1. Toll-like receptor 4 (TLR4) activation in response to pathogens or injury may promote atherosclerosis through stimulating neointima formation and plaque development. 2. Adventitial fibroblasts express TLR4 and TLR4 activation in these cells can induce neointima formation and early plaque development through increased migration and proliferation of smooth muscle cells. 3. Both luminal and adventitial TLR4 activation may contribute to atherosclerosis, though the direction of cell migration and their interactions require further study.

1. Submitted by:
Dominique P.V. de Kleijn, PhD
Associate Professor
Laboratory of Experimental Cardiology, Vascular Biology
University Medical Center, Utrecht, The Netherlands
Toll-Like Receptor 4 and
Atherosclerosis
Editorial Slides
VP Watch - August 21, 2002 - Volume 2, Issue 33

2. Toll-like receptor-4 (Tlr-4)
Cytokines
Chemokines
Injury
Stress/Necrosis
Infection
EDA
Fibronectin
III
Hsp 60
Lipopoly-
saccharides
Tlr-4
NfkB
Cell Migration
Cell Proliferation
+ +
+
+
+ +
+
+
+ +
Attraction inflammatory cells
Stimulation matrix turn-over
Stimulation adhesion molecules

3. Toll-Like Receptor-4 Polymorhism and Atherogenesis
Kiechl et al., N. Eng. J. Med. 2002; 347:185-92
Asp299Gly Tlr-4 polymorphism attenuates receptor signaling
Attenuated Tlr-4 receptor signaling is
associated with decreased atherosclerosis

4. What is between Tlr-4 activation
and atherosclerosis?
Can animal models help us to identify a possible
pathway?

5. Mouse models (I)
• Supporting: C3H mouse has a deficient Tlr-4
ApoE KO/C3H mouse gives less atherosclerotic
plaque compared to ApoE KO/B6 mouse. (Grimditch et al., 2000)
• Not supporting: C57Bl/10ScN has a deletion incl. the Tlr-4 gene
ApoE KO/ C57Bl/10ScN and ApoE KO/B6 do not differ in
arterial cholesterol levels. Plaque area not measured.
(Wright et al., 2000)
• Real Tlr-KO mouse models in comparable atherosclerotic
backgrounds are necessary to study Tlr-4 function in
atherosclerosis

6. Which cells express Tlr-4?
Well known:
Bone marrow derived inflammatory cells like:
Monocytes/macrophages
Lymphocytes

7. Role of Tlr-4 in bone marrow (BM) derived cells in
atherosclerotic mouse models (Shi et al., 2002; Van Eck et al., 2000)
Mouse models (I)
BM
Accep-
torBM
Donor
Tlr-4 deficient
C3H
Tlr-4 wildtype
B6
ApoE KO
Tlr4 wt
B6
ApoE wt
Tlr4 wt
B6
Minor
Atherosclerosis
Minor
Atherosclerosis
Minor
Atherosclerosis
Major
Atherosclerosis
•Pointing to a major role of Tlr-4 in arterial wall cells

8. Arterial wall cells expressing Tlr-4
Endothelial cells
Smooth muscle cells
Adventitial fibroblasts
Adventitial fibroblasts are relatively unknown

9. hTLR4 Vimentin
Tlr-4 expression
in primary human adventitial fibroblasts
Vink et al Circulation 2002 in press

10. Fibroblasts, Tlr-4 activation and
plaque/neointima formation
• LPS application in adventitia induces plaque formation in
hypercholesterolaemic rabbit model (Engelmann et al.
2000 Abstract)
• Adventitial fibroblasts play a role in neointima formation
(Shi et al., 2000)
• LPS application stimulates migration of mouse embryonic
fibroblasts in in vitro assay (De Kleijn et al., 2002)

11. Cuff
Femoral
artery
Mouse Femoral Cuff Model (III)
Gelatin ± LPS
Right femoral
artery

12. Adventitial LPS Application
in Tlr-4 Deficient Balb/C
and wt Balb/C Mouse
Relative amount of neointima formation with and without LPS application in mouse cuff model
Gelatin
Gelatin
+ LPS
Balb/C Wildtype Balb/C Tlr-4 deficient
100 % 41 %
•Tlr-4 is involved in neointima formation
•Atherosclerosis might not be initiated only at the luminal
side but also at the advential side.
26 % 14 %
Vink et al., Circulation 2002 in press

