BIOFENOLI ED INIBIZIONE DELL UPTAKE DI oxldl DA PARTE DI CELLULE MACROFAGICHE UMANE E MURINE Roberta Di Benedetto Centro Nazionale per la Qualità degli Alimenti e per i Rischi Alimentari Istituto Superiore di Sanità
Cardiovascular Disease (CVD) Main type of CVD is Atherosclerosis (AS) Endothelial dysfunction is one of earliest changes in AS Mechanical, chemical, inflammatory mediators can trigger endothelial dysfunction: - High blood pressure -Smoking - Inflammatory stimuli - Hyperlipidemia
What is atherosclerosis? A process that clogs arteries with deposits of cholesterol and lipids. Growth of the fibrous plaque results in vascular remodeling,, progressive luminal narrowing, blood-flow abnormalities,, and compromised oxygen supply to the target organ. Rupture of lesions can release particulate matter that blocks other blood vessels
Atherogenesis
LDL Oxidation and Atherosclerosis Brown and Goldstein were the first to propose that LDL must be oxidized before it could be taken up by macrophages and ultimately be incorporated into foam cells (Brown, 1983). Foam cells are the building blocks of the fatty streaks that form in the human artery in the beginning stages of atherosclerosis (Witztum( Witztum, 1991). Oxidized LDL is the beginning of the cascade.
Modification of LDL Apo B-100 LDL Derivatization: Aldehydes Glucosylation eg.. diabetes Oxidation: PUFA s are oxidized and trigger oxidation of apob 100 protein Derivatized LDL Oxidized LDL
The Scavenger Receptor How macrophages deal with oxidized or modified LDL The scavenger receptor recognizes modified and/or oxidized LDL and internalizes the modified LDL. Accumulation of these modified LDL in the cell leads to the accumulation of cholesterol droplets in the macrophage and the formation of foam cells.
The Scavenger Receptor, CD36 CD36 is a membrane glycoprotein expressed by several cell types and classified as class B scavenger receptor. Several functions, all depending on the molecular being expressed at the cell surface, have been ascribed to CD36. The CD36 cdna predicts a polypeptide of 53-kDa with 10 potential N-linked glycolsylation sites. CD36 has: two short intracellular domains a large extracellular domain is heavily N-glycosylated
BACKGROUND In previous studies we demonstrated that extra-virgin olive oil polyphenols, i.e. protocatechuic acid and oleuropein,, inhibit LDL oxidation mediated by murine J774 A.1 macrophage-like cells and improve antioxidative cellular defenses through a direct effect on DNA transcription of GSH-related enzymes. (Masella( et al., 2004 J Nutr) We could hypothesize that extra-virgin olive oil polyphenols,, i.e. protocatechuic acid and oleuropein,, can interfere in an other crucial event of the pathogenesis of atherosclerosis such as the uptake of oxidized LDL by macrophages. AIM OF THE STUDY Aim of the study was to investigate the capacity of protocatechuic acid and oleuropein to inhibit the uptake of oxldl by macrophage. We also assessed whether the antioxidants added to the cells had any direct effect on CD36 receptor.
Protocol for oxldl uptake in human macrophages Macrophage Incubation (24 hrs) oxldl (0.2 mg/ml) 14 C oleate-bsa 0.1mM (200`000 d.p.m.) LIPID EXTRACTION n- exane / isopropane 3:2, v/v; 30min. [Ac.prot.]=[oleu.]=0.025 mm NaOH 0.1M Thin Layer Chromatography (TLC) n- exane/diethyether/ acetic acid 80:20:1 v/v Protein determination Cholesterol ester fraction radioactivity (d.p.m.) d.p.m./ mg cell protein
14 C-oleate incorporation in human macrophages oxldl 3000 oxldl + Oleu. oxldl + Prot.Ac. d.p.m./mg cell protein 2500 2000 1500 1000 500-57% -37% 0 P<0,05
Protocol for oxldl uptake in J774.A1 macrophages LDL + 10µM CuSO 4 + 12µg ml -1 DiI 3hrs, 37 C DiIoxLDL Calibration curve + J774.A1 [Dil- oxldl]=0.1mg ml -1 [Ac.Prot.]=[Oleu]=0.025mM Incubation (24h) Absorbance (Em 563nm, Ex 523nm) Washing (2 x PBS/0.4% BSA) (2 x PBS) Cells counting FI ng -1 DiI-oxLDL ng Dil-oxLDL / 10 6 cells
DiI-oxLDL uptake in J774.A1 50 oxldl oxldl + Oleu. oxldl + Prot.Ac. µg DiI-oxLDL/10 6 cells 40 30 20 10-31% -29% 0 P<0,05
DiI-oxLDL uptake in J774.A1 (flow( cytometry method) To evaluate whether preloading the cells with the biophenols may have an effect on oxldl uptake, protocatechuic acid and tyrosol were added to the cells for 2 hours and then removed from the culture medium before the DiI-oxLDL addition (pre( pre-oleo. and pre-prot.ac Prot.Ac.) 120 100 nldl oxldl+ pre-oleu. oxldl + pre-prot.ac. oxldl oxldl + Oleu. oxldl + Prot.Ac. 80 60 40 20 0 P<0,05
Detection of CD36 on J774.A1 surface J774.A1 18hrs oxldl (0,1mg/mL) AbCD36 AbIgA-FITC Washing Flow cytometer
FITC-AbCD36 in J774.A1 macrophages ctr nldl Oleu. Prot.Ac. oxldl oxldl + Oleu. 300 oxldl + Prot.Ac. 250 * % respect of control 200 150 100 50 * * 0 * P <0,05
CD36 Western Blot Analysis in J774.A1 macrophages 150 100% % respect of control 100 50 * -37% * -36% 0 * P <0,05 vs oxldl P <0,05 vs ctr ctr Oleu. Prot.Ac. oxldl oxldl + oxldl + Oleu. Prot.Ac. ctr Oleu. Prot.Ac. oxldl oxldl + Oleu. oxldl + Prot.Ac. CD36 actin
CD36 mrna expression in J774.A1 macrophages % respect of control 180 160 140 120 100 80 60 40 20 0 ctr Oleu. Prot.Ac. oxldl oxldl + oxldl + Oleu. Prot.Ac. P < 0,05
Conclusions Preliminary results demonstrate that Protocatechuic Acid and Oleuropein: Decrease the uptake of oxldl by human and murine macrophages probably by counteracting the increase of CD36 surface expression induced by oxldl. Do not affect total CD36 protein expression although they increase CD36 mrna expression.
THE END Thanks to Dr Massimo Sanchez (Dip. BCN-Terapia Genica e Cellulare, Istituto Superiore di Sanità) for his helpful assistance in flow cytometry analysis.