© 1995 American Heart Association, Inc.
Articles |
Adenovirus-Mediated Gene Transfer In Vivo to Cerebral Blood Vessels and Perivascular Tissue
From the Departments of Internal Medicine, Physiology 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.
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Abstract |
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Abstract Gene transfer to blood vessels in vivo generally requires interruption of blood flow. Thus, gene transduction to cerebral blood vessels in vivo has not yet been achieved. In this study, we injected replication-deficient adenovirus into cerebrospinal fluid in an attempt to transduce genes to cerebral blood vessels. Recombinant adenovirus (1x109 infectious units) expressing nuclear-targeted bacterial ß-galactosidase driven by the cytomegalovirus promoter was injected into the cisterna magna of Sprague-Dawley rats. The brains were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside 1 to 7 days after injection of adenovirus. Leptomeningeal cells overlying the major arteries were efficiently transduced, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. ß-Galactosidase was expressed on days 1 and 3 after injection but was undetectable by day 7. Expression of the gene was `targeted' by altering the position of the head. When viral suspension was injected while the rat was in a nose-down position, the reporter gene was expressed extensively on the ventral surface of the brain, especially along the circle of Willis. When the position was changed to the nose-up or lateral position, the inferior or lateral region of the brain was stained primarily. Administration of the virus into the lateral ventricle provided extensive expression in ependymal cells and leptomeninges with some transduction to cerebral blood vessels. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, cisternal delivery may target specific brain regions by positioning of the head. This approach may be useful for studies of cerebral vascular biology and cerebral vascular gene therapy.
Key Words: gene therapy • adenovirus • cerebral blood vessel • ß-galactosidase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gene transfer is an attractive intervention for studies of basic mechanisms of vascular biology and therapy for vascular disease. A promising vector for gene transfer is replication-deficient adenovirus.1 2 3 4 By use of this vector, gene transduction has been achieved in several blood vessels, including common carotid,5 6 7 8 coronary,9 10 and iliofemoral11 12 arteries. However, there are major limitations in the transduction of genes to cerebral blood vessels in vivo after intravascular injection. First, to infect vessels efficiently, it is necessary with current approaches to interrupt blood flow transiently or to use a catheter that produces transduction that is limited to the vessel underlying the catheter. Second, it is likely that the blood-brain barrier will attenuate the infection of cerebral vessels beyond the endothelium after intravascular injection of viral vectors. Recent studies have shown that the injection of viral vectors directly into the cerebral vasculature does not result in endothelial transduction.13 To circumvent these obstacles to gene transfer to intracranial cerebral blood vessels, we have injected recombinant adenovirus into cerebrospinal fluid. Replication-deficient adenovirus carrying the gene for bacterial ß-galactosidase was administered, and gene expression was assessed by histochemical analysis.
Our first goal was to determine whether injection into cerebrospinal fluid would provide an alternative to intravascular injection for gene transfer to cerebral blood vessels. We adopted two approaches for the delivery of virus: injection into the cisterna magna and into the lateral ventricle. We explored whether these approaches would accomplish gene transfer to cerebral blood vessels and perivascular areas. Perivascular areas might be useful targets when genes that express diffusible substances are transferred. Second, we attempted to deliver adenovirus to specific regions of the brain. Adenovirus vectors were injected in 20% sucrose solution of PBS, which is more dense than cerebrospinal fluid. We changed the position of the head so that if the viral suspension is delivered to the most dependent part of the intracranial cavity, this approach might be used to target specific regions.
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Materials and Methods |
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Adenovirus Vectors
We used two different replication-deficient recombinant adenoviruses: Ad2/CMV-ßGal as a reporter virus and Ad2/CFTR-2 as a negative control, which were based on adenovirus serotype 2 (Ad2). Construction of these viruses has been described previously.14 15 The DNA constructs comprise a full-length copy of the adenovirus genome of
37.5 kb, from which the early
region 1 (E1) genes have been replaced by either cDNA for bacterial
ß-galactosidase (ßGal) gene preceded by a simian virus 40 (SV40)
nuclear localization signal in Ad2/CMV-ßGal (where CMV is
cytomegalovirus) or by the cDNA for the cystic fibrosis transmembrane
conductance regulator (CFTR) in Ad2/CFTR-2. Recombinant viruses were
grown in human embryonic kidney (293) cells that complement the E1
early viral promoters.14 Virus titer (infectious unit) was
determined by using anti-adenovirus antibody, and the virus was
suspended in PBS with 20% sucrose and was kept at -70°C until
use.
