Pathology That Underlies Chd

Published Date: 02 Nov 2017

infl ammatory fi bro - proliferative response to multiple forms of endothelial injury.

The response - to - injury hypothesis was proposed by Russell Ross and colleagues

over 30 years ago [8] , and has been refi ned and developed since.

The normal artery wall is composed of two organized layers: intima and media

(Figure 1.1 a). The intima is made up of a single layer of endothelial cells that are

seated on basement membrane and then the internal elastic lamina ( IEL ). Beneath

the IEL is the medial layer, comprising vascular smooth muscle cell s ( VSMC s)

surrounded by basement membrane and embedded in interstitial extracellular

matrix. The boundary of the media is marked by the external elastic lamina ( EEL ).

All infants have focal thickening of the coronary artery intima due to VSMC proliferation

[9] . Although focal thickening is an important hallmark of the developing

atherosclerotic plaque, this is considered to be an adaptive response to turbulent

blood fl ow rather than pathological.

Endothelial dysfunction initiated by the risk factors already described permits

the entry of lipids and infl ammatory cells into the artery wall (Figure 1.1 b). Once

in the artery, monocytes differentiate into macrophages which take up the lipid

and become foam cell macrophages. This results in the formation of lesions

termed " fatty streaks, " recognized as the onset of atherosclerosis (Figure 1.1 c).

Fatty streaks are small, slightly raised lesions caused by focal collections of foam

cell macrophages in the intima. They may be precursors of larger atherosclerotic

lesion occurs due to the formation of a necrotic core and a fi brous cap

(Figure 1.1 d).

Foam cell macrophages, engorged with lipid, begin to die and release their

contents, which contributes to the formation of a necrotic core. The release of the

cytoplasmic contents of the foam cells leads to the accumulation of extracellular

lipids and growth factors which induce infl ammation. The occurrence of VSMC

migration and proliferation results in the formation of a fi brous cap. VSMCs

migrate into the intima where they proliferate and deposit extracellular matrix.

The increase in cell number and presence of matrix causes augmentation of the

bulk of the plaque, which now protrudes into the lumen. This is termed a stable

advanced plaque (Figure 1.1 d). The size and composition of the plaque determine

its outcome.

Classifi cation schemes have been devised to categorize the various plaque types

[10 – 13] . A plaque with a large necrotic core, high content of infl ammatory cells,

and thin fi brous cap is termed an " unstable plaque " (Figure 1.1 e), and is more

prone to rupture than a plaque with a smaller necrotic core, lower content of

infl ammatory cells, and thick fi brous cap, termed a " stable plaque " (Figure 1.1 d).

Rupture of the plaque leads to thrombus formation, which can occlude the lumen

and cause the symptoms of MI or stroke (Figure 1.1 f). However, plaque rupture

does not always lead to occlusion of the artery and the plaque may restabilize and

heal over. This is at a cost since the " healed plaque " is larger [14] and repeated

episodes of plaque rupture and healing is associated with a greater incidence of a

fatal event [15] .

The aim of this book is to discuss the key molecules that drive the processes

underlying the pathogenesis of atherosclerosis. A greater understanding of the

molecules involved in atherosclerosis will no doubt lead to potential clinical targets

and design of new therapies. The subsequent chapters focus on particular key

molecules, however to serve as an introduction to these chapters we will briefl y

outline the key cellular processes in atherosclerosis.

1.4

Initiation of Plaque Formation – Endothelial Dysfunction

Atherosclerosis is thought to be initiated by damage to the endothelium, resulting

in altered endothelial function (Figure 1.1 b). The risk factors outlined above are

generally the cause of this damage through one or more of the following pathways:

high levels of oxidized low - density lipoprotein ( LDL ); free radicals such as reactive

oxygen species ( ROS ); genetic variations; elevated plasma homocysteine concentrations;

infectious microorganisms such as herpes virus or Chlamydia pneumoniae

; shear stress in areas of turbulent blood fl ow or endogenous infl ammatory

signals such as cytokines [16] .

Loss of normal nitric oxide ( NO ) production from the endothelium of the vessel

wall occurs during endothelial dysfunction. NO regulates many aspects of cardiovascular

homeostasis, including blood pressure and fl ow, smooth muscle contraction,

infl ammation, and platelet activation, and is a well - known atheroprotective

factor. The molecular mechanisms behind this loss of NO remain unclear, however

recent studies suggest that changes in the activity and regulation of endothelial

NO synthase by its cofactor tetrahydrobiopterin ( BH4 ) is an important contributor

[17] . See Chapter 14 by Cunnington and Channon for more detail.

