Chapter

Chapter General Introduction

1

Filip M.E. Segers 1. Cardiovascular Disease and Atherosclerosis 2.1 Plaque initiation 2.2 Plaque progression 2.3 Plaque stability and rupture

3.1 Macrophages 3.2 T Lymphocytes

! 4.1 Scavenger Receptor Class A 4.2 CD40/CD40L Pathway 4.3 Chemokine and Chemokine Receptors

5. Imaging in Atherosclerosis "# $ % % & '

10

General Introduction

1. Cardiovascular Disease and Atherosclerosis

2. Pathogenesis of Atherosclerosis 2.1 PLAQUE INITIATION

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Chapter 1

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0 % - % / % %% - % in medium and large sized arteries at predisposed sites and characterized +/ % - 8+ % %+ 0". In the past decades, % % % :

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,,/ % 7% - / -

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% + % / accumulated macrophages- "- &

%0 % $ %8% ,0% / (* - : +/ - % +/ !7 : / %7 % ! /

, %B/ : J % / % / + + (8 ==0X* 2.2 PLAQUE

PROGRESSION

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%+ -/ 7 % 0 12


B

A

Endothelial permeability

Leukocyte migration

Endothelial adhesion

Leukocyte adhesion

Smooth muscle migration

Foam cell formation

T-cell activation

Adherence and aggregation of platelets

Adherence and entry of leukocytes

D

C

Macrophage accumulation

Necrotic core formation

Fibrous cap formation

Plaque rupture

Thinning of fibrous cap

Hemorrhage from plaque microvessels

Figure 1.1.3 , (*K % %/ (X*Q/

: (*

% %- % (* 2 +-0 (%% #

#4K O$%=>>>*

13

Chapter 1

! / % 0 7% % % % %+ %7% 6, & Q 0 % 0 ( % - 0 %

*'7%;% - 7%;% : +/ - % +/ 7%;% % + % % lesions28 3$J ,% % , /, % %77 ( *- 8+ 7 % :8+ 2 % % - / %- : + +/ / 8+ cap. Moreover, due to a process called outward remodelling, the vessel is still + ; % ; ,

% + %! ,29(8 ==*

2.3 PLAQUE

STABILITY AND RUPTURE

[ / ,% %

%- % % % + %

-% + %! , %

%7 8+ - %/ Activated macrophages, T cells and other immune cells have been shown % % % atherosclerotic lesions&-]. Furthermore, activated macrophages, T cells, dendritic % + + % %-% -

+

=0. %: / + % +/ % 3$J , % % N % P- " . In addition these cells and in particular macrophages are also responsible % % 7% % 7 ($$*&K /$$0=-$$0L-$$0> %$$0= +

%, +/L0=. - / 7 % / : % 0 % %% +/ ! / , - , / % - , %% +/

! / ,% % 7 ,, % % + +, B8+ ^ J- % / % % +/ % % : 8+ ' %/ 7% %/% % +/ -8+ % + % % + % % , % %+ % + %-,/ / - - % , / -,, + % +/ %

/ / % % :](8 ==* As described above, immune responses during plaque initiation, progression % / % / % : /

+ , -, 8/0 %% % % % + 7 % % %N %% %/ % 0 + : /

+ - ,

7

%+/ %

- %,+%

% detail below.

14


3. Immune Cells in Atherosclerotic Lesion Development

3.1 MONOCYTES/MACROPHAGES + %% +/ +:

,: ('N'*, K0N0 mice. Lesion prone ApoE0N0, %

atherosclerosis&. 2 % % -

/ % % - / %% + + % / , - + / / H %

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Chapter 1

%% - + / +

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%%- % %/ , : % / % +/P&. In this thesis %:/ ,+ %

+ %%

3.2 T LYMPHOCYTES

47 - + atherosclerotic lesions is the T cell. T cell presence in human atherosclerotic , %/ % +% , %% P. Antigen presenting cells / % 8 % / % ! ( * : % % % % , , 8 $6 -+ X + helper and CD8+/ 7

