© 1995 American Heart Association, Inc.
Articles |
Endothelium-Specific In Vivo Gene Transfer
From the Molecular Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health (A.H.S., G.D., K.D.N., D.A.D.), Bethesda, Md, and the Division of Cardiovascular Pathology, Armed Forces Institute of Pathology (R.V.), Washington, DC.
Correspondence to David A. Dichek, MD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco CA 94141-9100. E-mail david_dichek@quickmail.ucsf.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Targeted expression of genetic material within the vascular endothelium is potentially a powerful tool for the investigation of endothelial cell (EC) biology. We developed, optimized, and characterized an efficient somatic transgenic model of EC-specific gene transfer. Rat carotid arteries were infused with adenovirus expressing a ß-galactosidase (ß-gal) gene. The level and cell-type specificity of recombinant gene expression were measured by assaying ß-gal activity in vessel extracts and by counting transduced cells in histological sections. Toxicity was evaluated by counting total ECs (3 days) and by measuring neointimal formation (14 days). Effects of transduction on the proliferation of vascular cells were measured with bromodeoxyuridine and [3H]thymidine. Maximum recombinant gene expression resulted from infusion of 1x1010 to 1x1011 plaque-forming units (pfu) per milliliter;
35%
of luminal ECs were transduced. A high degree of EC specificity (90%
to 98% of total transduced cells) was maintained over this range of
virus concentrations. More highly concentrated virus resulted in loss
of ß-gal expression and a large decrease in luminal EC number (97%
decrease, P<.001). Gene transfer at 4x1010
pfu/mL was efficient, preserved EC integrity, and caused minimal
neointimal formation. After gene transfer, there were early
(3-day) increases in both EC and smooth muscle cell proliferation. At
14 days, only EC proliferation remained elevated (18% versus 1.4% in
vehicle-infused arteries, P=.005). This animal model permits
efficient highly EC-specific gene transfer. Vascular toxicity is
minimal, although the EC proliferative index is elevated. This model
will be useful in experiments that elucidate the biological role of EC
gene products and define pathways of EC gene regulation and signal
transduction in vivo.
Key Words: endothelium • gene transfer • adenovirus • carotid arteries
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The vascular endothelium plays a major role in the regulation of important biological processes, including hemostasis,1 vasomotion,2 3 and the initiation of atherosclerosis.4 The molecular mechanisms through which the endothelium participates in these diverse biological processes are, however, incompletely understood.
Transfer and expression of genetic material within the vascular endothelium in vivo represents a powerful tool for the investigation of EC biology. Overexpression of specific genes within the endothelium could permit elucidation of the biological roles of their protein products. In addition, in vivo introduction of genetic regulatory sequences spliced to reporter genes would allow the definition of DNA sequences involved in the regulation of EC gene expression after physiological stimulation. This general strategy has been accomplished in other tissues in vivo5 6 but never in endothelium. Moreover, EC-specific expression of either dominant negative or constitutively active components of signal transduction pathways7 could help clarify pathways of endothelial gene regulation in vivo.
The execution of informative in vivo experiments using targeted gene expression in endothelium is dependent on the development of an animal model system of reproducible, efficient, EC-specific gene transfer. With such a system, the level of expression of transferred genes would be easily quantified, and the biological consequences of recombinant gene expression would be confidently and specifically attributed to alterations in EC physiology. Previous work has suggested that adenovirus-mediated gene transfer might be suitably efficient and sufficiently EC specific to permit the execution of biological experiments predicated on EC-specific gene transfer8 9 10 ; however, this has never been demonstrated quantitatively. In addition, the question of whether adenovirus-mediated gene transfer per se perturbs the phenotype of a normal artery (a critical issue in the design and interpretation of endothelial gene transfer experiments) has not been addressed.
In the present study, we describe the development of an animal model system of somatic EC-specific gene transfer using an adenovirus vector. The system is optimized to allow the use of lower concentrations of adenovirus, and effects of virus exposure and gene transfer on vessel phenotype are defined.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Viruses and Plasmids
Av1LacZ4, a recombinant replication-defective adenovirus, contains a nuclear targeted Escherichia coli ß-gal gene under the control of the Rous sarcoma virus long terminal repeat promoter. The construction of this vector (initially provided by Bruce Trapnell, Genetic Therapy Inc) has been described in detail.11 Exposure of a cell transduced with this vector to X-Gal results in a histochemical reaction that stains the nucleus blue. To construct AdSV40Luc, an expression cassette containing the simian virus 40 early promoter driving expression of a firefly luciferase gene, was ligated into the Bgl II site of pAdBglII (a plasmid containing the adenovirus type 5 sequences 0 to 1 and 9.2 to 16.1 map units; kindly provided by Dr Blake Roessler, University of Michigan). The resulting plasmid was linearized by digestion with Nhe I and cotransfected into 293 cells along with the large Cla I fragment of DNA prepared from type 5 adenovirus strain dl327.12 AdHS-1.2, which contains a cDNA for biologically active hirudisin,13 was constructed by methodology essentially identical to that used to construct Av1LacZ4 and AdSV40Luc. Recombinant virus was plaque-purified and propagated on 293 cells. Concentrated stocks of viruses were prepared by cesium banding and dialyzed against 10 mmol/L Tris-HCl (pH 7.5) and 1 mmol/L MgCl2 containing 10% glycerol. Aliquots of 10 to 50 µL of virus stock were stored over liquid nitrogen until use. The titers of concentrated virus stocks (1 to 2x1011 pfu/mL for Av1LacZ4, 5x1010 pfu/mL for AdSV40Luc, and 5x1011 pfu/mL for AdHS-1.2) were determined by plaque titration on 293 cells. The plasmid pCMVLuc,14 containing the human cytomegalovirus immediate-early promoter driving expression of a firefly luciferase gene, was used in cointernalization studies (see below).
Animals
All animal procedures were approved by the Animal Care and Use
Committee of the National Heart, Lung, and Blood Institute. Adult male
Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing
350 to 450 g were used. Rats were anesthetized with
intraperitoneal injections of ketamine
(Fort Dodge Laboratories, Inc) and xylazine (Miles Inc) in doses of 100
and 10 mg/kg, respectively. After surgery, animals were allowed water
ad libitum but were not allowed food until 8 to 12 hours later in order
to minimize the risk of aspiration.
In Vivo Gene Transfer Into Vessel Segments
To perform in vivo arterial gene transfer, the left
carotid system was surgically exposed, proximal and distal control was
obtained, and an arteriotomy was made on the external carotid artery. A
1-cm segment of common carotid artery was isolated. A 24-gauge
polytetrafluoroethylene catheter was introduced through the external
carotid arteriotomy, and the isolated vessel segment was flushed with
1 mL medium 199 (Biofluids).
Aliquots of adenovirus were thawed, placed on wet ice, and used within 2 hours. Medium 199 containing rat serum albumin (1 mg/mL, Sigma Chemical Co) was used as needed to dilute virus stocks. For each rat, 50 µL of adenovirus-containing or control solution were drawn up into a 24-gauge polytetrafluoroethylene catheter mounted on a tuberculin syringe. The catheter was inserted into the external carotid arteriotomy and secured with a silk tie, and the solution was infused into the isolated carotid segment, resulting in distension of the common carotid artery. The solution was allowed to dwell in the vessel segment for 20 minutes, during which time the carotid segment remained distended. The solution was then withdrawn into the catheter, the catheter was removed, and external carotid artery was ligated. Blood flow was reestablished through the common and internal carotid arteries.
