From the Departments of Internal Medicine and Pharmacology,
Cardiovascular Center and Center on Aging, University of Iowa College of
Medicine, and Veterans Administration Medical Center, Iowa City.
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
Methods—We injected replication-deficient recombinant adenovirus
expressing nuclear targeted ß-galactosidase, driven by either the
Rous sarcoma virus promoter (AdRSV-ßGal) or the cytomegalovirus
promoter (AdCMV-ßGal), into the cisterna magna of
anesthetized ICR and C57BL/6 strains of mice. The brains were
examined from 1 to 21 days after injection by chemiluminescent enzyme
activity assay or histochemical staining.
Results—After injection of AdRSV-ßGal, expression of
ß-galactosidase in ICR mice peaked on day 7 and returned to basal by
day 14. Expression of ß-galactosidase in C57BL/6 mice was maximal on
days 7 to 14 and was minimal by day 21 after injection of AdRSV-ßGal.
After injection of AdCMV-ßGal in C57BL/6 mice, peak expression of
transgene occurred on day 1 and was greatly diminished by day 3.
Transgene expression was observed primarily on the ventral surface of
the brain, with preferential expression in
leptomeninges and adventitia along the major cerebral
arteries of that region.
Conclusions—Injection of recombinant adenovirus in the cisterna
magna resulted in transgene expression in leptomeninges
and perivascular tissue of cerebral blood vessels in two strains of
mice. The CMV promoter elicited rapid but short-lived expression of the
transgene, while the RSV promoter elicited slower, more sustained
transgene expression. Expression of AdRSV transgene was prolonged in
C57BL/6 mice compared with ICR mice. This approach for gene transfer
may be useful to study cerebral vascular biology in genetically altered
strains of mice.
The major goal of this study was to achieve gene transfer to
cerebral blood vessels and perivascular tissue in mice by
administration of a viral vector into the cerebrospinal fluid. Gene
transfer in mice is attractive because it allows studies of vascular
mechanisms in genetically altered strains.
The time course of transgene expression appears to be dependent on host
immune responses to either the vector10 or the transgene product.11 We examined
transgene expression in ICR mice, a common outbred strain of
heterogeneous background, and in C57BL/6 mice, an inbred
strain that is commonly used as a background for genetically altered
strains.
The time course of transgene expression also may be dependent on the
promoter used in the vector. The CMV promoter, for example, provides
strong constitutive expression, and the RSV promoter generally provides
slower transgene expression. We assessed the time course of expression
using vectors driven by either the RSV or the CMV promoter.
Animals and Surgical Procedure
Histochemical Analysis for ß-Galactosidase
Chemiluminescent Assay
Statistical Analysis
ß-Galactosidase Activity in ICR and C57BL/6 Mice
Inflammatory Response
Gene Transfer to Mice
Gene transfer to cerebral blood vessels in mice is an attractive
possibility because genetically altered mice are available.
Administration of a vector to deliver a functional transgene to one of
these genetically altered strains could offer a new means of studying
vascular biology.
Determinants of Transgene Expression
Inbred animals (eg, C57BL/6 mice) are genetically more homozygous than
outbred animals (eg, ICR mice). Inbred and genetically altered strains
are useful for investigating specific questions that require distinct
genetic alterations, and outbred strains are useful for
screening.25 The C57BL/6 mouse appears to be more
susceptible to bacterial infection than outbred mice such as
ICR.25 26 Peak expression of the transgene in
C57BL/6 mice occurred at a time when expression had virtually
disappeared in ICR mice, and the duration of expression was longer in
C57BL/6 mice. Duration of expression after gene transfer is affected by
the host immune response to the viral proteins10
and the transgene product.11 Both cellular
and humoral immune responses play a role in the response to these
foreign proteins in brain27 28 and
peripheral tissues.29 30 31 32
Immunocompetence of the host, therefore, can greatly affect the time
course of transgene expression.25 26 We speculate
that differences in the immune status between the two strains of mice
used in this study may contribute to the difference in time course of
transgene expression.
