Discrimination between minimally modified LDL and fully oxidized LDL using monoclonal antibodies.

Low density lipoprotein (LDL) can be oxidized in a stepwise process that leads to the production of minimally modified low density lipoprotein (mm-LDL), in which only the lipid component is oxidized, and then of fully oxidized LDL (oxLDL), in which both the lipids and the protein are oxidized. The thiobarbituric acid-reactive substances (TBARS) assay is a recognized method for determination of oxidized LDL, however this method is unable to distinguish between mm-LDL and oxLDL. In this study, seven specific monoclonal antibodies (mAbs) against human LDL were generated and selectively bound to the apolipoprotein B-100 (apoB-100) component of LDL. Oxidized LDL was produced by incubation of human LDL with 10 µM CuSO4 for various times. The TBARS assay revealed that the optimal incubation time to achieve maximal lipid oxidation was 9 h. Indirect ELISA using the newly generated mAbs was implemented to differentiate between mm-LDL and oxLDL and it was found that binding of the mAbs to oxLDL was significantly decreased after 48 h of incubation, reflecting the oxidative modification of apoB-100. Our results suggest that the optimal times for incubation of LDL with CuSO4 for generation of mm-LDL and oxLDL were 9 h and 48 h, respectively.

A Method for In Vitro Measurement of Oxidized Low-Density Lipoprotein in Blood, Using Its Antibody, Fluorescence-Labeled Heptapeptide and Polyethylene Glycol.

Two oxidized forms of low-density lipoprotein (LDL), oxidized (Ox-LDL) and minimally modified (MM-LDL), and the immune complexes (LDL-ICs) that they form with their corresponding antibodies, play a major role in the pathogenesis of atherosclerosis. Recently, we reported that the heptapeptide KP6 (Lys-Trp-Tyr-Lys-Asp-Gly-Asp) coupled through its ε-amino group present on the N-terminal Lys to fluorescein isothiocyanate (FITC)- (FITC)KP6- binds specifically to Ox-LDL and MM-LDL, but not to native LDL. Here, to develop a novel method for measuring the levels of oxidatively modified LDL in blood, using (FITC)KP6, we analyzed the latter's binding with MM-LDL-IC and Ox-LDL-IC. Polyacrylamide gel electrophoresis analysis revealed that (FITC)KP6 could efficiently and specifically bind to polyethylene glycol (PEG)-precipitated MM-LDL-IC and Ox-LDL-IC in a dose-dependent manner with high sensitivity in plasma and serum. Our results indicate that the above method for measuring the levels of PEG-precipitated, oxidatively modified LDL-ICs, formed by the addition of anti-Ox-LDL antibody to blood, using (FITC)KP6, can aid the diagnosis of atherosclerosis.

A Fluorescence-Labeled Heptapeptide, (FITC)KP6, as an Efficient Probe for the Specific Detection of Oxidized and Minimally Modified Low-Density Lipoprotein.

Two oxidized forms of low-density lipoprotein (LDL), oxidized LDL (ox-LDL) and minimally modified LDL (MM-LDL), are believed to play a major role in the pathogenesis of atherosclerosis. Recently, we reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminus Lys to fluorescein isothiocyanate, (FITC)KP6, bound to ox-LDL but not to LDL. In the present study, we investigated whether (FITC)KP6 could be used as a fluorescent probe for the specific detection of MM-LDL and ox-LDL. Results from polyacrylamide gel electrophoresis and surface plasmon resonance proved that (FITC)KP6 could efficiently bind to MM-LDL as well as ox-LDL in a dose-dependent manner and with high affinity (K D = 3.16 and 3.54 ng/mL protein for MM-LDL and ox-LDL, respectively). (FITC) KP6 bound to lysophosphatidylcholine and oxidized phosphatidylcholine, both present abundantly in ox-LDL and MM-LDL, respectively. In vitro, (FITC)KP6 was detected on the surface and/or in the cytosol of human THP-1-derived macrophages incubated with ox-LDL and MM-LDL, but not LDL. These results suggest that (FITC)KP6 could be an efficient fluorescent probe for the specific detection of ox-LDL and MM-LDL and can therefore contribute to the identification, diagnosis, prevention, and treatment of atherosclerosis.

