Published online before print April 27, 2001, doi: 10.1161/hh0901.089884
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© 2001 American Heart Association, Inc.
Article |
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
From the Division of Hematology-Oncology (S.R., S.D., S.M., K.H.), Division of Pulmonary and Critical Care Medicine (R.R., F.F., S.W., R.G.C.), Belfer Gene Therapy Core Facility (N.R.H., R.G.C.), and Institute of Genetic Medicine (R.G.C.), Cornell University Medical College, New York, NY.
Correspondence to Shahin Rafii, MD, Cornell University Medical College, 1300 York Ave, Room D601, New York, NY 10021. E-mail srafii{at}mail.med.cornell.edu
Abstract
Abstract—Intravascular introduction of replication-deficient adenoviral vectors (Advectors) provides an ideal model of delivery of transgenes for the treatment of various vascular abnormalities. On the basis of the knowledge that Advectors can induce inflammatory responses after intravascular administration, we speculated that cellular activation by Advector infection could directly modulate the endothelial cell (EC) adhesion molecule/chemokine expression repertoire. Infection of human umbilical vein ECs or bone marrow microvascular ECs with an E1-E4+ Advector resulted in the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD34, but not E-selectin, P-selectin, CD36, CD13, CD44, HLA-DR or PECAM. Upregulation of ICAM-1, VCAM-1, and CD34 was apparent 12 hours after infection and persisted for weeks after infection. Selective induction of adhesion molecules was mediated by the presence of the E4 gene in the Advector, because infection of ECs with an E1-E4- Advector had no effect on adhesion molecule expression. ECs infected with E1-E4+ Advector, but not those infected with E1-E4- Advector, supported the adhesion of leukocytes. Monoclonal antibodies to ICAM-1 and VCAM-1 inhibited adhesion of leukocytes to E1-E4+-infected ECs. Infection of the ECs with E1-E4+ Advector, but not E1-E4- Advector, resulted in downregulation of expression of chemocytokines, including interleukin-8, MCP-1, RANTES, and GM-CSF. Nonetheless, a large number of leukocytes migrated through ECs infected with E1-E4+, but not those infected with E1-E4-, in response to exogenous chemokines. These results demonstrate that infection of ECs with E1-E4+ Advectors, but not E1-E4- Advectors, may directly augment inflammatory responses by upregulating expression of adhesion molecules and enhancing migration through Advector-infected ECs and suggest that E1-E4- Advectors may be a better choice for gene-transfer strategies directed to the ECs.
Key Words: endothelial activation • adhesion molecules • E4+ adenoviral vectors • leukocyte adhesion
Endothelial cells
(ECs) are an ideal target for gene therapy because they are readily
accessible to gene therapy vectors via the circulation and play a
critical role in the progression of disease processes including
inflammation, atherosclerosis, and tumor angiogenesis.
Advectors that could infect quiescent ECs provide ideal vectors to
introduce genes into vascular endothelium as well as
neointimal cells with high efficiency and low
toxicity.1 2 3 4 5 6
Advectors expressing the genes for
anti–
1-antitrypsin,7
catalase8 and NO
synthetase,9 simian virus-40
T antigen,10 and many other
genes have successfully been delivered to ECs in vitro or in vivo.
