p27-p16 Fusion Gene Inhibits Angioplasty-Induced Neointimal Hyperplasia and Coronary Artery Occlusion
Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury–induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 1012 viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus–treated vessels. The average intimal area of the AV-W9–treated group 10 days after balloon injury and treatment was 0.42±0.36 mm2, whereas the AV-null group demonstrated an intimal area of 0.70±0.52 mm2. At day 10 the average intimal thickness of the AV-W9–treated vessels was 9.1 μm (n=5, ×20 magnification) compared with 21.2 μm (n=5, ×20 magnification) in control virus–treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9–treated vessels demonstrated an average intimal thickness of 4.7 μm (n=8, ×20 magnification) compared with 13.3 μm (n=3, ×20 magnification) in control virus–treated vessels and 7.3 μm (n=5, ×20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty–induced intimal hyperplasia in the coronary artery.
Vascular smooth muscle cell (SMC) proliferation is induced in response to the overextension of coronary and peripheral arteries occurring during balloon angioplasty. This hyperproliferative response is believed to be a critical event in the therapy-associated complication of vessel reocclusion resulting from restenosis. Manipulation of the normal cell cycle control mechanisms in vascular SMCs at the angioplasty site has been suggested as a means of preventing the restenosis.1–12 The negative regulators of cell cycle progression, the cyclin-dependent kinase (CDK) inhibitors (CDKis), fall into the following two structurally distinct families: the INK4 family comprising p15, p16, p18, and p19, and the CIP/KIP family comprising p21, p27, and p57.13–17 Members of the INK4 and CIP/KIP families normally act in concert to regulate cell proliferation. We combined representative members of each family to create more potent cytostatic agents.
A series of p27KIP1 and p16INK4b fusion genes were created and introduced into the E1 region of an E1-deleted replication-deficient adenoviral vector (ΔE1-AV) under the transcriptional control of the cytomegalovirus (CMV) promoter enhancer. As expected, the chimeric p27-p16 molecules inhibited the kinase activity of the CDK4/cyclin D1, CDK2/cyclin E, and CDK2/cyclin B complexes in vitro. In addition, transduction of cells with the AV encoding the chimeric p27-p16 molecules inhibited cell cycle progression at multiple points in the cell cycle.18 One chimeric CDKi, termed W9, was selected from a series of p27-p16 fusion molecules for further development because of its superior antiproliferative activity in vascular SMCs18 and a rabbit carotid artery vascular injury model.19 W9 comprises a severely truncated derivative of p27 (amino acids 25 to 93) fused to p16. To assess its potential utility in preventing coronary restenosis, 1×1012 viral particles (vp) of AV-W9 were delivered using the Cordis Crescendo catheter to porcine coronary arteries immediately after balloon injury. Although we observed that only 5% of the vessel sections on average were transduced, AV-W9 had a pronounced impact on coronary intimal hyperplasia. Fifty to sixty-two percent of the injury-induced intimal thickening was inhibited by AV-W9 with no apparent toxicity.
Materials and Methods
Primary human SMCs were obtained from Clonetics. Primary pig coronary artery SMCs were isolated from pig and tested in transduction efficiency of adenovirus. SMCs were plated and infected with multiplicity of infection ranging from 1 to 100. Two days later, cells were harvested and loaded with fluorescein di-β-d-galactopyranoside substrate (Molecular Probes) by hypotonic shock for 1 minute. The reaction was stopped with 300 μmol/L chloroquine and cells were analyzed by flow cytometry. The functional titer was calculated on the basis of the following equation: [(% infected cells−% negative)×total cell count×dilution factor]/inoculum volume×100.
In Vivo Gene Transfer to Injured Artery
The study was approved by the Mayo Clinic Animals and Use Committee. The generation of the β-galactosidase (β-gal) and W9 AVs encoding the various transgenes has been described previously.18,20 In brief, W9 was created when a truncated p27 gene encoding amino acids 25 to 93 was generated by PCR and fused to the full-length p16, lacking the initiating ATG. The CDKis were expressed under the control of the CMV promoter/enhancer and the SV40 poly(A) signal. The AV-W9 and AV-null vectors used first-generation E1-deleted vector backbone (ΔE1-AV), whereas the AV–β-gal vector used both ΔE1-AV and second-generation E1- and E4-deleted vector backbone (ΔE1/ΔE4-AV). The AV-W9, AV-null, and AV–β-gal virus stocks were produced and purified by double-cesium chloride gradient centrifugation and then dialyzed against and stored in 5 mmol/L Tris and 3% sucrose at pH 7.4. Virus particle titers were determined by optical density reading at 260 nm on a Beckman DU 450 spectrophotometer. Infectious virus titers were determined by titration on 293 cells and measurement of hexon expression after infection. The final concentrations for the ΔE1/ΔE4 CMV LacZ was 2.5×1012 vp/mL and for ΔE1 AV-CMV-W9 and ΔE1 AV-CMV-W9 were 1.5×1012 vp/mL. The total viral particle–to–infectious particle ratios for the viruses was very similar and ranged from 100 to 250.
