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Cardiovascular Research 2001 50(3):538-546; doi:10.1016/S0008-6363(01)00269-3
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Copyright © 2001, European Society of Cardiology

Effect of cytomegalovirus immediate early gene products on endothelial cell gene activity

Esther Guettaa,*, Eleonora M Scarpatib and Paul E DiCorletoc

aDepartment of Human Genetics and Molecular Medicine, Sackler School of Medicine Tel Aviv University, Tel Aviv, Israel
bBIEX, Inc., Dublin, CA, USA
cDepartment of Cell Biology, Research Institute of the Cleveland Clinic Foundation, Cleveland, OH, USA

* Corresponding author. Present address: Danek Gertner Institute of Human Genetics Sheba Medical Center, Tel-Hashomer, 52621 Israel. Tel.: +972-3-530-3974; fax: +972-3-530-2914 guetta{at}post.tau.ac.il

Received 27 June 2000; accepted 5 February 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Cytomegalovirus (CMV) infection or reactivation from latency in vascular cells have been shown to contribute to atherosclerosis. CMV-infected endothelial cells (ECs) exhibit enhanced adhesion and procoagulant properties, changes compatible with processes observed in atherogenesis. The major immediate early promoter drives immediate early gene transcription. Immediate early (IE) gene products, IE72 and IE84, function as transcription factors and thereby influence expression of cellular genes, in permissive cells as well as in abortive infections, in which viral activity is limited to immediate early expression. ECs have been shown to harbor latent CMV, support abortive CMV infection and, under certain conditions, are permissive to productive viral infection. The objective of this study was to determine whether immediate early expression alone (in the absence of further progression of the virus life-cycle) results in the activation of EC genes associated with atherogenesis. Methods: The study was conducted in an in vitro transient transfection system in human and bovine vascular ECs, with CMV immediate early gene expression vectors and plasmids containing promoter sequences of adhesion molecule, growth factor and viral promoters driving the transcription of reporter genes. Results: CMV immediate early gene expression resulted in an increase in monocyte adhesion to ECs and in the relative promoter activities of cellular growth factor and adhesion molecule genes. In addition, the viral major immediate early promoter was regulated in EC by thrombin and the immediate early gene products. Conclusion: These results infer the possible existence of a positive feedback mechanism in the developing atherosclerotic lesion, in which enhanced immediate early gene expression leads to subsequent activation of EC genes, which might in turn result in further activation of CMV activity.

KEYWORDS Atherosclerosis; Cell culture/isolation; Endothelial factors; Gene expression; Infection/inflammation

This article is referred to in the Editorial by M. Levi (pages 432–433) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Cytomegalovirus (CMV) infection and reactivation from latency are possible contributing factors to the development of atherosclerosis [1]. Several lines of evidence support this concept: in studies on human-derived tissues, CMV DNA sequences were detected in the wall of atherosclerotic vessels and accelerated coronary atherosclerosis is more common in cardiac transplant patients exposed to CMV than in those who have had no such exposure [2–5]. Infection with CMV and other herpes viruses induces changes that are associated with atherosclerosis, the related process of restenosis, and transplantation-associated accelerated atherosclerosis. In animal models CMV infection results in increased smooth muscle cell (SMC) proliferation, neointimal response to vascular injury and lipid accumulation [6–9]. Infection of SMC in vitro leads to an increase in proliferation, elevated platelet-derived growth factor (PDGF) B-chain receptor expression, an increased uptake of oxidized low-density lipoprotein and elevated intracellular levels of reactive oxygen intermediates [10–12]. Infection of endothelial cells (ECs) results in procoagulant changes, as well as increased leukocyte adhesion [13–15].

Several aspects of CMV biology, highlighted below, support the concept of CMV involvement in atherogenesis. Following infection, which is usually subclinically manifested, CMV is harbored for life in a latent state; viral genome persists in cells, without viral gene expression [16]. Although CMV causes serious disease in immunocompromised subjects, such as neonates, patients with AIDS and transplant recipients, it is generally accepted that, with the exception of a mononucleosis-like syndrome, CMV does not cause disease in healthy immunocompetent individuals. An important exception to this paradigm is the proposed scheme, emerging from a growing body of evidence, in which activation of CMV in the vessel wall, or in monocytes recruited to the vessel wall, results in a subsequent contribution to the development of atherosclerotic lesions [1,17].