However, expression of genes by Advectors has been hampered by
infiltration of inflammatory cells and intravascular activation of
neointimal
cells.11
Organ- and site-specific activation of ECs plays a seminal role in the regulation of inflammatory processes. Inappropriate cellular activation of ECs secondary to viral infection have also been associated with pathogenesis of various life-threatening clinical conditions such as intravascular thrombosis.12 Several studies have shown that infection of ECs with various types of viruses results in upregulation of adhesion molecules. Cytomegalovirus (CMV) has been shown to upregulate intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1).13 14 HIV-1 infection has been shown to result in upregulation of VCAM-1 and E-selectin.15 Flavivirus and certain strains of measles virus have also been shown to upregulate ICAM-1 expression.16 17
Exposure of normal arteries to E1-E4+ Advectors has resulted in pronounced infiltration of T cells throughout the arterial wall and upregulation of ICAM-1 and VCAM-1 in arterial smooth muscle cells, ultimately leading to neointimal hyperplasia.11 These findings were present both 10 and 30 days after gene transfer, with no evidence of a decline in severity over time. The significance of ICAM-1 upregulation by Advectors in mediation in inflammatory response was demonstrated in a study in which injection of blocking monoclonal antibody (mAb) to leukocyte function–associated antigen-1 (LFA-1) inhibited the infiltration of neutrophils and T cells into the muscle cells that were infected with first-generation E1-E4+ Advectors.18 However, the exact mechanism for this long-term activation of neointimal cells and nonspecific infiltration of inflammatory cells by Advectors is not known. Whether endothelial activation by E1-E4+ Advectors contributes to neointimal infiltration with inflammatory cells is not known and is the subject of the studies reported in this paper.
On the basis of these studies, we speculated that Advector-mediated upregulation of adhesion molecules on ECs may play a direct role in aberrant induction and infiltration of inflammatory cells into neointima. In this report, we demonstrate that E1-E4+ Advectors induce long-term expression of ICAM-1 and VCAM-1 and restoration of CD34 expression on primary cultured ECs, resulting in augmentation of adhesion and transendothelial migration of leukocytes through the E1-E4+-infected ECs. Induction of these adhesion molecules and modulation of chemokine expression by the Advectors are mediated by the E4 region, since E1-E4- adenoviruses fail to alter ICAM-1, CD34, VCAM-1, or chemokine expression. These studies suggest that intravascular gene delivery by E1-E4- Advectors may provide an alternative mechanism to decrease the anti-Advector host immune response.
Materials and Methods
Preparation of ECs
Primary human umbilical vein ECs (HUVECs) and bone
marrow ECs (BMECs) were obtained as previously
described.19 ECs were grown
in endothelial growth medium (M199, 20% FCS, 10 ng/mL
vascular endothelial growth factor [VEGF; Peprotech],
5 ng/mL basic fibroblast growth factor [bFGF (FGF-2); Peprotech], and
1 U/mL heparin sulfate) at 37°C. Cells from passages 2 to 3 were used
in all of the experiments. Where indicated, studies were carried out
using serum-free growth factor–free medium (X-Vivo;
Bio-Whittaker).
Construction of Advectors
The Advectors used in this study included the
following: (1)
E1-E4+ AdNull
(E1-, E3-,
and E4+, CMV early/intermediate
promoter/enhancer, no transgene in the expression cassette); (2)
E1-E4+ Adßgal
(E1-, E3-,
and E4+, CMV promoter driving the
Escherichia coli
ß-galactosidase [ßgal] gene); (3)
E1-E4-
Adßgal (same as Adßgal, but with a complete deletion of the E4
gene, using the E. coli
ß-glucuronidase gene, in a nonexpressing configuration, as a spacer
in the E1 region).20
Advector stocks were purified by cesium chloride
centrifugation and quantified as plaque-forming units
(pfu) in 293 cells as previously
described.21 22
All Advectors had a particle/pfu ratio of 100 and were free of
replication-competent
Advectors.22
Flow Cytometry
ECs were infected with different multiplicities of
infection (MOI) of
E1-E4+ or
E1-E4-
Adßgal. Subsequently, the cells were harvested with PBS containing
2 mmol/L EDTA at 4°C. The cells were washed and incubated with
1.5 µL of FITC-conjugated mAbs to E-selectin (BioSource
International), ICAM-1 (Immunotech), VCAM-1 (Immunotech), and
VE-cadherin (BV9 clone, ImClone Systems) and a phycoerythrin (PE, red
fluorescence)–conjugated mAb to CD34
(Becton-Dickinson [BD]), platelet
endothelial cell adhesion molecule (PECAM; BD),
P-selectin (BD), CD44 (BD), human leukocyte antigen (HLA)–DR (BD),
CD13 (BD), and CXC chemokine receptor-4 (CXCR4; Pharmingen) for 20
minutes. The number of positive cells was compared with IgG isotype
control (FITC) and determined using a Coulter Elite flow cytometer
(Coulter). Nonviable cells identified by propidium iodide staining were
excluded.
