Toll-like receptors in Toll-like receptors in cardiovascular diseasecardiovascular disease

Experimental Cardiology, UtrechtExperimental Cardiology, Utrecht

AEHA, New Orleans 2004

Toll-like receptorsToll-like receptors

• Innate immune systemInnate immune system• First line of defenseFirst line of defense• Receptors for pathogen-associated patternsReceptors for pathogen-associated patterns• Family of 10 receptors in humanFamily of 10 receptors in human• Toll-like receptor 2 and 4 most attention in Toll-like receptor 2 and 4 most attention in

the cardiovascular fieldthe cardiovascular field

                                                                                                      

                                             

This This PathPathwayway

::

                                                                  

OthOther er

SpeSpecies:cies:

      

                                                      

Toll-like receptor pathway

Toll-like receptor 2Toll-like receptor 2

• Ligands associated with gram-positive Ligands associated with gram-positive bacteriabacteria

• Peptidoglycan.Peptidoglycan.• Peptidoglycan is associated with an Peptidoglycan is associated with an

unstable plaque phenotype unstable plaque phenotype (Laman et al Am. J. (Laman et al Am. J. Cardiol. 2002)Cardiol. 2002)

Toll-like Receptor 4Toll-like Receptor 4

• Toll-like receptor 4 is the receptor of exogenous LPS and Toll-like receptor 4 is the receptor of exogenous LPS and endogenous EDA (fibronectin) and Hsp60.endogenous EDA (fibronectin) and Hsp60.

• Activation results: Activation results: Cytokines and chemokinesCytokines and chemokines

Matrix metallo proteases, elastasesMatrix metallo proteases, elastases

• Endogenous ligands highly expressed in artritic and Endogenous ligands highly expressed in artritic and oncological specimens (Inflammation & matrix turn-over)oncological specimens (Inflammation & matrix turn-over)

Infection

Injury

Ischemia

Oxidative stress

Exo. Ligands

Endo. Ligands

Toll-like Receptor

2&4

Matrix turn-over &Inflammation

Atherosclerosis*

Plaque stability

Neointima formation*

Aneurysms

Heart Infarction

Heart dysfunction

remodeling*

Hypothetical pathway with a central role for Tlr2 & 4

Evidence atherosclerosisEvidence atherosclerosis

• Present in atherosclerotic lesions Present in atherosclerotic lesions (Edfeldt et al. Circulation 2002)(Edfeldt et al. Circulation 2002)

• TLR4 polymorphism is associated with carotid intima TLR4 polymorphism is associated with carotid intima thickness in humans thickness in humans (Kiechl et al. NEJM 2002)(Kiechl et al. NEJM 2002)

• TLR4 is involved in neointima formation TLR4 is involved in neointima formation in vivoin vivo (Vink et al. Circulation (Vink et al. Circulation 2002)2002)

• TLR4 is involved in outward remodeling TLR4 is involved in outward remodeling (Hollestelle et al, Circulation 2004)(Hollestelle et al, Circulation 2004)

TLRs, ligands and atherosclerosisTLRs, ligands and atherosclerosis

• Peptidoglycan Peptidoglycan • ED-AED-A

Peptidoglycan (PGN)Peptidoglycan (PGN)

• bacterial wall antigen (gram + bacteria)bacterial wall antigen (gram + bacteria)• mucosal sites (intestinal flora)mucosal sites (intestinal flora)• promotes chronic inflammation at non mucosal sites promotes chronic inflammation at non mucosal sites

(functional analog of LPS)(functional analog of LPS)• stimulates production of proinflammatory cytokines and stimulates production of proinflammatory cytokines and

matrix metalloproteinases, can activate complement matrix metalloproteinases, can activate complement and upregulates adhesion molecules on endothelial cellsand upregulates adhesion molecules on endothelial cells

• Furthermore, IgM, IgA and IgG antibodies specific for Furthermore, IgM, IgA and IgG antibodies specific for PGN have been found in human seraPGN have been found in human sera

PeptidoglycanPeptidoglycanBacterium

Cytoplasmic membrane

Peptidoglycan

Capsule

Immune response against peptidoglycan in atherosclerotic patients

PeptidoglycanPeptidoglycan

Presence of PGN is associated with Presence of PGN is associated with vulnerable plaque phenotypevulnerable plaque phenotype

Macrophages Macrophages Smooth muscle cells Smooth muscle cells Atheroma Atheroma

PGN PGN

J.D. Laman et al. Am J Cardiol. 2002 Jul 15;90(2):119-23.

