Jerzy-Roch Nofer

High Density Lipoproteins and Arteriosclerosis Role of Cholesterol Efflux and Reverse Cholesterol Transport

High density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease, and HDL exerts various potentially antiatherogenic properties, including the mediation of reverse transport of cholesterol from cells of the arterial wall to the liver and steroidogenic organs. Enhancement of cholesterol efflux and of reverse cholesterol transport (RCT) is considered an important target for antiatherosclerotic drug therapy. Levels and composition of HDL subclasses in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes, lipid transfer proteins, receptors, and cellular transporters. In vitro experiments as well as genetic family and population studies and investigation of transgenic animal models have revealed that HDL cholesterol plasma levels do not necessarily reflect the efficacy and antiatherogenicity of RCT. Instead, the concentration of HDL subclasses, the mobilization of cellular lipids for efflux, and the kinetics of HDL metabolism are important determinants of RCT and the risk of atherosclerosis.

HDL and arteriosclerosis: beyond reverse cholesterol transport

The inverse correlation between serum levels of high density lipoprotein (HDL) cholesterol and the risk of coronary heart disease, the protection of susceptible animals from atherosclerosis by transgenic manipulation of HDL metabolism, and several potentially anti-atherogenic in vitro-properties have made HDL metabolism an interesting target for pharmacological intervention in atheroslcerosis. We have previously reviewed the concept of reverse cholesterol transport, which describes both the metabolism and the classic anti-atherogenic function of HDL (Arterioscler. Thromb. Vasc. Biol. 20 2001 13). We here summarize the current understanding of additional biological, potentially anti-atherogenic properties of HDL. HDL inhibits the chemotaxis of monocytes, the adhesion of leukocytes to the endothelium, endothelial dysfunction and apoptosis, LDL oxidation, complement activation, platelet activation and factor X activation but also stimulates the proliferation of endothelial cells and smooth muscle cells, the synthesis of prostacyclin and natriuretic peptide C in endothelial cells, and the activation of proteins C and S. These anti-inflammatory, anti-oxidative, anti-aggregatory, anti-coagulant, and pro-fibrinolytic activities are exerted by different components of HDL, namley apolipoproteins, enzymes, and even specific phospholipids. This complexity further emphasizes that changes in the functionality of HDL rather than changes of plasma HDL-cholesterol levels determine the anti-atherogenicity of therapeutic alterations of HDL metabolism.

Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR

Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)‐induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC) ‐γ1 due to c‐erbB‐2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long‐term PLC‐γ1 tyrosine phosphorylation and sustained levels of inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c‐erbB‐2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short‐term PLC‐γ1 tyrosine phosphorylation and short‐term IP3 and DAG turnover. A third cell line expressing a point‐mutated c‐erbB‐2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC‐γ1 activation was attributed to this structure. Neither PLC‐γ1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC‐γ1 activation time course by c‐erbB‐2 signaling. Induction of cell migration was solely observable in the c‐erbB‐2‐positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D‐ collagen lattice. In summary, c‐erbB‐2 up‐regulation switches on the cell migration program by modulating the time course of PLC‐γ1 activation.

HDL-Associated Lysosphingolipids Inhibit NAD(P)H Oxidase–Dependent Monocyte Chemoattractant Protein-1 Production

Objectives—High-density lipoprotein (HDL) levels are inversely proportional to the risk of atherosclerosis, but mechanisms of HDL atheroprotection remain unclear. Monocyte chemoatractant protein-1 (MCP-1) constitutes an early component of inflammatory response in atherosclerosis. Here we investigated the influence of HDL on MCP-1 production in vascular smooth muscle cells (VSMCs) and rat aortic explants. Methods and Results—HDL inhibited the thrombin-induced production of MCP-1 in a concentration-dependent manner. The HDL-dependent inhibition of MCP-1 production was accompanied by the suppression of reactive oxygen species (ROS), which regulate the MCP-1 production in VSMCs. HDL inhibited NAD(P)H oxidase, the preponderant source of ROS in the vasculature, and prevented the activation of Rac1, which precedes NAD(P)H-oxidase activation. The HDL capacity to inhibit MCP-1 production, ROS generation, and NAD(P)H-oxidase activation was emulated by sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), two lysosphingolipids present in HDL, but not by apolipoprotein A-I. HDL-, S1P-, and SPC-induced inhibition of MCP-1 production was attenuated in VSMCs pretreated with VPC23019, an antagonist of lysosphingolipid receptors S1P1 and S1P3, but not by JTE013, an antagonist of S1P2. In addition, HDL, S1P, and SPC failed to inhibit MCP1 production and ROS generation in aortas from S1P3- and SR-B1–deficient mice. Conclusion—HDL-associated lysosphingolipids inhibit NAD(P)H oxidase-dependent ROS generation and MCP-1 production in a process that requires coordinate signaling through S1P3 and SR-B1 receptors.

