1-Dyslipidemia_Module-1-Pathophysiology (1)

Module 1: Pathophysiology of Dyslipidaemia in Relation to Atherosclerosis.

Learning Objectives • Review the role and function of lipids, lipoproteins, and apolipoproteins in cholesterol synthesis and absorption • Describe the pathophysiology of atherosclerosis, including the role of LDL, mechanisms of plaque formation, and atheroma evolution • Discuss gaps in knowledge on the pathophysiology of atherosclerosis LDL, low-density lipoprotein. 2.

What do we know is true? 3.

Lipids Adapted from Fahy E, et al. J Lipid Res 2009;50:S9-S14. 4 Main classes of interest in lipidology • Sterols: Cholesterol • Apolipoproteins • Triglycerides • Phospholipids Fatty acids Saturated Monounsaturated Polyunsaturated Triglycerides Glycerol Fatty acids Triglyceride = 1 glycerol + 3 fatty acids Unsaturation bonds Carbon Hydrogen.

Cholesterol Synthesis Pathway: “The Easy Way” ATP, adenosine triphosphate; CoA, coenzyme a; HMG CoA, hydroxymethylglutaryl-CoA synthase. Adapted from “Cholesterol: synthesis, metabolism, and regulation.” The Medical Biochemistry Page website. Accessed June 2020. 5 - HMG CoA reductase Cholesterol 7-α hydroxylase ATP citrate lyase - 7-α-hydroxycholesterol Mevalonic acid HMG CoA Cholesterol Acetyl CoA Citrate Biliary acids Bempedoic acid Statins.

Cholesterol Synthesis: “The Hard Way” CoA, coenzyme a; DHCR, dehydrocholesterol reductase; FPPS, farnesyl-pyrophosphate synthase; GGPPS, Geranylgeranyl pyrophosphate synthase; HMG CoA, hydroxymethylglutaryl-CoA; HMGCS, HMG CoA synthase; HNGCR, HMG-CoA reductase; HSD, hydroxysteroid dehydrogenase; IDI, isopentenyl-diphosphate delta isomerase; LBR, lamin-B receptor; LDM, lanosterol 14-demethylase; MK, mevalonate kinase; MVD, diphosphomevalonate decarboxylase; NSDHL, NAD(P)-dependent steroid dehydrogenase-like; PMK, phosphomevalonate kinase; SC4MOL, sterol-C4-methyl oxidase-like; SC5D, sterol-C5-desaturase; SM, squalene monooxygenase; SQS, squalene synthase. Sharpe LJ, Brown AJ. J Biol Chem 2013;288(26):18707-15. 6 Understanding the regulation of cholesterol synthesis in cells is important to understand cholesterol homeostasis of the whole organism.

Lipoproteins • Lipids are insoluble in water • Lipoproteins are soluble macromolecules consisting of lipids and proteins transported in plasma • Lipid fraction • Free and esterified cholesterol, triglycerides, and phospholipids • Protein fraction • Apolipoprotein Adapted from “Lipoproteins, blood lipids, and lipoprotein metabolism.” The Medical Biochemistry Page website. Accessed June 2020. 7 Esterified cholesterol Apolipoprotein.

Major Lipoproteins Apo, apolipoprotein. Adapted from Authors/Task Force Members, et al. Atherosclerosis 2019;290:140-205. 8 CM Chylomicrons VLDL Very low- density lipoprotein IDL Intermediate- density lipoprotein Lp(a) Lipoprotein(a) LDL Low-density lipoprotein Apo(a) B100 HDL High-density lipoprotein B100 12-16% 16-24% 20-35% 22-26% 6-15% 7-11% 55% 10-20% 35-46% 17-24% 6-9% Unesterified cholesterol Phospholipids Cholesteryl esters Triglycerides.

Apolipoproteins Apo, apolipoprotein; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; Lp(a); lipoprotein(a); VLDL, very low-density lipoprotein. Adapted from Remasamy I. Clin Chem Lab Med 2014;52(12):1695-727. 9 Apolipoprotein Molecular weight (daltons) Lipoproteins Chylomicrons HDL VLDL IDL LDL Lp(a) ApoAI 28,016 √ √ ApoAII 17,414 √ √ ApoIV 46,465 √ √ ApoAV - √ √ √ ApoB48 264,000 √ ApoB100 540,000 √ √ √ √ ApoCI 6630 √ √ √ √ ApoCII 8900 √ √ √ √ ApoCIII 8800 √ √ √ √ ApoE 34,145 √ √ √ √ Apo(a) 250,000-800,000 √.