Male New Zealand White rabbits (2 to 2.5 kg) were maintained on a normal diet. Before balloon injury, rabbits were fed a 1% cholesterol diet for 6 weeks. Rabbits were placed under appropriate anesthesia. A midline incision was made and the external carotid artery was exposed. An arteriotomy was performed, and a 2F Fogarty balloon catheter was inserted and inflated (50 to 60 μL saline to fill the balloon) and passed five times. The catheter was then removed and the animals were allowed to recover. Three days later, rabbits underwent gene transfer to both carotid arteries via arteriotomy. The vessel was clamped and an arteriotomy performed. Virus solution (100 μL) was infused per vessel, which is sufficient to expand the vessel to physiological dimensions. The virus was allowed to dwell for 15 minutes and the arteriotomy closed. The clamps were removed and circulation restored. High-titer viral stock was diluted with DMEM/virus storage medium at a 1:1 ratio (final viral particle titer 2.5×1012 vp/mL) to ensure equal composition of virus solutions at different viral titers. Sham infections were carried out using DMEM/virus storage medium alone.
The Crescendo and E-Med Microfuse catheters are intramural pressure-driven catheters. The Crescendo balloon size was 3.25 mm and the balloon length was 18 mm. This catheter is a dual-balloon catheter made of Duralyn, a nylon-based material. The catheter structure consists of an internal shaft containing two 60-μm pores and an outer balloon containing >1500 8-μm pores over the 18-mm balloon length. The Microfuse balloon size was 3.00 mm, and the balloon length was 20 mm. The catheter structure consists of a polyethylene balloon containing >10 000 0.6-μm pores distributed randomly over the length of the balloon.
The porcine coronary artery studies used 25- to 30-kg juvenile crossbred domestic female pigs. After anesthesia with intramuscular injection of ketamine (3 mg/kg) and xylazine (30 mg/kg), the carotid artery was exposed and 8F sheath advanced over a guidewire, ensuring proper hemostasis. A custom-curved 8F sheath was advanced to the aortic root and the left main coronary artery was engaged under fluoroscopic guidance. Of three coronary arteries (left anterior descending artery, right coronary artery, and left circumflex artery), two were used per animal. A 15-mm-length angioplasty balloon with a balloon injury:artery ratio of 1.2:1 expanded in the artery accomplished the injury. The balloon was inflated for 30 seconds at 8 atm and then removed after deflation. The infusion catheter was then passed into the same segment of coronary as the balloon injury under fluoroscopic guidance and delivering the recombinant AV according to the specifications of the catheter device. One milliliter of virus solution at 1 to 2×1012 vp/mL was delivered to the vessel in 30 seconds. The two infusion catheters used were the Crescendo and the Microfuse catheters. One infusion catheter was used per injured vessel to deliver the virus. After virus infusion, the arteriotomy was repaired and the animal allowed to recover.
Vessel Harvesting and Analysis
Rabbit vessels were harvested at 14 days after gene transfer. The animals were anesthetized and heparinized (700 IU IV), and the carotid arteries were dissected free. The animals were then given 100 mg/kg pentobarbital IV. A 2- to 2.5-cm section of the carotid artery was immediately excised and washed briefly in PBS. The vessel was cut into six smaller segments and subsequently either fresh-frozen or formalin-fixed for further analyses.
Pig vessels were harvested at 3, 10, or 28 days after gene transfer. Briefly, animals were euthanized on the harvest day, using standard commercial “Sleepaway,” and the heart was removed immediately and washed in PBS. Each of the coronary vessels was carefully dissected out. The excised segments were ≈2.0 to 2.5 cm total and were cut into six to eight segments. The tissues were fresh-frozen and further processed for LacZ expression and hematoxylin and eosin staining. For the efficacy study, the segments were formalin-fixed and paraffin-embedded. They were further processed and stained with Masson’s trichrome stain to assess neointimal formation. Vessels harvested on 3, 10, and 25 days after treatment were also evaluated for transgene expression by p16 immunohistochemistry.
