Preclinical Evaluation of Coronary Artery Stents.

Coronary artery disease is a major contributor to morbidity and mortality worldwide. While lifestyle changes and medication are the cornerstones of treatment, coronary artery balloon angioplasty and stenting are routinely performed on patients with acute coronary syndromes and chronic coronary artery disease who remain symptomatic with optical medical treatment. Several generations of coronary stents have been developed over recent decades. Balloon angioplasty and stenting are supported by the use of pharmaceutical agents applied onto balloons and the stent surface, either to advance the healing properties of the artery post-intervention or to prevent the formation of restenosis. New devices need to be rigorously tested for safety and efficacy before acceptance into clinical practice; thus, there is a continuing need for reliable and reproducible preclinical methods of stent evaluation. We describe here a pig coronary artery model as well as an atherosclerotic rabbit model for coronary artery stent research and describe basic steps in intravascular imaging and stent histology.

Evaluation of Biodegradable Stent Graft Coatings in Pig and Rabbit Models.

AIMS: Percutaneous coronary intervention is routinely performed to treat occlusive coronary artery disease. Coronary perforation is a potential complication and can be treated with a stent graft. Current stent grafts are associated with high restenosis rates. We tested the safety and feasibility of biodegradable stent grafts in pig and rabbit models. METHODS AND RESULTS: Stent grafts were examined in pig coronaries with repeated OCT imaging for 42 days. Novel biodegradable coatings were applied on a bare metal stent by either an electrospinning (ES) or dip coating (DC) method. A completely biodegradable system was made by ES coating a magnesium-based stent. A commercially available stent graft served as a control. ES devices showed less restenosis (44.3 ± 8.8 vs. 59.1 ± 11.1% in controls, p < 0.05) and smaller reduction in minimum lumen area (44.3 ± 13.4 vs. 64.4 ± 13.6% in controls, p < 0.05) at day 42. DC devices occluded during follow-up. ES devices showed recanalization through the graft wall at day 42. Feasibility of the ES and DC devices was evaluated in pig coronary aneurysms and rabbit aortic perforation models and sealed aneurysms and perforations without complications. CONCLUSIONS: Recanalization of the graft wall improves biocompatibility. Biodegradable stent grafts may present an alternative to permanent implants by showing reduced restenosis at day 42.

Improved endothelialization of small-diameter ePTFE vascular grafts through growth factor therapy.

BACKGROUND: Prosthetic vascular grafts in humans characteristically lack confluent endothelialization regardless of the duration of implantation. Use of high-porosity grafts has been proposed as a way to induce endothelialization through transgraft capillarization, although early experiments failed to show increased healing in man. OBJECTIVES: We hypothesized that transduction of tissues around the prosthetic conduit with vectors encoding VEGF receptor-2 (VEGFR2) ligands would augment transinterstitial capillarization and induce luminal endothelialization of high-porosity ePTFE grafts. METHODS: Fifty-two NZW rabbits received 87 ePTFE uni- or bilateral end-to-end interposition grafts in carotid arteries. Rabbits were randomized to local therapy with adenoviruses encoding AdVEGF-A165, AdVEGF-A109 or control AdLacZ and analyzed at 6 and 28 days after surgery by contrast-enhanced ultrasound and histology. RESULTS: AdVEGF-A165 and AdVEGF-A109 dramatically increased perfusion in perigraft tissues at 6 days (14.2 ± 3.6 or 16.7 ± 2.6-fold increases, P < 0.05 and P < 0.01). At 28 days, the effect was no longer significantly higher than baseline. At 6 days, no luminal endothelialization was observed in any of the groups. At 28 days, AdVEGF-A109- and AdVEGF-A165-treated animals showed enhanced ingrowth of transinterstitial capillaries (66.0 ± 13.7% and 77.4 ± 15.7% of graft thickness vs 44.7 ± 24.4% in controls, P < 0.05) and improved luminal endothelialization (11.2 ± 26.3% and 11.4 ± 22.2%, AdVEGF-A109 and AdVEGF-A165 vs 0% in controls, P < 0.05). No increased stenosis was observed in the treatment groups as compared to LacZ controls. CONCLUSIONS: This study suggests that transient local overexpression of VEGFR2 ligands in the peri-implant tissues at the time of graft implantation is a novel strategy to increase endothelialization of high-porosity ePTFE vascular grafts and improve the patency of small-diameter vascular prostheses.

