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(Stroke. 1998;29:2181-2188.)
© 1998 American Heart Association, Inc.

Original Contributions

Cationic Polymer and Lipids Enhance Adenovirus-Mediated Gene Transfer to Rabbit Carotid Artery

Kazunori Toyoda, MD; Hiroaki Ooboshi, MD; Yi Chu, PhD; Al Fasbender, PhD¹; Beverly L. Davidson, PhD; Michael J. Welsh, MD; Donald D. Heistad, MD

From the Departments of Internal Medicine, Physiology and Biophysics, and Pharmacology, Cardiovascular Center and Center on Aging (K.T., H.O., Y.C., B.L.D., D.D.H.), and the Howard Hughes Medical Institute (A.F., M.J.W), University of Iowa College of Medicine, and Veterans Administration Medical Center, Iowa City, Iowa.

Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail donald-heistad{at}uiowa.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Background and Purpose—Improvement of efficiency of gene transfer to endothelium could be useful for several applications. We tested the hypothesis that cationic nonviral molecules augment adenovirus-mediated gene transfer to blood vessels, perhaps by alteration of the surface charge of adenovirus and facilitation of binding to endothelium.

Methods—Carotid arteries from rabbits were incubated in vitro for 0.5 to 2 hours with an adenoviral vector alone or noncovalent complexes of adenovirus with poly-L-lysine (a cationic polymer) or lipofectin (a cationic lipid). Binding of adenovirus to the vessels was evaluated immediately after incubation with virus, and assay of transgene (ß-galactosidase) activity and histochemistry were performed 24 hours after gene transfer. To determine whether cationic molecules can be used to augment alteration of vascular function by adenovirus-mediated gene transfer, we also examined effects on gene transfer of endothelial nitric oxide synthase.

Results—Assay of ß-galactosidase activity indicated that both cationic molecules increased transgene expression in vessels by 5- to 6-fold. In contrast, when endothelium was removed from the vessels after gene transfer, poly-L-lysine and lipofectin did not significantly increase transgene activity. Histochemistry for ß-galactosidase also suggested that the adenovirus-cationic molecule complexes augmented transgene expression mainly in the endothelium. In addition, we found that complexing adenovirus with cationic molecules increased binding of adenovirus to the vessels. After gene transfer with recombinant adenovirus containing endothelial nitric oxide synthase, calcium ionophore (A23187) produced greater relaxation of vessels treated with adenovirus complexed with poly-L-lysine or lipofectin than those treated with adenovirus alone.

Conclusions—Cationic molecules improve the efficiency of adenovirus-mediated gene transfer to blood vessels.

Key Words: endothelium • gene expression • nitric oxide synthase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Replication-deficient recombinant adenovirus is a useful vector for gene transfer to blood vessels and potentially for gene therapy of vascular disease.^{1 2 3} The vascular targets of gene transfer depend in part on the method of administration. Endothelial and smooth muscle cells can be transduced by intraluminal injection into intact or balloon-injured vessels, respectively,^{4 5 6 7} and adventitial cells can be targeted by perivascular administration.^{8 9} Adenovirus, however, binds to and transduces endothelial and smooth muscle cells poorly compared with several cell lines. The poor binding of adenovirus may be due to low levels of expression of the fiber receptor for Ad5 and Ad2, serotypes that are used in most studies.¹⁰ It would be useful to overcome this limitation and improve the efficiency of adenovirus-mediated gene transfer to allow a reduction of viral titer or exposure time to the vessels.

Positively-charged nonviral vectors appear to interact with the negatively charged cell surface and enter into target cells through endocytosis pathways or fusion with cell membranes.^{11 12 13 14 15} One might expect, therefore, that association of cationic gene transfer agents with adenovirus could increase the efficiency of adenovirus-mediated gene transfer into target cells and vessels. Recently, we reported that noncovalent complexing of adenovirus-encoding transgene with poly-L-lysine, a cationic polymer, or several cationic lipids increased adenoviral uptake and transgene expression in several cell lines and airway epithelium.¹⁶ The rationale was that the cationic molecules would charge-associate with negatively charged adenovirus particles and facilitate attachment to the negatively charged cell membrane. The first goal of this study was to examine the hypothesis that cationic molecules will increase viral uptake and transgene expression by adenovirus-mediated gene transfer to blood vessels. We complexed adenovirus that expresses ß-galactosidase reporter gene with poly-L-lysine or the cationic lipid lipofectin and examined the efficiency of gene transfer by these complexes.