LDL-C Has the Main Role in Atherosclerosis • LDL-C is catabolised by 2 pathways: • A receptor-dependent pathway in the liver and various organs • A receptor-independent pathway in non-hepatic tissues • Imbalance in the 2 pathways is clinically significant: • Results in accumulation of deposited LDL-C • Key event in fatty streak development and atherosclerosis progression • LDL-C particles can influence the properties of the arterial wall, including: • Altering contractility and VSMC phenotype • Reducing endothelial NO synthesis • Activating pro-inflammatory cytokines following arterial injury LDL-C, low-density lipoprotein cholesterol; NO, nitric oxide; VSMC, vascular smooth muscle cell. Helkin A, et al. Vasc Endovascular Surg 2016;50(2) 107-18. Borén J, et al. Eur Heart J 2020;41:2313-30. 10 LDL-C.

Atherosclerosis Pathophysiology LDL-C, low-density lipoprotein cholesterol; MMP, matrix metalloproteinase; VCAM-1,vascular cell-adhesion molecule-1. Adapted from Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Sakakura K, et al. Heart Lung Circ 2013;22(6):399-411. 11 Monocyte Macrophage Foam cell Arterial lumen Intima Cytokines Adhesion molecules Oxidised LDL LDL-C VCAM-1 Selectins MMPs Collagen break down Smooth muscle cell migration Endothelium.

Atheroma Evolution: Early Lesions, PIT, and Fibroatheroma EL, extracellular lipid; NC, necrotic core; PIT, pathologic intimal thickening. Adapted from Virmani R, et al. Arterioscler Thromb Vasc Biol 2000;20(5):1262-75. Sakakura K, et al. Heart Lung Circ 2013;22(6):399-411. 12 Preatherosclerotic coronary lesions Intimal thickening Fatty streak Pathologic intimal thickening Fibroatheroma ? Progressive lesions if not treated Smooth muscle cells Proteoglycan matrix Variable lipid pool Macrophage infiltrate Acellular necrotic core Thick fibrous cap Stable lesions Lesions may regress Smooth muscle cells Macrophage foam cells Extracellular lipid Cholesterol clefts Necrotic core Calcified plaque Haemorrhage Thrombus Healed thrombus Collagen Illustration key.

Atheroma Evolution: The Vulnerable Plaque and Complex Lesions FC, fibrous cap; NC, necrotic core; SMC, smooth muscle cells; Th, thrombosis. Adapted from Virmani R, et al. Arterioscler Thromb Vasc Biol 2000;20(5):1262-75. Sakakura K, et al. Heart Lung Circ 2013;22(6):399-411. 13 Thin cap fibroatheroma “Vulnerable plaque” Thin fibrous cap ≤ 65 µm Rupture Erosion de-endothelialisation and platelet aggregation Calcified nodule Acute thrombus in direct contact with the intima, rich in SMCs and proteoglycan matrix with an absence of endothelial lining; poor lipid core; media often intact; inflammatory infiltrate minimal Rare; eruptive fragments of calcium that protrude into the lumen, causing a thrombotic event Extensive necrotic cholesterol core; macrophage and lymphocyte infiltrate ++; media often disrupted Smooth muscle cells Macrophage foam cells Extracellular lipid Cholesterol clefts Necrotic core Calcified plaque Haemorrhage Thrombus Collagen Illustration key.

Reassessing the Mechanisms of Acute Coronary Syndromes NET, neutrophil extracellular trap; STEMI, ST-elevation myocardial infarction. Adapted from Libby P, et al. Circ Res 2019;124:150-60. 14 Inflammation promotes a decreased synthesis and increased breakdown of collagen in plaque fibrous cap Associated with non-STEMI; platelet-rich thrombus anticoagulation Associated with STEMI Urgent, invasive revascularisation Gold standard therapy Ruptured Plaque • Thin fibrous cap • Collagen-poor fibrous cap • Large lipid core • Many macrophages • Fibrin-rich thrombus Eroded Plaque • Proteoglycan, glycosaminoglycan rich • Little or no lipid core • Neutrophils and NETs • Many smooth muscle cells • Platelet-rich thrombus Media Adventitia Media Adventitia.

