-induced neuronal migration. Furthermore, chondroitin sulfate C but not chondroitin sulfate A inhibited pleiotrophin-induced neuronal migration, in good accordance with our previous findings that chondroitin sulfate constitutes a part of the pleiotrophin-binding site of PTPζ, and PTPζ-pleiotrophin binding is inhibited by chondroitin sulfate C but not by chondroitin sulfate A. Immunocytochemical analysis indicated that the transmembrane forms of PTPζ are expressed on the migrating neurons especially at the lamellipodia along the leading processes. These results suggest that PTPζ is involved in the neuronal migration as a neuronal receptor of pleiotrophin distributed along radial glial fibers. PMID:9660874

  20. Increased expression of fibroblast growth factors in a rabbit skeletal muscle model of exercise conditioning.

    PubMed Central

    Morrow, N G; Kraus, W E; Moore, J W; Williams, R S; Swain, J L

    1990-01-01

    Increased tonic contractile activity from exercise or electrical stimulation induces a variety of changes in skeletal muscle, including vascular growth, myoblast proliferation, and fast to slow fiber type conversion. Little is known about the cellular control of such changes, but pleiotropic biochemical modulators such as fibroblast growth factors (FGFs) may be involved in this response and thus may be regulated in response to such stimuli. We examined the regulation of FGF expression in an in vivo model of exercise conditioning previously shown to exhibit vascular growth and fast to slow fiber conversion. FGFs were extracted by heparin-affinity chromatography from extensor digitorum longus muscles of adult rabbits subjected to chronic motor nerve stimulation at 10 Hz. Growth factor activity (expressed in growth factor units [GFUs]) of muscle stimulated for 3 and 21 d was assayed by [3H]thymidine incorporation in 3T3 fibroblasts and compared with that present in the contralateral unstimulated muscle. A small increase in heparin-binding mitogenic activity was observed as early as 3 d of stimulation, and by 21 d mitogenic activity increased significantly when normalized to either wet weight (stimulated, 287 +/- 61 GFU/g; unstimulated, 145 +/- 39 GFU/g) or to protein (stimulated, 5.3 +/- 1.1 GFU/mg; unstimulated, 2.2 +/- 0.6 GFU/mg) (+/- SE, P less than 0.05). Western analysis demonstrated increased amounts of peptides with immunological identity to acidic and basic FGFs in stimulated muscle. The increase in FGF content observed in this study is synchronous with neovascularization, myoblast proliferation, and fast to slow fiber type conversion previously shown in this model. These results demonstrate that increased expression of FGFs is associated with motor nerve stimulation and increased tonic contractile activity of skeletal muscle, and suggests that these proteins may play a regulatory role in the cellular changes that occur during exercise conditioning. Images

  1. Heparin-like synthetic polymers, named RGTAs, mimic biological effects of heparin in vitro.

    PubMed

    Rouet, Vincent; Meddahi-Pellé, Anne; Miao, Hua-Quan; Vlodavsky, Israel; Caruelle, Jean-Pierre; Barritault, Denis

    2006-09-15

    A family of biopolymers engineered to protect and stabilize heparin binding growth factors (HBGFs) show remarkable properties as wound healing agents in several in vivo tissue repair models to the extend that damaged tissues would recover almost its initial aspect and properties. These polymers where named RGTA for regenerating agents and proposed to act in vivo by enhancing the bioavailability of HBGFs at the site of the injury. To provide support for this hypothesis, we studied interaction of RGTA with FGF-2, taken as the paradigm of HBGFs, and its high- and low-affinity receptors as well as its ability to inhibit heparanase activity. We show that RGTA is comparable to heparin as it favors FGF-2 binding to FGFR-1 and FGF-2 dimerization and potentiates FGF-2-induced mitogenic activity. Furthermore, we show that RGTA inhibits the release of FGF-2 from its extracellular matrix storage sites by heparanase. Our data provide new evidence to support that RGTA may act in vivo both by enhancing HBGF activity and preserving HBGF availability by protecting the matrix low affinity heparan sulfates from rapid heparanase degradation. 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006.

  2. The effect of a stromal cell-derived factor-1α /heparin coating of a biodegradable vascular graft on recruitment of both endothelial and smooth muscle progenitor cells and accelerated healing

    PubMed Central

    Yu, Jian; Wang, Aijun; Tang, Zhenyu; Henry, Jeffrey; Lee, Benjamin; Zhu, Yiqian; Yuan, Failei; Huang, Fengping; Li, Song

    2012-01-01

    Small-diameter synthetic vascular grafts have high failure rate and tissue-engineered blood vessels are limited by the scalability. Here we engineered bioactive materials for in situ vascular tissue engineering, which recruits two types of endogenous progenitor cells for the regeneration of blood vessels. Heparin was conjugated to microfibrous vascular grafts to suppress thrombogenic responses, and stromal cell-derived factor-1α (SDF-1α) was immobilized onto heparin to recruit endogenous progenitor cells. Heparin-bound SDF-1α was more stable than adsorbed SDF-1α under both static and flow conditions. Microfibrous grafts were implanted in rats by anastomosis to test the functional performance. Heparin coating improved the short-term patency, and immobilized SDF-1α further improved the long-term patency. SDF-1α effectively recruited endothelial progenitor cells (EPCs) to the luminal surface of the grafts, which differentiated into endothelial cells (ECs) and accelerated endothelialization. More interestingly, SDF-1α increased the recruitment of smooth muscle progenitor cells (SMPCs) to the grafts, and SMPCs differentiated into smooth muscle cells (SMCs) in vivo and in vitro. Consistently, SDF-1α-immobilized grafts had significantly higher elastic modulus. This work demonstrates the feasibility of simultaneously recruiting progenitor cells of ECs and SMCs for in situ blood vessel regeneration. This in situ tissue engineering approach will have broad applications in regenerative medicine. PMID:22884813

  3. Growth factor gene therapy for Alzheimer disease.

    PubMed

    Tuszynski, Mark H; U, Hoi Sang; Alksne, John; Bakay, Roy A; Pay, Mary Margaret; Merrill, David; Thal, Leon J

    2002-11-15

    The capacity to prevent neuronal degeneration and death during the course of progressive neurological disorders such as Alzheimer disease (AD) would represent a significant advance in therapy. Nervous system growth factors are families of naturally produced proteins that, in animal models, exhibit extensive potency in preventing neuronal death due to a variety of causes, reversing age-related atrophy of neurons, and ameliorating functional deficits. The main challenge in translating growth factor therapy to the clinic has been delivery of growth factors to the brain in sufficient concentrations to influence neuronal function. One means of achieving growth factor delivery to the central nervous system in a highly targeted, effective manner may be gene therapy. In this article the authors summarize the development and implementation of nerve growth factor gene delivery as a potential means of reducing cell loss in AD.

  4. Effect of molecular composition of heparin and cellulose sulfate on multilayer formation and cell response.

    PubMed

    Aggarwal, Neha; Altgärde, Noomi; Svedhem, Sofia; Zhang, Kai; Fischer, Steffen; Groth, Thomas

    2013-11-12

    Here, the layer-by-layer method was applied to assemble films from chitosan paired with either heparin or a semisynthetic cellulose sulfate (CS) that possessed a higher sulfation degree than heparin. Ion pairing was exploited during multilayer formation at pH 4, while hydrogen bonding is likely to occur at pH 9. Effects of polyanions and pH value during layer formation on multilayers properties were studied by surface plasmon resonance ("dry layer mass"), quartz crystal microbalance with dissipation monitoring ("wet layer mass"), water contact angle, and zeta potential measurements. Bioactivity of multilayers was studied regarding fibronectin adsorption and adhesion/proliferation of C2C12 myoblast cells. Layer growth and dry mass were higher for both polyanions at pH 4 when ion pairing occurred, while it decreased significantly with heparin at pH 9. By contrast, CS as polyanion resulted also in high layer growth and mass at pH 9, indicating a much stronger effect of hydrogen bonding between chitosan and CS. Water contact angle and zeta potential measurements indicated a more separated structure of multilayers from chitosan and heparin at pH 4, while CS led to a more fuzzy intermingled structure at both pH values. Cell behavior was highly dependent on pH during multilayer formation with heparin as polyanion and was closely related to fibronectin adsorption. By contrast, CS and chitosan did not show such dependency on pH value, where adhesion and growth of cells was high. Results of this study show that CS is an attractive candidate for multilayer formation that does not depend so strongly on pH during multilayer formation. In addition, such multilayer system also represents a good substrate for cell interactions despite the rather soft structure. As previous studies have shown specific interaction of CS with growth factors, multilayers from chitosan and CS may be of great interest for different biomedical applications.

  5. Prevention of deep vein thrombosis after hip replacement: randomised comparison between unfractionated heparin and low molecular weight heparin.

    PubMed Central

    Leyvraz, P F; Bachmann, F; Hoek, J; Büller, H R; Postel, M; Samama, M; Vandenbroek, M D

    1991-01-01

    OBJECTIVE--To evaluate the efficacy and safety of two subcutaneous prophylactic regimens for postoperative deep vein thrombosis after total hip replacement. DESIGN--Prospective open randomised multicentre trial. SETTING--28 European departments of orthopaedic surgery. INTERVENTION--All patients had bilateral phlebography 10 days after surgery. 31 patients receiving low molecular weight heparin and 29 receiving unfractionated heparin were excluded from the efficacy analysis for various reasons. PATIENTS--349 patients undergoing total hip replacement between September 1988 and May 1989. 174 patients received subcutaneously a low molecular weight heparin (Fraxiparine) with anti-factor Xa activity of 41 IU/kg/day for three days, then 62 IU/kg/day from day 4 to day 10. 175 patients received subcutaneous unfractionated heparin at intervals of eight hours; doses were adjusted to maintain the activated thromboplastin time at two to five seconds above control values. MAIN OUTCOME MEASURE--Total incidence of deep vein thrombosis and incidence of proximal deep vein thrombosis on bilateral phlebography. RESULTS--The total incidence of deep vein thrombosis was 16% in patients receiving unfractionated heparin and 12.6% in patients receiving low molecular weight heparin (p = 0.45), and the incidence of thrombosis of the proximal veins was 13.1% and 2.9% respectively (p less than 0.001). Four patients receiving unfractionated heparin and one receiving low molecular weight heparin developed pulmonary embolism. The incidence of bleeding complications was low and comparable in the two groups. CONCLUSION--Low molecular weight heparin is at least as effective as unfractionated heparin in preventing deep vein thrombosis and is more effective at preventing thrombosis of the proximal veins in patients undergoing hip replacement. Low molecular weight heparin is not more likely to cause bleeding complications and is simpler to give than unfractionated heparin. PMID:1655136

  6. Growth factors from genes to clinical application

    SciTech Connect

    Sara, V.R. ); Hall, K.; Low, H. )

    1990-01-01

    The last decade has witnessed an explosion in the identification of growth factors and their receptors. This has been greatly facilitated by recombinant DNA technology, which has provided the tools not only to identify these proteins at the gene level but also to produce recombinant proteins for evaluating their biological activities. With the help of such techniques, we are moving toward an understanding of the biosynthesis of growth factors and their receptors, structure-function relationships, as well as mechanisms for intracellular signal transmission. The possibility of modifying these factors has opened new fields of clinical application. In this paper, four major areas of growth factor research are presented: the characterization of growth factor genes and their protein products, growth factor receptors and signal transduction by the receptors to mediate biological action, the biological actions of the various growth factors, and the role of growth factors in health and disease and their possible clinical application. Some of the topics covered include: structure of the IGFs and their variants; isoforms of PDGF receptor types; tyrosine kinase activation; structure of G-proteins in biological membranes; possible therapeutic application of NGF in the treatment of Parkinson's and Alzheimer's diseases; PDGF's possible role in the development of several fibroproliferative diseases and its therapeutic application in wound healing; and the possible use of angiogenic inhibitors in tumor treatment.

  7. Spatially Directed Guidance of Stem Cell Population Migration by Immobilized Patterns of Growth Factors

    PubMed Central

    Miller, Eric D.; Li, Kang; Kanade, Takeo; Weiss, Lee E.; Walker, Lynn M.; Campbell, Phil G.

    2011-01-01

    We investigated how engineered gradients of exogenous growth factors, immobilized to an extracellular matrix material, influence collective guidance of stem cell populations over extended time (>1 day) and length (>1 mm) scales in vitro. Patterns of low-to-high, high-to-low, and uniform concentrations of heparin-binding epidermal growth factor-like growth factor were inkjet printed at precise locations on fibrin substrates. Proliferation and migration responses of mesenchymal stem cells seeded at pattern origins were observed with time-lapse video microscopy and analyzed using both manual and automated computer vision-based cell tracking techniques. Based on results of established chemotaxis studies, we expected that the low-to-high gradient would most effectively direct cell guidance away from the cell source. All printed patterns, however, were found to direct net collective cell guidance with comparable responses. Our analysis revealed that collective “cell diffusion” down a cell-to-cell confinement gradient originating at the cell starting lines and not the net sum of directed individual cell migration up a growth factor concentration gradient is the principal driving force for directing mesenchymal stem cell population outgrowth from a cell source. These results suggest that simple uniform distributions of growth factors immobilized to an extracellular matrix material may be as effective in directing cell migration into a wound site as more complex patterns with concentration gradients. PMID:21272933

  8. [Transforming growth factor of beta-type].

    PubMed

    Stoĭka, R S

    1988-01-01

    Recent data about the structure and properties of the beta-type transforming growth factor as well as evidence about its influence on different target cells are presented. The regulatory action of the factor is shown to depend mainly on the type of tested cells, conditions of their culturing and the presence of other bioregulators of cell proliferation in the medium. The prospects of the beta-type transforming growth factor use in practice are considered.

  9. Heparin use in a rat hemorrhagic shock model induces biologic activity in mesenteric lymph separate from shock.

    PubMed

    Qin, Yong; Prescott, Lauriston M; Deitch, Edwin A; Kaiser, Vicki L

    2011-04-01

    Experimental data have shown that mesenteric lymph from rats subjected to trauma-hemorrhagic shock (THS) but not trauma-sham shock induces neutrophil activation, cytotoxicity, decreased red blood cell (RBC) deformability, and bone marrow colony growth suppression. These data have led to the hypothesis that gut factors produced from THS enter the systemic circulation via the mesenteric lymphatics and contribute to the progression of multiple organ failure after THS. Ongoing studies designed to identify bioactive lymph agents implicated factors associated with the heparin use in the THS procedure. We investigated if heparin itself was responsible for reported toxicity to human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cell toxicity was not induced by lymph when alternate anticoagulants (citrate and EDTA) were used in THS. Human umbilical vein endothelial cell toxicity was induced by lymph after heparin but not saline or citrate injection into trauma-sham shock and naive animals and was dose dependent. Activities of both heparin-releasable lipases (lipoprotein and hepatic) were detected in the plasma and lymph from THS and naive animals receiving heparin but not citrate or saline. Lymph-induced HUVEC toxicity correlated with lymph lipase activities. Finally, incubation of HUVECs with purified lipoprotein lipase added to naive lymph-induced toxicity in vitro. These data show that heparin, not THS, is responsible for the reported lymph-mediated HUVEC toxicity through its release of lipases into the lymph. These findings can provide alternative explanations for several of the THS effects reported in the literature using heparin models, thus necessitating a review of previous work in this field.

  10. Heparin use in a rat hemorrhagic shock model induces biologic activity in mesenteric lymph separate from shock

    PubMed Central

    Qin, Yong; Prescott, Lauriston M.; Deitch, Edwin A.; Kaiser, Vicki L.

    2011-01-01

    Experimental data has shown that mesenteric lymph from rats subjected to trauma-hemorrhagic shock (THS) but not trauma-sham shock (TSS) induces neutrophil activation, cytotoxicity, decreased red blood cell deformability and bone marrow colony growth suppression. These data have lead to the hypothesis that gut factors produced from THS enter the systemic circulation via the mesenteric lymphatics and contribute to the progression of Multiple Organ Failure (MOF) following THS. Ongoing studies designed to identify bioactive lymph agents implicated factors associated with the heparin use in the THS procedure. We investigated if heparin itself was responsible for reported toxicity to human umbilical vein endothelial cells (HUVECs). HUVEC toxicity was not induced by lymph when alternate anti-coagulants (citrate and EDTA) were used in THS. HUVEC toxicity was induced by lymph after heparin but not saline or citrate injection into TSS and naïve animals and was dose dependent. Activities of both heparin-releasable lipases (lipoprotein (LPL) and hepatic (HL)) were detected in the plasma and lymph from THS and naïve animals receiving heparin but not citrate or saline. Lymph-induced HUVEC toxicity correlated with lymph lipase activities. Finally, incubation of HUVECs with purified LPL added to naïve lymph induced toxicity in vitro. These data show that heparin, not THS, is responsible for the reported lymph-mediated HUVEC toxicity through its release of lipases into the lymph. These findings can provide alternative explanations for several of the THS effects reported in the literature using heparin models thus necessitating a review of previous work in this field. PMID:21063238

  11. Growth factors for the treatment of ischemic brain injury (growth factor treatment).

    PubMed

    Larpthaveesarp, Amara; Ferriero, Donna M; Gonzalez, Fernando F

    2015-04-30

    In recent years, growth factor therapy has emerged as a potential treatment for ischemic brain injury. The efficacy of therapies that either directly introduce or stimulate local production of growth factors and their receptors in damaged brain tissue has been tested in a multitude of models for different Central Nervous System (CNS) diseases. These growth factors include erythropoietin (EPO), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor (IGF-1), among others. Despite the promise shown in animal models, the particular growth factors that should be used to maximize both brain protection and repair, and the therapeutic critical period, are not well defined. We will review current pre-clinical and clinical evidence for growth factor therapies in treating different causes of brain injury, as well as issues to be addressed prior to application in humans.

  12. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, H. H.

    1987-01-01

    Muscle tissue culture techniques were developed to grow skeletal myofibers which differentiate into more adult-like myofibers. Mechanical simulation studies of these muscle cells in a newly developed mechanical cell simulator can now be performed to study growth processes in skeletal muscle. Conditions in the mechanical cell simulator were defined where mechanical activity can either prevent muscle wasting or stimulate muscle growth. The role of endogenous and exogenous growth factors in tension-induced muscle growth is being investigated under the defined conditions of tissue culture.

  13. A systems biology approach for the investigation of the heparin/heparan sulfate interactome.

    PubMed

    Ori, Alessandro; Wilkinson, Mark C; Fernig, David G

    2011-06-03

    A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.

  14. Angiogenic growth factors in preinvasive breast disease.

    PubMed

    Heffelfinger, S C; Miller, M A; Yassin, R; Gear, R

    1999-10-01

    Recently, we showed that preinvasive breast pathologies, such as usual hyperplasia, atypical hyperplasia, and carcinoma in situ, have an increased vascularity when compared with normal breast tissue (S. C. Heffelfinger et al., Clinical Cancer Res., 2: 1873-1878, 1996). To understand the mechanism of this increased vascularity, we examined by immunohistochemistry each of these pathological lesions for the expression of angiogenic growth factors. These studies showed that normal breast tissue contains numerous angiogenic agents, particularly vascular endothelial cell growth factor and basic fibroblast growth factor. At the transition from normal epithelium to proliferative breast disease, insulin-like growth factor (IGF) II expression was increased, primarily in the stroma and infiltrating leukocytes. However, among proliferative tissues, IGF I decreased with increasing vascularity. Finally, both epithelial vascular endothelial growth factor and epithelial and leukocytic platelet-derived endothelial cell growth factor increased at the transition to carcinoma in situ, whereas stromal and leukocytic basic fibroblast growth factor were elevated only in invasive carcinoma. Therefore, during histological progression there is also a complex progression of angiogenic growth factors. For CIS, two forms of vascularity are found: stromal microvascular density (MVD), and vascularity associated with the epithelial basement membrane (vascular score). There was 35% discordance between these two measurement systems. Among carcinoma in situ cases, decreases in stromal IGF II were associated with increasing vascular scores but not MVD, and increases in platelet-derived endothelial cell growth factor were associated with increasing MVD but not the vascular score. The presence of discordance and differential association with specific angiogenic agents suggests that these two forms of vascularity may be differentially regulated.

  15. Bridging endometrial receptivity and implantation: network of hormones, cytokines, and growth factors.

    PubMed

    Singh, Mohan; Chaudhry, Parvesh; Asselin, Eric

    2011-07-01

    The prerequisite of successful implantation depends on achieving the appropriate embryo development to the blastocyst stage and at the same time the development of an endometrium that is receptive to the embryo. Implantation is a very intricate process, which is controlled by a number of complex molecules like hormones, cytokines, and growth factors and their cross talk. A network of these molecules plays a crucial role in preparing receptive endometrium and blastocyst. Furthermore, timely regulation of the expression of embryonic and maternal endometrial growth factors and cytokines plays a major role in determining the fate of embryo. Most of the existing data comes from animal studies due to ethical issues. In this study, we comprehend the data from both animal models and humans for better understanding of implantation and positive outcomes of pregnancy. The purpose of this review is to describe the potential roles of embryonic and uterine factors in implantation process such as prostaglandins, cyclooxygenases, leukemia inhibitory factor, interleukin (IL) 6, IL11, transforming growth factor-β, IGF, activins, NODAL, epidermal growth factor (EGF), and heparin binding-EGF. Understanding the function of these players will help us to address the reasons of implantation failure and infertility.

  16. Platelet Activating Factor: A Growth Factor for Breast Cancer

    DTIC Science & Technology

    2006-09-01

    Factor for Breast Cancer PRINCIPAL INVESTIGATOR: Larry W. Daniel, Ph.D. CONTRACTING ORGANIZATION: Wake Forest University...A Growth Factor for Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-04-1-0682 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Larry W...Relevance: If PAF is found to be a growth and angiogenic factor for breast cancer cells, these studies can be followed up by in vivo studies in nude

  17. [Role of heparin in the formation of food lipemia].

    PubMed

    Sokolik, V V; Bozhko, G Kh

    2005-01-01

    Influence of endogenic and exogenic heparin in vivo on the basic forms of serum lipids content: cholesterol ethers, triacylglycerols, free fatty acids; as well as that glycosaminoglycan effect in vivo and in vitro on total lipoproteine lipase (LPL) activity and lecithin-cholesterol acyltransferase (LCAT) activity of human blood serum were investigated on food lipidemia model. The decrease of intercell reserve heparin content and increase of the background and post-heparin levels of blood serum LPL activity were indicated after two hours food load. The role of two factors, endogenic heparin being one of them, in the increase of postprandial LPL activity of blood serum were discussed. At the same time, some inhibition of blood serum LCAT activity two hours after food reception (evidently, as a result of endogenic heparin action) and to a considerable extent inhibition of cholesterol etherification under the action of exogenic heparin in vivo were ascertain. Heparin in vitro (50 U/ml of blood serum) did not influence LCAT and total LPL activities. It was summarised that endogenic heparin is a factor, taking part in lipolysis processes regulation.

  18. The role of fibroblast growth factors in tumor growth.

    PubMed

    Korc, M; Friesel, R E

    2009-08-01

    Biological processes that drive cell growth are exciting targets for cancer therapy. The fibroblast growth factor (FGF) signaling network plays a ubiquitous role in normal cell growth, survival, differentiation, and angiogenesis, but has also been implicated in tumor development. Elucidation of the roles and relationships within the diverse FGF family and of their links to tumor growth and progression will be critical in designing new drug therapies to target FGF receptor (FGFR) pathways. Recent studies have shown that FGF can act synergistically with vascular endothelial growth factor (VEGF) to amplify tumor angiogenesis, highlighting that targeting of both the FGF and VEGF pathways may be more efficient in suppressing tumor growth and angiogenesis than targeting either factor alone. In addition, through inducing tumor cell survival, FGF has the potential to overcome chemotherapy resistance highlighting that chemotherapy may be more effective when used in combination with FGF inhibitor therapy. Furthermore, FGFRs have variable activity in promoting angiogenesis, with the FGFR-1 subgroup being associated with tumor progression and the FGFR-2 subgroup being associated with either early tumor development or decreased tumor progression. This review highlights the growing knowledge of FGFs in tumor cell growth and survival, including an overview of FGF intracellular signaling pathways, the role of FGFs in angiogenesis, patterns of FGF and FGFR expression in various tumor types, and the role of FGFs in tumor progression.

