High Molecular Weight Dextran Sulfate Research Articles

Low-adhesion culture selection for human iPS cell-derived cardiomyocytes

Despite progress in generating cardiomyocytes from pluripotent stem cells, these populations often include non-contractile cells, necessitating cardiomyocyte selection for experimental purpose. This study explores a novel cardiomyocyte enrichment mechanism: low-adhesion culture selection. The cardiac cells derived from human induced pluripotent stem cells were subjected to a coating-free low-adhesion culture using bovine serum albumin and high molecular weight dextran sulfate. This approach effectively increased the population of cardiac troponin T-positive cardiomyocytes. Similar results were obtained with commercially available low-adhesion culture dishes. Subsequently, we accessed the practicality of selection of cardiomyocytes using this phenomenon by comparing it with established methods such as glucose-free culture and selection based on puromycin resistance genes. The cardiomyocytes enriched through low-adhesion culture selection maintained autonomous pulsation and responsiveness to beta-stimuli. Moreover, no significant differences were observed in the expression of genes related to subtype commitment and maturation when compared to other selection methods. In conclusion, cardiomyocytes derived from pluripotent stem cells were more low-adhesion culture resistant than their accompanying non-contractile cells, and low-adhesion culture is an alternative method for selection of pluripotent stem cell-derived cardiomyocytes.

Metal‐HisTag coordination for remote loading of very small quantities of biomacromolecules into PLGA microspheres

Challenges to discovery and preclinical development of long‐acting release systems for protein therapeutics include protein instability, use of organic solvents during encapsulation, specialized equipment and personnel, and high costs of proteins. We sought to overcome these issues by combining remote‐loading self‐healing encapsulation with binding HisTag protein to transition metal ions. Porous, drug‐free self‐healing microspheres of copolymers of lactic and glycolic acids with high molecular weight dextran sulfate and immobilized divalent transition metal (M2+) ions were placed in the presence of proteins with or without HisTags to bind the protein in the pores of the polymer before healing the surface pores with modest temperature. Using human serum albumin, insulin‐like growth factor 1, and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), encapsulated efficiencies of immunoreactive protein relative to nonencapsulation protein solutions increased from ~41%, ~23%, and ~9%, respectively, without Zn2+ and HisTags to ~100%, ~83%, and ~75% with Zn2+ and HisTags. These three proteins were continuously released in immunoreactive form over seven to ten weeks to 73%–100% complete release, and GM‐CSF showed bioactivity >95% relative to immunoreactive protein throughout the release interval. Increased encapsulation efficiencies were also found with other divalent transition metals ions (Co2+, Cu2+, Ni2+, and Zn2+), but not with Ca2+. Ethylenediaminetetraacetic acid was found to interfere with this process, reverting encapsulation efficiency back to Zn2+‐free levels. These results indicate that M2+‐immobilized self‐healing microspheres can be prepared for simple and efficient encapsulation by simple mixing in aqueous solutions. These formulations provide slow and continuous release of immunoreactive proteins of diverse types by using a amount of protein (e.g., <10 μg), which may be highly useful in the discovery and early preclinical development phase of new protein active pharmaceutical ingredients, allowing for improved translation to further development of potent proteins for local delivery.

Non-coating method for non-adherent cell culture using high molecular weight dextran sulfate and bovine serum albumin

Non-adherent cell culture surface has been widely used for producing cell spheroids and cell aggregates. The purpose of this study was to formulate a new method for non-adherent cell culture without coating or surface-modification that has been needed. We found that high-molecular-weight dextran sulfate (DS) and bovine serum albumin (BSA) synergistically prevented cell adhesion in media supplemented with no or low serum. This method worked on tissue culture-treated polystyrene surfaces as well as on commercially available low-attachment- and untreated polystyrene surfaces. Further investigation revealed that BSA may mediate the adsorption of DS to the surface. In addition, as the adsorption of fluorescently labeled fibronectin was inhibited by BSA alone, it appears that protein adsorption and cell adhesion do not always correlate. Finally, we demonstrated the successful formation of HepG2 spheroids and cardiomyocyte aggregates using this method. In conclusion, cell adhesion can be effectively suppressed by simply adding DS and BSA to the culture medium without coating or surface modification, and it may be useful for generating cell spheroids and aggregates.

