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Chronic kidney disease (CKD) is associated with high mortality rates, mainly due to cardiovascular diseases (CVD). Uremia has been considered a relevant risk factor for CVD in CKD patients, since uremic toxins (UTs) promote systemic and vascular inflammation, oxidative stress and senescence. Here, we demonstrate that uremic toxins indoxyl sulfate (IxS), p-cresyl sulfate (pCS) and indole acetic acid (IAA) are incorporated by human endothelial cells and inhibit the autophagic flux, demonstrated by cellular p62 accumulation. Moreover, isolated and mixed UTs impair the lysosomal stage of autophagy, as determined by cell imaging of the mRFP-GFP-LC3 protein. Endothelial cells exposed to UTs display accumulation of carbonylated proteins and increased sensitivity to hydrogen peroxide. Rapamycin, an autophagy activator which induces both autophagosome formation and clearance, prevented these effects. Collectively, our findings demonstrate that accumulation of oxidized proteins and enhanced cell sensitivity to hydrogen peroxide are consequences of impaired autophagic flux. These data provide evidence that UTs-induced impaired autophagy may be a novel contributor to endothelial dysfunction.

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Two sulfated polysaccharides (SPs), F2 and F3, isolated from Codium isthmocladum were found to contain galactose, sulfate, and pyruvate. The apparent molecular weights of F2 and F3 were determined to be 62 and 61 kDa, respectively. NMR spectroscopy combined with chemical analysis showed that F2 and F3 have the same structural features. However, F3 showed higher sulfate/sugar ratio (1/2.6) than F2 (1/4). F2 and F3 are essentially (1 → 3)-ß-D-galactans with some branching at C6. Pyruvylation occurs at O3 and O4, forming 3,4-O-(1-carboxyethylidene)-ß-D-Galp residues; some of these pyruvylated residues contain sulfate groups at C6. Some non-branching residues contain sulfate at C4. None of the SPs exhibited antioxidant activity. MTT results indicated that 1 mg/mL of both SPs about 40% of PANC-1 cell viability. At 10 µg/mL, F2 and F3 had 1.7-fold longer clotting times compared to that of Clexane® at the same concentration. The higher sulfate content of F3 is not a determining factor for pharmacological activities of galactans, considering that both F2 and F3 exerted the effects.

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Endothelial dysfunction in uremia can result in cell-to-cell junction loss and increased permeability, contributing to cardiovascular diseases (CVD) development. This study evaluated the impact of the uremic milieu on endothelial morphology and cell junction's proteins. We evaluated (i) serum levels of inflammatory biomarkers in a cohort of chronic kidney disease (CKD) patients and the expression of VE-cadherin and Zonula Occludens-1 (ZO-1) junction proteins on endothelial cells (ECs) of arteries removed from CKD patients during renal transplant; (ii) ECs morphology in vitro under different uremic conditions, and (iii) the impact of uremic toxins p-cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) as well as of total uremic serum on VE-cadherin and ZO-1 gene and protein expression in cultured ECs. We found that the uremic arteries had lost their intact and continuous endothelial morphology, with a reduction in VE-cadherin and ZO-1 expression. In cultured ECs, both VE-cadherin and ZO-1 protein expression decreased, mainly after exposure to Pi and uremic serum groups. VE-cadherin mRNA expression was reduced while ZO-1 was increased after exposure to PCS, IS, Pi, and uremic serum. Our findings show that uremia alters cell-to-cell junctions leading to an increased endothelial damage. This gives a new perspective regarding the pathophysiological role of uremia in intercellular junctions and opens new avenues to improve cardiovascular outcomes in CKD patients.