Previous studies have demonstrated alteration of vasomotor function by overexpression of type III nitric oxide synthase (NOS) (endothelial nitric oxide synthase) (eNOS) using gene transfer approaches.^{17 18 19} The second goal of this study was to determine whether cationic molecules enhance alteration of vascular function by adenovirus-mediated expression of eNOS.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Adenoviral Vectors
We used 2 replication-deficient recombinant adenoviruses serotype 5–encoding nuclear-targeted ß-galactosidase (Adßgal)^{20 21} and eNOS (AdeNOS), both driven by a cytomegalovirus (CMV) promotor. AdeNOS (AdCMVeNOS) was prepared according to our method for construction of AdRSVeNOS.¹⁷ Recombinant adenoviruses were plaque purified and virus titer determined by plaque assay on 293 cells. After purification, the virus was suspended in PBS with 3% sucrose and kept at -80°C until use. All amplifications and purifications were done by the University of Iowa Gene Transfer Vector Core.

Activity of NOS expressed after infection with AdeNOS was determined by measuring the conversion of L-[³H]arginine to L-[³H]citrulline 2 days after transfection of COS 1 cells with the virus. The measurement showed that 10 and 100 plaque-forming units (pfu)/cell of AdCMVeNOS increased formation of L-[³H]citrulline by 26- and 186-fold, respectively, and the increase was completely inhibited in the absence of calcium or in the presence of a NOS inhibitor.^21A

Complexes of Adenovirus-Cationic Molecules
Polymers of poly-L-lysine hydrochloride with an average molecular mass of 37.0 kDa (corresponding to 177 lysine residues, Sigma), and lipofectin (Gibco), which was formulated in a 1:1 weight ratio of cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) to dioleoyl phosphatidylethanolamine¹³ were used as cationic molecules. Complexes of adenovirus and cationic molecules were made by adding viral vectors and poly-L-lysine or lipofectin to PBS with 3% sucrose and mixing by gentle pipette tip aspiration. Final titer of Adßgal was 3x10⁹ pfu/mL (1.0x10¹¹ particles/mL), and that of AdeNOS was 2x10¹⁰ pfu/mL (1.0x10¹² particles/mL). The ratio of poly-L-lysine was expressed as a calculated average number of poly-L-lysine molecules per adenovirus particle, which varied from 30 to 3000 molecules per particle. The ratio of lipofectin, expressed as a calculated average number of DOTMA molecules per adenovirus particle, varied from 3x10⁴ to 1x10⁷ molecules per particle. The complexes were incubated for 15 minutes at room temperature, unless otherwise noted, before application to vessel rings.

Gene Transfer to Vessels
Adult male New Zealand White rabbits (weight, 2.4 to 3.0 kg) were studied. Rabbits were euthanized by an overdose of pentobarbital (100 mg/kg), and the common carotid arteries were removed. Vessels were placed in Krebs' bicarbonate solution (mmol/L): NaCl 118, KCl 4.7, KH₂PO₄ 1.2, MgSO₄·7H₂O 1.2, D-glucose 11.1, NaHCO₃ 25.0, CaCl₂·H₂O 2.54. Loose connective tissue was gently removed, without disruption of adherent adventitia, and vessels were cut into rings 2.5 mm in length. Each vessel ring was incubated with 100 µL of viral suspension, which contained adenovirus, complexes of adenovirus-cationic molecules, or vehicle (3% sucrose in PBS) for 30 minutes or 2 hours at 37°C, unless otherwise noted. Vessels were rinsed with PBS and incubated with Eagle's modified essential medium with 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 5% fetal bovine serum for 24 hours for the following analyses, except for Southern blotting, which was evaluated immediately after incubation of vessels with viral suspension.

