BIOFENOLI ED INIBIZIONE DELL’UPTAKE BIOFENOLI ED INIBIZIONE DELL’UPTAKE DI oxLDL DA PARTE DI CELLULE DI oxLDL DA PARTE DI CELLULE

MACROFAGICHE UMANE E MURINE MACROFAGICHE UMANE E MURINE

Roberta Di BenedettoRoberta Di BenedettoCentro Nazionale per la Qualità degli Alimenti e per i Rischi AlCentro Nazionale per la Qualità degli Alimenti e per i Rischi Alimentariimentari

Istituto Superiore di SanitàIstituto Superiore di Sanità

Cardiovascular Disease (CVD)Cardiovascular Disease (CVD)• Main type of CVD is Atherosclerosis (AS)

• Endothelial dysfunction is one of earliest changesin AS

• Mechanical, chemical, inflammatory mediators can trigger endothelial dysfunction:

- High blood pressure

- Smoking

- Inflammatory stimuli

- Hyperlipidemia

What is atherosclerosis?What is atherosclerosis?A process that clogs arteries with deposits of cholesterol A process that clogs arteries with deposits of cholesterol and lipids.and lipids.

GrowthGrowth of the of the fibrousfibrous plaqueplaque resultsresults in in vascularvascularremodelingremodeling, progressive luminal , progressive luminal narrowingnarrowing, , bloodblood--flowflowabnormalitiesabnormalities, and , and compromisedcompromised oxygenoxygen supplysupply toto the the target target organorgan..

Rupture of lesions can release particulate matter that Rupture of lesions can release particulate matter that blocks other blood vesselsblocks other blood vessels

AtherogenesisAtherogenesis

LDL Oxidation and AtherosclerosisLDL Oxidation and Atherosclerosis

Brown and Goldstein were the first to propose that Brown and Goldstein were the first to propose that LDL must be oxidized before it could be taken up by LDL must be oxidized before it could be taken up by macrophages and ultimately be incorporated into macrophages and ultimately be incorporated into foam cells (Brown, 1983). foam cells (Brown, 1983).

Foam cells are the building blocks of the fatty Foam cells are the building blocks of the fatty streaks that form in the human artery in the streaks that form in the human artery in the beginning stages of atherosclerosis (beginning stages of atherosclerosis (WitztumWitztum, , 1991).1991).

Oxidized LDL is the beginning of the cascade.Oxidized LDL is the beginning of the cascade.

Modification of LDL

DerivatizationDerivatization::AldehydesAldehydesGlucosylationGlucosylationegeg. diabetes. diabetes

Oxidation:Oxidation:PUFA’sPUFA’s are oxidized are oxidized and trigger oxidation of and trigger oxidation of apoBapoB100100 proteinprotein

LDLLDL

Apo B-100

DerivatizedDerivatizedLDLLDL

OxidizedLDL

The Scavenger ReceptorThe Scavenger ReceptorHow macrophages deal with oxidized or modified LDLHow macrophages deal with oxidized or modified LDL

The scavenger receptor recognizes modified The scavenger receptor recognizes modified and/or oxidized LDL and internalizes the and/or oxidized LDL and internalizes the modified LDL.modified LDL.

Accumulation of these modified LDL in the Accumulation of these modified LDL in the cell leads to the accumulation of cholesterol cell leads to the accumulation of cholesterol droplets in the macrophage and the formation droplets in the macrophage and the formation of foam cells.of foam cells.

The Scavenger Receptor, CD36The Scavenger Receptor, CD36CD36 CD36 isis a membrane a membrane glycoproteinglycoprotein expressedexpressed byby severalseveral cellcell typestypes and and

classifiedclassified asas class B scavenger class B scavenger receptorreceptor..

The CD36 The CD36 cDNAcDNA predictspredicts a a polypeptidepolypeptideof 53of 53--kDa kDa withwith 10 10 potentialpotential NN--linkedlinkedglycolsylationglycolsylation sitessites..

CD36 CD36 hashas::•• twotwo short short intracellularintracellular domainsdomains•• a a largelarge extracellularextracellular domaindomain•• isis heavilyheavily NN--glycosylatedglycosylated

SeveralSeveral functionsfunctions, , allall dependingdepending on the on the molecularmolecularbeingbeing expressedexpressed at the at the cellcell surfacesurface, , havehave beenbeenascribedascribed toto CD36.CD36.

BACKGROUNDBACKGROUNDIn previous studies we demonstrated that extraIn previous studies we demonstrated that extra--virgin olive oil virgin olive oil polyphenolspolyphenols, , i.e. i.e. protocatechuicprotocatechuic acid and acid and oleuropeinoleuropein, inhibit LDL oxidation mediated by , inhibit LDL oxidation mediated by murinemurine J774 A.1 macrophageJ774 A.1 macrophage--like cells and improve like cells and improve antioxidativeantioxidative cellular cellular defenses through a direct effect on DNA transcription of GSHdefenses through a direct effect on DNA transcription of GSH--related related enzymes. (enzymes. (MasellaMasella et al., 2004 et al., 2004 J J NutrNutr))

We could hypothesize that extraWe could hypothesize that extra--virgin olive oil virgin olive oil polyphenolspolyphenols, i.e. , i.e. protocatechuicprotocatechuic acid and acid and oleuropeinoleuropein, can interfere in an other crucial event , can interfere in an other crucial event of the pathogenesis of atherosclerosis such as the uptake of oxiof the pathogenesis of atherosclerosis such as the uptake of oxidized LDL dized LDL by macrophages.by macrophages.