  19. A supersulfated low-molecular-weight heparin (IK-SSH) increases plasma levels of free and total tissue factor pathway inhibitor after intravenous and subcutaneous administration in humans.

    PubMed

    Kaiser, B; Glusa, E; Hoppensteadt, D A; Breddin, H K; Amiral, J; Fareed, J

    1998-09-01

    Unfractionated as well as low-molecular-weight heparins (LMWH) are known to cause an increase in blood levels of tissue factor pathway inhibitor (TFPI). To study the effect of a newly developed supersulfated LMWH (IK-SSH, Iketon Farmaceutici) on TFPI concentrations in human plasma, the compound was injected into volunteers at doses of 0.14, 0.33 and 0.66 mg/kg intravenously or 0.33, 0.66 and 1.0 mg/kg subcutaneously. At certain known times blood was drawn and plasma levels of both total and free TFPI were measured using enzyme-linked immunosorbent assay methodology. Baseline plasma concentrations of TFPI were 72.2+/-3.1 ng/ml for total and 10.8+/-0.8 ng/ml for free TFPI. Intravenous or subcutaneous injection of IK-SSH led to a strong and long-lasting rise in TFPI levels which were increased more than 5-fold for total TFPI and more than 30-fold for free TFPI. Maximum TFPI levels were reached 5-10 min after intravenous and 60 min after subcutaneous administration. IK-SSH caused prolongation of ex-vivo clotting times in the APTT and Heptest assay, whereas thrombin time was not affected. Anticoagulant actions of IK-SSH showed a significant correlation to plasma concentrations of TFPI and they are thought to be based at least partially on the release of TFPI from vascular sites.

  20. An unnatural PIP simulates growth factor signaling.

    PubMed

    Swan, Laura

    2009-11-25

    In this issue of Chemistry & Biology, Laketa et al. describe the synthesis of a membrane permeant phosphoinositide lipid that acts to stimulate PI(3,4,5)P(3)-dependent signaling without the need of growth factor stimulation.

  1. New Clue Found to Growth Factor Action.

    ERIC Educational Resources Information Center

    Hoffman, Michelle

    1991-01-01

    Discussed is the discovery which may help to explain epidermal growth factor effects on the cell skeleton. The role of a protein called profilin in the regulation of the microfilament system is described. (CW)

  2. New Clue Found to Growth Factor Action.

    ERIC Educational Resources Information Center

    Hoffman, Michelle

    1991-01-01

    Discussed is the discovery which may help to explain epidermal growth factor effects on the cell skeleton. The role of a protein called profilin in the regulation of the microfilament system is described. (CW)

  3. The function of vascular endothelial growth factor.

    PubMed

    Nieves, Bonnie J; D'Amore, Patricia A; Bryan, Brad A

    2009-01-01

    Vascular endothelial growth factor (VEGF) is considered the master regulator of angiogenesis during growth and development, as well as in disease states such as cancer, diabetes, and macular degeneration. This review details our current understanding of VEGF signaling and discusses the benefits and unexpected side effects of promising anti-angiogenic therapeutics that are currently being used to inhibit neovacularization in tumors.

  4. Placental growth factor and vascular endothelial growth factor receptor-2 in human lung development.

    PubMed

    Janér, Joakim; Andersson, Sture; Haglund, Caj; Karikoski, Riitta; Lassus, Patrik

    2008-08-01

    We examined the pulmonary expression of 2 proangiogenic factors, namely, placental growth factor and vascular endothelial growth factor receptor-2, during lung development and acute and chronic lung injury in newborn infants. Six groups were included in an immunohistochemical study of placental growth factor and vascular endothelial growth factor receptor-2, that is, 9 fetuses, 4 preterm and 8 term infants without lung injury who died soon after birth, 5 preterm infants with respiratory distress syndrome of <2 days and 7 with respiratory distress syndrome of >10 days, and 6 with bronchopulmonary dysplasia. Placental growth factor concentrations in tracheal aspirate fluid were measured in 70 samples from 20 preterm infants during the first postnatal week. In immunohistochemical analyses, placental growth factor staining was seen in bronchial epithelium and macrophages in all groups. Distal airway epithelium positivity was observed mostly in fetuses and in preterm infants who died soon after birth. Vascular endothelial growth factor receptor-2 staining was seen in vascular endothelium in all groups and also in lymphatic endothelium in fetuses. Vascular endothelial growth factor receptor-2 staining in arterial endothelium was associated with higher and staining in venous endothelium with lower gestational age. In capillaries, less vascular endothelial growth factor receptor-2 staining was seen in bronchopulmonary dysplasia. The mean placental growth factor protein concentration in tracheal aspirate fluid during the first postnatal week was 0.64 +/- 0.42 pg/mL per IgA-secretory component unit. Concentrations during the first postnatal week were stable. Lower placental growth factor concentrations correlated with chorioamnionitis and lactosyl ceramide positivity. The vascular endothelial growth factor receptor-2 staining pattern seems to reflect ongoing differentiation and activity of different endothelia. Lower vascular endothelial growth factor receptor-2 expression

  5. Cancer cells. 3: Growth factors and transformation

    SciTech Connect

    Feramisco, J.; Ozanne, B.; Stiles, C.

    1985-01-01

    This book contains over 50 papers. Some of the titles are: Structure of Human Epidermal Growth Factor and Expression of Normal and Variant mRNAs in Epdermoid Carcinoma Cells; Tyrosine Kinase Activity Associated with the v-erb-B Gene Product; Cloning and Characterization of Human Epidermal Growth Factor-Receptor Gene Sequences in A431 Carcinoma Cells; Anti-oncogenes and the Suppression of Tumor Formation; and Normal Human sis/PDGF-2 Gene Expression Induces Cellular Transformation.

  6. Growth factors and growth factor receptors in the hippocampus. Role in plasticity and response to injury.

    PubMed

    Nieto-Sampedro, M; Bovolenta, P

    1990-01-01

    Various growth factors are present in the hippocampal formation and appear responsible for the prominent plasticity of this brain area. Although hormone-like growth-promoting polypeptides are the best known, recent studies emphasize the importance in the growth response of molecules such as laminin proteoglycans, neurotransmitters and growth inhibitors. The progress and problems in the study of these substances are reviewed.

  7. Insulin-like growth factor 1 and hair growth.

    PubMed

    Su, H Y; Hickford, J G; Bickerstaffe, R; Palmer, B R

    1999-11-01

    Insulin-like growth factor 1 (IGF-1) has been identified as an important growth factor in many biological systems.[1] It shares considerable structural homology with insulin and exerts insulin-like effects on food intake and glucose metabolism. Recently it has been suggested to play a role in regulating cellular proliferation and migration during the development of hair follicles. [2,3] To exert its biological effects, the IGF-1 is required to activate cells by binding to specific cell-surface receptors. The type I IGF receptor (IGF-1R) is the only IGF receptor to have IGF-mediated signaling functions.[1] In circulation, this growth factor mediates endocrine action of growth hormone (GH) on somatic growth and is bound to specific binding proteins (BPs). The latter control IGF transport, efflux from vascular compartments and association with cell surface receptors.[4] In tissues, IGF-1 is produced by mesenchymal type cells and acts in a paracrine and autocrine fashion by binding to the IGF-1R. This binding activates the receptor tyrosine kinase (RTK) that triggers the downstream responses and finally stimulates cell division.[5] IGF-1 may therefore be able to stimulate the proliferation of hair follicle cells through cellular signaling pathways of its receptors. Local infusion of IGF-1 into sheep has been reported to be capable of stimulating protein synthesis in the skin.[6] It may also increase the production of wool keratin. Recently, transgenic mice overexpressing IGF-1 in the skin have been shown to have earlier hair follicle development than controls.[7] In addition, this growth factor plays an important role in many cell types as a survival factor to prevent cell death.[8] This anti-apoptotic function of IGF-1 may be important to the development of follicle cells as follicles undergo a growth cycle where the regressive, catagen phase is apoptosis driven. In this review, the effects of IGF-1 on follicle cell proliferation and differentiation are discussed. In

  8. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    SciTech Connect

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-08-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

  9. Molecular level interaction of the human acidic fibroblast growth factor with the antiangiogenic agent, inositol hexaphosphate .

    PubMed

    Kumar, Sriramoju M; Wang, Han-Min; Mohan, Sepuru K; Chou, Ruey-Hwang; Yu, Chin

    2010-12-21

    Acidic fibroblast growth factor (FGF1) regulates a wide array of important biological phenomena such as angiogenesis, cell differentiation, tumor growth, and neurogenesis. Generally, FGFs are known for their strong affinity for the glycosaminoglycan heparin, as a prerequisite for recognition of a specific tyrosine kinase on the cell surface and are responsible for the cell signal transduction cascade. Inositol hexaphosphate (IP6) is a natural antioxidant and is known for its antiangiogenic role, in addition to its ability to control tumor growth. In the present study, we investigated the interaction of IP6 with the acidic fibroblast growth factor (FGF1) using various biophysical techniques including isothermal calorimetry, circular dichroism, and multidimensional NMR spectroscopy. Herein, we have reported the three-dimensional solution structure of the FGF1-IP6 complex. These data show that IP6 binds FGF1 and enhances its thermal stability. In addition, we also demonstrate that IP6 acts as an antagonist to acidic fibroblast growth factor by inhibiting its receptor binding and subsequently decreasing the mitogenic activity. The inhibition likely results in the ability of IP6 to antagonize the angiogenic and mitogenic activity of FGF1.

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

    PubMed

    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.

  11. Immobilization of heparin oligosaccharides onto radiofrequency plasma modified pyrolytic carbon-coated graphite.

    PubMed

    Yuan, S; Cai, W; Szakalas-Gratzl, G; Kottke-Marchant, K; Tweden, K; Marchant, R E

    1995-01-01

    Heparin oligosaccharides with different anticoagulant activities were prepared and immobilized onto pyrolytic carbon coated graphite (PC) heart valve materials commonly used in mechanical heart valve prostheses. Prior to immobilization, PC surfaces were modified by radiofrequency plasma polymerized N-vinyl-2-pyrrolidone (PPNVP) thin films (approximately 100 nm) and derivatized to provide surface hydroxyl groups. Cleaved, low affinity heparin (C-heparin) with factor Xa inhibition activity of 107 to 130 IU/mg, was prepared by partial deaminative cleavage of commercial crude heparin, and high-affinity heparin (HA-heparin) with factor Xa inhibition activity of 550 to 1000 IU/mg was prepared by fractionation of C-heparin using agarose-ATIII affinity chromatography. C-heparin and HA-heparin were immobilized to surface modified PC by reductive amination. Anticoagulant activity of the heparin immobilized surfaces was determined by chromogenic assay for the inhibition of factor Xa. Highest surface anticoagulant activity was measured on C-heparin immobilized surfaces (64.0 +/- 7.3 mIU/cm2) compared with HA-heparin immobilized surfaces (27.2 +/- 12.2 mIU/cm2), suggesting higher binding of C-heparin than HA-heparin on the modified PC surfaces. Immobilized surfaces were evaluated under dynamic flow conditions, by subjecting samples to shear stress of up to 206 dyn/cm2 in the presence of 5% albumin solution or human plasma. Anticoagulant activity of the immobilized heparin was retained, although reduced, and the modified surfaces showed evidence for protein resistance.

  12. An empirical phase diagram approach to investigate conformational stability of “second-generation” functional mutants of acidic fibroblast growth factor-1

    PubMed Central

    Alsenaidy, Mohammad A; Wang, Tingting; Kim, Jae Hyun; Joshi, Sangeeta B; Lee, Jihun; Blaber, Michael; Volkin, David B; Middaugh, C Russell

    2012-01-01

    Acidic fibroblast growth factor-1 (FGF-1) is an angiogenic protein which requires binding to a polyanion such as heparin for its mitogenic activity and physicochemical stability. To evaluate the extent to which this heparin dependence on solution stability could be reduced or eliminated, the structural integrity and conformational stability of 10 selected FGF-1 mutants were examined as a function of solution pH and temperature by a series of spectroscopic methods including circular dichroism, intrinsic and extrinsic fluorescence spectroscopy and static light scattering. The biophysical data were summarized in the form of colored empirical phase diagrams (EPDs). FGF-1 mutants were identified with stability profiles in the absence of heparin comparable to that of wild-type FGF-1 in the presence of heparin while still retaining their biological activity. In addition, a revised version of the EPD methodology was found to provide an information rich, high throughput approach to compare the effects of mutations on the overall conformational stability of proteins in terms of their response to environmental stresses such as pH and temperature. PMID:22113934

  13. Heparan sulfate proteoglycans mediate the angiogenic activity of the vascular endothelial growth factor receptor-2 agonist gremlin.

    PubMed

    Chiodelli, Paola; Mitola, Stefania; Ravelli, Cosetta; Oreste, Pasqua; Rusnati, Marco; Presta, Marco

    2011-12-01

    Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs. Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N-, 2-O, and 6-O-sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase(1/2) and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O-sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo. HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist.

  14. Synthesis and detection of N-sulfonated oversulfated chondroitin sulfate in marketplace heparin.

    PubMed

    Mans, Daniel J; Ye, Hongping; Dunn, Jamie D; Kolinski, Richard E; Long, Dianna S; Phatak, Nisarga L; Ghasriani, Houman; Buhse, Lucinda F; Kauffman, John F; Keire, David A

    2015-12-01

    N-sulfonated oversulfated chondroitin sulfate (NS-OSCS), recently reported as a potential threat to the heparin supply, was prepared along with its intermediate derivatives. All compounds were spiked into marketplace heparin and subjected to United States Pharmacopeia (USP) identification assays for heparin (proton nuclear magnetic resonance [(1)H NMR], chromatographic identity, % galactosamine [%GalN], anti-factor IIa potency, and anti-factor Xa/IIa ratio). The U.S. Food and Drug Administration (FDA) strong-anionic exchange high-performance liquid chromatography (SAX-HPLC) method resolved NS-OSCS from heparin and OSCS and had a limit of detection of 0.26% (w/w) NS-OSCS. The %GalN test was sensitive to the presence of NS-OSCS in heparin. Therefore, current USP heparin monograph tests (i.e., SAX-HPLC and %GalN) detect the presence of NS-OSCS in heparin.

  15. THE EFFECT OF HEPARIN ON THE EARLY STAGES OF BLOOD COAGULATION

    PubMed Central

    O'Brien, J. R.

    1960-01-01

    From experiments reported it is concluded that heparin combines with and inactivates Christman factor (C.F.) to form reversibly a heparin-C.F. complex. Apart from the effect of heparin on C.F. and on thrombin, heparin in moderate concentrations was not shown to inactivate any other coagulation factors. “Available” heparin is defined as heparin in such a state that it can delay some clotting systems. Heparin was rendered “unavailable” or inactive by plasma C.F. and especially by serum C.F., by platelet protein (but not intact platelets), and by platelet-like activity of serum (P.L.A.S.). These three proteins uniquely among all the plasma and serum proteins inactivate heparin. If an appropriate concentration of heparin is added to whole blood the clotting time is prolonged, due presumably to the inactivation of C.F. by heparin: “available” heparin disappears during clotting and none is found in the serum. The interactions summarized above probably account for these findings. The disappearance of available heparin would also account for the normal thrombin generation and prothrombin consumption observed when heparinized blood clots. Some properties of P.L.A.S. are reported and its possible role in normal coagulation is considered. Heparin is known to become attached preferentially to one protein rather than another. Some plasma and serum fractions can be arranged in order of their increasing affinity for heparin thus: β Lipoproteins

  16. Engineering growth factors for regenerative medicine applications.

    SciTech Connect

    Mitchell, Aaron C.; Briquez, Priscilla S.; Hubbell, Jeffrey A.; Cochran, Jennifer R.

    2016-01-15

    Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.

  17. Placenta Growth Factor in Diabetic Wound Healing

    PubMed Central

    Cianfarani, Francesca; Zambruno, Giovanna; Brogelli, Laura; Sera, Francesco; Lacal, Pedro Miguel; Pesce, Maurizio; Capogrossi, Maurizio C.; Failla, Cristina Maria; Napolitano, Monica; Odorisio, Teresa

    2006-01-01

    Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process. PMID:17003476

  18. FACTORS WHICH CONTROL MAXIMAL GROWTH OF BACTERIA

    PubMed Central

    Sinclair, N. A.; Stokes, J. L.

    1962-01-01

    Sinclair, N. A. (Washington State University, Pullman) and J. L. Stokes. Factors which control maximal growth of bacteria. J. Bacteriol. 83:1147–1154. 1962.—In a chemically defined medium containing 1% glucose and 0.1% (NH4)2SO4, both of these compounds are virtually exhausted by the growth of Pseudomonas fluorescens. If these carbon, energy, and nitrogen sources are added back to the culture filtrate, maximal growth to the level of the original culture is obtained. This process can be repeated several times with the same results. Eventually, however, the supply of minerals in the culture limits growth. When the nutrient levels are raised to 3% glucose and 0.3% (NH4)2SO4, lack of oxygen and low pH limit growth before the supply of nutrients is exhausted. There is no evidence that specific autoinhibitory substances are produced either in chemically defined or complex nitrogenous media or that physical crowding of the cells limits growth. The results with Escherichia coli are similar to those with P. fluorescens. However, after a few growth cycles aerobically and after only one growth cycle anaerobically, inhibitory substances, probably organic acids, accumulate and limit growth. PMID:13913264

  19. [The insulin-like growth factor IGFBP-1--specific marker for preterm delivery in pregnant women with clinical symptoms].

    PubMed

    Kolev, N; Ivanov, S; Kovachev, E; Slavchev, S

    2014-01-01

    The insulin-like growth factor IGFBP-1 is a binding protein (IBP-1), also known as placental protein (PP12), is encoded in people as IGFBP-1 gene. This gene encodes a protein in the domain of IGFBP-1 and domain thyroglobulin. During the last years highly phos-phorylated versions of IGFBP-1 (IGFBP-1 pM) have been found in decidual cells--a marker of threat for preterm birth. The quantity analysis of the insulin-like growth factor in the serum or heparinized plasma is used to locate diseases related to growth. Its levels in the plasma can scarcely be determined after birth and steadily rise with age while they reach their maximum during puberty. These levels rise constantly during pregnancy.

  20. Structural and functional characterization of oversulfated chondroitin sulfate/dermatan sulfate hybrid chains from the notochord of hagfish. Neuritogenic and binding activities for growth factors and neurotrophic factors.

    PubMed

    Nandini, Chilkunda D; Mikami, Tadahisa; Ohta, Mitsuhiro; Itoh, Nobuyuki; Akiyama-Nambu, Fumiko; Sugahara, Kazuyuki

    2004-12-03

    Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of D-glucuronic acid (GlcUA)- and L-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765-9776), suggest physiological roles of the hybrid chains in the development of the brain.

  1. PROSPECT - GROWTH FACTOR CONTROL OF BONE MASS

    PubMed Central

    Canalis, Ernesto

    2010-01-01

    Bone formation is determined by the number and function of osteoblasts. Cell number is governed by factors that regulate the replication and differentiation of pre-osteoblasts and factors that regulate osteoblastic cell death. Cell function is controlled by signals acting on the mature osteoblast. Platelet derived and fibroblast growth factors are bone cell mitogens. Bone morphogenetic proteins (BMP) and Wnt induce the differentiation of mesenchymal cells toward osteoblasts, and insulin-like growth factor (IGF)-I stimulates the function of mature osteoblasts and prevents their death. The activity of BMP, Wnt and IGF-I is modulated by extracellular antagonists or binding proteins. Changes in growth factor synthesis and activity may play a role in the pathogenesis of selected forms of osteoporosis, and alterations in the expression or binding of the extracellular antagonists can be associated with changes in bone mass. Current approaches to bone anabolic therapies for osteoporosis include the administration of a growth factor, such as IGF-I, or the neutralization of an antagonist. Ideally, the targeting of an anabolic agent should be specific to bone to preclude non-skeletal unwanted side effects. Clinical trials are needed to determine the long-term effectiveness and safety of novel anabolic agents for the management of osteoporosis. PMID:19718659

  2. Multifunctional silk-heparin biomaterials for vascular tissue engineering applications

    PubMed Central

    Seib, F. Philipp; Herklotz, Manuela; Burke, Kelly A.; Maitz, Manfred F.; Werner, Carsten; Kaplan, David L.

    2013-01-01

    Over the past 30 years, silk has been proposed for numerous biomedical applications that go beyond its traditional use as a suture material. Silk sutures are well tolerated in humans, but the use of silk for vascular engineering applications still requires extensive biocompatibility testing. Some studies have indicated a need to modify silk to yield a hemocompatible surface. This study examined the potential of low molecular weight heparin as a material for refining silk properties by acting as a carrier for vascular endothelial growth factor (VEGF) and improving silk hemocompatibility. Heparinized silk showed a controlled VEGF release over 6 days; the released VEGF was bioactive and supported the growth of human endothelial cells. Silk samples were then assessed using a humanized hemocompatibility system that employs whole blood and endothelial cells. The overall thrombogenic response for silk was very low and similar to the clinical reference material polytetrafluoroethylene. Despite an initial inflammatory response to silk, apparent as complement and leukocyte activation, the endothelium was maintained in a resting, anticoagulant state. The low thrombogenic response and the ability to control VEGF release support the further development of silk for vascular applications. PMID:24099708

  3. Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

    PubMed Central

    Sehgal, I; Bailey, J; Hitzemann, K; Pittelkow, M R; Maihle, N J

    1994-01-01

    Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways. Images PMID:8049525

  4. Heparin-induced thrombocytopenia (HIT II) in liver transplant recipients: a retrospective multivariate analysis of prognostic factors.

    PubMed

    Hüser, Norbert; Aßfalg, Volker; Reim, Daniel; Novotny, Alexander; Thorban, Stefan; Cheng, Zhangjun; Kornberg, Arno; Friess, Helmut; Büchler, Peter; Matevossian, Edouard

    2012-07-01

    We investigated the prevalence of HIT II in liver transplant recipients and analysed associated factors. In recipients with clinically suspected HIT II in the 4Ts pretest clinical scoring system HIPA-assay was performed. Next, 37 clinical variables were analysed retrospectively for their association with HIT II. Factors significantly correlated to our findings in univariate analysis were included in a multivariate model and binary logistic regression analysis. Among 46 recipients 21 patients were suspicious in the 4Ts pretest and 14 of them (30.4%) were diagnosed HIT-antibody positive. Patient's age (P = 0.001), postoperative dialysis (P = 0.028), and postoperative hospital stay (P = 0.035) were significantly associated with development of HIT-antibodies in univariate analysis. Postoperative dialysis and postoperative hospital stay turned out as epiphenomena of patient's age, the only independent predictor (P = 0.021). Using multiple χ(2) -testing, a cut-off could be calculated, assigning patients younger than 59 years to a low risk group and patients of 59 years and older to a high risk group. High incidence of peri-operative HIT II seroconversion in liver transplant recipients is not associated with factors known to induce thrombocyte activation, like blood products or cell-saver. Only patients' age was identified as independent predictor.

