Heparanase is an endo-B-glucuronidase that is capable of degrading heparan sulfate chains of prot... more Heparanase is an endo-B-glucuronidase that is capable of degrading heparan sulfate chains of proteoglycans, generating a variety of bioactive molecules such as growth factors and chemotactic and angiogenic agents. The expression of heparanase was investigated in the peripheral blood mononuclear cell fraction (PBMC) of 30 patients with breast cancer and 20 healthy control women by reverse transcription -polymerase chain reaction (RT-PCR) and immunocytochemistry. PBMC samples from all breast cancer patients at study entry showed the expression of heparanase, whereas no expression was observed for healthy women. Immunocytochemistry analysis demonstrated that heparanase was expressed in lymphocytes of breast cancer PBMC. Throughout follow-up, heparanase expression by RT-PCR decreased significantly after surgery in patients treated with neoadjuvant chemotherapy (P = .002) and after tamoxifen treatment (P = .040), whereas it increased significantly with the advent of systemic metastasis (P = .027). In vitro, either serum from breast cancer patients or a medium originated from coculture experiments of MCF-7 cells and lymphocytes from healthy women stimulated heparanase expression in normal lymphocytes. The results suggest that there is a tumor-inducing effect on heparanase expression by lymphocytes present in the PBMCs of breast cancer patients, which depends, in turn, on the interaction between a tumor and normal lymphocytes. Neoplasia (2007) 9, 504 -510
Our goal was to demonstrate the in vivo tumor 19 specific accumulation of crotamine, a natural pe... more Our goal was to demonstrate the in vivo tumor 19 specific accumulation of crotamine, a natural peptide from the 20 venom of the South American rattlesnake Crotalus durissus 21 terrif icus, which has been characterized by our group as a cell 22 36
In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of m... more In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca 2+ signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 μM Gm increases the cytosolic Ca 2+ and induces membrane permeabilization after 30 min of treatment. Direct Ca 2+ measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca 2+ depletion, which in turn resulted in oscillatory mitochondrial Ca 2+ signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix mit Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca 2+ -dependent pathway involving Gm translocation into the cell, ER Ca 2+ depletion and disruption, mitochondrial Ca 2+ overload and oxidative stress.
The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but ... more The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca 2+ -heparin, in which a-L-iduronate-2-O-sulfate residues adopt 1 C 4 conformation preferentially, is a substrate, while Na + -heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates b-elimination by abstracting the C5 proton of the a-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.
The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sul... more The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]2-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and tri-sulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluorescence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.
Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of bot... more Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of both parasite and target cell. We have previously demonstrated the role of heparan sulfate proteoglycan in the attachment and invasion of T. cruzi in cardiomyocytes. Herein, we have isolated the T. cruzi heparin-binding proteins (HBP-Tc) and investigated the nature of cardiomyocyte heparan sulfate (HS)binding site to the parasite surface ligand. Two major heparin-binding proteins with molecular masses of 65.8 and 59 kDa were observed in total extract of amastigote and trypomastigote forms of T. cruzi. Hydrophobic [S 35 ]methionine labeled proteins eluted from heparin-sepharose affinity chromatography also revealed both proteins in trypomastigotes but only the 59 kDa is strongly recognized by biotin-conjugated glycosaminoglycans. Competition assays were performed to analyze the role of sulfated proteoglycans, including heparin, keratan sulfate and both acetylated and highly sulfated domains of heparan sulfate, in the recognition and invasion process of T. cruzi. Significant inhibitions of 84% and 35% in the percentage of infection were revealed after treatment of the parasites with heparin and the N-acetylated/ N-sulfated heparan sulfate domain, respectively, suggesting the important role of the glycuronic acid and NS glucosamine domain of the HS chain in the recognition of the HBP-Tc during the T. cruzi-cardiomyocyte interaction. r
Thermal stabilisation of native antithrombin-III (AT), determined using differential scanning flu... more Thermal stabilisation of native antithrombin-III (AT), determined using differential scanning fluorimetry, correlated with the anticoagulant activity of heparin and heparin-related saccharides, while similar conformational changes were induced in native AT by a variety of active and inactive heparin-related
Heparin and its derivatives are known to regulate a variety of pathophysiological events related ... more Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis.