Leukocyte Transendothelial
Migration
Peripheral blood mononuclear cells
(PBMNCs) were obtained from normal donors in a heparin-coated syringe.
PBMNCs were isolated by Ficoll density gradient and washed 3 times in
HBSS. Subsequently, 2x105 cells were placed
on the upper chamber of transwell plates coated with confluent
monolayers of ECs. The integrity of the ECs was documented by
125I-labeled albumin exclusion as
previously described.23
Chemokines (in ng/mL: regulated on activation normal T expressed and
secreted [RANTES] 150 [R&D Systems], monocyte
chemoattractant protein-1 [MCP-1] 20 [R&D Systems], or
stromal-derived factor-1 [SDF-1] 200 [PeproTech]) were placed in
the lower chamber, and the number and phenotype of the
migrating cells were determined by flow cytometry as previously
described.23 Monocytes were
identified by a PE-conjugated mAb to CD14 (Becton Dickinson; BD); T
cells by a combination of PE-conjugated mAbs to CD3, CD4, and CD8; and
dendritic cells by mAbs to CD1a (BD) and CD83 (BD). Total leukocyte
migration was evaluated by staining with a PE-conjugated mAb to
common leukocyte antigen CD45 (BD and Immunotech). Contaminating ECs
were excluded by staining with VE-cadherin (clone BV9) or VEGF
receptor-2 (clone 6.64, ImClone Systems).
Adhesion Studies
Confluent monolayers of ECs were infected with 50 MOI
of E1-E4+ or
E1-E4-
Adßgal. After removal of the Advectors, the cells were placed in
either serum-free medium or endothelial growth medium.
On day 3 of Advector infection, ECs were also stimulated with
interleukin (IL)–1ß (10 U/mL) for 16 hours. Where indicated, PBMNCs
(105 cells/well) were incubated with
Adßgal stimulated by IL-1ß (10 U/mL) or with uninfected ECs in HBSS
supplemented with 2 mmol/L divalent cations, in the presence of
blocking mAbs to ICAM-1 (10 µg/mL; R&D Systems), E-selectin (10
µg/mL; R&D Systems), or VCAM-1 (10 µg/mL; Immunotech) or CD34 (10
µg/mL, R&D Systems). After an incubation period of 1 hour at 37°C,
the nonadherent population was removed. The attached cells bound to ECs
were recovered by EDTA treatment, and the number and phenotype
of attached cells were quantified by flow cytometry as described
above.23
Chemokine Determination
Early-passage ECs were placed in 24-well
Costar plates and were infected with either 50
MOI of E1-E4+
or E1-E4-
Adßgal. After removal of the Advectors, the cells were placed in
serum-free X-Vivo medium supplemented either with FGF-2 (2 ng/mL) or
with VEGF (10 ng/mL) to keep Advector-uninfected cells alive
during the conditioned medium collection period. After 3 days, the
conditioned media were collected and the amount of chemokines was
determined by commercially available ELISA assays (R&D Systems) through
a central core laboratory (Cytokine Core Laboratory, Baltimore,
MD).
Statistical Analysis
Data are expressed as mean±SEM of 3 to 5 independent
experiments. To detect differences between migrating and nonmigrating
cells, the paired Student t
test was applied. A P<0.05 was
considered statistically
significant.
