Sònia Benítez

Electronegative low-density lipoprotein

Purpose of review The occurrence in blood of an electronegatively charged LDL was described in 1988. During the 1990s reports studying electronegative LDL (LDL(-)) were scant and its atherogenic role controversial. Nevertheless, recent reports have provided new evidence on a putative atherogenic role of LDL(-). This review focuses on and discusses these new findings. Recent findings In recent years, LDL(-) has been found to be involved in several atherogenic features through its action on cultured endothelial cells. LDL(-) induces the production of chemokines, such as IL-8 and monocyte chemotactic protein 1, and increases tumor necrosis factor-α-induced production of vascular cell adhesion molecule 1, with these molecules being involved in early phases of leukocyte recruitment. LDL(-) from familial hypercholesterolemic patients also decreases DNA synthesis and intracellular fibroblast growth factor 2 production, which may contribute to impaired angiogenesis and increased apoptosis. In addition, the preferential association of platelet-activating factor acetylhydrolase with LDL(-) has been reported, suggesting a proinflammatory role of this enzyme in LDL(-). Summary Recent findings suggest that LDL(-) could contribute to atherogenesis via several mechanisms, including proinflammatory, proapoptotic and antiangiogenesis properties. Further studies are required to define the role of LDL(-) in atherogenesis more precisely and to clarify mechanisms involved in endothelial cell activation.

Electronegative LDL From Normolipemic Subjects Induces IL-8 and Monocyte Chemotactic Protein Secretion by Human Endothelial Cells

The presence in plasma of an electronegative LDL subfraction [LDL(−)] cytotoxic for endothelial cells (ECs) has been reported. We studied the effect of LDL(−) on the release by ECs of molecules implicated in leukocyte recruitment [interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1)] and in the plasminogen activator inhibitor-1 (PAI-1). LDL(−), isolated by anion-exchange chromatography, differed from nonelectronegative LDL [LDL(+)] in its higher triglyceride, nonesterified fatty acid, apoprotein E and apoprotein C-III, and sialic acid contents. No evidence of extensive oxidation was found in LDL(−); its antioxidant and thiobarbituric acid–reactive substances contents were similar to those of LDL(+). However, conjugated dienes were increased in LDL(−), which suggests that mild oxidation might affect these particles. LDL(−) increased, in a concentration-dependent manner, the release of IL-8 and MCP-1 by ECs and was a stronger inductor of both chemokines than oxidized LDL (oxLDL) or LDL(+). PAI-1 release increased slightly in ECs incubated with both LDL(−) and oxLDL but not with LDL(+). However, no cytotoxic effects of LDL(−) were observed on ECs. Actinomycin D inhibited the release of IL-8 and MCP-1 induced by LDL(−) and oxLDL by up to 80%, indicating that their production is mediated by protein synthesis. Incubation of ECs with N-acetyl cysteine inhibited production of IL-8 and MCP-1 induced by LDL(−) and oxLDL by >50%. The free radical scavenger butylated hydroxytoluene slightly inhibited the effect of oxLDL but did not modify the effect of LDL(−). An antagonist (BN-50730) of the platelet-activating factor receptor inhibited production of both chemokines by LDL(−) and oxLDL in a concentration-dependent manner. Our results indicate that LDL(−) shows proinflammatory activity on ECs and may contribute to early atherosclerotic events.

Electronegative LDL of FH subjects: chemical characterization and induction of chemokine release from human endothelial cells.

Electronegative LDL (LDL(-)) constitutes a plasma subfraction of LDL with proinflammatory properties. Its proportion is increased in familial hypercholesterolemia (FH); however, the characteristics of LDL(-) isolated from FH subjects have not been previously studied. In this work, the composition, oxidative status, and inflammatory capacity on endothelial cells of LDL(-) from FH and normolipemic (NL) subjects were evaluated. LDL(-) from FH was relatively enriched in esterified and free cholesterol and triglyceride, and had lower apoB and phospholipid content compared with the non-electronegative fraction (LDL(+)). LDL(-) also contained increased amounts of apoE, apoC-III, sialic acid, and non-esterified fatty acids (NEFAs). The same was observed in NL subjects, except that esterified cholesterol and phospholipid were similar in LDL(-) and LDL(+). No difference was observed between the two fractions concerning malondialdehyde, fatty acid hydroxides, and antioxidants, thereby indicating the absence of increased oxidation of LDL(-) compared with LDL(+). When LDL(-) (100 mg/l) from NL and FH subjects was incubated for 24 h with human umbilical vein endothelial cells (HUVECs), interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) increased twofold in the culture medium compared with LDL(+). Vascular cell adhesion molecule 1 (VCAM-1) expression was not increased by LDL(-). Our data indicate that LDL(-) from FH or NL subjects shows no evidence of increased oxidative modification compared to LDL(+); however, LDL(-) induces twofold the release of chemokines by endothelial cells. This effect, which may contribute to leukocyte recruitment and promote atherogenesis, may be greater in FH subjects in which LDL(-) can be up to eightfold higher than in NL subjects.

Platelet-Activating Factor Acetylhydrolase Is Mainly Associated With Electronegative Low-Density Lipoprotein Subfraction

Background Electronegative LDL [LDL(−)], a modified subfraction of LDL present in plasma, induces the release of interleukin‐8 and monocyte chemotactic protein‐1 from cultured endothelial cells. Methods and Results We demonstrate that platelet‐activating factor acetylhydrolase (PAF‐AH) is mainly associated with LDL(−). LDL(−) had 5‐fold higher PAF‐AH activity than the nonelectronegative LDL subfraction [LDL(+)] in both normolipemic and familial hypercholesterolemic subjects. Western blot analysis after SDS‐PAGE confirmed these results, because a single band of 44 kDa corresponding to PAF‐AH appeared in LDL(−) but not in LDL(+). Nondenaturing polyacrylamide gradient gel electrophoresis demonstrated that PAF‐AH was bound to LDL(−) regardless of LDL size. In accordance with the above findings, nonesterified fatty acids, a cleavage product of PAF‐AH, were increased in LDL(−) compared with LDL(+). Conclusions The high PAF‐AH activity observed in LDL(−) could be related to the proinflammatory activity of these lipoproteins toward cultured endothelial cells. (Circulation. 2003;108:92‐96.)