  5. A density gradient of basic fibroblast growth factor guides directional migration of vascular smooth muscle cells.

    PubMed

    Wu, Jindan; Mao, Zhengwei; Han, Lulu; Zhao, Yizhi; Xi, Jiabin; Gao, Changyou

    2014-05-01

    The migration of vascular smooth muscle cells (VSMCs) is an important process in many physiological events. It is of paramount importance to control the migration rate and direction of VSMCs by biomaterials. In this paper, a density gradient of basic fibroblast growth factor (bFGF) was fabricated using an injection method and the bio-conjugation between heparin and bFGF. The density of bFGF gradually increased with a slope of 17 ng/cm(2)/mm. Adhesion and migration of VSMCs were studied on the bFGF gradient. The VSMCs exhibited preferential orientation and an enhanced directional migration behavior on the gradient surface. Up to 70% cells migrated towards the region with a higher density of bFGF on the gradient. However, the bFGF gradient had no effect on the cell migration rate. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. [Acidic fibroblast growth factor promotes the growth of skeletal muscle satellite cells: experiment with rabbits].

    PubMed

    Dong, Shao-hong; Zang, Peng; Wen, Jun-min; Gao, Hong; Pang, Xin-li

    2007-05-08

    To explore if human recombinant acidic fibroblast growth factor (rhaFGF) can promote the proliferation of skeletal muscle satellite cells (MSCs). MSCs were obtained from rabbits and cultured, and divided into 2 groups: rhaFGF group, treated with rhaFGF of the concentrations of 0, 5, 10, 20, 40, 60, and 80 microg/L, and rhaFGF + low molecular weight heparin (LMWH) group, treated with rhaFGF of different concentrations and LMWH of the terminal concentration of 10 mg/L. MTT method was used to observe the proliferation of the MSCs so as to determine the appropriate concentration to be used in the next experiment. Other MSCs were cultured and treated with the rhaFGF of the appropriate concentration and then LMWH of the terminal concentration of 10 mg/L was added to be co-cultured for 3 days. Flow cytometry was used to observe the cell cycle of the MSCs. rhaFGF of the concentrations 20 approximately 60 ng/ml promoted the proliferation of MSCs, and 40 ng/ml was selected as the best concentration to be used in the next experiment. Treated with the rhaFGF of the concentration of 40 ng/ml and LMWH for 3 days, the proportion of MSCs at the stage G(0)/G(1) was significantly lower and those at the stage S significantly higher in comparison with the control group (both P < 0.01), however, not significantly different from those of the rhaFGF group. rhaFGF promotes the proliferation of MSCs.

  7. Unfractionated heparin: a nursing dilemma.

    PubMed

    Oertel, Lynn B

    2004-08-01

    Nurses face challenges in all aspects of their practice, especially with administering and monitoring drugs in a safe, effective manner. Key factors known to affect drug administration include general drug knowledge, formal nurse education, continuing education needs, clinical experience, and the nationwide nursing shortage. Other factors are advances in technologic aids and quality improvement initiatives. Emphasis on patient safety is growing, especially as it relates to drug therapy and high-alert drugs such as unfractionated heparin (UFH). Specific interventions related to UFH administration can enhance patient care management. Because nurses are at the site of direct patient care, they are often in an opportune position for identifying medication errors. At the same time, and most important, nurses need to collaborate with other health care professionals to actively develop solutions to minimize these errors. Adopting a systems approach and working collaboratively with an interdisciplinary team can result in improved patient outcomes.

  8. Protamine — antagonist to heparin

    PubMed Central

    Jaques, L. B.

    1973-01-01

    Protamine is used for titration of heparin in vitro for diagnosis of hemorrhagic states and for neutralization of heparin in vivo to terminate heparinization. The protamine equivalent varies with the heparin preparation, conditions of testing and, in vivo, with the amount of heparin present in the circulation. The latter depends on time after administration and the hemodynamic and metabolic state of the patient. Protamine, when injected rapidly, will release histamine and agglutinate platelets. Bleeding (spontaneous hemorrhage) demonstrates a multiple breakdown of hemostatic mechanisms due to surgical stress, drugs, exposure of the blood to foreign surfaces, etc. Simple rules for the amount of protamine required for an individual patient based on clinical judgement will be satisfactory in most cases. When hemostasis is not achieved, it must be appreciated that heparin and protamine are only part of a complex deteriorating situation. PMID:4122234

  9. Heparin: Past, Present, and Future

    PubMed Central

    Oduah, Eziafa I.; Linhardt, Robert J.; Sharfstein, Susan T.

    2016-01-01

    Heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. A contamination crisis in 2007–2008 increased the impetus to provide non-animal-derived sources of heparin, produced under cGMP conditions. In addition, recent studies suggest that heparin may have significant antineoplastic activity, separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin in treating inflammation, infertility, and infectious disease. A variety of strategies have been proposed to produce a bioengineered heparin. In this review, we discuss several of these strategies including microbial production, mammalian cell production, and chemoenzymatic modification. We also propose strategies for creating “designer” heparins and heparan-sulfates with various biochemical and physiological properties. PMID:27384570

  10. Expression of basic fibroblast growth factor in intact and ulcerated human gastric mucosa

    PubMed Central

    Hull, M; Brough, J; Powe, D; Carter, G; Jenkins, D; Hawkey, C

    1998-01-01

    Background—Basic fibroblast growth factor (bFGF) promotes angiogenesis and healing of gastric ulcers in rats, and bFGF expression is up regulated in such ulcers. However, little is known about expression of bFGF in human gastric mucosa. 
Aims—To investigate bFGF expression in intact human gastric mucosa and gastric ulcers and to determine whether low bFGF content or altered binding by mucosa is associated with ulceration. 
Subjects—Endoscopy outpatients, gastrectomy patients, and organ donors. 
Methods—bFGF was isolated by heparin affinity chromatography and characterised by western blotting and endothelial cell bioassay. bFGF was measured by immunoassay and its distribution defined by immunohistochemistry and in situ hybridisation. Binding of bFGF by heparan sulphate proteoglycans was investigated by sodium chloride and heparin extraction. 
Results—Bioactive bFGF (19 kDa) was detected in normal mucosa but bFGF mRNA was not found. bFGF expression was up regulated in granulation tissue endothelial cells, mononuclear cells, and epithelial cells at the ulcer rim. Gastric ulcer patients had constitutively low bFGF concentrations in intact antral mucosa which were not explained by changes in binding to heparan sulphate proteoglycans. 
Conclusions—bFGF expression is up regulated in human gastric ulcers. Low intact mucosal bFGF content is associated with gastric ulceration. 

 Keywords: basic fibroblast growth factor; gastric mucosa; heparan sulphate proteoglycan; peptic ulceration PMID:9824581

  11. Fibroblast-derived matrix (FDM) as a novel vascular endothelial growth factor delivery platform.

    PubMed

    Du, Ping; Hwang, Mintai P; Noh, Yong Kwan; Subbiah, Ramesh; Kim, In Gul; Bae, Soon Eon; Park, Kwideok

    2014-11-28

    Vascular endothelial growth factor (VEGF) is one of the most important signaling cues during angiogenesis. Since many delivery systems of VEGF have been reported, the presentation of VEGF using a more physiologically relevant extracellular matrix (ECM), however, has yet to be thoroughly examined. In this study, we propose that fibroblast-derived extracellular matrix (FDM) is a novel platform for angiogenic growth factor delivery and that FDM-mediated VEGF delivery can result in an advanced angiogenic response. The FDMs, activated by EDC/NHS chemistry, were loaded with varying amounts of heparin. Different doses of VEGF were subsequently immobilized onto the heparin-grafted FDM (hep-FDM); 19.6 ± 0.6, 39.2 ± 3.2, and 54.8 ± 8.9 ng of VEGF were tethered using 100, 300, and 500 ng of initial VEGF, respectively. VEGF-tethered FDM was found chemoattractive and VEGF dose-dependent in triggering human umbilical vein endothelial cells (ECs) migration in vitro. When hep-FDM-bound VEGF (H-F/V) was encapsulated into alginate capsules (A/H-F/V) and subjected to release test for 28 days, it exhibited a significantly reduced burst release at early time point compared to that of A/V. The cell proliferation results indicated a substantially extended temporal effect of A/H-F/V on EC proliferation compared to those treated with soluble VEGF. For a further study, A/H-F/V was transplanted subcutaneously into ICR mice for up to 4 weeks to assess its in vivo effect on angiogenesis; VEGF delivered by hep-FDM was more competitive in promoting blood vessel ingrowth and maturation compared to other groups. Taken together, this study successfully engineered an FDM-mediated VEGF delivery system, documented its capacity to convey VEGF in a sustained manner, and demonstrated the positive effects of angiogenic activity in vivo as well as in vitro.

  12. Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

    PubMed Central

    Thomas, Owain; Lybeck, Emanuel; Strandberg, Karin; Tynngård, Nahreen; Schött, Ulf

    2015-01-01

    Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is

  13. Heparin treatment in abruptio placentae

    PubMed Central

    Martin, T. R.; Read, H. C.; Fraser, M. E.

    1974-01-01

    Two cases of abruptio placentae with disseminated intravascular coagulation (DIC) were treated with heparin, and coagulation was monitored by thromboelastography as well as the usual hematology tests. The cases demonstrated the vagaries of DIC and both showed decreased overt hemorrhage after heparin treatment was started. Heparin may be indicated for the management of abruptio placentae where delivery is not imminent, where significant disseminated intravascular coagulation exists, and when adequate serial coagulation studies are available. ImagesFIG. 1 PMID:4829841

  14. Postpartum bone mineral density in women treated for thromboprophylaxis with unfractionated heparin or LMW heparin.

    PubMed

    Pettilä, Ville; Leinonen, Pekka; Markkola, Antti; Hiilesmaa, Vilho; Kaaja, Risto

    2002-02-01

    Venous thromboembolism remains an important cause of maternal mortality. In a randomised open study, 44 pregnant women with confirmed previous or current thromboembolism were randomised to receive either low-molecular-weight heparin, dalteparin (N = 21) once daily subcutaneously or unfractionated sodium heparin (UF heparin, N = 23) twice daily subcutaneously for thromboprophylaxis during pregnancy and puerperium. Bone mineral density (BMD) in the lumbosacral spine was measured with dual X-ray absorptiometry (DEXA) 1, 6, 16, 52 weeks and, if possible, 3 years after delivery. BMD values were also compared with those of healthy, delivered women (N = 19). Mean BMD of the lumbar spine was significantly lower in the unfractionated heparin group compared with the dalteparin and with the control groups (repeated measures ANOVA p = 0.02). BMD in the dalteparin group did not differ from BMD of healthy delivered women. Multiple logistic regression analysis revealed that therapy was the only independent factor influencing BMD at weeks 16 and 52. Therefore we recommend use of dalteparin instead of UF heparin for long-term thromboprophylaxis during and after pregnancy.

  15. Epidermal Growth Factor and Intestinal Barrier Function

    PubMed Central

    Liu, Hu; Yang, Shufen; Li, Zuohua; Zhong, Jinfeng

    2016-01-01

    Epidermal growth factor (EGF) is a 53-amino acid peptide that plays an important role in regulating cell growth, survival, migration, apoptosis, proliferation, and differentiation. In addition, EGF has been established to be an effective intestinal regulator helping to protect intestinal barrier integrity, which was essential for the absorption of nutrients and health in humans and animals. Several researches have demonstrated that EGF via binding to the EGF receptor and subsequent activation of Ras/MAPK, PI3K/AKT, PLC-γ/PKC, and STATS signal pathways regulates intestinal barrier function. In this review, the relationship between epidermal growth factor and intestinal development and intestinal barrier is described, to provide a better understanding of the effects of EGF on intestine development and health. PMID:27524860

  16. Role of hematopoietic growth factors in angiogenesis.

    PubMed

    Ribatti, D; Vacca, A; De Falco, G; Ria, R; Roncali, L; Dammacco, F

    2001-01-01

    In early ontogeny, hematopoiesis is closely associated with angiogenesis. This article reviews recent studies of the effect of hematopoietic growth factors on several endothelial cell functions together with recent findings about angiogenesis and antiangiogenic therapies in hematopoietic malignancies such as leukemia, lymphoma and myeloma. Copyright 2001 S. Karger AG, Basel

  17. Shed Syndecan-1 Translocates to the Nucleus of Cells Delivering Growth Factors and Inhibiting Histone Acetylation

    PubMed Central

    Stewart, Mark D.; Ramani, Vishnu C.; Sanderson, Ralph D.

    2015-01-01

    The heparan sulfate proteoglycan syndecan-1 is proteolytically shed from the surface of multiple myeloma cells and is abundant in the bone marrow microenvironment where it promotes tumor growth, angiogenesis, and metastasis. In this study, we demonstrate for the first time that shed syndecan-1 present in the medium conditioned by tumor cells is taken up by bone marrow-derived stromal cells and transported to the nucleus. Translocation of shed syndecan-1 (sSDC1) to the nucleus was blocked by addition of exogenous heparin or heparan sulfate, pretreatment of conditioned medium with heparinase III, or growth of cells in sodium chlorate, indicating that sulfated heparan sulfate chains are required for nuclear translocation. Interestingly, cargo bound to sSDC1 heparan sulfate chains (i.e. hepatocyte growth factor) was transported to the nucleus along with sSDC1, and removal of heparan sulfate-bound cargo from sSDC1 abolished its translocation to the nucleus. Once in the nucleus, sSDC1 binds to the histone acetyltransferase enzyme p300, and histone acetyltransferase activity and histone acetylation are diminished. These findings reveal a novel function for shed syndecan-1 in mediating tumor-host cross-talk by shuttling growth factors to the nucleus and by altering histone acetylation in host cells. In addition, this work has broad implications beyond myeloma because shed syndecan-1 is present in high levels in many tumor types as well as in other disease states. PMID:25404732

  18. Dalteparin, a low-molecular-weight heparin, promotes angiogenesis mediated by heparin-binding VEGF-A in vivo.

    PubMed

    Norrby, Klas; Nordenhem, Arvid

    2010-12-01

    Tumors are angiogenesis dependent and vascular endothelial growth factor-A (VEGF-A), a heparin-binding protein, is a key angiogenic factor. As chemotherapy and co-treatment with anticoagulant low-molecular-weight heparin (LMWH) are common in cancer patients, we investigated whether angiogenesis in vivo mediated by VEGF-A is modulated by metronomic-type treatment with: (i) the LMWH dalteparin; (ii) low-dosage cytostatic epirubicin; or (iii) a combination of these two drugs. Using the quantitative rat mesentery angiogenesis assay, in which angiogenesis was induced by intraperitoneal injection of very low doses of VEGF, dalteparin sodium (Fragmin(®) ) and epirubicin (Farmorubicin(®) ) were administered separately or in combination by continuous subcutaneous infusion at a constant rate for 14 consecutive days. Dalteparin was administered at 27, 80, or 240 IU/kg/day, i.e., doses that reflect the clinical usage of this drug, while epirubicin was given at the well-tolerated dosage of 0.4 mg/kg/day. While dalteparin significantly stimulated angiogenesis in an inversely dose-dependent manner, epirubicin did not significantly affect angiogenesis. However, concurrent treatment with dalteparin and epirubicin significantly inhibited angiogenesis. The effect of dalteparin is the first demonstration of a proangiogenic effect of any LMWH in vivo. The fact that co-treatment with dalteparin and epirubicin significantly inhibited angiogenesis suggests a complex drug effect.

  19. Post Treatment With an FGF Chimeric Growth Factor Enhances Epithelial Cell Proliferation to Improve Recovery From Radiation-Induced Intestinal Damage

    SciTech Connect

    Nakayama, Fumiaki; Hagiwara, Akiko; Umeda, Sachiko; Asada, Masahiro; Goto, Megumi; Oki, Junko; Suzuki, Masashi; Imamura, Toru; Akashi, Makoto

    2010-11-01

    Purpose: A fibroblast growth factor (FGF) 1-FGF2 chimera (FGFC) was created previously and showed greater structural stability than FGF1. This chimera was capable of stimulating epithelial cell proliferation much more strongly than FGF1 or FGF2 even without heparin. Therefore FGFC was expected to have greater biologic activity in vivo. This study evaluated and compared the protective activity of FGFC and FGF1 against radiation-induced intestinal injuries. Methods and Materials: We administered FGFC and FGF1 intraperitoneally to BALB/c mice 24 h before or after total-body irradiation (TBI). The numbers of surviving crypts were determined 3.5 days after TBI with gamma rays at doses ranging from 8 to 12 Gy. Results: The effect of FGFC was equal to or slightly superior to FGF1 with heparin. However, FGFC was significantly more effective in promoting crypt survival than FGF1 (p < 0.01) when 10 {mu}g of each FGF was administered without heparin before irradiation. In addition, FGFC was significantly more effective at promoting crypt survival (p < 0.05) than FGF1 even when administered without heparin at 24 h after TBI at 10, 11, or 12 Gy. We found that FGFC post treatment significantly promoted 5-bromo-2'-deoxyuridine incorporation into crypts and increased crypt depth, resulting in more epithelial differentiation. However, the number of apoptotic cells in FGFC-treated mice decreased to almost the same level as that in FGF1-treated mice. Conclusions: These findings suggest that FGFC strongly enhanced radioprotection with the induction of epithelial proliferation without exogenous heparin after irradiation and is useful in clinical applications for both the prevention and post treatment of radiation injuries.

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

    PubMed

    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.

  1. Incorporation of heparin-binding proteins into preformed dextran sulfate-chitosan nanoparticles

    PubMed Central

    Zaman, Paula; Wang, Julia; Blau, Adam; Wang, Weiping; Li, Tina; Kohane, Daniel S; Loscalzo, Joseph; Zhang, Ying-Yi

    2016-01-01

    Incorporation of proteins into dextran sulfate (DS)-chitosan (CS) nanoparticles (DSCS NPs) is commonly performed using entrapment procedures, in which protein molecules are mixed with DS and CS until particle formation occurs. As DS is an analog of heparin, the authors examined whether proteins could be directly incorporated into preformed DSCS NPs through a heparin binding domain-mediated interaction. The authors formulated negatively-charged DSCS NPs, and quantified the amount of charged DS in the outer shell of the particles. The authors then mixed the DSCS NPs with heparin-binding proteins (SDF-1α, VEGF, FGF-2, BMP-2, or lysozyme) to achieve incorporation. Data show that for DSCS NPs containing 100 nmol charged glucose sulfate units in DS, up to ~1.5 nmol of monomeric or ~0.75 nmol of dimeric heparin-binding proteins were incorporated without significantly altering the size or zeta potential of the particles. Incorporation efficiencies of these proteins were 95%–100%. In contrast, serum albumin or serum globulin showed minimal incorporation (8% and 4%, respectively) in 50% physiological saline, despite their large adsorption in water (80% and 92%, respectively). The NP-incorporated SDF-1α and VEGF exhibited full activity and sustained thermal stability. An in vivo aerosolization study showed that NP-incorporated SDF-1α persisted in rat lungs for 72 h (~34% remaining), while free SDF-1α was no longer detectable after 16 h. As many growth factors and cytokines contain heparin-binding sites/domains, incorporation into preformed DSCS NPs could facilitate in vivo applications of these proteins. PMID:27920522

  2. Incorporation of heparin-binding proteins into preformed dextran sulfate-chitosan nanoparticles.

    PubMed

    Zaman, Paula; Wang, Julia; Blau, Adam; Wang, Weiping; Li, Tina; Kohane, Daniel S; Loscalzo, Joseph; Zhang, Ying-Yi

    Incorporation of proteins into dextran sulfate (DS)-chitosan (CS) nanoparticles (DSCS NPs) is commonly performed using entrapment procedures, in which protein molecules are mixed with DS and CS until particle formation occurs. As DS is an analog of heparin, the authors examined whether proteins could be directly incorporated into preformed DSCS NPs through a heparin binding domain-mediated interaction. The authors formulated negatively-charged DSCS NPs, and quantified the amount of charged DS in the outer shell of the particles. The authors then mixed the DSCS NPs with heparin-binding proteins (SDF-1α, VEGF, FGF-2, BMP-2, or lysozyme) to achieve incorporation. Data show that for DSCS NPs containing 100 nmol charged glucose sulfate units in DS, up to ~1.5 nmol of monomeric or ~0.75 nmol of dimeric heparin-binding proteins were incorporated without significantly altering the size or zeta potential of the particles. Incorporation efficiencies of these proteins were 95%-100%. In contrast, serum albumin or serum globulin showed minimal incorporation (8% and 4%, respectively) in 50% physiological saline, despite their large adsorption in water (80% and 92%, respectively). The NP-incorporated SDF-1α and VEGF exhibited full activity and sustained thermal stability. An in vivo aerosolization study showed that NP-incorporated SDF-1α persisted in rat lungs for 72 h (~34% remaining), while free SDF-1α was no longer detectable after 16 h. As many growth factors and cytokines contain heparin-binding sites/domains, incorporation into preformed DSCS NPs could facilitate in vivo applications of these proteins.

  3. A comparison of red blood cell transfusion utilization between anti-activated factor X and activated partial thromboplastin monitoring in patients receiving unfractionated heparin.

    PubMed

    Belk, K W; Laposata, M; Craver, C

    2016-11-01

    Essentials Anti-activated factor X (Anti-Xa) monitoring is more precise than activated partial thromboplastin (aPTT). 20 804 hospitalized cardiovascular patients monitored with Anti-Xa or aPTT were analyzed. Adjusted transfusion rates were significantly lower for patients monitored with Anti-Xa. Adoption of Anti-Xa protocols could reduce transfusions among cardiovascular patients in the US. Background Anticoagulant activated factor X protein (Anti-Xa) has been shown to be a more precise monitoring tool than activated partial thromboplastin time (aPTT) for patients receiving unfractionated heparin (UFH) anticoagulation therapy. Objectives To compare red blood cell (RBC) transfusions between patients receiving UFH who are monitored with Anti-Xa and those monitored with aPTT. Patients/Methods A retrospective cohort study was conducted on patients diagnosed with acute coronary syndrome (ACS) (N = 14 822), diagnosed with ischemic stroke (STK) (N = 1568) or with a principal diagnosis of venous thromboembolism (VTE) (N = 4414) in the MedAssets data from January 2009 to December 2013. Anti-Xa and aPTT groups were identified from hospital billing details, with both brand and generic name as search criteria. Propensity score techniques were used to match Anti-Xa cases to aPTT controls. RBC transfusions were identified from hospital billing data. Multivariable logistic regression was used to identify significant drivers of transfusions. Results Anti-Xa patients had fewer RBC transfusions than aPTT patients in the ACS population (difference 17.5%; 95% confidence interval [CI] 16.4-18.7%), the STK population (difference 8.2%; 95% CI 4.4-11.9%), and the VTE population (difference 4.7%; 95% CI 3.3-6.1%). After controlling for patient age and gender, diagnostic risks (e.g. anemia, renal insufficiency, and trauma), and invasive procedures (e.g. cardiac catheterization, hemodialysis, and coronary artery bypass graft), Anti-Xa patients were less likely to have a transfusion while

  4. Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins.

    PubMed

    Mauri, Lucio; Boccardi, Giovanni; Torri, Giangiacomo; Karfunkle, Michael; Macchi, Eleonora; Muzi, Laura; Keire, David; Guerrini, Marco

    2017-03-20

    An NMR HSQC method has recently been proposed for the quantitative determination of the mono- and disaccharide subunits of heparin and low molecular weight heparins (LMWH). The focus of the current study was the validation of this procedure to make the 2D-NMR method suitable for pharmaceutical quality control applications. Pre-validation work investigated the effects of several experimental parameters to assess robustness and to optimize critical factors. Important experimental parameters were pulse sequence selection, equilibration interval between pulse trains and temperature. These observations were needed so that the NMR method was sufficiently understood to enable continuous improvement. A standard validation study on heparin then examined linearity, repeatability, intermediate precision and limits of detection and quantitation; selected validation parameters were also determined for LMWH.

