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Cardiovascular Research 2005 67(1):151-160; doi:10.1016/j.cardiores.2005.03.012
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Copyright © 2005, European Society of Cardiology

HCMV infection of human vascular smooth muscle cells leads to enhanced expression of functionally intact PDGF β-receptor

Barbara Reinhardtb, Thomas Mertensb, Ulrike Mayr-Beyrlea, Hedwig Franka, Anke Lüskeb, Karina Schierlingb and Johannes Waltenbergera,c,*

aDepartment of Internal Medicine II, Ulm University Medical Center, Ulm, Germany
bDepartment of Virology, Ulm University Medical Center, Ulm, Germany
cDepartment of Cardiology, University of Maastricht, Maastricht, The Netherlands

* Corresponding author. Department of Cardiology, University Hospital Maastricht, P. Debyelaan 25, PO Box 5800, Maastricht 6202 AZ, The Netherlands. Tel.: +31 43 387 5106; fax: +31 43 387 5104. Email address: j.waltenberger{at}cardio.azm.nl

Received 23 July 2004; revised 12 March 2005; accepted 15 March 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Background: Cytomegaloviruses have been shown to promote atherogenesis in animal models. In humans, several epidemiological and clinical studies suggest involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. HCMV is suspected to be associated with an enhanced restenosis rate and the occurrence of vasculopathies after solid organ transplantation. However, knowledge about the cellular and molecular bases of these findings is very limited.

Methods and results: Human coronary artery smooth muscle cells (HCASMC) were successfully infected with HCMV in vitro. Infection of HCASMC with all HCMV strains analyzed resulted in a substantial upregulation of the β-receptor of platelet-derived growth factor (PDGFR-β) expression as demonstrated by immunohistochemistry, immunofluorescence, FACS, and Western blot analysis. The amount of PDGFR-β protein present in HCASMC rapidly increased after 12 h of infection and this difference persisted for 72 h post-infection. We showed by quantitative FACS analysis that the extent of PDGFR-β upregulation differed significantly between the HCMV strains TB40E, Toledo, and AD169. The expression of insulin-like growth factor receptors as well as hepatocyte growth factor receptors, however, was downmodulated in HCMV-infected HCASMC. Most importantly, the HCMV-associated upregulation of PDGFR-β protein resulted in functionally intact receptors. A significantly higher increase of proliferative activity following stimulation with PDGF-BB was observed in HCMV-infected HCASMC compared to the uninfected control.

Conclusions: Our data suggest that HCMV directly activates the PDGF system, which could promote atherogenesis and restenosis by activation of smooth muscle cell proliferation and neointima formation.

KEYWORDS Growth factors; Atherosclerosis; Vascular smooth muscle cells; Cytomegalovirus; Platelet-derived growth factor


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The activation of coronary artery smooth muscle cells is a central process during the induction and progression of both atherogenesis and restenosis [1,2]. The activation of smooth muscle cells reflects a phenotypic change and involves stimulation of cell cycle progression, proliferation, migration, and production of extracellular matrix [3].

Various direct and indirect stimuli have been identified to either trigger or to promote smooth muscle cell activation. The indirect stimuli include endothelial dysfunction, which by itself can be triggered by a variety of factors [4], while direct stimuli include elevated levels of cholesterol and immunological responses such as complement attack and infection [5]. While the role of Chlamydia pneumoniae and Helicobacter pylori in atherogenesis is still under discussion, the human cytomegalovirus (HCMV) has clearly been shown to be associated with an enhanced rate of restenosis [6] and vasculopathy following solid organ transplantation [7]. Additionally, serological studies indicate a link between HCMV and atherosclerosis [8,9]; however, these data are still inconclusive [10]. A possible explanation may be the influence of the "infectious burden," which has been described to be a risk factor for atherosclerosis [5]. Furthermore, the presence of HCMV-DNA has been documented in atherosclerotic lesions [11] by DNA in situ hybridization, suggesting that HCMV can persist in the vessel wall in a latent state with unknown functional consequences [12].

