© 1997 American Heart Association, Inc.
Articles |
Augmented Adenovirus-Mediated Gene Transfer to Atherosclerotic Vessels
From the Departments of Internal Medicine and Pharmacology, Cardiovascular Center and Center on Aging, University of Iowa College of Medicine, and the Veterans Administration Medical Center, Iowa City, Iowa 52242.
Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Vascular endothelium is an important target for gene transfer in atherosclerosis. In this study, we examined gene transfer to normal and atherosclerotic blood vessels from two species, using an organ culture method. Using normal aorta, we determined optimal dose, duration of exposure to adenovirus, and duration of incubation of vessels in tissue culture. Aortas from normal and atherosclerotic monkeys were cut into rings and incubated for 2 hours with a recombinant adenovirus, carrying the reporter gene for ß-galactosidase driven by a cytomegalovirus (CMV) promoter. After 20 hours of incubation, transgene expression was assessed with a morphometric method after histochemical staining and a chemiluminescent assay of enzyme activity. Expression of ß-galactosidase after histochemical staining, expressed as percentage of total cells, was similar in adventitial cells of normal monkeys (21±4%, mean±SE%) and atherosclerotic monkeys (25±12%). Transgene expression in endothelium was higher in atherosclerotic than in normal vessel (53±3% versus 27±7%, P<.05). Chemiluminescent assay indicated greater ß-galactosidase activity (2.5±0.6 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.6±0.2 mU/mg of protein, P<.05). Aortas from normal (n=6) and atherosclerotic (n=5) rabbits also were examined. Transgene expression (after histochemical staining) in endothelium was much greater in atherosclerotic than normal rabbits (39±3% versus 9±2%, P<.05) and expression in adventitial cells was similar (normal 23±2%, atherosclerotic 24±4%). Chemiluminescent assay indicated greater ß-galactosidase activity (1.2±0.4 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.2±0.1 mU/mg protein, P<.05). These findings suggest that an adenoviral vector with a CMV promoter provides similar transgene expression in adventitia of both normal and atherosclerotic vessels. Gene transfer to the endothelium was much more effective in atherosclerotic than in normal vessels. Thus it may be possible to achieve greater transgene expression in atherosclerotic than in normal arteries.
Key Words: atherosclerosis • adenovirus • gene transfer • endothelium • ß-galactosidase
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Adenovirus-mediated gene transfer has been achieved to several blood vessels in vivo, including common carotid,1 2 3 4 5 6 7 coronary,8 9 iliofemoral,4 5 10 11 12 13 14 15 and pulmonary arteries.16 17 In these studies, gene transfer to blood vessels provided up to ~20-30% transfection of endothelial cells.18 19 Most of these studies have been performed with vessels from normal animals.
Several groups have accomplished gene transfer to atherosclerotic vessels.9 20 21 22 These previous studies9 20 21 22 have examined efficiency of gene transfer to atherosclerotic arteries after balloon injury, which typically denudes or damages endothelium.18 Although gene transfer with a recombinant adenovirus after balloon injury appears to be less efficient in atherosclerotic than normal vessels,22 effects of atherosclerosis on gene transfer have not been examined in undamaged vessels.
The goal of this study was to compare the efficiency of adenoviral gene transfer to endothelium of normal and atherosclerotic vessels. We describe a new method to accomplish gene transfer to vessels in vitro and have determined optimal conditions for gene transfer. We then examined gene transfer to aortas from monkeys and rabbits with diet-induced atherosclerosis. Vessels were incubated in tissue culture to accomplish gene transfer, and transgene expression was assessed both with a histochemical method and assay of enzyme activity.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
We obtained aorta from normal rabbits (n=5) to determine the time course of transgene expression, dose-response relationships, and effects of duration of exposure to virus in tissue culture. To compare transgene expression in normal and atherosclerotic vessels, we studied adult male cynomolgus monkeys and New Zealand White rabbits. Monkeys and rabbits were housed in the University of Iowa Animal Care Unit. Control monkeys (n=6) were fed Purina monkey chow that contains about 4% fat and is virtually free of cholesterol. Atherosclerotic monkeys (n=5) were fed a semisynthetic atherogenic diet containing 43% fat and 0.7% cholesterol for 17-32 months. Normal rabbits (n=6) were fed rabbit diet that contains about 2.5% fat and is virtually free of cholesterol. Atherosclerotic rabbits (n=5) were fed an atherogenic diet containing 9.5% fat and 2% cholesterol for 2 weeks, then 1% cholesterol for 3 months.