  5. Characterization of FGF family growth factors concerning branching morphogenesis of mouse lung epithelium.

    PubMed

    Goto, Asami; Yamazaki, Naohiro; Nogawa, Hiroyuki

    2014-05-01

    Mouse lung rudiments express eight members of fibroblast growth factor (FGF) family genes from embryonic day 10 (E10) to E13. Some of these are expressed in either the epithelium or mesenchyme, while others are expressed in both. Incorporating the results of our previous study, we characterized the branch-inducing activities of all of FGFs expressed in the early lung rudiment. Of these, FGF1, FGF2, FGF7, FGF9 and FGF10 induced branching morphogenesis in Matrigel-embedded E11 epithelium, and their effective concentrations varied (10 nM, 10 nM, 3 nM, 1 nM, and 100 nM, respectively). Whereas shaking culture dishes containing medium supplemented with FGF7 or FGF10 showed reduced branching morphogenesis, those supplemented with FGF1, FGF2, or FGF9 did not, suggesting the involvement of autocrine growth factor(s) in branching morphogenesis induced by FGF7 or FGF10. In the presence of heparin, a well-known activator of FGF signaling, cystic morphology with lumen expansion was observed in cultures containing FGF1, FGF7, or FGF10, but growth arrest was observed in cultures containing FGF2 or FGF9. These results indicate that several paracrine and autocrine FGFs function during branching morphogenesis of lung epithelium.

  6. Dual growth factor-immobilized microspheres for tissue reinnervation: in vitro and preliminary in vivo studies.

    PubMed

    Kim, Tae Ho; Oh, Se Heang; An, Dan Bi; Lee, Ji Youl; Lee, Jin Ho

    2015-01-01

    Growth factors (GFs) (basic fibroblast growth factor (bFGF) and/or nerve growth factor (NGF))-immobilized polycaprolactone (PCL)/Pluronic F127 microspheres were prepared using an isolated particulate-melting method and the sequential binding of heparin and GFs onto the microspheres. The GFs immobilized on the microspheres were released in a sustained manner over 28 days, regardless of GF type. From the in vitro culture of muscle-derived stem cells, it was observed that the NGF-immobilized microspheres induced more neurogenic differentiation than the bFGF-immobilized microspheres, as evidenced by a quantitative real-time polymerase chain reaction using specific neurogenic markers (Nestin, GFAP, β-tubulin, and MAP2) and Western blot (Nestin and β-tubulin) analyses. The dual bFGF/NGF-immobilized microspheres showed better neurogenic differentiation than the microspheres immobilized with single bFGF or NGF. From the preliminary animal study, the dual bFGF/NGF-immobilized microsphere group also showed effective nerve regeneration, as evaluated by immunocytochemistry using a marker - β-tubulin. The dual bFGF/NGF-immobilized PCL/Pluronic F127 microspheres may be a promising candidate for nerve regeneration in certain target tissues (i.e. muscles) leading to sufficient reinnervation.

  7. Changes in heparin dose response slope during cardiac surgery: possible result in inaccuracy in predicting heparin bolus dose requirement to achieve target ACT.

    PubMed

    Ichikawa, Junko; Mori, Tetsu; Kodaka, Mitsuharu; Nishiyama, Keiko; Ozaki, Makoto; Komori, Makiko

    2017-09-01

    The substantial interpatient variability in heparin requirement has led to the use of a heparin dose response (HDR) technique. The accuracy of Hepcon-based heparin administration in achieving a target activated clotting time (ACT) using an HDR slope remains controversial. We prospectively studied 86 adult patients scheduled for cardiac surgery requiring cardiopulmonary bypass. The total dose of calculated heparin required for patient and pump priming was administered simultaneously to achieve a target ACT of 450 s for HDR on the Hepcon HMS system. Blood samples were obtained after the induction of anesthesia, at 3 min after heparin administration and after the initiation of CPB to measure kaolin ACT, HDR slope, whole-blood heparin concentration based on the HDR slope and anti-Xa heparin concentration, antithrombin and complete blood count. The target ACT of 450 s was not achieved in 68.6% of patients. Compared with patients who achieved the target ACT, those who failed to achieve their target ACT had a significantly higher platelet count at baseline. Correlation between the HDR slope and heparin sensitivity was poor. Projected heparin concentration and anti-Xa heparin concentration are not interchangeable based on the Bland-Altman analysis. It can be hypothesized that the wide discrepancy in HDR slope versus heparin sensitivity may be explained by an inaccurate prediction of the plasma heparin level and/or the change in HDR of individual patients, depending on in vivo factors such as extravascular sequestration of heparin, decreased intrinsic antithrombin activity level and platelet count and/or activity.

  8. Haemocompatibility of paediatric membrane oxygenators with heparin-coated surfaces.

    PubMed

    Wendel, H P; Scheule, A M; Eckstein, F S; Ziemer, G

    1999-01-01

    Extracorporeal circulation (ECC) in paediatric patients with heparin-coated oxygenation systems is rarely investigated. The objective of this study was to evaluate, preclinically, the haemocompatibility of paediatric membrane oxygenators with heparin-coated surfaces. We compared 16 paediatric membrane oxygenators (Minimax, Medtronic) in an in vitro heart-lung machine model with fresh human blood. Eight of these oxygenation systems had a covalent heparin coating (Carmeda bioactive surface). After 90 min simulated ECC, the heparin-coated systems showed significantly higher platelet count, lower platelet-factor 4 release, reduced contact activation (factor XIIa and kallikrein), and lower neutrophil elastase levels (p < 0.05), compared to the noncoated oxygenator group. More biocompatible materials for paediatric operations may ameliorate the various postperfusion syndromes arising from ECC procedures, particularly unspecific inflammation, hyperfibrinolysis and blood loss.

  9. Nerve Growth Factor and Diabetic Neuropathy

    PubMed Central

    Vinik, Aaron

    2003-01-01

    Neuropathy is one of the most debilitating complications of both type 1 and type 2 diabetes, with estimates of prevalence between 50–90% depending on the means of detection. Diabetic neuropathies are heterogeneous and there is variable involvement of large myelinated fibers and small, thinly myelinated fibers. Many of the neuronal abnormalities in diabetes can be duplicated by experimental depletion of specific neurotrophic factors, their receptors or their binding proteins. In experimental models of diabetes there is a reduction in the availability of these growth factors, which may be a consequence of metabolic abnormalities, or may be independent of glycemic control. These neurotrophic factors are required for the maintenance of the neurons, the ability to resist apoptosis and regenerative capacity. The best studied of the neurotrophic factors is nerve growth factor (NGF) and the related members of the neurotrophin family of peptides. There is increasing evidence that there is a deficiency of NGF in diabetes, as well as the dependent neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) that may also contribute to the clinical symptoms resulting from small fiber dysfunction. Similarly, NT3 appears to be important for large fiber and IGFs for autonomic neuropathy. Whether the observed growth factor deficiencies are due to decreased synthesis, or functional, e.g. an inability to bind to their receptor, and/or abnormalities in nerve transport and processing, remains to be established. Although early studies in humans on the role of neurotrophic factors as a therapy for diabetic neuropathy have been unsuccessful, newer agents and the possibilities uncovered by further studies should fuel clinical trials for several generations. It seems reasonable to anticipate that neurotrophic factor therapy, specifically targeted at different nerve fiber populations, might enter the therapeutic armamentarium. PMID:14668049

  10. Transforming growth factor alpha and epidermal growth factor levels in bladder cancer and their relationship to epidermal growth factor receptor.

    PubMed Central

    Mellon, J. K.; Cook, S.; Chambers, P.; Neal, D. E.

    1996-01-01

    We have examined levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in neoplastic and non-neoplastic bladder tissue using a standard radioimmunoassay technique. Tumour samples had much higher TGF-alpha levels compared with EGF and TGF-alpha levels in malignant tissue were significantly higher than in benign bladder samples. There was, in addition, a difference in mean EGF levels from 'normal' bladder samples from non-tumour bearing areas of bladder in patients with bladder cancer compared with 'normal' bladder tissue obtained at the time of organ retrieval surgery. Levels of EGF and TGF-alpha did not correlate with levels of EGF receptor (EGFR) as determined by a radioligand binding method but levels of TGF-alpha > 10 ng gm-1 of tumour tissue did correlate with EGFR positivity defined using immunohistochemistry. These data suggest that TGF-alpha is the likely ligand for EGFR in bladder tumours. PMID:8605103

  11. Growth Factors and Tension-Induced Skeletal Muscle Growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.

    1994-01-01

    The project investigated biochemical mechanisms to enhance skeletal muscle growth, and developed a computer based mechanical cell stimulator system. The biochemicals investigated in this study were insulin/(Insulin like Growth Factor) IGF-1 and Steroids. In order to analyze which growth factors are essential for stretch-induced muscle growth in vitro, we developed a defined, serum-free medium in which the differentiated, cultured avian muscle fibers could be maintained for extended periods of time. The defined medium (muscle maintenance medium, MM medium) maintains the nitrogen balance of the myofibers for 3 to 7 days, based on myofiber diameter measurements and myosin heavy chain content. Insulin and IGF-1, but not IGF-2, induced pronounced myofiber hypertrophy when added to this medium. In 5 to 7 days, muscle fiber diameters increase by 71 % to 98% compared to untreated controls. Mechanical stimulation of the avian muscle fibers in MM medium increased the sensitivity of the cells to insulin and IGF-1, based on a leftward shift of the insulin dose/response curve for protein synthesis rates. (54). We developed a ligand binding assay for IGF-1 binding proteins and found that the avian skeletal muscle cultures produced three major species of 31, 36 and 43 kD molecular weight (54) Stretch of the myofibers was found to have no significant effect on the efflux of IGF-1 binding proteins, but addition of exogenous collagen stimulated IGF-1 binding protein production 1.5 to 5 fold. Steroid hormones have a profound effect on muscle protein turnover rates in vivo, with the stress-related glucocorticoids inducing rapid skeletal muscle atrophy while androgenic steroids induce skeletal muscle growth. Exercise in humans and animals reduces the catabolic effects of glucocorticoids and may enhance the anabolic effects of androgenic steroids on skeletal muscle. In our continuing work on the involvement of exogenrus growth factors in stretch-induced avian skeletal muscle growth, we

  12. Characterization of a multilayer heparin coating for biomolecule presentation to human mesenchymal stem cell spheroids

    PubMed Central

    Lei, J.; McLane, L. T.; Curtis, J. E.; Temenoff, J. S.

    2014-01-01

    Mesenchymal stem cells therapies have the potential to treat many pathologies, however, controlling cell fate after implantation remains challenging. We have used a multilayer technology to graft a range of 5 μg/mL – 5 mg/mL heparin onto the surface of MSC aggregates. Heparin coating does not affect cell viability (seen through LIVE/DEAD staining), cell anti-inflammatory properties (seen through co-culture with activated monocytes)and facilitates sequestration by coated cells of a growth factor (TGF-β1) that remains bioactive. This system can maximize therapeutic potential of MSC-based treatments because the cell surface-loaded protein could both signal to the cells to influence transplanted cell fate and be released into the surrounding environment to help repair injured tissue. PMID:25126416

  13. Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling.

    PubMed

    Tatsinkam, Arnold Junior; Mulloy, Barbara; Rider, Christopher C

    2015-08-15

    Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers.

  14. Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor.

    PubMed

    Tolonen, Jukka; Grönblad, Mats; Vanharanta, Heikki; Virri, Johanna; Guyer, Richard D; Rytömaa, Tapio; Karaharju, Erkki O

    2006-05-01

    Degenerated intervertebral disc has lost its normal architecture, and there are changes both in the nuclear and annular parts of the disc. Changes in cell shape, especially in the annulus fibrosus, have been reported. During degeneration the cells become more rounded, chondrocyte-like, whereas in the normal condition annular cells are more spindle shaped. These chondrocyte-like cells, often forming clusters, affect extracellular matrix turnover. In previous studies transforming growth factor beta (TGFbeta) -1 and -2, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) have been highlighted in herniated intervertebral disc tissue. In the present study the same growth factors are analysed immunohistochemically in degenerated intervertebral disc tissue. Disc material was obtained from 16 discs operated for painful degenerative disc disease. Discs were classified according to the Dallas Discogram Description. Different disc regions were analysed in parallel. As normal control disc tissue material from eight organ donors was used. Polyclonal antibodies against different growth factors and TGFbeta receptor type II were used, and the immunoreaction was detected by the avidin biotin complex method. All studied degenerated discs showed immunoreactivity for TGFbeta receptor type II and bFGF. Fifteen of 16 discs were immunopositive for TGFbeta-1 and -2, respectively, and none showed immunoreaction for PDGF. Immunopositivity was located in blood vessels and in disc cells. In the nucleus pulposus the immunoreaction was located almost exclusively in chondrocyte-like disc cells, whereas in the annular region this reaction was either in chondrocyte-like disc cells, often forming clusters, or in fibroblast-like disc cells. Chondrocyte-like disc cells were especially prevalent in the posterior disrupted area. In the anterior area of the annulus fibrosus the distribution was more even between these two cell types. bFGF was expressed in the anterior annulus

  15. Expression and effects of epidermal growth factor on human periodontal ligament cells.

    PubMed

    Teramatsu, Yoko; Maeda, Hidefumi; Sugii, Hideki; Tomokiyo, Atsushi; Hamano, Sayuri; Wada, Naohisa; Yuda, Asuka; Yamamoto, Naohide; Koori, Katsuaki; Akamine, Akifumi

    2014-09-01

    Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.

  16. Heparin suppresses the induction of c-fos and c-myc mRNA in murine fibroblasts by selective inhibition of a protein kinase C-dependent pathway.

    PubMed Central

    Wright, T C; Pukac, L A; Castellot, J J; Karnovsky, M J; Levine, R A; Kim-Park, H Y; Campisi, J

    1989-01-01

    Heparin is a complex glycosaminoglycan that inhibits the proliferation of several cell types in culture and in vivo. To begin to define the mechanism(s) by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the expression of the growth factor-inducible protooncogenes c-fos and c-myc. We show that heparin suppressed the induction of c-fos and c-myc mRNA by serum in murine (BALB/c) 3T3 fibroblasts. Using purified mitogens, we further show that suppression was most marked when protooncogene expression was induced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. By contrast, there was little or no suppression when the cells were stimulated by epidermal growth factor, which, in these cells, utilizes a protein kinase C-independent pathway for the induction of gene expression. Heparin also inhibited the change in cell morphology induced by the phorbol ester but had no effect on the morphological change induced by epidermal growth factor and agents that raise intracellular cAMP. Heparin did not inhibit intracellular protein kinase C activity, phorbol ester-induced down-regulation of protein kinase C, or phosphorylation of the 80-kDa intracellular protein kinase C substrate. These results suggest that heparin inhibits a protein kinase C-dependent pathway for cell proliferation and suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the kinase. Images PMID:2541434

  17. Nerve growth factor promotes human hemopoietic colony growth and differentiation.

    PubMed Central

    Matsuda, H; Coughlin, M D; Bienenstock, J; Denburg, J A

    1988-01-01

    Nerve growth factor (NGF) is a neurotropic polypeptide necessary for the survival and growth of some central neurons, as well as sensory afferent and sympathetic neurons. Much is now known of the structural and functional characteristics of NGF, whose gene has recently been cloned. Since it is synthesized in largest amounts by the male mouse submandibular gland, its role exclusively in nerve growth is questionable. NGF also causes histamine release from rat peritoneal mast cells in vitro, and we have shown elsewhere that it causes significant, dose-dependent, generalized mast cell proliferation in the rat in vivo when administered neonatally. Our experiments now indicate that NGF causes a significant stimulation of granulocyte colonies grown from human peripheral blood in standard hemopoietic methylcellulose assays. Further, NGF appears to act in a relatively selective fashion to induce the differentiation of eosinophils and basophils/mast cells. Depletion experiments show that the NGF effect may be T-cell dependent and that NGF augments the colony-stimulating effect of supernatants from the leukemic T-cell (Mo) line. The hemopoietic activity of NGF is blocked by polyclonal and monoclonal antibodies to NGF. We conclude that NGF may indirectly act as a local growth factor in tissues other than those of the nervous system by causing T cells to synthesize or secrete molecules with colony-stimulating activity. In view of the synthesis of NGF in tissue injury, the involvement of basophils/mast cells and eosinophils in allergic and other inflammatory processes, and the association of mast cells with fibrosis and tissue repair, we postulate that NGF plays an important biological role in a variety of repair processes. PMID:3413109

  18. Spontaneous heparin-induced thrombocytopenia syndrome without any proximate heparin exposure, infection, or inflammatory condition: Atypical clinical features with heparin-dependent platelet activating antibodies.

    PubMed

    Okata, Takuya; Miyata, Shigeki; Miyashita, Fumio; Maeda, Takuma; Toyoda, Kazunori

    2015-01-01

    Recent studies suggest that a thromboembolic disorder resembling heparin-induced thrombocytopenia (HIT), so-called spontaneous HIT syndrome, can occur in patients without any history of heparin exposure. It is likely due to anti-platelet factor 4 (PF4)/polyanion antibodies induced by other polyanions, such as bacterial surfaces and nucleic acids. We describe an atypical case of spontaneous HIT syndrome. A 70-year-old man suddenly presented with acute cerebral sinus thrombosis (CST). Soon after the initiation of unfractionated heparin (UFH) for the treatment of CST, his platelet count fell precipitously and he developed deep vein thrombosis, a clinical picture consistent with rapid-onset HIT but without any proximate episodes of heparin exposure, infection, trauma, surgery, or other acute illness. Antigen assays and a washed platelet activation assay indicated that the patient already possessed anti-PF4/heparin IgG antibodies with heparin-dependent platelet activation properties on admission. Cessation of UFH and initiation of argatroban resulted in prompt recovery of his platelet count without further thromboembolic events. We identified two similar cases in the literature. However, these patients do not meet the recently proposed criteria for spontaneous HIT syndrome. Even in atypical cases, however, inappropriate or delayed diagnosis of HIT appears to be associated with worse outcomes. We propose that these atypical cases should be included in the category of spontaneous HIT syndrome.

  19. Influencing hematopoietic differentiation of mouse embryonic stem cells using soluble heparin and heparan sulfate saccharides.

    PubMed

    Holley, Rebecca J; Pickford, Claire E; Rushton, Graham; Lacaud, Georges; Gallagher, John T; Kouskoff, Valerie; Merry, Catherine L R

    2011-02-25

    Heparan sulfate proteoglycans (HSPG) encompass some of the most abundant macromolecules on the surface of almost every cell type. Heparan sulfate (HS) chains provide a key interaction surface for the binding of numerous proteins such as growth factors and morphogens, helping to define the ability of a cell to respond selectively to environmental cues. The specificity of HS-protein interactions are governed predominantly by the order and positioning of sulfate groups, with distinct cell types expressing unique sets of HS epitopes. Embryos deficient in HS-synthesis (Ext1(-/-)) exhibit pre-gastrulation lethality and lack recognizable organized mesoderm and extraembryonic tissues. Here we demonstrate that embryonic stem cells (ESCs) derived from Ext1(-/-) embryos are unable to differentiate into hematopoietic lineages, instead retaining ESC marker expression throughout embryoid body (EB) culture. However hematopoietic differentiation can be restored by the addition of soluble heparin. Consistent with specific size and composition requirements for HS:growth factor signaling, chains measuring at least 12 saccharides were required for partial rescue of hematopoiesis with longer chains (18 saccharides or more) required for complete rescue. Critically N- and 6-O-sulfate groups were essential for rescue. Heparin addition restored the activity of multiple signaling pathways including bone morphogenic protein (BMP) with activation of phospho-SMADs re-established by the addition of heparin. Heparin addition to wild-type cultures also altered the outcome of differentiation, promoting hematopoiesis at low concentrations, yet inhibiting blood formation at high concentrations. Thus altering the levels of HS and HS sulfation within differentiating ESC cultures provides an attractive and accessible mechanism for influencing cell fate.

  20. Epidermal growth factor receptor signaling in tissue

    SciTech Connect

    Shvartsman, Stanislav; Wiley, H. S.; Lauffenburger, Douglas A.

    2004-08-01

    Abstract: A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptor's cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.

  1. Laboratory control of heparin therapy

    PubMed Central

    O'Shea, M. J.; Flute, P. T.; Pannell, G. M.

    1971-01-01

    The effect of heparin therapy was followed in 50 patients treated for thrombo-embolic disease. Individual response to a standard dose of 40,000 units of heparin daily showed a considerable variation and the effect was not constant on subsequent days. Five of the 50 patients developed a serious haemorrhage. It is proposed that to ensure the adequacy of treatment detectable levels of heparin should be obtained but because of the high risk of bleeding these levels should not be excessive. The results suggest that control of heparin therapy can be based on the thrombin clotting time. Using this test it is advised that treatment is monitored daily in order to achieve a plasma heparin level of up to 1 mg per 100 ml. PMID:5094686

  2. Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells*

    PubMed Central

    Adase, Christopher A.; Borkowski, Andrew W.; Zhang, Ling-juan; Williams, Michael R.; Sato, Emi; Sanford, James A.

    2016-01-01

    A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment. PMID:27048655

  3. Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells.

    PubMed

    Adase, Christopher A; Borkowski, Andrew W; Zhang, Ling-Juan; Williams, Michael R; Sato, Emi; Sanford, James A; Gallo, Richard L

    2016-05-27

    A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment.

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

    PubMed

    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. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  5. Heparin in malignant glioma: review of preclinical studies and clinical results.

    PubMed

    Schnoor, Rosalie; Maas, Sybren L N; Broekman, Marike L D

    2015-09-01

    Glioblastoma multiforme (GBM) is the most common primary brain tumor that is invariably lethal. Novel treatments are desperately needed. In various cancers, heparin and its low molecular weight derivatives (LMWHs), commonly used for the prevention and treatment of thrombosis, have shown therapeutic potential. Here we systematically review preclinical and clinical studies of heparin and LMWHs as anti-tumor agents in GBM. Even though the number of studies is limited, there is suggestive evidence that heparin may have various effects on GBM. These effects include the inhibition of tumor growth and angiogenesis in vitro and in vivo, and the blocking of uptake of extracellular vesicles. However, heparin can also block the uptake of (potential) anti-tumor agents. Clinical studies suggest a non-significant trend of prolonged survival of LMWH treated GBM patients, with some evidence of increased major bleedings. Heparin mimetics lacking anticoagulant effect are therefore a potential alternative to heparin/LMWH and are discussed as well.

  6. Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult arterial smooth muscle cells.

    PubMed Central

    Chen, J K; Hoshi, H; McKeehan, W L

    1987-01-01

    Myo-intimal proteoglycan metabolism is thought to be important in blood vessel homeostasis, blood clotting, atherogenesis, and atherosclerosis. Human platelet-derived transforming growth factor type beta (TGF-beta) specifically stimulated synthesis of at least two types of chondroitin sulfate proteoglycans in nonproliferating human adult arterial smooth muscle cells in culture. Stimulation of smooth muscle cell proteoglycan synthesis by smooth muscle cell growth promoters (epidermal growth factor, platelet-derived growth factor, and heparin-binding growth factors) was less than 20% of that elicited by TGF-beta. TGF-beta neither significantly stimulated proliferation of quiescent smooth muscle cells nor inhibited proliferating cells. The extent of TGF-beta stimulation of smooth muscle cell proteoglycan synthesis was similar in both nonproliferating and growth-stimulated cells. TGF-beta, which is a reversible inhibitor of endothelial cell proliferation, had no comparable effect on endothelial cell proteoglycan synthesis. These results are consistent with the hypothesis that TGF-beta is a cell-type-specific regulator of proteoglycan synthesis in human blood vessels and may contribute to the myo-intimal accumulation of proteoglycan in atherosclerotic lesions. Images PMID:3474655

  7. Effect of heparin on the biological properties and molecular signature of human mesenchymal stem cells

    PubMed Central

    Ling, Ling; Camilleri, Emily T.; Helledie, Torben; Samsonraj, Rebekah M.; Titmarsh, Drew M.; Chua, Ren Jie; Dreesen, Oliver; Dombrowski, Christian; Rider, David A.; Galindo, Mario; Lee, Ian; Hong, Wanjin; Hui, James H.; Nurcombe, Victor; van Wijnen, Andre J.; Cool, Simon M.

