Atherosclerosis is characterized by chronic inflammation of an injured intima. The term atherosclerosis is derived from athero-(meaning porridge) referring to the soft lipid-rich material in the centre of atheroma, and sclerosis (scarring) referring to connective tissue in the plaques. This term describes the cholesterol deposits and scarred portion in an arterial plaque or atheroma. Atherosclerosis is the commonest and the most important of the arterial diseases.

1. Pathophysiology
of
atherosclerosis
Nikita Kharal
M.Sc. Clinical Biochemistry
2nd year

2. Contents..
Atherosclerosis
Normal endothelial cell
Risk Factors
Hypothesis of Atherosclerosis
Pathophysiology of Atherosclerosis
LDL receptor Vs Scavenger receptor
Fate of atherosclerotic plaque
Hereditary Disorders of Lipid Metabolism and
Atherosclerosis
Treatment

3. Atherosclerosis
•Atherosclerosis is characterized by chronic inflammation of an
injured intima.
•The term atherosclerosis is derived from
•athero-(meaning porridge) referring to the soft lipid-rich
material in the centre of atheroma, and
•sclerosis (scarring) referring to connective tissue in the plaques.
•This term describes the cholesterol deposits and scarred portion
in an arterial plaque or atheroma.
•Atherosclerosis is the commonest and the most important of the
arterial diseases

4. Atherosclerosis Vs Arteriosclerosis
Atherosclerosis is a disease of large- and medium-sized
elastic and muscular arteries and results in the progressive
accumulation within the intima of inflammatory cells, smooth
muscle cells, lipids, and connective tissue.
Arteriosclerosis is a disease of the walls of smaller arteries
and arterioles which become thicker and less elastic, generally
as the result of chronic hypertension

5. Histological layers
The arterial wall itself consists of three layers:
1. The intima (Innermost layer; subendothelial layer)
2. The media (Middle layer; which contains VSMC)
3. Tunica adventitia (Outermost layer; composed of
looser connective tissue and containing relevant nerves)

6. Nature of endothelial cell
Normal endothelium has anticoagulant and antiadhesion
properties that repels cells floating in plasma.
Three of these characteristics are:
• The presence of heparin on their surface which exerts an
anticoagulant effect.
• The capacity to produce prostaglandins, which inhibit
platelet aggregation.
•The capacity to produce or activate enzymes (e.g. plasmin,
urokinase) that break down clots once they have begun to form

7. Non modifiable risk factors
i) Genetics: Family history is the most important independent
risk factor for atherosclerosis. Defect in LDL receptor results
familial hypercholesterolemia.
ii) Age: between ages 40 and 60, the incidence of myocardial
infarction increases fivefold.
iii) Gender: Premenopausal women are relatively protected
against atherosclerosis and its consequences compared with
age-matched men.

8. Modifiable Major Risk Factors
i) Hyperlipidemia/ hypercholesterolemia— is a major risk factor for
atherosclerosis; dietary(omega-3 fatty acids ; abundant in fish oils) and
pharmacologic interventions (Statins) can lower LDL or total serum
cholesterol
The mechanisms by which hyperlipidemia contributes to atherogenesis is
by impairing EC function that include:
•Increasing local reactive oxygen species production causing membrane and
mitochondrial damage,
•Oxygen free radicals also accelerate NO decay, dampening its vasodilator
activity.

9. ii) Hypertension: causes damage to endothelial cells due to the
shear stress as the blood flows through the arteries, especially
at positions where arteries branch.
iii) Cigarette smoking: causes damage to endothelial cells due
to free radicals present in tobacco smoke. It is estimated that
each puff of a cigarette produces 10^14 free radicals. Smoking
cessation reduces that risk substantially.
iv) Diabetes mellitus: induces hypercholesterolemia and
markedly increases the risk of atherosclerosis.

10. Additional Risk Factors
More than 75% of cardiovascular events can occur in healthy
women with LDL cholesterol levels below 160 mg/dL may be due to
i) Inflammation: Inflammation is present during all stages of
atherogenesis and is intimately linked with atherosclerotic
plaque formation and rupture
ii) Personality: Subjects exhibiting type A personality behaviour,
that include ‘ambition, competitive drive, aggressiveness, a
strong sense of time urgency’ are more likely to suffer from
atherosclerosis than type B personalities, who are more relaxed
and unhurried.