Adenovirus Vector
We used a replication-deficient adenovirus, Ad2/CMV-ßGal, as a
reporter virus. The recombinant virus was constructed at Genzyme
Corporation as described previously23 24 and replicated in
the University of Iowa Vector Core. The DNA constructs comprise a
full-length copy of the adenovirus genome of approximately 37.5 kb from
which the early region 1 genes (E1) have been replaced by a
cytomegalovirus (CMV) promoter and cDNA for bacterial ß-galactosidase
gene preceded by a nuclear localization signal for simian virus 40
large T antigen. Recombinant viruses were grown in human embryonic
kidney (293) cells that complement the E1 early viral promoters.
Ex Vivo Infection
Animals were euthanized with pentobarbital and aortae were
removed. The thoracic aorta from rabbits and abdominal aorta from
monkeys were used for ex vivo infection with replication-deficient
adenovirus to examine the efficiency of gene transfer. Each vessel was
cut into rings 3 mm in length and incubated with viral vectors
(1x109 plaque-forming U/mL) in phosphate-buffered saline
(PBS) with 3% sucrose at 37°C. After 2 hours of incubation with
virus, aortic rings from normal and atherosclerotic monkeys were
removed, rinsed with PBS to remove nonadherent virus, and incubated in
culture medium (Eagle's minimum essential medium with 100
µg/mL of penicillin, 100 units/mL of streptomycin, and 1%
bovine serum albumin) in a chamber aerated with 95%
O2 and 5% CO2. In preliminary experiments, we
found that transgene expression in rabbits was greater when vessels
were incubated in Krebs' solution than in Eagle's medium. We
therefore used Krebs' solution for incubation of rings of thoracic
aorta from normal and atherosclerotic rabbits after incubation of
vessels with virus for 2 hours. We performed studies of ex vivo gene
transfer with aortic rings from normal rabbits by changing several
parameters: viral titer
(1x108-1010 plaque-forming units (pfu)/mL)
duration of exposure to virus (5 minutes to 4 hours), or periods of
incubation (1-3 days). Following incubation with adenoviral vectors,
expression of ß-galactosidase was determined with histochemical
(morphometric) examination and chemiluminescent assay.
Histochemical and Morphometric Analysis of Gene
Expression
After exposure to virus for 2 hours and incubation for 20 hours,
vessels were rinsed with PBS and fixed with 2%
paraformaldehyde and 0.2%
glutaraldehyde in PBS for 10 minutes. The vessels were
washed thoroughly with PBS and incubated in
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal,
Sigma) solution for 3 hours at room temperature. Incubation with X-Gal
was limited to 3 hours to prevent staining endogenous
ß-galactosidase, which may be seen in the cytosol after longer (>4
hours) periods of incubation.25 The vessels were postfixed
with 4% formaldehyde in PBS. The fixed tissue was processed for
paraffin embedding, and sections (6 µm thick) were cut from the
block with a microtome, placed on slides, and counterstained with
nuclear fast red. Vessel sections were examined for positive staining
of ß-galactosidase (blue nuclei) by light microscopy.
Efficiency of transgene expression in blood vessels was assessed by morphometric analysis of histochemically stained sections. In each cross-section of vessel, transgene expression was assessed by counting stained nuclei versus total nuclei of the same cell layer (intima, media, and adventitia). More than 150 nuclei were counted in each region of interest in each vessel.