Local adventitial anti-angiogenic gene therapy reduces growth of vasa-vasorum and in-stent restenosis in WHHL rabbits.

BACKGROUND: Antiproliferative drugs in drug eluting stents (DES) are associated with complications due to impaired re-endothelialization. Additionally, adventitial neovascularization has been suggested to contribute to in-stent restenosis (ISR). Since Vascular Endothelial Growth Factors (VEGFs) are the key mediators of angiogenesis, we investigated feasibility and efficacy of local gene therapy for ISR utilizing soluble decoy VEGF receptors to reduce biological activity of adventitial VEGFs. METHOD: Sixty-nine adult WHHL rabbit aortas were subjected to endothelial denudation. Six weeks later catheter-mediated local intramural infusion of 1.5x10e10 pfu adenoviruses encoding soluble VEGF Receptor-1 (sVEGFR1), sVEGFR2, sVEGFR3 or control LacZ and bare metal stent implantation were performed in the same aortic segment. Marker protein expression was assessed at 6d in LacZ cohort. Immunohistochemistry, morphometrical analyses and angiography were performed at d14, d42 and d90. RESULTS: Transgene expression was localized to adventitia. All decoy receptors reduced the size of vasa-vasorum at 14d, AdsVEGFR2 animals also had reduced density of adventitial vasa-vasorum, whereas AdsVEGFR3 increased the density of vasa-vasorum. At d42, AdsVEGFR1 and AdsVEGFR2 reduced ISR (15.7 ±â€¯6.9% stenosis, P < 0.01 and 16.5 ±â€¯2.7%, P < 0.05, respectively) vs. controls (28.3 ±â€¯7.6%). Moreover, AdsVEGFR-3 treatment led to a non-significant trend in the reduction of adventitial lymphatics at all time points and these animals had significantly more advanced neointimal atherosclerosis at 14d and 42d vs. control animals. CONCLUSIONS: Targeting adventitial neovascularization using sVEGFR1 and sVEGFR2 is a novel strategy to reduce ISR. The therapeutic effects dissipate at late follow up following short expression profile of adenoviral vectors. However, inhibition of VEGFR3 signaling accelerates neoatherosclerosis.

Aluminum fluoride-18 labeled folate enables in vivo detection of atherosclerotic plaque inflammation by positron emission tomography.

Inflammation plays an important role in the development of atherosclerosis and its complications. Because the folate receptor ß (FR-ß) is selectively expressed on macrophages, an FR targeted imaging agent could be useful for assessment of atherosclerotic inflammation. We investigated aluminum fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate (18F-FOL) for the detection of atherosclerotic plaque inflammation. We studied atherosclerotic plaques in mice, rabbits, and human tissue samples using 18F-FOL positron emission tomography/computed tomography (PET/CT). Compound 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) was used as a comparison. Firstly, we found that the in vitro binding of 18F-FOL co-localized with FR-ß-positive macrophages in carotid endarterectomy samples from patients with recent ischemic symptoms. We then demonstrated specific accumulation of intravenously administered 18F-FOL in atherosclerotic plaques in mice and rabbits using PET/CT. We noticed that the 18F-FOL uptake correlated with the density of macrophages in plaques and provided a target-to-background ratio as high as 18F-FDG, but with considerably lower myocardial uptake. Thus, 18F-FOL PET/CT targeting of FR-ß-positive macrophages presents a promising new tool for the in vivo imaging of atherosclerotic inflammation.

Biodegradable coronary scaffolds: their future and clinical and technological challenges.

