Electronegative low-density lipoprotein. A link between apolipoprotein B misfolding, lipoprotein aggregation and proteoglycan binding.

PURPOSE OF REVIEW: Subendothelial retention of lipoproteins is considered the first step in the development of atherosclerosis, but the molecular mechanisms involved are poorly understood. Recent findings on the atherogenic properties of a minor electronegative fraction of LDL (LDL(-)) could contribute to a better understanding of this process. RECENT FINDINGS: Circular dichroism, Trp-fluorescence and two-dimensional nuclear magnetic resonance have shown that apolipoprotein B (apoB) in LDL(-) has an abnormal, misfolded conformation. Immunochemical analysis revealed a different conformation, mainly in the N-terminal and C-terminal extremes. These alterations contribute to the high susceptibility to aggregation of LDL(-). Moreover, LDL(-) can seed the aggregation of native LDL, suggesting an amyloidogenic character that has been attributed to the amphipathic helix cluster in the α2-domain. A phospholipase C (PLC)-like activity associated to LDL(-) seems to play a major role in the LDL(-)-induced aggregation. The aggregation of LDL(-) increases its binding to proteoglycans because of the abnormal conformation of the N-terminal extreme of apoB. SUMMARY: LDL(-) could play a relevant role in atherogenesis by acting as a priming factor that stimulates lipoprotein aggregation. This process, which appears to be mediated by a PLC-like activity intrinsic to LDL(-), increases the binding of LDL to proteoglycans and could promote subendothelial retention of these lipoproteins.

Immunochemical analysis of the electronegative LDL subfraction shows that abnormal N-terminal apolipoprotein B conformation is involved in increased binding to proteoglycans.

Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-).

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles.

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

Postprandial lipidemia is normal in non-obese type 2 diabetic patients with relatively preserved insulin secretion.

To assess postprandial lipidemia in normotriglyceridaemic type 2 diabetic patients treated with diet only, 12 non-obese patients (8 males, hemoglobin A(1c) [HbA(1c)] 6.80 +/- 0.67%) and 14 controls of similar age, body mass index (BMI), and fasting triglyceride (Tg) were given a test meal (58 g fat, 100,000 IU vitamin A). Fasting low-density lipoprotein (LDL) cholesterol (LDLc), high-density lipoprotein (HDL) cholesterol (HDLc), free fatty acids, and apolipoprotein B (apoB), and fasting and postprandial Tg, retinylpalmitate (RP), LDL size, glucose, and insulin were measured. The homeostasis assessment model (HOMA) index and lipoprotein (Lpl) and hepatic (HL) lipase activities were estimated. Patients showed lower fasting HDLc (1.12 +/- 0.26 v 1.40 +/- 0.28 mmol/L, P =.02) and a trend towards smaller LDL particles, which was significant 4 hours postprandially (25.86 +/- 0.40 v 26.16 +/- 0.30 nm, P =.04). The area under the curve of Tg (AUC-Tg) and RP, and Lpl were similar, but HL was higher in patients (156.63 +/- 23.89 v 118 +/- 43.27 U/L, P =.011). HL correlated inversely with LDL size and directly with the HOMA index. In conclusion, normotriglyceridemic type 2 diabetic patients with insulin resistance but relatively preserved insulin secretion show low fasting HDLc and increased HL, but normal postprandial lipidemia.

Early intervention in the 3xTg-AD mice with an amyloid ß-antibody fragment ameliorates first hallmarks of Alzheimer disease.