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INTRODUCTION: p-cresol (PC) and p-cresyl sulfate (PCS) are responsible for many of the uremia clinical consequences, such as atherosclerosis in Chronic Kidney Disease (CKD) patients. OBJECTIVES: We investigate the in vitro impact of PC and PCS on monocyte chemoattractant protein-1 (MCP-1) expression via NF-kappa B (NF-κB) p65 in VSMC. METHODS: PCS was synthesized by PC sulfatation. VSMC were extracted by enzymatic digestion of umbilical cord vein and characterized by immunofluorescence against α-actin antibody. The cells were treated with PC and PCS at their normal (n), uremic (u) and maximum uremic concentrations (m). Cell viability was assessed by MTT. MCP-1 expression was investigated by ELISA in cells supernatants after toxins treatment with or without the NF-κB p65 inhibitor. RESULTS: There was no significant difference in cell viability after toxins treatment for all concentrations tested. There was a significant increase in MCP-1 expression in cells treated with PCu and PCm (p < 0.001) and PCSn, PCSu and PCSm (p < 0.001), compared with the control. When VSMC were treated with the NF-κB p65 inhibitor plus PCu and PCm, there was a significant decrease in MCP-1 production (p < 0.005). This effect was not observed with PCS. CONCLUSIONS: VSMC are involved in atherosclerosis lesion formation and production of MCP-1, which contributes to the inflammatory response initiation. Our results suggest that PC mediates MCP-1 production in VSMC, probably through NF-κB p65 pathway, although we hypothesize that PCS acts through a different subunit pathway since NF-κB p65 inhibitor was not able to inhibit MCP-1 production.

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RESUMO Introdução: p-cresol (PC) e p-cresil sulfato (PCS) são responsáveis por muitas das consequências clínicas uremia, tais como a aterosclerose em pacientes com Doença Renal Crônica (DRC). Objetivos: No presente trabalho, investigamos in vitro o impacto de PC e PCS na expressão da quimiocina monocyte chemoattractant protein-1 (MCP-1) via NF-kappa B (NF-κB) p65 em VSMC. Métodos: O PCS foi sintetizado por sulfatação do PC. As VSMC foram extraídas por digestão enzimática da veia do cordão umbilical e caracterizadas por imunofluorescência através do anticorpo α-actina. As células foram tratadas com PC e PCS em suas concentrações normal (n), urêmica (u) e urêmica máxima (m). A viabilidade celular foi avaliada pelo ensaio de MTT. A expressão de MCP-1 foi investigada por ELISA em sobrenadantes de células após o tratamento com as toxinas, com ou sem o inibidor de NF-κB p65. Resultados: Não houve diferença significativa na viabilidade das células após o tratamento com toxinas para todas as concentrações testadas. Houve um aumento significativo na expressão de MCP-1 em células tratadas com PCu e PCm (p < 0,001) e PCSn, PCSu e PCSm (p < 0,001), em comparação com o controle. Quando as VSMC foram tratadas com o inibidor de NF-κB p65 mais PCu e PCm, houve uma diminuição significativa na produção de MCP-1 (p < 0,005). Este efeito não foi observado com PCS. Conclusões: VSMC estão envolvidas na formação da lesão aterosclerótica e produção de MCP-1, o que contribui para o início da resposta inflamatória. Os nossos resultados sugerem que a PC medeia a produção de MCP-1 em VSMC, provavelmente através da via NF-κB p65 e que PCS atue através de uma subunidade diferente da via, uma vez que o inibidor da porção p65 não foi capaz de inibir a produção de MCP-1.

ABSTRACT Introduction: p-cresol (PC) and p-cresyl sulfate (PCS) are responsible for many of the uremia clinical consequences, such as atherosclerosis in Chronic Kidney Disease (CKD) patients. Objectives: We investigate the in vitro impact of PC and PCS on monocyte chemoattractant protein-1 (MCP-1) expression via NF-kappa B (NF-κB) p65 in VSMC. Methods: PCS was synthesized by PC sulfatation. VSMC were extracted by enzymatic digestion of umbilical cord vein and characterized by immunofluorescence against α-actin antibody. The cells were treated with PC and PCS at their normal (n), uremic (u) and maximum uremic concentrations (m). Cell viability was assessed by MTT. MCP-1 expression was investigated by ELISA in cells supernatants after toxins treatment with or without the NF-κB p65 inhibitor. Results: There was no significant difference in cell viability after toxins treatment for all concentrations tested. There was a significant increase in MCP-1 expression in cells treated with PCu and PCm (p < 0.001) and PCSn, PCSu and PCSm (p < 0.001), compared with the control. When VSMC were treated with the NF-κB p65 inhibitor plus PCu and PCm, there was a significant decrease in MCP-1 production (p < 0.005). This effect was not observed with PCS. Conclusions: VSMC are involved in atherosclerosis lesion formation and production of MCP-1, which contributes to the inflammatory response initiation. Our results suggest that PC mediates MCP-1 production in VSMC, probably through NF-κB p65 pathway, although we hypothesize that PCS acts through a different subunit pathway since NF-κB p65 inhibitor was not able to inhibit MCP-1 production.