Red Algal Sulfated Galactan Binds and Protects Neural Cells from HIV-1 gp120 and Tat.

The potential neuroprotective capacity of four different sulfated glycans: Botryocladia occidentalis-derived sulfated galactan (BoSG) (MW > 100 kDa), Lytechinus variegatus-derived sulfated fucan (LvSF) (MW~90 kDa), high-molecular weight dextran sulfate (DxS) (MW 100 kDa), and unfractionated heparin (UFH) (MW~15 kDa), was assessed in response to the HIV-1 proteins, R5-tropic glycoprotein 120 (gp120) and/or trans-activator of transcription (Tat), using primary murine neurons co-cultured with mixed glia. Compared to control-treated cells in which HIV-1 proteins alone or combined were neurotoxic, BoSG was, among the four tested sulfated glycans, the only one capable of showing significant concentration-dependent neuroprotection against Tat and/or gp120, alone or combined. Surface plasmon resonance-based data indicate that BoSG can bind both HIV-1 proteins at nM concentrations with preference for Tat (7.5 × 10−8 M) over gp120 (3.2 × 10−7 M) as compared to UFH, which bound gp120 (8.7 × 10−7 M) over Tat (5.7 × 10−6 M). Overall, these data support the notion that sulfated glycan extracted from the red alga B. occidentalis, BoSG, can exert neuroprotection against HIV-1 Tat and gp120, potentially via direct molecular interactions.

Post-loading of proangiogenic growth factors in PLGA microspheres

Active self-encapsulation (ASE) is a recently developed post-loading method based on absorption of (positively charged) proteins in microporous PLGA microspheres loaded with negatively charged polysaccharides (trapping agents). The aim of this study was to investigate ASE for simultaneous loading and controlled release of multiple growth factors. For this purpose, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF) were loaded in microspheres containing high molecular weight dextran sulfate (HDS) as trapping agent; loading was performed in a concentrated growth factor solution of low ionic strength and of pH 5 under conditions at which the proteins are positively charged. Subsequent pore closure was induced by incubation of the growth factor-loaded microspheres at 42.5 °C, i.e. above the Tg of (hydrated) PLGA (~30 °C). A 1:1:1 combination of VEGF, FGF and IGF was loaded with high loading (4.3%) and loading efficiency (91%). The in vitro release kinetics and bioactivity of loaded growth factors were studied for 4 weeks using ELISA and an endothelial cell proliferation assay, respectively. While IGF was released quickly, VEGF and FGF were continuously released for 4 weeks in their bioactive form, whereby a growth factor combination had a synergistic angiogenic effect. Therefore, ASE is a suitable method for co-loading growth factors which can provide sustained release profiles of bioactive growth factors, which is attractive for vascularization of biomaterial implants.

Structural studies on the interaction of lysozyme with dextran sulfate

The complexation between hen egg white lysozyme (Lys) and high molecular weight dextran sulfate sodium salt (DSS) was investigated by means of dynamic light scattering (DLS), phase analysis, optical microscopy and UV–vis absorption measurements at different pH and univalent electrolyte concentrations. The specific pH values where soluble complex formation is initiated (pHc) and where phase separation occurs (pHϕ) were strongly dependent on the ionic strength (I), that suggest that complexation is governed by electrostatic interaction. The effect of I on complexation has nonmonotonic character displaying maximum in complex formation at I equal 0.18–0.20. The solution behavior, structure, effective charge of the formed complexes, protein structure within the complex proved to be dependent on the pH, and the [An−]/[Cat+] charge ratio. Complexation in dilute solutions leads to spectacular stabilization of lysozyme within the complexes against thermal aggregation, whereas in semidiluted solutions its results in a considerable decrease (20°) in the temperature of denaturation, suggesting that DSS has a higher affinity for the unfolded state than the native state of lysozyme.

