Structural features in heparin that interact with VEGF165 and modulate its biological activity.
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The 165 amino acid form of vascular endothelial growth factor (VEGF165) is a heparin-binding growth factor with mitogenic activity for vascular endothelial cells. We examined activities of various heparin derivatives toward their interactions with VEGF165 using an enzyme-linked immunosorbent assay and elucidated the structural features in heparin for the interactions. Native heparin interacted with VEGF165, whereas N-desulfated, N-acetylated (N-DS, N-Ac-) heparin, and 6-O-desulfated (6-O-DS-) heparin did not. The 2-O-desulfated (2-O-DS-) heparin retained the ability for the interaction with VEGF165. In contrast, the 2-O-DS-heparin exhibited no ability for the interaction with FGF-2 and HGF. Thus, structural requirements in heparin for the specific interaction with VEGF165 are distinct from those with FGF-2 and HGF which require a high content of 2-O-sulfate groups. In a cell proliferation assay, native heparin and 2-O-DS-heparin exhibited inhibitory abilities for VEGF165-induced proliferation of human umbilical vein endothelial cells (HUVECs) with their high concentrations (more than 64 microg/ml), while only native heparin could enhance the proliferation of the chlorate-treated cells. These results suggested that a high content of 2-O-sulfate groups is not required for the specific interaction with VEGF165alone, although it is essential for the mitogenic activity of the growth factor. (+info)
Enhanced channelling of sulphate through a rapidly exchangeable sulphate pool in response to stimulated glycosaminoglycan synthesis in pancreatic epithelial cells.
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The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP. (+info)
Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer of glycosaminoglycans to the nucleus.
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Molecules secreted by potential target cells may interfere with cationic lipid-mediated gene transfer. This has been studied using human lung fibroblasts and human epidermoid lung cancer cells. Secreted cell medium components caused a substantial decrease both in the uptake of cationic lipid-DNA complexes (2-4-fold) and in reporter gene expression (100-1000-fold). Metabolic labeling of the cell medium showed that especially [35S]sulfate-labeled macromolecules competed with DNA for binding to the cationic lipid. Release of DNA from the cationic lipid by cell medium components was demonstrated by an ethidium bromide intercalation assay. In the presence of the cationic lipid, the secreted macromolecules were internalized by the cells. By enzymatic digestions, it was shown that the competing macromolecules consist of chondroitin/dermatan sulfate and heparan sulfate proteoglycans and that the effects on transfection were mediated by the polyanionic glycosaminoglycan portion of the proteoglycan. Accordingly, pretreatment of cell medium with the polycationic peptide protamine sulfate abrogated the inhibitory effects on gene transfer. Fluorescence microscopy studies revealed that heparan sulfate, internalized as a complex with cationic lipids, accumulated in the cell nuclei. These results support the view that the lack of specificity of this type of gene transfer vehicle is a major hindrance to efficient and safe in vivo administration. (+info)
Characterization of [3H]-heparin binding in human vascular smooth muscle cells and its relationship to the inhibition of DNA synthesis.
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1. The glycosaminoglycan heparin inhibits vascular smooth muscle cell (VSMC) proliferation and migration, but the mechanism of its antiproliferative action remains unclear. Heparin has been reported to bind to high affinity cell surface sites on animal VSMC before undergoing receptor mediated endocytosis resulting in signal transduction into the cytoplasm and modulation of genes involved in proliferation. In this study, we have characterized the binding of [3H]-heparin to human saphenous vein-derived VSMC and examined whether there is any relationship between the affinity of [3H]-heparin binding and the inhibitory effect of heparin and its structural analogues on DNA synthesis. 2. At 4 degrees C [3H]-heparin binding to human VSMC occurred in a specific, time and concentration-dependent manner and was not influenced by the removal of calcium ions. Binding of the ligand appeared to occur to the cell surface and was both saturable and reversible. Kinetic and steady state data indicated a single class of binding sites. 3. The pharmacology of [3H]-heparin binding was examined in displacement studies using unlabelled heparin and structural analogues. A comparison of the rank potencies of heparin, heparan sulphate fraction II, low molecular weight heparin and trehalose octasulphate showed that there was a marked discrepancy between their estimated affinities in the binding assays and their effect on DNA synthesis. 4. In summary, we have characterized the heparin binding site on human saphenous vein-derived VSMC. Our findings suggest that the action of heparin and its analogues on DNA synthesis does not simply reflect an interaction with the cell-associated heparin binding site defined in these studies, but may also be determined by the internalization and metabolism of the glycosaminoglycan(s). (+info)
The CC-chemokine RANTES increases the attachment of human immunodeficiency virus type 1 to target cells via glycosaminoglycans and also activates a signal transduction pathway that enhances viral infectivity.
