CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 4, May 2012
228
AFRICA
of ADMA is catalysed by protein arginine methyltransferases
(PRMTs) and its degradation is catalysed by dimethylarginine
dimethylaminohydrolases (DDAH).
75
DDAH levels are often decreased in a variety of cardiovascular
diseases, which leads to the upregulation of ADMA. For
example, treatment of endothelial cells with TNF-
α
, ox-LDL and
glucose has been reported to diminish the activity of DDAH.
74,75
Furthermore, PRMTs and DDAH are ROS-sensitive; the activity
of the DDAH is impaired, whereas the activity of the PRMTs is
increased in conditions of oxidative stress.
75
Indeed, increased plasma levels of ADMA have been
documented in patients with conditions such as hyperlipidaemia,
hypertension, coronary artery disease, stroke and end-stage renal
disease.
75
Furthermore, ADMA was found to be significantly
elevated in patients with unstable angina, and reduced plasma
levels of ADMA at six weeks post-percutaneous coronary
intervention was found to be possibly indicative of a reduced risk
of recurrent cardiovascular events.
76
A recent study investigated the prognostic value of ADMA
with regard to cardiovascular disease and death (fatal or non-fatal
myocardial infarction, coronary insufficiency, angina pectoris,
stroke or TIA, intermittent claudication or heart failure) in
Framingham Offspring study participants.
77
Although ADMA
was significantly associated with all-cause mortality in this
population, the study could not find an association between
ADMA and cardiovascular disease incidence.
Circulating endothelial cells and endothelial
microparticles
Circulating endothelial cells (CECs), which are mature cells
that have detached from the endothelium, represent a novel
biomarker of endothelial injury.
78,79
In a healthy person, the
endothelium is constantly refurbished at a replication rate of
<
1% and levels of CECs are very low. Studies using a flow-
activated cell sorter (FACS) isolation technique reported CECs
ranging from 50–7 900 cells/ml in healthy individuals and up to
39 100 cells/ml in individuals with vascular diseases.
79
Potential mechanisms underlying endothelial cell detachment
may be mechanical injury, action of proteases and/or cytokines,
defective endothelial cell adhesion to the extracellular matrix,
cellular apoptosis, and injurious actions of cardiovascular risk
factors, such as occur during the induction of ED. Increased
levels of CECs are associated with ED, cardiovascular diseases
and a variety of other diseases.
78,79
Apoptotic CECs expressing
surface marker (CD146) have been reported to be increased in
patients with cardiovascular disease.
65
Other circulating cellular markers of endothelial injury
include endothelial microparticles (EMPs), which are small cell
membrane vesicles released into the circulation by activated or
apoptotic cells.
5,65
Patients with hypertension and coronary artery
disease have been reported to demonstrate high levels of EMPs,
65
and according to Tramontano
et al.
, statins can diminish the
release of EMPs in cultured coronary endothelial cells.
Nitrotyrosine upregulation
In addition to its ability to directly uncouple the eNOS enzyme,
which can lead to ED, peroxynitrite undergoes protonation to form
peroxynitrous acid (ONOOH), or it can combine with carbon
dioxide (CO
2
) to form nitroso-peroxocarboxylate (ONOOCO
2
–
),
both of which yield tyrosine-nitrating compounds.
80,81
Via
formation of these compounds, peroxynitrite leads to nitration
(addition of a NO
2
group) of tyrosine residues of proteins,
leading to formation of nitrotyrosine.
82
Under normal conditions, low levels of free or protein-bound
nitrotyrosine are detectable, which may indicate low levels of
oxidants and nitrating species produced during physiological
processes. However, significant nitrotyrosine upregulation is
observed in conditions that are associated with nitroxidative
stress such as inflammation, cardiovascular disease (including
ED and atherosclerosis) and neurodegenerative disorders.
80
Tyrosine nitration may modify the structure and function of
proteins, leading to alterations in catalytic activity of enzymes,
production of antigenic epitopes, and impaired cell signal
transduction.
82
It has recently been proposed that nitrotyrosine levels
can be clinically detected in urine samples using a surface
plasmon resonance (SPR) sensor
83
or high-performance liquid
chromatography.
84
However, nitrotyrosine measurements in the
context of ED research remain confined to the experimental
laboratory setting.
Other biomarkers of ED/vascular injury
Recently, the European Society of Cardiology Working Group on
Peripheral Circulation published a position statement on methods
for evaluating endothelial function.
85
In this comprehensive
review, several biochemical markers and assays that are used to
examine different aspects of endothelial function were discussed.
The working group mooted plasma ADMA levels as a potential
biomarker of endothelial function; however the authors cautioned
that currently, direct endothelial function measurements remain a
superior indicator and should not be replaced by plasma ADMA
level measurements due to inconsistent prognostic data obtained
with the latter.
Another biomarker with potential clinical application is
oxidised LDL levels; however this biomarker also presents with
some limitations. It is difficult to determine ox-LDL levels
in
vivo
and the ability of elevated plasma ox-LDL to independently
predict the development of coronary heart disease is still
equivocal.
In a recent study on a model of rat carotid injury, proteomic
analysis of blood proteins showed significantly differential
expression of vitamin D binding protein (VDBP), aldolase A
(aldo A) and apolipoprotein E (ApoE) two weeks after injury.
86
Reduced circulating levels of all three of these plasma markers
were associated with the presence of vascular injury and may
represent novel markers of ED; however, further research is
necessary.
Summary of assessment of endothelial function
With the development of an ever-increasing number of
measurement techniques of endothelial function (both direct
mechanical endothelial function assessment and measurement
of biomarkers of endothelial function), most authors agree
that more and larger human-based studies are necessary to
validate their clinical usefulness. The overall objective of such
studies should ultimately be to establish standardised protocols
allowing for the clinical diagnosis of ED, and quantification of
cardiovascular risk, followed by the reversal of ED by means of