Limitations of the Method
Conclusions and Speculation
In summary, the present study demonstrates successful use of an
adenoviral vector to induce transgene expression in perivascular
tissues near cerebral blood vessels. The onset and duration of
transgene expression are dependent on the promoter that is used and the
strain of mice. This in vivo technique may provide a useful tool to
evaluate the role of selected enzymes or receptors in normal and
genetically altered mice.
Received November 11, 1997;
revision received March 16, 1998;
accepted April 15, 1998.
CNS
Growth Factor Research Laboratory,
Massachusetts General Hospital,
Boston, Massachusetts
Points to be made from this study include the following: (1)
Viral vectors were introduced through direct intracisternal injection.
This is a relatively easy approach with which to deliver drugs or other
agents to rodents and, indeed, to humans (through a lateral cervical
puncture or direct injection into the cisterna magna). (2) Cells
successfully transfected included meningeal cells and cells of the
vascular adventitia. Thus, this method would not be appropriate for
circumstances in which transfection of cells on the cortical surface
(neurons and glia) or vascular media or endothelial cells is desired.
(3) Gene expression was transient and thus not appropriate for
circumstances in which ongoing gene expression is desired. (4) An
inflammatory response occurs, which, as the authors note, may limit the
application of this method.
In spite of such limitations, however, the transfection method
described is likely to be useful for studies in which transient local
gene expression in adventitial and meningeal cells is desired.
Moreover, in the case of genes encoding secreted proteins, gene
products expressed locally in these cells may diffuse to affect
adjacent cells in the cerebral vasculature and cortical surface.
Received November 11, 1997;
revision received March 16, 1998;
accepted April 15, 1998.
© 1998 American Heart Association, Inc.
Original Contributions
Adenovirus-Mediated Gene Transfer In Vivo to Cerebral Blood Vessels and Perivascular Tissue in Mice
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Background and Purpose—Gene transfer
to cerebral blood vessels has been accomplished in rats and dogs by
injection of replication-deficient adenovirus into cerebrospinal fluid.
In this study we examined transgene expression after injection of
adenovirus into the cerebrospinal fluid of mice. Responses were
observed in ICR mice and C57BL/6 mice, which are outbred and inbred
strains, respectively.
Key Words: adenovirus • cerebral arteries • gene transfer • promoter regions (genetics) • mice
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Gene transfer to
blood vessels offers a new tool to study vascular physiology, with
potential for treatment of vascular disease. Adenoviral vectors have
been used to achieve gene transfer in several types of
vessels.1 2 3 Gene transfer to blood vessels
usually has been accomplished by intraluminal administration of a
vector.4 5 6 This approach, however, is generally
limited by the need to transiently interrupt blood flow or to use a
double-balloon catheter that produces expression limited to a small
segment of vessel. Alternative approaches have been developed recently
that circumvent these obstacles by perivascular administration of
adenovirus to the carotid arteries of monkeys7
and to cerebral blood vessels of rats8 and
dogs.9
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Adenoviral Vector
We used two different replication-deficient recombinant
adenoviruses (serotype 5) encoding the reporter gene for bacterial
ß-galactosidase driven by either the Rous sarcoma virus promoter
(AdRSV-ßGal) or the immediate early cytomegalovirus promoter
(AdCMV-ßGal). These vectors were constructed according to methods
similar to those described previously.12 13
Briefly, these replication-deficient adenoviral vectors have been
deleted of sequences in the E3, E1A, and E1B regions, impairing the
ability of the virus to replicate. Either the RSV or CMV promoter was
used to drive transcription of lacZ, the gene for
Escherichia coli ß-galactosidase, with a simian virus 40
polyadenylation sequence cloned downstream from this reporter gene.
Recombinant viruses were grown in human embryonic kidney (293) cells
that complement the E1 early viral promoters. After purification, the
virus was suspended in PBS with 3% sucrose and kept at -80°C until
used.