Chemiluminescent Assay for ß-Galactosidase
To quantitate enzyme activity of the transgene product,
segments of vessels 3 mm long were studied. Adventitia was removed
so that transgene expression could be assessed in intima and media. The
aorta was minced with a scalpel blade and lysed with 150 µL of lysis
buffer containing 0.2% Triton X-100 and 100 mmol/L of
potassium phosphate, pH 7.8. The suspension of aorta was
centrifuged at 10 000g for 10 minutes, and the
supernatant was assayed for ß-galactosidase activity using the
Galacto-Light Plus assay kit (Tropix). Light emission was measured with
a Monolight 2010 luminometer (Analytical Luminescence Laboratory) and
calibrated with a standard curve generated using purified
Escherichia coli ß-galactosidase (Boehringer
Mannheim). Protein concentrations were determined using a Bio-Rad DC
Protein Assay (Bio-Rad) and normalized ß-galactosidase activity was
expressed as mU of ß-galactosidase per milligram of protein.
Background values for chemiluminescence measured in rings from normal
rabbits that were not transfected with virus was very low (0.17±0.03
mU/mg of protein, n=4). Values for each group were calculated using at
least two rings from each animal, then averaging the values from
separate animals.
Statistical Analysis
Data are presented as mean±SE. Differences in transgene
expression between normal and atherosclerotic vessels in the
morphometric examination were analyzed by
nonparametric Mann-Whitney U test. Differences
in enzyme activity between normal and atherosclerotic vessels measured
by chemiluminescent assay were analyzed by unpaired
t test. Differences in enzyme activity among several
treatment groups in normal vessels were analyzed by ANOVA
followed by Bonferroni's corrected t-test.
P<.05 was considered a significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Expression of Transgene in Rabbit Aorta
In normal rabbits, transgene expression was dependent on duration of exposure of the aorta to Ad2/CMV-ßGal (Fig 1A
). Maximal transgene expression
occurred after incubation of vessels in tissue culture for 2 days,
although the amount of transgene expression was not significantly
different after incubation for 1 and 2 days (Fig 1B). Transgene
expression increased as the titer of virus was increased from
1x108 to 1x1010 pfu/mL (Fig 1C). Based on
these findings, we have used a viral titer of 1x109 pfu/mL
(Fig 1C), 2 hours for duration of exposure to the virus, and incubation
for 1 day to compare transgene expression in normal and atherosclerotic
aorta.
|
Expression of Transgene in Normal and Atherosclerotic Monkey
Aorta
In normal monkeys, after exposure of the aorta to Ad2/CMV-ßGal,
there was prominent expression of transgene in cells in the adventitia
(Fig 2A and B), with only patchy
expression of the reporter gene visible on the luminal surface
(endothelium). There was no apparent staining of cells
in the medial layer.
|
In atherosclerotic monkeys, morphologic changes were similar to those
described previously.26 27 There was dense fibrofatty
intimal thickening, with focal intimal necrosis in the aorta. There was
prominent expression of transgene in adventitia of atherosclerotic
monkeys (Fig 2C
). Transgene expression in adventitial cells was similar
in normal and atherosclerotic monkeys (Fig 3). In contrast to observations of normal
monkeys, marked transgene expression was observed on the luminal
surface (endothelium) of atherosclerotic vessels (Fig 2C and D). The number of cells that were positive for ß-galactosidase
in endothelium was greater in atherosclerotic than
normal monkeys (Fig 3).
|
Activity of the transgene product was compared in aorta from normal
and atherosclerotic monkeys using a chemiluminescent assay. Enzyme
activity of ß-galactosidase in the aorta was about four-fold higher
in atherosclerotic than in normal monkeys (Fig 4).
|
Expression of Transgene in Normal and Atherosclerotic Rabbit
Aorta
In normal rabbits, after exposure of the aorta to Ad2/CMV-ßGal,
transgene expression in adventitial cells was prominent and transgene
expression in endothelial cells was modest (Fig 5A and
B). No staining of medial cells was
observed.
