-
[show abstract]
[hide abstract]
ABSTRACT: Endothelial cells are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways. J. Cell. Physiol. © 2011 Wiley-Liss, Inc.
Journal of Cellular Physiology 09/2011; · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Alpha5beta1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha5beta1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha5beta1 integrin also supports that integrin is a proteoglycan. Also, cells grown with xyloside adhered on fibronectin with no alteration in alpha5beta1 integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha5beta1 integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha5beta1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha5beta1 integrin are involved in cellular migration on fibronectin.
Biochemistry and Cell Biology 08/2009; 87(4):677-86. · 2.67 Impact Factor
-
Edvaldo S. Trindade,
Constance Oliver,
Maria C. Jamur, Hugo A.O. Rocha,
Célia R.C. Franco,
Rodrigo I. Bouças,
Thais R. Jarrouge,
Maria A.S. Pinhal,
Ivarne L.S. Tersariol,
Tiago C. Gouvêa,
Carl P. Dietrich,
Helena B. Nader
[show abstract]
[hide abstract]
ABSTRACT: Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent KD of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM. J. Cell. Physiol. 217: 328–337, 2008. © 2008 Wiley-Liss, Inc.
Journal of Cellular Physiology 10/2008; 217(2):328 - 337. · 3.87 Impact Factor
-
Edvaldo S Trindade,
Constance Oliver,
Maria C Jamur, Hugo A O Rocha,
Célia R C Franco,
Rodrigo I Bouças,
Thais R Jarrouge,
Maria A S Pinhal,
Ivarne L S Tersariol,
Tiago C Gouvêa,
Carl P Dietrich,
Helena B Nader
[show abstract]
[hide abstract]
ABSTRACT: Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.
Journal of Cellular Physiology 07/2008; 217(2):328-37. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.
Journal of Cellular Physiology 07/2008; 217(2):360-6. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The green algae of the genus Codium have recently been demonstrated to be an important source of sulfated galactans from the marine environment. Here, a sulfated galactan was isolated from the species Codium isthmocladum and its structure was studied by a combination of chemical analyses and NMR spectroscopy. Two fractions (SG 1, approximately 14 kDa, and SG 2, approximately 20 kDa) were derived from this highly polydisperse and heterogeneous polysaccharide. Both exhibited similar structures in (1)H 1D NMR spectra. The structural features of SG 2 and its desulfated derivative were analyzed by COSY, TOCSY, DEPT-HSQC, HSQC, and HMBC. This sulfated galactan is composed preponderantly of 4-sulfated, 3-linked beta-D-galactopyranosyl units. In minor amounts, it is sulfated and glycosylated at C-6. Pyruvate groups are also found, forming five-membered cyclic ketals as 3,4-O-(1'carboxy)-ethylidene-beta-D-galactose residues. A comparison of sulfated galactans from different marine taxonomic groups revealed similar backbones of 3-beta-D-Galp-1.
Glycobiology 04/2008; 18(3):250-9. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Geastrum saccatum a mushroom, native to Brazil, is produced under natural conditions in the unexplored reserve of Mata da Estrela-RN. This species has curative properties for eye infections and diseases such as asthma. The tissues of this mushroom contain carbohydrates, proteins, lipids, moisture and ashes in amounts of 42.3%, 37.05%, 9.01, 1.4% and 10.2%, respectively. An extract from this mushroom was characterized by chemical analyses and (13)C and (1)H NMR spectroscopy. It contains high amount of glucose and traces of galactose. The signal appearing at 103.5 ppm was assigned to C1 of beta-glucose. The signals observed between 20 and 40 ppm suggest the presence of a glucan-protein compound. This glucan inhibited the lipid peroxidation at the dose of 0.27 mg/mL (59.1%) and it can protect cells against oxidative stress by scavenging of the hydroxyl (77%) and superoxide (88.4%) radicals at 0.27 mg/mL. The glucan (30 mg/kg) reduces the polymorphonuclear cell migration (57.6%). The ear edema induced by croton oil was inhibited by glucan (60.4% at 10 mg/kg) and by its association with diclofenac (5 mg/kg) (89.2%) or L-NAME (60 mg/kg) (86.23%). Histological analyses of the ear edema induced by croton oil in the presence of glucan (10, 30 or 50 mg/kg) showed a reduced degree of the polymorphonuclear cell migration. We concluded that the glucan has antioxidant, and antiinflammatory properties as well as its antiinflammatory effect are mediated by inhibition of both nitric oxide synthase (NOS) and cyclooxygenase (COX).
