Condorelli

Lecture 3 – Atherosclerosis

●Atherosclerosis is a progressive pathologic process able to start at early age, with clinical manifestations
in adult life.

This disease is the first cause of death in occidental countries, and it is a multifactorial process, involving
large arteries (coronary and carotids ones).

It represents the primary cause of heart disease and stroke and it is the underlying
cause of most cardiovascular diseases, such as coronary artery disease, ischemic
gangrene, abdominal aortic aneurysm. It’s a progressive disease with an
accumulation of lipids and fibrous elements into the blood vessels: fatty streak,
indeed, is one of the first visible alterations, leading to the formation of
atherosclerotic plaque.

If this process occurs at the level of the

coronaries it can cause ischemic heart
attack;

if it occurs at the levels of carotids it can

cause ischemic stroke.

The symptoms are related to the lack of blood in some body districts, and generally the coronaries are the
most involved vessels.
 A normal artery is made of:

●intima, with endothelial cells;

●media, with smooth muscle cells,

elastic connective matrix, collagen I and
III, vitronectin, laminin and
elastinfibrillis (help the vase to
increase/decrease its size);

●adventitia, with fibroblast, nerve cells

and collagens.

The atherosclerotic plaque is a focal thickening of the inner-most layer of the artery, the intima.
We can think of Atherosclerosis as an endothelial damage and we have to imagine an excessive
proliferation of smooth muscle cells that spread to the intima.

Major components of plaque:

- Lipid = Cholesterol
- Cells (SMC, macrophages and other WBC)
- Extracellular Matrix (collagen, elastin, and PGs)
- Often calcification

Frequently, the plaques locate at the

biforcations of the arteries.
Atherosclerosis risk factors

Not modifiable:

-age

-sex the males are more subjected to this

disease whereas females are “protected”
until menopause, because of their high
Estrogens levels which induce an increase of
HDL.

Modifiable:

-Dislipidemia

- Hypertension

-smoke

-Diabetes

-Obesity

-Sedentary life

●In the atherosclerotic plaque, the increase of cholesterol-LDL leads to damage of endothelial cells.
Cholesterol may itself induce damage through its deposition in the arterial wall (initial sign of
inflammation). The consequences are multiple, like narrowing/occlusion, rupture, emboli…these can lead
to myocardial and cerebral infarcts and aortic aneurysm.

*aneurysm= An aneurysm is a bulge in a blood vessel caused by a weakness in the blood vessel wall,
usually where it branches.
As blood passes through the weakened blood vessel, the blood pressure causes a small area to bulge
outwards like a balloon.

Aneurysms can develop in any blood vessel in the body, but the 2 most common places are:

 the artery that transports blood away from the heart to the rest of the body (the abdominal aorta)
 the brain

Cholesterol may itself induce damage and its deposition in the artery wall may be a an initial signal of
inflammation.

The Atherosclerosis has been described as a chronic

inflammation process that may start ad an endothelial
damage response.

Damage is caused by Hypertension, smoke, diabetes and the

accumulation of cholesterol oxLDL…
●Inflammation Participates in all phases of atherothrombotic disease

-lesion initiation

-lesion progression

-Thrombotic complications

●The early lesions (fatty streaks) of atherosclerosis consist of subendothelial accumulations of cholesterol-
engorged macrophages, called ‘foam cells’.

Fatty streaks are not clinically significant, but they are the precursors of more advanced
lesions.
  this leads to the
formation of FOAM CELLS.

-LDLs penetrate through the endothelial barrier and bind to proteoglycans via ApoB100 to retain in the
subendothelial space.

LDLs induce the formation of several pro-inflammatory mediators: the intercellular adhesion molecule-1
(ICAM-1) and vascular-cell adhesion molecule-1 (VCAM-1) that increase monocyte and inflammatory cell
adhesion on the endothelium (LDLs are often oxidized by ROS, thus converting into macrophages).

The most important molecules in this process are cytokines, like proinflammatory ones.

Monocytes roll on the vascular surface to adhere to the site of activation and then migrate into the intima
attracted by chemokines from inside.
In the development of atherosclerotic plaque, the first change is the accumulation of LDL that are trapped
in the subendothelial matrix (intima).

Accumulation of oxidatively modified LDL in the intima contributes significantly to monocyte

recruitment and foam-cell formation and blood-derived inflammatory cells, particularly
monocytes/macrophages, have a key role in atherogenesis.

-Monocytes differentiate into macrophages and express scavenger receptors (SRs), cluster of
differentiation 36 (CD36), LOX-1, and Toll-like receptors (TLRs).

OxLDL– CD36 interaction induces macrophage activation and retention, macrophage SRs increase ox-LDL
uptake and foam-cell formation.

-As a result of activation, the macrophages release pro-inflammatory mediators, including ROS, nitrogen
species, cytokines, vasoactive molecules and proteases.

