(Circulation. 1999;99:1776-1779.)
© 1999 American Heart Association, Inc.
Brief Rapid Communications |
Adenovirus-Mediated Delivery of Fas Ligand Inhibits Intimal Hyperplasia After Balloon Injury in Immunologically Primed Animals
From the Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass (Z.L., M.S., T.N., K.W.), and Genzyme Corp, Framingham, Mass (J.M.K, G.Y.A).
Correspondence to Dr Kenneth Walsh, Division of Cardiovascular Research, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail kwalsh{at}opal.tufts.edu
Abstract
Background—Adenoviral constructs have been used for studies of injury-induced vascular hyperplasia in immunologically naive laboratory animals, but their usefulness for intra-arterial gene therapy may be limited by the prevalence of preexisting immunity to adenovirus in the patient population. Here, we explored the efficacy of adenovirus-mediated transfer of Fas ligand, a cytotoxic gene with immunomodulatory properties, in inhibiting injury-induced vascular lesion formation in both naive and immunologically primed animals.
Methods and Results—Lesion formation was evaluated in balloon-injured carotid arteries of naive and adenovirus-immunized rats that were infected with adenoviral constructs expressing Fas ligand (Ad-FasL), the cyclin-dependent kinase inhibitor p21 (Ad-p21), or ß-galactosidase (Ad-ßgal). In naive rats, Ad-FasL induced apoptosis in medial vascular smooth muscle cells and inhibited intimal hyperplasia by 60% relative to Ad-ßgal–treated vessels (P<0.05), whereas the cytostatic agent Ad-p21 decreased lesion size by 58% (P<0.05). In animals preimmunized with an adenoviral vector containing no transgene, Ad-FasL significantly inhibited neointima formation (73% reduction, P<0.05), but Ad-p21 failed to inhibit neointima formation relative to controls. Immunologically primed rats displayed robust T-cell infiltration in Ad-p21– and Ad-ßgal–treated vessels, but T-cell infiltration was markedly attenuated in Ad-FasL–treated vessels.
Conclusions—Our data demonstrate that adenovirus-mediated Fas ligand delivery can inhibit intimal hyperplasia in both immunologically primed and naive animals, whereas the efficacy of an adenovirus-mediated p21 delivery is limited to immunologically naive animals. This study documents, for the first time, the therapeutic efficacy of intravascular adenoviral gene transfer in animals with preexisting immunity to adenovirus.
Key Words: restenosis • genes • viruses • lymphocytes
Adenoviral vectors are useful for studies of injury-induced vascular hyperplasia because of their high transduction efficiency. These properties have resulted in optimistic predictions that adenoviral vectors may be used to deliver therapeutic genes to prevent postangioplasty restenosis.1 However, a limitation for adenovirus-mediated gene therapy in humans is the prevalence of preexisting immunity to adenovirus, which might lead to the destruction of adenovirus-transduced cells.2 Immune responses to adenoviral vectors are typically characterized by mononuclear cell infiltration, transgene elimination, and the inability to readminister the vector.3 4
The Fas-FasL system has been implicated in the regulation of physiological cell turnover, particularly during the downregulation of an immune response.5 Fas (CD95/Apo-1) is a death receptor expressed on many cell types, but expression of its ligand (FasL) is highly restricted. FasL expression occurs in immune-privileged tissues, where it is believed to function by killing Fas-bearing inflammatory cells.6 Constitutive FasL expression has also been detected in tumors, where it may function to induce apoptotic cell death in Fas-expressing immune cells when they attempt to enter the tumor.7 Recently, we reported that FasL is also expressed on the vascular endothelium, where it can function as a negative regulator of leukocyte extravasation.8
Potential adenovirus-mediated gene therapy strategies to treat proliferative vessel disorders include delivery of antithrombotic, antimigratory, cytostatic, or cytotoxic genes.1 These models of vascular gene therapy have all used immunologically naive laboratory animals. Because a large portion of the human patient population has developed immunity to several adenovirus serotypes, we evaluated the efficacy of adenovirus-mediated gene transfer in the rat carotid model of balloon injury in naive and immunologically primed animals. Adenovirus-mediated FasL gene transfer was examined because it efficiently induces apoptosis in smooth muscle cells9 and can function to eliminate peripheral T cells after an immune response.5 For comparison, adenovirus-mediated gene transfer was performed with cdk inhibitor p21, a negative regulator of smooth muscle cell proliferation10 that has no known immunomodulatory function. FasL gene delivery was found to inhibit neointima formation in both naive and adenovirus-immunized animals, whereas p21 inhibits neointima formation only in naive animals. These data suggest that FasL gene transfer to the vessel wall can overcome the limitations imposed by host immune responses to repeated adenovirus delivery, leading to an inhibition of pathological cell proliferation in animals with preexisting immunity to adenovirus.