The anti - infl ammatory enzyme heme oxygenase - 1 ( HO - 1 ) has also been reported

to have cytoprotective effects during endothelial dysfunction. HO - 1 is the

form of the heme oxygenase system and its importance in vasculoprotection is

demonstrated by the severe and persistent endothelial damage observed in human

HO - 1 defi ciency and Hmox1 −/ −mice (reviewed in [18] ). HO - 1 exerts a potent protective

effect against atherogenesis, cardiac ischemia/reperfusion injury, and both

graft rejection and accelerated arteriosclerosis post - transplantation. HO - 1 protects

tissues during infl ammatory stress through degradation of pro - oxidative heme,

production of bilirubin and carbon monoxide, and regulation of cellular iron [19] .

It has been proposed that these vasculoprotective effects are through multiple

mechanisms, including regulation of the cell cycle and angiogenesis, redox homeostasis,

and the modulation of complement system. More detail on this topic is

provided in Chapter 15 by Mason and Ali.

ROS are produced by enzyme systems present in the vascular wall, including

NADPH oxidase s ( NOX ), xanthine oxidase, and NO synthase, which are increased

in association with risk factors. The NOX family, which is expressed by neutrophils,

monocytes, VSMCs, endothelial cell s ( EC s), fi broblasts, macrophages,

and mast cells, is a particularly important source of ROS in the vessel wall. Abnormal

NOX activity is seen during endothelial dysfunction and results in the overproduction

of ROS, including superoxide (O 2 −), which promotes further endothelial

dysfunction through a number of effects including endothelial cell apoptosis,

vasoconstriction, lipid peroxidation, cell proliferation, and isoprostane formation.

O 2 −also reacts with NO, negating its cardioprotective effects and further ROS

production in the form of peroxynitrite. ROS modulate many processes involved

in atherosclerosis, including infl ammation, apoptosis, VSMC replication, angiogenesis,

and matrix turnover and therefore increased expression and/or activity of

NADPH oxidases has been proposed to play an important role in atherosclerosis

(see review by [20] ). For further detail see Chapter 13 by Jeremy and colleagues.

1.5

Infl ammation

Platelets adhere to dysfunctional endothelial cells at lesion - prone sites such as

the carotid artery bifurcation prior to leukocyte invasion and atherosclerotic

plaque formation [21] , indicating their involvement in atherogenesis (see

Chapter 17 by Harper and colleagues for more details). Platelet adherence and

aggregation is stimulated by integrins, P - selectin, fi brin, thromboxane A 2 , and

tissue factor [16] . Once adherent the platelets become activated and release, or

expose on their cell surface, multiple infl ammatory factors, proteases, and vasoactive

substances, which further promote endothelial cell dysfunction and infl ammation

[22] . This leads to recruitment and adhesion of neutrophils and monocytes

to the area. In addition, platelets directly bind leukocytes and therefore act as

bridging cells between the endothelium and plaque - forming cells [23] . Platelets

also release platelet factor 4 ( PF4 ) in atherosclerotic plaques [24] and promote

monocyte differentiation into macrophages and uptake of oxidized LDL by macrophages

[25] .

1.5 Infl ammation 11

Endothelial cell dysfunction also directly results in leukocyte invasion, leading to

the formation of a fatty streak (Figure 1.1 b). Leukocyte recruitment occurs through

a series of events involving adhesion molecules. First, leukocytes roll along the

endothelial surface of the vessel wall, which is mediated by endothelial expression

of selectins, including P - selectin. Second, leukocytes adhere tightly to the endothelium

and transmigrate through by extravasation, and these processes are mediated

by integrins and immunoglobulin superfamily members, such as vascular cell

adhesion molecule 1 ( VCAM - 1 ) and intercellular adhesion molecule 1 ( ICAM - 1 )

(see review by [26] ). More detail is provided in Chapter 3 by Wang and Huo.