Animals and Surgical Procedure
All animal procedures were approved by the Animal Care and Use
Review Committee at the University of Iowa. Male
Sprague-Dawley rats (n=25) that weighed 340 to 430 g were
used for the present study. Before injection of the virus, the rat
was anesthetized with pentobarbital (50 mg/kg IP), and the head was
shaved and placed in a stereotaxic apparatus.
Recombinant virus was injected into the cisterna magna (Ad2/CMV-ßGal, n=16; Ad2/CFTR-2, n=2), and vehicle (20% sucrose in PBS) alone was injected in two rats. For injection into the cisterna magna, skin was incised from the occipital to nuchal region, and the occipital bone was cleared of muscular tissue to expose the atlantooccipital membrane. A 27-gauge needle and syringe were mounted on the manipulating arm of a stereotaxic device, and the needle was inserted into the cisterna magna. After 100 µL of cerebrospinal fluid was withdrawn, 125 µL of viral suspension (0.8x1010 infectious units/mL) or vehicle was infused for 5 minutes, and the nuchal muscle and skin were sutured. The rats were injected with the head in the following three positions. In the nose-down position, the superior plane of the parietal bone was tilted forward by 30°. All rats that received Ad2/CFTR-2 or vehicle were injected in this position. In the nose-up position, the superior plane of the parietal bone was tilted backward by 30°. In the right lateral position, the heads and bodies of rats were turned to the right by 90°. Each position was maintained for 30 minutes after the injection.
For injection into the lateral ventricle, the midsagittal scalp was incised, and a small burr hole was made in the parietal region (1.0 mm posterior and 1.5 mm lateral to the bregma) with a dental drill. A needle on a Hamilton syringe was stereotactically inserted into the right lateral ventricle (4.0 mm in depth), and 100 µL of viral suspension (1x1010 infectious units/mL) was injected over 30 minutes. The superior plane of the parietal bone was kept horizontal during the injection. The burr hole was then covered with bone wax, and the scalp was sutured. After injection of adenovirus, the rats were housed for 1, 3, or 7 days. Rectal temperature was measured before and 1 day after intracisternal injection of Ad2/CMV-ßGal (n=6) or vehicle (n=2).
Histochemical Analysis of Gene Expression
After the designated survival periods, the rats were
anesthetized with pentobarbital (50 mg/kg IP) and perfused
transcardially with 2% paraformaldehyde and 0.2% glutaraldehyde in
PBS. The brain was removed and washed thoroughly with PBS. The whole
brain or coronal sections (thickness, 2 mm) were incubated in
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
(X-Gal, Sigma Chemical Co) staining solution for 2 hours at room
temperature, rinsed in PBS, and postfixed with 2% paraformaldehyde and
0.2% glutaraldehyde in PBS. The fixed brain was processed for
cryosection or paraffin embedding, and microtome sections (thickness, 6
to 14 µm) were cut from the block, placed on slides, and
counterstained with nuclear fast red.
Morphometric Analysis
Efficiency of transgene expression to the blood vessels or the
leptomeninges overlying vessels was assessed in rats, which were killed
1 day after injection of Ad2/CMV-ßGal. Among the sections that
contained the basilar artery or proximal part of the right middle
cerebral artery, three cross sections at 2-mm intervals were examined
for positive staining of ß-galactosidase (blue nuclei) by light
microscopy. In each section, transgene expression to the vessel was
assessed by counting stained nuclei versus total nuclei of the same
cell layer (intima, media, and adventitia). Expression to the
leptomeninges overlying the basilar artery or middle cerebral artery
was also estimated by counting positive nuclei in the meninges directly
overlying the arteries for the same length as the vessel diameter. More
than 80 nuclei were counted in each region of interest in each rat.
Statistical Analysis
Data are presented as mean±SEM. Differences in transgene
expression among three different positions of the head were analyzed by
a nonparametric Kruskal-Wallis test followed by Bonferroni's post hoc
t test.
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Results |
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Injection Into Cisterna Magna
Four rats, in which Ad2/CMV-ßGal was injected into the cisterna magna in the nose-down position, were killed on the following day. Extensive expression of bacterial ß-galactosidase was seen on the ventral surface of the brain in all rats, especially along major cerebral arteries (Fig 1A
and Table 1).