    2017-01-01

    Chronic use of heparin as an anti-coagulant for the treatment of thrombosis or embolism invokes many adverse systemic events including thrombocytopenia, vascular reactions and osteoporosis. Here, we addressed whether adverse effects might also be directed to mesenchymal stem cells that reside in the bone marrow compartment. Harvested human bone marrow-derived mesenchymal stem cells (hMSCs) were exposed to varying doses of heparin and their responses profiled. At low doses (<200 ng/ml), serial passaging with heparin exerted a variable effect on hMSC proliferation and multipotentiality across multiple donors, while at higher doses (≥100 µg/ml), heparin supplementation inhibited cell growth and increased both senescence and cell size. Gene expression profiling using cDNA arrays and RNA-seq analysis revealed pleiotropic effects of low-dose heparin on signaling pathways essential to hMSC growth and differentiation (including the TGFβ/BMP superfamily, FGFs, and Wnts). Cells serially passaged in low-dose heparin possess a donor-dependent gene signature that reflects their altered phenotype. Our data indicate that heparin supplementation during the culturing of hMSCs can alter their biological properties, even at low doses. This warrants caution in the application of heparin as a culture supplement for the ex vivo expansion of hMSCs. It also highlights the need for careful evaluation of the bone marrow compartment in patients receiving chronic heparin treatment. PMID:26484394

  8. Effect of heparin on the biological properties and molecular signature of human mesenchymal stem cells.

    PubMed

    Ling, Ling; Camilleri, Emily T; Helledie, Torben; Samsonraj, Rebekah M; Titmarsh, Drew M; Chua, Ren Jie; Dreesen, Oliver; Dombrowski, Christian; Rider, David A; Galindo, Mario; Lee, Ian; Hong, Wanjin; Hui, James H; Nurcombe, Victor; van Wijnen, Andre J; Cool, Simon M

    2016-01-15

    Chronic use of heparin as an anti-coagulant for the treatment of thrombosis or embolism invokes many adverse systemic events including thrombocytopenia, vascular reactions and osteoporosis. Here, we addressed whether adverse effects might also be directed to mesenchymal stem cells that reside in the bone marrow compartment. Harvested human bone marrow-derived mesenchymal stem cells (hMSCs) were exposed to varying doses of heparin and their responses profiled. At low doses (<200 ng/ml), serial passaging with heparin exerted a variable effect on hMSC proliferation and multipotentiality across multiple donors, while at higher doses (≥ 100 μg/ml), heparin supplementation inhibited cell growth and increased both senescence and cell size. Gene expression profiling using cDNA arrays and RNA-seq analysis revealed pleiotropic effects of low-dose heparin on signaling pathways essential to hMSC growth and differentiation (including the TGFβ/BMP superfamily, FGFs, and Wnts). Cells serially passaged in low-dose heparin possess a donor-dependent gene signature that reflects their altered phenotype. Our data indicate that heparin supplementation during the culturing of hMSCs can alter their biological properties, even at low doses. This warrants caution in the application of heparin as a culture supplement for the ex vivo expansion of hMSCs. It also highlights the need for careful evaluation of the bone marrow compartment in patients receiving chronic heparin treatment.

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

    PubMed

    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.

  10. Insulin-like growth factor 1: common mediator of multiple enterotrophic hormones and growth factors

    PubMed Central

    Bortvedt, Sarah F.; Lund, P. Kay

    2013-01-01

    Purpose of review To summarize recent evidence that IGF1 mediates growth effects of multiple trophic factors and discuss clinical relevance. Recent findings Recent reviews and original reports indicate benefits of growth hormone (GH) and long-acting glucagon-like peptide 2 (GLP2) analogues in short bowel syndrome and Crohn’s disease. This review highlights evidence that biomarkers of sustained small intestinal growth or mucosal healing and evaluation of intestinal epithelial stem cell biomarkers may improve clinical measures of intestinal growth or response to trophic hormones. Compelling evidence that IGF1 mediates growth effects of GH and GLP2 on intestine or linear growth in preclinical models of resection or Crohn’s disease is presented, along with a concept that these hormones or IGF1 may enhance sustained growth if given early after bowel resection. Evidence that SOCS protein induction by GH or GLP2 in normal or inflamed intestine, may limit IGF1-induced growth, but protect against risk of dysplasia or fibrosis is reviewed. Whether IGF1 receptor mediates IGF1 action and potential roles of insulin receptors are addressed. Summary IGF1 has a central role in mediating trophic hormone action in small intestine. Better understanding of benefits and risks of IGF1, receptors that mediate IGF1 action, and factors that limit undesirable growth are needed. PMID:22241077

  11. Heparin bridge therapy and post-polypectomy bleeding.

    PubMed

    Kubo, Toshiyuki; Yamashita, Kentaro; Onodera, Kei; Iida, Tomoya; Arimura, Yoshiaki; Nojima, Masanori; Nakase, Hiroshi

    2016-12-07

    To identify risk factors for post-polypectomy bleeding (PPB), focusing on antithrombotic agents. This was a case-control study based on medical records at a single center. PPB was defined as bleeding that occurred 6 h to 10 d after colonoscopic polypectomy and required endoscopic hemostasis. As risk factors for PPB, patient-related factors including anticoagulants, antiplatelets and heparin bridge therapy as well as polyp- and procedure-related factors were evaluated. All colonoscopic hot polypectomies, endoscopic mucosal resections and endoscopic submucosal dissections performed between January 2011 and December 2014 were reviewed. PPB occurred in 29 (3.7%) of 788 polypectomies performed during the study period. Antiplatelet or anticoagulant agents were prescribed for 210 (26.6%) patients and were ceased before polypectomy except for aspirin and cilostazol in 19 cases. Bridging therapy using intravenous unfractionated heparin was adopted for 73 patients. The univariate analysis revealed that anticoagulants, heparin bridge, and anticoagulants plus heparin bridge were significantly associated with PPB (P < 0.0001) whereas antiplatelets and antiplatelets plus heparin were not. None of the other factors including age, gender, location, size, shape, number of resected polyps, prophylactic clipping and resection method were correlated with PPB. The multivariate analysis demonstrated that anticoagulants and anticoagulants plus heparin bridge therapy were significant risk factors for PPB (P < 0.0001). Of the 29 PPB cases, 4 required transfusions and none required surgery. A thromboembolic event occurred in a patient who took anticoagulant. Patients taking anticoagulants have an increased risk of PPB, even if the anticoagulants are interrupted before polypectomy. Heparin-bridge therapy might be responsible for the increased PPB in patients taking anticoagulants.

  12. Heparin bridge therapy and post-polypectomy bleeding

    PubMed Central

    Kubo, Toshiyuki; Yamashita, Kentaro; Onodera, Kei; Iida, Tomoya; Arimura, Yoshiaki; Nojima, Masanori; Nakase, Hiroshi

    2016-01-01

    AIM To identify risk factors for post-polypectomy bleeding (PPB), focusing on antithrombotic agents. METHODS This was a case-control study based on medical records at a single center. PPB was defined as bleeding that occurred 6 h to 10 d after colonoscopic polypectomy and required endoscopic hemostasis. As risk factors for PPB, patient-related factors including anticoagulants, antiplatelets and heparin bridge therapy as well as polyp- and procedure-related factors were evaluated. All colonoscopic hot polypectomies, endoscopic mucosal resections and endoscopic submucosal dissections performed between January 2011 and December 2014 were reviewed. RESULTS PPB occurred in 29 (3.7%) of 788 polypectomies performed during the study period. Antiplatelet or anticoagulant agents were prescribed for 210 (26.6%) patients and were ceased before polypectomy except for aspirin and cilostazol in 19 cases. Bridging therapy using intravenous unfractionated heparin was adopted for 73 patients. The univariate analysis revealed that anticoagulants, heparin bridge, and anticoagulants plus heparin bridge were significantly associated with PPB (P < 0.0001) whereas antiplatelets and antiplatelets plus heparin were not. None of the other factors including age, gender, location, size, shape, number of resected polyps, prophylactic clipping and resection method were correlated with PPB. The multivariate analysis demonstrated that anticoagulants and anticoagulants plus heparin bridge therapy were significant risk factors for PPB (P < 0.0001). Of the 29 PPB cases, 4 required transfusions and none required surgery. A thromboembolic event occurred in a patient who took anticoagulant. CONCLUSION Patients taking anticoagulants have an increased risk of PPB, even if the anticoagulants are interrupted before polypectomy. Heparin-bridge therapy might be responsible for the increased PPB in patients taking anticoagulants. PMID:28018108

  13. High heparin content surface-modified polyurethane discs promote rapid and stable angiogenesis in full thickness skin defects through VEGF immobilization.

    PubMed

    McLuckie, Michelle; Schmidt, Christian A; Oosthuysen, Anel; Sanchez-Macedo, Nadia; Merker, Hannes; Bezuidenhout, Deon; Hoerstrup, Simon P; Lindenblatt, Nicole

    2017-09-01

    Three-dimensional scaffolds have the capacity to serve as an architectural framework to guide and promote tissue regeneration. Parameters such as the type of material, growth factors, and pore dimensions are therefore critical in the scaffold's success. In this study, heparin has been covalently bound to the surface of macroporous polyurethane (PU) discs via two different loading methods to determine if the amount of heparin content had an influence on the therapeutic affinity loading and release of (VEGF165 ) in full thickness skin defects. PU discs (5.4 mm diameter, 300 µm thickness, and interconnected pore size of 150 µm) were produced with either a low (2.5 mg/g) or high (6.6 mg/g) heparin content (LC and HC respectively), and were implanted into the modified dorsal skin chamber (MDSC) of C57BL/6 J mice with and without VEGF. Both low- and high-content discs with immobilized VEGF165 (LCV and HCV, respectively) presented accelerated neovascularization and tissue repair in comparison to heparin discs alone. However, the highest angiogenetic peak was on day 7 with subsequent stabilization for HCV, whereas other groups displayed a delayed peak on day 14. We therefore attribute the superior performance of HCV due to its ability to hold more VEGF165, based on its increased heparin surface coverage, as also demonstrated in VEGF elution dynamics. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2543-2550, 2017. © 2017 Wiley Periodicals, Inc.

  14. The role of transforming growth factor-beta, insulin-like growth factor I, and basic fibroblast growth factor in distraction osteogenesis of the mandible.

    PubMed

    Farhadieh, R D; Dickinson, R; Yu, Y; Gianoutsos, M P; Walsh, W R

    1999-01-01

    Distraction osteogenesis is a viable method for regenerating large amounts of bone. In contrast to fracture healing, the mode of bone formation in distraction osteogenesis is primarily intramembranous ossification. The basic biology of the process is still not well understood. The growth factor cascade is likely to play an important role in distraction. This study examines the growth factor cascade in a lengthened ovine mandible model. Twenty-four animals were divided into four groups with varying rates of distraction (1, 2, 3, and 4 mm/day). A unilateral distractor at the angle of the mandible was used. The mandibles were lengthened to 24 mm and fixed for a period of 5 weeks, after which the animals were killed. The sections were probed for transforming growth factor-beta, basic fibroblast growth factor, and insulin-like growth factor I. The growth factors studied were present in all four groups. Transforming growth factor-beta, basic fibroblast growth factor, and insulin-like growth factor I were present in both the bony matrix of the sections and the cytoplasm of the cells, osteoblasts, and a small number of mesenchymal cells. The sections obtained from groups distracted at faster rates showed stronger presence of the growth factors examined by more intense staining. In fracture healing, the localization of transforming growth factor-beta in stage I of healing corresponded with the precise region of intramembranous ossification in stage II. Diffuse presence of transforming growth factor-beta throughout the lengthened region corresponded with the process of intramembranous ossification observed in distraction. In fracture healing, insulin-like growth factor I and basic fibroblast growth factor have been shown to promote proliferation and differentiation of osteoblasts from precursor cells. The intense presence of insulin-like growth factor I and basic fibroblast growth factor in the distracted region may account for osteoblast proliferation and formation from

  15. Engineered heparins as new anticoagulant drugs.

    PubMed

    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.

  16. Engineered heparins as new anticoagulant drugs

    PubMed Central

    Vaidyanathan, Deepika; Williams, Asher; Dordick, Jonathan S.; Koffas, Mattheos A.G.

    2016-01-01

    Abstract 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. PMID:28516163

  17. Expression of transforming growth factor alpha, epidermal growth factor receptor and epidermal growth factor in precursor lesions to gastric carcinoma.

    PubMed Central

    Filipe, M. I.; Osborn, M.; Linehan, J.; Sanidas, E.; Brito, M. J.; Jankowski, J.

    1995-01-01

    Epidermal growth factor (EGF), its related peptide transforming growth factor (TGF-alpha) and their common receptor (EGFR) have been implicated in the control of cell proliferation and differentiation in the gastrointestinal epithelium and may play an important role in gastric carcinogenesis. We compared the immunohistochemical expression and topographic distribution of these peptides using Western blot analysis in gastric carcinoma precursor lesions and in non-cancer tissue. We observed: (i) increased and extended expression of TGF-alpha in normal mucosa and hyperplasia in carcinoma fields compared with non-cancer controls; (ii) increased expression of EGFR in intestinal metaplasia (IM) from carcinoma fields compared with controls; (iii) EGF expression was not detected in normal mucosa and only weakly in IM; (iv) coexpression of TGF-alpha/EGFR and EGF/EGFR was higher in intestinal metaplasia in carcinoma fields than in non-cancer controls. We conclude that altered expression of TGF-alpha/EGFR is associated with morphological changes during gastric carcinogenesis. In this regard increased expression of TGF-alpha is a very early event which is subsequently followed by up-regulation of EGFR and this has important biological and clinical implications. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7819044

  18. Replica-exchange molecular dynamics simulation of basic fibroblast growth factor adsorption on hydroxyapatite.

    PubMed

    Liao, Chenyi; Zhou, Jian

    2014-06-05

    The adsorption of basic fibroblast growth factor (bFGF) on the hydroxyapatite (001) surface was investigated by a combination of replica-exchange molecular dynamics (REMD) and conventional molecular dynamics (CMD) methods. In CMD, the protein cannot readily cross the surface water layer, whereas in REMD, the protein can cross the adsorption barrier from the surface water layer and go through weak, medium, then strong adsorption states with three energetically preferred configurations: heparin-binding-up (HP-up), heparin-binding-middle (HP-middle), and heparin-binding-down (HP-down). The HP-middle orientation, with the strongest adsorption energy (-1149 ± 40 kJ·mol(-1)), has the largest adsorption population (52.1-52.6%) and exhibits the largest conformational charge (RMSD of 0.26 ± 0.01 nm) among the three orientations. The HP-down and HP-up orientations, with smaller adsorption energies of -1022 ± 55 and -894 ± 70 kJ·mol(-1), respectively, have smaller adsorption populations of 27.4-27.7% and 19.7-20.5% and present smaller RMSD values of 0.21 ± 0.01 and 0.19 ± 0.01 nm, respectively. The convergent distribution indicates that nearly half of the population (in the HP-middle orientation) will support both FGF/FGFR and DGR-integrin signaling and another half (in the HP-up and HP-down orientations) will support DGR-integrin signaling. The major population (~80%) has the protein dipole directed outward. In the strong adsorption state, there are usually 2 to 3 basic residues that form the anchoring interactions of 210-332 kJ·mol(-1) per residue or that are accompanied by an acidic residue with an adsorption energy of ~207 kJ·mol(-1). Together, the major bound residues form a triangle or a quadrilateral on the surface and stabilize the adsorption geometrically, which indicates topologic matching between the protein and HAP surfaces.

  19. Delivery of basic fibroblast growth factor (bFGF) from photoresponsive hydrogel scaffolds.

    PubMed

    Andreopoulos, Fotios M; Persaud, Indushekhar

    2006-04-01

    Exogenous growth factor therapy has shown a notable promise in accelerating the healing of acute and chronic wounds. However, their susceptibility to enzymatic degradation and short contact time with the wound bed warrant the use of sophisticated delivery vehicles that stabilize the encapsulated peptides and control their rate of release. Herein, we describe the synthesis of a nitrocinnamate-derived polyethylene glycol (PEG-NC) hydrogel system and study the release kinetics of basic fibroblast growth factor (bFGF) as a function of hydrogel properties. Long-wave ultraviolet irradiation (365 nm) was used to alter the physical properties of the gel scaffold (i.e. degree of swelling) and consequently control the release rates of the encapsulated bFGF. The degree of swelling (DS) decreased from 10.7 to 8 as the length of irradiation increased from 5 to 30 min. Similarly, the DS decreased from 17.5 to 11.5 by increasing the initial PEG-NC concentration from 10 to 30 w/v% while keeping the crosslinking irradiation at 10 min. Radiolabeled I(125) studies were used to monitor the release of bFGF from PEG-NC hydrogels with variable swellabilities. By increasing the length of irradiation from 2 to 10 min the rate of bFGF release from PEG-NC gel scaffolds was decreased by 29% due to the enhanced crosslinking density. The bFGF-releasing PEG-NC hydrogels were not cytotoxic to human neonatal fibroblast cells and the released growth factor maintained its activity and induced fibroblast proliferation and collagen production in vitro. The addition of heparin within the gel scaffolds further increased the growth factor's activity.

  20. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

  1. Molecular basis for the Kallmann syndrome-linked fibroblast growth factor receptor mutation

    SciTech Connect

    Thurman, Ryan D.; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy; Kumar, Thallapuranam K. Suresh

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer The structural basis of the Kallmann syndrome is elucidated. Black-Right-Pointing-Pointer Kallmann syndrome mutation (A168S) induces a subtle conformational change(s). Black-Right-Pointing-Pointer Structural interactions mediated by beta-sheet G are most perturbed. Black-Right-Pointing-Pointer Ligand (FGF)-receptor interaction(s) is completely abolished by Kallmann mutation. Black-Right-Pointing-Pointer Kallmann mutation directly affects the FGF signaling process. -- Abstract: Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associated KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. {sup 1}H-{sup 15}N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR.

  2. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

  3. Transforming growth factor beta in Alzheimer's disease.

    PubMed Central

    Chao, C C; Hu, S; Frey, W H; Ala, T A; Tourtellotte, W W; Peterson, P K

    1994-01-01

    Alzheimer's disease (AD) has been hypothesized to be an inflammatory condition. We hypothesized that anti-inflammatory cytokines, such as transforming growth factor beta (TGF-beta), counteract the inflammatory process. In the present study, we found that TGF-beta levels were elevated in both cerebrospinal fluid and serum samples obtained from AD patients < 6 h after death. Serum TGF-beta levels were also markedly elevated before death. These results suggest that elevated TGF-beta levels in AD may represent a protective host response to immunologically mediated neuronal injury. PMID:7496909

  4. Characterization of currently marketed heparin products: adverse event relevant bioassays.

    PubMed

    Sommers, Cynthia D; Montpas, Nicolas; Adam, Albert; Keire, David A

    2012-01-01

    The polyanion oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in heparin products and was associated with severe hypotensive responses and other symptoms in patients receiving the drug. The OSCS associated adverse reactions were attributed to activation of the contact system via the plasma mediator, activated factor XII (FXIIa), which triggers kallikrein (KK) activity. Unlike heparin alone, OSCS, is able to activate FXII in plasma and stably bind to FXIIa enhancing plasma KK activity and the induction of vasoactive mediators such as bradykinin (BK), C3a and C5a. Similarly OSCS can interfere with heparin neutralization by the polycationic drug protamine. Here, we assess heparin (heparin sodium, dalteparin, tinzaparin or enoxaparin)-protamine complex formation and plasma based bioassays of KK, BK and C5a in a 96-well plate format. We establish the normal range of variation in the optimized bioassays across multiple lots from 9 manufacturers. In addition, because other oversulfated (OS) glycosaminoglycans (GAGs) besides OSCS could also serve as possible economically motivated adulterants (EMAs) to heparin, we characterize OS-dermatan sulfate (OSDS), OS-heparan sulfate (OSHS) and their native forms in the same assays. For the protamine test, OS-GAGs could be distinguished from heparin. For the KK assay, OSCS and OSDS were most potent followed by OSHS, and all had similar efficacies. Finally, OSDS had a greater efficacy in the C5a and BK assays followed by OSCS then OSHS. These data established the normal range of response of heparin products in these assays and the alteration in the responses in the presence of possible EMAs.

  5. [Heparin-induced thrombocytopenia type II: reexposure to heparin].

    PubMed

    Matthies, B; Bürger, T; Koch, B; Böck, M

    1999-10-29

    At the age of 55 years a now 70-year-old man had his aortic valve replaced by a prosthetic (Björk-Shiley) valve, and 11 years later a VDD pacemaker had been implanted. 18 months before the latest admission he had been hospitalized for treatment of staphylococcal endocarditis involving the aortic prothesis. At that time thrombocytopenia developed during heparin administration, diagnosed clinically and with the heparin-induced platelet activity (HIPA) test as type II heparin induced thrombocytopenia. His latest admission was for the diagnosis and treatment of peripheral arterial disease of the right leg (Fontaine stage IIb). Right popliteal and pedal pulses were not palpable. He was able to walk pain-free for only 70 m. Doppler sonography demonstrated an arm-leg index on the right of 0.7. Angiography revealed marked stenosis in the right superficial femoral artery and a filiform stenosis in the right popliteal artery. Both stenoses were relieved by percutaneous transluminal balloon angioplasty, in the course of which 5000 IU heparin were administered as a bolus intraarterially. Postoperative anticoagulation was maintained for 2 days with recombinant hirudin. There was no evidence of platelet reduction or heparin-induced antibodies despite the renewed infusion of heparin. Single re-administration of heparin in a patient who had developed a type II heparin-induced thrombocytopenia several years before does not necessarily lead to a booster of antibodies and thus to a reduction of platelets in the peripheral blood. It is a moot point whether the course in this case was an exception or the rule.

  6. Heparin-coated extracorporeal circulation in combination with low dose systemic heparinization reduces early postoperative blood loss in cardiac surgery.

    PubMed

    Mirow, N; Zittermann, A; Koertke, H; Maleszka, A; Knobl, H; Coskun, T; Kleesiek, K; Koerfer, R

    2008-04-01

    According to a recently performed meta-analysis, heparin-bonded circuits do not reduce blood loss in cardiac surgery patients compared to nonheparin-bonded circuits within the first 24 h postoperatively. We investigated the effects of heparin-coated circuits in combination with a reduced systemic heparin dose on early postoperative blood loss (first 12 h), platelet function, and postoperative complications. Patients who underwent their first coronary artery bypass graft surgery were included in a randomized prospective study. Group A (n=149) was perfused with an uncoated extracorporeal circulation (ECC)-set and groups B (n=152) and C (n=149) with heparin-coated ECC-sets. In groups A and B, conventional dose systemic heparin was given, whereas group C received low dose systemic heparin. Blood loss was assessed within the first 12 h postoperatively. Moreover, biochemical parameters of pro-coagulant activity and immunological function were measured. None of the pro-coagulant activity markers and immunological parameters measured differed preoperatively or postoperatively between study groups. However, intraoperative platelet counts and maximal intraoperative concentrations of platelet factor 4, ss-thromboglobulin, and poly-morpho-nuclear (PMN)-elastase were lowest in group C, whereas group C also had the highest concentrations of thrombin-antithrombin complex (P<0.018-0.001). Blood loss within the first 12 h postoperatively was 457 +/- 204 mL in group A, 431 +/- 178 mL in group B, and 382 +/- 188 mL in group C (P<0.01). Complication rates and 30-day mortality did not differ between study groups. The combined use of heparin-coated circuits and low dose systemic heparinization is able to reduce early postoperative blood loss without enhancing the risk of complications.