All animal procedures were approved by the Institutional Animal
Care and Use Review Committee. Male ICR mice (Harlan Sprague; weight,
16 to 26 g) (n=36) and C57BL/6 mice (Harlan Sprague; weight, 16 to
24 g) (n=64) were used. Mice were anesthetized with
pentobarbital (
75 mg/kg IP) or ketamine (40 mg/kg SC) and
acepromazine (1.5 mg/kg SC). The head was placed in a
stereotaxic apparatus with the nose
approximately 30° below horizontal. With the use of aseptic
techniques, the skin was incised, and muscle was cleared to expose the
atlanto-occipital membrane. A 30-gauge needle and Hamilton syringe were
mounted on the arm of the stereotaxic device, and the
needle was inserted 1.0 mm into the cisterna magna. Twenty
microliters of viral suspension (3x1010
plaque-forming units per milliliter) was infused over 20 minutes. The
needle was withdrawn 10 minutes after the infusion was completed, and
the nuchal muscle and skin were closed with sutures. Approximately 95%
of mice survived the procedure.
After 1 to 21 days, the mice were killed with pentobarbital
(120 mg/kg IP) and perfused transcardially with heparinized
(100 mU/mL) normal saline. The brain was removed and rinsed
thoroughly with normal saline. For histochemical staining of
ß-galactosidase activity, the brains were placed in 2%
paraformaldehyde and 0.2%
glutaraldehyde in PBS (<1 hour), then rinsed with PBS.
The whole brain or coronal sections were incubated in
5-bromo-4-chloro-indolyl-ß-D-galactopyranoside (X-Gal)
solution (2 hours at 24°C), rinsed in normal saline solution, and
then postfixed with 7% buffered formalin. The fixed tissue was then
processed for paraffin embedding, and microtome sections were cut from
the block, placed on slides, and counterstained with hematoxylin and
eosin or nuclear fast red. Brain sections were examined for positive
staining of ß-galactosidase (blue nuclei) by light microscopy.
To quantify ß-galactosidase activity, the ventral region of
the brain, corresponding to the brain stem (including basilar artery)
and circle of Willis, was sectioned for chemiluminescent enzyme assay.
The section of tissue was homogenized and lysed with 150
µL of lysis buffer containing 0.2% Triton X-100 and 100 mmol/L
potassium phosphate, pH 7.8. The suspension was centrifuged at
10 000g for 10 minutes, and the supernatant was assayed for
ß-galactosidase activity with the use of the Galacto-Light Plus assay
kit. Light emission was measured with a Monolight 2010 luminometer and
calibrated with a standard curve generated with the use of purified
E coli ß-galactosidase. Protein concentrations were
determined with a BioRad DC Protein Assay, and normalized
ß-galactosidase activity was expressed as milliunits
ß-galactosidase per milligram protein.
Data are presented as mean±SEM. A natural log
transformation of the data was used in the analysis to
normalize the variance among the treatment groups. To compare
ß-galactosidase activity with control value, ANOVA was performed,
followed by the Dunnett test for multiple comparisons. For comparisons
between groups (ICR versus C57BL/6, AdRSV-ßGal versus AdCMV-ßGal),
a two-way ANOVA was performed, with treatment and day as the two
factors of analysis. Treatment groups were then compared at
each day with P values adjusted by Bonferroni's method.
P<0.05 was considered a significant difference.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Histochemical Analysis of Gene Expression for
ß-Galactosidase
Staining for ß-galactosidase was observed primarily on the
ventral surface of the brain, especially along the major cerebral
arteries (Figure 1
). Transgene expression
was also observed on the medial dorsal surface of the cerebellum and
brain stem near the injection site. The dorsal cortical surface had few
positively stained cells. Nuclei were well transduced in the
leptomeningeal cells, especially those overlying cerebral blood vessels
on the ventral surface (Figure 2A and 2B). Medial and intimal cells did not demonstrate staining, and
adventitial cells were often stained.