Interplay Between Dyslipidaemia and Hypertension in the Development of Atherosclerosis NO, nitric oxide; RAAS, renin-angiotensin-aldosterone system. Adapted from Hurtubise J, et al Curr Atheroscler Rep 2016;18:82. Borghi C, et al. Nutr Metab Cardiovasc Dis 2017;27(2):115-20. 15 Dyslipidaemia Hypertension RAAS activation Oxidative stress Lipid peroxidation Endothelial dysfunction Decreased NO bioavailability Pro-inflammatory activity Impaired vascular contractility Atherosclerosis Antioxidants NO.

Myth or reality? LDL-C is the basis of atherosclerosis, not a surrogate marker. LDL-C, low-density lipoprotein cholesterol. 16.

Gap in Knowledge of Cellular Mechanisms •A comprehensive understanding of the cellular mechanisms underlying the development of atherosclerosis secondary to dyslipidaemia remains a gap in our knowledge Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Authors/Task Force Members, et al. Atherosclerosis 2019;290:140-205. Borén J, et al. Eur Heart J 2020;41:2313-30. Ference BA, et al. Eur Heart J 2017;38:2459-72. 17.

Is LDL-C the Basis of Atherosclerosis or Only a Surrogate Marker of the Disease? •What are the real consequences of the protective effects of HDL-C, the role of triglycerides, and modified lipid proteins or chylomicrons remnants? HDL-C, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol. Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Authors/Task Force Members, et al. Atherosclerosis 2019;290:140-205. Borén J, et al. Eur Heart J. 2020;41:2313-30. Ference BA, et al. Eur Heart J. 2017;38:2459-2472. 18 HDL-C Modified lipid proteins Triglycerides Chylomicron remnants Reality: Many other components play a role in vascular disease.

Other Influences on Atherosclerosis Development •Genetic background, lifestyle, healthcare access, comorbidities, and drugs all influence atherosclerosis development •How can we improve our understanding and act on these health determinants? Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Authors/Task Force Members, et al. Atherosclerosis 2019;290:140-205. 19.

Where do we go from here? 20.

Potential Actions to Prevent Atherosclerosis Progression and Reduce Cardiovascular Events LDL-C, low-density lipoprotein cholesterol. 21 How can we further prevent atherosclerosis progression and reduce cardiovascular events? LDL-C reduction With combined antihypertensive and antidyslipidaemia therapy? With combined statin or non-statin therapy earlier? Polygenetic risk scores or inflammation status Or explore the pleotropic effects of other drugs? LDL-C reduction Should we go deeper, or lower? Should we start sooner? Should we address multiple risk factors? Should we explore other mechanisms? Should we use anti-inflammatory or antithrombotic agents?.

Polling Question 1 Do you think we understand the mechanisms of atherosclerosis? A. Yes B. No C. I don’t know 22.

Polling Question 2 Which do you think is mainly responsible for progression of atherosclerotic plaque? A. Inflammation B. LDL-C C. Perhaps both LDL-C, low-density lipoprotein cholesterol. 23.

Polling Question 3 Which lipoprotein is of greatest concern to you? A. Lipoprotein(a) B. Low-density lipoprotein (LDL) C. Chylomicrons 24.

Key Take-Home Points (1/2) • Events that lead to development of atherosclerotic plaque: • LDLs and oxLDLs injure endothelial cells, promote monocyte adhesion, muscle cell migration, and platelet activation • All ApoB-containing lipoproteins < 70 nm can cross the endothelial barrier • Especially in the presence of endothelial dysfunction • They can become trapped after interaction with extracellular structures such as proteoglycans ApoB, apolipoprotein B; LDL, low-density lipoprotein; oxLDL, oxidised low-density lipoprotein. Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Authors/Task Force Members, et al. Atherosclerosis 2019;290:140-205. 25 LDL oxLDL Esterified cholesterol Apolipoprotein.