Fixation and Removal of Vessel Segments
Animals were killed by overdosing with pentobarbital (600
mg/kg IP). At the time of death, none of the animals had clinical
evidence of wound infection, and all common carotid arteries were
patent. For experiments involving histological
examination of vessels, perfusion fixation was carried out 3, 7, or 14
days after gene transfer. After retrograde cannulation of the abdominal
aorta and division of the left jugular vein, the arterial
tree was cleared of blood by perfusion with PBS (pH 7.4) at a pressure
of 100 mm Hg. After clearing, vessels were perfusion-fixed in situ
with 2% formaldehyde (Mallinckrodt) and 0.2%
glutaraldehyde (Sigma) in PBS (pH 7.2). Fixative was
perfused through the arterial cannula at 100 mm Hg for 5
minutes. The perfusion-fixed vessels were then excised and incubated in
fixative for an additional 2 hours. Other arteries were not
perfusion-fixed but were removed and immediately frozen for subsequent
measurement of recombinant ß-gal or luciferase activity.
Evaluation of Recombinant ß-gal and Luciferase Gene
Expression
Recombinant ß-gal expression was evaluated 3 days after gene
transfer by two methods: (1) histochemical staining of excised fixed
arteries with X-Gal and subsequent identification of blue-stained
nuclei, both grossly and on histological sections, and
(2) detergent extraction of excised arteries and measurement of ß-gal
activity in the tissue extracts. At 7 and 14 days after gene transfer,
ß-gal expression was evaluated by histochemical staining alone. For
histochemical staining, vessels were washed in PBS and incubated in
X-Gal for 2 hours, as described previously.11 The arteries
were then divided into segments of 2 to 4 mm in length, and these
segments (2 to 5 per vessel) were embedded in paraffin blocks. Sections
(5 µm thick) were cut from the blocks and counterstained with nuclear
fast red. For Av1LacZ4-exposed vessels, both total and X-Gal–stained
ECs (identified by characteristic morphology, luminal location, and
specific staining; see below) per histological cross
section were counted. The number of sections counted per vessel ranged
from 4 to 12. Initially, cells were counted in 8 to 12 randomly spaced
sections per vessel; however, the variability of both total and
X-Gal–stained cells between sections was so low (data not shown) that
in later experiments only 4 sections per vessel were counted. The
vessel cross sections were always spaced a minimum of 50 µm apart to
avoid counting the same cells twice. From the data obtained from
individual cross sections, the mean transduced cell number per section
was calculated for each artery. A mean percentage of transduced ECs per
vessel section was also calculated for each artery by dividing the mean
number of transduced ECs per section by the mean number of total ECs
per section.
Recombinant ß-gal or luciferase activity in vessel extracts was
measured after mincing each vessel into small pieces in lysis buffer
(200 µL per vessel; 0.2% Triton X-100 in 100 mmol/L potassium
phosphate [pH 7.8] for ß-gal measurement, and 1% Triton X-100, 100
mmol/L potassium phosphate, 1 mmol/L dithiothreitol, and 2 mmol/L EDTA
[pH 7.8] for luciferase measurement). Minced pieces were further
homogenized in a Polytron (Brinkman), and aliquots of
vessel lysate were assayed for ß-gal or luciferase activity. ß-gal
activity was measured with the substrate
3-(4-methoxyspirol[1,2-dioxetane-3,2'-tricyclo-[3.3.1.13,7]decan]-4-yl)phenyl-ß-D-galactopyranoside
(Galacto-Light, Tropix). Luciferase activity was measured with the
substrate
2-(6'-hydroxy-2'-benzothiazolyl)-
2-thiazoline-4-carboxylic
acid (D-luciferin, Analytical Luminescence Laboratory).
Light emission for both assays was measured with a Monolight 2010
luminometer (Analytical Luminescence Laboratory). For Av1LacZ4,
standard curves were generated by using purified E coli
ß-gal (specific activity, 300 U/mg; Boehringer Mannheim).
Luciferase activity in AdSV40Luc-transduced cells was quantified in
RLU. All samples were assayed in duplicate. Total protein in vessel
extracts was measured with the BCA protein assay (Pierce), with bovine
serum albumin used as a standard.
Evaluation of the EC Specificity of Gene Transfer
The EC specificity of in vivo gene transfer was assessed by
examination of histological sections taken from animals
killed 3 days after gene transfer. The same
histological sections used to calculate the number and
percentage of transduced ECs (see above) were examined for the presence
of blue-stained nuclei in the vascular media. Medial cells were
identified as smooth muscle cells by characteristic spindle morphology
and by immunohistochemical staining of companion sections by use of an
antibody to smooth muscle cell actin (Sigma Chemical) at a dilution of
1:10 000. For each vessel, the mean number of smooth muscle cells with
blue nuclei per histological section was calculated. EC
specificity of transduction was then determined for each vessel by the
following formula: mean X-Gal–positive (ie, having blue nuclei) ECs
per section/(mean X-Gal–positive ECs per section+mean X-Gal–positive
smooth muscle cells per section)x100.
Evaluation of EC Number in Vessel Sections
To evaluate the potential acute toxicity of the Av1LacZ4 vector,
we counted total luminal ECs present in
histological sections of control and virus-infused
vessels harvested 3 days after gene transfer. Vessel sections used for
this analysis included those described above (taken from
arteries exposed to Av1LacZ4) as well as identically prepared sections
taken from arteries exposed to control solutions (see below). Total
luminal ECs were again counted in a minimum of four randomly chosen
microscopic cross sections per vessel, separated by at least 50 µm.
To confirm the identification of luminal cells as ECs, additional
vessel cross sections were cut from the same paraffin blocks used to
provide sections for cell counting and were processed for
immunohistochemical detection of von Willebrand factor.
Staining was performed by use of an antibody to von Willebrand
factor (Dako Corp) at a dilution of 1:3200. Bound antibody was detected
with a secondary antibody conjugated to horseradish peroxidase.
Two groups of control vessels were used for the biochemical and histological analyses described above. One group was infused with medium 199 containing 1 mg/mL rat serum albumin, the medium used to dilute virus stocks and hereafter referred to as "viral diluent." This group served as the control for studies involving the evaluation of recombinant ß-gal gene expression, both in tissue extracts and in X-Gal–stained cross sections. An additional group of control vessels was used for studies involving analysis of potential acute (3 days after gene transfer) endothelial loss after exposure to Av1LacZ4. This second series of control vessels was exposed to 10 mmol/L Tris-HCl (pH 7.5) and 1 mmol/L MgCl2 containing 10% glycerol, the vehicle used in storing virus stocks and hereafter referred to as "virus storage buffer." By controlling for both the virus storage buffer and diluent, any differences in EC number between virus-treated and control vessels could be attributed to exposure to adenovirus.
Scanning Electron Microscopy
Carotid arteries were perfusion-fixed and stained with X-Gal as
described above. Vessels were then divided in half longitudinally,
dehydrated, critical point–dried with liquid carbon dioxide, coated
with gold palladium in a plasma coater, and visualized with a Jeol
JSM35 scanning electron microscope.15
Evaluation of EC and Smooth Muscle Cell Proliferation
We investigated the effect of adenovirus exposure on EC and
smooth muscle cell proliferation by using the proliferation markers
BrdU and [3H]thymidine. EC proliferation was assessed 3,
7, and 14 days after transduction (with BrdU); medial smooth muscle
cell proliferation was assessed 3 and 14 days after transduction (with
BrdU). In addition, the ability of the transduced EC population to
undergo mitosis 3 days after transduction was investigated by combined
X-Gal staining and [3H]thymidine
autoradiography. At 17, 9, and 1 hour before death,
animals were given either BrdU by subcutaneous administration (30 mg/kg
body wt per dose, Sigma) or [3H]thymidine by
intraperitoneal injection (50 mg/kg body wt per
dose, New England Nuclear). Animals were killed, and carotid vessels
were excised, X-Gal–stained, processed, and embedded in paraffin as
described above. In addition to the left carotid artery, the right
carotid artery (as a control for baseline EC mitosis in the animal) and
a segment of proximal duodenum (as a positive control for cell mitosis)
were harvested from each animal and processed in the same manner as the
left carotid artery. Because of a concern (for animals treated with
BrdU) that the nuclear localized X-Gal staining might interfere with
binding or detection of anti-BrdU antibody, vessel (and
positive-control duodenal) sections of these animals were immersed in
xylene overnight to extract the blue reaction product. Anti-BrdU
staining was then performed by using a specific antibody (Sigma) at a
dilution of 1:100. Bound antibody was detected with a secondary
antibody conjugated to horseradish peroxidase. The BrdU proliferative
index of ECs was calculated for each artery from two cross sections per
vessel from the following formula: mean BrdU-positive ECs per cross
section/mean total ECs per cross sectionx100.
