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(Circulation Research. 2001;88:903.)
© 2001 American Heart Association, Inc.

Molecular Medicine

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

Shahin Rafii, Sergio Dias, Sarah Meeus, Koichi Hattori, Ramalingam Ramachandran, Fred Feuerback, Stefan Worgall, Neil R. Hackett, Ronald G. Crystal

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

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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^l-, 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

	Introduction

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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

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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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

View larger version (61K): [in this window] [in a new window]   Figure 1. Infection of endothelial monolayers with E1-E4+ but not E1-E4- Advectors resulted in activation of ECs. HUVECs were infected with 50 MOI of either E1-E4+ Adßgal or E1-E4-Adßgal. Three days after infection, cells were fixed and expression of ß-gal was quantified. E1-E4- Adßgal–infected cells (B, x200) assume the typical cobblestone morphology of naive uninfected ECs (A, x200). Compared with E1-E4- Adßgal–infected cells (B), E1-E4+ Adßgal–infected cells (C, x200) become elongated and spindle-shaped, which is a morphology reminiscent of cytokine-activated endothelium. This activated morphology persisted for several weeks after infection.

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.

View larger version (33K): [in this window] [in a new window]   Figure 2. E1-E4+ infection of ECs results in upregulation of CD34, ICAM-1, and VCAM-1 expression. Confluent monolayers of BMECs were infected with 10 MOI of either E1-E4+ Advector or E1-E4- Adßgal in serum-free conditions. Three days after infection, expression of adhesion molecules was assessed by 2-color flow cytometry. Infection of ECs with E1-E4+ but not E1-E4- Adßgal vectors resulted in upregulation of CD34, ICAM-1, and VCAM-1 (n=4, P<0.01, E1-E4- compared with E1-E4+). There was no change in expression of E-selectin, PECAM, or P-selectin. As control, thrombin activation of ECs resulted in upregulation of P-selectin. IL-1ß activation of ECs resulted in induction of ICAM-1, VCAM-1, and E-selectin. Identical results were obtained for HUVECs (not shown).

View larger version (47K): [in this window] [in a new window]   Figure 3. Expression of CD34 and ICAM-1 and VCAM-1 by E1-E4+ Advector–infected ECs. HUVECs were infected with a single dose of 50 MOI of E1-E4+ Adßgal vector. After 12 hours, remaining Advectors were removed and level of expression of adhesion molecules was quantified on days 1 and 20 after infection by 2-color flow cytometry. Although resting uninfected HUVEC monolayers did not express ICAM-1, VCAM-1, or CD34, there was robust upregulation of CD34, ICAM-1, and VCAM-1 expression detectable on days 1 and 20 after infection (n=5, P<0.05). There were no changes in expression of PECAM, E-selectin, or P-selectin. BMECs show activation pattern similar to that of HUVEC monolayers (not shown).

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.

View this table: [in this window] [in a new window]   Table 1. Modulation of Surface Expression of Receptors by E1-E4+- and E1-E4--Infected ECs

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.

View larger version (19K): [in this window] [in a new window]   Figure 4. Time course of CD34, VCAM-1, and ICAM-1 expression by HUVECs infected with 50 MOI of E1-E4+ Advectors. Expression level of different adhesion molecules was determined at different time points by 2-color flow cytometry (n=3). There was rapid upregulation of ICAM-1, VCAM-1, and CD34 followed by persistent stable expression for several weeks.

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.

View larger version (31K): [in this window] [in a new window]   Figure 5. E1-E4+ Advector–infected HUVECs support adhesion of leukocytes. PBMNCs were incubated in the presence and absence of blocking mAbs to VCAM-1, ICAM-1, and CD34 with unstimulated HUVECs or HUVECs that were infected with either E1-E4+ or E1-E4- Adßgal. After 2 hours, nonadherent cells were removed and the adherent leukocyte population (CD45+, CD14+, CD15+, CD3+) was quantified by flow cytometry and phase-contrast microscopy. Blocking mAbs to ICAM-1 or ICAM-1+VCAM-1 inhibited the binding of leukocytes to ECs (n=4, P<0.05 for ICAM-1+VCAM-1 conditions).

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.

View this table: [in this window] [in a new window]   Table 2. Chemokine Production by E1-E4+- and E1-E4--Infected ECs

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).

View larger version (23K): [in this window] [in a new window]   Figure 6. Transmigration of leukocytes is enhanced through E1-E4+ Advector–infected HUVECs. PBMNCs were placed on the upper chamber of 5-µm transwell plates that were coated with confluent monolayers of unstimulated, IL-1ß–activated, E1-E4+ Adßgal–, or E1-E4- Adßgal–infected HUVECs. Immediately the following was added to the lower chamber (in ng/mL): SDF-1 200, MCP-1 20, or RANTES 150. After 3 hours of incubation at 37°C, the number and phenotype of migrating cells were assessed by 2-color flow cytometry using mAbs to monocytic (CD14 and CD15) and lymphocytic (CD3, CD4, and CD8) markers. For leukocytes migrating in response to SDF-1, the total number of migrating cells was quantified by determination of CD45+ cells (n=3). In all conditions, monocyte or lymphocyte migration in response to chemokines was enhanced through E1-E4+ HUVEC monolayers (n=3, P<0.05). There was no statistical significance in number of leukocytes migrating through unstimulated HUVECs (n=3, P>0.05).

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 tissue^{35 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.

	References

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