ATHEROSCLEROSIS

• Definition, histology, risks

• Role of the endothelium

• Molecular mechanisms of atherogenesis

• Therapies
 Definition
‘Athera’ = porridge/gruel
‘Sclerosis’ = hardening

• Disease of large & medium-sized

arteries

• ‘Furring’ or ‘Hardening’ of blood

vessels

• Atheromas (plaques) develop in

vessel wall - process is
atherogenesis.
 Architecture of arteries
Adventitia: Connective tissue

Intima: Endothelial monolayer

overlying loose connective tissue

Media: Smooth muscle cells

Atheromatous plaques
Atheromatous plaques
Rupture of plaque

Thrombus
formation

Blood supply restricted

Heart Brain

Heart attack Stroke

 Facts and Figures
• Atherosclerosis is the leading cause of death in the
developed world

• Predicted to become leading cause in developing

world

• Mortality rate is falling in the UK

• Highest European prevalence found in Eastern Europe

• Highest UK prevalence found in West of Scotland.

Causes of
mortality in
under 75s,
2014
 Cardiovascular disease (CVD) death rates per 100,000, by sex, selected
countries 2008
Ukraine
Russian Federation
Kazakhstan
Latvia
Lithuania
Estonia
Slovakia
Hungary
Poland
Czech Republic
Finland
Slovenia
Greece Women
Germany Men
Austria
Sweden
Ireland
United Kingdom
Portugal
Norway
Italy BHF Heart Statistics
Netherlands
Spain
France
Israel

800 600 400 200 0 200 400 600 800 1,000 1,200
 Age-standardised death rates from CHD/stroke in men
and women under 75, per 100,000, by local
authority, United Kingdom 2010/12

CHD Stroke
 Risk Factors

• Hyperlipidaemia elevated LDL cholesterol

low HDL cholesterol
high circulating triglycerides
• Hypertension
• Diabetes Mellitus & Obesity
• Lifestyle Smoking & Exercise
• Chronic Inflammatory Disease
• Hyperhomocysteinaemia
• Age & Sex ~10 yr delay in onset in females
• Family History/Genetics
Atherosclerosis is asymptomatic

• Often no symptoms before event

(heart attack/stroke)

• Cannot give definitive figure for

number with atherosclerosis
Early stage of atherosclerosis is
the ‘fatty streak’
Progression of Atheroma
The arterial endothelium
 Endothelial nitric oxide (NO) production
Blood endothelium Smooth
vessel muscle
lumen

relaxation
Shear stress
Acetylcholine
Bradykinin eNOS cGMP
Insulin/IGF-I
VEGF
NO NO
 Production of NO by endothelial Nitric
Oxide Synthase (Prof. Leiper’s session)

Shear stress
Acetylcholine
Bradykinin L-Arg, O2, NADPH
IGF-I
VEGF eNOS
Citrulline, NADP+, H+
+NO
 Regulation of eNOS

N Oxidase Reductase C
myr
palm
palm

L-Arginine Ca2+/CaM FAD

BH4 FMN
NADPH

Also: Phosphorylation at Ser1177

Dimerisation/Monomerisation
Association with hsp90, caveolin, GPCRs, others
Gene expression
Proposed arrangement of eNOS
Fluid shear stress
is an important
regulator of NO
synthesis in vivo.
 Shear stress (blood flow) stimulates eNOS
phosphorylation

32P-eNOS

Gallis, B. et al. J. Biol. Chem. 1999;274:30101-30108

 Figure 2
eNOS S1177 phosphorylation lowers
sensitivity of eNOS to Ca2+

Current Biology 1999 9, S1-848DOI: (10.1016/S0960-9822(99)80371-6)

 Stimulation of NO synthesis
Acetylcholine
Bradykinin Ca2+ CaM

P Ser1177eNOS NO
Shear
stress

P PKB

VEGF
IGF-1 PI3K
INSULIN
 Effects of NO: Vasodilation

NO

Haem
GTP
Guanylyl cyclase
cGMP +PPi

Phosphorylation of cGMP-dependent
smooth muscle protein kinase
contractile proteins
Superoxide sequesters NO, reducing
bioavailability
 Sources of superoxide