The rate of medial smooth muscle cell proliferation (as measured by the BrdU index) was determined on the same histological sections used to assess EC proliferation. Smooth muscle cell proliferation was measured at 3 and 14 days after gene transfer. The BrdU index of medial smooth muscle cells in unmanipulated right carotid arteries, harvested from the same animals and processed identically to the left sided vessels, was also determined and used as the baseline mitotic rate of smooth muscle cells in carotid artery media. The BrdU index of smooth muscle cells was calculated for each artery from one cross section per vessel from the following formula: BrdU positive smooth muscle cells per cross section/total smooth muscle cells per cross sectionx100.
The ability of adenovirus-transduced ECs to undergo mitosis at 3 days
after gene transfer was evaluated with [3H]thymidine
instead of BrdU, since X-Gal staining was less likely to interfere with
autoradiography than with anti-BrdU
immunohistochemistry. Sections of 5 µm thickness were dipped in Kodak
NTB-2 emulsion, stored at 4°C for 3 weeks, and developed with Kodak D
19 developer. Slides were then counterstained with nuclear fast red.
ECs with at least 10 overlying silver grains were identified
"[3H]thymidine positive" and therefore as
proliferative. The proliferation index of ECs was calculated for each
vessel from the mean of two cross sections (the variability in
proliferative indices between two sections taken from a particular
vessel was typically 
25%). In addition to the overall EC
proliferative index, [3H]thymidine indices were also
calculated individually for both the transduced (X-Gal–positive) and
untransduced EC populations.
Heat Inactivation of Av1LacZ4
To achieve low temperature heat inactivation,16
aliquots of virus were incubated at 45°C for 15 minutes, cooled at
4°C for 10 minutes, and then used immediately. Cos-7, a simian virus
40–transformed African green monkey kidney cell line, and 293 cells
were grown in improved MEM supplemented with 10% fetal bovine serum, 1
mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and
0.25 µg/mL amphotericin (all from Biofluids). Experiments in Cos-7
and 293 cells were carried out in 24- and 96-well tissue culture
plates, respectively.
Experiments to determine the effect of heat inactivation on the ability of Av1LacZ4 to mediate endosomal lysis and plasmid-mediated transfection were carried out in Cos-7 cells. Cos-7 cells (1x105 cells per well) were washed with Opti-MEM1 (GIBCO/BRL) and then further incubated in the same medium (225 µL per well) for 2 hours. pCMVLuc (5.0 µg diluted to a volume of 20 µL with Opti-MEM1) was added to each well, followed immediately by the addition of Av1LacZ4 (2.5x107 pfu diluted to 25 µL in Opti-MEM1). After 2 hours at 37°C, an additional 240 µL of Opti-MEM1 was added to each well. Twenty-two hours later, the medium was changed to improved MEM containing 10% calf serum and 1 mmol/L glutamine. After an additional 24-hour incubation, the cells were washed twice with PBS, lysed, and assayed for luciferase activity as described above. Experiments to determine the effect of heat inactivation on the ability of Av1LacZ4 to transfer a functional ß-gal gene were carried out in 293 cells. 293 cells (3x104 cells per well) were infected with Av1LacZ4 at mutiplicities of infection of 10, 100, and 1000 in a volume of 100 µL. Twenty-four hours later, the cells were washed with PBS, fixed, and stained with X-Gal as described above.
Statistics
For each group of vessels receiving the same treatment in
individual experiments, mean values were calculated from the values for
the individual vessels constituting the group (eg, total ECs, total and
percentage X-Gal–positive ECs, X-Gal–positive smooth muscle cells,
BrdU-positive ECs, BrdU-positive and total smooth muscle cells, and
[3H]thymidine-positive ECs). Means of experimental groups
were compared with means of control groups, and differences were
evaluated for significance by Student's unpaired t test.
Results are reported as mean±SD unless indicated otherwise. Not all
possible pairwise tests were performed; differences were considered to
be significant at P<.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In Vivo Gene Transfer and Expression of ß-gal
Seventy-one rat carotid arteries were incubated in vivo with solutions containing the Av1LacZ4 vector. Fifty-two vessels were fixed, stained with X-Gal, and then sectioned and used for histological studies; 17 vessels were homogenized and used for ß-gal activity studies; and 2 vessels were fixed and then processed en bloc for scanning electron microscopy to evaluate intimal integrity. Several of the vessels that were histologically sectioned were used for more than one assay; eg, recombinant gene expression was measured (by counting blue nuclei) and proliferative rate was determined (by counting BrdU-stained cells) on sections from the same vessel. For this reason, the total n value for assays on Av1LacZ4-infused vessels was >71. Twenty-four vessels were infused with control solutions, as detailed further below.
Efficiency and Specificity of EC Gene Transfer
To determine both the efficiency of EC gene transfer and the
percentage of transduced cells that were ECs, 20 vessels were
transduced at concentrations of Av1LacZ4 ranging from
1x109 to 2x1011 pfu/mL, fixed, stained with
X-Gal, and sectioned. Vessels were exposed to virus at concentrations
of 1x109 pfu/mL (n=2), 2x109 pfu/mL (n=3),
1x1010 pfu/mL (n=2), 2x1010 pfu/mL (n=3),
4x1010 pfu/mL (n=2), 5x1010 pfu/mL (n=2),
1x1011 pfu/mL (n=3), and 2x1011 pfu/mL (n=3).
Controls (n=3) received the viral diluent only.
Three days after transduction, arteries were harvested, stained with
X-Gal, and sectioned. The vast majority of X-Gal–positive cells seen
in tissue sections were identified as ECs (see "Materials and
Methods"). The number of transduced ECs was determined by counting
X-Gal–positive ECs in cross sections of vessels (Fig 1
). Recombinant gene expression was found in those
vessels exposed to concentrations of Av1LacZ4 from 2x109
to 1x1011 pfu/mL. Control vessels and vessels exposed to
1x109 pfu/mL Av1LacZ4 showed no X-Gal–stained cells;
vessels exposed to 2x109 pfu/mL Av1LacZ4 showed only very
few stained cells (mean, three per section; range, one to six).
Infusion of Av1LacZ4 in the range of 1x1010 to
1x1011 pfu/mL resulted in recombinant gene expression
within 42±20 (range, 19 to 91) ECs per cross section. More
specifically, the mean number of X-Gal–stained ECs at
1x1010 pfu/mL was 31 (range, 29 to 32) per cross section.