A biomimetic approach to active self-microencapsulation of proteins in PLGA

A biomimetic approach to organic solvent-free microencapsulation of proteins based on the self-healing capacity of poly (dl)-lactic-co-glycolic acid (PLGA) microspheres containing glycosaminoglycan-like biopolymers (BPs), was examined. To screen BPs, aqueous solutions of BP [high molecular weight dextran sulfate (HDS), low molecular weight dextran sulfate (LDS), chondroitin sulfate (CS), heparin (HP), hyaluronic acid (HA), chitosan (CH)] and model protein lysozyme (LYZ) were combined in different molar and mass ratios, at 37°C and pH7. The BP-PLGA microspheres (20–63μm) were prepared by a double water–oil–water emulsion method with a range of BP content, and trehalose and MgCO3 to control microclimate pH and to create percolating pores for protein. Biomimetic active self-encapsulation (ASE) of proteins [LYZ, vascular endothelial growth factor165 (VEGF) and fibroblast growth factor (FgF-20)] was accomplished by incubating blank BP-PLGA microspheres in low concentration protein solutions at ~24°C, for 48h. Pore closure was induced at 42.5°C under mild agitation for 42h. Formulation parameters of BP-PLGA microspheres and loading conditions were studied to optimize protein loading and subsequent release. LDS and HP were found to bind >95% LYZ at BP:LYZ>0.125 w/w, whereas HDS and CS bound >80% LYZ at BP:LYZ of 0.25–1 and <0.33, respectively. HA-PLGA microspheres were found to be not ideal for obtaining high protein loading (>2% w/w of LYZ). Sulfated BP-PLGA microspheres were capable of loading LYZ (~2–7% w/w), VEGF (~4% w/w), and FgF-20 (~2% w/w) with high efficiency. Protein loading was found to be dependent on the loading solution concentration, with higher protein loading obtained at higher loading solution concentration within the range investigated. Loading also increased with content of sulfated BP in microspheres. Release kinetics of proteins was evaluated in-vitro with complete release media replacement. Rate and extent of release were found to depend upon volume of release (with non-sink conditions observed <5ml release volume for ~18mg loaded BP-PLGA microspheres), ionic strength of release media and loading solution concentration. HDS-PLGA formulations were identified as having ideal loading and release characteristics. These optimal microspheres released ~73–80% of the encapsulated LYZ over 60days, with >90% of protein being enzymatically active. Nearly 72% of immunoreactive VEGF was similarly released over 42days, without significant losses in heparin binding affinity in the release medium.

Zinc-dependent contact system activation induces vascular leakage and hypotension in rodents

Contact to polyanions induces autoactivation of the serine protease factor XII that triggers the kallikrei-kinin system. Recent studies indicate that polysaccharide-induced autoactivation of factor XII has a role in allergy-related vascular leakage, and angioedema. Here, we characterize in vivo effects of the synthetic polysaccharide dextran sulfate in human plasma and in rodent models. Minute amounts of high-molecular-weight dextran sulfate-initiated factor XII-autoactivation and triggered formation of the inflammatory mediator bradykinin via plasma kallikrein-mediated cleavage of high-molecular-weight kininogen. High-molecular-weight kininogen fragments, containing the HKH20 sequence in domain D5H, blocked dextran sulfate-initiated bradykinin-generation by depleting plasma Zn2+ ions. Topical application of high molecular weight dextran sulfate increased leakage in murine skin microvessels, in a bradykinin-dependent manner. Intravital laser scanning microscopy showed a greater than two-fold elevated and accelerated fluid extravasation in C1 esterase inhibitor deficient mice that lack the major inhibitor of factor XII, compared to wild-type controls. Intra-arterial infusion of dextran sulfate induced a rapid transient drop in arterial blood pressure in rats and preinjection of kinin B2 receptor antagonists or HKH20 peptide blunted dextran sulfate-triggered hypotensive reactions. The data characterize dextran sulfate as a potent in vivo activator of factor XII with implications for bradykinin-mediated vascular permeability and blood pressure control.

