Preparation of VEGF/lentivirus
VEGF has been documented to inhibit neointimal hyperplasia in vivo (6
). Therefore, we chose to use this gene to demonstrate that MR-mediated RF heating would enhance expression of this gene in target vascular cells and, in doing so, effectively decrease the amount of neointimal hyperplasia associated with stent placement in a cholesterolemic animal. Lentivirus-based gene therapy has been evaluated in Phase I Clinical Trials (VIRxSYS-Corporation. Phase I clinical trial using VRX496 lentiviral vector. November 2004).
Thus, we used a lentivirus vector to carry a therapeutic gene, vascular endothelial growth factor (VEGF165
) gene (GeneCopoeia, Frederick, MD), and a marker gene, red fluorescent protein (RFP) gene. In the present study, the titers of viral supernatants were in the range of 1 X 106
to 6 X 106
transducing units (TUs) per milliliter of supernatants.
The primary portion of the MR/RF-heating system was an 0.014-inch (0.35-mm), copper-based intravascular MR imaging-heating-guidewire (MRIHG), which consisted of a coaxial cable, 5 feet (152.4-cm) in length with a 4.5-cm extension of the inner conductor (Microstock, Inc., West Point, PA). The MRIHG was connected to a custom external 180-MHz RF generator to deliver RF thermal energy to the target vessels.
Prior to applying it in the present study, the functionality of the MR-imaging/RF-heating system had been validated in vivo. By comparing different input powers, we established an optimized MR/RF heating protocol, which enabled us to achieve a temperature increase up to approximately 41°C from 37°C in the target femoral arteries of pigs by operating the 180-MHz RF generator at 4 watts through the MRIHG (Qiu B, et al. unpublished data).
This in vivo study was performed in 10 (five-paired) bilateral femoral-iliac arteries of five cholesterolemic pigs, approximately 20 kg in weight (Archar, Baltimore, MD). All animals were treated according to the “Principles of Laboratory Animal Care” of the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 80-23, revised 1985). The Animal Care and Use Committee at our institution approved the experimental protocol.
Gene delivery and stent placement
We used a gene delivery balloon catheter (Remedy; Scimed/Boston Scientific, Maple Grove, Minn) to locally deliver the VEGF/lentivirus into the target vessel wall using a previously-established gene delivery protocol (12
). Through a surgical cutdown in the carotid artery, we placed a 7F introducer into the upper abdominal aorta. Heparin (Elkins-Sinn, Inc., Cherry Hill, NJ,) was intravenously administered (100IU/kg) to initiate anticoagulation. We then obtained a digital subtraction angiography (DSA) of the pelvic and bilateral femoral-iliac arteries in two perpendicular planes. Based on the results of the DSA, we selected the gene-targeted femoral-iliac artery segments that did not include branches. Then, via the gene delivery balloon, we delivered 1.0–2.0-mL VEGF/lentivirus into each of the bilateral arteries at a flow of 10mL/hour. The gene infusion was maintained using a digital syringe pump (Harvard, Holliston, MA). The amounts of transferred gene/vectors were exactly the same in both sides of each animal, and was precisely controlled by the same infusion time using the same infusion parameters set with the digital pump. The duration of the balloon inflation with gene infusion was 2 minutes for four times with a 2-minute interval to restore blood flow.
Before gene delivery, the 0.014-inch MRIHG was placed into the guidewire channel of the catheter and was connected to the external RF generator. From the beginning of gene delivery, we used the previously established MR/RF heating protocol to locally heat the unilateral gene-targeted artery segments up to approximately 41°C for 20 minutes, while the contralateral gene-targeted arteries were not heated to serve as controls.
Subsequently, we placed 10 identical, stainless steel, balloon-expandable stents (BeStentTM, Minneapolis, Minn) into the 10 VEGF-targeted bilateral femoral-iliac arteries. presents the experimental design and steps in the in vivo validation studies. All stents were primarily used as inductive devices to facilitate the formation of neointimal hyperplasia, i.e., in-stent stenosis. For each of the artery segments, the diameter ratios between the targeted artery and the stent were approximately 2.5mm/3.0mm, or 3.0mm/3.5mm. Immediately after the stent placement, we obtained DSA to confirm the success of the procedure.
Figure 1 Experimental design for MR/RF-enhanced VEGF gene therapy to prevent in-stent neointimal hyperplasia, including a) initiation of cholesterol diet; b) delivery of VEGF genes to bilateral femoral-iliac arteries with local MR/RF heat on the left side; c) (more ...)
After gene/stent interventions, we kept the pig alive, with a continuation of the high cholesterol diet (Modified Laboratory Mini-Pig Diet Grower 5081, TestDiet, Richmond, IN) for an additional two months. At day 60 after the gene/stent interventions, we obtained DSA again to examine the formation of in-stent stenosis in bilateral targeted arteries. Then, we euthanized the animals and harvested the bilateral, gene-targeted and/or stented artery segments for pathology correlation and confirmation. Since this study focused on a technical development, we evaluated only the short-term therapeutic effect of VEGF (two-months after gene/stent interventions) with no attempt to evaluate the long-term functional period of VEGF gene expression in the arterial tissues.
All of the stent-containing artery specimens were fixed with 10% buffered formalin, embedded in methylmethacrylate, and sectioned at 5-μm-thick slides at three levels of the proximal and distal ends, as well as the middle portions of the stented artery segment using a laser microtome (Division of Charles River Laboratories, Inc., Frederick, MD). The histological slides were then stained with hematoxylin and eosin (HE).
With microscopy, we photographed 30 histological slides (10 artery segments x 3 slides per segment = 30 slides), cross-sectionally viewed at 13.6-times magnification. On each of the 30 photographs, we recorded the average thickness of the in-stent neointimal hyperplasia by measuring the shortest distance from the inner margin of the stent to the endothelial layer of neointimal hyperplasia. Thus, a total of 249 measurements were recorded, including 123 measurements for VEGF+RF-arteries and 126 measurements for VEGF-only-arteries. Unequal numbers of two measurements were due to the variable number of stent structures that appeared in different histology slides/photographs. An unpaired Student’s t test was used to compare the differences in the average thickness of in-stent neointimal hyperplasia in the arteries between the VEGF-RF and the VEGF-only treatments. The data were given as mean ± standard error and considered significantly different at the level of p < 0.05.