Endothelial damage increases endothelium permeability and therefore once

bound, leukocytes can migrate through the endothelium and into the intima in

response to the various chemokines released by intimal cells and damaged

endothelium [27] . Chemokines are presented on the endothelial surface by glycosaminoglycan

( GAG ) binding or as a membrane - bound molecules, for example

CX 3 CL1 (fractalkine) [28] . This permits strong binding of the monocyte via the

cognate chemokine receptor. In the intima, the monocytes and T lymphocytes

mature and release chemoattractant cytokines (or chemokines), for example

Chemokin (C - C motif) ligand 2 (CCL - 2 also known as MCP - 1), which establishes

a chemokine gradient for migration and further amplifi es the infl ammatory

response. Interestingly, chemokines also regulate retention of macrophages in the

plaque [29] .

T cells also accumulate in the atherosclerotic plaque in response to chemokines

chemokine (C - X - C motif) ligand 9, 10 and 11 (CXCL9, CXCL10, and CXCL11).

Direct evidence for the involvement of CCL2 and its receptor CCR2 in atherogenesis

come from ApoE −/ −mice that lack either CCL2 or CCR2 expression, as these

mice have dramatically less macrophage recruitment and atherosclerotic plaque

formation [30, 31] . Chapter 2 by White, Channon, and Greaves provides further

details regarding the role of chemokines in atherosclerosis. Infl ammation continues

throughout atherosclerotic plaque development, and mediates a myriad of

proinfl ammatory cytokines, including interferon - ( IFN - ), tumor necrosis factor -

( TNF - ), interleukin - 1 ( IL - 1 ), IL - 2, and IL - 18 (see review by [32] ). Chapter 4 by

Tedgui, Ait - Oufella, and Mallat provides further details. It is of note that some

cytokines, such as IL - 10 and transforming growth factor - (TGF - ) are atheroprotective,

through the induction of T reg cells (see review by [33] ).

The importance of immunity in atherosclerosis is not restricted to the adaptive

immune system: innate immunity is also important. Toll - like receptor s ( TLR s) are

pathogen - associated molecular pattern receptors which respond to invading

microorganisms, as well as modifi ed LDL and " danger signals " released from

damaged tissues, by initiating an infl ammatory response. For example, TLR4 is

responsible for cellular activation after recognition of bacterial lipopoly saccharide s

( LPS ), as well as microbial and eukaryotic heat shock protein - 60 ( HSP - 60 ). TLR4

is expressed in human atherosclerotic plaques and interestingly, polymorphisms

in the TLR4 gene have varying effects on risk of acute coronary syndromes [34]

and TLR4 deletion in ApoE −/ −mice reduced atherosclerosis [35] . Further evidence

implicating TLRs in atherosclerosis comes from studies sing TLR2 −/ −mice crossed

12 1 Pathogenesis of Atherosclerosis

with LDLR −/ −mice, which developed smaller atherosclerotic lesions [36, 37] . See

Chapter 5 by Yan, Edfeldt, and Lundberg for additional information.

Furthermore, the classic short pentraxin, C - reactive protein ( CRP ), which is used

as a prognostic marker for ischemic heart disease, is a component of the innate

immune system. The mechanisms behind the involvement of CRP in heart disease

are unknown, as experimentation has been made diffi cult due to a genetic difference

between mouse and human CRP. However, a recent study has shown that

the long pentraxin PTX3, which is evolutionally conserved, provides a cardioprotective

effect in mice via modulation of the complement pathway. Levels of PTX3

are elevated in myocardial ischemia in both mouse and human, therefore PTX3

could also have potential as a prognostic marker [38] . See Chapter 7 by Mantovani

and colleagues for additional details.

Peroxisome proliferator – activated receptor s ( PPAR s) expressed by endothelial

cells in the vessel wall are an important family of nuclear receptors that regulate

monocyte recruitment, adhesion, and transmigration as well as having anti -

infl ammatory properties and the ability to reduce oxidative stress (see [39] and

Chapter 6 by Gizard and Pineda - Torra for more detailed information). PPARs are

also expressed by monocytes and macrophage and T cells and are associated with

atheroprotective processes including inhibition of infl ammatory molecules and

increased cholesterol effl ux.

1.6

Foam Cell Formation

Increased permeability of the endothelium also results in infi ltration of LDL into

the blood vessel wall. Once in the blood vessel wall LDL is modifi ed and is more

readily taken up by cells [40] . In the intima, growth factors ( monocyte colony -

stimulating factor , M - CSF ) and cytokines (TNF - and IFN - ) are released and

cause monocytes to differentiate into macrophages and mature into active macrophages.