Improved drug loading and antibacterial activity of minocycline-loaded PLGA nanoparticles prepared by solid/oil/water ion pairing method

BackgroundLow drug entrapment efficiency of hydrophilic drugs into poly(lactic-co-glycolic acid) (PLGA) nanoparticles is a major drawback. The objective of this work was to investigate different methods of producing PLGA nanoparticles containing minocycline, a drug suitable for periodontal infections.MethodsDifferent methods, such as single and double solvent evaporation emulsion, ion pairing, and nanoprecipitation were used to prepare both PLGA and PEGylated PLGA nanoparticles. The resulting nanoparticles were analyzed for their morphology, particle size and size distribution, drug loading and entrapment efficiency, thermal properties, and antibacterial activity.ResultsThe nanoparticles prepared in this study were spherical, with an average particle size of 85–424 nm. The entrapment efficiency of the nanoparticles prepared using different methods was as follows: solid/oil/water ion pairing (29.9%) > oil/oil (5.5%) > water/oil/water (4.7%) > modified oil/water (4.1%) > nano precipitation (0.8%). Addition of dextran sulfate as an ion pairing agent, acting as an ionic spacer between PEGylated PLGA and minocycline, decreased the water solubility of minocycline, hence increasing the drug entrapment efficiency. Entrapment efficiency was also increased when low molecular weight PLGA and high molecular weight dextran sulfate was used. Drug release studies performed in phosphate buffer at pH 7.4 indicated slow release of minocycline from 3 days to several weeks. On antibacterial analysis, the minimum inhibitory concentration and minimum bactericidal concentration of nanoparticles was at least two times lower than that of the free drug.ConclusionNovel minocycline-PEGylated PLGA nanoparticles prepared by the ion pairing method had the best drug loading and entrapment efficiency compared with other prepared nanoparticles. They also showed higher in vitro antibacterial activity than the free drug.

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.

Phosphatidylserine (PS)-Positive Erythrocytes Bind to Both Immobilized and Soluble Thrombospondin-1 (TSP-1) Via Its Heparin Binding Domain.

Phosphatidylserine (PS)-dependent erythrocyte adhesion to both cultured endothelial cells and the components of sub-endothelial matrix appears to be mediated in part via thrombospondin-1 (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP has not been identified. Since a cell-binding domain for anionic heparin is located at the amino-terminal domain of TSP, we hypothesized that anionic PS-positive (PS+ve) red cells bind to this domain. In a recent preliminary study, using a flow adhesion system and PS+ve erythrocytes (prepared by treating control AA red cells with A23187), we have demonstrated that heparin inhibited PS+ve erythrocyte adhesion to immobilized TSP in a concentration-dependent manner with 58 to 77% inhibition noted at concentrations between 1 and 50 U/ml (n=9, P<0.001). Other anionic polysaccharides including high molecular weight dextran sulfate and chondroitin sulfate A also inhibited PS+ve erythrocyte adhesion to immobilized TSP with the magnitude of the inhibitory effects comparable to heparin. These results suggested that the heparin-binding domain on TSP may be involved in PS-mediated red cell adhesion to immobilized TSP. We have extended these studies to characterize the PS-binding site on TSP using monoclonal antibodies directed against specific cell-binding domains on the molecule and also using specific TSP peptides. We demonstrate that pre-incubation of immobilized TSP with an antibody directed against the heparin-binding domain on TSP (TSP-Ab9, Lab Vision) blocked PS-mediated red cell adhesion to immobilized TSP (80% inhibition compared to an isotype-matched negative control antibody, n=7, P<0.001), whereas an antibody that recognizes the collagen-binding domain on TSP (TSP-Ab4) did not affect this process. In addition, incubation of PS+ve erythrocytes with a TSP peptide containing the specific heparin-binding motif, KKTRG, inhibited PS-mediated red cell adhesion by 55% (P<0.001, n=6), whereas a peptide lacking the binding motif had no effect. Since protein confirmation of immobilized TSP appears to be different from that of soluble TSP, we next investigated whether soluble TSP, like immobilized TSP, also interacted with PS+ve erythrocytes. Erythrocytes containing 8 to 10% PS+ve cells were incubated in the absence or the presence of increasing concentrations of soluble TSP (0.1 to 10 μg/ml), and then analyzed by flow cytometry for surface bound TSP using both adhesion blocking (TSP-Ab9) and non-blocking (TSP-Ab4) anti-TSP antibodies. We demonstrate that soluble TSP binds to PS+ve erythrocytes in a concentration-dependent manner with 3 to 11% TSP-positive (TSP+ve) red cells measured at soluble TSP concentrations between 1 to 10 μg/ml (n=4). In addition, TSP binding could be detected only with the non-adhesion blocking antibody TSP-Ab4, which recognizes the collagen-binding domain on TSP. The adhesion blocking antibody TSP-Ab9 that interacts with the heparin binding domain, failed to detect any TSP+ve red cells. No TSP+ve erythrocytes were detected when PS-negative control red cells were evaluated in binding assays. In parallel adhesion experiments, soluble TSP inhibited PS+ve erythrocyte adhesion to immobilized TSP at concentrations at which significant TSP binding to erythrocytes occurred (43% and 44% inhibition at 5 and 10 μg of soluble TSP per ml, n=4). These results conclusively demonstrate that PS-positive erythrocytes interact with both immobilized and fluid phase TSP through the heparin-binding domain, and that heparin blocks this interaction.

