Alternative approaches to the prevention of coronary In-stent restenosis

Robertson, Keith Euan (2014) Alternative approaches to the prevention of coronary In-stent restenosis. PhD thesis, University of Glasgow.

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Abstract

Cardiovascular disease (CVD) has become the most common cause of mortality worldwide, accounting for approximately 30% of all deaths. The primary pathological process that underlies it is atherosclerosis. Atherosclerosis leads to the development of flow-limiting lesions that accumulate over years to decades under the stimulus of genetic and environmental factors. These contribute to both impaired tissue oxygen delivery with resultant clinical symptoms and acute coronary syndromes carrying a significant burden of morbidity and mortality. Within the UK population, 28% of premature deaths in men and 20% in women are attributable to CVD carrying an estimated £30bn annual economic cost. Myocardial revascularisation in the form of coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) are established therapies for both acute coronary syndromes and symptomatic chronic disease refractory to pharmacological therapy. CABG remains the standard for patients with complex multi-vessel disease however in patients with less complex disease PCI is a reasonable alternative, and remains the gold-standard for patients with simple, focal CAD. The long-term results of CABG are limited by the failure of conduit grafts leading to recurrent symptoms, MI and/or repeat revascularization that carries with it a significant excess risk. Despite advancements in surgical technique and adjunctive medical therapy, the commonly used saphenous vein grafts are constrained by a cumulative graft failure rate of 50% at 10 years. Intracoronary stents, used for PCI are limited by the restenosis or thrombosis of stented vessel segments, phenomena that also confer significant morbidity and mortality. Vein graft disease (VGD) and in-stent restenosis (ISR)/thrombosis share similar pathological features related to the response to vessel injury, principally thrombosis, the development of neointimal hyperplasia, impaired endothelialisation and accelerated atherosclerosis. Gene-therapy remains a potential alternative to the use of pharmacotherapy for the prevention and treatment of these important clinical entities. Preclinical studies have provided a greater understanding of the underlying mechanistic pathways in both VGD and ISR and have provided insight into potential pathophysiological targets for manipulation with gene therapy. However, as yet, translation to the clinical setting has been less successful. Although stent technology and associated clinical outcomes continue to improve, preclinical studies suggest that gene therapy with a safe and stable vector expressing endogenous proteins that restore normal vessel physiology can be an intuitive alternative to cytotoxic drugs for the prevention of stent complications. The principal aim of this PhD was to investigate alternative approaches to the prevention of coronary in-stent restenosis. It was initially planned to do this using a gene therapy approach with development and delivery of therapeutic viral vectors to a large animal model of ISR. Subsequently an interest was taken in the role of microRNAs in the development of neointimal hyperplasia and the potential for their therapeutic modulation in the prevention of ISR and use as cardiovascular biomarkers. Initial experiments focused on the assessment of adenoviral and lentiviral vectors for the transduction of human coronary artery smooth muscle cells (HCASMC). The dedifferentiation of vascular smooth muscle cells (VSMC) to a proliferative and migratory phenotype from their differentiated contractile state is a key process in the development of neointimal hyperplasia. Unmodified adenoviral (Ad5) and second-generation lentiviral vectors were used to transduce HCASMC in vitro. Successful gene expression was achieved with both vectors, however, in keeping with previous studies significant transduction with Ad5 could only be obtained at higher viral concentrations. Lentivirus was much more efficient, showing significant cell transduction at low viral doses. It was initially planned to take this vector forward to an in vivo model but it unfortunately proved time consuming and prohibitively expensive to produce in large enough titres. Adenoviral vectors were therefore used for in vivo delivery. A local delivery catheter approach, for the delivery of an adenoviral vector was tested ex vivo and successful viral transduction of explanted porcine coronary arteries was obtained. The Clearway RXTM (Atrium Medical) perfusion balloon catheter was designed to deliver a therapeutic agent directly to intra-coronary lesions. It was therefore hypothesized that it may be able to effectively deliver a gene-delivery vector. A porcine model of coronary stent delivery and overexpansion has been shown to develop consistent vessel injury and the development of neointima. This was therefore used to assess the ability of the Clearway RX to deliver an adenovirus expressing β-galactosidase. Measurable viral transduction of the arterial wall was not obtained most likely due to the Clearway being unable to maintain viral contact with the vessel for long enough to achieve significant transduction. Similar issues affected the only other commercially available