Results
Infection With
E1-E4+
Advectors, but Not
E1-E4-
Advectors, Results in Activation of ECs
We have previously shown that infection of HUVECs with
E1-E4+
Advectors results in the generation of a unique state in which ECs do
not proliferate or undergo
apoptosis.24 During
this period, ECs can survive independently of
endothelial growth factors. However, this state is
associated with profound morphological changes. Within 24 hours of
infection of HUVECs with 50 pfu of
E1-E4+ Adßgal
(Figure 1C
), ECs become spindle-shaped and elongated,
reminiscent of cytokine (ie, IL-1ß)–activated
endothelium. However,
E1-E4-
Adßgal–infected ECs maintain the original cobblestone morphology
typical of ECs in culture
(Figures 1A and 1B) and remain dependent on VEGF and FGF-2 for
survival.
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Infection With
E1-E4+
Advectors, but Not
E1-E4-
Advectors, Results in Upregulation of ICAM-1 and VCAM-1 and Restoration
of CD34 Expression on ECs
It is well established that stimulation of resting ECs
with inflammatory cytokines such as IL-1ß or tumor necrosis
factor (TNF)– results in transient upregulation of ICAM-1, VCAM-1,
and E-selectin.25 Although
CD34 is expressed invariably by various types of ECs in
vivo,26 27 28
cultivation of ECs in vitro results in a rapid downregulation of CD34
expression.29 In addition,
stimulation of ECs with inflammatory cytokines such as IL-1ß,
TNF, or lipopolysaccharides results in rapid downregulation of
CD34 mRNA or protein
expression.29
Infection of unstimulated ECs with either 10 or 50 MOI of
E1-E4+ Adßgal
resulted in upregulation of ICAM-1 and VCAM-1 and restoration of CD34
expression
(Figures 2 and 3). Infection of ECs with
E1-E4-
Adßgal vectors had no effect on the expression of VCAM-1, ICAM-1, or
E-selectin, suggesting that expression of the AdE4 gene is the
principal mediator of adhesion molecule upregulation on ECs induced by
E1- Advectors.
E1-E4-
Adßgal–infected ECs had the same activation profile as that of the
uninfected naive ECs.
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There were no changes in the expression of
endothelial adhesion molecules, including E-selectin,
P-selectin, PECAM, and CD44, with either
E1-E4+ or
E1-E4-
Adßgal vectors. Moreover,
E1-E4+
infection of ECs did not affect the expression of other surface
molecules such as CD36 (GPIV), CD13, and HLA-DR
(Figures 2
and 3,
Table 1). However, there was downregulation of CXCR4 by
infection with
E1-E4+ Adßgal
vector.
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Time Course of Activation of CD34, ICAM-1, and
VCAM-1 by
E1-E4+
Advectors
E1-E4+
Adßgal or AdNull infection of ECs resulted in induction of CD34,
ICAM-1, and VCAM-1 within 24 hours of infection and reached maximum
levels 48 hours after infection
(Figure 4). Although stimulation of ECs with
inflammatory cytokines such as IL-1ß resulted in transient
upregulation of adhesion molecules, E4-mediated upregulation of
adhesion molecules persisted for several weeks to months. This
prolonged activation of endothelial adhesion molecules
was not due to the factors released by the
E1-E4+
Adßgal– or AdNull–infected HUVECs, given that conditioned medium
obtained from
E1-E4+
Adßgal– or AdNull–infected HUVECs failed to induce adhesion
molecule expression (not shown). Advector infection of the ECs was
performed in the presence of polymyxin to block the activation of ECs
by any contaminating endotoxin. However, because transient exposure of
ECs to Advector preparations for 12 hours resulted in long-term
activation of ECs, these data strongly suggest that upregulation of
adhesion molecules is mediated by introduction and long-term expression
of the E4 gene.
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Leukocyte Adhesion to ECs Is Enhanced by
E1-E4+
Infection of ECs
Upregulation of adhesion molecules by
inflammatory cytokines has been shown to mediate adhesion of
leukocytes to
ECs.30 31
Similarly, infection of ECs with
E1-E4+ Adßgal
vectors resulted in increased adhesion of
CD45+ leukocytes. The majority of adherent
leukocytes (80±10%) were
CD15+CD14+
myeloid cells and, to a smaller degree, CD3+
lymphocytes (9±4%)
(Figure 5). Infection of ECs with
E1-E4-
Adßgal vectors did not enhance the binding of leukocytes to the ECs.
