Adenovirus-Mediated Gene Transfer to Normal and Atherosclerotic Arteries
A Novel Approach
Abstract Previous studies of gene transfer to blood vessels in vivo have relied on intraluminal, catheter-based methods for delivery of adenoviral and other vectors. In this study, topical application of a replication-deficient adenoviral vector was used as an alternative method of gene transfer to the vessel wall. We administered recombinant adenovirus (1.0 to 1.5×1010 pfu/mL) containing the nuclear targeted bacterial β-galactosidase gene topically to arteries in normal and atherosclerotic cynomolgus monkeys. Topical administration was achieved by injection of adenoviral suspension within the periarterial sheath. Segments of femoral and carotid arteries were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside 1 day after treatment with the adenovirus. After topical administration of virus, β-galactosidase activity was observed in ≈20% of cells in the adventitia in both normal and atherosclerotic arteries. There was no detectable β-galactosidase activity in cells of the intima or media. Thus, topical application provides an alternative method for gene transfer to blood vessels in vivo. This approach, which does not require interruption of blood flow and does not disrupt the endothelium, may be useful for studies of vascular biology and perhaps gene therapy in both normal and atherosclerotic vessels.
- Received January 18, 1995.
- Accepted October 4, 1995.
Gene transfer to blood vessels in vivo is currently accomplished by intraluminal administration of adenoviral or other vectors.1 2 3 4 5 6 7 8 9 There are important limitations to this approach. First, to achieve significant transduction of cells in the vessel wall, blood flow is usually either stopped briefly or for several minutes.2 3 4 5 6 7 8 9 Second, intraluminal administration results in low efficiency of gene transfer beyond the endothelium.2 Significant transfection of cells in the media or adventitia generally is achieved only when the endothelium is denuded or damaged by balloon injury.4 9 This limitation is particularly relevant for studies of vascular reactivity where an intact endothelium is important for normal vascular function.
Recently, several investigators have demonstrated that delivery of adenovirus vector by a double balloon catheter, sometimes with increased intraluminal pressure, enhances gene transfer to the vascular smooth muscle layer in short segments of vessel.8 10 It is not clear whether normal endothelial function is preserved in the face of elevated intraluminal pressures. In atherosclerotic arteries, the thickened neointima may be an additional barrier to effective gene transfer to the vessel wall after intraluminal delivery.
These limitations led us to seek an alternative method for delivery of the vector. It has been demonstrated that topical application of drugs or growth factors to the adventitial surface of arterial segments in vivo can alter vascular function.11 12 13 14 15 Furthermore, periadventitial application of antisense oligonucleotides can suppress intimal proliferation after endothelial injury.16 Topical delivery of vectors eliminates the need to interrupt blood flow or disrupt the endothelium and may prove useful in studies in whichgene transfer is used to examine vascular function. Therefore, in this study, we injected recombinant replication-deficient adenovirus within the carotid and femoral sheaths to accomplish gene transfer by topical application to the vessel wall.
We tested the hypothesis that application of adenoviral vector to the adventitial surface would result in gene transfer to the arterial wall. Our first goal was to characterize in arteries of normal monkeys the location and extent of transgene expression after in vivo topical delivery of the adenoviral vector. Second, we determined whether this approach would accomplish significant gene transfer to arteries of atherosclerotic monkeys.
We used two replication-deficient recombinant adenoviruses that were based on adenovirus serotype 2: Ad2/CMV-βgal as a reporter virus and Ad2/CFTR-6 as a negative control. Construction of these viruses has been described previously.17 18 The DNA constructs comprise a full-length copy of the adenovirus genome of ≈37.5 kb from which the early region 1 genes (E1) have been replaced by either cDNA for bacterial β-galactosidase gene preceded by an SV40 nuclear localization signal in Ad2/CMV-βgal or by the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) gene in Ad2/CFTR-6. Recombinant viruses were grown in human embryonic kidney (293) cells that complement the E1 early viral promoter.17 The virus was suspended in phosphate-buffered saline (PBS) with 3% sucrose and stored at −70°C until used.