Erythrocyte adhesion is modified by alterations in cellular tonicity and volume

We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.

Modulation of Erythrocyte Adhesion by Changes in Cellular Tonicity and Volume.

The sickle red blood cell (SS-RBC) membrane manifests many abnormal properties, including oxidative damage of membrane proteins and lipids, disruption of the lipid bilayer, abnormal clustering of surface proteins, dehydration, and increased adhesive properties. Conditions that alter the RBC membrane may modify or abnormally expose its native components and contribute to the adhesive properties of the RBC. We have previously reported that sickle RBCs have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. Since a significant proportion of sickle RBCs are inherently dehydrated, in part due to intracellular K+ loss via the Gardos and K-Cl co-transport channels, we test the hypothesis that dehydration-induced alterations in membrane organization or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilized TSP under flow conditions. Expanding on our previous preliminary results, we found that sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (p<0.005), suggesting that cellular dehydration may contribute to its adhesive phenotype. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (p<0.01). Furthermore, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides chondroitin sulfate A and high molecular weight dextran sulfate, but not by the anionic polysaccharide chondroitin sulfate C. TSP adhesion of sickle and dehydrated normal RBCs in our in vitro system was also not inhibited by competitors of CD47-, band 3-, or RBC phosphatidylserine-meditaed adhesion. Increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers; this adhesion was also inhibited by chondroitin sulfate A and high molecular weight dextran sulfate but not by chondroitin sulfate C. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs, as compared to untreated normal murine RBCs, to kidney capillaries following reinfusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, and suggest that changes in volume are more important than changes in intracellular tonicity in the adhesion of RBCs to TSP. There are striking similarities in the adhesive properties of sickle and dehydrated normal RBCs, supporting the theory that adhesion of sickle RBCs arises as the result of exposure or modification of normal erythroid membrane components. Characterization of the membrane epitope(s) responsible for adhesion of dehydrated normal erythrocytes to TSP may thus provide a parallel avenue to identify reagents that can inhibit RBC adhesion and resultant vasoocclusion in SCD.

Characterization of Sugar Binding by Osteoclast Inhibitory Lectin

Osteoclast inhibitory lectin (OCIL) is a membrane-bound C-type lectin that blocks osteoclast differentiation and, via binding to its cognate receptor NKRP1D, inhibits natural killer cell-mediated cytotoxicity. OCIL is a member of the natural killer cell receptor C-type lectin group that includes CD69 and NKRP1D. We investigated carbohydrate binding of soluble recombinant human and mouse OCIL in enzyme-linked immunosorbent assay-based assays. OCIL bound immobilized high molecular weight sulfated glycosaminoglycans, including fucoidan, lambda-carrageenan, and dextran sulfate, but not unsulfated dextran or sialated hyaluronic acid. Carbohydrate binding was Ca(2+)-independent. Binding of immobilized low molecular weight glycosaminoglycans, including chondroitin sulfate (A, B, and C forms) and heparin, was not observed. However, the soluble forms of these low molecular weight glycosaminoglycans competed for OCIL binding of immobilized fucoidan (as did soluble fucoidan, dextran sulfate, and lambda-carrageenan), indicating that OCIL does recognize these carbohydrates. Inhibition constants for chondroitin sulfate A and heparin binding were 380 and 5 nm, respectively. Immobilized and soluble monosaccharides did not bind OCIL. The presence of saturating levels of fucoidan, dextran sulfate, and lambda-carrageenan did not affect OCIL inhibition of osteoclast formation. The fucoidan-binding lectins Ulex europaeus agglutinin I and Anguilla anguilla agglutinin did not block osteoclast formation or affect the inhibitory action of OCIL. Although the osteoclast inhibitory action of OCIL is independent of sugar recognition, we have found that OCIL, a lectin widely distributed, but notably localized in bone, skin, and other connective tissues, binds a range of physiologically important glycosaminoglycans, and this property may modulate OCIL actions upon other cells.