local delivery catheter, the GENIETM which forms a therapeutic drug reservoir between two occlusive balloons. Again, long enough incubation times to mediate unmodified Ad5 arterial transduction could not be obtained and the pigs tolerated the procedures poorly. To overcome these issues a virus-coated stent was developed using the spray-coatable YukonTM stent system and a Poloxamer 407 gel with thermoreversible properties. Poloxamer 407 has previously been used to successfully augment adenoviral transduction in small animal models. This system was therefore tested in the porcine model. No viral transduction of the vessel wall was obtained and analysis of distal organ sites confirmed off-target viral sequestration. Interest turned to the investigation of the role of microRNAs (miRs) in the development of in-stent restenosis. miRs are short, non-coding ribonucleic acids that act to control gene expression at a posttranscriptional level. Several miRs have been shown to play key roles in establishing smooth muscle and endothelial cell fate, tissue homeostasis and are now implicated in the complex regulation of the phenotype of cell types involved in vascular remodelling and the development of ISR. Using in vitro models of VSMC proliferation and migration, the dynamic expression of regulatory miRs implicated in the control of VSMC phenotype was assessed. These experiments suggested that miR-21, miR-146a, miR-221 and miR-365 overexpression may contribute to the promotion of a dedifferentiated VSMC phenotype in the development of human neointimal lesions. The results for miR-143 and miR-145 were less clear however. It was hypothesised that expression patterns of these regulatory miRs in the vasculature would alter in response to stent-induced injury. All previously reported studies in this area have used in-vitro or in-vivo rodent models of vascular injury. A more clinically relevant large animal (porcine) model of in-stent restenosis was therefore utilised to assess the expression levels of miRs previously reported to play a role in VSMC regulation as well as novel targets. RNA was extracted from vessels snap frozen at the time of sacrifice and changes in miR expression within the vessel wall were determined using quantitative real time PCR (qRT-PCR). Using an electrolysis method to achieve stent dissolution it was possible to use in-situ hybridization to co-localise dysregulated miRs within neointimal lesions. Both miR-21 and the miR-143/miR-145 cluster appear to play a key role in the regulation of VSMC phenotypic switch and are shown to be both dynamic and significantly upregulated in developing neointima following stent implantation. This localised upregulation in the miR-143/miR-145 cluster, despite a consistent overall “whole-vessel” downregulation, is particularly novel and differs from previously published findings. This has important implications towards future therapeutic manipulation. Additionally, and consistent with suggestions that microRNAs operate in regulatory networks, a panel of novel miRs, including miR-142, have been identified that may play an important role in the development of ISR and merit future investigation. Finally, experiments were performed to investigate the potential role of circulating miRs as biomarkers in patients undergoing CABG surgery. RNA was extracted from serum pre- and post-surgery and the dynamic and temporal expression of a panel of miRs associated with both acute and stable chronic coronary artery disease determined using qRT-PCR. Correlation analysis was performed with classical biomarkers and peri-operative factors. Dynamic changes were seen in circulating levels of miR-133a (which was seen to correlate with the biomarker troponin T), miR-122, miR-92a and miR-126. Although patient numbers were small, and this data is therefore observational, it is the first to investigate a temporal serum profile of dynamic miRs in the setting of uncomplicated CABG and show early potential for their use as biomarkers. It also adds to the growing evidence for the presence of a circulating microRNA network for cardiovascular disease that may hold significant future potential for therapeutic manipulation in specific pathological settings. Although the initial aims of developing a therapeutic gene therapy vector for the prevention of in-stent restenosis and its delivery to and testing in a large animal in vivo model were not achieved, this thesis adds to the understanding of the developing and increasingly complex role played by microRNAs in the setting of vascular remodelling following coronary stent implantation. It is hoped that the therapeutic modulation of miR levels could become an effective approach for the prevention of ISR and optimisation of patient outcomes in the clinical setting.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: coronary artery disease, in-stent restenosis, gene therapy, adenovirus, microRNA, porcine
Subjects: R Medicine > RZ Other systems of medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Funder's Name: UNSPECIFIED
Supervisor's Name: Baker , Professor Andrew H and Oldroyd, Professor Keith G and Kennedy , Dr Simon
Date of Award: 2014
Depositing User: Dr Keith E Robertson
Unique ID: glathesis:2014-5193
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 20 Jun 2014 12:38
Last Modified: 20 Jun 2014 12:40
URI: http://theses.gla.ac.uk/id/eprint/5193

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