Abdominal aortic aneurysms (AAAs) are permanent dilations of the aorta that exceed 50% of the original size of the vessel or that are greater than 3cm in maximum diameter. After a period of asymptomatic expansion, rupture of AAA can occur with an associated mortality rate as high as 90% in developed countries. AAAs are characterized by chronic inflammation, extracellular matrix (ECM) protein remodeling, intravascular hematomas and atherosclerosis. The ECM proteins, collagen and elastin, are the most abundant structural proteins of the arterial wall. They provide tensile strength and elastic recoil to the arterial wall. Turnover of the ECM proteins, and particularly, the balance between degradation and synthesis is vital for preserving the structural integrity of the aortic wall. Deregulation of ECM synthesis and degradation is thought to be crucial for the development and outcome of AAAs. Collagen is a major ECM protein involved in AAA remodeling. Following vascular injury, myofibroblasts and other cell types synthesize pro-collagens, mostly type 1 and type 3. Pro-collagen proteinases initially convert these precursor molecules into collagen fibers which subsequently become cross-linked into collagen fibrils by lysyl-oxidase. In parallel, upregulation of matrix metalloproteinases (MMPs) causes ECM degradation that not only facilitates adaptive changes (including removal of cellular debris by inflammatory cells and migration of myofibroblasts) but also contributes to weakening of the myocardium and ventricular dilatation. Thus, a fine tuned balance of collagen turnover is crucial for AAA remodeling and vascular integrity.
This project will investigate the role of collagen I and III in the evolution and instability of AAA and the potential impact of therapies that aim in modulating the fibrotic response using in vivo molecular MRI, SPECT-PET and ex vivo techniques.
Year 1: In this project we will initially use a commercially available collagen I-binding contrast agent to image AAA remodelling in an Angiotensin–II infused murine model using a clinical 3T MR scanner. The temporal evolution of the biological processes that occur in this animal model are well known and hence the choice of those particular imaging time points proposed in the project. Animals will be scanned at days d0-d7-d14 and d21 post infusion of Angio-II after injection of the contrast agent. Subsequently, tissues will be collected for ex vivo histological, molecular and biochemical analysis.
Year 2: We have already identified new peptides that could potentially have selectivity towards collagens I and III and we have performed preliminary in vitro binding essays. The peptides carry a DOTA chelate and thus have the flexibility to be labelled with lanthanides (europium or gadolinium) or radioisotopes for SPECT and/ or PET. In year 2, the student will expand the in vitro binding essays and subsequently use radiolabelled peptides for initial biodistribution and biological studies in control and diseased using PET and SPECT imaging. The most promising probe will then be conjugated with gadolinium for in vivo MRI experiments described in Year 3. For this part of the work we collaborate with experienced chemists within our Division (Dr Rafael Torres, Dr Michelle Ma and Prof. Phil Blower who have been added to the supervisory team). There is also an on going collaboration with Dr Lacerda at the CNRS (France) who has performed similar work in the past and she agreed to provide training and expertise as needed.
Year 3: The best peptides for collagen I and III identified in year 2, will be used for in vivo imaging of collagen remodelling in the same Angio-II murine model used in Year 1.
- Generation, genotyping and maintenance of murine colonies, animal handling, tail injections, anesthesia, tissue harvesting
- Surgical skills: implantation of mini-osmotic pumps
- Peptide chemistry for the co-ordination of lanthanides (e.g., gadolinium, Europium) or radioactive compounds to peptides
- In vitro and in vivo PET/SPECT imaging experiments
- In vitro and in vivo vessel wall MRI protocols including late-gadolinium enhancement, T1 mapping protocols and blood flow measurements
- Ex vivo binding assays of radioactive isotopes or Europium labelled peptides (DELFIA assays)
- Ex vivo histology, immunohistochemistry, proteomics, western blotting, ELISA, PCR, FACS, ICP-MS of extracted tissues or cell lines
- Image processing and analysis software
- Statistical analysis and software
All of the infrastructure, equipment, and animal models are currently available within the School of Biomedical Engineering and Imaging Sciences at KCL that has a multidisciplinary team of supervisors who have been working closely together on several projects. We will ensure that the student will be trained and supervised on all of the required techniques by experienced members of the Division.