CMV replication in permissive host cells is accomplished by a three-stage sequence of viral gene expression [18]. A different group of viral proteins is expressed in each stage. The immediate early genes, transcribed from the major immediate early promoter are the first viral genes expressed. The immediate early proteins, IE72 and IE84 regulate the expression of the second group of viral genes, the early genes, which in turn regulate the expression of the late genes. Under certain conditions an abortive infection is established in which viral expression is limited to the immediate early stage, without additional viral activities [19]. The immediate early gene products function as transcription factors and thereby influence expression of cellular genes, even in the absence of a productive infection (in which the entire viral life cycle is completed) [19,20]. Since the first step in viral reactivation is the expression of the immediate early genes, driven by the major immediate early promoter, the activity of this promoter is crucial not only in productive CMV infection, but also in restrictive virus–host interactions and in reactivation from latency.

Vascular ECs harbor CMV DNA in vivo, and cultured ECs are permissive hosts for endothelial-passaged clinical isolates of CMV [21,22]. Infection of ECs with these strains of virus results in virus production and development of cytopathic effects, whereas infection with fibroblast-passaged laboratory strains of CMV leads to a limited infection, without cytopathic effects, and restricted immediate early expression [22]. It is unknown which mode of viral activity is pertinent in vivo. It is possible that there is a heterogeneous response to CMV among the ECs lining the vessel wall, resulting in abortive infection in some cells, and in other cells exhibiting a permissive phenotype, a productive infection takes place. In both schemes, activation of the viral major immediate early promoter and subsequent immediate early expression are accomplished.

In an earlier study, we showed that the activity of the CMV major immediate early promoter increases in monocytes as a result of co-culture with ECs, SMCs and exposure to oxidized low-density lipoprotein [23]. In addition, the virus could be transmitted from monocytes that harbored the virus in a non-active state, to vascular ECs and SMCs present in the co-culture system. We suggested that this in vitro model might represent one of the mechanisms underlying CMV reactivation and enhanced viral activity in the vessel wall, which in turn, might contribute to the progression of the atherosclerotic process. In the present report, this model has been expanded in order to examine the effect of this activation of CMV immediate early gene expression on the promoter activity of EC genes and on the auto-regulation of major immediate early promoter in ECs.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Fetal bovine serum was purchased from Whittaker Bioproducts (Walkersville, MD, USA). Tissue culture media were purchased from Irvine Scientific (Santa Anna, CA, USA). Lipofectin and Optimem I reduced serum transfection medium were purchased from Gibco (Gaithursburg, MD, USA). [14C] chloramphenicol, from Amersham (Piscataway, NJ, USA), [51Cr] chromate from DuPont NEN (Boston, MA, USA), and bovine {alpha}-thrombin from Alexis Biochemicals, (San Diego, CA, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA). Tissue culture plasticware was purchased from Costar (Cambridge, MA, USA). All reagents used in cell culture were tested for endotoxin contamination using the Limulus endotoxin detection assay kit from BioWhittaker. The lack of significant endotoxin contamination was ruled out by the absence of sensitivity to polymyxin B in treated samples versus those that were not treated.

2.2 Cells
Human umbilical vein ECs (HUVECs) were isolated from umbilical veins by collagenase treatment and cultured in MCDB 107 medium supplemented with heparin (90 µg/ml), 15% fetal bovine serum (FBS, v/v) and ECs growth supplement (150 µg/ml). Culture plates were coated with fibronectin (1 µg/cm2). Cells were used between passages 2 and 4 [24].

Bovine aortic ECs (BAECs) were cultured in DME–F12 medium, supplemented with 5% FBS, and used up to passage 10 [24].

ECs were identified according to their morphology and positive staining with anti-von-Willebrand antibodies.

The procurement of human and animal tissues for primary cell preparation was carried out according to the principals of the Declaration of Helsinki and The Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health, respectively.

The human microvascular EC line, immortalized with SV-40 virus large T antigen, HMEC-1, was a gift from E.W. Ades (Biological Products Branch, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA, USA). Cells were cultured as described, in MCDB-131 medium supplemented with 15% fetal bovine serum, 10 ng/ml epidermal growth factor, 1 µg/ml hydrocortisone [25].

