SHAPE Society

1. Toll-like receptors inToll-like receptors in
cardiovascular diseasecardiovascular disease
Experimental Cardiology, UtrechtExperimental Cardiology, Utrecht
AEHA, New Orleans 2004

2. 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 inToll-like receptor 2 and 4 most attention in
the cardiovascular fieldthe cardiovascular field

3. ThisThis
PathPath
wayway
::
OthOth
erer
SpeSpe
ciescies
::
Toll-like receptor pathway

4. Toll-like receptor 2Toll-like receptor 2
• Ligands associated with gram-positiveLigands associated with gram-positive
bacteriabacteria
• Peptidoglycan.Peptidoglycan.
• Peptidoglycan is associated with anPeptidoglycan is associated with an
unstable plaque phenotypeunstable plaque phenotype (Laman et al Am. J.(Laman et al Am. J.
Cardiol. 2002)Cardiol. 2002)

5. Toll-like Receptor 4Toll-like Receptor 4
• Toll-like receptor 4 is the receptor of exogenous LPS andToll-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 andEndogenous ligands highly expressed in artritic and
oncological specimens (Inflammation & matrix turn-over)oncological specimens (Inflammation & matrix turn-over)

6. 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

7. Evidence atherosclerosisEvidence atherosclerosis
• Present in atherosclerotic lesionsPresent in atherosclerotic lesions (Edfeldt et al. Circulation 2002)(Edfeldt et al. Circulation 2002)
• TLR4 polymorphism is associated with carotid intimaTLR4 polymorphism is associated with carotid intima
thickness in humansthickness in humans (Kiechl et al. NEJM 2002)(Kiechl et al. NEJM 2002)
• TLR4 is involved in neointima formationTLR4 is involved in neointima formation in vivoin vivo
(Vink et al. Circulation(Vink et al. Circulation 2002)2002)
• TLR4 is involved in outward remodelingTLR4 is involved in outward remodeling
(Hollestelle et al, Circulation 2004)(Hollestelle et al, Circulation 2004)

8. TLRs, ligands and atherosclerosisTLRs, ligands and atherosclerosis
• PeptidoglycanPeptidoglycan
• ED-AED-A

9. 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 sitespromotes chronic inflammation at non mucosal sites
(functional analog of LPS)(functional analog of LPS)
• stimulates production of proinflammatory cytokinesstimulates production of proinflammatory cytokines
and matrix metalloproteinases, can activateand matrix metalloproteinases, can activate
complement and upregulates adhesion molecules oncomplement and upregulates adhesion molecules on
endothelial cellsendothelial cells
• Furthermore, IgM, IgA and IgG antibodies specificFurthermore, IgM, IgA and IgG antibodies specific
for PGN have been found in human serafor PGN have been found in human sera

10. PeptidoglycanPeptidoglycan
Bacterium
Cytoplasmic membrane
Peptidoglycan
Capsule
Immune response against peptidoglycan in atherosclerotic patients

11. PeptidoglycanPeptidoglycan

12. Presence of PGN is associated withPresence of PGN is associated with
vulnerable plaque phenotypevulnerable plaque phenotype
MacrophagesMacrophages ↑↑
Smooth muscle cellsSmooth muscle cells ↓↓
AtheromaAtheroma ↑↑
PGNPGN
J.D. Laman et al. Am J Cardiol. 2002 Jul 15;90(2):119-23.

13. 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
IgM IgG IgA
ratio(patientserum/standardserum)
control patients
atherosclerotic patients
*
*: P=0.02
OudeNijhuis et al Atherosclerosis 2004

14. Peptidoglycan andPeptidoglycan and intimaintima-media thickness-media thickness
*: p=0.02
**:p=0.004
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1 2 3 4
IMT in quartiles
IgMlevelsagainstPGN
(patientserum/standardserum)
*
**
peptidoglycan and atherosclerotic disease

15. • Immunoglobulin M Type of Autoantibodies toImmunoglobulin M Type of Autoantibodies to
Oxidized Low-Density Lipoprotein Has an InverseOxidized Low-Density Lipoprotein Has an Inverse
Relation to Carotid Artery AtherosclerosisRelation to Carotid Artery Atherosclerosis
(Karvonen et al Circulation 2003)(Karvonen et al Circulation 2003)
• Antibodies to oxidized LDL in relation to carotidAntibodies to oxidized LDL in relation to carotid
atherosclerosis, cell adhesion molecules, andatherosclerosis, cell adhesion molecules, and
phospholipase A.phospholipase A.
(Hulthe et al. ATVB 2002)(Hulthe et al. ATVB 2002)

16. Cuff experiment in Apo E-/- miceCuff experiment in Apo E-/- mice
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
intima/mediaratio
gelatin gelatin + peptidoglycan

17. Flow Chamber Model
VCR
Video Recorder
Pump
37 C Incubation Chamber
Heater
Camera
Flow Chamber
Cell Reservoir
Microscope
Monitor

18. TLR2 stimulation decreases totalTLR2 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
#adherentmonocytes/mm2
0
100
200
300
400
500
600
700
800
#adherentmonocytes/mm2
*
*
*
Shear rate 0.8 dyn/cm2
*
**
TNFα-activated
HUVEC
E-selectin and
ICAM-1
expressing L-cells
*: p<0.05

19. Monocyte-endothelium interactions
Endothelium
Monocyte
Rolling
adhesion
Firm adhesion
Transmigration
Activation
Selectins
Integrins
Chemoattractants

20. TLR2 stimulation increases firmTLR2 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%firmlyadherentmonocytes
40
60
80
100
120
mlyadherentmonocytes
* * *
*: p<0.05

21. Migration assayMigration assay
Boyden
chamber
Monocytes
Migration filter =>
staining
Stop filter
Chemoattractant
Cell count per
10 µm
170 µm

22. Monocyte migration
0
5
10
15
20
25
30
35
40
45
50
HEPES3+ C5a 10e-8 C5a 10e-9 PAF 10e-8
meandistance(um)
Control
PGN

23. ED-AED-A
• Fibronectin variants are generated from aFibronectin variants are generated from a
single gene by alternative RNA splicing ofsingle gene by alternative RNA splicing of
the V, EIIIA, and EIIIB segments, whichthe V, EIIIA, and EIIIB segments, which
are also known as CS-1, ED-A, and ED-Bare also known as CS-1, ED-A, and ED-B
segments, respectively.segments, respectively.
• ED-A is an endogenous ligand for Toll LikeED-A is an endogenous ligand for Toll Like
receptor 4.receptor 4.

24. EDA and Hsp60 upregulated 30 days after the
ligation

25. 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
EDA(pg)
apo 5
apo 11
apo 15
apo25
apo 40

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

27. ConclusionsConclusions
• TLR ligation can induce an inflammatoryTLR ligation can induce an inflammatory
response and subsequently acceleratedresponse 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 forendogenous and exogenous ligands for
TLRs may be considered as targets forTLRs may be considered as targets for
interventionintervention

28. 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