Lysosomal oxidation of LDL alters lysosomal pH, induces senescence and increases secretion of pro-inflammatory cytokines in human macrophages

Objective We have shown that aggregated low density lipoproteins (LDL) is internalised by macrophages and oxidised in lysosomes by redox-active iron. We have now investigated if the lysosomal oxidation of LDL impairs lysosomal function and if a lysosomotropic antioxidant can prevent these alterations. Approach and Results LDL aggregated by sphingomyelinase (SMase-LDL) caused increased lysosomal lipid peroxidation in human monocyte-derived macrophages or THP-1 macrophage-like cells, as shown by a fluorescent probe, Foam-LPO. The pH of the lysosomes was increased considerably by lysosomal LDL oxidation as shown by Lysosensor Yellow/Blue and LysoTracker Red. SMase-LDL induced senescence-like properties in the cells as shown by β-galactosidase staining and levels of p53 and p21. Inflammation plays a key role in atherosclerosis. SMase-LDL treatment increased the LPS-induced secretion of TNF-α, IL-6 and MCP-1. The lysosomotropic antioxidant, cysteamine inhibited all of the above changes. Conclusions Targeting lysosomes with antioxidants, such as cysteamine, to prevent the intralysosomal oxidation of LDL might be a novel therapy for atherosclerosis

Multi-fluid simulations of chromospheric magnetic reconnection in a weakly ionized reacting plasma

We present results from the first self-consistent multi-fluid simulations of chromospheric magnetic reconnection in a weakly ionized reacting plasma. We simulate two dimensional magnetic reconnection in a Harris current sheet with a numerical model which includes ion-neutral scattering collisions, ionization, recombination, optically thin radiative loss, collisional heating, and thermal conduction. In the resulting tearing mode reconnection the neutral and ion fluids become decoupled upstream from the reconnection site, creating an excess of ions in the reconnection region and therefore an ionization imbalance. Ion recombination in the reconnection region, combined with Alfv\'{e}nic outflows, quickly removes ions from the reconnection site, leading to a fast reconnection rate independent of Lundquist number. In addition to allowing fast reconnection, we find that these non-equilibria partial ionization effects lead to the onset of the nonlinear secondary tearing instability at lower values of the Lundquist number than has been found in fully ionized plasmas.These simulations provide evidence that magnetic reconnection in the chromosphere could be responsible for jet-like transient phenomena such as spicules and chromospheric jets.Comment: 8 Figures, 32 pages tota

Low density lipoprotein oxidation by ferritin at lysosomal pH

Oxidation of low density lipoprotein (LDL) has been proposed to be involved in the pathogenesis of atherosclerosis. We have previously shown that LDL can be oxidised by iron in lysosomes. As the iron-storage protein ferritin might enter lysosomes by autophagy, we have investigated the ability of ferritin to catalyse LDL oxidation at lysosomal pH. LDL was incubated with ferritin at 37 °C and pH 4.5 and its oxidation monitored spectrophotometrically at 234 nm by the formation of conjugated dienes and by measuring oxidised lipids by HPLC or a tri-iodide assay. Iron released from ferritin was measured using the ferrous iron chelator bathophenanthroline and by ultrafiltration followed by atomic absorption spectroscopy. LDL was oxidised effectively by ferritin (0.05–0.2 μM). The oxidation at lysosomal pH (pH 4.5) was much faster than at pH 7.4. Ferritin increased cholesteryl linoleate hydroperoxide, total lipid hydroperoxides and 7-ketocholesterol. Iron was released from ferritin at acidic pH. The iron chelators, diethylenetriaminepentaacetate and EDTA, and antioxidant N,N ׳-diphenyl-p-phenylenediamine inhibited the oxidation considerably, but not entirely. The antioxidant tempol did not inhibit the initial oxidation of LDL, but inhibited its later oxidation. Cysteamine, a lysosomotropic antioxidant, inhibited the initial oxidation of LDL in a concentration-dependent manner, however, the lower concentrations exhibited a pro-oxidant effect at later times, which was diminished and then abolished as the concentration increased. These results suggest that ferritin might play a role in lysosomal LDL oxidation and that antioxidants that accumulate in lysosomes might be a novel therapy for atherosclerosis. 1. Introduction The oxidation of low density lipoprotein (LDL) has been proposed to occur in the extracellular space of the arterial wall and lead to the formation of foam cells and atherosclerosis (Steinberg, 2009). The oxidation of LDL by cells requires the presence of micromolar concentrations of the transition metals copper or iron in the medium (Steinbrecher et al., 1984; Leake and Rankin, 1990). Free copper or iron are not readily available in the plasma or interstitial fluid because they exist in a tightly bound form. A number of mechanisms have been proposed to be involved in the oxidation of LDL in vivo, but at present, there is no consensus on the predominant mechanism by which LDL is modified in vivo. Cultured macrophages have been shown, however, to take up aggregated or acetylated LDL quickly and oxidise it in lysosomes (Wen and Leake, 2007). Cholesterol crystals derived from oxidised LDL in lysosomes have been reported to rupture these organelles in macrophages and activate the NLRP3 inflammasome (Duewell et al., 2010). This might be important as atherosclerosis is a chronic inflammatory disease an

Iron released from transferrin at acidic pH can catalyse the oxidation of low density lipoprotein

AbstractLow density lipoprotein (LDL) oxidation within the arterial wall may contribute to the disease of atherosclerosis. We have investigated the conditions under which transferrin (the major iron-carrying protein in plasma) may release iron ions to catalyse the oxidation of LDL. Transferrin that had been incubated at pH 5.5 released approximately 10% of its bound iron in 24 h, as measured by ultrafiltration and atomic absorption spectroscopy. Furthermore, transferrin co-incubated with LDL and l-cysteine at pH 5.5 resulted in the oxidation of the LDL as measured by thiobarbituric acid-reactive substances and electrophoretic mobility. This effect was observed at transferrin concentrations as low as 40% of its average plasma concentration. The release of iron from transferrin in atherosclerotic lesions due to a localised acidic pH may help to explain why LDL oxidation occurs in these lesions

Structural Examination of Au/Ge(001) by Surface X-Ray Diffraction and Scanning Tunneling Microscopy

The one-dimensional reconstruction of Au/Ge(001) was investigated by means of autocorrelation functions from surface x-ray diffraction (SXRD) and scanning tunneling microscopy (STM). Interatomic distances found in the SXRD-Patterson map are substantiated by results from STM. The Au coverage, recently determined to be 3/4 of a monolayer of gold, together with SXRD leads to three non-equivalent positions for Au within the c(8x2) unit cell. Combined with structural information from STM topography and line profiling, two building blocks are identified: Au-Ge hetero-dimers within the top wire architecture and Au homo-dimers within the trenches. The incorporation of both components is discussed using density functional theory and model based Patterson maps by substituting Germanium atoms of the reconstructed Ge(001) surface.Comment: 5 pages, 3 figure