%, % % + % % % % ! /% 58. H / - + +/59. A %8 / + T cells"]0"% %% 0 ' %- + %8

%N % , % 60. + X % / : 8- + helper T cells can be ;% , +

` = (=* % (* $ + = +/ = % 0 ! / % 0 / : (Q40T % 4Q0R* % + "-"Q40T % % , % 7

% % % 0 ! // : and chemokines, and proteases. This process leads to renewed T cell recruitment , , +/59, 65 6 , 0= 0N0 % %%Q40T7

atherosclerotic lesions and in reduced lesion development66Th2 responses are % % 0 % "-". In contrast with =- %% 0 % % 0 / : 0-0P-0>-0=]-0= %0 % , VQ0U"&0% % ;%

% % 0P68-0=]">0& %VQ0U&0&" ,% / : 6 ,- ! %7 0 % +% % H 0 % / , &&,0%8 / K0N0 mice resulted in reduced atherosclerotic lesion development as well&L /-0

%, 16


4. Targeting the Unstable Atherosclerotic Plaque 4.1 SCAVENGER

RECEPTOR CLASS A (SR-A) =>&>-V % %X , ,8 % + (3#J * % % /,+ + % % ; %8% , % / (* +%+/ - % / :/ culprits in macrophage cholesterol accumulationLP0L&3#/ L % +

(

0V*, + % % - - %8% %+ %/ %8% % 0 % - % - % %/ % and pathogen components88. Macrophages in atherosclerotic lesions have + , 7

7 / %

17

Chapter 1

% $$ % - , % +/&>. % +/ + - / ;% %+/ + =N ,% 0 ! / = /80. As mentioned earlier, CD8+ T cells are also present in atherosclerotic lesions, +% %- ' %/, + + / L+%8 / % K0N0 mice %8 81- , %/ , % L+ T cell activation82. V - %% / % % , 0 % % % , + % 8 - % + - 0 8 %% +/ (#*, ,%0$6 7 % %+/ 0 / -,7

% on the antigen presenting cell plasma membrane and bind to receptors on the - /, +/#L. The best ;% 0 / L0X& % ]0 ] L = - + 0+ % ] % % Q40T- / + % ] % Q40T 3/ + %7

0 ! / % ! 0

/ - % % 0 + / , 0 / / % 0 / ,/ / 7 0%% % ,, ]0],/ % %

: %8% - , 3#0%%: %8% + - ,%/+% / 0 ! / / lesional macrophages89 ,, /

' %

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Structure, function and expression

(3#0*/ % +/ L91, 92 3#0 / + / / - 3#0- 3#0 % 3#0 /- , % / 0% %N :% + %+ % >. K "%% % ` 40 / - transmembrane, spacer, R0 %0 - % /0 8 + 7/ >0>"/3#0 / % ==] % , % / >&, whereas

18


19

Chapter 1

/3#0 0 %+ 7/0 % /3#0 % / 0% % + , dominant negative properties> %0+ % 3#0 % % / % % - %% /

/

% 898 3#0 7

% % % , %0+ % /> 3#0 7+ / + %- + ,0%8 %- %0+ % 8/ / % / % / %+ % domain99 % / / %8% ( '7 %*+ /+ % ( / % / /%*- / % ( %7 % % *- ( / / % / %* %%)A=]]0=]&. / % / 7

% +/ % %

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0 8 / 109 K7

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, $ /N$ / 3 Q ($03Q*115, 116, %V ,Q (VQ*==& and phorbol esters==& have been shown % % / % 3#0 7

4 Q R (4Q0R*118 V , Q U (VQ0U*119- T (Q40T*98- / % (3*120 % V / $ /3 Q (V$03Q*121 have been shown to decrease

7

3 3#0 % / %8% (*-+ 7-+ , 3#07

+ #4 % =- =. There is also evidence that pathogens 3#07

6 / (6$)* 3$J %

3# % + 7 :=- , + V % 3#0 7

0%%% (f * % macrophages125.