Animals and Surgical Procedures
All animal procedures were approved by the Animal Care and Use Review Committee at the University of Iowa. Five normal and seven atherosclerotic cynomolgus monkeys were used for this study. Normal monkeys (mean±SEM weight, 7.2±2.0 kg) were fed commercial laboratory chow (Purina monkey chow, Ralston Purina Co). Atherosclerotic monkeys (weight, 6.3±1.0 kg) were fed an atherogenic diet for 18 to 22 months. The atherogenic diet contained 41% of total calories as fat and 0.8% cholesterol. Total plasma cholesterol was 98±19 mg/dL in normal monkeys and 541±164 mg/dL in atherosclerotic monkeys.
Animals were sedated with ketamine HCl (10 mg/kg IM) and anesthetized with pentobarbital (25 to 30 mg/kg IV). A 1- to 2-cm segment of the carotid or femoral artery was surgically exposed. Topical application was achieved by injection of 0.3 to 1.0 mL of the adenovirus suspension (1.0 to 1.5×1010 pfu/mL) within the femoral or carotid sheath using a 26-gauge needle. Five arteries from four normal monkeys and eight arteries from seven atherosclerotic monkeys were treated by topical application of Ad2/CMV-βgal. As a control, we used an adenovirus suspension containing Ad2/CFTR-6 (1.6×1010 pfu/mL). We performed intraluminal delivery of Ad2/CMV-βgal (0.3 to 0.5 mL) to the carotid and femoral arteries in two monkeys (one normal and one atherosclerotic). For intraluminal administration of virus, a 22-gauge catheter was introduced into the vessel lumen, and blood flow within the vessel segment was stopped by ligatures placed around the catheter and 1- to 1.5-cm distal to the catheter tip. After aspiration of the blood into the lumen, 0.5 to 1 mL of adenovirus suspension containing Ad2/CMV-βgal (1.0 to 1.5×1010 pfu/mL) in 3% sucrose was introduced into the vessel lumen and allowed to dwell for 15 minutes. The ligatures were then released and blood flow was reestablished. One day after administration of virus the animals were euthanatized, and the vessel segments were removed and analyzed for expression of β-galactosidase.
Histochemical and Morphometric Analysis of Gene Expression
Transgene expression in blood vessels was assessed in monkeys that were euthanatized 1 day after topical or intraluminal administration of Ad2/CMV-βgal. Excised segments of vessel were washed thoroughly with PBS, then incubated in X-Gal (Sigma Chemical Co) staining solution for 1 hour at room temperature. Incubation with X-Gal was limited to 1 hour to prevent staining endogenous β-galactosidase, which may be seen in cytosol after longer periods (>4 hours) of incubation.19 The segments were then rinsed in PBS and fixed in 2% paraformaldehyde and 0.2% glutataldehyde in PBS. The tissues were embedded in paraffin, and microtome sections (5 μm thick) were counterstained with hemotoxylin and eosin.
We performed histological examination on a segment of artery approximately 1 cm long. Two or three sections from each vessel segment were examined for positive staining of β-galactosidase (blue nuclei) by light microscopy. In each section, transgene expression to the vessel was estimated by counting stained and unstained nuclei of the same cell layer (intima, media, and adventitia). More than 100 nuclei were counted in each region of interest in each vessel segment.
Data are presented as mean±SEM. Differences in transgene expression between normal and atherosclerotic arteries after topical application of the adenovirus suspension were analyzed by a nonparametric Kruskal-Wallis test followed by Bonferroni’s post hoc t test.
Topical Application in Normal Vessels
In five vessel segments (three carotid and two femoral arteries) from four normal monkeys treated with topical application of Ad2/CMV-βgal, gene transfer to the vessel wall was observed in all five arterial segments. Expression of bacterial β-galactosidase was observed in cells of the adventitia but not the intima or media (Fig 1a⇓ and 1b⇓). Morphometric analysis indicated that 24±3% of nuclei in the adventitia were transduced (Fig 1c⇓). There was no detectable evidence of an inflammatory cell infiltrate in the vessel wall. In arteries of normal monkeys treated topically with Ad2/CFTR-6 there was no expression of β-galactosidase in the vessel wall.
