Angiogenesis and Fibrosis in Aortic Stenosis
Angiogenesis and Fibrosis in Aortic Stenosis
Angiogenesis and fibrosis lie at the heart of a number of fundamental processes responsible
 for cardiovascular disease. In this proposal, the investigators intend to build upon a highly
 successful programme of studies exploring the cardiovascular applications of positron
 emission tomography. Specifically, the investigators will explore the potential role of a
 novel radiotracer, 18F-fluciclatide, which is a highly selective ligand for the αvβ3 and αvβ5
 integrin receptors that are up regulated during angiogenesis, and tissue fibrosis and
 remodelling. This tracer has been successfully used to assess angiogenesis in metastatic
 tumours and its uptake is suppressed by anti-angiogenic therapies. The investigators here
 propose to describe the pattern of uptake of 18F-fluciclatide in cardiovascular diseases,
 specifically aortic stenosis and aortic atherosclerosis. The investigators will correlate
 18F-fluciclatide uptake with in vivo measures of angiogenesis and fibrosis as well as ex vivo
 histological characterisation of tissue. If successful, this novel radiotracer could provide
 an extremely important non-invasive method of assessing in vivo angiogenesis, plaque
 vulnerability, and tissue remodelling as well as potential applications in developing stem
 cell therapies.
Integrins

 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.

 1.1.2.1 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.

 1.1.2.2 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.

 1.1.3 Fluciclatide

 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.

 1.1.4 Aims

 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 will:

 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
 fibrosis.

 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
 disease.
Inclusion Criteria

 -  asymptomatic mild (peak valve velocity of 2-5-3.0 m/s; n=10), moderate (peak valve
 velocity of 3.0-4.0 m/s; n=10) or severe aortic stenosis (peak valve velocity of >4.0
 m/s; n=10) and 10 patients with severe aortic stenosis proceeding to aortic valve
 replacement.

 -  Healthy control subjects (n=10) will have no past medical history of ischaemic heart
 disease or valvular heart disease and have a structurally normal heart on
 echocardiography.

 Exclusion Criteria:

 -  Atrial fibrillation

 -  Hepatic failure (Childs-Pugh grade B or C)

 -  Renal failure (estimated glomerular filtration rate <25 mL/min)

 -  Women of child-bearing potential

 -  Contraindication to magnetic resonance imaging

 -  Inability to undergo scanning

 -  Ochronosis and those with any form of collagen-vascular disease.