  7. Silver nanoparticles and growth factors incorporated hydroxyapatite coatings on metallic implant surfaces for enhancement of osteoinductivity and antibacterial properties.

    PubMed

    Xie, Chao-Ming; Lu, Xiong; Wang, Ke-Feng; Meng, Fan-Zhi; Jiang, Ou; Zhang, Hong-Ping; Zhi, Wei; Fang, Li-Ming

    2014-06-11

    Research on incorporation of both growth factors and silver (Ag) into hydroxyapatite (HA) coatings on metallic implant surfaces for enhancing osteoinductivity and antibacterial properties is a challenging work. Generally, Ag nanoparticles are easy to agglomerate and lead to a large increase in local Ag concentration, which could potentially affect cell activity. On the other hand, growth factors immobilization requires mild processing conditions so as to maintain their activities. In this study, bone morphology protein-2 (BMP-2) and Ag nanoparticle contained HA coatings were prepared on Ti surfaces by combining electrochemical deposition (ED) of Ag and electrostatic immobilization of BMP-2. During the ED process, chitosan (CS) was selected as the stabilizing agent to chelate Ag ions and generate Ag nanoparticles that are uniformly distributed in the coatings. CS also reduces Ag toxicity while retaining its antibacterial activity. Afterwards, a BMP/heparin solution was absorbed on the CS/Ag/HA coatings. Consequently, BMP-2 was immobilized on the coatings by the electrostatic attraction between CS, heparin, and BMP-2. Sustained release of BMP-2 and Ag ions from HA coatings was successfully demonstrated for a long period. Results of antibacterial tests indicate that the CS/Ag/HA coatings have high antibacterial properties against both Staphylococcus epidermidis and Escherichia coli. Osteoblasts (OB) culture reveals that the CS/Ag/HA coatings exhibit good biocompatibility. Bone marrow stromal cells (BMSCs) culture indicates that the BMP/CS/Ag/HA coatings have good osteoinductivity and promote the differentiation of BMSCs. Ti bars with BMP/CS/Ag/HA coatings were implanted into the femur of rabbits to evaluate the osteoinductivity of the coatings. Results indicate that BMP/CS/Ag/HA coatings favor bone formation in vivo. In summary, this study presents a convenient and effective method for the incorporation of growth factors and antibacterial agents into HA coatings. This

  8. Heparin versus low molecular weight heparin K 2165 in chronic hemodialysis patients: a randomized cross-over study.

    PubMed

    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.

  9. Postoperative Bleeding After Change in Heparin Supplier: A Cardiothoracic Center Experience.

    PubMed

    Bojan, Mirela; Fischer, Andreas; Narayanasamy, Ashok; Yea, Paul; Dunnett, Eleanor; Kelleher, Andrea

    2017-02-16

    Unfractionated heparin is a mixture of glycosaminoglycans with different pharmacologic and pharmacokinetic properties. The literature suggests that blood loss after cardiac surgery is related to both elevated postoperative heparin concentrations and the potency of different heparin brands. An audit of the observed increase in the incidence of cardiac surgery-related bleeding after change in heparin supplier. Patient characteristics were compared between groups before and after a change in heparin brands. Tertiary cardiothoracic center. All patients undergoing cardiac surgery between August 1, 2011, and April 30, 2012. None. Two hundred eighty patients underwent surgery before a change in heparin brands and 216 after a change. Their preoperative and intraoperative characteristics were similar. Postoperative chest tube drainages and blood transfusions were significantly greater after the change in heparin brands (postoperative chest drainage 476.8 ± 393.1 v 344.8 ± 323.2 mL/6 h and 1,062.2 ± 738.8 v 841.8 ± 567.4 mL/24 h, respectively; both p < 0.001) despite the administration of larger amounts of protamine, fresh frozen plasma/platelet transfusions, and cryoprecipitate. Heparin recirculation within 24 hours of bypass was noted in about 70% of the samples tested using either anti-factor X activity or the thromboelastography ratio between nonheparinase R and heparinase-modified R and was not associated with the heparin brand. The likelihood ratio chi-square test for nested models identified an added predictive value of the heparin brand when included as a predictor of bleeding (chest drainage >800 mL/6 h) in a model comprising recirculation, assessed using either an elevated anti-factor X activity or ratio between nonheparinase R and heparinase-modified R. It is likely that the observed increase in postoperative bleeding was related to the pharmacologic properties of the new heparin brand rather than a higher incidence of heparin recirculation. Copyright © 2017

  10. Autologous Growth Factor Injections in Chronic Tendinopathy

    PubMed Central

    Sandrey, Michelle A.

    2014-01-01

    Reference: de Vos RJ, van Veldhoven PLJ, Moen MH, Weir A, Tol JL. Autologous growth factor injections in chronic tendinopathy: a systematic review. Br Med Bull. 2010;95:63–77. Clinical Question: The authors of this systematic review evaluated the literature to critically consider the effects of growth factors delivered through autologous whole-blood and platelet-rich–plasma (PRP) injections in managing wrist-flexor and -extensor tendinopathies, plantar fasciopathy, and patellar tendinopathy. The primary question was, according to the published literature, is there sufficient evidence to support the use of growth factors delivered through autologous whole-blood and PRP injections for chronic tendinopathy? Data Sources: The authors performed a comprehensive, systematic literature search in October 2009 using PubMed, MEDLINE, EMBASE, CINAHL, and the Cochrane library without time limits. The following key words were used in different combinations: tendinopathy, tendinosis, tendinitis, tendons, tennis elbow, plantar fasciitis, platelet rich plasma, platelet transfusion, and autologous blood or injection. The search was limited to human studies in English. All bibliographies from the initial literature search were also viewed to identify additional relevant studies. Study Selection: Studies were eligible based on the following criteria: (1) Articles were suitable (inclusion criteria) if the participants had been clinically diagnosed as having chronic tendinopathy; (2) the design had to be a prospective clinical study, randomized controlled trial, nonrandomized clinical trial, or prospective case series; (3) a well-described intervention in the form of a growth factor injection with either PRP or autologous whole blood was used; and (4) the outcome was reported in terms of pain or function (or both). Data Extraction: All titles and abstracts were assessed by 2 researchers, and all relevant articles were obtained. Two researchers independently read the full text of

  11. Autologous growth factor injections in chronic tendinopathy.

    PubMed

    Sandrey, Michelle A

    2014-01-01

    de Vos RJ, van Veldhoven PLJ, Moen MH, Weir A, Tol JL. Autologous growth factor injections in chronic tendinopathy: a systematic review. Br Med Bull. 2010;95:63-77. The authors of this systematic review evaluated the literature to critically consider the effects of growth factors delivered through autologous whole-blood and platelet-rich-plasma (PRP) injections in managing wrist-flexor and -extensor tendinopathies, plantar fasciopathy, and patellar tendinopathy. The primary question was, according to the published literature, is there sufficient evidence to support the use of growth factors delivered through autologous whole-blood and PRP injections for chronic tendinopathy? The authors performed a comprehensive, systematic literature search in October 2009 using PubMed, MEDLINE, EMBASE, CINAHL, and the Cochrane library without time limits. The following key words were used in different combinations: tendinopathy, tendinosis, tendinitis, tendons, tennis elbow, plantar fasciitis, platelet rich plasma, platelet transfusion, and autologous blood or injection. The search was limited to human studies in English. All bibliographies from the initial literature search were also viewed to identify additional relevant studies. Studies were eligible based on the following criteria: (1) Articles were suitable (inclusion criteria) if the participants had been clinically diagnosed as having chronic tendinopathy; (2) the design had to be a prospective clinical study, randomized controlled trial, nonrandomized clinical trial, or prospective case series; (3) a well-described intervention in the form of a growth factor injection with either PRP or autologous whole blood was used; and (4) the outcome was reported in terms of pain or function (or both). All titles and abstracts were assessed by 2 researchers, and all relevant articles were obtained. Two researchers independently read the full text of each article to determine if it met the inclusion criteria. If opinions differed on

  12. Disruption of cell-matrix interactions by heparin enhances mesenchymal progenitor adipocyte differentiation

    SciTech Connect

    Luo Weijun; Shitaye, Hailu; Friedman, Michael; Bennett, Christina N.; Miller, Joshua; MacDougald, Ormond A.; Hankenson, Kurt D.

    2008-11-01

    Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0. 1 {mu}g/ml when applied before day 3 during the induction course. Heparin's effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.

  13. Transforming growth factor beta regulates thyroid growth. Role in the pathogenesis of nontoxic goiter.

    PubMed Central

    Grubeck-Loebenstein, B; Buchan, G; Sadeghi, R; Kissonerghis, M; Londei, M; Turner, M; Pirich, K; Roka, R; Niederle, B; Kassal, H

    1989-01-01

    The production and growth regulatory activity of transforming growth factor beta were studied in human thyroid tissue. As estimated by its mRNA expression in fresh tissue samples, transforming growth factor beta was produced in normal and in diseased thyroid glands. Transforming growth factor beta mRNA was mainly produced by thyroid follicular cells and in lesser quantities by thyroid infiltrating mononuclear cells. The concentrations of transforming growth factor beta mRNA were lower in iodine-deficient nontoxic goiter than in Graves' disease and normal thyroid tissue. Transforming growth factor beta protein secretion by cultured thyroid follicular cells was also low in nontoxic goiter, but could be increased by addition of sodium iodide (10 microM) to the culture medium. Recombinant transforming growth factor beta did not affect basal tritiated thymidine incorporation in cultured thyroid follicular cells, but inhibited, at a concentration of 10 ng/ml, the growth stimulatory influence of insulin-like growth factor I, epidermal growth factor, transforming growth factor alpha, TSH, and partly that of normal human serum on cultured thyroid follicular cells. This inhibition was greater in Graves' disease than in nontoxic goiter. These results suggest that transforming growth factor beta may act as an autocrine growth inhibitor on thyroid follicular cells. Decreased transforming growth factor beta production and decreased responsiveness to transforming growth factor beta may be cofactors in the pathogenesis of iodine-deficient nontoxic goiter. Images PMID:2921318

  14. Recombinant expression and purification of heparin binding proteins: midkine and pleiotrophin from Escherichia coli.

    PubMed

    Singh, Priyo K; Srivastava, Vivek

    2012-10-01

    Midkine (MDK) and Pleiotrophin (PTN) belong to a class of heparin-binding growth factors and are highly expressed in a number of cancers. Bioactive and recombinant MDK and PTN are critical reagent for cancer drug discovery studies. MDK and PTN belong to a newly evolving family of secreted neurotrophic and developmentally regulated heparin-binding molecules. PTN is related to MDK with 45% sequence identity and both proteins have been shown to be involved in promoting neurite outgrowth. MDK is a cysteine-rich 13kDa protein containing five disulfide bonds and PTN is 19kDa protein containing ten disulphide bonds. In this study, we expressed recombinant human MDK (rhMDK), mouse MDK (rmMDK) and human pleiotrophin (rhPTN) in Escherichia coli BL21(DE3)pLysS strain. Soluble rhMDK, rmMDK and rhPTN were expressed at a high-level in this strain and the protein was purified (∼90%) by a one-step purification using heparin affinity chromatography. A total of 4mg purified MDK and 7mg of purified PTN were obtained with the overall yield from 1L of bacterial culture. Activity of purified rhMDK and rhPTN was confirmed by a cell proliferation assay using NIH3T3 cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Lactoferrin – A Novel Bone Growth Factor

    PubMed Central

    Naot, Dorit; Grey, Andrew; Reid, Ian R; Cornish, Jillian

    2005-01-01

    Lactoferrin is an iron-binding glycoprotein that belongs to the transferrin family. It is present in breast milk, in epithelial secretions, and in the secondary granules of neutrophils. In healthy subjects lactoferrin circulates at concentrations of 2–7 x 10−6 g/ml. Lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Recently, we have shown that lactoferrin can also promote bone growth. At physiological concentrations, lactoferrin potently stimulates the proliferation and differentiation of primary osteoblasts and also acts as a survival factor inhibiting apoptosis induced by serum withdrawal. Lactoferrin also affects osteoclast formation and, in murine bone marrow culture, lactoferrin potently inhibits osteoclastogenesis. In vivo, local injection of lactoferrin above the hemicalvaria of adult mice results in substantial increases in the dynamic histomorphometric indices of bone formation and bone area. The mitogenic effect of lactoferrin in osteoblast-like cells is mediated mainly through LRP1, a member of the family of low-density lipoprotein receptor-related proteins that are primarily known as endocytic receptors. Using confocal laser scanning microscopy, we demonstrated that fluorescently labeled lactoferrin is endocytosed and can be visualized in the cytoplasm of primary osteoblastic cells. Lactoferrin also induces activation of p42/44 MAPK signaling in primary osteoblasts, but the two pathways seem to operate independently as activation of MAPK signaling, but not endocytosis, is necessary for the mitogenic effect of lactoferrin. We conclude that lactoferrin may have a physiological role in bone growth and healing, and a potential therapeutic role as an anabolic factor in osteoporosis. PMID:16012127

  16. An engineered heparin-binding form of VEGF-E (hbVEGF-E). Biological effects in vitro and mobilizatiion of precursor cells.

    PubMed

    Heil, Matthias; Mitnacht-Krauss, Rita; Issbrücker, Katja; van den Heuvel, Joop; Dehio, Christoph; Schaper, Wolfgang; Clauss, Matthias; Weich, Herbert A

    2003-01-01

    Vascular endothelial growth factor (VEGF-A) is the founding member of a family of angiogenic proteins with various binding abilities to three cognate VEGF receptors. Previously, a gene encoding from the genome of parapox orf virus (OV) with about 25% amino acid identity to mammalian VEGF-A was named VEGF-E and shown to bind and specifically activate the vascular endothelial growth factor receptor VEGFR-2 (KDR/flk-1). Here, we have generated a novel heparin-binding form of VEGF-E by introducing the heparin-domain of the human VEGF-A(165) splice variant into the viral VEGF-E protein. Recombinant heparin-binding VEGF-E (hbVEGF-E) is shown to stimulate proliferation and sprout formation of macro- and microvascular endothelial cells to a similar extent as the parental OV-VEGF-E but fails to activate peripheral mononuclear cells. However, hbVEGF-E is more potent in binding competition assays with primary human endothelial cells when compared to the OV-VEGF-E. This can be explained by our finding that binding of hbVEGF-E but not of parental OV-VEGF-E to the VEGFR-2 is strongly increased by the addition of neuropilin-1 (NP-1), a cognate co-receptor for VEGF-A. The engineered hbVEGF-E was compared with the VEGFR-1 selective and also heparin-binding form of placenta growth factor (PlGF-2) in vivo. Both heparin-binding homologues induced mobilization of endothelial progenitor cells from the bone marrow and gave rise to similar colony numbers of myeloic cells in a colony-forming assay. These findings suggest that both VEGFR-1 and VEGFR-2 are involved in stem cell mobilization.

  17. Bioengineered heparins and heparan sulfates.

    PubMed

    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.

  18. Enhanced regenerative healing efficacy of a highly skin-permeable growth factor nanocomplex in a full-thickness excisional mouse wound model

    PubMed Central

    Bae, Il-Hong; Park, Jin Woo; Kim, Dae-Yong

    2014-01-01

    Exogenous administration of growth factors has potential benefits in wound healing; however, limited percutaneous absorption, inconsistent efficacy, and the need for high doses have hampered successful clinical use. To overcome these restrictions, we focused on the development of a topical formulation composed of highly skin-permeable multimeric nanocomplex of growth factors. In the present study, we fused low-molecular-weight protamine (LMWP) with epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-I), and platelet-derived growth factor A ligand (PDGF-A) (producing recombinant [r]LMWP-EGF, rLMWP-IGF-I, and rLMWP-PDGF-A, respectively) via genetic modification. Then, we used in vitro cell proliferation studies to assess the biological activity and the benefits of the combination. The LMWP-conjugated growth factors were complexed with low-molecular-weight heparin (LMWH) and formulated with Poloxamer 188 as a delivery vehicle. After confirming the enhanced skin permeability, in vivo studies were performed to assess whether the LMWP-conjugated growth factor nanocomplex formulations accelerated the healing of full-thickness wounds in mice. The LMWP-conjugated growth factors were biologically equivalent to their native forms, and their combination induced greater fibroblast proliferation. rLMWP-EGF showed significantly enhanced permeability and cumulative permeation, and the rates for rLMWP-IGF-I and rLMWP-PDGF-A, across excised mouse skin, were 124% and 164% higher, respectively, than for the native forms. The LMWP-fused growth factors resulted in formation of nanocomplexes (23.51±1.12 nm in diameter) in combination with LMWH. Topical delivery of growth factors fused with LMWP accelerated wound re-epithelialization significantly, accompanied by the formation of healthy granulation tissue within 9 days compared with a free–growth factor complex or vehicle. Thus, the LMWP-conjugated growth factor nanocomplex can induce rapid, comprehensive healing and may

  19. Customized Ca–P/PHBV nanocomposite scaffolds for bone tissue engineering: design, fabrication, surface modification and sustained release of growth factor

    PubMed Central

    Duan, Bin; Wang, Min

    2010-01-01

    Integrating an advanced manufacturing technique, nanocomposite material and controlled delivery of growth factor to form multifunctional tissue engineering scaffolds was investigated in this study. Based on calcium phosphate (Ca–P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocomposite microspheres, three-dimensional Ca–P/PHBV nanocomposite scaffolds with customized architecture, controlled porosity and totally interconnected porous structure were successfully fabricated using selective laser sintering (SLS), one of the rapid prototyping technologies. The cytocompatibility of sintered Ca–P/PHBV nanocomposite scaffolds, as well as PHBV polymer scaffolds, was studied. For surface modification of nanocomposite scaffolds, gelatin was firstly physically entrapped onto the scaffold surface and heparin was subsequently immobilized on entrapped gelatin. The surface-modification improved the wettability of scaffolds and provided specific binding site between conjugated heparin and the growth factor recombinant human bone morphogenetic protein-2 (rhBMP-2). The surface-modified Ca–P/PHBV nanocomposite scaffolds loaded with rhBMP-2 significantly enhanced the alkaline phosphatase activity and osteogenic differentiation markers in gene expression of C3H10T1/2 mesenchymal stem cells. Together with osteoconductive nanocomposite material and controlled growth factor delivery strategies, the use of SLS technique to form complex scaffolds will provide a promising route towards individualized bone tissue regeneration. PMID:20504805

  20. Customized Ca-P/PHBV nanocomposite scaffolds for bone tissue engineering: design, fabrication, surface modification and sustained release of growth factor.

    PubMed

    Duan, Bin; Wang, Min

    2010-10-06

    Integrating an advanced manufacturing technique, nanocomposite material and controlled delivery of growth factor to form multifunctional tissue engineering scaffolds was investigated in this study. Based on calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocomposite microspheres, three-dimensional Ca-P/PHBV nanocomposite scaffolds with customized architecture, controlled porosity and totally interconnected porous structure were successfully fabricated using selective laser sintering (SLS), one of the rapid prototyping technologies. The cytocompatibility of sintered Ca-P/PHBV nanocomposite scaffolds, as well as PHBV polymer scaffolds, was studied. For surface modification of nanocomposite scaffolds, gelatin was firstly physically entrapped onto the scaffold surface and heparin was subsequently immobilized on entrapped gelatin. The surface-modification improved the wettability of scaffolds and provided specific binding site between conjugated heparin and the growth factor recombinant human bone morphogenetic protein-2 (rhBMP-2). The surface-modified Ca-P/PHBV nanocomposite scaffolds loaded with rhBMP-2 significantly enhanced the alkaline phosphatase activity and osteogenic differentiation markers in gene expression of C3H10T1/2 mesenchymal stem cells. Together with osteoconductive nanocomposite material and controlled growth factor delivery strategies, the use of SLS technique to form complex scaffolds will provide a promising route towards individualized bone tissue regeneration.

  1. Augmented production of heparin-binding mitogenic proteins by preadipocytes from massively obese persons.

    PubMed Central

    Teichert-Kuliszewska, K; Hamilton, B S; Deitel, M; Roncari, D A

    1992-01-01

    Basic fibroblast growth factor (bFGF) stimulates the replication of preadipocytes and inhibits their differentiation. In this study we explored whether the same or related polypeptides were produced locally and acted by paracrine/autocrine mechanisms in adipose tissue. Omental preadipocytes from 7 lean and 10 massively obese (> 170% reference) subjects were grown to confluence in subculture. Total RNA was hybridized with a synthetic deoxynucleotide for human bFGF. In the case of all cell strains, there was expression of two major bFGF transcripts, 7.0 and 3.7 kb. Although there was considerable variation in the degree of expression, preadipocytes from massively obese subjects revealed much greater expression than did cells from the lean (P < 0.001). In studies of conditioned media prepared with preadipocytes, the presence of proteins belonging to the heparin-binding (fibroblast) growth factor family was indicated by Western blot analysis, for a 66-kD protein with anti-(1-24)bFGF, and for a 32-kD protein with anti-(40-63)bFGF antibodies. The relative quantity of the 66-kD protein correlated with body mass index at r = 0.72. bFGF-related proteins probably function normally to maintain an appropriate complement of adipocyte precursors. The augmented expression of heparin-binding growth factors in preadipocytes from some massively obese people probably contributes to the excessive cellularity of their fat depots. Images PMID:1401060

  2. [Growth Hormone-Insulin Growth Factor I (GH-IGF-I) axis and growth].

    PubMed

    Castell, A-L; Sadoul, J-L; Bouvattier, C

    2013-10-01

    Normal human linear growth results from an evolutionary process expressing the sum effect of multiple genes. The growth hormone (GH) - insulin like growth factor (IGF)-I axis is one of the main actors in the growth process. Defects in this axis can be responsible for short or tall stature. Short stature is defined as smaller than - 2 standard deviations (SD). It is a very common reason for consultation in pediatrics; indeed, 2.5 % of children are concerned. Multiple causes make diagnosis difficult. In this article, we detail the most common constitutional causes of small size, including those related to a defect in the GH-IGF-I axis. Then, we report, the first results of the clinical and genetic study conducted on 213 patients with gigantism. Tall stature is defined by a height superior to 2 SD. Finally, recent work linking epigenetics and growth - via signaling pathways of GH-IGF-I axis - will be presented.

  3. Insulin-like growth factor and epidermal growth factor signaling in breast cancer cell growth: focus on endocrine resistant disease.

    PubMed

    Voudouri, Kallirroi; Berdiaki, Aikaterini; Tzardi, Maria; Tzanakakis, George N; Nikitovic, Dragana

    2015-01-01

    Breast cancer is the most common type of cancer for women worldwide with a lifetime risk amounting to a staggering total of 10%. It is well established that the endogenous synthesis of insulin-like growth factor (IGF) and epidermal growth factor (EGF) polypeptide growth factors are closely correlated to malignant transformation and all the steps of the breast cancer metastatic cascade. Numerous studies have demonstrated that both estrogens and growth factors stimulate the proliferation of steroid-dependent tumor cells, and that the interaction between these signaling pathways occurs at several levels. Importantly, the majority of breast cancer cases are estrogen receptor- (ER-) positive which have a more favorable prognosis and pattern of recurrence with endocrine therapy being the backbone of treatment. Unfortunately, the majority of patients progress to endocrine therapy resistant disease (acquired resistance) whereas a proportion of patients may fail to respond to initial therapy (de novo resistance). The IGF-I and EGF downstream signaling pathways are closely involved in the process of progression to therapy resistant disease. Modifications in the bioavailability of these growth factors contribute critically to disease progression. In the present review therefore, we will discuss in depth how IGF and EGF signaling participate in breast cancer pathogenesis and progression to endocrine resistant disease.

