Atherosclerosis

Established therapies

Statins Asparin β-blockers Renin-angiotensin system inhibitors

Grades of atherosclerosis

1 - Foam cells 2- Fatty streak 3 - Extracellular fatty streak 4- Lipid core 5- Atherosclerotic plaque lipid core embedded in fibrosis 6- Complicated atherosclerotic plaque (plaque rupture, thrombosis, haemorrhage).

Experimental Systems

1. Epidemiological studies 2. Clinical Trials 3. Experiments on samples from patients (e.g. gene expression profiling on unstable plaques) 4. Cell culture model systems 5. Animal Models: Diet-induced atherosclerosis in apolipoprotein E- or LDL Receptor-deficient mice.

Atherosclerosis as an inflammatory disorder 1-4

1. Markers of inflammation (e.g. interleukin-6, C-reactive protein) are elevated in individuals with cardiovascular disease 2. Epidemiological studies have demonstrated increased vascular risk in individuals with elevated levels of certain cytokines (IL-6, IL-1, TNF-), cell adhesion molecules (e.g. intercellular adhesion molecule-1)) and acute-phase proteins (e.g. C-reactive protein, fibrinogen, serum amyloid A) 3. Presence of immune and inflammatory cells (e.g. T-lymphocytes, macrophages) in atherosclerotic lesions 4. Increased expression of pro-inflammatory cytokines in atherosclerotic lesions

Atherosclerosis as an inflammatory disorder 5 - 9

5. Immune-cell recruitment initiates atherosclerotic-plaque formation 6. ApoE-/- or LDL-R-/- mice that are also deficient for chemokines [e.g.monocyte chemoattractiant protein-1 (MCP-1)), chemokine receptors (e.g. MCP-1 receptor) or adhesion molecules (e.g. E-selectin, P-selectin, vascular cell adhesion molecule-1) have reduced severity of atherosclerosis 7. Reduced atherosclerosis in hypercholesterolaemic mice lacking proinflammatory cytokines (e.g.IFN-, IL-12, IL-18) or their receptors (e.g.IFN- receptor) 8. Increased atherosclerosis in hypercholesterolaemic mice injected with IFN- 9. Increased atherosclerosis in hypercholesterolaemic mice lacking anti-inflammatory cytokines such as TGF- or IL-10

Progression of fatty streak to fibrous plaque

A chronic inflammatory response due to cytokines produced by macrophages and T-lymphocytes Migration of smooth muscle cells from the media to the intima Proliferation of smooth muscle cells Formation of smooth muscle cell-derived foam cells Secretion of extracellular matrix proteins.

Statins

About 75% of total cholesterol pool derived from de nono synthesis Statins inhibit HMG-CoA reductase, a rate limiting step in the biosynthesis of cholesterol Produce about 50-60% reduction in LDL-cholesterol Also inhibit the inflammatory response Numerous clinical trials have supported the protective role of statins in the development of atherosclerosis.

Progression of normal artery to fatty streak

Accumulation of LDL and its oxidation to oxLDL Initial damage to ECs by oxLDL and other atherogenic factors Expression of adhesion proteins and cytokines by ECs Infiltration of monocytes and T-lymphocytes Macrophage differentiation and expression of SRs Uptake of oxLDL by macrophages to form foam cells

Peroxisome-proliferator activated receptors

Anti-atherogenic and anti-inflammatory in mouse model systems TZD (glitazones) used in type II diabetes and fibrates effectively lower triglyceride levels Clinical trials have been largely disappointing A non-agonist PPARγ ligand has been identified that blocks phosphorylation of this receptor associated with obesity Selective PPAR modulators

Structure of Lipoproteins

Apolipoprotein Free cholesterol Cholesteryl ester Triglyceride Phospholipid

Progression of fibrous plaque to plaque rupture

Apoptosis and necrosis of macrophage and smooth muscle cell-derived foam cells Accumulation of extracellular cholesterol Secretion of matrix metalloproteinases by macrophages leading to instability of the fibrous plaque.

Atherosclerosis definition

Atherosclerosis is a chronic inflammatory response in the walls of arteries, in large part due to deposition of lipoproteins (plasma proteins that carry cholesterol and triglycerides).

Lifestyle changes

Cessation of smoking. Reduce uptake of saturated fats and salt. Eat five servings of fruit and vegetables per day. At least 30 minutes of moderate exercise on five or more days per week. Combating detrimental psychosocial factors such as stress, depression and anxiety.

HDL treatments

Cholesterol ester transfer protein (CETP) inhibitors: torcetrapib failed in clinic (off target effects?); delcetrapib and anacetrapib have entered clinical evaluation. Apolipoprotein A-I (ApoA-I): apoA-I milano and various peptide mimetics (some success), manipulation of transcription more difficult. Inhibitors of cholesterol ester transfer protein (CETP) as its deficiency in humans is characterised by increased HDL levels and slightly reduced LDL levels. Clinical tials with torcetrapib failed; others in progress. miRNA (e.g. miR-33a/b antagonists raise plasma HDL and lower VLDL triglycerides in non-human primates.