Key Take-Home Points (2/2) • Atherosclerosis: • Is the sequelae of chronic arterial inflammation secondary to prolonged exposure to oxidative stressors • Involves multiple cell types and cellular mediators • The size of the total atherosclerotic plaque burden is determined by: • The concentration of circulating LDL-C and other ApoB-containing lipoproteins • The total duration of exposure to these lipoproteins ApoB, apolipoprotein B; LDL-C, low-density lipoprotein cholesterol. Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Authors/Task Force Members, et al. Atherosclerosis 2019;290:140-205. 26.

Appendix slides 27.

Exogenous (Intestinal) Lipid Transport Pathway Chylomicrons (CMs) • Formed in the enterocytes and contain CE and TG (> 90%) • Acquire CE from HDL in exchange of TG • Also exchange ApoA-I and ApoA-IV for ApoC and ApoE • Formed by re-esterification of FFA • Carried to the peripheral tissues, including muscles and adipose tissue Free fatty acids (FFAs) • Released by the action of activated LPL • Undergo β-oxidation to be used as energy source or stored as fat in the adipose tissues ApoCII • Required for the activation of the LPL ApoE • Required for the recognition of the CM remnants by the liver’s receptors Apo, apolipoprotein; CE, cholesterol esters; CETP, cholesterol ester transfer protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LPL, lipoprotein lipase; TG, triglycerides. Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Ramasamy I. Clin Chem Lab Med 2014;52(12):1695-727. 28.

NPC1L1 and ABCG5/G8 Cholesterol Absorption Regulation and CM Synthesis ABCG5/G8, ATP-binding cassette sub-family G5/G8; ACAT, acyl-CoA:cholesterol; B48, apolipoprotein-B48; CE, cholesteryl ester; CM, chylomicron; FA, fatty acids; FATP4, fatty acid binding protein 4; FC, free cholesterol; MTP, microsomal transfer protein; NPC1L1, Niemann-Pick C1-like 1 protein; TG, triglyceride. Adapted from de Bari O, et al. J Lipids. 2012;2012:302847. 29 MTP Immature CM ABCG5/G8.

Endogenous Lipid Transport Pathway • VLDLs are synthesised by the liver and are largely responsible for transport of endogenous triglycerides • ApoB is the main apolipoprotein of VLDL and LDL • ApoCII and ApoE are acquired from HDL, where ApoCII activates LPL • As TGs are removed, density of the particle increases and particle transitions to an IDL • Particles are removed from circulation or go on to become LDLs after further removal of TGs Apo, apolipoprotein; CETP, cholesterol ester transfer protein; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; LPL, lipoprotein lipase; SR-BI, scavenger receptor class B type I; TG, triglyceride; VLDL, very low-density lipoprotein. Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Ramasamy I. Clin Chem Lab Med 2014;52(12):1695-727. 30.

LDL, low-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; LDL-R, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9. Horton JD, et al. J Lipid Res 2009;50:S172-7. Ramasamy I. Clin Chem Lab Med 2014;52(12):1695-727. 31 LDL LDL-R LDL Endocytosis PCSK9 Lysosome LDL-R recycling PCSK9 inhibitor PCSK9 prevents LDL-R recycling Endocytosis PCSK9 and LDL-R Recycling.

HDL-C and Reverse Cholesterol Transport • HDL-C mediates RCT in the transport of cholesterol from peripheral tissues to the liver, either as FC or bile acids • Enterocytes, hepatocytes, and macrophages are responsible for HDL-C lipidation • Nascent HDL-C can be esterified by LCAT to mature HDL-C • In the direct pathway, CE and FC from HDL are selectively taken up by the liver via SR-BI • HDL-CE can also be transferred to ApoB-containing lipoproteins via CETP and disposed in the liver ABCA1, adenosine triphosphate-binding cassette transporter; ApoB, apolipoprotein B; CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; EL, endothelial lipase; FC, free cholesterol; HDL-C, high-density lipoprotein cholesterol; HL, hepatic lipase; LCAT, lecithin-cholesterol acyltransferase; RCT, reverse cholesterol transport; SR-BI, scavenger receptor class B type I. Helkin A, et al. Vasc Endovascular Surg 2016;50(2):107-18. Ramasamy I. Clin Chem Lab Med 2014;52(12):1695-727. 32.