Aim: Several noninvasive markers are being used to assess the structural liver damage in patients... more Aim: Several noninvasive markers are being used to assess the structural liver damage in patients with chronic hepatitis C (CHC). We evaluated the capacity of serum hyaluronic acid (HA), aspartate aminotransferase (AST)/ALT ratio, the AST to platelet ratio index (APRI) and g-glutamyltransferase (GGT) levels to predict the intensity of hepatic fibrosis in patients with CHC. Patients and methods: In a total of 206 hepatitis C virus RNA-positive biopsied patients, AST, ALT, GGT levels, platelet count and serum HA concentration were determined. The APRI was calculated as the ratio of AST to platelets. Results: HA levels were best correlated with disease stage (r 5 À 0.694; Po0.001). In the diagnosis of significant fibrosis (F2-F4), HA levels [AUC 5 0.879, 95% CI (0.832-0.927)] and APRI [AUC 5 0.824 (0.772-0.903)] were the markers with the best diagnostic accuracy. These parameters also best identified the presence of cirrhosis (F4), with an AUC of 0.908 (0.868-0.949) for HA and of 0.837 (0.772-0.903) for APRI. Conclusion: Serum HA was the parameter that alone presented the best diagnostic accuracy in the assessment of hepatic fibrosis in CHC. The APRI showed a better diagnostic sensitivity than GGT levels or the AST/ALT ratio. Its simple determination and low cost make this index a valid alternative for the noninvasive staging of CHC.
Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-rel... more Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin-like compound with striking anti-inflammatory action and negligible anticoagulant and hemorrhagic activities. Objectives: To investigate the role of this novel heparin-like compound in angiogenic processes. Methods and Results: The anti-angiogenic effect of this heparinoid in laser-induced CNV and in vitro models is reported. The compound binds to growth factors (FGF-2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2-D network organization in capillary-like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF-b1 in the choroidal tissue. The low content of 2-O-sulfate groups in this heparinoid may explain its potent anti-angiogenic effect. Conclusions: The properties of the shrimp heparinoid, such as potent antiangiogenic and anti-inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti-angiogenic effect of the heparinoid is proposed.
Journal of Biochemical and Biophysical Methods, 2003
Benzhydrylamine-resin (BHAR), a copolymer of styrene-divinylbenzene containing phenylmethylamine ... more Benzhydrylamine-resin (BHAR), a copolymer of styrene-divinylbenzene containing phenylmethylamine groups, used as solid support for peptide synthesis, was examined regarding physicochemical and anion exchanger chromatographic properties. The greater the ionic strength of the medium the poorer the solvation of beads. This effect is less pronounced the higher the amino group content of BHAR. The BHAR chromatographic behavior was compared with commercial cationic resins in columns of constant cation binding capacity. Three negatively charged heparan sulfate disaccharides were successfully purified in a 2.4 mmol/g BHAR that showed as good or better anion exchange performance than classical tertiary or quaternary amino group-containing resins. The BHAR chromatographic resin exclusion limit was estimated to be 30 kDa based on purification experiments of heparins of different molecular weight. D
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.co... more A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombo... more 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 378C, 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. Telephone Fax C1 REPRINT BILLING DEPARTMENT • • 111 RIVER STREET • • HOBOKEN, NJ 07030 FAX: (201) 748-7670
Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates t... more 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.
Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfat... more Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfate secreted by cultured endothelial cells. The increase in synthetic rate takes place immediately after exposure of the cells to heparin, affects only heparan sulfate, and i s specific for the endothelial cell. No stimulation by other glycosaminoglycans was observed. Analysis of the disaccharide products formed by the action of heparitinases reveals a higher degree of sulfation of the uronic acid residues in the heparan sulfate of cells exposed to heparin.
Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretor... more Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to rnonensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [ 3 5 S]-s~lfate/[~H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there i s a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that rnonensin affects the hexosarnine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.
Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and develop... more Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3b during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cellsubstratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering. J. Cell. Biochem. 109: 957-966,
Previous studies have shown that heparin induces vascular relaxation via integrin-dependent nitri... more Previous studies have shown that heparin induces vascular relaxation via integrin-dependent nitric oxide (NO)-mediated activation of the muscarinic receptor. The aim of this study was to identify the structural features of heparin that are necessary for the induction of vasodilatation. To address this issue, we tested heparin from various sources for their vasodilatation activities in the rat aorta ring. Structural and chemical characteristics of heparin, such as its molecular weight and substitution pattern, did not show a direct correlation with the vasodilation activity. Principal component analysis (PCA) of circular dichroism (CD), 1 H-nuclear magnetic resonance (NMR) and vasodilation activity measurements confirmed that there is no direct relationship between the physico-chemical nature and vasodilation activity of the tested heparin samples. To further understand these observations, unfractionated heparin (UFH) from bovine intestinal mucosa, which showed the highest relaxation effect, was chemically modified. Interestingly, non-specific O-and N-desulfation of heparin reduced its anticoagulant, antithrombotic, and antihemostatic activities, but had no effect on its ability to induce vasodilation. On the other hand, chemical reduction of the carboxyl groups abolished heparin-induced vasodilation and reduced the affinity of heparin toward the extracellular matrix (ECM). In addition, dextran and dextran sulfate (linear non-sulfated and highly sulfated polysaccharides, respectively) did not induce significant relaxation, showing that the vasodilation activity of polysaccharides is neither charge-dependent nor backbone unspecific. Our results suggest that desulfated heparin molecules may be used as vasoactive agents due to their low side effects.
N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate ... more N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. In this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. The phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. The peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. The peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. The discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis.
Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus duris... more Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.
Heparanase is an endo-B-glucuronidase that is capable of degrading heparan sulfate chains of prot... more Heparanase is an endo-B-glucuronidase that is capable of degrading heparan sulfate chains of proteoglycans, generating a variety of bioactive molecules such as growth factors and chemotactic and angiogenic agents. The expression of heparanase was investigated in the peripheral blood mononuclear cell fraction (PBMC) of 30 patients with breast cancer and 20 healthy control women by reverse transcription -polymerase chain reaction (RT-PCR) and immunocytochemistry. PBMC samples from all breast cancer patients at study entry showed the expression of heparanase, whereas no expression was observed for healthy women. Immunocytochemistry analysis demonstrated that heparanase was expressed in lymphocytes of breast cancer PBMC. Throughout follow-up, heparanase expression by RT-PCR decreased significantly after surgery in patients treated with neoadjuvant chemotherapy (P = .002) and after tamoxifen treatment (P = .040), whereas it increased significantly with the advent of systemic metastasis (P = .027). In vitro, either serum from breast cancer patients or a medium originated from coculture experiments of MCF-7 cells and lymphocytes from healthy women stimulated heparanase expression in normal lymphocytes. The results suggest that there is a tumor-inducing effect on heparanase expression by lymphocytes present in the PBMCs of breast cancer patients, which depends, in turn, on the interaction between a tumor and normal lymphocytes. Neoplasia (2007) 9, 504 -510
Our goal was to demonstrate the in vivo tumor 19 specific accumulation of crotamine, a natural pe... more Our goal was to demonstrate the in vivo tumor 19 specific accumulation of crotamine, a natural peptide from the 20 venom of the South American rattlesnake Crotalus durissus 21 terrif icus, which has been characterized by our group as a cell 22 36
In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of m... more In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca 2+ signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 μM Gm increases the cytosolic Ca 2+ and induces membrane permeabilization after 30 min of treatment. Direct Ca 2+ measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca 2+ depletion, which in turn resulted in oscillatory mitochondrial Ca 2+ signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix mit Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca 2+ -dependent pathway involving Gm translocation into the cell, ER Ca 2+ depletion and disruption, mitochondrial Ca 2+ overload and oxidative stress.