At 2x1010 pfu/mL, 31 (range, 19 to 38) X-Gal–stained ECs
per section were present; at 4x1010 pfu/mL, 50 (range,
36 to 64) X-Gal–stained ECs per section were present; at
5x1010 pfu/mL, 31 (range, 25 to 37) X-Gal–stained ECs per
section were present; and at 1x1011 pfu/mL, 62 (range,
36 to 91) X-Gal–stained ECs per section were present. This range
of total transduced ECs per section represents recombinant
gene expression in 34±15% of total luminal ECs (mean±SD of all
vessels transduced with Av1LacZ4 concentrations ranging from
1x1010 to 1x1011 pfu/mL, n=12). Strikingly,
exposure to Av1LacZ4 at the highest concentration (2x1011
pfu/mL, undiluted virus stock) resulted in complete absence of X-Gal
staining both macroscopically and in histological
vessel cross sections (n=3). This unanticipated finding was supported
by a concomitant lack of ß-gal activity in detergent extracts of two
additional vessels transduced at 2x1011 pfu/mL (see
below).
|
Although the vast majority of X-Gal–positive cells in vessels exposed
to Av1LacZ4 were identified as ECs (Fig 2A), occasional
X-Gal–positive cells were identified as smooth muscle cells (see
"Materials and Methods"; Fig 2B). The relation between virus
concentration and EC specificity of transduction was determined by
counting total transduced cells as well as transduced ECs in sections
taken from vessels transduced with viral solutions containing
2x109 to 1x1011 pfu/mL (Fig 3). With exposure to Av1LacZ4 at 2x109
pfu/mL, 90% of X-Gal–stained cells were ECs (n=3; range, 75% to
100%). At 1x1010 pfu/mL, 96% of X-Gal–stained cells
were ECs (n=2; range, 95% to 96%); at 2x1010 pfu/mL,
95% of X-Gal–stained cells were ECs (n=3; range, 92% to 97%); at
4x1010 pfu/mL, 95% of X-Gal–stained cells were ECs (n=2;
range, 94% to 96%); at 5x1010 pfu/mL, 98% of
X-Gal–stained cells were ECs (n=2; range, 98% to 99%); and at
1x1011 pfu/mL, 95% of X-Gal–stained cells were ECs (n=3;
range, 90% to 100%). Thus, a very high degree of EC specificity
(means, 90% to 98%) was maintained throughout the entire range of
virus concentrations at which gene transfer occurred. No adventitial
staining was observed in any of the vessels.
|
|
Because we found a relatively constant level of EC transduction over
vector concentrations ranging from 1x1010 to
1x1011 pfu/mL and because our results showed a loss of
recombinant gene expression at the highest vector concentration
(2x1011 pfu/mL), we performed additional experiments to
aid in the definition of ideal vector concentration. Arteries were
exposed to Av1LacZ4 at concentrations of 1x109 pfu/mL
(n=2), 2x109 pfu/mL (n=3), 4x109 pfu/mL
(n=3), 1x1010 pfu/mL (n=2), 4x1010 pfu/mL
(n=2), 6x1010 pfu/mL (n=1), 1x1011 pfu/mL
(n=2), and 2x1011 pfu/mL (n=2). Controls (n=3) received
viral diluent. Three days after transduction, arteries were harvested,
and ß-gal activity was measured from tissue extracts (Fig 4). Extracts of vessels exposed to 1x109
and 2x109 pfu/mL had mean ß-gal activities of 1.3
(range, 1.1 to 1.4) µU/µg total vessel protein and 0.8 (range, 0.5
to 1.2) µU/µg, respectively. These values were virtually identical
to those of control vessels (0.8 [range, 0.4 to 1.3] µU/µg total
vessel protein). Incubation of the arterial segments with
higher concentrations of Av1LacZ4 in the range of 4x109 to
1x1011 pfu/mL resulted in an increase in ß-gal activity.
Mean ß-gal activity at 4x109 pfu/mL was 7.9 (range, 6.4
to 10.4) µU/µg total vessel protein; at 1x1010 pfu/mL,
activity was 12.1 (range, 11.5 to 12.7) µU/µg total vessel protein;
at 4x1010 pfu/mL, activity was 8.4 (range, 7.4 to 9.3)
µU/µg total vessel protein; at 6x1010 pfu/mL, activity
was 21.1 µU/µg total vessel protein (n=1); and at
1x1011 pfu/mL, activity was 12.5 (range, 8.5 to 16.5)
µU/µg total vessel protein. Exposure to Av1LacZ4 at a concentration
of 2x1011 pfu/mL resulted in a dramatic decrease in
ß-gal activity in tissue extracts (1.6 [range, 1.5 to 1.7]
µU/µg total vessel protein), approximately equivalent to control
values. When combined with the data from X-Gal–stained vessels (Fig 1
), these results indicate that maximal levels of both transduced ECs
and recombinant gene expression are achieved at virus concentrations
(5x109 to 5x1010 pfu/mL) significantly below
those maximally attainable. Therefore, we performed most of the
remaining studies at a virus concentration of 4x1010
pfu/mL. The loss of recombinant gene expression with undiluted virus
(2x1011 pfu/mL), as seen both in vessel extracts and with
X-Gal staining (Figs 1 and 4), was likely due to acute toxicity of the
viral infusate, as we have reported previously for injured
vessels,17 and as will be discussed later.
|
Duration of Recombinant ß-gal Gene Expression
To define further the ability of this gene transfer system to
permit the study of recombinant gene expression in normal uninjured
vessels, we performed a series of time-course studies. Our goals were
first to examine the duration of recombinant gene expression and then
to define the effects (if any) of the gene transfer protocol on the
arterial phenotype during the time period of
maximal recombinant gene expression. We began by studying the duration
of recombinant gene expression in 20 rat carotid arteries transduced
with Av1LacZ4 at a concentration of 4x1010 pfu/mL.
Arteries were harvested at 3 days (n=10), 7 days (n=6), and 14 days
(n=4) after gene transfer, X-Gal–stained, and sectioned. Control
vessels (n=5 at each time point) were infused with viral diluent and
were processed identically to the virus-infused vessels. Three days
after gene transfer, 45±26 transduced ECs per
histological section were present (range, 20 to 98
stained cells per section; mean, 34% of total ECs; Fig 5). At 7 days, 47±10 transduced ECs per section were
present (range, 37 to 66 stained cells per section; mean, 39% of
total ECs); at 14 days, 20±23 transduced ECs were present (range,
4 to 53 stained cells per section; mean, 17% of total ECs). Thus,
significant recombinant gene expression was detectable for up to 2
weeks after gene transfer but at a level reduced at 14 days when
compared with that found at 3 to 7 days. No X-Gal staining was observed
in control vessels.
|
Vessel Morphology After Adenovirus Gene Transfer
We next assessed whether gene transfer into uninjured arteries
resulted in alterations in vessel morphology. Vessel morphology was
evaluated histologically by assessing
endothelial integrity, by evaluating the presence of
acute medial necrosis and acute inflammatory cell infiltrates, and by
examining vessels for the development of a neointima and
the presence of chronic inflammatory infiltrates at a later time point
(14 days).
We assessed the acute effects of gene transfer on
endothelial integrity 3 days after gene transfer by
using the same histological sections of rat carotid
arteries that were used for the study of dose responsiveness and EC
specificity (Figs 1 and 3, n=19). Therefore, the concentrations of
Av1LacZ4 ranged from 1x109 to 2x1011 pfu/mL.
Both viral diluent (n=3) and virus storage buffer (n=3) were included
as controls.
Light microscopic analysis of histological
sections revealed that vessels infused with either control solution and
vessels infused with Av1LacZ4-containing solutions (from
1x109 to 1x1011 pfu/mL) had an intact luminal
endothelial layer (Fig 6A). In contrast,
infusion of Av1LacZ4 at a concentration of 2x1011 pfu/mL
resulted in virtually complete denudation of the
endothelium (Fig 6B).
|
The relation between virus concentration and EC number was addressed in
a more quantitative manner by counting ECs in stained tissue sections.
After infusion of Av1LacZ4 at a concentration of 2x1011
pfu/mL, EC number was significantly decreased to 3% of control values
(3±4 cells per section versus 139±12 cells with infusion of virus
storage buffer control, P<.001, Fig 7).