View larger version (110K):
[in a new window]
Figure 1. Expression of ß-galactosidase on the ventral
surface of the brain of a C57BL/6 mouse 1 day after intracisternal
injection of AdCMV-ßGal. There was dark blue staining of
leptomeninges near the basilar artery, circle of
Willis, middle cerebral artery, and smaller arteries.
View larger version (114K):
[in a new window]
Figure 2. Expression of ß-galactosidase in the brain of a
C57BL/6 mouse 1 day after intracisternal injection of AdCMV-ßGal.
Brain sections were stained with X-Gal and counterstained with
hematoxylin and eosin. High-power views (x320) show expression of
ß-galactosidase in the basilar artery (A) and the communicating
artery (B). There was dark blue staining of
leptomeninges and adventitia. In both panels,
leukocytes can be seen around transfected tissues.
In ICR mice, peak expression of ß-galactosidase was observed 7
days after injection of AdRSV-ßGal (Figure 3). Transgene expression virtually
disappeared 14 days after injection of the virus. In C57BL/6 mice,
after injection of AdRSV-ßGal, peak expression was observed at 7 to
14 days (at a time when expression had virtually disappeared in ICR
mice) and diminished (but was not gone) by 21 days (Figure 3). After
injection of AdCMV-ßGal in C57BL/6 mice, peak expression of transgene
was observed at 1 day and was greatly diminished by 3 to 7 days (Figure 4).
View larger version (16K):
[in a new window]
Figure 3. Expression of ß-galactosidase on ventral brain
stem and cerebrum after intracisternal injection of AdRSV-ßGal in ICR
and C57BL/6 mice. Each value indicates mean±SEM of 3 to 7 mice.
*P<0.05 vs day 0; 
P<0.05 vs ICR
mice.
View larger version (17K):
[in a new window]
Figure 4. Expression of ß-galactosidase on ventral brain
stem and cerebrum after intracisternal injection of AdRSV-ßGal or
AdCMV-ßGal in C57BL/6 mice. Each value indicates the mean±SEM of 3
to 8 mice. *P<0.05 vs day 0; P<0.05
vs AdRSV-ßGal (Figure 3
).
Polymorphonuclear and mononuclear leukocytes were observed in
vascular adventitia and surrounding leptomeninges on
the ventral and lateral surfaces of the brain after injection of
AdCMV-ßGal (Figure 2) or AdRSV-ßGal at day 1 and day 7.
Vehicle-injected mice had few or no leukocytes. No animals had head
tilts, paralysis, or detectable behavioral changes for up to 21
days.
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
The major finding of this study was that administration of
replication-deficient adenovirus into the cisterna magna of mice
produced expression of the transgene, which demonstrates that the
method is feasible in mice. The gene product was expressed in a
time-dependent manner on the ventral surface of the brain stem and
cerebrum, especially in leptomeningeal cells along the major cerebral
blood vessels and in adventitial cells. Transgene expression appeared
to persist longer in C57BL/6 mice than ICR mice. Finally, we found that
peak expression was greater, occurred much sooner, and diminished
earlier with the use of an adenoviral vector driven by a CMV immediate
early promoter than with the use of an adenoviral vector driven by an
RSV promoter.
Gene transfer to blood vessels is generally accomplished by
intraluminal delivery with the use of
perforated,14 15
double-balloon,6 14 16 or hydrogel-coated
balloon6 15 catheters or by direct
injection4 5 14 into blood vessels. For gene
transfer to cerebral blood vessels, this approach is limited by the
apparent need to interrupt cerebral blood flow and to disrupt the
blood-brain barrier.8 As an alternative approach,
two groups have accomplished gene transfer to cerebral blood vessels in
rats8 and dogs9 17 by
intracisternal injection. In this study transgene expression was
observed in perivascular tissue and adventitia of cerebral arteries in
both strains of mice. As in previous
studies,8 9 17 transgene expression was not
observed in media, endothelium, or neurons.
