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 Current Model of HDL-C Metabolism: Reverse Cholesterol Transport Bile ABCG1 Mature HDL-C SR-BI A-1 CE FC CE LCAT CE SR-BI FC HL, EL LDL Receptor CETP A-1 Liver FC Nascent HDL Macrophage Net transfer of cholesterol CE B VLDL/LDL-C Cuchel M et al. ATVB. 2003;23:1710–1712; Assmann G et al. Circulation. 2004;109(23 suppl 1):III-8–III-14. 2 • An appropriate model of cholesterol efflux from the atherosclerotic plaque should reflect the complex structure of the lesion itself. • What is the predominant form of cholesterol present in the vulnerable atherosclerotic plaque ? Lipid accounts for 40% of surface area of vulnerable plaques Relation of Plaque Lipid Composition and Morphology to the Stability of Human Aortic Plaques. C.V. Felton et al, ATVB 1997;17:1337-1345 In what physical forms is cholesterol present within vulnerable plaques ? 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) FC -rich microdomains are present in regions of human aortic tissue with lipid-containing foam cells FOAM CELLS ORO Haematoxylin Mab Mab58B1 58B1 + DAPI Autofluorescence Control W/O FOAM CELLS Ong DS et al J Lipid Res. 2010 51:2303-13 Incubation of human monocyte-derived macrophage cultures with AcLDL generates extracellular unesterified cholesterol-rich microdomains 1 Day + AcLDL 2 Days + AcLDL + Mab 58B1 Cell nuclei: DAPI Control no AcLDL Control Mab Ong DS et al J Lipid Res. 2010 51:2303-13 Unesterified cholesterol-rich microdomains can be removed by cholesterol acceptors HDL apoAI Ong DS et al J Lipid Res. 2010 51:2303-13 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) a-HDL SR-BI Endothelium ABCG1 1 SR-BI a-HDL 16 LA, AA, 12SHETE AGEs ABCG1 a-HDL MPO MDA AGEs SR-BI insulin Atherosclerotic Lesion Golgi ER TLR CE 7-KC-3-sulfate IRF LXR gene Glucose 17 PPARa PPARg 7-KC SR-BI gene AGEs, Hypoxia Oxidative stress Zhao et al, Curr Opin Lipidol 2010 RXR LXR 19 G P S 2 12 FC FC Endosome /lysosme 14 CRP, Glucose Oxidative stress Nucleus 7-KC 4 a-HDL SR-BI 7 ACAT NCEH FC 10 8 StAR CE Lipid droplet 9 27-OHC CYP27 5 a-HDL Aqueous diffusion FC FC FC MPO MDA AGEs FC 7-KC 6 11 ABCG1 gene ABCG1 FC PL FC 13 18 2 ABCG1 HMG-CoA Reductase a-HDL 3 Endosome /lysosme 15 5 FC Mitochondria Macrophage HDL-Mediated Cholesterol Efflux From Cholesterol-Rich Macrophages Two main pathways: Rader DJ. J Clin Invest. 2006;116:3090–3100. Lipid-poor nascent pre-β-HDL apo A-1 – ABCA1 transporter Mature HDL apo E – SR-BI receptor – ABCG1 transporter Does this pathway comprehensively account for all forms of cholesterol which may potentially undergo efflux from the atherosclerotic plaque ? 14 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 ABCG1 IS HIGHLY EXPRESSED IN HUMAN FOAM CELLS UPON STIMULATION BY LXR AGONISTS Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930 CELLULAR CHOLESTEROL EFFLUX TO HDL IN HUMAN MACROPHAGES IS ABCG1 INDEPENDENT 120 ABCG1 KD Ctrl 50nM siRNA % of ABCG1 100 80 60 40 20 0 mRNA Prot Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930 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) HMDM Larrede S., Arterioscler Thromb Vasc Biol. 2009, 29:1930 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 IN HUMAN MACROPHAGES 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 Cholesterol efflux studies should be conducted in human macrophages and not in mouse macrophages!!! HDL: New Dimensions QUALITY QUANTITY Particle structure HDL-C / Apo AI Lipidome, Proteome Functionality Kontush A, Chapman MJ. Pharmacol Rev. 2006. 21 22 Mechanisms of Cellular Cholesterol Efflux to HDL particles Extracellular space ABCA1 Lipid-poor ApoA-I Diffusion Discoidal HDL LCAT SR-B1 Diffusion Small spherical HDL LCAT Larger spherical HDL Cell membrane SR-B1 ABCG1 Diffusion SR-B1 ABCG1 FC FC FC FC 22 Proteolytic inactivation of pre-HDL impairs ABCA1-dependent initiation of reverse cholesterol transport in the atherosclerotic plaque Lee-Rueckert and Kovanen, Curr Opin Lipidol 2011 SUMMARY • 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 • What is the evidence that HDL/apoAI mediate cholesterol efflux from the plaque? 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 26 • HDL Infusions : • apoAI Milano/PL • Reconstituted HDL (apoAI/PL/detergent?) • Recombinant apoAI/PL (LPS?) • ……. Impact of HDL on Plaque Evolution : Experimental Evidence for Plaque Regression 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 Reconstituted HDL (CSL111) reduced atheroma in man • Single infusion of CSL111 reduced plaque lipid >60% in femoral arteries (Circ. Res. 2008) Reduction of plaque lipid by CSL111 80000 Oil Red O Lesion Area (µm2) • Four infusions of our prototype, CSL111, reduced the volume of coronary atheroma in ERASE (JAMA 2007) 60000 40000 * 20000 0 *P<0.05 from placebo 29 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 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 Impact of rHDL infusion on atherosclerotic lesions in the SFA Lipid content Inflammation -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 ? Can the cholesterol cargo in HDL potentially contribute to plaque cholesterol accumulation and thence progression ? apoE-HDL and Reverse Cholesterol Transport Mahley et al., JCI, 2006,116:1226-29 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 • Are these observations relevant to emerging therapeutic approaches to raise HDL-C and normalise its function in dyslipidemic, high risk metabolic states ? HDL metabolism: Impact of CETP inhibition Macrophage FC apoB ABCA1 PL FC VLDL A-I PreßHDL apoB CE LCAT HDL LDL-R LDL CE TG Liver CETP Peripheral tissues CE,FC FC E CE CE CE CE AI AI E HDL HDL Chapman et al, Eur Heart J 2009 E SR-BI Bile acids Cholesterol