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Based on considered achievements of the last 25 years, specific combinations of sulfation patterns and glycosylation types have been proved to be key structural players for the anticoagulant activity of certain marine glycans. These conclusions were obtained from comparative and systematic analyses on the structure-anticoagulation relationships of chemically well-defined sulfated polysaccharides of marine invertebrates and red algae. These sulfated polysaccharides are known as sulfated fucans (SFs), sulfated galactans (SGs) and glycosaminoglycans (GAGs). The structural combinations necessary for the anticoagulant activities are the 2-sulfation in α-L-SGs, the 2,4-di-sulfation in α-L-fucopyranosyl units found as composing units of certain sea-urchin and sea-cucumber linear SFs, or as branching units of the fucosylated chondroitin sulfate, a unique GAG from sea-cucumbers. Another unique GAG type from marine organisms is the dermatan sulfate isolated from ascidians. The high levels of 4-sulfation at the galactosamine units combined with certain levels of 2-sulfation at the iduronic acid units is the anticoagulant structural requirements of these GAGs. When the backbones of red algal SGs are homogeneous, the anticoagulation is proportionally dependent of their sulfation content. Finally, 4-sulfation was observed to be the structural motif required to enhance the inhibition of thrombin via heparin cofactor-II by invertebrate SFs.

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The structures of two sulfated xylomannans extracted from the red alga Nemalion helminthoides were determined. These two fractions plus a sulfated mannan, isolated from the same alga and whose structure was previously reported, were subjected to chemical modification. The mannan was oversulfated with SO(3)-pyridine in dimethyl sulfoxide at 60 °C during two and three hours and the xylomannans were subjected to Smith degradation in order to eliminate xylose side-chains. Structural analysis of all derivatives was carried out by methylation analysis and (13)C NMR spectroscopy. Antiviral activity against herpes simplex virus type 1 and 2, and dengue virus type 2 of native and modified mannans and xylomannans was estimated. Anticoagulant effect of the active fractions was also determined.

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Nemalion helminthoides, collected in the Argentine South Atlantic coast, was extracted with hot water and the crude product fractionated using cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration and seven fractions were separated and analyzed. Structural analysis of the main fractions, those soluble in 3.0 and 4.0 M NaCl (yields 21.0% and 13.8%, respectively) and those insoluble in 4.0 M NaCl (yield 20.0%), indicated that this seaweed biosynthesizes (1-->3)-linked alpha-D-mannans that are sulfated at positions 4 and 6. Three mannan fractions comprising considerable amounts of xylose were also isolated in very low total yield (2.0%). The fractions that were soluble in 3.0 and 4.0 M NaCl showed low antiherpetic activity whereas this activity was considerable for the fraction solubilized in 2.0 M NaCl (yield 0.5%) which contained single stubs of beta-D-xylose. A xylan, soluble in cetrimide solution, containing (1-->3, 1-->4)-linked beta-D-xylose residues, was also isolated in minor amount.