|
In atherosclerotic rabbits, there was diffuse thickening of the
aorta and the lesions consisted largely of foam cells. There was
prominent expression of the reporter gene in the adventitia, which was
similar in normal and atherosclerotic rabbits (Fig 6). In contrast to observations in normal
rabbits, transgene expression was prominent in the luminal surface
(endothelium) of atherosclerotic rabbits (Fig 5C and
D), consistent with results observed in atherosclerotic
monkeys. The number of cells positive for histochemical staining of
ß-galactosidase in endothelial cells was four-fold
higher in atherosclerotic than normal rabbit aorta (Fig 6). Enzyme
activity for ß-galactosidase was five-fold higher in vessels from
atherosclerotic rabbits than in normal vessels (Fig 7).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The major finding of this study is that, after adenovirus-mediated gene transfer to the aorta ex vivo, transgene expression is greater in atherosclerotic vessels than in normal vessels. Both a histochemical method and chemiluminescent assay indicate greater transgene expression in endothelium of atherosclerotic than normal vessels in both monkeys and rabbits.
Gene Transfer to Atherosclerotic Vessels
Previous studies demonstrated gene transfer to vessels from
atherosclerotic rabbit using several vectors, including
liposomes20 and recombinant
adenoviruses.9 21 22 In contrast to our findings, an
important study22 indicated that adenovirus-mediated
transgene expression was less efficient in atherosclerotic iliac
arteries than normal vessels. A likely explanation for differences in
findings is that the previous study delivered the adenoviral vectors by
intravascular injection after balloon injury. Intravascular adenoviral
gene delivery after balloon angioplasty may denude most of the
endothelium,18 which presumably prevents
observation of augmented transgene expression in the
endothelium.
Another possible explanation for different findings in this and a
previous study22 is that a different promoter was used to
drive transgene expression after infection. The previous study used a
long terminal repeat of Rous sarcoma virus as a promoter and our virus
was driven by a CMV promoter. This CMV promoter has several repeated
sequences, some of which are known to bind nuclear factor (NF) 
B and
cyclic AMP response element-binding (CREB) protein, resulting in
enhancement of binding of RNA polymerase II.28 29 It is
likely that transgene expression under control of CMV promoter is
augmented when these sequences are stimulated in the transfected
tissue, because we and others30 31 have observed that
expression of the reporter gene transfected with recombinant
adenoviruses is augmented with phorbol 12-myristate 13-acetate
or forskolin that presumably stimulates the CMV promoter.
Atherosclerotic lesions contain macrophages and neutrophils
that release proinflammatory cytokines that may
activate NFB.32 Reactive oxygen species, which
are present in increased amounts in atherosclerotic
tissue,33 may also activate NFB.34
A recent report suggested that NFB is activated in
endothelium from atherosclerotic but not
nonatherosclerotic vessels.35 Furthermore, oxidized LDL
may activate a cyclic AMP pathway in
endothelium.36 Thus activity of the CMV
promoter may be enhanced in atherosclerotic endothelium
compared with normal endothelium. A recent preliminary
report suggested that augmented gene transfer was observed in an
atherosclerotic region of human arteries that contained clusters of
macrophages.37
Augmented expression of transgene in endothelium of atherosclerotic arteries could be due in part to an increase in infection by the adenoviral vectors. Upregulation of adenovirus-binding proteins, including integrins,38 in atherosclerotic endothelium could contribute to augmented expression in this study. Another recent study,39 however, indicated that expression of integrins in the endothelium was similar in normal and atherosclerotic arteries.
Experimental Model
In this study, we demonstrated that ex vivo gene transfer to blood
vessels is feasible, and we determined optimal conditions for transgene
expression. Nevertheless, there are several potential limitations in
this study. First, we should consider the possibility that transgene
expression occurs more slowly in normal than in atherosclerotic
vessels, which could account for less transgene expression in normal
vessels. Our studies with normal rabbit aorta, however, indicate that
almost maximal expression is observed within 1 day after infection when
Ad2/CMV-ßgal is used. Therefore, a slower time course of transgene
expression in normal vessels could not explain less expression of
transgene in normal than atherosclerotic vessels.