Southern Blot Analysis for Adenoviral DNA
We evaluated the relative quantity of adenovirus that bound or entered vessels after 30 minutes or 2 hours of incubation with virus using Southern blot analysis for adenoviral DNA. Vessels were incubated with viral suspension of Adßgal alone, Adßgal plus 300 molecules per particle of poly-L-lysine, Adßgal plus 3x10⁵ molecules per particle of lipofectin, or vehicle. Vessels then were rinsed with PBS, and adenoviral DNA was isolated from vessels following the method described previously.²² These ratios of poly-L-lysine and lipofectin were selected on the basis of our findings that these ratios of cationic molecules produced maximal augmentation of transgene expression with the use of the chemiluminescent assay described below.

Adenoviral DNA was blotted onto nitrocellulose membrane (Life Technologies) and identified by hybridization of the immobilized DNA with a ß-galactosidase–specific probe labeled with [³²P]dCTP (Amersham). Nitrocellulose membranes were exposed to Kodak Biomax film at -80°C. Autoradiographs were scanned by QCS³²⁰⁰ flatbed scanner (Imapro) and analyzed for density of bands of adenoviral DNA (areaxmean) with Volume Trace Motif version 1.21 (University of Iowa Image Analysis Facility).

To evaluate the quantity of adenovirus that binds to the vessel surface but is not internalized, we also performed the experiments at 4°C.

Chemiluminescent Assay for ß-Galactosidase
ß-Galactosidase activity was assayed to quantify enzyme activity of the transgene product, as described previously in detail.²³ Briefly, vessels were incubated with viral suspension of Adßgal alone, Adßgal plus 30 to 3000 molecules per particles of poly-L-lysine, Adßgal plus 3x10⁴ to 1x10⁷ molecules per particle of lipofectin, or vehicle for 30 minutes or 2 hours and then incubated with media for 24 hours. In some vessels, endothelium was denuded by gently rubbing the intimal surface after incubation of vessels for 24 hours. Vessels were minced and lysed with 100 µL of lysis buffer, and the supernatant was assayed with a Galacto-Lite Plus assay kit (Tropix Inc) and a luminometer (Monolight 2010, Analytical Luminescence Laboratory). We quantified the ß-galactosidase activity based on a standard curve generated using purified ß-galactosidase (Boehringer Mannheim) and expressed activity as milliunits ß-galactosidase per milligram protein. All assays were performed with 2 vessel rings from each animal, and values were averaged.

Histochemistry for ß-Galactosidase
X-gal staining was performed for gene expression of ß-galactosidase, as previously described in detail.^{17 23} Briefly, after incubation of vessels with viral suspension or vehicle for 2 hours and after further incubation of vessels with media for 24 hours, vessels were prefixed with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS, incubated in 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal, Sigma), and postfixed with 4% formaldehyde in PBS at room temperature, with a thorough rinse with PBS before each step. Incubation with X-gal was limited to 3 hours to prevent staining endogenous ß-galactosidase, which may be seen after >4 hours of incubation.²⁴ The fixed tissue was processed for paraffin embedding, and serial 6-µm sections were counterstained with nuclear fast red. Vessel sections were chosen in a blinded manner and examined for positive staining of ß-galactosidase by light microscopy.

Vascular Function
Isometric tension was recorded to assess alteration of vascular reactivity after gene transfer of eNOS, as previously described in detail.^{17 25 26} Vessels were incubated with viral suspension of AdeNOS alone, AdeNOS plus 300 molecules per particle of poly-L-lysine, Adßgal plus 1x10⁵ molecules per particle of lipofectin, or vehicle for 30 minutes or 2 hours and then incubated in media for 24 hours. The ratios of cationic molecules were chosen on the basis of experiments that demonstrated preliminary measurements of isometric tension using different ratios of the molecules. In separate experiments, we incubated vessels for 30 minutes in 3% sucrose in PBS with similar ratios of poly-L-lysine or lipofectin, without adenovirus vector, and then incubated the vessels in media for 24 hours.