The molecular and cellular bases for the pathogenic effects of HCMV are partially based on its influence on the pattern of host cell gene expression [13,14]. Some of the effects of HCMV have even been shown to be strain-specific [15].

Different molecules have been identified to be mediating the HCMV-induced changes of the cellular response including cytokines [16] and growth factors [17]. Peptide growth factors such as platelet-derived growth factors are known to be important molecular triggers for smooth muscle cell activation. Moreover, peptide growth factors have been shown to be causally involved in the processes of atherogenesis and restenosis [3].

Our study was designed to characterize molecular consequences of HCMV infection of HCASMC in vitro. We analyzed the expression of the β-receptor of platelet-derived growth factor (PDGFR-β) using immunofluorescence, FACS analysis, and Western blot analysis, and found evidence for HCMV-induced upregulation of PDGFR-β. Interestingly, the degree of upregulation differed between HCMV isolates. Moreover, we demonstrated that the ligand-dependent stimulation of the upregulated PDGFR-β protein is fully intact and consequently leads to a significant increase of proliferation in HCMV-infected cells compared to uninfected HCASMC.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Cells and viruses
Primary human coronary artery smooth muscle cells (HCASMC) and human umbilical cord endothelial cells (HUVEC) were purchased from Clonetics (BioWhittaker, Walkersville, MA) and cultured in smooth muscle cells basal medium supplemented with 5% FCS and growth factors (SmGM BulletKit; BioWhittaker, Walkersville, MA). HUVEC were cultured in endothelial cell basal medium supplemented with 5% FCS and growth factors (EGM-MV single quotes; BioWhittaker, Walkersville, MA). HCASMC and HUVEC were used in passage 5 or 6 for infection. Human foreskin fibroblasts (HFF) were produced in our laboratory and used between passages 15 and 18. In general, the study conforms to the Declaration of Helsinki.

The human cytomegalovirus (HCMV) laboratory strain AD169 and the clinical isolate Toledo were obtained from the American Type Culture Collection (ATCC; Rockville, MA). The endotheliotropic clinical isolate TB40E was kindly provided by Dr. Ch. Sinzger (University of Tübingen). Virus stocks were produced on HFF. With a cytopathic effect (CPE) of 90%, virus-containing supernatants were harvested, centrifuged to remove cellular debris, and afterwards concentrated by ultracentrifugation to ensure high-titered virus stocks. Virus stocks were stabilized by sucrose phosphate and stored at –70 °C. Infectivity was determined by plaque titration of stock virus on HFF. Tenfold dilutions of virus stocks were performed in quadruplicate. After staining for viral late antigen (monoclonal antibody AAC10; DAKO, Glostrup, Denmark), viral infectivity was determined by plaque counting and was expressed as plaque-forming units (PFU/ml).

2.2. Infection of HCASMC
For immunofluorescence experiments, HCASMC were grown on tissue culture chamber slides (Becton Dickinson, Franklin Lakes, NJ) and, for FACS analysis, cells were cultured in six-well plates (Greiner, Nürtingen, Germany). For immunoblot and phosphotyrosine blot analysis, HCASMC were grown in 75-cm2 flasks (Greiner, Nürtingen, Germany). At a confluency of 80%, HCASMC were infected with a multiplicity of infection (MOI) of one. After 24 h, cultures were washed and new medium was added. In parallel, equal amounts of PFU of the identical virus stock were added to HCASMC cultures after UV inactivation. Additionally, infections were performed in the presence of 100 µM ganciclovir (GCV, Cymeven®; Hoffmann-La Roche, Grenzach-Wyhlen, Germany) or 250 mg/ml phosphonoacetic acid (PAA; Sigma, Deisenhofen, Germany). Conditioned media were produced on TB40E-infected HCASMC, non-infected HCASMC, and HUVEC. An MOI of 10 was used to infect HUVEC cultures. After 72 h postinfection (p.i.), media were harvested and centrifuged at 3000 rpm for 10 min. Following UV inactivation, conditioned media were stored at –70 °C.