Second, it is not certain that findings based on gene transfer to vessels in vitro is predictive of gene transfer in vivo. We suggest, however, that this ex vivo model is a meaningful step between cell culture experiments and gene transfer in vivo. Furthermore, our ex vivo model has several advantages compared with studies in vivo. First, conditions can be optimized by using multiple vascular rings, instead of multiple animals. A second advantage is that intravascular administration of virus in vivo (especially at high doses) produces an inflammatory response and may cause damage to the endothelium, which can be avoided with ex vivo transfection. Another advantage of the ex vivo approach is that transgene expression is observed in adventitia as well as endothelium. Perivascular expression of transgene may prove to be an important strategy for vascular gene transfer,40 and the ex vivo approach allows characterization of transgene expression in both endothelium and adventitia.
A third limitation of this study is that we examined only one titer of virus (1x109 pfu/mL) in the comparison between normal and atherosclerotic vessels. At higher viral titers in vivo (eg, >1011 pfu/mL),6 19 transgene expression reaches a plateau or may even decrease, probably because very high titer may decrease transgene expression by producing inflammation and tissue damage. The titer we used is within a range for titer-dependent increases in transgene expression, as observed in our experiment and others,6 19 without tissue damage, and seems suitable for comparisons in this study.
In conclusion, an adenoviral vector with a CMV promoter provides effective transgene expression in adventitia in both normal and atherosclerotic vessels. Gene transfer to the endothelium was much more effective in atherosclerotic than in normal vessels. Thus, we speculate that it may be possible to achieve relatively preferential transgene expression in atherosclerotic arteries.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by NIH grants NS 24621, HL 16066, HL 14388, AG 10269, research funds from the Veterans Administration, and funds from the Carver Trust of the University of Iowa. We thank Donald J. Piegors, Pamela K. Tompkins, and D. Dean Potter for their excellent technical assistance, and Arlinda LaRose for secretarial assistance. B. L. Davidson is a fellow of the Roy J. Carver charitable trust. F. M. Faraci is an established investigator of the American Heart Association. We also thank Alan E. Smith, Genzyme, for the gift of Ad2/CMV-ßGal, and the University of Iowa Gene Transfer Vector Core and Richard D. Anderson for preparation of virus.
Received July 19, 1996; accepted February 5, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Lemarchand P, Jaffe HA, Danel C, Cid MC, Kleinman HK, Stratford-Perricaudet LD, Perricaudet M, Pavirani A, Lecocq J-P, Crystal RG. Adenovirus-mediated transfer of a recombinant human
1-antitrypsin cDNA to human endothelial
cells. Circ Res.. 1993;72:1132-1138.
2.
Guzman RJ, Lemarchand P, Crystal RG, Epstein SE,
Finkel T. Efficient and selective adenovirus-mediated gene
transfer into vascular neointima.
Circulation.. 1993;88:2838-2848.
3.
Lee SW, Trapnell BC, Rade JJ, Virmani R, Dichek DA.
In vitro adenoviral vector-mediated gene transfer into
balloon-injured rat carotid arteries. Circ Res.. 1993;73:797-807.
4. Rome JJ, Shayani V, Newman KD, Farrell S, Lee SW, Virmani R, Dichek DA. Adenoviral vector-mediated gene transfer into sheep arteries using a double-balloon catheter. Hum Gene Ther.. 1994;5:1249-1258.[Medline] [Order article via Infotrieve]
5.
Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel GJ,
Nabel EG, Parmacek MS, Leiden JF. Cytostatic gene therapy for
vascular proliferative disorders with a constitutively active form of
the retinoblastoma gene product. Science.. 1995;267:518-522.
6.
Schulick AH, Newman KD, Virmani R, Dichek DA.
In vivo gene transfer into injured carotid arteries:
optimization and evaluation of acute toxicity.
Circulation.. 1995;91:2407-2414.
7. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest.. 1995;96:2260-2268.
8. Barr E, Carroll J, Kalynych AM, Tripathy SK, Kozarsky K, Wilson JM, Leiden JM. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Gene Ther.. 1994;1:51-58.[Medline] [Order article via Infotrieve]
9.
French BA, Wojciech M, Ali NM, Geske RS, Finnigan JP,
Rodgers GP, Roberts R, Raizner AE. Percutaneous
transluminal in vitro gene transfer by recombinant adenovirus in normal
porcine coronary arteries, atherosclerotic arteries, and two
models of coronary restenosis.
