Unsaturated lipid peroxidation-derived aldehydes activate autophagy in vascular smooth-muscle cells

Proteins modified by aldehydes generated from oxidized lipids accumulate in cells during oxidative stress and are commonly detected in diseased or aged tissue. The mechanisms by which cells remove aldehyde-adducted proteins, however, remain unclear. Here, we report that products of lipid peroxidation such as 4-HNE (4-hydroxynonenal) and acrolein activate autophagy in rat aortic smooth-muscle cells in culture. Exposure to 4-HNE led to the modification of several proteins, as detected by anti-protein-4-HNE antibodies or protein-bound radioactivity in [3H]4-HNE-treated cells. The 4-HNE-modified proteins were gradually removed from cells. The removal of 4-HNE-modified proteins was not affected by the oxidized protein hydrolase inhibitor, acetyl leucine chloromethyl ketone, or lactacystin, although it was significantly decreased by PSI (proteasome inhibitor I), the lysosome/proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-leucinal), insulin or the autophagy inhibitor 3-MA (3-methyladenine). Pre-incubation of cells with rapamycin accelerated the removal of 4-HNE-modified proteins. Treatment with 4-HNE, nonenal and acrolein, but not nonanal or POVPC (1-palmitoyl-2-oxovaleroyl phosphatidyl choline), caused a robust increase in LC3-II (microtubule-associated protein 1 light chain 3-II) formation, which was increased also by rapamycin, but prevented by insulin. Electron micrographs of 4-HNE-treated cells showed extensive vacuolization, pinocytic body formation, crescent-shaped phagophores, and multilamellar vesicles. Treatment with 3-MA and MG-132, but not proteasome-specific inhibitors, induced cell death in 4-HNE-treated cells. Collectively, these results show that lipid peroxidation-derived aldehydes stimulate autophagy, which removes aldehyde-modified proteins, and that inhibition of autophagy precipitates cell death in aldehyde-treated cells. Autophagy may be an important mechanism for the survival of arterial smooth-muscle cells under conditions associated with excessive lipid peroxidation.     

Myocardial ischaemia inhibits mitochondrial metabolism of 4-hydroxy-trans-2-nonenal

Myocardial ischaemia is associated with the generation of lipid peroxidation products such as HNE (4-hydroxy-trans-2-nonenal); however, the processes that predispose the ischaemic heart to toxicity by HNE and related species are not well understood. In the present study, we examined HNE metabolism in isolated aerobic and ischaemic rat hearts. In aerobic hearts, the reagent [(3)H]HNE was glutathiolated, oxidized to [(3)H]4-hydroxynonenoic acid, and reduced to [(3)H]1,4-dihydroxynonene. In ischaemic hearts, [(3)H]4-hydroxynonenoic acid formation was inhibited and higher levels of [(3)H]1,4-dihydroxynonene and [(3)H]GS-HNE (glutathione conjugate of HNE) were generated. Metabolism of [(3)H]HNE to [(3)H]4-hydroxynonenoic acid was restored upon reperfusion. Reperfused hearts were more efficient at metabolizing HNE than non-ischaemic hearts. Ischaemia increased the myocardial levels of endogenous HNE and 1,4-dihydroxynonene, but not 4-hydroxynonenoic acid. Isolated cardiac mitochondria metabolized [(3)H]HNE primarily to [(3)H]4-hydroxynonenoic acid and minimally to [(3)H]1,4-dihydroxynonene and [(3)H]GS-HNE. Moreover, [(3)H]4-hydroxynonenoic acid was extruded from mitochondria, whereas other [(3)H]HNE metabolites were retained in the matrix. Mitochondria isolated from ischaemic hearts were found to contain 2-fold higher levels of protein-bound HNE than the cytosol, as well as increased [(3)H]GS-HNE and [(3)H]1,4-dihydroxynonene, but not [(3)H]4-hydroxynonenoic acid. Mitochondrial HNE oxidation was inhibited at an NAD(+)/NADH ratio of 0.4 (equivalent to the ischaemic heart) and restored at an NAD(+)/NADH ratio of 8.6 (equivalent to the reperfused heart). These results suggest that HNE metabolism is inhibited during myocardial ischaemia owing to NAD(+) depletion. This decrease in mitochondrial metabolism of lipid peroxidation products and the inability of the mitochondria to extrude HNE metabolites could contribute to myocardial ischaemia/reperfusion injury.     

Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria

In Gram-negative bacteria, the envelope is a sophisticated barrier protecting the cell against external toxic compounds. Membrane transporters, e.g., porins or efflux pumps, are main filters regulating the internal accumulation of various hydrophilic molecules. Regarding bacterial susceptibility towards antibacterial agents, membrane permeability is part of the early bacterial defense. The bacterium manages the translocation process, influx and efflux, to control the intracellular concentration of various molecules. Antibiotics and biocides are substrates of these mechanisms and the continuing emergence of multidrug resistant isolates is a growing worldwide health concern. Different strategies could be proposed to bypass the bacterial membrane barrier, comprising influx and efflux mechanisms, in order to restore the activity of antibiotics against resistant bacteria.     

Reductive metabolism increases the proinflammatory activity of aldehyde phospholipids

The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1β, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A(2) (PLA(2)) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.     

Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone ('core' aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte-endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C(16:0-20:4) phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C(16:0-20:4) phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.     

Complete Genome Sequence of the Multiresistant Taxonomic Outlier Pseudomonas aeruginosa PA7

Pseudomonas aeruginosa PA7 is a non-respiratory human isolate from Argentina that is multiresistant to antibiotics. We first sequenced gyrA, gyrB, parC, parE, ampC, ampR, and several housekeeping genes and found that PA7 is a taxonomic outlier. We report here the complete sequence of the 6,588,339 bp genome, which has only about 95% overall identity to other strains. PA7 has multiple novel genomic islands and a total of 51 occupied regions of genomic plasticity. These islands include antibiotic resistance genes, parts of transposons, prophages, and a pKLC102-related island. Several PA7 genes not present in PAO1 or PA14 are putative orthologues of other Pseudomonas spp. and Ralstonia spp. genes. PA7 appears to be closely related to the known taxonomic outlier DSM1128 (ATCC9027). PA7 lacks several virulence factors, notably the entire TTSS region corresponding to PA1690-PA1725 of PAO1. It has neither exoS nor exoU and lacks toxA, exoT, and exoY. PA7 is serotype O12 and pyoverdin type II. Preliminary proteomic studies indicate numerous differences with PAO1, some of which are probably a consequence of a frameshift mutation in the mvfR quorum sensing regulatory gene.     

Aldose reductase-catalyzed reduction of aldehyde phospholipids

Oxidation of unsaturated phospholipids results in the generation of aldehyde side chains that remain esterified to the phospholipid backbone. Such "core" aldehydes elicit immune responses and promote inflammation. However, the biochemical mechanisms by which phospholipid aldehydes are metabolized or detoxified are not well understood. In the studies reported here, we examined whether aldose reductase (AR), which reduces hydrophobic aldehydes, metabolizes phospholipid aldehydes. Incubation with AR led to the reduction of 5-oxovaleroyl, 7-oxo-5-heptenoyl, 5-hydroxy-6-oxo-caproyl, and 5-hydroxy-8-oxo-6-octenoyl phospholipids generated upon oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC). The enzyme also catalyzed the reduction of phospholipid aldehydes generated from the oxidation of 1-alkyl, and 1-alkenyl analogs of PAPC, and 1-palmitoyl-2-arachidonoyl phosphatidic acid or phosphoglycerol. Aldose reductase catalyzed the reduction of chemically synthesized 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POVPC) with a K(m) of 10 mum. Addition of POVPC to the culture medium led to incorporation and reduction of the aldehyde in COS-7 and THP-1 cells. Reduction of POVPC in these cells was prevented by the AR inhibitors sorbinil and tolrestat and was increased in COS-7 cells overexpressing AR. Together, these observations suggest that AR may be a significant participant in the metabolism of several structurally diverse phospholipid aldehydes. This metabolism may be a critical regulator of the pro-inflammatory and immunogenic effects of oxidized phospholipids.