1. HDL mediated efflux of cholesterol from the arterial wall: Can HDL promote removal of cholesterol from atherosclerotic lesions? Prof. John Chapman INSERM, Hôpital de la Pitié Paris, France

2. 2 Current Model of HDL-C Metabolism: Reverse Cholesterol Transport B CE Bile SR-BI LDL Receptor Liver FC CE LCAT A-1 CE HL, EL Macrophage Mature HDL-C Cuchel M et al. ATVB. 2003;23:1710–1712; Assmann G et al. Circulation. 2004;109(23 suppl 1):III-8–III-14. FC Nascent HDL CETP VLDL/LDL-C Net transfer of cholesterol FC ABCA1 SR-BI ABCG1 CE

3. An appropriate model of cholesterol efflux from the atherosclerotic plaque should reflect the complex structure of the lesion itself.

4. What is the predominant form of cholesterol present in the vulnerable atherosclerotic plaque ?

5. Relation of Plaque Lipid Composition and Morphology to the Stability of Human Aortic Plaques. C.V. Felton et al, ATVB 1997;17:1337-1345 Lipid accounts for 40% of surface area of vulnerable plaques

6. In what physical forms is cholesterol present within vulnerable plaques ?

7. Cholesterol is differentially distributed between plaque components Cholesterol is present in distinct physical forms : - in extracellular cholesterol-rich microdomains - in monocyte-derived macrophage foam cells (CE > FC) - in apoptotic cellular debris, primarily within the necrotic core (FC > CE)

8. FC -rich microdomains are present in regions of human aortic tissue with lipid-containing foam cells Ong DS et al J Lipid Res. 2010 51:2303-13 Control ORO Haematoxylin Mab 58B1 + DAPI Autofluorescence FOAM CELLS W/O FOAM CELLS

10. Incubation of human monocyte-derived macrophage cultures with AcLDL generates extracellular unesterified cholesterol-rich microdomains Ong DS et al J Lipid Res. 2010 51:2303-13 1 Day + AcLDL + Mab 58B1 2 Days + AcLDL Cell nuclei: DAPI Control no AcLDL Control Mab

11. Unesterified cholesterol-rich microdomains can be removed by cholesterol acceptors Ong DS et al J Lipid Res. 2010 51:2303-13 HDL apoAI

12. Cholesterol is differentially distributed between plaque components Cholesterol is present : - in extracellular cholesterol-rich microdomains - in monocyte-derived macrophage foam cells (CE > FC) - in apoptotic cellular debris, primarily within the necrotic core (FC > CE)

13. Zhao et al, Curr Opin Lipidol 2010

14. 14 HDL-Mediated Cholesterol Efflux From Cholesterol-Rich Macrophages Two main pathways:  Lipid-poor nascent pre-β-HDL –ABCA1 transporter  Mature HDL –SR-BI receptor –ABCG1 transporter Rader DJ. J Clin Invest. 2006;116:3090–3100. apo E apo A-1 Does this pathway comprehensively account for all forms of cholesterol which may potentially undergo efflux from the atherosclerotic plaque ?

15. STIMULATION OF CELLULAR CHOLESTEROL EFFLUX BY LXR AGONISTS IN HUMAN MACROPHAGES Question: Does the induction of cholesterol efflux to HDL by LXR agonists result from stimulation of ABCG1 expression? Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930

16. ABCG1 IS HIGHLY EXPRESSED IN HUMAN FOAM CELLS UPON STIMULATION BY LXR AGONISTS Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930

17. CELLULAR CHOLESTEROL EFFLUX TO HDL IN HUMAN MACROPHAGES IS ABCG1 INDEPENDENT 50nM siRNA Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930

18. STIMULATION OF CHOLESTEROL EFFLUX TO HDL BY LXR AGONISTS REQUIRES THE ABCA1 TRANSPORTER IN HUMAN MACROPHAGES TD: Tangier Disease Macrophage (Exon 34, 1536 Ser/Phe) Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930 HMDM

19. CELLULAR CHOLESTEROL EFFLUX TO HDL IN HUMAN MACROPHAGES REQUIRES THE CLA-1 RECEPTOR NB: Non-Blocking Cla-1 antibody B: Blocking Cla-1 antibody Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930

20. 1- ABCG1 does not promote FC efflux to HDL  mice 2- SR-BI/Cla-1 participates in free FC efflux to HDL  mice 3- Stimulation of FC efflux by LXR is an ATP-dependent transport mediated by ABCA1 independently of ABCG1 IN HUMAN MACROPHAGES Cholesterol efflux studies should be conducted in human macrophages and not in mouse macrophages!!!