International Immunopharmacology 10/2007; 7(9):1160-9. · 2.38 Impact Factor
-
Adriana M Nakahata,
Norlene R Bueno, Hugo A O Rocha,
Célia R C Franco,
Roger Chammas,
Clovis R Nakaie,
Miriam G Jasiulionis,
Helena B Nader,
Lucimeire A Santana,
Misako U Sampaio,
Maria Luiza V Oliva
[show abstract]
[hide abstract]
ABSTRACT: Purified from Bauhinia rufa seeds, BrTI is a Kunitz proteinase inhibitor that contains the RGD sequence. BrTI inhibits trypsin (K(iapp) 2.9 nM) and human plasma kallikrein (K(iapp) 14.0 nM) but not other related enzymes. The synthetic peptide YLEPVARGDGGLA-NH(2) (70 microM) inhibited the adhesion to fibronectin of B16F10 (high-metastatic B16 murine mouse melanoma cell line) and of Tm5 (murine melanoma cell lines derived from a non-tumorigenic lineage of pigmented murine melanocytes, melan-a). YLEPVARGEGGLA-NH(2) in which Asp(9) was changed into Glu does not affect the cell attachment. Moreover, this peptide was functional only when the sequence present in the native protein was preserved, since YLIPVARGDGGLA-NH(2) in which Glu(3) was changed into Ile does not interfere with B16F10 and was less effective on Tm5 cell line adhesion. Neither YLEPVARGDGGLA-NH(2), YLIPVARGDGGLA-NH(2) or YLEPVARGEGGLA-NH(2) inhibit the interaction of RAEC (endothelial cell line from rabbit aorta) with fibronectin.
International Journal of Biological Macromolecules 01/2007; 40(1):22-9. · 2.45 Impact Factor
-
Hugo A O Rocha,
Fábio A Moraes,
Edvaldo S Trindade,
Célia R C Franco,
Ricardo J S Torquato,
Silvio S Veiga,
Ana P Valente,
Paulo A S Mourão,
Edda L Leite,
Helena B Nader,
Carl P Dietrich
[show abstract]
[hide abstract]
ABSTRACT: The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked beta-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked alpha-fucose and one or two nonsulfated, 4-linked beta-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.
Journal of Biological Chemistry 01/2006; 280(50):41278-88. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The correlation between structure, anticlotting, antithrombotic and hemorrhagic activities of heparin, heparan sulfate, low molecular weight heparins and heparin-like compounds from various sources that are in used in clinical practice or under development is briefly reviewed. Heparin-like molecules composed exclusively of iduronic acid 2-O-sulfate residues have weak anticlotting activities, whereas molecules that contain both iduronic acid 2-O sulfate, iduronic acid and small amounts of glucuronic acid, such as heparin, or mixed amounts of glucuronic and iduronic acids (mollusk heparins) possess high anticlotting and anti-Xa activities. These results also suggest that a proper combination of these elements might produce a strong antithrombotic agent. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate derived from bovine pancreas and a sulfated fucan from brown algae have a potent antithrombotic activity in arterial and venous thrombosis model "in vivo" with a negligible activity upon the serine-proteases of the coagulation cascade "in vitro". These and other results led to the hypothesis that antithrombotic activity of heparin and other antithrombotic agents is due at least in part by their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate. All the antithrombotic agents derived from heparin and other heparinoids have hemorrhagic activity. Exceptions to this are a heparan sulfate from bovine pancreas and a sulfated fucan derived from brown algae, which have no hemorrhagic activity but have high antithrombotic activities "in vivo". Once the structure of these compounds are totally defined it will be possible to design an ideal antithrombotic.
Current Pharmaceutical Design 02/2004; 10(9):951-66. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Geastrum saccatum a mushroom, native to Brazil, is produced under natural conditions in the unexplored reserve of Mata da Estrela-RN. This species has curative properties for eye infections and diseases such as asthma. The tissues of this mushroom contain carbohydrates, proteins, lipids, moisture and ashes in amounts of 42.3%, 37.05%, 9.01, 1.4% and 10.2%, respectively. An extract from this mushroom was characterized by chemical analyses and 13C and 1H NMR spectroscopy. It contains high amount of glucose and traces of galactose. The signal appearing at 103.5 ppm was assigned to C1 of β-glucose. The signals observed between 20 and 40 ppm suggest the presence of a glucan–protein compound. This glucan inhibited the lipid peroxidation at the dose of 0.27 mg/mL (59.1%) and it can protect cells against oxidative stress by scavenging of the hydroxyl (77%) and superoxide (88.4%) radicals at 0.27 mg/mL. The glucan (30 mg/kg) reduces the polymorphonuclear cell migration (57.6%). The ear edema induced by croton oil was inhibited by glucan (60.4% at 10 mg/kg) and by its association with diclofenac (5 mg/kg) (89.2%) or l-NAME (60 mg/kg) (86.23%). Histological analyses of the ear edema induced by croton oil in the presence of glucan (10, 30 or 50 mg/kg) showed a reduced degree of the polymorphonuclear cell migration. We concluded that the glucan has antioxidant, and antiinflammatory properties as well as its antiinflammatory effect are mediated by inhibition of both nitric oxide synthase (NOS) and cyclooxygenase (COX).
International Immunopharmacology.