The inflammatory properties of electronegative low-density lipoprotein from type 1 diabetic patients are related to increased platelet-activating factor acetylhydrolase activity

Aims/hypothesisChemical and biological characteristics of LDL(−) from type 1 diabetic subjects were analysed. The diabetic patients were studied during poor and optimised glycaemic control.Materials and methodsTotal LDL was subfractionated into electropositive LDL(+) and electronegative LDL(−) by anion exchange chromatography and the lipid and protein composition of the two determined.ResultsLDL(−) differed from LDL(+) in that it had higher triglyceride, non-esterified fatty acids, apoE, apoC-III and platelet-activating factor acetylhydrolase (PAF-AH), as well as lower apoB relative content. No evidence of increased oxidation was observed in LDL(−). LDL(−) increased two-fold the release of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in endothelial cells, suggesting an inflammatory role. Optimisation of glycaemic control after insulin therapy decreased the proportion of LDL(−), but did not modify the composition of LDL subfractions, except for a decrease in PAF-AH activity in LDL(−). The possibility that LDL(−) could be generated by non-enzymatic glycosylation was studied. Fructosamine and glycated LDL content in LDL subfractions from type 1 diabetic patients was greater than in LDL subfractions isolated from normoglycaemic subjects, and decreased after glycaemic optimisation in both subfractions. However, no difference was observed between LDL(+) and LDL(−) before and after insulin therapy.Conclusions/interpretationThese results provide evidence that LDL(−) is not produced by glycosylation. Nevertheless, LDL(−) from diabetic patients displays inflammatory potential reflected by the induction of chemokine release in endothelial cells. This proatherogenic effect could be related to the high PAF-AH activity in LDL(−).

Novel phospholipolytic activities associated with electronegative low-density lipoprotein are involved in increased self-aggregation.

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.

Proteomic analysis of electronegative low-density lipoprotein

Low density lipoprotein is a heterogeneous group of lipoproteins that differs in lipid and protein composition. One copy of apolipoprotein (apo)B accounts for over 95% of the LDL protein, but the presence of minor proteins could disturb its biological behavior. Our aim was to study the content of minor proteins in LDL subfractions separated by anion exchange chromatography. Electropositive LDL [LDL(+)] is the native form, whereas electronegative LDL [LDL(−)] is a minor atherogenic fraction present in blood. LC-ESI MS/MS analysis of both LDL fractions identified up to 28 different proteins. Of these, 13 proteins, including apoB, were detected in all the analyzed samples. LDL(−) showed a higher content of most minor proteins. Statistical analysis of proteomic data indicated that the content of apoE, apoA-I, apoC-III, apoA-II, apoD, apoF, and apoJ was higher in LDL(−) than in LDL(+). Immunoturbidimetry, ELISA, or Western blot analysis confirmed these differences. ApoJ and apoF presented the highest difference between LDL(+) and LDL(−) (>15-fold). In summary, the increased content of several apolipoproteins, and specifically of apoF and apoJ, could be related to the physicochemical characteristics of LDL(−), such as apoB misfolding, aggregation, and abnormal lipid composition.

Aggregated Electronegative Low Density Lipoprotein in Human Plasma Shows a High Tendency toward Phospholipolysis and Particle Fusion

Aggregation and fusion of lipoproteins trigger subendothelial retention of cholesterol, promoting atherosclerosis. The tendency of a lipoprotein to form fused particles is considered to be related to its atherogenic potential. We aimed to isolate and characterize aggregated and nonaggregated subfractions of LDL from human plasma, paying special attention to particle fusion mechanisms. Aggregated LDL was almost exclusively found in electronegative LDL (LDL(−)), a minor modified LDL subfraction, but not in native LDL (LDL(+)). The main difference between aggregated (agLDL(−)) and nonaggregated LDL(−) (nagLDL(−)) was a 6-fold increased phospholipase C-like activity in agLDL(−). agLDL(−) promoted the aggregation of LDL(+) and nagLDL(−). Lipoprotein fusion induced by α-chymotrypsin proteolysis was monitored by NMR and visualized by transmission electron microscopy. Particle fusion kinetics was much faster in agLDL(−) than in nagLDL(−) or LDL(+). NMR and chromatographic analysis revealed a rapid and massive phospholipid degradation in agLDL(−) but not in nagLDL(−) or LDL(+). Choline-containing phospholipids were extensively degraded, and ceramide, diacylglycerol, monoacylglycerol, and phosphorylcholine were the main products generated, suggesting the involvement of phospholipase C-like activity. The properties of agLDL(−) suggest that this subfraction plays a major role in atherogenesis by triggering lipoprotein fusion and cholesterol accumulation in the arterial wall.

CD14 and TLR4 mediate cytokine release promoted by electronegative LDL in monocytes

AIMS Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes. METHODS AND RESULTS Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells. CONCLUSIONS CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations.

Electronegative low-density lipoprotein subfraction from type 2 diabetic subjects is proatherogenic and unrelated to glycemic control

The physicochemical and biological characteristics of electronegative low‐density lipoprotein (LDL) (LDL(−)) from type 2 diabetic patients (DM2), before and after insulin therapy, were studied.