AIM OF THE STUDYAIM OF THE STUDYAim of the study was to investigate the capacity of Aim of the study was to investigate the capacity of protocatechuicprotocatechuic acid acid and and oleuropeinoleuropein to inhibit the uptake of to inhibit the uptake of oxLDLoxLDL by macrophage. We also by macrophage. We also assessed whether the antioxidants added to the cells had any dirassessed whether the antioxidants added to the cells had any direct ect effect on CD36 receptor.effect on CD36 receptor.

Protocol for oxLDL uptake in human macrophages

MacrophageIncubation (24 hrs)

oxLDL (0.2 mg/ml)14C oleate-BSA 0.1mM (200`000 d.p.m.)[Ac.prot.]=[oleu.]=0.025 mM

LIPID EXTRACTION

n- exane / isopropane3:2, v/v; 30min.

NaO

H0.

1M Thin Layer Chromatography

(TLC)n- exane/diethyether/acetic acid 80:20:1 v/v

Cholesterol ester fraction radioactivity (d.p.m.)

Protein determination

d.p.m./ mg cell protein

1414CC--oleate oleate incorporationincorporation in in humanhuman macrophagesmacrophagesd.

p.m

./mg

cell

prot

ein

0

500

1000

1500

2000

2500

3000

oxLDL

oxLDL + Prot.Ac.oxLDL + Oleu.

P<0,05

--57%57%

--37%37%

ProtocolProtocol forfor oxLDL uptake in J774.A1 oxLDL uptake in J774.A1 macrophagesmacrophages

FI ng-1 DiI-oxLDL

Absorbance(Em 563nm, Ex 523nm)

Cells counting

Washing(2 x PBS/0.4% BSA)

(2 x PBS)Incubation (24h)

[Dil- oxLDL]=0.1mg mL-1

[Ac.Prot.]=[Oleu]=0.025mM

+ J774.A1DiI-

oxLDL

LDL

3hrs, 37°C+ 10µM CuSO4

+12µg mL-1 DiI

Cal

ibra

tion

curv

e

ng Dil-oxLDL / 106 cells

DiIDiI--oxLDLoxLDL uptake in J774.A1uptake in J774.A1µg

DiI-

oxLD

L/10

6 ce

lls

0

10

20

30

40

50

P<0,05

oxLDL

oxLDL + Prot.Ac.oxLDL + Oleu.

--31%31% --29%29%

DiIDiI--oxLDLoxLDL uptake in J774.A1 (uptake in J774.A1 (flowflow cytometrycytometry methodmethod))

To evaluate whether preloading the cells with the To evaluate whether preloading the cells with the biophenolsbiophenols may have an effect on may have an effect on oxLDLoxLDL uptake, uptake, protocatechuicprotocatechuic acid and acid and tyrosoltyrosol were added to the cells for 2 hours were added to the cells for 2 hours and then removed from the culture medium before the and then removed from the culture medium before the DiIDiI--oxLDLoxLDL addition (addition (prepre--Oleo.Oleo.and and prepre--Prot.AcProt.Ac..))

0

20

40

60

80

100

120 oxLDL oxLDL + Prot.Ac.oxLDL + Oleu.nLDL oxLDL+ pre-Oleu. oxLDL + pre-Prot.Ac.

P<0,05

Detection of CD36 on J774.A1 Detection of CD36 on J774.A1 surfacesurface

J774.A1

18hrsoxLDL oxLDL (0,1mg/mL)(0,1mg/mL)

AbCD36AbCD36 AbIgAAbIgA--FITCFITC

Washing

Flow cytometer

FITCFITC--AbCD36 in J774.A1 AbCD36 in J774.A1 macrophagesmacrophages

oxLDLoxLDL + Oleu.oxLDL + Prot.Ac.

ctr Oleu.Prot.Ac.nLDL

0

50

100

150

200

250

300

* P <0,05

*

* *

% re

spec

tof c

ontro

l

CD36 Western CD36 Western BlotBlot AnalysisAnalysis in J774.A1 in J774.A1 macrophagesmacrophages

0

50

100

150

-37%-36%

100%

ctr Oleu. Prot.Ac. oxLDL oxLDL + Prot.Ac.

oxLDL + Oleu.* P <0,05 vs oxLDL

§ P <0,05 vs ctr

% re

spec

tof c

ontro

l

actin

CD36

ctr Oleu. Prot.Ac. oxLDLoxLDL + Prot.Ac.

oxLDL + Oleu.

**

§

§

CD36 mRNA CD36 mRNA expressionexpression in J774.A1 in J774.A1 macrophagesmacrophages

0

2040

60

80100

120

140160

180

ctr Oleu. Prot.Ac. oxLDL oxLDL + Prot.Ac.

oxLDL + Oleu.

% re

spec

tof c

ontro

l

P < 0,05

ConclusionsConclusions

Preliminary results demonstrate that Preliminary results demonstrate that ProtocatechuicProtocatechuic Acid Acid and and OleuropeinOleuropein::

•• Decrease the uptake of Decrease the uptake of oxLDLoxLDL by human and by human and murinemurinemacrophages probably by counteracting the increase macrophages probably by counteracting the increase of CD36 surface expression induced by of CD36 surface expression induced by oxLDLoxLDL..

•• Do not affect total CD36 protein expression although Do not affect total CD36 protein expression although they increase CD36 mRNA expression.they increase CD36 mRNA expression.

THE ENDTHE END

ThanksThanks toto Dr Massimo Dr Massimo SanchezSanchez ((DipDip. . BCNBCN--TerapiaTerapia Genica e Cellulare, Istituto Genica e Cellulare, Istituto Superiore di Sanità) Superiore di Sanità) forfor hishis helpfulhelpful assistanceassistance in in flowflow cytometrycytometry analysisanalysis..