The dorsal surface was relatively devoid of positive staining of
ß-galactosidase. Nuclei in the leptomeningeal cells, especially in
those overlying the cerebral blood vessels on the ventral surface (Fig 1C), were well transduced, with 33% of the cells stained in the
meninges overlying the proximal middle cerebral artery (Table 2).
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Adventitial cells of cerebral vessels, including the basilar or middle
cerebral artery, were occasionally stained (Figs 1B
and 2B and Table 2). Although positive staining was not
observed in the medial or intimal cell layers of arteries or veins,
smaller vessels (arterioles and venules) in the subarachnoid space
occasionally revealed blue-stained nuclei (Fig 1D). Diameter of the
vessels with positive staining of smooth muscle cells was 50 to 100
µm, and positive staining was observed in <2% of the small vessels.
Because we limited X-Gal staining to 2 hours, endogenous
ß-galactosidase, which might be seen in cytosol after longer (>4
hours) periods of staining,16 was not observed. One day
after injection of Ad2/CFTR-2 (109 infectious units, n=2)
or vehicle alone (n=2) into the cisterna magna, expression of
ß-galactosidase was not detected in nuclei or cytosol (Fig 1E).
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Transgene expression was observed 3 days after injection of
Ad2/CMV-ßGal, but the intensity tended to be less than at 1 day after
injection (Table 1). After 7 days, no expression of ß-galactosidase
was detected in the brain, including the leptomeninges and cerebral
blood vessels (Table 1).
Polymorphonuclear leukocytes were observed in the subarachnoid space
(Figs 1C, 1E, and 2B) 1 day after injection of either recombinant
adenovirus, but there was no infiltration of inflammatory cells into
the brain parenchyma. After injection with vehicle alone, inflammatory
cells were not observed in the brain on the day following injection.
The cellular response on days 3 and 7 after injection of Ad2/CMV-ßGal
involved mainly mononuclear cells, and the number of cells was less on
day 7. Rectal temperature was 37.0±0.2°C before and 37.3±0.3°C
(P<.05, paired t test) 1 day after injection of
Ad2/CMV-ßGal (n=6). Injection of vehicle alone did not alter
temperature.
Effect of Position of Head
Ad2/CMV-ßGal was also injected in different head positions to
"target" brain regions. One day after we injected adenovirus with
the head in the right lateral position, expression of ß-galactosidase
was located mainly along the right middle cerebral artery and its
branches, and there was almost no staining on the left side of the
brain (Fig 2A). When adenovirus was injected in the nose-up position,
expression in the leptomeninges overlying the brain stem was augmented
(Fig 2C). Transgene expression in the leptomeninges overlying the
basilar artery after injection in the nose-up position (48±12%) was
significantly higher than after injection in the lateral position
(11±2%, P<.05) and the nose-down position (21±2%,
P<.05). In contrast, gene transduction to the meninges
overlying the right middle cerebral artery or to the adventitial cells
of the artery tended to be higher in the right lateral position than in
the other positions (Table 2 and Fig 2B).
Injection Into Lateral Ventricle
Ad2/CMV-ßGal was administered into the lateral ventricle
in five rats. One day after injection, extensive transduction to the
ependymal cells that line the ventricles was observed (Fig 2D), in
contrast to absence of ependymal transduction after injection into the
cisterna. Subependymal cells were not transduced. Expression of
ß-galactosidase was also observed on the ventral surface of the
brain, and positive nuclei were located mainly in leptomeningeal cells
overlying the vessels with some expression in the adventitia; 35±5%
of cells in leptomeninges overlying the proximal middle cerebral artery
were stained. Gene transduction tended to be less on day 3 and was not
observed on day 7 after injection into the ventricle (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have demonstrated that administration of replication-deficient adenovirus into cerebrospinal fluid produces extensive expression of the transgene encoding bacterial ß-galactosidase. The gene product was expressed on the surface of the cerebrum, especially along major cerebral blood vessels. Leptomeningeal cells overlying the major vessels were extensively transduced, and adventitial cells in large arteries and smooth muscle cells in small vessels also expressed the foreign gene. When the position of the head was altered during the injection of virus, transduction was achieved mainly in the most dependent part of the brain.
Several groups have accomplished gene transfer to blood vessels in vivo or ex vivo with cationic lipids17 18 19 20 21 22 23 24 or retroviruses.25 26 27 28 29 Efficiency of transgene expression, however, was low. Recently, adenovirus-mediated gene transfer has been accomplished in peripheral vessels, including common carotid, coronary, and iliofemoral arteries, by using perforated,8 10 double-balloon,8 11 12 or hydrogel-coated balloon10 12 catheters or by direct injection5 6 7 8 9 into blood vessels. The apparent need for interruption of cerebral blood flow and for disruption of the blood-brain barrier is a major limitation in gene transfer to cerebral blood vessels.