Vessel rings were suspended in organ baths filled with Krebs' bicarbonate solution maintained at 37°C and bubbled with 95% O₂/5% CO₂ and connected to force transducers. Resting tension of vessels was increased to 3 g, and vessels were contracted twice with 100 mmol/L of KCl. After vasocontraction with 1 to 3 µmol/L of phenylephrine (corresponding to 70% of 100 mmol/L KCl contraction), concentration-response curves for sodium nitroprusside (10^-9 to 10^-5 mol/L) and calcium ionophore A23187 (10^-9 to 10^-6 mol/L) were generated. Results were expressed as percent relaxation of contraction produced by phenylephrine.

L-Phenylephrine hydrochloride (Sigma) and sodium nitroprusside (Sigma) were dissolved in saline, and A23187 (Sigma) was dissolved in dimethyl sulfoxide (final concentration <0.1%).

Statistical Analysis
Values are expressed as mean±SEM. One-way ANOVA was used for comparison among groups. Two-way repeated-measures ANOVA was used for comparison of responses to A23187 and sodium nitroprusside. Post hoc analysis for significance was made with Fisher's protected least significant difference test. P<0.05 was accepted as statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Southern Blot Analysis for Adenoviral DNA
To determine whether incorporation of adenovirus into a cationic complex would increase binding to vessels, we measured adenoviral DNA in vessel rings. In all vessels that were incubated with Adßgal plus cationic molecules for either 30 minutes or 2 hours at 37°C, adenoviral DNA was greater than in vessels treated with Adßgal alone (n=4, Figures 1 and 2). Adenoviral DNA was >2-fold greater in vessels incubated with adenovirus plus poly-L-lysine (0.05<P<0.1) and >3-fold greater in vessels incubated with adenovirus plus lipofectin (P<0.05). An increase in adenoviral DNA bound to vessels was also observed in vessels incubated with Adßgal plus cationic molecules at 4°C (n=2; Figure 1).

View larger version (26K): [in this window] [in a new window]   Figure 1. Southern blot analysis of adenoviral DNA of rings from a rabbit carotid artery after 30 minutes or 2 hours of infection with Adßgal (3x109pfu/mL, 1x1012 particles/mL) with or without cationic molecules at 4°C (top lanes) and 37°C (bottom lanes). BG indicates background (no Adßgal used); V, Adßgal alone; PL, Adßgal with 300 molecules of poly-L-lysine per adenovirus particle; and LF, Adßgal with 3x105 molecules of lipofectin per adenovirus particle.

View larger version (12K): [in this window] [in a new window]   Figure 2. Adenoviral DNA quantified from Southern blot analysis of rings of carotid arteries (n=4 rabbits) after 30 minutes or 2 hours of incubation with Adßgal (3x109pfu/mL) with or without cationic molecules at 37°C. BG indicates background (no Adßgal used); Ad, Adßgal alone; Ad+PL, Adßgal with 300 molecules of poly-L-lysine per adenovirus particle; and Ad+LF, Adßgal with 3x105 molecules of lipofectin per adenovirus particle. Bars show mean±SEM. *P<0.05 vs Ad.

Chemiluminescent Assay for ß-Galactosidase
We complexed adenovirus with cationic molecules to examine effects on transgene expression in blood vessels. Transgene expression (ß-galactosidase activity) was significantly increased by incubation of vessels with Adßgal plus poly-L-lysine, compared with incubation with Adßgal alone, for either 30 minutes or 2 hours (n=6, Figure 3). The maximum increase in activity by poly-L-lysine was 5-fold after 30 minutes of incubation (1000 molecules per particle of poly-L-lysine; P<0.0001) and 6-fold after 2 hours of incubation (300 molecules per particle of poly-L-lysine; P<0.0001). Complexing the virus with an excessively high ratio of poly-L-lysine (3000 molecules per particle) did not augment transgene activity.