The vessels were excised, cut into six sections, and either frozen in OCT compound or formalin-fixed and paraffin-embedded. For the delivery studies, the vessels were first cut lengthwise to confirm site of injury. Frozen tissues were used with β-gal stain for the delivery studies, or for immunohistochemistry to detect transgene expression. To evaluate neointimal formation, formalin-fixed and paraffin-embedded tissue was cut into 5-μm sections and stained with Masson’s trichrome (Sigma).
Frozen sections were fixed in 0.5% glutaraldehyde for 5 minutes. Sections were incubated in 5-bromo 4-chloro 3-indolyl β-d-galactopyranoside for 12 to 15 hours. Sections were then washed and lightly counterstained with hematoxylin.
Transgene expression was assessed by immunostaining on paraffin-embedded sections using the rabbit polyclonal p16 antibody (Santa Cruz Biotechnologies). The secondary antibody used was a biotinylated rabbit IgG-B followed by incubation in horseradish peroxidase–streptavidin complex. Positive staining was visualized using 3′,3′-diaminobenzidine chromogen. Sections were counterstained lightly with hematoxylin.
Sections were analyzed under ×100 magnification. At least three sections per group and three to four fields per section were analyzed. Neointimal thicknesses were measured using a micrometer. Intima areas were determined by digital planimetry using Spot RT digital camera software version 3.0 (Diagnostic Instruments). LacZ or W9 transgene expression was determined using a semiquantitative immunohistochemical assay. The intensity and quantity of positive staining pattern was scored on a percentage scale for LacZ expression. p16 immunohistochemistry was scored on a 4+ scale.
Sera were collected from animals before or at various time points after gene delivery. Serum glutamine pyruvate transaminase (sGPT), alkaline phosphatase (AP), glutamic oxaloacetate transferase (sGOT), creatine phosphokinase, and creatinine assays were performed using kits obtained from Sigma.
Inhibition of Neointimal Hyperplasia in a Rabbit Balloon Injury Model
AV-W9 has been shown to be a more potent antiproliferative than the parental p16 and p27 molecules.18,19,21 Before testing the virus in pigs, we compared the impact of the AV-W9 virus to a control AV vector that encoded no transgene (AV-null). New Zealand White rabbit carotid arteries were balloon-injured and 3 days later exposed to 1011 vp of AV-W9 or AV-null (Figure 1). The intimal thickness of the AV-W9–treated vessels (6.0 μm, n=4) was ≈5-fold lower than untreated balloon-injured vessels (25.2 μm, n=4) and 3-fold lower than AV-null–treated vessels (16.8 μm, n=4). AV-W9 clearly inhibited intimal hyperplasia in this vascular balloon injury model.
Transduction of Human and Pig Arterial SMCs
We next tested the relative ease of transducing human and porcine arterial SMCs with AVs. This was done by culturing primary human and porcine SMC cultures with increasing doses of AV-β-gal and then assessing the relative transduction efficiencies 2 days later. Five- to 10-fold higher doses of both first-generation ΔE1-AV and second-generation ΔE1/ΔE4-AV vectors were necessary to transduce the primary porcine arterial SMCs compared with primary human arterial SMCs (Figure 2). This finding suggests that similar or even lower doses of vector will be required to transduce human SMCs to achieve levels of gene transfer similar to those in the porcine vascular models.
Catheter-Mediated Delivery of AV to Balloon-Injured Coronary Arteries
The efficiency of virus delivery to the vessel injury site is a significant challenge for cytostatic gene therapy. AVs are compatible with many types of infusion catheters. We have shown that there is no apparent loss in AV particles or infectious titers after prolonged incubation in several standard infusion catheters.22
We selected two infusion catheters, the Crescendo and the Microfuse, to determine the optimal conditions for AV delivery to porcine coronary arteries. With both catheters significant gene transfer was achieved in balloon-injured vessels by delivering 1012 vp of the second-generation ΔE1/ΔE4–AV-β-gal vector and using a 30- to 35-second infusion time. Staining of the vessels 3 days after gene therapy indicated punctated β-gal expression in the neointimal and adjacent medial regions of the treated vessels using either the Crescendo catheter (Figure 3, CC) or the Microfuse catheter (Figure 3, EM). No staining was observed in control injured vessels (Figure 3, Neg). The average transduction efficiency determined by microscopic quantification of β-gal staining in three sections from each of three ΔE1/ΔE4–AV-β-gal–transduced vessels was essentially equivalent using either the Crescendo or the Microfuse (Figure 3). The average transduction efficiency of the ΔE1/ΔE4–AV-β-gal–transduced vessels was 4.4±4.5% of the Crescendo catheter vessel sections and 4.5±4.5% of the Microfuse catheter vessel sections. The maximal transduction efficiency was also similar for each of these catheters, as follows: 16% of the vessel section with the Crescendo catheter and 15% of the vessel section with the Microfuse catheter. Minimal transduction of uninjured coronary arteries was achieved using either of the catheters (data not shown).
