HDL3 stimulates paraoxonase 1 antiatherogenic catalytic and biological activities in a macrophage model system: in vivo and in vitro studies.

We analyzed in-vivo and in-vitro high density lipoprotein (HDL) effects on paraoxonase 1 (PON1) antiatherogenic properties in serum and in macrophages. Intraperitoneal injection to C57BL/6 mice of recombinant PON1 (rePON1) + HDL, in comparison to HDL or to rePON1 alone, significantly increased serum PON1 arylesterase activity (by 20%), and serum-mediated cholesterol efflux from J774A.1 macrophages (by 18%). Similarly, in peritoneal macrophages (MPM) harvested from mice injected with HDL + rePON1 versus rePON1 alone, we observed reduction in oxidative stress (by 11%), increase in cellular PON1 activity (by 14%) and in HDL-mediated cholesterol efflux (by 38%). Incubation of serum or HDL with rePON1, substantially increased PON1 arylesterase activity, two-fold more than the expected additive values. HDL2 and HDL3 increased PON1 activity by 199% or 274%, respectively. Macrophage (J774A.1) cholesterol efflux rate significantly increased by HDL3 + rePON1 versus HDL3 alone (by 19%), but not by HDL2 + rePON1 versus HDL2 alone. Oxidation of HDL3 reduced its ability to induce macrophage cholesterol efflux, and abolished HDL3 stimulatory effects on rePON1. Addition of exogenous polyphenol quercetin (60 µM), but not phosphatidylcholine or apolipoprotein A1, to HDL + rePON1 increased PON1 activity (by 404%), increased the ability to reduce oxidative stress in J774A.1 macrophages (by 53%) and to stimulate macrophage cholesterol efflux (by 14%). Upon adding the hypocholesterolemic drug simvastatin (15 µg/mL) to HDL + rePON1, PON1 activity and the ability to induce macrophage cholesterol efflux increased, in comparison to HDL + rePON1. We thus concluded that HDL (mostly HDL3), stimulates PON1 antiatherogenic activities in macrophages, and these PON1 activities were further stimulated by quercetin, or by simvastatin.

HDL inhibits endoplasmic reticulum stress by stimulating apoE and CETP secretion from lipid-loaded macrophages.

The role of HDL in the modulation of endoplasmic reticulum (ER) stress in macrophage-derived foam cells is not completely understood. Therefore, we aimed to investigate whether HDL may inhibit ER stress in correlation with the secretion of apoE and CETP from lipid-loaded macrophages. To this purpose, THP-1 macrophages were loaded with lipids by incubation with human oxidized LDL (oxLDL) and then exposed to human HDL3. ER stress signaling markers, protein kinase/Jun-amino-terminal kinase (SAPK/JNK p54/p46) and eukaryotic initiation factor-2α (eIF2α), as well as the secreted apoE and CETP, were evaluated by immunoblot analysis. Out of the many different bioactive lipids of oxLDL, we tested the effect of 9-hydroxy-octadecadienoic acid (9-HODE) and 4-hydroxynonenal (4-HNE) on ER stress. Tunicamycin was used as positive control for ER stress induction. Results showed that oxLDL, 9-HODE and 4-HNE induce ER stress in human macrophages by activation of eIF-2α and SAPK/JNK (p54/p46) signaling pathways. OxLDL stimulated apoE and CETP secretion, while tunicamycin determined a reduction of the secreted apoE and CETP, both in control and lipid-loaded macrophages. The addition of HDL3 to the culture medium of tunicamycin-treated cells induced: (i) the reduction of ER stress, expressed as decreased levels of eIF-2α and SAPK/JNK, and (ii) a partial recovery of the secreted apoE and CETP levels in lipid-loaded macrophages. These data suggest a new mechanism by which HDL3 diminish ER stress and stimulate cholesterol efflux from lipid-loaded macrophages.

Impaired antioxidant action of high density lipoprotein in patients with type 1 diabetes with normoalbuminuria and microalbuminuria.