Angioplasty and stenting are standard treatment options for both stabile occlusive coronary artery disease and acute myocardial infarctions. Over the last years, several biodegradable stent systems have entered pre-clinical and clinical evaluation and into clinical practice. A strong supporting scaffold is necessary after angioplasty to prevent elastic recoil of the vessel but in the long term a permanent metallic stent will only impair normal physiology of the artery wall. Thus, the main advantage of a resorbable system is the potential for better vessel recovery and function in the long term. The new stent systems differ from traditional stents in size and biological responses and questions have risen regarding their mechanical strength and increased risk of stent thrombosis. Here, we present current treatment options with biodegradable scaffolds, discuss further key areas for improvements and review novel technological advances in the context of all up-to-date clinical trial information. New material choices are also covered as well as special considerations for pre-clinical testing.

Ultrasound imaging with bolus delivered contrast agent for the detection of angiogenesis and blood flow irregularities.

Highly increased blood flow and vascularity after angiogenic gene therapy have raised concerns of shunting and hemangioma-like blood pool formation that might decrease effective perfusion and ruin the beneficial effects of the therapy. Contrast enhanced ultrasound is a promising noninvasive tool for studying skeletal muscle perfusion. The objectives of the present study were to test bolus and infusion administrations of ultrasound microbubble contrast media in imaging vascular growth in skeletal muscle and assess the functionality of vessels grown with angiogenic gene therapy. Contrast enhanced ultrasound was used to study changes in skeletal muscle perfusion in normal and gene-transduced rabbit hindlimbs 6 days after gene transfer. Adenoviral gene transfer of VEGF (10e(9)-10e(11) viral particles) or ß-galactosidase control gene (10e(11) viral particles) was done under anesthesia and induced up to 16-fold increases in relative tissue perfusion. Contrast intensity versus time curves were plotted and analyzed for contrast kinetics. Bolus administration of the contrast media was highly feasible in analyzing skeletal muscle blood flow and its kinetics. Maximal signal intensity of the bolus signal reflected relative changes in both blood flow and volume equally to the infusion method. Flow irregularities were detected after angiogenic gene therapy. In conclusion, bolus delivery of ultrasound contrast agent is highly feasible for the relative analysis of both quantity and quality of blood flow after angiogenic gene therapy. The kinetics of blood flow can and should be studied more extensively in both preclinical and clinical trials of angiogenic gene therapy since there is increasing evidence of flow irregularities in angiogenic vessels.

Capillary enlargement, not sprouting angiogenesis, determines beneficial therapeutic effects and side effects of angiogenic gene therapy.

AIMS: Currently, it is still unclear which mechanisms drive metabolic benefits after angiogenic gene therapy. The side-effect profile of efficient angiogenic gene therapy is also currently incompletely understood. In this study, the effects of increasing doses of adenoviral (Ad) vascular endothelial growth factor-A (VEGF-A) were evaluated on vascular growth, metabolic benefits, and systemic side effects. METHODS AND RESULTS: Adenoviral vascular endothelial growth factor-A or AdLacZ control was injected intramuscularly (10(9)-10(11) vp/mL) or intra-arterially (5 × 10(11) vp/mL) into rabbit (n = 102) hindlimb muscles and examined 6 or 14 days later. Blood flow, tissue oedema, metabolic benefits, and the structure of angiogenic vessels were assessed using ultrasound imaging, modified Miles assay, arterial blood gas and metabolite analyses, and light and confocal microscopy, respectively. Safety analyses included cardiac ultrasound, electrocardiograms, and blood and tissue samples. Sprouting angiogenesis was already induced with low AdVEGF-A concentrations, whereas higher concentrations were needed to reach efficient capillary enlargement and increases in target muscle perfusion. Interestingly, metabolic benefits, such as improved aerobic energy metabolism and decreased metabolic acidosis during exercise, after AdVEGF-A administration were highly correlated to the level of capillary enlargement but not to sprouting angiogenesis. Several systemic dose-dependent side effects, including transient increases in liver, kidney, and pancreatic enzymes, and signs of cardiac effects were observed. CONCLUSION: Efficient capillary enlargement leading to significant increases in tissue perfusion is needed to gain metabolic benefits after angiogenic gene therapy. However, the risk of systemic side effects can increase as the efficiency of angiogenic gene therapy is improved. Importantly, the unstable wall structure of the newly formed vessels seems not to compromise the metabolic benefits.