HDL3-Mediated Inhibition of Thrombin-Induced Platelet Aggregation and Fibrinogen Binding Occurs via Decreased Production of Phosphoinositide-Derived Second Messengers 1,2-Diacylglycerol and Inositol 1,4,5-tris-Phosphate

We demonstrate that physiological concentrations of HDL3 inhibit the thrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL3-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate turnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation, and intracellular calcium mobilization. The inhibitory effects of HDL3 on inositol 1,4,5-tris-phosphate formation and intracellular calcium mobilization were abolished after covalent modification of HDL3 with dimethylsuberimidate. Furthermore, they could be blocked by calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated protein kinase C inhibitors. However, the inhibitory effects of HDL3 were not blocked by H89, a protein kinase A inhibitor. In addition, HDL3 failed to induce cAMP formation but stimulated the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL3-mediated inhibition of thrombin-induced inositol 1,4,5-tris-phosphate formation, intracellular calcium mobilization, and fibrinogen binding and the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. Taken together, these findings indicate that the HDL3-mediated inhibition of thrombin-induced fibrinogen binding and aggregation occurs via inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and formation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Protein kinase C may be involved in this process.

Low-density lipoproteins inhibit the Na+/H+ antiport in human platelets : A novel mechanism enhancing platelet activity in hypercholesterolemia

BACKGROUND LDL have been reported to augment platelet activation, and increased platelet reactivity has been observed in familial hypercholesterolemia. However, the underlying mechanisms of this putatively atherogenic effect is unknown. Because intracellular pH (pHi) may play an important role in platelet function, we examined the influence of LDL on pHi and Na+/H+ antiport activity in human platelets and compared it with the effect of [3-methylsulfonyl-4-piperidinobenzoyl] guanidine hydrochloride (HOE 694), a selective Na+/H+ antiport inhibitor. METHODS AND RESULTS Using a fluorescent dye technique, we demonstrated that incubation of platelets with physiological concentrations of LDL or with HOE 694 decreased pHi. In addition, both LDL and HOE 694 inhibited the Na+/H+ antiport in platelets treated with sodium propionate or thrombin. The inhibitory effect of LDL was observed both in normal and in glycoprotein (GP)IIb/IIIa-as well as in GPIIIb (CD36)-deficient platelets and was not influenced by the covalent modification of apolipoprotein B lysine residues, suggesting that specific LDL binding sites were not involved. Thrombin-induced phosphoinositide breakdown, diacylglycerol formation, and Ca2+ mobilization, as well as platelet aggregation and granule secretion, were potentiated by both LDL and HOE 694. pHi and Na+/H+ antiport activity were significantly reduced in platelets from patients with familial hypercholesterolemia. Both parameters were normalized after normalization of LDL levels by apheresis treatment. CONCLUSIONS LDL inhibits the Na+/H+ antiport most likely via receptor-independent mechanisms, thereby augmenting platelet reactivity. This novel mechanisms explains increased platelet reactivity in patients with familial hypercholesterolemia and may contribute to the atherogenic potential of LDL.

c-erbB-2/EGFR as dominant heterodimerization partners determine a motogenic phenotype in human breast cancer cells

Separate mechanisms for oncogenesis and metastasis have been postulated. We show here that prolonged and invasive cell migration, a key mechanism in cancer metastasis, is linked to c‐erbB‐2 signaling. Cell lines with c‐erbB‐2 and EGFR expression and transphosphorylation activity display a high transendothelial invasiveness in an endothelial‐extra‐cellular matrix model mimicking a capillary vessel wall in vitro. Tyrosine‐phosphorylated c‐erbB‐2 receptors and EGFR are localized predominantly in areas of the cell with high membrane extension activity. On the molecular level, there is a subtle cross talk between the transmembrane signaling molecule c‐erbB‐2 and the actin cytoskeleton at multiple levels, including the generation of the second messenger PIP2 and the mobilization of the actin‐regulatory protein gelsolin. Our data strongly suggest that c‐erbB‐2, especially in a heterodimer with EGFR, is closely involved in signaling pathways, inducing alterations in cell morphology that are required for a human breast cancer cell to become motile and conceivably metastatic.—Brandt, B. H., Roetger, A., Dittmar, T., Nikolai, G., Seeling, M., Merschjann, A., Nofer, J.‐R., Dehmer‐Möller, G., Junker, R., Assmann, G., Zaenker. K. S. c‐erbB‐2/EGFR as dominant heterodimerization partners determine a motogenic phenotype in human breast cancer cells. FASEB J. 13, 1939–1949 (1999)

High-density lipoproteins, platelets and the pathogenesis of atherosclerosis.