Adenovirus vectors have been used to transfer genes to the brain by direct administration into the parenchyma, and several cell types, including neurons and astrocytes, were transduced.30 31 32 33 34 Studies have shown prominent gene transduction to ependymal cells by injection of recombinant adenoviruses into the lateral ventricle,33 35 but gene transfer to cerebral blood vessels was not described. In the present study, we have demonstrated that administration of recombinant adenovirus provides gene transfer to cerebral blood vessels and, with higher efficiency, to the leptomeningeal cells overlying cerebral blood vessels.
Preferential expression in the leptomeninges overlying the major cerebral vessels was observed in the present study. One possible reason for the unique topology relates to the anatomic location of the vessels. The grooves on the surface of the brain that contain vessels are relatively large and may allow greater access by the viral suspension to the meninges overlying the vessels. A second possibility is that the meninges overlying the vessels have a greater susceptibility to infection.
Another novel observation in the present study was that gene targeting could be accomplished by altering the position of the head. When intracranial gene transfer is accomplished, the vector or the gene product may produce adverse effects in a variety of nonvascular tissues, including neurons, the pituitary, and regions that affect autonomic and other functions. It is important, therefore, to target specific regions for gene transfer. In the present study, the viral vector preferentially transduced the reporter gene in the most dependent part of the brain. Using a 293 cell assay, we have confirmed that recombinant virus alone does not settle in solution 30 minutes after the virus suspension is well mixed with PBS (data not shown). It is likely that the high specific gravity (1.21 g/mL) of the sucrose suspension coupled with a slow rate of flow of cerebrospinal fluid provided selective delivery of the virus to the most dependent part of the subarachnoid space. Although our gene targeting is regional and not tissue specific, we were able to express transgene in perivascular tissue and adventitia of the middle cerebral artery only in the right, not in the left, hemisphere when suspension of virus was injected with the animal in the right lateral position. Thus, by use of an adenovirus vector, it is possible to transfer genes to blood vessels in relatively limited regions of the brain.
Transfer of the reporter gene to the endothelium was not observed in the present study, and overall, transfection efficiency to media and adventitia of major cerebral arteries was low. We cannot exclude the possibility that recombinant viruses infected these cells but that the reporter gene was not expressed. Nevertheless, there are several reasons to be optimistic that alteration of cerebral vascular function may be achieved by this approach. First, X-Gal staining likely underestimates (up to 1/10) the number of cells that have been transduced,15 and higher sensitivity may be obtained by using other methods, such as fluorescein di-(ß-D-galactopyranoside) assay or immunostaining of the enzyme.31 34 In addition, transduction of a portion of cells in vessels may be sufficient to alter several important functions of vessels. Functional effects of a biochemical product, such as growth hormone,36 may be greater than expected on the basis of the efficiency of transgene expression to the target tissue. Finally, gene transfer to leptomeningeal cells overlying vessels may produce effects on the function of cerebral blood vessels, especially when diffusible mediators are considered. When viral vectors contain genes that encode enzymes (such as nitric oxide synthase), which produce highly diffusible substances, it is likely that release of the vasoactive products from the meninges may affect the underlying blood vessels.
Expression of ß-galactosidase was relatively short in the present study compared with other reports.5 7 10 11 12 The brief duration of expression may be based on loss of viral genome, transcriptional downregulation, or rapid turnover of target cells.4 9 10 Brief expression of the gene, however, has some advantages that can be of use in physiological and pathophysiological studies of cerebral vessels. For example, administration of a replication-deficient virus into cerebrospinal fluid may offer a therapeutic approach to the prevention of vasospasm after subarachnoid hemorrhage. Because vasospasm usually does not occur until several days after subarachnoid hemorrhage, it may be possible to prevent vasospasm with short-term expression of a gene that encodes a potent vasodilator substance. Candidate genes for that purpose include genes encoding nitric oxide synthase, because nitric oxide is highly diffusible and produces potent relaxation of cerebral blood vessels.37 38
Before gene therapy can be used for cerebral (or other) vessels, there are many limitations that will need to be overcome. First, expression of transgenes after the administration of adenovirus does not last sufficiently long for many therapeutic applications, even when different promoters are used.5 7 10 11 12 Although brief expression is optimal for the treatment of abnormalities of brief duration, longer expression may be necessary for the treatment of chronically diseased vessels. A second limitation concerns the safety of central injection of adenovirus. Although >10 million people have received live oral tablets as a viral vaccine with no detectable toxicity,3 the pathological effects after direct injection of recombinant adenovirus into cerebrospinal fluid will need to be analyzed carefully. In the present study, we observed a transient leukocytosis in cerebrospinal fluid and a slight increase in body temperature after injection of adenovirus. Third, expression of a functional transgene product will need to be achieved at a sufficient concentration in blood vessels without toxicity for other tissues.