21. 21 HDL: New Dimensions QUANTITY HDL-C / Apo AI QUALITY Particle structure Lipidome, Proteome Functionality Kontush A, Chapman MJ. Pharmacol Rev. 2006.

22. 22 Mechanisms of Cellular Cholesterol Efflux to HDL particles Extracellular spaceCell membrane FC ABCA1 Diffusion SR-B1 Diffusion SR-B1 ABCG1 Diffusion SR-B1 ABCG1 Lipid-poor ApoA-I Discoidal HDL Small spherical HDL Larger spherical HDL LCAT

23. Lee-Rueckert and Kovanen, Curr Opin Lipidol 2011 Proteolytic inactivation of pre-HDL impairs ABCA1-dependent initiation of reverse cholesterol transport in the atherosclerotic plaque

24. The potential efficacy of HDL / apoAI to efflux arterial cholesterol is dependent upon : the specific apoAI / HDL particle ligand(s) ligand quantity and quality (functionality) plaque structure and compartmentalisation plaque PG and GAG composition plaque protease activities relative expression levels of cholesterol efflux proteins in macrophage foam cells SUMMARY

25. What is the evidence that HDL/apoAI mediate cholesterol efflux from the plaque?

26. 26 Long history of HDL infusion reducing atheroma burden 1989: infusion of HDL reduced rabbit athero Repeated in rabbits and mice 1990s: transgenic studies showed ApoAI to be the active agent

27. HDL Infusions : apoAI Milano/PL Reconstituted HDL (apoAI/PL/detergent?) Recombinant apoAI/PL (LPS?) …….

28. Infusion of Recombinant Apo AI Milano/Phospholipid complexes over 5 weeks produced significant regression of coronary atherosclerosis as estimated on the basis of atheroma volume by IVUS in patients with acute coronary syndromes. Nissen et al., JAMA, 2003, 290 : 2292-2300 These findings suggest that elevation of HDL/Apo AI may enhance cholesterol efflux from plaque tissue and may therefore be a critical component of atherosclerotic plaque regression Impact of HDL on Plaque Evolution : Experimental Evidence for Plaque Regression

29. 29 Reconstituted HDL (CSL111) reduced atheroma in man Four infusions of our prototype, CSL111, reduced the volume of coronary atheroma in ERASE (JAMA 2007) Single infusion of CSL111 reduced plaque lipid >60% in femoral arteries (Circ. Res. 2008) Oil Red O Lesion Area (µm 2 ) *P<0.05 from placebo * Reduction of plaque lipid by CSL111

30. Impact of Reconstituted HDL infusion on Peripheral Arterial disease Recruited patients (n=10) with symptomatic PAD and lesions in SFA on Duplex US amenable to percutaneous revascularisation. Excluded those with Cr >0.25mmol/l or other co-morbidities such as hx of organ transplantation, malignancy with expected survival < 12 months Baseline Investigations : ABI, Fasting Plasma for Inflammatory Markers, Lipid profile Randomised to IV Placebo or IV CSL 111 (80mg/Kg) given over 4 hr infusion 5-7 days following infusion patients admitted to hospital for revascularisation procedureInvolving atherectomy (Foxhollow Atherectomy ® ) ± Angioplasty/Stenting

31. Figure 3. Accumulation of lipids and macrophage size in the lesions. Shaw J A et al. Circulation Research 2008;103:1084-1091 Copyright © American Heart Association

32. Lipid content Inflammation Impact of rHDL infusion on atherosclerotic lesions in the SFA -45% intra-plaque VCAM-1 expression -40% lipid content -40% macrophage cell size -50% circulating monocyte activation (CD11b) +15% cellular cholesterol efflux capacity Enhanced RCT ?

33. Can the cholesterol cargo in HDL potentially contribute to plaque cholesterol accumulation and thence progression ?

34. apoE-HDL and Reverse Cholesterol Transport Mahley et al., JCI, 2006,116:1226-29

35. ApoE- HDL -is specifically bound and retained in arterial matrix by extracellular PGs, notably biglycan O’Brien et al, Atherosclerosis 2004: 177 O’Brien et al, Circulation 1998: 98 Ollin et al, ATVB 2001: 21

36. Are these observations relevant to emerging therapeutic approaches to raise HDL-C and normalise its function in dyslipidemic, high risk metabolic states ?

37. apoB Macrophage VLDL SR-BI Liver CE,FC ABCA1 A-I PLFC LDLLCAT CE apoB Peripheral tissues Bile acids Cholesterol CETP HDL metabolism: Impact of CETP inhibition CE LDL-R FC CE HDL TG CE E E E HDL AI Preß- HDL Chapman et al, Eur Heart J 2009