1 Abteilung Virologie, Universitätsklinikum Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany
2 Roche Diagnostics GmbH, Nonnenwald 2, 82372 Penzberg, Germany
3 Department of Plastic Surgery, University Hospital Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany
Correspondence
Thomas Mertens
thomas.mertens{at}medizin.uni-ulm.de
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ABSTRACT |
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INTRODUCTION |
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Cytokines and various growth factors, e.g. vascular endothelial growth factor (VEGF), are important regulators of vascular functions and integrity (Waltenberger, 1997). The VEGF protein family influences endothelial cell survival, angiogenesis and vascular permeability (Zachary & Gliki, 2001
). In the pathology of herpetic stromal keratitis, herpes simplex virus-induced VEGF-associated angiogenesis has been shown to occur in ocular lesions (Zheng et al., 2001
). Human herpesvirus 8 is known to be associated with the development of malignancies such as primary effusion lymphomas and Kaposi's sarcoma, which are interlaced with newly formed vessels. Viral cytokine-induced VEGF expression that increased tumour cell growth was demonstrated in primary effusion lymphoma cell lines (Liu et al., 2001
). Our study was designed to identify consequences of HCMV infection on peptide growth factor expression. We have demonstrated HCMV-induced modulation of VEGF production and shown that Sp1 sites on the VEGF promoter are involved in the activation of VEGF gene expression. This HCMV-induced effect was shown in fibroblasts, a commonly used cell type that is susceptible to permissive HCMV infection, as well as in smooth muscle cells (SMC), a relevant cell type in plaque development in atherosclerosis. The mitogenic activity of the released VEGF protein on endothelial cells was shown. This HCMV-induced paracrine effect might be relevant in vasculopathies.
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METHODS |
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RNA extraction and Northern blotting.
Total RNA was extracted and 10 µg total RNA was separated as described previously (Minisini et al., 2003). RNA samples were obtained from uninfected cells as well as from cells infected with viable virus, UV-inactivated virus and GFP-expressing virus, pre-incubated with HCMV hyperimmunoglobulin (Flebogamma; Grifols). RNA was transferred onto a nylon membrane and immobilized by incubation for 2 h at 60 °C. For hybridization, a DNA probe specific for VEGF was amplified by PCR. Membranes were stripped for 20 min with 0·1x SSC and 0·1 % SDS for rehybridization with a probe specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Probes were labelled with [32P]dCTP by using a random-priming kit (Pharmacia). Hybridization was carried out at 42 °C for 18 h in the presence of 50 % formamide. Filters were washed to a stringency of 0·2x SSC, 0·1 % SDS at 65 °C. Intensity of RNA signals was quantified by densitometry. Ratios of specific VEGF RNA levels were standardized according to the signals obtained with the GAPDH probe.
Plasmids and transient expression.
For transient expression, constructs consisting of different 5' regions of the VEGF promoter linked to the luciferase reporter gene were used as described previously (Finkenzeller et al., 1997) and the dual luciferase reporter assay system (Promega) was used for internal standardization. Briefly, HFF were grown on 12-well plates and transfected with 30 µg of each of the indicated plasmids by calcium phosphate precipitation as described by Sambrook et al. (1989)
, followed by glycerol shock. Cells were harvested 24 h post-transfection and resuspended in 100 µl lysis buffer. Luciferase activity was determined with a Lumat LB 9507 luminometer (Berthold). For transient-expression experiments in mock- and virus-infected cultures, cells were infected with viable or UV-inactivated HCMV 2 h before transfection.
Analysis of VEGF protein by Western blotting.
Western blot analysis for detection of VEGF expressed in mock- or HCMV-infected cells and cells incubated with UV-inactivated virus was performed as described previously (Michel et al., 1996). Cell lysates were extracted and separated by SDS-PAGE (12 % acrylamide). VEGF proteins were detected with the antiserum sc152 (Santa Cruz) and visualized by enhanced chemiluminescence (Amersham Biosciences).
Immunofluorescence.
SMC or HFF cultured on chamber slides were washed three times with PBS and fixed with ice-cold methanol. Primary antibodies were directed against the HCMV immediate-early antigen (Argene Biosoft, clone E13, exon 2, major IE) or the VEGF protein (Santa Cruz). Antibodies were added at a dilution of 1 : 50 in PBS supplemented with 1 % BSA (Sigma). Slides were incubated for 45 min at 37 °C. After extensive washing, a fluorescein isothiocyanate-labelled donkey anti-rabbit antibody (Dianova) was added at a dilution of 1 : 50 and a tetramethylrhodamine isothiocyanate-labelled goat anti-mouse antibody (Dianova) was used at a dilution of 1 : 30 in PBS/BSA. As antibody controls, we used irrelevant primary antibodies (DAKO) or the primary antibodies were omitted. After 45 min incubation at 37 °C, slides were washed and mounted with glycerol gelatin. Analysis was performed by using a fluorescence microscope (Zeiss).
Production of conditioned media.
Cell-free supernatants were produced in parallel on non-infected and TB40E-infected SMC and on SMC incubated with UV-inactivated virus. Cultures were washed and cultured in SMC basal medium supplemented with 1 % BSA and 1 % FCS. After 48 and 72 h, supernatants were harvested and centrifuged at 3000 r.p.m. for 10 min. Following UV inactivation, supernatants were stored at 70 °C.
