Andrew D. Watson

Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics.

The clinical events resulting from atherosclerosis are directly related to the oxidation of lipids in LDLs that become trapped in the extracellular matrix of the subendothelial space. These oxidized lipids activate an NF kappa B-like transcription factor and induce the expression of genes containing NF kappa B binding sites. The protein products of these genes initiate an inflammatory response that initially leads to the development of the fatty streak. The progression of the lesion is associated with the activation of genes that induce arterial calcification, which changes the mechanical characteristics of the artery wall and predisposes to plaque rupture at sites of monocytic infiltration. Plaque rupture exposes the flowing blood to tissue factor in the lesion, and this induces thrombosis, which is the proximate cause of the clinical event. There appear to be potent genetically determined systems for preventing lipid oxidation, inactivating biologically important oxidized lipids, and/or modulating the inflammatory response to oxidized lipids that may explain the differing susceptibility of individuals and populations to the development of atherosclerosis. Enzymes associated with HDL may play an important role in protecting against lipid oxidation in the artery wall and may account in part for the inverse relation between HDL and risk for atherosclerotic clinical events.

Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein.

Our group has previously demonstrated that oxidized phospholipids in mildly oxidized LDL (MM-LDL) produced by oxidation with lipoxygenase, iron, or cocultures of artery wall cells increase monocyte-endothelial interactions and this sequence of events is blocked by HDL. To obtain further insight into the mechanism by which HDL abolishes the activity of MM-LDL we investigated the effect of the HDL-associated ester hydrolase paraoxonase (PON). Treatment of MM-LDL with purified PON significantly reduced the ability of MM-LDL to induce monocyte-endothelial interactions. Inactivation of PON by pretreating HDL with heat or EDTA reduced the ability of HDL to inhibit LDL modification. HPLC analysis of phospholipids isolated from MM-LDL before and after treatment with purified PON showed that the 270 nm absorbance of phospholipids was decreased, while no effect was observed on 235 nm absorbance. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and specific fractions of Ox-PAPC isolated by HPLC induced the same monocyte-endothelial interactions as did MM-LDL. Biologically active and inactive HPLC fractions of Ox-PAPC were compared by fast atom bombardment-mass spectrometry which revealed that active fractions possessed ions with a mass to charge [correction of change] ratio greater than native PAPC by multiples of 16 D suggesting the addition of 3 and 4 oxygen atoms to PAPC. Comparison of Ox-PAPC by fast atom bombardment-mass spectrometry before and after PON treatment showed that PON destroyed these multi-oxygenated molecules found in biologically active fractions of Ox-PAPC. These results suggest that PON in HDL may protect against the induction of inflammatory responses in artery wall cells by destroying biologically active lipids in mildly oxidized LDL.

The Yin and Yang of Oxidation in the Development of the Fatty Streak A Review Based on the 1994 George Lyman Duff Memorial Lecture

Recent data support the hypothesis that the fatty streak develops in response to specific phospholipids contained in LDL that become trapped in the artery wall and become oxidized as a result of exposure to the oxidative waste of the artery wall cells. The antioxidants present within both LDL and the microenvironments in which LDL is trapped function to prevent the formation of these biologically active, oxidized lipids. Enzymes associated with LDL and HDL (eg, platelet activating factor acetylhydrolase) or with HDL alone (eg, paraoxonase) destroy these biologically active lipids. The regulation and expression of these enzymes are determined genetically and are also significantly modified by environmental influences, including the acute-phase response or an atherogenic diet. The balance of these multiple factors leads to an induction or suppression of the inflammatory response in the artery wall and determines the clinical course.

Lipid Oxidation Products Have Opposite Effects on Calcifying Vascular Cell and Bone Cell Differentiation A Possible Explanation for the Paradox of Arterial Calcification in Osteoporotic Patients

Atherosclerotic calcification and osteoporosis often coexist in patients, yielding formation of bone mineral in vascular walls and its simultaneous loss from bone. To assess the potential role of lipoproteins in both processes, we examined the effects of minimally oxidized low-density lipoprotein (MM-LDL) and several other lipid oxidation products on calcifying vascular cells (CVCs) and bone-derived preosteoblasts MC3T3-E1. In CVCs, MM-LDL but not native LDL inhibited proliferation, caused a dose-dependent increase in alkaline phosphatase activity, which is a marker of osteoblastic differentiation, and induced the formation of extensive areas of calcification. Similar to MM-LDL, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) and the isoprostane 8-iso prostaglandin E2 but not PAPC or isoprostane 8-iso prostaglandin F2 alpha induced alkaline phosphatase activity and differentiation of CVCs. In contrast, MM-LDL and the above oxidized lipids inhibited differentiation of the MC3T3-E1 bone cells, as evidenced by their stimulatory effect on proliferation and their inhibitory effect on the induction of alkaline phosphatase and calcium uptake. These results suggest that specific oxidized lipids may be the common factors underlying the pathogenesis of both atherosclerotic calcification and osteoporosis.

Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein.