The maturation process causes macrophages to increase their expression

of various scavenger receptors, including scavenger receptors A and B1, and CD36,

which can bind and internalize modifi ed LDL (reviewed in [41] ). The relative

importance of scavenger receptors in atherosclerotic plaque development is controversial.

Currently, it is thought that while scavenger receptor A is pro - atherogenic

(primarily as a consequence of uptake of minimally oxidized LDL by

macrophages in the arterial wall), scavenger receptor B is protective against atherosclerosis

at later timepoints (primarily because of its function in the removal of

high - density lipoprotein ( HDL ) - cholesterol esters in the liver) [42] . Lipid ingestion,

which is likely to be benefi cial at the onset, results in the accumulation of fatty

droplets within the macrophages, which take on a foamy appearance under microscopy

and are therefore termed foam cells [43] . Foam cells release growth factors

and cytokines, which are involved in lesion progression, as well as matrix -

degrading metalloproteinase s ( MMP s), which stimulate matrix degradation.

See Chapter 12 by Moore and Rayne for further information. Together with T

lymphocytes, foam cells form the fatty streak.

1.7 Vascular Smooth Muscle Cell Migration and Proliferation 13

The proteolytic enzymes chymase and tryptase derived from the mast cells have

been shown to profoundly modify the composition and function of HDL particles

and chymase proteolytically modifi es LDL [44] . Stimulated mast cells therefore can

accelerate foam cell formation in the intimal areas in which mast cells and macrophages

coexist, and are surrounded with intimal fl uid enriched with plasma -

derived LDL and HDL particles. Further details are available in Chapter 11 by

Kovanen.

As well as being taken up by macrophages, cholesterol can also be effl uxed via

transporters, such as ABCA - 1 and ABCG - 1, to extracellular HDL - based acceptors,

such as apolipoprotein E. This process is known as reverse cholesterol transport

( RCT ). After effl ux to HDL, cholesterol may be esterifi ed in the plasma by the

enzyme lecithin:cholesterol acyltransferase and is ultimately transported from

HDL to the liver, either directly via the scavenger receptor BI or after transfer to

apolipoprotein B - containing lipoproteins by the cholesteryl ester transfer protein.

Cholesterol effl ux is thought to be atheroprotective by removing the cholesterol

accumulation and therefore reducing foam cell formation [45] . Promotion of macrophage

RCT is a potential therapeutic approach to preventing or regressing

atherosclerotic vascular disease, but robust measures of RCT in humans will be

needed in order to confi dently advance RCT - promoting therapies in clinical development

(reviewed in [46] ).

Monocytes and macrophages display a high level of heterogeneity to allow them

to specialize in particular functions. Many macrophage phenotypes have been

identifi ed and at least two of these (M1 and M2) have been shown to be present

in atherosclerotic plaques. The phenotype expressed by a macrophage depends on

the chemical signals used to induce macrophage differentiation. Th1 cytokines,

such as IFN - , IL - 1 and lipopolysaccharide, induce a " classical " activation profi le

called M1. On the other hand, Th2 cytokines, such as IL - 4 and IL - 13, induce an

" alternative " activation program called M2. The difference in the activity of these

two subtypes is extreme, while M1 macrophages are proinfl ammatory, M2 are

anti - infl ammatory [47] . Macrophages can change from one phenotype to another

and therefore the modulation of this phenotypic change is an interesting therapeutic

strategy. Macrophage phenotypes are discussed in detail in Chapter 18 by

Johnson and Jenkins.

1.7

Vascular Smooth Muscle Cell Migration and Proliferation

As fatty streaks develop into more complex, advanced atherosclerotic plaques, they

develop a fi brous cap, consisting of VSMCs, which form a protective layer between

the lesion and the lumen of the vessel. In the development from a fatty streak to

an advanced plaque, VSMCs migrate from the media into the intima, where they

undergo proliferation and lay down extracellular matrix (Figure 1.1 d). During

these processes, VSMCs undergo phenotypic modulation from contractile to a

synthetic phenotype resulting in VSMC de - differentiation. See Chapter 16 by Coen

and Bochaton - Piallat for further detail. Fibrous cap formation