Endothelial Dysfunction in Atherogenesis

Question 1

How can the endothelium be thought of on an organismal level?

Answer 1

On a whole organism level, the endothelium can be considered an organ in itself, weighing approximately 1kg and accounting for between 1 and 6x10^13 cells.

Question 2

What is the structure of the endothelium?

Answer 2

These cells inside the blood vessels are simple squamous epithelial cells, with a monolayer of flat and polygonal cells and sitting on a basement membrane above some connective tissue.

This structure is found lining not only the blood vessels and heart but also the lympatic vessels.

Question 3

What are the roles of the endothelium?

Answer 3

• Barrier
o Selective permeability
o Active transport

• Vascular tone
o Regulation of the SMCs through paracrine signalling
 NO, prostacyclin, endothelin
 Thus regulates blood pressure

• Haemostasis
o Regulation of thrombosis
 Provision of anti-thrombotic surface
 Expression of assorted clotting/anti-clotting factors, eg heparin

• Angiogenesis
o Formation of tip cells that migrate up VEGF gradients leads to formation of new blood vessels
o Responds to variety of (anti)angiogenic factors

Question 4

What is the role of VEGF wrt the endothelium?

Answer 4

Aside from its angiogenic function, VEGF also increases blood flow and reduces pressure in animal models. This is mediated by its stimulation of NO and prostacyclin.

It is required for microvasculature maintenance in many tissues, and for adult function in organs such as the kidneys.

Question 5

What does VEGF knockout cause?

Answer 5

Lee et al, 2007 showed that knocking out VEGF only in endothelial cell lines in mice led to progressive endothelial degradation and sudden death.

Question 6

How is VEGF being targeted in therapy?

Answer 6

VEGF pathway stimulants are being tested as a treatment for ischemic heart disease in the hope it will produce new vasculature. Though largely safe and well tolerated, it does also lead to the hypotension VEGF causes.

It is also worth noting that VEGF is targeted in cancer treatments (avastin), but downregulation by these drugs can increase hypertension and risk of CVD.

Question 7

What is contradictory about the role of VEGF in atherosclerosis?

Answer 7

Despite its role in prevention of atherosclerosis it has also been implicated in promoting the growth and pathological progression of existing lesions.

VEGF is produced in response to the hypoxia within atherosclerotic plaques, and is associated with the formation of plexiform bodies found in the most vulnerable plaques.

Question 8

How does one assess endothelial dysfunction?

Answer 8

Flow-mediated Dilation is used to measure activation, and works by measurement of arterial dilation before, during and after the removal of a sphygmomanometer cuff.

Lack of relaxation of the blood vessels in the fingers is indicative of endothelial activation and thus CVD.

Question 9

What factors can trigger endothelial activation?

Answer 9

Infection
Tobacco Smoke
Homocysteine
Altered Biomechanical Forces
NF-κB
Hypercholesterolaemia

Question 10

How does infection trigger endothelial activation?

Answer 10

Several pathogens have been identified in lesions or correlated with the incidence of atherosclerosis, but as of yet no causative links have been drawn.

Infection agents linked include bacteria such as Chlamydiae pneumoniae, viruses such as Herpes simplex and even distant infections such as periodontitis.

Question 11

How does Tobacco Smoke trigger endothelial activation?

Answer 11

A very large number of the chemicals, including but not limited to nicotine, CO and free radicals, in tobacco smoke are linked to endothelial dysfunction in a variety of ways.

In rats subjected to tobacco smoke, the endothelium became more disorganised leading to greater adhesion of both platelets and white blood cells.

Question 12

What is the role of homocysteine in the endothelium?

Answer 12

This is a synthetic amino acid produced from methionine, which stimulates many cellular functions via a number of cell signalling pathways, including DNA damage via p53, ER stress and Ca2+ influx.

Question 13

How does homocysteine trigger endothelial activation?

Answer 13

It is toxic to endothelial cells, as well as decreasing NO production and increasing collagen production, adding to its pro-thrombotic capacity.