2.3. Fluorescence-activated cell sorting analysis
For FACS experiments, cells were harvested 48 h or 72 h p.i. Non-infected as well as TB40E-infected HCASMC cultures were analyzed in parallel in all experiments, studying the effect of inactivated virus, conditioned media, or viral polymerase inhibitors.

Cells were detached and separated using trypsin (trypsin and trypsin-neutralizing solution; BioWhittaker, Walkersville, MA). Single cell suspensions were washed with PBS and fixed for 30 min with 2% paraformaldehyde at 4 °C. Cells were 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, Barcelona, Spain) and 0.1% saponin. An irrelevant rabbit anti-mouse antibody (DAKO, Glostrup, Denmark), a specific polyclonal antibody directed against an intracellular epitope of the PDGFR-β protein (Upstate Biotechnology, Lake Placid, NY), insulin-like growth factor receptor β (IGFR-β), or the hepatocyte growth factor receptor (c-Met) (Santa Cruz, Santa Cruz, CA) was applied. Cells were washed thoroughly after an incubation of 30 min on ice. A fluorescein isothiocyanate (FITC)-labeled donkey anti-rabbit secondary antibody (Dianova, Hamburg, Germany) was added in FACS buffer. Cells were washed thoroughly following 30-min incubation on ice. Quantitative analysis was performed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using the Cellquest research software. A total of 10 x 104 events per sample was collected and analyzed.

2.4. Immunohistochemistry
HCASMC cultured on chamber slides were used for analysis. Immunoreactivity of PDGFR-β was performed by immunohistochemistry [18] using a specific polyclonal rabbit antiserum (UBI 06-498; UBI). Detection was performed using the Vectastain kit (Vector Burlingame, CA) including the AEC substrate. Several control experiments were performed in order to prove specificity: leaving out the primary antibody performed negative controls for each mode of infection. Blocking of the specific antibody was achieved using the peptide QPNEGDNDYIIPLA corresponding to aa 1013–1025 of the human PDGF receptor-β containing a carboxy-terminal alanine residue (UBI). This peptide had been used for immunization to generate the antiserum UBI 06-498. An irrelevant peptide served as negative control. Photographs were taken using a Zeiss microscope (primary magnification: 40 x).

2.5. Immunofluorescence
HCASMC cultured on chamber slides were extensively washed with PBS and fixed with ice-cold methanol. Slides were air-dried and stored at –20 °C until use. The primary antibodies were directed against the viral immediate early antigen (Argene Biosoft, Varilhes, France), viral early and late antigen (clone CCH2 and ACC10, DAKO, Glostrup, Denmark), or the PDGFR-β protein (Upstate Biotechnology, Lake Placid, NY). Specific as well as irrelevant control antibodies (Sigma, Deisenhofen, Germany) were added at a dilution of 1:50 in PBS supplemented with 1% BSA (Sigma, Deisenhofen, Germany). Slides were incubated for 45 min at 37 °C. After washing with PBS for three times, the FITC-labeled secondary donkey anti-rabbit antibody was added at a dilution of 1:50 and the TRITC-labeled secondary goat anti-mouse antibody was used at a dilution of 1:30 in PBS/BSA. After 45-min incubation at 37 °C, slides were washed three times with PBS, mounted, and viewed with a fluorescence microscope (Zeiss, Jena, Germany). To evaluate the percentage of cells expressing viral gene products, cells were counterstained with DAPI.

2.6. Western blot analysis and phosphotyrosine blot analysis
HCASMC were infected with various HCMV strains (see above) at an MOI of 1 and were stimulated with 50 ng/ml PDGF-BB for 7 min in the case of phosphotyrosine analysis. The assay was performed 48 h p.i. as previously described [19]. In brief, cells were solubilized in lysis buffer. Lysates were used for immunoprecipitation of PDGFR-β using a specific polyclonal antiserum (Santa Cruz, Santa Cruz, CA). PDGFR-β protein or tyrosine-phosphorylated proteins were detected by immunoblotting using either the PDGFR-β-specific antiserum (Santa Cruz) or the anti-phosphotyrosine antibody PY99 (Transduction Laboratories) followed by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG or goat-anti-rabbit IgG and application of the CDP Star System (Tropix). Quantitation of specific bands was performed using the LAS-1000 imager (Fuji).