• NADH/NADPH oxidase
• Xanthine oxidase
• Lipoxygenase
• Cycloxygenase
• P-450 monooxygenase
• Oxidative phosphorylation
• Uncoupled eNOS - low BH4
Vascular NADPH oxidase (Dr.
Montezano’s session)
 Antioxidant defences

• Superoxide dismutase (SOD)

.
O2-  H2O2 + O2

• Catalase H2O2  H2O + O2

• Glutathione (provides reducing –SH),

• Ascorbic acid (Vitamin C – transfers e-)

 Glutathione
reduced

oxidised
 Oxidative Stress

Imbalance between oxidants and antioxidants in

favour of the oxidants, potentially leading to damage
Vascular ROS and NO bioavailability
 Endothelial dysfunction

• Characterised by reduction in bioavailability of vasodilators

(including NO) and increased vasoconstrictors

• Leads to impaired endothelium-dependent vasodilation

• Also get endothelial ‘activation’ - proinflammatory,

proliferative, procoagulatory
 Endothelial dysfunction: Vessel
responses to acetylcholine in subjects
with coronary artery disease

NO donor
Thanyasiri, P. et al. Am J Physiol Heart Circ Physiol 289: H513-H517 2005;
doi:10.1152/ajpheart.01086.2004
 What do we know for certain

• Enhanced LDL-cholesterol deposition in

subendothelial space

• Circulating leukocytes stick to the endothelium,

migrate into the arterial wall and differentiate into
macrophages

• Macrophages take up LDL-cholesterol, forming

‘foam cells’

• Inflammation is major facet of atherogenesis.

Recruitment of Blood Monocytes by Endothelial
Cell Adhesion Molecules

Monocyte Rolling Vessel Lumen

Sticking

Transmigration

E-Selectin VCAM-1 Endothelium

ICAM-1
MCP-1

Intima
Stimulation of Endothelial Cell Adhesion Molecule
Expression via NFkB
TNFa
IL-1b,
IFNg, Ubiquitinylation (K48)
LPS, Degradation P
Palmitate IkBa
others
IKK

P
IkBa
p50 p65 ICAM-1
VCAM-1
p50 p65 E-selectin
nucleus MCP-1
cytosol
 E-Selectin, VCAM-1, ICAM-1

• Adhesion molecules on endothelial plasmamembrane

• Mediate binding of leukocyte to endothelium

VCAM = Vascular cell adhesion molecule-1

ICAM = Intercellular cell adhesion molecule-1

E-Selectin, VCAM-1, ICAM-1

E-selectin VCAM-1, ICAM-1

Leukocyte adhesion and adhesion molecule
expression is reduced by nitric oxide donors

NO donors
 MCP-1, M-CSF

• Cytokines produced in inflammation

MCP-1 = monocyte chemoattractant protein-1

Stimulates migration of monocytes

M-CSF = macrophage colony stimulating factor

Stimulates differentiation & division of

monocytes into macrophages
 Leukocyte migration

VCAM-1

MCP-1 M-CSF
 Reduced Lipid Deposition in MCP-1–Deficient
Atherosclerotic Mice

LDL-R –/–
MCP-1 +/+

LDL-R –/–
MCP-1 –/–

Gu L et al. Mol Cell 1998;2:275-281.

Migration & MCP-1 expression is reduced
by nitric oxide donors
 Risk Factors

• Hyperlipidaemia elevated LDL cholesterol

low HDL cholesterol
high circulating triglycerides
• Hypertension
• Diabetes Mellitus & Obesity
• Lifestyle Smoking & Exercise
• Chronic Inflammatory Disease
• Hyperhomocysteinaemia
• Age & Sex ~10 yr delay in onset in females
• Family History/Genetics
Cholesterol and Mortality
 Lipoproteins & Cholesterol

• Lipoproteins are high molecular mass complexes of

specific proteins and lipids

• Lipoproteins function to transport lipids in the blood.