13. Cytokines
Chemokines
Hypothetical adventitial Tlr-4 dependent pathway
Injury
Stress/Necrosis
Infection
EDA
Fibronectin
III
Hsp 60
Lipopoly-
saccharides
Tlr-4
NfkB
Adventitial
Fibroblast
Medial Smooth Muscle Cell
Migration
Proliferation
Neointima Formation
Plaque formation
Luminal factors
e.a.
shear stress,
lipids
+ +
+
+
+ +
+
+
+
+
+
+
+ +
+?

14. Neointima formation
as the soil for
atherosclerosis
Adventitial Tlr-4 activation
e.a. injury, infection, stress
Luminal factors
e.a. shear stress, lipids, endothelial dysfunction and activation
? ? Direction of cell migration
Most likely:
Both luminal and adventitial side are important

15. Conclusion
Preclinical data pointed to a role of Tlr-4 in
atherosclerosis
Now, also clinical data show
that Tlr-4 is associated with atherosclerosis
(Kiechl et al., N. Eng. J. Med. 2002; 347:185- 92)

16. Questions
•What is the role of the adventitial endothelial cell?
•Does adventitial Tlr-4 activation affects the luminal endothelial cell?
•Is plaque matrix turn-over affected by Tlr-4 activation?
•Is plaque inflammation affected by Tlr-4 activation?
•Are their Tlr-4 antagonists?
•Can we use Tlr-4 as a (local) target of intervention?
•Should we target the adventitial layer?

17. References
de Kleijn DP, Smeets MB, Kemmeren PP, Lim SK, Van Middelaar BJ, Velema E, Schoneveld A,
Pasterkamp G, Borst C. Acute-phase protein haptoglobin is a cell migration factor involved in
arterial restructuring.FASEB J. 2002 Jul;16(9):1123-5.
Grimsditch DC, Penfold S, Latcham J, Vidgeon-Hart M, Groot PH, Benson GM.
C3H apoE(-/-) mice have less atherosclerosis than C57BL apoE(-/-) mice despite
having a more atherogenic serum lipid profile. Atherosclerosis. 2000 Aug;151(2):389-97.
Kiechl S, Lorenz E, Reindl M, Wiedermann CJ, Oberhollenzer F, Bonora E, Willeit J,
Schwartz DA. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med.
2002 Jul 18;347(3):185-92.
Li G, Chen SJ, Oparil S, et al. Direct in vivo evidence demonstrating neointimal migration of
adventitial fibroblasts after balloon injury of rat carotid arteries. Circulation 2000;101:1362-5.
Shi W, Wang X, Tangchitpiyanond K, Wong J, Shi Y, Lusis AJ. Atherosclerosis in
C3H/HeJ mice reconstituted with apolipoprotein E-null bone marrow. Arterioscler Thromb
Vasc Biol. 2002 Apr 1;22(4):650-5.
Van Eck M, Herijgers N, Vidgeon-Hart M, Pearce NJ, Hoogerbrugge PM, Groot PH,
Van Berkel TJ. Accelerated atherosclerosis in C57Bl/6 mice transplanted with Apo
E-deficient bone marrow. Atherosclerosis. 2000 May;150(1):71-80.
Vink A., Schoneveld, van der Meer J, Middelaar BJ. , Sluijter JPG, Smeets MB,
Quax PHA, Lim SK, Borst C, Pasterkamp G, De Kleijn, DPV. In Vivo Evidence
for a Role of Toll-like Receptor 4 in the Development of Intimal Lesions Circulation 2002 in press
Wright SD, Burton C, Hernandez M, Hassing H, Montenegro J, Mundt S, Patel S, Card DJ,
Hermanowski-Vosatka A, Bergstrom JD, Sparrow CP, Detmers PA, Chao YS. Infectious agents
are not necessary for murine atherogenesis. J Exp Med. 2000 Apr 17;191(8):1437-42.