2.7. Cell proliferation assay
Using a commercially available assay (Promega, Madison, WI, USA), we determined the number of viable cells in proliferation following stimulation with PDGF-BB (Peprotech) according to the manufacturer's instructions. In brief, HCASMC were cultured in 12-well plates (Greiner, Nürtingen, Germany). Four hours post-infection, mock-infected and TB40E-infected HCASMC were put in starvation medium containing 1% FCS. Cells were stimulated with PDGF-BB (15 ng/ml) for 12 h or were left unstimulated, and Owen's reagent was added 24 h post-infection to stimulated and unstimulated HCASMC. The bioreduction of the color compound was stopped after 2 h by adding SDS. The absorbance was recorded at 490 nm. The assay was performed in duplicate, and HCASMC from five different donors were evaluated.

2.8. Statistical analysis
The Wilcoxon signed rank test or the paired t-test was used for statistical analysis of the data.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. All HCMV strains used are equally capable of infecting and replicating in HCASMC
Since we wanted to include the two well characterized and widely used HCMV strains AD169 and Toledo in our study, as well as a wild-type strain (TB40E), we first analyzed the replication of the three virus variants in HCASMC. This was important since it was already known that the highly fibroblast-adapted AD169 strain exhibits a restricted cell tropism and is unable to infect and replicate in endothelial cells. HCASMC were infected with the HCMV strains at an MOI of 1 and the presence of viral antigens representative of the distinct sequential replication phases was determined quantitatively after 48 h. The percentage of infected cells after 48 h in all experiments was very close to 100. As can be seen further from Table 1, the replication of all three viruses was comparable.


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  		Table 1 Percentage of human coronary artery smooth muscle cells expressing the indicated viral antigens

 
3.2. The upregulation of PDGFR-β in HCASMC is induced by three distinct HCMV strains
HCMV infection of HCASMC induces the upregulation of PDGFR-β expression, which could be observed as early as 24 h postinfection (p.i.) using immunohistochemistry and immunofluorescence staining (Fig. 1). The two techniques exhibited different sensitivities and were thereby helpful with different respects. Immunohistochemistry (Fig. 1A, Panels A–D) was more sensitive than immunofluorescence (Fig. 1A, Panels E and F). PDGFR-β was well detectable by immunohistochemistry in non-infected cells (Fig. 1A, Panel A) as compared to the control without specific antibody (Fig. 1A, Panel B). A clear upregulation of PDGFR-β was visible 48 h after infection (Fig. 1A, Panel C), but not in the mock-infected control; Fig. 1A, Panel D). It has to be noticed that the magnification in Fig. 1A–D was identical, although the infected cells look bigger ("cytomegalo") due to the virus-induced cytopathic effect.


Figure 1
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  		Fig. 1 HCMV strain-dependent induction of PDGFR-β expression. Infection of subconfluent HCASMC (80%) with the HCMV strains AD169, TB40E, and Toledo using an MOI of 1 leads to increased expression of PDGFR-β. (A) The upper panels (A–D) show PDGFR-β expression using immunohistochemistry 72 h after infection. Mock-infected HCASMC (A and B) and TB40E-infected SMC (C and D) were stained with (A and C) and without (B and D) the primary antibody recognizing the PDGFR-β (magnification, x 400). In addition, immunofluorescence (panels E–H) was performed 48 h p.i. (magnification, x 200). The PDGFR-β protein is visualized by green cytoplasmic fluorescence and the IE-Ag of HCMV is visualized by red nuclear staining. In cells showing a strong PDGFR-β expression, the nuclear staining impresses yellow. Mock-infected HCASMC (E) show a faint baseline PDGFR-β expression, when compared to a control staining, where the primary antibody was omitted (insert in E). Differences in fluorescence intensities after staining for the PDGFR-β could be observed between HCASMC infected with TB40E (F), Toledo (G), or with the laboratory strain AD169 (H). (B) The kinetics of PDGFR-β expression in HCASMC following TB40E infection was assessed using immunohistochemistry. While there was no mock-related upregulation of PDGFR-β expression, TB40E resulted in a strong induction with a maximal effect between 48 h and 72 h postinfection. (C) Specificity control experiment. Blocking of the specific antibody was achieved using the peptide QPNEGDNDYIIPLA corresponding to aa 1013–1025 of human PDGF receptor-β. This peptide had been used for immunization to generate the antiserum UBI 06-498. An irrelevant peptide served as negative control. Primary magnification, x 40.