View larger version (31K): [in this window] [in a new window]   Figure 3. Chemiluminescent assay for ß-galactosidase (ß-gal) activity in rings of carotid arteries (n=6 rabbits) 24 hours after treatment with Adßgal (3x109 pfu/mL, 30 minutes or 2 hours of infection) plus different ratios of poly-L-lysine (top) or lipofectin (bottom) to adenovirus. BG indicates background (no Adßgal used). Values are mean±SEM. *P<0.05 vs 0 molecules of poly-L-lysine (or lipofectin) per adenovirus particle (Adßgal alone).

Adßgal plus lipofectin incubated for 30 minutes or 2 hours also significantly augmented ß-galactosidase activity compared with treatment with Adßgal alone (n=6; Figure 3). Maximum enzyme activity increased by 6-fold after incubation for 30 minutes (1x10⁶ molecules per particle of lipofectin; P<0.0001) and by 5-fold for 2 hours of incubation (1x10⁶ molecules per particle of lipofectin; P<0.0001). An excessively high ratio of lipofectin (1x10⁷ molecules per particle), however, attenuated enzyme activity.

In vessels exposed to adenovirus complexed with cationic molecules, denudation of endothelium after incubation in medium for 24 hours greatly reduced transgene expression compared with intact vessels (n=6; Table). This finding suggests that the increase in gene transfer was primarily to endothelial cells.

View this table: [in this window] [in a new window]   Table 1. ß-Galactosidase Activity in Carotid Arteries With and Without Endothelium

Histochemistry for ß-Galactosidase
We used a histochemical method to determine the site of transgene expression after gene transfer with complexes of adenovirus with cationic molecules. There was no detectable expression of ß-galactosidase in vessels incubated with vehicle. In vessels treated with Adßgal alone, positive staining for ß-galactosidase was observed in some cells, both in the endothelium and in adventitial cells (Figure 4, top panel). In vessels treated with Adßgal and 300 molecules per particle of poly-L-lysine or 3x10⁵ molecules per particle of lipofectin, transgene expression in endothelium appeared to be greatly augmented (Figure 4, middle and bottom panels).

View larger version (71K): [in this window] [in a new window]   Figure 4. X-gal staining of carotid arteries 24 hours after treatment with Adßgal (3x109 pfu/mL, 2 hours of exposure) plus cationic molecules. Blue color shows positive stain for ß-galactosidase. Top, Treated with Adßgal alone; middle, Adßgal plus 300 molecules of poly-L-lysine per adenovirus particle; bottom, Adßgal plus 3x105 molecules of lipofectin per adenovirus particle. Bar=200 µm.

Vasomotor Function
We examined effects of cationic molecules on alteration of vascular function by adenovirus-mediated expression of eNOS. Relaxation of vessels to A23187 was similar after treatment for 30 minutes with AdeNOS alone (maximum relaxation [R_max], 89±2.3%; EC₅₀ [log] [mol/L], -7.05±0.05) and vehicle (R_max, 84±5.4%; EC₅₀, -7.03±0.08) (n=8). A23187 (10^-8 to 10^-7 mol/L) produced greater relaxation in vessels treated for 30 minutes with AdeNOS plus poly-L-lysine (R_max, 89±3.4%; EC₅₀, -7.28±0.03 [P<0.0005 versus AdeNOS alone]) or lipofectin (R_max, 90±2.0%; EC₅₀, -7.24±0.04 [P<0.005 versus AdeNOS alone]) than vessels treated with AdeNOS alone (n=7; Figure 5, left panel). Relaxation of vessels to nitroprusside was similar after each treatment (n=7; Figure 5, right panel). Treatment of vessels with poly-L-lysine or lipofectin alone (without adenovirus) did not alter vasorelaxation to A23187 and nitroprusside compared with vessels treated with vehicle (n=3, data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 5. Response to calcium ionophore (A23187) (left) and sodium nitroprusside (right) in carotid arteries 24 hours after treatment with AdeNOS (2x1010 pfu/mL, 30 minutes of exposure) with or without cationic molecules (n=7 rabbits). AdeNOS indicates AdeNOS alone; AdeNOS+PL, AdeNOS plus 300 molecules of poly-L-lysine per adenovirus particle; and AdeNOS+LF, AdeNOS plus 1x105 molecules of lipofectin per adenovirus particle. Values (mean±SEM) are expressed as percent relaxation of contraction produced by phenylephrine. Responses differ significantly in vehicle vs AdeNOS+PL, vehicle vs AdeNOS+LF, AdeNOS alone vs AdeNOS+PL, and AdeNOS alone vs AdeNOS+LF (P<0.05).