Antibody response against peptidoglycanAntibody response against peptidoglycan

Immune response against peptidoglycan in atherosclerotic patients

0,0

0,2

0,4

0,6

0,8

1,0

1,2

I gM I gG I gAratio

(pat

ient

ser

um/s

tand

ard

seru

m) control patients

atherosclerotic patients

*

*: P=0.02OudeNijhuis et al Atherosclerosis 2004

Peptidoglycan and Peptidoglycan and intimaintima-media thickness-media thickness

*: p=0.02**:p=0.004

00,10,20,30,40,50,60,70,80,9

1 2 3 4IMT in quartiles

IgM

leve

ls ag

ains

t PGN

(p

atie

nt s

erum

/sta

ndar

d se

rum

)

***

peptidoglycan and atherosclerotic disease

• Immunoglobulin M Type of Autoantibodies to Oxidized Immunoglobulin M Type of Autoantibodies to Oxidized Low-Density Lipoprotein Has an Inverse Relation to Low-Density Lipoprotein Has an Inverse Relation to Carotid Artery Atherosclerosis Carotid Artery Atherosclerosis

(Karvonen et al Circulation 2003)(Karvonen et al Circulation 2003)

• Antibodies to oxidized LDL in relation to carotid Antibodies to oxidized LDL in relation to carotid atherosclerosis, cell adhesion molecules, and atherosclerosis, cell adhesion molecules, and phospholipase A. phospholipase A.

(Hulthe et al. ATVB 2002)(Hulthe et al. ATVB 2002)

Cuff experiment in Apo E-/- miceCuff experiment in Apo E-/- mice

0,00,10,20,30,40,50,60,70,80,9

intim

a/m

edia

ratio

gelatin gelatin + peptidoglycan

Flow Chamber Model

VCR

Video Recorder

Pump

C Incubation Chamber

Heater

Camera

Flow ChamberCell ReservoirMicroscope

Monitor

TLR2 stimulation decreases total TLR2 stimulation decreases total monocyte adhesion (2)monocyte adhesion (2)

Control Pam3Cys-SK4 PGN Anti L-selectin Ab

0

100

200

300

400

500

600

700

800

# ad

here

nt m

onoc

ytes

/mm

2

0

100

200

300

400

500

600

700

800

# ad

here

nt m

onoc

ytes

/mm

2

**

*

Shear rate 0.8 dyn/cm2

***

TNFα-activated HUVEC

E-selectin and ICAM-1 expressing L-cells

*: p<0.05

Monocyte-endothelium interactions

Endothelium

Monocyte

Rollingadhesion

Firm adhesionTransmigration

Activation

SelectinsIntegrins

Chemoattractants

TLR2 stimulation increases firm TLR2 stimulation increases firm monocyte adhesionmonocyte adhesion

Control Pam3Cys-SK4 PGN Anti L-selectin Ab

E-selectin and ICAM-1 expressing L-cells

0

20

40

60

80

100

120%

firm

ly a

dher

ent m

onoc

ytes

40

60

80

100

120

% fi

rmly

adh

eren

t mon

ocyt

es

* * *

*: p<0.05

Migration assayMigration assay

Boyden chamber

MonocytesMigration filter => stainingStop filterChemoattractant

Cell count per 10 m

170 m

Monocyte migration

0

5

10

15

20

25

30

35

40

45

50

HEPES3+ C5a 10e-8 C5a 10e-9 PAF 10e-8

mea

n di

stan

ce (u

m)

Control

PGN

ED-AED-A

• Fibronectin variants are generated from a Fibronectin variants are generated from a single gene by alternative RNA splicing of single gene by alternative RNA splicing of the V, EIIIA, and EIIIB segments, which the V, EIIIA, and EIIIB segments, which are also known as CS-1, ED-A, and ED-B are also known as CS-1, ED-A, and ED-B segments, respectively.segments, respectively.

• ED-A is an endogenous ligand for Toll Like ED-A is an endogenous ligand for Toll Like receptor 4. receptor 4.

EDA and Hsp60 upregulated 30 days after the ligation

The natural history of atherosclerosis in the Apo E KO mouse.The natural history of atherosclerosis in the Apo E KO mouse.EDA in apoE

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0 1 2 3 4 5 6

group

ED

A (

pg)

apo 5

apo 11

apo 15

apo25

apo 40

Tan MH, Blood. 2004 Jul 1;104(1):11-8.

ConclusionsConclusions

• TLR ligation can induce an inflammatory TLR ligation can induce an inflammatory response and subsequently accelerated response and subsequently accelerated plaque formation and intima formationplaque formation and intima formation

• (The adaptive immune reponse upon) (The adaptive immune reponse upon) endogenous and exogenous ligands for endogenous and exogenous ligands for TLRs may be considered as targets for TLRs may be considered as targets for intervention intervention

AcknowkedgementsAcknowkedgements

• Many people but in particular:Many people but in particular:– Dominique de Klein (project leader TLRs)Dominique de Klein (project leader TLRs)– Manon OudeNijhuisManon OudeNijhuis– Arjan SchoneveldArjan Schoneveld