11. iii) Hyperhomocysteinemia: Serum homocysteine levels
correlate with coronary atherosclerosis, peripheral vascular
disease, stroke, and venous thrombosis.
•Elevated circulating homocysteine (>100 µmol/L) and is
associated with premature vascular disease.
•Homocysteine oxidises spontaneously to form thiolactone
which can damage cell membrane.

12. Lipoprotein a [Lp(a)]: secreted by the liver.
• A high plasma level of Lp(a) interferes with the conversion of
plasminogen to plasmin, the role of which is to break down
blood clots and even disperse small clots.
•Hence, Lp(a) inhibits the breakdown of fibrin clots so
contributing to blockage of arteries.

13. Hypothesis of Atherosclerosis
A number of theories regarding the origin and progression of lesion of
atherosclerosis have been proposed ;
i) Insudation hypothesis: The concept hypothesised by Virchow in 1856
that atherosclerosis is a form of cellular proliferation of the intimal cells
resulting from increased upkate of lipids from the blood which was named
as ‘lipid theory’.
ii) Encrustation hypothesis: The proposal put forth by Rokitansky in 1852
that atheroma consist of blood forming thrombi that composed of platelets,
fibrin and leucocytes, was named as ‘encrustation theory’ or
‘thrombogenic theory’.

14. Currently, pathogenesis of atherosclerosis is explained on the
basis of the following two theories:
1. RESPONSE-TO-INJURY HYPOTHESIS
i) The original response to injury hypothesis: first described
in 1973 according to which the initial event in atherogenesis
was considered to be endothelial injury followed by smooth
muscle cell proliferation so that the early lesions consist of
smooth muscle cells mainly.
ii) The modified response-to-injury hypothesis: implicates
lipoprotein entry into the intima as the initial event followed
by lipid accumulation in the macrophages (foam cells now)
according to modified theory, foam cells are believed to be the
dominant cells in early lesions.

15. 2. MONOCLONAL HYPOTHESIS
As per this hypothesis proliferation of smooth muscle cells is
the primary event and this proliferation is monoclonal as
proliferated smooth muscle cells in atheromatous plaques has only
one of the two forms of glucose-6-phosphate dehydrogenase
(G6PD) isoenzymes, suggesting monoclonality in origin.
The monoclonal proliferation of smooth muscle cells in
atherosclerosis may be initiated by mutation caused by
exogenous chemicals (e.g. cigarette smoke),
endogenous metabolites (e.g. lipoproteins) and some viruses.

16. Pathophysiology of Atherosclerosis
Atherosclerosis progression is best explained by response-to-
injury hypothesis that include the following sequence:
1. Endothelial injury and dysfunction, causing (among other
things) increased vascular permeability, leukocyte adhesion,
and thrombosis.
2. Accumulation of lipoproteins (mainly LDL and its oxidized
forms) in the vessel wall.
3. Monocyte adhesion to the endothelium, followed by
migration into the intima and transformation into
macrophages and foam cells.
4. Platelet adhesion.

17. 5. Recruitment of T cells
6. Factor release from activated platelets, macrophages, and vascular
wall cells, inducing SMC recruitment, either from the media or
from circulating precursors.
7. SMC proliferation, ECM production
8. Lipid accumulation both extracellularly and within cells
(macrophages and SMCs).
9. Calcification of ECM and necrotic debris late in the pathogenesis.

18. 1) Endothelial injury and dysfunction
 Most important causes of endothelial dysfunction is
hemodynamic disturbances
 Normally, there is a balance between synthetic programs
controlled by two sets of transcription factors acting as
transcriptional integrators.
 Kruppel-like factors (KLF2, KLF4) control the anti
atherogenic/anti inflammatory program, and
 NFκB controls the proinflammatory program.
 In the dysfunctional endothelium, the KLF factors become
suppressed, and the NFκB predominates.