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Novel compounds presenting anticoagulant activity, such as sulfated polysaccharides, open new perspectives in medicine. Elucidation of the molecular mechanism behind this activity is desirable by itself, as well as because it allows for the design of novel compounds. In the present study, we investigated the action of an algal sulfated galactan, which potentiates alpha-thrombin inactivation by antithrombin. Our results indicate the following: 1) both the sulfated galactan and heparin potentiate protease inactivation by antithrombin at similar molar concentrations, however they differ markedly in the molecular size required for their activities; 2) this galactan interacts predominantly with exosite II on alpha-thrombin and, similar to heparin, catalyzes the formation of a covalent complex between antithrombin and the protease; 3) the sulfated galactan has a higher affinity for alpha-thrombin than for antithrombin. We propose that the preferred pathway of sulfated galactan-induced inactivation of alpha-thrombin by antithrombin starts with the polysaccharide binding to the protease through a high-affinity interaction. Antithrombin is then added to the complex and the protease is inactivated by covalent interactions. Finally, the antithrombin-alpha-thrombin covalent complex dissociates from the polysaccharide chain. This mechanism resembles the action of heparin with low affinity for antithrombin, as opposed to heparin with high affinity for serpin.

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The effects of the alkylaminoalkanethiosulfuric acids (AAATs), new schistosomicidal drugs, on Schistosoma mansoni ATP diphosphohydrolase isoforms, members of the NTPDase family, were analyzed. Pre-incubation of worm adult tegument with AAATs derivatives, with small apolar alkyl groups and an apolar alkane portion of 6 or 8 carbon atoms linked to the amino group, inhibited ATPase activity with a Ki 100-1000 microM. Little inhibition (20%) was observed when ADP was the substrate. The 2-[(tert-butyl)amino]-1-ethanethiosulfuric acid (100 microM) which has a less lipophilic structure, inhibited 28% ATPase and 12% ADPase activities, suggesting that the lipophilicity, although important, is not the only requisite for enzyme activity inhibition. The N-(sec-butyl)-2-bromo-1-octanaminium bromide, which contains a bromide atom instead of thiosulphate, inhibited <10% of the enzyme activity, suggesting the involvement of cysteine residue(s) from S. mansoni ATP diphosphohydrolase isoforms in a mixed disulfide formation. Treatment of parasite tegument with 5 mM iodoacetamide or 1 mM dithiothreitol protected ATPase and ADPase activities against inhibition by AAATs, corroborating the participation of disulfide interchange in the AAATs mechanism. Since S. mansoni ATP diphosphohydrolase isoforms and potato apyrase share structural similarities, the latter enzyme was also tested. ADPase activity from potato apyrase was inhibited by 55%, showing a higher sensitivity to 1 mM AAATs than that shown by ADPase activity from the tegument, while the ATPase activities from both samples showed similar inhibition levels. Furthermore, sulfhydryl reagents protected potato apyrase activity. Therefore, it is possible that both soluble S. mansoni ATP diphosphohydrolase and membrane-associated isoforms are targets for the AAATs.

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The aim of this article included the development and evaluation of the capacity of nanoemulsions to improve the activity of the novel schistosomicidal drug-2-(butylamino)-1-phenyl-1-ethanethiosulfuric acid (BphEA). BphEA is a compound with a poor solubility in water, which makes its application as a drug difficult. Nanoemulsion formulations presenting anionic (NANOSTOA, NANOST and NANOLP) and cationic (NANOSTE) interfacial charges were prepared to encapsulate BphEA. These formulations were characterized by the encapsulation rate, diameter, and zeta potential. NANOSTOA, NANOST, and NANOLP presented an entrapment efficiency and zeta potential of 18.7+/-1.8% and -33.6+/-1.2 mV; 20.5+/-3.0% and -31.5+/-5.7 mV; as well as 33.8+/-7.2% and -62.6+/-1.3 mV, respectively. NANOSTE presented an entrapment efficiency of 51.8+/-5.0% and a zeta potential of 25.7+/-3.9 mV. The mean droplet size (between 200 and 252 nm) and polydispersity index (between 0.158 and 0.294) were similar for all formulations. The stability study showed no alteration in these formulations' zeta potential and size. The in vitro schistosomicidal activity of BphEA was higher with the use of NANOSTE than with free BphEA. In addition, release studies revealed a good stability of NANOSTE containing BphEA in a biological medium. These results indicate that cationic nanoemulsions can represent an interesting delivery system for the pharmaceutical formulation of BphEA.