Infusion of Av1LacZ4 at concentrations below 2x1011 pfu/mL
did not affect the number of luminal ECs.
|
To achieve a more complete assessment of the effect of gene transfer
(at submaximal virus concentration) on the integrity of the luminal
endothelium, we performed scanning electron microscopy
of two vessels that had been exposed to 4x1010 pfu/mL
Av1LacZ4. Imaging of the entire luminal surface established that the
integrity of the luminal endothelium was not
compromised 3 days after gene transfer (Fig 8).
|
Because we previously observed medial necrosis and neutrophilic medial infiltrates in balloon-injured carotid arteries transduced at high virus concentrations,17 we examined sections of vessels transduced with Av1LacZ4 from 1x109 to 2x1011 pfu/mL for these features. No evidence of medial necrosis or inflammatory infiltrates was found in the examination of hematoxylin and eosin–stained sections taken at 3, 7, or 14 days after gene transfer.
We also examined histological sections of vessels of
animals killed 14 days after gene transfer for additional evidence of
vessel injury. Evidence for vessel injury was obtained by examination
for the presence of a neointima (defined as one or more
cell layers between the internal elastic lamina and the luminal
endothelium). Arteries were harvested from seven rats
exposed to Av1LacZ4 at 4x1010 pfu/mL and five rats exposed
to viral diluent only. Only small areas of neointima were
present in five of seven vessels exposed to Av1LacZ4 (15 of 28
total cross sections contained neointima);
neointima was absent in the other two vessels. Among the
diluent-exposed vessels, two of five vessels (5 of 20 total cross
sections) contained neointima. When present, the
neointima generally encompassed <20% of the luminal
circumference and in all cases was limited to one to two cell layers in
thickness (Fig 9). Thus, little evidence of vessel
injury could be found 14 days after virion infusion. We conclude from
these morphological studies that significant vessel toxicity,
manifested by endothelial denudation, results from
exposure to highly concentrated virus. At lower virus concentrations,
there is only very slight evidence of vascular injury at times up to 14
days after gene transfer.
|
To determine if the loss of ECs with exposure to virus stock was specific for Av1LacZ4, we investigated the effects of AdHS-1.2 infusion. After infusion of undiluted AdHS-1.2 stock (5x1011 pfu/mL, n=6), EC number was significantly decreased to 37% of control values (51±21 cells per section versus 139±12 cells with infusion of virus storage buffer control, P<.001). Infusion of AdHS-1.2 at 1x1011 pfu/mL resulted in a smaller decrease in luminal ECs (114±11 cells per section, n=2, P=.10).
Infusion of Heat-Inactivated Adenovirus
Heat inactivation of adenovirus at 45°C blocks endosome
disruption but not virus binding or internalization; the result is a
failure to achieve recombinant gene expression in cells transduced with
heat-inactivated adenovirus vectors.16
Aliquots of Av1LacZ4 were heat-inactivated and tested both
for their ability to enhance plasmid-mediated gene transfer and their
ability to transfer a functional ß-gal gene. Heat inactivation
resulted in a 270-fold decrease in the ability of Av1LacZ4 to enhance
transfer of a luciferase-expressing plasmid (pCMVLuc) into Cos-7 cells
and a 4 log decrease in the efficiency of ß-gal gene transfer into
293 cells (data not shown). We infused three arteries with aliquots of
heat-inactivated Av1LacZ4 at a concentration of
2x1011 pfu/mL. Infusion resulted in no detectable ß-gal
expression (by X-Gal stain) yet a significant decrease in EC number
compared with arteries infused with virus storage buffer control (2±1
versus 139±12 cells per section, P<.001).
EC and Smooth Muscle Cell Proliferation After Exposure to
Av1LacZ4
To characterize further the effect of gene transfer on the
arterial phenotype, we measured the proliferative
rate of both ECs and smooth muscle cells in transduced
arterial segments. Twenty-one rat carotid arteries were
exposed to Av1LacZ4 at a concentration of 4x1010 pfu/mL
and were harvested at 3 (n=8), 7 (n=6), and 14 (n=7) days after
transduction. For each of the three time points, we also examined five
control vessels that were infused with viral diluent alone. Vessels
exposed to Av1LacZ4 and harvested 3 days after gene transfer had an EC
BrdU proliferation index of 18.1±7.3%, nearly 10-fold greater than
that measured in the right carotid arteries (2±1.1%,
P<.001, n=5). However, this elevated proliferation index
was not significantly different from that calculated for left carotid
arteries receiving diluent only (13.5±8.1%, P=.31, Fig 10). Seven days after gene transfer, the EC BrdU index
for virus-treated vessels was 14.5±13.1% versus 3.5±1.5% for
diluent-treated vessels. This 4-fold difference between the indices was
not significant (P=.12). Fourteen days after gene transfer,
the EC BrdU index of Av1LacZ4-exposed vessels remained elevated at
17.7±9.8%, a significant 13-fold increase over the value of
1.4±1.6% for diluent-treated vessels (P=.005). Therefore,
adenovirus transduction resulted in a significant increase in EC
proliferation at 14 days.
|
The same anti-BrdU–stained cross sections from arteries exposed to Av1LacZ4 and harvested 3 (n=8) and 14 (n=7) days after gene transfer were used to analyze the effect of the gene transfer protocol on medial smooth muscle cell proliferation. Three days after gene transfer, the proliferative index of medial smooth muscle cells in virus-exposed vessels was essentially equal to that of vessels infused with viral diluent (9.1±8.0% versus 7.4±4.6%, P=.68). Fourteen days after gene transfer, the BrdU index of medial smooth muscle cells in virus-exposed vessels decreased but remained essentially identical to the index of diluent-exposed vessels (0.9±0.8% versus 0.6±0.6%, P=.40). The proliferative index of medial smooth muscle cells in the unmanipulated right carotid arteries was 0.3±0.3% at 3 days and 0.4±0.4% at 14 days. Therefore, medial smooth muscle cell proliferative rates increased early after the surgical procedure but were not specifically affected by gene transfer.
Proliferation of the Transduced EC Population
Because the EC proliferative index was increased in transduced
vessels, we sought to determine whether the proliferating cells were
specifically transduced or untransduced cells. Combined
[3H]thymidine autoradiography and
X-Gal staining was performed on sections of four rat carotid arteries
that had been exposed to Av1LacZ4 at a concentration of
4x1010 pfu/mL and harvested 3 days after transduction (Fig 11). The [3H]thymidine index for the
X-Gal–positive (transduced) population of ECs was 11.3±3.4%,
essentially identical to that of the X-Gal–negative (untransduced) EC
population (15.3±4.7%, P=.21). Therefore, the transduced
EC population includes mitotically active cells, with no difference in
mitotic activity specifically attributable to gene transfer.
|
In Vivo Gene Transfer and Expression of Luciferase
We considered the possibility that certain of our results, such as
the plateau of recombinant gene expression and the range of virus
concentration over which this plateau occurred, might be features that
were unique to the Av1LacZ4 virus. Therefore, we carried out additional
experiments with the AdSV40Luc vector, which has both a different
promoter and a different transgene than Av1LacZ4. Seventeen rat carotid
arteries were exposed to AdSV40Luc, then removed after 3 days, and
assayed for recombinant luciferase activity. The arteries were exposed
to diluted AdSV40Luc at concentrations of 8x107 pfu/mL
(n=2), 4x108 pfu/mL (n=3), 2x109 pfu/mL
(n=3), and 1x1010 pfu/mL (n=4) and to undiluted virus
stock at 5x1010 pfu/mL (n=5). Controls (n=3) received
viral diluent. Three days after gene transfer, arteries were harvested,
and luciferase activity was quantified in tissue extracts (Fig 12). Luciferase activity was undetectable in extracts
of vessels exposed to control solution (<1 RLU/µg total vessel
protein). Extracts of arteries exposed to AdSV40Luc at
8x107 and 4x108 pfu/mL had low levels of
luciferase activity, mean values of 300 (range, 120 to 470) and 220
(range, 49 to 520) RLU/µg total vessel protein, respectively.