As we reported previously,¹⁷ vasorelaxation to A23187 was enhanced after 2 hours of incubation with AdeNOS alone compared with vehicle treatment. In contrast to 30 minutes of incubation with AdeNOS, however, treatment with adenovirus plus cationic molecules for 2 hours improved responses to A23187 only modestly (not significant) compared with treatment with AdeNOS alone (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
In this study we examined effects of cationic molecules on efficiency of vascular gene transfer with recombinant adenovirus. There are 4 major findings in the present study. First, association of adenovirus with an optimal ratio of poly-L-lysine or lipofectin enhanced binding of adenovirus to blood vessels. Second, these complexes augmented transgene expression in vessels, as assessed by enzyme activity and histochemistry. Augmentation of expression was observed in intact vessels but not after endothelial denudation, which indicates that the cationic molecules increase efficiency of gene transfer primarily to endothelium. Third, by using these complexes, the time of exposure of vessels to adenoviral vectors could be reduced to accomplish similar efficacy. This conclusion is based on the findings that transgene expression after exposure of vessels to adenoviral vectors with cationic molecules for 30 minutes is greater than that observed after exposure of vessels to adenovirus alone for 2 hours (Figures 3 and 4). Fourth, complexes of adenovirus and cationic molecules augmented vasorelaxation to A23187 after gene transfer of eNOS. Thus, association of adenoviral vector and cationic molecules improves the efficiency of adenovirus-mediated vascular gene transfer ex vivo, resulting in increased transgene expression and augmented alteration of vascular function.

Mechanisms for Enhanced Gene Transfer by Cationic Molecules
Several processes are required for transduction of target cells, including viral binding to the cell, internalization, escape from the endosome, and entry into the nucleus. Both nonviral and viral vectors have advantages for gene transfer. Cationic nonviral vectors have the advantage that they facilitate binding to the cell surface. Adenoviral vectors have the advantage that viral proteins facilitate steps subsequent to binding, such as escape from the endosome and entry into the nucleus. Thus, association of cationic molecules with an adenovirus may facilitate gene transfer by enhancing charge association of adenovirus with the cell surface and by permitting subsequent virus-mediated steps. We previously processed these complexes for transmission electron microscopy using a negative stain technique.¹⁶ A lamellar appearance of the cationic lipid was observed surrounding the adenovirus particles, which suggests that cationic molecules are directly linked to adenoviral particles.

Several investigators have used adenovirus and poly-L-lysine to facilitate plasmid-mediated gene transfer.^{27 28 29 30} With that approach, poly-L-lysine is used to couple plasmid bearing the transgene to the adenovirus, and in some cases to a ligand. Adenoviral proteins, then, facilitate escape of plasmid DNA from endosomes. Plasmid DNA complexed to cationic lipid (like lipofectin¹³ and lipofectamine³¹) and adenovirus also has been used to enhance gene transfer.^{32 33 34} In those previous studies, however, transgene was not encoded in the adenoviral DNA. In contrast, we used adenovirus that encodes a transgene and noncovalently coupled poly-L-lysine or lipofectin with the virus. This method has the advantage that viral proteins facilitate multiple steps in gene transfer, including not only escape from endosome but also transport and entry of DNA into the nucleus.¹⁶ An important advantage of this method is that these charge-associated complexes are easy to prepare.¹⁶