Topical Application in Atherosclerotic Vessels
In eight atherosclerotic vessels from seven monkeys, extensive transduction of cells in the adventitial layer was observed after topical application of Ad2/CMV-βgal (Fig 2a⇓ and 2b⇓). Seventeen percent (±6%) of the nuclei in the adventitia stained positive for expression of β-galactosidase (Fig 2c⇓). There was no significant difference between normal and atherosclerotic vessels in the percentage of adventitial cells expressing β-galactosidase after topical application of Ad2/CMV-βgal. As in normal vessels cells in the intima and adventitia were not transduced, and no inflammatory response was observed. Atherosclerotic arteries treated topically with Ad2/CFTR-6 showed no expression of β-galactosidase.
In two vessels from one normal and one atherosclerotic monkey, expression of β-galactosidase 1 day after intraluminal administration of Ad2/CMV-βgal was minimal and limited to <1% of the endothelial cells (Fig 3⇓). There was no expression of β-galactosidase in the media or adventitia. We did not detect β-galactosidase activity in any layer of the vessel wall in arteries treated intraluminally with Ad2/CFTR-6 (n=2).
There are two major findings in this study. First, topical delivery of replication-deficient adenovirus by injection within the periarterial sheath results in expression of the transgene encoding bacterial β-galactosidase in the arterial wall. Second, atherosclerotic and normal arteries are both effectively transduced after topical delivery of replication-deficient adenovirus. Expression is substantial but limited to cells in the mid-portion of the adventitia, with approximately 20% of the nuclei staining positive. Cells of the intima and media are not transduced after topical application of the vector.
Intraluminal Approach for Gene Transfer
Previous studies of gene transfer to blood vessels in vivo have relied on intraluminal, catheter-based methods for delivery of lipid and retroviral or adenoviral vectors.2 3 4 5 6 7 8 9 20 These techniques generally require vessel occlusion to achieve significant gene transfer to the vessel wall. Despite arterial occlusion, however, efficiency of gene transfer to endothelium and media is variable. Efficient gene transfer generally is observed only after disruptive techniques. For instance, in vessels with an intact intima, transfection efficiency to the endothelium after intraluminal delivery of adenoviral vector has ranged from 2% to 30%, with minimal (<1%) transfection of the media.21 22 In contrast, in vessels where the endothelium has been denuded or damaged by balloon injury, transfection efficiency to the media ranges from 0.18% to 22%.21 23 Furthermore, although transient vascular occlusion poses little risk for gene transfer to peripheral vessels, interruption of blood flow in the cerebral or coronary circulation for even short periods of time results in ischemia with potentially serious consequences.
We performed studies of intraluminal administration via an indwelling catheter (in two monkeys) and confirmed that in uninjured normal and atherosclerotic arteries, transgene expression was limited to a few cells in the endothelium. However, because the number of animals studied was small, we cannot exclude the possibility that greater expression might sometimes occur after intraluminal administration.
Recently, several investigators have demonstrated that delivery of adenovirus at higher pressure enhances the efficiency of gene transfer to vascular smooth muscle.8 9 It is likely, however, that elevation of intravascular pressure to 0.5 to 1.0 atmosphere will impair normal endothelial function. With the use of topical delivery of adenovirus, we were able to achieve gene transfer to the adventitia that is comparable to the 2% to 30% transfection efficiency to the endothelium/media reported by other investigators after intraluminal delivery.24 Furthermore, topical delivery does not require stopping blood flow or disrupting the endothelium.
Gene Transfer to Atherosclerotic Blood Vessels
Several investigators have examined gene transfer to atherosclerotic arteries in vivo.7 21 25 In those studies atherosclerotic lesions were induced by endothelial damage followed by a high cholesterol diet for up to 8 weeks, and gene transfer was accomplished in the setting of balloon injury. Gene transfer to atherosclerotic vessels in these studies7 25 was limited to the neointima. In a recent study, intraluminal delivery of adenoviral vector to the iliac artery of rabbits fed a high cholesterol diet for 7 weeks resulted in transduction of 0.2% of medial and neointimal cells, compared with 2% of medial cells in arteries of normal rabbits.21 A high cholesterol diet fed for less than 8 weeks in rabbits generally induces only early atherosclerotic lesions. Nevertheless, the results of this study21 suggest that atherosclerotic vessels may be resistant to gene transfer by intraluminal delivery of vector.