LDL uptake, ic trafficking and efflux modification

Clinical trials with acyl coenzyme A:cholesterol acyltransferase have been disappointing More studies on other targets, particularly those that promote cholesterol efflux (ABCA1, apoE).

LDL modification

Clinical trials with antioxidant vitamins have failed to reveal reduction in cardiovascular events Members of phospholipase A2 family (PLA2) associate with lipoprotein particles to modify phospholipids and generate atherogenic moieties Potential of inhibitors sPLA2 (Verespladib) and Lp-PLA2 (Darapladib) have proved disappointing (VISTA-16; SOLID TIMA-52)

1986 world wide deaths

Coronary disease 7.2 million. Cancer 6.3. Cerebrovascular 4.6.

Clinical manifestations of atherosclerosis

Coronary heart disease (CHD) - angina pectoris, MI, sudden cardiac death, congestive heart failure & arrythmias. Cerebrovascular disease - stroke. Peripheral vascular disease - gangrene, cold feet, painful feet.

Animal models

Diet-induced atherosclerosis in apolipoprotein E- or LDL Receptor-deficient mice - Use of pharmaceutical modifiers Double knock-out mice (e.g. apoE-/-, gene X-/-) Bone marrow transplantation of irradiated apoE-/- or LDL-receptor-/- with that from wild-type or homozygous/heterozygous gene X-deficient mice

Dual and dietary control

Dual control of cholesterol synthesis by statins and uptake of dietary cholesterol using ezetimibe, inhibitor of intestinal cholesterol transporter Niemann-Pick C1-like protein 1 Uptake of dietary cholesterol can also inhibited by consumption of sitostanol/sitosterol margarines or using bile acid sequestrants (e.g. colesevelam)

Cell culture model systems

Effect of extracellular factors on uptake or efflux of cholesterol in primary macrophages; migration of labelled monocytes to endothelial cells; smooth muscle cell proliferation

PPAR gamma

Expression: Fat cells, large intestine, macrophages Ligands: Certain polyunsaturated fatty acids, TZDs Functions: Increases FA storage through adipocyte differentiation, decreases hyperglycemia, increases insulin sensitivity, direct anti-atherosclerotic vascular effects.

Farnesoid-X-Receptor (FXRs)

Expression: Liver, intestine Ligands: Bile acids, farnesol Functions: Regulates the expression of a number of genes implicated in the synthesis, transport and detoxification of bile acids. Also, control lipid metabolism.

Retinoid-X-Receptor (RXR)

Expression: Liver, kidney, heart, macrophages Ligands: Retinoic acid Functions: Facilitates the action of other nuclear receptors.

PPAR alpha

Expression: Liver, kidney, heart, muscle, macrophages Ligands: Certain polyunsaturated fatty acids, fibrates Functions: Increases FFA uptake and oxidation, reduces TG levels, direct anti-atherosclerotic vascular effects, increases HDL-C levels

Liver-X-Receptors (LXRs)

Expression: Liver, macrophages Ligands: Oxysterols Functions: Increases cholesterol catabolism to bile acids, increases peripheral cholesterol transport, decreases net choleaterol absorption, increases fatty acid uptake and synthesis.

PPAR delta

Expression: Most tissues Ligands: Certain polyunsaturated fatty acids, fibrates Functions: Increases fatty acid oxidation, decreases TG levels, direct anti-atherosclerotic vascular effects, increases HDL-C levels, decreases body weight.

Nuclear receptors

Fibrates (e.g. fenofibrate) lower circulating triglycerides Polyunsaturated fatty acids have a protective effect against atherosclerosis Thiazolidinediones (TZDs) improve insulin sensitivity in type II diabetes.

Grades with clinical symptoms

Graded V and VI

HDL Cholesterol

HDL has a protective effect for risk of atherosclerosis. Epidemiological studies show that the lower the HDL-C level, the higher the risk for atherosclerosis. HDL-C tends to be low when triglycerides are high. HDL-C is lowered by smoking, obesity and physical inactivity. apoA-I is the major apolipoprotein in HDL and an elevated ApoA-I is linked to reduced CVD risk.

Atherosclerosis

Hardening or furring of arteries leading to heart attacks, strokes and gangrene of the extremeties.

Neimann-Pick C1-like protein (NPLC1L1)

Inhibition of intestinal NPLC1L1 by ezetimibe can reduce LDL-C levels by almost 20% in individuals treated with statins Many previous clinical trials failed to show that combined statin and ezetimibe treatment reduces cardiovascular event rate than those seen by statin alone IMPROVE-IT (18,144 subjects) and PRECISE-IVUS (229 subjects) showed ezetimibe plus statin resulted in further lowering of LDL cholesterol and improved cardiovascular outcomes

M-CSF

Macrophage colony-stimulating factor A potent monocyte activator and co-mitogen. Produced by endothelial and smooth muscle cells. Localizes in human and experimental atheroma.