U937 cells, originally derived from a human histiocytic lymphoma (ATCC, Rockville, MD, USA) were propagated in suspension culture in RPMI-1640 medium containing 5% FBS.

All cell cultures were maintained at 37°C and 5% CO2.

2.3 Plasmids
The plasmids pHD101CAT3 [26], containing a 2.1-kilobase region of the human CMV major immediate early promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, and pRC/RSV IE284, an IE84 expression vector [27] were gifts of E.S. Huang, Department of Immunology and Microbiology, University of North Carolina, Chapel Hill, NC, USA. The IE72 expression vector, pRSV72 [28] was a gift of J.A. Nelson, Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA. The pRC/RSV plasmid (Promega, Madison, WI, USA), or pRC/RSV-LUC (the pRC/RSV plasmid into which the firefly luciferase cDNA was cloned downstream to the RSV promoter), were used for negative controls in co-transfection experiments. The p400CAT plasmid, contains the 400 base-pair (bp) region of the PDGF B-chain gene, upstream to the transcription initiation site, fused to the CAT reporter gene [29]. The E-selectin promoter–reporter construct, p510LUC, contains the E-selectin promoter (510 bp sequence 5' to the transcription initiation site) fused to the firefly luciferase reporter gene (original vector: pGL3 vector, Promega). The VCAM–CAT promoter–reporter construct was a gift from M.F. Iademarco, Washington University School of Medicine, St. Louis, MO, USA [30].

2.4 Transfection
Cells were plated at a density of 3x105 cells/well, in six-well cluster plates, allowed to reach 50–70% confluency and then transfected. In experiments with the PDGF B-chain promoter–reporter plasmid, co-transfection mixtures contained 0.25 µg of IE72 or IE84 plasmid DNA, mixed with 2.5 µg of the p400CAT reporter vector in 100 µl Optimem I medium. Lipofectin (15 µg) in 100 µl Optimem I medium was added to the plasmid mixtures, mixed and incubated at room temperature according to the manufacturer's recommendations. An 800-µl volume of Optimem I was added to the Lipofectin-plasmid DNA mixture and the final mixture was added to each well. The cells were incubated at 37°C for 4 h, rinsed in Optimem, and refed with growth medium.

Transfection efficiency was measured with a β-gal expression vector, in separate samples, since the various co-transfecting vectors had varying effects on β-gal expression. In bovine cells the efficiency was 3% and in human ECs, between 0.5 and 1%.

2.5 Monocyte adhesion assay
Adhesion of BAECs transfected with immediate early expression vectors to U937 cells was measured as previously described [31]. Briefly, U-937 cells were labeled for 90 min at 37°C with 51Cr (100 µCi, sodium chromate) in culture medium. The labeled cells were washed and 106 radiolabeled viable cells were added to each well of BAECs. After binding occurred for 1 h, at 4°C, the wells were washed and the cells lysed with 1% Triton X-100. An aliquot of the lysate was removed for gamma radiation counting. The number of U-937 cells bound per well was calculated from the initial specific activity (counts per min per cell) of the monocytic cell preparation. Spontaneous release of Cr from the monocytic cells during the assay was <5% of the total count.

2.6 CAT assay
The cells, harvested 48 h post-transfection by scraping, were lysed by three freeze–thaw cycles. The CAT assay was performed using [14C] chloramphenicol and Tris buffer as previously described [29]. Determinations were made within the linear range of the assay. Presented data represent the mean of triplicate wells, and are normalized to protein content per well, as determined by the BCA assay (microtiter plate protocol) (Pierce, Rockford, IL, USA).

2.7 Luciferase assay
Twenty-four hours after transfection cells were washed and harvested by scraping in 200 µl of lysis buffer (Promega). Cell debris was pelleted by centrifugation, and the supernatant was assayed for luciferase activity. Presented data represent the mean of triplicate wells, and are normalized to protein content per well, as determined by the BCA assay.