However, IL-1ß activation of ECs with
E1-E4-
Adßgal vectors resulted in enhanced adhesion of leukocytes to
ECs.
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The adhesion of leukocytes (CD45+ cells) to the E1-E4+ Adßgal ECs could be partially blocked by neutralizing mAbs to ICAM-1 and, to a smaller extent, VCAM-1 but not CD34. However, a combination of neutralizing mAbs to ICAM-1 and VCAM-1 decreased binding of leukocytes to E1-E4+ Adßgal–infected ECs by 55±5%. These data suggest that other, as-yet-unrecognized, adhesion molecules may mediate adhesion of leukocytes to E1-E4+ Adßgal–infected ECs.
Infection of HUVECs With
E1-E4+
Advectors Results in Profound Downregulation of Vascular-Derived
Chemokines
Transendothelial migration of
leukocytes into inflammatory sites is not only dependent on the
upregulation of adhesion molecules but is also influenced by the
regional release of chemokines. Chemokines are produced by
different cells, including leukocytes, endothelial and
stromal cells. Infection of ECs with
E1-E4+ Adßgal
resulted in profound downregulation of inducible chemokines, including
RANTES, IL-8, and MCP-1
(Table 2). In addition, there was a decrease in expression
of chemocytokines such as GM-CSF. Of note, suppression of
chemokine production by
E1-E4+ Adßgal
vectors could be overridden by stimulation of the
E1-E4+
Adßgal–infected ECs with IL-1ß. There was no significant change in
chemokine expression by
E1-E4-
Adßgal–infected ECs. These data suggest that downregulation of
chemokines by
E1-E4+ Advector
gene transfer vectors may play a minor role in the regulation of the
leukocyte trafficking, because upregulation of inflammatory
cytokines such as IL-1ß during
E1-E4-Adßgal
infection may override
E1-E4+ Adßgal
suppression of chemokine expression.
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Chemokine-Induced Migration of Leukocytes Is
Enhanced Through
E1-E4+-Infected
ECs
Combinatorial interaction between adhesion molecules
and chemokines is critical for successful
transendothelial migration of leukocytes to the
inflammatory sites. To assess the capacity of
E1-E4+
Advector–infected ECs to mediate migration of PBMNCs, intact
monolayers of HUVECs infected with either
E1-E4+ or
E1-E4-
Adßgal were placed on the upper transwell of 5-µm transwell
plates, and the capacity of MCP-1, SDF-1, or RANTES to induce migration
of leukocytes was evaluated
(Figure 6).
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CXCR4, the chemokine receptor for SDF-1, is expressed on
almost all leukocytes including monocytes and T
cells.32 33
Compared with
E1-E4-
Advector–infected HUVECs, migration of leukocytes
(CD45+ cells) in response to SDF-1 through
E1-E4+
Advector–infected HUVECs was enhanced by 48±7%
(Figure 6). Similarly,
E1-E4+
Adßgal–infected HUVECs supported the migration of significantly more
monocytes and T cells in response to MCP-1 and RANTES, respectively.
These data suggest that collective modulation of known and
as-yet-unidentified adhesion molecules on ECs by E4 gene enhances the
migration of leukocytes.
Discussion
Intravascular administration of transgenes by first-generation Advector E1-E4+ vectors is associated with infiltration of vascular bed with inflammatory cells, which may curtail the gene delivery of adenoviruses and therapeutic efficacy.11 In this report we suggest a mechanism for induction of this inflammatory process by demonstrating that E1-E4+ but not E1-E4- Advectors can directly induce activation of ECs by upregulation of ICAM-1 and VCAM-1 and restore CD34 expression. We also demonstrate that infection of ECs with E1-E4+ Advector but not with E1-E4- Advector modulates chemokine production by ECs, resulting in a significant augmentation of leukocyte adhesion and transendothelial migration of inflammatory cells. Collectively, these results strongly suggest that infection of ECs with E1-E4+ Advectors may induce nonspecific infiltration of inflammatory cells, in part by direct modulation of endothelial adhesion molecule and chemokine expression, and that this process can be circumvented by using E1-E4- Advector gene transfer vectors.