The thickened neointima of atherosclerotic vessels may be a barrier limiting effective gene transfer to the vessel wall, and thus diffusion of gene products, after intraluminal delivery. We used monkeys that were fed an atherogenic diet for 1.5 to 2 years. The monkeys developed characteristic arterial atherosclerotic lesions consisting of dense fibrofatty intimal thickening with focal intimal necrosis in large arteries.26 We found no difference between normal and atherosclerotic vessels in percentage of adventitial cells that stained positive after topical delivery of Ad/CMV-βgal. Thus, in atherosclerotic vessels when the goal is to deliver a gene product to vascular smooth muscle to affect vasomotor function, adventitia may be an appropriate target for gene transfer.
Limitations of Topical Delivery
Topical delivery of the adenoviral vector resulted in gene transfer to cells in the mid-portion of the adventitia but not to cells in the media or intima. A possible explanation for this pattern of transgene expression in adventitia is that because the outer portion of the adventitia is relatively acellular there are few targets for the adenoviral vector. The external elastic laminae, matrix, and adventitial cells in the inner portion of the adventitia may form a physical barrier that prevents the adenovirus from penetrating into the medial layer.10
There are several potential limitations to the use of this approach to affect vascular function. One concern is that the number of cells infected, and consequently, gene expression, may not be sufficient to alter vascular function. Previous work has demonstrated, however, that histological identification of successful gene transfer may underestimate the functional effect of bioactive products expressed by the transgene.27 This is particularly relevant if the transgene encodes for a secretable protein product because high expression by a few cells may be sufficient to affect biological function. Furthermore, X-Gal staining used in our study to identify successfully transduced cells is not maximally sensitive. Up to 10% of cells expressing β-galactosidase may not stain positively with X-Gal.18
Another concern is whether gene transfer limited to cells in the adventitia will be able to affect vascular function. This will most likely depend on the ability of the transgene product expressed in the adventitia to reach the smooth muscle cells of the media. Transport of labeled albumin has been demonstrated from the adventitia into the arterial media,28 and delivery of antiproliferative agents, such as heparin and antisense oligonucleotides from the adventitia to the media, has also been shown to be effective.16 Therefore, it is likely that overexpression in the adventitia of genes that encode enzymes that produce highly diffusible substances will affect the underlying vascular smooth muscle.
We did not perform studies to determine peak β-galactosidase expression after topical delivery of the adenoviral vector. However, previous work in our laboratory has shown that in rat brain29 and dog pericardium (unpublished data, 1995) the number of cells expressing β-galactosidase is maximal 1 day after administration of virus and markedly decreased by day 7. Loss of expression may be due in part to host immune responses to recombinant proteins expressed by transduced cells, loss of viral genome, or transcriptional downregulation.6 7 30 Brief expression is not a major limitation to the use of gene transfer for studying vascular biology but is a significant obstacle to potential therapeutic applications of gene transfer.
In this study, histological examination of vessels 1 day after treatment revealed expression of the transgene with no detectable inflammatory cell infiltrate. Absence of inflammatory cells is surprising. In previous studies in our laboratory, tissues examined 3 and 7 days after administration of adenovirus in brain29 and pericardium (authors’ unpublished data, 1995) showed a significant mononuclear cell infiltrate. It is possible that the site of application of adenovirus (the adventitia) results in an attenuated inflammatory response.
Topical delivery of adenoviral vector by injection into the periarterial sheath results in gene transfer to the adventitial cells of both normal and atherosclerotic vessels and provides an alternative method for gene transfer to blood vessels in vivo. This approach, which does not require interruption of blood flow and does not disrupt the endothelium, may be useful for studies of vascular biology and perhaps for gene therapy in both normal and atherosclerotic vessels.
This work was supported by NIH grants HL 14388, NS 24621, HL 16066, AG 10269, and NS 34568; by research funds from the Veterans Administration; and by funds from the Carver Trust of the University of Iowa. Dr Davidson is a fellow of the Roy J. Carver charitable trust. We thank Richard D. Anderson (University of Iowa Vector Core), Pamela Tompkins, and Lisa DeBerg for their expert technical assistance and Drs Frank M. Faraci, Michael J. Welsh, and Ronald E. Haskell for critical review of this manuscript. We also thank Alan E. Smith (Genzyme, Cambridge, Mass) for the gift of Ad2/CMV-βgal and Ad2/CFTR-2.
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