Exposure of vessels to higher concentrations of AdSV40Luc resulted in
an increase in luciferase activity. Mean luciferase activity at
2x1010 pfu/mL was 3100 (range, 1500 to 5100) RLU/µg
total vessel protein; at 1x1010 pfu/mL, activity was 5300
(range, 1400 to 15000) RLU/µg total vessel protein; and at
5x1010 pfu/mL, activity was 3600 (range, 720 to 9300)
RLU/µg total vessel protein. Thus, a plateau of recombinant gene
expression as virus concentration approaches 1010 pfu/mL
appears to be a constant feature of EC-specific gene transfer in the
rat carotid artery. Whether this same concentration of virus achieves a
plateau of recombinant gene expression in other species will require
specific experimentation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The present study demonstrates the validation of a model of in vivo EC-specific arterial gene transfer with adenovirus vectors. Specifically, the major findings are as follows: (1) Recombinant gene expression is
95% EC specific over a wide range of
virus concentrations. (2) Approximately 35% of luminal ECs express the
recombinant gene. (3) The level of recombinant gene expression is
easily quantified in vessel extracts. (4) Peak levels of gene
expression can be achieved at submaximal virus concentrations,
permitting conservation of virus stocks and avoiding acute toxicity.
(5) At submaximal virus concentrations, the phenotype of a
transduced rat carotid artery is minimally altered by gene transfer,
with increased EC (but not smooth muscle cell) proliferation, only
slight neointimal formation, and no evidence of EC loss or
inflammatory infiltrates. (6) Toxicity resulting in luminal EC loss
occurs only with exposure to very high concentrations of
adenovirus. Several groups have reported animal models of high-efficiency adenovirus vector–mediated gene transfer into peripheral arteries. Lemarchand et al8 described what appeared to be largely endothelial gene transfer in sheep carotid arteries, but the extent of EC specificity was not reported. Moreover, the level of gene transfer was not quantified or optimized, and no data were presented concerning the phenotype of the transduced arteries. In two previous studies from our laboratory, adenovirus-mediated gene transfer into sheep carotid arteries resulted in significant recombinant gene expression in both the luminal endothelium and the adventitia, with access of virions to the adventitia through branches of vasa vasorum that bypass the vascular media.9 10 Interestingly, Guzman et al,18 using an uninjured rat carotid artery model similar to that in the present study, found the endothelium to be largely resistant to adenovirus-mediated gene transfer. Therefore, despite the existence of several previous studies of adenovirus-mediated gene transfer into normal arteries,8 9 10 18 19 20 the present quantitative description and characterization of a model of highly EC-specific gene transfer is novel.
The EC specificity that we observed likely results from the properties
of both the adenovirus and the rat carotid artery. Our
group9 and others19 20 have found that the
intact endothelium of elastic arteries is a barrier to
the penetration of adenovirus vectors. If the
endothelium is damaged, smooth muscle cell transduction
occurs.9 20 Moreover, in arterial segments
with branches, high levels of gene transfer are found in the
adventitia.9 It is notable that the rat common carotid
artery has no branches21 and that the
endothelium remains intact despite manipulation of the
vessel at the time of gene transfer (Figs 2 and 8). As a result,
surgical isolation of the common carotid artery followed by infusion of
adenovirus vectors results in highly EC-specific gene transfer. Rat
carotid smooth muscle cells are clearly susceptible to high-efficiency
in vivo gene transfer with the Av1LacZ4 vector if the
endothelium is removed.17 The rare
transduced smooth muscle cell seen in the present study (Fig 2B)
likely results from a very low level of nondenuding
endothelial injury caused by surgical manipulation.
We found significant vascular toxicity at high virus titers, manifested by endothelial denudation and loss of recombinant gene expression. This observation is similar to our findings in a model of gene transfer into balloon-injured rat arteries17 and again suggests that the ability of adenovirus vectors to achieve high levels of recombinant gene expression is limited by associated toxicity. The finding of significant EC loss after infusion of both Av1LacZ4 and AdHS-1.2 implies that toxicity is a general effect of highly concentrated adenovirus. That heat-inactivated Av1LacZ4 also caused significant cell loss despite loss of an ability to mediate endosomal lysis implicates adenovirus binding and/or internalization in the observed toxicity. Relevant to this observation, adenovirus penton-base protein is known to cause adherent cells in culture to "round up" and detach,22 an effect mediated through an RGD amino acid sequence in the penton base that is also found in a number of extracellular matrix molecules. Normally, the RGD sequence of adhesion molecules mediates cell-matrix adhesion via binding to cell surface integrins.23 It is possible that RGD-mediated cell detachment (as a result of competitive displacement of adhesion molecules by adenovirus) is causing the EC loss that we observed; however, further experiments will be necessary to confirm this. We also considered the possibility that something copurifying with the adenovirus might be the toxic agent. However, the requirement that this toxic agent be of equivalent buoyant density to adenovirus and nondialyzable argues against this possibility. Finally, the existence of a plateau of recombinant gene expression at virus concentrations at which toxicity was not found indicates that more "efficient" (ie, fewer virions, same expression) gene transfer can be achieved at submaximal virus concentrations. Virus delivery at concentrations along this plateau will minimize virus-related vascular toxicity and will permit conservation of adenovirus stocks.
To define the biological substrate on which we may study recombinant
gene expression in ECs with this animal model, we investigated several
aspects of the phenotype of the transduced arteries. Both the
endothelium and the media were intact and free from
inflammatory infiltrates 3 days after gene transfer. However, the
proliferative indices of both transduced and untransduced ECs as well
as that of underlying medial smooth muscle cells were somewhat
elevated. This increase in proliferation at 3 days is apparently a
consequence of the surgical procedure rather than of gene transfer per
se (Fig 10). At 14 days after gene transfer, inflammatory infiltrates
are still absent, smooth muscle cell proliferation returns to baseline,
but EC proliferation remains elevated, at this time as a direct result
of the gene transfer protocol. Thus, at both 3 and 14 days, although
recombinant gene expression is not studied in a uniformly quiescent
normal artery, the large majority (82%) of ECs are not proliferating.
The modestly elevated endothelial proliferative rate at
14 days (a time at which recombinant gene expression is declining) may
be due to immune-mediated rejection and replacement of transduced cells
by neighboring untransduced cells, as described elsewhere for
adenovirus-transduced hepatocytes.24 Use of
second generation adenovirus vectors might permit a further decrease in
EC proliferation at 14 days and would appear to be particularly useful
if prolongation of recombinant gene expression were
desired.25 26 Prolongation of gene expression was not,
however, an objective of the present study; as shown by others in
both the myocardium5 and
peripheral nervous system,6 prolonged
recombinant gene expression is not necessary to support the utility of
a somatic gene transfer system to study mechanisms of gene regulation
in vivo.
Despite the potential limitation related to increased cellular
proliferation and the small amount of gene transfer into smooth muscle
cells of the vascular media (representing 5% of total
transduced cells), this animal model of EC-specific gene transfer is
likely to be useful for the study of in vivo EC biology.
Cell-type–specific targeting of recombinant gene expression, primarily
through germ-line transmission of transgenes in mice, has yielded
significant biological insights elsewhere in the
cardiovascular system.27 Extensive efforts
to use germ-line targeting to achieve EC-specific expression in adult
animals28 29 have not yet been successful. Even if
specific germ-line targeting of endothelium is
achieved, the present somatic gene-transfer system will retain
significant advantages. First, the present system is site specific,
with the potential to study local rather than systemic effects of gene
expression and the possibility to use other vessels in the same animal
as controls. Second, the system offers the possibility of testing
individual (or multiple) genetic constructs without generating separate
lines of transgenic animals.