Role of SR-A during pathogenesis of atherosclerosis Q -+/ %8% + %

3#0 6 , % % , 3#0 7

LP- L&- = ; % %0% % plaque development. 3 % % % Research , %8% ( 7* lesions="-=&-7

3#0

, / %%% atherogenesis=L0=]- % , 7% 7% % , % ,

== =>>&- 3;:+ %% % 3#0/ % +/ 3#0%8 an ApoE0N0 background. These mice showed higher plasma cholesterol levels and a 60% decrease in average atherosclerotic lesions size compared to normal ApoE0 N0 mice26. 37 % +: % , % %%3#0%8 / 0N0 mice results in a minor reduction in % %% / % 0 %%

/ : "- "L % $#'=. 6 , 3#0 %8 / Ky% - %/ K

%,%/ + =-=, showed

8 / =P 3+ / X+ % % %8 / % 0 8 %8 / 3#0(+ / %* % % % % / L{ % "]{ % 7 =". Q + , % % K %8 +: % ,% 7

3#0 %% % =&-=L ]]-f H ,% " % 3#0 % + : : + + % %8%:+/ % % + =>. Moore et al=] % %+/3;:26 and reported that ApoE0N0 mice %% 3#0 " % % %:+ % ;f+ al.==7 % % %+/ %/ + % + % : : : 3#0 %" K%8 +: % + % / 0 "- + , + % %% 3#0% -7 + 3#0 % 3% +/ K: %8 / ,% 0 3#0 X,: + 0 > %=,: , / %% =3+ / / / 3#0 7 % , % %

20


4.2 CD40/CD40L PATHWAY

3- %7

] / + (&:- & % * - + (4Q#* /=-7

% 7 =K7

] %%+/ 0 ! / 4Q0R-Q40T and also interleukins (0=-0- %0*=P0=& -0 /, "0= % ] 7

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+ % % / X % -+ - - / % - (* % % % / % 0 (KJ - 3$J % 8+ + *=L. % % ] =P ] %-+ R / / + /+7

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, 158, dendritic - - % 0 ( % and smooth muscle cells and epithelial cells=L ]- =P /7

% 0% %

% % 8 - 0 ! // : 0 =-0=-4Q0R %Q40T and glucocorticoids=P>0="= %7

=P 0/ / 0=(0=* % % (4Q0*="0=".

21

Chapter 1

/ /7 3#0 %% - , % % : ,%

+ /- % $ + % 7

3#0 % ! % % 8 0%% % / + % + 8 3#0 / % ! + % %% %/ 3 %% +

% 7 : , 3#0 + In chapter 2, we will describe the % % ; / =P0 3#00 8 % % chapter 5,

/ % + %

# / + ]( ]* + %165. Platelets are the ] % + >P{ ] 166. 3 +]-=L:- % ]- ++ / % +/ + %] 8 ="&. Soluble ] % 07

%] + 7 168. Some suggest that associations with

07

% ] % ]169 but it is becoming / % =&].

The CD40/CD154 pathway

]0=P -]+ % % ;%=&=. Upon - + ^

% /(#Q *% %+ % ( * / #Q + % % % , ,/ =& 2 , 7 + #Q/+ % 8%- ,#Q=-#Q-#Q % #Q" %/ % #QP %/

, ] % , ,/, +%+/#Q-#Q-#Q P % #Q"- 4Q0~X ,/- , + 0 0 ! / % 0 =&-=&'] ,/ % +/ #Q L 0% : ($f*- 0O 40 : (O4f* % K#f=N=&P0=&> ] , 4Q0 ~X- 0= % 4Q0- , / % =P7

K% ]

%+: - ,+/ ] %] , , 7

="&-=L]. Impact of CD40 signalling on atherosclerosis at all stages

/ -]N=P,/ % % %/ % % 7

% K% % , , ] 7

% K 7 0 ! // : ( 4Q0R-0= %Q40T* %% +/ % % 181 $ 7 +

, %=P7

K 182Q -0 % (K0 -$0= %)$0=* + , K ] % =P- , % !7 ! / cells=L=- =L0=L" ] % =P % % , /- % 0==L&, and % , % 188. As mentioned earlier, 4Q0 ~X- 0= % 3 % ! /