 Classes of Lipoproteins

• Chylomicrons

• Very Low Density Lipoproteins (VLDL)

• Intermediate Density Lipoproteins (IDL)

• Low Density Lipoproteins (LDL)

• High Density Lipoproteins (HDL)

 Chylomicrons

• Formed in intestine from dietary TG and cholesterol

• Deliver TG to peripheral tissues (primarily adipose)

• Remaining cholesterol primarily delivered to liver

 VLDL

• VLDL is synthesised in the liver from TG and cholesterol

• Deliver TG to peripheral tissues

• Density increases as lose more TG & cholesterol

VLDL IDL LDL

 LDL
• formed as lipids lost by VLDL

• Principal constituent is cholesterol

• Principal transporter of cholesterol to peripheral

tissues

• Is recognised by LDL receptor in liver (clathrin-

mediated endocytosis)

• LDL receptor downregulated by intracellular hepatic

cholesterol (high cholesterol diet therefore increases
plasma LDL)
 HDL

• Enriched in protein relative to lipid (therefore

most dense)

• Produced by liver & intestine

• Remove cholesterol from peripheral tissues

• Transport cholesterol to liver (reverse

cholesterol transport)
 Classes of Lipoproteins

CM VLDL LDL HDL

Density (g/mL) <0.95 <1.006 1.019-1.063 1.063-1.210
Diameter (nm) 75-1200 30-80 20-25 5-12
% protein (wt) 2 10 23 43-55
% PL (wt) 9 18 20 25-30
% chol (wt) 1 7 8 3-5
% CE (wt) 3 12 37 16-20
% TAG (wt) 85 50 10 1-2

apoproteins A1, A2 A1, A2

B-48 B-100 B-100
C1, C2, C3 C1, C2, C3 C1, C2, C3
E E E
Lipoproteins and risk of atherosclerosis

• High LDL cholesterol

• Low HDL cholesterol

 Early events:
Uptake of LDL/oxLDL by macrophages

Vessel Lumen
LDL

Endothelium
LDL Hydrolysis of Phosphatidylcholine
to Lysophosphatidylcholine

Macrophage Oxidation of Lipids and ApoB

Other Chemical Modifications

Modified LDL
Intima
Foam Cell
Modified LDL are Proinflammatory
 Early events:
Uptake of LDL/oxLDL by macrophages
LDL Modified LDL

LDL receptor
Scavenger receptor
SR-AI, SRA-II, CD36
Macrophage

Foam Cell
 HDL and Reverse Cholesterol Transport
Bile

FC CE
CE
SR-BI FC
ABCA1
Liver
A-I Macrophage

LCAT A-I
FC
CE
FC
Nascent
Mature HDL
HDL
 ATP-binding cassette, sub-family A
member 1 (ABCA1)

Cholesterol efflux pump in the cellular lipid removal

pathway
Mutations in this gene have been associated with
Tangier's disease and familial high-density lipoprotein
deficiency
 Tangier’s Disease

• Autosomal codominant disorder due to mutations in both

alleles of ABC1 gene

• Extremely marked reduction in HDL-C and apoA-I

• Cholesterol accumulation in macrophages and other cells of

reticulo-endothelial system

• Increased risk of premature atherosclerotic vascular disease

• Heterozygotes have moderately reduced HDL-C and apoA-I

levels and increased risk of premature atherosclerotic
vascular disease
 HDL Metabolism in Tangier Disease

A-I Nascent HDL

A-I
LCAT CE
FC FC
ABC1
Macrophage
Rapid
catabolism
 Cholesterol

•Vital constituent of cellular membranes

•Precursor of steroids & bile salts
•Component of diet and synthesised endogenously
• Synthesise ~ 1g/day in skin, liver, intestine
• Consume/Absorb ~ 0.3g/day
 Cholesterol synthesis
• Multi-step process
– Starting point is acetyl CoA

• Rate limiting step is catalysed by

HMG CoA reductase

Hydroxymethylglutaryl CoA 
Mevalonate
 Regulation of HMGR
• Phosphorylation/Dephosphorylation in
response to hormones

• Feedback inhibition by cholesterol

– inhibits HMGR gene expression
Due to reduced activation of the transcription
factor - Sterol Response Element Binding Protein
(SREBP)
– stimulates HMGR degradation
 Regulation of HMGR by
ATP
phosphorylation

ADRENALINE AMP-activated protein kinase

GLUCAGON
P
cAMP active inactive
PKA
+
P Protein phosphatase
PPI-1
Phosphoprotein phosphatase inhibitor-1
 Hypotheses of atherogenesis

• ‘Response to injury’ initial step is injury to

endothelium, promoting
proinflammatory response

• ‘Response to retention’ initial step is lipoprotein

retention by arterial wall.

