Differential Trafficking of Oxidized LDL and Oxidized LDL Immune Complexes in Macrophages: Impact on Oxidative Stress

Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCgamma receptor I (FCgamma RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC.Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B'

Sphingosine 1-Phosphate Distribution in Human Plasma: Associations with Lipid Profiles

The physiological significance of sphingosine 1-phosphate (S1P) transport in blood has been debated. We have recently reported a comprehensive sphingolipid profile in human plasma and lipoprotein particles (VLDL, LDL, and HDL) using HPLC-MS/MS (Hammad et al., 2010). We now determined the relative concentrations of sphingolipids including S1P in the plasma subfraction containing lipoproteins compared to those in the remaining plasma proteins. Sphingomyelin and ceramide were predominantly recovered in the lipoprotein-containing fraction. Total plasma S1P concentration was positively correlated with S1P concentration in the protein-containing fraction, but not with S1P concentration in the lipoprotein-containing fraction. The percentage of S1P transported in plasma lipoproteins was positively correlated with HDL cholesterol (HDL-C) concentration; however, S1P transport in lipoproteins was not limited by the concentration of HDL-C in the individual subject. Thus, different plasma pools of S1P may have different contributions to S1P signaling in health and disease

Decreased plasma levels of select very long chain ceramide species are associated with the development of nephropathy in type 1 diabetes.

OBJECTIVE: Sphingolipid metabolism is altered in diabetes and we analyzed the plasma concentrations of sphingolipid species to investigate their association with the development of albuminuria in type 1 patients with diabetes. MATERIALS AND METHODS: Samples were collected from 497 type 1 diabetic patients during their enrollment into the Diabetes Control and Complications Trial (DCCT). We determined plasma concentrations of multiple ceramide species and individual sphingoid bases and their phosphates using high performance liquid chromatography-tandem mass spectrometry and investigated their association with the development of albuminuria during 14-20 years of follow-up. RESULTS: Patients exhibited normal albumin excretion rates (AER/24h) at the time of plasma sampling. Although the majority of patients (N = 291; 59%) exhibited normal levels of albuminuria throughout follow-up, 141 patients (28%) progressed to microalbuminuria (40 mg/24h ≤ AER/24h), while 65 (13%) progressed to macroalbuminuria (AER ≥ 300 mg/24h). To test the association of log transformed plasma sphingolipid level with the development of albuminuria, generalized logistic regression models were used where normal, micro- and macroalbuminuria were the outcomes of interest. Models were adjusted for DCCT treatment group, baseline retinopathy, gender, baseline HbA1c %, age, AER, lipid levels, diabetes duration, and the use of ACE/ARB drugs. Increased plasma levels of very long, but not long chain ceramide species measured at DCCT baseline were associated with decreased odds to develop macroalbuminuria during the subsequent nineteen years (DCCT Baseline to EDIC year 8). CONCLUSION: These studies demonstrate, prospectively, that decreased plasma levels of select ceramide species are associated with the development of macroalbuminuria in type 1 diabetes

Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in lysosomal compartment.

<p>U937 cells were treated with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h, with lysosomal marker (Lyso Tracker Green DND-26, 50 nM) applied for the last 30 min of incubation. Cells were treated with labeled oxLDL and oxLDL-IC at 18 µg/ml and 24 µg/ml, respectively. Live cells were washed with DPBS then suspended in sealed capillaries and visualized using confocal microscopy. Arrows point at co-localization of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC with lysosomal compartment.</p

Characterization of oxLDL labeling and uptake by U937 cells.

<p>(<b>A</b>) Migration of fluorescently labeled oxLDL analyzed by agarose gel electrophoresis: lane 1: native LDL, lane 2: oxLDL, lane 3: DiI-oxLDL, lane 4: DiO-oxLDL, lane 5: Alexa 546-oxLDL, lane 6: LPDS. (<b>B</b>) Uptake of fluorescently labeled oxLDL. U937 cells were treated with labeled oxLDL: DiO-oxLDL, DiI-oxLDL, or Alexa 546-oxLDL (24 µg/ml) for 5 h then fixed with 4% formaldehyde and visualized in sealed capillaries using confocal microscopy. (<b>C</b>) FACS analysis showing dose-dependent uptake of labeled oxLDL 90 min post treatment in U937 cells. Each data point represents the mean ± range of duplicate determinations (1×10⁴ cells/determination), and data presented are representative of four independent experiments.</p

Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in endosomal vesicles.

<p>U937 cells were treated with endosomal marker (Alexa 488-transferrin, green) and with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h. Cells were treated with Alexa 488-transferrin (5 µg/ml) and with either labeled oxLDL (24 µg/ml) or labeled oxLDL-IC (32 µg/ml), fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 laser scanning confocal microscope. Arrows point at co-localization of lipid and apolipoprotein moities of oxLDL and oxLDL-IC with Alexa 488-transferrin.</p

Lipid moieties of oxLDL and oxLDL-IC co-localize when administered sequentially but not simultaneously.

<p>U937 cells were incubated with DiI-oxLDL-IC (red) and DiO-oxLDL (green) (<b>A</b>) sequentially and (<b>B</b>) in parallel in U937 cells. They were incubated for a total of 5 h. Sequential experiment involved 2 h incubation of oxLDL-IC (32 µg/ml), prior to addition of oxLDL (24 µg/ml) for 3 h. Lyso Tacker Blue DND-22, (50 nM) was used and applied for the last 30 min of incubation. Cells were fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope. Arrows point at co-localization of oxLDL in the lysosomal compartment in the upper panel, and the co-localization of oxLDL and oxLDL-IC in the lower panel.</p

Lipid moiety of oxLDL-IC but not oxLDL co-localizes with induced HSP70/70B' in endosomal vesicles.

<p>RAW 264.7 cells were transfected with HSP70-GFP (<b>A</b>) or HSP70B'-GFP (<b>B</b>), then treated with DiI-oxLDL (24 µg/ml), DiI-oxLDL-IC (32 µg/ml), or DPBS vehicle in serum-free DMEM for 3 h. Alexa 633-transferrin (10 µg/ml) was then added for an additional 2 h. Cells were then fixed with 4% formaldehyde, washed with DPBS, and visualized using confocal microscopy.</p