  4. Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism.

    PubMed

    Koland, J G; Cerione, R A

    1988-02-15

    The mechanism by which the protein kinase activity of the epidermal growth factor (EGF) receptor is activated by binding of growth factor was investigated. Detergent-solubilized receptor in monomeric form was isolated by sucrose density gradient centrifugation and both its kinase and autophosphorylation activities monitored. In a low ionic strength medium and with MnCl2 as an activator, the activity of the monomeric receptor was EGF-independent. However, with 0.25 M ammonium sulfate present, the MnCl2-stimulated kinase activity was strikingly EGF-dependent. In contrast, the kinase activity expressed in the presence of MgCl2 showed growth factor control in the absence of added salt. Under the conditions of these experiments there was apparently little tendency for growth factor to induce aggregation of the receptor, indicating that the allosteric activation of the receptor kinase by EGF occurred via an intramolecular mechanism. Whereas detergent-solubilized receptor was the subject of these studies, the kinase activity of cell surface receptors might also be controlled by an intramolecular mechanism. These results indicate that an individual receptor molecule has the potential to function as a transmembrane signal transducer.

  5. Nerve growth factor: neurotrophin or cytokine?

    PubMed

    Bonini, S; Rasi, G; Bracci-Laudiero, M L; Procoli, A; Aloe, L

    2003-06-01

    Nerve growth factor (NGF) is a neutrophin exerting an important role in the development and functions of the central and peripheral nervous system. However, it has recently been documented that several immune cells - such as mast cells, lymphocytes and eosinophils - produce, store and release NGF. Moreover, NGF high and low affinity receptors are widely expressed in the immune system, thus indicating the potential of responding to this neurotrophin through an autocrine mechanism. In fact, NGF influences development differentiation, chemotaxis and mediator release of inflammatory cells as well as fibroblast activation through a complex network influenced by other pro-inflammatory cytokines. Finally, NGF is increased in biological fluids of several allergic, immune and inflammatory diseases. Data reviewed suggest, therefore, that NGF might also be viewed as a (Th2?) cytokine with a modulatory role in allergic inflammation and tissue remodeling. Copyright 2003 S. Karger AG, Basel

  6. The Fibroblast Growth Factor signaling pathway

    PubMed Central

    Ornitz, David M; Itoh, Nobuyuki

    2015-01-01

    The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc. PMID:25772309

  7. Neuropeptides as lung cancer growth factors.

    PubMed

    Moody, Terry W; Moreno, Paola; Jensen, Robert T

    2015-10-01

    This manuscript is written in honor of the Festschrift for Abba Kastin. I met Abba at a Society for Neuroscience meeting and learned that he was Editor-in-Chief of the Journal Peptides. I submitted manuscripts to the journal on "Neuropeptides as Growth Factors in Cancer" and subsequently was named to the Editorial Advisory Board. Over the past 30 years I have published dozens of manuscripts in Peptides and reviewed hundreds of submitted manuscripts. It was always rewarding to interact with Abba, a consummate professional. When I attended meetings in New Orleans I would sometimes go out to dinner with him at the restaurant "Commanders Palace". When I chaired the Summer Neuropeptide Conference we were honored to have him receive the Fleur Strand Award one year in Israel. I think that his biggest editorial contribution has been the "Handbook of Biologically Active Peptides." I served as a Section Editor on "Cancer/Anticancer Peptides" and again found that it was a pleasure working with him. This review focuses on the mechanisms by which bombesin-like peptides, neurotensin and vasoactive intestinal peptide regulate the growth of lung cancer. Published by Elsevier Inc.

  8. Can heparin immobilized surfaces maintain nonthrombogenic activity during in vivo long-term implantation?

    PubMed

    Nojiri, C; Kido, T; Sugiyama, T; Horiuchi, K; Kijima, T; Hagiwara, K; Kuribayashi, E; Nogawa, A; Ogiwara, K; Akutsu, T

    1996-01-01

    The authors previously demonstrated that heparin immobilized surfaces showed excellent nonthrombogenic properties for extracorporeal membrane oxygenation experiments as long as 168 hr. The characteristics of the heparin immobilized surfaces include high heparin bioactivity and prevention of platelet adhesion and complement activation. However, it is not known whether the heparin immobilized surfaces would be effective for in vivo long-term implantation. Heparin bioactivity may be lost because of complete degradation or blocking of binding sites on heparin by adsorbed proteins. This study attempted to elucidate the in vivo long-term fate of heparin immobilized surfaces. The blood contacting surfaces of the ventricular assist device (VAD) made from polyurethane was modified with heparin immobilization and evaluated in a long-term sheep left VAD (LVAD) model for as long as 3 months. After removal of the VAD, heparin bioactivity was measured by Factor Xa assay. The blood contacting surfaces were analyzed with a scanning electron microscope, and the adsorbed proteins on the surfaces of the diaphragm were analyzed by SDS-PAGE and Western blotting. The thickness of adsorbed proteins on the surfaces also was measured by a confocal laser microscope. For the control ventricular assist devices, thrombus formation was observed within 1 month, whereas heparin immobilized VADs were able to operate thrombus free for periods as long as 3 months. The control surfaces demonstrated a thick adsorbed protein layer on thin surfaces, whereas heparin immobilized surfaces maintained thinner adsorbed proteins on thin surfaces. Anti Factor Xa activity of the heparinized surfaces disappeared after 15 days, but the surfaces remained nonthrombogenic even after heparin bioactivity was completely lost. The protein composition analyzed by SDS-PAGE showed an albumin dominant pattern on the heparinized surfaces. The band of 110 kD corresponding to C3b was detected only on the control surfaces, which

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Fluid shear stress differentially modulates expression of genes encoding basic fibroblast growth factor and platelet-derived growth factor B chain in vascular endothelium.

    PubMed Central

    Malek, A M; Gibbons, G H; Dzau, V J; Izumo, S

    1993-01-01

    Fluid shear stress has been shown to be an important regulator of vascular structure and function through its effect on the endothelial cell. We have explored the effect of shear stress on the expression of the heparin-binding growth factors platelet-derived growth factor B chain (PDGF-B) and basic fibroblast growth factor (bFGF) in bovine aortic endothelial cells using a purpose-built cone-plate viscometer. Using morphometric analysis, we have mimicked the endothelial cell shape changes encountered in vivo in response to shear stress and correlated these with changes in gene expression. Steady laminar shear stress of 15 and 36 dyn/cm2 both resulted in endothelial cell shape change, but the higher shear stress induced greater and more uniform alignment in the direction of flow and nuclear protrusion after 24 h. Steady laminar shear stress of both 15 and 36 dyn/cm2 induced a significant 3.9- and 4.2-fold decrease, respectively, in PDGF-B mRNA at 9 h. In contrast, steady laminar shear of 15 dyn/cm2 induced a mild and transient 1.5-fold increase in bFGF mRNA while shear of 36 dyn/cm2 induced a significant 4.8-fold increase at 6 h of shear which remained at 2.9-fold at 9 h. Pulsatile and turbulent shear stress showed the same effect as steady laminar shear stress (all at 15 dyn/cm2 time-average magnitude) on PDGF-B and bFGF mRNA content. Cyclic stretch (20% strain, 20/min) of cells grown on silicone substrate did not significantly affect either PDGF-B or bFGF mRNA levels. These results suggest that expression of each peptide growth factor gene is differentially regulated by fluid shear stress in the vascular endothelial cell. These results may have implications on vascular structure and function in response to hemodynamic forces and present a model for the study of transduction of mechanical stimuli into altered gene expression. Images PMID:8408655

  11. [Regulation of uterine cellular proliferation with estrogens and growth factors].

    PubMed

    Alvarez-Rodríguez, C; Baiza-Guzmán, L A

    1996-09-01

    In this paper the role of estrogen and growth factors in the uterine cellular proliferation is analyzed. The evidences indicate that the estradiol-stimulate cell division is associated with the induction of expression of a variety of growth factors from the all major uterine cell types (epithelia, stroma and myometrium). These growth factors amplify the estrogen proliferation signal in autocrine and/or paracrin fashion. The best-studied growth factors in the uterine response to estradiol are epidermal growth factor (EGF) and insulin-like growth factor (IGF-1). Uterine cell proliferation is a complex process that involves interactions of several growth factors, ovarian steroids hormones action and cell to cell signaling.

  12. Reduced Expression of the Epidermal Growth Factor Signaling System in Preeclampsia

    PubMed Central

    Armant, D. Randall; FRITZ, Rani; KILBURN, Brian A.; KIM, Yeon Mee; NIEN, Jyh Kae; MAIHLE, Nita J.; ROMERO, Roberto; LEACH, Richard E.

    2014-01-01

    Introduction The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. Methods Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunocytochemistry in the trophoblast of placentas (N=76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. Results Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p<0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p<0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p< 0.05). Discussion Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia. PMID:25589361

  13. Reduced expression of the epidermal growth factor signaling system in preeclampsia.

    PubMed

    Armant, D R; Fritz, R; Kilburn, B A; Kim, Y M; Nien, J K; Maihle, N J; Romero, R; Leach, R E

    2015-03-01

    The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. The angiogenic growth factors HGF and VEGF in serum and plasma from neuroblastoma patients.

    PubMed

    Sköldenberg, Erik G; Larsson, Anders; Jakobson, Ake; Hedborg, Fredrik; Kogner, Per; Christofferson, Rolf H; Azarbayjani, Faranak

    2009-08-01

    To determine whether concentrations of the angiogenic growth factors hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGF-A) correlate with clinical and genetic markers in samples taken at diagnosis in children with neuroblastoma (NB). Heparin plasma (P-) and serum (S-) samples of healthy controls (n=73, mean age +/- SD 3.5+/-2.1; females/males: 23/50) and patients with NB (n=62; 2.2+/-1.8; 26/36) were collected between 1988 and 1999. Clinical data included age at diagnosis, gender, stage, outcome, amplification of the oncogene MYCN, loss of heterozygosity at the short arm of chromosome 1 (1p LOH) and ploidy. HGF and S-VEGF-A were elevated in NB as compared to controls (38/62 patients, p<0.0001 and p<0.05, Mann-Whitney U test). HGF concentrations were higher in high-stage (stage 3-4) as compared to low-stage (stage 1-2) disease (p<0.01). P-HGF was elevated in patients with 1p LOH (p<0.01), MYCN amplification (p<0.001) and di- or tetraploidy (p<0.001). S-HGF concentration was elevated in patients MYCN-amplified tumors only. Plasma and S-HGF concentrations were higher in the deceased group (p<0.05), but not P or S-VEGF-A. This study showed that concentrations of HGF and S-VEGF-A are elevated in patients with NB. Furthermore, HGF and S-VEGF-A concentrations correlate to higher stage disease and HGF correlates to genetic markers known to indicate a poor outcome. These observations imply that HGF and VEGF-A have biological roles in NB and suggest the possibility of interference with HGF or VEGF-A signaling as a therapeutic strategy.

  15. Hepatocyte growth factor, vascular endothelial growth factor, glial cell-derived neurotrophic factor and nerve growth factor are differentially affected by early chronic ethanol or red wine intake.

    PubMed

    Fiore, Marco; Mancinelli, Rosanna; Aloe, Luigi; Laviola, Giovanni; Sornelli, Federica; Vitali, Mario; Ceccanti, Mauro

    2009-08-10

    Ethanol intake during pregnancy and lactation induces severe changes in brain and liver throughout mechanisms involving growth factors. These are signaling molecules regulating survival, differentiation, maintenance and connectivity of brain and liver cells. Ethanol is an element of red wine which contains also compounds with antioxidant properties. Aim of the study was to investigate differences in hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in brain areas and liver by ELISA of 1-month-old male mice exposed perinatally to ethanol at 11 vol.% or to red wine at same ethanol concentration. Ethanol was administered before and during pregnancy up to pups' weaning. Ethanol per se elevated HGF in liver and cortex, potentiated liver VEGF, reduced GDNF in the liver and decreased NGF content in hippocampus and cortex in the offspring. We did not find changes in HGF or NGF due to red wine exposure. However, we revealed elevation in VEGF levels in liver and reduced GDNF in the cortex of animals exposed to red wine but the VEGF liver increase was more marked in animals exposed to ethanol only compared to the red wine group. In conclusion the present findings in the mouse show differences in ethanol-induced toxicity when ethanol is administered alone or in red wine that may be related to compounds with antioxidant properties present in the red wine.

  16. Design of Growth Factor Sequestering Biomaterials

    PubMed Central

    Belair, David G.; Le, Ngoc Nhi; Murphy, William L.

    2014-01-01

    Growth factors (GFs) are major regulatory proteins that can govern cell fate, migration, and organization. Numerous aspects of the cell milieu can modulate cell responses to GFs, and GF regulation is often achieved by the native extracellular matrix (ECM). For example, the ECM can sequester GFs and thereby control GF bioavailability. In addition, GFs can exert distinct effects depending on whether they are sequestered in solution, at two-dimensional interfaces, or within three-dimensional matrices. Understanding how the context of GF sequestering impacts cell function in the native ECM can instruct the design of soluble or insoluble GF sequestering moieties, which can then be used in a variety of bioengineering applications. This Feature Article provides an overview of the natural mechanisms of GF sequestering in the cell milieu, and reviews the recent bioengineering approaches that have sequestered GFs to modulate cell function. Results to date demonstrate that the cell response to GF sequestering depends on the affinity of the sequestering interaction, the spatial proximity of sequestering in relation to cells, the source of the GF (supplemented or endogenous), and the phase of the sequestering moiety (soluble or insoluble). We highlight the importance of context for the future design of biomaterials that can leverage endogenous molecules in the cell milieu and mitigate the need for supplemented factors. PMID:25182455

  17. [Endogenous heparin-like syndrome: analysis of clinical observations].

    PubMed

    Bulanov, A Iu; Iatskov, K V; Shulutko, E M; Glukhova, T E; Andreĭchenko, S A

    2012-01-01

    One of the reasons for non-surgical bleeding is heparin-like syndrome (HLS), under which is understanded presence of heparin effect in the absence of it's exogenous application. The role of endogenous heparins perform glycosaminoglycans -- biologically active substances. HLS is accompanied by endothelium damage and discussed in the network of the systemic inflammatory response syndrome (SIRS). HLS is described in liver future, sepsis, pregnancy and a number of hemoblastosis. Hypocoagulation effect of endogenous heparin localizates in X coagulation factor. The main method of diagnosis - thromboelastography. The use of a specific heparin antidote - Protamine sulfate has not confirmed clinical efficacy. Priority direction in the therapy of - methods of "shunt hemostasis". In this paper, we present the analysis of observations of 4 patients with developed endogenous HLS. In 2 cases (combination of sepsis with hepatic failure in one patient and initial thrombophilia in other) HLS has been accompanied by massive bleeding (massive hemothoraxc with haemorrhagic shock, a massive intraoperative blood loss). For HLS relief in these cases was used prothrombine complex concentrate (PCC) (in the 1st case), recombinant VIIa factor (in the 2nd case). In other cases, HLS (in a patient with multiple myeloma and childbirth in the postpartum period), haemorrhagic syndrome was not so expressed, the treatment was carried out with FFP transfusion.

  18. A reevaluation of heparin requirements for cardiopulmonary bypass.

    PubMed

    Cardoso, P F; Yamazaki, F; Keshavjee, S; Schaefers, H J; Hsieh, C M; Wang, L S; Glynn, M F; Patterson, G A; Cooper, J D

    1991-01-01

    We wished to determine if reduction in the standard heparin administration for cardiopulmonary bypass could be accomplished safely with the use of membrane oxygenators. An experimental study was designed to evaluate two different heparin administration protocols for cardiopulmonary bypass with hollow-fiber membrane oxygenators. Two groups of six pigs were submitted to hypothermic cardiopulmonary bypass (28 degrees C) for 3 hours, then rewarmed, decannulated, and reassessed after 1 hour. In group I (control) heparin was administered to maintain the activated clotting time in excess of 450 seconds; in group II activated clotting time was maintained between 250 and 300 seconds. The mean total heparin administered was 41,000 units in group I and 25,000 units in group II. Concentration of coagulation factors II, V, and VIII, fibrinogen, and platelet count were determined before, during, and 1 hour after bypass. No significant difference in any of these coagulation parameters was observed between the groups. The performance of the oxygenators was similar in both groups, with no evidence of thrombosis. Thus reduced heparin administration, enough to keep activated clotting time between 250 and 300 seconds, was not related either to major coagulation factors and platelet consumption or to derangements in the oxygenator's performance.

  19. The isolation and characterization of growth regulatory factors produced by a herpes simplex virus Type 2 transformed mouse tumor cell line, H238

    SciTech Connect

    Stagg, R.B.

    1988-01-01

    This study was performed in an attempt to associate HSV-2-transformation with specific growth factors in order to develop a testable model for HSV-2-transformation. We report here the isolation and characterization of four growth regulatory factors produced by H238, an HSV-2-transformed mouse tumor cell line. These factors were separated from the H238-CM by heparin-sepharose affinity chromatography into three peaks of mitogenic activity and a fourth containing inhibitory activity for splenocytes. The three peaks of mitogenic activity have been identified based on physiochemical characteristics: the first supported the anchorage-independent growth of EGF treated NRK-c-49 cells and resembles transforming growth factor-{beta} (TGF-{beta}); the second bound to lectin-coated sepharose beads and was sensitive to trypsin, neuroaminidase, and the reducing agent dithiothreitol (DTT) and, resembled a platelet-derived growth factor (PDGF)-like factor; and the third displaced ({sup 125}I)-labeled basic fibroblast growth factor (bFGF) in a dose-dependent fashion when tested with a radioimmune assay. The fourth peak was inhibitory for a variety of splenocyte function assays. A model for the interaction of these factors in vivo is presented with an emphasis on testability.

  20. Heterogeneity of the chondroitin sulfate portion of phosphacan/6B4 proteoglycan regulates its binding affinity for pleiotrophin/heparin binding growth-associated molecule.

    PubMed

    Maeda, Nobuaki; He, Jue; Yajima, Yuki; Mikami, Tadahisa; Sugahara, Kazuyuki; Yabe, Tomio

    2003-09-12

    PTP zeta is a receptor-type protein-tyrosine phosphatase that is synthesized as a chondroitin sulfate proteoglycan and uses pleiotrophin as a ligand. The chondroitin sulfate portion of this receptor is essential for high affinity binding to pleiotrophin. Here, we purified phosphacan, which corresponds to the extracellular domain of PTP zeta, from postnatal day 7 (P7) and P12 rat cerebral cortex (PG-P7 and PG-P12, respectively) and from P20 rat whole brain (PG-P20). The chondroitin sulfate of these preparations displayed immunologically and compositionally different structures. In particular, only PG-P20 reacted with the monoclonal antibody MO-225, which recognizes chondroitin sulfate containing the GlcA(2S)beta 1-3GalNAc(6S) disaccharide unit (D unit). Analysis of the chondroitinase digestion products revealed that GlcA beta 1-3GalNAc(4S) disaccharide unit (A unit) was the major component in these preparations and that PG-P20 contained 1.3% D unit, which was not detected in PG-P7 and PG-P12. Interaction analysis using a surface plasmon resonance biosensor indicated that PG-P20 had approximately 5-fold stronger affinity for pleiotrophin (dissociation constant (KD) = 0.14 nM) than PG-P7 and PG-P12, although all these preparations showed similar low affinity binding to pleiotrophin after chondroitinase ABC digestion (KD = 1.4 approximately 1.6 nM). We also found that shark cartilage chondroitin sulfate D containing approximately 20% D unit bound to pleiotrophin with moderate affinity (KD = 2.7 nM), whereas whale cartilage chondroitin sulfate A showed no binding to this growth factor. These results suggest that variation of chondroitin sulfate plays important roles in the regulation of signal transduction in the brain.

  1. Report of successful use of argatroban as an alternative anticoagulant during coronary stent implantation in a patient with heparin-induced thrombocytopenia and thrombosis syndrome.

    PubMed

    Lewis, B E; Iaffaldano, R; McKiernan, T L; Rao, L; Donkin, J; Wallenga, J M

    1996-06-01

    Heparin-induced thrombocytopenia and thrombosis syndrome (HITTS) is a severe complication of heparin caused by an antibody response to the heparin-platelet factor 4 complex which results in severe thrombosis. Heparin rechallenge in HITTS patients carries a high risk of inducing thrombosis. Antithrombin agents represent treatment alternatives in HITTS patients who require anticoagulation. We report successful coronary stent implantation in a HITTS patient using the antithrombin agent argatroban.

  2. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman W.

    1987-01-01

    New muscle tissue culture techniques were developed to grow embryonic skeletal myofibers which are able to differentiate into more adultlike myofibers. Studies on mechanical simulation of cultured muscle cell growth will now be more directly applicable to mechanically-induced growth in adult muscle, and lead to better models for understanding muscle tissue atrophy caused by disuse in the microgravity of space.

  3. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    SciTech Connect

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-05-01

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 {mu}M triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.

  4. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation.

    PubMed

    Martínez-García, Eva; Irigoyen, Marta; Ansó, Elena; Martínez-Irujo, Juan José; Rouzaut, Ana

    2008-05-01

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 muM triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.

  5. Synthesis of multilayered alginate microcapsules for the sustained release of fibroblast growth factor-1

    PubMed Central

    Khanna, Omaditya; Moya, Monica L; Opara, Emmanuel C; Brey, Eric M

    2010-01-01

    Alginate microcapsules coated with a permselective poly-L-ornithine (PLO) membrane have been investigated for the encapsulation and transplantation of islets as a treatment for type 1 diabetes. The therapeutic potential of this approach could be improved through local stimulation of microvascular networks in order to meet mass transport demands of the encapsulated cells. Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor with optimal effect occurring when it is delivered in a sustained manner. In this paper, a technique is described for the generation of multilayered alginate microcapsules with an outer alginate layer that can be used for the delivery of FGF-1. The influence of alginate concentration and composition (high mannuronic acid (M) or guluronic acid (G) content) on outer layer size and stability, protein encapsulation efficiency, and release kinetics was investigated. The technique results in a stable outer layer of alginate with a mean thickness between 113–164 µm, increasing with alginate concentration and G-content. The outer layer was able to encapsulate and release FGF-1 for up to thirty days, with 1.25% of high G alginate displaying the most sustained release. The released FGF-1 retained its biologic activity in the presence of heparin, and the addition of the outer layer did not alter the permselectivity of the PLO coat. This technique could be used to generate encapsulation systems that deliver proteins to stimulate local neovascularization around encapsulated islets. PMID:20725969

  6. The use of de-differentiated chondrocytes delivered by a heparin-based hydrogel to regenerate cartilage in partial-thickness defects.