In summary, we have described, characterized, and optimized an animal model of EC-specific recombinant gene expression. Future experiments will define the utility of this system in the study of mechanisms of endothelial gene regulation30 and signal transduction in vivo.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
Dr Schulick was funded by the Pharmacology Research Associates Training program of the National Institute of General Medical Sciences, National Institutes of Health. We thank Dr Jeffrey J. Rade for providing the AdHS-1.2 vector and Eleonora Dorfman for help in the preparation of virus stocks.
Received December 23, 1994; accepted June 6, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Gimbrone MA. Vascular Endothelium in Hemostasis and Thrombosis. Edinburgh, Scotland: Churchill Livingstone; 1986.
2. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1994;332:411-415.
3.
Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G.
Endothelium-derived relaxing factor produced and
released from artery and vein is nitric oxide. Proc Natl
Acad Sci U S A. 1994;84:9265-9269.
4. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
5.
Aoyagi T, Izumo T. Mapping of the pressure
response element of the c-fos gene by direct DNA injection into beating
hearts. J Biol Chem. 1993;268:27176-27179.
6.
Bessereau JL, Stratford-Perricaudet LD, Piette J, Le
Poupon C, Changeux JP. In vivo and in vitro analysis of
electrical activity-dependent expression of muscle acetylcholine
receptor genes using adenovirus. Proc Natl Acad Sci
U S A. 1994;91:1304-1308.
7.
Abdellatif M, MacLellan WR, Schneider MD. p21
Ras as a governor of global gene expression. J
Biol Chem. 1994;269:15423-15426.
8.
Lemarchand P, Jones M, Yamada I, Crystal RG. In
vivo gene transfer and expression in normal uninjured blood vessels
using replication-deficient recombinant adenovirus vectors.
Circ Res. 1993;72:1132-1138.
9.
Rome JJ, Shayani V, Flugelman MY, Newman KD, Farb A,
Virmani R, Dichek DA. Anatomic barriers influence the
distribution of in vivo gene transfer into the arterial
wall. Arterioscler Thromb. 1994;14:148-161.
10. 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]
11.
Lee SW, Trapnell BC, Rade JJ, Virmani R, Dichek DA.
In vivo adenoviral vector–mediated gene transfer into
balloon-injured rat carotid arteries. Circ
Res. 1993;73:797-807.
12. Graham FL, Prevec L. Manipulation of adenovirus vectors. In: Murray EJ, ed. Methods in Molecular Biology: Gene Transfer and Expression Protocols. Clifton, NJ: The Humana Press Inc; 1991;7:109-128.
13.
Knapp A, Degenhardt T, Dodt J. Hirudisins:
hirudin-derived thrombin inhibitors with disintegrin
activity. J Biol Chem. 1992;267:24230-24234.
14. Liu JM, Fujii H, Green SW, Komatsu N, Young NS, Shimada T. Indiscriminate activity from the B19 parvovirus P6 promoter in nonpermissive cells. Virology. 1991;182:361-364. [Medline] [Order article via Infotrieve]
15. Prevosti LG, Leon MB, Smith PD, Dodd JT, Bonner RF, Robinowitz M, Clark RE, Virmani R. Early and late healing responses of normal canine artery to excimer laser irradiation. J Thorac Cardiovasc Surg. 1988;96:150-156. [Abstract]
16.
Seth P, Rosenfeld M, Higginbotham J, Crystal RG.
Mechanism of enhancement of DNA expression consequent to
cointernalization of a replication-deficient adenovirus and unmodified
plasmid DNA. J Virol. 1994;68:933-940.
17.
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.
18.
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.
19.
Willard JE, Landau C, Glamann DB, 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.
20.
Steg PG, Feldman LJ, Scoazec JY, 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.
21. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327-333. [Medline] [Order article via Infotrieve]
22.
Bai M, Harfe B, Freimuth P. Mutations that alter
an arg-gly-asp (RGD) sequence in the adenovirus type 2 penton base
protein abolish its cell-rounding activity and delay virus
reproduction in flat cells. J
Virol. 1993;67:5198-5205.
23. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11-25. [Medline] [Order article via Infotrieve]
24.
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E,
Wilson JM. Cellular immunity to viral antigens limits E1-deleted
adenoviruses for gene therapy. Proc Natl Acad Sci
U S A. 1994;91:4407-4411.
25. Yang Y, Nunes FA, Berencsi K, Gonczol E, Engelhardt JF, Wilson JM. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet. 1994;7:362-369. [Medline] [Order article via Infotrieve]
26.
Engelhardt JF, Ye X, Doranz B, Wilson JM.
Ablation of E2A in recombinant adenoviruses improves transgene
persistence and decreases inflammatory response in mouse liver.
Proc Natl Acad Sci U S A. 1994;91:6196-6200.
27.
Hunter JJ, Zhu H, Lee KJ, Kubalak S, Chien KR.
Targeting gene expression to specific
cardiovascular cell types in transgenic mice.
Hypertension. 1993;22:608-617.
28. Hilkert RJ, de la Monte S, Lee M, Yun JS, Wagner TE, Quertermous T. Transgene expression directed by the endothelin-1 promoter is cell-type-specific but integration-site dependent. J Vasc Med Biol. 1993;4:138-147.
29. Harats D, Kurihara H, Belloni P, Oakley H, Ziober A, Ackley D, Cain G, Kurihara Y, Lawn R, Sigal E. Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter. J Clin Invest. 1995;95:1335-1344.
30. Dong G, Dichek DA. Regulated gene expression in endothelial cells following adenoviral gene transfer. Circulation. 1994;90(suppl I, pt 2):I-140. Abstract.