The effect of high molecular weight dextran sulfate on the production of interleukin-8 in monocyte cell culture

It has been demonstrated that high molecular weight dextran sulfate (HMDS) is involved in the activation of immune cells. We have shown that HMDS increases the concentration of interleukin (IL)-8 in the medium of monocyte cell culture, in a dose-dependent fashion, whereas under the same conditions, low molecular weight dextran sulfate (LMDS) does not exhibit any effect on IL-8 biosynthesis. The effect of HMDS on IL-8 production is additive to that of IL-1β and tumor necrosis factor-α (TNFα). Flow cytometric analysis revealed the biosynthesis of IL-8 in monocytes incubated in the presence of the HMDS. We hereby postulate that HMDS induces IL-8 biosynthesis in monocyte cell culture.

Specificity of glycosaminoglycan suppression of endothelin-1 production by human umbilical vein endothelial cells

Endothelin-1 (ET-1) is the most potent vasoconstrictor peptide found in nature. Its production is stimulated by thrombin. By inhibiting thrombin we have previously shown that heparin, a highly negatively-charged glycosaminoglycan (GAG), suppresses the production of ET-1 by cultured human umbilical vein endothelial cells (HUVEC). The purpose of our study is to determine the effect of other GAGs and related compounds on ET-1 production. The GAGs and related compounds used in the study were: chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, fucoidin, low molecular weight dextran sulfate, high molecular weight dextran sulfate, and hyaluronan. HUVEC were incubated for 48 hr with media containing these GAGs and related compounds and with media without GAG as control. ET-1 levels were measured by radioimmunoassay. GAGs and related molecules with higher sulfate content, heparin, chondroitin sulfate B, low and high molecular weight dextran sulfates significantly suppressed ET-1 production by HUVEC. Fucoidin also suppressed ET-1 production despite its lower sulfate content, probably because of its structural similarity to heparin. These compounds may be useful for future in vivo studies.

Anionic Polysaccharides Inhibit Adhesion of Sickle Erythrocytes to the Vascular Endothelium and Result in Improved Hemodynamic Behavior

AbstractThe abnormal adherence of sickle red blood cells (SS RBC) to vascular endothelium may play an important role in vasoocclusion in sickle cell anemia. Thrombospondin (TSP), unusually large molecular weight forms of von Willebrand factor, and laminin are known to enhance adhesion of SS RBC. Also, these endothelial proteins bind to sulfated glycolipids and this binding is inhibited by anionic polysaccharides. Reversible sickling may expose normally cryptic membrane sulfatides that could mediate this adhesive interaction. In this study, we have investigated the effect of anionic polysaccharides, in the presence or absence of TSP, on SS RBC adhesion to the endothelium, using cultured human umbilical vein endothelial cells (HUVEC) (for the adhesion assay) and the ex vivo mesocecum of the rat (for hemodynamic evaluation). The baseline adhesion (ie, without added TSP) of SS RBC to HUVEC was most effectively inhibited by high molecular weight dextran sulfate (HDS), whereas low molecular weight dextran sulfate (LDS) and the glycosaminoglycan chondroitin sulfate A (CSA) also had significant inhibitory effects. Heparin was mildly effective whereas other glycosaminoglycans (chondroitin sulfates B and C, heparan sulfate, and fucoidan) were ineffective. Similarly, HDS and CSA resulted in an improved hemodynamic behavior of SS RBC. Soluble TSP caused significant increases in SS RBC adhesion and in the peripheral resistance. Both HDS and CSA prevented TSP-enhanced adhesion and hemodynamic abnormalities. Thus, anionic polysaccharides can inhibit SS RBC-endothelium interaction in the presence or absence of soluble TSP. These agents may interact with RBC membrane component(s) and prevent TSP-mediated adhesion of SS RBC to the endothelium.