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We have studied the mechanisms by which the CC-chemokine RANTES can enhance the infectivities of human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses, when present at concentrations in excess of 500 ng/ml in vitro. Understanding the underlying mechanisms might throw light on fundamental processes of viral infection, in particular for HIV-1. Our principal findings are twofold: firstly, that oligomers of RANTES can cross-link enveloped viruses, including HIV-1, to cells via glycosaminoglycans (GAGs) present on the membranes of both virions and cells; secondly, that oligomers of RANTES interact with cell-surface GAGs to transduce a herbimycin A-sensitive signal which, over a period of several hours, renders the cells more permissive to infection by several viruses, including HIV-1. The enhancement mechanisms require that RANTES oligomerize either in solution or following binding to GAGs, since no viral infectivity enhancement is observed with a mutant form of the RANTES molecule that contains a single-amino-acid change (glutamic acid to serine at position 66) which abrogates oligomerization. (+info)
Oligomeric self-association of basic fibroblast growth factor in the absence of heparin-like glycosaminoglycans.
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Basic fibroblast growth factor (FGF-2) represents a class of heparin-binding growth factors that are stored in the extracellular matrix attached to heparin-like glycosaminoglycans (HLGAGs). It has been proposed that cell surface HLGAGs have a central role in the biological activity of FGF-2, presumably by inducing dimers or oligomers of FGF-2 and leading to the dimerization or oligomerization of FGF receptor and hence signal transduction. We have previously proposed that FGF-2 possesses a natural tendency to self-associate to form FGF-2 dimers and oligomers; HLGAGs would enhance FGF-2 self-association. Here, through a combination of spectroscopic, chemical cross-linking and spectrometric techniques, we provide direct evidence for the self-association of FGF-2 in the absence of HLGAGs, defying the notion that HLGAGs induce FGF-2 oligomerization. Further, the addition of HLGAGs seems to enhance significantly the FGF-2 oligomerization process without affecting the relative percentages of FGF-2 dimers, trimers or oligomers. FGF-2 self-association is consistent with FGF-2's possessing biological activity both in the presence and in the absence of HLGAGs; this leads us to propose that FGF-2 self-association enables FGF-2 to signal both in the presence and in the absence of HLGAGs. (+info)
Glycosaminoglycans mediate the coacervation of human tropoelastin through dominant charge interactions involving lysine side chains.
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Following cellular secretion into the extracellular matrix, tropoelastin is transported, deposited, and cross-linked to make elastin. Assembly by coacervation was examined for an isoform of tropoelastin that lacks the hydrophilic domain encoded by exon 26A. It is equivalent to a naturally secreted form of tropoelastin and shows similar coacervation performance to its partner containing 26A, thereby generalizing the concept that splice form variants are able to coacervate under comparable conditions. This is optimal under physiological conditions of temperature, salt concentration, and pH. The proteins were examined for their ability to interact with extracellular matrix glycosaminoglycans. These negatively charged molecules interacted with positively charged lysine residues and promoted coacervation of tropoelastin in a temperature- and concentration-dependent manner. A testable model for elastin-glycosaminoglycan interactions is proposed, where tropoelastin deposition during elastogenesis is encouraged by local exposure to matrix glycosaminoglycans. Unmodified proteins are retained at approximately 3 microM dissociation constant. Following lysyl oxidase modification of tropoelastin lysine residues, they are released from glycosaminoglycan interactions, thereby permitting those residues to contribute to elastin cross-links. (+info)
The influence of tissue cross-talking on OA progression: role of nonsteroidal antiinflammatory drugs.
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Osteoarthritis is increasingly recognized as a complex illness in which interrelationships between the different tissues of the joint are important. We are still some way from a complete understanding of the pathophysiologic and temporal relationships between bone, synovial tissue and cartilage. Recent evidence points to a significant role for cytokines and growth factors in osteoarthritis that leads to a preponderance of catabolic processes in the joint. In-vitro culture of human cartilage has been used as a model to measure the effects of drugs used in the treatment of osteoarthritis on anabolic and catabolic processes. On this basis, the nonsteroidal antiinflammatory drugs can be categorized into one of three classes depending on whether they are inhibitory (e.g., indomethacin and naproxen), neutral (e.g., diclofenac, aspirin and piroxicam) or stimulatory (e.g., aceclofenac, tenidap and tolmetin) of glycosaminoglycan synthesis in chondrocytes. The marked differences between these nonsteroidal antiinflammatory drugs suggest that a mechanism other than cyclooxygenase inhibition is involved in their effects on glycosaminoglycan synthesis. Inhibition of IL-1beta and the stimulation of growth factors are suggested as possible mechanisms. Although the significance of these properties of nonsteroidal antiinflammatory drugs awaits confirmation in in-vivo and clinical situations, they do provide the clinician with a new parameter with which to choose therapy in osteoarthritis. (+info)