In conclusion, adenovirus injected into cerebrospinal fluid provided gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to leptomeningeal cells overlying cerebral blood vessels. This approach, which does not require interruption of blood flow and circumvents the blood-brain barrier, may be useful in the study of cerebral vascular biology and in cerebral vascular gene therapy.
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Acknowledgments |
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This study was supported by National Institutes of Health grants NS-24621, HL-16066, HL-14388, AG-10269, and NS-34568; the Howard Hughes Medical Institute; research funds from the Veterans Administration; and funds from the Carver Trust of the University of Iowa. Dr Welsh is an Investigator of the Howard Hughes Medical Institute. Dr Davidson is a fellow of the Roy J. Carver charitable trust. We thank Pamela Tompkins, Lisa DeBurg, and Richard Anderson for their excellent assistance and Dr Frank M. Faraci for critical review of this manuscript. We also thank Alan E. Smith, Genzyme, for the gift of Ad2/CMV-ßGal and Ad2/CFTR-2.
Received January 31, 1995; accepted April 3, 1995.
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 V. G. Khurana, L. A. Smith, T. A. Baker, D. Eguchi, T. O'Brien, and Z. S. Katusic Protective Vasomotor Effects of In Vivo Recombinant Endothelial Nitric Oxide Synthase Gene Expression in a Canine Model of Cerebral Vasospasm Stroke, March 1, 2002; 33(3): 782 - 789. [Abstract] [Full Text] [PDF]
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 J. Ai Kim, C. C. Hedrick, Dangci Xie, and M. J. Fisher Adenoviral-Mediated Transfer of Tissue Plasminogen Activator Gene into Brain Capillary Endothelial Cells In Vitro Angiology, September 1, 2001; 52(9): 627 - 634. [Abstract] [PDF]
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M. A. Yenari, M. Minami, G. H. Sun, T. J. Meier, D. M. Kunis, J. R. McLaughlin, D. Y. Ho, R. M. Sapolsky, and G. K. Steinberg Calbindin D28K Overexpression Protects Striatal Neurons From Transient Focal Cerebral Ischemia Stroke, April 1, 2001; 32(4): 1028 - 1035. [Abstract] [Full Text] [PDF]
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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]
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H. Nakane, Y. Chu, F. M. Faraci, L. W. Oberley, D. D. Heistad, and P. H. Chan Gene Transfer of Extracellular Superoxide Dismutase Increases Superoxide Dismutase Activity in Cerebrospinal Fluid Editorial Comment Stroke, January 1, 2001; 32(1): 184 - 189. [Abstract] [Full Text] [PDF]
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 K. Toyoda, F. M. Faraci, Y. Watanabe, T. Ueda, J. J. Andresen, Y. Chu, S. Otake, and D. D. Heistad Gene Transfer of Calcitonin Gene-Related Peptide Prevents Vasoconstriction After Subarachnoid Hemorrhage Circ. Res., October 27, 2000; 87(9): 818 - 824. [Abstract] [Full Text] [PDF]
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 K. M. Channon, H. Qian, and S. E. George Nitric Oxide Synthase in Atherosclerosis and Vascular Injury : Insights From Experimental Gene Therapy Arterioscler Thromb Vasc Biol, August 1, 2000; 20(8): 1873 - 1881. [Abstract] [Full Text] [PDF]
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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]
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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]
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J. J. Zhang, L. Chao, J. Chao, Y. Chu, and D. D. Heistad Adenovirus-Mediated Kallikrein Gene Delivery Reduces Aortic Thickening and Stroke-Induced Death Rate in Dahl Salt-Sensitive Rats • Editorial Comment Stroke, September 1, 1999; 30 (9): e1925 - 1932. [Abstract] [Full Text] [PDF]
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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]
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I. J. Kullo, R. D. Simari, and R. S. Schwartz Vascular Gene Transfer : From Bench to Bedside Arterioscler Thromb Vasc Biol, February 1, 1999; 19(2): 196 - 207. [Full Text] [PDF]