1. Prospective and interventional studies demonstrate an inverse relationship between plasma high‐density lipoprotein (HDL)–cholesterol and the incidence of coronary artery disease. Although the atheroprotective effects of HDL are usually attributed to the reverse cholesterol transport, in which HDL shuttles cholesterol from cells in the arterial wall to the liver, other mechanisms are also under investigation.

Branched Chain Fatty Acids Induce Nitric Oxide-dependent Apoptosis in Vascular Smooth Muscle Cells

Clinical observations in patients with peroxisomal disorders and studies employing corresponding mouse models have shown that supraphysiological concentrations of dietary branched chain fatty acids (BCFAs) are associated with a high level of toxicity, which is poorly understood at present. Here we show that phytanic and pristanic acid, two BCFAs that are metabolized in peroxisomes, promote apoptosis in cultured vascular smooth muscle cells of human, rat, and porcine origin. Under the conditions used, the apoptosis-promoting effect of BCFAs was neither shared by saturated or unsaturated straight chain fatty acids nor by artificial peroxisome proliferators, which, like phytanic and pristanic acid, have been shown to activate the peroxisome proliferator-activated receptor α (PPARα). We could demonstrate, however, that BCFA induced tumor necrosis factor α (TNFα) activation and secretion, which is an obligatory step required for induction of apoptosis by BCFAs. Furthermore, incubation of VSMCs with BCFA increased inducible nitric-oxide synthase (iNOS) mRNA and protein concentrations markedly within 2 h of treatment. Correspondingly, apoptosis was significantly reduced when the cells were co-treated with the competitive NOS inhibitors monomethyl-l-arginine monoacetate and aminoguanidine. Moreover, co-incubation with TGFβ1, previously shown to destabilize iNOS mRNA, also abolished apoptosis. These results establish a new signaling cascade in which natural BCFA induced NO-dependent apoptosis, which is apparently triggered by autocrine secretion of TNFα in cultured VSMCs.

HDL3 Stimulates Multiple Signaling Pathways in Human Skin Fibroblasts

The influence of HDL3 on phospholipid breakdown was examined in human skin fibroblasts. HDL3 elicited phosphatidylcholine (PC) and phosphatidylinositol (PI) turnover and activated multiple phospholipases. In [14C]lyso-PC-labeled or [14C]choline (Cho)-labeled cells, a biphasic activation of PC-specific phospholipase D (PLD) with peak maxima 30 to 60 seconds and 5 to 7 minutes after stimulation with 20 micrograms/mL HDL3 was shown by (1) a 1.5- to 3-fold increase in Cho release, and (3) transphosphatidylation of PC to phosphatidylbutanol in the presence of 0.3% butanol. Activation of PC-specific PLD was paralleled by an activation of PC-specific phospholipase C (PLC). A significant increase in [14C]diacylglycerol (DG) was seen from 2 minutes after stimulation onward and remained for at least 2 hours. By means of butanol, the PA-phosphohydrolase (PPH) inhibitor propranolol, and the PC-PLC inhibitor D609, we demonstrated that the initial PC-derived DG formation occurred primarily by a coupled PLD/PPH pathway and that a major part of the sustained DG formation was derived directly from PC by PC-PLC. By down-regulating protein kinase C (PKC) we demonstrated that PKC activates PC-PLC and desensitizes PC-PLD at no longer incubation times. The sustained PC hydrolysis as well as HDL3-mediated PI turnover and PC resynthesis was observed on stimulation with 5 to 75 micrograms/mL HDL3, whereas the rapid activation of PC-PLD/PPH was detected only on stimulation with HDL3 at concentrations of between 10 and 75 micrograms/mL. Only the latter response could be mimicked by apolipoprotein A-I and apolipoprotein A-II proteoliposomes, and only this response was inducible by cholesterol loading. The HDL3-mediated second-messenger responses were inhibited by modification of HDL3 by tetranitromethane and could not be mimicked by protein-free liposomes. These data suggest that HDL3-induced cell signaling in human skin fibroblasts is mediated by specific protein-receptor interaction and that more than one agonist activity may be involved.