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Disintegrins represent a group of disulfide-rich peptides ranging in size from 41 to over 80 residues and are antagonists of several integrin receptors. Disintegrins containing an RGD or KGD sequence are potent inhibitors of platelet aggregation as they block the binding of fibrinogen to alpha(IIb)beta(3) integrin. The high affinity binding to alpha(IIb)beta(3) in comparison to short linear peptides has been attributed to the localisation of the RGD or KGD sequence within a defined three-dimensional structure. Cystine knot microproteins are members of another family of small disulfide-rich peptides that consist of only 28-40 amino acid residues. They display numerous biological activities depending on the peptide sequence of loop regions that are fixed on a structural scaffold that is stabilised by three knot-forming disulfide bonds. In the present study we grafted RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin. The engineered proteins were much more potent to inhibit the fibrinogen binding, alpha(IIb)beta(3) activation and platelet aggregation when compared to the grafted peptides. Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in human macrophages (Mphi), a significant feature in atherogenesis. We found that induction of apoptosis in Mphi by oxLDL, C2-ceramide, tumor necrosis factor alpha (TNF-alpha), and hydrogen peroxide (H2O2) was associated with enhanced expression of manganese superoxide dismutase (MnSOD) and p53. Treatment of cells with p53 or MnSOD antisense oligonucleotides prior to stimulation with oxLDL, C2-ceramide, TNF-alpha, or H2O2 caused an inhibition of the expression of the respective protein together with a marked reduction of apoptosis. Exposure to N-acetylcysteine before treatment with oxLDL, C2-ceramide, TNF-alpha, or H2O2 reversed a decrease in cellular glutathione concentrations as well as the enhanced production of p53 and MnSOD mRNA and protein. In apoptotic macrophages of human atherosclerotic plaques, colocalization of MnSOD and p53 immunoreactivity was found. These results indicate that in oxLDL-induced apoptosis, a concomitant induction of p53 and MnSOD is critical, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ceramide pathway.

The sphingomyelin (SM) pathway is an ubiquitous and evolutionarily conserved signaling system in which ceramide (CA), generated from SM by the action of various isoforms of sphingomyelinases (SMases) functions as an important second messenger. Recent evidence suggests that branching pathways of sphingolipid metabolism mediate either apoptotic or mitogenic responses depending on cell type and the nature of the stimulus. Events involving SM metabolites and CA in particular include proliferation, differentiation and growth arrest as well as the induction of apoptosis. An improved understanding of SMase-dependent signaling may afford relevant insights into the pathogenesis of diseases and provide novel strategies and selective targets for a therapeutic intervention e.g. in cancer, cardiovascular and neurodegenerative diseases, HIV and septic shock. This article briefly summarizes the role of SMases in signaling pathways, its potential contribution in the development and maintenance of various pathobiological states and analyzes the perspective of a potentially isotype-specifc inhibition of SMases as a novel therapeutic concept.

An aqueous extract of propolis and the phenolic component of propolis, propol, were assayed for antioxidative and antiapoptotic properties. Both additions inhibited Cu(2+)-initiated low density lipoprotein (LDL) oxidation as characterized by a reduction of the lag time, reduced the increase of relative electrophoretic mobility during oxidation and markedly diminished apoptosis of human macrophages exposed to minimally modified (mmLDL). Moreover, aqueous propolis extract and propol blocked the mmLDL-induced decrease of glutathione (GSH) and the activation of the transcription factor NF-kappa B in these cells. The potent phenolic antioxidant propol thus expands the capability of cells to neutralize oxidative stress and to prevent apoptosis and is therefore suggested to significantly contribute to the antiinflammatory and antioxidative effects of propolis.

Phosphatidylcholines (1-O-alcoxy-2-amino-2-desoxy-phosphocholines and 1-pyrene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-acetylhydrolase (PAF-AH), an enzyme purified from plasma, and with macrophage-like U937 cells. Novel phosphatidylcholines containing a sn-2-carbamoylester group such as 1-O-hexadecyl-2-desoxy-2-amino-methylcarbamoyl-2-methyl-rac-glycer o-3-phosphocholine 11 were found to act as site-specific irreversible enzyme inhibitors with Ki-values up to 83 (K(irev)) and 177 (Ki(inact)) microm. The compounds exhibit only marginal inhibition of Ca2+-dependent phospholipases. Kinetic data show that phosphocholines carrying a terminal sn-1-pyrene moiety inhibit PAF-AH activity with an effectivity similar to analogs with an aliphatic chain. 1-O-Decyloxy-[10-(4-pyrenyl)-butoxy]-2-desoxy-2-amino-carbamoyl-me thyl-rac(-glycero-3-phosphocholine 13 could be used for enzyme labeling and to demonstrate an inhibitor-enzyme stoichiometry of 0.7:1. At 8 degrees C, the compound accumulated in the membranes of U937 cells, at 37 degrees C it was internalized into intracellular compartments. Structure activity studies in a mixed micelle assay indicated that the inhibition power of reversible and irreversible inhibitors increases along with the (sn)-1-chain length similar to the structure-dependent binding of ether phospholipids to the PAF-receptor. Unlike the situation at the (sn)-1-position, increasing chain length at the sn-2-position, or an alkyl branching of the glycerol backbone significantly reduced the inhibitory potency.