Circulation.. 1994;90:2402-2413.
10.
Willard JE, Landau C, Glamann B, Burns D, Jessen ME,
Pirwitz MJ, Gerard RD, Meidell RS. Genetic modification of the
vessel wall: comparison of surgical and catheter-based techniques for
delivery of recombinant adenovirus. Circulation.. 1994;89:2190-2197.
11.
Ohno T, Gordon D, San G, Pompili VJ, Imperiale MJ,
Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell
proliferation after arterial injury.
Science.. 1994;265:781-784.
12.
Steg PG, Feldman LJ, Scoazec J-Y, Tahlil O, Barry JJ,
Boulechfar S, Ragot T, Isner JM, Perricaudet M.
Arterial gene transfer to rabbit
endothelial and smooth muscle cells using
percutaneous delivery of an adenoviral vector.
Circulation.. 1994;90:1648-1656.
13. Li J-J, Ueno H, Tomita H, Yamamoto H, Kanegae Y, Saito I, Takeshita A. Adenovirus-mediated arterial gene transfer does not require prior injury for submaximal gene expression. Gene Ther.. 1995;2:351-354.[Medline] [Order article via Infotrieve]
14.
Ueno H, Li J-J, Tomita H, Yamamoto H, Pan Y, Kanegae Y,
Saito I, Takeshita A. Quantitative analysis of repeat
adenovirus-mediated gene transfer into injured canine femoral
arteries. Arterioscler Thromb Vasc Biol.. 1995;15:2246-2253.
15. Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R, Sukhova G, Libby P, Dichek DA. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointima hyperplasia. J Clin Invest.. 1995;96:2955-2965.
16.
Muller DWM, Gordon D, San H, Zhiyong Y, Pompili VJ,
Nabel G, Nabel EG. Catheter-mediated pulmonary vascular
gene transfer and expression. Circ Res.. 1994;75:1039-1049.
17.
Schachtner SK, Rome JJ, Hoyt RFJ, Newman KD, Virmani R,
Dichek DA. In vivo adenovirus-mediated gene transfer via the
pulmonary artery of rats. Circ Res.. 1995;76:701-709.
18.
Rome JJ, Shayani V, Flugelman MY, Newman KD, Farb A,
Virmani R, Dichek D. Anatomic barriers influence the
distribution of in vivo gene transfer into the arterial
wall: modeling with microscopic tracer particles and verification with
a recombinant adenoviral vector. Arterioscler
Thromb.. 1994;14:148-161.
19.
Schulick AH, Dong G, Newman KD, Virmani R, Dichek DA.
Endothelium-specific in vivo gene
transfer. Circ Res.. 1995;77:475-485.
20. Leclerc G, Gal D, Takeshita S, Nikol S, L W, Isner JM. Percutaneous arterial gene transfer in a rabbit model: efficiency in normal and balloon-dilated atherosclerotic arteries. J Clin Invest.. 1992;90:936-944.
21. Landau C, Pirwitz MJ, Willard MA, Gerard RD, Meidell RS, Willard JE. Adenoviral mediated gene transfer to atherosclerotic arteries after balloon angioplasty. Am Heart J.. 1995;129:1051-1057.[Medline] [Order article via Infotrieve]
22. Feldman LJ, Steg PG, Zheng LP, Chen D, Kearney M, McGarr SE, Barry JJ, Dedieu J, Perricaudet M, Isner JM. Low-efficiency of percutaneous adenovirus-mediated gene transfer in atherosclerotic rabbit. J Clin Invest.. 1995;95:2662-2671.
