1.4.3.1 Stent Models
With the expanding and widespread clinical use of stents the development of true preclinical ISR, rather than vessel injury, models has become of increasing relevance. The use of a coated stent to deliver gene therapy is attractive as it is site-specific, potentially helping to avoid the distal spread of therapeutic agents and viral vectors, thus minimising systemic toxic effects. The first progress in the field of gene therapy to prevent ISR was made by Dichek et al, who successfully seeded stainless steel stents with sheep endothelial cells that had been modified to express markers using retroviral gene transfer (Dichek et al., 1989). Much later the efficiency of this process was improved using endothelial cells expressing VEGF (Koren et al., 2006). The first successful use of a “gene-eluting stent” in- vivo was performed in a pig coronary angioplasty model. The stent polymer eluted plasmid DNA for green fluorescent protein (GFP) allowing detection of arterial transfection (Klugherz et al., 2000). The same group also reported successful transfection of porcine coronary arteries using a collagen coated stent with covalently-bonded monoclonal antibodies to adenovirus. This allowed binding of adenoviral particles to the stent allowing a degree of controlled site-specific local release (Klugherz et al., 2002). Another group used a different approach, seeding SMC harvested from porcine jugular veins onto fibronectin-coated stents, transducing them to express GFP and inserting them into porcine coronary arteries (Panetta et al., 2002).
Adding to the controversy surrounding the role of VEGF, Walter et al inserted human VEGF-2 plasmid coated polymer stents into the iliac arteries of normocholesterolemic and hypercholesterolemic rabbits. In their study VEGF accelerated endothelialisation compared to control stents (98% vs. 79% endothelial cover at 10 days) and also inhibited NIH as assessed by intravascular ultrasound with an 87% increase in lumenal cross-sectional area and a 54% reduction in cross-sectional narrowing (Walter et al., 2004). In a similar study however, it had been reported that VEGF-eluting stents tested in a rabbit model significantly reduced thrombosis but did not promote endothelialization or reduce NIH (Swanson et al., 2003). It is possible that differences in stent types used (polymer coated with plasmid DNA vs. radiolabelled absorption of VEGF) could explain the differences seen in these rabbit iliac studies.
Unsurprisingly, with such noted similarities in their pathologies, many targets thought to have potential in vein graft failure have also been tested in stent models. Johnson et al coated stents with an adenovirus expressing TIMP-3 and applied them to porcine coronary arteries (Johnson et al., 2005). A 40% reduction in neointimal area compared to bare metal stents at 28 days was conferred. Stents coated with a biocompatible polymer expressing 7ND-MCP-1 reduced neointimal area in rabbits (~60% reduction at 28 days post-stenting) and monkeys (25-30% reduction out to 6 months) (Egashira et al., 2007). This provides evidence that long-term clinical benefit may be achievable with the use of a gene-eluting stent.
The above studies have all used polymer coated stents to elute the genes of therapeutic interest, much in the same way that DES deliver their active compound. This polymer component however has been shown to cause a local inflammatory response which contributes to delayed healing and late complications such as thrombosis (van der Giessen et al., 1996, Garg and Serruys, 2010a). Levy’s group in Philadelphia has therefore investigated alternatives to polymer coatings. The first method tested was gene delivery via bisphosphonate binding to the metallic stent surface. Following in vitro optimization they delivered Ad expressing iNOS to a rat carotid stenting model achieving a significant reduction in NIH (Fishbein et al., 2006). Furthermore, to enable better control of vector stability and delivery kinetics they developed a synthetic complex allowing adenoviral particles to reversibly bind to the stent through a hydrolysable ester bond. Again this method achieved a reduction in NIH using AdiNOS in the rat (Fishbein et al., 2008).
Not all attempts to manipulate growth factors have been successful. Adenoviral delivery of an antagonist to TGF-β, shown to be effective in reducing ECM formation in the VGD models, did not reduce stent-induced NIH in porcine coronaries despite a small decrease in ECM deposition. It did appear to increase vascular inflammation leading the authors to conclude that it may have a detrimental effect on lesion progression (Chung et al., 2010).
These encouraging results in some pre-clinical models of restenosis have been demonstrated despite overall low percentages (typically <10%) of transduced VSMC at the site of treatment (Sharif et al., 2006).
1.4.3.2 Thrombosis
Although rare, stent thrombosis remains an important complication of PCI. Best assessments of risk with DES suggest an ongoing incidence of 0.3-0.6% per annum with a mortality rate of 10-30% (Garg and Serruys, 2010b). A significant reduction in risk has been obtained by improvement in stent technology and methods of assessing adequate deployment as well as the use of prolonged dual anti-platelet therapy. Incomplete stent strut endothelialisation and polymer-induced inflammation have been heavily implicated in the aetiology of stent thrombosis. The potential of a gene-eluting stent to accelerate endothelialisation, thereby reducing the potential for thrombosis as well as inhibiting NIH makes it an intuitive competitor for the DES. To maximise effect however, delivery would have to be as biocompatible as possible, both from the perspective of any polymer used to bind the vector to the stent, as well as the delivery vector/transgene of choice. As previously discussed in the vectors section, local inflammation is a recognised issue. Although new-generation vectors may markedly reduce this in terms of systemic effects once delivered to the patient, even low-grade local inflammation may be enough to contribute to stent thrombosis. This approach requires testing in suitable preclinical models to identify safety and efficacy before moving on to randomized, controlled clinical trials with long term follow-up.