19. 2. Accumulation of lipoproteins (mainly LDL
and its oxidized forms) in the vessel wall
Damaged vascular endothelium has an increased permeability to
circulating lipids
Prolonged residence of LDL in the site of lesion involves a local
alteration in proteoglycan (heparan sulfate) composition endothelial
cells.

20. Exposure of the polar segments of apoB-100, containing lysine and
arginine residues has high affinity for arterial wall proteoglycans
which promote the retention of LDL in the subendothelial region of
the intima that heightened susceptibility to oxidative modification.
Because in the plasma, the LDL particles are protected against
oxidation by antioxidants such as vitamin C and β-carotene. Once
they lodge in the intima, this protection is removed.

21. Fatty acids and phospholipids in the LDL are subject to
oxidation by macrophages expressing a range of oxidizing
enzymes, including lipoxygenases, myeloperoxidase, NADPH
oxidases and free radicals generated by the auto-oxidation of
homocysteine.

22. Oxidized LDL is toxic to endothelial cells, causing additional
intimal damage and subsequent retention of cholesterol-rich
lipoproteins
Oxidized LDL further stimulates expression of VCAM-1 and
MCP-1 in the endothelium, maintaining the influx of
leukocytes are neutrophils and monocytes into the intima.
Oxidized LDL is not recognized by the B-100 receptors
which normally exist in cells.

23. 3. Monocyte adhesion to the endothelium, followed by migration into
the intima and transformation into macrophages and foam cells.
The injured endothelial cells increased expression of vascular cell
adhesion molecule-1 (VCAM-1), P-selectin, and increased levels of
ICAM-1.
Adhesion molecules cause monocytes to move out of the
bloodstream and adhered to the endothelium,

24. Endothelium, macrophage, and smooth muscle cells when
appropriately stimulated releases monocyte chemoattractant
protein-1 (MCP-1), which act as a potential chemotactic stimulus for
mononuclear phagocytes and T lymphocytes to penetrate into the
intima
Monocytes are then transformed into macrophages
Oxidized LDL is then taken up by scavenger receptors in
macrophages in the arterial wall, because phosphorylcholine group
found in oxidized LDL is also a component of the capsular
polysaccharide of bacteria which act as antigen

25. Eventually macrophage becomes foam cells
laden with cholesterol.

26. LDL receptor and regulation
 When LDL concentrations are high
The receptor is downregulated, that is, fewer receptors appear
on the cell surface, so LDL uptake is inhibited
Cholesterol synthesis is inhibited i.e. HMG coenzyme A
reductase is inhibited.
 When the concentration of cholesterol in the cell is low,
both uptake and synthesis are increased

28. Macrophage receptor and Oxidized LDL
Macrophages expressed a scavenger receptor denoted MARCO that bind
with oxidatively modified lipoprotein particles.
Macrophages expressed a scavenger receptor including CD36 and
macrosialin/CD68.
Macrophage-colony stimulating factor (M-CSF), a cytokine found
within plaques, can augment scavenger receptor expression by human
macrophages.
Hence, in contrast to LDL receptor, macrophage receptor donot have
downregulation resulting in formation of foam cells.

30. Foam cells
Foam cells play a harmful role in lesion progression,
including the production of pro-inflammatory signals
Foam cells causes activation of inflammasomes and the NF-
κB pathway
An accumulation of these foam cells in the subendothelial
space of blood vessels form the earliest gross evidence of a
developing atherosclerotic plaque known as a fatty streak.

31. 4. Platelet adhesion
Platelets adhere to damaged endothelium.
 Platelets induce inflammatory responses in both leukocytes and
endothelial cells.
The presence of a large number of platelets produce
thromboxanes, in comparison with the decreased number of
endothelial cells producing prostacyclins that results in an increase
in the local concentration ratio, thromboxane/prostacyclin, which
favours platelet aggregation and hence clot formation.

32. 5. Recruitment of lymphocytes
 As the cycle of endothelial cell damage progresses,
endothelial cells within the plaque releases a stream of
cytokines, such as
 interleukin (IL)-1 and
tumor necrosis factor-α which activates T and B lymphocytes
and macrophages.