Methods
Rat Carotid Artery Balloon-Injury Model, Histology, and
Immunohistochemistry
The left carotid arteries of male Sprague-Dawley rats (400 to
500 g) were injured with 2F embolectomy catheters (Baxter Edwards
Healthcare) as described.9 Vessels were treated with the
indicated viral solution (1x108 pfu) or saline
for 15 minutes. Serotype 5 adenoviral vectors expressing murine FasL,
human p21, bacterial ß-galactosidase, or no transgene are deleted in
E1 and E3 regions and express transgenes under the control of
cytomegalovirus promoter. Rats were preimmunized by the
intravenous administration of 1x109
pfu of the transgene-null adenoviral vector via the penile vein 2 weeks
before balloon injury. Vessels harvested 1, 3, or 14 days after balloon
injury were typically fixed in 100% methanol and embedded in paraffin.
Sections were stained with hematoxylin and eosin, Richardson's
combination elastic tissue trichrome stain, Hoechst 33258, or
TUNEL.8 Intimal and medial areas were measured with a
computerized sketching program. For immunohistochemistry, sections were
rehydrated, blocked with 5% goat serum and 0.01% Triton X-100 in PBS,
and incubated with the T-cell marker antibodies anti-CD3 (Sigma) or
anti-CD8 (Serotech) or the monocyte/macrophage marker anti-CD68
(Servtec) for 1 hour. After they had been rinsed with PBS, biotinylated
antibody to rabbit IgG or mouse IgG was applied for 20 minutes,
followed by alkaline phosphatase–conjugated streptavidin.
Antibody complexes were detected by the Fast Red system (BioGenex)
followed by counterstaining with Mayer's hematoxylin.
Adenovirus Serotype 5–Specific Antibody Measurement
Sera collected from naive and immunized rats at different time
points were analyzed by ELISA essentially as
described.11 Briefly, serial 2-fold dilutions of rat sera
were added to wells coated with wild-type adenovirus 5. Bound
virus-specific antibodies were detected by the addition of rabbit
anti-rat IgG (Jackson ImmunoResearch Laboratories), followed by
horseradish peroxidase–conjugated goat anti-rabbit IgG (Cappel-Organon
Teknika) was used as the secondary antibody. The serum titer was
defined as the reciprocal of the highest dilution that produced an
optical density at 490 nm 
0.1.
Data and Statistical Analysis
All data were evaluated with 2-tailed, unpaired Student's
t test or compared by 1-way ANOVA followed by Fisher's
t test and were expressed as the mean±SEM. A value of
P<0.05 was considered statistically significant.
Results
Animal Model
Two experimental groups of rats were analyzed in the
study. In group 1, carotid arteries of immunologically naive rats
(n=30) underwent balloon injury, immediately followed by the localized
delivery of either saline (vehicle), Ad-ß-gal
(1x108 pfu), Ad-FasL
(1x108 pfu), or Ad-p21
(1x108 pfu). In group 2, 38 rats were immunized
against human adenovirus by intravenous injection of
replication-defective adenoviral vector containing no insert
(1x109 pfu). Animals were uniformly seronegative
for adenovirus 5–specific antibodies at the time of immunization, and
antibody titers of 955±437 (range, 400 to 1600) developed 2 weeks
after immunizations when balloon injury was performed. Two weeks after
preimmunization, left common carotid arteries of 20 immunized rats were
balloon-injured and immediately treated with either saline (vehicle),
Ad-ß-gal, Ad-FasL, or Ad-p21 at the same viral titers as used in
group 1. Most animals were euthanized at 14 days after injury to assess
intimal hyperplasia. In group 1, 10 animals were euthanatized at 24
hours after injury to assess apoptosis and cell density. At the
time of harvest, all vessels were patent, and the medial layers
appeared normal with regard to size and cellularity (Figure 1
).
|
Efficacy of Adenovirus-Mediated Gene Transfer
Intimal hyperplasia in the naive rats (group 1) was greatest in
vessels treated with saline or Ad-ßgal with intima/media ratios of
1.3 (Figure 1B
through D). In this group, both Ad-FasL and
Ad-p21 inhibited intimal hyperplasia by 60% and 58%, respectively
(Figure 1K). No difference in medial area was detected in the
Ad-FasL– or Ad-p21–treated vessels compared with saline- or
Ad-ßgal–treated vessels. Ad-FasL–treated vessels harvested at 24
hours after injury revealed 5-fold more TUNEL-positive nuclei
than in the Ad-ßgal control vessels (P<0.01). Many
TUNEL-positive nuclei appeared pyknotic (Figure 1A). At this
time point, medial cellularity was reduced by 41% (P<0.05)
in the Ad-FasL–treated vessels relative to the Ad-ßgal control
vessels, but medial cellularity returned to normal by 14 days.