• ‘Oxidative modification’ oxidation of lipoproteins is

initial step.

All hypotheses have central role for lipoproteins

 Proposed mechanism of atherosclerosis

Monocyte Vessel Lumen

LDL

Adhesion Endothelium
MCP-1
Molecules LDL
Intima
Modified
Cytokines LDL Growth Factors
Metalloproteinases

Cell Proliferation
Macrophage
Foam Cell Matrix Degradation

Ross R. N Engl J Med 1999;340:115-126.

 Risk Factors

• Hyperlipidaemia elevated LDL cholesterol

low HDL cholesterol
high circulating triglycerides
• Hypertension
• Diabetes Mellitus & Obesity
• Lifestyle Smoking & Exercise
• Chronic Inflammatory Disease
• Hyperhomocysteinaemia
• Age & Sex ~10 yr delay in onset in females
• Family History/Genetics
 Atherosclerosis & Immune cells
1859 - Virchow describes cellular inflammatory
lesions in atherosclerosis - thought they were
primary in the cause of disease

1913 Anichkov – fed cholesterol to rabbits and

drew foam cell macrophages he saw in the
atherosclerotic plaques

1993 Ross - “Response to injury theory”

2004 Wick - “Auto-immune concept of

atherosclerosis”
 Genetic associations between
inflammation and atherosclerosis?

Family history is important independent risk factor

for CAD

•Well known associations with genes related to

lipid metabolism.

•Recent findings also link genes related to

inflammation and immunity with CAD
•CXCL12 (stromal cell-derived factor-1 – chemokine)
•HLA (human leukocyte antigen)
•IL-6R (interleukin-6 receptor)
•IL-5 (interleukin-5 – cytokine)
•OX-40L (TNF family-related ligand for CD134)
 Cells of the immune system

Haematopoietic stem cell

Common lymphoid progenitor Common myeloid progenitor

B cell T cell
progenitor NK cell progenitor Neutrophil Eosinophil Monocyte Basophil

Memory B cell CD4 Th cell CD8 Tc cell

Plasma cell Dendritic cell Macrophage

 What happens during an immune
response?

Surveillance has Troops mobilised

detected a problem

Target eliminated
Clean up

Depart and return to base

 How do immune cells communicate?
Cytokines

Broad and loose category of approx 50-100 small proteins (5-20kDa)

Includes – chemokines, interleukins, interferons, tumour necrosis factors,

adipokines

Classification and nomenclature is complex

 Interleukins – an expanding
family... (IL-1 to IL-37)
Structural Homology Related Function

IL-1 family (11 ligands) Pro-inflammatory/killing bacteria (Th1)

IL-1α, IL-1β, IL-1Ra, IL- IL-1a
18, IL-1F5 (IL-36Ra), IL-1b
IL-1F6 (IL-36α), IL-1F7 IL-6
(IL-37), IL-1F8(IL-36β), IL-12
IL-1F9 (IL-36γ), IL-1F10
and IL-33

IL-12 family Anti-inflammatory/killing parasites (Th2)

IL-12 (p35/p40) IL-4
IL-23 (p19/p40) IL-5
IL-27 (p28/EBI3) IL-13
IL-35 (p35/EBI3) IL-10
IL-33
Pro-IL-1 family members – common
structural motifs
IL-1β receptor signalling
Evidence for a role of IL-1β in atherosclerosis
IL-1R1 deficiency reduces advanced Atherosclerosis is reduced
atherosclerotic plaque size and outward re- in ApoE-/- mice treated with
modelling at the aortic root. an antibody against IL-1b

J Clin Invest. 2012;122(1):70–79

Atherosclerosis. 2011;216:313-320
Evidence for a role of IL-1β in atherosclerosis
CANTOS Trial
 Early Atherosclerosis
Contributing factors:
High plasma lipids E-selectin
Low/Oscillatory shear stress  ICAM-1/VCAM-1/MCP-1
Endothelial dysfunction  NO
Bifurcations

Activated endothelial
cells express
adhesion
molecules and
recruit inflammatory
cells, predominantly
monocytes by
secreting
chemotactic factors
lipid
Intermediate Atherosclerosis

