Submitted by:

Dominique P.V. de Kleijn, PhDAssociate Professor

Laboratory of Experimental Cardiology, Vascular BiologyUniversity Medical Center, Utrecht, The Netherlands

Toll-Like Receptor 4 and Atherosclerosis

Editorial Slides VP Watch - August 21, 2002 - Volume 2, Issue 33

Toll-like receptor-4 (Tlr-4)

CytokinesChemokines

Injury Stress/NecrosisInfection

EDA FibronectinIII

Hsp 60Lipopoly-saccharides

Tlr-4

NfkB

Cell MigrationCell Proliferation

+ +++

+ +

+

++ +

Attraction inflammatory cellsStimulation matrix turn-overStimulation adhesion molecules

Toll-Like Receptor-4 Polymorhism and AtherogenesisKiechl 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

What is between Tlr-4 activation and atherosclerosis?

Can animal models help us to identify a possible pathway?

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

Which cells express Tlr-4?

Well known:Bone marrow derived inflammatory cells like:

Monocytes/macrophagesLymphocytes

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 deficientC3H

Tlr-4 wildtypeB6

ApoE KOTlr4 wtB6

ApoE wtTlr4 wtB6

MinorAtherosclerosis

MinorAtherosclerosis

MinorAtherosclerosis

MajorAtherosclerosis

•Pointing to a major role of Tlr-4 in arterial wall cells

Arterial wall cells expressing Tlr-4

Endothelial cellsSmooth muscle cellsAdventitial fibroblasts

Adventitial fibroblasts are relatively unknown

hTLR4 Vimentin

Tlr-4 expression in primary human adventitial fibroblasts

Vink et al Circulation 2002 in press

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 embryonicfibroblasts in in vitro assay (De Kleijn et al., 2002)

Cuff

Femoralartery

Mouse Femoral Cuff Model (III)

Gelatin LPS

Right femoralartery

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

CytokinesChemokines

Hypothetical adventitial Tlr-4 dependent pathway

Injury Stress/NecrosisInfection

EDA FibronectinIII

Hsp 60Lipopoly-saccharides

Tlr-4

NfkB

AdventitialFibroblast

Medial Smooth Muscle CellMigrationProliferation

Neointima Formation

Plaque formation

Luminal factorse.a. shear stress,lipids

+ +++

+ +

+

++

+

++

+ +

+?

Neointima formation as the soil foratherosclerosis

Adventitial Tlr-4 activatione.a. injury, infection, stress

Luminal factorse.a. shear stress, lipids, endothelial dysfunction and activation

? ? Direction of cell migration

Most likely:Both luminal and adventitial side are important

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)

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?

Referencesde 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 despitehaving 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 ApoE-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.