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Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca(2+) by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K(+) leading to an enhancement of the Ca(2+) transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca(2+) at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K(+) but not by Li(+) and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K(+). These data suggest that the regulation of the SERCA pump by K(+) and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.

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Two new sulfated polyhydroxylated steroidal xylosides, minutosides A (1) and B (2), together with the known pycnopodioside B (3), have been isolated from the brine shrimp active fraction of the ethanolic extract of the starfish Anasterias minuta. The structures have been elucidated by extensive 1D and 2D NMR as well as FABMS analysis and chemical methods. Compound 2 is the first example of a polyhydroxylated steroidal xyloside containing an amide function in the aglycon side chain. The three xylosides exhibited antifungal activity against Cladosporium cucumerinum and Aspergillus flavus.

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Aqueous extraction of gametophytic Schizymenia binderi afforded a polysaccharide composed of galactose and sulfate groups in a molar ratio of 1.0:0.89 together with uronic acids (6.8 wt%) and minor amounts of other neutral sugars. Alkali-treatment of the polysaccharide afforded a polysaccharide devoid of 3,6-anhydrogalactose. 13C NMR spectroscopy of the desulfated alkali-treated polysaccharide showed a backbone structure of alternating 3-linked beta-D-galactopyranosyl and 4-linked alpha-galactopyranosyl units that are predominantly of the D-configuration and partly of the L-configuration. Methylation, ethylation and NMR spectroscopic studies of the alkali-treated polysaccharide indicated that the sulfate groups are located mainly at positions O-2 of 3-linked beta-D-galactopyranosyl residue and at position O-3 of 4-linked-alpha-galactopyranosyl residues, the latter is partially glycosylated at position O-2. The sulfated galactan from S. binderi exhibited highly selective antiviral activity against Herpes simplex virus types 1 and 2, with selectivity indices (ratio cytotoxicity/antiviral activity) >1000 for all assayed virus strains. This compound was shown to interfere with the initial adsorption of viruses to cells.

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Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3',4'-tetrasulphate (QTS) and quercetin 3-acetyl-7,3',4'-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 microM, final concentration), and the platelet aggregation was induced by using adenosine 5'-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 microM) and Qc (250 microM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 microM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA2) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B2 (TxB2) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA2, as judged by TxB2 formation, in stimulated WP and a marked interference on the TxA2 receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.

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Marine alga is an abundant source of sulfated polysaccharides with potent anticoagulant activity. However, several attempts to identify the specific structural features in these compounds, which confer the biological activity, failed due to their complex, heterogeneous structure. We isolated and characterized several sulfated alpha-L-galactans and sulfated alpha-L-fucans from marine invertebrates. In contrast to the algal fucans and galactans, these invertebrate polysaccharides have a simple structure, composed of well-defined units of oligosaccharides. We employed two of these compounds to elucidate their structure-anticoagulant action relationship. Our results indicate that a 2-sulfated, 3-linked alpha-L-galactan, but not an alpha-L-fucan, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaces thrombin. Thus, the anticoagulant activity of sulfated galactan and sulfated fucan is not merely a consequence of their charge density. The interaction of these polysaccharides with coagulation cofactors and their target proteases are specific. Identification of specific structural requirements in sulfated galactans and sulfated fucans necessary for interaction with coagulation cofactors is an essential step for a more rational approach to develop new anticoagulant and antithrombotic drugs.

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We describe a variety of sulfated polysaccharides with regular and well-defined structures which are useful tools for elucidating structure/biological function relationship. Several of these compounds have anticoagulant and antithrombotic activities. The most studied and promising polysaccharide is a fucosylated chondroitin sulfate, composed of a chondroitin sulfate-like backbone, substituted at position 3 of the beta-D-glucuronic acid with heavily sulfated fucose side chains. The anticoagulant activity of this polysaccharide is mediated by both antithrombin and heparin cofactor II; it has antithrombotic activity when targeted at the intrinsic coagulation pathway.

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The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p -2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.

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