Biochemistry of

Atherosclerosis
 Atherosclerosis
• Atherosclerosis is a disease of large and medium-sized
muscular arteries and is characterized by:
– endothelial dysfunction
– vascular inflammation
– the buildup of lipids, cholesterol, calcium, and cellular debris
within the intima of the vessel wall
• It leads to the narrowing of arteries or complete
blockage.
• Its main components are endothelial disfunction, lipid
deposition, inflammatory reaction in the vascular wall.
• Remodeling of vessel wall.
 Atherosclerosis
• Intense cross-talk between EC, VSMC, plasma-derived
inflammatory cells, lymphocytes (involves array of
chemokines, cytokines, growth factors).

• Attraction of cells to the sites of atherosclerotic lesion.

• Migration, proliferation, apoptosis, excess production of

extracellular matrix.
 Normal Blood Vessel Wall

Three well-defined concentric layers

a) Tunica intima
(1) Endothelium
(2) Subendothelial layer
b)  Tunica media
(1) Smooth muscle
(2) Elastin
c) Tunica adventitia (externa)
(1) CT(Connective tissue)
surrounding TM(Tunica Media)
(2) Arterioles in larger vessels

4
 Normal Blood Vessel Wall
• The lumen of healthy arterial wall is lined by confluent
layer of endothelial cells.
• Arterial endothelium repels cells and inhibits blood clotting.
• Endothelium controls important function:
1. the ability of blood vessels to dilatate (vasodilatation)
2. the ability of blood vessels to constrict (vasoconstriction)
• Endothelium regulates tissue and organ blood flow,
releases variety substances to control vasomotor tone:
– prostacyclines
– hyperpolarizing factor
– endothelin
– NO
 Normal Blood Vessel Wall
• In the case of intact endothelium, the stimulus for
vasodilatation are:
– mechanical stimulation by  blood flow
– catecholamines, bradykinin, platelets-released serotonin
stimulate specific receptors
• In the case of endothelium disfunction:
– direct vasoconstrictor action of the stimuli on the VSMC
outweighs the endothelium-dependent vasodilatator effect
– this action leads to paradoxial vasoconstriction

(Hypercholesterolemia and other cardiovascular risk factors are

associated with endothelial disfunction).
 Shear Stress
• Exercise is an important
mechanical stimulus
mediated by shear stress to
increased blood flow.
• Shear stress () is a
biomechanical lateral force
that is determined by blood
flow, vessel geometry and
fluid viscosity.
• The parallel frictional drag
force of shear stress is one of
the important blood flow-
induced mechanical stresses
acting on the vessel wall.

Patel, A., Honoré, E.: Polycystins and renovascular mechanosensory transduction.

Nature Reviews Nephrology6:530, 2010
 Shear Stress
• Shear stress activates the endothelium and induces
nitric oxide release, which causes vasorelaxation.
• Maintenance of a physiologic, laminar shear stress is
crucial for normal vascular functioning,
• Shear stress regulates vascular caliber, inhibits
proliferation, thrombosis and inflammation of the vessel
wall.
• Thus, shear stress is atheroprotective.
 Nitric Oxide (NO)
• Factor which controls vasodilatation: endothelium-derived relaxing factor
(EDRF)

• The activity of eNOS is controlled by intracellular Ca2+.

• eNOS – endothelial constitutive enzyme (continually expressed) and iNOS,
inducible, from VSMC.
• NO cause vasodilatation via stimulation guanylate cyclase and cGMP
production (second messenger for many important cellular functions, particularly for
signalling smooth muscle relaxation).
The Development of Atherosclerosis
• The key event – damage to the endothelium
caused by excess of lipoproteins, hypertension,
diabetes, components of cigarette smoke.
• Endothelium becomes more permeable to
lipoproteins.
• Lipoproteins move below the endothelial layer (to
intima).
• Endothelium loses its cell-repelent quality.
• Inflammatory cells move itno the vascular wall.
 Response-to-injury hypothesis of atherosclerosis as proposed by Ross (781).