 
The upregulation of PDGFR-β protein expression in infected HCASMC was time-dependent and started 12 h p.i. The maximal expression was seen at 48–72 h p.i. (Fig. 1B). With further progression of permissive infection, a reduction of PDGFR-β expression was observed due to the virus-induced cytopathic effect. Specificity controls for the PDGFR-β antiserum have been performed using the specific peptide against which the antiserum was raised in rabbits, as well as an irrelevant peptide to inhibit immunohistochemical staining (Fig. 1C).

3.3. The different virus strains exhibit differences in upregulation of PDGFR-β expression in infected cells
Interestingly, the extent of PDGFR-β protein upregulation differed between the phenotypically distinct HCMV-isolates. To better analyze this finding, we used the immunofluorescence technique, which in our hands proved to be less sensitive but allowed better differentiation between intensity levels of PDGFR-β protein expression. Intense green fluorescence signals could be detected after infection with the endotheliotropic, less fibroblast adapted HCMV isolates TB40E and Toledo (Fig. 1A, Panels F and G). The green fluorescence observed in AD169-infected cells was substantially fainter (Fig. 1A, Panel H), indicating a lower level of PDGFR-β protein present in HCASMC infected with the laboratory strain AD169. Non-infected HCASMC did not show a signal for PDGFR-β, indicating an expression level below the limit of detection by immunofluorescence. However, when using FACS analysis (Fig. 2) or Western blot analysis (Fig. 3A), a low level of baseline PDGFR-β expression in non-infected HCASMC could be demonstrated.


Figure 2
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  		Fig. 2 FACS analysis of PDGFR-β expression in HCMV-infected and non-infected SMC. HCASMC were infected with the HCMV strains AD169, TB40E, or Toledo at an MOI of 1. Cells were detached and analysed by FACS 48 h after infection. In all histograms, the filled peaks represent the irrelevant antibody control. The strong black line represents the fluorescence intensity of the PDGFR-β from non-infected HCASMC and the strong gray line the fluorescence intensity of TB40E-infected HCASMC. Panel A: Addition of viable virus is necessary to trigger PDGFR-β upregulation. The thin gray line shows the fluorescence intensity after incubation of the cells with UV-inactivated TB40E. Panel B: Inhibition of viral DNA replication does not prevent HCMV-induced upregulation of the PDGFR-β. The thin gray and the broken line represent the fluorescence intensity of the PDGFR-β in TB40E-infected HCASMC incubated with PAA or GCV, respectively. Panel C: Incubation of HCASMC with conditioned media from TB40E-infected HCASMC or HUVEC does not influence PDGFR-β expression. The thin gray and the broken line show the fluorescence intensity of the PDGFR-β in non-infected HCASMC after incubation with conditioned media from HCMV-infected HCASMC or HUVEC, respectively. Identical results were obtained by addition of media from non-infected HCASMC or HUVEC (data not shown). Panel D: Quantitative differences in upregulation of PDGFR-β expression by the indicated HCMV strains. Ratios of mean fluorescence intensity of infected to non-infected HCASMC assessed within the same experiment. FACS analysis was performed 48 h after infection. Mean ratios ± SD are shown (n=6 for TB40E and Toledo, n=5 for AD169). HCASMC from three different lots (donors) have been used. The extent of PDGFR-β upregulation following infection with the laboratory strain AD169 was significantly lower compared to the upregulation following infection with the more "wild-type"-like HCMV strain TB40E (Wilcoxon signed rank test, p=0.017). Panel E: Influence of HCMV infection on the expression of the PDGFR-β, IGFR-β, and c-Met on HCASMC (passage 5). Ratios of mean fluorescence intensity of infected to non-infected HCASMC assessed within the same experiment. FACS analysis was performed 48 h after infection. Mean ratios ± SD are shown (n=3). HCASMC from three different lots (donors) have been used.