In the present study Southern blot analysis showed that complexing adenovirus with poly-L-lysine or lipofectin increased adenoviral binding and/or internalization to vessels compared with adenovirus alone. (In all 4 vessels studied, this effect was observed.) Similar results obtained at 4°C and 37°C suggest enhanced binding because internalization of virus is blocked at 4°C. Although we did not evaluate the specific vessel layers of increased adenoviral binding, it is likely that endothelium is an important site for binding because the consequential enhancement in transgene overexpression was demonstrated there. Endothelium has relatively few receptors for adenoviral fiber,¹⁰ and it seems unlikely that enhancement of viral binding occurs through these receptors. We reported that addition of excess fiber protein or a neutralizing antibody to fiber did not affect the efficiency of gene transfer by adenovirus with polylysine.¹⁶ We also demonstrated, using transmission electron microscopy, that endosomes in cells treated with adenovirus and polylysine were larger and contained more adenoviral particles than endosomes in cells treated with adenovirus alone.¹⁶ Thus, these cationic complexes seem to act primarily through receptor-independent pathways to facilitate entry into cells.

Blood Vessel and Complexes of Adenovirus and Cationic Molecules
Blood vessels are potential candidates for gene therapy. Complexes of adenovirus and cationic molecules have been reported to enhance efficiency of plasmid-mediated^{33 35} or adenovirus-mediated³⁶ gene transfer to vascular endothelium and muscle in primary cell cultures. These complexes also enhanced efficiency of plasmid-mediated gene transfer to jugular veins³⁷ and femoral arteries,³⁵ which was demonstrated by histomorphometry after X-gal staining.

In contrast to most earlier studies, transgene in our approach was encoded by adenovirus, which retains intact viral functions for gene transfer. Moreover, the present study extended previous findings in several ways. Effects of poly-L-lysine and lipofectin on adenovirus-mediated vascular gene transfer were evaluated with the use of an assay of enzyme activity, which is more sensitive than histomorphometry after X-gal staining. Assay of enzyme activity in vessels with intact and denuded endothelium indicated that complexes of adenovirus and cationic molecules increased transgene expression primarily in endothelium. Histochemistry also suggested that transgene expression was augmented primarily in endothelium. Cationic molecules tended to increase transgene expression in adventitia, but the effect was not statistically significant.

Poloxamer 407, a viscous biocompatible polyol, increases efficiency of adenovirus-mediated gene transfer to vascular smooth muscles in cell cultures³⁸ and in vivo.³⁹ The viscosity of the poloxamer may result in entrapment of viral vector around the target cells and may enhance the efficiency of gene transfer. In this study we used different molecules and extended the previous findings³⁹ by evaluation of adenoviral binding using Southern blot analysis, examination of the role of endothelium in transgene expression, and using complexes of adenovirus with cationic molecules to transfer eNOS.

In this study an excess ratio of poly-L-lysine and lipofectin to adenovirus decreased the efficiency of vascular gene transfer. Electron microscopy indicates that high ratios of poly-L-lysine produce aggregation of adenovirus particles.¹⁶ We speculate that reduction of efficiency of transgene expression at a high ratio of cationic molecules may be related to aggregation of virus, which may inhibit internalization of adenovirus to vessels. Thus, determination of the optimal ratio of cationic molecules is essential for potential uses of adenovirus-cationic molecule complexes.

Implications
Current approaches to vascular gene transfer have several limitations. For example, intraluminal delivery of vectors generally requires interruption of blood flow.^{4 5 6 7} Thus, reduction of exposure time of adenovirus to vessels is an important goal for gene transfer to vessels. The present and previous studies showed that infection of adenoviral vector alone for 30 minutes produced much less transgene expression than 2 hours of infection.²³ Viral binding and transgene expression was comparable, after 30 minutes of infection with complexes of adenovirus and cationic molecules, to the level observed after 2 hours of infection by adenovirus alone. Alternatively, it may be possible to reduce by severalfold the amount of adenovirus that is required for gene transfer with the use of these complexes.

