The Identification of In Vivo Angiogenesis and Fibrosis in Aortic Stenosis Using Positron Emission Tomography
Integrins are a group of molecules responsible for intercellular adhesion and signalling.
They comprise a superfamily of heterodimeric receptors that are composed of 18 different α
and β subunits. In combination, they can generate 24 different receptor subtypes with a
range of physiological and pathophysiological functions. The αvβ3 receptor is an integrin
that is found at low levels on mature endothelial cells but is markedly up regulated on
endothelial cells of actively growing blood vessels. It was previously known as the
vitronectin receptor although it was subsequently found to bind many other ligands including
fibrinogen, fibronectin, laminin, thrombospondin, von Willebrand factor, and certain
collagen subtypes. These features are also seen with the αvβ5 integrin receptor, with both
receptors recognising the arginine-glycine-aspartate (RGD) motif present on these ligands.
1.1.2 Role of αvβ3 and αvβ5 Integrins in Cardiovascular Disease
The expression of αvβ3 and αvβ5 receptors is up regulated in a number of diseased states and
this has been particularly well characterised in the angiogenesis associated with tumour
growth and metastases. However, there are many potential roles for this integrin pathway in
cardiovascular disease including myocardial infarction, atherosclerosis, restenosis, aortic
stenosis and aneurysm disease that have been relatively unexplored.
18.104.22.168 Aortic Stenosis
Aortic stenosis is characterized by extensive valvular thickening due to accumulation of
fibrous tissue and remodeling of the extracellular matrix. In all three layers of the valve,
abundant fibroblast-like cells are found and are commonly referred to as valvular
interstitial cells. A sub-population of these cells become activated by the inflammatory
activity within the valve and differentiate into myofibroblasts. Whilst fibroblasts control
the synthesis of collagen in the normal valve, myofibroblasts are responsible for the
accelerated fibrosis observed within stenotic valves. In addition, matrix metalloproteinases
are secreted by myofibroblasts and inflammatory cells, and have an important and complex
role in the restructuring of the valve leaflet matrix. As already indicated, activation and
differentiation of fibroblasts into myofibroblasts are dependent on αvβ3 and αvβ5 receptor
expression. In addition, mirroring the situation in carotid atherosclerosis, patients with
severe aortic stenosis have a high incidence (78%) of intraleaflet haemorrhage and this is
associated with angiogenesis and more rapid disease progression.
Histopathological studies have confirmed fibrosis to be an integral part of the left
ventricular hypertrophic process in aortic stenosis. Myofibroblasts infiltrate the
myocardium and secrete extracellular matrix proteins including collagen types I and III.
Areas of fibrosis are observed to co-localize with areas of myocyte apoptosis and it has
been suggested that fibrosis occurs as a form of scarring after myocyte death and injury. As
with fibrosis in the valve, the renin-angiotensin system, transforming growth factor-beta
and an imbalance in matrix metalloproteinase and their tissue inhibitor activity have all
been implicated in this process. A mid-wall pattern of fibrosis has been observed in the
myocardium of up to 38% of patients with moderate or severe aortic stenosis and has been
associated with a more advanced hypertrophic response. Importantly, there is also an 8-fold
increase in mortality associated with mid-wall fibrosis.
22.214.171.124 Atherosclerosis and Restenosis
The development of atherosclerosis is due to a complex interplay of oxidised lipid,
inflammatory cell infiltration, and smooth muscle cell migration in the arterial wall. Once
established, atherosclerotic plaques may progress and rupture leading to the clinical
presentations of acute myocardial infarction and stroke. Features associated with plaque
rupture include a thin fibrous cap, lipid-rich pool and intraplaque haemorrhage. Indeed,
plaque rupture is particularly associated with plaque neovascularisation and
vascular-endothelial growth factor expression suggesting that instability may be induced by
angiogenesis. Thus, up regulation of αvβ3 and αvβ5 receptors may represent a novel marker
of, and potential therapeutic target for, plaque vulnerability.
Fluciclatide is a RGD-containing cyclic peptide that has recently been developed as an
18F-radiotracer to detect tumour angiogenesis by positron emission tomography. It is highly
selective for the αvβ3 and αvβ5 receptors with affinities (EC50) of 11.1 and 0.1 nM
respectively with minimal cross reactivity with the αIIbβ3 receptor (EC50 281 nM).
Pre-clinical tumour work has demonstrated that 18F-fluciclatide is taken up by glioblastomas
and that this is suppressed by the anti-angiogenic tyrosine kinase inhibitor, sunitinib,
confirming the specificity of fluciclatide for areas of angiogenesis. It has been assessed
in phase I clinical trials and found to be safe and well tolerated.
To date, there have been many preclinical studies examining the application of radiotracers
targeting the αvβ3 and αvβ5 integrin receptors. The clinical application of these tracers
has been largely limited to oncology as a method of assessing angiogenesis within tumours.
