Heparin has long been known to slow the growth of vascular smooth muscle cells. However, the mechanism(s) by which heparin acts has yet to be resolved. The identification of a putative heparin receptor in endothelial cells with antibodies that blocked heparin binding to the cells provided the means to further examine the possible involvement of a heparin receptor in smooth muscle cell responses to heparin. Immunoprecipitation of a smooth muscle cell protein with the anti-heparin receptor antibodies provided evidence that the protein was present in smooth muscle cells. Experiments with the anti-heparin receptor antibodies indicate that the antibodies can mimic heparin in decreasing PDGF induced thymidine and BrdU incorporation. The anti-heparin receptor antibodies were also found to decrease MAPK activity levels after activation similarly to heparin. These results support the identification of a heparin receptor and its role in heparin effects on vascular smooth muscle cell growth.
The binding of heparin or heparan sulphate to a variety of cell types results in specific changes in cell function. Endothelial cells treated with heparin alter their synthesis of heparan sulphate proteoglycans and extracellular matrix proteins. In order to identify a putative endothelial cell heparin receptor that could be involved in heparin signalling, anti-(endothelial cell) monoclonal antibodies that significantly inhibit heparin binding to endothelial cells were prepared. Four of these antibodies were employed in affinity-chromatographic isolation of a heparin-binding protein from detergent-solubilized endothelial cells. The heparin-binding protein isolated from porcine aortic endothelial cells using four different monoclonal antibodies has an M(r) of 45,000 assessed by SDS/PAGE. The 45,000-M(r) heparin-binding polypeptide is isolated as a multimer. The antibody-isolated protein binds to heparin-affinity columns as does the pure 45,000-M(r) polypeptide, consistent with its identification as a putative endothelial heparin receptor.
Decay accelerating factor (DAF) is a cell-surface phosphatidylinositol-anchored protein that protects the cell from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have measured DAF on human umbilical vein endothelial cells (HUVEC) by immunoradiometric assay after its removal by phosphatidylinositol-specific phospholipase C or Nonidet P-40 detergent extraction and have previously demonstrated that DAF synthesis can be stimulated by phorbol ester activation of protein kinase C. We now report that although stimulation (4-48 h) of HUVEC with various cytokines, including TNF, IL-1, and IFN-gamma, did not alter DAF levels, wheat germ agglutinin (WGA) (5-50 micrograms/ml), a lectin specific for binding N-acetyl neuraminic acid and N-acetyl glucosamine residues, increased DAF levels fivefold when incubated with HUVEC for 12 to 24 h. The lectins Con A and PHA also stimulated DAF expression twofold, whereas a number of others including Ulex europaeus, Bandeiraea simplicifolia lectin I, and Ricinus communis agglutinin I, which bind to endothelial cells, were inactive. The increase in DAF by WGA was inhibited by N-acetyl glucosamine (10-50 mM) but by neither N-acetyl neuraminic acid nor removal of surface N-acetyl neuraminic acid with neuraminidase. However, succinylated WGA, which has unaltered affinity for N-acetyl glucosamine but not longer binds N-acetyl neuraminic acid, was inactive. These data suggest that the binding of WGA to sugar residues alone is not sufficient to trigger DAF expression and that occupation of additional, specific sites are required. The increase in DAF levels on HUVEC was blocked by inhibitors of RNA and protein synthesis. We conclude that continuous occupation by WGA of specific binding sites on HUVEC triggers events leading to DAF synthesis. This unique, long term stimulation of endothelial cells by lectins may be relevant to cell:cell interactions at the endothelium.
A number of cell-surface proteins are anchored by a phosphatidylinositol (PI)-glycan moiety. These proteins can be released by PI-specific phospholipases C (PI-PLC). Decay-accelerating factor (DAF) is such a cell-surface protein that protects cells from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have studied the regulation of DAF synthesis in human umbilical vein endothelial cells (HUVEC), a cell that has the highest level of surface DAF among those human cells that have been studied. HUVEC DAF was measured by immunoradiometric assay of detergent extracts and of cell supernatants after treatment of cells with a bacterial (Bacillus thuringiensis) PI-PLC. Eighty percent of the HUVEC DAF (4 to 8 x 10(5) molecules/cell) was released by exogenously added PI-PLC, indicating that it is predominantly PI-anchored. The level of PI-PLC-sensitive HUVEC DAF was increased three- to fourfold by overnight treatment of cultures with the protein kinase C activators, PMA (1 to 10 nM), phorbol-12,13-dibutyrate (10 to 100 nM), and teleocidin A (1 to 10 nM) under conditions where cell number, protein, and lactate dehydrogenase remain unchanged. This DAF synthesis was blocked by the protein kinase C inhibitor K-252a in a dose-dependent manner (ED50 = 0.06 microM). The biologically inactive phorbols, 4-alpha-phorbol-12 myristate-13-acetate (1 microM) and 4-alpha-phorbol-12, 13-didecanoate (1 microM) did not increase DAF levels. The newly expressed DAF in PMA-stimulated cells was still largely PI-anchored. In contrast, another PI-anchored protein, alkaline phosphatase, was not altered by PMA treatment, demonstrating that the PMA effect is not uniform among all surface proteins. The increased expression of DAF only was evident 8 h after PMA addition and was blocked by the RNA and protein synthesis inhibitors, actinomycin D and cycloheximide, indicating that both transcription and translation are required for DAF synthesis induced by phorbol esters. It is concluded that protein kinase C activators cause selective induction of endothelial cell DAF and that DAF synthesis involves protein kinase C activation.
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