 

Figure 3
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  		Fig. 3 (A) Western blot analysis for PDGFR-β expression in HCASMC following infection with HCMV strain TB40E (MOI 1). Infection resulted in a strong upregulation of PDGFR-β protein expression in a time period of up to 72 h, while mock infection did not increase receptor levels. Anti-PDGFR-β antibodies were used for immunoprecipitation as well as for immunoblotting. Quantitation of the receptor band is indicated below the blot image. (B) PDGF-BB-induced tyrosine phosphorylation of PDGFR-β in HCASMC. Cells were infected with HCMV strain TB40E or mock-infected for 48 h (MOI 1) and subsequently stimulated with PDGF-BB (50 ng/ml) for 7 min, followed by SDS polyacrylamide gel electrophoresis and phosphotyrosine blot analysis. Quantitation of the tyrosine-phosphorylated band is provided below the blot image.

 
To better quantify the differences in PDGFR-β protein expression, FACS analysis (specific mean fluorescence intensity) was performed. At 48 h p.i., the strongest increase of PDGFR-β protein was observed for the endotheliotropic, wild-type isolate TB40E with a 6.2 ± 3.6-fold upregulation compared to mock-infected HCASMC (n=6, p=0.0027) (Fig. 2D). In the same setting, infection with the laboratory-adapted strain AD169 resulted in a 3.4 ± 2.2-fold (n=5, p=0.0039) upregulation of PDGFR-β protein expression in HCASMC, while infection with the Toledo isolate enhanced the fluorescence intensity by 4.3 ± 3.5 (n=6, p=0.0028) as compared to non-infected HCASMC (Fig. 2D). In a more extensive analysis combining the 48 h and 72 h time points postinfection, the difference in the upregulation of PDGFR-β expression between the endothelial cell-adapted TB40E and the fibroblast-adapted isolate AD169 was significant (n=9, p=0.017, Wilcoxon signed rank test). The number of infected cells as well as the velocity of viral replication were similar following infection with different HCMV isolates (see also Table 1).

3.4. Viral gene expression is essential for PDGFR-β upregulation
To differentiate whether viral gene expression is required for HCMV-induced upregulation of PDGFR-β expression or whether the mere contact of HCASMC with viral proteins is sufficient, the cells were incubated with UV-inactivated virus. Following incubation with UV-inactivated virus, the level of PDGFR-β protein expression remained unchanged compared to non-infected HCASMC. Thus, viral structural proteins introduced into the HCASMC culture by addition of UV-inactivated virus suspensions were not sufficient to induce enhanced PDGFR-β expression (Fig. 2A). Rather, viral gene products were necessary for induction of PDGFR-β gene expression.

Furthermore, infection was performed in the presence of two distinct inhibitors of viral DNA synthesis. The addition of these substances in the used concentrations prevents the expression of viral late genes without being toxic [20]. Interestingly, inhibition of viral DNA replication by addition of effective but non-toxic concentrations of either GCV or PAA did not prevent HCMV-associated upregulation of PDGFR-β. This clearly indicates that viral immediate early genes or viral early genes are responsible for HCMV-induced changes of the expression of PDGFR-β (Fig. 2B).

Since HCMV is known to induce the secretion of stimulatory molecules such as IL-6 and IL-8 [21,22], HCASMC were additionally incubated with supernatants of either infected or non-infected HCASMC. However, incubation of non-infected HCASMC with conditioned media from HCMV-infected HCASMC or HUVEC did not influence PDGFR-β expression. Therefore, HCMV-induced paracrine stimulation of PDGFR-β expression can be excluded in non-infected bystander cells (Fig. 2C).