23. Rich DP, Couture LA, Guiggio VM, Armentano D, Espino PC, Hehir K, Welsh MJ, Smith AE, Gregory RJ. Development and analysis of recombinant adenovirus for gene therapy of cyctic fibrosis. Hum Gene Ther.. 1993;4:461-476.[Medline] [Order article via Infotrieve]
24. Zabner J, Couture LA, Smith AE, Welsh MJ. Correction of cAMP-stimulated fluid secretion in cystic fibrosis airway epithelia: efficacy of adenovirus-mediated gene transfer in vitro. Hum Gene Ther.. 1994;5:585-593.[Medline] [Order article via Infotrieve]
25. Lal B, Cahan MA, Couraud P-O, Goldstein GW. Development of endogenous ß-galactosidase and autofluorescence in rat brain microvessels: implication for cell tracking and gene transfer studies. J Histochem Cytochem.. 1994;42:953-956.[Abstract]
26. Armstrong ML, Heistad DD, Marcus ML, Piegors DJ, Abboud FM. Hemodynamic sequelae of regression of experimental atherosclerosis. J Clin Invest.. 1983;71:104-113.
27.
Armstrong ML, Heistad DD, Marcus ML, Megan MB, Peigors
DJ. Structural and hemodynamic responses of
peripheral arteries of macaque monkeys to atherogenic
diet. Arteriosclerosis.. 1985;5:336-346.
28. Boshart M, Weber F, Jahn G, Dorsch-Häsler K, Fleckenstein B, Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell.. 1985;41:521-530.[Medline] [Order article via Infotrieve]
29.
Sambutti LC, M CJ, Wilkinson GWG, Mocarski ES.
NFB activation of the cytomegalovirus enhancer is mediated by
a viral transactivator and by T cell stimulation.
EMBO J.. 1989;8:4251-4258.[Medline]
[Order article via Infotrieve]
30. Christenson SD, Ooboshi H, Faraci F, Davidson BL, Heistad DD. Approaches to enhance expression after adenovirus-mediated gene transfer to carotid artery. FASEB J.. 1996;10:A276 (abstract).
31.
Wilkinson GWG, Akrigg A. Constitutive and
enhanced expression from the CMV major IE promoter in a defective
adenovirus vector. Nucleic Acids Res.. 1992;20:2233-2239.
32.
Thanos D, Maniatis T. NFB: a lesson in family
values. Cell.. 1995;80:529-532.[Medline]
[Order article via Infotrieve]
33. Smith C, Mitchinson MJ, Aruoma OI, Halliwell B. Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions. Biochem J.. 1992;286:901-905.
34. Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J.. 1996;10:709-720.[Abstract]
35. Brand K, Page S, Rogler G, Bartsch A, Brandle R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest.. 1996;97:1715-1722.[Medline] [Order article via Infotrieve]
36. Parhami F, Fang ZT, Fogelman AM, Andalibi A, Territo MC, Berliner JA. Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J Clin Invest.. 1993;92:471-478.
37. Rekhter MD, Simari RD, Work C, Nabel EG, Gordon D. Adenovirus-mediated gene transfer into human coronary arteries and rabbit aorta. Circulation.. 1995;92:I-502 (abstract).
38.
Wickham TJ, Mathias P, Cheresh DA, Nemerow GR.
Integrin vß3 and vß5 promote adenovirus
internalization but not virus attachment. Cell.. 1993;73:309-319.[Medline]
[Order article via Infotrieve]
39.
Hoshiga M, Alpers CE, Smith LL, Giachelli CM, Schwartz
SM. vß3 integrin expression in normal and atherosclerotic
artery. Circ Res.. 1995;77:1129-1135.
40.
Rios CD, Ooboshi H, Piegors D, Davidson BL, Heistad DD.
Adenovirus-mediated gene transfer to normal and atherosclerotic
arteries: a novel approach. Arterioscler Thromb Vasc
Biol.. 1995;15:2241-2245.