    PubMed

    Kim, Mihye; Kim, Se Eun; Kang, Seong Soo; Kim, Young Ha; Tae, Giyoong

    2011-11-01

    Partial-thickness cartilage defects, with no subchondral bone injury, do not repair spontaneously, thus there is no clinically effective treatment for these lesions. Although the autologous chondrocyte transplantation (ACT) is one of the promising approaches for cartilage repair, it requires in vitro cell expansion to get sufficient cells, but chondrocytes lose their chondrogenic phenotype during expansion by monolayer culture, leading to de-differentiation. In this study, a heparin-based hydrogel was evaluated and optimized to induce cartilage regeneration with de-differentiated chondrocytes. First, re-differentiation of de-differentiated chondrocytes encapsulated in heparin-based hydrogels was characterized in vitro with various polymer concentrations (from 3 to 20 wt.%). Even under a normal cell culture condition (no growth factors or chondrogenic components), efficient re-differentiation of cells was observed with the optimum at 10 wt.% hydrogel, showing the complete re-differentiation within a week. Efficient re-differentiation and cartilage formation of de-differentiated cell/hydrogel construct were also confirmed in vivo by subcutaneous implantation on the back of nude mice. Finally, excellent cartilage regeneration and good integration with surrounding, similar to natural cartilage, was also observed by delivering de-differentiated chondrocytes using the heparin-based hydrogel in partial-thickness defects of rabbit knees whereas no healing was observed for the control defects. These results demonstrate that the heparin-based hydrogel is very efficient for re-differentiation of expanded chondrocytes and cartilage regeneration without using any exogenous inducing factors, thus it could serve as an injectable cell-carrier and scaffold for cartilage repair. Excellent chondrogenic nature of the heparin-based hydrogel might be associated with the hydrogel characteristic that can secure endogenous growth factors secreted from chondrocytes, which then can promote

  7. Significance of thrombin-receptors of thrombocytes for the interaction of heparins and low-molecular-weight heparin in human whole blood clotting.

    PubMed

    Harenberg, J; Schuler, M; Zimmermann, R; Heptner, W

    1988-01-01

    We describe in the present paper the results of the influence of normal and low-molecular-weight heparin on the interaction of human fibrinogen and thrombocytes in human whole blood cotting ex vivo. During the coagulation process sequential measurements of fibrinopeptide A reflect fibrin formation and determination of platelet factor 4 indicate activation of thrombocytes. The data show that low-molecular-weight heparin inhibits plasma thrombin generation in vivo for longer than normal heparin and it affects the fibrinogen platelet binding less. There is good evidence that a lonely factor Xa inhibition mediates this anticoagulant mechanism. Therefore, these data favor the hypothesis that antifactor Xa activity prevents indeed blood clotting.

  8. Growth factors and their relationship to neoplastic and paraneoplastic disease.

    PubMed

    Badawi, R A; Birns, J; Watson, T; Kalra, L

    2005-04-01

    Growth factors are extracellular signaling molecules that act in an autocrine and paracrine fashion to regulate growth, proliferation, differentiation, and survival of cells. Dysregulation of the growth factor networks is intimately related to the molecular pathogenesis of neoplastic and paraneoplastic disease. Increasing knowledge of the molecular mechanisms underlying growth factors and their actions on cell cycling, cell division, and cell death is shedding light on new therapeutic avenues for molecular targeting of tumors. Epidermal growth factor and vascular endothelial growth factor both offer examples of how growth factor biology and its relationship to cancer can be harnessed to create effective clinical therapeutic tools such as monoclonal antibodies. This approach heralds a future in which rational molecular oncological therapy may increasingly become the norm.

  9. Kinetics of epidermal growth factor in saliva.

    PubMed

    Ino, M; Ushiro, K; Ino, C; Yamashita, T; Kumazawa, T

    1993-01-01

    Human epidermal growth factor (hEGF) stimulates the growth and differentiation of various tissues. We measured EGF levels in saliva (n = 128), urine (n = 94), and serum (n = 99) with radioimmunoassay in order to study the kinetics of hEGF in saliva of normal subjects and patients with oral disease. Salivary EGF levels showed an apparent diurnal rhythm related to the taking of meals. Urinary and serum EGF levels showed no obvious diurnal rhythm. There was no significant correlation between salivary and urinary EGF levels, nor between salivary and serum EGF levels. Salivary EGF levels were significantly lower in the younger group (0-9 years old, 3.06 +/- 0.32 ng/ml, p < 0.05) than in the elder group (10-79 years old, 4.78 +/- 3.5 ng/ml), but did not correlate with age in the elder group. There was no significant difference between males and females between EGF levels in saliva, urine or serum. The relative proportion of EGF levels in submandibular gland saliva, parotid saliva, and whole saliva was 1:6:4. The positive rate of immunohistochemical EGF showed no significant differences between submandibular gland, parotid gland, sublingual gland or minor salivary gland. Salivary EGF levels were markedly low in patients with oral inflammations (stomatitis aphthosa, or peritonsillar abscess) or head and neck tumors (squamous cell carcinoma of the tongue, oral cavity, hypopharynx or larynx). These findings may be significant pathophysiologically. Low salivary EGF levels may reduce the capacity of oral mucosal defense mechanisms to fight against injury by physiochemical agents.

  10. Endorsement of Growth Factors in Experiential Training Groups

    ERIC Educational Resources Information Center

    Kiweewa, John; Gilbride, Dennis; Luke, Melissa; Seward, Derek

    2013-01-01

    The purpose of this study was to identify student growth factors during a semester long Master's level group counseling class. Results indicated that 12 growth factors accounted for 86% of the total number of critical incidents that participants reported as influencing their personal growth and awareness during the group experience. Two other…

  11. Endorsement of Growth Factors in Experiential Training Groups

    ERIC Educational Resources Information Center

    Kiweewa, John; Gilbride, Dennis; Luke, Melissa; Seward, Derek

    2013-01-01

    The purpose of this study was to identify student growth factors during a semester long Master's level group counseling class. Results indicated that 12 growth factors accounted for 86% of the total number of critical incidents that participants reported as influencing their personal growth and awareness during the group experience. Two other…

  12. Gene expression of growth factors and growth factor receptors for potential targeted therapy of canine hepatocellular carcinoma.

    PubMed

    Iida, Gentoku; Asano, Kazushi; Seki, Mamiko; Sakai, Manabu; Kutara, Kenji; Ishigaki, Kumiko; Kagawa, Yumiko; Yoshida, Orie; Teshima, Kenji; Edamura, Kazuya; Watari, Toshihiro

    2014-03-01

    The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.

  13. Photo induced surface heparin immobilization.

    PubMed

    Nakayama, Y; Matsuda, T

    1993-01-01

    This paper describes a novel method providing durable layering of heparin immobilized hydrogels on fabricated devices. The preparation method is based on photochemistry of a dithiocarbamate group that is dissociated into a highly reactive radical pair upon ultraviolet (UV) irradiation. By taking advantage of characteristics of the photo generated radicals, hydrogel formation and its fixation onto a substrate surface were attained. The immobilization of heparin onto poly(ethylene terephtalate) was demonstrated. First, a mixed aqueous solution containing a photoreactive water soluble poly(N,N-dimethylacrylamide-covinylbenzyl N,N-diethyldithiocarbamate) and heparin was coated on the substrate. Subsequent UV irradiation resulted in the simultaneous formation of a heparin immobilized hydrogel and its chemical fixation onto the substrate. No delamination was found after vigorous washing with water. Significant inhibition of platelet adhesion and markedly prolonged blood coagulation times were observed, which are apparently derived from the surface hydrogel, and from released and chemically fixed surface heparin. Thus, it is expected that the photochemical method developed here provides potent antithrombogenicity to artificial organs.

  14. Encapsulation of basic fibroblast growth factor by polyelectrolyte multilayer microcapsules and its controlled release for enhancing cell proliferation.

    PubMed

    She, Zhen; Wang, Chunxia; Li, Jun; Sukhorukov, Gleb B; Antipina, Maria N

    2012-07-09

    Basic fibroblast growth factor (FGF2) is an important protein for cellular activity and highly vulnerable to environmental conditions. FGF2 protected by heparin and bovine serum albumin was loaded into the microcapsules by a coprecipitation-based layer-by-layer encapsulation method. Low cytotoxic and biodegradable polyelectrolytes dextran sulfate and poly-L-arginine were used for capsule shell assembly. The shell thickness-dependent encapsulation efficiency was measured by enzyme-linked immunosorbent assay. A maximum encapsulation efficiency of 42% could be achieved by microcapsules with a shell thickness of 14 layers. The effects of microcapsule concentration and shell thickness on cytotoxicity, FGF2 release kinetics, and L929 cell proliferation were evaluated in vitro. The advantage of using microcapsules as the carrier for FGF2 controlled release for enhancing L929 cell proliferation was analyzed.

  15. [The effect of preoperative administration of heparin on the heparin resistance development].

    PubMed

    Brzek, V; Lonský, V; Jiska, S; Kubícek, J; Nováková, D; Valentová, P; Bímová, J; Volt, M

    2009-03-01

    Heparin resistance is relatively frequent problem in cardio surgery. We were try to determine real occurrence heparin resistance before operation. Purpose of the project--to find the real frequency of heparin resistance in patients who will undergo a cardio surgical operation. To find out the dependence between the pre-operational application of heparin and the development of heparin resistance. We recorded pre-operative administration heparin in patients. If the dose of heparin was 5 mg/kg and more then we insert patients to the group heparin resistant. In our collection was heparin resistance in 203 patients from 624, it was 32.5%. Test agreement relative frequency with 22% was throw out--p < 0.001--heparin resistance in our group statistically different from 22% heparin resistance was higher than hypothesis. Heparin before operation was administrate 181 patients, which make to 29%. For administration of heparin was hypothesis of independence thrown (p < 0.001). Results of our works confirmed statistically significant occurrence of heparin resistance in patients that was administration heparin pre-operative. Heparin resistance occurred against presumption 22% in 32.5% in our group. It is statistic significant difference.

  16. Enzymatically in situ shell cross-linked micelles composed of 4-arm PPO-PEO and heparin for controlled dual drug delivery.

    PubMed

    Kim, Bae Young; Bae, Jin Woo; Park, Ki Dong

    2013-12-10

    We report a controlled dual drug delivery system using heparinized 4-arm poly(propylene oxide) (PPO)-poly(ethylene oxide) (PEO) micelles (cHTM) that are sterically stabilized by enzymatic shell cross-linking (SCL). Tyramine (TA) was chemically conjugated to 4-arm PPO-PEO (Tetronic) and heparin, resulting in Tetronic-TA (Tet-TA) and heparin-TA (Hep-TA), respectively. To prepare a series of cHTM, different amounts of Hep-TA were added to a micellar solution of Tet-TA, followed by addition of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) to trigger SCL between TA groups at the micellar surfaces. Increasing the feed amount of Hep-TA led to increased heparin content of cHTM, thereby resulting in increased micelle size with more negatively charged surfaces. All SCL micelles were found to be highly stable over 4weeks, showing negligible changes in their sizes and zeta potentials. Dual drug-loaded cHTM containing indomethacin (IMC) and basic fibroblast growth factor (bFGF) were prepared via a one-pot procedure. With favorable IMC loading, the loading efficiencies of bFGF into cHTM were much higher than those in the controls due to the presence of heparin on the micellar surface. After bFGF was added to IMC loaded cHTM the surface of HTM became less negative with an increase in size, suggesting successful binding of positively charged bFGF to heparinized micelle surfaces. In vitro release data clearly showed more sustained release of IMC and bFGF as compared with non-cross-linked micelles. Based on these results, we suggest that cHTM can be used as a new drug delivery platform for controlled dual drug release.

  17. [Stem cells and growth factors in wound healing].

    PubMed

    Pikuła, Michał; Langa, Paulina; Kosikowska, Paulina; Trzonkowski, Piotr

    2015-01-02

    Wound healing is a complex process which depends on the presence of various types of cells, growth factors, cytokines and the elements of extracellular matrix. A wound is a portal of entry for numerous pathogens, therefore during the evolution wound healing process has formed very early, being critical for the survival of every individual. Stem cells, which give rise to their early descendants progenitor cells and subsequently differentiated cells, play a specific role in the process of wound healing. Among the most important cells which take part in wound healing the following cells need to be distinguished: epidermal stem cells, dermal precursor of fibroblasts, adipose-derived stem cells as well as bone marrow cells. The activity of these cells is strictly regulated by various growth factors, inter alia epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF). Any disorders in functioning of stem cells and biological activity of growth factors may lead to the defects in wound healing, for instance delayed wound healing or creation of hypertrophic scars. Therefore, knowledge concerning the mechanisms of wound healing is extremely essential from clinical point of view. In this review the current state of the knowledge of the role of stem cells and growth factors in the process of wound healing has been presented. Moreover, some clinical aspects of wound healing as well as the possibility of the therapy based on stem cells and growth factors have included.

  18. Heparin-induced thrombocytopenia: a general review.

    PubMed

    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).

  19. Transforming growth factor alpha and epidermal growth factor levels in normal human gastrointestinal mucosa.

    PubMed Central

    Cartlidge, S. A.; Elder, J. B.

    1989-01-01

    Acid soluble proteins from 23 samples of normal human gastrointestinal mucosa derived from four normal adult organ donors were extracted and subjected to specific radiommunoassays for transforming growth factor alpha (TGF alpha) and urogastrone epidermal growth factor (URO-EGF). All tissues were found to contain immunoreactive TGF alpha and levels ranged from 57 to 4,776 pg-1 wet weight of tissue. Although levels varied between tissue donors, the distribution of TGF alpha throughout the gastrointestinal tract appeared similar in all cases. URO-EGF levels were much lower (0-216 pg g-1 wet weight). TGF alpha levels in extracts of gastrointestinal mucosa from a 7-year-old female donor were higher and the observed distribution was markedly different from adult levels. URO-EGF was not detected in mucosal or submucosal tissue extracts from this patient. Further studies in juveniles are indicated. PMID:2803941

  20. Immune heparin-induced thrombocytopenia resulting from preceding exposure to heparin catheter flushes.

    PubMed

    Muslimani, Alaa A; Ricaurte, Basma; Daw, Hamed A

    2007-07-01

    Immune heparin-induced thrombocytopenia (HIT) is a life-threatening adverse effect of heparin. It can result from any type of heparin exposure and by any route of administration; however only a few cases are reported after exposures to small quantities of heparin from catheter flushes. The major clinical problem associated with HIT is thrombosis. Early detection and institution of alternative, non-heparin anticoagulation are important. We report a patient with HIT associated with use of therapeutic-dose unfractionated heparin in whom immune sensitization to heparin was triggered by two 500-unit exposure to UFH associated with intravascular catheter flushing for antineoplastic chemotherapy in a patient with colon adenocarcinoma.

  1. Novel cystogenic role of basic fibroblast growth factor in developing rodent kidneys.

    PubMed

    Li, Zhuangwu; Jerebtsova, Marina; Liu, Xue-Hui; Tang, Pingtao; Ray, Patricio E

    2006-08-01

    Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor that is accumulated in human dysplastic and cystic renal diseases. Previous studies have shown that bFGF can modulate the growth of developing renal tubules; however, its role in the pathogenesis of renal cyst formation is not clearly understood. Here, we tested the hypothesis that overexpression of bFGF in developing rodent kidneys induces cyst formation in vivo. We used two different adenoviral-mediated gene-transferring approaches to overexpress bFGF in developing rodent kidneys. Initially, metanephric kidney (MK) explants harvested from embryonic day 15 Sprague-Dawley rats were infected with adenoviral vectors (rAd) encoding human bFGF or LacZ genes and transplanted under the renal capsule of adult female rats. Subsequently, to determine whether bFGF could induce renal cysts in developing kidneys with an intact renal collecting system, we injected rAd-bFGF or LacZ vectors in the retroorbital plexus of newborn mice. Basic FGF induced a more efficient integration of the MK explants into the host kidneys and increased the vascularization and proliferation of developing tubules, leading to tubular dilatation and rapid formation of renal cysts. In addition, we successfully expressed human bFGF in the kidney of newborn mice in vivo and induced tubular dilatation and renal cysts. In contrast, mice injected with rAd-lacZ did not develop tubular dilatation or renal cysts. To the best of our knowledge, these experiments show for the first time that overexpression of bFGF in developing rodent kidneys can induce the formation of renal cysts in vivo.

  2. Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6.

    PubMed

    Volz, Yvonne; Koschut, David; Matzke-Ogi, Alexandra; Dietz, Marina S; Karathanasis, Christos; Richert, Ludovic; Wagner, Moritz G; Mély, Yves; Heilemann, Mike; Niemann, Hartmut H; Orian-Rousseau, Véronique

    2015-06-29

    CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs.

  3. [Growth factors in human tooth development].

    PubMed

    Bellone, C; Barni, T; Pagni, L; Balboni, G C; Vannelli, G B

    1990-03-01

    Our research concerns the immunohistochemical localization of EGF and IGF-I receptors in the tooth germ, using monoclonal antibodies. The results show that in the early phases of human tooth development EGF and IGF-I receptors are present. At bud stage both receptors are localized at dental laminae level, in some epithelial cells of the tooth bud and in some mesenchymal cells. At cap stage the receptors are present in the outer and inner enamel epithelium, and in some cells of stellate reticulum. As far as concerns the mesenchymal cells, some cells of dental papilla in contact with enamel organ, are intensely positive. The immunopositivity is present also in some mesenchymal cells at follicular level. At late cap stage and at early bell stage receptors are not present at inner enamel epithelium level but they can be detectable in the mesenchyma of dental papilla and in some cells of the follicle. On the basis of these results it may be hypothesized that EGF and IGF-I can act as growth factors in the modulation of cellular proliferation and differentiation during the human tooth morphogenesis. Moreover, it is possible that these substances can play a role in the mesenchymal-epithelial interaction in the developing human tooth.

  4. Transforming growth factor beta1 and aldosterone

    PubMed Central

    Matsuki, Kota; Hathaway, Catherine K.; Chang, Albert S.; Smithies, Oliver; Kakoki, Masao

    2016-01-01

    Purpose of review It is well established that blocking renin-angiotensin II-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. Recent findings TGFbeta1 suppresses adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps from 10% to 300% normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10 % hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. Summary These results identify TGFbeta signaling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism as well as assist in the development of novel therapeutic strategies for hypertension. PMID:25587902

  5. Vascular Endothelial Growth Factor in Eye Disease

    PubMed Central

    Penn, J.S.; Madan, A.; Caldwell, R.B.; Bartoli, M.; Caldwell, R.W.; Hartnett, M.E.

    2012-01-01

    Collectively, angiogenic ocular conditions represent the leading cause of irreversible vision loss in developed countries. In the U.S., for example, retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration are the principal causes of blindness in the infant, working age and elderly populations, respectively. Evidence suggests that vascular endothelial growth factor (VEGF), a 40 kDa dimeric glycoprotein, promotes angiogenesis in each of these conditions, making it a highly significant therapeutic target. However, VEGF is pleiotropic, affecting a broad spectrum of endothelial, neuronal and glial behaviors, and confounding the validity of anti-VEGF strategies, particularly under chronic disease conditions. In fact, among other functions VEGF can influence cell proliferation, cell migration, proteolysis, cell survival and vessel permeability in a wide variety of biological contexts. This article will describe the roles played by VEGF in the pathogenesis of retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the rationales for targeting other VEGF-related modulators of angiogenesis. PMID:18653375

  6. Nerve growth factor actions on the brain

    SciTech Connect

    Martinez, H.J.

    1989-01-01

    We examined the effect of the trophic protein, nerve growth factor (NGF), on cultures of fetal rat neostriatum and basal forebrain-medial septal area (BF-MS) to define its role in brain development. Treatment of cultures with NGF resulted in an increase in the specific activity of the cholinergic enzyme choline acetyltransferase (CAT) in both brain areas. CAT was immunocytochemically localized to neurons. In the BF-MS, NGF treatment elicited a marked increase in staining intensity and an apparent increase in the number of CAT-positive neurons. Moreover, treatment of BF-MS cultures with NGF increased the activity of acetylcholinesterase, suggesting that the cholinergic neuron as a whole was affected. To begin defining mechanisms of action of NGF in the BF-MS, we detected NGF receptors by two independent methods. Receptors were localized to two different cellular populations: neuron-like cells, and non-neuron-like cells. Dissociation studies with ({sup 125}I)NGF suggested that high affinity receptors were localized to the neuron-like population. Only low-affinity receptors were localized to the non-neuron-like cells. Moreover, employing combined immunocytochemistry and ({sup 125}I)NGF autoradiography, we detected a subpopulation of CAT-containing neutrons that exhibited high-affinity binding. Unexpectedly, a gamma-aminobutyric acid (GABA)-containing cell group also expressed high affinity binding. However, only subsets of cholinergic or GABA neurons expressed high-affinity biding, suggesting that these transmitter populations are composed of differentially response subpopulations.

  7. Nerve growth factor enhances sleep in rabbits.

    PubMed

    Takahashi, S; Krueger, J M

    1999-04-02

    Nerve growth factor (NGF) elicits rapid-eye-movement sleep (REMS) in cats. Removal of NGF receptor-positive cholinergic basal forebrain neurons inhibits REMS in rats. The aim of the present study was to determine the effects of NGF on sleep and brain temperature (Tbr) in rabbits. Male rabbits were implanted with electroencephalograph (EEG) electrodes, a brain thermistor and an intraventricular (i.c.v.) guide cannula. Rabbits received human beta-NGF i.c.v. (0.01, 0.1, 1.0 or 10 microg] and on a separate day, 25 microl pyrogen-free saline i.c.v. as control. EEG and Tbr were recorded for 23 h after injections. The highest two doses of NGF increased both non-REMS and REMS across the 23-h recording period. REMS was enhanced dose-dependently. Tbr was not affected by any dose of NGF. These results suggest that NGF is involved in both REMS and non-REMS regulation.

  8. [Epidermal growth factor, innovation and safety].

    PubMed

    Esquirol Caussa, Jordi; Herrero Vila, Elisabeth

    2015-10-05

    Bioidentical recombinant human epidermal growth factor (rhEGF) is available in concentrations and purity suitable for therapeutic use in long time stable formulations. Beneficial effects in several skin pathologies and lesions have been reported (traumatic and surgical wound healing, laser induced wounds, abnormal scars, keloids, radiation or chemotherapy induced dermatitis, post inflammatory hyperpigmentation or for skin aging damage repairing) and also may be considered for the treatment of several oropharingeal and high gastroesophageal tract mucosa diseases (mouth sores, pharyngeal fistulas, ulcers), and several corneal or conjunctive mucosa lesions. rhEGF has not shown any important side or collateral effects in humans or in laboratory experimentation animals, showing optimal tolerability and safety with continuous use for months. Compounding gives advantages of versatility, individualization, personalization, molecular stability, safety and effectiveness in ideal conditions, showing good tissue penetration, both on intact skin and skin lesions that expose the lower planes to the surface. rhEGF compounds can be considered for prevention or as a treatment of diverse skin and mucosa diseases and conditions through compounding preparations.

  9. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity.

    PubMed

    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li

    2016-04-22

    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  10. Low molecular weight heparins and heparinoids.

    PubMed

    Eikelboom, John W; Hankey, Graeme J

    2002-10-07

    Several low molecular weight (LMW) heparin preparations, including dalteparin, enoxaparin and nadroparin, as well as the heparinoid danaparoid sodium, are approved for use in Australia. LMW heparins are replacing unfractionated heparin for the prevention and treatment of venous thromboembolism and the treatment of non-ST-segment-elevation acute coronary syndromes. The advantages of LMW heparins over unfractionated heparin include a longer half-life (allowing once-daily or twice-daily subcutaneous dosing), high bioavailability and predictable anticoagulant response (avoiding the need for dose adjustment or laboratory monitoring in most patients), and a low risk of heparin-induced thrombocytopenia and osteoporosis. Laboratory monitoring of LMW heparin therapy should be considered in newborns and children, patients with renal impairment, those who are pregnant, and those at the extremes of bodyweight (eg, < 40 kg or > 100 kg). LMW heparins should: be avoided or used with caution in patients undergoing neuraxial anaesthesia, owing to the potential for epidural haematoma formation; not be used (ie, are contraindicated) in patients with immune heparin-induced