188, 189 + %

-] % , =P 8 - , Q40T- % % / : 0%% = -

22


Targeting the CD40/CD154 dyad

%

% - ]N=P %/% :/ / + % %% 3 %8 / 0 :%/0 $ / % +

%, ] =P209 %% -+ 8 + % % + %/0%% ]N=P 7 0 % 0 %% - -

0 0 % % =&-=L=-=]. + %/0%% + :% ]N=P ,/ + %% +/ %0N0 %(=,: *, =P + % %% ;(P>{* ,% (&>{*Q - ,% 8 /, N/ / %%% 7

)$0= %$$ - % 211 %/ + %- % % %% %

- + / %% %

$ / - % + /-

23

Chapter 1

atherosclerosis59 %% - =P %% % % 0 ! / : ( Q40T-0"-0= %0=P*=LL0 190 + Q - ] K -3$ - %/ / -

% % : ( 0L- ! / ($*0=R-#4K3-3Q0=R %$0=*, % % / / % -

! =>=0=>P. [ + %

+/ % -]N=P%/% + ! / %

: 7

% - K % 3$ , 7

: - #0= ( $0=R %#4K3*-#0( $0=*-#0P( $0=R %#4K3* %#0( 3Q0=R*=>"0=>L. ] + % -

+ ,-

% +/ atherosclerotic plaques199, 200] K % 7

% , ()KVQ*- % 0=P %/ 7/ 0( 70*]=0]. % % - ] % 7 7 % % - +/ +/ ] 7

K -3$ % %+ 7 /($$ *=L-=L= , %

(Q* 7

% % + % ]0]L Q /- ] ( %

+/ ]*/ :/ + +/ : +, - ! - + - % 168.

- N/ 8+ + % 8+ :

, / / - % - ! / / : % ,% %=-=$%8 + K %=P-%%% ; % % +/ (%0 - %% N / / % 0*=. These % , ]N=P , % % - % +/ % + 8 ]N=P+ :%- % H 215, 216 using humanized 0=P + % ( ;% PL* % +/

% + 0+ + :% + %

- / %+ % +/ : N % =&- =L %0 / % / + %/ + % -

7 + / %% -, /- % + atherosclerotic lesions. In chapter 3,,% +% / ] + % % % , % % ! /

4.3 CHEMOKINES

: / % !

%

47 / 219 and adhesion molecules220, recent studies have +%:/

8 +

/ : , : / - : 221, 222. 3 - 7/ P] : + % +% X % 40 / % -/+ ;% , H ( % * % , ( %*/. Chemokines interact via chemokine -V0 % (V# * & + % - 7

% : / ' ] %/+ % % % ,%/% / % : ; 8 / % -, / : + % 7 % % / : % . Chemokines are known + % 7 -% %% : / - % % % : /0 % V / % 7

: - % : % ! / - %% % % : ! /

X - , % ] : + 225 V 0 24


25

Chapter 1

8 % K0N0 mice, revealed 0% % 0 $0= % 0P- $0=R- $0=U- : %

- / % , %+/ $0= %$0P 226. # / / , % %+, : % / % %%+/+ 0+ % : - + / + : ( Q 0 (Q0 * %#4K3(P** %% % : % on the endothelium221. $0=- / 0=-+ %B /J : / % + + %% 7 $0= % +/ / (K - % 3$ * % % / / & $0= 7

% 0

% % % + % lesions21, 2283% 7 % 8% $0= % (#* 19, 229, ] - , ! % +;