Furthermore, we could show by FACS analysis that the HCMV-induced upregulation of the PDGFR-β in HCASMC is not a generalized virus-induced effect on growth factor receptors. On the contrary, IGFR-β and c-Met were even slightly downmodulated by HCMV (Fig. 2E).

3.5. Enhanced level of PDGFR-β expression and enhanced PDGF-BB-inducible activation of PDGFR-β following infection with HCMV
In order to further characterize the changes in PDGFR-β expression in HCASMC following infection with HCMV, we performed Western blot analysis. Infection of HCASMC with TB40E resulted in a 5.6-fold upregulation of PDGFR-β protein expression after 24 h, which further increased to 9.2-fold after 48 h p.i. (Fig. 3A). After 72 h of infection, the level of PDGFR-β expression was elevated 8.2-fold. In mock-infected cells, PDGFR-β expression level remained low and did not increase (Fig. 3A). In a series of five independent experiments, maximal upregulation of PDGFR-β was observed within 48 h of infection with TB40E (mean increase to 546% expression, p=0.009). In contrast to the PDGFR-β protein, the protein expression level of the IGF-I receptor did not significantly change at 48 h after infection (n=3, n.s.; data not shown).

Moreover, we have performed phosphotyrosine blot analysis using these samples to detect the level of tyrosine phosphorylation of the PDGFR-β in the absence of exogenous ligand. The level of receptor activity correlated well with the level of protein expression (Fig. 3B).

PDGF-BB-induced tyrosine phosphorylation of PDGFR-β in HCASMC was quantitatively assessed using phosphotyrosine blot analysis. Cells were infected with the HCMV strains TB40E for 48 h and subsequently stimulated with PDGF-BB (50 ng/ml) for 7 min prior to electrophoresis and phosphotyrosine blot analysis. The intensity of the tyrosine-phosphorylated band representing the activated PDGFR-β was quantitated. This evaluation clearly demonstrates that the PDGFR-β proteins are functionally intact following HCMV infection. Moreover, a significant increase of activated PDGFR-β levels to an average of 214% (n=6, p<0.05) could be found within 48 h following infection with TB40E (Fig. 3B).

Further, we assessed PDGF-BB-induced proliferation of TB40E-infected HCASMC using a colorimetric method. As demonstrated in Fig. 4, a statistically significant (p<0.0085 using paired t-test, n=5) increase in proliferative activity could be observed in HCMV-infected cells following PDGF-BB stimulation compared to mock-infected HCASMC. These data clearly demonstrate a functional consequence of HCMV-induced up-regulation of the PDGFR-β.


Figure 4
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  		Fig. 4 PDGF-BB-induced increase in HCASMC cell proliferation is significantly enhanced in HCMV-infected cells. Cells were infected with the TB40E strain and were cultivated in starvation medium (1% FCS). 12 h p.i., untreated, mock-infected, and TB40E-infected HCASMC were stimulated with PDGF-BB for 12 h followed by incubation with Owen's reagent for 2 h (n=5). The absorbance was read at 490 nm. Checkered bars indicate the increase following PDGF-BB stimulation compared to unstimulated cells (black bars, mean ± S.E.M.). Infected HCASMC showed a significantly stronger stimulation following ligand stimulation compared to mock-infected HCASMC (paired t-test, p=0.0085).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
We have investigated potential pro-atherogenic effects of HCMV and have found an elevated expression of PDGFR-β protein in HCASMC following infection with various strains of HCMV. Since the activation of PDGFR-β protein has been closely linked to a pro-atherogenic phenotype, our data provide a molecular basis for the pro-atherogenic action of HCMV in human SMC.

It is well established [3] that the upregulation of PDGFR-β in vascular smooth muscle cells occurs during atherogenesis [18,23] and during the process of restenosis [24]. Moreover, inhibition of PDGF activity by various strategies including PDGF-neutralizing antibodies [25], antisense approaches [26], or the use of PDGFR kinase inhibitors [24,27,28] results in significant reduction of arterial remodelling. In turn, enhanced expression and activity of PDGFR-β protein in vascular smooth muscle cells indicate an enhanced effect of PDGF and suggest a pro-atherogenic action.