The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but ... more The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca 2+ -heparin, in which a-L-iduronate-2-O-sulfate residues adopt 1 C 4 conformation preferentially, is a substrate, while Na + -heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates b-elimination by abstracting the C5 proton of the a-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.
The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sul... more The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]2-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and tri-sulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluorescence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.
Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of bot... more Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of both parasite and target cell. We have previously demonstrated the role of heparan sulfate proteoglycan in the attachment and invasion of T. cruzi in cardiomyocytes. Herein, we have isolated the T. cruzi heparin-binding proteins (HBP-Tc) and investigated the nature of cardiomyocyte heparan sulfate (HS)binding site to the parasite surface ligand. Two major heparin-binding proteins with molecular masses of 65.8 and 59 kDa were observed in total extract of amastigote and trypomastigote forms of T. cruzi. Hydrophobic [S 35 ]methionine labeled proteins eluted from heparin-sepharose affinity chromatography also revealed both proteins in trypomastigotes but only the 59 kDa is strongly recognized by biotin-conjugated glycosaminoglycans. Competition assays were performed to analyze the role of sulfated proteoglycans, including heparin, keratan sulfate and both acetylated and highly sulfated domains of heparan sulfate, in the recognition and invasion process of T. cruzi. Significant inhibitions of 84% and 35% in the percentage of infection were revealed after treatment of the parasites with heparin and the N-acetylated/ N-sulfated heparan sulfate domain, respectively, suggesting the important role of the glycuronic acid and NS glucosamine domain of the HS chain in the recognition of the HBP-Tc during the T. cruzi-cardiomyocyte interaction. r
Thermal stabilisation of native antithrombin-III (AT), determined using differential scanning flu... more Thermal stabilisation of native antithrombin-III (AT), determined using differential scanning fluorimetry, correlated with the anticoagulant activity of heparin and heparin-related saccharides, while similar conformational changes were induced in native AT by a variety of active and inactive heparin-related
Heparin and its derivatives are known to regulate a variety of pathophysiological events related ... more Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis.
Aim: Several noninvasive markers are being used to assess the structural liver damage in patients... more Aim: Several noninvasive markers are being used to assess the structural liver damage in patients with chronic hepatitis C (CHC). We evaluated the capacity of serum hyaluronic acid (HA), aspartate aminotransferase (AST)/ALT ratio, the AST to platelet ratio index (APRI) and g-glutamyltransferase (GGT) levels to predict the intensity of hepatic fibrosis in patients with CHC. Patients and methods: In a total of 206 hepatitis C virus RNA-positive biopsied patients, AST, ALT, GGT levels, platelet count and serum HA concentration were determined. The APRI was calculated as the ratio of AST to platelets. Results: HA levels were best correlated with disease stage (r 5 À 0.694; Po0.001). In the diagnosis of significant fibrosis (F2-F4), HA levels [AUC 5 0.879, 95% CI (0.832-0.927)] and APRI [AUC 5 0.824 (0.772-0.903)] were the markers with the best diagnostic accuracy. These parameters also best identified the presence of cirrhosis (F4), with an AUC of 0.908 (0.868-0.949) for HA and of 0.837 (0.772-0.903) for APRI. Conclusion: Serum HA was the parameter that alone presented the best diagnostic accuracy in the assessment of hepatic fibrosis in CHC. The APRI showed a better diagnostic sensitivity than GGT levels or the AST/ALT ratio. Its simple determination and low cost make this index a valid alternative for the noninvasive staging of CHC.
Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-rel... more Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin-like compound with striking anti-inflammatory action and negligible anticoagulant and hemorrhagic activities. Objectives: To investigate the role of this novel heparin-like compound in angiogenic processes. Methods and Results: The anti-angiogenic effect of this heparinoid in laser-induced CNV and in vitro models is reported. The compound binds to growth factors (FGF-2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2-D network organization in capillary-like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF-b1 in the choroidal tissue. The low content of 2-O-sulfate groups in this heparinoid may explain its potent anti-angiogenic effect. Conclusions: The properties of the shrimp heparinoid, such as potent antiangiogenic and anti-inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti-angiogenic effect of the heparinoid is proposed.
Journal of Biochemical and Biophysical Methods, 2003
Benzhydrylamine-resin (BHAR), a copolymer of styrene-divinylbenzene containing phenylmethylamine ... more Benzhydrylamine-resin (BHAR), a copolymer of styrene-divinylbenzene containing phenylmethylamine groups, used as solid support for peptide synthesis, was examined regarding physicochemical and anion exchanger chromatographic properties. The greater the ionic strength of the medium the poorer the solvation of beads. This effect is less pronounced the higher the amino group content of BHAR. The BHAR chromatographic behavior was compared with commercial cationic resins in columns of constant cation binding capacity. Three negatively charged heparan sulfate disaccharides were successfully purified in a 2.4 mmol/g BHAR that showed as good or better anion exchange performance than classical tertiary or quaternary amino group-containing resins. The BHAR chromatographic resin exclusion limit was estimated to be 30 kDa based on purification experiments of heparins of different molecular weight. D
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.co... more A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombo... more 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 378C, 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. Telephone Fax C1 REPRINT BILLING DEPARTMENT • • 111 RIVER STREET • • HOBOKEN, NJ 07030 FAX: (201) 748-7670
Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates t... more 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.
Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfat... more Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfate secreted by cultured endothelial cells. The increase in synthetic rate takes place immediately after exposure of the cells to heparin, affects only heparan sulfate, and i s specific for the endothelial cell. No stimulation by other glycosaminoglycans was observed. Analysis of the disaccharide products formed by the action of heparitinases reveals a higher degree of sulfation of the uronic acid residues in the heparan sulfate of cells exposed to heparin.
Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretor... more Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to rnonensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [ 3 5 S]-s~lfate/[~H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there i s a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that rnonensin affects the hexosarnine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.
Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and develop... more Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3b during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cellsubstratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering. J. Cell. Biochem. 109: 957-966,
Previous studies have shown that heparin induces vascular relaxation via integrin-dependent nitri... more Previous studies have shown that heparin induces vascular relaxation via integrin-dependent nitric oxide (NO)-mediated activation of the muscarinic receptor. The aim of this study was to identify the structural features of heparin that are necessary for the induction of vasodilatation. To address this issue, we tested heparin from various sources for their vasodilatation activities in the rat aorta ring. Structural and chemical characteristics of heparin, such as its molecular weight and substitution pattern, did not show a direct correlation with the vasodilation activity. Principal component analysis (PCA) of circular dichroism (CD), 1 H-nuclear magnetic resonance (NMR) and vasodilation activity measurements confirmed that there is no direct relationship between the physico-chemical nature and vasodilation activity of the tested heparin samples. To further understand these observations, unfractionated heparin (UFH) from bovine intestinal mucosa, which showed the highest relaxation effect, was chemically modified. Interestingly, non-specific O-and N-desulfation of heparin reduced its anticoagulant, antithrombotic, and antihemostatic activities, but had no effect on its ability to induce vasodilation. On the other hand, chemical reduction of the carboxyl groups abolished heparin-induced vasodilation and reduced the affinity of heparin toward the extracellular matrix (ECM). In addition, dextran and dextran sulfate (linear non-sulfated and highly sulfated polysaccharides, respectively) did not induce significant relaxation, showing that the vasodilation activity of polysaccharides is neither charge-dependent nor backbone unspecific. Our results suggest that desulfated heparin molecules may be used as vasoactive agents due to their low side effects.
N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate ... more N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. In this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. The phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. The peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. The peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. The discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis.
Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus duris... more Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.
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Papers by Helena Nader