Several reports have shown that infection of A594 cells,34 vascular neointimal cells,11 or pulmonary tissue35 36 37 with E1-E4+ Advectors induces upregulation of ICAM-1. In this report, we demonstrate that E1-E4+ Advectors, but not E1-E4- Advectors, induce long-term induction of ICAM-1 on the ECs. Upregulation of ICAM-1 contributes to adhesion of leukocytes to ECs, enhancing infiltration of inflammatory cells. This nonspecific inflammatory process may act independently of immune response to Advector antigens to interfere with persistent gene expression by Advectors.
Upregulation of VCAM-1 on ECs and coactivation of its ligand VLA-4 on leukocytes play a critical role in adhesion of leukocytes to ECs. In the present study, E1-E4+ Advector infection of the ECs resulted in sustained expression of VCAM-1, and neutralizing mAbs to ICAM-1 and VCAM-1 substantially decrease the binding of leukocytes to E1-E4+ Advector–infected ECs. However, because the combination of neutralizing mAbs to VCAM-1 and ICAM-1 did not completely block the binding of leukocytes to ECs, there may be an as-yet-unidentified adhesion molecule(s) that mediates adhesion of leukocytes to E1-E4+ Advector–infected ECs.
The significance of CD34 expression in the modulation of leukocyte adhesion to ECs is not known. L-Selectin expressed on leukocytes mediates adhesion and migration of these cells to CD34 expressed on high endothelial venules.38 39 Based on the observations in the present study, it is possible that restoration or upregulation of CD34 expression by inflamed ECs after E1-E4+ Advector infection may enhance transendothelial migration of leukocytes in organ-specific vascular beds such as lymph nodes.
Chemokines modulate the inflammatory process by providing
directional cues for leukocytes to migrate into the inflamed
microenvironment.
E1-E4+ Advector
infection of ECs results in significant augmentation of migration of
leukocytes in response to MCP-1, RANTES, and SDF-1. On the other hand,
E1-E4+ Advector
infection of endothelium resulted in a profound
decrease in MCP-1, IL-8, and RANTES production. It is
intriguing that IL-1ß activation of Advector-infected
endothelium restores expression of MCP-1, IL-8, and
RANTES to the levels of IL-1ß–activated uninfected ECs. The
role of IL-1ß and TNF- in induction of inflammation by
E1-E4+
Advectors was underscored by the demonstration that IL-1ß
production by ECs after
E1-E4+ Advector
infection played a key role in induction of inflammation and activation
of antivector and antitransgene immune responses that curtail the gene
delivery and therapeutic
efficacy.40 41 In
this context, IL-1ß and TNF- produced as a result of
E1-E4+-induced
inflammation may restore local chemokine production to the
inflammatory levels. Intravascular introduction of
E1-E4+
Advectors may upregulate ICAM-1 and VCAM-1, facilitating infiltration
of activated T cells and monocytes that may in turn release
inflammatory cytokines such as IL-1ß and TNF. This chain of
events promotes an inflammatory response and may ultimately augment a
humoral immune response to Advectors.
We have previously observed that introduction of the E4 gene into ECs results in a state of "suspended animation" whereby ECs do not divide or undergo apoptosis.24 Given that E1-E4+ Advectors, but not the E1-E4- Advectors, induced prolonged survival of infected cells, it is logical to hypothesize that Advector E4 gene products may play a key role not only in the E1-E4+ Advector–mediated survival but also in upregulation of adhesion molecules. Furthermore, conditioned medium from E1-E4+-infected ECs failed to induce the expression of adhesion molecules, which lends credence to the possibility that intracellular interaction of E4 genes with the transcription of endogenous factors may be responsible for the altered phenotype of the ECs after infection with E1-E4+ Advectors.
