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Enhanced Plasma Levels of Oxidized Low-Density Lipoprotein Increase Circulating Nuclear Factor-Kappa B Activation in Patients With Unstable Angina

Luciano Cominacini, MD^_*,_*, Maurizio Anselmi, MD, Ulisse Garbin, MD^_*, Anna Fratta Pasini, MD^_*, Chiara Stranieri, BSc, PhD^_*, Massimiliano Fusaro, MD, Cristina Nava, MD^_*, Pierfrancesco Agostoni, MD, Dritan Keta, MD, Pietro Zardini, MD, Tatsuya Sawamura, MD, PhD and Vincenzo Lo Cascio, MD^_*

^_* Department of Biomedical and Surgical Sciences, Section of Internal Medicine, University of Verona, Verona, Italy
Section of Cardiology, University of Verona, Verona, Italy
Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan

View larger version (14K): [in a new window]   Figure 1 (A) Concentrations of plasma oxidized low-density lipoprotein (Ox-LDL) in unstable angina (UA) and stable angina (SA) patients and in control subjects. *p < 0.001 vs. control subjects; p < 0.01 vs. SA patients. (B) Concentrations of circulating nuclear factor-kB (NF-kB) in UA and SA patients and in control subjects. The NF-kB was extracted from peripheral blood mononuclear cells derived from UA and SA patients and control subjects. *p < 0.001 vs. control subjects; p < 0.01 vs. SA patients.

View larger version (23K): [in a new window]   Figure 2 Correlation between the concentrations of plasma oxidized low-density lipoprotein (Ox-LDL) and the circulating levels of nuclear factor-kB (NF-kB) in all subjects considered in the study.

View larger version (20K): [in a new window]   Figure 3 Concentrations of circulating nuclear factor-kB (NF-kB) in separated monocytes and lymphocytes derived from unstable angina (UA) and stable angina (SA) patients and control subjects. The NF-kB was extracted from the separated monocytes and lymphocytes of a subgroup of UA, SA, and control subjects. *p < 0.001 vs. lymphocytes; p < 0.001 vs. control subjects; p < 0.01 vs. SA patients.

View larger version (21K): [in a new window]   Figure 4 (A) Basal lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) messenger ribonucleic acid (mRNA) expression in pools of separated lymphocytes (1 to 3) and monocytes (4 to 6), derived from control subjects (1 and 4) and stable (2 and 5) and unstable (3 and 6) angina patients. The LOX-1 mRNA was analyzed by reverse-transcriptase polymerase chain reaction. The LOX-1 mRNA levels were normalized to the levels of beta-actin mRNA. Data illustrated on the bar graph represent the mean ± SD of six different experiments. *p < 0.001 vs. lymphocytes (1 to 3). (B) The LOX-1 protein expression in pools of separated lymphocytes (1 to 3) and monocytes (4 to 6) derived from control subjects (1 and 4) and stable (2 and 5) and unstable (3 and 6) angina patients. Lymphocytes and monocytes were separated as described in the Methods section. The LOX-1 protein expression was analyzed by flow cytometry with a specific anti-LOX-1 monoclonal antibody. Results are expressed as mean fluorescence intensity (MFI) and are means ± SD of experiments performed in triplicate on six separate occasions. *p < 0.001 vs. lymphocytes (1 to 3).

View larger version (27K): [in a new window]   Figure 5 Oxidized low-density lipoprotein (Ox-LDL)-dependent activation of nuclear factor-kB (NF-kB) in monocytes and lymphocytes. The purified monocytes and lymphocytes (3 x 105/ml, 200 µl/well) were cultured in 96-well trays for 20 h at 37°C with increasing amounts of Ox-LDL in the presence of control mouse immunoglobulin G (IgG) (50 µg/ml) or anti-LOX-1 monoclonal antibody (mAb) (20 µg/ml). The NF-kB was measured in cellular extract as described in the Methods section. Results are means ± SD of experiments performed in triplicate on six separate occasions. *p < 0.001 vs. control value (ox-LDL = 0 mg/ml); p < 0.001 vs. anti-LOX-1 mAb and lymphocytes. White bars = Ox-LDL + monocytes + IgG; black bars = Ox-LDL + monocytes + anti-LOX-1 mAb; ruled bars = Ox-LDL + lymphocytes + IgG.

View larger version (18K): [in a new window]   Figure 6 Plasma-dependent activation of nuclear factor-kB (NF-kB) in monocytes. Monocytes from healthy volunteers were evaluated for NF-kB activation after they were cultured for 20 h in medium supplemented with either 40% serum from 10 unstable angina (UA) patients with the highest oxidized low-density lipoprotein (ox-LDL) plasma concentrations (range from 41 to 62 µg/ml) or 40% serum from 10 control subjects with the lowest ox-LDL levels (range from 4 to 7 µg/ml). Monocytes were also incubated with the corresponding lipoprotein-depleted serum (LPDS). In some experiments, anti-lectin-like ox-LDL receptor-1 (LOX-1) monoclonal antibody (mAb) (20µg/ml) or control mouse immunoglobulin G (50 µg/ml) was also added to the cell culture. Results are means ± SD of experiments performed in triplicate on six separate occasions. *p < 0.001 vs. serum from control subjects (C); p < 0.001 vs. serum UA + anti-LOX-1 mAb; p < 0.001 vs. LPDS UA; p < 0.01 vs. LPDS C.