Monocytes migrate into

intima, differentiate into
macrophages and ingest
lipid to form foam cells

lipid
 scavenger receptors
 chemokines
 cytokines
 free radicals/ROS
 MMPs, cathepsins
 CD4+ T cells
 Stable Atherosclerotic Plaque

Many intimal smooth

muscle cells
(repair phenotype)

Thick fibrous cap

(smooth muscle cells & matrix) Compensatory
Few
vessel
Macrophages,
enlargement
T cells

Small free lipid core

SMC
 Unstable Thin Cap Fibroatheroma
Intimal SMCs become
Activated MF induce intimal
senescent and apoptotic
SMC death and degrade matrix
EC Leukocyte in the fibrous cap (matrix
transmigration metalloproteinases)

CD4+ T cells secrete

Platelets aggregate Th1 cytokines which
at site of erosion activate MF

Large free
lipid core
Calcification
(RANK/OPG)

SMC
 Ruptured Atherosclerotic Plaque

Thrombus forms and extends

into the lumen and the plaque

Inflammatory
response at site
of rupture
thrombus

What defines rupture?

Large free
lipid core
-Torn fibrous cap

-Not endothelial erosion

SMC
Size Doesn’t Matter! Inflammation Does!

‘safe’ plaque ‘unsafe’ plaque

ANGIOGRAPHIC APPEARANCE
Summary – atherosclerotic plaque stability

ox-LDL
genetic susceptibility
? Infection
? Inflammatory disease
smooth muscle cells

repair inflammation

plaque stability

plaque instability
 Monocyte-macrophage
subsets and plaque stability
Pro-inflammatory
Anti-inflammatory
Plaque Progression
Plaque Regression
Damage

Repair
Ly6Chi
Ly6Clow

IFNy
IL-4

M1
M2

IL-1b TNFa
IL-10 IL-12 iNOS
Endocytic Arginase
Other immune cells in
atherosclerosis?
 T cell cytokines and plaque inflammation
IL-4
Th2/Tregs IL-6 Th1
PROTECTIVE
Th17/IL-17 PRO-ATHEROGENIC
repair
IL-1ra IL-1b
IL-5 inflammation
IL-2
IL-10 Stable IL-12
TGFb IL-18
IL-25 TNFa
IL-33 IFNg
Unstable

Hansson & Libby (2006) Nat Rev Immunol 6, 508-519

 B-cell responses in atherosclerosis
Few B cells are found in the intima of atherosclerotic plaques, BUT patients have high
titres of IgM auto-antibodies against plaque antigens eg HSP70, ox-LDL

Ait-Oufella H et al. Circulation Research. 2014;114:1640-1660

 Distinct immune cell infiltrates in plaques
and adventitia.

Mohanta S K et al. Circulation Research. 2014;114:1772-1787

 How do we treat atherosclerosis?

• Coronary artery bypass graft

• Angioplasty
• Stent
Coronary Artery Bypass Graft
Angioplasty
Stent
How do we reduce atherogenesis?
 Anti-atherogenic therapies

• Xenical/Orlistat & Benecol

•Statins

• Fibrates

• Antioxidants

• IL-1β sequestration

• Diet & Exercise

 Inhibition of lipid digestion & absorption

•Orlistat (Xenical) inhibits pancreatic lipases,

reduces digestion and absorption of dietary lipids
•Steatorrhea
• Reduces LDL, as more LDL receptor

•Benecol inhibits dietary cholesterol absorption

•Plant stanol esters stop cholesterol entry into
micelles during digestion/absorption.
 Statins inhibit HMG-CoA Reductase
Hydroxymethylglutaryl-Coenzyme A Reductase

• Catalyses first step of sterol (cholesterol and steroid) synthesis

 Statin Chemical Structures
HO HO
Lovastatin HO O Simvastatin O Pravastatin COONa
O OH
O
O
O H O H