AV-W9 Inhibits Intimal Thickening in the Coronary Artery
To assess the inhibition of intimal hyperplasia by AV-W9 in the porcine coronary artery, two of the three coronary arteries were injured with a 15-mm balloon catheter and either were left untreated or were treated with 1012 vp of either AV-W9 or a control null virus. AV-W9 treatment significantly prevented intimal thickening compared with the control balloon-injured vessels that received either the AV-null virus or no virus treatment. Typical sections from three different AV-W9, AV-null, and control injury vessels at 10 days stained with Masson’s trichrome stain are shown at ×5 magnification (Figure 4A). Neointima can be observed in both AV-W9– and AV-null–treated vessels (Figure 4A, middle and bottom panels); however, the severity of intimal thickening is greatly reduced in the AV-W9–treated vessels. In addition to demonstrating a thicker neointima, the AV-null–treated vessels demonstrated an appreciable atrophy in the medial layer. Quantification of intimal and medial thickness indicated that intima/media (I/M) ratios in AV-W9–treated vessels were on average 62% lower than in AV-null–treated vessels (P=0.001), in vessels harvested 10 days after gene transfer (Figure 4A). The AV-W9–treated group demonstrated an I/M ratio of 16 (n=5), whereas the AV-null group demonstrated an I/M ratio of 7 (n=5). AV-W9 treatment resulted in 40% lower intimal areas for the AV-W9 treatment group compared with the AV-null group. The intimal area of the AV-W9–treated group was 0.42±0.36 mm2, whereas the AV-null group demonstrated an intimal area of 0.70±0.52 mm2. At day 10 the average intimal thickness of the AV-W9–treated vessels was 9.1 μm (n=5, ×20 magnification) compared with 21.2 μm (n=5, ×20 magnification) in control virus–treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer in which AV-W9–treated vessels demonstrated I/M ratios that were 54% lower than those of sham-treated vessels (P=0.001) and 48% lower than AV-null treated vessels (P=0.02). The average intimal thickness of the AV-W9–treated vessels was 4.7 μm (n=8, ×20 magnification) compared with 13.3 μm (n=3, ×20 magnification) in control virus–treated vessels and 7.3 μm (n=5, ×20 magnification) in the sham-treated vessels. These results clearly demonstrate that AV-W9 treatment can significantly impact neointimal hyperproliferation and vascular occlusion in this coronary artery balloon injury model.
The degree of W9 gene transfer was measured in vessels 3, 10, and 28 days after gene transfer. W9 expression was determined by immunohistochemistry using an anti-p16 antibody with a scale of “−” to “+4,” where “+4” represents the maximal p16 staining (Figure 5). Peak W9 expression was seen at day 3 followed by a rapid dropoff in expression to undetectable levels by day 28. No W9 expression was detected in vessels that received the null vector. This kinetics of W9 expression is consistent with the biology of first-generation viruses in the vasculature.23,24,24a This also suggests that the antiproliferative effects of AV-W9 occur within 10 days of gene transfer and balloon injury.
Safety of AV-W9 Vascular Gene Therapy
To assess the systemic implications of coronary delivery of AVs, serum transaminase, AP, and creatinine levels were measured in serum samples obtained from pigs 3 days after treatment. AVs at high doses have been shown to mediate hepatocellular toxicity after vascular administration. We have observed in mice and rabbits19 and Hackett et al25 have observed in pigs that the majority of virus that is delivered to the vasculature ends up in secondary organs. In rodents the primary target is the liver followed by the lung,19 and in pigs the reverse is the case. Hackett et al25 have reported that administration of 1012 vp of a first-generation AV to the coronary artery of 28- to 30-kg swine resulted in 74% of the vector going to the lung, 16% of the vector going to the liver, and ≈8% of the vector remaining in the heart. Many groups have reported that virus-mediated hepatitis is typically observed 3 to 10 days after vector administration. To assess this in the gene-modified pigs, serum samples were collected 3 days after balloon injury and virus delivery (Figures 6A through 6C). No indication of systemic toxicity was detected, and serum enzyme levels for both vector-treated and untreated animals were within the normal range and were indistinguishable from the pretreatment values.