The retention of ox-LDL leads to foam cell apoptosis and inflammatory progression.
Ox-LDLs increases the expression of growth factors, PDGF for migration and bFGF for proliferation, on
SMCs.

The inflammatory cells also produce growth factors, causing the migration of smooth muscle cells from the
media to the intima, leading to the proliferation and production of extracellular matrix.

The smooth muscle cells also change their phenotype, switching from a contractive phenotype to a
secretory one, becoming able to synthesize vasoactive and inflammatory mediators and are involved in the
binding and internalization of oxLDL through the scavenger receptor.

To summarize: The primary event of atherosclerosis is endothelial damage.

Endothelial damage causes expression of adhesion molecules on cellular surface with consequent
recruitment and adhesion of inflammatory cells.

These cells produce growth factors that stimulate the migration of SMC from the media to the intima.

SMC proliferate and produce components of the extracellular matrix that take part to the plaque.

SMC proliferation contributes to the thickening of atherosclerotic plaques and formation of a necrotic
core. Several leukocyte adhesion molecules and chemokines govern the recruitment of blood monocytes
which become macrophage once entered the tissue.
●The atherosclerotic plaque initially grows towards the adventitia until a critical point, after which begins
to expand outwards on the lumen.

The anatomy of the plaque consists of: lipid core at the center; on the top a fibrous cap (acts as a barrier)
and on the right a shoulder.

We can have 2 types of plaques:

- Stable plaque made of a very big fibrous cap (Extracellular matrix and fibroblast, very few
inflammatory cell)
- Vulnerable plaque  is unstable, with thin fibrous cap, lipid pools and macrophages, it may go
into rupture and thrombus formation.

A vulnerable plaque, often not stenotic, has a high likelihood of becoming disrupted and forming a
thrombogenic focus after exposure to an acute risk factor (functional definition);

the histologic definition is a plaque containing very thin fibrous cap, lipids and few inflammatory
cells.
●A patient with a vulnerable plaque is also prone to ischemic diseases; the rupture of the plaque it’s
followed by platelets and coagulation process.

This process completely interrupts the blood flow into the lumen, causing necrosis of the tissue.

*The term “vulnerable” is defined as “susceptible to injury or susceptible to attack”.

Macrophages, T cells and mast cells respond to pro-inflammatory stimuli by producing proteases,
cytokines, and prothrombotic factors.

Plaque rupture exposes blood components to tissue factor, initiating coagulation, the recruitment of
platelets, and the formation of a thrombus.

●Role of diabetes
Diabetes is an important factor of risk because chronic Hyperglicemy leads to blood accumulation of
glycated proteins leading to a higher vascular permeability and oxLDL levels;

Smoke, on the other hand, enhances the formation of free radicals that increase oxLDL levels and
production of cytokines, and nicotin also induces direct cytotoxicity.

●Role of somatic mutations of blood cells

Many aged individual present somatic mutations in blood cells.

These mutations bring to clonal expansion of mutated clones but not cancer.

Clonal hematopoiesis of indeterminate potential (CHIP)

Clonal hematopoiesis it’s a term most commonly used to refer to a

population of myeloid cells with an acquired gene mutation (often linked
to myelodysplastic syndromes and leukemia).

Chip associated mutations Genes most often mutated in CH are also

commonly mutated in hematologic malignancies (myelodysplastic
syndromes, acute myeloid leukemia) "leukemia-associated genes" or
"leukemia driver genes.“

They reflect a broad array of cellular functions (transcription factors,

chromatin modifiers, DNA repair, signal transduction, regulators of
cellular metabolism) and give proliferative or survival advantage to affected cells, which enables the
expansion of the clone.

In some cases, these mutations are found in individuals who have no detectable hematologic malignancy.
In such cases it may be referred to as clonal hematopoiesis of indeterminate potential (CHIP); the people
with these mutations may have higher risk of developing atherosclerosis.
The occurring mutations are referred to as leukemia-associated genes, all linked to proliferating
components.

The most common affected genes are DNMT3A, JAK2, TET2 and ASXL1.

For example, in mice, TET2 deficiency promotes IL-1B overproduction in macrophages.

Role of TET2

-The first of these genes reported to exhibit somatic mutations in blood cells in individuals with clonal
hematopoiesis without blood cancer was TET2.

More than 130 different mutations have been reported in this gene in blood cells of cancer-free individuals

 TET2 is a transcriptional regulator that can facilitate both transcriptional activation and repression
depending on the molecular/cellular context.

It is able to catalyze the conversion of 5-methylcytosine into 5- hydroxymethylcytosine, a process that

facilitates DNA demethylation and transcriptional activation.

TET2 loss-of-function–driven clonal hematopoiesis accelerates atherosclerosis in hyperlipidemic mice in

vitro and in vivo studies suggest that accelerated atherosclerosis in conditions of TET2 loss-of-function–
driven clonal hematopoiesis is mainly because of the exacerbated expression of proinflammatory cytokines
and chemokines in TET2-deficient macrophages.