Avaliação da aterosclerose subclínica e de níveis plasmáticos de LDL minimamente modificada em pacientes com espondilite anquilosante e sua correlação com a atividade da doença / Evaluation of sub-clinical atherosclerosis and plasma levels of minimally modified LDL in patients with ankylosing spondylitis and its correlation with disease activity

INTRODUÇÃO: A aterosclerose acelerada foi demonstrada em algumas doenças autoimunes, principalmente lúpus eritematoso sistêmico e artrite reumatóide. Embora a alta prevalência do uso de corticosteróides possa ser um fator complicador, por causa de seus efeitos prejudiciais em diversos fatores de risco, acredita-se que, nesses pacientes, a inflamação sistêmica per se desempenhe papel importante na aterogênese. MÉTODOS: Avaliamos a aterosclerose subclínica e os níveis plasmáticos de LDL eletronegativa circulante em pacientes com espondilite anquilosante (EA). Catorze pacientes que atendiam aos critérios de Nova York modificados para EA foram comparados com 13 controles equiparados. Avaliamos a espessura da íntima-média (EIM) na carótida por ultrassonografia bilateral da artéria carótida comum, artéria carótida interna e na bifurcação. Os grupos foram homogêneos, no que tange a fatores de risco cardiovasculares. Apenas um paciente no grupo de EA estava sendo medicado com corticosteróide. RESULTADOS: A presença de inflamação ativa foi demonstrada por BASDAI elevado e níveis mais elevados de PCR em pacientes versus controles (12,36 vs. 3,45 mg/dl, P=0,002). Não observamos diferença na EIM da carótida entre os dois grupos, em qualquer local da artéria. A média de EIM (6 mensurações em 3 locais pré-especificados, bilateralmente) foi 0,72 ± 0,28 no grupo de EA e 0,70 ± 0,45 mm nos controles (P=0,91). Também não observamos diferença significativa na LDL minimamente modificada entre pacientes e controles (14,03 ± 17,40 vs. 13,21 ± 10,21; P=0,88). CONCLUSÕES: Pacientes com EA não demonstraram aumento na EIM da carótida, em comparação com controles. Do mesmo modo, os níveis plasmáticos circulantes de LDL(-) não diferiram significativamente nos dois grupos.

INTRODUCTION: Accelerated atherosclerosis has been shown in some autoimmune diseases, mainly in Systemic Lupus Erythematosus and Rheumatoid Arthritis. Although high prevalence of corticosteroids use may be a confounding factor due to their detrimental effects on several risk factors, systemic inflammation per se is supposed to play an important role in atherogenesis in these patients. METHODS: We have evaluated sub-clinical atherosclerosis and plasma levels of circulating electronegative LDL, which represents the fraction of LDL that is minimally modified, in patients with ankylosing spondylitis (AS). Fourteen patients who fulfilled the modified New York criteria for AS were compared with 13 paired controls. Carotid intimal-media thickness (IMT) was assessed by ultrasonography bilaterally in common carotid artery, internal carotid artery and in the bifurcation. Groups were homogeneous regarding cardiovascular risk factors. Only a single patient in AS group was in use of corticosteroid. RESULTS: The presence of active inflammation was demonstrated by elevated BASDAI and higher CRP levels and in patients versus controls (12.36 vs. 3.45 mg/dl, P = 0.002). No difference was found in carotid IMT between both groups, in any site of artery. Averaged IMT (6 measurements, at 3 pre-specified sites bilaterally) was 0.72 ± 0.28 in AS group and 0.70 ± 0.45 mm in controls (P = 0.91). Minimally modified LDL did not differ significantly either between patients and controls (14.03 ± 17.40 vs. 13.21 ± 10.21; P = 0.88). CONCLUSIONS: Patients with AS did not show increased carotid IMT in comparison to controls. In the same way, circulating plasma levels of LDL (-), did not differ significantly in both groups.

Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25 percent cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm²). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.