P #4K3(regulated on activation, normal T cell e7

% %s%* %+/ -/ / -K -3$ % % + % : (#=-# %#P* ,/ -7

#4K3 +%% -, 7

0 ; , %%/ / =. In %% 7

+/ -P also be secreted via R0 +/% / % -,/ : /%

%! ,0. == K 7 7

% 3$ % +/ : # 7

% / / - % % - % + == , / % +% % / + %/ - ==, + % % # 7

% 0 P. Q - ! %7 % , / "0L. ! / ($*0= : +/ + - , % - , % capacities> : %% +/ + % / - % -% % - %/ / %+ % $0=R7

+ + % - / 0 % =>"- = $0=R % +/ % % 221 Q /- , : - #= % #P X 7

% / % % - + 7

%

6 ,% $0=R - % % %% +; :/ 3 0%% (3Q*0=R =-8 % +% 0%% - % + %+/ : 7

%+/ - % % - + / - % , #- + % =>. Q 3Q0=R / 7

% % , / recruit mononuclear and progenitor cells in vivo]. In chapter 4-,%7

: % : %

0 %% 0 N0 % % 8%

+ : ( * %% in vivo.

Figure 1.3.% ,

+ % % % (%% %/4# /]]``>=0>P*

26


' % % % % ()* % % %+/ / % % % % 4 , % % -

% : % % / % / or thrombotic occlusions. In particular as recent insights seem to suggest that +/ % % ; + : %/ % ,% %

- % %8 ;=-. ' ; 0/ / 6 ,- % , % - - %% % / % , / severe outcome' , %/ + %

% /(3K* %

/(K*-,+ ; % + % 8 /% %% %

X 3K %K% / -

/-+ /: %( +* % , $ ($#*

, ,( ,* / % 3K % K- + % / (=]0=P 3K- 0 K-=]] $# %= * /- : % -$# /%% + % % -: ; % %+/ % 0 $# + % 7 P- 8/ 0 treatments" % % 8 N % % (8+ -% - % *&- L 6 ,- ! /

, % +/ % K7 + %: % % / % +- , : +- : B J + + : -,%+ / , %,!+

%

(, % + * V /- + % + ;% % % 8/ ; % + / /

+ %% / + : - 8 / % 8/ % , 7/Q = % %% , 27

Chapter 1

5. Imaging and Atherosclerosis

% % + imaging and applicable imaging modalities. The probe needs to be conjugated to a contrast enhancing carrier. Magnetic : % 0+ % - , % %

% - /,

>,

7% + $# 8 - 7% (3'*- (/% %/ =]0=L] * % % 7% / , - %7 3' /

% / %/ % +/ 0 % / (#K3*in vivo. %

2 3 '7% (23'*- 7 23' ; 3' (=P0] * %:- %7 % % 7+ / , + % +/#K3 in vivo+ 3' %23', % #K3- % ( %%, characterization250*- % /: 23' + Q -,, 23' $ 0 8 23': %/,% %- 7 / %8 / 23' ;- %// 7 % % : / : / - , : 0%% % / (, ; &] % % +/ # 251* % / P- P % % - 7 0=]- ,

, +: %% % / % macrophages252 % % / %/ :P /23':+/

%, % 23' , + + % %+ , 0 / 7/ in vitro and in vivo + % 23'P-PP. V + % / plaque shoulder region, that macrophage content is correlated with plaque +/- % 23' - %% 2 ,- % % % %

- 0 %%- -, %/ :+/ + / % plaques. Ruehm et al.256 , 8 , % $# , /% ++ %/ 23' H Similar results

28


6. Research Models for Targeting and Imaging Atherosclerosis % /- 8 % +% +/ 3 ", is an established technique % / % + % % / % , % % + % 8 % , + % / % % - % + % -, 8 / % 8/ / + % % /+ "0"". %

% B+ J- + %N +/% /%+/ + + ,- +;%- / + - 0+ % % + % % % + %% %- % %8% + % % 7 /' % + %

29

Chapter 1

were obtained in atherosclerotic plaques in atherosclerotic lesion prone ApoE0 N0 miceP& and even in human carotid arteries> %% - /