2.8 Statistical analysis
All values are expressed as the mean±standard deviation. One-way ANOVA and Dunnett post-hoc test were applied to all values with the following two exceptions: the effect of IE expression on PDGF B-chain promoter activity in HUVECs was analyzed with a one-sample t-test and the interaction between TNF{alpha} treatment and IE expression was analyzed by two-way ANOVA. Log transformation of values was applied where appropriate in order to approach normal distribution.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Immediate early expression in ECs augments monocyte adhesion
To determine the effect of immediate early gene expression on monocyte adherence to ECs, BAECs were transfected with IE72 and IE84 expression vectors. Initially, an attempt was made to isolate colonies of stable transfectant cell clones expressing the CMV immediate early genes. However, since the expression of these genes could not be continuously maintained in the isolated colonies, and was lost after 3–4 passages of the expanded colonies in culture, a transient transfection system was employed. Transcription of immediate early mRNA was detected by Northern blotting using RNA from pools of transiently transfected ECs (data not shown). These pools were used for adhesion assays. Monocyte adhesion to ECs expressing IE72 and IE84 gene products, increased by 1.9- and 2.4-fold, respectively, compared to pooled ECs transfected with control plasmid (P=0.043 and 0.015, respectively) (Fig. 1). Stimulation of ECs with TNF-{alpha} caused a 4.5-fold increase in monocyte adhesion in control cultures; whereas, the combination of TNF-{alpha} and IE72 or IE84 expression led to a greater increase in monocyte adhesion, 7.9- and 13.5-fold, respectively (P=0.05 and P<0.01, respectively) (Fig. 1). Analysis of the interaction between TNF{alpha} and IE expression did not reveal a statistically significant increase in monocyte adhesion induced by TNF-{alpha} on IE transfected cells in comparison to the TNF-{alpha} effect on control cells (P=0.498).


Figure 1
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  		Fig. 1 Effect of CMV immediate early expression in BAEC cells on U-937 cell adhesion. Non-stimulated BAECs transiently transfected with pRC, pIE72 and pIE84 expression vectors (black bars), and after 4-h treatment with TNF-{alpha} (10 ng/ml) (dotted bars). Data represent one of two similar experiments and are reported as mean±standard deviation from triplicate wells.

 
3.2 Effect of immediate early expression on E-selectin and VCAM-1 promoter activity in EC
We investigated the effect of CMV immediate early gene expression on E-selectin and VCAM-1 promoter activity in EC from several different sources. In HUVECs, co-transfection of the E-selectin promoter–reporter vector with CMV IE72 and IE84 expression vectors led to a 26- and 25-fold increase in E-selectin promoter activity, respectively (P<0.001) (Fig. 2A). A stimulatory effect was also observed in HMEC-1 cells, with a 53-fold increase for IE72 and a 23-fold increase for IE84 in E-selectin promoter–reporter activity (P<0.001) (Fig. 2B).


Figure 2
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  		Fig. 2 Effect of immediate early expression on E-selectin promoter activity in human and bovine ECs. HUVECs (A), HMECs (B) and BAECs (C) were co-transfected with pRC, pIE72 and pIE84 expression plasmids and E-selectin promoter-driven luciferase reporter gene construct. Data represent one of three similar experiments and are reported as mean±standard deviation from triplicate wells.

 
Co-transfection of BAECs with an IE72 expression plasmid and E-selectin promoter–reporter plasmid, containing a 510-bp sequence of the promoter driving the expression of the fire-fly luciferase reporter gene, led to a 3-fold increase in E-selectin promoter activity. Co-transfection with IE84 had no significant effect on E-selectin promoter activity (P<0.001) (Fig. 2C).

VCAM-1 promoter activity was not changed by immediate early gene expression in co-transfected ECs (data not shown).

3.3 Effect of immediate early expression on PDGF B-chain promoter activity in ECs
The effect of IE72 and IE84 expression on PDGF B-chain promoter activity was investigated. HUVECs were co-transfected with immediate early expression vectors and a plasmid containing a 400-bp sequence of the c-sis promoter driving the CAT reporter gene. Co-transfection of IE72 construct with PDGF promoter–reporter construct, led to a modest increase of 2.3±0.8 fold (P=0.09, n=3) in promoter activity whereas IE84 expression did not result in increased activity (Fig. 3A). Co-transfection of HMEC-1 cells, with the IE72 expression vector and the PDGF promoter–reporter construct, led to a 5.4-fold increase in reporter gene activity (P=0.001), whereas IE84 expression did not cause any significant effect (Fig. 3B). Interestingly, in BAECs, the basal activity of the PDGF B-chain promoter was inhibited as a result of CMV IE72 and IE84 expression (P<0.001) (Fig. 3C).