We have recently demonstrated that NO-mediated relaxation of rabbit carotid arteries was augmented after gene transfer of AdeNOS for 2 hours.¹⁷ In the present study, using the same methods, we confirmed our previous finding¹⁷ but failed to augment vasomotor function after a shorter period (30 minutes) of infection with AdeNOS alone. Thus, exposure of adenovirus alone to vessels for 30 minutes may not be sufficient for eNOS gene transduction in our conditions.

Association of AdeNOS with cationic molecules significantly increased the alteration of function by eNOS. Thus, the complexes of adenovirus and cationic molecules produce sufficient augmentation of expression of transgene to affect vascular function. The finding that responses to a nitric oxide donor, nitroprusside, were similar among all groups suggests that the augmented relaxation to acetylcholine was specific for stimulation of eNOS and was not related to nonspecific changes in vascular smooth muscle. Poly-L-lysine itself can alter basal vascular tone and endothelium-dependent vasomotor function of cerebral vessels.⁴⁰ In the present study, however, poly-L-lysine was removed 24 hours before vasomotor function tests, and therefore it was unlikely that polylysine affected concentration-response curves to A23187. Furthermore, treatment of vessel rings with poly-L-lysine or lipofectin alone, without adenovirus, did not alter vascular function.

In conclusion, association of adenovirus-encoding transgene with cationic molecules significantly improves efficiency of vascular gene transfer of a reporter gene and, to a lesser extent, eNOS. We speculate that gene transfer of eNOS may prove to be useful for gene therapy. Because the eNOS mechanism may be impaired in several disease states (including atherosclerosis, diabetes, and hypertension), it is possible that gene transfer of eNOS may be useful in improving vascular function in these diseases. It is likely, however, that gene transfer of eNOS will prove to more useful for transient expression, for example, early after cerebral ischemia or perhaps to transiently inhibit adherence of platelets or leukocytes to endothelium. For these purposes, complexes of adenovirus with cationic molecules may be promising tools for augmentation of gene transfer to blood vessels.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL 16066, NS 24621, and HL 14388; research funds from the Veterans Administration; and funds from the Carver Trust of the University of Iowa. Dr Davidson is a fellow of the Roy J. Carver charitable trust. We thank Frank M. Faraci for his advice, Robert M. Brooks and Pamela K. Tompkins for their technical assistance, and Arlinda LaRose for typing the manuscript. We also thank the University of Iowa Gene Transfer Vector Core, supported in part by the Carver Foundation, and Richard D. Anderson for preparation of virus.

	Footnotes

 
¹ Dr Fasbender passed away February 6, 1998.

Received March 9, 1998; revision received May 27, 1998; accepted June 24, 1998.

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Editorial Comment

Gary K. Steinberg, MD, PhD, Guest Editor

Department of Neurosurgery Stanford Stroke Center Stanford University School of Medicine Stanford, California

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Although gene therapy for vascular disease appears to hold great promise for the future, poor adenoviral uptake and low efficiency of adenovirus-mediated gene transfer are major limitations of current gene transfer techniques to blood vessels. In this study, the authors have demonstrated that two different cationic, nonviral molecules (poly-L-lysine and lipofectin) significantly increase transgene activity by approximately 5 to 6 times. An important question is whether complexing the cationic molecule to adenovirus will alter the ability of the transfected virus to produce the desired gene product. However, Toyoda et al have shown convincingly that gene transfer using cationic molecules associated with recombinant adenovirus encoding eNOS augments relaxation to A23187. In fact, treatment with AdeNOS plus cationic molecules for 30 minutes produced greater relaxation than those treated with AdeNOS alone. It is unclear why a 2-hour incubation with AdeNOS complex to cationic molecules does not significantly enhance relaxation to A23187 compared with AdeNOS alone. Also, it is not certain why an excess ratio of poly-L-lysine and lipofectin to adenovirus decreases the efficiency of vascular gene transfer. These unexpected results emphasize the multiple parameters that must be optimized for this approach with cationic molecules to be successful. The authors should be congratulated for pursuing an innovative strategy that may have important practical implications for gene therapy of blood vessels.

Received March 9, 1998; revision received May 27, 1998; accepted June 24, 1998.

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