(A) Early stage - endothelial injury or

dysfunction is characterized by enhanced
endothelial permeability and low-density
lipoprotein (LDL) deposition in the
subendothelial space followed by leukocyte
adhesion and transmigration across the
endothelium.
(B) Intermediate stages atherosclerosis -
foam cell formation and an inflammatory
response including T-cell activation, the
adherence and aggregation
of platelets, and further entry of leukocytes
into the arterial wall along with migration of
smooth muscle cells into the intima.
(C) Advanced atherosclerosis - macrophage
accumulation continue, fibrous cap, and
necrosis in the core of the lesion is formed.
Stocker R , and Keaney J F Physiol Rev 2004;84:1381-1478

©2004 by American Physiological Society

 The Role of Oxidative Stress in
Atherosclerosis
• The oxidative modification of low density lipoproteins (LDL) in the
arterial wall by reactive oxygen species (ROS) (O2.-, OH-, ONOO-)
• Increased production of free ROS in hypercholesterolemia, diabetes
mellitus (DM), arterial hypertension, smoking, age
• LDL retained in the intima (in part by binding to proteoglycan)
undergoes oxidative modification.
• Lipid hydroperoxides, lysophospholipides, carbonyl compounds
localize in the lipid fraction.
• Oxygen free-radicals inactivate NO rapidly.
• NO + superoxide (O2.-)  peroxynitrite (ONOO-).
• NO has no longer vasoprotective function.
 NADPH oxidase
 growth factors/cytokines
NADPH + 2O2 ↔ NADP+ + 2·O2- + H+  vascular peptide
 mechanical stretch

 FFA


? glucose

e-
SOD catalase/peroxidase
O2 ·O 2
-
H2O2 H2O + ½ O2

Signal molecules
MAPK, PTK, PTP, Ca2+, MMPs, ino channels

Vascular
Cell growth/apoptosis fibrosis contraction/dilatation inflammation remodeling
 Interplay between NADPH oxidase and other sources of ROS

• O2•− generated from NADPH oxidase can potentially influence ROS production by
other enzymatic sources of O2•− . For example, xanthine dehydrogenase is
converted into O2•− -generating xanthine oxidase through oxidation, which may
result in augmentation of O2•− production. Similarly, mitochondria are susceptible to
oxidative damage which can result in enhanced mitochondrial ROS production.
Finally, O2•− or ONOO•− can degrade the essential NOS cofactor H4B and thereby
promote NOS uncoupling, which leads to O2•− generation by the enzyme.
 Scheme of the domain structure of the NOS dimer, showing cofactor and substrate binding
sites.

Andrew P J , and Mayer B Cardiovasc Res 1999;43:521-

531

Copyright © 1999, European Society of Cardiology

 Stages of NOS dimer assembly.

Andrew P J , and Mayer B Cardiovasc Res 1999;43:521-531

Copyright © 1999, European Society of Cardiology

 Schematic illustrating the role of BH4 in regulating eNOS activity in vascular disease.

A. In healthy vascular endothelium, BH4 availability is not

limiting. eNOS production of NO is appropriate for the
regulation of multiple antiatherogenic biological effects.
Superoxide production by various oxidases acts
predominantly in a signaling capacity. Peroxynitrite formed
by interaction between NO and superoxide is minimal.

B. In vascular disease states such as diabetes, hypertension, or

hypercholesterolemia, superoxide production by oxidases is
markedly increased. NO production may remain unaffected
initially, but NO bioavailability is reduced because of
scavenging interactions with superoxide, forming increased
peroxynitrite.

C. Peroxynitrite and other reactive oxygen species oxidize

BH4, via the BH3 radical to BH2 and biopterin, which
reduces the bioavailability of BH4 and promotes eNOS
uncoupling. eNOS now generates superoxide rather than NO,
which contributes to vascular oxidative stress and further
reduces NO bioavailability. Ascorbate may protect against
oxidative degradation of BH4 by directly reducing the BH3
radical to BH4.