Figure 3
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  		Fig. 3 Effect of immediate early expression on PDGF B-chain promoter activity in human and bovine ECs. HUVECs (A), HMECs (B) and BAECs (C), were co-transfected with pRC, pIE72 and pIE84 expression plasmids and PDGF promoter-driven CAT reporter gene construct. Data for HUVECs represent mean fold increase for three separate experiments, and for HMECs and BAECs, one of three similar experiments.

 
3.4 Regulation of major immediate early promoter activity in ECs
The effect of thrombin treatment on major immediate early promoter activity in ECs was examined. Cells transfected with a major immediate early promoter reporter-plasmid, and treated with thrombin showed a three-fold increase in major immediate early promoter activity in both bovine and human EC, compared with baseline major immediate early promoter promoter activity (Fig. 4).


Figure 4
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  		Fig. 4 CMV major immediate early promoter response to thrombin in ECs. ECs were transfected with major immediate early promoter-CAT plasmid and treated with thrombin (10 units/ml), 18 h after transfection for 6 h. Data represent one of three similar experiments and are reported as mean±standard deviation from triplicate wells.

 
The auto-regulation of major immediate early promoter activity was investigated in human ECs. IE84 expression resulted in a 7-fold decrease (P<0.001) in major immediate early promoter activity, whereas IE72 expression did not alter major immediate early promoter activity.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results presented here demonstrate that CMV immediate early gene products might contribute to the activation of EC genes that are associated with development of the atherosclerotic lesion. These changes may help to explain the broader observation of an association between cytomegalovirus infection and atherosclerosis [1].

Transient transfection of bovine ECs with CMV immediate early expression vectors led to an increase in monocyte adhesion in uninduced and in TNF-{alpha} activated ECs. CMV immediate early gene products activate cellular and viral promoters through the activation of nuclear transcription factors [27,32] and might also induce adhesion of monocytes to ECs through this mechanism, as the promoters of adhesion molecule genes encode transcription factor binding sites [33,34]. The immediate early gene-mediated induction of monocyte adhesion to ECs could also be through a redox-sensitive mechanism since increased intracellular levels of oxygen free radicals lead to an increase in monocyte adhesion and CMV immediate early gene activity results in increased intracellular ROS levels [35,12,36].

The adhesion response in immediate early gene-expressing ECs might represent elevated adhesion molecule gene activity, as evidenced by increased E-selectin promoter activity in ECs co-transfected with IE72 or IE84 expression plasmids and the E-selectin promoter–reporter vector. Both IE72 and IE84 activated the E-selectin promoter in EC of human origin, whereas in bovine ECs, only IE72 generated a response. This difference between ECs of human and bovine origins might be due to divergent regulation mechanisms — transcription factors expressed in human cells that are necessary for the IE84-mediated activation might be absent from the bovine cells.

CMV-mediated increases in adhesion molecule activity were demonstrated by Shahgasempour et al., who reported an increase in leukocyte adhesion as early as 6 h post-infection with CMV in HUVECs, a time course coinciding with immediate early gene expression in infected cells [37]. Conversely, Sedmak et al. did not detect an induction of E-selectin expression in CMV-infected ECs [38]. This discordance might be due to the different stages of infection at which the adhesion molecule expression was measured — early in infection [37], versus late in infection [38]. In our system immediate early expression did not lead to increased promoter activity of adhesion molecule VCAM-1, contrary to previous results showing that infection of HUVECs with CMV leads to an increase in VCAM-1 expression [38]. The induction of VCAM-1 might require additional CMV related factors that are provided upon infection with viral particles, as opposed to transfection-mediated expression of selected genes.

Elevated expression of several growth factors has been linked to atherogenesis [39]. PDGF BB, secreted by EC stimulates SMC proliferation, and is expressed in atherosclerotic lesions [40]. CMV infection leads to an augmentation of transplant-associated accelerated atherosclerosis in a rat aortic allograft model system, with a concomitant increase in PDGF BB mRNA levels in the affected vascular tissues of the infected animals [41]. Non-stimulated ECs have constitutive activity of the PDGF B-chain promoter in vitro, which is activated by the binding of transcription factors to response elements present in the 400 bp region 5' to the transcription initiation site in response to various agonists [29,42]. We examined the possibility that in vascular ECs, CMV immediate early expression leads to an increase in PDGF BB expression, by stimulating promoter activity. In human ECs, IE72 expression led to an increase in PDGF B-chain promoter activity, whereas, IE84 expression had no effect. In cells of bovine origin, immediate early expression led to a decrease in PDGF B-chain promoter activity, possibly due to the interaction of immediate early gene products with negative regulating elements in these cells. In a previous report, infection of ECs with CMV did not result in increased secretion of PDGF BB, however, the possibility of an increase in intracellular PDGF could not be ruled out, since only secreted PDGF levels were measured in this study [43].