33. 6. Smooth muscle cell recruitment,
proliferation
Interactions between T cells and foam cells promote a
chronic inflammatory state and help recruit smooth muscle
cells into the intima.
 Lymphocytes and endothelial cells releases growth factors,
such as :
platelet-derived growth factor, fibroblast growth factor, and
tissue growth factor-α,
which stimulate smooth muscle cells proliferation and/or
migration to move from the middle tunica media into the
intima.

34. Cytokines released from T cells, such as interferon-γ, exert
pro-inflammatory and pro-atherogenic effects on macrophages
and smooth muscle cells.
Smooth muscle cells are induced to secrete extracellular
matrix such as collagen, elastin, and proteoglycans that
accumulate within fibrous atherosclerotic plaques which gives
stability and strength to the plaque and fix the plaque firmly in
the vessel wall.
As the plaque grows, the lumen of the artery narrows.
7. ECM production

36. 8. Lipid accumulation both
extracellularly and within cells
The cells that have accumulated lipids die and release
their cholesterol esters content forming necrotic debris.

37. 9. Calcification of ECM and necrotic
debris
As an atheroma grows
older, scar tissue builds up
and calcium is deposited in
the lesion making atheroma
hard. This is where the term
"hardening of the arteries“

38. Characteristics of atherosclerosis
1)Fibrous plaques: The plaques have a central core of
cholesterol and cholesterol esters; lipid-laden macrophages, or
foam cells; calcium; and necrotic debris.
(2) Fibrous cap: The core is covered by a subendothelial
fibrous cap, which is made up of smooth muscle cells, foam
cells, fibrin and other coagulation proteins, as well as
extracellular matrix material, such as collagen, elastin,
glycosaminoglycans, and proteoglycans.

39. Fate of atherosclerotic plaques
Stable plaques
Has few inflammatory cells and a thick fibrous cap narrow the lumen
of the artery, but are less likely to cause acute complications.
 Cause symptoms of reversible ischaemia in the supplied organ, e.g.
angina pectoris, chronic lower limb ischaemia
Unstable/ Vulnurable plaques
 Has more inflammatory cells have a thin fibrous cap vulnerable to
erosion, cracking, or rupture.
 Cause acute ischaemic events, e.g. acute coronary syndromes, stroke,
acute lower limb ischaemia..

41. Complication of Atheroma
Macrophage comprised of enzymes such as collagenases,
gelatinases, and stromelysin as well as non-metalloenzymes such as
the cysteine proteinases cathepsins S and K can degrade
macromolecules of the extracellular matrix
Especially, collagen which is the major structural component of the
fibrous cap and accounts for its mechanical strength and stability
A net increase in collagen degradation and reduced collagen
synthesis, destabilizing the mechanical integrity of the fibrous cap.

42. Thrombolism: When the plaque wall ruptures, blood comes in
contact with the thrombotic factors in the interior forming
thrombus that completely occludes the artery.
Embolism: These thrombi may get dislodged to become
emboli which can travel to and block smaller arteries.

43. Type of Embolism
Organs that suffer most from arterial
thromboembolism include:
• Brain: Emboli cause ischemic necrosis of brain
tissue (strokes).
• Intestine: Mesenteric emboli cause bowel
infarction; ensuing acute abdomen necessitates
immediate surgery.
• Lower extremity: Embolism of a leg artery
leads to pain, absence of pulses, and a cold limb.
• Kidney: Renal artery embolism may infarct the
entire kidney

44. Aneurysm: Blockage of an artery can also cause irreversible
dilation by causing changes in media include atrophy and
thinning of the media and fragmentation of internal elastic
lamina.

48. Hereditary Disorders of Lipid
Metabolism and Atherosclerosis

49. i) Familial Hypercholesterolemia
 Autosomal dominant disease, which results from mutations
in the LDL receptor gene.
The mutations lead to excessively high levels of plasma
cholesterol and cause early onset ischemic heart disease.
 In addition to the accelerated accumulation of cholesterol in
the arteries, LDL cholesterol also deposits in the skin and
tendons to form xanthomas.