This article has been cited by other articles:
 |
|
 |
|
  J. W. Elrod, M. R. Duranski, W. Langston, J. J.M. Greer, L. Tao, T. R. Dugas, C. G. Kevil, H. C. Champion, and D. J. Lefer eNOS Gene Therapy Exacerbates Hepatic Ischemia-Reperfusion Injury in Diabetes: A Role for eNOS Uncoupling Circ. Res., July 7, 2006; 99(1): 78 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. CONDORELLI, J. K. AYCOCK, G. FRATI, and C. NAPOLI Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidation-sensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice FASEB J, October 1, 2001; 15(12): 2162 - 2170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Ooboshi, S. Ibayashi, J. Takada, H. Yao, T. Kitazono, and M. Fujishima Adenovirus-Mediated Gene Transfer to Ischemic Brain : Ischemic Flow Threshold for Transgene Expression Stroke, April 1, 2001; 32(4): 1043 - 1047. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chu, D. D. Heistad, M. I. Cybulsky, and B. L. Davidson Vascular Cell Adhesion Molecule-1 Augments Adenovirus-Mediated Gene Transfer Arterioscler Thromb Vasc Biol, February 1, 2001; 21(2): 238 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.Y. Alexander, M.J. Brosnan, C. A. Hamilton, J. P. Fennell, E. C. Beattie, E. Jardine, D. D. Heistad, and A. F. Dominiczak Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP Cardiovasc Res, August 18, 2000; 47(3): 609 - 617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Lund, F. M. Faraci, F. J. Miller Jr, and D. D. Heistad Gene Transfer of Endothelial Nitric Oxide Synthase Improves Relaxation of Carotid Arteries From Diabetic Rabbits Circulation, March 7, 2000; 101(9): 1027 - 1033. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Toyoda, F. M. Faraci, A. F. Russo, B. L. Davidson, and D. D. Heistad Gene transfer of calcitonin gene-related peptide to cerebral arteries Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H586 - H594. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Nakane, F. J. Miller Jr, F. M. Faraci, K. Toyoda, and D. D. Heistad Gene Transfer of Endothelial Nitric Oxide Synthase Reduces Angiotensin II-Induced Endothelial Dysfunction Hypertension, February 1, 2000; 35(2): 595 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. I. M. Campbell, M. A. Kuliszewski, and D. J. Stewart Cell-Based Gene Transfer to the Pulmonary Vasculature . Endothelial Nitric Oxide Synthase Overexpression Inhibits Monocrotaline-Induced Pulmonary Hypertension Am. J. Respir. Cell Mol. Biol., November 1, 1999; 21(5): 567 - 575. [Abstract] [Full Text]
|
|
|
|
|
|
D. D. Gutterman Adventitia-dependent influences on vascular function Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1265 - H1272. [Full Text] [PDF]
|
|
|
|
|
|
K. D. Lake-Bruse, F. M. Faraci, E. G. Shesely, N. Maeda, C. D. Sigmund, and D. D. Heistad Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H770 - H776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. D. Lund, F. M. Faraci, H. Ooboshi, B. L. Davidson, D. D. Heistad, and C. D. Kontos Adenovirus-Mediated Gene Transfer Is Augmented in Basilar and Carotid Arteries of Heritable Hyperlipidemic Rabbits • Editorial Comment Stroke, January 1, 1999; 30(1): 120 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ooboshi, K. Toyoda, F. M. Faraci, M. G. Lang, and D. D. Heistad Improvement of Relaxation in an Atherosclerotic Artery by Gene Transfer of Endothelial Nitric Oxide Synthase Arterioscler Thromb Vasc Biol, November 1, 1998; 18(11): 1752 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Toyoda, H. Ooboshi, Y. Chu, A. Fasbender, B. L. Davidson, M. J. Welsh, D. D. Heistad, and G. K. Steinberg Cationic Polymer and Lipids Enhance Adenovirus-Mediated Gene Transfer to Rabbit Carotid Artery • Editorial Comment Stroke, October 1, 1998; 29(10): 2181 - 2188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Rekhter, R. D. Simari, C. W. Work, G. J. Nabel, E. G. Nabel, and D. Gordon Gene Transfer Into Normal and Atherosclerotic Human Blood Vessels Circ. Res., June 29, 1998; 82(12): 1243 - 1252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Miller, D. D. Gutterman, C. D. Rios, D. D. Heistad, and B. L. Davidson Superoxide Production in Vascular Smooth Muscle Contributes to Oxidative Stress and Impaired Relaxation in Atherosclerosis Circ. Res., June 29, 1998; 82(12): 1298 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Morishita Lessons From Human Arteries : How to Design a Gene Therapy Strategy for Treatment of Cardiovascular Disease Circ. Res., June 29, 1998; 82(12): 1349 - 1351. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||