Alp N J , and Channon K M Arterioscler Thromb Vasc Biol

Copyright © American Heart Association 2004;24:413-420
The scheme of LDL a HDL
Difference between OxLDL and MM-LDL. LDL is thought to be modified in a stepwise manner
during the generation of OxLDL. In the initial phase of modification, the lipid components react
with oxidation reagents, resulting in radical chain reactions that produce many types of lipid
oxidation products. Then, the lipid oxidation products react with the apoB protein to generate
adducts and cross-links. Radicals can attack the apoB protein directly, resulting in oxidative
changes of amino acid side chains and the cleavage of peptide bonds. MM-LDL may contain lipid
oxidation products without extensive protein modification, because it binds to LDL receptor rather
than scavenger receptors. As modification on the apoB protein proceeds, it loses the affinity to
LDL receptor, and, in turn, it becomes a ligand of scavenger receptors.
 Lipoprotein(a) or Lp(a)
• Lp(a) - LDL like particle with a molecule of
Apolipoprotein B-100 linked by a disulphide
bridge to Apolipoprotein (a).
• The apo(a) component is similar to plasminogen
in terms of structure and competes with
plasminogen for binding sites resulting in reduced
fibrinolysis and the formation of blood clots.
• Lp(a) is also thought to speed up the process of
atherosclerosis by binding LDL, calcium and
other components into an atherosclerotic plaque
on the blood vessel wall.
• This dual action of Lp(a) explains its role in the
promotion of cardiovascular disease.
 The development of atherosclerosis
• Disfunctional endothelium express adhesion molecules – selectins,
mediated the „rolling“ interaction of cells.
• The key molecule - vascular cell addhesion molecule-1 (VCAM-
1) promotes monocytes adhesion (precursors of macrophages).
• Addhering cells are stimulated by monocyte chemoattractant
protein-1 (MCP-1).
• Monocytes cross the endothelium, settle down in the intima.
 NO inactivation due to oxidative stress

• Angiotensin II (a key mediator

of oxidative stress in vascular
wall) stimulates macrophages
NADH/NADPH oxidase to
produce of O2.-
• In the early stage of process,
ROS are released from
endothelium.
• In the later atherogenetic
process, ROS are produced by
macrophages in thickening
vessel wall.

Schächinger V., Zeiher A.M.: Nephrol Dial Transplant (2002): 2055

 Triggers for inflammation in
atherosclerosis
• Reduced NO bioactivity, increased oxidative stress leads
to tyrosine nitration of protein in vessel wall.
• Nitrotyrosine and oxidized LDL activate transcriptional
factor NF-B (nuclear factor kappa-light-chain-enhancer of
activated B cells).
• NF-B increases proliferation of VSMC
• Reduced NO bioactivity and enhanced oxidative stress
stimulate cytokine production (interleukins, TNF, MCP-
1, interferons) and monocytes attraction.
 Inflammation in atherosclerosis
(mononuclear cells)
Endothelial cells undergo inflammatory activation, produce different leukocyte
adhesion molecules (VCAM-1). Monocytes penetrate into tunica intima. Their
receptor CCR2 interact with monocyte chemoattractant protein 1 (MCP-1)

• Resident monocytes
acquire characteristics of
tissue macrophages.
• Macrophages express
scavenger receptors for
oxidized LDL,
internalized lipoprotein
particles. Macrophages
change to foam cells
(lipid droplets are
accumulated within the
cytoplasm).
• Foam cells secrete pro-
inflammatory cytokines.
• This process amplify
local inflammation and
ROS.
Fom the review article: Libby P.: Inflammation in atherosclerosis. Nature 420, 2002: 868-874
 Inflammation in atherosclerosis (T-
lymhocytes)
T-lymphocytes enter the intima facilitated by VCAM-1 and trio chemokines- IP-10
(inducible protein-1), Mig (monokine induced by interferon-and I-TAC
(interferon-inducibleT-cells -chemoattractant). Trio chemokines bind to CXCR3
chemokine receptor expressed by T-cells in the atherogenic lesion.

T-cells are activated by

antigens (Ox-LDL, heat-
shock proteins),
produce cytokines.
Cytokines influence
behaviour of other cells
present in the
atheroma.
CD154 (protein
expressed on activated
T-cells) binding to CD40
ligand expressed on
macrophages induces
the epression of other
cytokines, MMPs, tissue
factor.
Fom the review article: Libby P.: Inflammation in atherosclerosis. Nature 420, 2002: 868-874
 Inflammation in atherosclerosis (mast
cells)
Mast cells infiltrate to the intima. Chemoattractant eotaxin mediate migration of
mast cells and interacts with the chemokine receptor CCR3. Resident mast cells in
the intima degranulate, release TNF-, heparin, and enzymes activating
proMMPs.

