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

  2. 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

  3. Toll-like receptor pathway

  4. 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)

  5. 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)

  6. Hypothetical pathway with a central role for Tlr2 & 4 Infection Exo. Ligands Atherosclerosis* Plaque stability Neointima formation* Aneurysms Heart Infarction Heart dysfunction remodeling* Injury Ischemia Oxidative stress Toll-like Receptor 2&4 Matrix turn-over & Inflammation Endo. Ligands

  7. 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)

  8. TLRs, ligands and atherosclerosis • Peptidoglycan • ED-A

  9. 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

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

  11. Peptidoglycan

  12. 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 15;90(2):119-23.

  13. Immune response against peptidoglycan in atherosclerotic patients Antibody response against peptidoglycan *: P=0.02 OudeNijhuis et al Atherosclerosis 2004

  14. peptidoglycan and atherosclerotic disease Peptidoglycan and intima-media thickness *: p=0.02 **:p=0.004

  15. 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)

  16. Cuff experiment in Apo E-/- mice

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

  18. Control Pam3Cys-SK4 PGN Anti L-selectin Ab 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 TNFα-activated HUVEC * 400 # adherent monocytes/mm2 300 200 100 0 *: p<0.05 Shear rate 0.8 dyn/cm2

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

  20. Control Pam3Cys-SK4 PGN Anti L-selectin Ab 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 *: p<0.05 40 20 0 Shear rate 0.8 dyn/cm2

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

  22. 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.

  23. EDA and Hsp60 upregulated 30 days after the ligation

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

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

  26. 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

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