Anionic Polysaccharides Inhibit Adhesion of Sickle Erythrocytes to the Vascular Endothelium and Result in Improved Hemodynamic Behavior

The abnormal adherence of sickle red blood cells (SS RBC) to vascular endothelium may play an important role in vasoocclusion in sickle cell anemia. Thrombospondin (TSP), unusually large molecular weight forms of von Willebrand factor, and laminin are known to enhance adhesion of SS RBC. Also, these endothelial proteins bind to sulfated glycolipids and this binding is inhibited by anionic polysaccharides. Reversible sickling may expose normally cryptic membrane sulfatides that could mediate this adhesive interaction. In this study, we have investigated the effect of anionic polysaccharides, in the presence or absence of TSP, on SS RBC adhesion to the endothelium, using cultured human umbilical vein endothelial cells (HUVEC) (for the adhesion assay) and the ex vivo mesocecum of the rat (for hemodynamic evaluation). The baseline adhesion (ie, without added TSP) of SS RBC to HUVEC was most effectively inhibited by high molecular weight dextran sulfate (HDS), whereas low molecular weight dextran sulfate (LDS) and the glycosaminoglycan chondroitin sulfate A (CSA) also had significant inhibitory effects. Heparin was mildly effective whereas other glycosaminoglycans (chondroitin sulfates B and C, heparan sulfate, and fucoidan) were ineffective. Similarly, HDS and CSA resulted in an improved hemodynamic behavior of SS RBC. Soluble TSP caused significant increases in SS RBC adhesion and in the peripheral resistance. Both HDS and CSA prevented TSP-enhanced adhesion and hemodynamic abnormalities. Thus, anionic polysaccharides can inhibit SS RBC-endothelium interaction in the presence or absence of soluble TSP. These agents may interact with RBC membrane component(s) and prevent TSP-mediated adhesion of SS RBC to the endothelium.

High molecular weight dextran sulfate increases the activity of NF-kappaB-regulated promoter in monocyte-derived macrophages.

It is known that sulfated polysaccharides can mimic the action of common T-cell mitogens. To investigate the molecular basis of the mitogenic effect of high molecular weight dextran sulfate (HMDS), monocyte-derived macrophages were transfected with recombinant plasmid containing chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) promoter, which is regulated by transcription factor NF-kappaB. We observed that HMDS, similar to bacterial lipopolysaccharide (LPS), increases the expression of CAT reporter gene suggesting increased activity of NF-kappaB. The activation of NF-kappaB correlated with the increased expression of B7.1 molecules. It was postulated that this NF-kappaB-regulated promoter might play a role in the activation of the accessory cells as well as the rate of replication of HIV-1 in monocyte-derived macrophages.

Enhanced human immunodeficiency virus infection in macrophages by high-molecular-weight dextran sulfate is associated with conformational changes of gp120 and expression of the CCR5 receptor.

High-molecular-weight dextran sulfate (HMDS) inhibits infection of CD4+ lymphocytes by T-cell (T)-tropic human immunodeficiency virus (HIV) isolates, but augments replication of macrophage (M)-tropic isolates in primary human macrophages and phorbol myristate acetate (PMA)-differentiated THP-1 monocytic cells. To address the mechanism responsible for HMDS-mediated increases in HIV replication in macrophages, we analyzed the interaction between HMDS and functional domains of gp120 on the surface of PMA-differentiated THP-1 cells infected with M-tropic HIV isolates. Immunofluorescence staining of the infected cells revealed that HMDS inhibited the binding of monoclonal antibodies (mAbs) directed to the V3 and C4 domains of gp120, but augmented the binding of three neutralizing antibodies directed to the V2 region of gp120. The extent of HMDS-mediated changes within the V2 loop of gp120 was associated with increased virus binding and replication in PMA-differentiated THP-1 cells and primary macrophages. The effect was dependent on expression of the CCR5 receptor and was inhibited by the beta-chemokine RANTES. Results of this study suggest that HMDS-mediated increases in HIV infection in macrophages are associated with conformational changes within the V2 region of gp120 and enhanced interaction between gp120 and the CCR5 coreceptor on the target cell.