Here we wish to explore the role of the αvβ3 and αvβ5 receptor radiotracer,
18F-fluciclatide, to assess angiogenesis and fibrosis in patients with aortic stenosis as a
measure of both valvular and myocardial fibrosis. This patient group will have co-existent
aortic atheroma and this will provide us with an opportunistic assessment of tracer uptake
in atherosclerosis. We feel it is important to assess a range of cardiovascular conditions
to determine whether αvβ3 and αvβ5 integrin receptor expression is particular to certain
disease processes. If successful, these preliminary data will permit the more detailed
exploration of specific disease areas and novel therapeutic interventions. At present,
fluciclatide is not licensed or approved for clinical use and is being used here as an
Investigational Agent to explore the pathophysiology of aortic stenosis.
1.2 ORIGINAL HYPOTHESES
We hypothesise that 18F-fluciclatide can identify the expression of the αvβ3 and αvβ5
integrin receptors in vivo in man in two major cardiovascular disease areas: aortic
atherosclerosis and aortic stenosis. Specifically, we hypothesise that 18F-fluciclatide
1. Be taken up into aortic atherosclerotic plaque.
2. Show demonstrable uptake in the aortic valve and myocardium of patients with aortic
stenosis that will correlate with the degree of active angiogenesis and fibrosis.
6.1 ANGIOGENESIS AND FIBROSIS IN AORTIC STENOSIS
Aortic stenosis is associated with substantial left ventricular hypertrophy and consequent
myocardial fibrosis with the latter predicting prognosis. Left ventricular hypertrophy and
associated fibrosis is also a major risk factor for adverse cardiovascular events in a
number of other conditions including essential hypertension. Cardiac magnetic resonance
imaging is the gold-standard method of assessing for the presence of myocardial fibrosis but
it does not necessarily indicate the on going activity of the fibrotic process. In this
study, we will assess the uptake of 18F-fluciclatide in patients with aortic stenosis as a
model of pressure overload left ventricular hypertrophy. We will also seize the opportunity
to determine whether there is any uptake of 18F-fluciclatide in the aortic valve given that
this has been shown to have areas of fibrosis and angiogenesis.
All study patients and healthy volunteers will undergo blood sampling, echocardiogram,
positron emission and computed tomography scans with 18F-fluciclatide as well as cardiac
magnetic resonance imaging with assessment of gadolinium late enhancement. Following
injection of 18F-fluciclatide, patients will be monitored using our standard clinical
approach, including observation of haemodynamic parameters, and this will continue
throughout their study visit until departure. In patients undergoing aortic valve
replacement surgery, aortic valve tissue will be retained and a 3-mm tru-cut biopsy of left
ventricular myocardium obtained with which to compare the findings from the scans.
Healthy volunteer patients will not undergo repeat assessment. After a period of one to two
years from their initial scan, patients with Aortic Stenosis will return for repeat blood
sampling, cardiac magnetic resonance imaging with assessment of late gadolinium enhancement
and echocardiogram. Those patients who have undergone an aortic valve replacement will
undergo repeat positron emission and computed tomography scans with 18F-fluciclatide six
months after their operation, prior to their second cardiac MRI scan.
Blood samples will be assessed using standard clinical biochemical and haematological
profiles such as full blood count and urea and electrolytes. In addition, markers of cardiac
ischaemia, fibrosis and angiogenesis will be assessed. Additional serum, plasma and DNA will
be stored at -80 degrees Celsius for future potential analyses.
6.1.2 Study Interpretation
We anticipate that myocardial uptake of 18F-fluciclatide will be increased in patients with
aortic stenosis and left ventricular hypertrophy. We expect the degree of myocardial uptake
to correlate with cardiac magnetic resonance imaging assessment of fibrosis as well as the
histological measures of fibrosis and αvβ3 and αvβ5 integrin receptor expression. We expect
the degree of myocardial uptake to predict cardiac magnetic resonance imaging assessment of
fibrosis following a period of one to two years. In exploratory analyses, we will also take
the opportunity to assess the extent of 18F-fluciclatide uptake within the aortic valve
itself, and if successful, correlate this with histological measures of angiogenesis and
6.2 ANGIOGENESIS IN AORTIC ATHEROSCLEROSIS
Patients with aortic stenosis will have a high prevalence of concomitant aortic
atherosclerosis. In Dr Dweck's Fellowship, we were able to exploit this association and
undertake secondary analyses of 18F-sodium fluoride uptake in aortic and coronary
atherosclerosis. This generated some highly innovative findings that informed our
understanding of atherosclerosis and the role of calcification.
6.2.1 Study Schedule
We will use the datasets obtained from the patients above to explore the uptake of
18F-fluciclatide within the thoracic aorta. Atherosclerosis will be identified using
computed tomography and magnetic resonance images obtained of the thorax at the time of the
study scans. No additional image acquisition will be required. This will provide pilot data
to inform subsequent dedicated studies focused on acutely inflamed atherosclerotic plaques,
such as patients with recent transient ischaemic attacks or strokes attributable to carotid
Observational Model: Cohort, Time Perspective: Prospective
The mean and maximum standardised uptake values (SUV) of fluciclatide for the myocardium and its correlation with the severity of aortic stenosis determined echocardiographically.
1 - 2 years
David E Newby, MBChB PhD
University of Edinburgh
United Kingdom: Research Ethics Committee