Exposure of human blood monocytes derived macrophages to modified (oxidized or acetylated) LDL induced a ∼40% elevation (60 pmol/106 cells) of the endogenous level of the sphingolipid ceramide. A rise of both neutral and acidic SMase activity was found after treatment with oxidized LDL (250 and 80%), while addition of acLDL stimulated only the neutral enzyme (280%). Sphingo(phospho)lipids from LDL were transferred to the cell membrane and distributed into intracellular compartments as observed with acLDL containing BODIPY-FL-C5-SM. Quantitation of ceramide after the addition of [3H-N-acetyl]- or BODIPY-FL-C5-SM-labeled modified LDL (27 μg/ml) to the cell culture medium indicated that approximately 210 pmol CA/106 cells was generated from exogenous (ox/acLDL) SM. These results demonstrate a stimulation of the sphingomyelin-ceramide pathway by modified LDL utilizing primarily exogenous (LDL-derived) substrate and suggest that the effects of modified LDL are at least partially due to an increased level of the messenger ceramide.©1997 Federation of European Biochemical Societies.

Treatment of P388D1 macrophage-like cells with oxLDL enhanced protein kinase C (PKC) activity in cell extracts. Similar effects were induced by acetylated LDL (acLDL) and maleylated albumin (malBSA). Treatment with oxLDL, acLDL and malBSA was also accompanied by increased production of prostaglandins as well as by an enhanced level of prostaglandin H synthase 2 (cyclooxygenase 2, COX 2). Modified (lipo)proteins differentially affected the levels of individual cytosolic PKC-isoenzymes. Effects of oxLDL on PKC activity/expression were abrogated by indometacin, by pre-exposure to the dual lipoxygenase/cyclooxygenase inhibitor ML 3000 and by treatment with N-(2-cyclohexy-loxy-4-nitrophenyl)methane sulfonamide (NS-398). Thesse results suggest a predominantly COX 2-dependent and isotype-specific effect of modified (lipo)proteins on PKC.

Mitogen activated protein kinase in extracts of U-937 macrophage-like cells was stimulated by LDL and oxLDL. A maximum value (161% of the basal phosphotransferase activity) was obtained after 6 min exposure to oxidized LDL (27 μ/ml) using APRTPGGRR peptide substrate. The activatory effect was more pronounced (LDL 181%, oxLDL 201%) when MAPK of stimulated cells was immunoprecipitated with anti-p42MAPK antibodies and phosphotransferase activity was assayed in immune complexes. Stimulation produced by oxLDL was inhibited by poly I, fucoidan, dextran sulfate and by the MAPKK inhibitor PD 098059 but not by PMA-mediated depletion of PKC or by pre-treatment with chloroquine or with pertussis toxin. These results suggest a direct mitogenic effect of LDL which, in the case of oxLDL, is dependent on scavenger receptor ligation but not on G-protein mediated or PKC-dependent signal transduction.

Treatment of cells with LPS-free oxLDL significantly enhanced protein kinase C (PKC) activity in cell extracts from P388D1 macrophage-like cells as determined by phosphorylation of histone H1 or Ac-MBP[4-14] substrate peptide. This effect was abolished by the PKC inhibitors H-7 and bisindolylmaleimide I while pertussis toxin failed to block stimulation. The phosphotransferase activity was also increased by acetylated LDL (acLDL) and maleylated albumin (malBSA), the oxLDL effect was inhibited by chloroquine which also blocked oxLDL-induced stimulation of tyrosine kinase activity. Marginal stimulation of PKC activity was observed when lipid extracts from oxLDL were used, indicating that uptake via scavenger receptors (SR) is mandatory. Polyinosinic acid (poly I) exhibited a concentration-dependent inhibition of the oxLDL-induced effect suggesting that SR II/I but not CD36 interactions are critical to PKC activation. Modified (lipo)proteins increased the concentration of diacylglycerol and differentially affected the levels of individual PKC isoenzymes predominantly in the cytosolic fraction. Changes of activity induced by oxLDL could be primarily assigned to alterations of the activities and levels of the isoenzymes beta and delta. Treatment with oxLDL, acLDL, and malBSA was also accompanied by increased production of prostaglandins as well as by an enhanced level of cyclooxygenase 2 (COX 2) as determined by Western blot analysis. Effects (correction) of oxLDL on PKC activity/expression was suppressed by the cyclooxygenase, 2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,2-dihydro-1H-pyrrolizine-5- ylacetic acid (ML 3000), and by treatment with the specific COX 2-inhibitor N-(2-cyclohexyloxy-4-nitrophenyl) methane-sulfonamide (NS-398). These results indicate that oxLDL, acLDL, and malBSA exhibit a COX 2-dependent and isotype specific effect on PKC in P388D1 cells following uptake via SR II/I and subsequent lysosomal degradation.