Promoters are an important determinant of the level and time
course of expression after gene transfer.18 19 20
Promoters have different levels of constitutive activity and different
response elements that induce or inhibit activity. In this study we
used adenovirus vectors driven by CMV or RSV promoters. The CMV major
immediate early promoter/enhancer drives constitutive expression, and
response elements within the enhancer allow inducible expression
through binding of active transcription factors, such as cAMP response
element binding protein and nuclear factor-
B. The CMV, but not RSV,
promoter is associated with inflammatory response elements, nuclear
factor-B, and cAMP response element binding
protein.21 22 23 24 We observed that peak expression
was reached much sooner (day 1 versus day 7 to 14) and appeared to be
greater with the CMV promoter than with the RSV promoter. Thus,
different intensity and duration of transgene expression in cerebral
vessels and perivascular tissue can be provided on the basis of choice
of viral promoters.
Although this method offers promise for study of cerebral vascular
function, it has important limitations. First, the duration of
transgene expression was only 2 to 3 weeks when the RSV promoter was
used, and longer expression may be desirable for some studies and
therapeutic approaches. Second, transient inflammation, with
leukocytosis produced by injection of adenovirus into the cerebrospinal
fluid, may affect studies of vascular function. For studies of vascular
biology, the inflammatory responses can be addressed with appropriate
controls. Inflammation, nevertheless, will present a major obstacle
to gene therapy in which adenoviral vectors are used.
The duration of expression noted in this study is relatively short
(<1 and 3 weeks for CMV and RSV promoters, respectively). This
duration is too brief for some therapeutic applications for chronically
diseased vessels. The duration may be useful, however, when applied to
physiological studies and
pathophysiological applications. For example, an
adenovirus that encodes for endothelial nitric oxide
synthase has been demonstrated to be functional in
carotid33 and cerebral blood
vessels.9 Transient gene replacement may be
feasible in cerebral vessels of endothelial nitric
oxide synthase–deficient mice.34 In relation to
pathophysiology, we speculate that prevention of vasospasm may be
feasible after subarachnoid
hemorrhage.35 36 37 38
Selected Abbreviations and Acronyms
AdCMV-ßGal
=
adenovirus expressing nuclear targeted ß-galactosidase driven by
cytomegalovirus promoter
AdRSV-ßGal
=
adenovirus expressing nuclear targeted ß-galactosidase driven by Rous
sarcoma virus promoter
CMV
=
cytomegalovirus
PBS
=
phosphate-buffered saline
RSV
=
Rous sarcoma virus
Acknowledgments
This study was supported by National Institutes of Health grants
NS-24621, HL-16066, and HL-14388, research funds from the Veterans
Administration, and funds from the Carver Trust of the University of
Iowa. Dr Lake is supported by institutional training grant DK-07690. Dr
Davidson is a fellow of the Roy J. Carver Charitable Trust. Dr
Faraci is an established Investigator of the American Heart
Association. We thank Pamela K. Tompkins and D. Dean Potter for
technical assistance and Arlinda LaRose for secretarial assistance. We
also thank the University of Iowa Gene Transfer Vector Core and Richard
D. Anderson for preparation of virus.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
In this article, the authors show that intracisternal injection of
adenovirus containing the lacZ gene results in successful
transfection of cerebral blood vessels and meningeal cells. There were
differences in the length of gene expression depending on the viral
promoter and strain of recipient mice. The authors note that this
strategy may be useful as a tool to study cerebrovascular function and
may even represent a method for therapeutic gene delivery.
Selected Abbreviations and Acronyms
AdCMV-ßGal
=
adenovirus expressing nuclear targeted ß-galactosidase driven by
cytomegalovirus promoter
AdRSV-ßGal
=
adenovirus expressing nuclear targeted ß-galactosidase driven by Rous
sarcoma virus promoter
CMV
=
cytomegalovirus
PBS
=
phosphate-buffered saline
RSV
=
Rous sarcoma virus
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