HCMV-seropositive heart transplant patients do have a substantially higher risk of suffering from a coronary artery occlusion with subsequent graft loss or rejection as compared to the CMV-seronegative control group [29]. With the availability of potent immunosuppressive therapies for the prevention of acute rejection, vasculopathy has developed a major limitation of long-term allograft survival. Therefore, the perception of the importance of HCMV infection following organ transplantation has increased substantially. The PDGF system plays an important pathogenic role in allograft vasculopathy [30,31]. Our data clearly suggest an enhanced PDGF responsiveness of HCASMC due to an increased expression of the PDGFR-β in infected cells.

Pathogenic effects of HCMV can be based on its influence on the pattern of host cell gene expression [13,14] or, as it has been shown recently by stimulation of SMC, through the virally encoded G-protein receptor homologue US28 [32,33]. HCMV is strictly species-specific and, although only one serotype has been defined, a number of virus variants have been described with genotypic and phenotypic differences. Virus strains that are propagated on a particular cell type, such as endothelial cells or fibroblasts, retain tropism for that cell type and often fail to replicate efficiently on other cell types [34,35]. TB40E as well as the Toledo strain are regarded to be HCMV wild-type strains. In contrast, AD169 as well as the Towne strain [17] are not able to infect and replicate in endothelial cells. The existence of large deletions relative to clinical strains calls into question its de facto role as a prototype. Therefore, another more "wild-type"-like HCMV strain should be used in parallel when studying HCMV-induced modulations of atherosclerosis-relevant cellular genes and their molecular basis.

Recently, the induction of PDGFR-β by HCMV infection of smooth muscle cells with the laboratory-adapted Towne strain has been shown to occur in rat smooth muscle cells in vitro [17]. Proliferation of infected rat smooth muscle cells was increased to 150% compared to uninfected cells. However, this study was limited since the expression of viral genes in this system is not defined. Further, the observed stimulation of rat cells might be artificial, since cells were incubated with an MOI of 200; thus an unnaturally high number of viral particles and regulatory viral proteins [36] were introduced into the system. In contrast, our homologous system naturally supports the species-specific infection. This allows insight into how HCMV might trigger early steps of atherogenesis in humans. Furthermore, it is well known that permissive in vitro animal cell cultures studying a rat cytomegalovirus infection in rat endothelial cells directly induced an increased expression of HLA class II molecules [37], whereas this could not be observed after infection of human endothelial cells with HCMV [38]. These publications clearly indicate that even potentially comparable permissive animal and human in vitro systems do not necessarily identify similar virus-induced modulations of cellular gene expression. By inhibiting viral late gene expression in standard blocking experiments, we could show that HCMV gene expression is mandatory for the virus-induced increase in PDGFR-β protein and that this effect is dependent on viral "immediate early" or "early" gene products. So far, we have not determined whether transcriptional or posttranscriptional virus-induced effects are responsible for the observed PDGFR-β upregulation. The previously published data using non-permissive rat SMC infected with HCMV are in concordance with our findings obtained in a human experimental system [17].

HCMV infection of human smooth muscle cells in vitro is a lytic infection leading to reduced cellular viability and to a reduction of cell number within 72 h of infection. Nevertheless, FACS analysis clearly identified the enhanced expression of PDGFR-β on living HCASMC, suggesting the pro-atherogenic phenotype of HCMV-infected cells. Despite potential limitations, our data on the significant functional upregulation of PDGFR-β protein in response to infection with various HCMV strains clearly exceed all information that had previously been available on this topic. Our novel data obtained in a human system are nicely complemented by most recent in vivo data: using a rat model of CMV infection (RCMV), significant RCMV-triggered up-regulation of PDGF ligand and PDGF receptors was observed in inflammatory and SMC-like cells in tracheal allografts [39]. This was associated with the development of obliterative bronchiolitis.


   Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft project SFB451/A2 to J.W. and T.M., by a Heisenberg scholarship (Wa734/5-1) to J.W., and by the Cardiovascular Research Institute Maastricht (CARIM).


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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