Mutations in the enzymes involved in its metabolism, specifically MTHFR and CBS, lead to accumulation of homocysteine. These individuals commonly die due to CVD by age 20, providing proof of its atherogenic role.

Question 14

What effect does shear stress have on the endothelium?

Answer 14

Shear stress caused by the force of the blood on the endothelium is linked to increased endothelial stability as it activates the mechanical receptors in the cells, causing them to increase production of NO by eNOS as well as anti-inflammatory agents such as COX-2, antioxidant SODs and anti-proliferative p21 family proteins. It also leads to inhibition of the angiotensin 2 receptor which reduces blood pressure.

This is particularly true of unidirectional laminar flow and pulsatile shear stress.

Question 15

How can altered biomechanical forces trigger endothelial activation?

Answer 15

Regions of disturbed flow, such as artery bifurcations, branch points and regions of high curvature in the artery reduce the shear stress and have high risk of atherogenesis.

This acts through upregulation of inflammatory pathways and atherogenic factors, including ROS, NF-κB, Rho, Ras, VCAM-1 and MCP-1.

Question 16

What atherogenic components stimulate NF-kB?

Answer 16

• Oxidised lipids/lipoproteins

• Cytokines
o IL-1β
o TNF-α

• Altered Biomechanical forces
o Decreased shear stress
o Increased strain

• Free radicals
• Infection

Question 17

Which of the effects of NF-kB are involved in lesion progression?

Answer 17

• Pro-inflammatory cytokines
o IL-1
o IL-6
o TNF

• Increased adhesion molecule expression
o VCAM-1
o ICAM-1
o E-selectin

• Chemokine production
o MCP-1
o IL-8

• Colony stimulating factors
o M-CSF
o GM-CSF

Question 18

What mediates endothelial activation by hypercholesterolaemia?

Answer 18

Elevated LDL leads to easier penetration of the lipoproteins into the intima where they may be oxidised and lead to damage and inflammation, as well as increased oxidative stress.

Question 19

How do oxLDLs lead to endothelial activation?

Answer 19

The complete list of oxLDL effects is largely similar to those of NF-κB, including production of adhesion molecules, chemokines (i.e. chemoattractant cytokines), proliferation, NO inhibition etc.

They also stimulate the phenotypic change of the invading monocytes into macrophages, and alter their gene expression via interaction with PPARs and ABCA1.

Question 20

What are the main biomarkers of endothelial dysfunction?

Answer 20

There are many different molecules of varying origin that act as biomarkers for this process, one of the most notable being myeloperoxidase which predicted the heart attack risk of 85% of angina patients.

Another is the N-terminal fragment of the brain natriuretic pro-protein which correlates with mortality in stable CHD patients.

Question 21

Why are blood-borne biomarkers of endothelial activation useful? What is their primary disadvantage?

Answer 21

Such blood-borne biomarkers are highly useful as they allow a cheap non invasive test that provides much information:

• Assessment of risk in healthy subjects
• Prediction of future cardiac events
• Monitoring of disease progression

Many of these are tests of inflammation, a more indirect measurement as it is effectively measuring one risk factor, albeit one that results from many others.

Question 22

What biomarker is a direct measurement of endothelial activation?

Answer 22

The presence of soluble CAMs (composed of the extracellular domains) are directly indicative of endothelial damage, as their presence indicates that they have been shed from the endothelium due to activation, potentially as a result of MMP action.

Their levels in the circulation increase in response to many diseases however.

Question 23

How is the presence of soluble CAMs measured?

Answer 23

Two are particularly important; sVCAM-1 plasma concentration correlates well with VCAM-1 expression in lesions (as well as intima thickness), something which is very difficult to directly measure.

It is hence a dynamic surrogate marker of EC activation and independent predictor of CVD risk. The other, slCAM-1 is an early marker, being found in normolipidaemic children from families with a history of high CVD risk.