Mildly oxidized low density lipoprotein (MM-LDL) produced by oxidative enzymes or cocultures of human artery wall cells induces endothelial cells to produce monocyte chemotactic protein-1 and to bind monocytes. HDL prevents the formation of MM-LDL by cocultures of artery wall cells. Using albumin treatment and HPLC we have isolated and partially characterized bioactive oxidized phospholipids in MM-LDL. Platelet activating factor-acetylhydrolase (PAF-AH), a serine esterase, hydrolyzes short chain acyl groups esterified to the sn-2 position of phospholipids such as PAF and particular oxidatively fragmented phospholipids. Treatment of MM-LDL with PAF-AH (2-4 x 10(-2) U/ml) eliminated the ability of MM-LDL to induce endothelial cells to bind monocytes. When HDL protected against the formation of MM-LDL by cocultures, lysophosphatidylcholine was detected in HDL; whereas when HDL was pretreated with diisopropyl fluorophosphate, HDL was no longer protective and lysophosphatidylcholine was undetectable. HPLC analysis also revealed that the active oxidized phospholipid species in MM-LDL had been destroyed after PAF-AH treatment. In addition, treatment of MM-LDL with albumin removed polar phospholipids that, when reisolated, induced monocyte binding to endothelial cells. These polar phospholipids, when treated with PAF-AH, lost biological activity and were no longer detected by HPLC. These results suggest that PAF-AH in HDL protects against the production and activity of MM-LDL by facilitating hydrolysis of active oxidized phospholipids to lysolipids, thereby destroying the biologically active lipids in MM-LDL.

Thematic review series: Systems Biology Approaches to Metabolic and Cardiovascular Disorders. Lipidomics: a global approach to lipid analysis in biological systems

Lipids are water-insoluble molecules that have a wide variety of functions within cells, including: 1) maintenance of electrochemical gradients; 2) subcellular partitioning; 3) first- and second-messenger cell signaling; 4) energy storage; and 5) protein trafficking and membrane anchoring. The physiological importance of lipids is illustrated by the numerous diseases to which lipid abnormalities contribute, including atherosclerosis, diabetes, obesity, and Alzheimers disease. Lipidomics, a branch of metabolomics, is a systems-based study of all lipids, the molecules with which they interact, and their function within the cell. Recent advances in soft-ionization mass spectrometry, combined with established separation techniques, have allowed the rapid and sensitive detection of a variety of lipid species with minimal sample preparation. A “lipid profile” from a crude lipid extract is a mass spectrum of the composition and abundance of the lipids it contains, which can be used to monitor changes over time and in response to particular stimuli. Lipidomics, integrated with genomics, proteomics, and metabolomics, will contribute toward understanding how lipids function in a biological system and will provide a powerful tool for elucidating the mechanism of lipid-based disease, for biomarker screening, and for monitoring pharmacologic therapy.

Structural Identification of a Novel Pro-inflammatory Epoxyisoprostane Phospholipid in Mildly Oxidized Low Density Lipoprotein

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytesin vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A2 and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product ofm/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.

Evidence for a Role of Phospholipid Oxidation Products in Atherogenesis

There is growing evidence for the accumulation of phospholipid oxidation products (some of which can also be formed enzymatically) in several chronic disease processes including atherosclerosis. There also is considerable evidence that enzymes involved in hydrolysis of these phospholipids (present in both lipoproteins and cells) may be important in regulation of atherogenesis. In vitro studies suggest that these lipids can activate vascular wall cells to states that contribute to the atherosclerotic process. This review focuses on two types of bioactive phospholipids: phosphatidyl cholines in which the sn-2 fatty acid has been modified by oxidation and lysophosphatidic acid in which both the sn-2 and sn-3 positions have been modified. The mechanism by which these phospholipid oxidation products activate cells has revealed the presence of several different receptors and signal transduction pathways.

EGFR Signaling Through an Akt-SREBP-1–Dependent, Rapamycin-Resistant Pathway Sensitizes Glioblastomas to Antilipogenic Therapy

Inhibitors of fatty acid signaling promote apoptosis in glioblastoma cells with highly active EGFR signaling. Inhibiting Lipid Metabolism to Combat Glioblastoma Glioblastoma, the most common form of brain cancer, is frequently lethal. Glioblastoma is often associated with increased signaling through the epidermal growth factor receptor (EGFR); however, therapeutic efforts focused on inhibiting EGFR signaling have been disappointing. Guo et al. analyzed tumor tissue removed from glioblastoma patients before and during treatment with the EGFR inhibitor lapatinib and found that EGFR signaling activated sterol regulatory element–binding protein 1 (SREBP-1), a key regulator of lipid metabolism, and increased the cellular concentrations of fatty acids. Intriguingly, inhibiting fatty acid synthesis promoted apoptosis in glioblastoma cells with substantial EGFR signaling both in vitro and when transplanted into immunodeficient mice, but not in glioblastoma cells with little EGFR signaling. Thus, inhibition of fatty acid synthesis may represent a new avenue toward treating glioblastomas driven by EGFR signaling. Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element–binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1, possibly explaining rapamycin’s poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl coenzyme A reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated prosurvival metabolic pathway and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.

Role of group II secretory phospholipase A2 in atherosclerosis. 2. Potential involvement of biologically active oxidized phospholipids

Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.