This article has been cited by other articles:
 |
|
 |
|
  G. Otsuka, R. Agah, A. D. Frutkin, T. N. Wight, and D. A. Dichek Transforming Growth Factor Beta 1 Induces Neointima Formation Through Plasminogen Activator Inhibitor-1-Dependent Pathways Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 737 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Watanabe and D. D. Heistad Targeting cerebral arteries for gene therapy Exp Physiol, May 1, 2005; 90(3): 327 - 331. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-W. Park, D.-H. Kim, H.-J. You, J.-J. Sir, S.-I. Jeon, S.-W. Youn, H.-M. Yang, C. Skurk, Y.-B. Park, K. Walsh, et al. Activated Forkhead Transcription Factor Inhibits Neointimal Hyperplasia After Angioplasty Through Induction of p27 Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 742 - 747. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Chandiwal, V. Balasubramanian, Z. K. Baldwin, M. S. Conte, and L. B. Schwartz Gene Therapy for the Extension of Vein Graft Patency: A Review Vascular and Endovascular Surgery, January 1, 2005; 39(1): 1 - 14. [Abstract] [PDF]
|
|
|
|
|
|
L. G. Melo, M. Gnecchi, A. S. Pachori, D. Kong, K. Wang, X. Liu, R. E. Pratt, and V. J. Dzau Endothelium-Targeted Gene and Cell-Based Therapies for Cardiovascular Disease Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1761 - 1774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. S. Katusic, N. M. Caplice, and K. A. Nath Nitric Oxide Synthase Gene Transfer as a Tool to Study Biology of Endothelial Cells Arterioscler Thromb Vasc Biol, November 1, 2003; 23(11): 1990 - 1994. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. T. Grey, C. Longo, T. Shukri, V. I. Patel, E. Csizmadia, S. Daniel, M. B. Arvelo, V. Tchipashvili, and C. Ferran Genetic Engineering of a Suboptimal Islet Graft with A20 Preserves {beta} Cell Mass and Function J. Immunol., June 15, 2003; 170(12): 6250 - 6256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Scherpereel, J. J. Rome, R. Wiewrodt, S. C. Watkins, D. W. Harshaw, S. Alder, M. Christofidou-Solomidou, E. Haut, J.-C. Murciano, M. Nakada, et al. Platelet-Endothelial Cell Adhesion Molecule-1-Directed Immunotargeting to Cardiopulmonary Vasculature J. Pharmacol. Exp. Ther., March 1, 2002; 300(3): 777 - 786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wen, R. M. Driscoll, D. B. Schneider, and D. A. Dichek Inclusion of the E3 Region in an Adenoviral Vector Decreases Inflammation and Neointima Formation After Arterial Gene Transfer Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1777 - 1782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. DeYoung, C. Tom, and D. A. Dichek Plasminogen Activator Inhibitor Type 1 Increases Neointima Formation in Balloon-Injured Rat Carotid Arteries Circulation, October 16, 2001; 104(16): 1972 - 1971. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.’i. Sato, T. Mohacsi, A. Noel, C. Jost, P. Gloviczki, G. Mozes, Z. S. Katusic, T. O’Brien, and W. G. Mayhan In Vivo Gene Transfer of Endothelial Nitric Oxide Synthase to Carotid Arteries From Hypercholesterolemic Rabbits Enhances Endothelium-Dependent Relaxations • Editorial Comment Stroke, April 1, 2000; 31(4): 968 - 975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Schneider, G. Vassalli, S. Wen, R. M. Driscoll, A. B. Sassani, M. B. DeYoung, R. Linnemann, R. Virmani, and D. A. Dichek Expression of Fas Ligand in Arteries of Hypercholesterolemic Rabbits Accelerates Atherosclerotic Lesion Formation Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 298 - 308. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Vassalli, R. Agah, R. Qiao, C. Aguilar, and D. A. Dichek A Mouse Model of Arterial Gene Transfer : Antigen-Specific Immunity Is a Minor Determinant of the Early Loss of Adenovirus-Mediated Transgene Expression Circ. Res., October 29, 1999; 85 (9): e25 - e32. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Aschner, N. Kovacs, J. V. Perciaccante, J. P. Figueroa, N. Thrikawala, G. S. Robins, and D. W. Busija Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries Am J Physiol Heart Circ Physiol, July 1, 1999; 277(1): H371 - H379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S.C. Weber, G. Draude, W. Erl, R. de Martin, and C. Weber Monocyte Arrest and Transmigration on Inflamed Endothelium in Shear Flow Is Inhibited by Adenovirus-Mediated Gene Transfer of Ikappa B-alpha Blood, June 1, 1999; 93(11): 3685 - 3693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Mann, G. H. Gibbons, H. Hutchinson, R. S. Poston, E. G. Hoyt, R. C. Robbins, and V. J. Dzau Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues PNAS, May 25, 1999; 96(11): 6411 - 6416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
J. M. Waugh, E. Yuksel, J. Li, M. D. Kuo, M. Kattash, R. Saxena, R. Geske, S. N. Thung, S. M. Shenaq, and S. L. C. Woo Local Overexpression of Thrombomodulin for In Vivo Prevention of Arterial Thrombosis in a Rabbit Model Circ. Res., January 22, 1999; 84(1): 84 - 92. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Rekhter, N. Shah, R. D. Simari, C. Work, J.-S. Kim, G. J. Nabel, E. G. Nabel, and D. Gordon Graft Permeabilization Facilitates Gene Therapy of Transplant Arteriosclerosis in a Rabbit Model Circulation, September 29, 1998; 98(13): 1335 - 1341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Mozes, T. Mohacsi, P. Gloviczki, S. Menawat, I. Kullo, D. Spector, J. Taylor, T. B. Crotty, and T. O'Brien Adenovirus-Mediated Gene Transfer of Macrophage Colony Stimulating Factor to the Arterial Wall In Vivo Arterioscler Thromb Vasc Biol, July 1, 1998; 18(7): 1157 - 1163. [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]
|
|
|
|
|
|
K. M. Channon, H. Qian, S. A. Youngblood, E. Olmez, G. A. Shetty, V. Neplioueva, M. A. Blazing, and S. E. George Acute Host-Mediated Endothelial Injury After Adenoviral Gene Transfer in Normal Rabbit Arteries : Impact on Transgene Expression and Endothelial Function Circ. Res., June 29, 1998; 82(12): 1253 - 1262. [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]
|
|
|
|
|
|
A. H. Schulick, A. J. Taylor, W. Zuo, C.-b. Qiu, G. Dong, R. N. Woodward, R. Agah, A. B. Roberts, R. Virmani, and D. A. Dichek Overexpression of transforming growth factor beta 1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia PNAS, June 9, 1998; 95(12): 6983 - 6988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. Ooboshi, C. D. Rios, Y. Chu, S. D. Christenson, F. M. Faraci, B. L. Davidson, and D. D. Heistad Augmented Adenovirus-Mediated Gene Transfer to Atherosclerotic Vessels Arterioscler Thromb Vasc Biol, September 1, 1997; 17(9): 1786 - 1792. [Abstract] [Full Text]
|
|
|
|
 |
|
 I. J. Kullo, G. Mozes, R. S. Schwartz, P. Gloviczki, M. Tsutsui, Z. S. Katusic, and T. O'Brien Enhanced Endothelium-Dependent Relaxations After Gene Transfer of Recombinant Endothelial Nitric Oxide Synthase to Rabbit Carotid Arteries Hypertension, September 1, 1997; 30(3): 314 - 320. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. J Feldman and G. Steg Optimal techniques for arterial gene transfer Cardiovasc Res, September 1, 1997; 35(3): 391 - 404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. H Baker, D. Mehta, S. J George, and G. D Angelini Prevention of vein graft failure: potential applications for gene therapy Cardiovasc Res, September 1, 1997; 35(3): 442 - 450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Vassalli and D. A Dichek Gene therapy for arterial thrombosis Cardiovasc Res, September 1, 1997; 35(3): 459 - 469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J.M Roks, Y. M Pinto, M. Paul, F. Pries, M. Stula, T. Eschenhagen, H.-D. Orzechowski, S. Gschwendt, J. Wilschut, and W. H van Gilst Vectors based on Semliki Forest virus for rapid and efficient gene transfer into non-endothelial cardiovascular cells: comparison to adenovirus Cardiovasc Res, September 1, 1997; 35(3): 498 - 504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M Channon, G. J Fulton, J. L Gray, B. H Annex, G. A Shetty, M. A Blazing, K. G Peters, P.-O. Hagen, and S. E George Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels Cardiovasc Res, September 1, 1997; 35(3): 505 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Maeda, U. Ikeda, Y. Ogasawara, M. Urabe, T. Takizawa, T. Saito, P. Colosi, G. Kurtzman, K. Shimada, and K. Ozawa Gene transfer into vascular cells using adeno-associated virus (AAV) vectors Cardiovasc Res, September 1, 1997; 35(3): 514 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 G. Dong, A. H. Schulick, M. B. DeYoung, and D. A. Dichek Identification of a cis-Acting Sequence in the Human Plasminogen Activator Inhibitor Type-1 Gene That Mediates Transforming Growth Factor-beta 1 Responsiveness in Endothelium in Vivo J. Biol. Chem., November 22, 1996; 271(47): 29969 - 29977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
S. Rafii, S. Dias, S. Meeus, K. Hattori, R. Ramachandran, F. Feuerback, S. Worgall, N. R. Hackett, and R. G. Crystal Infection of Endothelium With E1-E4+, but Not E1-E4-, Adenovirus Gene Transfer Vectors Enhances Leukocyte Adhesion and Migration by Modulation of ICAM-1, VCAM-1, CD34, and Chemokine Expression Circ. Res., May 11, 2001; 88(9): 903 - 910. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||