Tolllike receptors in cardiovascular disease

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Toll-like receptors in cardiovascular disease

• Experimental Cardiology, Utrecht

AEHA, New Orleans 2004
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Toll-like receptors

• Innate immune system
• First line of defense
• Receptors for pathogen-associated patterns
• Family of 10 receptors in human
• Toll-like receptor 2 and 4 most attention in the
  cardiovascular field

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Toll-like receptor pathway
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Toll-like receptor 2

• Ligands associated with gram-positive bacteria
• Peptidoglycan.
• Peptidoglycan is associated with an unstable
  plaque phenotype (Laman et al Am. J. Cardiol.
  2002)

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Toll-like Receptor 4

• Toll-like receptor 4 is the receptor of
  exogenous LPS and endogenous EDA (fibronectin)
  and Hsp60.
• Activation results Cytokines and chemokines
• Matrix metallo proteases, elastases
• Endogenous ligands highly expressed in artritic
  and oncological specimens (Inflammation matrix
  turn-over)

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Hypothetical pathway with a central role for Tlr2
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Infection
Exo. Ligands
Atherosclerosis Plaque stability Neointima
formation Aneurysms Heart Infarction Heart
dysfunction remodeling
Injury Ischemia Oxidative stress
Toll-like Receptor 24
Matrix turn-over Inflammation
Endo. Ligands
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Evidence atherosclerosis

• Present in atherosclerotic lesions (Edfeldt et
  al. Circulation 2002)
• TLR4 polymorphism is associated with carotid
  intima thickness in humans (Kiechl et al. NEJM
  2002)
• TLR4 is involved in neointima formation in vivo
• (Vink et al. Circulation 2002)
• TLR4 is involved in outward remodeling
• (Hollestelle et al, Circulation 2004)

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TLRs, ligands and atherosclerosis

• Peptidoglycan
• ED-A

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Peptidoglycan (PGN)

• bacterial wall antigen (gram bacteria)
• mucosal sites (intestinal flora)
• promotes chronic inflammation at non mucosal
  sites (functional analog of LPS)
• stimulates production of proinflammatory
  cytokines and matrix metalloproteinases, can
  activate complement and upregulates adhesion
  molecules on endothelial cells
• Furthermore, IgM, IgA and IgG antibodies specific
  for PGN have been found in human sera

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Peptidoglycan

Immune response against peptidoglycan
in atherosclerotic patients
Bacterium
Cytoplasmic membrane
Peptidoglycan
Capsule
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Peptidoglycan
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Presence of PGN is associated with vulnerable
plaque phenotype

• Macrophages ?
• Smooth muscle cells ?
• Atheroma ?

PGN
J.D. Laman et al. Am J Cardiol. 2002 Jul
1590(2)119-23.
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Antibody response against peptidoglycan

Immune response against peptidoglycan
in atherosclerotic patients
P0.02
OudeNijhuis et al Atherosclerosis 2004
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Peptidoglycan and intima-media thickness


peptidoglycan and atherosclerotic disease
p0.02 p0.004
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• Immunoglobulin M Type of Autoantibodies to
  Oxidized Low-Density Lipoprotein Has an Inverse
  Relation to Carotid Artery Atherosclerosis
• (Karvonen et al Circulation 2003)
• Antibodies to oxidized LDL in relation to carotid
  atherosclerosis, cell adhesion molecules, and
  phospholipase A.
• (Hulthe et al. ATVB 2002)

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Cuff experiment in Apo E-/- mice
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Flow Chamber Model
Heater
Monitor
Camera
Video Recorder
Microscope
Cell Reservoir
Flow Chamber
Pump
37 C Incubation Chamber
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TLR2 stimulation decreases total monocyte
adhesion (2)
800
700
600


E-selectin and ICAM-1 expressing L-cells
500

400
adherent monocytes/mm2
300
200
100
0
800
700
600


500
TNFa-activated HUVEC

400
adherent monocytes/mm2
300
200
100
0
plt0.05
Shear rate 0.8 dyn/cm2
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Monocyte-endothelium interactions
Chemoattractants
Integrins
Selectins
Monocyte
Activation
Firm adhesion
Rolling adhesion
Transmigration
Endothelium
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TLR2 stimulation increases firm monocyte adhesion
E-selectin and ICAM-1 expressing L-cells
120



100
80
firmly adherent monocytes
60
40
20
0
120
100
80
60
firmly adherent monocytes
plt0.05
40
20
0
Shear rate 0.8 dyn/cm2
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Migration assay
Boyden chamber
Monocytes Migration filter gt staining Stop
filter Chemoattractant
Cell count per 10 ?m
170 ?m
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(No Transcript)
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ED-A

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

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EDA and Hsp60 upregulated 30 days after the
ligation
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The natural history of atherosclerosis in the Apo
E KO mouse.
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Tan MH, Blood. 2004 Jul 1104(1)11-8.
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Conclusions

• TLR ligation can induce an inflammatory response
  and subsequently accelerated plaque formation and
  intima formation
• (The adaptive immune reponse upon) endogenous and
  exogenous ligands for TLRs may be considered as
  targets for intervention

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Acknowkedgements

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