% 7 0=](23'*- , 23'0 %%

carotid atheromatous plaques258, 25947

- % active targeting usingV%0+ % :%, 8 + %/ 3#0N26047 - + 7%;% : % % + %/ $ % %%/% ($*0/ epitopes261 % 7 ,V% (V%*0 H % , '70 8 + %

8 % % / 7% 0 lesions262 47 % '7- % - ; -77 %$$J +

/ % V%0+ %$# 23'0 + %$# +

23'0+ %$# % H

8% , % Q 7 0 23' + %% +: % 7 %% % 3 % 23' + : 8 % 7

/ :/

% , ! - / 8/ ". In chapter 5 , % % % % % 0+ % 23' / / 8 % + % /7

% - -,

+ % + %/% / 0 0 ! /

- %% % % % Q / + / + % N ,+ + % % , % % / +

/%"&-"L# /-

% % 8 % 0 +/ % /">- &] 3/- % , 8/ K0 - $0=&=, '0=&-$0=&-$$0-$$0>& and various cathepsins&P%/+ % 8% These s8% +;% % % %/&" ( +* "&.

%% 8 , /+ ,%+/ 8 /-

8/ % / in vitro % 3+ / %%%,+ % in vivo models, as in vitro models lack 7/ %

0 / % 7/ % / % % % %/ % % %% % %/ + studied in several species, such as rats, rabbits&&-&L, swine&> % 0 human primates280, 281 6 , ( * % % +/ ;- , % / - %

,%0/ / % %/ %,0+:`$% , + % % (Q ==* 0 P&XN" /% / :0: , % 0/ 0 diet282 / %: : + %/ N / % - K0N0 L- L- Ky % 285 and the Ldlr0N0 mice286 /% % - prone to lesion development. [ 8 % / % %- 7 - , cholesterol diet. ApoE0N0 mice develop lesions throughout the arterial tree which ;%+/ %% -8+ - % L&. Ldlr0N0% %! % ,/ - /% ,/ % / % less progressed than in ApoE0N0 Ky% - / % % % %/ + - , % /% - / ) % %% % %% % aortic valve area and in carotid arteries285.

30


Chapter 1

% % - , %- , - % %

+ % +- ,7 %, % ,! , +%+/ 0 + % / 288, 289 % 7 % 0" ,: - % % / /

% % ! /% 47 % - %: : % + % % %%

% %

% % %+;% % - L0> +:

0 (P&XN"* % %+/ +/ 290 + %0

- ,B: : J /+/ /- 0+ %/: : + + =>>P-, + , ,

/ % % % >=0>. K % + , +%+/% 3+ / H -%+ , : : % (, %

+: %* % + , % : / - , + 7

+%% % % +, % 0 7

%

+ + /%%

%>0>". in vivo % ,%/% % %/% , %

6 ,- + % % % ,/ +/7 %

% 7 ,/ % % / % %% % % ' %%

% chapter 6.

31

7. Thesis Outline % +% , % , + subdivided into two parts. Part I 8 % + 6, / % / % % 8 , % + %N / ! /

% %

Chapter 2 % + 8- 8 / % % (=*

-, / %% Chapter 3% + % %% 4=-%, 8/ + % % , ] 0/ / : /

+ % , , , + ! / % atherosclerosis. In chapter 4-,, 0/ / % / 7

N : % : % 0 %% % %% plaque homing potential in vivo % % % In Part II- ,+ % ( %* most promising targets described in Part I. In chapter 5, we develop a magnetic resonance imaging contrast agent, coated with our scavenger receptor peptide % 0 0 % % "% + % %% % , % plaques. Q / chapter 7- , + % Q % % % % ,+%

% % %

,+%%

%

32


REFERENCES /%0O - % #- $- 3 V- Q X- Q f- Q % K- Q f-V -V %f-6 4-6 3-6 $-6 ,%)-f

X-f 3-: % - +-$-$ $-$

O-$ ; -4 V-'J -# )-# %[-3 #-3 -3 %#-3 + O- -[

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