AIMS: Patients with type 1 diabetes, in the absence of chronic complications, have serum concentrations of high density lipoprotein cholesterol (HDL-C) similar to the general population. However, their HDL particles may be dysfunctional. We aimed to evaluate the antioxidant effect of HDL2 and HDL3 obtained from Caucasian males with type 1 diabetes with normoalbuminuria and microalbuminuria. METHODS: Twenty Caucasian men with type 1 diabetes (10 with normoalbuminuria and 10 with microalbuminuria) and 10 healthy Caucasian men participated in the study. Lipoproteins were obtained by density gradient ultracentrifugation. The antioxidant effect of HDL was assessed by measuring lipid hydroperoxide (LOOH) concentration after 3h of pooled LDL oxidation catalyzed by 5µM CuSO4 in the absence or presence of HDL2 or HDL3. RESULTS: The control, normoalbuminuria, and microalbuminuria groups had similar HDL-C concentration and estimated glomerular filtration rate. Glycemic control was similar between diabetes groups (HbA1c 8.1±0.9% and 8.3±0.7%, P=0.70), but estimated glucose disposal rate was lower in patients with microalbuminuria (8.0±0.6 and 4.5±1.1mg/kg/min, P<0.01). The relative antioxidant effect of HDL2 from control, normoalbuminuria, and microalbuminuria groups were 92.8±2.4%, 85.4±1.7%, and 74.2±4.6%, respectively (P<0.01), and the HDL3 effect were 95.0±2.2%, 86.4±4.4%, and 75.3±4.2%, respectively (P<0.01). CONCLUSION: Both HDL2 and HDL3 inhibited LOOH formation in copper-catalyzed oxidation of LDL in vitro. Overall, this antioxidant effect was lower in Caucasian men with type 1 diabetes, and was further compounded in those with microalbuminuria.

Mutual self-defence: the trypanolytic factor story.

Around 1900 Laveran and Mesnil discovered that African trypanosomes (prototype: Trypanosoma brucei brucei) do not survive in the blood of some primates and humans. The nature of the trypanolytic factor present in these sera has been the focus of a long-standing debate between different groups, but recent developments have allowed the proposal of a coherent model incorporating most seemingly divergent views and providing an interesting example of the complex interplay that continuously occurs between hosts and parasites. Possibly as an adaptation to their natural environment, great African apes and humans have acquired a new member of the apolipoprotein-L family, termed apoL1. This protein is the only one of the family to be secreted in the blood, where it binds to a subset of HDL particles that also contain another human-specific protein, haptoglobin-related protein or Hpr. T. b. brucei possesses a specific surface receptor for the haptoglobin-hemoglobin (Hp-Hb) complex, as a way to capture heme into hemoproteins that contribute to cell growth and resistance to the oxidative stress of the host. As this receptor does not discriminate between Hp and Hpr, Hpr-containing HDL particles of human serum are efficiently taken up by the parasite, leading to the simultaneous internalization of apoL1, Hpr and Hb-derived heme. Once in the lysosome, apoL1 is targeted to the lysosomal membrane, where its colicin-like anionic pore-forming activity triggers an influx of chloride ions from the cytoplasm. Osmotic effect linked to this ionic flux leads to uncontrolled swelling of the lysosome, ultimately causing the death of the parasite. Two T. brucei clones, termed Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense, have managed to resist this lysis mechanism and, therefore, cause sleeping sickness in humans. While the mechanism of this resistance is still not known in the case of T. b. gambiense, the dominant factor responsible for resistance of T. b. rhodesiense has been identified. This protein, named SRA for Serum Resistance-Associated, is a truncated version of the major and variable surface antigen of the parasite, the Variant Surface Glycoprotein or VSG. Presumably due to its defective nature, SRA is not targeted to the plasma membrane as do regular VSGs, but ends up in the late endosomal compartment. In this location SRA is thought to neutralize apoL1 through coiled-coil interactions between alpha-helices. We discuss the potential of these discoveries in terms of fight against the disease.

The 15-lipoxygenase-modified high density lipoproteins 3 fail to inhibit the TNF-alpha-induced inflammatory response in human endothelial cells.

Endothelial dysfunction represents one of the earliest events in vascular atherogenesis. Proinflammatory stimuli activate endothelial cells, resulting in an increased expression of adhesion molecules and chemoattractants that mediate leukocyte and monocyte adhesion, migration, and homing. High density lipoproteins (HDL) inhibit endothelial cell expression of adhesion molecules in response to proinflammatory stimuli. In the present work, we demonstrate that the modification of HDL(3) (the major and the most antiatherogenic HDL subfraction) by 15-lipoxygenase (15-LO), an enzyme overexpressed in the atherosclerotic lesions, impairs the anti-inflammatory activity of this lipoprotein. The 15-LO-modified HDL(3) failed to inhibit TNF-alpha-mediated mRNA and protein induction of adhesion molecules and MCP-1 in several models of human endothelial cells, and promoted inflammatory response by up-regulating the expression of such mediators of inflammation and by increasing monocyte adhesion to endothelial cells. Moreover, 15-LO-modified HDL(3) were unable to contrast the formation of reactive oxygen species in cells incubated with TNF-alpha, and increased the reactive oxygen species content in unstimulated cells. Activation of NF-kappaB and AP-1 was mainly involved in the expression of adhesion molecules and MCP-1 induced by 15-LO-HDL(3). Altogether, these results demonstrate that enzymatic modification induced by 15-LO impaired the protective role of HDL(3), generating a dysfunctional lipoprotein endowed with proinflammatory characteristics.