50. ii) Tangier disease
recessive inherited disorder,
Cells have a defective transport protein, known as ATP-cassette binding
protein A1 (ABCA1).
ABCA1 transfer cholesterol, cholesteryl esters, and other lipids from
the inner to the outer leaflet of the plasma membrane, from which they
can be captured by apolipoprotein A-I to form HDL
 Hence, no transport cholesterol out of cells to be picked up by HDL.

51. Treatment
At least three treatments are used to decrease the development
of atherosclerosis: diet, drugs and surgery
i) Diet
Protective factors for atherosclerosis are
a. fish oils,
b. antioxidants, and
c. garlic

52. omega-3 fatty acid Vs omega-6 fatty acid
a) Fish oil
omega-3 fatty acid in fish oil contains five
double bonds which is eicosapentaenoic acid,
it gives rise to prostacyclins and thromboxanes
of the three series
Thromboxane A3 has much less of a
thrombolytic effect
prostacyclin I3 has more of an antithrombotic
activity
Thus, the risk of formation of a thrombus is
decreased when omega-3 fatty acids are the
substrate for the cyclooxygenase

53. omega-6 fatty acid, arachidonic acid contain only four
double bond.
it gives rise to the two series, thromboxane A2 and
prostacyclin I2
Thromboxane A2 has more of a thrombolytic effect
prostacyclin I3 has much less of an antithrombotic activity

54. b) Antioxidants
These are naturally occurring compounds that have the
ability to lower the levels of free radicals:
They include vitamins C and E, the carotenoids and the
flavonoids.
Large clinical trials of antioxidants (vitamin E or β-carotene)
in human showed no effect on cardiovascular outcomes
demonstrated by meta-analyses.
This finding may be due to the wrong antioxidant being
chosen, the wrong dose, the wrong subjects or the wrong stage
of atherogenesis being targeted: foam cell formation is the
earliest stage of the atheromatous plaque, and cardiovascular
outcomes are noted with advanced plaque formation, generally
several decades later.

55. c) Ingestion of garlic, onion or other tubers,
 protects against coronary artery disease.
 The active agents in these tubers are the allyl sulphides.
(e.g. allylcysteine sulphoxide, CH2=CH · SO ·
CH2CH(NH2)COOH).
• increase in the NADH/ NAD+ and NADPH/NADP+
concentration ratios particularly in endothelial cells.
• This results in an increased level of reduced glutathione

56. Drugs
Drugs that lower the blood levels of cholesterol are frequently
used as part of the treatment; these include:
a. Oral bile acid binding exchange resins: Resins such as
cholestyramine are effective because, when taken by mouth,
they prevent the reabsorption of bile acids in the lower small
intestine, so that they are excreted in the faeces.
• Since bile acids are formed in the liver from cholesterol,
synthesis of more acids requires more cholesterol uptake by
the liver from the blood, which occurs via LDLcholesterol,
so that the concentration of the latter is decreased.

57. (b) HMG-CoA reductase inhibitors. The enzyme HMGCoA
reductase is the rate-limiting enzyme in synthesis of cholesterol
and hence drugs that inhibit this enzyme are particularly
effective in lowering the blood cholesterol level. These drugs
are known as statins (e.g. simvastatin, provastatin).
b) Aspirin: inhibits cyclooxygenase production of
thromboxanes (e.g. Thromboxane A2), which inhibits platelet
aggregation and further blocking of blood vessels

58. Surgery
Surgical intervention, e.g. coronary artery bypass
or balloon angioplasty, is frequently used.

60. Reference
• Molecular_Basis_of_Cardiovascular_Disease_A_Companion
_to_Braunwald's.pdf
• Lippincott’s Illustrated Reviews: Biochemistry Sixth Edition
• Lehninger Principles Of Biochemistry Fourth Edition
• Harper’s Illustrated Biochemistry, 30th edition
• Michael Lieberman, Marks’ Essentials of Medical
Biochemistry- A clinical approach, 2nd edition
• Tietz Textbook of Clinical Chemistry and Molecular
Diagnostics, 5th edition
• Vasudevan DM, S S, Vaidyanathan K. Textbook of
Biochemistry for Medical Student, 6th edition.