The prevention of arterial restenosis using endovascular photodynamic therapy

Atherosclerosis is the commonest aetiology in deaths arising from cardiovascular disease. It is characterised by the build up of an intraluminal plaque leading to arterial stenosis. Balloon angioplasty offers a minimally invasive method of dilating such stenoses in both peripheral and coronary arteries. However, despite very favourable immediate results, 15-40% of arteries restenose within 3-6 months following angioplasty with obvious clinical and resource implications. Restenosis is caused by a combination of neointimal hyperplasia (NIH) and negative geometric remodelling, the combined effect of which results in luminal narrowing. The aim of this thesis was to investigate a method of inhibiting restenosis using photodynamic therapy (PDT). PDT involves the interaction of light of a specific wavelength with a pre-administered photosensitiser to produce cell death by oxygen-dependent cytotoxic mediators. Experimental project Preliminary studies established the pharmacokinetics of the chosen photosensitiser 5-aminolaevulinic acid (ALA) in a swine model. From these experiments the optimum drug-light interval was calculated and in a second study, PDT was applied to normal porcine iliac and coronary arteries using an endovascular light source. Depletion of medial vascular smooth muscle cells (VSMC) was seen at 3 and 14 days and was found to be partially dependent on the drug-light interval. Finally, iliac and coronary arteries were balloon injured and then treated with PDT or sham illumination. Histomorphometric studies following harvest at 28 days showed less NIH and less negative remodelling in the group treated with PDT. Clinical project A clinical pilot study of angioplasty with adjuvant PDT was commenced. Patients deemed to be at high risk of restenosis underwent femoral angioplasty followed by endovascular PDT and were followed up by duplex and digital subtraction angiography at 6 months. The results of this small study would suggest that PDT is successful in inhibiting restenosis, but this now needs to be confirmed in a randomised controlled trial

Linking quantitative radiology to molecular mechanism for improved vascular disease therapy selection and follow-up

Objective: Therapeutic advancements in atherosclerotic cardiovascular disease have improved the prevention of ischemic stroke and myocardial infarction. However, diagnostic methods for atherosclerotic plaque phenotyping to aid individualized therapy are lacking. In this thesis, we aimed to elucidate plaque biology through the analysis of computed-tomography angiography (CTA) with sufficient sensitivity and specificity to capture the differentiated drivers of the disease. We then aimed to use such data to calibrate a systems biology model of atherosclerosis with adequate granularity to be clinically relevant. Such development may be possible with computational modeling, but given, the multifactorial biology of atherosclerosis, modeling must be based on complete biological networks that capture protein-protein interactions estimated to drive disease progression. Approach and Results: We employed machine intelligence using CTA paired with a molecular assay to determine cohort-level associations and individual patient predictions. Examples of predicted transcripts included ion transporters, cytokine receptors, and a number of microRNAs. Pathway analyses elucidated enrichment of several biological processes relevant to atherosclerosis and plaque pathophysiology. The ability of the models to predict plaque gene expression from CTAs was demonstrated using sequestered patients with transcriptomes of corresponding lesions. We further performed a case study exploring the relationship between biomechanical quantities and plaque morphology, indicating the ability to determine stress and strain from tissue characteristics. Further, we used a uniquely constituted plaque proteomic dataset to create a comprehensive systems biology disease model, which was finally used to simulate responses to different drug categories in individual patients. Individual patient response was simulated for intensive lipid-lowering, anti-inflammatory drugs, anti-diabetic, and combination therapy. Plaque tissue was collected from 18 patients with 6735 proteins at two locations per patient. 113 pathways were identified and included in the systems biology model of endothelial cells, vascular smooth muscle cells, macrophages, lymphocytes, and the integrated intima, altogether spanning 4411 proteins, demonstrating a range of 39-96% plaque instability. Simulations of drug responses varied in patients with initially unstable lesions from high (20%, on combination therapy) to marginal improvement, whereas patients with initially stable plaques showed generally less improvement, but importantly, variation across patients. Conclusion: The results of this thesis show that atherosclerotic plaque phenotyping by multi-scale image analysis of conventional CTA can elucidate the molecular signatures that reflect atherosclerosis. We further showed that calibrated system biology models may be used to simulate drug response in terms of atherosclerotic plaque instability at the individual level, providing a potential strategy for improved personalized management of patients with cardiovascular disease. These results hold promise for optimized and personalized therapy in the prevention of myocardial infarction and ischemic stroke, which warrants further investigations in larger cohorts

The role of thrombin and antithrombin-therapy in interventional cardiology

Thrombin and thrombus. The plasma coagulation system is activated in response to vascular injury and, within several minutes, thrombin is generated by a series of linked proteolytic reactions that take place on cell surfaces. These reactions are balanced by naturally occurring inhibitory and procoagulant molecules. Thrombin has three major actions which are it's powerfull stimulus for platelet aggregation and activation; fibrinogen conversion to insoluble fibrin strands in the final common pathwa

Development of a Stem Cell-Based Tissue Engineered Vascular Graft

Limited autologous vascular graft availability and poor patency rates of synthetic grafts for small-diameter revascularization (e.g., coronary artery bypass, peripheral bypass, arteriovenous graft for hemodyalisis access, etc.) remain a concern in the surgical community. A tissue engineering vascular graft (TEVG), including suitable cell source, scaffold, seeding, and culture methods can potentially solve these limitations. Muscle-derived stem cells (MDSCs) are multipotent cells, with long-term proliferation and self-renewal capabilities, which represent a valid candidate for vascular tissue engineering applications due to their plasticity/heterogeneity. The poly(ester urethane) urea (PEUU) is also an attractive potential candidate for use as a TEVG due to its elasticity and tunable mechanical and degradation properties. We hypothesized that a novel scaffold optimally seeded with stem cells, acutely cultured and stimulated in vitro, and ultimately implanted in vivo will remodel into a functional vascular tissue. To test this hypothesis, we developed an innovative, multidisciplinary framework to fabricate and culture a TEVG in a timeframe compatible with clinical practice. In this approach, MDSCs were incorporated into a newly-designed and characterized PEUU-based scaffold via a novel seeding device, which was tested quantitatively for cell seeding uniformity and viability. The seeded TEVGs were acutely cultured in dynamic conditions and assessed for cell phenotype, proliferation, and spreading. The conduits were then implanted systemically in a small and a large animal model and assessed, at different time points, for patency rate, remodeling, and cellular engraftment and phenotype. The seeding technology demonstrated a rapid, efficient, reproducible, and quantitatively uniform seeding without affecting cell viability. The PEUU scaffold that was developed is suitable for arterial applications, exhibiting appropriate strength, compliance, and suture retention properties. The dynamic culture resulted in cell proliferation and spreading within the 3D scaffold environment. Rat preclinical studies suggested a role of the seeded MDSCs in the maintenance of patency and in the remodeling of the TEVG toward a native-like structure. Pig studies were inconclusive due to a poor pre-implantation cell density. Future work should address this and other issues encountered during the large animal study, and should test longer time points in both models. Finally, this approach might benefit from a more readily available cell source such as the bone marrow