The single-chain variable fragment, scFv-h3D6, has been shown to prevent in vitro toxicity induced by the amyloid ß (Aß) peptide in neuroblastoma cell cultures by withdrawing Aß oligomers from the amyloid pathway. Present study examined the in vivo effects of scFv-h3D6 in the triple-transgenic 3xTg-AD mouse model of Alzheimer disease. Prior to the treatment, five-month-old female animals, corresponding to early stages of the disease, showed the first behavioral and psychological symptoms of dementia -like behaviors. Cognitive deficits included long- and short-term learning and memory deficits and high swimming navigation speed. After a single intraperitoneal dose of scFv-h3D6, the swimming speed was reversed to normal levels and the learning and memory deficits were ameliorated. Brain tissues of these animals revealed a global decrease of Aß oligomers in the cortex and olfactory bulb after treatment, but this was not seen in the hippocampus and cerebellum. In the untreated 3xTg-AD animals, we observed an increase of both apoJ and apoE concentrations in the cortex, as well as an increase of apoE in the hippocampus. Treatment significantly recovered the non-pathological levels of these apolipoproteins. Our results suggest that the benefit of scFv-h3D6 occurs at both behavioral and molecular levels.

Methionine-induced hyperhomocysteinemia impairs the antioxidant ability of high-density lipoproteins without reducing in vivo macrophage-specific reverse cholesterol transport.

SCOPE: High plasma homocysteine concentrations have been associated with increased risk of cardiovascular disease both in humans and experimental animal models, whereas plasma HDL-cholesterol concentration is inversely correlated with such disorders. This work aimed to study the impact of methionine-induced hyperhomocysteinemia (HHcy) on two major antiatherogenic functions of HDL, namely their capacity to prevent LDL oxidation and induce in vivo macrophage-specific reverse cholesterol transport. METHODS AND RESULTS: Methionine-induced HHcy in mice resulted in an approximately 20% decreased concentration of HDL-cholesterol and HDL main protein component, apolipoprotein A-I. The HDL potential to resist oxidation as well as to prevent LDL oxidative modification was impaired in hyperhomocysteinemic mice. Activities of paraoxonase-1 and platelet activation factor acetylhydrolase, two of the main HDL-associated enzymes with antioxidant activity, were reduced. The ability of HDL to efflux cholesterol from macrophages was decreased in hyperhomocysteinemic mice; however, the in vivo macrophage-specific reverse cholesterol transport measured as the output of labeled cholesterol into feces did not significantly differ between groups. CONCLUSION: Our data indicate that the HDL from methionine-induced hyperhomocysteinemic mice was more prone to oxidation and displayed lower capacity to protect LDL against oxidative modification than that of control mice, highlighting a mechanism by which a diet-induced HHcy may facilitate progression of atherosclerosis.

Effect of improving glycemic control in patients with type 2 diabetes mellitus on low-density lipoprotein size, electronegative low-density lipoprotein and lipoprotein-associated phospholipase A2 distribution.

The aim of this study was to determine the effect of intensified hypoglycemic therapy in patients with type 2 diabetes mellitus on the distribution of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity between high-density lipoprotein and low-density lipoprotein (LDL) and its relation with the lipid profile and other qualitative properties of LDL. Forty-two patients with type 2 diabetes on the basis of poor glycemic control and normal or near normal LDL cholesterol were recruited. Lifestyle counseling and pharmacologic hypoglycemic therapy were intensified to improve glycemic control, but lipid-lowering therapy was unchanged. At 4 ± 2 months, glycosylated hemoglobin had decreased by a mean of 2.1%, but the only effect on the lipid profile were statistically significant decreases in nonesterified fatty acids and apolipoprotein B concentration. LDL size increased and the proportion of electronegative LDL decreased significantly. In parallel, total Lp-PLA2 activity decreased significantly, promoting a redistribution of Lp-PLA2 activity toward a higher proportion in high-density lipoprotein. Improvements in glycemic control led to more marked changes in Lp-PLA2 activity and distribution in patients with diabetes who had not received previous lipid-lowering therapy. In conclusion, optimizing glycemic control in patients with type 2 diabetes promotes atheroprotective changes, including larger LDL size, decreased electronegative LDL, and a higher proportion of Lp-PLA2 activity in high-density lipoprotein.