O
O O H3 C H
H3C H H3C H H3 C
H3C H3C H H
H H CH3 H CH3
CH3 CH3

HO
H3C H3C

CH3 CH3 CH3 CH3

OH OH O O OH OH
O O

O* Ca++ O* N NHC
NHC N

F F
Atorvastatin F

CH3 CH3

OH OH O CH3 N CH3 O

O*Na+
CH3OCH2 O*Na++
N CH3
OH OH

CH3 Fluvastatin
Cerivastatin
F
Inhibition of HMG CoA reductase in liver
reduces LDL in plasma

chol

LDLR

LDL
 Simvastatin in Hyperlipidemia
Changes in Lipids
20 16
13
10
2 3
0
% change

-10 -4

-20
Placebo
-30 -28
-29 Simvastatin 40 mg
-33
-40 -36 Simvastatin 80 mg

-50
LDL-C* HDL-C* TG†
*Mean; †median

n = 119–121 per treatment group

P  0.05 for between-treatment comparisons
Stein E et al. Am J Cardiol 2000;86:406-411
 First Acute Major Coronary Event:
AFCAPS/TexCAPS
AFCAPS/ TexCAPS
0.07

0.06

37% Risk Reduction

Cumulative Incidence

0.05
Placebo (p = 0.00008)
0.04

0.03
Lovast atin
0.02

0.01

0.00

0 1 2 3 4 5 5+ Years
Years of Follow-up

Downs et a l, JAMA 1 998; 27 9:16 15-22.

 Fibrate Chemical Structures

O CH3 CH3

Cl C O C COO CH

CH3
Fenofibrate CH3
CH3

Cl O C COOC2H5

Clofibrate CH3
CH3 CH3

O CH2 CH2 CH2 CH2 C COOH

CH3
CH3 Gemfibrozil
 Fibrates activate PPARa

•Peroxisome proliferator-activated
receptor-a

•Transcription factor that forms

homodimer with the retinoic acid
receptor (RXR)

• Upregulates expression of enzymes

concerned with FA transport,
synthesis and oxidation including
ABCA1
 HDL and Reverse Cholesterol Transport
Bile

FC CE
CE
SR-BI FC
ABCA1
Liver
A-I Macrophage

LCAT A-I
FC
CE
FC
Nascent
Mature HDL
HDL
 Fibrates stimulate Cholesterol Efflux

Fibrates

PPARs
A-I

FC LXR/RXR
ABCA1
 Major Coronary Events in Gemfibrozil vs.
Placebo Groups
25
Cumulative Incidence (%)

20 Placebo
–22% reduction
15
P = 0.006
10 Gemfibrozil

0
0 1 2 3 4 5 6
Year

Rubins HB et al. N Engl J Med 1999;341:410-418.

 Antioxidant therapy

• Oxidative stress implicated in atherogenesis

• Trials with antioxidants (Vitamin E) in humans have

shown no beneficial effect.

• Casts doubt on causative relationship between

oxidative stress and atherosclerosis

• May have not used relevant antioxidant therapy - our

knowledge of oxidants involved in atherosclerosis is
incomplete
Emerging anti-inflammatory therapies in
clinical atherosclerosis

Antagonists of IL-1 superfamily

Klingenberg, R. et al. Eur Heart J 2009 30:2838-2844

 Immunization against atherosclerosis

• If B cells and auto-antibodies are protective can you vaccinate

against atherosclerosis?

• Immunization with LDL has been shown to reduce

atherosclerosis in cholesterol-fed rabbits (Palinski et al, PNAS
1995)

• Problem – which antigen to choose? (oxLDL, HSP, chlamydia?)

• Problem – you don’t want to induce B2a cells and harmful
antigen-specific T cells (instead try to induce B1a and Tregs?)
• Problem – when to vaccinate people?
 Canukinumab (CANTOS Trial)

• ~10,000 patients that had previously had a heart attack

• Canukinumab – human monoclonal antibody targetting IL-1β

• Sequesters IL-1β

• Reduced future cardiovascular events vs placebo

• Independent of blood lipid levels

 Diet
• Reduce dietary cholesterol

• Mediterranean diet (less saturated fat) rather than

alpine diet

• Fish oils reduce plasma TG

• Nuts, Soy, Phytosterols, Garlic been proposed to

have beneficial effects on blood lipids
 Exercise

7.5 • Improves insulin sensitivity Lean = <17% body fat

Normal = 17-25% body fat
6 Obese = >25% body fat
RR of CVD mortality

4.5
Fit
Unfit Fit = top 80% in maximal
3
treadmill exercise test

1.5 Unfit = lowest 20% in

maximal treadmill exercise
0
Lean Normal Obese test

22,000 men 30-83yr

Lee, Blair, Jackson, Am J Clin Nutr 1999