Fluorescence-activated cell sorting (FACS) analysis.
HCAEC were harvested after 48 h incubation with conditioned media from HCMV-infected and non-infected SMC. Cells were detached and separated by using trypsin (BioWhittaker). Cells were fixed with 4 % paraformaldehyde and washed with PBS containing 3 % heat-inactivated FCS, 0·1 % sodium azide and 10 mM HEPES (FACS buffer). To minimize non-specific staining and to permeabilize the membrane, cells were incubated for 45 min at 4 °C with FACS buffer supplemented with 10 % human immunoglobulin (Flebogamma, 5 %; Grifols) and 0·1 % saponin. An irrelevant antibody (DAKO) or a specific mAb directed against an intracellular epitope of the VEGF receptor-2 (VEGFR-2) protein (Santa Cruz) was applied. After washing, a phycoerythrin (PE)-labelled goat anti-mouse secondary antibody (DAKO) was added. Cells were washed thoroughly following 30 min incubation on ice. Quantitative analysis was performed on a FACScan flow cytometer (Becton Dickinson) using the CellQuest research software. In total, 1x105 events per sample were collected and analysed.
Endothelial cell proliferation assay.
HUVEC were seeded at a confluence of 50 % on 96-well plates (Greiner). Before adding the conditioned media produced on SMC, HUVEC were starved overnight. Starvation media were removed and conditioned media were added. Each sample was analysed in six replicates. To identify the mitogenic protein, neutralizing mAbs directed against either VEGF (RD Systems) or an irrelevant antigen were added to aliquots of conditioned media 1 h prior to the proliferation assay, according to the manufacturer's instructions. HUVEC were stimulated by conditioned media for 24 h. During the final 12 h incubation, 1 µCi (37 kBq) [3H]thymidine per well (Amersham Biosciences) was added. After the stimulation period, medium was removed and cells were lysed and detached with 100 µl 0·1 % saponin and trypsin (5 g l1) in PBS. The lysates were transferred to a filter and scintillation counting was performed by using a Betaplate 96-well harvester (Pharmacia). For statistical analysis, the MannWhitney unpaired non-parametric test was used.
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RESULTS |
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DISCUSSION |
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Recently, immunohistochemical studies of human coronary artery tissues indicated that advanced atherosclerotic lesions contained a statistically significant number of intimal blood vessels, as well as higher numbers of VEGF-expressing cells within the intima (Chen et al., 1999). By double immunostaining, the majority of VEGF-expressing cells were identified as being SMC (Couffinhal et al., 1997
; Chen et al., 1999
). Within advanced lesions, VEGF could even be detected in intraplaque microvasculature. These authors suggested that, in vivo, VEGF induces neointimal angiogenesis and intimal hyperplasia; therefore, VEGF might promote progression of vasculopathies (Inoue et al., 1998
; Moulton et al., 1999
, 2003
). Supporting data were published by Celletti et al. (2001)
, who demonstrated that the application of recombinant human VEGF increased the rate and degree of plaque development in an animal model. Additionally, a strong correlation has been found between intragraft VEGF protein expression and the development of intimal thickening in a cardiac allograft model (Lemström et al., 2002
). Further, VEGF induces increased expression of adhesion molecules on the surface of endothelial cells (Keck et al., 1989
) and attracts and activates monocytes (Clauss et al., 1990
; Barleon et al., 1996
). Thus, by increasing VEGF release, HCMV infection might enhance chemoattraction of monocytes, thus promoting inflammatory processes within the vessel wall. Neovascularization, expression of adhesion molecules and recruitment of leukocytes to plaques are all factors that contribute to vascular disease (de Boer et al., 1999
). As no animal model for HCMV infection exists, due to its strict species specificity, direct evidence of HCMV-induced VEGF expression in vivo is not feasible. However, in humans, potential involvement of HCMV in the pathogenesis of vasculopathies is supported by the detection of viral antigen and nucleic acids in SMC cultured from coronary artery plaque material (Melnick et al., 1983
). HCMV nucleic acid could be identified in DNA extracted from atherosclerotic femoral arteries and abdominal aortas (Hendrix et al., 1991
). Viral DNA was detected in 90 % of severe and in 53 % of minimal atherosclerotic lesions. By using an artery organ-culture model for HCMV infection, we could demonstrate the presence of HCMV-infected SMC in the vascular media and intima (Reinhardt et al., 2003
), thus establishing SMC as a viral target cell within the vessel wall.
In conclusion, we have demonstrated a previously unknown HCMV-induced modulation of the cellular VEGF gene. An increased amount of functional VEGF protein is produced and released by HCMV-infected SMC. This induced a paracrine stimulation of endothelial-cell activation and proliferation. Concerning the pathophysiological relevance of this virus-induced change of the SMC phenotype, we can only hypothesize that increased VEGF protein levels in the environment of HCMV-infected SMC may enhance inflammatory processes or potentially promote plaque angiogenesis, finally supporting the development of virus-induced vasculopathy.
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ACKNOWLEDGEMENTS |
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Received 28 May 2004;
accepted 5 October 2004.
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