It is conceivable that this E4-mediated locked-in state may
result in transduction of signaling pathways that promotes
stabilization of the expression of CD34 and upregulation of VCAM-1 and
ICAM-1. The E4 region of Advector contains 7 open reading frames
(ORFs) that regulate different aspects of cellular regulatory
functions. E4ORF4 binds and activates protein phosphatase
2A42 and may play a role in
the regulation of DNA synthesis and activator protein-1
(AP-1) transcription factor
activity.42 43
Given that expression of adhesion molecules is partially dependent on
AP-1 activation, it is conceivable that long-term activation of ICAM-1
and VCAM-1 and long-term restoration of CD34 expression may be mediated
through E4ORF4 expression. The promoters of the ICAM-1 and VCAM-1 genes
contain recognition sequences for the inducible nuclear transcription
factor 
B (NF-B).43 It
is possible, therefore, that gene products produced by E4 gene may
interact irreversibly with NF-B resulting in an activated
state whereby ECs are reprogrammed to constitutively express VCAM-1,
ICAM-1, and CD34.
ECs are exclusively sensitive to endotoxins. Endotoxins derived from bacterial cell membrane or other sources of lipopolysaccharides could induce significant activation of ICAM-1, VCAM-1, and E-selectin. However, long-term activation of adhesion molecules by E1-E4+ Advectors observed in the present study did not result from endotoxin contamination for several reasons. First, E1-E4- Advectors prepared in a manner similar to that of E1-E4+ Advectors did not activate adhesion molecules on ECs. Second, endotoxins result in a transient activation of adhesion molecules on ECs, whereas introduction of the E4 gene in ECs resulted in a sustained expression of ICAM-1 and VCAM-1 for several weeks to months. Finally, differential long-term upregulation of ICAM-1 and VCAM-1, but not E-selectin, by E4 gene products strongly suggests that specific E1--driven cellular signaling pathways, not the nonspecific activation observed with endotoxin, regulate E1-E4+ Advector–mediated adhesion molecule expression.
Expression of transgenes appears to be prolonged by the absence of the E4 region due to a reduced immune response.44 Moreover, several studies have shown that deletion of the E4 gene may result in diminished CMV transcription and transgene expression.45 46 47 48 On the basis of the data presented, lack of E4 gene does not seem to diminish transgene expression in the ECs. In fact, ECs infected with the same MOI of either E4+ Adßgal or E1- Adßgal express similar levels of LacZ, suggesting that at least under these in vitro conditions transgene expression is not altered by E4 deletion. These results suggest that expression of transgenes in the ECs may be regulated by a set of as-yet-unrecognized factors that are not significantly influenced by the lack of E4 genes.
Elucidating the molecular mediators of inflammatory and immune responses to adenoviruses injected intravascularly or into the tissues may allow for designing strategies to inhibit inflammatory reactions, thereby reducing toxicity and vector clearance and, therefore, enhancing the clinical efficacy of Advector-mediated gene therapy. On the basis of the data in the present study, the use of E1-E4- Advectors for gene delivery may provide an alternative strategy to dampen initial phases of the destructive inflammatory process and partially diminish immune response.
Acknowledgments
S.R. is supported by Grants R01 HL-58707 and R01 HL-61849, Program Project HL-66952 (Project 2), and Pilot Project P01 HL-59312, all from the NHLBI; the Dorothy Rodbell Foundation for Sarcoma Research; and the Rich Foundation. R.G.C. was supported in part by grants from the Will Rogers Memorial Fund (Los Angeles, Calif), Gen Vec, Inc (Gaithersburg Md), and National Heart, Lung, and Blood Institute (NHLBI) Grant R01 HL 57318.
Footnotes
Original received January 3, 2001; revision received March 8, 2001; accepted March 8, 2001.
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