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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]
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 K.-F. Lin, J. Chao, and L. Chao Atrial Natriuretic Peptide Gene Delivery Reduces Stroke-Induced Mortality Rate in Dahl Salt-Sensitive Rats Hypertension, January 1, 1999; 33(1): 219 - 224. [Abstract] [Full Text] [PDF]
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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]
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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]
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H. Onoue, M. Tsutsui, L. Smith, A. Stelter, T. O'Brien, Z. S. Katusic, and F. M. Faraci Expression and Function of Recombinant Endothelial Nitric Oxide Synthase Gene in Canine Basilar Artery After Experimental Subarachnoid Hemorrhage • Editorial Comment Stroke, September 1, 1998; 29(9): 1959 - 1966. [Abstract] [Full Text] [PDF]
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M. Tsutsui, A. F. Y. Chen, T. O'Brien, T. B. Crotty, and Z. S. Katusic Adventitial Expression of Recombinant eNOS Gene Restores NO Production in Arteries Without Endothelium Arterioscler Thromb Vasc Biol, August 1, 1998; 18(8): 1231 - 1241. [Abstract] [Full Text] [PDF]
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S. D. Christenson, K. D. Lake, H. Ooboshi, F. M. Faraci, B. L. Davidson, D. D. Heistad, and S. P. Finklestein Adenovirus-Mediated Gene Transfer In Vivo to Cerebral Blood Vessels and Perivascular Tissue in Mice • Editorial Comment Stroke, July 1, 1998; 29(7): 1411 - 1416. [Abstract] [Full Text] [PDF]
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 F. M. FARACI and D. D. HEISTAD Regulation of the Cerebral Circulation: Role of Endothelium and Potassium Channels Physiol Rev, January 1, 1998; 78(1): 53 - 97. [Abstract] [Full Text] [PDF]
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 A. F. Y. Chen, S.-W. Jiang, T. B. Crotty, M. Tsutsui, L. A. Smith, T. O'Brien, and Z. S. Katusic Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries PNAS, November 11, 1997; 94(23): 12568 - 12573. [Abstract] [Full Text] [PDF]
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I. J. Kullo, G. Mozes, R. S. Schwartz, P. Gloviczki, T. B. Crotty, D. A. Barber, Z. S. Katusic, and T. O'Brien Adventitial Gene Transfer of Recombinant Endothelial Nitric Oxide Synthase to Rabbit Carotid Arteries Alters Vascular Reactivity Circulation, October 7, 1997; 96(7): 2254 - 2261. [Abstract] [Full Text]
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S. S. Meyrelles, H. Z. Mao, D. D. Heistad, and M. W. Chapleau Gene Transfer to Carotid Sinus In Vivo : A Novel Approach to Investigation of Baroreceptors Hypertension, September 1, 1997; 30(3): 708 - 713. [Abstract] [Full Text]
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 G. Vassalli and D. A Dichek Gene therapy for arterial thrombosis Cardiovasc Res, September 1, 1997; 35(3): 459 - 469. [Abstract] [Full Text] [PDF]
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M. G. Muhonen, H. Ooboshi, M. J. Welsh, B. L. Davidson, and D. D. Heistad Gene Transfer to Cerebral Blood Vessels After Subarachnoid Hemorrhage Stroke, April 1, 1997; 28(4): 822 - 829. [Abstract] [Full Text]
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A. F.Y. Chen, T. O'Brien, M. Tsutsui, H. Kinoshita, V. J. Pompili, T. B. Crotty, D. J. Spector, and Z. S. Katusic Expression and Function of Recombinant Endothelial Nitric Oxide Synthase Gene in Canine Basilar Artery Circ. Res., March 1, 1997; 80(3): 327 - 335. [Abstract] [Full Text]
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D. D. Heistad and F. M. Faraci Gene Therapy for Cerebral Vascular Disease Stroke, September 1, 1996; 27(9): 1688 - 1693. [Abstract] [Full Text]
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C. D. Rios, H. Ooboshi, D. Piegors, B. L. Davidson, and D. D. Heistad Adenovirus-Mediated Gene Transfer to Normal and Atherosclerotic Arteries : A Novel Approach Arterioscler Thromb Vasc Biol, December 1, 1995; 15(12): 2241 - 2245. [Abstract] [Full Text]
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