Intimal hyperplasia in immunized animals (group 2) treated with saline
or Ad-ßgal was similar to that in vessels observed in naive animals
(Figure 1E through J). In this group, Ad-FasL treatment resulted
in an intima/media ratio of 0.34±0.11, a 72% reduction compared with
the Ad-ßgal control group (P<0.05) (Figure 1L).
However, Ad-p21 was ineffective at inhibiting intimal hyperplasia in
immunized rats.
Inflammation in Vessels of Immunized Animals
To test whether ectopic FasL expression can modulate the cellular
immune response to adenoviral antigen, we performed immunohistochemical
staining for CD3-positive T cells on sections of arteries harvested
from immunized rats 3 or 14 days after injury and local gene delivery.
Robust T-cell infiltration was observed in Ad-p21– and
Ad-ßgal–treated vessels, but T-cell infiltration was markedly
attenuated in Ad-FasL–treated vessels (Figure 2A through D). Surprisingly, T-cell
accumulation occurred predominantly in the medial layers at the 14-day
time point (Figure 2E). Ad-FasL–treated vessels had 76%
(P<0.05) and 73% (P<0.05) fewer T cells than
Ad-ßgal– and Ad-p21–treated vessels, respectively. T-cell
infiltration in the Ad-FasL–treated vessels was not significantly
different from that in the saline-treated control vessels. Staining for
CD8-positive T cells also revealed marked infiltration in the media of
Ad-p21– and Ad-ßgal–treated vessels, but CD8-positive cell
infiltration was markedly reduced in Ad-FasL–treated vessels.
Monocytes/macrophages were rarely observed in any vessels.
|
Discussion
A limitation of adenovirus-mediated gene therapy is the immune response that induces the destruction of genetically altered cells.3 4 Repeat administration of recombinant adenoviral vectors can be associated with confounding immune responses and low gene transfer efficiency, and high doses can promote an inflammatory response and intimal hyperplasia when administered locally to normal vessels of seronegative laboratory animals.2 12
In this study, we compared the efficacy of 2 genes, FasL and p21,
for their ability to inhibit intimal hyperplasia in balloon-injured
carotid arteries of rats that were either naive or immunized with an
empty adenoviral vector. Both the cytostatic agent Ad-p21 and the
cytotoxic agent Ad-FasL were effective at inhibiting intimal
hyperplasia in naive rats, consistent with previous
reports.9 10 However, only Ad-FasL inhibited
neointima formation in immunized rats (Figure 1).
Administration of recombinant adenovirus constructs elicits cellular
and humoral responses to viral and foreign transgene
products.13 In our study, animals were immunized with
an adenovirus construct expressing no transgene to eliminate the immune
response to the transgene-encoded proteins after the second adenovirus
administration. Prior immunization resulted in the production
of anti-adenovirus antibodies, and robust T-cell infiltration was noted
in arteries that were subsequently reinfected with Ad-ßgal or Ad-p21.
In contrast, T-cell infiltration was markedly reduced in the immunized
rats that received Ad-FasL (Figure 2), whereas levels of
anti-adenovirus antibodies were unaffected (unpublished data). These
data suggest that local delivery of FasL has 2 effects on the vessel
wall: it induces apoptosis in Fas-bearing smooth muscle cells,
and it suppresses the destructive response of T lymphocytes toward
cells expressing viral proteins.
FasL is naturally expressed on the vascular endothelium, where it appears to function as an inhibitor of inflammatory cell infiltration.8 The data presented here suggest that local delivery of FasL to the denuded vessel wall essentially confers this anti-inflammatory property of the endothelium to the smooth muscle cells of the media. Therefore, the abilities of FasL to inhibit intimal hyperplasia and augment the endogenous immunosuppressive properties of the vessel wall make it uniquely suited for gene therapy of inflammatory-fibroproliferative disorders.
Acknowledgments
This work was supported by NIH grants AG-15052, HL-50692, and AR-40197 to Dr Walsh.
Received December 8, 1998; revision received February 2, 1999; accepted February 11, 1999.
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