Inflammatory therapeutic approaches

Small molecule inhibitors of inflammatory agent or key components of intracellular signalling pathway. Neutralizing monoclonal antibodies. Soluble decoy receptors. Manipulating production/action of certain classes of immune cells (e.g. Tregs)

Risk factors for CVD

Modifiable - Smoking, dyslipidaemia, raised BP, DM, obesity, dietary factors, thrombogenic factors, lack of excercise, alcohol. Non-modifiable - Family history of CVD, Personal history of CVD, Age, Gender.

Familial Hypercholesterolaemia

Most common genetic disorder in Europe and USA. Caused by a mutation of the LDL receptor. Increases risk of cardiovascular disease. Two types of FH. Heterozygous FH: One LDL-receptor gene affected; affects about 1 in 500 people; TC ~360-560mg/dL in adulthood. Homozygous FH: both LDL-receptor gene affected; rare, about 1 in million people; TC ~600-1200mg/dL in adulthood.

19th Century work on Atherosclerosis

Most of the pathologists of the 19th century considered atherosclerosis as a degenerative process with intimal proliferation of connective tissue and calcification.

Cholesterol

Plasma cholesterol at levels >200 mg/dL cause 4.4 million deaths a year. Incidence of plasma cholesterol >200 mg/dL in 51% (107 million) adults in the USA. 10% reduction in plasma cholesterol results in 15% reduction in mortality from coronary heart disease. LDL-C is a major target to prevent CHD.

Dyslipidaemias

Raised LDL-C Low HDL-C Raised triglycerides

HDLs

Reverse cholesterol transport; Anti-inflammatory actions Low levels of HDL are common in obese individuals. Increasing circulating HDL levels is an important avenue to limit atherosclerosis Complex biology, interaction with numerous proteins and heterogeneity Nicotinic acid increases HDL levels and shown some reduction in cardiovascular events in clinical trials but tolerability issues.

LDL-C and statins

Several large clinical trials and meta-analysis have demonstrated the impact of LDL-C lowering by statins in the reduction of incidence of cardiovascular events. Statins have actions beyond lowering LDL-C (pleiotropic effects). Statins reduce cardiovascular mortality by ~30%. A number of trials have highlighted the significance of residual cardiovascular risk after treatment of LDL-C to target levels. Some patients fail to attain recommended LDL-C target levels with maximal doses of statins whereas many can't tolerate existing agents due to various side effects. Need to understand the disease in more detail and target other factors such as low HDL-C, high triglyceride levels, oxidative stress and the inflammatory response

Brown and Goldstein 19th

Showed LDL receptor is not involved in macrophage foam cell formation and proposed that a macrophage receptor that recognises acetylated LDL plays a key role.

Ignatowski 1908

Showed atherosclerosis is induced in rabbits by feeding them a diet of milk and egg york. Adding pure cholesterol to rabbit food has a similar effect. Gave rise to the lipid theory of atherosclerosis.

LDL Cholesterol

Strongly associated with atherosclerosis and cardiovascular disease. 10% increase results in an approximate 20% increase in coronary heart disease risk. Most of the cholesterol in plasma is found in LDL particles.

Steinberg 1980s

Suggested a central role for oxidised LDL in atherosclerosis. A number of scavenger receptor mediating their uptake were subsequently identified.

Types of Lipoproteins

Triglyceride-rich lipoproteins: Chylomicrons, Very low-density lipoproteins (VLDL). Cholesterol-rich lipoproteins: Low-density lipoproteins (LDL), High-density lipoproteins (HDL). Intermediate-density lipoproteins (IDL) or remnants are produced by processing of chylomicrons or VLDL.

LDL uptake regulation

Uptake of LDL via LDL receptor is under negative feed-back regulation ↑ Uptake, ↓ LDL receptor expression LDL, particularly modified LDL (e.g. oxidised LDL, glycated LDL) is also taken up by a number of so called scavenger receptors (SRs) SRs are not under negative fee-back regulation. Leads to formation of lipid-loaded foam cells.

Alternative therapies

Vaccination using components of LDL/modified LDL and other antigens that mount cellular and humoral immune response in atherosclerosis (e.g. HSP60). Infusion of anti-LDL antibodies Inhibit the action of pro-atherogenic cytokines (e.g. IL-1 receptor antagonist, human antibody against IL-1β, low doses of methotrexate) CIRT: Cardiovascular Inflammation Reduction Trial CANTOS: Canakinumab Anti-inflammatory Thrombosis Outcomes Study Stimulate the action of anti-atherogenic cytokines or cells that produce them (e.g Treg)