Fom the review article: Libby P.: Inflammation in atherosclerosis. Nature 420, 2002: 868-874
The process of atherogenesis – an
overview
 The process of atherogenesis
• Lipid entry into the arterial wall is a key process in
atherogenesis.
• Hypercholesterolemia – factor for VCAM-1 and MCP-1
induction.
• LDL and VLDL are most atherogenic, enter vascular wall
more easily.
• LDL – in plasma are protected against oxidation by vit.
E, ubiquinon, plasma antioxidants (-carotene, vit. C).
• Out of plasma, LDL phospholipides and fatty acids
oxidize.
 The process of atherogenesis
• Activated macrophages produce enzymes –
lipoxygenases, myeloperoxidase, NADPH oxidase.
• Oxidized LDL are cytotoxic to endothelial cells, mitogenic
for macrophages.
• Oxidized LDL apolipoprotein apoB100 bind to the
scavenger receptor.
• Scavenger receptors are not subjected to regulation by
intracellular cholesterol level.
• Macrophages take up oxidized LDL, overload with lipids.
 The process of atherogenesis
• Macrophages changed to foam cells.
• Foam cells ruptured (apoptosis).
• Lipid release to intima and their accumulation becomes centre of
atherosclerotic plaques.
• Plaque growth – periodically accelerated by a cycle of plaque
rupture and thrombosis.
• The lipid center and fibrous cap
are the main parts of a mature
atherosclerotic plaque.
• Plaque emerges from the
structurally changed vascular
wall.
•Macrophages secrete enzymes
degrade extracellular matrix of
the cap
• So-called vulnerable plaque
ruptures easily.
•The thrombus formed at the
rupture site.
The process of atherogenesis
 The process of atherogenesis
• IFN- induces macrophage MMP expression.
• IFN- inhibits VSMC proliferation and collagen synthesis which
further weakening the cap.
• VSMC undergo apoptosis.
• After plaque rupture the area is exposed its interior to the blood.
• The interior of the plaque is highly thrombogenic – the small-
molecular-weight glycoprotein (tissue factor) initiates the extrinsic
clotting cascade.
• Tissue factor complexes with factor VII/VIIa, factor IX and X are
activated.
• Platelets are activated and thrombus forms quickly on the surface of
a ruptured plaque.
• Thrombus completely occludes the arterial lumen. It cause tissue
necrosis (myocardial infarction or brain stroke).
 Cardiovascular risk and its assesment
• Plasma concentration of lipoproteins (LDL-cholesterol,
HDL-cholesterol, and triacylglycerols) –  5.2 mmol/L
(200 mg/dL) increases the risk.

• Optimal level of LDL-cholesterol - 2.6 mmol/L (100

mg/dL)
 Cardiovascular risk and its assesment
The risk of atherosclerotic event can be decreased by:
– cholesterol low diet
– omega-3 fatty acids (abundant in fish oils)
– exercise
– smoking cessation
– control of high pressure
– drugs statins, fibrates (fibric acid), ezetimibe, antioxidants

Statins – inhibit intracellular cholesterol synthase (HMG-CoA

reductase).
Fibrates - lower plasma cholesterol by stimulating LPL, decreasing
TAG concentration, and increasing HDL.
Ezetimibe – inhibitor of intestinal cholesterol transporter.
carotene, -tocoferol, vitamin C (such as these contained in fruits or
their combinations) have preventive benefit, protect LDL against
oxidation.
 Summary
• Atherosclerosis is an intima-based lesion organized into a fibrous
cap and an atheromatous (gruel-like) core and composed of SMCs,
ECM, inflammatory cells, lipids, and necrotic debris.

• Atherogenesis is driven by an interplay of inflammation and injury to

vessel wall cells.

• Atherosclerotic plaques accrue slowly over decades but may

acutely cause symptoms due to rupture, thrombosis, hemorrhage,
or embolization.

• Risk factor recognition and reduction can reduce the incidence and
severity of atherosclerosis-related disease.

35
Figure 1. Production of nitric oxide (NO) by endothelial cells.
NO is produced by the action of eNOS on L-arginine. This reaction requires a
number of cofactors, including tetrahydrobiopterin (BH4) and NADPH). Increased
intercellular Ca in response to vasodilator agonists or shear stress displaces the
inhibitor caveolin from CaM, activating eNOS. NO diffuses to vascular smooth
muscle and causes relaxation by activating GC, thereby increasing
intracellular cyclic guanosine monophosphate (cGMP).
Figure 2. Endothelial nitric oxide synthase (eNOS) signaling.
eNOS is composed of 2 globular protein modules (reductase
and oxygenase domains) connected via a flexible protein strand.