This analysis was extended to AV-W9–treated pigs with serum samples obtained 10 days after balloon angioplasty and administration of AV-W9 or AV-null (Figures 6D through 6G). The sGPT, sGOT, AP, creatine kinase, and creatinine levels in the AV-W9–treated animals were within the normal range and were indistinguishable from the pretreatment values. There was a slight elevation in creatinine levels in the AV-null–treated animal, although outwardly the animals appeared to be healthy. It is not clear why the delivery of 1012 vp of AV–β-gal and AV-W9 had no impact on serum enzyme levels and the same dose of AV-null did. All values were back within the normal range by day 21. Hematoxylin and eosin–stained sections from AV-W9–treated vessels revealed no increase in inflammatory cell infiltrate compared with sections from sham-treated vessels (data not shown). Overall these findings demonstrate that catheter delivery of AV-W9 to angioplasty-treated porcine coronary arteries was effective in inhibiting neointimal hyperplasia and was well tolerated.
Recognizing that the INK and CIP/KIP families of CDKi normally work together in controlling cell proliferation and predicting that together these molecules would demonstrate increased potency, we combined the activities of these molecules in a chimeric p27-p16 fusion molecule named W9. AVs encoding W9 were more effective than the parental molecules at inhibiting the proliferation of human SMCs in vitro.18 In a rabbit model of intimal hyperplasia, neointimal growth was 3- to 5-fold greater in untreated or control virus–treated vessels compared with the AV-W9–treated vessels. AV-W9 also suppressed 50% to 60% of the neointimal hyperproliferation in a coronary balloon injury model in pigs. This was achieved with a single administration of AV-W9 using an infusion catheter with infusion times of <35 seconds in duration.
Although we achieve a relatively low efficiency of gene transfer (maximally 16% of the vessel area with an average over the treatment area of 4.4±4.5% of the vessel area), we observe a significant impact on intimal thickening. One mechanism that might explain this result is that those SMCs that contribute to the intimal hyperplasia may be “exposed” to the virus after the injury and therefore may be more readily infected. The punctated pattern of transgene expression observed with the β-gal viruses does indeed suggest that specific sections of the vessels are transduced. These vessel sections that are more prone to adenovirus transduction may also be a significant source of the SMCs that participate in vessel occlusion. Alternatively, the proliferative SMCs may be preferentially infected by the virus, perhaps as a result of the upregulation of the receptor required for virus binding, coxsackie adenovirus receptor (CAR),26 or the receptor required for virus internalization, αvβ3 integrin.27
The potent antiproliferative activity of W9 appears to be a relatively unique property of this particular p27-p16 fusion molecule. Alternative p27-p16 fusion molecules with less extensive deletions in the p27 gene have been shown to be less active than W9. This hierarchy of activities was observed not only in primary human SMCs and endothelial cells18 but also in a series of human tumor-derived cell lines.21 The reason for the increased activity of W9 is unclear. In a series of in vitro kinase inhibition assays, W9 could not be distinguished biochemically from p27. From the standpoint of protein stability, W9 did demonstrate an increased protein half-life in a subset of tumor-derived cell lines; however, this was not the case in either proliferating or quiescent SMCs. The subcellular localization of W9 does appear to be distinct from that of p27. W9 protein localizes to both the nucleus and perinucleus, whereas p27 localizes predominantly in the nucleus.18 This may allow W9 to redirect the localization of its targets, although this has not been formally shown.
One of the attractive features of AV-W9 therapy as a means of controlling intimal hyperplasia is that this approach essentially mimics the natural process of cell cycle control. Quiescent cells that take up AV-W9 appear to be unaffected in vitro, and AV-W9 therapy does not appear to be associated with any systemic toxicity in the two animal models we have used to date. This is an important consideration, as most of the virus will collect in the lung and liver tissues. Despite the relatively low efficiency of gene transfer to the target vessel, balloon injury–induced intimal hyperplasia was significantly inhibited with AV-W9 therapy using infusion catheters and delivery conditions compatible for coronary artery gene therapy. The ability of a single administration of AV-W9 to suppress intimal thickening despite low levels of gene transfer may reflect the enhanced adenovirus transduction of activated SMCs over quiescent SMCs. This may be mediated by an upregulation of the receptors for adenovirus. These activated SMCs are likely the cells that contribute to vessel reocclusion after angioplasty and therefore regulating their proliferation may be sufficient to impact vascular occlusion. We conclude that AV-W9 therapy at the time of angioplasty may increase the efficacy of this commonly used therapy for coronary vascular occlusive disease.
This research was supported by Cell Genesys Inc (Foster City, Calif).
Original received January 19, 2001; revision received June 14, 2001; accepted June 14, 2001.
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