The CMV major immediate early promoter, studied in vascular SMC and transformed cell lines of fibroblast and leukocyte origins, is regulated by cytokines, oxidative stress, and by the gene products transcribed from this promoter [12,23,44–46]. The IE72 product activates the major immediate early promoter, whereas, IE84 inhibits promoter activity [45,46]. In the present study, major immediate early promoter activity in ECs was repressed by IE84 expression, while IE72 expression did not result in any change.

Thrombin regulation of major immediate early promoter activity was also investigated. Several endothelial genes linked to atherosclerotic changes are responsive to thrombin [29,47]. For example, the PDGF-B chain promoter in EC is activated by thrombin through the binding of a thrombin-inducible nuclear factor to a thrombin response element identified in the promoter [29]. Examination of the CMV major immediate early promoter revealed two potential thrombin response elements CCACCC at –395 and –150 bp upstream to the transcription initiation site, within the enhancer region, and one site at –40 bp, outside the enhancer region [48]. In this study, major immediate early promoter activity increased in ECs in response to thrombin. Since CMV infection induces procoagulant changes in infected cells, it is conceivable that a positive feedback mechanism exists through which CMV infection induces pro-coagulant changes, and the resulting increase in thrombin activity induces the major immediate early promoter and promoters of thrombin-sensitive EC genes.

Thrombin-mediated induction of CMV major immediate early promoter might also act through activation of nuclear transcription factor NF-{kappa}B. Stimulation of the thrombin receptor leads to activation of NF-{kappa}B, generating subsequent cellular responses, and, in parallel, the CMV major immediate early promoter activity is positively regulated by NF-{kappa}B [49–51].

The objective of this study was to examine the effect of CMV immediate early expression on cellular gene activity. In order to prove that the promoter transactivation we observed corresponds to an increase in the endogenous levels of the respective gene products, gene expression assays need to be carried out. In our hands, we could not demonstrate an increase in E-selectin mRNA nor PDGF-BB mRNA in RNA samples extracted from cells transiently transfected with immediate early expression vectors by Northern blotting technique. This result most likely reflects the low transfection efficiency of these cells; i.e. only a small percentage of the cells in the culture were expressing immediate early proteins. Preliminary results with the more sensitive technique of RNAse protection assay, indicated an increase in E-selectin mRNA levels in IE72 and IE84 expressing cells, compared with control transfected cells. However, transient expression of immediate early genes in HUVECs did not induce E-selectin surface expression, measured by immunocytochemistry and radioimmunoassay techniques. Once again, low transfection efficiency in HUVECs [52], combined with the higher sensitivity of the promoter–reporter systems relative to the expression assays employed, might together provide an explanation for the discrepant results: that is promoter activation in the absence of increased protein expression.

The method of gene delivery as well as the characteristics of the transfected cells both influence the likelihood of observing cellular changes at the protein level induced by CMV immediate early gene expression. Such changes were observed in VSMCs in which virus-mediated gene transfer methods were applied yielding high efficiency of gene delivery, and, in tumor-derived cell lines [53–56].

In conclusion, thrombin activates the CMV major immediate early promoter resulting in immediate early gene expression. This leads to increased activity of the promoters of the PDGF B-chain and E-selectin genes, suggesting the possibility of a link between viral immediate early genes and increased monocyte adhesion and smooth muscle cell growth.

Time for primary review 21 days.


   Acknowledgements
 
We wish to thank Dr. Robert Rossero for having constructed the E-selectin promoter–reporter vector that was used in this study. We also thank Professor Jacov Tal for critical reading of this manuscript. This work was supported in part by US National Institutes of Health grants HL 29582 and HL 34727 (Dr. P.E. DiCorleto).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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